Anti-semaphorin 3A antibodies and uses thereof for treating eye or eye diseases

Abstract

To provide antibodies and fragments thereof that target semaphorin 3A (Sema3A).SOLUTION: More specifically, anti-semaphorin 3A antibodies and methods of use for the treatment of various diseases or disorders are disclosed.SELECTED DRAWING: None

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention generally relates to antibodies and fragments thereof targeting Semaphorin 3A (Sema3A). More specifically, anti-Semaphorin 3A antibodies and methods of use for treating various diseases or disorders are disclosed. Pharmaceutical compositions and kits containing anti-Semaphorin 3A antibodies are also disclosed.

[0002] 2. Background of the Invention Ischemic retinopathy is characterized by the reduction or dysfunction of retinal vasculature, which leads to reduced blood flow and hypoxia.Retinal ischemia upregulates pro-angiogenic growth factors that promote retinal neovascularization, which can lead to blindness.However, when there is strong pathological neovascularization in the vitreous, which is a normally avascular eye area, revascularization of the ischemic retina does not occur.

[0003] The growth of these abnormal new blood vessels creates most of the threat to vision, since they may leak, bleed, or scar, which may result in retinal detachment. Current treatments for ischemic retinopathy seek to stop the pathological blood vessel growth, but do not address the underlying ischemia that drives that growth. Furthermore, standard treatments for diabetic retinopathy include laser destruction of portions of the retina in an attempt to stop the growth of new blood vessels and preserve central vision. However, these treatments are somewhat inadequate. Although some patients are able to maintain stable vision for many years, a high percentage of patients with retinopathy eventually become completely blind.

[0004] As a result, there remains an unmet need for new therapeutic approaches to effectively treat ocular or retinal diseases.

[0005] Summary of the Invention Semaphorin 3A is an endogenously secreted protein belonging to the class 3 semaphorin family (Sema3) that was initially identified as an axon guidance molecule and was involved in vascular pathfinding and network formation. Neurofilins 1 and 2 (Nrp1 and Nrp2) and A / D-type plexins (Plxns) act as ligands and their signaling subunits that bind to the semaphorin 3 receptor complex on the endothelial cell (EC) surface. As a special member of the semaphorin 3 family, semaphorin 3A first binds exclusively to neurofilin 1 and then to plexins A1-4 as a complex (Nrp1 / PlexA1-4). In this receptor complex, neurofilin 1 acts as the binding component, whereas plexins A1-4 act as the signaling components.

[0006] Human semaphorin 3A is a protein as disclosed in SEQ ID NO: 22 and available under the NCBI reference sequence NP_006071.1. Furthermore, human semaphorin 3A is encoded by gene ID: 10371 (NCBI).

[0007] Although semaphorin 3A has been studied for many years in tumor angiogenesis and metastasis, its effect on retinal neovascularization remains unclear. The inventors have demonstrated that semaphorin 3A is secreted by hypoxic retinal ganglion cells and acts as a signal of vascular repulsion. Semaphorin 3A drives away neovessels from ischemic regions by inducing the collapse of the cytoskeleton in these cells. Without wishing to be bound by theory, the inventors hypothesize that this may explain why revascularization of ischemic regions does not occur, and instead pathological neovascularization occurs in vitreous regions due to the upregulation of semaphorin 3A.

[0008] Semaphorin 3A is secreted by hypoxic neurons in the ischemic / avascular retina, thereby inhibiting retinal revascularization and enhancing pathological preretinal neovascularization.

[0009] The present inventors have addressed this pathological situation by developing antibodies that target semaphorin 3 A. Accordingly, the present invention provides monoclonal antibodies that specifically bind to semaphorin 3 A, preferably human semaphorin 3 A.

[0010] In a first aspect, the present invention provides a method for producing a composition comprising the steps of: - a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:1 (H-CDR1); the amino acid sequence of SEQ ID NO:2 (H-CDR2); and the amino acid sequence of SEQ ID NO:3 (H-CDR3); and - a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4 (L-CDR1); the amino acid sequence of SEQ ID NO: 5 (L-CDR2); and the amino acid sequence of SEQ ID NO: 6 (L-CDR3); The present invention provides an anti-semaphorin 3A antibody or an antigen-binding fragment thereof, comprising:

[0011] In another embodiment, the present invention provides a method for producing a composition comprising: - a heavy chain variable region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10; and a light chain variable region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13. The present invention provides an anti-semaphorin 3A antibody or an antigen-binding fragment thereof, comprising: Where: - the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:1 (H-CDR1); the amino acid sequence of SEQ ID NO:2 (H-CDR2); and the amino acid sequence of SEQ ID NO:3 (H-CDR3); and the light chain variable region comprises the amino acid sequence of SEQ ID NO:4 (L-CDR1); the amino acid sequence of SEQ ID NO:5 (L-CDR2); and the amino acid sequence of SEQ ID NO:6 (L-CDR3).

[0012] In another embodiment, the present invention provides a method for producing a composition comprising: - a heavy chain variable region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10; and a light chain variable region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13. The present invention provides an anti-semaphorin 3A antibody or an antigen-binding fragment thereof, comprising:

[0013] In yet another embodiment, the present invention provides a method for producing a composition comprising: - a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10; and a light chain variable region comprising the amino acid sequence of SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13; The present invention provides an anti-semaphorin 3A antibody or an antigen-binding fragment thereof, comprising:

[0014] In another embodiment, the present invention provides a method for producing a composition comprising: a. a heavy chain variable region and a light chain variable region comprising the amino acid sequences of SEQ ID NO:7 and SEQ ID NO:11, respectively; b. a heavy chain variable region and a light chain variable region comprising the amino acid sequences of SEQ ID NO:8 and SEQ ID NO:11, respectively; c. a heavy chain variable region and a light chain variable region comprising the amino acid sequences of SEQ ID NO:9 and SEQ ID NO:12, respectively; or d. A heavy chain variable region and a light chain variable region comprising the amino acid sequences of SEQ ID NO:10 and SEQ ID NO:13, respectively. The present invention provides an anti-semaphorin 3A antibody or an antigen-binding fragment thereof, comprising:

[0015] In yet another embodiment, the present invention provides a method for producing a composition comprising: a heavy chain comprising, preferably consisting of, the amino acid sequence of SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:19; and a light chain comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 18 or SEQ ID NO: 20; The present invention provides an anti-semaphorin 3A antibody or an antigen-binding fragment thereof, comprising:

[0016] In a particular embodiment, the present invention provides a method for producing a composition comprising the steps of: a. a heavy chain comprising the amino acid sequence of SEQ ID NO:14 and a light chain comprising the amino acid sequence of SEQ ID NO:15; b. a heavy chain comprising the amino acid sequence of SEQ ID NO:16 and a light chain comprising the amino acid sequence of SEQ ID NO:15; c. a heavy chain comprising the amino acid sequence of SEQ ID NO: 17 and a light chain comprising the amino acid sequence of SEQ ID NO: 18; or d. An anti-semaphorin 3A antibody or an antigen-binding fragment thereof, comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and a light chain comprising the amino acid sequence of SEQ ID NO: 20.

[0017] In certain preferred embodiments, the anti-semaphorin 3A antibody is a humanized anti-semaphorin 3A antibody.

[0018] In a second aspect, the present invention provides an anti-semaphorin 3A antibody or an antigen-binding fragment thereof that binds to at least one amino acid residue within the amino acid region 370 to 382 of human semaphorin 3A as shown in SEQ ID NO:22.

[0019] In one embodiment, the present invention provides an anti-semaphorin 3A antibody, or antigen-binding fragment thereof, that binds to at least one amino acid residue within the amino acid region set forth in SEQ ID NO: 21 (DSTKDLPDDVITF). In a preferred embodiment, the present invention provides an anti-semaphorin 3A antibody, or antigen-binding fragment thereof, that binds to the amino acid region as set forth in SEQ ID NO: 21.

[0020] In a third aspect, the present invention provides an anti-semaphorin 3A antibody, or an antigen-binding fragment thereof, for use as a medicament.

[0021] In one embodiment, the invention provides anti-Semaphorin 3A antibodies or antigen-binding fragments for inhibiting the vascular degenerative effects of Semaphorin 3A and / or for improving retinal revascularization.

[0022] In one embodiment, the invention provides an anti-semaphorin 3A antibody, or an antigen-binding fragment thereof, for use in treating or preventing a retinal or ocular disease.

[0023] In a fourth aspect, the present invention provides an anti-semaphorin 3A antibody, or an antigen-binding fragment thereof, for use in the treatment or prevention of a disease selected from the group consisting of retinopathy, ischemic retinopathy, diabetic retinopathy (including proliferative diabetic retinopathy and non-proliferative diabetic retinopathy), diabetic macular edema, diabetic macular ischemia, age-related macular degeneration, retinitis pigmentosa, inherited retinal dystrophies, myopic degeneration, retinal artery occlusion, endophthalmitis, uveitis, cystoid macular edema, choroidal neovascular membrane secondary to any retinal disease, optic neuropathy, glaucoma, retinal detachment, toxic retinopathy, radiation retinopathy, traumatic retinopathy, drug-induced retinal vasculopathy, retinal neovascularization, polypoidal choroidal vasculopathy, retinal vasculitis, retinal microaneurysm, Fuchs' dystrophy, macular telangiectasia, Usher syndrome, and Stargardt's disease.

[0024] In another embodiment, the present invention provides an anti-semaphorin 3A antibody or antigen-binding fragment thereof for use in the treatment or prevention of a disease selected from the group consisting of diabetic retinopathy (including proliferative diabetic retinopathy and non-proliferative diabetic retinopathy), ischemic retinopathy, diabetic macular edema, diabetic macular ischemia, age-related macular edema, retinal neovascularization, glaucoma, and choroidal neovascularization. Preferably, the disease is diabetic macular edema and / or diabetic macular ischemia.

[0025] In a preferred embodiment, the invention provides an anti-semaphorin 3A antibody, or an antigen-binding fragment thereof, for use in the treatment of diabetic macular ischemia by promoting revascularization (revascularization) in the ischemic retina and preventing pathological neovascularization in the vitreous region of the eye.

[0026] In another preferred embodiment, the invention provides an anti-Semaphorin 3A antibody, or antigen-binding fragment thereof, for use in the treatment of diabetic macular edema by reducing the permeability of the blood-retinal barrier.

[0027] In another preferred embodiment, the present invention provides an anti-semaphorin 3A antibody or an antigen-binding fragment thereof for inhibiting semaphorin 3A-induced blood-retinal barrier permeability and / or semaphorin 3A-induced vascular regression from an ischemic area.

[0028] In a fifth aspect, the present invention provides a pharmaceutical composition comprising an anti-semaphorin 3A antibody or an antigen-binding fragment thereof and a pharma- ceutically acceptable carrier.

[0029] In one embodiment, the present invention provides an anti-semaphorin 3A antibody or an antigen-binding fragment thereof, or a pharmaceutical composition comprising an anti-semaphorin 3A antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is administered by a parenteral, intravenous, intravitreal, or subcutaneous administration route, preferably by the intravitreal route.

[0030] In a sixth aspect, the present invention provides a method for producing a composition comprising the steps of: - a heavy chain as set forth in SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:19, or a sequence encoding a heavy chain variable region as set forth in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10; and a light chain as set forth in SEQ ID NO: 15, SEQ ID NO: 18, or SEQ ID NO: 20, or a sequence encoding a light chain variable region as set forth in SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13; The present invention provides an isolated polynucleotide or group of polynucleotides comprising:

[0031] In one embodiment, the invention provides an expression vector comprising an isolated polynucleotide or polynucleotides comprising a sequence encoding a heavy chain as set forth in SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:19, or a heavy chain variable region as set forth in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10; and a light chain as set forth in SEQ ID NO:15, SEQ ID NO:18, or SEQ ID NO:20, or a light chain variable region as set forth in SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13.

[0032] In one embodiment, the invention provides a viral vector comprising an isolated polynucleotide or polynucleotides comprising a heavy chain as set forth in SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:19, or a sequence encoding a heavy chain variable region as set forth in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10; and a light chain as set forth in SEQ ID NO:15, SEQ ID NO:18, or SEQ ID NO:20, or a sequence encoding a light chain variable region as set forth in SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13.

[0033] In one embodiment, the invention provides a host cell comprising an expression vector or isolated polynucleotide or polynucleotides comprising a heavy chain as set forth in SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:19, or a sequence encoding a heavy chain variable region as set forth in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10; and a light chain as set forth in SEQ ID NO:15, SEQ ID NO:18, or SEQ ID NO:20, or a sequence encoding a light chain variable region as set forth in SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13.

[0034] In one embodiment, the invention provides a method for producing an anti-semaphorin 3A antibody or antigen-binding fragment thereof, comprising the steps of obtaining a host cell comprising an expression vector or isolated polynucleotide or polynucleotides comprising a heavy chain as set forth in SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:19, or a sequence encoding a heavy chain variable region as set forth in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10; and a light chain as set forth in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10, or a sequence encoding a light chain variable region as set forth in SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13; and culturing the host cell.

