阅读说明：本技术 抗-sema3a抗体及其用于治疗眼或眼部疾病的用途 (anti-SEMA 3A antibodies and their use for treating ocular or ocular diseases ) 是由 L·托马斯 R·R·巴雷特 K·L·博瓦特 R·加内桑 P·古普塔 F·韩 D·刘 J 于 2020-05-08 设计创作，主要内容包括：本发明是关于靶向信号素3A(semaphorin 3A,Sema3A)的抗体及其片段。更特定而言,公开抗-Sema3A抗体及用于治疗各种疾病或病症的方法。(The present invention relates to antibodies and fragments thereof that target semaphorin 3A (Sema 3A). More particularly, anti-Sema 3A antibodies and methods for treating various diseases or disorders are disclosed.)

1. An anti-Sema 3A antibody or antigen-binding fragment thereof, comprising:

a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:1 (H-CDR1), the amino acid sequence of SEQ ID NO:2 (H-CDR2), and the amino acid sequence of SEQ ID NO:3 (H-CDR 3); and

the 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-CDR 3).

2. An anti-Sema 3A antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises:

a heavy chain variable region comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9 or SEQ ID No. 10; and

a light chain variable region comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO 11, SEQ ID NO 12, or SEQ ID NO 13.

3. An anti-Sema 3A antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises:

a heavy chain variable region comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9 or SEQ ID No. 10; and

a light chain variable region comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO 11, SEQ ID NO 12, or SEQ ID NO 13;

wherein:

the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:1 (H-CDR 1); the amino acid sequence of SEQ ID NO:2 (H-CDR 2); and the amino acid sequence of SEQ ID NO 3 (H-CDR 3); and is

The light chain variable region comprises the amino acid sequence of SEQ ID NO 4 (L-CDR 1); the amino acid sequence of SEQ ID NO. 5 (L-CDR 2); and the amino acid sequence of SEQ ID NO. 6 (L-CDR 3).

4. An anti-Sema 3A antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises:

a heavy chain variable region comprising the amino acid sequence of SEQ ID NO 7,8, 9 or 10, and

a light chain variable region comprising the amino acid sequence of SEQ ID NO 11, SEQ ID NO 12 or SEQ ID NO 13.

5. An anti-Sema 3A antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises:

a. a variable heavy chain and a variable light chain comprising the amino acid sequences of SEQ ID NO 7 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 8 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 9 and SEQ ID NO 12, respectively; or

d. A variable heavy chain and a variable light chain comprising the amino acid sequences of SEQ ID NO 10 and SEQ ID NO 13, respectively.

6. An anti-Sema 3A antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises:

a heavy chain comprising, preferably consisting of, the amino acid sequence of SEQ ID NO 14, 15, 17 or 19; and

a light chain comprising, preferably consisting of, the amino acid sequence of SEQ ID NO 15, 18 or 20.

7. An anti-Sema 3A antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises:

a. a heavy chain comprising the amino acid sequence of SEQ ID NO. 14 and a light chain comprising the amino acid sequence of SEQ ID NO. 15;

b. a heavy chain comprising the amino acid sequence of SEQ ID NO 16 and a light chain comprising the amino acid sequence of SEQ ID NO 15;

c. a heavy chain comprising the amino acid sequence of SEQ ID NO 17 and a light chain comprising the amino acid sequence of SEQ ID NO 18; or

d. A heavy chain comprising the amino acid sequence of SEQ ID NO 19 and a light chain comprising the amino acid sequence of SEQ ID NO 20.

8. An anti-Sema 3A antibody or antigen-binding fragment thereof that binds to at least one amino acid residue within amino acid region 370-382 of human Sema3A as set forth in SEQ ID NO 22.

9. An anti-Sema 3A antibody or antigen-binding fragment thereof that binds to SEQ ID No. 21.

10. An antibody or antigen-binding fragment according to any one of claims 1 to 9 for use as a medicament.

11. The antibody or antigen-binding fragment for use according to claim 10, for use in inhibiting the vasosuppressive effect (vasosuppressive effect) of SemaA 3.

12. The antibody or antigen-binding fragment for use according to claim 10 for use in improving retinal revascularization.

13. The antibody or antigen-binding fragment of any one of claims 1 to 9 for use in the treatment or prevention of an ocular or retinal disease.

14. The antibody or antigen-binding fragment for use according to claim 13, wherein the disease is selected from the group consisting of: retinopathy, ischemic retinopathy; diabetic retinopathy including proliferative diabetic retinopathy and nonproliferative diabetic retinopathy; diabetic macular edema, diabetic macular ischemia, age-related macular degeneration, retinitis pigmentosa, hereditary retinal dystrophy, myopic degeneration, retinal artery occlusion, endophthalmitis, uveitis, cystoid macular edema, choroidal neovascular membranes secondary to any retinal disease, optic nerve disease, glaucoma, retinal detachment, toxic retinopathy, radiation retinopathy, traumatic retinopathy, drug-induced retinal vasculopathy, retinal neovascularization, polypoidal choroidal vasculopathy, retinal vasculitis, retinal microaneurysms, Foster's dystrophy (Fuch's dystrophy), macular telangiectasia, Eustachian syndrome (usher syndrome), and Stargardt disease (Stargardt disease).

15. The antibody or antigen-binding fragment for use according to claim 13 or 14, wherein the disease is selected from the group consisting of: diabetic retinopathy including proliferative diabetic retinopathy and nonproliferative diabetic retinopathy; ischemic retinopathy, diabetic macular edema, diabetic macular ischemia, age-related macular edema, retinal neovascularization, glaucoma, and choroidal neovascularization.

16. The antibody or antigen-binding fragment for use according to claims 13 to 15, wherein the disease is diabetic macular edema and/or diabetic macular ischemia.

17. The antibody or antigen-binding fragment for use according to claims 13 to 15, wherein:

the disease is diabetic macular ischemia, and

the antibody or antigen binding fragment promotes angiogenesis (revascularization) within the ischemic retina and prevents pathological neovascularization of the vitreous region of the eye.

18. The antibody or antigen-binding fragment for use according to claim 13 or 15, wherein:

the disease is diabetic macular edema, and

the antibody or antigen binding fragment reduces the permeability of the blood retinal barrier.

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

20. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of claims 1 to 19 and a pharmaceutically acceptable carrier.

21. The antibody or antigen-binding fragment thereof of any one of claims 1-19, or the pharmaceutical composition of claim 20, wherein the antibody or antigen-binding fragment thereof is administered by a parenteral route of administration, an intravenous route of administration, an intravitreal route of administration, or a subcutaneous route of administration.

22. The antibody or antigen-binding fragment thereof of any one of claims 1 to 19, or the pharmaceutical composition of claim 20, wherein the antibody or antigen-binding fragment thereof is administered by the intravitreal route.

23. One or more isolated polynucleotides comprising:

a sequence encoding the heavy chain as shown in SEQ ID NO 14, 16, 17 or 19 or the heavy chain variable region as shown in SEQ ID NO 7,8, 9 or 10; and

a sequence encoding the light chain as set forth in SEQ ID NO 15, 18 or 20 or the light chain variable region as set forth in SEQ ID NO 11, 12 or 13.

24. An expression vector comprising one or more isolated polynucleotides according to claim 23.

25. A host cell comprising one or more isolated polynucleotides according to claim 23 or an expression vector according to claim 24.

26. A method of producing an anti-Sema 3A antibody or antigen-binding fragment thereof, comprising:

a. obtaining a host cell according to claim 25; and

b. the host cell is cultured.

27. The method of claim 26, further comprising recovering and purifying the antibody or antigen-binding fragment thereof.

[ technical field ] A method for producing a semiconductor device

The present invention relates generally to antibodies and fragments thereof that target semaphorin 3A (semaphorin 3A, Sema 3A). More particularly, anti-Sema 3A antibodies and methods of use for treating various diseases or disorders are disclosed. Pharmaceutical compositions and kits comprising the anti-Sema 3A antibodies are also disclosed.

[ Prior Art ]

Ischemic retinopathy is characterized by loss or dysfunction of the retinal vasculature, which leads to reduced blood flow and hypoxia. Retinal ischemia results in the up-regulation of pro-angiogenic growth factors that promote retinal neovascularization, which can lead to blindness. However, revascularization of ischemic retina does not occur when there is robust pathological neovascularization into the vitreous (i.e., the normally avascular region of the eye).

The growth of these abnormal new blood vessels poses a major threat to vision, as they can leak, cause bleeding, or cause scarring, which can ultimately lead to retinal detachment. Current treatments for ischemic retinopathy attempt to stop the growth of pathological blood vessels, but do not address the underlying ischemia that drives their growth. In addition, the standard treatment for diabetic retinopathy involves destroying a portion of the retina with a laser in an attempt to stop new blood vessel growth and preserve central vision. However, such treatments are somewhat inefficient. While some patients can maintain stable vision for many years, a high percentage of patients suffering from retinopathy eventually suffer from complete vision loss.

Thus, there remains an unmet need for new therapeutic approaches that are effective in treating ocular or retinal diseases.

[ summary of the invention ]

Sema3A is an endogenous secreted protein belonging to the family of class 3 semaphorins (Sema3), which was originally identified as an axon-directing molecule and involved in vascular pathway formation and network formation. Cyclins 1 and 2(Nrp1 and Nrp2) and plexin a/D (Plxn) function as ligand binding and signal transduction subunits of the Sema3 receptor complex on the surface of Endothelial Cells (EC). As a special member of the Sema3 family, Sema3A first binds exclusively to Nrp1 and then combines with plexin a1-4 as a complex (Nrp1/PlexA 1-4). In this receptor complex, Nrp1 served as the binding module, while PlexA1-4 served as the signal transduction module.

