Anti-Nrp1A antibody and its use for treating eye or ocular diseases

Abstract

The present invention relates to antibodies and fragments thereof that target the A domain of Neuropilin-1 (Nrp1A). More specifically, anti-Nrp1A antibodies and methods for the treatment of a number of diseases or disorders are disclosed.

Description

Technical Field

[0001] The present invention generally relates to antibodies and fragments thereof that target neuropilin-1 (Nrp1), more precisely, the A domain of Nrp1 (Nrp1A). More specifically, anti-Nrp1A antibodies and methods for treating various diseases or disorders are disclosed. Pharmaceutical compositions containing the anti-Nrp1A antibodies are also disclosed.

Background Art

[0002] Diabetic retinopathy is one of the most debilitating diabetic complications. Despite significant advances in understanding the etiology of this disease and the effectiveness of current treatments, diabetic retinopathy remains the leading cause of new-onset blindness among working-age individuals.

[0003] Diabetic retinopathy is characterized by abnormal progression occurring at the vascular, glial, and neuronal levels. One of the vascular complications is the reduction of small capillaries that leads to retinal ischemia. Ischemic retinopathy is characterized by the disappearance and dysfunction of the retinal vascular system, causing a reduction in blood flow and hypoxia. The dropout of capillaries often appears around the foveal avascular zone (FAZ), expanding its size, and this condition is called diabetic macular ischemia (DMI). Retinal ischemia simultaneously causes an increase in proangiogenic growth factor and an increase in vasorepulsion factor, misleading the direction of angiogenesis. While revascularization of the ischemic retina does not occur, robust pathological neovascularization occurs within the vitreous (the area of the eye where there are usually no blood vessels). The abnormal growth of these new blood vessels in proliferative retinopathy can cause bleeding or leakage that can lead to scars that can ultimately result in retinal detachment, creating the majority of the threats to vision. Current treatments for proliferative retinopathy attempt to destroy the existing pathological blood vessels but do not address the underlying ischemia that drives the growth of such blood vessels. Currently, the standard treatment for proliferative retinopathy is to destroy part of the retina with a laser and attempt to stop the growth of new blood vessels and maintain central vision. However, these treatments are somewhat ineffective. Some patients can maintain stable vision for years, but a high percentage of patients with retinopathy will ultimately go completely blind.

[0004] Retinopathy may also be characterized by an increase in retinal vascular leakage, leading to macular edema. Currently, patients with diabetic macular edema are treated with compounds that target vascular endothelial growth factor A (VEGF-A), a growth factor that promotes both angiogenesis and vascular permeability. This treatment approach may not be sufficient for the treatment of patients suffering from both macular ischemia and macular edema.

[0005] Accordingly, there remains a need for therapeutic means for treating patients who can benefit from the angiogenesis-promoting properties of VEGF-A, particularly patients suffering from both macular ischemia and macular edema. As a result, the need for new therapeutic means for efficiently treating eye diseases and retinal diseases remains unmet.

SUMMARY OF THE INVENTION

[0006] Neuropilins (NRPs) are transmembrane glycoprotein receptors that play important roles in the development of the nervous and vascular systems as receptors for members of the class 3 semaphorin family (SEMAs), which are axon guidance factors, and members of the vascular endothelial growth factor (VEGF) family, which are angiogenesis factors. Two neuropilin proteins, neuropilin-1 (Nrp1) and neuropilin-2 (Nrp2), have been identified. Their extracellular regions contain three domains: two CUB homology domains as Sema3 ligand-binding domains (domain A of Nrp1, also referred to herein as "Nrp1A"), two coagulation factor V / VIII homology domains as VEGF-binding domains (domain B of Nrp1, also referred to herein as "Nrp1B"), and a MAM domain (c) involved in the dimerization of Nrp1. Nrp1 can bind to VEGF-A165, VEGF-B, VEGF-E, PlGF, Sema3A, Sema3B, and Sema3C, whereas Nrp2 binds to VEGF-A165, VEGF-A145, VEGF-C, VEGF-D, SEMA3B, Sema3C, Sema3F, and Sema3G. The binding site for the VEGF ligand is located on the B domain of Nrp1, whereas the binding to semaphorin is located on the A domain of Nrp1.

[0007] The sequence of human Nrp1 is available online, and the precursor of Nrp1 isoform A is shown in SEQ ID NO: 26 and is available based on the sequence of the reference protein NP_003864. Nrp1 has been studied for many years regarding tumor angiogenesis and metastasis, but its effect on retinal revascularization and neovascularization is not fully understood. The inventors have shown herein that targeting Nrp1, particularly the A domain of Nrp1, is an effective means for treating eye diseases and retinal diseases.

[0008] In a first aspect, the present invention provides 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-Nrp1A antibody or an antigen-binding fragment thereof.

[0009] In one embodiment, the present invention provides 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: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17, 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, an anti-Nrp1A antibody or an antigen-binding fragment thereof.

[0010] In one embodiment, the present invention provides 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: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17, comprising a heavy chain variable region, 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, comprising a light chain variable region, 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). 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). Provided is an anti-Nrp1A antibody or an antigen-binding fragment thereof.

[0011] In yet another embodiment, the present invention A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17, And a light chain variable region comprising the amino acid sequence of SEQ ID NO: 11. Provided is an anti-Nrp1A antibody or an antigen-binding fragment thereof.

[0012] In another embodiment, the present invention a. A variable heavy chain and a variable light chain comprising the amino acid sequences of SEQ ID NO: 10 and SEQ ID NO: 11, respectively, b. A variable heavy chain and a variable light chain comprising the amino acid sequences of SEQ ID NO: 12 and SEQ ID NO: 11, respectively, c. A variable heavy chain and a variable light chain comprising the amino acid sequences of SEQ ID NO: 13 and SEQ ID NO: 11, respectively, d. A variable heavy chain and a variable light chain comprising the amino acid sequences of SEQ ID NO: 14 and SEQ ID NO: 11, respectively, e. A variable heavy chain and a variable light chain comprising the amino acid sequences of SEQ ID NO: 15 and SEQ ID NO: 11, respectively, f. A variable heavy chain and a variable light chain comprising the amino acid sequences of SEQ ID NO: 16 and SEQ ID NO: 11, respectively, or g. An anti-Nrp1A antibody or an antigen-binding fragment thereof, comprising a variable heavy chain and a variable light chain comprising the amino acid sequences of SEQ ID NO: 17 and SEQ ID NO: 11, respectively.

[0013] In yet another embodiment, the present invention A heavy chain comprising the amino acid sequence of SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25, preferably a heavy chain consisting of the amino acid sequence of SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25, and A light chain comprising the amino acid sequence of SEQ ID NO: 19, preferably a light chain consisting of the amino acid sequence of SEQ ID NO: 19, an anti-Nrp1A antibody or an antigen-binding fragment thereof.

[0014] In a specific embodiment, the present invention a. A heavy chain comprising the amino acid sequence of SEQ ID NO: 18 and a light chain comprising the amino acid sequence of SEQ ID NO: 19, b. A heavy chain comprising the amino acid sequence of SEQ ID NO: 20 and a light chain comprising the amino acid sequence of SEQ ID NO: 19, c. A heavy chain comprising the amino acid sequence of SEQ ID NO: 21 and a light chain comprising the amino acid sequence of SEQ ID NO: 19, d. A heavy chain comprising the amino acid sequence of SEQ ID NO: 22 and a light chain comprising the amino acid sequence of SEQ ID NO: 19, e. A heavy chain comprising the amino acid sequence of SEQ ID NO: 23 and a light chain comprising the amino acid sequence of SEQ ID NO: 19, f. A heavy chain comprising the amino acid sequence of SEQ ID NO: 24 and a light chain comprising the amino acid sequence of SEQ ID NO: 19, or g. An anti-Nrp1A antibody or an antigen-binding fragment thereof, comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 25 and a light chain comprising the amino acid sequence of SEQ ID NO: 19.

[0015] In a specific preferred embodiment, the anti-Nrp1A antibody is a humanized anti-Nrp1A antibody.

[0016] In a second aspect, the present invention provides an anti-Nrp1A antibody or an antigen-binding fragment thereof that binds to at least one amino acid residue within amino acid region 68 - 77 of human Nrp1 shown in SEQ ID NO: 26.

[0017] In one embodiment, the present invention provides an anti-Nrp1A antibody or an antigen-binding fragment thereof that binds to at least one amino acid residue within the amino acid region shown in SEQ ID NO: 26. In a preferred embodiment, the present invention provides an anti-Nrp1A antibody or an antigen-binding fragment thereof that binds to the amino acid region shown in SEQ ID NO: 27.

[0018] In a third aspect, the present invention provides an anti-Nrp1A antibody or an antigen-binding fragment thereof for use as a medicament.

[0019] In one embodiment, the present invention provides an anti-Nrp1A antibody or antigen-binding fragment thereof for suppressing the vasorepulsive effect of Sema3A and / or improving retinal revascularization. In a further embodiment, the present invention provides an anti-Nrp1A antibody or antigen-binding fragment thereof for suppressing the permeability of the blood-retinal barrier (BRB) induced by Sema3A and suppressing the permeability of the blood-retinal barrier induced by VEGF, particularly VEGF-A.

[0020] In yet another embodiment, the present invention redirects the direction of angiogenesis towards the ischemic region to improve retinal revascularization, prevents pathological angiogenesis in the vitreous region, prevents the breakdown of the blood-retinal barrier induced by Sema3A, Provided is an anti-Nrp1A antibody or antigen-binding fragment thereof for preventing disruption of the blood-retinal barrier induced by VEGF-A.

[0021] In one embodiment, the present invention provides an anti-Nrp1A antibody or antigen-binding fragment thereof for treating or preventing a retinal disease or an eye disease.

[0022] In another embodiment, the present invention relates to a method for treating one or more retinal diseases or eye diseases, the method comprising administering a pharmaceutically effective amount of an antibody or antigen-binding fragment to a patient in need of treatment.

[0023] In a fourth aspect, the present invention provides an anti-Nrp1A antibody or antigen-binding fragment thereof for treating or preventing a disease selected from the group consisting of retinopathy, proliferative retinopathy (PR) such as retinopathy of prematurity and ischemic retinopathy, proliferative diabetic retinopathy (PDR), and diabetic retinopathy (DR) including non-proliferative diabetic retinopathy, diabetic macular edema (DME), diabetic macular ischemia (DMI), age-related macular degeneration, retinitis pigmentosa, hereditary retinal dystrophy, myopic degeneration, retinal vein occlusion, retinal artery occlusion, endophthalmitis, uveitis, cystoid macular edema, choroidal neovascular membrane following any retinal disease, optic neuropathy, glaucoma, retinal detachment, toxic retinopathy, radiation retinopathy, traumatic retinopathy, drug-induced retinal vasculopathy, retinal angiogenesis, polypoidal choroidal vasculopathy, retinal vasculitis, retinal capillary hemangioma, Fuchs dystrophy, macular telangiectasia, Ascher syndrome, and Stargardt disease.

[0024] In another embodiment, the present invention provides an anti-Nrp1A antibody or antigen-binding fragment thereof for treating or preventing 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 degeneration, retinal angiogenesis, glaucoma, and choroidal neovascularization. Preferably, the disease is diabetic macular edema and / or diabetic macular ischemia.

[0025] In a preferred embodiment, the present invention provides an anti-Nrp1A antibody or an antigen-binding fragment thereof for the treatment of diabetic macular ischemia by promoting angiogenesis (revascularization) in the ischemic retina and reducing pathological angiogenesis in the vitreous region of the eye. In one embodiment, the antibody of the present invention does not inhibit angiogenesis induced by VEGF, preferably angiogenesis induced by VEGF-A.

[0026] In another preferred embodiment, the present invention provides an anti-Nrp1A antibody or an antigen-binding fragment thereof for the treatment of diabetic macular edema by reducing, preferably preventing, the permeability of the blood-retinal barrier induced by Sema3A and reducing, preferably preventing, the permeability of the blood-retinal barrier induced by VEGF-A.

[0027] In a fifth aspect, the present invention provides a pharmaceutical composition comprising an anti-Nrp1A antibody or an antigen-binding fragment thereof and a pharmaceutically acceptable carrier.

[0028] In one embodiment, the present invention provides an anti-Nrp1A antibody or an antigen-binding fragment thereof or a pharmaceutical composition comprising an anti-Nrp1A antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof is administered by parenteral route, intravenous route, intravitreal route or subcutaneous route, preferably by intravitreal route.

[0029] In a sixth aspect, the present invention one or more isolated polynucleotides comprising a sequence encoding a heavy chain shown in SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25, or a sequence encoding a heavy chain variable region shown in SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17, and a sequence encoding a light chain shown in SEQ ID NO: 19 or a sequence encoding a light chain variable region shown in SEQ ID NO: 11.

[0030] In one embodiment, the present invention provides an expression vector comprising one or more isolated polynucleotides, which comprise a sequence encoding a heavy chain shown in SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25, or a sequence encoding a heavy chain variable region shown in SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17, and a sequence encoding a light chain shown in SEQ ID NO: 19 or a sequence encoding a light chain variable region shown in SEQ ID NO: 11.

[0031] In one embodiment, the present invention provides a viral vector comprising one or more isolated polynucleotides, which comprise a sequence encoding a heavy chain shown in SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25, or a sequence encoding a heavy chain variable region shown in SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17, and a sequence encoding a light chain shown in SEQ ID NO: 19 or a sequence encoding a light chain variable region shown in SEQ ID NO: 11.

[0032] In one embodiment, the present invention provides a host cell comprising an expression vector or one or more isolated polynucleotides, wherein the expression vector or one or more isolated polynucleotides comprise a sequence encoding a heavy chain shown in SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25, or a sequence encoding a heavy chain variable region shown in SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17, and a sequence encoding a light chain shown in SEQ ID NO: 19 or a sequence encoding a light chain variable region shown in SEQ ID NO: 11.

[0033] In one embodiment, the present invention provides a method for producing an anti-Nrp1A antibody or an antigen-binding fragment thereof, the method comprising obtaining a host cell comprising an expression vector or one or more isolated polynucleotides, and culturing the host cell, wherein the expression vector or one or more isolated polynucleotides comprise a sequence encoding a heavy chain shown in SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25, or a sequence encoding a heavy chain variable region shown in SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17, and a sequence encoding a light chain shown in SEQ ID NO: 19 or a sequence encoding a light chain variable region shown in SEQ ID NO: 11.

[0034] In one embodiment, the method for producing the anti-Nrp1A antibody or an antigen-binding fragment thereof further comprises recovering and purifying the anti-Nrp1A antibody or an antigen-binding fragment thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0035]

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Figure 7

Modes for Carrying Out the Invention

[0036] Definitions The general structures of antibodies and immunoglobulins are well known to those skilled in the art, and these molecules are typically glycoproteins of heterotetramers of approximately 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is covalently bound to the heavy chain by one disulfide bond to form a heterodimer, and the heterotrimer molecule is formed via a covalent disulfide bond between the two identical heavy chains of the heterodimer. The light chain and the heavy chain are bound to each other by one disulfide bond, but the number of disulfide bonds between the two heavy chains varies depending on the immunoglobulin isotype. Each heavy chain and each light chain also have intra-chain disulfide bridges at regular intervals. Each heavy chain has a variable domain (V H = variable heavy chain) at the amino terminus, followed by three or four constant domains (C H1 , C H2 , C H3 and C H4 ), and a hinge region between C H1 and C H2 . Each light chain has two domains, a variable domain (V L = variable light chain) at the amino terminus and a constant domain (C L ) at the carboxy terminus. The V L domain is non-covalently associated with the V H domain, while the C L domain is usually covalently bound to the C H1 domain via a disulfide bond. Certain 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).