[0035] In one embodiment, the method for producing an anti-semaphorin 3A antibody or antigen-binding fragment thereof further comprises recovering and purifying the anti-semaphorin 3A antibody or antigen-binding fragment thereof. [Brief description of the drawings]

[0036] [Figure 1] Localization of Semaphorin 3A in the Human Eye. This figure shows the localization of Semaphorin 3A in the human eye in pre-identified retinal samples from human donors with a history of diabetic retinopathy or primary open angle glaucoma (POAG) compared to age-matched controls (age controls) and subjects with diabetes but no ocular pathology (diabetic controls). Semaphorin 3A was found in the vessel walls of retinal blood vessels. Additionally, unidentified but distinct fluorescent objects of Semaphorin 3A were observed in the retinal ganglion cell layer. [Diagram 2] Efficacy in cell permeability assay. Fluorescein isothiocyanate (FITC)-dextran permeability in human retinal microvascular endothelial cells is measured by transwell assay. Anti-TNP antibody is a control antibody against trinitrophenol. Anti-Semaphorin 3A antibody is the antibody described in the present invention (clone I). Significance against recombinant human Semaphorin 3A is shown. [Diagram 3]Cytoskeleton collapse (Xcelligence) in human retinal microvascular endothelial cells. Semaphorins 3A-F all induce cytoskeleton collapse in human retinal endothelial cells. The antibody of the present invention (clone I) is specific for semaphorin 3A and prevents only the collapse induced by semaphorin 3A. [Figure 4A] Efficacy on edge cell density and avascular area in vivo. (A) and (C) Edge cell density and avascular area were investigated in an oxygen-induced retinopathy pup mouse model. Animals were exposed to 75% oxygen from day 7 to day 12 and received one intravitreal antibody injection after returning to normoxia on day 12. Anti-TNP antibody is a control antibody against trinitrophenol. Anti-semaphorin 3A antibody is the antibody described in the present invention (clone I). On postnatal day 17, retinal flat mounts were prepared and stained with isolectin B4 and used for edge cell counting and retinal avascular area size determination. (B) The correlation between edge cell density and avascular area is shown. [Figure 4B] Efficacy on edge cell density and avascular area in vivo. (A) and (C) Edge cell density and avascular area were investigated in an oxygen-induced retinopathy pup mouse model. Animals were exposed to 75% oxygen from day 7 to day 12 and received one intravitreal antibody injection after returning to normoxia on day 12. Anti-TNP antibody is a control antibody against trinitrophenol. Anti-semaphorin 3A antibody is the antibody described in the present invention (clone I). On postnatal day 17, retinal flat mounts were prepared and stained with isolectin B4 and used for edge cell counting and retinal avascular area size determination. (B) The correlation between edge cell density and avascular area is shown. [Figure 4C]Efficacy on edge cell density and avascular area in vivo. (A) and (C) Edge cell density and avascular area were investigated in an oxygen-induced retinopathy pup mouse model. Animals were exposed to 75% oxygen from day 7 to day 12 and received one intravitreal antibody injection after returning to normoxia on day 12. Anti-TNP antibody is a control antibody against trinitrophenol. Anti-semaphorin 3A antibody is the antibody described in the present invention (clone I). On postnatal day 17, retinal flat mounts were prepared and stained with isolectin B4 and used for edge cell counting and retinal avascular area size determination. (B) The correlation between edge cell density and avascular area is shown.

[0037] Detailed Description of the Invention definition The general structure of an antibody or immunoglobulin is well known to those skilled in the art; these molecules are typically heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is covalently linked to a heavy chain by one disulfide bond to form a heterodimer, and the heterotetrameric molecule is formed through covalent disulfide bonds between the two identical heavy chains of the heterodimer. The light and heavy chains are linked to each other by one disulfide bond, while the number of disulfide bonds between the two heavy chains varies depending on the immunoglobulin isotype. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain contains a variable domain (V) at the amino terminus. H = heavy chain variable domain), followed by three or four constant domains (C H1 , C H2 , C H3 and C H4 ), and a hinge region between CH1 and CH2. Each light chain contains an amino-terminal variable domain (V L = light chain variable domain) and the carboxy-terminal constant domain (C L ) has two domains. L The domain is non-covalently linked to V H domain, whereas C L The domain is generally CH1 The light-chain variable domain is covalently linked to the heavy-chain variable domain via disulfide bonds. Particular amino acid residues are thought to form an interface between the light-chain variable domain and the heavy-chain variable domain (Chothia et al., 1985, J. Mol. Biol. 186:651-663).

[0038] Certain domains within the variable domains vary widely between different antibodies, i.e., are "hypervariable". These hypervariable domains contain residues that are directly involved in the binding and specificity of each particular antibody to its specific antigenic determinant. The hypervariability in both the light and heavy variable domains is concentrated in three segments known as complementarity determining regions (CDRs) or hypervariable loops (HVLs). CDRs are defined by sequence comparison as described by Kabat et al., 1991, In: Sequences of Proteins of Immunological Interest, 5th ed., U.S. Public Health Service, National Institutes of Health, Bethesda, Missouri, while HVLs are structurally defined according to the three-dimensional structure of the variable domain as described by Chothia and Lesk, 1987, J. Mol. Biol. 196: 901-917. While these two methods result in slightly different identifications of CDRs, the structural definition is preferred. As defined by Kabat, CDR-L1 is located at approximately residues 24-34 within the light chain variable domain, CDR-L2 is located at approximately residues 50-56, and CDR-L3 is located at approximately residues 89-97; CDR-H1 is located at approximately residues 31-35 within the heavy chain variable domain, CDR-H2 is located at approximately residues 50-65, and CDR-H3 is located at approximately residues 95-102. Thus, the CDR1, CDR2, and CDR3 of the heavy and light chains define the unique and functional properties specific to a given antibody.

[0039] The three CDRs in each of the heavy and light chains are separated by framework regions (FRs), which contain sequences that tend to be less variable. From the amino terminus to the carboxy terminus of the heavy and light chain variable domains, the FRs and CDRs are arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The predominantly β-sheet conformation of the FRs brings the CDRs in each chain into close proximity with each other and with the CDRs of the other chain. The resulting conformation contributes to the antigen-binding site (see Kabat et al., 1991, NIH Publ. No. 91-3242, Vol. I, pages 647-669), although not all CDR residues are directly involved in binding to the antigen.

[0040] FR residues and immunoglobulin constant domains are not directly involved in antigen binding, but contribute to antigen binding and / or mediate antibody effector functions. Some FR residues are believed to have a significant effect on antigen binding in at least three ways: by directly binding non-covalently to the epitope, by interacting with one or more CDR residues, and by affecting the interface between the heavy and light chains. The constant domains are not directly involved in antigen binding, but mediate various immunoglobulin effector functions, such as antibody participation in antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cellular phagocytosis (ADCP).

[0041] The light chains of vertebrate immunoglobulins are assigned to one of two clearly distinct classes, kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains. In contrast, the heavy chains of mammalian immunoglobulins are assigned to one of five major classes according to the sequence of their constant domains: IgA, IgD, IgE, IgG, and IgM. IgG and IgA are further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2, respectively. The heavy chain constant domains corresponding to the various classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the classes of native immunoglobulins are well known.

[0042] The terms "antibody," "anti-semaphorin 3A antibody," "humanized anti-semaphorin 3A antibody," and "mutated humanized anti-semaphorin 3A antibody" are used herein in the broadest sense and specifically include monoclonal antibodies (including full-length monoclonal antibodies), multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, such as variable domains and other portions of antibodies, that exhibit a desired biological activity, such as binding to semaphorin 3A.

[0043] The term "monoclonal antibody" (mAb) refers to an antibody from a substantially homogeneous antibody population; that is, the individual antibodies within the population are identical except for naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic determinant or "epitope." Thus, the modifier "monoclonal" indicates a substantially homogeneous antibody population directed against the same epitope, and is not to be construed as requiring production of the antibodies by any particular method. It should be understood that the monoclonal antibodies may be made by any technique or method known in the art, including, for example, the hybridoma method (Kohler et al., 1975, Nature 256:495) or recombinant DNA methods known in the art (see, e.g., U.S. Pat. No. 4,816,567), or isolation of recombinantly produced monoclonal antibodies using phage antibody libraries using the techniques described in Clackson et al., 1991, Nature 352: 624-628, and Marks et al., 1991, J. Mol. Biol. 222: 581-597.

[0044] A chimeric antibody consists of the variable regions of the heavy and light chains of an antibody from one species (e.g., a non-human mammal, e.g., a mouse) and the constant regions of the heavy and light chains of an antibody from another species (e.g., a human), and can be obtained by linking a DNA sequence encoding the variable region of an antibody from a first species (e.g., a mouse) to a DNA sequence of the constant region of an antibody from a second species (e.g., a human) and transforming a host with an expression vector containing the linked sequences to enable it to produce the chimeric antibody. Alternatively, a chimeric antibody can also be one in which one or more regions or domains of the heavy and / or light chains are identical to, homologous to, or variants of corresponding sequences in a monoclonal antibody from another immunoglobulin class or isotype, or from a consensus or germline sequence. A chimeric antibody can also include fragments of such antibodies. However, such antibody fragments retain the desired biological activity of the parent antibody, e.g., binding to the same epitope (see, e.g., U.S. Pat. No. 4,816,567; and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81: 6851-6855).

[0045] The terms "antibody fragment", "antigen-binding fragment", "anti-semaphorin 3A antibody fragment", "humanized anti-semaphorin 3A antibody fragment", and "mutated humanized anti-semaphorin 3A antibody fragment" refer to a portion of a full-length anti-semaphorin 3A antibody, in which the variable region or functional capability, such as binding to a specific semaphorin 3A epitope, is retained. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fd, Fv, scFv, and scFv-Fc fragments, diabodies, linear antibodies, single-chain antibodies, minibodies, diabodies formed from antibody fragments, and multispecific antibodies formed from antibody fragments.

[0046] Useful antibody fragments can be generated by treating full-length antibodies with enzymes such as papain or pepsin. Papain digestion is used to generate two identical antigen-binding antibody fragments, called "Fab" fragments (each with one antigen-binding site), and a residual "Fc" fragment. The Fab fragment also contains the constant domains of the light chain and the C domain of the heavy chain. H1 Treatment with pepsin yields an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0047] The Fab' fragment is H1 They differ from Fab fragments by the presence of additional residues at the C-terminus of the domain, including one or more cysteines from the antibody hinge region. F(ab')2 antibody fragments are pairs of Fab' fragments linked by cysteine ​​residues in the hinge region. Other chemical linkages of antibody fragments are also known.

[0048] An "Fv" fragment contains a complete antigen recognition and binding site consisting of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. In this configuration, the three CDRs of each variable domain interact to form the V H -V L Together, the six CDRs define an antigen-binding site on the surface of the dimer and confer antigen-binding specificity to the antibody.

[0049] "Single-chain Fv" or "scFv" antibody fragments are fragments of the V H Domain and V L and a single chain Fv variant comprising a V domain, wherein the domains are present in a single polypeptide chain. The single chain Fv is capable of recognizing and binding to an antigen. The scFv polypeptide also optionally comprises a V H Domain and V LThey may contain a polypeptide linker located between the domains, which promotes the formation of the desired three-dimensional structure for binding of the scFv to the antigen (see, e.g., Pluckthun, 1994, In The Pharmacology of monoclonal Antibodies, Vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315).

[0050] Other recognized antibody fragments include a pair of tandem Fd segments (V H -C H1 -V H -C H1 ) to form a pair of antigen-binding regions. These "chain antibodies" may be bispecific or monospecific, e.g., as described in Zapata et al. 1995, Protein Eng. 8(10):1057-1062.

[0051] A humanized antibody or humanized antibody fragment is a special type of chimeric antibody that can bind to a predetermined antigen and includes an immunoglobulin amino acid sequence variant or a fragment thereof that includes one or more FRs that have substantially the amino acid sequence of a human immunoglobulin and one or more CDRs that have substantially the amino acid sequence of a non-human immunoglobulin. This non-human amino acid sequence, often referred to as an "import" sequence, is typically taken from an "import" antibody domain, particularly a variable domain. Generally, a humanized antibody includes at least the CDRs or HVLs of a non-human antibody inserted between the FRs of a human heavy or light chain variable domain.

[0052] The present invention describes certain humanized anti-Semaphorin 3A antibodies that contain CDRs derived from a murine or chimeric antibody inserted between the FRs of human germline sequence heavy and light chain variable domains. It will be understood that certain murine FR residues may be important for the function of the humanized antibody, and therefore certain human germline sequence heavy and light chain variable domain residues are altered to be identical to the corresponding murine sequence residues.

[0053] As used herein, the terms "antibody of the invention" and "anti-semaphorin 3A antibody of the invention" refer to an anti-semaphorin 3A antibody or an antigen-binding fragment thereof described herein. Preferably, the terms refer to any antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:1 (H-CDR1), the amino acid sequence of SEQ ID NO:2 (H-CDR2), and the amino acid sequence of SEQ ID NO:3 (H-CDR3), and a light chain variable region comprising the amino acid sequence of SEQ ID NO:4 (L-CDR1), the amino acid sequence of SEQ ID NO:5 (L-CDR2), and the amino acid sequence of SEQ ID NO:6 (L-CDR3).

[0054] In one embodiment, the humanized anti-Semaphorin 3A antibody comprises substantially all of at least one, typically two, variable domains (e.g., as contained in Fab, Fab', F(ab')2, Fabc, and Fv fragments), in which all or substantially all of the CDRs correspond to the CDRs of a non-human immunoglobulin, specifically herein the CDRs are murine sequences and the FRs are human immunoglobulin consensus or germline sequence FRs. In another embodiment, the human anti-Semaphorin 3A antibody also comprises at least a portion of an immunoglobulin Fc region, typically that of a human immunoglobulin. Usually, the antibody will contain at least the variable domains of both light chains, as well as the heavy chain. The antibody will also contain the CDRs of the heavy chain, if appropriate. H1 Area, hinge area, C H2 area, C H3 Area and / or C H4 It may include one or more of the regions.

[0055] The humanized anti-semaphorin 3A antibody may be selected from any class of immunoglobulin, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. For example, the constant domain may be a complement-binding constant domain, in which case it is desired that the humanized antibody exhibit cytotoxic activity, and the isotype is typically IgG1. If such cytotoxic activity is not desired, the constant domain may be another isotype, for example IgG2. Alternative humanized anti-semaphorin 3A antibodies may include sequences from more than one immunoglobulin class or isotype, and it is within the skill of the art to select a particular constant domain to optimize the desired effector function. In a specific embodiment, the invention provides antibodies that are IgG1 antibodies, more particularly IgG1 antibodies characterized by reduced effector function.

[0056] Preferably, the anti-semaphorin 3A antibody of the invention is a humanized antibody formatted as IgG1KO.