Human semaphorin 3A is the protein as disclosed in SEQ ID NO:22 and obtainable with the NCBI reference sequence NP-006071.1. Furthermore, human Sema3A is encoded by gene ID 10371 (NCBI).

Sema3A has been studied for many years in tumor angiogenesis and metastasis, but its effect on retinal neovascularization is still unclear. The inventors have demonstrated that semaphorin 3A is secreted by hypoxic retinal ganglion cells and serves as a vascular repulsion cue. Sema3A repels new blood vessels away from the ischemic area by inducing cytoskeletal collapse in these cells. Without wishing to be bound by theory, the inventors have hypothesized that this would be explained by how revascularization of the ischemic area does not occur, and in contrast upregulation of Sema3A leads to pathological neovascularization into the vitreous region.

Semaphorin 3A is secreted by hypoxic neurons in ischemic/avascular retinas, thereby inhibiting retinal revascularization and enhancing pathological preretinal neovascularization.

The inventors addressed this pathological condition by developing antibodies targeting Sema 3A. Accordingly, the present invention provides monoclonal antibodies that specifically bind to Sema3A, preferably human Sema 3A.

In a first aspect, the present invention provides an anti-Sema 3A antibody or antigen-binding fragment thereof, comprising:

-a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:1 (H-CDR1), the amino acid sequence of SEQ ID NO:2 (H-CDR2), and the amino acid sequence of SEQ ID NO:3 (H-CDR 3); 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-CDR 3).

In another embodiment, the invention provides an anti-Sema 3A antibody or antigen-binding fragment thereof, comprising:

-a heavy chain variable region comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9 or SEQ ID NO 10; and

-a light chain variable region comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO 11, SEQ ID NO 12 or SEQ ID NO 13;

wherein:

-the heavy chain variable region comprises the amino acid sequence of SEQ ID NO 1 (H-CDR 1); the amino acid sequence of SEQ ID NO:2 (H-CDR 2); and the amino acid sequence of SEQ ID NO 3 (H-CDR 3); and is

-the light chain variable region comprises the amino acid sequence of SEQ ID NO 4 (L-CDR 1); the amino acid sequence of SEQ ID NO. 5 (L-CDR 2); and the amino acid sequence of SEQ ID NO. 6 (L-CDR 3).

In another embodiment, the invention provides an anti-Sema 3A antibody or antigen-binding fragment thereof, comprising:

-a heavy chain variable region comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9 or SEQ ID NO 10; and

-a light chain variable region comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID No. 11, SEQ ID No. 12 or SEQ ID No. 13.

In another embodiment, the invention provides an anti-Sema 3A antibody or antigen-binding fragment thereof, comprising:

-a heavy chain variable region comprising the amino acid sequence of SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9 or SEQ ID NO 10; and

-a light chain variable region comprising the amino acid sequence of SEQ ID NO 11, SEQ ID NO 12 or SEQ ID NO 13.

In another embodiment, the invention provides an anti-Sema 3A antibody or antigen-binding fragment thereof, comprising:

a. a variable heavy chain and a variable light chain comprising the amino acid sequences of SEQ ID NO 7 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 8 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 9 and SEQ ID NO 12, respectively; or

d. A variable heavy chain and a variable light chain comprising the amino acid sequences of SEQ ID NO 10 and SEQ ID NO 13, respectively.

In another embodiment, the invention provides an anti-Sema 3A antibody or antigen-binding fragment thereof, comprising:

-a heavy chain comprising, preferably consisting of, the amino acid sequence of SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 17 or SEQ ID No. 19; and

-a light chain comprising, preferably consisting of, the amino acid sequence of SEQ ID NO 15, 18 or 20.

In certain embodiments, the invention relates to an anti-Sema 3A antibody or antigen-binding fragment thereof, comprising:

a. a heavy chain comprising the amino acid sequence of SEQ ID NO. 14 and a light chain comprising the amino acid sequence of SEQ ID NO. 15;

b. a heavy chain comprising the amino acid sequence of SEQ ID NO 16 and a light chain comprising the amino acid sequence of SEQ ID NO 15;

c. a heavy chain comprising the amino acid sequence of SEQ ID NO 17 and a light chain comprising the amino acid sequence of SEQ ID NO 18; or

d. A heavy chain comprising the amino acid sequence of SEQ ID NO 19 and a light chain comprising the amino acid sequence of SEQ ID NO 20.

In a particularly preferred embodiment, the anti-Sema 3A antibody is a humanized anti-Sema 3A antibody.

In a second aspect, the present invention provides anti-Sema 3A antibodies or antigen-binding fragments thereof that bind to at least one amino acid residue within the amino acid region 370-382 of human Sema3A as depicted in SEQ ID NO: 22.

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

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

In one embodiment, the present invention provides anti-Sema 3A or an antigen-binding fragment for use in inhibiting the angiostatic effect (vasopressive effect) of SemaA and/or for improving retinal revascularization.

In one embodiment, the present invention provides an anti-Sema 3A antibody or antigen-binding fragment thereof for use in the treatment or prevention of a retinal or ocular disease.

In a fourth aspect, the invention provides an anti-Sema 3A antibody or antigen-binding fragment thereof for use in the treatment or prevention of a disease selected from the group consisting of: retinopathy, ischemic retinopathy, diabetic retinopathy (including proliferative and nonproliferative diabetic retinopathy), diabetic macular edema, diabetic macular ischemia, age-related macular degeneration, retinitis pigmentosa, hereditary trophism atrophy, myopic degeneration, retinal artery occlusion, endophthalmitis, uveitis, cystoid macular edema, choroidal neovascular membranes secondary to any retinal disease, optic nerve disease, glaucoma, retinal detachment, toxic retinopathy, radiation retinopathy, traumatic retinopathy, drug-induced retinal vasculopathy, retinal neovascularization, polypoidal choroidal vasculopathy, retinal vasculitis, retinal microaneurysms, fusch's dystrophy, macular telangiectasia, retinal vascular diseases, retinal diseases, and the like, retinal diseases, Usser syndrome and Stargardt disease.

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

In a preferred embodiment, the present invention provides an anti-Sema 3A antibody or antigen-binding fragment thereof for use in treating diabetic macular ischemia by promoting ischemic intraretinal vascular regeneration (revascularization) and preventing pathological neovascularization of the vitreous region of the eye.

In another preferred embodiment, the present invention provides an anti-Sema 3A antibody or antigen-binding fragment thereof for use in treating diabetic macular edema by decreasing the permeability of the blood retinal barrier.

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

In a fifth aspect, the invention provides a pharmaceutical composition comprising an anti-Sema 3A antibody or antigen-binding fragment thereof and a pharmaceutically acceptable carrier.

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

In a sixth aspect, the invention provides one or more isolated polynucleotides comprising:

-a sequence encoding the heavy chain as shown in SEQ ID NO 14, 16, 17 or 19 or the heavy chain variable region as shown in SEQ ID NO 7,8, 9 or 10; and

-a sequence encoding the light chain as shown in SEQ ID NO 15, 18 or 20 or the light chain variable region as shown in SEQ ID NO 11, 12 or 13.

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

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

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

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

In one embodiment, the method of producing an anti-Sema 3A antibody or antigen-binding fragment thereof further comprises recovering and purifying the anti-Sema 3A antibody or antigen-binding fragment thereof.

[ description of the drawings ]

FIG. 1: sema3A positioning in human eyes

This figure shows the localization of Sema3A in human eyes in pre-designated retinal samples from human donors with a history of diabetic retinopathy or Primary Open Angle Glaucoma (POAG) compared to age-matched controls (age ctrl) and individuals with diabetes but no ocular condition (DM ctrl). Sema3A is found in the vascular system wall of retinal vessels. In addition, unidentified but unique Sema3A fluorescent objects were observed in the retinal ganglion cell layer.

FIG. 2: potency in cell Permeability assays

The penetration of FITC-polydextrose into human retinal microvascular endothelial cells was measured in a transwell assay. anti-TNP is a control antibody against trinitrophenol. anti-Sema 3A is an antibody of the invention (pure line I). Showing significance relative to recombinant human Sema 3A.

FIG. 3: cytoskeletal collapse in HRMEC (xcelligence)

Sema3A-F induced cytoskeletal collapse in human retinal endothelial cells. The antibody of the invention (clone I) is specific for Sema3A and only prevents Sema 3A-induced collapse.

FIG. 4: effect on tip cell density and avascular zone in vivo

(A) And (C) studying apical cell density and avascular zones in a model of oxygen-induced retinopathy in mouse pups. Animals were exposed to 75% oxygen from P7 to P12 and received a single intravitreal injection of antibody after P12 returned to normoxic levels. anti-TNP is a control antibody against trinitrophenol. anti-Sema 3A is an antibody of the invention (pure line I). At P17, retinal plating was prepared, stained with isolectin B4 and tip cells were counted and the size of the avascular region of the retina was determined.

(B) A correlation between tip cell density and avascular zone is shown.