[0037] Certain domains within the variable domain vary significantly between different antibodies (i.e., are "hypervariable"). These hypervariable domains contain residues that are directly involved in the binding and specificity of the antibody for the specific epitope of each particular antibody. The hypervariability in both the light-chain variable domain and the heavy-chain variable domain is concentrated in three segments known as complementarity-determining regions (CDRs) or hypervariable loops (HVLs). CDRs are defined by sequence comparison in Kabat et al., 1991, In: Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., while HVLs are structurally defined based on the three-dimensional structure of the variable domain as described in Chothia and Lesk, 1987, J. Mol. Biol. 196: 901-917. When 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 in the light-chain variable domain, CDR-L2 at approximately residues 50-56, CDR-L3 at approximately residues 89-97; CDR-H1 is located at approximately residues 31-35 in the heavy-chain variable domain, CDR-H2 at approximately residues 50-65, and CDR-H3 at approximately residues 95-102. Thus, CDR1, CDR2, and CDR3 of the heavy and light chains define the unique and functional properties specific to a given antibody.

[0038] The three CDRs in each heavy and light chain are separated by framework regions (FRs), which contain sequences that tend to be more variable. The FRs and CDRs are arranged from the amino terminus to the carboxy terminus of the heavy and light chain variable domains in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The predominantly beta-sheet conformation of the FRs brings the CDRs in each chain into close proximity with each other, as well as with the CDRs from the other chain. Although not all CDR residues are directly involved in antigen binding, the resulting conformation contributes to the antigen-binding site (see Kabat et al., 1991, NIH Publ. No. 91-3242, Vol. I, pages 647-669).

[0039] FR residues and Ig 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 significant effects on antigen binding in at least three ways: by directly non-covalently binding to the epitope, by interacting with one or more CDR residues, and by influencing the interface between the heavy and light chains. The constant domains are not directly involved in antigen binding, but mediate antibody participation in various Ig effector functions, such as antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cellular phagocytosis (ADCP).

[0040] Vertebrate immunoglobulin light chains 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, mammalian immunoglobulin heavy chains are assigned to one of five major classes, IgA, IgD, IgE, IgG, and IgM, based on the sequence of their constant domains. IgG and IgA are each further divided into subclasses (isotypes), e.g., IgG 1 , IgG 2 , IgG 3 , IgG 4 , IgA 1 , and IgA 2It is divided into. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structure and three-dimensional configuration of natural immunoglobulin classes are well known.

[0041] The terms "antibody", "anti-Nrp1A antibody", "humanized anti-Nrp1A antibody", and "humanized anti-Nrp1A antibody variant" 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 (e.g., variable domains, other parts of the antibody representing the desired biological activity (e.g., binding to Nrp1A)).

[0042] The term "monoclonal antibody" (mAb) refers to an antibody of a substantially homogeneous population of antibodies. That is, the individual antibodies in this population are identical except for naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific antibodies against an "epitope", which is a single antigenic determinant. Thus, the modifier "monoclonal" indicates a substantially homogeneous population of antibodies against the same epitope and should not be construed as requiring antibody production by any specific method. Monoclonal antibodies can be produced by any technique or methodology known in the art (e.g., the hybridoma method (Kohler et al., 1975, Nature 256:495), recombinant DNA methods known in the art (see, e.g., U.S. Patent No. 4,816,567), 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, including methods for isolating recombinantly produced monoclonal antibodies).

[0043] A chimeric antibody consists of a heavy chain variable region and a light chain variable region of an antibody derived from one species (e.g., a non-human mammal such as a mouse), and a heavy chain constant region and a light chain constant region of an antibody of another species (e.g., a human). A DNA sequence encoding the variable region of an antibody derived from the first species (e.g., a mouse) is ligated to a DNA sequence for the constant region of an antibody derived from the second species (e.g., a human), and a host is transformed with an expression vector containing the ligated sequence to enable the production of a chimeric antibody, thereby obtaining it. Alternatively, a chimeric antibody may also be one in which one or more regions or domains of the heavy chain and / or light chain are identical, homologous, or variants of the corresponding sequences in a monoclonal antibody derived from another immunoglobulin class or isotype, or from a consensus sequence or germline sequence. A chimeric antibody may include a fragment of such an antibody as long as the fragment of the antibody represents the desired biological activity (e.g., binding to the same epitope) of its parent antibody (see, for example, U.S. Patent No. 4,816,567; and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81: 6851-6855).

[0044] The terms "antibody fragment", "antigen-binding fragment", "anti-Nrp1A antibody fragment", "humanized anti-Nrp1A antibody fragment", "humanized anti-Nrp1A antibody fragment variant" refer to a part of a full-length anti-Nrp1A antibody (where the variable region or functional ability (e.g., specific Nrp1 epitope binding) is retained). Examples of antibody fragments include Fab, Fab’, F(ab’) 2 , Fd, Fv, scFv and scFv-Fc fragments, diabodies, linear antibodies, single-chain antibodies, minibodies, bispecific antibodies formed from antibody fragments, and multispecific antibodies formed from antibody fragments, but are not limited thereto.

[0045] Full-length antibodies can be treated with enzymes such as papain and pepsin to generate useful antibody fragments. Papain digestion is used to produce two identical antigen-binding antibody fragments called "Fab" fragments, each having a single antigen-binding site, and the remaining "Fc" fragment. The Fab fragment also contains the constant domain of the light chain and the C H1 domain of the heavy chain. Pepsin treatment yields an F(ab’) 2 fragment that has two antigen-binding sites and can cross-link antigens.

[0046] The Fab’ fragment differs from the Fab fragment by the presence of additional residues containing one or more cysteines derived from the antibody hinge region at the C-terminus of the C H1 domain. The F(ab’) 2 antibody fragment is a pair of Fab’ fragments linked by cysteine residues in the hinge region. Other chemical linkages of antibody fragments are also known.

[0047] The "Fv" fragment contains a complete antigen recognition binding site consisting of a dimer of one heavy chain variable domain and one light chain variable domain in a tight non-covalent association. In this conformation, the three CDRs of each variable domain interact to define the antigen-binding site on the surface of the V H -V L dimer. The six CDRs together confer antigen-binding specificity to the antibody.

[0048] The "single-chain Fv" or "scFv" antibody fragment is a single-chain Fv variant containing the V H domain and the V L domain (the domains are present in a single polypeptide chain). Single-chain Fv can recognize and bind antigens. The scFv polypeptide also contains a linker between the V H domain and the V LIt may contain a polypeptide linker placed between domains (see, for example, Pluckthun, 1994, In The Pharmacology of monoclonal Antibodies, Vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315).

[0049] Other recognized antibody fragments include antibody fragments comprising a pair of tandem Fd segments (V H -C H1 -V H -C H1 ) for forming a pair of antigen-binding regions. These "linear antibodies" can be bispecific or monospecific, for example, as described in Zapata et al. 1995, Protein Eng. 8(10):1057-1062.

[0050] A "humanized antibody" or "humanized antibody fragment" is a specific type of chimeric antibody or its fragment that contains immunoglobulin amino acid sequence variants, can bind to a given antigen, and includes one or more FRs substantially having the amino acid sequence of a human immunoglobulin and one or more CDRs substantially having the amino acid sequence of a non-human immunoglobulin. This non-human amino acid sequence, often referred to as the "transferred" sequence, is typically obtained from a "transferred" antibody domain, specifically the variable domain. Generally, a humanized antibody contains at least the CDR or HVL of a non-human antibody inserted between the FRs of a human heavy chain variable domain or a human light chain variable domain.

[0051] The present invention describes a specific humanized anti-Nrp1A antibody containing CDRs derived from mouse or chimeric antibodies inserted between the FRs of the heavy chain variable domain and the FRs of the light chain variable domain of human germline sequences. Since certain mouse FR residues may be important for the function of the humanized antibody, it will be understood that residues of certain human germline sequences in the heavy chain variable domain and the light chain variable domain are modified to be the same as the residues of the corresponding mouse sequences.

[0052] As used herein, the expressions "the antibody of the present invention" and "the anti-Nrp1A antibody of the present invention" refer to an antibody against Nrp1 or an antigen-binding fragment thereof described herein, preferably an antibody against the A domain of Nrp1 or an antigen-binding fragment thereof. Preferably, the antibody of the present invention comprises a heavy chain variable region comprising the amino acid sequences of SEQ ID NO: 1 (H-CDR1), SEQ ID NO: 2 (H-CDR2), and SEQ ID NO: 3 (H-CDR3), and a light chain variable region comprising the amino acid sequences of SEQ ID NO: 4 (L-CDR1), SEQ ID NO: 5 (L-CDR2), and SEQ ID NO: 6 (L-CDR3).

[0053] In one embodiment, the present invention relates to a humanized anti-Nrp1A antibody. The humanized antibody comprises substantially all of at least one, typically two variable domains (e.g., contained in Fab, Fab’, F(ab’) 2 , Fabc, Fv fragments, etc.). As used herein, all or substantially all of the CDRs correspond to the CDRs of non-human immunoglobulins. In particular, as used herein, the CDRs are mouse sequences and the FRs are the consensus sequences or germline sequences of human immunoglobulins. In another aspect, the humanized anti-Nrp1A antibody also comprises at least a portion of the Fc region of an immunoglobulin, typically a portion of the Fc region of a human immunoglobulin. Usually, the antibody will contain both a light chain and at least the variable domain of the heavy chain. The antibody may also optionally contain one or more C H1 , hinge, C H2 , C H3 , and / or CH4 It may include a region.

[0054] The humanized anti-Nrp1A antibody can be selected from any class of immunoglobulins including IgM, IgG, IgD, IgA, and IgE, as well as IgG 1 , IgG 2 , IgG 3 , IgG 4 , IgA 1 , and IgA 2 and any isotype including. For example, when it is desired for the humanized antibody to exhibit cytotoxic activity, the constant domain can be a complement fixing constant domain, and the isotype is typically IgG 1 . When such cytotoxic activity is not desired, the constant domain may be another isotype (e.g., IgG 2 ). Alternative humanized anti-Nrp1A antibodies can include sequences from multiple immunoglobulin classes or isotypes, and selecting a specific constant domain to optimize the desired effector function is routine art in the art. In certain embodiments, the invention provides IgG 1 antibodies, more specifically, IgG 1 antibodies characterized by reduced effector function.

[0055] In a preferred embodiment, the anti-Nrp1A antibody of the invention is a humanized antibody formatted like IgG1KO.

[0056] The FRs, and CDRs or HVLs of a humanized anti-Nrp1A antibody need not exactly correspond to the parental sequences. For example, one or more residues in the grafted CDR or HVL, or in the consensus FR sequence or germline FR sequence, may be altered (e.g., mutated) by substitution, insertion, or deletion such that the resulting amino acid residue is no longer identical to the original residue at the corresponding position in any of the parental sequences, yet the antibody retains its binding function to Nrp1. Such alterations will typically not be extensive and will be conservative. Usually, at least 75% of the residues of the humanized antibody, more frequently at least 90% of the residues of the humanized antibody, and most frequently more than 95%, more than 98%, or more than 99% of the residues of the humanized antibody will correspond to the residues of the parental consensus FR or germline FR and the grafted CDR sequences.

[0057] The interface (the "V L -V H interface") between the heavy chain variable region and the light chain variable region is the residue of the immunoglobulin that affects the proximity or orientation of the two strands to each other. Certain residues that can participate in the interchain interaction include residues 34, 36, 38, 44, 46, 87, 89, 91, 96, and 98 of V L and residues 35, 37, 39, 45, 47, 91, 93, 95, 100, and 103 of V H (using the numbering system shown in Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1987)). U.S. Patent No. 6,407,213 also discusses that residues such as residues 43 and 85 of V L and residues 43 and 60 of V H may also participate in this interaction. These residues are shown only for human IgG, but the residues are applicable across species. Important antibody residues that are reasonably predicted to participate in the interchain interaction are selected for substitution to the consensus sequence.

[0058] The terms "consensus sequence" and "consensus antibody" refer to an amino acid sequence that contains the amino acid residue that most frequently occurs at each position in all immunoglobulins (e.g., human immunoglobulin variable domains) of any particular class, isotype, or subunit structure. The consensus sequence may be based on immunoglobulins of a particular species or many species. A "consensus" sequence, structure, or antibody, as described in certain embodiments, encompasses a consensus human sequence and is understood to refer to an amino acid sequence that contains the amino acid residue that most frequently occurs at each position in all immunoglobulins of any particular class, isotype, or subunit structure. Thus, a consensus sequence contains an amino acid sequence having at each position the amino acids present in one or more known immunoglobulins, but need not exactly replicate the entire amino acid sequence of any single immunoglobulin. The consensus sequence of the variable region is not obtained from any naturally occurring antibody or immunoglobulin (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.), and variants thereof. The FRs of the consensus sequence of the heavy chain and the FRs of the consensus sequence of the light chain, and variants thereof, provide useful sequences for the preparation of humanized anti-Nrp1A antibodies. See, for example, U.S. Patent Nos. 6,037,454 and 6,054,297.

[0059] Human germline sequences are found naturally in the human population. Combinations of these germline genes give rise to antibody diversity. The germline antibody sequences of the light chains of antibodies are derived from the conserved human germline kappa v-genes and j-genes, or lambda v-genes and j-genes. Similarly, the heavy chain sequences are derived from the germline v-genes, d-genes, and j-genes (LeFranc, M-P, and LeFranc, G, “The Immunoglobulin Facts Book” Academic Press, 2001).

[0060] An “isolated” antibody is one that has been identified, separated, and / or recovered from the components of its natural environment. Contaminant components of the antibody's natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and can include enzymes, hormones, or other proteinaceous or non-proteinaceous solutes. In one embodiment, the antibody will be purified to an isolate greater than at least 95% of antibody weight.

[0061] The term “antibody performance” refers to factors / characteristics that contribute to antibody recognition of an antigen or the effectiveness of an antibody in vivo. In a preferred embodiment, antibody performance refers to the ability of an antibody to prevent cytoskeletal disruption in retinal cells. Changes in the amino acid sequence of an antibody can affect antibody properties (e.g., folding), and can affect physical factors (e.g., 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 (Kd) of the antibody for the antigen, the three-dimensional structure of the antibody, protein stability, and the half-life of the antibody).

[0062] As used herein, the term "identical" or "% identical" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that are identical, or two or more sequences or subsequences in which a particular percentage of nucleotide or amino acid residues are identical, when compared and aligned to maximize correspondence. To determine % identity, sequences are aligned for optimal comparison (e.g., gaps can be introduced into the sequence of the first amino acid or nucleic acid sequence for optimal alignment with the second amino acid or nucleic acid sequence). Then the amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are 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 the same at that position. The % identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions (e.g., overlapping positions) × 100). In some embodiments, after introducing gaps into the sequences as necessary, the lengths of the two sequences being compared are the same (e.g., excluding additional sequences that extend beyond the sequences being compared). For example, when comparing variable region sequences, leader sequences and / or constant domain sequences are not taken into account. For sequence comparison between two sequences, "corresponding" CDRs refer to CDRs at the same position in both sequences (e.g., CDR-H1 of each sequence).

[0063] The determination of % identity or % similarity between two arrays can be accomplished using a mathematical algorithm. Preferred examples of mathematical algorithms used for comparing two arrays include, but are not limited to, 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. BLAST nucleotide searches can be performed with the NBLAST program (score = 100, wordlength = 12) to obtain nucleotide sequences homologous to the nucleic acid encoding the protein of interest. BLAST protein searches can be performed with the XBLAST program (score = 50, wordlength = 3) to obtain amino acid sequences 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 to detect remote relationships between molecules (ibid). When using the BLAST program, gapped BLAST program, and PSI-Blast program, the default parameters of each program (e.g., XBLAST and NBLAST) can be used. Another preferred example of a mathematical algorithm used for comparing sequences includes, but is not limited to, 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 using the ALIGN program to compare 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 and include ADVANCE and ADAM described in Torellis and Robotti, 1994, Comput. Appl. Biosci. 10:3-5, and FASTA 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 both sequences being compared are found by looking at aligned residue pairs, and when ktup = 1, single aligned residues are examined. Ktup may be set to 2 or 1 for protein sequences and to 1 to 6 for DNA sequences. If ktup is not specified, the default is 2 for proteins and 6 for DNA. Alternatively, protein sequence alignment may be performed using the CLUSTAL W algorithm as described in Higgins et al., 1996, Methods Enzymol. 266:383-402.

