Anti-FLT-1 antibodies for treatment of duchenne muscular dystrophy

Abstract

The present invention provides, inter alia, anti-Flt-1 antibodies and methods for the treatment of muscular dystrophy, in particular Duchenne muscular dystrophy (DMD). In some embodiments, a method according to the invention comprises administering to an individual suffering from or susceptible to DMD an effective amount of an anti-Flt-1 antibody or antigen binding protein thereof such that at least one symptom or feature of DMD is reduced in intensity, severity, or frequency or delayed onset.

Description

[0001] This application is a divisional application of a Chinese patent application with the application number 202210037246.0, the filing date of April 7, 2016, and the invention title of "Anti-FLT-1 Antibody for the Treatment of Duchenne Muscular Dystrophy". The application with the application number 202210037246.0 is a divisional application of a Chinese patent application with the application number 201680032814.0, the filing date of April 7, 2016, and the invention title of "Anti-FLT-1 Antibody for the Treatment of Duchenne Muscular Dystrophy". The original application is the Chinese national phase application of a PCT international application with the international application number PCT / US2016 / 026352. This PCT international application claims the priority of US Provisional Application Serial Numbers 62 / 144,251 and 62 / 307,645 filed on April 7, 2015 and March 14, 2016, respectively.

[0002] Related Applications

[0003] This application claims the priority of US Provisional Application Serial Number 62 / 144,251 filed on April 7, 2015 and US Provisional Application Serial Number 62 / 307,645 filed on March 14, 2016. The entire disclosures of the respective provisional applications are hereby incorporated by reference. Background

[0005] Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder that affects an estimated 1 in 3,600 male births, with an estimated 50,000 affected individuals worldwide. The disorder is characterized by the progressive wasting of muscle, and affected children are wheelchair-dependent by the age of 13. Affected individuals typically present symptoms at age 3, and the median survival of such individuals is between 25 and 30 years. Respiratory failure due to diaphragmatic muscle weakness and cardiomyopathy is a common cause of death.

[0006] DMD is caused by mutations in the dystrophin gene. The dystrophin gene is located on the X chromosome and encodes the protein dystrophin. Dystrophin is responsible for connecting the contractile machinery of muscle fibers (actin-myosin complex) to the surrounding extracellular matrix through the dystroglycan complex. Mutations in the dystrophin gene result in altered or absent dystrophin and abnormal sarcolemal membrane function. Although both males and females can carry mutations in the dystrophin gene, females are rarely affected by DMD.

[0007] One feature of DMD is ischemia in affected tissues. Ischemia is a restriction or reduction in the blood supply to a tissue or organ, resulting in insufficient oxygen and nutrient requirements for cellular metabolism. Ischemia is typically caused by constriction or blockage of blood vessels, leading to damage or dysfunction of the tissue or organ. Treatment of ischemia aims to increase blood flow to the affected tissue or organ.

[0008] Currently, DMD is incurable. Several treatment approaches have been investigated, including gene therapy and corticosteroid administration, yet there remains a need for alternatives for DMD patients. Summary of the Invention

[0010] The present invention particularly provides improved methods and compositions for treating muscular dystrophy, specifically Duchenne muscular dystrophy (DMD) and / or Becker muscular dystrophy, based on anti-Flt-1 antibody therapy. As described in the examples below, the present invention is in part based on the discovery that an anti-Flt-1 antibody or an antigen-binding fragment thereof is capable of inhibiting the binding of VEGF and other ligands to the Flt-1 receptor, thereby increasing the amount of VEGF and / or other ligands available for binding to the VEGF receptor. Increasing the availability of VEGF promotes angiogenesis, along with an increase in blood flow to the muscle to counteract functional ischemia and result in improvement of the structural and functional features of DMD. Indeed, as shown in the examples of the present invention, the inventors have demonstrated that administration of the anti-Flt-1 antibody improves measures of muscle pathology (e.g., improved angiogenesis, reduced fibrosis, reduced necrosis). Accordingly, the present invention provides a safe and effective antibody-based therapeutic agent for treating DMD.

[0011] On the one hand, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to human Flt-1, the antibody or the antigen-binding fragment thereof comprising one or more complementarity-determining regions (CDRs), the complementarity-determining regions selected from the group consisting of: variable light (VL) chain CDR1, which is defined by an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 19 to 21; VL CDR2, which is defined by an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 22 to 24; VL CDR3, which is defined by an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 25 to 34; variable heavy (VH) chain CDR1, which is defined by an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 1 to 4; VH CDR2, which is defined by an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 5 to 14; and VH CDR3, which is defined by an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 15 to 18.

[0012] In some embodiments, the one or more CDRs include VL CDR3, which is defined by an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 25 to 34; and VH CDR3, which is defined by an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 15 to 18.

[0013] In another embodiment, the one or more CDRs include VL CDR1, which is defined by an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 19 to 21; VL CDR2, which is defined by an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 22 to 24; and VL CDR3, which is defined by an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 25 to 34. In a specific embodiment, the VL chain comprises VL CDR1, VL CDR2, and VL CDR3 defined by the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 22, and SEQ ID NO: 25, respectively. In another embodiment, the VL chain comprises VL CDR1, VL CDR2, and VL CDR3 defined by the amino acid sequences of SEQ ID NO: 20, SEQ ID NO: 23, and SEQ ID NO: 25, respectively. In another embodiment, the VL chain comprises VL CDR1 and VL CDR2 defined by the amino acid sequences of SEQ ID NO: 21 and SEQ ID NO: 24, respectively, and VL CDR3 defined by the amino acid sequence of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 34. In a specific embodiment, the VL chain comprises VL CDR1, VL CDR2, and VL CDR3 defined by the amino acid sequences of SEQ ID NO: 21, SEQ ID NO: 24, and SEQ ID NO: 32, respectively.

[0014] In other embodiments, the one or more CDRs include VH CDR1, which is defined by an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 1 to 4; VH CDR2, which is defined by an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 5 to 14; and VH CDR3, which is defined by an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 15 to 18. In a specific embodiment, the VH chain comprises VH CDR1, VH CDR2, and VH CDR3 defined by the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO: 15, respectively. In another embodiment, the VH chain comprises VH CDR1, VH CDR2, and VH CDR3 defined by the amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 6, and SEQ ID NO: 16, respectively. In another embodiment, the VH chain comprises VH CDR1, VH CDR2, and VH CDR3 defined by the amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 10, and SEQ ID NO: 18, respectively. In another embodiment, the VH chain comprises VH CDR1 and VH CDR3 defined by the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 17, respectively, and VH CDR2 defined by the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 13, or SEQ ID NO: 14. In another embodiment, the VH chain comprises VH CDR1 and VH CDR3 defined by the amino acid sequences of SEQ ID NO: 3 and SEQ ID NO: 17, respectively, and VH CDR2 defined by the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 12. In another embodiment, the VH chain comprises VH CDR1, VH CDR2, and VH CDR3 defined by the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 9, and SEQ ID NO: 17, respectively. In a specific embodiment, the VH chain comprises VH CDR1, VH CDR2, and VH CDR3 defined by the amino acid sequences of SEQ ID NO: 3, SEQ ID NO: 12, and SEQ ID NO: 17, respectively.

[0015] On the other hand, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to human Flt-1, the antibody or the antigen-binding fragment thereof comprising: (i) a light chain variable (VL) region comprising an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 49 to 61; and / or (ii) a heavy chain variable (VH) region comprising an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 35 to 48. In a specific embodiment, the VL region comprises the amino acid sequence of SEQ ID NO: 60, and the VH region comprises the amino acid sequence of SEQ ID NO: 45.

[0016] In some embodiments, the antibody further comprises a heavy chain constant region comprising an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 87 to 89.

[0017] On the other hand, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to human Flt-1, the antibody or the antigen-binding fragment thereof comprising: (i) a light chain comprising an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 75 to 86; and / or (ii) a heavy chain comprising an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 62 to 74. In a specific embodiment, the light chain comprises the amino acid sequence of SEQ ID NO: 76, and the heavy chain comprises the amino acid sequence of SEQ ID NO: 71.

[0018] In another embodiment, the antibody or the antigen-binding fragment thereof is selected from the group consisting of: IgG, F(ab')2, F(ab)2, Fab', Fab, ScFv, diabody, triabody, and tetrabody. In one embodiment, the antibody or the antigen-binding fragment thereof is IgG. In another embodiment, the antibody or the antigen-binding fragment thereof is IgG1. In another embodiment, the antibody or the antigen-binding fragment thereof is a monoclonal antibody. In a specific embodiment, the antibody is a humanized monoclonal antibody. In another embodiment, the humanized monoclonal antibody comprises a human Fc region. In some embodiments, the Fc region contains one or more mutations that enhance the binding affinity between the Fc region and the FcRn receptor such that the in vivo half-life of the antibody is prolonged. In another embodiment, the Fc region contains one or more mutations at positions corresponding to Leu 234, Leu 235, and / or Gly 237 of human IgG1.

[0019] In one embodiment, the antibody or antigen-binding fragment thereof does not bind to VEGFR2 and / or VEGFR3. In another embodiment, the antibody or antigen-binding fragment thereof does not bind to murine or simian Flt-1.

[0020] On the other hand, the present invention provides an isolated antibody or antigen-binding fragment thereof that recognizes a peptide or fragment thereof comprising an amino acid sequence corresponding to positions 139 to 148, positions 139 to 153, positions 178 to 206, positions 199 to 204, and positions 128 to 138 of SEQ ID NO: 90. In one embodiment, the peptide consists of an amino acid sequence corresponding to positions 130 to 138, positions 141 to 148, positions 141 to 153, and positions 193 to 206 of SEQ ID NO: 90.

[0021] On the other hand, the present invention provides an isolated antibody or antigen-binding fragment thereof that competes with any anti-Flt-1 antibody or antigen-binding fragment thereof.

[0022] On the other hand, the present invention provides a pharmaceutical composition comprising an anti-Flt-1 antibody or antigen-binding fragment thereof and a pharmaceutically acceptable carrier.

[0023] On the other hand, the present invention provides a polynucleotide encoding a CDR, VL region, VH region, light chain, and / or heavy chain of the antibody or antigen-binding fragment thereof of the present invention. In one embodiment, the present invention provides an expression vector comprising the polynucleotide. In another embodiment, the present invention provides a host cell comprising the polynucleotide or the expression vector. In a specific embodiment, the present invention provides a method for preparing an antibody or antigen-binding fragment thereof that specifically binds to human Flt-1, the method comprising culturing the host cell. In another embodiment, the hybridoma cell produces the antibody or antigen-binding fragment thereof.

[0024] On the other hand, the present invention provides a method for treating an Flt-1-mediated disease, disorder, or condition, the method comprising administering an anti-Flt-1 antibody or antigen-binding fragment thereof to a subject in need of treatment. In a specific embodiment, the Flt-1-mediated disease, disorder, or condition is Duchenne muscular dystrophy, Becker muscular dystrophy, bronchopulmonary dysplasia, preeclampsia, or chronic kidney disease.

[0025] On the other hand, the method provides a method for treating Duchenne muscular dystrophy (DMD), the method comprising administering to a subject having or at risk of DMD an effective amount of an anti-Flt-1 antibody or an antigen-binding fragment thereof, such that at least one symptom or feature of DMD is reduced or the onset is delayed in terms of intensity, severity or frequency. In one embodiment, the method further comprises administering to the subject one or more additional therapeutic agents. In a specific embodiment, the additional therapeutic agent is selected from the group consisting of prednisone, deflazacort, follistatin, RNA modulating therapeutic agents, exon-skipping therapeutic agents and gene therapy.

[0026] In one embodiment, the antibody or antigen-binding fragment thereof is administered parenterally. In some embodiments, parenteral administration is selected from intravenous, intradermal, intrathecal, inhalation, transdermal (topical), intraocular, intramuscular, subcutaneous and / or transmucosal administration. In a specific embodiment, the parenteral administration is intravenous administration. In another embodiment, the parenteral administration is subcutaneous administration.

[0027] In some embodiments, the antibody or antigen-binding fragment thereof is administered once daily, twice weekly, once weekly or once monthly. In a specific embodiment, the antibody or antigen-binding fragment is administered twice weekly.

[0028] In another embodiment, the effective amount of the antibody or antigen-binding fragment thereof is a dose of about 1 mg / kg to 50 mg / kg. In a specific embodiment, the dose is about 1 mg / kg, 3 mg / kg or 10 mg / kg.

[0029] In one embodiment, the administration of the antibody or antigen-binding fragment thereof results in reduced fibrosis and / or necrosis relative to a control. In another embodiment, the administration of the antibody or antigen-binding fragment thereof results in improved angiogenesis in the muscle of the subject relative to a control. In another embodiment, the improved angiogenesis is reflected by increased blood flow in muscle pathology, increased VEGF levels in serum, decreased creatine kinase (CK) levels in serum, increased CD31 score measured by IHC and / or decreased sFlt-1 levels in serum. In another embodiment, the administration of the antibody or antigen-binding fragment thereof results in improved muscle function relative to a control. In another embodiment, the improved muscle function is reflected by improved muscle strength and / or fatigue resistance.

[0030] On the other hand, the present invention provides a method for treating tissue fibrosis, the method comprising administering to a subject in need thereof an effective amount of an anti-Flt-1 antibody or an antigen-binding fragment thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The teachings described herein will be more fully understood from the following description of various illustrative embodiments when read in conjunction with the accompanying drawings. It is to be understood that the drawings described below are for illustrative purposes only and are not intended to limit the scope of the teachings in any way.

[0033] Figure 1A Exemplary results showing clearance of the anti-Flt-1 antibody 13B4 following intravenous administration to mice at a dose of 10 mg / kg.

[0034] Figure 1B Exemplary results showing clearance of the anti-Flt-1 antibody 10G12 following intravenous administration to mice at a dose of 10 mg / kg.

[0035] Figure 2A Exemplary results showing peak serum anti-Flt-1 antibody levels. Figure 2B Exemplary results showing trough serum anti-Flt-1 antibody levels.

[0036] Figure 3 Exemplary results showing reduced levels of free soluble Flt-1 (sFlt-1) in serum following administration of anti-Flt-1 antibody (13B4 or 10G12), isotype control antibody, commercial antibody (Angio), or vehicle alone to mdx mice.

[0037] Figure 4 Exemplary results showing increased serum VEGF levels following administration of anti-Flt-1 antibody (13B4 or 10G12), isotype control antibody, commercial antibody (Angio), or vehicle alone to mdx mice.

[0038] Figures 5A - 5D Exemplary results showing CD31 staining of tissue sections obtained from the diaphragms of mdx mice administered anti-Flt-1 antibody (13B4 or 10G12), isotype control antibody, or commercial antibody (Angio).

[0039] Figure 6A Exemplary results showing quantification of CD31-positive area as a percentage of the total stained area in tissue sections obtained from the diaphragm. Figure 6B Exemplary results showing quantification of CD31-positive area as a percentage of the total stained area in tissue sections obtained from the tibialis anterior (TA) muscle.

[0040] Figure 6B Exemplary results of the quantification of the CD31 positive area shown as a percentage of the total stained area in tissue sections obtained from the tibialis anterior (TA) muscle of mdx mice administered with anti-Flt-1 antibody.

[0041] Figure 7 Exemplary results depicting the binding of anti-Flt-1 antibody to recombinant sFlt-1 as determined by ELISA.

[0042] Figure 8 Exemplary results depicting the inhibition of the binding of sFlt-1 to VEGF by anti-Flt-1 antibody in a competitive ELISA assay.

[0043] Figure 9 Exemplary results depicting the rescue of VEGF R2 phosphorylation by anti-Flt-1 antibody. Human primary venous endothelial cells (HUVEC) were treated with VEGF in the presence of sFlt-1 and anti-Flt-1 antibody and the level of VEGF R2 phosphorylation was measured. The rescue percentage indicates the level of VEGF R2 phosphorylation relative to the level of VEGF R2 phosphorylation when HUVEC were treated with VEGF and sFlt-1 alone (i.e., without anti-Flt-1 antibody).

[0044] Figure 10 Exemplary results depicting the inhibition of the binding of soluble Flt-1 to VEGF by anti-Flt-1 antibody in a competitive ELISA assay.

[0045] Figure 11 Exemplary results of the clearance of anti-Flt-1 antibody from serum within 672 hours after intravenous administration of anti-Flt-1 antibody to mice at a dose of 10 mg / kg.

[0046] Figures 12A - 12C Exemplary results of CD31 staining of tissue sections from the diaphragm. Figures 12D - 12F Exemplary results of CD31 staining of tissue sections obtained from the tibialis anterior muscle.

[0047] Figures 13A - 13C Exemplary biodistribution of the antibodies in the diaphragm, tibialis and gastrocnemius muscles of mice over a 256-hour time course after administration of anti-Flt-1 antibodies 27H9, 13B4 and 21B3.

[0048] Figure 14A Exemplary results depicting the peak anti-Flt-1 antibody 21B3 level at peak exposure. Figure 14B Exemplary results depicting the trough anti-Flt-1 antibody 21B3 level.

[0049] Figure 15 Shows exemplary results depicting free sFlt-1 following administration of anti-Flt-1 antibody 21B3 to mdx mice.

[0050] Figure 16 Shows exemplary results depicting VEGF levels following administration of anti-Flt-1 antibody 21B3 to mdx mice.

[0051] Figures 17A - 17E Shows exemplary results of CD31 staining of tissue sections obtained from the diaphragms of mdx mice administered anti-Flt-1 antibody 21B3 or isotype control antibody.

[0052] Figure 18 Shows exemplary results of quantification of the percentage of CD31 positivity normalized in tissue sections obtained from the diaphragms of mdx mice administered anti-Flt-1 antibody 21B3 or isotype control antibody.

[0053] Figures 19A - 19E Shows exemplary results of CD31 staining of tissue sections obtained from the tibialis anterior muscles of mdx mice administered anti-Flt-1 antibody 21B3 or isotype control antibody.

[0054] Figure 20 Shows exemplary results of quantification of the percentage of CD31 positivity normalized in tissue sections obtained from the tibialis anterior muscles of mdx mice administered anti-Flt-1 antibody 21B3 or isotype control antibody.

[0055] Figure 21A Shows exemplary results of reverse-phase liquid chromatography / mass spectrometry (RP-LC / MS) analysis for determination of the molecular weight of deglycosylated 21B3 antibody. Figure 21B Shows exemplary results of analysis of the glycosylation pattern of the heavy chain.

[0056] Figure 22A and 22B Shows exemplary results depicting rescue of phosphorylation of VEGFR2 by anti-Flt-1 antibody. Human primary venous endothelial cells (HUVECs) were treated with VEGF in the presence of sFlt-1 and anti-Flt-1 antibody and the level of VEGFR2 phosphorylation was determined. The rescue percentage indicates the level of VEGFR2 phosphorylation relative to the level of VEGFR2 phosphorylation when HUVECs were treated with VEGF and sFlt-1 alone (i.e., without anti-Flt-1 antibody).

[0057] Figure 23 Shows exemplary results depicting binding of anti-Flt-1 antibody to recombinant sFlt-1 by ELISA.

[0058] Figure 24Exemplary results showing serum levels of free anti-Flt-1 antibody 21B3 and isotype control antibody in mdx mice are presented.

[0059] Figure 25 Exemplary results showing serum levels of free sFlt-1 in mdx mice treated with anti-Flt-1 antibody 21B3 or isotype control antibody are presented.

[0060] Figure 26 Exemplary results showing serum levels of VEGF in mdx mice treated with anti-Flt-1 antibody 21B3 or isotype control antibody are presented.

[0061] Figures 27A - 27H Exemplary results of CD31 staining of tissue sections obtained from the diaphragm of mdx mice administered anti-Flt-1 antibody 21B3 or vehicle control for 6 (27A - 27D) or 12 (27E - 27H) weeks are presented.

[0062] Figures 28A - 28H Exemplary results of CD31 staining of tissue sections obtained from the gastrocnemius muscle of mdx mice administered anti-Flt-1 antibody 21B3 or vehicle control for 6 weeks (28A - 28D) or 12 weeks (28E - 28H) are presented.

[0063] Figures 29A - 29H Exemplary results of CD31 staining of tissue sections obtained from the tibialis muscle of mdx mice administered anti-Flt-1 antibody 21B3 or vehicle control for 6 weeks (29A - 29D) or 12 weeks (29E - 29H) are presented.

[0064] Figures 30A - 30C Exemplary results of quantification of the percentage of positive CD31 staining in tissue sections obtained from the diaphragm, gastrocnemius muscle, and tibialis muscle of mdx mice administered anti-Flt-1 antibody 21B3 or vehicle control antibody for 6 weeks or 12 weeks are presented.