[0057] The FR and CDR or HVL of the humanized anti-semaphorin 3A antibody need not correspond exactly to the parent sequence. For example, one or more residues in the import CDR or HVL or the FR sequence of the consensus or germline sequence may be modified (e.g., mutagenized) by substitution, insertion, or deletion, so that the resulting amino acid residue is no longer identical to the original residue at the corresponding position of either parent sequence, but the antibody nevertheless retains the function of binding to semaphorin 3A. Such modifications will typically not be extensive, but conservative modifications. Usually, at least 75%, more frequently at least 90%, and most frequently more than 95%, or more than 98%, or more than 99% of the humanized antibody residues will correspond to the residues of the FR and import CDR sequences of the parent consensus or germline sequence.

[0058] The interface between the heavy and light chain variable regions ("V L -VH Immunoglobulin residues that affect the "interface" are those that affect the proximity or orientation of the two chains with respect to each other. Specific residues that may be involved in interchain interactions include the V L Residues 34, 36, 38, 44, 46, 87, 89, 91, 96 and 98, and V H These include residues 35, 37, 39, 45, 47, 91, 93, 95, 100, and 103 (using the numbering system set out in Kabat et al., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Missouri, 1987). L Residues 43 and 85, and V H Residues such as residues 43 and 60 are also considered to be potentially involved in this interaction. Although these residues are shown only for human IgG, they are applicable across species. Key antibody residues that are reasonably expected to be involved in interchain interactions are selected for substitution in the consensus sequence.

[0059] The terms "consensus sequence" and "consensus antibody" refer to an amino acid sequence that includes the most frequently occurring amino acid residue at each position in all immunoglobulins of any particular class, isotype, or subunit structure, such as human immunoglobulin variable domains. A consensus sequence may be based on immunoglobulins of a particular species or of many species. A "consensus" sequence, structure, or antibody is understood to include a consensus human sequence as described in certain embodiments, and refers to an amino acid sequence that includes the most frequently occurring amino acid residue at each position in all human immunoglobulins of any particular class, isotype, or subunit structure. Thus, a consensus sequence contains an amino acid sequence that has an amino acid at each position that is present in one or more known immunoglobulins, but may not exactly duplicate the complete amino acid sequence of any single immunoglobulin. The consensus sequence of the variable region is not derived from any naturally occurring antibody or immunoglobulin (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th ed., U.S. Public Health Service, National Institutes of Health, Bethesda, Missouri) and variants thereof. The FRs of the heavy and light chain consensus sequences, and variants thereof, provide useful sequences for the preparation of humanized anti-Semaphorin 3A antibodies, see, for example, U.S. Patent Nos. 6,037,454 and 6,054,297.

[0060] Human germline sequences are naturally found in the human population. The combination of such germline genes generates antibody diversity. Germline antibody sequences for antibody light chains are derived from conserved human germline kappa or lambda v- and j-genes. Similarly, heavy chain sequences are derived from germline v-, d-, and j-genes (LeFranc, MP, and LeFranc, G, "The Immunoglobulin Facts Book" Academic Press, 2001).

[0061] An "isolated" antibody is one that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of the antibody's natural environment are materials that may interfere with diagnostic or therapeutic uses of the antibody, such as enzymes, hormones, or other proteinaceous or nonproteinaceous solutes. In one embodiment, the antibody will be isolated and purified to at least 95% by weight of the antibody.

[0062] The term "antibody performance" refers to factors / properties that contribute to the recognition of an antigen by an antibody or the in vivo effectiveness of an antibody. In a preferred embodiment, it refers to the ability of an antibody to prevent the breakdown of the cytoskeleton in retinal cells. Changes in the amino acid sequence of an antibody can affect antibody properties such as folding and the initial rate of binding of the antibody to the antigen (k a ), the dissociation constant of the antibody from the antigen (k d ), the affinity constant of the antibody for the antigen (K d ), can affect physical factors such as antibody conformation, protein stability, and antibody half-life.

[0063] As used herein, the term "identical" or "percent identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that have the same or a certain percentage of the same nucleotides or amino acid residues when compared and aligned for maximum correspondence. To determine the percent identity, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced into the first amino acid or nucleic acid sequence for optimal alignment with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity = number of identical positions / total number of positions (e.g., overlapping positions) x 100). In some embodiments, the two sequences being compared are the same length after gaps are introduced into the sequences, where appropriate (e.g., to exclude additional sequences extending beyond the sequences being compared). For example, when comparing variable region sequences, leader and / or constant domain sequences are not considered. For sequence comparison between two sequences, a "corresponding" CDR refers to the CDR at the same position in both sequences (e.g., CDR-H1 of each sequence).

[0064] The determination of the percent identity or similarity between two sequences can be achieved using a mathematical algorithm. A preferred non-limiting example of the mathematical algorithm used for comparing two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403-410. A BLAST nucleotide search can be performed using the NBLAST program, score=100, word length=12, to obtain the nucleotide sequence that is homologous to the nucleic acid that codes for the protein of interest. Protein searches by BLAST can be performed using the XBLAST program, score=50, word length 3, to obtain amino acid sequences that are homologous to the protein of interest. To obtain gapped alignments for comparison purposes, gapped BLAST can be used as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules (ibid.). When using BLAST, gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. Another preferred, non-limiting example of a mathematical algorithm used for sequence comparison is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package.When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Additional algorithms for sequence analysis are known in the art, including ADVANCE and ADAM, as described in Torellis and Robotti, 1994, Comput. Appl. Biosci. 10:3-5; and FASTA, as described in Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444-8. In FASTA, ktup is a control option that sets the sensitivity and speed of the search. When ktup=2, similar regions in the two sequences being compared are found by examining aligned residue pairs; when ktup=1, single aligned amino acids are examined. For protein sequences, ktup can be set to 2 or 1, or for DNA sequences, 1-6. If ktup is not specified, it defaults to 2 for proteins and 6 for DNA. Alternatively, protein sequence alignments can be performed using the CLUSTAL W algorithm as described by Higgins et al., 1996, Methods Enzymol. 266:383-402.

[0065] As used herein, the expressions "cell," "cell line," and "cell culture" are used synonymously and all such designations include the progeny thereof. Thus, "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom without regard for the number of transfers.

[0066] The term "mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sport, or pet animals, such as dogs, horses, cats, cows, etc. Preferably, the mammal is a human.

[0067] As used herein, a "disease" or "disorder" is any condition that would benefit from treatment with a humanized anti-Semaphorin 3A antibody described herein, including chronic and acute disorders or diseases, including those conditions that predispose a mammal to the disorder in question.

[0068] The term "intravitreal injection" has its ordinary meaning in the art and refers to the introduction of an anti-semaphorin 3A antibody, or an antigen-binding fragment thereof, into the vitreous of a patient.

[0069] The term "subcutaneous administration" refers to the introduction of an anti-Semaphorin 3A antibody or antigen-binding fragment thereof, by relatively slow and sustained delivery from a drug depot, under the skin of an animal or human patient, preferably into a pocket between the skin and the underlying tissue. The pocket can be created by pinching the skin or by pulling the skin up and away from the underlying tissue.

[0070] The term "subcutaneous infusion" refers to the introduction of a drug under the skin of an animal or human patient, preferably into a pocket between the skin and the underlying tissue, by relatively slow and sustained delivery from a drug reservoir, over a period of time, including but not limited to, 30 minutes or less, or 90 minutes or less. Optionally, the infusion can be performed by subcutaneous implantation of a drug delivery pump implanted under the skin of the animal or human patient, where the pump delivers a predetermined amount of drug over a predetermined period of time, for example, 30 minutes, 90 minutes, or over the length of the treatment regimen.

[0071] The term "subcutaneous bolus" refers to administration of a drug under the skin of an animal or human patient, where the bolus drug delivery is within about 15 minutes; in another embodiment, within 5 minutes, and in yet another embodiment, within 60 seconds. In still yet another embodiment, administration is within a pocket between the skin and the underlying tissue, where the pocket can be created by pinching or pulling the skin up and away from the underlying tissue.

[0072] The term "therapeutically effective amount" is used to refer to an amount of an anti-Semaphorin 3A antibody or antigen-binding fragment thereof that relieves or ameliorates one or more symptoms of the disorder being treated. It is the amount that, upon treatment, has a beneficial patient outcome. Efficacy can be measured in a conventional manner, depending on the condition to be treated. For example, in ocular / retinal diseases or disorders characterized by cells expressing Semaphorin 3A, efficacy can be measured by determining the response rate, e.g., recovery of vision, or by assessing the delay time until the disease progresses.

[0073] As used herein, the terms "treatment" and "therapy" and the like are meant to include therapeutic measures as well as preventative or suppressive measures for a disease or disorder that produce any clinically desirable or beneficial effect, including, but not limited to, alleviation or mitigation of one or more symptoms, regression, slowing, or cessation of a disease or disorder. Thus, for example, the term treatment includes administration of an anti-semaphorin 3A antibody or an antigen-binding fragment thereof before or after the onset of symptoms of a disease or disorder, thereby preventing or eliminating one or more signs of a disease or disorder. As another example, the term includes combating symptoms of a disease by administering an anti-semaphorin 3A antibody or an antigen-binding fragment thereof after clinical signs of the disease. In addition, administration of an anti-semaphorin 3A antibody or an antigen-binding fragment thereof after onset and after the development of clinical symptoms (where administration affects clinical parameters of a disease or disorder, regardless of whether the treatment results in remission of the disease) includes "treatment" or "therapy" as used herein. Furthermore, so long as a composition of the present invention, alone or in combination with another therapeutic agent, reduces or ameliorates at least one symptom of the disorder being treated compared to the symptom in the absence of the anti-semaphorin 3A antibody composition or antigen-binding fragment thereof, the result should be deemed to be an effective treatment of the underlying disorder, regardless of whether all symptoms of the disorder are alleviated.

[0074] The term "package insert" is used to refer to instructions customarily included in the commercial packaging of therapeutic products that contain information regarding the indications, uses, administration, contraindications, and / or warnings concerning the use of such therapeutic products.

[0075] Antibodies of the Invention

[0076] In a first aspect, the present invention relates to an anti-semaphorin 3A antibody or an antigen-binding fragment thereof, preferably a humanized anti-semaphorin 3A antibody, more preferably a humanized monoclonal anti-semaphorin 3A antibody.

[0077] In a first characterization, an antibody library targeting semaphorin 3A mutants was generated by placing the CDRs of mouse antibodies into the FRs of the human consensus heavy and light chain variable domains and further engineering the FRs with different modifications.

[0078] This resulted in humanized antibodies directed against semaphorin 3A with enhanced properties, as disclosed herein. The sequences of the antibodies of the invention are shown in Table 1 below.

[0079] [Table 1] TIFF0007638332000002.tif218169 TIFF0007638332000003.tif254169

[0080] In one embodiment, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: - a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:1 (H-CDR1); the amino acid sequence of SEQ ID NO:2 (H-CDR2); and the amino acid sequence of SEQ ID NO:3 (H-CDR3); and - a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4 (L-CDR1); the amino acid sequence of SEQ ID NO: 5 (L-CDR2); and the amino acid sequence of SEQ ID NO: 6 (L-CDR3); The present invention provides an anti-semaphorin 3A antibody or an antigen-binding fragment thereof, comprising:

[0081] In another embodiment, the present invention provides a method for producing a composition comprising: - a heavy chain variable region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10; and a light chain variable region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13. The present invention provides an anti-semaphorin 3A antibody or an antigen-binding fragment thereof, comprising:

[0082] In another embodiment, the present invention provides a method for producing a composition comprising: - a heavy chain variable region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10; and a light chain variable region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13. Including, - the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:1 (H-CDR1); the amino acid sequence of SEQ ID NO:2 (H-CDR2); and the amino acid sequence of SEQ ID NO:3 (H-CDR3); and - an anti-semaphorin 3A antibody or an antigen-binding fragment thereof, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 4 (L-CDR1); the amino acid sequence of SEQ ID NO: 5 (L-CDR2); and the amino acid sequence of SEQ ID NO: 6 (L-CDR3).

[0083] In yet another embodiment, the present invention provides a method for producing a composition comprising: - a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10; and a light chain variable region comprising the amino acid sequence of SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13; The present invention provides an anti-semaphorin 3A antibody or an antigen-binding fragment thereof, comprising:

[0084] In a preferred embodiment, the present invention provides a method for producing a composition comprising the steps of: - a heavy chain variable region and a light chain variable region comprising the amino acid sequences of SEQ ID NO: 7 and SEQ ID NO: 11, respectively; - a heavy chain variable region and a light chain variable region comprising the amino acid sequences of SEQ ID NO: 8 and SEQ ID NO: 11, respectively; - a heavy chain variable region and a light chain variable region comprising the amino acid sequences of SEQ ID NO: 9 and SEQ ID NO: 12, respectively; or - a heavy chain variable region and a light chain variable region comprising the amino acid sequences of SEQ ID NO: 10 and SEQ ID NO: 13, respectively; The present invention provides an anti-semaphorin 3A antibody or an antigen-binding fragment thereof, comprising:

[0085] In yet another embodiment, the present invention provides a method for producing a composition comprising: a heavy chain comprising, preferably consisting of, the amino acid sequence of SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:19; and a light chain comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 18 or SEQ ID NO: 20; The present invention provides an anti-semaphorin 3A antibody or an antigen-binding fragment thereof, comprising:

[0086] In a particular embodiment, the present invention provides a method for producing a composition comprising the steps of: a. a heavy chain comprising the amino acid sequence of SEQ ID NO: 14 and a light chain comprising the amino acid sequence of SEQ ID NO: 15, said antibody being designated "Clone I"; b. a heavy chain comprising the amino acid sequence of SEQ ID NO: 16 and a light chain comprising the amino acid sequence of SEQ ID NO: 15, said antibody being designated "Clone II"; c. a heavy chain comprising the amino acid sequence of SEQ ID NO: 17 and a light chain comprising the amino acid sequence of SEQ ID NO: 18, said antibody being designated "Clone III"; or d. a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and a light chain comprising the amino acid sequence of SEQ ID NO: 20, said antibody being designated "Clone IV" The present invention relates to an anti-semaphorin 3A antibody or an antigen-binding fragment thereof, comprising:

[0087] The IgG1-KO mutants were generated by introducing mutations into the Fc region. Mutations that reduce or inhibit effector functions are well known to those skilled in the art and are fully disclosed in the prior art, for example in Wang et al, Protein Cell 2018, 9(1):63-73 and Stewart et al. Journal for ImmunoTherapy of Cancer 2014, 2:29. Typically, a non-limiting list of mutations introduced into the Fc region of IgG1 to reduce Fc effector functions includes: -L234A and L235A; -L234A, L235A, and N297Q; -L234A, L235A, and P329G; or -L234A, L235A, and D265A where residues are numbered according to the EU index of Kabat.