[0064] As used herein, the terms "cell", "cell line", and "cell culture" are used interchangeably, and all such names include their progeny. Thus, "transformants" and "transformed cells" include the primary subject cells and cultures derived therefrom, regardless of the number of passages.

[0065] The term "mammal" for therapeutic purposes refers to any animal classified as a mammal, including humans, domestic animals, and zoo, sports, or pet animals (e.g., dogs, horses, cats, cows, etc.). Preferably, the mammal is a human.

[0066] As used herein, "disease" or "disorder" is any condition that would benefit from treatment with the humanized anti-Nrp1A antibody described herein. This condition includes chronic and acute disorders, or diseases, such as pathological conditions that predispose a mammal to the disorder in question.

[0067] The term "intravitreal injection" has its ordinary meaning in the art and refers to the introduction of an anti-Nrp1A antibody or an antigen-binding fragment thereof into the vitreous of a patient.

[0068] The term "subcutaneous administration" refers to the introduction of an anti-Nrp1A antibody or an antigen-binding fragment thereof into a pocket between the skin and the underlying tissue of an animal or human patient, preferably, by relatively slow sustained delivery from a drug container. A pocket can be created by pinching or lifting the skin away from the underlying tissue.

[0069] The term "subcutaneous infusion" refers to the introduction of a drug into a pocket between the skin and the underlying tissue of an animal or human patient, preferably, by relatively slow sustained delivery from a drug container over time, where the time can be 30 minutes or less, or 90 minutes or less, but is not limited thereto. The infusion can be effected by subcutaneous implantation of a drug delivery pump implanted subcutaneously in the animal or human patient, where the pump delivers a predetermined amount of the drug over a predetermined time (e.g., 30 minutes, 90 minutes) or over a period spanning the length of a treatment regimen.

[0070] The term "subcutaneous bolus" refers to the administration of a drug under the skin of an animal or human patient, where the bolus drug delivery is less than about 15 minutes, in another aspect less than 5 minutes, and in yet another aspect less than 60 seconds. In yet another aspect, the administration is within a pocket between the skin and the underlying tissue, where the pocket can be created by pinching or lifting the skin away from the underlying tissue.

[0071] The term "therapeutically effective amount" is used to refer to the amount of an anti-Nrp1A antibody or antigen-binding fragment thereof that reduces or restores one or more of the symptoms of the disorder being treated, such that this amount has a beneficial result for the patient. Efficacy can be measured by conventional methods, depending on the condition to be treated. For example, in an eye / retinal disease or disorder characterized by cells expressing Nrp1A, efficacy can be measured by confirming the response rate (e.g., visual recovery) or by assessing the delay time to disease progression.

[0072] Terms such as "treatment" and "therapy" as used herein are meant to include therapeutic, prophylactic or suppressive treatment for a disease or disorder that leads to any clinically desirable or beneficial effect, such as alleviation or relief of one or more symptoms, regression, delay or arrest of disease or disorder progression, but are not limited thereto. Thus, for example, the term "treatment" includes administration of an anti-Nrp1A antibody or 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 the disease or disorder. As another example, the term includes administration of an anti-Nrp1A antibody or antigen-binding fragment thereof after the clinical signs of a disease to combat the symptoms of the disease. Also, administration of an anti-Nrp1A antibody or antigen-binding fragment thereof after onset and after clinical symptoms have appeared (wherein the administration affects the clinical parameters of the disease or disorder, whether or not the treatment leads to recovery of the disease) is included in "treatment" or "therapy" as used herein. Further, as long as the composition of the present invention, alone or in combination with another therapeutic agent, alleviates or restores at least one symptom of the disorder being treated as compared to the symptoms when an anti-Nrp1A antibody composition or antigen-binding fragment thereof is not used, the result should be considered an effective treatment of the underlying disorder, regardless of whether all symptoms of the disorder are alleviated.

[0073] The term "package insert" is used to refer to the instructions customarily included in the commercial package of a therapeutic product, and said package insert contains information about indications, usage, administration, contraindications and / or precautions regarding the use of such therapeutic product.

[0074] The antibody of the present invention In a first aspect, the present invention relates to an anti-Nrp1A antibody or an antigen-binding fragment thereof. Preferably, said antibody is a humanized anti-Nrp1A antibody, and more preferably, a humanized monoclonal anti-Nrp1A antibody. In initial characterization, the CDRs of mouse antibodies or human antibodies derived from a phage library were placed in the FRs of human consensus heavy-chain variable domain and light-chain variable domain, and further the FRs were manipulated with different mutations to create a library of antibodies targeting Nrp1A variants. Thereby, a humanized antibody against Nrp1A having enhanced properties as disclosed herein was obtained. The sequences of the antibodies of the present invention are shown in Table 1 below.

[0075] [Table 1] TIFF0007691976000002.tif221170 TIFF0007691976000003.tif58170

[0076] In one embodiment, the present invention - 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), and provides an anti-Nrp1A antibody or an antigen-binding fragment thereof.

[0077] In another embodiment, the present invention - 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: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17, 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, and provides an anti-Nrp1A antibody or an antigen-binding fragment thereof.

[0078] In yet another embodiment, the present invention - a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17, and - a light chain variable region comprising the amino acid sequence of SEQ ID NO: 11, and provides an anti-Nrp1A antibody or an antigen-binding fragment thereof.

[0079] In a preferred embodiment, the present invention a. a variable heavy chain and a variable light chain each comprising the amino acid sequences of SEQ ID NO: 10 and SEQ ID NO: 11, b. a variable heavy chain and a variable light chain each comprising the amino acid sequences of SEQ ID NO: 12 and SEQ ID NO: 11, c. a variable heavy chain and a variable light chain each comprising the amino acid sequences of SEQ ID NO: 13 and SEQ ID NO: 11, d. a variable heavy chain and a variable light chain each comprising the amino acid sequences of SEQ ID NO: 14 and SEQ ID NO: 11, e. a variable heavy chain and a variable light chain each comprising the amino acid sequences of SEQ ID NO: 15 and SEQ ID NO: 11, f. a variable heavy chain and a variable light chain each comprising the amino acid sequences of SEQ ID NO: 16 and SEQ ID NO: 11, or g. a variable heavy chain and a variable light chain each comprising the amino acid sequences of SEQ ID NO: 17 and SEQ ID NO: 11, and provides an anti-Nrp1A antibody or an antigen-binding fragment thereof.

[0080] In yet another embodiment, the present invention - a heavy chain comprising the amino acid sequence of SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25, preferably a heavy chain consisting of the amino acid sequence of SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25, and - a light chain comprising the amino acid sequence of SEQ ID NO: 19, preferably a light chain consisting of the amino acid sequence of SEQ ID NO: 19, to provide an anti-Nrp1A antibody or an antigen-binding fragment thereof.

[0081] In yet another embodiment, the present invention a. a heavy chain comprising the amino acid sequence of SEQ ID NO: 18 and a light chain comprising the amino acid sequence of SEQ ID NO: 19 (the antibody is referred to as "Clone I"), b. a heavy chain comprising the amino acid sequence of SEQ ID NO: 20 and a light chain comprising the amino acid sequence of SEQ ID NO: 19 (the antibody is referred to as "Clone II"), c. a heavy chain comprising the amino acid sequence of SEQ ID NO: 21 and a light chain comprising the amino acid sequence of SEQ ID NO: 19 (the antibody is referred to as "Clone III"), d. a heavy chain comprising the amino acid sequence of SEQ ID NO: 22 and a light chain comprising the amino acid sequence of SEQ ID NO: 19 (the antibody is referred to as "Clone IV"), e. a heavy chain comprising the amino acid sequence of SEQ ID NO: 23 and a light chain comprising the amino acid sequence of SEQ ID NO: 19 (the antibody is referred to as "Clone V"), f. a heavy chain comprising the amino acid sequence of SEQ ID NO: 24 and a light chain comprising the amino acid sequence of SEQ ID NO: 19 (the antibody is referred to as "Clone VI"), or g. comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 25 and a light chain comprising the amino acid sequence of SEQ ID NO: 19 (the antibody is referred to as "Clone VII") to provide an anti-Nrp1A antibody or an antigen-binding fragment thereof.

[0082] The IgG1-KO variant is created by introducing mutations into the Fc region. Mutations that reduce or suppress effector functions are well known to those skilled in the art and are fully disclosed in the prior art, for example, 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 its effector function includes -L234A and L235A, -L234A, L235A, and N297Q, -L234A, L235A, and P329G, or -L234A, L235A, and D265A, wherein said residues are numbered according to the Kabat EU index. In a preferred embodiment, the antibody of the present invention contains two mutations (L234A and L235A) in the Fc region to reduce effector function.

[0083] The CDRs shown in SEQ ID NOs: 1-6 disclosed herein are shown in Table 2 below according to CCG (Chemical Computing Group as described in Almagro et al., Proteins 2011; 79:3050-3066, and Maier et al, Proteins 2014; 82:1599-1610).

Table 2

[0084] An additional numbering system based on Kabat numbering is summarized in Table 3 below.

Table 3

[0085] An additional numbering system based on Chothia is shown in Table 4 below.

Table 4

[0086] Thus, in certain embodiments, the present invention - 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), or - a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7 (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), or - relates to an anti-Nrp1A antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8 (H-CDR1), the amino acid sequence of SEQ ID NO: 9 (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).

[0087] The anti-Nrp1A antibody of the present invention binds to human Nrp1A with high affinity. In embodiments related to this aspect, the anti-Nrp1A antibody of the present invention has a K D < 50 nM and binds to human Nrp1A. In another embodiment, as exemplified in Example 8, the anti-Nrp1A antibody of the present invention has a K D < 15 nM and binds to human Nrp1A. The anti-Nrp1A antibody of the present invention also binds to cynomolgus monkey Nrp1A, mouse Nrp1A, rat Nrp1A, and Syrian hamster Nrp1A. The high binding affinity of the antibodies of the present invention contributes to extending the neutralization time of Nrp1A after intravitreal injection and further enables a reduction in the injection frequency. The higher binding affinity also enables administration of a lower dose, thereby limiting potential side effects. The improved binding affinity and reduced injection frequency significantly improve the effectiveness of treatment for patients in need thereof. It also provides valuable benefits to patients, particularly improved medication observance and compliance.

[0088] The anti-Nrp1A antibodies of the present invention prevent the disruption of the cytoskeleton in retinal cells induced by Sema3A with a functional potency of less than 130 pM, preferably less than 110 pM, more preferably less than 100 pM. In a preferred embodiment, as exemplified in Example 4, the anti-Nrp1 antibody of the present invention prevents the disruption of the cytoskeleton in retinal cells induced by Sema3A with a functional potency of 98 pM. This result demonstrates the effectiveness of the antibodies of the present invention in suppressing the vascular repulsion induced by Sema3A.

[0089] The anti-Nrp1A antibodies of the present invention also suppress the permeability of the blood-retinal barrier induced by VEGF-A with a functional potency of less than 4 nM, preferably less than 3 nM, more preferably less than 1 nM. In a preferred embodiment, as exemplified in Example 5, the anti-Nrp1A antibody of the present invention prevents the loss of retinal cell integrity induced by VEGF with a functional potency of 0.74 nM. This result demonstrates the effectiveness of the antibodies of the present invention in suppressing the permeability of the blood-retinal barrier induced by VEGF-A.

[0090] In a further aspect, as described in Example 9, the anti-Nrp1A antibody of the present invention has been demonstrated to have a low immunogenic risk. This depends on the in silico prediction of the immunogenicity of the antibody. The immunogenic risk is typically evaluated by various well-known methods, for example, computer algorithms for predicting T cell epitopes, which are the major immunogenic influencing factors. It has actually been reported that by using an algorithm based on a computer matrix approach (available under the name EpiMatrix (manufactured by EpiVax)), it is possible to predict the sequences containing T cell epitopes present in the target protein. Those skilled in the art may refer to Van Walle et al., Expert Opin Biol Ther. 2007 March; 7(3): 405-18 and Jawa and al., Clin Immunol. 2013 Dec;149(3):534-55.

[0091] The antibody of the present invention is different from the therapeutic approach based on targeting Sema3. In fact, the antibody of the present invention suppresses the permeability of the blood-retinal barrier induced by VEGF, preferably the permeability of the blood-retinal barrier induced by VEGF-A, and this effect is not seen with compounds targeting Sema3A. Therefore, the anti-Nrp1A therapeutic approach based on the antibody of the present invention has the following advantages: - Suppressing the repulsive action of Sema3A on blood vessels, - Suppressing the permeability of the blood-retinal barrier induced by Sema3A, and - Suppressing the permeability of the blood-retinal barrier induced by VEGF-A.

[0092] The antibody of the present invention is different from the therapeutic approach based on VEGF inhibition. In fact, the antibody of the present invention suppresses the binding of Sema3A to Nrp1, thereby resulting in the suppression of the vascular repulsion induced by Sema3A and improving blood reperfusion (especially in patients suffering from DMI). As described in Example 5, the inventors compared the efficacy of the antibody of the present invention with that of Avastin®, Eylea® and Lucentis® in particular in a VEGF-induced cell integrity loss assay. Avastin®, Eylea® and Lucentis® all target VEGF, whereas the antibody of the present invention targets the A domain of Nrp1. It should be noted that the efficacy of the antibody of the present invention in the VEGF-induced cell integrity loss assay is similar to that of Avastin® and Eylea®, and better than that of Lucentis®. Therefore, the inventors - prevent the binding of Nrp1 and Sema3A so as to inhibit the induction of blood-retinal barrier permeability and repulsive vascular responses by Sema3A, and - surprisingly, affect the permeability of the blood-retinal barrier induced by VEGF-A developed an antibody.

[0093] The inventors have demonstrated that the antibody of the present invention exhibits even more advantageous properties compared to other antibodies or fragments targeting Nrp1 described in the prior art, as described in Example 3 herein. The other antibodies target different epitopes on Nrp1. The inventors compared the properties of the antibody of the present invention with - an antibody against the A domain of Nrp1 (YW64.3), and - an antibody against the B domain of Nrp1 (YW107.4.87). The inventors have shown that the antibody of the present invention is effective in a cell skeleton disassembly assay induced by Sema3A, while YW107.4.87 is not effective (Example 4). Such results explain that the antibody of the present invention suppresses the repulsive vascular responses induced by Sema3A, thereby providing improved properties for revascularization (especially in patients suffering from DMI).

[0094] The inventors also showed that the antibodies of the present invention have improved thermal stability compared to YW64.3, as evaluated by DSC (Example 11). Such results explain that the antibodies of the present invention remain in their native and active conformations at physiological temperature. It should be noted that a higher thermal transition midpoint (T m ) reflects improved stability of the protein at lower temperatures. Thus, the inventors have shown that the antibodies of the present invention exhibit the characteristic of improved thermal stability, which contributes to showing improved therapeutic efficacy and enables a reduced injection volume and injection frequency for patients. Note that T m , when it is a therapeutic product, means a longer shelf life and improved stability over time.

[0095] Humanized and amino acid sequence variants Further variant anti-Nrp1A antibodies and antibody fragments can be engineered based on a set of CDRs identified by the sequences shown in SEQ ID NOs: 1-6. It should be understood that in the said variant anti-Nrp1A antibodies and antibody fragments, the amino acid sequences of the CDRs are invariant, but the surrounding regions, such as the FR regions, can be engineered. Amino acid sequence variants of the anti-Nrp1A antibody can be prepared by introducing appropriate nucleotide changes into the DNA of the anti-Nrp1A antibody or by peptide synthesis. Such variants include, for example, deletions from residues within the amino acid sequence of the anti-Nrp1A antibody of the examples herein, and / or insertions into such residues, and / or substitutions of such residues. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, as long as the final construct has the desired characteristics. Amino acid changes can also alter the post-translational processes of the humanized or variant anti-Nrp1A antibody (e.g., changing the number and position of glycosylation sites). Another type of amino acid variant of the antibody may involve altering the original glycosylation pattern of the antibody. As used in this context, the term "altering" means removing one or more carbohydrate moieties found in the antibody and / or adding one or more glycosylation sites that were not previously present in the antibody.