[0065] Figures 31A - 31H Exemplary results of immunohistochemical staining of type I collagen of tissue sections obtained from the diaphragm of mdx mice administered anti-Flt-1 antibody 21B3 or vehicle control for 6 weeks (31A - 31D) or 12 weeks (31E - 31H) are presented.

[0066] Figures 32A - 32H Exemplary results of immunohistochemical staining of type I collagen of tissue sections obtained from the gastrocnemius muscle of mdx mice administered anti-Flt-1 antibody 21B3 or vehicle control for 6 weeks (32A - 32D) or 12 weeks (32E - 32H) are presented.

[0067] Figures 33A - 33HExemplary results of immunohistochemical staining of type I collagen in tissue sections obtained from the tibialis muscles of mdx mice administered anti-Flt-1 antibody 21B3 or vehicle control for 6 weeks (33A - 33D) or 12 weeks (33E - 33H).

[0068] Figures 34A - 34C Exemplary results of quantification of the percentage of positive type I collagen staining in tissue sections obtained from the diaphragm, gastrocnemius, and tibialis muscles of mdx mice administered anti-Flt-1 antibody 21B3 or vehicle control antibody for 6 weeks or 12 weeks.

[0069] Figure 35A and 35B Exemplary results of the percentage of necrosis in the gastrocnemius muscles of mdx mice administered anti-Flt-1 antibody 21B3 or vehicle control antibody for 6 weeks or 12 weeks.

[0070] Figure 36 Exemplary results of a differential heat map depicting the comparison of hydrogen / deuterium exchange of human sFlt-1 alone with that of a mixture of human sFlt-1 and anti-Flt-1 antibody (21B3). Gray: no deuterium protection; blue: deuterium protection after Fab binding.

[0071] Figure 37 Exemplary results of a differential heat map depicting the comparison of hydrogen / deuterium exchange of human sFlt-1 alone with that of a mixture of human sFlt-1 and anti-Flt-1 antibody (21C6). Gray: no deuterium protection; blue: deuterium protection after Fab binding.

[0072] Figures 38A - 38E Exemplary results of MS / MS spectra of peptides containing the identification of amino acid residues from the epitope region.

[0073] Definition

[0074] For easier understanding of the present disclosure, certain terms are first defined below. Additional definitions for the following terms and other terms are listed throughout the specification.

[0075] Affinity: As is known in the art, "affinity" is a measure of the tightness with which a particular ligand binds to its cognate. In some embodiments, the ligand or cognate is Flt-1. In some embodiments, the ligand or cognate is soluble Flt-1. In some embodiments, the ligand or cognate is recombinant Flt-1. In one specific embodiment, the ligand or cognate is human sFlt-1. In one specific embodiment, the ligand or cognate is recombinant sFlt-1. In other embodiments, the ligand or cognate is an anti-Flt-1 antibody. Affinity can be measured in different ways. In some embodiments, affinity is measured by a quantitative assay. In some such embodiments, the concentration of the binding cognate can be fixed to exceed the ligand concentration to mimic physiological conditions. Alternatively or additionally, in some embodiments, the concentration of the binding cognate and / or the ligand concentration can be varied. In some such embodiments, the affinity can be compared to a reference under comparable conditions (e.g., concentration).

[0076] Affinity maturation (or affinity matured antibody): As used herein, the term "affinity maturation" or "affinity matured antibody" refers to an antibody that has one or more changes in one or more of its CDRs, which changes result in an increase in the affinity of the antibody for an antigen compared to the parental antibody that does not have those changes. In some embodiments, an affinity matured antibody will have a nanomolar or even picomolar affinity for the target antigen. Affinity matured antibodies can be generated by any of a variety of procedures known in the art. Mark et al., BioTechnology 10:779-783 (1992) describe affinity maturation by V H and V L domain shuffling. Random mutagenesis of CDR and / or framework residues is described by: Barbas et al., Proc. Nat. Acad. Sci. U.S.A. 91:3809-3813 (1994); Schier et al., Gene 169:147-155 (1995); Yelton et al., J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al., J. Mol. Biol. 226:889-896 (1992).

[0077] Improve: As used herein, the term "improve" refers to the prevention, alleviation, or mitigation of a subject's condition or an improvement of the condition. Improvement includes, but does not require, complete recovery or complete prevention of a disease symptom.

[0078] Animal: As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to a human at any stage of development. In some embodiments, "animal" refers to a non-human animal at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cow, primate, and / or pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In some embodiments, the animal can be a transgenic animal, a genetically engineered animal, and / or a clone.

[0079] Antibody: As used herein, the term "antibody" refers to any immunoglobulin, whether natural or produced in whole or in part synthetically. All derivatives thereof that maintain the ability to specifically bind are also included within the term. The term also encompasses any protein having a binding domain that is homologous or substantially homologous to an immunoglobulin binding domain. Such proteins can be derived from natural sources or produced in whole or in part synthetically. Antibodies can be monoclonal or polyclonal. Antibodies can be members of any immunoglobulin isotype, including any human isotype: IgG, IgM, IgA, IgD, and IgE. In certain embodiments, the antibody can be a member of the IgG immunoglobulin class (e.g., IgG1, IgG2, IgG3, etc.). In some embodiments, the antibody can be a human antibody. In some embodiments, the antibody can be a humanized antibody.

[0080] As is known to those of ordinary skill in the art, antibodies produced in nature generally comprise four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy and light chain consists of a variable region (abbreviated herein as HCVR, VH, or V H and LCVR, VL, or V L ), respectively) and a constant region. The constant region of the heavy chain contains C H 1, C H 2, and C H 3 domains (and optionally, in the case of IgM and IgE, a C H 4 domain). The constant region of the light chain contains one domain, C L . The V H and V L regions also contain hypervariable regions called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each V H and V LIt consists of three CDRs and four FRs, which are arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The binding region of the heavy and light chains contains a binding domain that interacts with the antigen. The constant region of the antibody can mediate the binding of immunoglobulins to host tissues or factors, including different cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q).

[0081] Antigen-binding portion: As used herein, the term "antigen-binding portion" or "antigen-binding fragment" refers to one or more fragments or portions of an antibody that retain the ability to specifically bind to an antigen (e.g., Flt-1). Examples of antigen-binding portions include (i) Fab fragments, a monovalent fragment consisting of V H , V L , C H 1 and C L domains; (ii) F(ab')2 fragments, a bivalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region; (iii) Fd fragments consisting of V H and C H domains; (iv) Fv fragments consisting of V H and V L domains of a single arm of an antibody; (v) dAb fragments containing a single variable domain (Ward et al., (1989) Nature 341:544-546); (vi) isolated complementarity-determining regions (CDRs); (vii) Fab' fragments, which are essentially Fab with a part of the hinge region; (viii) nanobodies, heavy chain variable regions containing a single variable domain and two constant domains. In addition, although the two domains of the Fv fragment (V H and V L ) are encoded by separate genes, they can be joined by a synthetic linker using recombinant methods, and the linker can enable them to form a single protein chain in which the V H and V L regions are paired to form a monovalent molecule (referred to as single-chain Fv (scFv); see, for example, Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). The antigen-binding fragment of an antibody can optionally include a single-chain antibody fragment. Alternatively or additionally, the antigen-binding fragment of an antibody can include, for example, multiple chains linked together by disulfide bonds. The antigen-binding fragment of an antibody can optionally include a multimolecular complex. Functional antibody fragments generally contain at least about 50 amino acids, and more typically contain at least about 200 amino acids.

[0082] In some embodiments, an antibody fragment contains sufficient sequence of the parent antibody such that the antibody fragment binds the same antigen as the parent antibody; in some embodiments, the fragment binds to the antigen with an affinity comparable to that of the parent antibody and / or competes with the parent antibody for binding to the antigen.

[0083] Those skilled in the art will understand that the term “antibody fragment” does not mean and is not limited to any particular mode of production. Antibody fragments can be produced by any suitable method, including but not limited to cleavage of intact antibodies, chemical synthesis, and recombinant production. The utility of fragments is screened in the same manner as intact antibodies.

[0084] About or approximately: As used herein, when applied to one or more target values, the terms “about” or “approximately” refer to a value similar to the recited reference value. In certain embodiments, the terms “about” or “approximately” refer to a range of values (greater than or less than) within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction of the recited reference value, unless otherwise stated or otherwise apparent from the context (except in cases where such a numerical value would exceed 100% of a possible value).

[0085] Associated with: Two events or entities are “associated” with each other as the term is used herein if the presence, level, and / or form of one event or entity is related to the presence, level, and / or form of another event or entity. For example, if the presence, level, and / or form of a particular entity (e.g., a polypeptide) is related to the incidence and / or susceptibility of a particular disease, disorder, and / or condition (e.g., across relevant populations), then the particular entity is considered to be associated with the disease, disorder, or condition. In some embodiments, two or more entities are physically “associated” with each other if they interact directly or indirectly such that they are and remain in physical proximity to each other. In some embodiments, two or more entities that are physically associated with each other are covalently linked to each other; in some embodiments, two or more entities that are physically associated with each other are not covalently linked to each other but are non-covalently associated, for example, by hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0086] Carrier or diluent: As used herein, the terms “carrier” and “diluent” refer to pharmaceutically acceptable (e.g., safe and non-toxic for administration to humans) carrier or diluent substances useful in the preparation of pharmaceutical formulations. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), pH buffered solutions (e.g., phosphate buffered saline), sterile saline solutions, Ringer's solution, or glucose solutions.

[0087] CDR: As used herein, refers to the complementarity determining regions within the variable region of an antibody. There are three CDRs in each variable region of the heavy and light chains, which are designated CDR1, CDR2, and CDR3 of each variable region. "A set of CDRs" or "CDR set" refers to a group of three or six CDRs present in a single variable region capable of binding an antigen or the CDRs of homologous heavy and light chain variable regions capable of binding an antigen. Certain systems for defining CDR boundaries have been established in the art (e.g., Kabat, Chothia, etc.); those skilled in the art understand the differences between these systems and are able to understand CDR boundaries to the extent required for understanding and practicing the claimed invention.

[0088] Chimeric antibody: As used herein, refers to an antibody whose amino acid sequence contains V H and V L region sequences found in a first species and constant region sequences found in a second species different from the first species. In many embodiments, the chimeric antibody has murine V H and V L regions linked to a human constant region. In some embodiments, an antibody having human V H and V L regions linked to a non-human constant region (e.g., murine constant region) is referred to as an "inverse chimeric antibody".

[0089] Dosage form: As used herein, the terms "dosage form" and "unit dosage form" refer to physically discrete units of a therapeutic protein (e.g., an antibody) for a patient to be treated. Each unit contains a predetermined quantity of the active substance calculated to produce the desired therapeutic effect. However, it will be understood that the total dosage of the composition will be determined by the attending physician within the scope of reasonable medical judgment.

[0090] Dysfunction: As used herein, the term "dysfunction" refers to abnormal function. Dysfunction of a molecule (e.g., a protein) can be caused by an increase or decrease in the activity associated with such a molecule. Dysfunction of a molecule can be caused by a defect associated with the molecule itself or with other molecules that directly or indirectly interact with or regulate the molecule.

[0091] Epitope: As used herein, includes any portion specifically recognized by an immunoglobulin (e.g., an antibody, an antibody fragment thereof, a receptor) binding component. In some embodiments, an epitope consists of multiple chemical atoms or groups on an antigen. In some embodiments, such chemical atoms or groups are surface-exposed when the antigen adopts a relevant three-dimensional conformation. In some embodiments, such chemical atoms or groups are physically proximate to each other in space when the antigen adopts such a conformation. In some embodiments, when the antigen adopts an alternative conformation (e.g., linearized), at least some of such chemical atoms or groups are physically separated from each other.

[0092] Fc region: As used herein, the term "Fc region" refers to a dimer of two "Fc polypeptides", each "Fc polypeptide" comprising the constant region of an antibody excluding the first constant region immunoglobulin domain. In some embodiments, the "Fc region" comprises two Fc polypeptides linked by one or more disulfide bonds, a chemical linker, or a peptide linker. An "Fc polypeptide" refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and may also comprise part or all of the flexible hinge at the N-terminus of these domains. For IgG, the "Fc polypeptide" comprises the immunoglobulin domains Cgamma2 (Cγ2) and Cgamma3 (Cγ3), and the lower part of the hinge between Cgamma1 (Cγ1) and Cγ2. Although the boundaries of the Fc polypeptide may vary, the human IgG heavy chain Fc polypeptide is generally defined as comprising residues starting at T223 or C226 or P230 to its carboxyl terminus, where the numbering is according to the EU index in Kabat et al., (1991, NIH Publication 91-3242, National Technical Information Services, Springfield, VA). For IgA, the Fc polypeptide comprises the immunoglobulin domains Calpha2 (Cα2) and Calpha3 (Cα3), and the lower part of the hinge between Calpha1 (Cα1) and Cα2. The Fc region can be synthetic, recombinant, or derived from natural sources such as IVIG.

[0093] Framework or framework region: As used herein, refers to the sequence of the variable region minus the CDR. Since the CDR sequences can be determined by different systems, the framework sequences are similarly subject to corresponding different interpretations. The six CDRs also divide the framework regions on the heavy and light chains into four subregions (FR1, FR2, FR3, and FR4) on each chain, where CDR1 is located between FR1 and FR2, CDR2 is located between FR2 and FR3, and CDR3 is located between FR3 and FR4. Without designating a specific subregion as FR1, FR2, FR3, or FR4, other mentioned framework regions represent the combined FR within the variable region of a single naturally occurring immunoglobulin chain. As used herein, FR represents one of the four subregions, FR1 for example represents the first framework region closest to the amino terminus of the variable region and 5' relative to CDR1, and FRs represents two or more subregions that make up the framework region.

[0094] Half-life: As used herein, the term "half-life" is the time required for an amount such as protein concentration or activity to decline to half of the value measured at the start of a period of time.

[0095] High affinity: As used herein, when referring to IgG-type antibodies, the term "high affinity" means having a K -8 of 10 -9 M or less, more preferably 10 -10 M or less and even more preferably 10 D M or less for an antibody against the domain of Flt-1. However, for other antibody isotypes, "high affinity" binding can vary. For example, for IgM isotypes, "high affinity" binding means having a K -7 of 10 -8 M or less, more preferably 10 -9 M or less, even more preferably 10 D M or less for an antibody.

[0096] Human antibody: As used herein, is intended to include antibodies having variable and constant regions produced (or assembled) from human immunoglobulin sequences. In some embodiments, although the amino acid sequence of the antibody (or antibody component) contains residues or elements not encoded by human germline immunoglobulin sequences (e.g., containing sequence variations that may (initially) have been introduced by in vitro random or site-specific mutagenesis or in vivo somatic mutations), such as in one or more CDRs and particularly CDR3, the antibody (or antibody component) can be considered "human".

[0097] Human monoclonal antibody: As used herein, it is intended to refer to an antibody having variable regions and exhibiting a single binding specificity, wherein both the framework regions and the CDR regions are derived from human germline immunoglobulin sequences. In one embodiment, the human monoclonal antibody is produced by a hybridoma comprising B cells obtained from a transgenic non-human animal (e.g., a transgenic mouse) having a genome with a human heavy chain transgene and a light chain transgene fused to an immortalized cell.

[0098] Humanization: As is known in the art, the term "humanization" is generally used to refer to an antibody (or antibody component) whose amino acid sequence contains V H and V L region sequences from a reference antibody produced in a non-human species (e.g., mouse, llama), and includes modifications in those sequences relative to the reference antibody, intended to make them more "human-like", i.e., more similar to human germline variable sequences. In some embodiments, a "humanized" antibody (or antibody component) is an antibody (or antibody component) that immunospecifically binds to a target antigen and has a framework (FR) region with an amino acid sequence substantially like that of a human antibody and a complementarity determining region (CDR) with an amino acid sequence substantially like that of a non-human antibody (e.g., mouse, llama). A humanized antibody comprises substantially all of at least one and usually two variable domains (Fab, Fab’, F(ab’)2, FabC, Fv), wherein all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., the donor immunoglobulin) and all or substantially all of the framework regions are those of human immunoglobulin consensus sequences. In some embodiments, the humanized antibody also comprises at least a portion of the immunoglobulin constant region (Fc), usually at least a portion of a human immunoglobulin constant region. In some embodiments, the humanized antibody contains a light chain and at least the variable domain of the heavy chain. The antibody may also comprise C H 1, hinge, C H 2, C H 3 and optionally C H 4 regions. In some embodiments, the humanized antibody contains only humanized V L regions. In some embodiments, the humanized antibody contains only humanized V H regions. In some specific embodiments, the humanized antibody contains humanized V H and V L regions.

[0099] Hypertrophy: As used herein, the term "hypertrophy" refers to an increase in the volume of an organ or tissue due to the enlargement of its component cells.

[0100] Improvement, increase or decrease: As used herein, the terms "improvement", "increase" or "decrease" or grammatical equivalents thereof indicate a value relative to a baseline measurement, such as a measurement in the same individual before initiation of the treatment described herein, or in a control individual (or control individuals) in the absence of the treatment described herein. A "control individual" is an individual suffering from the same form of disease as the individual being treated, and is approximately the same age as the individual being treated (to ensure that the disease stage of the individual being treated is comparable to that of the control individual).

[0101] Inhibition: As used herein, the terms "inhibition", "inhibit" and "inhibiting" refer to a process or method of reducing or decreasing the activity and / or expression of a target protein or gene. Generally, inhibiting a protein or gene means reducing the expression or associated activity of the protein or gene by at least 10% or more, such as 20%, 30%, 40% or 50%, 60%, 70%, 80%, 90% or more, or reducing the expression or associated activity by more than 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 50-fold, 100-fold or more, as measured by one or more methods described herein or recognized in the art.

[0102] In vitro: As used herein, the term "in vitro" refers to events that occur in an artificial environment, such as in a test tube or reaction vessel, in cell culture, etc., rather than within a multicellular organism.

[0103] In vivo: As used herein, the term "in vivo" refers to events that occur within a multicellular organism, such as a human or non-human animal. In the context of a cell-based system, the term can be used to refer to events that occur within a living cell (as opposed to, for example, an in vitro system).

[0104] Isolated antibody: As used herein, the term "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to Flt-1). In addition, an isolated antibody can be substantially free of other cellular materials and / or chemicals.

[0105] K a : As used herein, refers to the association rate of a specific antibody-antigen interaction; and the term "K d " as used herein is intended to refer to the dissociation rate of a specific antibody-antigen interaction. As used herein, the term "K D " is intended to refer to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and is expressed as a molar concentration (M). The K D value of an antibody can be determined using well-established methods in the art. Methods for determining the K DThe preferred method is by using surface plasmon resonance, preferably using a biosensor system, such as system.

[0106] Light chain shuffling: As used herein, the term "light chain shuffling" is intended to refer to an affinity maturation step in which the heavy chain sequence remains constant and a library of light chain sequences is generated. The light chain library is screened against the heavy chain to identify antibodies with improved binding affinity. The improved binding affinity can be in the nanomolar or picomolar range.

[0107] Monoclonal antibody: As used herein, the term "monoclonal antibody" is intended to refer to a preparation of antibody molecules consisting of a single molecule. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope.

[0108] Pharmaceutically acceptable: As used herein, the term "pharmaceutically acceptable" refers to a substance that is applicable, within the scope of sound medical judgment, to contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0109] Polypeptide: As used herein, the term "polypeptide" refers to a continuous chain of amino acids joined together via peptide bonds. The term is used to refer to amino acid chains of any length, but one of ordinary skill in the art will understand that the term is not limited to long chains and can refer to the smallest chain containing two amino acids joined together via peptide bonds. As is known to those skilled in the art, polypeptides can be processed and / or modified.

[0110] Prevention: As used herein, the term "prevent" or "prevention" when used in connection with the occurrence of a disease, disorder, and / or condition is intended to mean reducing the risk of developing the disease, disorder, and / or condition. See the definition of "risk".

[0111] Protein: As used herein, the term "protein" refers to one or more polypeptides that act as discrete units. The terms "polypeptide" and "protein" can be used interchangeably if a single polypeptide is a discrete functional unit and does not require permanent or transient physical association with other polypeptides to form a discrete functional unit. If the discrete functional unit contains more than one polypeptide physically associated with each other, the term "protein" refers to the multiple polypeptides that are physically linked and act together as a discrete unit.