[ embodiment ] A method for producing a semiconductor device

Definition of

The general structure of antibodies or immunoglobulins is well known to those skilled in the art and these molecules are heterotetrameric glycoproteins, typically about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is covalently linked to a heavy chain by one disulfide bond to form a heterodimer, and the heterotrimeric molecule is formed via a covalent disulfide bond between two identical heavy chains of the heterodimer. Although the light and heavy chains are linked together by one disulfide bond, the number of disulfide bonds between the two heavy chains varies according to the immunoglobulin isotype. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable domain at the amino terminus (V)_HVariable heavy chain) followed by three or four constant domains (C)_H1、C_H2、C_H3And C_H4) And C_H1And C_H2The hinge region in between. Each light chain has two domains, the amino-terminal variable domain (V)_LVariable light chain) and carboxy-terminal constant domain (C)_L)。V_LDomains with V_HDomains associate non-covalently, and C_LDomains are usually linked to C via a disulfide bond_H1The domains are covalently linked. It is believed that particular amino acid residues form an interface between the light and heavy chain variable domains (Chothia et al, 1985, J.mol.biol.186: 651-.

Certain domains within a variable domain differ widely between different antibodies, i.e., "hypervariable". The hypervariable domains contain residues which are directly involved in the binding and specificity of each particular antibody for its specific antigenic determinant. Hypervariability in both light and heavy chain variable domains is concentrated in three segments called Complementarity Determining Regions (CDRs) or hypervariable loops (HVLs). The CDRs are defined by sequence comparison by Kabat et al, 1991 in: sequences of Proteins of Immunological Interest, 5 th edition, Public Health Service, National Institutes of Health, Bethesda, Md., and HVL is structurally defined according to the three-dimensional structure of variable domains as described by Chothia and Lesk,1987, J.mol.biol.196: 901-. In cases where the two methods result in slightly different identifications of the CDRs, the structural definition is preferred. CDR-L1 is located at about residues 24-34, CDR-L2 is located at about residues 50-56, and CDR-L3 is located at about residues 89-97 in the light chain variable domain as defined by Kabat; CDR-H1 is located at about residues 31-35 in the heavy chain variable domain, CDR-H2 is located at about residues 50-65, and CDR-H3 is located at about residues 95-102. Thus, the CDRs 1, 2, CDR3 of the heavy and light chains define unique and functional properties specific for a given antibody.

The three CDRs within each of the heavy and light chains are separated by Framework Regions (FRs) that contain sequences that tend to be less variable. From the amino-terminus to the carboxy-terminus of the heavy and light chain variable domains, the FRs and CDRs are arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR 4. The majority of the β -sheet conformation of the FRs brings the CDRs within each chain into close proximity with each other, as well as with CDRs from other chains. The resulting conformation contributes to the antigen binding site (see Kabat et al, 1991, NIH Publ. Nos. 91-3242, Vol. I, p.647-669), but not all CDR residues are necessarily involved directly in antigen binding.

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 thought to have a significant effect on antigen binding in at least three ways: by direct non-covalent binding to an epitope, by interaction with one or more CDR residues, and by affecting the interface between the heavy and light chains. The constant domains are not directly involved in antigen binding, but mediate various Ig effector functions, such as antibody involvement in antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent phagocytosis (ADCP).

Based on the amino acid sequence of the constant domains, light chains of vertebrate immunoglobulins are assigned to one of two distinctly different classes, kappa (κ) and lambda (lambda, λ). By comparison, the heavy chains of mammalian immunoglobulins are assigned to one of five major classes according to the sequence of the constant domains: IgA, IgD, IgE, IgG and IgM. IgG and IgA are further divided into subclasses (isotypes), e.g., IgG and IgA, respectively₁、IgG₂、IgG₃、IgG₄、IgA₁And IgA₂. The heavy chain constant domains corresponding to different immunoglobulin classes are called α, δ, ε, γ and μ, respectively. The subunit structure and three-dimensional conformation of the native immunoglobulin class are well known.

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

The term "monoclonal antibody" (mAb) refers to an antibody of a substantially homogeneous population of antibodies; that is, the individual antibodies in the population are identical except for naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic determinant, i.e., an "epitope". Thus, the modifier "monoclonal" indicates a substantially homogeneous population of antibodies directed to the same epitope, and is not to be construed as requiring production of the antibody by any particular method. It is understood that monoclonal antibodies can be made by any technique or method known in the art; including, for example, hybridoma methods (Kohler et al, 1975, Nature256:495), or recombinant DNA methods known in the art (see, e.g., U.S. Pat. No. 4,816,567), or isolation methods using recombinant generated monoclonal antibodies from phage antibody libraries, using techniques described in Clackson et al, 1991, Nature352: 624-.

Chimeric antibodies consist of the variable regions of the heavy and light chains of an antibody from one species (e.g., a non-human mammal, such as a mouse) and the constant regions of the heavy and light chains of an antibody from another species (e.g., a human), and can be obtained by ligating a DNA sequence encoding the variable region of an antibody from a first species (e.g., a mouse) with a DNA sequence encoding the constant region of an antibody from a second (e.g., a human) species and transforming a host with an expression vector containing the ligated sequence so that it produces a chimeric antibody. Alternatively, a chimeric antibody may also be an antibody in which one or more regions or domains of the heavy and/or light chain are identical, homologous, or variants thereof to corresponding sequences in a monoclonal antibody from another immunoglobulin class or isotype, or from consensus sequences or species. Chimeric antibodies can include fragments of such antibodies, provided that the antibody fragment exhibits the desired biological activity of its parent antibody, e.g., binding to the same epitope (see, e.g., U.S. Pat. No. 4,816,567; and Morrison et al, 1984, Proc. Natl. Acad. Sci. USA 81: 6851-.

The terms "antibody fragment", "antigen-binding fragment", "anti-Sema 3A antibody fragment", "humanized anti-Sema 3A antibody fragment", "variant humanized anti-Sema 3A antibody fragment" refer to a portion of a full-length anti-Sema 3A antibody in which the variable region or functional capacity is retained, e.g., specific Sema3A epitope binding. Examples of antibody fragments include, but are not limited to, Fab ', F (ab')2, Fd, Fv, scFv, and scFv-Fc fragments, diabodies, linear antibodies, single chain antibodies, minibodies, diabodies formed from antibody fragments, and multispecific antibodies formed from antibody fragments.

Can be treated with enzymes such as papain or pepsinFull length antibodies to produce useful antibody fragments. Papain digestion is used to produce two identical antigen-binding antibody fragments, referred to as "Fab" fragments (each having a single antigen-binding site) and a residual "Fc" fragment. Fab fragments also contain the constant domain of the light chain and the C of the heavy chain_H1A domain. Pepsin treatment produces F (ab') with two antigen binding sites and still cross-links the antigen₂And (3) fragment.

Fab' fragments differ from Fab fragments by the presence of additional residues, including C_H1One or more cysteines of the antibody hinge region at the C-terminus of the domain. F (ab')₂Antibody fragments are pairs of Fab' fragments joined by cysteine residues in the hinge region. Other chemical couplings of antibody fragments are also known.

"Fv" fragments contain the entire antigen recognition and binding site, which consists of a dimer of one heavy and one light chain variable domain in tight, non-covalent association. In this configuration, the three CDRs of each variable domain interact to define V_H-V_LAntigen binding sites on the surface of the dimer. The six CDRs collectively confer antigen binding specificity to the antibody.

"Single chain Fv" or "scFv" antibody fragments are V comprising an antibody_HAnd V_LSingle-chain Fv variants of a domain, wherein the domain is present in a single polypeptide chain. Single-chain Fv's are capable of recognizing and binding antigen. The scFv polypeptide may also optionally contain a site V_HAnd V_LPolypeptide linkers between domains to facilitate formation of The desired three-dimensional structure of scFv-bound antigens (see, e.g., Pluckthun,1994, In The Pharmacology of monoclonal Antibodies, Vol.113, edited by Rosenburg and Moore, Springer-Verlag, New York, p.269-315).

Other recognized antibody fragments include those comprising a pair of tandem Fd segments (V)_H-C_H1-V_H-C_H1) To form those antibody fragments of a pair of antigen binding regions. Such "linear antibodies" may be bispecific or monospecific, as described, for example, in Zapata et al 1995, Protein Eng.8(10): 1057-1062.

A humanized antibody or humanized antibody fragment is a specific type of chimeric antibody (which includes immunoglobulin amino acid sequence variants) or fragment thereof that is capable of binding to a predetermined antigen and that comprises one or more FRs having substantially the amino acid sequence of a human immunoglobulin and one or more CDRs having substantially the amino acid sequence of a non-human immunoglobulin. This non-human amino acid sequence, often referred to as an "import" sequence, is typically taken from an "import" antibody domain, in particular a variable domain. In general, humanized antibodies comprise at least the CDRs or HVLs of a non-human antibody inserted between the FRs of a human heavy or light chain variable domain.

Specific humanized anti-Sema 3A antibodies are described that contain CDRs derived from murine or chimeric antibodies inserted between the FRs of the heavy and light chain variable regions of the sequence of the human race. It will be appreciated that certain murine FR residues may be important for the function of humanized antibodies, and thus certain races are those in which the sequence heavy and light chain variable domain residues are modified to be identical to those of the corresponding murine sequences.

The expressions "antibody of the invention" and "anti-Sema 3A antibody of the invention" as used herein refer to the anti-Sema 3A antibody or antigen-binding fragment thereof described herein. Preferably, the expression refers to any antibody comprising: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:1 (H-CDR1), the amino acid sequence of SEQ ID NO:2 (H-CDR2), and the amino acid sequence of SEQ ID NO:3 (H-CDR 3); 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-CDR 3).