[0096] In some embodiments, the invention includes a nucleic acid molecule encoding an amino acid sequence variant of an anti-Nrp1A antibody described herein. Nucleic acid molecules encoding amino acid sequence variants of the anti-Nrp1A antibody are prepared by a variety of methods known in the art. Such methods include isolation from natural sources (in the case of naturally occurring amino acid sequence variants), or oligonucleotide-mediated (or site-directed) mutagenesis, mutagenesis by PCR, and cassette mutagenesis of previously prepared mutant or non-mutant versions of the anti-Nrp1A antibody, but are not limited thereto.

[0097] In certain embodiments, the anti-Nrp1A antibody is an antibody fragment. Techniques for generating antibody fragments have been developed. Fragments can be obtained by proteolytic digestion of intact antibodies (see, for example, Morimoto et al., 1992, Journal of Biochemical and Biophysical Methods 24:107-117; and Brennan et al., 1985, Science 229:81). Alternatively, fragments can be produced directly in recombinant host cells. For example, Fab’-SH fragments can be recovered directly from E. coli and chemically coupled to form F(ab’) 2 fragments (see, for example, Carter et al., 1992, Bio / Technology 10:163-167). By another approach, F(ab’) 2Fragments can be isolated directly from the culture of recombinant host cells. Other methods for producing antibody fragments will be apparent to those skilled in the art.

[0098] The anti-Nrp1A antibody and its antigen-binding fragments may include modifications. In certain embodiments, it may be desirable to use anti-Nrp1A antibody fragments rather than intact antibodies. It may be desirable to modify the antibody fragments to increase their serum half-life. This can be accomplished, for example, by incorporating salvage receptor binding epitopes into the antibody fragments. In one method, appropriate regions of the antibody fragment can be altered (e.g., by mutation), or the epitope can be incorporated into a peptide tag that fuses with the antibody fragment at the C-terminus or an intermediate position (e.g., by DNA synthesis or peptide synthesis). See, for example, WO96 / 32478. In another embodiment, the invention includes covalent modifications of the anti-Nrp1A antibody. Covalent modifications include modifications of cysteinyl residues, histidyl residues, lysinyl residues and amino-terminal residues, arginyl residues, tyrosyl residues, carboxyl side chains (aspartyl or glutamyl), glutaminyl residues and asparaginyl residues, or seryl residues, or threonyl residues. Another type of covalent modification includes chemically or enzymatically conjugating a glycoside to the antibody. Such modifications may, where applicable, be performed by chemical synthesis or by enzymatic or chemical cleavage of the antibody. Another type of covalent modification of the antibody can be introduced into the molecule by reacting the amino acid residues targeted by the antibody with an organic derivatization reagent capable of reacting with selected side chains or amino-terminal or carboxyl-terminal residues.

[0099] Removal of any carbohydrate moieties on the antibody can be achieved chemically or enzymatically. Chemical deglycosylation is described in Hakimuddin et al., 1987, Arch. Biochem. Biophys. 259:52 and Edge et al., 1981, Anal. Biochem., 118:131. Enzymatic cleavage of carbohydrate moieties on the antibody can be achieved by using various endoglycosidases and exoglycosidases as described in Thotakura et al., 1987, Meth. Enzymol 138:350. Another useful covalent modification involves coupling the antibody to a non-protein polymer (e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylene) by the methods described in one or more of U.S. Patent No. 4,640,835, U.S. Patent No. 4,496,689, U.S. Patent No. 4,301,144, U.S. Patent No. 4,670,417, U.S. Patent No. 4,791,192, and U.S. Patent No. 4,179,337.

[0100] Epitope binding In a second aspect, the invention relates to an antibody that recognizes a specific "Nrp1A epitope". Specifically, the antibodies of the invention bind to the human Nrp1A epitope shown in SEQ ID NO: 26. In one aspect, the invention is an anti-Nrp1A antibody or antigen-binding fragment thereof that binds to at least one amino acid residue within amino acid region 68 - 77 of human Nrp1A shown in SEQ ID NO: 26. In another aspect, the invention relates to an Nrp1A antibody or antigen-binding fragment thereof that binds to the sequence shown in SEQ ID NO: 27. The sequences shown in SEQ ID NOs: 26 and 27 are shown in Table 5 below.

[0101]

Table 5

[0102] As used herein, the term "Nrp1A epitope" refers to a molecule (e.g., a peptide) or a fragment of a molecule that can bind to an anti-Nrp1A antibody or an antigen-binding fragment thereof. Such term further includes, for example, an Nrp1A antigen determinant recognized by any antibody or antibody fragment of the present invention having a combination of a heavy chain CDR selected from SEQ ID NOs: 1-3 and a light chain CDR selected from SEQ ID NOs: 4-6. The Nrp1A antigen epitope can be included in proteins, protein fragments, peptides, etc. Epitopes are most commonly proteins, short oligopeptides, peptidomimetics (i.e., organic compounds that mimic the antibody-binding properties of the Nrp1 antigen), or combinations thereof. The antibodies of the present invention have been found to bind to specific epitopes of human Nrp1A. Preferably, the anti-Nrp1A antibody or an antigen-binding fragment thereof binds to at least one amino acid residue within the amino acid region 68-77 of the extracellular domain of human Nrp1A shown in SEQ ID NO: 26. In the context of epitope binding, the expression "binds to... within the amino acid region X-Y" means that the anti-Nrp1A antibody or an antigen-binding fragment thereof binds to at least one amino acid residue, preferably all amino acid residues, within the amino acid region specified in the sequence.

[0103] In another aspect, the anti-Nrp1A antibody or an 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 shown in SEQ ID NO: 27. Preferably, the anti-Nrp1A antibody or an antigen-binding fragment thereof binds to the amino acid sequence shown in SEQ ID NO: 27.

[0104] Therapeutic uses In a third aspect, the present invention relates to the use of an anti-Nrp1A antibody or an antigen-binding fragment thereof as a medicament. As described above, Sema3A is the natural ligand of NRP1. More precisely, Sema3A binds to the A domain of Nrp1. The inventors have shown by way of specific examples that semaphorin 3A is secreted by hypoxic retinal ganglion cells in ischemic / avascular retina and acts as a trigger for vascular repulsion. The inventors have confirmed that Sema3A actually repels new blood vessels from the ischemic area by inducing cytoskeletal disassembly in these cells, thereby suppressing retinal angiogenesis and promoting pathological preretinal neovascularization. By targeting Nrp1, preferably the A domain of Nrp1, the antibody of the present invention prevents the binding of Nrp1 and Sema3A. The inventors have shown that by modulating the vascular repulsive effect using the Nrp1A antibody, the number of tip cells increases and the direction of angiogenesis is redirected towards the ischemic area (e.g., the pathologically enlarged foveal avascular zone in humans suffering from diabetic macular ischemia) (Example 6).

[0105] In addition to preventing the binding of the A domain of Nrp1 and Sema3A, the antibody of the present invention exhibits an unexpected property of suppressing the retinal permeability induced by VEGF, preferably VEGF-A. As described above, VEGF-A is the natural ligand of the B domain of Nrp1. Although the antibody of the present invention targets the A domain of Nrp1, it does not specifically target the binding of Nrp1B and VEGF-A. However, the inventors have observed that the antibody of the present invention suppresses the permeability of the blood-retinal barrier induced by VEGF-A. Without wishing to be bound by theory, the inventors have hypothesized that the suppression of the permeability of the retinal barrier induced by VEGF-A by an antibody against the A domain of Nrp1, preferably the antibody of the present invention, is due to steric hindrance by a signal holoreceptor complex consisting of Nrp1, VEGF receptor 2 and further coreceptors.

[0106] VEGF-A is secreted, among other things, by hypoxic astrocytes. VEGF-A is an important factor in the progression of both proliferative DR and proliferative DME, and changes the permeability of retinal capillaries by regulating adherens junctions (e.g., VE-cadherin) and tight junctions (e.g., occludin). VEGF-A stimulates endothelial cells to release matrix metalloproteinase (MMP) and urokinase-type plasminogen activator, resulting in the degradation of the basement membrane and enabling cell migration. Therefore, the secretion of VEGF-A under hypoxic conditions contributes to retinal permeability and is a worsening factor that exacerbates macular edema. In addition, the inventors have shown that both Sema3A and VEGF-A promote vascular permeability and cause vascular leakage by binding to Nrp1, which contributes to macular edema. The inventors have developed an antibody targeting Nrp1A to address this pathological condition, - To improve retinal revascularization, redirect the direction of angiogenesis towards the ischemic area, - Prevent pathological angiogenesis in the vitreous region, - Prevent the breakdown of the blood-retinal barrier induced by Sema3A, and - Prevent the breakdown of the blood-retinal barrier induced by VEGF-A and have proven to be very useful for this.

[0107] Therefore, by combining two unexpected effects, the antibody of the present invention - Typically improves the revascularization of ischemic avascular areas in the retinas of patients with PDR, especially DMI, - Typically prevents vascular leakage induced by the secretion of Sema3A in patients with PDR, especially DME, and - Typically prevents vascular leakage induced by the secretion of VEGF-A in patients with PDR, especially DME and have proven to be very beneficial for this. As a result, the present invention provides an anti-Nrp1A antibody or an antigen-binding fragment thereof for treating or preventing retinal diseases or eye diseases.

[0108] It should be noted that, as shown in Example 12, the antibody of the present invention does not interfere with the binding of VEGF and Nrp1. The antibody of the present invention does not act on angiogenesis induced by VEGF (since the antibody of the present invention does not inhibit VEGF-A-induced endothelial cell proliferation), and only affects VEGF-A-induced retinal permeability. As shown in Example 13, the inventors of the present invention actually demonstrated that the antibody of the present invention does not inhibit endothelial cell proliferation. The inventors also showed that in the VEGF-induced network formation assay (Example 14) and laser-induced choroidal neovascularization (Example 15), the antibody of the present invention does not act on angiogenesis induced by VEGF. Therefore, the inventors of the present invention confirmed that the antibody of the present invention does not inhibit angiogenesis induced by VEGF-A.

[0109] As described throughout the disclosure of the present invention, the antibody of the present invention suppresses the repulsive action of Sema3A on blood vessels, making it possible to redirect the direction of angiogenesis towards the ischemic region. In addition, the antibody of the present invention prevents the breakdown of the blood-retinal barrier induced by both Sema3A and VEGF-A. Surprisingly, despite its inhibitory effect on the permeability of the blood-retinal barrier induced by VEGF-A, the antibody of the present invention does not affect angiogenesis induced by VEGF-A. In addition, the antibody of the present invention does not prevent blood reperfusion. Therefore, it is considered not to interfere with angiogenesis in the ischemic region. Thus, the antibody of the present invention is extremely useful when improving blood reperfusion in clinical situations where blood reperfusion is to be promoted, for example, in the ischemic avascular region (typically in the retina of patients suffering from PDR, especially DMI).

[0110] In a fourth aspect, the present invention relates to an anti-Nrp1A antibody or an antigen-binding fragment thereof for the treatment or prevention of diseases selected from the group consisting of retinopathy, proliferative retinopathy such as retinopathy of prematurity and 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, hereditary retinal dystrophy, myopic degeneration, retinal vein occlusion, retinal artery occlusion, endophthalmitis, uveitis, cystoid macular edema, choroidal neovascular membrane following any retinal disease, optic neuropathy, glaucoma, retinal detachment, toxic retinopathy, radiation retinopathy, traumatic retinopathy, drug-induced retinal vasculopathy, retinal angiogenesis, polypoidal choroidal vasculopathy, retinal vasculitis, retinal capillary hemangioma, Fuchs dystrophy, macular telangiectasia, Ascher syndrome, and Stargardt disease. The anti-Nrp1A antibody of the present invention is 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 edema, retinal angiogenesis, and choroidal angiogenesis. In a preferred embodiment, the disease is diabetic macular ischemia, and the antibody of the present invention promotes angiogenesis (revascularization) in the ischemic retina and prevents pathological angiogenesis in the vitreous region of the eye.

[0111] In another preferred embodiment, the disease is diabetic macular edema, and the antibody of the present invention reduces the permeability of the blood-retinal barrier induced by Sema3A and VEGF-A. In another preferred embodiment, the present invention provides an anti-Nrp1A antibody or an antigen-binding fragment thereof that inhibits vasoregression induced by Sema3A from ischemic regions, inhibits the permeability of the blood-retinal barrier induced by Sema3A, and inhibits the permeability of the blood-retinal barrier induced by VEGF-A.

[0112] As used herein, the expressions "inhibition of blood-retinal barrier permeability (BRB)", "inhibition of retinal permeability", and "inhibition of vascular permeability" are used interchangeably and mean disruption of the blood-retinal barrier that can cause vascular leakage. The vascular leakage can be induced, on the one hand, by Sema3A and, on the other hand, by VEGF, preferably VEGF-A. The inventors have now developed an antibody that can not only inhibit the permeability of BRB induced by Sema3A but also inhibit the permeability of BRB induced by VEGF-A. Therefore, the antibody of the present invention prevents disruption of the blood-retinal barrier and prevents loss of retinal cell integrity induced by Sema3A and / or VEGF-A.

[0113] In a preferred embodiment, the antibody of the present invention is useful for the treatment of diabetic macular edema (DME) and / or diabetic macular ischemia (DMI). In a preferred embodiment, the antibody of the present invention is useful for the treatment of patients suffering from DME and DMI. Preferably, the antibody of the present invention is used for the treatment of DMI defined by an expansion of the foveal avascular zone (FAZ) exceeding 15%, 20%, 25%, more preferably 30% of the foveal avascular zone (FAZ). Since the antibody of the present invention inhibits the retinal permeability induced by VEGF-A without affecting the pro-angiogenic effect that VEGF-A may have on angiogenesis in the ischemic retina, the present invention has proven to be extremely useful for patients suffering from both DMI and DME.

[0114] In a fifth aspect, the present invention provides a pharmaceutical composition comprising an anti-Nrp1A antibody or an antigen-binding fragment thereof and a pharmaceutically acceptable carrier. The anti-Nrp1A antibody or antigen-binding fragment thereof is administered by any suitable means, including intravitreal, oral, parenteral, subcutaneous, intraperitoneal, intralung, and intranasal. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In addition, the anti-Nrp1A antibody is preferably administered by pulse infusion (particularly with a reduction in antibody dosage). In one embodiment, dosing depends in part on whether it is administered over a short period or long term, but is preferably given by injection, most preferably by intravenous or subcutaneous injection. Preferably, the anti-Nrp1A antibody is administered intravitreally into the eye.

[0115] For the prevention or treatment of a disease, the appropriate dosage of the antibody will be determined by various factors, such as the type of disease to be treated as defined above, the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, previous treatments, the patient's medical history and response to the antibody, and the discretion of the attending physician. The antibody is preferably administered to the patient in a single dose or over a series of treatments. In a preferred embodiment, for a single injection, the dosage range of the antibody of the present invention that can be applied is usually 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. The term "suppression" is used herein in the same context as "recovery" and "mitigation" and means reducing or decreasing one or more features of a disease.

[0116] The antibody composition will be prepared, dosed, and administered in a manner consistent with good medical practice. Factors to consider 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 drug delivery, the method of administration, the dosing schedule, and other factors known to medical practitioners. The "therapeutically effective amount" of the antibody to be administered is determined by such considerations and is the minimum amount necessary to prevent, ameliorate, or treat the eye disease or retinal disease addressed by the antibody of the present invention. The antibody may be formulated with one or more agents that are commonly used, but not necessarily required, for the prevention or treatment of the disorder in question. The effective amount of such other agent is determined by the amount of anti-Nrp1A antibody present in the formulation, the type of disorder or treatment, and other factors considered above. Such other agents are generally used in the same amounts and by the same routes of administration as those used above, or in amounts of about 1 to 99% of the amounts used hitherto.