[0112] Risk: As will be understood from the context, a "risk" of a disease, disorder, and / or condition includes the likelihood that a particular individual will develop a disease, disorder, and / or condition (e.g., DMD). In some embodiments, the risk is expressed as a percentage. In some embodiments, the risk ranges from 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% up to 100%. In some embodiments, the risk is expressed as a risk relative to the risk associated with a reference sample or a set of reference samples. In some embodiments, the reference sample or set of reference samples has a known risk of a disease, disorder, condition, and / or event (e.g., DMD). In some embodiments, the reference sample or set of reference samples is from an individual comparable to the specific individual. In some embodiments, the relative risk is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.

[0113] Selective binding: As used herein, "selective binding", "selectively bind", "specific binding", or "specifically bind" refers to the preferential binding of a binding moiety to a target rather than to an entity that is not the target, with respect to the binding moiety and the target. Some degree of non-specific binding may occur between the binding moiety and non-targets. In some embodiments, the binding moiety selectively binds the target if the binding between the binding moiety and the target is greater than 2-fold, greater than 5-fold, greater than 10-fold, or greater than 100-fold compared to the binding between the binding moiety and the non-target. In some embodiments, the binding moiety selectively binds the target if the binding affinity is less than about 10 -5 M, less than about 10 -6 M, less than about 10 -7 M, less than about 10 -8 M or less than about 10 -9 M.

[0114] Striated muscle: As used herein, the term "striated muscle" refers to multinucleated muscle tissue having a regular arrangement of its intracellular contractile units, sarcomeres, which results in striations being visible using a microscope and under voluntary control. Generally, striated muscle can be cardiac muscle, skeletal muscle, and branchiomeric muscle.

[0115] Smooth muscle: As used herein, the term "smooth muscle" refers to non-voluntary, non-striated muscle, including single- and multi-unit muscle.

[0116] Subject: As used herein, the term "subject" refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate). Humans include pre-natal and post-natal forms. In many embodiments, the subject is a human. A subject can be a patient, which refers to a person sent to a healthcare provider for diagnosis or treatment of a disease. The term "subject" may be used interchangeably herein with "individual" or "patient". A subject may have or be susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder.

[0117] Substantially: As used herein, the term "substantially" refers to a qualitative situation that exhibits the target characteristic or property of an overall or near overall range or degree. One of ordinary skill in the biological arts will appreciate that biological and chemical phenomena rarely, if ever, achieve complete and / or proceed to completion or achieve or avoid absolute results. Accordingly, the term "substantially" is used herein to account for the inherent lack of potential completeness in many biological and chemical phenomena.

[0118] Substantial homology: The phrase "substantial homology" is used herein to refer to a comparison between amino acid sequences or nucleic acid sequences. As will be understood by one of ordinary skill in the art, two sequences are generally considered to be "substantially homologous" if they contain homologous residues at corresponding positions. Homologous residues can be identical residues. Alternatively, homologous residues can be non-identical residues that will have suitably similar structural and / or functional characteristics. For example, as is well known to one of ordinary skill in the art, certain amino acids are generally classified as "hydrophobic" or "hydrophilic" amino acids, and / or as having "polar" or "non-polar" side chains. Substitution of one amino acid for another of the same type is generally considered to be a "homologous" substitution.

[0119] As is well known in the art, amino acid sequences or nucleic acid sequences can be compared using any of a variety of algorithms, including those available in commercial computer programs such as BLASTN for nucleotide sequences and BLASTP, Gapped BLAST, and PSI-BLAST for amino acid sequences. Exemplary such programs are described in Altschul et al., basic local alignment search tool, J. Mol. Biol., 215(3):403-410, 1990; Altschul et al., Methods in Enzymology; Altschul et al., "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis et al., Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998; and Misener et al. (eds.), Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999. In addition to identifying homologous sequences, the above programs typically provide an indication of the degree of homology. In some embodiments, two sequences are considered to be substantially homologous if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the corresponding residues of the two sequences are homologous over the relevant segment of residues. In some embodiments, the relevant segment is the complete sequence. In some embodiments, the relevant segment is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more residues.

[0120] Substantial identity: The phrase "substantial identity" is used herein to refer to a comparison between amino acid sequences or nucleic acid sequences. As would be understood by one of ordinary skill in the art, two sequences are generally considered to be "substantially identical" if they contain the same residues at corresponding positions. As is well known in the art, amino acid sequences or nucleic acid sequences can be compared using any of a variety of algorithms, including those available in commercial computer programs such as BLASTN for nucleotide sequences and BLASTP, Gapped BLAST, and PSI-BLAST for amino acid sequences. Exemplary such programs are described in Altschul et al., Basic local alignment search tool, J. Mol. Biol., 215(3):403-410, 1990; Altschul et al., Methods in Enzymology; Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis et al., Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998; and Misener et al. (eds.), Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999. In addition to identifying identical sequences, the above programs typically provide an indication of the degree of identity. In some embodiments, two sequences are considered to be substantially identical if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the corresponding residues are identical over the relevant segment of residues. In some embodiments, the relevant segment is the complete sequence. In some embodiments, the relevant segment is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more residues.

[0121] Surface plasmon resonance: As used herein, refers to an optical phenomenon that permits real-time analysis of specific binding interactions by, for example, detecting changes in protein concentration within a biosensor matrix using, e.g., a Biacore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). For further description, see Jonsson, U. et al., (1993) Ann. Biol. Clin. 51:19-26; Jonsson, U. et al., (1991) Biotechniques 11:620-627; Johnsson, B. et al., (1995) J. Mol. Recognit. 8:125-131; and Johnnson, B., et al., (1991) Anal. Biochem. 198:268-277.

[0122] A subject having: A subject “having” a disease, disorder, and / or condition has been diagnosed as having or exhibiting one or more symptoms of the disease, disorder, and / or condition (such as, e.g., DMD).

[0123] A subject predisposed to: A subject “predisposed to” a disease, disorder, and / or condition has not been diagnosed as having the disease, disorder, and / or condition. In some embodiments, a subject predisposed to a disease, disorder, and / or condition may not exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, a subject predisposed to a disease, disorder, condition, or event (such as, e.g., DMD) may have one or more of the following characteristics: (1) a genetic mutation associated with development of the disease, disorder, and / or condition; (2) a genetic polymorphism associated with development of the disease, disorder, and / or condition; (3) an increase and / or decrease in the expression and / or activity of a protein associated with the disease, disorder, and / or condition; (4) a habit and / or lifestyle associated with development of the disease, disorder, condition, and / or event; (5) having undergone, planning to undergo, or in need of a transplant. In some embodiments, a subject predisposed to a disease, disorder, and / or condition will develop the disease, disorder, and / or condition. In some embodiments, a subject predisposed to a disease, disorder, and / or condition will not develop the disease, disorder, and / or condition.

[0124] Target tissue: As used herein, the term “target tissue” refers to any tissue affected by a disease to be treated such as DMD. In some embodiments, target tissue includes tissue that exhibits disease-related pathology, symptoms, or characteristics, including but not limited to muscle wasting, skeletal deformity, cardiomyopathy, muscle ischemia, cognitive impairment, and impaired respiratory function. In some embodiments, the target tissue is smooth muscle, striated muscle, or cardiac muscle.

[0125] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" of a therapeutic agent means an amount sufficient to treat, diagnose, prevent the symptoms of, and / or delay the onset of a disease, disorder, and / or condition in a subject afflicted with or susceptible to the disease, disorder, and / or condition. One of ordinary skill in the art will understand that a therapeutically effective amount is typically administered via a dosing regimen that includes at least one unit dose.

[0126] Treat: As used herein, the term "treat", "treatment", or "treating" refers to any method for partially or completely alleviating, ameliorating, mitigating, inhibiting, preventing one or more symptoms or characteristics of a particular disease, disorder, and / or condition (such as DMD), delaying its onset, reducing its severity, and / or reducing its incidence. For the purpose of reducing the risk of developing disease-related pathologies, treatment can be administered to subjects who do not exhibit signs of the disease and / or who exhibit only early signs of the disease. Detailed description

[0128] The present invention particularly provides methods and compositions for treating muscular dystrophy, including Duchenne muscular dystrophy (DMD) and / or Becker muscular dystrophy, based on the use of an anti-Flt-1 antibody or an antigen-binding fragment thereof as a therapeutic agent for treating muscular dystrophy. In some embodiments, the present invention provides a method for treating DMD, the method comprising administering a therapeutically effective amount of an anti-Flt-1 antibody or an antigen-binding fragment thereof to an individual afflicted with or susceptible to DMD, such that at least one symptom or characteristic of DMD is reduced in intensity, severity, or frequency or the onset is delayed.

[0129] Aspects of the present invention are described in detail in the following sections. The use of the sections is not meant to limit the present invention. Each section can be applicable to any aspect of the present invention. In this application, unless otherwise indicated, the use of "or" means "and / or".

[0130] Duchenne muscular dystrophy (DMD)

[0131] DMD is a disease characterized by the progressive deterioration of muscles and the loss of muscle-related functions throughout the body. It is contemplated that the present invention provides methods and compositions for slowing, delaying, or preventing muscle degeneration, regenerating muscle, and reversing, eliminating, delaying, preventing, or minimizing fibrosis, inflammation, and other symptoms or characteristics associated with DMD and other muscular dystrophies in various muscle tissues.

[0132] Muscle tissue

[0133] There are two main types of muscle tissue in animals, striated muscle and smooth muscle. As used herein, the term "striated muscle" refers to muscle tissue containing repeating sarcomeres. Striated muscle tends to be under voluntary control and is attached to bone. Striated muscle permits voluntary movement of the body and includes major muscle groups, including quadriceps, gastrocnemius, biceps, triceps, trapezius, deltoid, etc. Striated muscles tend to be very long, and many striated muscles can function independently. However, some striated muscles are not attached to bone, including those in the mouth, anus, heart, and upper part of the esophagus.

[0134] On the other hand, smooth muscle has a very different structure. Smooth muscle tends to be organized into continuous sheets with mechanical connections between smooth muscle cells rather than a series of long muscles with individual skeletal attachments. Smooth muscle is typically located in the walls of hollow organs and is generally not under voluntary control. The smooth muscle lining a particular organ must withstand the same load and contract simultaneously. Smooth muscle functions at least in part to handle changes in load in the hollow organ caused by movement and / or posture or pressure changes. This dual role means that smooth muscle must not only be able to contract like striated muscle but must also be able to contract tonically to maintain organ size against a continuous load. Examples of smooth muscle are those lining blood vessels, bronchioles, the bladder, and the gastrointestinal tract such as the rectum.

[0135] The strength of a muscle depends on the number and size of muscle cells and its anatomical structure. Increasing the diameter of muscle fibers by synthesizing new myofibrils (hypertrophy) and / or forming more muscle cells (hyperplasia) will increase the force - generating capacity of the muscle.

[0136] Muscles can also be grouped according to location or function. In some embodiments, an Flt - 1 antibody or an antigen - binding fragment thereof is targeted to one or more muscles of the face, one or more muscles used for chewing, one or more muscles of the tongue and neck, one or more muscles of the chest, one or more muscles of the shoulder girdle and arm, one or more muscles of the arm and shoulder, one or more ventral and dorsal forearm muscles, one or more muscles of the hand, one or more muscles of the erector spinae, one or more muscles of the pelvic girdle and leg, and / or one or more muscles of the front leg and foot.

[0137] In some embodiments, the muscles of the face include, but are not limited to, intraocular muscles such as the ciliary muscle, dilator pupillae, sphincter pupillae; muscles of the ear such as the auricular muscles, temporoparietal muscle, stapedius muscle, tensor tympani muscle; muscles of the nose such as the procerus muscle, nasalis muscle, dilator naris muscle, depressor septi nasi muscle, levator labii alaeque nasi muscle; muscles of the mouth such as the levator anguli oris muscle, depressor anguli oris muscle, orbicularis oris muscle, buccinator muscle, zygomaticus major and minor muscles, platysma muscle, levator labii superioris muscle, depressor labii inferioris muscle, risorius muscle, mentalis muscle, and / or corrugator supercilii muscle.

[0138] In some embodiments, the chewing muscles include, but are not limited to, the masseter, temporalis, medial pterygoid, and lateral pterygoid. In some embodiments, the muscles of the tongue and neck include, but are not limited to, the genioglossus, styloglossus, glossopalatinus, hyoglossus, digastric, stylohyoid, mylohyoid, geniohyoid, omohyoid, sternohyoid, sternothyroid, thyrohyoid, sternocleidomastoid, anterior scalene, middle scalene, and / or posterior scalene.

[0139] In some embodiments, the muscles of the chest, shoulder girdle, and arm include, but are not limited to, the subclavius, pectoralis major, pectoralis minor, rectus abdominis, external oblique, internal oblique, transversus abdominis, diaphragm, external intercostal, internal intercostal, serratus anterior, trapezius, levator scapulae, rhomboid major, rhomboid minor, latissimus dorsi, deltoid, subscapularis, supraspinatus, infraspinatus, teres major, teres minor, and / or coracobrachialis.

[0140] In some embodiments, the muscles of the arm and shoulder include, but are not limited to, the long head of the biceps brachii, short head of the biceps brachii, long head of the triceps brachii, lateral head of the triceps brachii, medial head of the triceps brachii, anconeus, pronator teres, supinator, and / or brachialis.

[0141] In some embodiments, the muscles of the ventral and dorsal forearm include, but are not limited to, the brachioradialis, flexor carpi radialis, flexor carpi ulnaris, palmaris longus, extensor carpi ulnaris, extensor carpi radialis longus, extensor carpi radialis brevis, extensor digitorum, and extensor digiti minimi.

[0142] In some embodiments, the muscles of the hand include, but are not limited to, the intrinsic muscles of the hand, such as the thenar muscles, abductor pollicis brevis, flexor pollicis brevis, opponens pollicis, hypothenar muscles, abductor digiti minimi, flexor digiti minimi, opponens digiti minimi, palmar interossei, dorsal interossei, and / or lumbricals.

[0143] In some embodiments, the erector spinae muscles include, but are not limited to, the cervical muscles, spinalis, longissimus, and / or iliocostalis.

[0144] In some embodiments, the muscles of the pelvic girdle and leg include, but are not limited to, the psoas major, iliacus, quadratus femoris, adductor longus, adductor brevis, adductor magnus, gracilis, sartorius, quadriceps femoris such as rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gastrocnemius, fibularis (peroneus) longus, soleus, gluteus maximus, gluteus medius, gluteus minimus, hamstrings: biceps femoris: long head, hamstrings: biceps femoris: short head, hamstrings: semitendinosus, hamstrings: semimembranosus, tensor fasciae latae, pectineus, and / or tibialis anterior.

[0145] In some embodiments, the muscles of the front legs and feet include, but are not limited to, the extensor digitorum longus, extensor hallucis longus, peroneus brevis, plantaris, posterior tibialis, flexor digitorum longus, extensor digitorum brevis, extensor hallucis brevis, abductor hallucis, flexor hallucis brevis, abductor digiti minimi, flexor digiti minimi brevis, opponens digiti minimi, extensor digitorum brevis, lumbricals of the foot, quadratus plantae or accessory flexor, flexor digitorum brevis, dorsal interossei, and / or plantar interossei.

[0146] Exemplary muscle targets are summarized in Table 1.

[0147] Table 1.

[0148]

[0149]

[0150] Muscular dystrophy

[0151] Muscular dystrophy is a group of inherited disorders that cause muscle degeneration, leading to weakness and impaired movement. The central feature of all muscular dystrophies is that they are progressive in nature. Muscular dystrophies include, but are not limited to: Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, and myotonic dystrophy types 1 and 2, including the congenital form of myotonic dystrophy type 1. Symptoms can vary depending on the type of muscular dystrophy, with some or all muscles being affected. Exemplary symptoms of muscular dystrophy include delayed development of muscle motor skills, difficulty using one or more muscle groups, difficulty swallowing, speaking, or eating, drooling, ptosis, frequent falls, loss of strength in adult muscles or muscle groups, loss of muscle size, gait disturbances due to physical weakness or altered biomechanics, and / or cognitive or behavioral disorders / mental retardation.

[0152] Although there is no known cure for muscular dystrophy, several supportive treatments are used, including symptomatic and disease-modifying therapies. Corticosteroids, ACE inhibitors, angiotensin receptor blockers, physical therapy, orthotic devices, wheelchairs, or other assistive medical devices for ADL and lung function are commonly used in muscular dystrophy. Cardiac pacemakers are used to prevent sudden death due to arrhythmias in myotonic dystrophy. Antimyotonic agents that improve myotonia (inability to relax) include mexilitine, and in some cases phenytoin, procainamide, and quinine.

[0153] Duchenne muscular dystrophy

[0154] Duchenne muscular dystrophy (DMD) is a recessive X-linked form of muscular dystrophy that causes muscle degeneration and eventual death. DMD is characterized by proximal muscle weakness, abnormal gait, gastrocnemius (calf) muscle hypertrophy, and elevated creatine kinase. Many DMD patients are diagnosed around 5 years of age, at which time symptoms / signs typically become more pronounced. Affected individuals usually stop walking around 10 - 13 years of age and die in or before their mid-20s due to respiratory complications and cardiomyopathy.

[0155] In individuals with DMD, serum creatine kinase levels can increase more than 10-fold compared to unaffected individuals. In some embodiments, administration of the provided composition to an affected individual results in a decreased serum creatine kinase level compared to the baseline level before treatment. Typically, the baseline level is measured immediately before treatment. In some embodiments, administration of the provided composition results in a decrease in serum creatine kinase level of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% compared to the baseline serum creatine kinase level just before treatment. In some embodiments, administration of the provided composition results in a serum creatine kinase level that is less than about 3500 IU / L, 3000 IU / L, 2500 IU / L, 2000 IU / L, 1500 IU / L, 1000 IU / L, 750 IU / L, 500 IU / L, 250 IU / L, 100 IU / L, 90 IU / L, 80 IU / L, 70 IU / L or 60 IU / L. In some embodiments, administration of the provided composition results in a decreased serum creatine kinase level compared to the serum creatine kinase level of untreated subjects.

[0156] The disorder DMD is caused by mutations in the dystrophin gene located on the human X chromosome, which encodes the protein dystrophin, an important structural component within muscle tissue that provides structural stability to the dystrophin-associated protein complex (DGC) of the cell membrane. Dystrophin links the internal cytoplasmic actin filament network to the extracellular matrix, thereby providing physical strength to muscle fibers. Thus, alterations or absence of dystrophin leads to abnormal sarcolemma tearing and necrosis of muscle fibers. Although both sexes can carry the mutation, females rarely exhibit severe disease symptoms.

[0157] The main symptoms of DMD are muscle weakness associated with muscle wasting, which typically first affects voluntary muscles, particularly those of the buttocks, pelvic region, thighs, shoulders, and calves. Muscle weakness also occurs in the arms, neck, and other areas. The calves are often enlarged. Signs and symptoms usually appear before age 6 and can be present as early as infancy. Cardiomyopathy typically occurs in DMD patients after age 18. Other physical symptoms include, but are not limited to, delayed ability to walk independently, progressive difficulty walking, stepping, or running, and eventual loss of the ability to walk (usually by age 12); frequent falls; fatigue; difficulty with motor skills (running, jumping, hopping); increased lumbar lordosis, leading to hip flexor shortening; impaired Achilles and hamstring tendon function, connective tissue fibrosis; muscle fiber abnormalities; pseudohypertrophy (enlargement) of the tongue and calf muscles caused by replacement of muscle tissue with fat and connective tissue; high risk of non-progressive weakness in neurobehavioral disorders (such as ADHD), learning disabilities (dyslexia), and specific cognitive skills (particularly short-term non-verbal memory); skeletal abnormalities (including scoliosis in some cases).

[0158] The muscle changes observed in DMD are accompanied by an increase in connective tissue (i.e., development of fibrosis) and are caused by responses or repair processes involving mechanical, humoral, and / or cytokines. Lack of functional dystrophin results in instability of the muscle fiber membrane and, as a result, cells are less resistant to mechanical shear and tend to have an excessive influx of electrolytes, leading to tissue damage. Because muscle tissue in DMD is damaged, the ability to recover is limited by satellite cell proliferation. This leads to necrosis, inflammation, fibrosis, and adipocyte replacement. The increase in connective tissue occurs early in the disease process because the conjunctive tissue that sheathes each muscle cell and covers the sarcolemma (i.e., endomysium) increases before observable muscle damage. The increase in collagen connective tissue is a factor in the muscle pathology of DMD, adversely affecting the nutritional supply to affected muscle cells and secondarily influencing muscle strength and the age at which mobility is lost.