In one aspect, the humanized anti-Sema 3A antibody comprises substantially all of at least one, and typically two, variable domains (e.g., the variable domains contained in Fab, Fab ', F (ab')2, Fabc, and Fv fragments), wherein all or substantially all of the CDRs correspond to those of a non-human immunoglobulin, and in particular herein, the CDRs are murine sequences and the FRs are those of a human immunoglobulin consensus sequence or species. In another aspect, the humanized anti-Sema 3A antibody also includes at least a portion of an immunoglobulin Fc region, typically a human immunoglobulin. Typically, the antibody will contain a light chain and at least a heavy chainA variable domain. If appropriate, the antibody may also comprise heavy chain C_H1Hinge, C_H2、C_H3And/or C_H4One or more of the zones.

The humanized anti-Sema 3A antibody may be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including IgG₁、IgG₂、IgG₃、IgG₄、IgA₁And IgA₂. For example, the constant domain may be a complement fixation constant domain, wherein the humanized antibody is expected to exhibit cytotoxic activity, and the isotype is typically an IgG₁. In cases where such cytotoxic activity is not desired, the constant domain may be of another isotype, e.g., an IgG₂. Alternative humanized anti-Sema 3A antibodies may comprise sequences from more than one immunoglobulin class or isotype, and it is within the ordinary skill in the art to select specific constant domains to optimize the desired effector function. In a specific embodiment, the invention provides an antibody that is an IgG1 antibody, more particularly an IgG1 antibody characterized by reduced effector function.

Preferably, the anti-Sema 3A antibody of the invention is a humanized antibody formatted as IgG1 KO.

The FR and CDR or HVL of the humanized anti-Sema 3A antibody need not correspond exactly to the parental sequences. For example, one or more residues in the CDR or HVL or consensus sequence or the FR sequence may be altered (e.g., mutagenized) by substitution, insertion or deletion such that the resulting amino acid residues are no longer identical to the original residues in the corresponding positions in either parent sequence, but the antibody still retains the function of binding to Sema 3A. The changes are generally not extensive and will be conservative changes. Typically, at least 75%, more typically at least 90%, and most often greater than 95%, or greater than 98%, or greater than 99% of the humanized antibody residues will correspond to those for which the parent consensus sequence or species is a FR and input CDR sequence.

Affecting the interface between the heavy and light chain variable regions ('V')_L-V_HInterfacial ") are those that affect the proximity or orientation of two chains to each other. Certain residues that may participate in interchain interactionsComprising V_LResidues 34, 36, 38, 44, 46, 87, 89, 91, 96 and 98 and V_HResidues 35, 37, 39, 45, 47, 91, 93, 95, 100 and 103 (using the numbering system described 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, for example, V_LResidues 43 and 85 and V_HResidues 43 and 60 may also be involved in this interaction. Although these residues are only applicable to human IgG, they are also applicable across species. Important antibody residues that are reasonably expected to participate in interchain interactions are selected for substitution into the consensus sequence.

The terms "consensus sequence" and "consensus antibody" refer to an amino acid sequence comprising the most frequently occurring amino acid residue at each position in all immunoglobulins of any particular class, isotype, or subunit structure, e.g., a human immunoglobulin variable domain. The consensus sequence may be based on immunoglobulins of a particular species or of a number of species. A "consensus" sequence, structure or antibody is understood to encompass a consensus human sequence as described in certain embodiments, and refers to an amino acid sequence comprising the most frequently occurring amino acid residue at each position in all human immunoglobulins of any particular class, isotype or subunit structure. Thus, a consensus sequence contains an amino acid sequence that has at each position an amino acid present in one or more known immunoglobulins, but which may not be completely repeated with the entire amino acid sequence of any single immunoglobulin. The variable region consensus sequence is not obtained from any naturally occurring antibody or immunoglobulin. Kabat et al, 1991, Sequences of Proteins of Immunological Interest, 5 th edition, Public Health Service, National Institutes of Health, Bethesda, Md., and variants thereof. The FR of the heavy and light chain consensus sequences and variants thereof provide useful sequences for the preparation of humanized anti-Sema 3A antibodies. See, for example, U.S. patent nos. 6,037,454 and 6,054,297.

The human species is the species in which the sequence naturally occurs in the human population. Those species are combinations of genes that create antibody diversity. The light chain species of the antibody is from the conserved human species of kappa or lambda V genes and j genes. Similarly, The heavy chain sequences are from The v-, d-and j-genes (LeFranc, M-P and LeFranc, G, "The Immunoglobulin proteins products Book" Academic Press, 2001).

An "isolated" antibody is one that has been identified and isolated and/or recovered from a component of its natural environment. Contaminating components of the natural environment of an antibody are those substances that may interfere with the diagnostic or therapeutic use of the antibody, and may be enzymes, hormones, or other proteinaceous or nonproteinaceous solutes. In one aspect, the antibody will be purified to at least greater than 95% isolation by weight of the antibody.

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

As used herein, the term "identical" or "percent identity," in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence. To determine the percent identity, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid for optimal alignment with a second amino acid sequence or a second nucleic acid sequence). The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percentage of 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, two sequences being compared have the same length after gaps are introduced in the sequences, if appropriate (e.g., excluding additional sequences that extend beyond the sequences being compared). For example, when comparing variable region sequences, leader and/or constant domain sequences are not considered. For sequence comparison between two sequences, a "corresponding" CDR refers to a CDR in the same position in both sequences (e.g., CDR-H1 for each sequence).

Determination of percent identity or percent similarity between two sequences can be accomplished using a mathematical algorithm. A preferred non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul,1990, Proc. Natl. Acad. Sci. USA 87: 2264-. The algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al, 1990, J.Mol.biol.215: 403-. BLAST nucleotide searches can be performed using NBLAST programs (score 100, word length 12) to obtain nucleotide sequences homologous to nucleic acids encoding proteins of interest. BLAST protein searches can be performed using the XBLAST program (score 50, word length 3) to obtain D amino acid sequences homologous to the protein of interest. To obtain a gapped alignment for comparison purposes, gapped BLAST can be utilized as described in Altschul et al, 1997, Nucleic Acids Res.25: 3389-. Alternatively, PSI-Blast can be used to perform an iterative search that detects remote relationships between molecules (supra). When utilizing BLAST, gapped BLAST, and PSI-BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. Another preferred non-limiting example of a mathematical algorithm for comparing sequences is the algorithm of Myers and Miller, CABIOS (1989). This algorithm was incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When comparing amino acid sequences using the ALIGN program, 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 as described in Torellis and Robotti,1994, Compout.Appl.biosci.10: 3-5; and FASTA as described in Pearson and Lipman,1988, Proc.Natl.Acad.Sci.USA 85: 2444-8. Within FASTA, ktup is a control option that sets the sensitivity and speed of the search. If ktup is 2, a similar region in the two sequences compared is found by observing the aligned residue pair; if ktup ═ 1, the single aligned amino acids are examined. ktup can be set to 2 or 1 for protein sequences or 1 to 6 for DNA sequences. If ktup is not specified, the default value is 2 for proteins and 6 for DNA. Alternatively, protein sequence alignment may be performed using the CLUSTAL W algorithm as described by Higgins et al, 1996, Methods enzymol.266: 383-402.

As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably and all such designations include progeny thereof. Thus, "transformants" and "transformed cells" include primary individual cells and cultures derived therefrom, regardless of the number of transfers.

For therapeutic purposes, the term "mammal" refers to any animal classified as a mammal, including humans, domestic and farm animals, as well as zoo, sports, or pet animals, such as dogs, horses, cats, cattle, and the like. Preferably, the mammal is a human.

As used herein, a "disease" or "disorder" is any condition that would benefit from treatment with the humanized anti-Sema 3A antibodies described herein. This includes chronic and acute disorders or diseases, including those pathological conditions that predispose a mammal to the disorder in question.

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

The term "subcutaneous administration" refers to the introduction of an anti-Sema 3A antibody or antigen-binding fragment thereof under the skin of an animal or human patient, preferably within the sac between the skin and underlying tissues, by relatively slow, sustained delivery from a drug container. Pinching or pulling the skin up and away from the underlying tissue can create a pocket.

The term "subcutaneous infusion" refers to the introduction of a drug under the skin of an animal or human patient, preferably within a pocket between the skin and underlying tissues, by relatively slow, sustained delivery from a drug container for a period of time (including but not limited to 30 minutes or less, or 90 minutes or less). Optionally, the infusion may be performed by subcutaneously implanting a drug delivery pump implanted under the skin of an animal or human patient, wherein the pump delivers a predetermined amount of drug over a predetermined period of time (e.g., 30 minutes, 90 minutes, or a period of time spanning the length of a treatment regimen).

The term "subcutaneous bolus" refers to the administration of a drug under the skin of an animal or human patient, wherein the bolus drug is delivered for 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 pouch between the skin and underlying tissue, wherein the pouch can be created by pinching or pulling the skin up and away from the underlying tissue.

The term "therapeutically effective amount" is used to refer to an amount of an anti-Sema 3A antibody or antigen-binding fragment thereof that reduces or ameliorates one or more symptoms of the disorder being treated. Which is an amount that has a beneficial patient outcome when alleviating or ameliorating one or more symptoms of the treated disorder. Depending on the condition to be treated, efficacy can be measured in a conventional manner. For example, in an ocular/retinal disease or condition characterized by cells expressing Sema3A, potency can be measured by determining the response rate, e.g., vision recovery or by assessing the delay time until disease progression.