[0117] Method of treatment In another aspect, the invention also encompasses any method for treating or preventing an eye disease or ocular disorder in a patient in need thereof, said method comprising administration of an anti-Nrp1A antibody of the invention. Preferably, the invention relates to a method for suppressing the vasorepressive effect of SemaA3 using an antibody of the invention. More preferably, the invention relates to said method for improving retinal revascularization.

[0118] Preferably, the present invention relates to a method for treating or preventing an eye disease or a retinal disease, the method comprising administering a pharmaceutically effective amount of an antibody of the present invention to a patient in need of treatment or prevention. Preferably, the disease includes retinopathy, proliferative retinopathy such as retinopathy of prematurity and 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, hereditary retinal dystrophy, myopic degeneration, retinal vein occlusion, retinal artery occlusion, endophthalmitis, uveitis, cystoid macular edema, choroidal neovascularization following any retinal disease, optic neuropathy, glaucoma, retinal detachment, toxic retinopathy, radiation retinopathy, traumatic retinopathy, drug-induced retinal vasculopathy, retinal angiogenesis, polypoidal choroidal vasculopathy, retinal vasculitis, retinal capillary hemangioma, Fuchs dystrophy, macular telangiectasia, Ascher syndrome, and Stargardt disease. More preferably, the disease is 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 angiogenesis, glaucoma, and choroidal neovascularization. In a further preferred embodiment, the disease is diabetic macular edema and / or diabetic macular ischemia. All of the disclosed technical features described herein are applicable to the treatment method.

[0119] Pharmaceutical Compositions and Their Administration A composition comprising an anti-Nrp1A antibody or an antigen-binding fragment thereof can be administered to a subject having an eye disease or a retinal disease or at risk of an eye disease or a retinal disease. The present invention further provides the use of an anti-Nrp1A antibody or an antigen-binding fragment thereof in the manufacture of a medicament for the prevention or treatment of a disease associated with Nrp1A. As used herein, the term "subject" means any mammalian patient to whom an anti-Nrp1A antibody or an antigen-binding fragment thereof can be administered, and examples include humans or non-human mammals (e.g., primates, rodents, and dogs). A human is an example of a subject specifically intended for treatment using the methods described herein. The anti-Nrp1A antibody or an antigen-binding fragment thereof can be administered alone or in combination with another composition. Various delivery systems are known and can be used to administer an anti-Nrp1A antibody or an antigen-binding fragment thereof. Routes of administration include, but are not limited to, intravitreal, topical, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The anti-Nrp1A antibody or an antigen-binding fragment thereof can be administered, for example, by infusion, bolus, or injection and can be administered together with other biologically active agents. Administration can be systemic or local. In a preferred embodiment, administration is by intravitreal injection. Such injectable formulations can be prepared, for example, in pre-filled syringes.

[0120] In a typical embodiment, the pharmaceutical composition is prepared as a pharmaceutical composition suitable for intravenous or subcutaneous administration to humans according to conventional methods. Typically, the composition for administration by injection is a solution in a sterile isotonic aqueous buffer. Optionally, the pharmaceutical composition may also contain a solubilizing agent and a local anesthetic (e.g., lidocaine) that alleviates pain at the injection site. Generally, these components are given separately or mixed and given in unit dosage form (e.g., as a lyophilized powder or anhydrous concentrate in a sealed container such as an ampoule or sachet indicating the amount of the active agent). When attempting to administer the pharmaceutical composition by infusion, the pharmaceutical composition can be dispensed in an infusion bottle containing sterile pharmaceutical-grade water or physiological saline. When administering the pharmaceutical composition by injection, an ampoule of sterile water for injection or physiological saline can be provided so that the components can be mixed before administration. Furthermore, the pharmaceutical composition can be provided as a pharmaceutical kit, which includes (a) a container containing the anti-Nrp1A antibody or an antigen-binding fragment thereof in lyophilized form and (b) a second container containing a pharmaceutically acceptable diluent for injection (e.g., sterile water). The pharmaceutically acceptable diluent can be used for the reconstitution or dilution of the lyophilized anti-Nrp1A antibody or an antigen-binding fragment thereof. Such containers may be accompanied by instructions in a form determined by a government agency that regulates the manufacture, use, or sale of pharmaceuticals and biological products, and the instructions reflect the approval by the agency for manufacture, use, or sale for human administration.

[0121] The amount of the anti-Nrp1A antibody or an antigen-binding fragment thereof effective for the treatment or prevention of an eye disease or a retinal disease can be determined by standard clinical techniques. In addition, in vitro assays may optionally be used to help confirm the range of optimal dosages. The exact dosage used in the formulation is also determined by the route of administration and the stage of the disorder and should be determined based on the judgment of medical personnel and the circumstances of each patient. The effective dosage can be estimated from the dose-response curve obtained from in vitro or animal model test systems. For example, the toxicity and therapeutic effect of the anti-Nrp1A antibody or an antigen-binding fragment thereof are ED 50It can be determined in cell cultures or experimental animals by standard pharmaceutical procedures for determining a therapeutically effective amount (in 50% of the population). Anti-Nrp1A antibodies or antigen-binding fragments thereof that exhibit a large therapeutic index are preferred.

[0122] Data obtained from cell culture assays and animal studies can be used to formulate a range of dosage amounts for use in humans. The dosage of the anti-Nrp1A antibody or antigen-binding fragment thereof is typically within a range of circulating concentration that is substantially non-toxic or non-toxic ED 50 The dosage can vary within this range depending on the dosage form used and the route of administration utilized. For any anti-Nrp1A antibody or antigen-binding fragment thereof used in the methods of the present invention, a therapeutically effective amount can first be estimated from cell culture assays. The dosage can be formulated in an animal model to achieve a range of circulating plasma concentrations that includes the IC 50 (i.e., the concentration of the test compound that achieves half of the maximum inhibition of symptoms). Such information can be used to more accurately determine a useful dosage in humans. Plasma levels can be measured, for example, by high performance liquid chromatography, ELISA, or the like.

[0123] For intravitreal injection of anti-Nrp1A antibodies, generally a longer treatment interval is preferred. The anti-Nrp1A antibodies of the present invention can be administered at longer intervals due to their improved efficacy. In one embodiment, the anti-Nrp1A 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-Nrp1A antibody of the present invention is administered once every 3 months.

[0124] Since the amount that can be administered to the eye is severely restricted, it is very important that the anti-Nrp1A antibody of the present invention can be prepared at a high concentration. Furthermore, the potency of the anti-Nrp1A antibody is particularly important because a potent antibody can exert its effect even at a lower dose, thereby not only enhancing the activity but also extending the treatment interval. The antibody of the present invention can be prepared at a very high concentration, and examples of such concentration include, but are 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 antibody of the present invention can be prepared in a liquid formulation of about 50 mg / mL. A typical dosage that can be administered to a patient is about 2.5 mg / eye. Typical buffer components that can be used in such a formulation include, for example, sodium acetate, PS20, and trehalose dihydrate. In one embodiment, the anti-Nrp1A antibody is prepared with 10 mM histidine buffer, 240 mM sucrose, 0.02 w / v% polysorbate 20 (pH 5.5), and the final protein concentration is 60 mg / mL.

[0125] In some embodiments, a pharmaceutical composition comprising an anti-Nrp1A antibody or an antigen-binding fragment thereof can further comprise a therapeutic agent that is bound or not bound to a binder. Regarding the treatment regimen for combinatorial administration, in certain embodiments, the anti-Nrp1A antibody or an antigen-binding fragment thereof is administered simultaneously with a therapeutic agent. In another specific embodiment, the therapeutic agent is administered from at least 1 hour to a maximum of several months before or after the administration of the anti-Nrp1A antibody or an antigen-binding fragment thereof (e.g., at least 1 hour, 5 hours, 12 hours, 1 day, 1 week, 1 month, or 3 months before or after the administration of the anti-Nrp1A antibody or an antigen-binding fragment thereof).

[0126] Polynucleotides, vectors, host cells, and recombinant methods In a sixth aspect, the present invention encompasses an isolated polynucleotide comprising a sequence encoding an anti-Nrp1A antibody, a vector, a host cell comprising the polynucleotide, and recombinant means for antibody production. The isolated polynucleotide can encode any desired form of the anti-Nrp1A antibody, and such forms include, for example, full-length monoclonal antibodies, Fab, Fab', F(ab') 2 , and Fv fragments, bispecific antibodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. A polynucleotide comprising a sequence encoding an anti-Nrp1A antibody, or a fragment or chain thereof, may be fused with one or more control or regulatory sequences known in the art and may be included in a suitable expression vector and host cell known in the art. Each polynucleotide molecule encoding a heavy-chain variable domain or a light-chain variable domain may independently be fused with a polynucleotide sequence encoding a constant domain (e.g., a human constant domain), thereby enabling the production of intact antibodies. Alternatively, the polynucleotides or portions thereof may be fused to each other, thereby providing a template for the production of single-chain antibodies. Regarding the production of recombinants, the polynucleotide encoding the antibody is inserted into a replicable vector for cloning (amplification of DNA) or expression. Many suitable vectors for expressing recombinant antibodies are available. The components of the vector generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0127] The anti-Nrp1A antibody may be produced as a fusion of polypeptides, and the antibody is fused with a heterologous polypeptide (e.g., a signal sequence), or another polypeptide having a specific cleavage site at the amino terminus of the mature protein or polypeptide. The selected heterologous signal sequence is typically one that is recognized and processed by the host cell (i.e., cleaved by signal peptidase). For prokaryotic host cells that do not recognize or process the signal sequence of the anti-Nrp1A antibody, the signal sequence may be replaced by a prokaryotic signal sequence. The signal sequence may be, for example, alkaline phosphatase, penicillinase, lipoprotein, the leader of heat-stable enterotoxin II, etc. For yeast secretion, the native signal sequence may be replaced by, for example, the yeast invertase α-factor (including the α-factor leaders of Saccharomyces and Kluyveromyces), acid phosphatase, the leader sequence obtained from Candida albicans glucoamylase, or the signal described in WO90 / 13646. In mammalian cells, mammalian signal sequences and viral secretion leaders (e.g., the gD signal of herpes simplex) can be used. The DNA of such a precursor region is ligated in-frame to the DNA encoding the humanized anti-Nrp1A antibody.

[0128] Expression vectors and cloning vectors contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells. Generally, in a cloning vector, this sequence enables the vector to replicate independently of the host chromosomal DNA and includes an origin of replication or an autonomous replication sequence. Such sequences are well known for various bacteria, yeasts, and viruses. The origin of replication from plasmid pBR322 is suitable for most Gram-negative bacteria, the origin of the 2-μm plasmid is suitable for yeast, and various viral origins (SV40, polyoma virus, adenovirus, VSV, and BPV) are suitable for cloning vectors in mammalian cells. Generally, an origin of replication component is not necessary for mammalian expression vectors (the SV40 origin can be used typically only for the reason that it contains an early promoter). Expression vectors and cloning vectors may contain a gene encoding a selectable marker that facilitates the identification of expression. Typical selectable marker genes are genes encoding proteins that confer resistance to antibiotics and other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline, or proteins that complement auxotrophic deficiencies, or in other options, proteins that supply specific nutrients not present in complex media (for example, a gene encoding Bacillus D-alanine racemase).

[0129] An example of a selection scheme is to use a drug to stop the growth of host cells. Cells successfully transformed with a heterologous gene survive the selection scheme because they produce a protein conferring drug resistance. Examples of such dominant selection use drugs such as neomycin, mycophenolic acid, and hygromycin. Common selectable markers for mammalian cells enable the identification of cells capable of taking up a nucleic acid encoding a humanized anti-Nrp1A antibody, for example, DHFR (dihydrofolate reductase), thymidine kinase, metallothionein-I and metallothionein-II (e.g., primate metallothionein genes), adenosine deaminase, ornithine decarboxylase, etc. Cells transformed with a DHFR selection gene are first identified by culturing all transformants in a medium containing methotrexate (Mtx, a competitive antagonist of DHFR). When using wild-type DHFR, a suitable host cell is a Chinese hamster ovary (CHO) cell line lacking DHFR activity (e.g., DG44). Alternatively, host cells (especially wild-type hosts containing endogenous DHFR) transformed or co-transformed with a DNA sequence encoding an anti-Nrp1 antibody, wild-type DHFR protein, and other selectable markers (e.g., aminoglycoside 3'-phosphotransferase (APH)) can be selected by cell growth in a medium containing a selection agent for the selectable marker (e.g., an aminoglycoside antibiotic such as kanamycin, neomycin, or G418). See, for example, U.S. Patent No. 4,965,199.

[0130] When producing recombinants 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 selectable marker. The TRP1 gene provides a selectable marker for yeast mutant strains lacking the ability to grow in tryptophan, such as for ATCC deposit number 44076 or PEP4-1 (Jones, 1977, Genetics 85:12). The presence of trp1 damage in the yeast host cell genome provides an effective environment for subsequently detecting transformation by growth in the absence of tryptophan. Similarly, Leu2p-deficient yeast strains such as ATCC 20,622 and 38,626 are complemented by known plasmids having the LEU2 gene. In addition, vectors derived from the 1.6 μm circular plasmid pKD1 can be used for the transformation of Kluyveromyces yeast. Alternatively, an expression system for the large-scale production of recombinant bovine chymosin has been reported for K. lactis (Van den Berg, 1990, Bio / Technology 8:135). A stable multi-copy expression vector for the 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).

[0131] Expression vectors and cloning vectors usually contain a promoter that is recognized by the host organism and operably linked to a nucleic acid molecule encoding the anti-Nrp1A antibody or a polypeptide chain thereof. Promoters suitable for use with prokaryotic hosts include the phoA promoter, the β-lactamase and lactose promoter systems, alkaline phosphatase, the tryptophan (trp) promoter system, and hybrid promoters (e.g., the tac promoter). Other known bacterial promoters are also suitable. The promoters used in bacterial systems also contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding the humanized anti-Nrp1A antibody. Many eukaryotic promoter sequences are known. Substantially all eukaryotic genes have an AT-rich region located approximately 25-30 bases upstream from the site where transcription is initiated. Another sequence found 70-80 bases upstream from the transcription start point of many genes is the CNCAAT region (where N can be any nucleotide). Most eukaryotic genes have an AATAAA sequence at their 3' end, which can be a signal for polyA tail addition to the 3' end of the coding sequence. All such sequences are appropriately inserted into eukaryotic expression vectors.

[0132] Examples of suitable promoting sequences for use with yeast hosts include promoters for 3-phosphoglycerate kinase and other glycolytic enzymes such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triose phosphate isomerase, phosphoglucose isomerase, and glucokinase. Inducible promoters have the added advantage of transcription being controlled by growth conditions. Examples of inducible promoters include promoters for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, derivative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for the utilization of maltose and galactose in yeast. Yeast enhancers are also advantageously used in conjunction with yeast promoters.

[0133] Transcription of the anti-Nrp1A antibody from the vector in mammalian host cells is controlled, for example, by a promoter obtained from the genome of a virus (e.g., polyoma virus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papillomavirus, Rous sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, and simian virus 40 (SV40)), a promoter obtained from a heterologous mammalian promoter (e.g., actin promoter or immunoglobulin promoter), or a promoter obtained from a heat shock promoter, provided that such promoter is compatible with the host cell line. The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the origin of replication of the SV40 virus. The immediate early promoter of human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment. An expression system for DNA in mammalian hosts using bovine papillomavirus as a vector is disclosed in U.S. Patent No. 4,419,446. Modifications of this expression system are described in U.S. Patent No. 4,601,978. See also Reyes et al., 1982, Nature 297:598-601, which discloses the expression of human p-interferon cDNA in mouse cells under the control of the thymidine kinase promoter from herpes simplex virus. Alternatively, the long terminal repeat of Rous sarcoma virus can be used as a promoter.