[0088] In a preferred embodiment, the antibody of the invention comprises two mutations in the Fc region, L234A and L235A, which reduce effector function.

[0089] The CDRs disclosed herein and shown in SEQ ID NOs: 1-6 are presented according to the Kabat numbering and are summarized in Table 2 below with their positions in Kabat.

[0090] [Table 2]

[0091] The anti-semaphorin 3A antibodies of the invention bind to human semaphorin 3A with high affinity. In one embodiment related to this aspect, the anti-semaphorin 3A antibodies of the invention bind to human semaphorin 3A with a K of less than 50 pM. D In another embodiment, the anti-semaphorin 3A antibody of the invention binds to human semaphorin 3A with a K of less than 35 pM, as exemplified in Example 4. D In a preferred embodiment, the anti-semaphorin 3A antibody of the present invention binds to human semaphorin 3A with a K of less than 30 pM. D Combine with.

[0092] The anti-semaphorin 3A antibody of the present invention also binds to cynomolgus monkey semaphorin 3A, mouse semaphorin 3A, rat semaphorin 3A, and rabbit semaphorin 3A.

[0093] The anti-semaphorin 3A antibodies of the invention prevent semaphorin 3A-induced cytoskeletal collapse in retinal cells with a functional potency of less than 100 pM, preferably less than 80 pM, and more preferably less than 70 pM. In a preferred embodiment, the anti-semaphorin 3A antibodies of the invention prevent semaphorin 3A-induced cytoskeletal collapse in retinal cells with a functional potency of 69 pM, as illustrated in Example 4.

[0094] In a further embodiment, the anti-semaphorin 3A antibodies of the present invention have been demonstrated to have low immunogenicity risk as described in Example 5. This is based on computational prediction of the immunogenicity of the antibody. The immunogenicity risk is typically assessed by various well-known methods, such as by computational algorithms to predict T-cell epitopes, which are the main immunogenicity-influencing factors.

[0095] Sequences containing T cell epitopes present in a protein of interest can be predicted by using an algorithm based on a computer matrix approach available under the name EpiMatrix (produced by the company EpiVax). Those skilled in the art can refer to Van Walle et al., Expert Opin Biol Ther. 2007 March; 7(3): 405-18 and Jawa and al., Clin Immunol. 2013 December; 149(3): 534-55.

[0096] The inventors have shown that the antibodies of the invention exhibit advantageous properties over other antibodies or fragments targeting semaphorin 3A described in the prior art and described herein.

[0097] The present inventors compared the binding affinity of an antibody targeting semaphorin 3A disclosed in WO 2014123186 (Kiome Biosciences, Inc.) with that of the antibody of the present invention. The antibody of WO 2014123186 is disclosed for use in treating Alzheimer's disease. Example 8 of the present invention shows that the antibody of the present invention was found to have a higher binding affinity for human semaphorin 3A than the prior art antibody disclosed by Kiome Biosciences, Inc.

[0098] The inventors also compared the properties of the antibody according to the present invention with scFv fragments as disclosed in WO2017074013 (Samsung). These fragments are disclosed for use in the treatment of various cancers. Example 9 of the present invention shows that the antibody of the present invention was found to have a higher binding affinity to human semaphorin 3A than the prior art antibody fragments disclosed by WO2017074013.

[0099] Higher binding affinity extends the neutralization time of semaphorin 3A after intravitreal injection of the antibody, allowing for less frequent injections. Higher binding affinity also allows for the administration of smaller doses, limiting possible side effects. Thus, the antibodies of the present invention provide technical advantages over prior art antibodies. The improved binding affinity and reduced injection frequency significantly improve the efficacy of patient treatment in patients in need thereof. The antibodies of the present invention also provide valuable benefits to patients, in particular improved drug compliance.

[0100] The inventors also compared the functional efficacy of the antibodies of the present invention and a commercially available antibody targeting semaphorin 3A as described in Example 11 of the present invention. The inventors showed that under the same conditions, the antibodies of the present invention prevented semaphorin-induced cytoskeletal collapse in retinal cells (Example 3), whereas the commercially available antibody did not (Example 11).

[0101] Humanized and Amino Acid Sequence Variants Still other mutated anti-semaphorin 3A antibodies and antibody fragments may be engineered based on the CDR sets identified under the sequences shown in SEQ ID NOs: 1-6. It is understood that the amino acid sequences of the CDRs in the mutated anti-semaphorin 3A antibodies and antibody fragments remain unchanged, but the surrounding regions, such as FR regions, may be engineered. Amino acid sequence variants of anti-semaphorin 3A antibodies may be prepared by introducing appropriate nucleotide changes into the DNA of the anti-semaphorin 3A antibody or by peptide synthesis. Such variants include, for example, deletions from, and / or insertions into, and / or substitutions of residues within the amino acid sequences of the anti-semaphorin 3A antibodies of the Examples herein. Any combination of deletions, insertions, and substitutions may be made to arrive at the final construct, provided that the final construct has the desired characteristics. Amino acid changes may also alter post-translational processes of the humanized or mutated anti-semaphorin 3A antibody, such as changing the number or position of glycosylation sites.

[0102] Another type of amino acid variant of an antibody involves altering the native glycosylation pattern of the antibody, with the term "altering" in this context meaning deleting one or more carbohydrate moieties found in the antibody and / or adding one or more glycosylation sites not previously present in the antibody.

[0103] In some embodiments, the present invention includes nucleic acid molecules encoding amino acid sequence variants of the anti-semaphorin 3A antibodies described herein. Nucleic acid molecules encoding amino acid sequence variants of anti-semaphorin 3A antibodies are prepared by various methods known in the art, including, but not limited to, isolation from natural sources (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of previously prepared mutant or non-mutant forms of anti-semaphorin 3A antibodies.

[0104] In certain embodiments, the anti-semaphorin 3A antibody is an antibody fragment. There are techniques developed for the production of antibody fragments. Fragments can be obtained through proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., 1992, Journal of Biochemical and Biophysical Methods 24:107-117; and Brennan et al., 1985, Science 229:81). Alternatively, the fragments can be produced directly in recombinant host cells. For example, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (see, e.g., Carter et al., 1992, Bio / Technology 10:163-167). According to another approach, F(ab')2 fragments can be directly isolated from recombinant host cell culture medium. Other techniques for producing antibody fragments will be apparent to those skilled in the art.

[0105] The anti-semaphorin 3A antibodies and antigen-binding fragments thereof may include modifications.

[0106] In certain embodiments, it may be desirable to use anti-semaphorin 3A antibody fragments rather than intact antibodies. It may be desirable to modify the antibody fragments to extend their serum half-life. This can be accomplished, for example, by incorporating a salvage receptor binding epitope into the antibody fragment. In one method, the appropriate region of the antibody fragment may be modified (e.g., mutated), or the epitope may be incorporated into a peptide tag, which is then fused to either end or the middle of the antibody fragment, for example, by DNA synthesis or peptide synthesis. See, for example, WO 96 / 32478.

[0107] In another embodiment, the present invention includes covalent modifications of anti-semaphorin 3A antibodies. Covalent modifications include modifications of cysteine ​​residues, histidine residues, lysine and amino-terminal residues, arginine residues, tyrosine residues, carboxyl side groups (asparagine or glutamine), glutaminine and asparagine residues, or serine residues, or threonine residues. Another type of covalent modification includes chemically or enzymatically coupling glycosides to the antibody. Such modifications may be performed by chemical synthesis or, if applicable, by enzymatic or chemical cleavage of the antibody. Other types of covalent modifications of the antibody may be introduced into the molecule by reacting targeted amino acid residues of the antibody with organic derivatizing agents capable of reacting with selected side chains, i.e., amino-terminal or carboxy-terminal residues.

[0108] Removal of any carbohydrate moieties present on the antibody can be accomplished chemically or enzymatically. Chemical deglycosylation is described by Hakimuddin et al., 1987, Arch. Biochem. Biophys. 259:52, and by Edge et al., 1981, Anal. Biochem., 118:131. Enzymatic cleavage of carbohydrate moieties on the antibody can be achieved by the use of various endo- and exoglycosidases as described by Thotakura et al., 1987, Meth. Enzymol 138:350.

[0109] Another type of useful covalent modification involves linking the antibody to one of a variety of nonproteinaceous polymers, such as polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, by methods set forth in one or more of U.S. Pat. Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, and 4,179,337.

[0110] Epitope binding In a second aspect, the present invention relates to an antibody that recognizes a specific “Semaphorin 3A antigen epitope” and “Semaphorin 3A epitope.” In particular, the antibody of the present invention binds to an epitope of human Semaphorin 3A having SEQ ID NO:22.

[0111] In one aspect, the present invention relates to an anti-semaphorin 3A antibody or an antigen-binding fragment thereof that binds to at least one amino acid residue within the amino acid region 370 to 382 of human semaphorin 3A as shown in SEQ ID NO:22.

[0112] In another aspect, the invention relates to an anti-semaphorin 3A antibody, or an antigen-binding fragment thereof, that binds to SEQ ID NO:21.

[0113] The sequences of SEQ ID NOs:21 and 22 are shown in Table 5 below.

[0114] [Table 3]

[0115] As used herein, the terms "semaphorin 3A antigenic epitope" and "semaphorin 3A epitope" refer to a molecule (e.g., a peptide) or a fragment of a molecule capable of binding to an anti-semaphorin 3A antibody or an antigen-binding fragment thereof. These terms further include a semaphorin 3A antigenic determinant recognized by any of the antibodies or antibody fragments of the invention having a combination of light chain CDRs and heavy chain CDRs selected, for example, from the heavy chain CDRs of SEQ ID NOs: 1-3 and the light chain CDRs of SEQ ID NOs: 4-6.

[0116] The semaphorin 3A antigen epitope may be contained in a protein, a fragment of a protein, a peptide, etc. The epitope is typically a protein, a short oligopeptide, an oligopeptide mimetic (i.e., an organic compound that mimics the antibody binding properties of the semaphorin 3A antigen), or a combination thereof.

[0117] The antibodies or antibody fragments of the invention have been found to bind to a unique epitope on human semaphorin 3A. Preferably, the anti-semaphorin 3A antibody or antigen-binding fragment thereof binds to at least one amino acid residue within the amino acid region 370-382 of the extracellular domain of human semaphorin 3A having SEQ ID NO:22. This epitope is located near the interface between semaphorin 3A and the Plexin A receptor. Binding of the antibody to this epitope inhibits the formation of a signaling halo-receptor complex of the ligand semaphorin 3A, the receptor Plexin A, and the co-receptor Nrp1, thereby interfering with the biological effects of such signaling.

[0118] With respect to binding to an epitope, the phrase "binds within amino acid region XY..." means that an anti-semaphorin 3A antibody or antigen-binding fragment thereof binds to at least one amino acid residue, and preferably all amino acid residues, within the amino acid region specified in the sequence.

[0119] In another embodiment, the anti-semaphorin 3A antibody or antigen-binding fragment thereof binds to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% of the amino acid sequence set forth in SEQ ID NO: 22. Preferably, the anti-semaphorin 3A antibody or antigen-binding fragment thereof binds to SEQ ID NO:22.

[0120] Therapeutic Use In a third aspect, the present invention relates to an anti-semaphorin 3A antibody, or an antigen-binding fragment thereof, for use as a medicament.

[0121] In one embodiment, the invention provides an anti-Semaphorin 3A antibody, or an antigen-binding fragment thereof, for inhibiting the vascular degenerative effects of Semaphorin A and / or for improving retinal revascularization.

[0122] Preferably, the present invention provides an anti-semaphorin 3A antibody or an antigen-binding fragment thereof for use in the treatment or prevention of retinal or ocular diseases. The present inventors have actually developed an antibody targeting semaphorin 3A, which is - redirecting angiogenesis towards ischemic areas to improve retinal revascularization; - preventing pathological neovascularization of the vitreous region; and -A process for preventing the breakdown of the blood-retinal barrier It will be extremely helpful for.

[0123] As previously described, semaphorin 3A is a vascular repulsion signal secreted by hypoxic retinal ganglion cells. By binding to neurofilin-1, semaphorin 3A activates intracellular signaling of plexin receptors on endothelial cells, resulting in dissociation of actin fibers. This leads to the collapse of the cytoskeleton in the filopodia of end cells, specialized endothelial cells that direct the growth of new blood vessels and prevent revascularization of ischemic areas in the retina. The inventors have shown that modulating vascular repulsion with a neutralizing semaphorin 3A antibody will increase the number of end cells and redirect angiogenesis toward ischemic areas, such as the pathologically enlarged foveal avascular zone in humans with diabetic macular ischemia.

[0124] Therefore, in a fourth aspect, the present invention relates to an anti-semaphorin 3A antibody, or an antigen-binding fragment thereof, for use in the treatment or prevention of a disease selected from the group consisting of retinopathy, ischemic retinopathy, diabetic retinopathy (including proliferative diabetic retinopathy and non-proliferative diabetic retinopathy), diabetic macular edema, diabetic macular ischemia, age-related macular degeneration, retinitis pigmentosa, inherited retinal dystrophies, myopic degeneration, retinal artery occlusion, endophthalmitis, uveitis, cystoid macular edema, choroidal neovascular membrane secondary to any retinal disease, optic neuropathy, glaucoma, retinal detachment, toxic retinopathy, radiation retinopathy, traumatic retinopathy, drug-induced retinal vasculopathy, retinal neovascularization, polypoidal choroidal vasculopathy, retinal vasculitis, retinal microaneurysm, Fuchs' dystrophy, macular telangiectasia, Usher syndrome, and Stargardt's disease.