[0159] In some embodiments, in vivo administration of an anti-Flt-1 antibody or an antigen-binding fragment thereof results in a reduction in fibrosis of muscle tissue. In some embodiments, the muscle is skeletal muscle. In specific embodiments, the muscle is cardiac muscle, diaphragm muscle, gastrocnemius muscle, and / or tibialis anterior (TA) muscle. In some embodiments, the reduced fibrosis is demonstrated by reduced collagen staining. In some embodiments, the collagen is type I collagen. In some embodiments, the reduced fibrosis can be measured, for example, by measuring the percentage of the collagen-positive area in the muscle of a mouse administered with an anti-Flt-1 antibody or an antigen-binding fragment thereof. For example, the percentage of the collagen-positive area in the diaphragm of a mouse administered with an anti-Flt-1 antibody or an antigen-binding fragment thereof can be at least about 3.0%, about 3.5%, about 4.0%, about 4.5%, about 5.0%, about 5.5%, about 6.0%, about 6.5%, about 7.0%, about 7.5%, about 8.0%, about 8.5%, or about 9.0% of the total tissue area. In a specific embodiment, the percentage of the collagen-positive area in the diaphragm of a mouse administered with an anti-Flt-1 antibody is significantly lower than the percentage of the collagen-positive area in the diaphragm of a mouse administered with an isotype control antibody.

[0160] In some embodiments, in vivo administration of an anti-Flt-1 antibody or an antigen-binding fragment thereof results in a reduction in necrosis of muscle tissue. In some embodiments, the muscle is skeletal muscle. In specific embodiments, the muscle is cardiac muscle, diaphragm muscle, gastrocnemius muscle, and / or tibialis muscle (TA). In some embodiments, the reduced necrosis can be measured, for example, by measuring the percentage of the necrosis-positive area in the muscle of a mouse administered with an anti-Flt-1 antibody or an antigen-binding fragment thereof. For example, the percentage of the necrosis-positive area in the gastrocnemius of a mouse administered with an anti-Flt-1 antibody or an antigen-binding fragment thereof can be at least about 0.5%, about 0.45%, about 0.4%, about 0.35%, about 0.3%, about 0.25%, about 0.2%, about 0.15%, about 0.1%, about 0.05%, or about 0.025% of the total tissue area. In a specific embodiment, the percentage of the necrosis-positive area in the gastrocnemius of a mouse administered with an anti-Flt-1 antibody is significantly lower than the percentage of the necrosis-positive area in the gastrocnemius of a mouse administered with an isotype control antibody.

[0161] In some embodiments, in vivo administration of an anti-Flt-1 antibody or an antigen-binding fragment thereof results in increased muscle strength and / or fatigue resistance.

[0162] Flt-1 receptor

[0163] The Flt-1 receptor (also known as vascular endothelial growth factor receptor 1 (VEGFR-1) or Flt-1) is a receptor encoded by the FLT1 gene and expressed on the cell membranes of endothelial cells and monocytes. The vascular endothelial growth factor (VEGF) family of signaling glycoproteins acts as a potential initiator of angiogenesis during embryogenesis and postnatal growth. Specifically, the binding of VEGF-A ligands to VEGF receptors has been shown to promote vascular permeability and also to trigger endothelial cell migration, proliferation, and survival, and the newly formed endothelial cells provide the basic structure of the new vascular system. The major VEGF signaling molecule for angiogenesis, VEGF-A, mediates its signal through VEGF receptor-1 (VEGFR-1, also known as Flt-1) and VEGF receptor-2 (VEGFR-2, also known as Flk-1). A soluble form of Flt-1 (sFlt-1) also exists, but lacks the intracellular signaling domain and is therefore thought to function only by chelating VEGF-A or other ligands that bind to it. sFlt-1 and other molecules containing Flt-1 binding sites that are not associated with intracellular signal transduction pathways are referred to as "decoy receptors". The Flt-1 and Flk-1 receptors contain extracellular VEGF-A binding domains and intracellular tyrosine kinase domains, and are both shown to be expressed during developmental stages and tissue regeneration in the hemangioblast and endothelial cell lineages. Compared to Flk-1, Flt-1 has approximately 10-fold greater binding affinity (K d ~2 -10 pM) for VEGF-A, but weaker tyrosine kinase activity indicates that angiogenesis signal transduction after VEGF-A binding to Flt-1 is weaker than that generated by VEGF-A binding to Flk-1. Thus, homozygous Flt-1 gene knockout mice die during embryogenesis from overproduction of endothelial cells and destruction of vascular tissue. In contrast, homozygous Flk-1 gene knockout mice die from defects in organized vascular development due to lack of yolk sac blood island formation during embryogenesis. Both the Flt-1 and Flk-1 receptors are required for normal development, but selective increases in VEGF-A concentration can permit greater binding to the Flk-1 receptor and induce an angiogenic effect that increases capillary density and promotes muscle regeneration, fibrosis, and reduction of inflammation, as well as alleviation of symptoms and features associated with DMD and other muscular dystrophies in various muscle tissues.

[0164] As used herein, the term "Flt-1 receptor" refers to both soluble and membrane-associated Flt-1 receptors or functional fragments thereof.

[0165] Anti-Flt-1 antibody

[0166] As used herein, the term "anti-Flt-1 antibody" refers to any antibody or antigen-binding fragment thereof that binds to the Flt-1 receptor (e.g., soluble or membrane-associated Flt-1 receptor). In some embodiments, anti-Flt-1 antibodies are produced that bind to the Flt-1 receptor with high affinity. Without wishing to be bound by theory, it is believed that anti-Flt-1 antibodies that bind to the Flt-1 receptor inhibit one or more endogenous ligands from binding to Flt-1, and thereby allow a greater amount of available ligand to associate with other VEGF receptors such as the Flk-1 receptor. Increasing the availability of VEGF promotes angiogenesis, along with an increase in blood flow to the muscle to counteract functional ischemia and produce improvements in the structural and functional characteristics of DMD. In some embodiments, antibodies that bind to the Flt-1 receptor increase the amount of VEGF available to bind to other VEGF receptors.

[0167] In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof comprises the sequences provided in Table 2.

[0168] Table 2.

[0169]

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179]

[0180] In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof comprises one or more complementarity determining regions (CDRs) selected from the group consisting of: VL CDR1, which is defined by an amino acid sequence having at least 60%, 70%, 80%, 90%, 95% or 98% identity to any one of SEQ ID NOs: 19 to 21; VL CDR2, which is defined by an amino acid sequence having at least 60%, 70%, 80%, 90%, 95% or 98% identity to any one of SEQ ID NOs: 22 to 24; VL CDR3, which is defined by an amino acid sequence having at least 60%, 70%, 80%, 90%, 95% or 98% identity to any one of SEQ ID NOs: 25 to 34; VH chain CDR1, which is defined by an amino acid sequence having at least 60%, 70%, 80%, 90%, 95% or 98% identity to any one of SEQ ID NOs: 1 to 4; VH CDR2, which is defined by an amino acid sequence having at least 60%, 70%, 80%, 90%, 95% or 98% identity to any one of SEQ ID NOs: 5 to 14; and VH CDR3, which is defined by an amino acid sequence having at least 60%, 70%, 80%, 90%, 95% or 98% identity to any one of SEQ ID NOs: 15 to 18. In some embodiments, VL CDR3 is not SEQ ID NO: 25. In some embodiments, VH CDR3 is not SEQ ID NO: 15.

[0181] In some embodiments, the one or more CDRs comprise VL CDR3, which is defined by an amino acid sequence having at least 60%, 70%, 80%, 90%, 95% or 98% identity to any one of SEQ ID NOs: 25 to 34; and VH CDR3, which is defined by an amino acid sequence having at least 60%, 70%, 80%, 90%, 95% or 98% identity to any one of SEQ ID NOs: 15 to 18. In some embodiments, VL CDR3 is not SEQ ID NO: 25. In some embodiments, VH CDR3 is not SEQ ID NO: 15.

[0182] In some embodiments, the one or more CDRs include VL CDR1, which is defined by an amino acid sequence having at least 60%, 70%, 80%, 90%, 95% or 98% identity to any one of SEQ ID NOs: 19 to 21; VL CDR2, which is defined by an amino acid sequence having at least 60%, 70%, 80%, 90%, 95% or 98% identity to any one of SEQ ID NOs: 22 to 24; and VL CDR3, which is defined by an amino acid sequence having at least 60%, 70%, 80%, 90%, 95% or 98% identity to any one of SEQ ID NOs: 25 to 34. In some embodiments, VL CDR3 is not SEQ ID NO: 25.

[0183] In some embodiments, the one or more CDRs include VH CDR1, which is defined by an amino acid sequence having at least 60%, 70%, 80%, 90%, 95% or 98% identity to any one of SEQ ID NOs: 1 to 4; VH CDR2, which is defined by an amino acid sequence having at least 60%, 70%, 80%, 90%, 95% or 98% identity to any one of SEQ ID NOs: 5 to 14; and VH CDR3, which is defined by an amino acid sequence having at least 60%, 70%, 80%, 90%, 95% or 98% identity to any one of SEQ ID NOs: 15 to 18. In some embodiments, VH CDR3 is not SEQ ID NO: 15.

[0184] In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof comprises a VL chain that contains VL CDR1, VL CDR2, and VL CDR3 defined by the amino acid sequences of SEQ ID NO:19, SEQ ID NO:22, and SEQ ID NO:25, respectively. In another embodiment, the anti-Flt-1 antibody or antigen-binding fragment thereof comprises a VL chain that contains VL CDR1, VL CDR2, and VL CDR3 defined by the amino acid sequences of SEQ ID NO:20, SEQ ID NO:23, and SEQ ID NO:25, respectively. In another embodiment, the anti-Flt-1 antibody or antigen-binding fragment thereof comprises a VL chain that contains VL CDR1 and VL CDR2 defined by the amino acid sequences of SEQ ID NO:21 and SEQ ID NO:24, respectively, and VL CDR3 defined by the amino acid sequence of SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, or SEQ ID NO:34. In a specific embodiment, the anti-Flt-1 antibody or antigen-binding fragment thereof comprises a VL chain that contains VL CDR1 defined by the amino acid sequence of SEQ ID NO:21, VL CDR2 defined by the amino acid sequence of SEQ ID NO:24, and VL CDR3 defined by the amino acid sequence of SEQ ID NO:32. In some embodiments, VL CDR3 is not SEQ ID NO:25.

[0185] In another embodiment, the anti-Flt-1 antibody or antigen-binding fragment thereof comprises a VH chain, and the VH chain comprises VH CDR1, VH CDR2 and VH CDR3 defined by the amino acid sequences of SEQ ID NO:1, SEQ ID NO:5 and SEQ ID NO:15, respectively. In another embodiment, the anti-Flt-1 antibody or antigen-binding fragment thereof comprises a VH chain, and the VH chain comprises VH CDR1, VH CDR2 and VH CDR3 defined by the amino acid sequences of SEQ ID NO:2, SEQ ID NO:6 and SEQ ID NO:16, respectively. In another embodiment, the anti-Flt-1 antibody or antigen-binding fragment thereof comprises a VH chain, and the VH chain comprises VH CDR1, VH CDR2 and VH CDR3 defined by the amino acid sequences of SEQ ID NO:2, SEQ ID NO:10 and SEQ ID NO:18, respectively. In another embodiment, the anti-Flt-1 antibody or antigen-binding fragment thereof comprises a VH chain, and the VH chain comprises VH CDR1 and VH CDR3 defined by the amino acid sequences of SEQ ID NO:2 and SEQ ID NO:17, respectively, and VH CDR2 defined by the amino acid sequence of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:13 or SEQ ID NO:14. In another embodiment, the anti-Flt-1 antibody or antigen-binding fragment thereof comprises a VH chain, and the VH chain comprises VH CDR1 and VH CDR3 defined by the amino acid sequences of SEQ ID NO:3 and SEQ ID NO:17, respectively, and VH CDR2 defined by the amino acid sequence of SEQ ID NO:9, SEQ ID NO:11 or SEQ ID NO:12. In another embodiment, the anti-Flt-1 antibody or antigen-binding fragment thereof comprises a VH chain, and the VH chain comprises VH CDR1, VH CDR2 and VH CDR3 defined by the amino acid sequences of SEQ ID NO:4, SEQ ID NO:9 and SEQ ID NO:17, respectively. In a specific embodiment, the anti-Flt-1 antibody or antigen-binding fragment thereof comprises a VH chain, and the VH chain comprises VH CDR1 defined by the amino acid sequence of SEQ ID NO:3, VH CDR2 defined by the amino acid sequence of SEQ ID NO:12, and VH CDR3 defined by the amino acid sequence of SEQ ID NO:17. In some embodiments, VH CDR3 is not SEQ ID NO:15.

[0186] In another embodiment, the anti-Flt-1 antibody or antigen-binding fragment thereof comprises a light chain VL region comprising an amino acid sequence having at least 60%, 70%, 80%, 90%, 95% or 98% identity to any one of SEQ ID NOs: 49 to 61; and / or a heavy chain VH region comprising an amino acid sequence having at least 60%, 70%, 80%, 90%, 95% or 98% identity to any one of SEQ ID NOs: 35 to 48. In a specific embodiment, the VL region comprises the amino acid sequence of SEQ ID NO: 60, and the VH region comprises the amino acid sequence of SEQ ID NO: 45. In another embodiment, the antibody further comprises a heavy chain constant region comprising an amino acid sequence having at least 60%, 70%, 80%, 90%, 95% or 98% identity to any one of SEQ ID NOs: 87 to 89. In some embodiments, the VL region is not SEQ ID NO: 49 or SEQ ID NO: 50. In some embodiments, the VH region is not SEQ ID NO: 35 or SEQ ID NO: 36.

[0187] In another embodiment, the anti-Flt-1 antibody or antigen-binding fragment thereof comprises a light chain comprising an amino acid sequence having at least 60%, 70%, 80%, 90%, 95% or 98% identity to any one of SEQ ID NOs: 75 to 86; and / or a heavy chain comprising an amino acid sequence having at least 60%, 70%, 80%, 90%, 95% or 98% identity to any one of SEQ ID NOs: 62 to 74. In a specific embodiment, the light chain comprises the amino acid sequence of SEQ ID NO: 76, and the heavy chain region comprises the amino acid sequence of SEQ ID NO: 71.

[0188] In some embodiments, the heavy chain of the anti-Flt-1 antibody or antigen-binding fragment thereof comprises the amino acid sequence

[0189] MGWSCIILFLVATATGVHSELQLVESGGGLVQPGGSLRLSCAASGF

[0190] TFSDYSASWVRQAPGKGLEWVSAISWSGDSTYYAESVKGRFTIFR

[0191] DNSKNTLYLQMNSLRAEDTAVYYCAKSWATPIESLYYYGSDYWG

[0192] QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV

[0193] TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC

[0194] NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPP

[0195] KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT

[0196] KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE

[0197] KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV

[0198] EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:108). In some embodiments, the heavy chain of the anti-Flt-1 antibody or its antigen-binding fragment comprises the amino acid sequence ELQLVESGGGLVQPGGSLRLSCAASGFTFSDYSASWVRQAPGKGLEWVSAISWSGDSTYYAESVKGRFTIFRDNSKNTLYLQMNSLRAEDTAVYYCAKSWATPIESLYYYGSDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:109).

[0199] In some embodiments, the light chain of the anti-Flt-1 antibody or its antigen-binding fragment comprises the amino acid sequence

[0200] MGWSCIILFLVATATGVHSSYELTQPLSVSVALRQAAKITCGGNNIG

[0201] SQTAQWYQQKPGQAPVLVIYANNRRPSGIPERFSGSKSGNTATLTIS

[0202] RAQAGDEADYYCQVWDASTQAIVFGGGTKLTVLGQPKAAPSVTL

[0203] FPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTT

[0204] PSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSX (SEQ ID NO:110).

[0205] In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof comprises a heavy chain of SEQ ID NO:108 or SEQ ID NO:109 and a light chain of SEQ ID NO:110 or SEQ ID NO:76.

[0206] In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof binds to human Flt-1 with an affinity greater than about 10 -7 M, greater than about 0.5x10 -7 M, greater than about 10 -8 M, greater than about 0.5x10 -8 M, greater than about 10 -9 M, greater than about 0.5x10 -9 M, greater than about 10 -10 M, greater than about 0.5x10 -10 M, greater than about 10 -11 M, greater than about 0.5x10 -11 M, greater than about 10 -12 M, or greater than about 0.5x10 -12 M. In other embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof binds to murine Flt-1 with an affinity greater than about 10 -7 M, greater than about 0.5x10 -7 M, greater than about 10 -8 M, greater than about 0.5x10 -8 M, greater than about 10 -9 M, greater than about 0.5x10 -9 M, greater than about 10 -10 M, greater than about 0.5x10 -10 M, greater than about 10 -11 M, greater than about 0.5x10 -11 M, greater than about 10 -12 M, or greater than about 0.5x10 -12 M. The affinity of the Flt-1 antibody can be measured, for example, in surface plasmon resonance assays such as BIACORE assays.

[0207] In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof is characterized by an IC 50Less than about 500 pM, less than about 400 pM, less than about 300 pM, less than about 200 pM, less than about 100 pM, less than about 50 pM, less than about 25 pM, less than about 10 pM, less than about 5 pM, or less than about 1 pM. In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof is characterized by an IC 50 Less than about 500 pM, less than about 400 pM, less than about 300 pM, less than about 200 pM, less than about 100 pM, less than about 50 pM, less than about 25 pM, less than about 10 pM, less than about 5 pM, or less than about 1 pM.

[0208] In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof inhibits the binding and / or activity of VEGF at the Flt-1 receptor. In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof is characterized by an IC for inhibition of the binding of VEGF to human Flt-1 in a competitive assay 50 Less than about 500 pM, less than about 400 pM, less than about 300 pM, less than about 200 pM, less than about 100 pM, less than about 50 pM, less than about 25 pM, less than about 10 pM, less than about 5 pM, or less than about 1 pM.

[0209] In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof competes with VEGF for binding to soluble Flt-1 and / or inhibits the binding of VEGF to soluble Flt-1. In other embodiments, said competition and / or inhibition is in a dose-dependent manner. In a specific embodiment, inhibition of the binding of VEGF to Flt-1 results in increased phosphorylation of VEGFR2. Without wishing to be bound by theory, binding of the anti-Flt-1 antibody or antigen-binding fragment thereof to Flt-1 inhibits the binding of VEGF to Flt-1. Unbound VEGF binds to VEGFR2, which can be demonstrated by measuring phosphorylation of VEGFR2. In a specific embodiment, the anti-Flt-1 antibody or antigen-binding fragment rescues VEGFR2 phosphorylation in a dose-dependent manner. For example, VEGFR2 phosphorylation can be rescued by at least about 100%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, or about 5%.

[0210] In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof provides a rescue of greater than about 95%, greater than about 90%, greater than about 85%, greater than about 80%, greater than about 75%, greater than about 70%, greater than about 65%, greater than about 60%, greater than about 55%, greater than about 50%, greater than about 45%, greater than about 40%, greater than about 35%, greater than about 30%, greater than about 25%, greater than about 20%, greater than about 15%, or greater than about 10% in a biological assay. In one specific embodiment, the biological assay comprises human primary venous endothelial cells (HUVECs) stimulated with VEGF in the presence of sFlt-1 and the anti-Flt-1 antibody or antigen-binding fragment thereof. VEGF-induced cell activation can be assayed by determining the phosphorylation status of the VEGF R2 receptor. In the presence of sFlt-1 alone (e.g., without the anti-Flt-1 antibody), the data can be expressed as the percentage of rescue of phosphorylation of the VEGF R2 receptor relative to the phosphorylation of the VEGF R2 receptor.

[0211] In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof has a half-life of greater than about 200 hours, greater than about 150 hours, greater than about 100 hours, greater than about 95 hours, greater than about 90 hours, greater than about 85 hours, greater than about 80 hours, greater than about 75 hours, greater than about 70 hours, greater than about 65 hours, greater than about 60 hours, greater than about 55 hours, greater than about 50 hours, or greater than about 45 hours and ranges therein. In some embodiments, the half-life is measured in mice.

[0212] In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof has a maximum serum concentration of greater than about 400 ug / mL, greater than 375 ug / mL, greater than about 350 ug / mL, greater than about 325 ug / mL, greater than about 300 ug / mL, greater than about 275 ug / mL, greater than about 250 ug / mL, greater than about 225 ug / mL, greater than about 200 ug / mL, greater than about 175 ug / mL, greater than about 150 ug / mL, greater than about 125 ug / mL, greater than about 100 ug / mL, greater than about 75 ug / mL, or greater than about 50 ug / mL and ranges therein. In some embodiments, the maximum serum concentration is measured in mice.

[0213] In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof selectively binds to Flt-1 and has minimal or no significant binding to other VEGF receptors. In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof selectively binds to Flt-1 and has minimal or no significant binding to VEGF R2 (Flk-1) and / or VEGF R3 (Flt-4).