[0134] Another useful element that can be used in recombinant expression vectors is an enhancer sequence, which is used to increase the transcription of DNA encoding an anti-Nrp1A antibody by higher eukaryotes. Many enhancer sequences from mammalian genes (such as globin, elastase, albumin, α-fetoprotein, and insulin) are currently known. However, typically, enhancers from eukaryotic viruses are used. Examples include the SV40 enhancer (bp100-270) on the late side of the origin of replication, the cytomegalovirus immediate early promoter enhancer, the polyomavirus enhancer on the late side of the origin of replication, and the adenovirus enhancer. See also Yaniv, 1982, Nature 297:17-18 for a description of enhancing elements for the activation of eukaryotic promoters. The enhancer can be spliced into the vector at the 5' or 3' position relative to the sequence encoding the anti-Nrp1A antibody, but is preferably located at the 5' position from the promoter. Expression vectors used in eukaryotic host cells (nucleated cells from yeast, fungi, insects, plants, animals, humans, or other multicellular organisms) can also contain sequences necessary for transcription termination and sequences necessary for mRNA stabilization. Such sequences are generally available from the 5' untranslated regions and, optionally, the 3' untranslated regions of eukaryotic or viral DNA or cDNA. Such regions contain nucleotide segments that are transcribed as polyadenylation fragments in the untranslated portion of the mRNA encoding the anti-Nrp1A antibody. One useful transcription termination component is the polyadenylation region of bovine growth hormone. See WO94 / 11026 and the expression vectors disclosed therein. In some embodiments, the anti-Nrp1A antibody can be expressed using the CHEF system (see, for example, U.S. Patent No. 5,888,809, the disclosure of which is incorporated herein by reference).

[0135] As used herein, suitable host cells for cloning or expressing DNA in vectors are cells of the prokaryotes, yeasts, or higher eukaryotes described above. Suitable prokaryotes for this purpose include eubacteria such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as the genus Escherichia (e.g., E. coli), the genus Enterobacter, the genus Erwinia, the genus Klebsiella, the genus Proteus, the genus Salmonella (e.g., Salmonella typhimurium), the genus Serratia (e.g., Serratia marcescans), and the genus Shigella, as well as the genus Bacillus such as Bacillus subtilis and Bacillus licheniformis (e.g., Bacillus licheniformis 41 P disclosed in DD266,710 published on April 12, 1989), the genus Pseudomonas such as Pseudomonas aeruginosa, and the genus Streptomyces. Other strains such as E. coli B, E. coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable, but one preferred E. coli cloning host is E. coli 294 (ATCC 31,446). These examples are illustrative and not limiting.

[0136] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi and yeasts are suitable cloning hosts or expression hosts for vectors encoding anti-Nrp1A antibodies. Saccharomyces cerevisiae, or baker's yeast, is the most commonly used among lower eukaryotic microbial hosts. However, many other genera, species, and strains are generally available and useful herein, for example, Schizosaccharomyces pombe; Kluyveromyces hosts such as Kluyveromyces lactis, Kluyveromyces fragilis (ATCC 12,424), Kluyveromyces bulgaricus (ATCC 16,045), Kluyveromyces wickeramii (ATCC 24,178), Kluyveromyces waltii (ATCC 56,500), Kluyveromyces drosophilarum (ATCC 36,906), Kluyveromyces thermotolerans, and Kluyveromyces marxianus; Yarrowia (EP402,226); Pichia pastors (EP183,070); Candida; Trichoderma reesia (EP244,234); Neurospora crassa; Schwanniomyces, such as Schwanniomyces occidentalis; and filamentous fungi such as Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as Aspergillus nidulans, Aspergillus niger.

[0137] Suitable host cells for the expression of the glycosylated anti-Nrp1A antibody are derived from multicellular organisms. Examples of invertebrate cells include plant cells and insect cells, and such insect cells include, for example, a number of baculovirus cell lines and mutants, and similar acceptable insect host cells, which are from hosts such as Spodoptera frugiperda (fall armyworm), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), Bombyx mori (silkworm), etc. Various virus strains for transfection are publicly available (e.g., the L-1 mutant of Autographa californica NPV, the Bm-5 strain of Bombyx mori NPV), and such viruses can be used particularly for the transfection of Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be used as hosts. The anti-Nrp1A antibody of the present invention can also be incorporated into a viral vector. That is, a polynucleotide encoding the anti-Nrp1A antibody or its antigen-binding fragment is introduced into a viral vector and expressed in the patient's body after infection with the virus.

[0138] In another aspect, expression of the anti-Nrp1A antibody is carried out in vertebrate cells. The growth of vertebrate cells in culture (tissue culture) is conventional and the techniques are widely available. Examples of useful mammalian host cell lines include simian kidney CV1 cell line transformed by SV40 (COS-7, ATCC CRL 1651), human embryonic kidney cell line (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 CCL 10), 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), simian kidney cells (CV1, ATCC CCL 70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical cancer cells (HELA, ATCC CCL 2), dog kidney cells (MDCK, ATCC CCL34), buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human hepatocytes (Hep G2, HB 8065), mouse mammary gland cancer cells (MMT 060562, ATCC CCL51), TR1 cells (Mather et al., 1982, Annals N.Y. Acad. Sci. 383: 44-68), MRC5 cells, FS4 cells, and human liver cancer cell line (Hep G2).

[0139] The host cells are transformed with the above-described expression vector or cloning vector for the production of the anti-Nrp1A antibody and cultured in a conventional nutrient medium that has been appropriately modified for induction of the promoter, selection of transformants, or amplification of the gene encoding the desired sequence. The host cells used for the production of the anti-Nrp1A antibodies described in this specification can be cultured in various media. Commercially available media such as Ham's F10 (Ham’s F10, Sigma-Aldrich Co., St. Louis, Mo.), Minimal Essential Medium ((MEM), (Sigma-Aldrich Co.), RPMI-1640 (Sigma-Aldrich Co.), and Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich Co.) are suitable for culturing the host cells. In addition, 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. Patent No. 4,767,704, U.S. Patent No. 4,657,866, U.S. Patent No. 4,927,762, U.S. Patent No. 4,560,655, U.S. Patent No. 5,122,469, WO90 / 103430, and WO87 / 00195 can be used as the culture medium for the host cells. Any of these media may be supplemented, if necessary, with hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium salts, magnesium salts, phosphates), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., gentamicin), trace elements (defined as inorganic compounds that are 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 that would be known to those skilled in the art. The culture conditions, such as temperature, pH, etc., are those previously used for the host cells selected for expression and will be apparent to those skilled in the art.

[0140] When using recombinant techniques, antibodies can be produced intracellularly or in the periplasmic space of the cell membrane, or secreted directly into the medium. When an antibody is produced intracellularly, as a first step, the cells can be disrupted to release the protein. The particulate debris (including the lysed host cell fragments) can be removed, for example, by centrifugation or ultrafiltration. Carter et al., 1992, Bio / Technology 10:163-167, describes procedures for isolating antibodies secreted into the periplasmic space of E. coli. Briefly, the cell paste is thawed over about 30 minutes in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF). The cell debris can be removed by centrifugation. When the antibody is secreted into the medium, generally a commercially available protein concentration filter (e.g., an Amicon or Millipore Pellicon ultrafiltration unit) is used to first concentrate the supernatant from such an expression system. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis, and an antibiotic may be included to prevent the growth of foreign contaminants. A variety of methods can be used to isolate the antibody from the host cell.

[0141] Antibody compositions prepared from cells can be purified, for example, using hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography (using affinity chromatography is a typical purification method). The suitability of protein A as an affinity ligand depends on the species and isotype of the Fc domain of any immunoglobulin present in the antibody. Protein A can be used for the purification of antibodies based on human γ1 heavy chain, human γ2 heavy chain, or human γ4 heavy chain (see, for example, Lindmark et al., 1983 J. Immunol. Meth. 62:1-13). Protein G is recommended for all mouse isotypes and human γ3 (see, for example, Guss et al., 1986 EMBO J. 5:1567-1575). The matrix to which the affinity ligand binds is most often agarose, but other matrices are also available. Mechanically stable matrices (e.g., controlled pore glass, poly(styrene-divinyl)benzene) allow for faster flow rates and shorter processing times compared to those achievable with agarose. If the antibody contains a C H3 domain, Bakerbond ABX™ resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification. Other methods for protein purification, such as fractionation by ion exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica, chromatography on heparin sepharose™, chromatography on anion exchange resins or cation exchange resins (e.g., polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation, are also available depending on the antibody to be recovered.

[0142] After any preliminary purification steps, the mixture containing the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer between about pH 2.5 to 4.5, typically at a low salt concentration (e.g., from about 0 to 0.25 M salt).

[0143] The present invention also includes nucleic acids that hybridize to all or a portion of a nucleotide sequence (e.g., a portion encoding a variable region), represented by one or more isolated polynucleotide sequences encoding an anti-Nrp1A or antibody fragment, under the low stringency conditions, medium stringency conditions, and high stringency conditions defined herein. The length of the hybridizing portion of the hybridizing nucleic acid is typically at least 15 (e.g., 20, 25, 30, or 50) nucleotides. 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 the sequence of all or a portion of a nucleic acid encoding an anti-Nrp1A polypeptide (e.g., a heavy chain variable region or a light chain variable region) or its complement. Nucleic acids that hybridize of the type described herein can be used, for example, as cloning probes, primers (e.g., PCR primers), or diagnostic probes.

[0144] In one embodiment, the present invention relates to one or more isolated polynucleotides comprising a sequence encoding a heavy chain shown in SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25, or a sequence encoding a heavy chain variable region shown in SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17, and a sequence encoding a light chain shown in SEQ ID NO: 19 or a sequence encoding a light chain variable region shown in SEQ ID NO: 11. It should be understood that in the anti-Nrp1A antibodies and antibody fragments, the nucleic acid sequences encoding the CDRs are invariant (being invariant with respect to the encoded amino acids, and equivalents of the DNA sequences due to codon degeneracy being possible), while the surrounding regions, such as the FR regions, can be manipulated.

[0145] Product In another aspect, it includes products containing materials useful for the treatment of the disorders described above. The product includes a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The container can be formed from various materials, such as glass or plastic. The container holds a composition effective for the treatment of the condition and may have a sterile access port. For example, the container may be a vial with an intravenous solution bag or stopper that can be pierced by a hypodermic needle. The active agent in the composition is an anti-Nrp1A antibody or its antigen-binding fragment. The label on the container or the label together with the container indicates that the composition is for use in the treatment of a selected condition. The product can further include a second container containing a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or a dextrose solution. It can further include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, syringes, and an accompanying document with instructions for use. The present invention will be further described in the following examples, which are not intended to limit the scope of the present invention.

Examples

[0146] Example 1: Increased expression of Sema3A in the vitreous of DME and PDR patients The expression of Sema3A in the retina of samples from human donors with a history of diabetic retinopathy was examined by immunohistochemistry. The protocol for immunostaining is as follows: 1. Thaw the slides and air-dry the samples at room temperature (RT) for 30 minutes. 2. Draw a pap pen box and let it dry. 3. At RT, antigen activation in 1% SDS (for 5 minutes), 4. Wash the slides 3 times with PBS (for 5 minutes each) 5. At RT, block the sections for 30 minutes in a 1% BSA / 0.3% Triton X-100 / PBS solution (blocking solution), 6. Dilute the rabbit anti-Sema3a (abcam, ab23393) primary antibody 1:200 with the blocking solution and incubate the sections on the slides overnight at RT, 7. Rinse the slides 3 times with PBS (for 5 minutes each), 8. Incubate with the secondary antibody, donkey anti-rabbit Alexa fluor 546 (invitrogen, A10040) diluted 1:400 in a DAPI / 0.3% Triton X-100 / PBS solution and incubate the sections on the slides for 3 hours at RT, 9. Rinse the slides 3 - 5 times with PBS (for 5 minutes each), 10. Cover the sections with a coverslip using an aqueous mounting medium (Aquamount) and air dry, 11. Image the sections at 40x magnification and rank the intensity.

[0147] A set of 3 sections per human donor was immunostained for Sema3A. Using a 5-point grading scheme (0 = no detection, 1 = low intensity, few spots, 2 = moderate intensity, several spots, 3 = bright intensity, extensive staining, and 4 = very bright intensity, numerous detections), the Sema3A labeling in each region was evaluated separately by an observer pre-trained for this particular task. The observer was unaware of the health status of the eye donors. In the retina, Sema3A was associated with the vascular walls of the retinal blood vessels. Compared to patients without eye lesions, the expression of Sema3A in the retinal vasculature and the parenchymal tissue of the retina was increased in patients with diabetic macular edema (Figure 1).

[0148] Example 2: Inhibition of permeability induced by VEGF Permeability was measured in the retina of BN rats. Recombinant human VEGF-A (250 ng / 2.5 μL) was intravitreally injected to induce enhanced permeability. Antibodies were injected simultaneously with VEGF. Twenty-four hours after VEGF treatment, Evans blue dye (45 mg / mL) was intravenously injected into the vena caudalis mediana (1 mL / kg). After 30 minutes, the eyes were enucleated and fixed in formalin. Retinal flat mounts were prepared and Evans blue fluorescence was measured at 620 nm using a confocal fluorescence microscope and image analysis software.

[0149] The results are shown in Figure 2. The antibody of the present invention completely suppressed the permeability induced by VEGF. This is similar to the result observed with aflibercept (Eylea®), a VEGF trap. Antibodies against semaphorin 3A, an Nrp1 ligand, do not suppress VEGF-A-induced permeability in the retina. This confirmed that the antibody of the present invention against the A domain of Nrp1 suppresses the effect via VEGF-A, and that this ability distinguishes the antibody of the present invention from antibodies against semaphorin 3A, an Nrp1 ligand. The inventors have shown that although the antibody against Sema3A completely suppresses the permeability induced by Sema3A, it does not suppress the permeability induced by VEGF-A. The inventors hypothesized that the suppression of VEGF-A-induced retinal permeability by the antibody against the A domain of Nrp1 is due to steric hindrance by a signal holoreceptor complex consisting of Nrp1, VEGF receptor 2, and additional coreceptors.

[0150] Example 3: Preparation of antibodies against Nrp1A and Nrp1B for comparison purposes For comparison purposes, the inventors developed antibodies against each of Npr1A and Nrp1B as disclosed in WO2008143666 and WO2007056470. Such antibodies are - an antibody against Nrp1A, called "YW64.3"; and - an antibody against Nrp1B, called "YW107.4.87". The anti-Nrp1A antibody is disclosed in WO2007056470 as clone "YW64.3" and has the following characteristics: - The heavy chain variable domain is the sequence of number 4 in WO2007056470, and - The light chain variable domain is the sequence of number 3 in WO2007056470. The anti-Nrp1B antibody is disclosed in WO2007056470 as clone "YW107.4.87" and has the following characteristics: - The heavy chain is the sequence of number 6 in WO2007056470, and - The light chain is the sequence of number 5 in WO2007056470. The sequences of YW64.3 and YW107.4.87 are summarized as follows in Table 6 below.

[0151]

Table 6

[0152] Example 4: Cytoskeleton disassembly assay induced by Sema3A - Efficacy of the antibody of the present invention in the decrease of impedance induced by Sema3A, and comparison of the antibody of the present invention with clone YW64.3 and clone YW107.4.87 The cell activity of the Nrp1 antibody was evaluated by measuring the disruption of the cytoskeleton in human retinal microvascular endothelial cells (HRMEC) using the XCELLigence system (Real Time Cell Analysis Instrument, manufactured by ACEA Biosciences). This system measures cell adhesion, confluence, and integrity based on the impedance of the cells. HRMEC endogenously express neuropilin-1 (Nrp1) and plexin, which are components of class 3 semaphorin receptors. Binding to this receptor complex induces semaphorin to trigger the disruption of F-actin fibers in the endothelium. In this functional assay, when recombinant Sema3A protein was added to the confluent layer of human retinal microvascular endothelial cells, the impedance of the cells decreased due to the disruption of the cytoskeleton and subsequent cell contraction (measured as a decrease in cell impedance).