The terms "treatment" and "therapy" and the like as used herein are meant to include both therapeutic as well as prophylactic or inhibitory measures of a disease or disorder that result in any clinically desirable or beneficial effect, including, but not limited to, alleviation or relief of one or more symptoms, regression of the disease or disorder, slowing or stopping the progression of the disease or disorder. Thus, for example, the term treating includes administering an anti-Sema 3A antibody or antigen-binding fragment thereof, either before or after the onset of symptoms of the disease or disorder, thereby preventing or removing one or more signs of the disease or disorder. As another example, the term includes administration of an anti-Sema 3A antibody or antigen-binding fragment thereof after clinical expression of the disease to combat the symptoms of the disease. In addition, administration of an anti-Sema 3A antibody or antigen-binding fragment thereof after onset and after clinical symptoms have developed (wherein the administration would still affect the clinical parameters of the disease or disorder regardless of whether the treatment resulted in amelioration of the disease) includes "treatment" or "therapy" as used herein. Furthermore, as long as the compositions of the invention alone or in combination with another therapeutic agent can alleviate or ameliorate at least one symptom of the condition being treated, the result should be considered effective in treating the underlying condition, regardless of whether all symptoms of the condition are alleviated, as compared to the symptoms when the anti-Sema 3A antibody composition or antigen-binding fragment thereof is not used.

The term "package insert" is used to refer to instructions typically included in commercial packaging for therapeutic products that contain information about the indications, usage, administration, contraindications, and/or warnings concerning the use of such therapeutic products.

Antibodies of the invention

In a first aspect, the invention relates to an anti-Sema 3A antibody or antigen-binding fragment thereof. Preferably, the antibody is a humanized anti-Sema 3A antibody, more preferably a humanized monoclonal anti-Sema 3A antibody.

In initial characterization, antibody libraries targeting Sema3A variants were generated by placing the CDRs of a murine antibody in the FRs of human consensus heavy and light chain variable domains and further by engineering the FRs with different alterations.

This resulted in humanized antibodies to Sema3A, which had enhanced properties as disclosed herein. The sequences of the antibodies of the invention are shown in table 1 below.

Table 1:

in one embodiment, the invention provides an anti-Sema 3A antibody or antigen-binding fragment thereof, comprising:

-a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:1 (H-CDR1), the amino acid sequence of SEQ ID NO:2 (H-CDR2), and the amino acid sequence of SEQ ID NO:3 (H-CDR 3); 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-CDR 3).

In another embodiment, the invention provides an anti-Sema 3A antibody or antigen-binding fragment thereof, comprising:

-a heavy chain variable region comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9 or SEQ ID NO 10; and

-a light chain variable region comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID No. 11, SEQ ID No. 12 or SEQ ID No. 13.

In another embodiment, the invention provides an anti-Sema 3A antibody or antigen-binding fragment thereof, comprising:

-a heavy chain variable region comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9 or SEQ ID NO 10; and

-a light chain variable region comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO 11, SEQ ID NO 12 or SEQ ID NO 13;

wherein:

-the heavy chain variable region comprises the amino acid sequence of SEQ ID NO 1 (H-CDR 1); the amino acid sequence of SEQ ID NO:2 (H-CDR 2); and the amino acid sequence of SEQ ID NO 3 (H-CDR 3); and is

-the light chain variable region comprises the amino acid sequence of SEQ ID NO 4 (L-CDR 1); the amino acid sequence of SEQ ID NO. 5 (L-CDR 2); and the amino acid sequence of SEQ ID NO. 6 (L-CDR 3).

In another embodiment, the invention provides an anti-Sema 3A antibody or antigen-binding fragment thereof, comprising:

-a heavy chain variable region comprising the amino acid sequence of SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9 or SEQ ID NO 10; and

-a light chain variable region comprising the amino acid sequence of SEQ ID NO 11, SEQ ID NO 12 or SEQ ID NO 13.

In a preferred embodiment, the present invention provides an anti-Sema 3A antibody or antigen-binding fragment thereof, comprising:

-a variable heavy chain and a variable light chain comprising the amino acid sequences of SEQ ID No. 7 and SEQ ID No. 11, respectively;

-a variable heavy chain and a variable light chain comprising the amino acid sequences of SEQ ID No. 8 and SEQ ID No. 11, respectively;

-a variable heavy chain and a variable light chain comprising the amino acid sequences of SEQ ID No. 9 and SEQ ID No. 12, respectively; or

-a variable heavy chain and a variable light chain comprising the amino acid sequences of SEQ ID NO 10 and SEQ ID NO 13, respectively.

In another embodiment, the invention provides an anti-Sema 3A antibody or antigen-binding fragment thereof, comprising:

-a heavy chain comprising, preferably consisting of, the amino acid sequence of SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 17 or SEQ ID No. 19; and

-a light chain comprising, preferably consisting of, the amino acid sequence of SEQ ID NO 15, 18 or 20.

In certain embodiments, the invention relates to an anti-Sema 3A antibody or antigen-binding fragment thereof, comprising:

a. a heavy chain comprising the amino acid sequence of SEQ ID NO. 14 and a light chain comprising the amino acid sequence of SEQ ID NO. 15, referred to as "germline I";

b. a heavy chain comprising the amino acid sequence of SEQ ID NO 16 and a light chain comprising the amino acid sequence of SEQ ID NO 15, referred to as "germline II";

c. a heavy chain comprising the amino acid sequence of SEQ ID NO 17 and a light chain comprising the amino acid sequence of SEQ ID NO 18, referred to as "clone III"; or

d. The heavy chain comprising the amino acid sequence of SEQ ID NO 19 and the light chain comprising the amino acid sequence of SEQ ID NO 20, this antibody is referred to as "pure line IV".

IgG1-KO mutants were prepared by introducing mutations in the Fc region. Mutations to reduce or inhibit effector function are well known to those skilled in the art and are well disclosed in the prior art, for example, in Wang et al, Protein Cell 2018,9(1):63-73 and Stewart et al, Journal for ImmunoTherapy of Cancer 2014,2: 29. Generally, a non-limiting list of mutations introduced in the IgG1 Fc region to reduce the effector function of Fc comprises:

L234A and L235A;

L234A, L235A and N297Q;

L234A, L235A and P329G; or

L234A, L235A and D265A;

wherein the residues are numbered according to the EU index of Kabat.

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

The CDRs disclosed herein and depicted in SEQ ID NOs 1 to 6 are presented according to Kabat numbering and are summarized in table 2 below with Kabat positions.

Table 2:

CDR	kabat sequence	Kabat position	SEQ ID NO:
				HCDR1	SYYMS	31-35	1
HCDR2	TIIKSGGYAYYPDSVKD	50-66	2
				HCDR3	GGQGAMDY	99-106	3
LCDR1	RASQSIGDYLH	24-34	4
				LCDR2	YASQSIS	50-56	5
LCDR3	QQGYSFPYT	89-97	6

The anti-Sema 3A antibodies of the invention bind to human Sema3A with high affinity. In an embodiment of this aspect, the anti-Sema 3A antibody of the invention is represented by K_D<50pM bound to human Sema 3A. In another embodiment, the anti-Sema 3A antibody of the invention is represented by K, as exemplified in example 4_D<35pM bound to human Sema 3A. In a preferred embodiment, the anti-Sema 3A antibody of the invention is represented by K_D<30pM bound to human Sema 3A.

The anti-Sema 3A antibodies of the invention also bind to cyno-Sema3A, mouse Sema3A, rat Sema3A and rabbit Sema 3A.

The anti-Sema 3A antibodies of the invention prevent Sema 3A-induced cytoskeletal collapse in retinal cells with a functional efficacy of less than 100pM, preferably less than 80pM, more preferably less than 70 pM. In a preferred embodiment, the anti-Sema 3A antibodies of the invention prevent Sema 3A-induced cytoskeletal collapse in retinal cells with a functional efficacy of 69pM, as exemplified in example 4.

In another aspect, the anti-Sema 3A antibodies of the invention demonstrate low risk of immunogenicity, as described in example 5. This relies on the computer to predict the immunogenicity of the antibody. Immunogenic risk is typically assessed by various well-known methods, such as by computer algorithms for predicting T cell epitopes, which are the major immunogenic contributors.

It has in fact been reported that sequences containing T cell epitopes present in a protein of interest can be predicted by using an algorithm based on the computational matrix method (available under the name EpiMatrix (manufactured by EpiVax)). Those skilled in the art can refer to Van Walle et al, Expert Opin Biol ther, 3 months 2007; 7(3) 405-18 and Jawa et al, Clin Immunol.2013 for 12 months; 149(3):534-55.

The inventors have shown that the antibodies of the invention exhibit more favorable properties than other antibodies or fragments targeting Sema3A mentioned in the prior art and described herein.

The inventors compared the binding affinity of the antibodies targeting Sema3A disclosed in WO2014123186 (chome Bioscience) with the affinity of the antibodies of the invention. Antibodies of publication WO2014123186 for use in the treatment of Alzheimer's disease. This example 8 shows that the antibodies of the invention demonstrate higher binding affinity to human Sema3A than the prior art antibodies disclosed by Chiome Bioscience.

The inventors also compared the properties of the antibodies of the invention with ScFv fragments disclosed in WO2017074013 (Samsung). Such fragments are disclosed for use in the treatment of various cancers. Example 9 shows that the antibodies of the invention demonstrate higher binding affinity for human Sema3A than the prior art antibody fragments disclosed in WO 2017074013.