[0125] The anti-semaphorin 3A antibodies of the present invention are particularly useful for treating or preventing diabetic retinopathy (including proliferative diabetic retinopathy and non-proliferative diabetic retinopathy), ischemic retinopathy, diabetic macular edema, diabetic macular ischemia, age-related macular degeneration, retinal neovascularization, and choroidal neovascularization.

[0126] In a preferred embodiment, the disease is diabetic macular ischemia and the antibodies of the invention promote revascularization within the ischemic retina (revascularization) and prevent pathological neovascularization of the vitreous region of the eye.

[0127] In another preferred embodiment, the disease is diabetic macular edema and the antibody of the invention reduces the permeability of the blood-retinal barrier.

[0128] In another preferred embodiment, the present invention provides an anti-semaphorin 3A antibody or an antigen-binding fragment thereof for inhibiting semaphorin 3A-induced blood-retinal barrier permeability and / or semaphorin 3A-induced vascular regression in ischemic areas.

[0129] In a preferred aspect, the antibodies of the invention are useful for treating diabetic macular edema (DME) and / or diabetic macular ischemia (DMI). In a preferred embodiment, the antibodies of the invention are useful for treating patients suffering from DME and DMI. Preferably, the antibodies of the invention are used to treat DMI, as defined by an enlargement of the foveal avascular zone (FAZ) of 15% or more, 20% or more, 25% or more, and more preferably 30% or more.

[0130] In a fifth aspect, the present invention provides a pharmaceutical composition comprising an anti-semaphorin 3A antibody or an antigen-binding fragment thereof and a pharma- ceutically acceptable carrier.

[0131] The anti-semaphorin 3A antibody or its antigen-binding fragment is administered by any suitable means, including intravitreal, oral, parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal. Parenteral injections include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Additionally, the anti-semaphorin 3A antibody is suitably administered by pulse infusion, particularly with tapering doses of the antibody. In one embodiment, the dosage is administered by injection, most preferably intravenous or subcutaneous injection, depending in part on whether the administration is brief or chronic. Preferably, the anti-semaphorin 3A antibody is administered through intravitreal injection into the eye.

[0132] For the prevention or treatment of disease, the appropriate dose of antibody will depend on a variety of factors, such as the type of disease to be treated, as defined above, the severity and history of the disease, whether the antibody is being administered for prophylactic or therapeutic purposes, previous treatments, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments.

[0133] In a preferred embodiment, the dose range of the antibody of the invention applicable per injection is typically 1 mg / eye to 10 mg / eye, preferably 1.5 mg / eye to 5 mg / eye, more preferably 2 mg / eye to 3 mg / eye, and even more preferably about 2.5 mg / eye.

[0134] The term "suppress" is used herein in the same context as "amelioration" and "relief" to mean a decrease or lessening of one or more characteristics of a disease.

[0135] The antibody compositions will be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to be considered in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the dosing schedule, and other factors known to medical practitioners. The "therapeutically effective amount" of the antibody to be administered will be governed by such considerations, and is the minimum amount necessary to prevent, ameliorate, or treat the ocular or retinal disease addressed by the antibody of the invention.

[0136] The antibody need not be, but is optionally, formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents will depend on the amount of anti-Semaphorin 3A antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These will generally be used in the same dosages and routes of administration as previously used herein, or at about 1-99% of the dosages previously used.

[0137] In another aspect, the present invention also relates to an anti-Semaphorin 3A antibody or antigen-binding fragment for use in the treatment or prevention of non-ophthalmic diseases, such as autoimmune arthritis, neuropathic pain, osteoporosis, and cancer.

[0138] Treatment In another aspect, the present invention also encompasses any method for treating or preventing the eye or an eye disease in a patient in need thereof, the method comprising administering an anti-semaphorin 3A antibody of the present invention.

[0139] Preferably, the present invention relates to the use of an antibody according to the invention for inhibiting the vasoregressive action of semaphorin 3A.More preferably, the present invention relates to said method for improving retinal revascularization.

[0140] Preferably, the present invention relates to a method for treating or preventing an ocular or retinal disease comprising administering to a patient in need thereof a pharma- tically effective amount of an antibody according to the present invention, preferably the disease is selected from the group consisting of retinopathy, ischemic retinopathy, diabetic retinopathy (including proliferative and non-proliferative diabetic retinopathy), diabetic macular edema, diabetic macular ischemia, age-related macular degeneration, retinitis pigmentosa, hereditary retinal dystrophies, myopic degeneration, retinal artery occlusion, endophthalmitis, uveitis, cystoid macular edema, choroidal neovascular membrane secondary to any retinal disease, optic neuropathy, glaucoma, retinal detachment, toxic retinopathy, radiation retinopathy, traumatic retinopathy, drug-induced retinal vasculopathy, retinal neovascularization, polypoidal choroidal vasculopathy, retinal vasculitis, retinal microaneurysm, Fuchs' dystrophy, macular telangiectasia, Usher syndrome, and Stargardt's disease. More preferably, the disease is selected from the group consisting of diabetic retinopathy (including proliferative and non-proliferative diabetic retinopathy), ischemic retinopathy, diabetic macular edema, diabetic macular ischemia, age-related macular edema, retinal neovascularization, glaucoma, and choroidal neovascularization. In a further preferred embodiment, the disease is diabetic macular edema and / or diabetic macular ischemia.

[0141] All disclosed technical features described herein are applicable to said treatment methods.

[0142] Pharmaceutical Compositions and Their Administration The composition comprising the anti-semaphorin 3A antibody or an antigen-binding fragment thereof may be administered to a subject having or at risk of having an ocular or retinal disease. The present invention further provides the use of the anti-semaphorin 3A antibody or an antigen-binding fragment thereof in the manufacture of a medicament for the prevention or treatment of having an ocular or retinal disease or a semaphorin 3A disease. All disclosed technical features described herein are applicable to said use of the anti-semaphorin 3A antibody or an antigen-binding fragment thereof in the manufacture of a medicament. As used herein, the term "subject" refers to any mammalian patient (including, for example, humans and non-human mammals, such as primates, rodents, and dogs) to which the anti-semaphorin 3A antibody or an antigen-binding fragment thereof can be administered. Subjects particularly targeted for treatment using the methods described herein include humans. The anti-semaphorin 3A antibody or an antigen-binding fragment thereof may be administered alone or in combination with other compositions.

[0143] Various delivery systems are known and can be used to administer anti-semaphorin 3A antibodies or antigen-binding fragments thereof. Introduction methods include, but are not limited to, intravitreal, ophthalmic solution, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. Anti-semaphorin 3A antibodies or antigen-binding fragments thereof can be administered, for example, by infusion, bolus, or injection, and may be administered together with other biologically active agents. Administration can be systemic or local. In a preferred embodiment, administration is by injection into the vitreous. Such a formulation for injection can be prepared, for example, in a prefilled syringe.

[0144] The anti-semaphorin 3A antibody or antigen-binding fragment thereof may be administered as a pharmaceutical composition comprising a therapeutically effective amount of the anti-semaphorin 3A antibody or antigen-binding fragment thereof and one or more pharma- ceutically compatible ingredients.

[0145] In a typical embodiment, the pharmaceutical composition is formulated according to the usual procedures as a pharmaceutical composition adapted for intravenous or subcutaneous administration to humans. Typically, compositions for administration by injection are solutions in sterile isotonic aqueous buffer. If necessary, the medicament may also contain a stabilizer and a local anesthetic, such as lignocaine, to ease pain at the injection site. Generally, the ingredients are supplied in unit dosage form, either separately or mixed together, in a sealed container, such as an ampoule or sachet indicating the amount of active agent, for example as a lyophilized dry powder or water-free concentrate. If the medicament is to be administered by injection, it can be dispensed using an infusion bottle containing sterile water or saline of pharmaceutical grade. If the medicament is to be administered by injection, an ampoule of sterile water for injection or saline is provided so that the ingredients can be mixed prior to administration.

[0146] Further, the pharmaceutical composition may be provided as a pharmaceutical kit, comprising (a) a container containing the anti-semaphorin 3A antibody or antigen-binding fragment thereof in lyophilized form, and (b) a second container containing a pharma- ceutically acceptable diluent for injection (e.g., sterile water). The pharma- ceutically acceptable diluent may be used for reconstitution or dilution of the lyophilized anti-semaphorin 3A antibody or antigen-binding fragment thereof. Optionally, such container(s) may be accompanied by a notice in a format prescribed by a governmental authority regulating the manufacture, use, or sale of pharmaceuticals or biological products, the notice reflecting approval by the authority for manufacture, use, or sale for administration to humans.

[0147] The amount of anti-semaphorin 3A antibody or its antigen-binding fragment that is effective for treating or preventing eye disease or retinal disease can be determined by standard clinical techniques. In addition, in vitro assays can be used in some cases to help identify optimal dosage ranges. The exact dosage to be used in the formulation also depends on the route of administration and the stage of the disorder, and should be determined according to the judgment of the practitioner and each patient's circumstances. Effective dosages can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0148] For example, the toxicity and therapeutic efficacy of an anti-semaphorin 3A antibody or antigen-binding fragment thereof can be determined in cell culture or experimental animals using ED 50 (the dose that is therapeutically effective in 50% of the population) can be determined by standard pharmaceutical procedures. Anti-semaphorin 3A antibodies or antigen-binding fragments thereof that exhibit large therapeutic indices are preferred.

[0149] The data obtained from cell culture assays and animal studies can be used in formulating a range of dosages for use in humans. The dose of the anti-Semaphorin 3A antibody or antigen-binding fragment thereof is typically within the ED range with little or no toxicity. 50 The circulating concentration of the antibody or antibody fragment thereof will generally lie within a range of concentrations including 0.1 to 0.5%. The dosage may vary within this range depending on the dosage form employed and the route of administration used. The therapeutically effective dose for any anti-semaphorin 3A antibody or antigen-binding fragment thereof used in the method can be estimated initially from cell culture assays. In animal models, the IC as determined in cell culture medium can be used to estimate the therapeutically effective dose. 50 Doses can be formulated to achieve a circulating plasma concentration range that includes (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms). Such information can be used to accurately determine useful doses in humans. Plasma levels can be measured, for example, by high performance liquid chromatography, ELISA, and the like.

[0150] For intravitreal injection of anti-semaphorin 3A antibodies, longer treatment intervals are generally preferred. Due to its improved binding affinity and potency, the anti-semaphorin 3A antibodies of the present invention can be administered at longer intervals.

[0151] In one embodiment, the anti-semaphorin 3A antibody is administered every 6 weeks, preferably every 7 weeks, preferably every 8 weeks, preferably every 9 weeks, preferably every 10 weeks, preferably every 11 weeks, more preferably every 12 weeks. In a further preferred embodiment, the anti-semaphorin 3A antibody of the present invention is administered once every 3 months.

[0152] The volume that can be administered to the eye is strictly limited, so it is very important that anti-semaphorin 3A antibody can be formulated to a high concentration.In addition, the efficacy of anti-semaphorin 3A antibody is very important, because a strong antibody can exert its effect even at a lower dose, thereby extending the activity and also the treatment interval.

[0153] The antibodies of the invention can be formulated up to very high doses, including but not limited to 20 mg / ml, 30 mg / ml, 40 mg / ml, 50 mg / ml, 60 mg / ml, 70 mg / ml, 80 mg / ml, 90 mg / ml, or 100 mg / ml. Preferably, the antibodies of the invention can be formulated in a liquid formulation of about 50 mg / ml.

[0154] A typical dose that can be administered to a patient is about 2.5 mg / eye. Typical buffer components that can be used for such formulations include, for example, sodium acetate, PS20, and trehalose dihydrate.

[0155] In one embodiment, the anti-semaphorin 3A antibody is formulated at a final concentration of 60 mg / ml using 10 mM histidine buffer, 240 mM sucrose, 0.02 w / v % polysorbate 20, pH 5.5.

[0156] In some embodiments, a pharmaceutical composition comprising an anti-semaphorin 3A antibody or antigen-binding fragment thereof may further comprise a therapeutic agent, which is either conjugated or unconjugated to the binding agent.

[0157] Regarding therapeutic regimens for combined administration, in certain embodiments, the anti-semaphorin 3A antibody or antigen-binding fragment thereof is administered simultaneously with the therapeutic agent. In another specific embodiment, the therapeutic agent is administered at least 1 hour to several months before or after administration of the anti-semaphorin 3A antibody or antigen-binding fragment thereof, for example, at least 1 hour, 5 hours, 12 hours, 1 day, 1 week, 1 month, or 3 months before or after administration of the anti-semaphorin 3A antibody or antigen-binding fragment thereof.

[0158] Polynucleotides, Vectors, Host Cells, and Recombinant Methods In a sixth aspect, the present invention encompasses isolated polynucleotides comprising sequences encoding anti-semaphorin 3A antibodies, vectors, and host cells containing the polynucleotides, as well as recombinant techniques for the production of the antibodies. The isolated polynucleotides can encode anti-semaphorin 3A antibodies in any desired form, including, for example, full-length monoclonal antibodies, Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0159] The polynucleotide(s) containing sequences encoding the anti-semaphorin 3A antibody or fragments thereof or chains thereof can be fused to one or more regulatory or control sequences as known in the art and can be included in a suitable expression vector or host cell as known in the art. Each polynucleotide molecule encoding a heavy or light chain variable domain can be independently fused to a polynucleotide sequence encoding a constant domain, such as a human constant domain, allowing for the production of an intact antibody. Alternatively, polynucleotides or portions thereof can be fused to each other, providing a template for the production of single chain antibodies.

[0160] For recombinant production, a polynucleotide encoding the antibody is inserted into a replicable vector for cloning (amplification of the DNA) or expression. Many vectors suitable for expressing recombinant antibodies are available. The vector components generally include one or more of the following, but are not limited to: a signal sequence, an origin of replication, one or more marker genes, an enhancer sequence, a promoter, and a transcription termination sequence.