[0153] Briefly described, the E-plate was coated with an adhesion factor. Cells were seeded at a density of 20,000 cells / well and grown to confluence overnight inside the XCELLigence device under normal cell growth conditions. In the presence of 3 mM CaCl 2 Sema3A in combination with a combination of Nrp1 antibodies and Sema3A not in combination with a combination of Nrp1 antibodies were added. The cell index was normalized to the time point before the addition of the substance. Calculations were performed 5 hours after stimulation. In the cytoskeleton disassembly assay, to determine the functional efficacy and comparison among exemplary antibodies of the present invention (clone I having the sequence shown in SEQ ID NO: 10 as VH and the sequence shown in SEQ ID NO: 11 as VL), clone YW64.3, clone YW107.4.87, and an antibody against Sema3A, an IC 50 shift experiment was performed by combining the Sema3A concentration-response curve with an increasing concentration of the antibody. A Gaddum - Schild plot was performed to calculate the pA 2 value (the negative logarithm of the antibody concentration required to shift the Sema3A concentration-response curve by a factor of 2). The potency in M was calculated from the pA 2 value (as potency (10;-X)) and is disclosed in Table 7 below.

[0154]

Table 7

[0155] Example 5: Cell integrity loss assay induced by VEGF - Efficacy of the antibody of the present invention in the decrease of impedance induced by VEGF - A, and comparison of the antibody of the present invention with clone YW64.3, clone YW107.4.87 and VEGF trap VEGF-A induces relaxation of cell-cell contact, which can be measured as a transient decrease in impedance in endothelial cells. The antibodies of the present invention prevent the decrease in impedance induced by functional VEGF-A. The cell activity of Nrp1 antibodies that prevent the loss of cell integrity induced by VEGF was evaluated by measuring the decrease in impedance in human retinal microvascular endothelial cells (HRMEC) using the XCELLigence system (a real-time cell analysis device, manufactured by ACEA Biosciences). HRMEC endogenously express neuropilin-1 (Nrp1) and VEGFR2, which are components of the VEGF holoreceptor. VEGF-A induces relaxation of cell-cell junctions between endothelial cells. In this functional assay, when recombinant VEGF-A protein was added to a confluent layer of human retinal microvascular endothelial cells, the impedance of the cells decreased due to loss of cell integrity.

[0156] Briefly, the E-plate was coated with adhesion factors. Cells were seeded at a density of 20,000 cells / well and grown to confluence overnight inside the XCELLigence device under normal cell growth conditions. Before the addition of VEGF-A and the antibody, the medium was changed to serum-free medium containing 0.5% BSA (for 3 hours). The cell index was normalized to the time point before the addition of the substance. Calculations were performed using the minimum impedance induced by VEGF (about 30 minutes after stimulation). To determine the functional potency, the EC50 of the antibody that prevents the loss of cell integrity induced by a fixed concentration of recombinant human VEGF-A was measured. The geometric mean value of the EC50 values of individual experiments was calculated. The results for an exemplary antibody of the present invention (Clone I having the sequence shown in SEQ ID NO: 10 as VH and the sequence shown in SEQ ID NO: 11 as VL) and several comparative molecules are summarized in Table 8 below.

[0157]

Table 8

[0158] Example 6: Effects of anti - VEGF treatment and anti - Nrp1A on tip cell density, avascular area and retinal plexus in the mouse OIR model In a mouse model of oxygen-induced retinopathy (OIR), the effect of an exemplary antibody of the present invention (clone I having the sequence shown in SEQ ID NO: 10 as VH and the sequence shown in SEQ ID NO: 11 as VL) on revascularization of the ischemic avascular region was examined. Litters of C57BL / 6J mice were exposed to an environment of 75% oxygen from postnatal day 7 to postnatal day 12. This caused vascular regression and the formation of an avascular region at the center of the retina. After returning to normal oxygen pressure, this region became ischemic. On postnatal day 12, mouse pups were subjected to a single intravitreal injection of 10 μg of the antibody in 0.5 μL of solution in each eye under anesthesia with isoflurane. On postnatal day 17, the animals were sacrificed and the eyes were enucleated. The eyes were fixed in formalin, retinal flat mounts were made, and the retinal blood vessels were stained with isolectin B4 there. The number of tip cells (specialized endothelial cells that initiate the formation of new blood vessels) in the avascular anterior part of the entire retina (the boundary between the vascularized peripheral region and the avascular central region of the retina) was counted. The cells were identified by their characteristic morphology indicative of the extension of filopodia. For analysis, the number of tip cells was normalized to the length of the avascular anterior part. The size of the avascular region was measured using a confocal microscope and image analysis software. The contralateral eye was used for histological sectioning of the eye cup, and the number of pre-retinal cell nuclei was counted.

[0159] The antibody of the present invention increased the tip cell density in the mouse OIR model (Figure 3A). Furthermore, the antibody of the present invention showed a decrease in the avascular region (Figure 3C). In contrast, aflibercept (Eylea®), a VEGF trap, neither increased the tip cell density nor decreased the avascular region. There was a negative correlation between the tip cell density and the size of the avascular region (Figure 3B), indicating the mechanistic dependence of these two parameters. Overall, the antibody of the present invention decreased the size of the ischemic avascular region in an animal model of oxygen-induced retinopathy and showed a beneficial effect on diabetic macular ischemia. Pathological angiogenesis in the vitreous was suppressed by aflibercept, whereas the antibody of the present invention showed a moderate reduction in this pathological condition, as demonstrated by pre-retinal cell nuclei (Figure 3D).

[0160] Example 7: Comparison of t1 / 2 of the antibody of the present invention and Avastin in rabbit eyes The inventors measured the half-life of an exemplary antibody of the present invention (Clone I having the sequence shown in SEQ ID NO: 10 as VH and the sequence shown in SEQ ID NO: 11 as VL) under various conditions. The results are summarized in Table 9 below.

Table 9

[0161] The half-lives calculated in the vitreous, retina, and aqueous humor were 4.8 days, 3.5 days, and 4.5 days, respectively. These half-lives are similar to those reported in a paper on Avastin (anti-VEGF, bevacizumab, Bakri et al., Opthalmology, 2007), a recombinant humanized monoclonal IgG1 antibody clinically used, and this was also experimentally confirmed in-house. The in-vitreous clearance of full-length IgG mainly depends on its molecular size, and since the antibody of the present invention and Avastin® are similar, such results were as expected. 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 human ocular half-life of Avastin® is 9.73 ± 1.48 days (Hutton-Smith, 2016).

[0162] Example 8: Binding affinity to human Nrp1A The inventors evaluated the binding affinity of an exemplary antibody of the present invention (Clone I having the sequence shown in SEQ ID NO: 10 as VH and the sequence shown in SEQ ID NO: 11 as VL). This experimental running buffer and all dilutions (except where stated otherwise) were performed in PBS-T-EDTA containing 0.01% Tween20 [100 μL of 100% Tween20 was added to 2 L of PBS-T-EDTA so that the final concentration of Tween20 was 0.01%]. The GLM sensor chip was normalized and pre-adjusted as per the manufacturer's recommendations. The sensor chip was activated horizontally with an equal mixture of EDC / s-NHS at a flow rate of 30 μL / min for 300 s. The sensor chip was immobilized horizontally with recombinant protein A / G (6 μg / mL in 10 mM acetate (pH 4.5)) at a flow rate of 30 μL / min for 300 s, thereby immobilizing ~4370 - 4875 RU of protein A / G on the surface. The sensor chip was deactivated horizontally with 1 M ethanolamine hydrochloride at a flow rate of 30 μL / min for 300 s. The sensor chip was stabilized horizontally three times and vertically three times with 0.85% phosphoric acid at a flow rate of 100 μL / min for 18 s.

[0163] The antibody of the present invention (0.6 μg / mL) was captured vertically on the surface of protein A / G at a flow rate of 30 μL / min for 300 s, achieving a capture level of ~1678 RU. PBS-T-EDTA was injected horizontally at a flow rate of 100 μL / min for 60 s and dissociation was carried out for 120 s to stabilize the baseline. On top of the captured antibody, the analyte was injected horizontally at a flow rate of 30 μL / min for 300 s and dissociation was carried out for 1800 s. The concentrations of the analyte were 0 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM, and 100 nM. The surface was regenerated by injecting 0.85% phosphoric acid horizontally once and vertically once at a flow rate of 100 μL / min for 18 s. PBS-T-EDTA was injected vertically once and horizontally once at a flow rate of 100 μL / min for 60 s.

[0164] Inter-spots (interactions with the sensor surface) and blanks (PBS-T-EDTA containing 0.01% Tween20 or 0 nM analyte) were subtracted from the raw data. Subsequently, the sensorgrams were globally fitted to a 1:1 Langmuir binding equation to provide on-rate values (ka), off-rate values (kd), and affinity values (K D ). The results are summarized in Table 10 below.

[0165]

Table 10

[0166] Example 9: Evaluation of the immunogenicity of the antibody of the present invention The inventors evaluated the predicted immunogenicity of an exemplary antibody of the present invention (Clone I having the sequence shown in SEQ ID NO: 10 as VH and the sequence shown in SEQ ID NO: 11 as VL). For this purpose, the inventors used an in silico tool (EpiMatrix from EpiVax) to predict such T cell epitopes. By screening the sequences of many human antibody isolates, EpiVax identified several highly conserved HLA ligands that are considered to be controllable. Experimental evidence suggests that many of these peptides are positively tolerogenic in most subjects. Such highly conserved regulatory and promiscuous T cell epitopes are known as Tregitopes (De Groot et al. Blood. 2008 Oct 15;112(8):3303-11). In the presence of a significant number of Tregitopes, the immunogenic potential of neoepitopes contained in humanized antibodies can be effectively controlled.

[0167] For the purpose of analyzing antibody immunogenicity, EpiVax includes a Tregitope-adjusted EpiMatrix score and a prediction corresponding to an anti-therapeutic antibody response. To calculate the Tregitope-adjusted EpiMatrix score, the Tregitope score was subtracted from the EpiMatrix protein score. The Tregitope-adjusted score has been shown to correlate well with the clinical immune responses observed for a set of 23 commercially available antibodies (De Groot et al. Clin Immunol. 2009 May;131(2):189-201). The results on the EpiMatrix scale are summarized in Table 11 below.

[0168] [Table 11]

[0169] The sequences of the antibodies of the present invention were scored at the lower end of the EpiMatrix scale. This indicates that the antibodies of the present invention have a more strongly restricted potential for immunogenicity. The EpiMatrix scale is well known to those skilled in the art and can be particularly seen in Figure 2 of the paper by Mufarrege et al. (Clin Immunol., 2017 Mar;176:31-41).

[0170] Example 10: Comparison of the binding affinity of the antibody of the present invention, YW64.3 and YW107.4.87 to human Nrp1 The inventors evaluated the binding affinity of not only the exemplary antibody of the present invention (Clone I having the sequence shown in SEQ ID NO: 10 as VH and the sequence shown in SEQ ID NO: 11 as VL) to human Nrp1, but also the binding affinity of YW64.3 and YW107.4.87 to human Nrp1. This experimental running buffer and all dilutions (except where stated otherwise) were performed in PBS-T-EDTA containing 0.01% Tween20 [100 μL of 100% Tween20 was added to 2 L of PBS-T-EDTA so that the final concentration of Tween20 was 0.01%]. The GLM sensor chip was normalized and pre-adjusted as per the manufacturer's recommendations. The sensor chip was activated horizontally with an equal mixture of EDC / s-NHS at a flow rate of 30 μL / min for 300 seconds. The sensor chip was immobilized horizontally with recombinant protein A / G (60 μg / mL in 10 mM acetate (pH 4.5)) at a flow rate of 30 μL / min for 300 seconds. The sensor chip was deactivated horizontally with 1 M ethanolamine hydrochloride at a flow rate of 30 μL / min for 300 seconds. The sensor chip was stabilized horizontally three times and vertically three times with 0.85% phosphoric acid at a flow rate of 100 μL / min for 18 seconds.

[0171] Exemplary antibodies of the present invention, YW64.3 and YW107.4.87, were captured on the surface of protein A / G across three of the six vertical channels. Human Nrp1A was prepared in PBS-T-EDTA buffer at concentrations of 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 0 nM. Injection of the PBS-T-EDTA buffer was used as a double reference for kinetic data analysis. Across the six horizontal channels, each human Nrp1A solution and the PBS-T-EDTA buffer were injected simultaneously at a flow rate of 40 μL / min for 5 minutes, followed by a dissociation period of 30 minutes. The surface was regenerated by injecting 0.85% phosphoric acid at a flow rate of 100 μL / min for 18 seconds, followed by PBS-T-EDTA at a flow rate of 100 μL / min for 60 seconds. From the raw data, the inter-spot (interaction with the sensor surface) and the blank (PBS-T-EDTA containing 0.01% Tween20 or 0 nM analyte) were subtracted. Subsequently, the sensorgram was globally fit to a 1:1 Langmuir binding model to provide the on-rate value (ka), off-rate value (kd), and affinity value (K D ) The kinetics and affinity data of the antibodies of the present invention, YW64.3 and YW107.4.87, are listed in Table 12 below, respectively.

[0172]

Table 12

[0173] Example 11: Comparison of the protein thermal stability of the antibody of the present invention and YW64.3 Differential scanning calorimetry (DSC) is a thermodynamic technique that measures heat capacity as a function of temperature and constitutes the most accurate method for evaluating the thermal stability of protein structures. DSC is widely used to evaluate the thermal stability and structural changes of proteins. The signal from the sample cell is compared to a reference cell lacking the protein in the same solution environment. As the temperature of the cell increases, the temperature difference between the reference cell and the sample cell is continuously measured and calibrated to units of power. This data channel is called the DP signal or the differential power between the reference cell and the sample cell. The DP signal is converted to heat capacity. The heat capacity is continuously recorded as a function of temperature. After subtracting the buffer and analyzing the resulting thermogram, the enthalpy and (apparent) temperature transition midpoint (T m ) for each transition can be obtained. The temperature (T m ) of protein unfolding is linked to the stability of the antibody, particularly aggregation and long-term stability during storage of therapeutic products. The temperature transitions of the CH2 and 3 domains of monoclonal antibodies are typically invariant for different antibodies within an isotype, and the unfolding of the CH2 domain precedes that of the CH3 domain.

[0174] The inventors compared the following T m : - The antibody of the present invention, more precisely, "Clone I" having the sequence shown in SEQ ID NO: 10 as VH and the sequence shown in SEQ ID NO: 11 as VL, and - Clone YW64.3 disclosed in Example 3 of this specification for comparison purposes. Automated capillary differential scanning calorimetry (MicroCal, LLC) was used to monitor the thermal unfolding and aggregation of 1 mg / mL of the lead compound and YW64.3 in 10 mM histidine (pH 6.0) from 25°C to 110°C at a scan rate of 60°C / hour. Data were analyzed using Origin 7.0 software (Origin-Lab). To obtain the apparent midpoint temperature (T m ) of unfolding, all thermograms were baseline corrected and fitted using a two-state model in Origin. The melting curves of the lead compound and YW64.3 are disclosed in Table 13 below.

[0175]

Table 13

[0176] For the antibody of the present invention in 10 mM histidine (pH 6.0), the first T m occurred at 68.33°C, which likely corresponds to the unfolding of the CH2 domain. Two additional T m , 83.23°C and 89.70°C, correspond to the CH3 domain and the Fab region, respectively. Similarly, for YW64.3, the CH2 domain unfolded at 68.5°C, followed by the CH3 at 81.96°C, and finally the Fab domain at 84.65°C. Thus, the unfolding temperature of the Fab of the antibody of the present invention is approximately 5°C higher than that of YW64.3. Higher T m values mean that there are fewer molecules in the unfolded state at a given temperature. Thus, higher T m values are beneficial for therapeutic protein pharmaceuticals in order to maintain an active structure at physiological temperature. m values.