[0214] In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof has a ka greater than about 1 x 10 -3 M -1 sec -1 、 greater than about 1 x 10 -4 M -1 sec -1 、 greater than about 1 x 10 -5 M -1 sec -1 、 greater than about 1 x 10 -6 M -1 sec -1 or greater than about 1 x 10 -7 M -1 sec -1 when binding to human Flt-1.

[0215] In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof has a kd greater than about 1 x 10 -3 sec -1 、 greater than about 1 x 10 -4 sec -1 、 greater than about 1 x 10 -5 sec -1 or greater than about 1 x 10 -6 sec -1 when binding to human Flt-1.

[0216] In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof has a K -8 greater than about 1 x 10 -9 M, greater than about 1 x 10 -10 M, greater than about 1 x 10 -11 M, greater than about 1 x 10 -12 M or greater than about 1 x 10 D M when binding to human Flt-1.

[0217] In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof binds to soluble Flt-1. In certain embodiments, the binding is dose-dependent, wherein higher concentrations of the antibody or antigen-binding fragment bind a greater amount of soluble Flt-1.

[0218] In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof has a percent human identity greater than about 99%, greater than about 98%, greater than about 97%, greater than about 96%, greater than about 95%, greater than about 94%, greater than about 93%, greater than about 92%, greater than about 91%, greater than about 90% or greater than about 80%.

[0219] In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof has a percent human homology greater than about 99%, greater than about 98%, greater than about 97%, greater than about 96%, greater than about 95%, greater than about 94%, greater than about 93%, greater than about 92%, greater than about 91%, greater than about 90% or greater than about 80%.

[0220] In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof binds to the Flt-1 protein. In some embodiments, the Flt-1 protein is a recombinant protein, such as recombinant sFlt-1. In one specific embodiment, the anti-Flt-1 antibody or antigen-binding fragment thereof binds to human Flt-1 isoform 1 (NP_002010.2 GI:156104876; SEQ ID NO:90) (Table 13). In another embodiment, the anti-Flt-1 antibody or antigen-binding fragment thereof binds to human Flt-1 isoform X1 (XP_011533316.1 GI:767977511; SEQ ID NO:91). In another embodiment, the anti-Flt-1 antibody or antigen-binding fragment thereof binds to human Flt-1 isoform 2 precursor (NP_001153392.1 GI:229892220; SEQ ID NO:92). In another embodiment, the anti-Flt-1 antibody or antigen-binding fragment thereof binds to human Flt-1 isoform 3 precursor (NP_001153502.1 GI:229892300; SEQ ID NO:93). In another embodiment, the anti-Flt-1 antibody or antigen-binding fragment thereof binds to human Flt-1 isoform 4 precursor (NP_001153503.1 GI:229892302; SEQ ID NO:94).

[0221] In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof binds to a specific epitope of the Flt-1 protein. For example, the anti-Flt-1 antibody or antigen-binding fragment thereof binds to the amino acid sequence provided in Table 3.

[0222] Table 3.

[0223]

[0224] In some embodiments, in vivo administration of an anti-Flt-1 antibody or an antigen-binding fragment thereof produces peak serum antibody levels of at least about 700 ug / mL, at least about 650 ug / mL, at least about 600 ug / mL, at least about 550 ug / mL, at least about 500 ug / mL, at least about 450 ug / mL, at least about 400 ug / mL, at least about 350 ug / mL, at least about 300 ug / mL, at least about 250 ug / mL, at least about 200 ug / mL, at least about 150 ug / mL, at least about 100 ug / mL, at least about 50 ug / mL, at least about 40 ug / mL, at least about 30 ug / mL, at least about 20 ug / mL, at least about 10 ug / mL, or at least about 5 ug / mL, and ranges therebetween. In some embodiments, the peak serum antibody levels are dose-dependent.

[0225] In some embodiments, in vivo administration of an anti-Flt-1 antibody or an antigen-binding fragment thereof produces trough serum antibody levels of at least about 450 ug / mL, at least about 400 ug / mL, at least about 350 ug / mL, at least about 300 ug / mL, at least about 250 ug / mL, at least about 200 ug / mL, at least about 150 ug / mL, at least about 100 ug / mL, at least about 50 ug / mL, or at least about 25 ug / mL, and ranges therebetween. In some embodiments, the trough serum antibody levels are dose-dependent.

[0226] In some embodiments, administration in vivo of an anti-Flt-1 antibody or an antigen-binding fragment thereof results in a reduced serum level of soluble Flt-1 compared to a baseline level or compared to the level in a subject administered a vehicle alone. Generally, the baseline level is measured just prior to administration. In some embodiments, administration of the anti-Flt-1 antibody or an antigen-binding fragment thereof results in a reduction in the serum level of soluble Flt-1 of at least about 95%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, or about 10% compared to the baseline serum level of soluble Flt-1 just prior to administration. In some embodiments, administration of the anti-Flt-1 antibody or an antigen-binding fragment thereof results in the serum level of soluble Flt-1 being reduced to less than about 4000 pg / mL, about 3500 pg / mL, about 3000 pg / mL, about 2500 pg / mL, about 2000 pg / mL, about 1750 pg / mL, about 1500 pg / mL, about 1250 pg / mL, about 1000 pg / mL, about 900 pg / mL, about 800 pg / mL, about 700 pg / mL, about 600 pg / mL, about 500 pg / mL, about 450 pg / mL, about 400 pg / mL, about 350 pg / mL, about 300 pg / mL, about 250 pg / mL, about 200 pg / mL, about 150 pg / mL, about 100 pg / mL, about 50 pg / mL, or about 10 pg / mL and ranges therein. In some embodiments, administration of the antibody or an antigen-binding fragment thereof results in a reduced serum level of soluble Flt-1 compared to the serum level of soluble Flt-1 in a subject not administered the anti-Flt-1 antibody or an antigen-binding fragment thereof. In some embodiments, the reduced serum level of soluble Flt-1 is dose-dependent.

[0227] In some embodiments, in vivo administration of an anti-Flt-1 antibody or antigen-binding fragment thereof results in an increase in the serum level of VEGF as compared to baseline levels or as compared to the levels in subjects treated with vehicle alone. Typically, baseline levels are measured immediately prior to treatment. In some embodiments, administration of an anti-Flt-1 antibody or antigen-binding fragment thereof results in an increase in the serum level of VEGF of at least about 95%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20% or about 10% as compared to the baseline serum level of VEGF just prior to administration. In some embodiments, administration of an anti-Flt-1 antibody or antigen-binding fragment thereof results in the serum level of VEGF increasing to more than about 500 pg / mL, about 450 pg / mL, about 400 pg / mL, about 350 pg / mL, about 300 pg / mL, about 250 pg / mL, about 200 pg / mL, about 150 pg / mL, about 100 pg / mL, about 50 pg / mL or about 25 pg / mL and ranges therein. In some embodiments, administration of an anti-Flt-1 antibody or antigen-binding fragment thereof results in an increased serum level of VEGF as compared to the serum level of VEGF in untreated subjects. In some embodiments, the increased serum level of VEGF is dose-dependent.

[0228] In some embodiments, in vivo administration of an anti-Flt-1 antibody or an antigen-binding fragment thereof results in increased angiogenesis in muscle tissue. In some embodiments, the muscle is skeletal muscle. In a specific embodiment, the muscle is the diaphragm, gastrocnemius, and / or tibialis anterior (TA) muscle. In some embodiments, the increased angiogenesis is demonstrated by increased CD31 staining of an endothelial cell marker such as CD31. In some embodiments, the increased staining can be measured, for example, by measuring the percentage of CD31-positive area in the muscle of a mouse administered with an anti-Flt-1 antibody or an antigen-binding fragment thereof. For example, the percentage of CD31-positive area in the diaphragm of a mouse administered with an anti-Flt-1 antibody or an antigen-binding fragment thereof can be at least about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, or about 2.5% of the total tissue area. In another example, the percentage of CD31-positive area in the TA muscle of a mouse administered with an anti-Flt-1 antibody or an antigen-binding fragment thereof can be at least about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, or about 1.0% of the total tissue area. In a specific embodiment, the percentage of CD31-positive area in the diaphragm or TA muscle of a mouse administered with an anti-Flt-1 antibody can be significantly higher than the percentage of CD31-positive area in the diaphragm or TA muscle of a mouse administered with an isotype control antibody.

[0229] In some embodiments, an increase in staining of an endothelial cell marker can be measured, for example, by measuring the normalized percentage of CD31 positivity in the muscle of a mouse administered an anti-Flt-1 antibody or an antigen-binding fragment thereof. In a specific embodiment, the increase in CD31 staining in the muscle of a mouse administered an anti-Flt-1 antibody or an antigen-binding fragment thereof is relative to the CD31 staining measured in the muscle of a mouse administered an isotype control antibody. For example, the normalized percentage of CD31 positivity in the diaphragm muscle of a mouse administered an anti-Flt-1 antibody or an antigen-binding fragment thereof can be at least about 200%, about 190%, about 180%, about 170%, about 160%, about 150%, about 140%, about 130%, about 120%, or about 110%. In another example, the normalized percentage of CD31 positivity in the TA muscle of a mouse administered an anti-Flt-1 antibody or an antigen-binding fragment thereof can be at least about 300%, about 290%, about 280%, about 270%, about 260%, about 250%, about 240%, about 230%, about 220%, about 210%, about 200%, about 190%, about 180%, about 170%, about 160%, about 150%, about 140%, about 130%, about 120%, or about 110% and ranges therein.

[0230] In some embodiments, the anti-Flt-1 antibody or an antigen-binding fragment thereof selectively binds to human Flt-1 and has minimal or no significant binding to other mammalian Flt-1 receptors (e.g., binding affinity less than 10 -7 M or 10 -6 M). In some embodiments, the anti-Flt-1 antibody or an antigen-binding fragment thereof selectively binds to human Flt-1 and does not bind to cynomolgus monkey Flt-1. In some embodiments, the anti-Flt-1 antibody or an antigen-binding fragment thereof selectively binds to human Flt-1 and does not bind to mouse Flt-1.

[0231] In some embodiments, the anti-Flt-1 antibody or an antigen-binding fragment thereof binds to human Flt-1 and cynomolgus monkey Flt-1. In some embodiments, the anti-Flt-1 antibody or an antigen-binding fragment thereof binds to cynomolgus monkey Flt-1. In some embodiments, the anti-Flt-1 antibody or an antigen-binding fragment thereof binds to human Flt-1 and mouse Flt-1.

[0232] In some embodiments, the anti-Flt-1 antibody or an antigen-binding fragment thereof is selected from the group consisting of IgG, F(ab’)2, F(ab)2, Fab’, Fab, ScFv, diabody, triabody, and tetrabody.

[0233] In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof is IgG. In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof is IgG1.

[0234] Engineered constant region

[0235] In some embodiments, a suitable anti-Flt-1 antibody contains an Fc domain or a portion thereof that binds to the FcRn receptor. As a non-limiting example, a suitable Fc domain can be derived from an immunoglobulin subclass such as IgG. In some embodiments, the suitable Fc domain is derived from IgG1, IgG2, IgG3, or IgG4. Particularly suitable Fc domains include those derived from human or humanized antibodies.

[0236] Improved binding between the Fc domain and the FcRn receptor is expected to result in an extended serum half-life. Thus, in some embodiments, a suitable Fc domain (SEQ ID NO: 104) contains one or more amino acid mutations that result in improved binding to FcRn. Various mutations within the Fc domain that affect improved binding to FcRn are known in the art and can be suitable for practicing the present invention. In some embodiments, the suitable Fc domain contains one or more mutations at one or more positions corresponding to Leu 234, Leu 235, Gly 237, Thr 250, Met 252, Ser 254, Thr 256, Thr 307, Glu 380, Met 428, His 433, and / or Asn 434 of human IgG1.

[0237] Some mutations in the Fc domain result in reduced binding of IgG to the FcRn receptor and thus inhibit effector function. In some embodiments, a suitable Fc domain (SEQ ID NO: 104) contains one or more mutations at one or more positions corresponding to Leu 234, Leu 235, and Gly 237 of human IgG1. In one specific embodiment, Leu 234 is mutated to Ala. In another embodiment, Leu 235 is mutated to Ala. In another embodiment, Gly 237 is mutated to Ala.

[0238] In some embodiments, the anti-FLT-1 antibody or antigen-binding fragment contains a spacer and / or is linked to another entity. In some embodiments, the linker or spacer contains GAP GGGGGAAAAAGGGGG GAP(SEQ ID NO:105) (GAG linker) has a sequence with at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identity. In some embodiments, the linker or spacer comprises a sequence that is G AP GGGGGAAAAAGGGGG GAP GGGGGAAAAAGGGGG GAP (SEQ ID NO:106) (GAG2 linker) has a sequence with at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identity. In some embodiments, the linker or spacer comprises a sequence that is GAP GGGGGAAAAAGGGGG GAP GGGGGAAAAAGGGGG GAP GG GGGAAAAAGGGGG GAP (SEQ ID NO:107) (GAG3 linker) has a sequence with at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identity.

[0239] Generation of anti-Flt-1 antibodies and antigen-binding fragments

[0240] The recombinant anti-Flt-1 antibodies or antigen-binding fragments suitable for the present invention can be produced by any available means. For example, the recombinant anti-Flt-1 antibodies or antigen-binding fragments can be recombinantly produced by using a host cell system engineered to express a nucleic acid encoding the recombinant anti-Flt-1 antibody or antigen-binding fragment.

[0241] Accordingly, the present invention further provides polynucleotide sequences encoding the various amino acid sequences described herein. In some embodiments, the present invention provides a polynucleotide sequence encoding the amino acid sequence of the heavy or light chain of an anti-Flt-1 antibody described herein (e.g., any one of SEQ ID NOs: 62-86 or SEQ ID NOs: 108-110). In some embodiments, the present invention provides a polynucleotide sequence encoding the variable region of the amino acid sequence of the heavy or light chain of an anti-Flt-1 antibody described herein (e.g., any one of SEQ NOs: 35-61). In some embodiments, the present invention provides a polynucleotide sequence encoding the CDR region of the amino acid sequence of the heavy or light chain of an anti-Flt-1 antibody described herein (e.g., any one of SEQ ID NOs: 1-34). In some embodiments, the present invention provides a polynucleotide sequence encoding the constant region amino acid sequence of an anti-Flt-1 antibody described herein (e.g., any one of SEQ ID NOs: 87-89). In some embodiments, the present invention provides a polynucleotide sequence encoding the Fc region amino acid sequence of an anti-Flt-1 antibody described herein (e.g., SEQ ID NO: 104). In some embodiments, the present invention provides a polynucleotide sequence encoding the linker amino acid sequence of an anti-Flt-1 antibody described herein (e.g., SEQ ID NOs: 105-107).

[0242] In some embodiments, the polynucleotide sequence encoding the amino acid sequence of the heavy chain, light chain, variable region, CDR region, Fc region or linker region of an anti-Flt-1 antibody further comprises a sequence encoding a signal peptide. As a non-limiting example, a suitable signal peptide comprises the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 111).

[0243] The various polynucleotide sequences described herein can be embodied in various vector systems for expressing a recombinant anti-Flt-1 antibody or an antigen-binding fragment thereof.

[0244] Any expression system can be used when producing an antibody recombinantly. By way of giving some examples, known expression systems include, for example, eggs, baculovirus, plants, yeast or mammalian cells.

[0245] In some embodiments, the recombinant anti-Flt-1 antibody or an antigen-binding fragment thereof suitable for the present invention is produced in mammalian cells. Non-limiting examples of mammalian cells that can be used according to the present invention include the BALB / c mouse myeloma line (NSO / l, ECACC No.: 85110503); human retinoblasts (PER.C6, CruCell, Leiden, The Netherlands); and the simian kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651).

[0246] In some embodiments, the present invention provides a recombinant anti-Flt-1 antibody or antigen-binding fragment thereof produced by human cells. In some embodiments, the present invention provides an anti-Flt-1 antibody or antigen-binding fragment thereof produced by CHO cells.

[0247] A pharmaceutical composition comprising the antibody of the present invention

[0248] The present invention further provides a pharmaceutical composition comprising a therapeutically active ingredient according to the present invention (such as an anti-Flt-1 antibody or antigen-binding fragment thereof) together with one or more pharmaceutically acceptable carriers or excipients. Such pharmaceutical compositions may optionally comprise one or more additional therapeutically active substances.

[0249] Although the description of the pharmaceutical compositions provided herein mainly relates to pharmaceutical compositions suitable for administration to humans on a doctor's prescription, those skilled in the art will understand that such compositions are generally suitable for administration to all kinds of animals. Altering a pharmaceutical composition suitable for administration to humans so that the composition is suitable for administration to various animals is well known, and an ordinary veterinary pharmacologist can design and / or carry out such alterations by merely ordinary experimentation, if any.

[0250] The formulations of the pharmaceutical compositions described herein can be prepared by any method known or hereafter developed in the field of pharmacology. Generally, such preparation methods include the steps of associating the active ingredient with a diluent or another excipient or carrier and / or one or more other auxiliary ingredients, and then, if necessary and / or desired, shaping and / or packaging the product into the desired single-dose or multi-dose unit.

[0251] The pharmaceutical compositions according to the present invention can be prepared, packaged, and / or sold in a single unit dose and / or in large batches of multiple single unit doses. As used herein, "unit dose" is a discrete amount of a pharmaceutical composition that contains a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dose of the active ingredient to be administered to a subject and / or a suitable fraction of such dose, such as half or one-third of such dose.

[0252] The relative amounts of the active ingredient, pharmaceutically acceptable excipient or carrier, and / or any additional ingredients in the pharmaceutical compositions according to the present invention will vary depending on the identity, size, and / or condition of the subject to be treated and further depending on the route by which the composition is to be administered. For example, the composition may contain between 0.1% and 100% (w / w) of the active ingredient.

[0253] The pharmaceutical preparation may additionally contain pharmaceutically acceptable excipients or carriers. Excipients or carriers as used herein include any and all solvents, dispersion media, diluents or other liquid vehicles, dispersing or suspending aids, surfactants, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, etc. that are suitable for the specific dosage form desired. Remington’s The Science and Practice of Pharmacy, 21st Edition, A.R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006; which is incorporated herein by reference) discloses various excipients for formulating pharmaceutical compositions and known techniques for their preparation. The use of such excipients or carriers is encompassed within the scope of the present invention, except for any conventional excipient medium or carrier that is incompatible with the substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a harmful manner with any other component of the pharmaceutical composition.

[0254] In some embodiments, the pharmaceutically acceptable excipient or carrier is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% pure. In some embodiments, the excipient or carrier is approved for human and veterinary use. In some embodiments, the excipient or carrier is approved by the U.S. Food and Drug Administration. In some embodiments, the excipient or carrier is of pharmaceutical grade. In some embodiments, the excipient or carrier meets the standards of the United States Pharmacopeia (USP), European Pharmacopeia (EP), British Pharmacopeia, and / or International Pharmacopeia.

[0255] Pharmaceutically acceptable excipients or carriers used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersants and / or granulating agents, surfactants and / or emulsifying agents, disintegrants, binders, preservatives, buffering agents, lubricants and / or oils. Such excipients or carriers may optionally be included in the pharmaceutical composition. Excipients or carriers such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening agents, flavoring agents, and / or aromatic agents may be present in the composition, depending on the judgment of the formulator.

[0256] Suitable pharmaceutically acceptable excipients or carriers include, but are not limited to, water, salt solutions (e.g., NaCl), saline, buffered saline, alcohols, glycerol, ethanol, gum arabic, vegetable oils, benzyl alcohol, polyethylene glycol, gelatin, carbohydrates (such as lactose, amylose or starch), sugars (such as mannitol, sucrose, or others), dextrose, magnesium stearate, talc, silicic acid, viscous paraffin, aromatic oils, fatty acid esters, hydroxypropylmethyl cellulose, polyvinylpyrrolidone, etc., and combinations thereof. If desired, the pharmaceutical formulation may be admixed with adjuvants (such as lubricants, preservatives, stabilizers, wetting agents, emulsifying agents, salts affecting osmotic pressure, buffers, coloring agents, flavoring agents and / or aromatic substances, etc.), which do not produce a harmful reaction with the active compound or interfere with its activity. In a preferred embodiment, a water-soluble carrier suitable for intravenous administration is used.

[0257] If desired, the suitable pharmaceutical composition or medicament may also contain small amounts of wetting agents or emulsifying agents or pH buffers. The composition may be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation or powder. The composition may also be formulated as a suppository with conventional binders and carriers such as triglycerides. Oral formulations may contain standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharin, cellulose, magnesium carbonate, etc.