[0161] The anti-semaphorin 3A antibody may also be produced as a fusion polypeptide, in which the antibody is fused to a heterologous polypeptide, such as a signal sequence or other polypeptide with a specific cleavage site at the amino terminus of the mature protein or polypeptide. The heterologous signal sequence selected is typically one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the signal sequence of the anti-semaphorin 3A antibody, the signal sequence may be replaced with a prokaryotic signal sequence. The signal sequence may be, for example, the leader sequence of alkaline phosphatase, penicillinase, lipoprotein, heat-stable enterotoxin II, etc. The native signal sequence for secretion in yeast may be substituted, for example, by leader sequences derived from yeast invertase alpha-factor (including the Saccharomyces and Kluyveromyces alpha-factor leader sequences), acid phosphatase, C. albicans glucoamylase, or the signal sequences described in WO 90 / 13646. In mammalian cells, mammalian signal sequences may be used as well as viral secretory leader sequences, such as the herpes simplex gD signal sequence. DNA for such a precursor region is ligated in reading frame to DNA encoding the humanized anti-Semaphorin 3A antibody.

[0162] Expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Typically, in cloning vectors, this sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and includes an origin of replication or an autonomously replicating sequence. Such sequences are well known for a variety of bacteria, yeast, and viruses. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2-v. plasmid origin is suitable for yeast, and various viral origins of replication (simian virus (SV) 40, polyoma virus, adenovirus, vesicular stomatitis virus, and bovine papilloma virus) are useful for cloning vectors in mammalian cells. Generally, the origin of replication component is not needed for mammalian expression vectors (the SV40 origin may typically be used simply because it contains the early promoter).

[0163] Expression and cloning vectors may contain a gene encoding a selectable marker to facilitate identification of expression. Typical selectable marker genes encode proteins that confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline, or alternatively complement auxotrophic deficiencies or, in other alternatives, supply specialized nutrients not present in complex media, such as genes encoding D-alanine racemase for Bacillus.

[0164] One example of a selection scheme utilizes drugs that stop the growth of host cells. Those cells that are successfully transformed with a heterologous gene produce a protein that confers drug resistance and thus survive the selection regimen. Examples of such dominant selection use the drugs neomycin, mycophenolic acid, and hygromycin. Common selection markers for mammalian cells are selection markers that allow the identification of cells that are competent to take up nucleic acid encoding a humanized anti-semaphorin 3A antibody, such as DHFR (dihydrofolate reductase), thymidine kinase, metallothionein I and II (e.g., primate metallothionein genes), adenosine deaminase, ornithine decarboxylase, etc. Cells transformed with the DHFR selection gene are first identified by culturing all the transformants in a culture medium containing methotrexate (Mtx), a competitive antagonist of DHFR. When wild-type DHFR is used, a suitable host cell is a Chinese hamster ovary (CHO) cell line deficient in DHFR activity (eg, DG44).

[0165] Alternatively, host cells (particularly wild-type hosts containing endogenous DHFR) transformed or co-transformed with a DNA sequence encoding an anti-semaphorin 3A antibody, a wild-type DHFR protein, and another selectable marker, such as an aminoglycoside 3'-phosphate transferase (APH), can be selected by growing the cells in medium containing a selection agent for the selectable marker, such as an aminoglycoside antibiotic, such as kanamycin, neomycin, or G418. See, e.g., U.S. Patent No. 4,965,199.

[0166] When recombinant production is carried out in yeast cells as host cells, the TRP1 gene present in the yeast plasmid YRp7 (Stinchcomb et al., 1979, Nature 282: 39) can be used as a selection marker. The TRP1 gene provides a selection marker for mutant strains of yeast lacking the ability to grow in tryptophan, e.g., ATCC No. 44076 or PEP4-1 (Jones, 1977, Genetics 85:12). Thus, the presence of the trp1 lesion in the yeast host cell genome provides an effective environment for detecting transformation by growth in the absence of tryptophan. Similarly, Leu2p-deficient yeast strains, e.g., ATCC 20,622 and 38,626, are complemented by known plasmids carrying the LEU2 gene.

[0167] Additionally, vectors derived from the 1.6 μm circular plasmid pKD1 can be used for transformation of Kluyveromyces yeast. Alternatively, an expression system for large-scale production of recombinant calf chymosin for Kluyveromyces lactis has been reported (Van den Berg, 1990, Bio / Technology 8:135). A stable multicopy expression vector for secretion of mature recombinant human serum albumin by industrial strains of Kluyveromyces has also been disclosed (Fleer et al., 1991, Bio / Technology 9:968-975).

[0168] Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to the nucleic acid molecule encoding the anti-Semaphorin 3A antibody or its polypeptide chain. Suitable promoters for use in prokaryotic hosts include the phoA promoter, β-lactamase and lactose promoter systems, alkaline phosphatase, tryptophan (trp) promoter system, and hybrid promoters such as the tac promoter. Other known bacterial promoters are also suitable. Promoters for use in bacterial systems will also contain a Shine-Dalgarno (SD) sequence operably linked to the DNA encoding the humanized anti-Semaphorin 3A antibody.

[0169] Many eukaryotic promoter sequences are known. Virtually all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated. Another sequence found 70-80 bases upstream from the transcription start site of many genes is a CNCAAT region, where N can be any nucleotide. At the 3' end of most eukaryotic genes is an AATAAA sequence, which can be a signal for addition of a poly A tail to the 3' end of the coding sequence. All of these sequences are appropriately inserted into eukaryotic expression vectors.

[0170] Examples of promoter sequences suitable for use in yeast hosts include the promoters of 3-phosphoglycerate kinase or other glycolytic enzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.

[0171] Inducible promoters have the added advantage of transcription being controlled by growth conditions. These include yeast promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, inducible enzymes related to nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes involved in maltose and galactose utilization. Vectors and promoters suitable for use in expression in yeast are further described in EP 73,657. Yeast enhancers are also advantageously used with yeast promoters.

[0172] Transcription of the anti-semaphorin 3A antibody from the vector in a mammalian host cell is controlled by a promoter derived from the genome of a virus such as polyoma virus, fowlpox virus, adenovirus (e.g., adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, and simian virus 40 (SV40), from a heterologous mammalian promoter, such as the actin promoter or an immunoglobulin promoter, or from a heat shock promoter, provided that such promoter is compatible with the host cell system.

[0173] The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment which also contains the SV40 viral origin of replication. The immediate early promoter of the human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment. A system for expressing DNA in mammalian hosts using bovine papilloma virus as a vector is disclosed in U.S. Pat. No. 4,419,446. A modification of this system is described in U.S. Pat. No. 4,601,978. See also Reyes et al., 1982, Nature 297:598-601, who disclosed expression of human p-interferon cDNA in mouse cells under the control of a thymidine kinase promoter derived from herpes simplex virus. Alternatively, the Rous sarcoma virus long terminal repeat can be used as a promoter.

[0174] Another useful sequence that can be used in a recombinant expression vector is an enhancer sequence, which is used to increase the transcription of DNA encoding an anti-semaphorin 3A antibody by higher eukaryotic cells. Many enhancer sequences are now known from mammalian genes (e.g., globin, elastase, albumin, α-fetoprotein, and insulin). Typically, however, enhancers from eukaryotic cell viruses are used. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. See also Yaniv, 1982, Nature 297:17-18 for a description of enhancing sequences for the activation of eukaryotic promoters. The enhancer can be spliced ​​to the 5' or 3' position of the anti-semaphorin 3A antibody-encoding sequence of the vector, but is preferably located at the 5' site of the promoter.

[0175] Expression vectors used in eukaryotic host cells (nucleated cells derived from yeast, fungi, insects, plants, animals, humans, or other multicellular organisms) may also contain sequences required for transcription termination and for stabilizing mRNA. Such sequences are generally available from the 5' and sometimes 3' untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments that are transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the anti-semaphorin 3A antibody. One useful transcription termination component is the bovine growth hormone polyadenylation region. See WO 94 / 11026 and the expression vectors disclosed therein. In some embodiments, anti-semaphorin 3A antibodies can be expressed using the CHEF system (see, e.g., U.S. Pat. No. 5,888,809; the disclosure of which is incorporated herein by reference).

[0176] Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryotic, yeast, or higher eukaryotic cells described above. Suitable prokaryotic cells for this purpose include eubacteria, including gram-negative and gram-positive organisms, such as Enterobacteriaceae, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as bacilli, e.g., B. subtilis, B. licheniformis (e.g., B. licheniformis 41, disclosed in German Patent No. 266,710 published April 12, 1989), Pseudomonas, e.g., P. aeruginosa, and Streptomyces. One preferred E. coli cloning host is E. coli 294 (ATCC 31,446), although other strains such as E. coli B, E. coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are also suitable. These examples are illustrative rather than limiting.

[0177] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for anti-Semaphorin 3A antibody-encoding vectors. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, many other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts, such as K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus (ATCC 36,906). marxianus); Yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces, such as Schwanniomyces occidentali; and filamentous fungi, such as Neurospora, Penicillium, Tolypocladium and Aspergillus, such as A. nidulans and A. niger.

[0178] Suitable host cells for the expression of glycosylated anti-semaphorin 3A antibodies are obtained from multicellular organisms. Examples of invertebrate cells include, for example, numerous baculovirus strains and mutants, as well as plant and insect cells, including corresponding permissive insect host cells derived from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori (silkworm). A wide variety of viral strains for transfection are publicly available, e.g., the L-1 mutant of Autographa californica nuclear polyhedrosis virus (NPV) and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used specifically for transfection of Spodoptera cells.

[0179] Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco may also be utilized as hosts.

[0180] The anti-semaphorin 3A antibody of the present invention may also be incorporated into a viral vector, i.e., a polynucleotide encoding the anti-semaphorin 3A antibody or an antigen-binding fragment thereof is introduced into a viral vector and then expressed in the patient's body after infection with the virus.

[0181] In another embodiment, expression of the anti-Semaphorin 3A antibody is in vertebrate cells. Propagation of vertebrate cells in culture (tissue culture) has become a routine procedure and the techniques are widely available. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL1651), human embryonic kidney lines (293 cells or 293 cells subcloned for growth in suspension culture), (Graham et al., 1977, J. Gen Virol. 36: 59), baby hamster kidney cells (BHK, ATCC CLL10), Chinese hamster ovary cells / -DHFR1 (CHO, Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77: 4216; e.g., DG44), mouse Sertoli cells (TM4, Mather, 1980, Biol. Reprod. 23:243-251), mouse kidney cells (CV1 ATCC CCL 70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical carcinoma cells (HELA, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), buffalo rat hepatocytes (BRL3A, ATCC CRL1442), human lung cells (W138, ATCC CCL 75), human hepatocytes (Hep G2, HB8065), mouse mammary tumor (MMT060562, ATCC CCL51), TR1 cells (Mather et al., 1982, Annals NY Acad. Sci. 383: 44-68), MRC5 cells, FS4 cells, and a human hepatoma line (Hep G2).

[0182] Host cells are transformed with the above-described expression vectors or cloning vectors for the production of anti-semaphorin 3A antibodies and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying genes encoding the desired sequences.

[0183] The host cells used to produce the anti-semaphorin 3A antibodies described herein can be cultured in a variety of media. Commercially available media, such as Ham's F10 (Sigma-Aldrich, St. Louis, MO), Minimal Essential Medium ((MEM), Sigma-Aldrich), RPMI-1640 (Sigma-Aldrich), and Dulbecco's Modified Eagle Medium ((DMEM), Sigma-Aldrich) are suitable for culturing the host cells. Furthermore, any of the media described in one or more of Ham et al., 1979, Meth. Enz. 58: 44, Barnes et al., 1980, Anal. Biochem. 102: 255, U.S. Pat. No. 4,767,704, U.S. Pat. No. 4,657,866, U.S. Pat. No. 4,927,762, U.S. Pat. No. 4,560,655, U.S. Pat. No. 5,122,469, WO 90 / 103430, and WO 87 / 00195 may be used as a culture medium for the host cells. Any of these media may be supplemented as necessary with hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphate), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., gentamicin), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Other supplements may also be included at appropriate concentrations, as would be known to one of skill in the art. Culture conditions, such as temperature, pH, etc., will be those previously used with the host cell selected for expression and will be apparent to one of skill in the art.

[0184] When using recombinant techniques, antibodies may be produced intracellularly in the periplasmic space or directly secreted into the medium. If antibodies are produced intracellularly, the cells may be disrupted as a first step to release the protein. Particulate debris, either host cells or lysed fragments, may be removed, for example, by centrifugation or ultrafiltration. Carter et al., 1992, Bio / Technology 10:163-167, describes a procedure for isolating antibodies secreted into the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for about 30 minutes. Cell debris may be removed by centrifugation. If antibodies are secreted into the medium, the supernatant of such expression systems is generally first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. Protease inhibitors, such as PMSF, may be included in any of the above steps to inhibit protein degradation and antibiotics may be included to prevent the growth of adventitious contaminants.A variety of methods can be used to isolate antibodies from host cells.

[0185] The antibody composition prepared from the cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being a typical purification technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human gamma 1, gamma 2, or gamma 4 heavy chains (see, e.g., Lindmark et al., 1983 J. Immunol. Meth. 62:1-13). Protein G is recommended for all mouse isotypes and human gamma 3 (see, e.g., Guss et al., 1986 EMBO J. 5:1567-1575). The matrix to which the affinity ligand is attached is most often agarose, although other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. If the antibody contains a CH3 domain, Bakerbond ABX™ resin (JT Baker, Phillipsburg, NJ) is useful for purification. Other techniques for protein purification, such as fractionation on ion exchange columns, ethanol precipitation, reversed-phase HPLC, silica chromatography, heparin chromatography, Sepharose™ chromatography on anion or cation exchange resins (e.g., polyaspartic acid columns), isoelectric focusing, SDS-PAGE, and ammonium sulfate precipitation, are also available depending on the antibody to be recovered.

[0186] After any prior purification step(s), the mixture containing the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography, typically performed at low salt concentration (e.g., about 0-0.25 M salt) and using an elution buffer at a pH of about 2.5-4.5.