Higher binding affinity extends the time for neutralization of Sema3A after intravitreal injection of the antibody and allows for reduced injection frequency. The higher binding affinity further allows lower doses to be administered, thereby limiting potential side effects. Thus, the antibodies of the present invention provide technical advantages over prior art antibodies. The improved binding affinity and reduced injection frequency significantly improve the therapeutic efficacy of a patient in need thereof. It also provides valuable benefits to patients, especially improved drug compliance and compliance.

The inventors also compared the functional efficacy of the antibodies of the invention and a commercially available antibody targeting Sema3A as described in example 11. The inventors have shown that under the same conditions, the antibodies of the invention prevent Sema 3A-induced cytoskeletal collapse in retinal cells (example 3), whereas the commercially available antibodies do not (example 11).

Humanized and amino acid sequence variants

Other variant anti-Sema 3A antibodies and antibody fragments can be engineered based on the set of CDRs identified under the sequences depicted in SEQ ID NOs 1-6. It will be appreciated that in such variant anti-Sema 3A antibodies and antibody fragments, the amino acid sequence of the CDRs remains unchanged, but surrounding regions (e.g., the FR regions) may be engineered. Amino acid sequence variants of the anti-Sema 3A antibody can be prepared by introducing appropriate nucleotide changes into the anti-Sema 3A antibody DNA, or by peptide synthesis. Such variants include, for example, deletions from, and/or insertions into, and/or substitutions of, residues within the amino acid sequence of the anti-Sema 3A antibodies of the examples herein. Any combination of deletions, insertions, and substitutions are made to arrive at the final construct, provided that the final construct possesses the desired characteristics. Amino acid changes can also alter post-translational processes of humanized or variant anti-Sema 3A antibodies, such as changing the number or position of glycosylation sites.

Another type of amino acid variant of an antibody involves altering the original glycosylation pattern of the antibody. The term "altering" in this context means deleting one or more carbohydrate moieties found in the antibody, and/or adding one or more glycosylation sites not previously present in the antibody.

In some aspects, the invention includes nucleic acid molecules encoding amino acid sequence variants of the anti-Sema 3A antibodies described herein. Nucleic acid molecules encoding amino acid sequence variants of the anti-Sema 3A antibody are prepared by a variety of methods known in the art. Such methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of a previously prepared variant or non-variant form of the anti-Sema 3A antibody.

In certain embodiments, the anti-Sema 3A antibody is an antibody fragment. Techniques for producing antibody fragments have been developed. Fragments can be derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al, 1992, Journal of Biochemical and Biophysical Methods24: 107-117; and Brennan et al, 1985, Science 229: 81). Alternatively, the fragments may be produced directly in a recombinant host cell. For example, Fab '-SH fragments can be recovered directly from E.coli and chemically coupled to form F (ab')₂Fragments (see, e.g., Carter et al, 1992, Bio/Technology10: 163-. By another method, F (ab') can be isolated directly from recombinant host cell cultures₂And (3) fragment. Other techniques for producing antibody fragments will be apparent to the skilled practitioner.

anti-Sema 3A antibodies and antigen-binding fragments thereof may include modifications.

In certain embodiments, it may be desirable to use anti-Sema 3A antibody fragments rather than whole antibodies. It may be desirable to modify antibody fragments to increase their serum half-life. This can be achieved, for example, by incorporating salvage receptor binding epitopes into the antibody fragment. In one approach, the appropriate region of the antibody fragment may be altered (e.g., mutated), or the epitope may be incorporated into a peptide tag, which is then fused to the antibody fragment at either end or in the middle, e.g., by DNA or peptide synthesis. See, for example, WO 96/32478.

In other embodiments, the invention encompasses covalent modifications of anti-Sema 3A antibodies. Covalent modifications include modification of cysteinyl residues, histidinyl residues, abscinyl and amino-terminal residues, arginyl residues, tyrosyl residues, pendant carboxyl groups (aspartyl or glutamyl), glutaminyl and aspartyl residues, or seryl, or threonyl residues. Another type of covalent modification involves chemical or enzymatic coupling of the glycoside to the antibody. Such modifications may be made by chemical synthesis or by enzymatic or chemical cleavage of the antibody, if applicable. Other types of covalent modifications of antibodies can be introduced into the molecule by reacting targeted amino acid residues of the antibody with organic derivatizing agents capable of reacting with selected side chains or amino or carboxy terminal residues.

Removal of any carbohydrate moieties present on the antibody can be accomplished chemically or enzymatically. Chemical deglycosylation is described by Hakimuddin et al, 1987, Arch.biochem.Biophys.259:52 and Edge et al, 1981, anal.biochem.,118: 131. Enzymatic cleavage of the carbohydrate moiety on antibodies can be achieved by using various endo-and exoglycosidases, as described in Thotakura et al, 1987, meth. enzymol 138: 350.

Another type of useful covalent modification comprises linking the antibody to one of a variety of non-protein polymers (e.g., polyethylene glycol, polypropylene glycol, or polyalkylene oxide) in a manner 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.

Epitope binding

In a second aspect, the invention relates to antibodies that recognize the specific "Sema 3A epitope" and "Sema 3A epitope". In particular, the antibodies of the invention bind to the epitope of human Sema3A having SEQ ID No. 22.

In one aspect, the invention relates to an anti-Sema 3A antibody or antigen-binding fragment thereof that binds to at least one amino acid residue within amino acid region 370-382 of human Sema3A as set forth in SEQ ID NO 22.

In another aspect, the invention relates to an anti-Sema 3A antibody or antigen-binding fragment thereof that binds to SEQ ID No. 21.

The sequences SEQ ID NO 21 and 22 are shown in Table 5 below.

TABLE 5

The terms "Sema 3A epitope" and "Sema 3A epitope" as used herein refer to a molecule (e.g., a peptide) or fragment of a molecule that is capable of binding to an anti-Sema 3A antibody or antigen-binding fragment thereof. The terms further include, for example, the Sema3A antigenic determinant recognized by any antibody or antibody fragment of the invention having a light chain and heavy chain CDR combination selected from the group consisting of the heavy chain CDRs of SEQ ID NOs 1-3 and the light chain CDRs of SEQ ID NOs 4-6.

The Sema3A epitope may be included in a protein, protein fragment, peptide, or the like. Epitopes are the most common proteins, short oligopeptides, oligopeptides (i.e., organic compounds that mimic the antibody binding properties of the Sema3A antigen), or combinations thereof.

It has been found that the antibodies or antibody fragments of the present invention bind to a unique epitope of human Sema 3A. Preferably, the anti-Sema 3A antibody or antigen-binding fragment thereof binds to at least one amino acid residue within amino acid region 370-382 of the extracellular domain of human Sema3A having SEQ ID NO 22. This epitope is located near the interface between Sema3A and the plexin a receptor. Binding of the antibody to this epitope inhibits formation of a signaling intact holoreceptor (holoreceptor) complex of the ligand Sema3A, the receptor plexin a, and the co-receptor Nrp1, resulting in a biological effect that interferes with this signaling.

In the context of epitope binding, the phrase "binds within amino acid region X-Y …" means that the anti-Sema 3A antibody or antigen-binding fragment thereof binds to at least one, and preferably all, amino acid residues within the indicated amino acid region in the sequence.

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

Therapeutic uses

In a third aspect, the invention relates to an anti-Sema 3A antibody or antigen-binding fragment thereof for use as a medicament.

In one embodiment, the invention provides anti-Sema 3A or an antigen-binding fragment for use in inhibiting the angiostatic effect of SemaA and/or for improving retinal revascularization.

Preferably, the present invention provides an anti-Sema 3A antibody or antigen-binding fragment thereof for use in the treatment or prevention of a retinal or ocular disease. Indeed, the present inventors have developed antibodies targeting Sema3A that are extremely helpful in:

-redirecting angiogenesis to the ischemic zone to improve revascularization of the retina;

-prevention of pathological neovascularization of the vitreous region; and

-prevention of blood retinal barrier disruption.

As mentioned previously, Sema3A is a vascular repulsion cue secreted by hypoxic retinal ganglion cells. By binding to cyclin-1, it activates intracellular signaling of the plexin receptor on endothelial cells, leading to disassembly of actin fibers. This results in cytoskeletal collapse in the filopodia of apical cells, specialized endothelial cells that direct new blood vessel growth and prevent revascularization of ischemic areas in the retina. The inventors have shown that modulation of vascular repulsion with the neutralizing Sema 3A-antibody increases the number of TIP cells and redirects angiogenesis to ischemic areas, such as the pathologically enlarged foveal avascular zone in humans with diabetic macular ischemia.

Accordingly, in a fourth aspect, the present invention relates to an anti-Sema 3A antibody or antigen-binding fragment thereof for use in the treatment or prevention of a disease selected from the group consisting of: retinopathy, ischemic retinopathy, diabetic retinopathy (including proliferative and nonproliferative diabetic retinopathy), diabetic macular edema, diabetic macular ischemia, age-related macular degeneration, retinitis pigmentosa, hereditary trophism atrophy, myopic degeneration, retinal artery occlusion, endophthalmitis, uveitis, cystoid macular edema, choroidal neovascular membranes secondary to any retinal disease, optic nerve disease, glaucoma, retinal detachment, toxic retinopathy, radiation retinopathy, traumatic retinopathy, drug-induced retinal vasculopathy, retinal neovascularization, polypoidal choroidal vasculopathy, retinal vasculitis, retinal microaneurysms, Foss trophism, macular telangiectasia, non-proliferative diabetic retinopathy, diabetic macular degeneration, age-related retinopathy, retinitis pigmentosa, hereditary trophism, retinal arterial atrophy, cystic fibrosis, retinal arterial obstruction, endophthalmitis, uveitis, retinal neovasculitis, retinal vasculitis, retinal microaneurysms, retinal neovascularisation, macular telangiectasia, and retinal vascular diseases, Ews' syndrome and stargardt disease.