[0177] Example 12: The antibody of the present invention does not interfere with the binding of Nrp1 to VEGF The inventors further showed that an exemplary antibody of the present invention (Clone I having the sequence shown in SEQ ID NO: 10 as VH and the sequence shown in SEQ ID NO: 11 as VL) can bind to human Nrp1 even in the presence of VEGF and bound to Nrp1 in a competitive assay. For this purpose, in a sample plate, each sensor was immersed in a 10 μg / mL biotinylated hVEGF165 solution prepared with 1x kinetic buffer (Molecular Devices) for 2 minutes to capture biotinylated human vascular endothelial growth factor-165 (hVEGF165) on a streptavidin sensor chip (Molecular Devices, LLC. San Jose CA). Next, the sensor was transferred to a well containing 1x kinetic buffer to wash away any unbound molecules (for 2 minutes). Next, 100 nM human Nrp1 prepared with 1x kinetic buffer was captured by hVEGF165 for 10 minutes. Finally, the sensor was immersed in the antibody of the present invention at various concentrations (100 nM and 400 nM) for 10 minutes.

[0178] Data from the active sensor was compared with several controls, including controls without capture of hVEGF165, without hNrp1, and without antibody. The data show that the antibody of the present invention can bind to human Nrp1 even in the presence of VEGF and in a state bound to Nrp1 (Figure 4). This represents that the antibody of the present invention does not interfere with the binding of VEGF and human Nrp1.

[0179] Example 13: The antibody of the present invention does not interfere with VEGF - A - induced endothelial cell proliferation VEGF-A is one of the most important growth factors for endothelial cells that induces proliferation. Endothelial cell proliferation in human retinal microvascular endothelial cells (HRMEC) was examined using the Incucyte system (Sartorius). In this functional assay, addition of recombinant VEGF-A protein to a sub-confluent layer of HRMEC induces the proliferation of HRMEC. Briefly described, 96-well plates were coated with gelatin. Cells were seeded at a density of 3000 cells / well and allowed to adhere in complete endothelial growth medium for 18 hours. The cells were washed once with endothelial basal medium supplemented with 2% FCS and cultured in the same medium for 8 hours. Antibodies containing VEGF-A and / or exemplary antibodies of the present invention (Clone I having the sequence shown in SEQ ID NO: 10 as VH and the sequence shown in SEQ ID NO: 11 as VL) were added, and the cells were grown inside an Incucyte device. Phase contrast pictures were taken every 4 hours for a total of 96 hours. The images were used to evaluate the cell number. The cell number was normalized to the time point before the addition of the substance. The area under the curve was calculated from the growth curve and subtracted from the baseline value (Figure 5). The inventors have shown that the antibody of the present invention does not prevent endothelial cell growth induced by 10 ng / mL of VEGF-A, whereas aflibercept (Eylea®), a VEGF trap, shows a dose-dependent decrease in VEGF-A-induced HRMEC growth.

[0180] Example 14: VEGF - induced network formation assay - Efficacy of the antibody of the present invention in the VEGF - A - induced formation of endothelial network - like structures in co - culture with fibroblasts, and comparison between the antibody of the present invention and VEGF trap VEGF-A is a major regulator of angiogenesis and strongly induces the growth of new blood vessels from existing blood vessels. When cultured on a fibroblast layer, angiogenesis can be measured in vitro as the ability of endothelial cells to arrange themselves in a network-like structure. Network formation can be quantified after staining with the endothelial cell marker CD31. The inventors have - an exemplary antibody of the present invention (Clone I having the sequence shown in SEQ ID NO: 10 as VH and the sequence shown in SEQ ID NO: 11 as VL), and - an anti-VEGF antibody (bevacizumab, Avastin®) evaluated and compared for their ability to prevent VEGF-induced endothelial network formation. More precisely, in co-culture with fibroblasts, the cell activity of the compound that inhibits VEGF-induced endothelial network formation was evaluated by the ability of the compound to inhibit the formation of the endothelial network structure induced by VEGF-A. HUVECs endogenously express neuropilin-1 (Nrp1) and VEGFR2, which are components of the VEGF holoreceptor. In this functional assay, the addition of recombinant VEGF-A protein to endothelial cells seeded on a confluent layer of fibroblasts increased the formation of endothelial networks.

[0181] Briefly, normal human dermal fibroblasts (NHDF) derived from juvenile foreskin were seeded at a density of 25,000 cells / well in an equal mixture of FGM-2 medium and EGM medium in a CellCarrier Ultra 96-well plate. NHDF were cultured for 7 days under normal growth conditions (with one medium change). The medium was removed, and human umbilical vein endothelial cells (HUVEC) were seeded on top of the NHDF at a density of 5,000 cells / well in a 1 / 10 EGM / EBM mixture. In the incubator, HUVEC were allowed to attach for 4 hours. The medium was removed, and the cells were then stimulated with a fixed concentration of recombinant human VEGF-A and concentration-response curve antibodies in 1 / 10 EGM / EBM medium. The cells were cultured for 7 days under normal culture conditions (with a change to freshly prepared stimulation medium on day 3).

[0182] The cells were fixed with 70% ethanol / H on ice 2Fixed with O for 30 minutes, followed by blocking with DPBS + 1% BSA for 30 minutes. Endothelial cells were stained with CD31 antibody (Miltenyi 130-108-038) at room temperature for 60 minutes. After washing three times with DPBS, 488-labeled secondary antibody (anti-mouse IgG PAb-A488 PLUS; Thermo A32723) and Hoechst were added and cultured at room temperature in the dark for 60 minutes. The cells were washed three times with DPBS. Using an Opera Phenix equipped with a 5x air objective lens, the plate was imaged in the channels for AF488 and Hoechst. The Hoechst staining of the nucleus is only for confirming that the cell layer is intact after the staining procedure and is not included in the image analysis. Harmony4.9 software was used to calculate the 488-positive network area per well.

[0183] To determine the functional potency, the IC 50 of the antibody that blocks endothelial network formation induced by a fixed concentration of recombinant human VEGF-A was measured. The network area induced by VEGF-A was calculated (= average value of the network area induced by VEGF-A - average value of the basal network area) and set to 100%. The results are shown in Figure 6. Data are represented as mean ± SD relative to the effect of VEGF-A. The geometric mean of the IC 50 values of individual experiments was calculated. The maximum efficacy was calculated as the percentage of suppression of the VEGF-induced network area at the highest antibody concentration, and the mean value was calculated. The results are summarized in Table 14.

[0184]

Table 14

[0185] The inventors have shown that the antibody of the present invention has no substantial effect on in vitro angiogenesis induced by VEGF-A. In contrast, the VEGF trap (bevacizumab, Avastin®) effectively and potently inhibited the network formation induced by VEGF-A. These results confirmed the surprising and unexpected property of the antibody of the present invention, which does not affect VEGF-A-induced angiogenesis while preventing the breakdown of the blood-retinal barrier induced by VEGF-A.

[0186] Example 15: Laser - induced choroidal neovascularization in BN rats - Efficacy of the antibody of the present invention against laser - induced choroidal neovascularization in BN rats, and comparison between the antibody of the present invention and VEGF trap VEGF-A is a major regulator of angiogenesis and strongly induces the growth of new blood vessels from existing blood vessels. Angiogenesis can be measured in vivo in the eye of a living organism as VEGF-A-dependent choroidal neovascularization after the occurrence of lesions in the retinal pigment epithelium (RPE) and Bruch's membrane by laser coagulation. Angiogenesis can be quantified after lesion staining by isolectin B4 staining in flat mounts of the RPE, choroid, and sclera. This experimental in vivo model perforates Bruch's membrane depending on laser damage, thereby resulting in subretinal vascular replenishment from the choroid. This has proven useful for testing test angiogenesis therapies. The inventors evaluated and compared the effects of - an exemplary antibody of the present invention (Clone I having the sequence shown in SEQ ID NO: 10 as VH and the sequence shown in SEQ ID NO: 11 as VL), and - the VEGF trap (aflibercept, Eylea®) on laser-induced choroidal neovascularization.

[0187] Male BN rats (BN / Crl) weighing 160 g to 180 g were obtained from Charles River Laboratories (Sulzfeld, Germany). Under anesthesia, the animals were placed in front of the fundus camera so that the optic nerve was centered in the image. Using a Micron IV system (Phoenix Research Laboratories, Pleasanton, CA), a laser treatment was performed with a green argon laser (Merilas) at a wavelength of 532 nm. The laser beam diameter was matched to the diameter of the optic nerve, and four lesions were created in each eye using laser pulses with an energy of 400 mW and a pulse width of 150 milliseconds. The lesions were placed between large blood vessels at a distance of approximately twice their diameter from the optic nerve. The success of Bruch's membrane disruption was identified by the formation of bubbles immediately after the laser beam and confirmed by OCT scan.

[0188] For intravitreal injection, the rats were anesthetized by intraperitoneal injection of ketamine (67 mg / kg) and xylazine (6.7 mg / kg). In addition to dilating the pupils by topical administration of Mydrum eye drops, the animals were given analgesic eye drops (Novesine 0.4%). Using a 34G Hamilton syringe, an intravitreal injection was performed at the ora serrata. Each eye received two intravitreal injections with a volume of 5 μL. The first intravitreal injection was performed immediately after the laser treatment on day 1 (in the same anesthesia), and the second intravitreal injection was performed on day 8. On the 14th day after laser treatment, the animals were sacrificed by cervical dislocation under anesthesia. The eyes were enucleated and cut along the ora serrata. The cornea, iris, lens, vitreous, and retina were removed, and the remaining eyecup (consisting of RPE, choroid, and sclera) was fixed in PFA (4%) at 4 °C for 1 hour and then transferred to PBS containing 0.1% Triton X100 (1 hour, 4 °C). Using FITC-labeled isolectin B4 (10 μg / mL in saline), the eyecup was stained overnight at room temperature in the dark and washed three times with PBS. The eyecup was transferred to a glass slide and cut four times to form a flat cloverleaf structure. The tissue was covered with a mounting medium (Vectashield H-1200 containing DAPI), and a coverslip was placed on top to obtain an RPE / choroid / sclera flat mount (with the RPE side up). Using an LSM700 confocal laser scanning microscope (Carl Zeiss, Jena), the samples were analyzed at a wavelength of 488 nm, and the lesion size was measured by image analysis.

[0189] The results are shown in Fig. 7. The antibody of the present invention had no effect on the lesion area, whereas aflibercept (registered trademark), a VEGF trap, reduced the lesion area by 24%. Therefore, the antibody of the present invention did not affect VEGF-A-dependent choroidal neovascularization in BN rats. From the above results, it was confirmed that the antibody of the present invention does not inhibit angiogenesis induced by VEGF-A. Thus, it was confirmed that the antibody of the present invention is extremely useful in clinical situations where it is intended to promote blood reperfusion (for example, in patients with diabetic macular ischemia in whom retinal blood reperfusion is effective). As described throughout the disclosure of the present invention, the antibody of the present invention suppresses the repulsive action of Sema3A on blood vessels, enabling the direction of angiogenesis to be redirected towards the ischemic region. In addition, the antibody of the present invention suppresses, on the one hand, the breakdown of the blood-retinal barrier induced by Sema3A and, on the other hand, the breakdown of the blood-retinal barrier induced by VEGF-A. Despite its inhibitory effect on the permeability of the blood-retinal barrier induced by VEGF-A, surprisingly, the antibody of the present invention does not affect angiogenesis induced by VEGF-A. In conclusion, as shown herein, the antibodies of the present invention do not prevent revascularization, indicating that they will not interfere with angiogenesis in the ischemic region. Thus, such results confirm that the antibodies of the present invention are very beneficial for improving revascularization in ischemic avascular regions, typically in the retina of patients suffering from PDR, particularly DMI.

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-Nrp1A antibody or an antigen-binding fragment thereof.

2. The antibody or an antigen-binding fragment thereof, A heavy chain variable region comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17, and A light chain variable region comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 11, the anti-Nrp1A antibody or an antigen-binding fragment thereof according to claim 1.

3. The antibody or an antigen-binding fragment thereof, A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17, and A light chain variable region comprising the amino acid sequence of SEQ ID NO: 11, the anti-Nrp1A antibody or an antigen-binding fragment thereof according to claim 1.

4. The antibody or an antigen-binding fragment thereof, A heavy chain comprising the amino acid sequence of SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25, and A light chain comprising the amino acid sequence of SEQ ID NO: 19, the anti-Nrp1A antibody or an antigen-binding fragment thereof according to claim 1.

5. The antibody or an antigen-binding fragment thereof, a. A heavy chain comprising the amino acid sequence of SEQ ID NO: 18 and a light chain comprising the amino acid sequence of SEQ ID NO: 19, b. A heavy chain comprising the amino acid sequence of SEQ ID NO: 20 and a light chain comprising the amino acid sequence of SEQ ID NO: 19, c. A heavy chain comprising the amino acid sequence of SEQ ID NO: 21 and a light chain comprising the amino acid sequence of SEQ ID NO: 19, d. A heavy chain comprising the amino acid sequence of SEQ ID NO: 22 and a light chain comprising the amino acid sequence of SEQ ID NO: 19, e. A heavy chain comprising the amino acid sequence of SEQ ID NO: 23 and a light chain comprising the amino acid sequence of SEQ ID NO: 19, f. A heavy chain comprising the amino acid sequence of SEQ ID NO: 24 and a light chain comprising the amino acid sequence of SEQ ID NO: 19, or g. The anti-Nrp1A antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 25 and a light chain containing the amino acid sequence of SEQ ID NO:

19.

6. A pharmaceutical composition comprising the antibody or antigen-binding fragment according to any one of claims 1 to 5.

7. A pharmaceutical composition comprising the antibody or antigen-binding fragment according to any one of claims 1 to 5 for treating or preventing an eye disease or a retinal disease.

8. The disease includes proliferative retinopathy such as retinopathy, retinopathy of prematurity, and 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, hereditary retinal dystrophy, myopic degeneration, retinal vein occlusion, retinal artery occlusion, endophthalmitis, uveitis, cystoid macular edema, choroidal neovascular membrane following any retinal disease, optic neuropathy, glaucoma, retinal detachment, toxic retinopathy, radiation retinopathy, traumatic retinopathy, drug-induced retinal vasculopathy, retinal angiogenesis, polypoidal choroidal vasculopathy, retinal vasculitis, retinal capillary hemangioma, Fuchs dystrophy, macular telangiectasia, Ascher syndrome, and Stargardt disease. The pharmaceutical composition according to claim 7, selected from the group consisting of

9. The disease is 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 degeneration, retinal angiogenesis, glaucoma, and choroidal neovascularization. The pharmaceutical composition according to claim 7 or 8.

10. The disease is diabetic macular edema and / or diabetic macular ischemia. The pharmaceutical composition according to any one of claims 7 to 9.

11. The disease is diabetic macular ischemia, The antibody or antigen-binding fragment promotes angiogenesis (revascularization) in the ischemic retina and prevents pathological angiogenesis in the vitreous region of the eye. The pharmaceutical composition according to any one of claims 7 to 9.

12. The antibody or antigen-binding fragment does not inhibit angiogenesis induced by VEGF-A. The pharmaceutical composition according to claim 11.

13. The disease is diabetic macular edema, The antibody or antigen-binding fragment reduces the permeability of the blood-retinal barrier induced by Sema3A, The pharmaceutical composition according to any one of claims 7 to 9, wherein the antibody or antigen-binding fragment reduces the permeability of the blood-retinal barrier induced by VEGF-A.

14. The pharmaceutical composition according to any one of claims 7 to 13, which is administered by parenteral, intravenous, intravitreal or subcutaneous administration.

15. The pharmaceutical composition according to any one of claims 7 to 13, which is administered by intravitreal administration.

16. A sequence encoding a heavy chain shown in SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25, or a sequence encoding a heavy chain variable region shown in SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17, and One or more isolated polynucleotides comprising a sequence encoding a light chain shown in SEQ ID NO: 19 or a sequence encoding a light chain variable region shown in SEQ ID NO:

11.

17. An expression vector comprising one or more isolated polynucleotides according to claim 16.

18. A host cell comprising one or more isolated polynucleotides according to claim 16 or an expression vector according to claim 17.

19. a. Obtaining the host cell according to claim 18, and b. Culturing the host cell, A method for producing an anti-Nrp1A antibody or an antigen-binding fragment thereof.

20. The method according to claim 19, further comprising recovering and purifying the antibody or antigen-binding fragment thereof.

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