[0258] The pharmaceutical composition or medicament may be formulated according to conventional procedures into a pharmaceutical composition suitable for administration to humans. For example, in some embodiments, the composition for intravenous administration is typically a solution in a sterile isotonic aqueous buffer. If necessary, the composition may also contain solubilizing agents and local anesthetics to alleviate the pain at the injection site. Generally, the ingredients are supplied separately or mixed together in unit dosage forms, such as in the form of dry lyophilized powders or anhydrous concentrates in airtight sealed containers such as ampoules or cartridges indicating the amount of the active agent. When the composition is to be administered by infusion, it may be dispensed in an infusion bottle containing sterile pharmaceutical grade water, saline or dextrose / water. When the composition is administered by injection, ampoules of sterile water for injection or saline may be provided so that the ingredients can be mixed before administration.

[0259] General considerations in the formulation and / or manufacture of medicaments can be found, for example, in Remington: The Science and Practice of Pharmacy, 21st Edition, Lippincott Williams & Wilkins, 2005 (incorporated herein by reference).

[0260] Route of Administration

[0261] The anti-Flt-1 antibody or antigen-binding fragment thereof described herein (or a composition or medicament comprising the anti-Flt-1 antibody or antigen-binding fragment thereof described herein) is administered by any suitable route. In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment protein or a pharmaceutical composition comprising the anti-Flt-1 antibody or antigen-binding fragment protein is administered parenterally. Parenteral administration can be intravenous, intradermal, intrathecal, inhaled, transdermal (topical), intraocular, intramuscular, subcutaneous, intramuscular and / or transmucosal administration. In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof or a pharmaceutical composition comprising the anti-Flt-1 antibody or antigen-binding fragment thereof is administered subcutaneously. As used herein, the term "subcutaneous tissue" is defined as the layer of loose, irregular connective tissue directly under the skin. For example, subcutaneous administration can be carried out by injecting the composition into an area including but not limited to the thigh area, abdominal area, buttock area or scapular area. In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof or a pharmaceutical composition comprising the anti-Flt-1 antibody or antigen-binding fragment thereof is administered intravenously. In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof or a pharmaceutical composition comprising the anti-Flt-1 antibody or antigen-binding fragment thereof is administered orally. If desired, more than one route can be used simultaneously.

[0262] In some embodiments, the administration produces only a local effect in the individual, while in other embodiments, the administration produces an effect in multiple parts of the individual, such as a systemic effect. Generally, the administration results in the delivery of the anti-Flt-1 antibody or antigen-binding fragment to one or more target tissues, including but not limited to the kidney, liver, brain, spinal cord, intestine, eye, lung, spleen, heart (including cardiac muscle, striated muscle and smooth muscle).

[0263] In some embodiments, the striated muscle is selected from the group consisting of: triceps, tibialis anterior, soleus, gastrocnemius, quadriceps and diaphragm.

[0264] In some embodiments, the smooth muscle is the muscle lining blood vessels, bronchioles, bladder and gastrointestinal tract such as the rectum.

[0265] Dosage Forms and Administration Regimens

[0266] In some embodiments, the composition is administered in a therapeutically effective amount and / or according to an administration regimen associated with a particular desired outcome (e.g., treating muscular dystrophy such as Duchenne muscular dystrophy or reducing the risk thereof).

[0267] The specific dose or amount to be administered according to the present invention can vary, for example, depending on the nature and / or extent of the desired result, depending on details of the route and / or timing of administration, and / or depending on one or more characteristics (such as body weight, age, personal history, genetic characteristics, lifestyle parameters, severity of heart defect and / or risk level of heart defect, etc., or a combination thereof). Such dose or amount can be determined by a person of ordinary skill in the art. In some embodiments, an appropriate dose or amount is determined according to standard clinical techniques. Alternatively or additionally, in some embodiments, an appropriate dose or amount is determined by using one or more in vitro or in vivo assays to assist in identifying a desirable or optimal dose range or amount to be administered.

[0268] In various embodiments, an anti-Flt-1 antibody or an antigen-binding fragment thereof is administered in a therapeutically effective amount. Generally, a therapeutically effective amount is sufficient to achieve a meaningful benefit to the subject (such as treating, modulating, curing, preventing, and / or improving a underlying disease or condition). In some specific embodiments, an appropriate dose or amount to be administered can be extrapolated from a dose-response curve derived from in vitro or animal model test systems.

[0269] In some embodiments, the provided composition is provided as a pharmaceutical formulation. In some embodiments, the pharmaceutical formulation is or comprises a unit dose for administration according to a dosing regimen related to achieving a reduction in the incidence or risk of muscular dystrophy such as Duchenne muscular dystrophy.

[0270] In some embodiments, a formulation comprising the anti-Flt-1 antibody or antigen-binding fragment described herein is administered as a single dose. In some embodiments, a formulation comprising the anti-Flt-1 antibody or antigen-binding fragment described herein is administered at regular time intervals. As used herein, administering at a certain "time interval" indicates administering a therapeutically effective amount periodically (as distinct from a single-dose). The time interval can be determined by standard clinical techniques. In some embodiments, a formulation comprising the anti-Flt-1 antibody or antigen-binding fragment described herein is administered once every two months, once a month, twice a month, once every three weeks, once every two weeks, once a week, twice a week, three times a week, once a day, twice a day, or once every six hours. The time interval for administration for an individual is not necessarily a fixed time interval, but can vary over time according to the needs of the individual. In a specific embodiment, the anti-Flt-1 antibody or its antigen-binding fragment is administered twice a week.

[0271] As used herein, the term "bi-monthly" means administered once every two months (i.e., once every two months); the term "monthly" means administered once every month; the term "every three weeks" means administered once every three weeks (i.e., once every three weeks); the term "every two weeks" means administered once every two weeks (i.e., once every two weeks); the term "weekly" means administered once every week; and the term "daily" means administered once every day.

[0272] In some embodiments, the formulations comprising the anti-Flt-1 antibodies or antigen-binding fragments described herein are administered at irregular time intervals. In some embodiments, the formulations comprising the anti-Flt-1 antibodies or antigen-binding fragments described herein are administered for a defined period of time at regular time intervals.

[0273] As described herein, the term "therapeutically effective amount" is largely determined based on the total amount of the therapeutic agent contained in the pharmaceutical composition of the present invention. The therapeutically effective amount is typically administered in a dosing regimen that may comprise multiple unit doses. For any particular composition, the therapeutically effective amount (and / or the appropriate unit dose within the effective dosing regimen) may vary, for example, depending on the route of administration or combination with other agents.

[0274] In some embodiments, the anti-Flt-1 antibody or its antigen-binding fragment is administered at a dose in the range of about 0.1 mg / kg to about 50 mg / kg. In other embodiments, the anti-Flt-1 antibody or its antigen-binding fragment is administered at a dose in the range of about 0.1 mg / kg to about 40 mg / kg, about 0.1 mg / kg to about 30 mg / kg, about 0.1 mg / kg to about 20 mg / kg, about 0.1 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 5 mg / kg, about 0.1 mg / kg to about 3 mg / kg, about 0.1 mg / kg to about 1 mg / kg, about 1.0 mg / kg to about 40 mg / kg, about 1.0 mg / kg to about 30 mg / kg, about 1.0 mg / kg to about 20 mg / kg, about 1.0 mg / kg to about 10 mg / kg, about 1.0 mg / kg to about 5 mg / kg, or about 1.0 mg / kg to about 3 mg / kg. In specific embodiments, the anti-Flt-1 antibody or its antigen-binding fragment is administered at a dose of about 1.0 mg / kg, about 3.0 mg / kg, about 10 mg / kg, or about 20 mg / kg.

[0275] In some embodiments, administration of an anti-Flt-1 antibody or an antigen-binding fragment thereof reduces the intensity, severity, or frequency of at least one DMD sign or symptom or delays its onset. In some embodiments, administration of an anti-Flt-1 antibody or an antigen-binding fragment thereof reduces the intensity, severity, or frequency of at least one DMD sign or symptom or delays its onset, and the DMD sign or symptom is selected from the group consisting of: muscle wasting, skeletal deformity, cardiomyopathy, muscle ischemia, cognitive impairment, and impaired respiratory function.

[0276] In some embodiments, administration of an anti-Flt-1 antibody or an antigen-binding fragment thereof improves clinical outcomes as measured by, for example, a 6-minute walk test, quantitative muscle strength tests, timed motor performance tests. Brooke and Vignos limb function scales, pulmonary function tests (forced vital capacity, forced expiratory volume in 1 second, peak expiratory flow rate, maximum inspiratory and expiratory pressures), health-related quality of life, knee and elbow flexors, elbow extensors, shoulder abduction, grip strength, time to rise from supine position, North Star Ambulatory Assessment, timed 10-meter walk / run, Egen-Klassification scale, Gowers score, Hammersmith motor ability, hand held myometry, range of motion, goniometry, hypercapnia, Nayley Scales of Infant and Toddler Development, and / or caregiver burden scale.

[0277] Combination therapy

[0278] In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof is administered in combination with one or more additional therapeutic agents. In one embodiment, the additional therapeutic agent is a corticosteroid, such as prednisone. In another embodiment, the additional therapeutic agent is a glucocorticoid, such as deflazacort. In another embodiment, the additional therapeutic agent is follistatin or a recombinant protein thereof. In another embodiment, the additional therapeutic agent is an RNA regulatory therapeutic agent. The RNA regulatory therapeutic agent can be an exon skipping therapeutic agent or a gene therapy. The RNA regulatory therapeutic agent can be, for example, Drispersen, PRO044, PRO045, Eteplirsen (AVI-4658), SRP-4053, SRP-4045, SRP-4050, SRP-4044, SRP-4052, SRP-4055, or SRP-4008. In some embodiments, the additional therapeutic agent is currently used to treat muscular dystrophy. In other embodiments, the additional therapeutic agent can also be used to treat other diseases or disorders. In some embodiments, the known therapeutic agent is administered according to its standard or approved dosing regimen and / or schedule. In some embodiments, the known therapeutic agent is administered according to a regimen that is altered compared to its standard or approved dosing regimen and / or schedule. In some embodiments, such an altered regimen is different from the standard or approved method of administration in that the amount of one or more unit doses is altered (e.g., decreased or increased), and / or in that the frequency of administration is altered (e.g., in that one or more time intervals between unit doses are extended, resulting in a lower frequency, or one or more time intervals between unit doses are decreased, resulting in a higher frequency).

[0279] Embodiment

[0280] The present invention can be carried out by the following embodiments:

[0281] Embodiment 1. An antibody or antigen-binding fragment thereof that specifically binds to human Flt-1, the antibody or antigen-binding fragment thereof comprising one or more complementarity determining regions (CDRs) selected from the group consisting of:

[0282] Variable light (VL) chain CDR1, which is defined by an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 19 to 21;

[0283] VL CDR2, which is defined by an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 22 to 24;

[0284] VL CDR3, which is defined by an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 25 to 34;

[0285] Variable heavy (VH) chain CDR1, which is defined by an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 1 to 4;

[0286] VH CDR2, which is defined by an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 5 to 14; and

[0287] VH CDR3, which is defined by an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 15 to 18.

[0288] Embodiment 2. The antibody or antigen-binding fragment thereof according to Embodiment 1, wherein one or more of the CDRs comprise the VL CDR3, which is defined by an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 25 to 34; and the VH CDR3, which is defined by an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 15 to 18.

[0289] Embodiment 3. The antibody or antigen-binding fragment thereof according to Embodiment 1 or 2, wherein one or more of the CDRs comprise the VL CDR1, which is defined by an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 19 to 21; the VL CDR2, which is defined by an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 22 to 24; and the VL CDR3, which is defined by an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 25 to 34.

[0290] Embodiment 4. The antibody or antigen-binding fragment thereof according to any one of the foregoing embodiments, wherein one or more of the CDRs comprise the VH CDR1, which is defined by an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 1 to 4; the VH CDR2, which is defined by an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 5 to 14; and the VH CDR3, which is defined by an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 15 to 18.

[0291] Embodiment 5. The antibody or antigen-binding fragment thereof according to any one of the foregoing embodiments, which comprises a VL chain, and the VL chain comprises the VL CDR1, the VL CDR2, and the VL CDR3, which are defined by the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 22, and SEQ ID NO: 25, respectively.

[0292] Embodiment 6. The antibody or antigen-binding fragment thereof according to any one of Embodiments 1-4, which comprises a VL chain, and the VL chain comprises the VL CDR1, the VL CDR2, and the VL CDR3, which are defined by the amino acid sequences of SEQ ID NO: 20, SEQ ID NO: 23, and SEQ ID NO: 25, respectively.

[0293] Embodiment 7. The antibody or antigen-binding fragment thereof according to any one of Embodiments 1-4, which comprises a VL chain, and the VL chain comprises the VL CDR1 and the VL CDR2, which are defined by the amino acid sequences of SEQ ID NO: 21 and SEQ ID NO: 24, respectively, and the VL CDR3, which is defined by the amino acid sequence of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 34.

[0294] Embodiment 8. The antibody or antigen-binding fragment thereof according to any one of Embodiments 1-4, wherein the VL chain comprises the VL CDR1, the VL CDR2, and the VL CDR3, which are defined by the amino acid sequences of SEQ ID NO: 21, SEQ ID NO: 24, and SEQ ID NO: 32, respectively.

[0295] Embodiment 9. An antibody or antigen-binding fragment thereof as described in any of the preceding embodiments, which comprises a VH chain, and the VH chain comprises the VH CDR1, the VH CDR2, and the VH CDR3 defined by the amino acid sequences of SEQ ID NO:1, SEQ ID NO:5, and SEQ ID NO:15, respectively.

[0296] Embodiment 10. An antibody or antigen-binding fragment thereof as described in any of Embodiments 1-8, which comprises a VH chain, and the VH chain comprises the VH CDR1, the VH CDR2, and the VH CDR3 defined by the amino acid sequences of SEQ ID NO:2, SEQ ID NO:6, and SEQ ID NO:16, respectively.

[0297] Embodiment 11. An antibody or antigen-binding fragment thereof as described in any of Embodiments 1-8, which comprises a VH chain, and the VH chain comprises the VH CDR1, the VH CDR2, and the VH CDR3 defined by the amino acid sequences of SEQ ID NO:2, SEQ ID NO:10, and SEQ ID NO:18, respectively.

[0298] Embodiment 12. An antibody or antigen-binding fragment thereof as described in any of Embodiments 1-8, which comprises a VH chain, and the VH chain comprises the VH CDR1 and the VH CDR3 defined by the amino acid sequences of SEQ ID NO:2 and SEQ ID NO:17, respectively, and the VH CDR2 defined by the amino acid sequence of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:13, or SEQ ID NO:14.

[0299] Embodiment 13. An antibody or antigen-binding fragment thereof as described in any of Embodiments 1-8, which comprises a VH chain, and the VH chain comprises the VH CDR1 and the VH CDR3 defined by the amino acid sequences of SEQ ID NO:3 and SEQ ID NO:17, respectively, and the VH CDR2 defined by the amino acid sequence of SEQ ID NO:9, SEQ ID NO:11, or SEQ ID NO:12.

[0300] Embodiment 14. An antibody or antigen-binding fragment thereof as described in any of Embodiments 1-8, which comprises a VH chain, and the VH chain comprises the VH CDR1, the VH CDR2, and the VH CDR3 defined by the amino acid sequences of SEQ ID NO:4, SEQ ID NO:9, and SEQ ID NO:17, respectively.

[0301] Embodiment 15. An antibody or antigen-binding fragment thereof as described in any one of Embodiments 1-8, which comprises a VH chain, and the VH chain comprises the VH CDR1, the VH CDR2, and the VH CDR3 defined by the amino acid sequences of SEQ ID NO:3, SEQ ID NO:12, and SEQ ID NO:17, respectively.

[0302] Embodiment 16. An antibody or antigen-binding fragment thereof that specifically binds to human Flt-1, which comprises: (i) a light chain variable (VL) region that comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NO:49 to SEQ ID NO:61; and / or (ii) a heavy chain variable (VH) region that comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NO:35 to SEQ ID NO:48.

[0303] Embodiment 17. The antibody or antigen-binding fragment thereof as described in Embodiment 16, wherein the VL region comprises the amino acid sequence of SEQ ID NO:60 and the VH region comprises the amino acid sequence of SEQ ID NO:45.

[0304] Embodiment 18. The antibody as described in any one of the foregoing embodiments, wherein the antibody further comprises a heavy chain constant region that comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NO:87 to SEQ ID NO:89.

[0305] Embodiment 19. An antibody or antigen-binding fragment thereof that specifically binds to human Flt-1, which comprises: (i) a light chain that comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NO:75 to SEQ ID NO:86; and / or (ii) a heavy chain that comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NO:62 to SEQ ID NO:74.

[0306] Embodiment 20. The antibody or antigen-binding fragment thereof as described in Embodiment 19, wherein the light chain comprises the amino acid sequence of SEQ ID NO:76 and the heavy chain comprises the amino acid sequence of SEQ ID NO:71.

[0307] Embodiment 21. The antibody or antigen-binding fragment thereof as described in any one of the foregoing embodiments, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of: IgG, F(ab’)2, F(ab)2, Fab’, Fab, ScFv, diabody, triabody, and tetrabody.

[0308] Embodiment 22. The antibody or antigen-binding fragment thereof as described in Embodiment 21, wherein the antibody or antigen-binding fragment thereof is IgG.

[0309] Embodiment 23. The antibody or antigen-binding fragment thereof as described in Embodiment 22, wherein the antibody or antigen-binding fragment thereof is IgG1.

[0310] Embodiment 24. The antibody or antigen-binding fragment thereof as described in any one of the foregoing embodiments, wherein the antibody or antigen-binding fragment thereof is a monoclonal antibody.

[0311] Embodiment 25. The antibody or antigen-binding fragment thereof as described in any one of the foregoing embodiments, wherein the antibody or antigen-binding fragment thereof is a humanized monoclonal antibody.

[0312] Embodiment 26. The antibody or antigen-binding fragment thereof as described in Embodiment 25, wherein the humanized monoclonal antibody comprises a human Fc region.

[0313] Embodiment 27. The antibody or antigen-binding fragment thereof as described in Embodiment 26, wherein the Fc region comprises one or more mutations that enhance the binding affinity between the Fc region and the FcRn receptor so as to prolong the in vivo half-life of the antibody.

[0314] Embodiment 28. The antibody or antigen-binding fragment thereof as described in any one of the foregoing embodiments, wherein the Fc region comprises one or more mutations at positions corresponding to Leu 234, Leu 235, and / or Gly 237 of human IgG1.

[0315] Embodiment 29. The antibody or antigen-binding fragment thereof as described in any one of the foregoing embodiments, wherein the antibody or antigen-binding fragment thereof does not bind to VEGFR2 and / or VEGFR3.

[0316] Embodiment 30. The antibody or antigen-binding fragment thereof as described in any one of the foregoing embodiments, wherein the antibody or antigen-binding fragment thereof does not bind to murine or simian Flt-1.

[0317] Embodiment 31. An isolated antibody or antigen-binding fragment thereof that recognizes a peptide or a fragment thereof comprising the following amino acid sequence, said amino acid sequence corresponding to positions 139 to 148, positions 139 to 153, positions 178 to 206, positions 199 to 204, and positions 128 to 138 of SEQ ID NO: 90.

[0318] Embodiment 32. The isolated antibody or antigen-binding fragment thereof according to Embodiment 31, wherein the peptide consists of the following amino acid sequence, said amino acid sequence corresponding to positions 130 to 138, positions 141 to 148, positions 141 to 153, and positions 193 to 206 of SEQ ID NO: 90.

[0319] Embodiment 33. An isolated antibody or antigen-binding fragment thereof that competes with the antibody or antigen-binding fragment thereof according to any one of the foregoing embodiments.

[0320] Embodiment 34. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of the foregoing embodiments and a pharmaceutically acceptable carrier.

[0321] Embodiment 35. A polynucleotide that encodes a CDR, VL region, VH region, light chain, and / or heavy chain of the antibody or antigen-binding fragment thereof according to any one of Embodiments 1-33.

[0322] Embodiment 36. An expression vector comprising the polynucleotide according to Embodiment 35.

[0323] Embodiment 37. A host cell comprising the polynucleotide according to Embodiment 35 or the expression vector according to Embodiment 36.

[0324] Embodiment 38. A method for preparing an antibody or antigen-binding fragment thereof that specifically binds to human Flt-1, said method comprising culturing the host cell according to Embodiment 37.