[0187] Also included are nucleic acids that hybridize to all or a portion (e.g., a portion encoding a variable region) of the nucleotide sequence set forth by the isolated polynucleotide sequence(s) encoding the semaphorin 3A antibody or antibody fragment under low, medium, and high stringency conditions as defined herein. The hybridizing portion of the hybridizing nucleic acid is typically at least 15 (e.g., 20, 25, 30, or 50) nucleotides in length. The hybridizing portion of the hybridizing nucleic acid is at least 80%, e.g., at least 90%, at least 95%, or at least 98% identical to a portion or the entire sequence of the nucleic acid encoding the anti-semaphorin 3A polypeptide (e.g., the variable region of the heavy or light chain) or its complement. Hybridizing nucleic acids of the type described herein can be used, for example, as probes, primers, e.g., PCR primers, or diagnostic probes for cloning.

[0188] In one embodiment, the invention relates to an isolated polynucleotide or group of polynucleotides comprising a heavy chain as set forth in SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:19, or a sequence encoding a heavy chain variable region as set forth in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10; and a light chain as set forth in SEQ ID NO:15, SEQ ID NO:18, or SEQ ID NO:20, or a sequence encoding a light chain variable region as set forth in SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13.

[0189] In the above-mentioned anti-semaphorin 3A antibodies and antibody fragments, the nucleic acid sequences encoding the CDRs remain unchanged (unchanged with respect to the amino acids they encode, although equivalent DNA sequences due to codon degeneracy are also possible), but the surrounding regions, e.g., the FR regions, may be engineered.

[0190] product In another embodiment, the article of manufacture includes a material useful for treating the above disorders. The article of manufacture includes a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers can be formed from a variety of materials, such as glass or plastic. The container holds a composition effective for treating a condition and can have a sterile access port. For example, the container can be an infusion bag or a vial with a stopper that can be pierced by a hypodermic needle. The active agent in the composition is an anti-semaphorin 3A antibody or an antigen-binding fragment thereof. A label on or attached to the container indicates that the composition is used to treat a selected condition. The article of manufacture can further include a second container containing a pharma- ceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution, and dextrose solution. It can also include other materials desirable from the standpoint of the manufacturer and user, such as other buffers, diluents, filters, needles, syringes, and package inserts containing instructions for use.

[0191] The present invention is further described in the following examples, which are not intended to limit the scope of the invention.

[0192] Working Example Example 1: Upregulation of Semaphorin 3A in the Vitreous of Patients with Diabetic Macular Edema and Proliferative Diabetic Retinopathy The expression of Semaphorin 3A in the retina of samples from human donors with a history of diabetic retinopathy was examined by immunohistochemistry. The immunostaining protocol was as follows: 1. Thaw the slides and allow the specimens to air dry at room temperature (RT) for 30 minutes; 2. Draw a line around the area with a pompom pen and let it dry; 3. Antigen retrieval in 1% SDS for 5 minutes at room temperature; 4. Wash slides with PBS three times for 5 min; 5. Block sections in 1% bovine serum albumin / 0.3% Tritonx 100 / PBS solution (blocking solution) for 30 min at room temperature; 6. Dilute rabbit anti-semaphorin 3a (Abcam, ab23393) primary antibody 1:200 in blocking solution. Incubate the sections on the slides overnight at room temperature; 7. Rinse slides 3 times with PBS for 5 minutes; 8. Perform a second antibody incubation with donkey anti-rabbit Alexa fluor546 (Invitrogen, A10040) at a dilution of 1:400 in DAPI / 0.3% Triton x 100 / PBS solution. Incubate the sections on the slides for 3 hours at room temperature; 9. Rinse slides 3-5 times with PBS for 5 min; 10. Coverslip the sections with Aquamount mounting medium and allow to air dry; 11. Sections are imaged at 40x magnification and the intensity is graded.

[0193] A set of three sections per each human donor was immunostained for Semaphorin 3A. Semaphorin 3A labeling in each of these regions was independently assessed using a five-point grading scheme (0 = not detected at all; 1 = low intensity, few spots; 2 = moderate intensity, few spots; 3 = bright intensity, widespread staining; and 4 = very bright intensity, abundant detection) by observers who were previously trained for this specific task. The observers were blinded to the health status of the ocular donors. Semaphorin 3A in the retina was bound to the vasculature walls of retinal blood vessels. Expression of Semaphorin 3A in the retinal vasculature and retinal parenchyma was increased in patients with diabetic macular edema compared to diabetics without ocular pathology (Figure 1).

[0194] Example 2: Efficacy in cell permeability assay Transcellular permeability was measured by permeation of FITC-dextran through monolayers of human retinal microvascular endothelial cells (HRMEC).

[0195] Briefly, in vitro endothelial permeability was measured using a Millipore kit ("In Vitro Vascular Permeability Assay" Cat. No. ECM642) and human retinal microvascular endothelial cells (HRMEC). The assay kit provides a 96-well receiver plate with cell culture inserts. The inserts contain 1 μm pores and are coated with rat tail collagen type I. HRMEC were seeded onto the inserts at a density of 25000 cells / well and the cells were allowed to grow into a monolayer for 3 days. The cells were treated overnight with recombinant VEGF-A, semaphorin 3A and the antibodies described in the present invention. A high molecular weight FITC-dextran solution provided in the kit was added to the inserts to allow the fluorescent molecules to pass through the monolayer of endothelial cells. In vitro permeability was determined by measuring the fluorescence of the solution in the receiver plate wells at 485 nm / 535 nm (excitation and absorption).

[0196] The inventors have tested an exemplary antibody according to the invention: Clone I. The antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:14 and a light chain comprising the amino acid sequence of SEQ ID NO:15.

[0197] The anti-semaphorin 3A antibody described in the present invention completely inhibited the permeability induced by semaphorin 3A, but not by VEGF-A (Figure 2).Importantly, the permeability effect of semaphorin 3A is independent of VEGF-A.The use of a control antibody directed against TNP confirms that the effect is due to the specific targeting of semaphorin 3A by the antibody of the present invention.

[0198] Example 3: Measurement of cytoskeletal disruption in a cellular assay The intracellular activity of an exemplary antibody (clone I) according to the present invention was assessed by measuring cytoskeleton disintegration in human retinal microvascular endothelial cells (HRMEC) using the XCELLigence system (a real-time cell analysis instrument as marketed by ACEA Biosciences). The system measures cell attachment and confluence via cellular impedance. HRMEC endogenously express neuropilin-1 (Nrp1) and plexins, components of the class 3 semaphorin holoreceptor. By binding to this receptor complex, semaphorins induce disintegration of F-actin fibers in the endothelium. In this functional assay, the addition of recombinant semaphorin 3A protein to a confluent layer of human retinal microvascular endothelial cells reduces cellular impedance due to cytoskeleton disintegration and subsequent cell shrinkage, measured as a decrease in cellular impedance.

[0199] Briefly, E-Plates View were coated with attachment factors. Cells were seeded at a density of 20,000 cells / well and then grown overnight to form a monolayer in the XCELLigence instrument under its normal growth conditions.

[0200] Semaphorin 3A (or other class 3 semaphorins) with and without anti-Semaphorin 3A antibodies according to the combination of the present invention were added in the presence of 3 mM CaCl2. The cell index was normalized to the time point before the addition of the substance. Calculations were performed 5 hours after stimulation.

[0201] Cytoskeletal collapse induced by semaphorin 3A can be completely prevented by the anti-semaphorin 3A antibody described in the present invention. Cytoskeletal collapse induced by other tested semaphorins (B, C, E and F) cannot be prevented by the anti-semaphorin 3A antibody described in the present invention, confirming the specificity of the antibody of the present invention for semaphorin 3A (Figure 3).

[0202] Example 4: Affinity and Cellular Potency A) Affinity The running buffer and all dilutions (unless otherwise noted) for this experiment were made with PBS-T-EDTA containing 0.01% Tween 20 [100 μl of 100% Tween 20 was added to 2 L of PBS-T-EDTA to make a final concentration of 0.01% Tween 20]. The GLM sensor chip was normalized and preconditioned according to the manufacturer's recommendations. The sensor chip was activated for 300 seconds with an equivalent mixture of EDC / s-NHS (sulfo-N-hydroxysuccinimide) in the horizontal orientation at a flow rate of 30 μl / min and immobilized for 300 seconds with human Fab binder (10 μg / ml in 10 mM acetate, pH 5.0) in the horizontal orientation at a flow rate of 30 μl / min, resulting in approximately 6739-7414 RU of human Fab binder on the surface. The sensor chip was inactivated with 1 M ethanolamine hydrochloride for 300 s in the horizontal direction at a flow rate of 30 μl / min. The sensor chip was stabilized with 10 mM glycine (pH 2.1) for 18 s at a flow rate of 100 μl / min once in the horizontal direction and once in the vertical direction.

[0203] We tested an exemplary antibody (clone I) described in the present invention. The antibody (0.5 μg / ml) was captured vertically on the human Fab binder surface for 300 seconds at a flow rate of 25 μl / min, resulting in a capture level of approximately 180 RU. The baseline was stabilized by injecting PBS-T-EDTA horizontally for 60 seconds at a flow rate of 40 μl / min. Analytes were injected vertically on the captured antibody at a flow rate of 40 μl / min for 600 seconds and allowed to dissociate for 7200 seconds. Analyte concentrations were 0 nM, 0.625 nM, 1.25 nM, 2.5 nM, 5 nM, and 10 nM. The surface was regenerated by injecting 10 mM glycine (pH 2.1) for 18 seconds at a flow rate of 100 μl / min, once horizontally and once vertically. PBS-T-EDTA was injected vertically once for 60 seconds at a flow rate of 25 μl / min.

[0204] Interspot (interaction with the sensor surface) and blank (PBS-T-EDTA containing 0.01% Tween 20 or 0 nM analyte) were subtracted from the raw data. Sensorgrams were then globally fitted to 1:1 Langmuir binding to determine the binding kinetics (k a ), dissociation rate (k d ), and affinity (K D ) was obtained.

[0205] B) Cellular potency For the determination of functional potency in the cytoskeleton disruption assay, the Semaphorin 3A concentration-response curve was combined with increasing concentrations of the antibody as in the IC50 shift experiment. A Gaddam's Schild plot was performed to calculate the pA2 value (negative logarithm of the concentration of antibody required to shift the Semaphorin 3A concentration-response curve by 2-fold). Potency (pM) was calculated from the pA2 value as = potency(10;-X).

[0206] The results are summarized in Table 6 below.

[0207] [Table 4]

[0208] Example 5: Evaluation of the immunogenicity of antibodies of the invention The inventors have evaluated the predicted immunogenicity of an exemplary antibody according to the present invention, namely, clone I. The antibody comprises heavy and light chains comprising the amino acid sequences of SEQ ID NO:14 and SEQ ID NO:15, respectively.

[0209] For this purpose, we used a computational tool to predict T cell epitopes (EpiMatrix, developed by the company EpiVax).

[0210] By screening the sequences of many human antibody isolates, EpiVax identified several highly conserved HLA ligands that appear to have regulatory potential. Experimental evidence suggests that many of these peptides are in fact actively tolerogenic in most subjects. These highly conserved, regulatory, and promiscuous T cell epitopes are now known as regulatory T cell epitopes (De Groot et al. Blood. 2008 Oct 15;112(8):3303-11). The immunogenicity of neoepitopes contained in humanized antibodies can be effectively controlled in the presence of a significant number of regulatory T cell epitopes.

[0211] For the purpose of antibody immunogenicity analysis, EpiVax has developed a T-cell epitope regulated EpiMatrix score and prediction of antibody response to the corresponding therapeutic. To calculate the T-cell epitope regulated EpiMatrix score, the T-cell epitope regulated score is inferred from the EpiMatrix protein score. The T-cell epitope regulated score has been shown to correlate well with the clinical immune response observed for a set of 23 commercially available antibodies (De Groot et al. Clin Immunol. 2009 May;131(2):189-201).

[0212] The results for the EpiMatrix scales are summarized in Table 7 below.

[0213] [Table 5]

[0214] The antibody sequences of the present invention receive the lowest score on the EpiMatrix scale, indicating that the antibodies of the present invention have very little potential for immunogenicity. The EpiMatrix scale is well known to those skilled in the art and can be found, inter alia, in Figure 2 of the publication Mufarrege et al. Clin Immunol. 2017 Mar;176:31-41.

[0215] Example 6: Efficacy on cell density and avascular area in vivo (oxygen-induced retinopathy (OIR) model) The effect of an exemplary antibody (clone I) described in the present invention on revascularization of ischemic avascular areas was investigated in an oxygen-induced retinopathy (OIR) mouse model. C57BI / 6J mouse littermates were exposed to an atmosphere of 75% oxygen from postnatal day 7 to postnatal day 12. This leads to vessel regression in the central retina and the formation of an avascular area. After returning to normoxia, this area becomes ischemic. On postnatal day 12, pups receive a single intravitreal injection of 10 μg of antibody in 0.5 μl of solution in each eye under anesthesia with isoflurane. On postnatal day 17, animals are sacrificed and ocular nuclei are removed. Eyes are fixed in formalin and retinal flat mounts are prepared in which retinal vessels are stained with isolectin B4. The number of edge cells (specialized endothelial cells that initiate the formation of new blood vessels) is counted at the avascular front along the entire retina (the border between the vascularized peripheral and avascular central areas of the retina).

[0216] Edge cells were identified by their special morphology, which shows filopodial extensions. For analysis, the number of edge cells is normalized to the length of the avascular front. The size of the avascular area is determined using a confocal microscope and image analysis software.

[0217] The anti-semaphorin 3A antibody of the present invention increases the density of edge cells in a mouse OIR model (Figure 4). Moreover, it shows a decrease in the avascular area. There is a negative correlation between edge cell density and the size of the avascular area, indicating that the two parameters are mechanistically dependent. The use of a control antibody directed against TNP confirms that the effect is due to the specific targeting of semaphorin 3A by the antibody of the present invention.