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

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

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

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

In a preferred aspect, the antibodies of the invention are useful for treating Diabetic Macular Edema (DME) and/or Diabetic Macular Ischemia (DMI). In a preferred embodiment, the antibodies of the invention are useful for treating patients suffering from DME and DMI. Preferably, the antibodies of the invention are used to treat DMI defined by greater than 15%, 20%, 25% and more preferably 30% enlargement of the Foveal Avascular Zone (FAZ).

In a fifth aspect, the invention provides a pharmaceutical composition comprising an anti-Sema 3A antibody or antigen-binding fragment thereof and a pharmaceutically acceptable carrier.

The anti-Sema 3A antibody or antigen-binding fragment thereof is administered by any suitable means, including intravitreal, oral, parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Furthermore, the anti-Sema 3A antibody is suitably administered by pulse infusion, in particular with a reduced dose of antibody. In one aspect, the dose is administered by injection, most preferably intravenous or subcutaneous injection, depending in part on the length of time of administration. Preferably, the anti-Sema 3A antibody is administered via intravitreal injection into the eye.

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

In preferred embodiments, the applicable dose range for each injection of the antibody of the invention is typically 1 mg/eye to 10 mg/eye, preferably between 1.5 mg/eye and 5 mg/eye, more preferably between 2 mg/eye and 3 mg/eye, and even more preferably about 2.5 mg/eye.

The term "inhibit" is used herein in the same context as "ameliorate" and "reduce" to mean reduce or reduce one or more features of a disease.

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

The antibody need not be, but is optionally, formulated with one or more agents currently used for the prevention or treatment of the disorder in question. The effective amount of these other agents will depend on the amount of anti-Sema 3A antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. Such other agents are generally used at the same dosages and routes of administration as used above or about 1% to 99% of the dosages used above.

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

Method of treatment

In another aspect, the invention also encompasses any method of treating or preventing an ocular or ocular disease in a patient in need thereof, comprising administering an anti-Sema 3A antibody of the invention.

Preferably, the invention relates to methods of inhibiting the angiostatic effect of SemaA3 using the antibodies of the invention. More preferably, the present invention relates to the method for improving retinal revascularization.

Preferably, the present invention relates to a method for treating or preventing an ocular or retinal disease comprising administering to a patient in need thereof a pharmaceutically effective amount of an antibody of the present invention. Preferably, the disease is selected from the group consisting of: retinopathy, ischemic retinopathy, diabetic retinopathy (including proliferative and nonproliferative diabetic retinopathy), diabetic macular edema, diabetic macular ischemia, age-related macular degeneration, retinitis pigmentosa, hereditary trophism atrophy, myopic degeneration, retinal artery occlusion, endophthalmitis, uveitis, cystoid macular edema, choroidal neovascular membranes secondary to any retinal disease, optic nerve disease, glaucoma, retinal detachment, toxic retinopathy, radiation retinopathy, traumatic retinopathy, drug-induced retinal vasculopathy, retinal neovascularization, polypoidal choroidal vasculopathy, retinal vasculitis, retinal microaneurysms, Foss trophism, macular telangiectasia, non-proliferative diabetic retinopathy, diabetic macular degeneration, age-related retinopathy, retinitis pigmentosa, hereditary trophism, retinal arterial atrophy, cystic fibrosis, retinal arterial obstruction, endophthalmitis, uveitis, retinal neovasculitis, retinal vasculitis, retinal microaneurysms, retinal neovascularisation, macular telangiectasia, and retinal vascular diseases, Ews' syndrome and stargardt disease. More preferably, the disease is selected from the group consisting of: diabetic retinopathy (including proliferative and nonproliferative diabetic retinopathy), ischemic retinopathy, diabetic macular edema, diabetic macular ischemia, age-related macular edema, retinal neovascularization, glaucoma, and choroidal neovascularization. In a further preferred embodiment, the disease is diabetic macular edema and/or diabetic macular ischemia.

All of the disclosed features described herein are applicable to this treatment.

Pharmaceutical compositions and administration thereof

A composition comprising an anti-Sema 3A antibody or antigen-binding fragment thereof can be administered to an individual having or at risk of having an eye or retinal disease. The invention further provides the use of an anti-Sema 3A antibody or antigen-binding fragment thereof for the manufacture of a medicament for the prevention or treatment of an ocular or retinal disease or a Sema3A disease. All of the disclosed features described herein are applicable to this use of an anti-Sema 3A antibody or antigen-binding fragment thereof in the manufacture of a medicament. The term "subject" as used herein means any mammalian patient to which an anti-Sema 3A antibody or antigen-binding fragment thereof can be administered, including, for example, humans and non-human mammals, such as primates, rodents, and dogs. Individuals specifically intended for treatment using the methods described herein include humans. The anti-Sema 3A antibody or antigen-binding fragment thereof can be administered alone or in combination with other compositions.

Various delivery systems are known and may be used to administer the anti-Sema 3A antibody or antigen-binding fragment thereof. Methods of introduction include, but are not limited to, intravitreal, eyedrop, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The anti-Sema 3A antibody or antigen-binding fragment thereof can be administered, for example, by infusion, bolus injection, or injection, and can be administered with other biologically active agents. Administration can be systemic or local. In a preferred embodiment, administration is by intravitreal injection. Formulations for such injections may be prepared, for example, in pre-filled syringes.

The anti-Sema 3A antibody or antigen-binding fragment thereof can be administered as a pharmaceutical composition comprising a therapeutically effective amount of an anti-Sema 3A antibody or antigen-binding fragment thereof and one or more pharmaceutically compatible ingredients.

In typical embodiments, the pharmaceutical compositions are formulated according to conventional procedures to be suitable for intravenous or subcutaneous administration to humans. Typically, compositions for administration by injection are solutions in sterile isotonic aqueous buffer. If desired, the pharmaceutical composition may also include a solubilizing agent and a local anesthetic (e.g., lidocaine) to reduce pain at the site of injection. Typically, the ingredients are supplied separately or mixed together in unit dosage form, e.g., as a dry lyophilized powder or water-free concentrate in an air-tight sealed container (e.g., ampoule or sachet) that indicates the amount of active agent. Where the pharmaceutical composition is to be administered by infusion, it may be dispensed in an infusion bottle containing sterile pharmaceutical grade water or saline. Where the pharmaceutical composition is administered by injection, an ampoule of sterile water or saline for injection may be provided to mix the ingredients prior to administration.

In addition, the pharmaceutical composition may be provided in the form of a pharmaceutical kit comprising (a) a container containing the anti-Sema 3A antibody or antigen-binding fragment thereof in lyophilized form; and (b) a second container containing a pharmaceutically acceptable diluent for injection (e.g., sterile water). A pharmaceutically acceptable diluent may be used to reconstitute or dilute the lyophilized anti-Sema 3A antibody or antigen-binding fragment thereof. Optionally, the containers may carry a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale.

The amount of anti-Sema 3A antibody or antigen-binding fragment thereof effective to treat or prevent an ocular or retinal disease can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration and the stage of the condition, and should be determined at the discretion of the attendant physician and in the light of the circumstances of each patient. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

For example, the toxicity and therapeutic efficacy of an anti-Sema 3A antibody or antigen-binding fragment thereof can be determined by standard pharmaceutical procedures in cell cultures or experimental animals for determining ED₅₀(a therapeutically effective dose in 50% of the population). An anti-Sema 3A antibody or antigen-binding fragment thereof that exhibits a large therapeutic index is preferred.

The data obtained from such cell culture assays and animal studies can be used to formulate a range of doses for use in humans. The dose of anti-Sema 3A antibody or antigen-binding fragment thereof is generally within the circulating concentration range, which includes ED₅₀With little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration employed. For any anti-Sema 3A antibody or antigen-binding fragment thereof used in the method, the therapeutically effective dose can be estimated initially from cell culture analysis. The dosage can be formulated in animal models to achieve a circulating plasma concentration range (including IC)₅₀I.e., the concentration of test compound that achieves half-maximal inhibition of symptoms) as determined in cell culture. This information can be used to more accurately determine useful doses in humans. The amount in plasma can be measured, for example, by high performance liquid chromatography, ELISA, and the like.

For intravitreal injection of anti-Sema 3A antibody, generally a longer interval between treatments is preferred. The anti-Sema 3A antibodies of the invention can be administered at longer intervals due to their improved binding affinity and efficacy.

In one embodiment, the anti-Sema 3A antibody is administered every 6 weeks, preferably every 7 weeks, preferably every 8 weeks, preferably every 9 weeks, preferably every 10 weeks, preferably every 11 weeks, and more preferably every 12 weeks. In yet another preferred embodiment, the anti-Sema 3A antibody of the present invention is administered every 3 months.

Because the volume that can be administered to the eye is severely limited, it is of paramount importance that the anti-Sema 3A antibody can be formulated in high concentrations. Moreover, the efficacy of the anti-Sema 3A antibody is of paramount importance, since potent antibodies can exert their effects at even lower doses and thereby prolong activity and also the interval between treatments.

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

A typical dose that can be administered to a patient is about 2.5 mg/eye. Typical buffer components that can be used in the formulation include, for example, sodium acetate, PS20, and trehalose dihydrate.