[0325] Embodiment 39. A hybridoma cell that produces the antibody or antigen-binding fragment thereof according to any one of Embodiments 1-33.

[0326] Embodiment 40. A method for treating an Flt-1-mediated disease, disorder, or condition, said method comprising administering to a subject in need thereof the antibody or antigen-binding fragment thereof according to any one of Embodiments 1-33.

[0327] Embodiment 41. The method according to Embodiment 40, wherein the Flt-1-mediated disease, disorder, or condition is Duchenne muscular dystrophy, Becker muscular dystrophy, preeclampsia, or chronic kidney disease.

[0328] Embodiment 42. A method for treating Duchenne muscular dystrophy (DMD), the method comprising:

[0329] administering to a subject having or at risk of DMD an effective amount of an antibody or antigen-binding fragment thereof as described in any one of Embodiments 1-35, such that at least one symptom or feature of DMD is reduced or the onset is delayed in terms of intensity, severity, or frequency.

[0330] Embodiment 43. The method according to Embodiment 42, wherein the method further comprises administering to the subject one or more additional therapeutic agents.

[0331] Embodiment 44. The method according to Embodiment 43, wherein the one or more additional therapeutic agents are selected from the group consisting of prednisone, deflazacort, follistatin, RNA regulatory therapeutic agents, exon skipping therapeutic agents, and gene therapy.

[0332] Embodiment 45. The method according to any one of Embodiments 42-44, wherein the antibody or antigen-binding fragment thereof is administered parenterally.

[0333] Embodiment 46. The method according to Embodiment 45, wherein the parenteral administration is selected from intravenous, intradermal, intrathecal, inhalation, transdermal (topical), intraocular, intramuscular, subcutaneous, and / or transmucosal administration.

[0334] Embodiment 47. The method according to Embodiment 46, wherein the parenteral administration is intravenous administration.

[0335] Embodiment 48. The method according to Embodiment 46, wherein the parenteral administration is subcutaneous administration.

[0336] Embodiment 49. The method according to any one of Embodiments 42-48, wherein the antibody or antigen-binding fragment thereof is administered once daily, twice weekly, once weekly, or once monthly.

[0337] Embodiment 50. The method according to Embodiment 49, wherein the antibody or antigen-binding fragment thereof is administered twice weekly.

[0338] Embodiment 51. The method according to any one of Embodiments 42-48, wherein the effective amount of the antibody or antigen-binding fragment thereof is a dose of about 1 mg / kg to 50 mg / kg.

[0339] Embodiment 52. The method according to Embodiment 51, wherein the dose is about 1 mg / kg.

[0340] Embodiment 53. The method according to embodiment 51, wherein the dose is about 3 mg / kg.

[0341] Embodiment 54. The method according to embodiment 51, wherein the dose is about 10 mg / kg.

[0342] Embodiment 55. The method according to any one of embodiments 42-54, wherein the administration of the antibody or its antigen-binding fragment results in reduced fibrosis and / or necrosis relative to a control.

[0343] Embodiment 56. The method according to any one of embodiments 42-54, wherein the administration of the antibody or its antigen-binding fragment results in improved angiogenesis in the muscle of the subject relative to a control.

[0344] Embodiment 57. The method according to embodiment 56, wherein the improved angiogenesis is reflected by an increase in blood flow in muscle pathology, an increase in VEGF levels in serum, a decrease in creatine kinase (CK) levels in serum, an increased CD31 score measured by IHC, and / or a decrease in sFlt-1 levels in serum.

[0345] Embodiment 58. The method according to any one of embodiments 42-54, wherein the administration of the antibody or its antigen-binding fragment results in improved muscle function relative to a control.

[0346] Embodiment 59. The method according to embodiment 58, wherein the improved muscle function is reflected by improved muscle strength and / or fatigue resistance.

[0347] Embodiment 60. A method of treating tissue fibrosis, the method comprising administering to a subject in need thereof an effective amount of an antibody or its antigen-binding fragment according to any one of embodiments 1-33. Example

[0348] Example 1. Generation and Characterization of a High-Affinity Anti-Flt-1 Antibody

[0349] Antibody production

[0350] Monoclonal antibodies against soluble Flt-1 were generated using the llama monoclonal antibody method. Briefly, llamas were immunized with recombinant human soluble Flt-1 (purchased from ABCAM) and sera were collected.

[0351] Antibody characterization

[0352] Antibodies that bind to human and murine Flt-1 were further characterized with respect to 1) VH family; 2) affinity for Flt-1; 3) IC50; 4) dissociation rate screening by Biacore assay; 5) cross-reactivity with cynomolgus monkey Flt-1 and 6) binding to VEGF R2 and VEGF R3. As shown in Table 4, candidate antibodies against human Flt-1 (hFl-1) and murine Flt-1 (mFlt-1) were characterized.

[0353] Table 4.

[0354]

[0355] The pharmacokinetic properties of antibodies 13B4 and 10G12 were studied in mice by intravenous administration of 10 mg / kg of each antibody (Table 5). The data demonstrated that antibody 10G12 could not be detected for more than 288 hours, while antibody 13B4 could be detected at 672 hours ( Figures 1A - 1B ).

[0356] Table 5.

[0357]

[0358] In vivo efficacy of antibody

[0359] Mdx mice (i.e., a mouse model of Duchenne muscular dystrophy) were treated with 20 mg / kg of antibody 13B4 or antibody 10G12 by intravenous administration twice a week starting at 4 weeks of age for one month. Control mice were treated with vehicle only, an isotype control antibody that does not bind to Flt-1, or a commercial anti-Flt-1 antibody called Flt-1:VEGF antagonist (Angio Proteomie, catalog number AP-MAB0702). To evaluate the serum antibody concentration at the trough exposure point, blood was collected 4 days after the fifth intravenous dose. To evaluate the serum antibody concentration at the peak exposure point, blood was collected 24 hours after the last dose. The peak and trough concentrations of antibodies 13B4 and 10G12 are shown in Figure 2A and 2B . The concentrations of free antibody 13B4 and free antibody 10G12 in blood at the peak and trough exposure time points were higher than those of the isotype control antibody and the commercial control antibody.

[0360] To evaluate serum free sFlt-1 levels and VEGF levels, blood was collected 24 hours after the fifth intravenous dose and before sacrifice. Administration of antibody 13B4, antibody 10G12, and the commercial control antibody significantly reduced the serum concentration of sFlt-1 compared to the isotype control antibody (p < 0.0001) ( Figure 3)。Administration of antibody 13B4 and antibody 10G12 resulted in a significant increase in serum VEGF levels compared to the isotype control antibody (p < 0.001)( Figure 4 )。Administration of the commercial control antibody also resulted in a significant increase in blood levels of VEGF compared to the isotype control antibody (p < 0.05)( Figure 4 )。

[0361] Histopathology

[0362] Mice were sacrificed at the end of the 30-day treatment period, and the diaphragm and tibialis anterior (TA) muscles were harvested and sectioned to determine whether the anti-Flt-1 antibodies induced angiogenesis in skeletal muscle. Muscle sections were stained with the endothelial cell marker CD31. A significant increase in capillary density was observed in the diaphragms of mice treated with antibody 13B4, 10G12, or the commercial control antibody compared to the diaphragms of mice treated with the isotype control antibody( Figures 5A - 5D )。Data were quantified using automated quantitative imaging software as shown in Figures 6A - 6B . The CD31-positive area was significantly increased in the diaphragms of mice treated with the commercial control antibody (p < 0.05), antibody 13B4 (p < 0.01), and antibody 10G12 (p < 0.0001) compared to the diaphragms of mice treated with the isotype control antibody. A significant increase in the CD31-positive area was also demonstrated in the tibialis anterior (TA) muscles of mice treated with antibody 10G12 compared to the tibialis anterior muscles of mice treated with the isotype control antibody (p < 0.01).

[0363] This study demonstrated that administration of Flt-1 antibodies (e.g., 10G12 and 13B4) to mdx mice resulted in a significant increase in endothelial cell proliferation, a decrease in soluble Flt-1 in serum, and an increase in serum VEGF concentration. These antibodies demonstrated binding affinities in the pM range for the Flt-1 target (see Table 4), an IC50 for Flt-1 binding of less than 100 pM (see Table 4), and greater than 50% rescue of VEGF signal transduction in a biological assay.

[0364] Example 2. Generation and Characterization of High-Affinity Anti-Flt-1 Antibodies

[0365] Additional anti-Flt-1 monoclonal antibodies were generated as described above. These antibodies were further characterized for their binding affinity to the sFlt-1 antigen (by ELISA and Biacore); competition for VEGF in an sFlt-1:VEGF competitive ELISA; and performance in a cell-based assay.

[0366] Antibody characterization - Binding to target

[0367] Determination of the binding of monoclonal anti-Flt-1 antibodies to recombinant sFlt-1 antigen in an ELISA assay( Figure 7 ). All antibodies demonstrated a dose-dependent increase in binding. The binding affinity of anti-Flt-1 antibodies for murine and human Flt-1 antigens was measured by surface plasmon resonance methodology (i.e., Biacore) (Table 6). Antibodies that bound to human Flt-1 in the nanomolar range together with antibody 11A11 demonstrated the highest binding affinity for human Flt-1. Biacore analysis also demonstrated that the antibodies did not cross-react with VEGF R2 or VEGF R3 (Table 6), however all antibodies did cross-react with cynomolgus monkey Flt-1.

[0368] Table 6.

[0369]

[0370] Antibody characterization - Competition / antagonism

[0371] To estimate the potency of the antibodies, the antibodies were assayed in a competitive ELISA using human sFlt-1 and VEGF. The antibody concentrations tested ranged from 0.1 mg / mL to 10,000 ng / mL. A commercial anti-Flt-1 antibody served as a control. All antibodies except 11A11 were able to block the binding of VEGF to sFlt-1( Figure 8 ).

[0372] Antibody characterization - Cell - based assay

[0373] Human primary venous endothelial cells (HUVEC) were stimulated with VEGF in the presence of sFlt-1 and monoclonal antibodies 02G07, 11A11 and 13B4. VEGF-induced cell activation was assayed by determining the phosphorylation status of the VEGF R2 receptor. In the presence of sFlt-1 alone (e.g., without anti-Flt-1 antibody), the data was expressed as the percentage rescue of phosphorylation of the VEGFR2 receptor relative to the phosphorylation of the VEGF R2 receptor. Monoclonal antibodies rescued cell activation (i.e., phosphorylation) by antagonizing soluble Flt-1( Figure 9 ).

[0374] Example 3. Characterization of high affinity anti-Flt-1 antibodies generated by light chain shuffling

[0375] Light chain shuffling of antibodies 18B6, 11A11 and 13B4 described in Example 2 was performed to improve the affinity and potency of the candidate antibodies.

[0376] Antibody characterization - Binding to target

[0377] The resulting antibodies exhibit increased affinity for the Flt-1 antigen. For example, the K D of antibody 21C6 is D approximately 10-fold higher than the K D of the parental antibody 11A11. Similarly, the K D of antibody 21B3 is approximately 5-fold higher than the K

[0378] of the parental antibody 13B4 (Table 7).

[0379]

[0380] Antibody characterization - Competition / antagonism

[0381] To estimate the potency of the antibodies, after light chain shuffling, the antibodies were assayed in a competitive ELISA using human sFlt-1 and VEGF. The antibody concentrations tested were in the range of 0.2 mg / mL to 200 ng / mL. The ability of the parental antibody 13B4 to competitively bind sFlt-1 was compared with that of the antibodies generated by light chain shuffling. All antibodies demonstrated dose-dependent inhibition of the binding of VEGF to human sFlt-1, with clone 21B3 being the most effective competitor ( Figure 10 ).

[0382] In vivo efficacy

[0383] To determine the serum half-life and pharmacokinetic characteristics of the light chain shuffled antibodies, single 10 mg / kg doses of the light chain shuffled antibodies 27H9 and 21B3 and the parental antibody 13B4 (each labeled with I 125 ) were administered to mice. Serum was collected at 0.083, 0.25, 0.5, 1, 4, 8, and 24 hours and at 3, 5, 7, 14, 21, and 28 days, and the serum concentration of the antibodies was determined. The serum half-life was reduced in the light chain shuffled antibodies compared to the parental antibody ( Figure 11 ). However, the light chain shuffled antibodies showed improved pharmacodynamic characteristics compared to the parental. For example, antibody 27H9 reached a maximum concentration of 222.4 μg / mL at 0.083 hours, while the parental antibody 13B4 reached a maximum concentration of 217 μg / mL at 0.5 hours (Table 8).

[0384] Table 8.

[0385]

[0386] Histopathology

[0387] To determine whether the light chain reshuffled antibodies and parental antibodies were able to induce endothelial cell proliferation, mdx mice were treated with 20 mg / kg antibody intravenously every two weeks for 4 weeks. At the end of the treatment period, the mice were sacrificed, and the diaphragm and tibialis anterior muscles were harvested and sectioned to determine whether the antibodies induced angiogenesis in skeletal muscle. Muscle sections were stained with the endothelial cell marker CD31. A significant increase in capillary density in the diaphragm was observed in mice treated with antibodies 13B4 and 21B3 compared to the diaphragm of mice treated with the isotype control antibody( Figures 12A - 12C ). In addition, a significant increase in capillary density in the tibialis anterior muscle from mice treated with the light chain reshuffled antibody 21B3 was observed compared to the tibialis anterior muscle from mice treated with the isotype control antibody( Figures 12D - 12F ).

[0388] The biodistribution of antibodies 27H9, 13B4, and 21B3 in the diaphragm, tibialis, and gastrocnemius muscles was determined using 125 I-labeled antibodies. The diaphragm showed the highest exposure to all antibodies over the time course( Figures 13A - 13C ).

[0389] These studies demonstrated that administration of Flt-1 antibodies (i.e., 13B4 and 21B3) to mdx mice resulted in a significant increase in endothelial cell proliferation.

[0390] Example 4. In Vivo Efficacy of High Affinity Anti-Flt-1 Antibody 21B3

[0391] Mdx mice were treated with 1, 3, 10, or 20 mg / kg antibody 21B3 or 20 mg / kg isotype control antibody by intravenous administration twice a week starting at 4 weeks of age for one month. To evaluate serum antibody concentrations at the trough exposure point, blood was collected 4 days after the fifth intravenous dose. To evaluate blood antibody concentrations at the peak exposure point, blood was collected 24 hours after the last dose. The peak and trough serum concentrations of antibody 21B3 and the isotype control antibody are shown in Figure 14A and 14B . The peak and trough levels of antibody 21B3 were dose-dependent and higher than those of the isotype control antibody.

[0392] To evaluate serum levels of free sFlt-1, blood was collected on days 0, 14, and 28. Administration of antibody 21B3 induced a dose-dependent decrease in serum free sFlt-1 levels. As observed on days 14 and 28, the response was more persistent at 10 and 20 mg / kg doses. However, a statistically significant decrease in free sFlt-1 levels was observed at 3, 10, and 20 mg / kg doses compared to free sFlt-1 levels in mice treated with vehicle alone( Figure 15 ).

[0393] To evaluate the serum levels of VEGF, blood was collected on day 0, day 14, and day 28. Administration of antibody 21B3 induced a dose-dependent increase in serum VEGF levels. As observed for free sFlt-1 levels, the response was more persistent at the 10 and 20 mg / kg doses and at days 14 and 28. Indeed, a statistically significant increase in serum VEGF was observed at the 10 and 20 mg / kg doses of antibody 21B3 compared to the serum VEGF levels in mice treated with vehicle alone( Figure 16 ).

[0394] Histopathology

[0395] At the end of the 30-day treatment period, the mice were sacrificed, and the diaphragm and tibialis anterior muscles were collected and sectioned to determine whether the anti-Flt-1 antibody induced angiogenesis in skeletal muscle. Muscle sections were stained for the endothelial cell marker CD31. A significant increase in capillary density was observed in the diaphragm of mice treated with antibody 21B3 compared to the capillary density in the diaphragm of mice treated with isotype control antibody( Figures 17A - 17E ). Data were quantified using the automated quantitative imaging software as shown in Figure 18 . A significant increase in the CD31-positive area was present in the diaphragm of mice treated with 10 mg / kg or 20 mg / kg compared to the CD31-positive area in the diaphragm of mice treated with isotype control antibody (p<0.0001).

[0396] A significant increase in capillary density was also observed in the tibialis anterior muscle of mice treated with antibody 21B3 compared to the tibialis anterior muscle in mice treated with isotype control antibody( Figures 19A - 19E ). Data were quantified using the automated quantitative imaging software as shown in Figure 20 . A significant increase in the CD31-positive area was present in the tibialis anterior muscle of mice treated with 10 mg / kg or 20 mg / kg compared to the CD31-positive area in the tibialis anterior muscle of mice treated with isotype control antibody (p<0.0001).

[0397] RP - LC / MS characterization

[0398] The molecular weight of deglycosylated 21B3 antibody was determined by reverse-phase liquid chromatography / mass spectrometry (RP-LC / MS)( Figure 21A ). After reduction, the molecular weights of the light and heavy chains were determined. The glycosylation pattern of the heavy chain was also determined( Figure 21B ).

[0399] These results demonstrate that administration of the Flt-1 antibody (i.e., 21B3) to mdx mice results in a significant increase in endothelial cell proliferation, a decrease in soluble Flt-1 in serum, and an increase in the concentration of VEGF in serum.

[0400] Example 5. Characterization of a Humanized High-Affinity Anti-Flt-1 Antibody

[0401] The light chain reshuffled antibodies described in Example 3 were further modified to introduce sequence variations in the CDR regions and / or the Fc effector regions. These antibodies were evaluated by surface plasmon resonance methods (e.g., Biacore) to determine the binding characteristics (Table 9). Antibody 27H9 NG / NAAAA exhibits approximately a 2-fold reduced binding affinity for Flt-1.

[0402] Table 9.

[0403]

[0404]

[0405] The ability of the antibodies to rescue cell activation by antagonizing sFlt-1 was also evaluated in a cell-based assay. Human primary venous endothelial cells (HUVECs) were stimulated with VEGF in the presence of sFlt-1 and monoclonal antibodies. VEGF-induced cell activation was determined by ascertaining the phosphorylation status of the VEGF R2 receptor. Monoclonal antibodies rescued cell activation (i.e., phosphorylation) by antagonizing soluble Flt-1 ( Figure 22A ), and antibody 27H9 NG / NAAAA (NA+AAA) had similar potency compared to the unmutated parental antibody (wt).

[0406] Example 6. Antibody Optimization

[0407] Candidate antibodies were analyzed to identify the closest human VH and VL germline sequences and to identify the different residues within the framework regions within the CDRs and within the oxidation / isomerization sites. A Fab library containing human and wild-type residues was constructed and fused to human constant domains. Phage display was applied to identify Fabs with the same or better dissociation rate as the parental antibody 21B3 (i.e., no loss of affinity). The Fabs with the desired dissociation rate were sequenced and compared to the human germline, and those with the highest identity (e.g., VH+VL identity > 95%) and homology (e.g., > 96% homology) were selected for conversion into human monoclonal antibodies. The Fabs were also analyzed for unwanted amino acids. Table 10 provides the Fabs ranked by the percentage of human identity, with some clones having up to 97.6% human identity and 98.8% homology.

[0408] Table 10.

[0409]

[0410] Example 7. Characterization of Anti-Flt-1 Monoclonal Antibodies

[0411] The heat resistance of monoclonal antibodies was analyzed using the Biacore method. At a concentration of 100 μg / mL, each monoclonal antibody was incubated in phosphate-buffered saline at different temperatures for 1 hour. After 1 hour of incubation, the antibody was slowly cooled to 25°C within two hours and then incubated overnight at 4°C. Then, the percentage of functional antibody was measured by binding to human Flt-1 using Biacore (see Table 11). The heat resistance of the wild-type antibody was consistent with previous experiments. However, mutations in the VH or VL regions reduced the melting temperature by approximately 2°C, except for the 27H6 DG / DA clone. The AAA mutation in the Fc region had no effect on the heat resistance of the antibody.

[0412] Table 11.

[0413]

[0414]

[0415] The binding affinity of humanized clones was analyzed by Biacore (Table 12). The ability of antibodies 27H4, 27H6, and 27H9 to rescue VEGF signaling was determined in a VEGF:sFlt-1 cell-based assay ( Figure 22B ). Briefly, human primary venous endothelial cells (HUVECs) were stimulated with VEGF in the presence of sFlt-1 and monoclonal antibodies 27H4, 27H6, and 27H9. VEGF-induced cell activation was determined by determining the phosphorylation status of the VEGF R2 receptor. In the presence of sFlt-1 alone (e.g., without anti-Flt-1 antibody), the data were expressed as the percentage of rescue of phosphorylation of the VEGF R2 receptor relative to the phosphorylation of the VEGF R2 receptor. The ability of antibodies 27H4, 27H6, and 27H9 to antagonize the binding of VEGF and sFlt-1 was also determined by ELISA ( Figure 23 ).