[0218] In conclusion, the anti-semaphorin 3A antibodies of the invention reduce the size of the ischemic avascular area in an animal model of oxygen-induced retinopathy, indicating a beneficial effect in the treatment of diabetic macular ischemia.

[0219] Example 7: Effects of anti-semaphorin 3A antibody and Avastin in rabbit eyes 1 / 2 Comparison. The results are summarized in Table 8 below.

[0220] [Table 6]

[0221] The calculated half-lives were 3.9 days, 4.1 days, and 3.3 days in the vitreous, retina, and aqueous humor, respectively. These half-lives are similar to those reported in the literature for the clinically used recombinant humanized monoclonal IgG1 antibody Avastin (anti-VEGF antibody, bevacizumab, Bakri et al., Ophthalmology, 2007), which was also experimentally confirmed by the present inventors. These results were as expected, because the intravitreal clearance of full-length IgG depends mainly on its molecular size, which is similar in the antibody of the present invention and Avastin. Therefore, the human PK (including the ocular half-life of the antibody of the present invention and Avastin) is expected to be similar. The reported half-life of Avastin in the human eye is 9.73 ± 1.48 days (Hutton-Smith, 2016).

[0222] Example 8: Comparison of binding affinity between the antibody of the present invention and chiome antibody For comparative purposes, the inventors have developed a humanized antibody directed against semaphorin 3A, as disclosed in WO2014123186 (Kiome Biosciences), which has the following features: the heavy chain is as depicted in SEQ ID NO: 11 of WO2014123186; - the light chain is as set forth in SEQ ID NO: 12 of WO2014123186.

[0223] The inventors have developed two forms of this antibody: - formatted on IgG1KO Fc, hereafter referred to as "Chiome Antibody A", and -formatted on IgG1KO-FcRn null, hereafter referred to as "Kiome Antibody B".

[0224] High surface density anti-human Fab antibodies (GE Healthcare) were immobilized onto six horizontal channels on a GLM chip (Bio-Rad) via direct amine coupling according to the Bio-Rad manufacturer's manual.

[0225] The antibody of the present invention (clone I) and chiome antibody were captured at the minimum surface density on 5 of 6 vertical channels on the surface of anti-human Fab antibody for dynamic binding assay. Human semaphorin 3A was prepared in PBS-T-EDTA buffer (Bio-Rad) at concentrations of 100, 50, 25, 12.5, 10, 6.25, 5, 2.5, 1.25, 0.625, and 0 nM. Injection of PBS-T-EDTA buffer was used as a double reference for dynamic data analysis. Each of the human semaphorin 3A solution and PBS-T-EDTA buffer was injected simultaneously on the 6 horizontal channels at a flow rate of 40 μL / min for 10 minutes, followed by a dissociation phase for 2 hours. The surface was regenerated by an 18 s injection of 10 mM glycine hydrochloride (pH 2.1) (GE Healthcare) at a flow rate of 100 μl / min, followed by a 60 s injection of PBS-T-EDTA at a flow rate of 25 μl / min. Binding sensorgrams were fitted to a 1:1 Langmuir model to calculate binding and dissociation rates and affinities.

[0226] Kinetic and affinity data for antibodies and chiome antibodies of the present invention binding to human semaphorin 3A are listed in Table 9 below.

[0227] [Table 7]

[0228] conclusion The results show that the antibodies of the present invention were found to have superior binding affinity to human semaphorin 3A than the prior art antibodies disclosed in International Publication No. 2014123186 (Kiome Biosciences, Inc.).

[0229] Example 9: Comparison of binding affinity between the antibody of the present invention and Samsung's scFv scFv fragments such as those disclosed in WO2017074013 (Samsung) are compared.

[0230] For comparative purposes, the inventors have developed three disclosed scFv fragments ("Samsung scFvs") having the features disclosed in Table 10 below.

[0231] [Table 8]

[0232] High surface density anti-His antibody (GE Healthcare) was immobilized on six horizontal channels on a GLM chip (Bio-Rad) via direct amine coupling according to Bio-Rad's manufacturer's manual. Samsung scFv antibody was captured at minimum surface density on five of six vertical channels on the anti-His antibody surface for dynamic binding assays. Human semaphorin 3A was prepared in PBS-T-EDTA buffer (Bio-Rad) at concentrations of 100, 50, 25, 12.5, 10, 6.25, 5, 2.5, 1.25, 0.625, and 0 nM. Injection of PBS-T-EDTA buffer was used as a double reference for dynamic data analysis. Human semaphorin 3A solution and PBS-T-EDTA buffer were each injected simultaneously on the six horizontal channels at a flow rate of 40 μL / min for 10 min, followed by a 1-h dissociation phase. The surface was regenerated by an 18 s injection of 10 mM glycine hydrochloride (pH 2.1) (GE Healthcare) at a flow rate of 100 μl / min, followed by a 60 s injection of PBS-T-EDTA at a flow rate of 25 μl / min. Binding sensorgrams were fitted to a 1:1 Langmuir model to calculate binding and dissociation rates and affinities.

[0233] Binding of the antibodies of the invention to human semaphorin 3A (clone I) was performed in a similar manner, but using goat anti-human IgG (Invitrogen) to capture the antibodies of the invention. Binding of the antibodies of the invention and Samsung scFv to cynomolgus monkey, mouse, rat, or rabbit semaphorin 3A was also performed using the same method.

[0234] The kinetic and affinity data of the antibodies of the invention and the Samsung scFv are listed in Table 11 below.

[0235] [Table 9]

[0236] conclusion The antibodies of the invention have higher binding affinity to human, cynomolgus monkey, mouse, or rabbit semaphorin 3A than the three Samsung scFvs as disclosed in WO2017074013.

[0237] Example 10: Comparison of the affinity of two antibodies according to the invention The present inventors have developed two antibodies having CDRs as shown in SEQ ID NOs: 1 to 6. The two antibodies have Fc regions that differ in the following respects: one antibody contains the combination of L234A and L235A (Antibody A), and The other antibody contains the mutation H435A (Antibody B). (The residues are numbered according to the EU index of Kabat).

[0238] No statistical difference was observed in the binding affinity of both antibodies to human semaphorin 3A having CDRs as shown in SEQ ID NOs: 1 to 6. Therefore, affinity to human semaphorin 3A is maintained in the antibodies directed against semaphorin 3A according to the present invention.

[0239] Example 11: Efficacy of commercially available antibodies directed against semaphorin 3A For comparison, we tested the cellular activity of a commercially available antibody targeting Semaphorin 3A, which is marketed by Creative Biolabs under the reference "Anti-Human SEMA3A Therapeutic Antibody, Humanized (Cat. No.: TAB-556CL)". Said commercially available antibody will be referred to as "Creative Biolabs antibody" in the following.

[0240] The cellular activity of Creative Biolabs antibodies was assessed by measuring cytoskeleton disintegration in human retinal microvascular endothelial cells (HRMEC) using the same protocol as disclosed in Example 3. Potency calculations and determinations were performed 5 hours after stimulation.

[0241] Under these conditions, Creative Biolabs antibodies showed no activity against Semaphorin 3A-induced cytoskeletal disruption, and indeed Semaphorin 3A-induced cytoskeletal disruption was not prevented by Creative Biolabs antibodies.

[0242] Therefore, it was found that the Creative Biolabs antibody does not prevent cytoskeletal collapse in retinal cells induced by Semaphorin 3A, whereas under the same conditions the antibody of the present invention does, confirming the surprising and unexpected effect of the antibody of the present invention.

Claims

1. a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:1 (H-CDR1); the amino acid sequence of SEQ ID NO:2 (H-CDR2); and the amino acid sequence of SEQ ID NO:3 (H-CDR3); and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4 (L-CDR1); the amino acid sequence of SEQ ID NO: 5 (L-CDR2); and the amino acid sequence of SEQ ID NO: 6 (L-CDR3); An anti-semaphorin 3A antibody or an antigen-binding fragment thereof comprising A pharmaceutical composition for the treatment or prevention of diabetic macular edema, comprising: The pharmaceutical composition, wherein the antibody or antigen-binding fragment reduces the permeability of the blood-retinal barrier.

2. In the manufacture of a medicament for treating or preventing diabetic macular edema, a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:1 (H-CDR1); the amino acid sequence of SEQ ID NO:2 (H-CDR2); and the amino acid sequence of SEQ ID NO:3 (H-CDR3); and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4 (L-CDR1); the amino acid sequence of SEQ ID NO: 5 (L-CDR2); and the amino acid sequence of SEQ ID NO: 6 (L-CDR3); An anti-semaphorin 3A antibody or an antigen-binding fragment thereof comprising The use of The antibody or antigen-binding fragment reduces the permeability of the blood-retinal barrier.

3. the antibody or antigen-binding fragment thereof a heavy chain variable region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10; and - comprising a light chain variable region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13; The pharmaceutical composition according to claim 1.

4. the antibody or antigen-binding fragment thereof a heavy chain variable region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10; and a light chain variable region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13; Including, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:1 (H-CDR1); the amino acid sequence of SEQ ID NO:2 (H-CDR2); and the amino acid sequence of SEQ ID NO:3 (H-CDR3); and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 4 (L-CDR1); the amino acid sequence of SEQ ID NO: 5 (L-CDR2); and the amino acid sequence of SEQ ID NO: 6 (L-CDR3); The pharmaceutical composition according to claim 1.

5. the antibody or antigen-binding fragment thereof a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10; and a light chain variable region comprising the amino acid sequence of SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13; Including, The pharmaceutical composition of claim 1.

6. the antibody or antigen-binding fragment thereof a. a heavy chain variable region and a light chain variable region comprising the amino acid sequences of SEQ ID NO:7 and SEQ ID NO:11, respectively; b. a heavy chain variable region and a light chain variable region comprising the amino acid sequences of SEQ ID NO:8 and SEQ ID NO:11, respectively; c. a heavy chain variable region and a light chain variable region comprising the amino acid sequences of SEQ ID NO:9 and SEQ ID NO:12, respectively; or d. A heavy chain variable region and a light chain variable region comprising the amino acid sequences of SEQ ID NO:10 and SEQ ID NO:13, respectively. Including, The pharmaceutical composition according to claim 1.

7. the antibody or antigen-binding fragment thereof a heavy chain comprising the amino acid sequence of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 19; and a light chain comprising the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 18, or SEQ ID NO: 20 Including, The pharmaceutical composition according to claim 1.

8. the antibody or antigen-binding fragment thereof a. a heavy chain comprising the amino acid sequence of SEQ ID NO: 14 and a light chain comprising the amino acid sequence of SEQ ID NO: 15; b. a heavy chain comprising the amino acid sequence of SEQ ID NO: 16 and a light chain comprising the amino acid sequence of SEQ ID NO: 15; c. a heavy chain comprising the amino acid sequence of SEQ ID NO: 17 and a light chain comprising the amino acid sequence of SEQ ID NO: 18; or d. A heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and a light chain comprising the amino acid sequence of SEQ ID NO: 20; The pharmaceutical composition according to claim 1.

9. the antibody or antigen-binding fragment thereof a heavy chain variable region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10; and - comprising a light chain variable region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13; 3. The use according to claim 2.

10. the antibody or antigen-binding fragment thereof a heavy chain variable region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10; and a light chain variable region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13; Including, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:1 (H-CDR1); the amino acid sequence of SEQ ID NO:2 (H-CDR2); and the amino acid sequence of SEQ ID NO:3 (H-CDR3); and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 4 (L-CDR1); the amino acid sequence of SEQ ID NO: 5 (L-CDR2); and the amino acid sequence of SEQ ID NO: 6 (L-CDR3); 3. The use according to claim 2.

11. the antibody or antigen-binding fragment thereof a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10; and a light chain variable region comprising the amino acid sequence of SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13; Including, 3. The use according to claim 2.

12. the antibody or antigen-binding fragment thereof a. a heavy chain variable region and a light chain variable region comprising the amino acid sequences of SEQ ID NO:7 and SEQ ID NO:11, respectively; b. a heavy chain variable region and a light chain variable region comprising the amino acid sequences of SEQ ID NO:8 and SEQ ID NO:11, respectively; c. a heavy chain variable region and a light chain variable region comprising the amino acid sequences of SEQ ID NO:9 and SEQ ID NO:12, respectively; or d. A heavy chain variable region and a light chain variable region comprising the amino acid sequences of SEQ ID NO:10 and SEQ ID NO:13, respectively. Including, 3. The use according to claim 2.

13. the antibody or antigen-binding fragment thereof a heavy chain comprising the amino acid sequence of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 19; and a light chain comprising the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 18, or SEQ ID NO: 20 Including, 3. The use according to claim 2.

14. the antibody or antigen-binding fragment thereof a. a heavy chain comprising the amino acid sequence of SEQ ID NO: 14 and a light chain comprising the amino acid sequence of SEQ ID NO: 15; b. a heavy chain comprising the amino acid sequence of SEQ ID NO: 16 and a light chain comprising the amino acid sequence of SEQ ID NO: 15; c. a heavy chain comprising the amino acid sequence of SEQ ID NO: 17 and a light chain comprising the amino acid sequence of SEQ ID NO: 18; or d. A heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and a light chain comprising the amino acid sequence of SEQ ID NO: 20; 3. The use according to claim 2.

15. The pharmaceutical composition of claim 1 , wherein the antibody or antigen-binding fragment inhibits semaphorin 3A-induced blood-retinal barrier permeability and / or semaphorin 3A-induced vascular regression from ischemic areas.

16. 10. The pharmaceutical composition of claim 1, which is administered by parenteral, intravenous, intravitreal, or subcutaneous administration route.

17. 17. The pharmaceutical composition of claim 16, which is administered by the intravitreal route.

18. The use according to claim 2, wherein the antibody or antigen-binding fragment inhibits semaphorin 3A-induced blood-retinal barrier permeability and / or semaphorin 3A-induced vascular regression from ischemic areas.

19. 3. The use according to claim 2, wherein the medicament is administered by parenteral, intravenous, intravitreal or subcutaneous administration route.

20. 20. The use according to claim 19, wherein the medicament is administered by the intravitreal route.

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