[0416] Table 12.

[0417] ka (1 / Ms) kd (1 / s) Rmax (RU) KD (M) 21B3 5.4E+05 1.7E-04 292 3.2E-10 27H4 3.4E+05 8.2E-05 305 .5E-10 27H9 3.3E+05 8.4E-05 323 2.6E-10 27H6 9.1E+05 1.4E-04 436 1.6E-10

[0418] Example 8. In Vitro Study of Anti-Flt-1 Antibodies on Muscle Pathology

[0419] Mdx mice were treated with the anti-Flt-1 antibody 21B3 at 1, 3, or 10 mg / kg or an IgG1 isotype control antibody at 10 mg / kg by intravenous administration twice a week starting at 3 weeks of age for 6 or 12 weeks.

[0420] To assess serum levels of free antibody concentration, blood was collected from mice 4 days after the intravenous doses administered at weeks 2, 4, 7, and 10. Terminal samples were collected 24 hours after the last dose ( Figure 24 ). At the 10 mg / kg dose, there were statistically significant differences in serum levels of free antibody at all time points compared to the serum levels of free antibody in mice receiving the isotype control antibody. At the 3 mg / kg dose, there were statistically significant differences in serum levels of free antibody at weeks 4, 7, and 10 and at terminal time compared to the serum levels of free antibody in mice receiving the isotype control antibody. At the 1 mg / kg dose, there was a statistically significant difference in serum levels of free antibody at terminal time compared to the serum levels of free antibody in mice receiving the isotype control antibody.

[0421] To assess serum levels of free sFlt-1, blood was collected from mice 4 days after the intravenous doses administered at weeks 2, 4, 7, and 10. Terminal samples were collected 24 hours after the last dose ( Figure 25 ). At the 10 mg / kg dose, there were statistically significant differences in serum levels of free sFlt-1 at all time points compared to the serum levels of free sFlt-1 in mice receiving the isotype control antibody. At the 3 mg / kg dose, there were statistically significant differences in serum levels of free sFlt-1 at weeks 4, 7, and 10 and at terminal time compared to the serum levels of free sFlt-1 in mice receiving the isotype control antibody. At the 1 mg / kg dose, there was a statistically significant difference in serum levels of free sFlt-1 at terminal time compared to the serum levels of free sFlt-1 in mice receiving the isotype control antibody.

[0422] To assess serum levels of VEGF, blood was collected from mice 4 days after the intravenous doses administered at weeks 2, 4, 7, and 10. Terminal samples were collected 24 hours after the last dose ( Figure 26) Administration of antibody 21B3 induced a dose-dependent increase in serum VEGF levels. At a dose of 10 mg / kg, there were statistically significant differences in serum VEGF levels at all time points compared to the serum levels of VEGF in mice receiving the isotype control antibody. At a dose of 3 mg / kg, there were statistically significant differences in serum VEGF levels at week 7 and at the time of sacrifice compared to the serum levels of VEGF in mice receiving the isotype control antibody. At a dose of 1 mg / kg, there was a statistically significant difference in serum VEGF levels at the time of sacrifice compared to the serum levels of VEGF in mice receiving the isotype control antibody.

[0423] Histopathology

[0424] Mice were sacrificed at weeks 6 and 12 of the treatment period, and the diaphragm, gastrocnemius, and tibialis anterior muscles were collected and sectioned to determine whether treatment with anti-Flt-1 antibody induced angiogenesis and prevented fibrosis and necrosis in skeletal muscle.

[0425] Angiogenesis

[0426] Muscle sections were stained for the endothelial cell marker CD31 ( Figures 27A - 27H , 28A-28H, and 29A-29H). A significant increase in capillary density was observed in all muscle groups studied in mice treated with antibody 21B3 compared to the muscles of mice treated with the isotype control antibody. Data were quantified using automated quantitative imaging software. There were statistically significant increases in the CD31-positive area in the diaphragm of mice treated with 3 mg / kg (p < 0.01) and 10 mg / kg (p < 0.0001) of antibody 21B3 at weeks 6 and 12 compared to the CD31-positive area in the diaphragm of mice treated with the isotype control antibody. There was a statistically significant increase in the CD31-positive area in the gastrocnemius of mice treated with 10 mg / kg of antibody 21B3 at weeks 6 and 12 (p < 0.05) compared to the CD31-positive area in the gastrocnemius of mice treated with the isotype control antibody. There were statistically significant increases in the CD31-positive area in the tibialis anterior muscle of mice treated with 3 mg / kg of antibody 21B3 at week 6 (p < 0.05) and week 12 (p < 0.0001) and in mice treated with 10 mg / kg (p < 0.0001) of antibody 21B3 at weeks 6 and 12 compared to the CD31-positive area in the tibialis anterior muscle of mice treated with the isotype control antibody. ( Figures 30A - 30C )。

[0427] Fibrosis

[0428] Sections of muscle were also stained immunohistochemically for type I collagen ( Figures 31A - 31H 32A - 32H and 33A - 33H). A significant decrease in type I collagen staining was observed in the diaphragm and gastrocnemius muscles of mice treated with antibody 21B3 compared to those of mice treated with isotype control antibody. Compared to type I collagen staining in the diaphragm of mice treated with isotype control antibody, there were statistically significant decreases in type I collagen staining in the diaphragm of mice treated with 1 mg / kg (p < 0.0001), 3 mg / kg (p < 0.001), and 10 mg / kg (p < 0.0001) of antibody 21B3 at 12 weeks. Compared to type I collagen staining in the gastrocnemius muscle of mice treated with isotype control antibody, there were statistically significant decreases in type I collagen staining in the gastrocnemius muscle of mice treated with 1 mg / kg (p < 0.01), 3 mg / kg (p < 0.05), and 10 mg / kg (p < 0.001) of antibody 21B3 at 12 weeks.( Figures 34A - 34C ).

[0429] Necrosis

[0430] The percentage of necrosis present in the gastrocnemius muscle of mice treated with 21B3 antibody was determined relative to the percentage of necrosis present in the gastrocnemius muscle of mice treated with isotype control antibody. A trend towards improved necrosis was observed( Figures 35A - 35B ).

[0431] Example 9. Mapping of epitopes on human sFlt - 1 targeted by anti - Flt - 1 antibodies 21B3 and 21C6

[0432] Peptide - level epitopes on human sFlt - 1 targeted by anti - human sFlt - 1 monoclonal antibodies (mAbs) 21B3 and 21C6 were mapped by hydrogen - deuterium exchange (HDX) mass spectrometry.

[0433] Pepsin digestion and LC - MS

[0434] For pepsin digestion, 10 μg of sFlt-1 or a mixture of sFlt-1 and antibody (21B3) (10 μg:20 μg) or a mixture of sFlt-1 and antibody (21C6) (10 μg:20 μg) was denatured in 0.365 M TCEP and 1.7 M guanidine hydrochloride (pH 2.5). The mixture was subjected to on-line pepsin digestion, and the resulting peptides were analyzed using a UPLC-MS system consisting of a Waters Acquity UPLC coupled to a MicroTOF-Q2 mass spectrometer (Bruker). The peptides were separated on a 50 mm x 1 mm C8 column with a 19-minute gradient of 5%-28.5% solvent B (0.1% formic acid in acetonitrile). Solvent A was 0.1% formic acid in water. The solvent mixing valve, injection valve, C8 column, and all connecting stainless steel tubes were immersed in a refrigerated circulating water bath maintained at 0 °C. Peptide identification was performed by searching the MS / MS data against the sFlt-1 sequence using Mascot. The mass tolerances for precursor and product ions were 0.1 Da and 0.2 Da, respectively.

[0435] Deglycosylation treatment

[0436] 200 μg of human sFlt-1 recombinant protein was incubated with 10 μl of PNGase F at 37 °C for 4 hours.

[0437] Fab preparation

[0438] Fabs were prepared from two anti-sFlt-1 mAbs (21B3 and 21C6) by papain digestion and protein A capture using a Pierce Fab Preparation Kit.

[0439] Size - exclusion chromatography (SEC)

[0440] To examine the binding between native or deglycosylated human sFlt-1 and two anti-human sFlt-1 mAbs (21B3 and 21C6) on SEC, 10 μg of sFlt-1 (native or deglycosylated) was mixed with 40 μg of anti-sFlt-1 mAb. Native or deglycosylated sFlt-1 alone, anti-sFlt-1 mAb alone, or the complex was injected into an SEC column with PBS as the mobile phase at a flow rate of 0.35 ml / min and the proteins were monitored at 280 nm. The Fabs generated from anti-sFlt-1 mAbs (21B3 and 21C6) and the binding between anti-sFlt-1 Fab and sFlt-1 were also evaluated using SEC.

[0441] HDX

[0442] Incubate 10 μL of human sFlt-1 (10 μg) or a mixture of sFlt-1 and mAb (21B3) (10 μg:20 μg) or a mixture of sFlt-1 and mAb (21C6) (10 μg:20 μg) with 90 μL of deuterium oxide-labeling buffer (50 mM phosphate, 100 mM sodium chloride, pH 7.4) for 0 s, 30 s, 2 min, 10 min, 1 h, or 4 h. Quench deuterium exchange by adding 100 μL of 3.4 M guanidine hydrochloride, 0.73 M TCEP buffer (final pH 2.5), and then perform the pepsin digestion and LC-MS analysis as described above. Record mass spectra in the MS-only mode. Process the raw MS data using HDExaminer software (Sierra Analytics, CA). Calculate the deuterium level using the average mass difference between the deuterated peptide and its native form (t0).

[0443] Results

[0444] To verify that glycan removal does not alter the binding of human sFlt-1 to the antibodies, native and deglycosylated sFlt-1 proteins were mixed with anti-human sFlt-1 IgG (21B3 and 21C6), and complex formation was monitored on size-exclusion chromatography. The data demonstrated that native human sFlt-1 bound fully to both anti-human sFlt-1 IgG (21B3 and 21C6), while deglycosylated human sFlt-1 did not bind fully to anti-human sFlt-1 mAb (21B3) or did not bind to anti-human sFlt-1 mAb (21C6), indicating that deglycosylation disrupted the interaction between human sFlt-1 and the antibodies. Therefore, native human sFlt-1 was selected for the HD exchange experiment. Due to the non-uniform glycosylation and high complexity of the 12 N-linked glycosylation sites, native human sFlt-1 initially showed poor sequence coverage. To improve the sequence coverage, the glycan masses at each glycosylation site were identified, and high sequence coverage (85.2%) was achieved for native human sFlt-1.

[0445] Native human sFlt-1 alone or in complex with anti-human sFlt-1 mAb (21B3) or anti-human sFlt-1 mAb (21C6) was incubated in deuterium oxide. Deuterium exchange was carried out at room temperature for 0 sec, 30 sec, 2 min, 10 min, 60 min, and 240 min. The exchange reaction was quenched by low pH and the protein was digested with pepsin. Deuterium levels at identified peptides were monitored from mass shifts on LC-MS. Deuterium accumulation curves over the exchange time were plotted for all peptides. Although most human sFlt-1 peptides exhibited the same or similar deuterium levels with or without anti-human sFlt-1 mAbs (21B3 and 21C6), several peptide segments had significantly reduced deuterium incorporation upon binding of mAb 21B3 or mAb 21C6. Residues 117 - 129 (corresponding to amino acids 141 - 153 of SEQ ID NO:90) and 169 - 182 (corresponding to amino acids 193 - 206 of SEQ ID NO:90) experienced strong deuterium protection upon binding to anti-human sFlt-1 mAb 21B3, while residues 106 - 114 (corresponding to amino acids 130 - 138 of SEQ ID NO:90) and 117 - 124 (corresponding to amino acids 141 - 148 of SEQ ID NO:90) experienced strong deuterium protection upon binding to anti-human sFlt-1 mAb 21C6. These strongly protected regions were designated as epitope peptides for anti-human sFlt-1 mAbs (21B3 and 21C6) and were highlighted in blue in the differential heat maps shown in Figure 36 and Figure 37 The MS / MS spectra of the identified peptides containing amino acid residues from the epitope regions are shown in Figures 38A - 38E Peptide 115 - 124 corresponds to amino acids 139 - 148 of SEQ ID NO:90; peptide 115 - 129 corresponds to amino acids 139 - 153 of SEQ ID NO:90; peptide 154 - 182 corresponds to amino acids 178 - 206 of SEQ ID NO:90; peptide 175 - 180 corresponds to amino acids 119 - 204 of SEQ ID NO:90; and peptide 104 - 114 corresponds to amino acids 128 - 138 of SEQ ID NO:90.

[0446] Conclusions

[0447] An 85.2% sequence coverage was achieved for human sFlt-1. Residues 117 - 129 and 169 - 180 experienced strong deuterium protection upon binding to anti-human sFlt-1 mAb 21B3, while residues 106 - 114 and 117 - 124 experienced strong deuterium protection upon binding to anti-human sFlt-1 mAb 21C6. These strongly protected regions were designated as epitope peptides for the respective anti-human sFlt-1 mAbs.

[0448] Equivalent Cases and Scope

[0449] Those skilled in the art will recognize or be able to determine many equivalent cases of the specific embodiments of the invention described herein using only routine experimentation. The scope of the present invention is not intended to be limited to the above description, but rather as set forth in the following claims.

[0450] Table 13.

[0451]

[0452]

[0453]

[0454] Table 14.

[0455]

Claims

1. A polynucleotide encoding an anti-Flt-1 antibody or an antigen-binding fragment thereof, characterized in that comprising complementarity determining regions (CDRs) selected from the group consisting of: (a) a variable light (VL) chain CDR1 defined by the amino acid sequence of SEQ ID NO:21, a VL CDR2 defined by the amino acid sequence of SEQ ID NO:24, a VL CDR3 defined by the amino acid sequence of SEQ ID NO:26, a variable heavy (VH) chain CDR1 defined by the amino acid sequence of SEQ ID NO:2, a VH CDR2 defined by the amino acid sequence of SEQ ID NO:6, and a VH CDR3 defined by the amino acid sequence of SEQ ID NO:16; (b) a VL CDR1 defined by the amino acid sequence of SEQ ID NO:21, a VL CDR2 defined by the amino acid sequence of SEQ ID NO:24, a VL CDR3 defined by the amino acid sequence of SEQ ID NO:27, a VH CDR1 defined by the amino acid sequence of SEQ ID NO:2, a VH CDR2 defined by the amino acid sequence of SEQ ID NO:7, and a VH CDR3 defined by the amino acid sequence of SEQ ID NO:17; (c) a VL CDR1 defined by the amino acid sequence of SEQ ID NO:21, a VL CDR2 defined by the amino acid sequence of SEQ ID NO:24, a VL CDR3 defined by the amino acid sequence of SEQ ID NO:26, a VH CDR1 defined by the amino acid sequence of SEQ ID NO:3, a VH CDR2 defined by the amino acid sequence of SEQ ID NO:12, and a VH CDR3 defined by the amino acid sequence of SEQ ID NO:17; (d) a VL CDR1 defined by the amino acid sequence of SEQ ID NO:21, a VL CDR2 defined by the amino acid sequence of SEQ ID NO:24, a VL CDR3 defined by the amino acid sequence of SEQ ID NO:28, a VH CDR1 defined by the amino acid sequence of SEQ ID NO:2, a VH CDR2 defined by the amino acid sequence of SEQ ID NO:8, and a VH CDR3 defined by the amino acid sequence of SEQ ID NO:17; or (e) a VL CDR1 defined by the amino acid sequence of SEQ ID NO:21, a VL CDR2 defined by the amino acid sequence of SEQ ID NO:24, a VL CDR3 defined by the amino acid sequence of SEQ ID NO:32, a VH CDR1 defined by the amino acid sequence of SEQ ID NO:3, a VH CDR2 defined by the amino acid sequence of SEQ ID NO:12, and a VH CDR3 defined by the amino acid sequence of SEQ ID NO:

17.

2. The polynucleotide according to claim 1, wherein the anti-Flt-1 antibody or antigen-binding fragment thereof comprises: The VL CDR1 defined by the amino acid sequence of SEQ ID NO:21, The VL CDR2 defined by the amino acid sequence of SEQ ID NO:24, The VL CDR3 defined by the amino acid sequence of SEQ ID NO:26, The VH CDR1 defined by the amino acid sequence of SEQ ID NO:2, The VH CDR2 defined by the amino acid sequence of SEQ ID NO:6, and The VH CDR3 defined by the amino acid sequence of SEQ ID NO:

16.

3. The polynucleotide according to claim 1, wherein the anti-Flt-1 antibody or its antigen-binding fragment comprises: The VL CDR1 defined by the amino acid sequence of SEQ ID NO:21, The VL CDR2 defined by the amino acid sequence of SEQ ID NO:24, The VL CDR3 defined by the amino acid sequence of SEQ ID NO:27, The VH CDR1 defined by the amino acid sequence of SEQ ID NO:2, The VH CDR2 defined by the amino acid sequence of SEQ ID NO:7, and The VH CDR3 defined by the amino acid sequence of SEQ ID NO:

17.

4. The polynucleotide according to claim 1, wherein the anti-Flt-1 antibody or its antigen-binding fragment comprises: The VL CDR1 defined by the amino acid sequence of SEQ ID NO:21, The VL CDR2 defined by the amino acid sequence of SEQ ID NO:24, The VL CDR3 defined by the amino acid sequence of SEQ ID NO:26, The VH CDR1 defined by the amino acid sequence of SEQ ID NO:3, The VH CDR2 defined by the amino acid sequence of SEQ ID NO:12, and The VH CDR3 defined by the amino acid sequence of SEQ ID NO:

17.

5. The polynucleotide according to claim 1, wherein the anti-Flt-1 antibody or its antigen-binding fragment comprises: The VL CDR1 defined by the amino acid sequence of SEQ ID NO:21, The VL CDR2 defined by the amino acid sequence of SEQ ID NO:24, The VL CDR3 defined by the amino acid sequence of SEQ ID NO:28, The VH CDR1 defined by the amino acid sequence of SEQ ID NO:2, The VH CDR2 defined by the amino acid sequence of SEQ ID NO:8, and The VH CDR3 defined by the amino acid sequence of SEQ ID NO:

17.

6. The polynucleotide according to claim 1, wherein The antibody or its antigen-binding fragment is selected from the group consisting of IgG, F(ab')2, F(ab)2, Fab', Fab, ScFvs, diabodies, triabodies and tetra-bodies, optionally wherein the antibody or its antigen-binding fragment is IgG, optionally wherein the antibody or its antigen-binding fragment is IgG1.

7. The polynucleotide according to claim 1, wherein The antibody or antigen-binding fragment thereof is a monoclonal antibody, optionally wherein the antibody is a humanized monoclonal antibody, optionally wherein the humanized monoclonal antibody comprises a human Fc region, optionally wherein the Fc region comprises one or more mutations that enhance the binding affinity between the Fc region and the FcRn receptor, thereby prolonging the half-life of the antibody in vivo, optionally wherein the Fc region contains one or more mutations at positions corresponding to Leu 234, Leu 235, and / or Gly 237 of human IgG1.

8. The polynucleotide according to claim 1, wherein The antibody or antigen-binding fragment thereof does not bind to VEGFR2 and / or VEGFR3.

9. The polynucleotide according to claim 1, wherein The antibody or antigen-binding fragment thereof does not bind to murine or simian Flt-1.

10. The polynucleotide according to claim 1, wherein the anti-Flt-1 antibody or antigen-binding fragment thereof comprises: (a) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 60 and a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 45, (b) a light chain comprising the amino acid sequence of SEQ ID NO: 76 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 71, (c) a light chain comprising the amino acid sequence of SEQ ID NO: 85 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 72, (d) a light chain comprising the amino acid sequence of SEQ ID NO: 84 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 71, (e) a light chain comprising the amino acid sequence of SEQ ID NO: 86 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 73, or (f) a light chain comprising the amino acid sequence of SEQ ID NO: 83 and a heavy chain comprising the amino acid sequence of SEQ ID NO:

70.

11. A pharmaceutical composition, characterized in that A polynucleotide encoding the anti-Flt-1 antibody or antigen-binding fragment thereof according to claim 1 and a pharmaceutically acceptable carrier.

12. An expression vector comprising the polynucleotide according to any one of claims 1 to 10.

13. An isolated cell comprising the polynucleotide according to any one of claims 1 to 10 or the expression vector according to claim 12.

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