In one embodiment, the anti-Sema 3A antibody is formulated with 10mM histidine buffer, 240mM sucrose, 0.02 w/v% polysorbate 20 at pH 5.5, with a final protein concentration of 60 mg/mL.

In some embodiments, the pharmaceutical composition comprising the anti-Sema 3A antibody or antigen-binding fragment thereof may further comprise a therapeutic agent conjugated or unconjugated to a binding agent.

With respect to the treatment regimen for the combined administration, in particular embodiments, the anti-Sema 3A antibody or antigen-binding fragment thereof is administered concurrently with the therapeutic agent. In another specific embodiment, the therapeutic agent is administered at least one hour and up to several months, e.g., at least 1 hour, 5 hours, 12 hours, one day, one week, one month, or three months, before or after the anti-Sema 3A antibody or antigen-binding fragment thereof is administered.

Polynucleotides, vectors, host cells and recombinant methods

In a sixth aspect, the invention encompasses isolated polynucleotides comprising sequences encoding anti-Sema 3A antibodies, vectors and host cells comprising the polynucleotides, and recombinant techniques for producing antibodies. The isolated polynucleotide may encode any desired form of anti-Sema 3A antibody, including, for example, full-length monoclonal antibodies, Fab ', F (ab')₂And Fv fragment, diabody, linear antibody, single-chain antibodyMolecules and multispecific antibodies formed from antibody fragments.

A polynucleotide comprising a sequence encoding an anti-Sema 3A antibody or fragment or chain thereof may be fused to one or more regulatory or control sequences, as is known in the art, and may be contained in a suitable expression vector or host cell, as is known in the art. Each of the polynucleotide molecules encoding the heavy or light chain variable domains can be independently fused to a polynucleotide sequence encoding a constant domain, e.g., a human constant domain, such that a complete antibody can be produced. Alternatively, the polynucleotides or portions thereof may be fused together to provide a template for the production of single chain antibodies.

For recombinant production, the polynucleotides encoding the antibodies are inserted into replicable vectors for cloning (amplification of the DNA) or expression. There are many suitable vectors available for the expression of recombinant antibodies. The carrier component typically includes (but is not limited to) one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer component, a promoter, and a transcription termination sequence.

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

Expression and cloning vectors contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells. Typically, in cloning vectors, this sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and includes an origin of replication or an autonomously replicating sequence. Such sequences are well known for a variety of bacteria, yeasts and viruses. The origin of replication of the plastid pBR322 is applicable to most Gram-negative (Gram-negative) bacteria, 2-upsilon. the plastid origin is applicable to yeast, and various viral origins (SV40, polyoma, adenovirus, VSV, and BPV) are useful as cloning vectors in mammalian cells. Typically, mammalian expression vectors do not require an origin of replication component (typically only the SV40 origin may be used, as it contains an early promoter).

Expression and cloning vectors may contain identified genes encoding selectable markers to facilitate expression. Typical selectable marker genes encode proteins that confer resistance to antibiotics or other toxins, such as ampicillin (ampicilin), neomycin (neomycin), methotrexate (methotrexate) or tetracycline, or alternatively, are complement auxotrophic deficient proteins, or in other alternatives supply specific nutrients not present in complex media, such as the gene encoding the D-alanine racemase for bacillus (bacillus).

One example of a selection scheme utilizes a drug to prevent growth of the host cell. Those cells that are successfully transformed with a heterologous gene produce a protein conferring drug resistance and therefore survive the selection regimen. The dominant selection example uses the drugs neomycin, mycophenolic acid and hygromycin. Common selectable markers for mammalian cells are those capable of identifying cells competent to take up nucleic acids encoding humanized anti-Sema 3A antibodies, such as DHFR (dihydrofolate reductase), thymidine kinase, metallothionein-I and-II (e.g., primate metallothionein genes), adenosine deaminase, ornithine decarboxylase, and the like. Cells transformed with the DHFR selection gene were first identified by culturing all the transformants in medium containing the amine methotrexate (Mtx), a competitive antagonist of DHFR. When wild-type DHFR is used, a suitable host cell line is a Chinese Hamster Ovary (CHO) cell line deficient in DHFR activity (e.g., DG 44).

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

When recombinant production is carried out in a yeast cell as a host cell, the TRP1 gene present in yeast plastid YRp7 (Stinchcomb et al, 1979, Nature 282:39) can be used as a selectable marker. The TRP1 gene provides a selection marker for yeast mutants lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1(Jones,1977, Genetics 85: 12). The presence of a trp1 lesion in the yeast host cell genome provides an effective environment for detecting transitions by growth in the absence of tryptophan. Similarly, Leu2 p-deficient yeast strains (e.g., ATCC 20,622 and 38,626) were supplemented with known plastids having the Leu2 gene.

In addition, vectors derived from the 1.6 μm circular plasmid pKD1 can be used for the transformation of yeast of the genus Kluyveromyces. Alternatively, expression systems for large-scale production of recombinant calf chymosin from Kluyveromyces lactis have been reported (Van den Berg,1990, Bio/Technology 8: 135). Stable multi-copy expression vectors for secretion of mature recombinant human serum albumin by industrial strains of the genus Kluyveromyces have also been disclosed (Fleer et al, 1991, Bio/Technology 9: 968-.

Expression and cloning vectors typically contain a promoter that is recognized by the host organism and operably linked to a nucleic acid molecule encoding an anti-Sema 3A antibody or polypeptide chain thereof. Promoters suitable for use in prokaryotic hosts include the phoA promoter, the beta-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 promoter used in the bacterial system also contained the Shine-Dalgamo (s.d.) sequence operably linked to DNA encoding the humanized anti-Sema 3A antibody.

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

Examples of suitable promoter sequences for use with yeast hosts include promoters for 3-phosphoglycerate kinase or other glycolytic enzymes (e.g., enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase).

Inducible promoters have the additional advantage that transcription is controlled by growth conditions. These promoters include the yeast promoter regions of alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, nitrogen metabolism-related derivatizing enzymes, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for yeast expression are further described in EP73,657. Yeast enhancers are also advantageously used with yeast promoters.

Transcription of the anti-Sema 3A antibody from the vector in mammalian host cells is under the control of the following promoters: for example, promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (e.g., adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus and Simian Virus 40(SV 40); from heterologous mammalian promoters, such as the actin promoter or immunoglobulin promoter, or from heat shock promoters, provided that the promoters are compatible with the host cell system.

The early and late promoter side of the SV40 virus is conveniently obtained as the SV40 restriction fragment, which also contains the SV40 viral origin of replication. The immediate early promoter of human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment. A system for expressing DNA in a mammalian host using bovine papilloma virus as a vector is disclosed in U.S. patent No. 4,419,446. Modifications of this system are described in U.S. Pat. No. 4,601,978. See also Reyes et al, 1982, Nature297: 598-. Alternatively, the Rous sarcoma virus (rous sarcoma) long terminal repeat can be used as the promoter.

Another useful component that can be used in recombinant expression vectors is an enhancer sequence, which is used to increase transcription of DNA encoding anti-Sema 3A antibodies by higher eukaryotes. Many enhancer sequences are now known from mammalian genes such as globin, elastase, albumin, alpha-fetoprotein, and insulin. However, typically an enhancer from a eukaryotic cell virus is used. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the viral replication origin, and adenovirus enhancers. See also Yaniv,1982, Nature297: 17-18 for a description of the enhancer modules used to activate eukaryotic promoters. The enhancer may be spliced into the vector at the 5' or 3' position of the anti-Sema 3A antibody coding sequence, but is preferably located at the 5' site of the promoter.

Expression vectors for use in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms) may also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are typically obtained from 5 'and occasionally 3' untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the anti-Sema 3A antibody. One useful transcription termination component is the bovine growth hormone polyadenylation region. See WO94/11026 and expression vectors disclosed therein. In some embodiments, the anti-Sema 3A antibody may be expressed using the CHEF system. (see, e.g., U.S. patent 5,888,809; the disclosure of which is incorporated herein by reference).

Suitable host cell lines for cloning or expressing the DNA in the vectors herein are the above mentioned prokaryotic, yeast or higher eukaryotic cells. Suitable prokaryotes for this purpose include eubacteria, such as gram-negative or gram-positive organisms, for example Enterobacteriaceae (Enterobacteriaceae), for example Escherichia (Escherichia), such as Escherichia coli (e.coli), Enterobacter (Enterobacter), Erwinia (Erwinia), Klebsiella (Klebsiella), Proteus (Proteus), Salmonella (Salmonella) (e.g. Salmonella typhimurium), Serratia (Serratia), such as Serratia marcescens (Shigella), and bacillus (bacillus) such as bacillus subtilis (b.subtilis) and bacillus (b.liciniformis) (e.g. Pseudomonas licheniformis (Pseudomonas sp) such as Pseudomonas licheniformis (Pseudomonas sp) disclosed in 4. 266,710 published in 1989), Pseudomonas (Pseudomonas sp.e.g. Pseudomonas aeruginosa). A preferred host for Escherichia coli cloning is Escherichia coli 294(ATCC 31,446), but other strains (e.g., Escherichia coli B, Escherichia coli X1776(ATCC 31,537), and Escherichia coli W3110(ATCC 27,325)) are also suitable. The examples are illustrative and not restrictive.

In addition to prokaryotes, eukaryotic microorganisms (e.g., filamentous fungi or yeast) are suitable hosts for cloning or expression of vectors encoding anti-Sema 3A antibodies. Saccharomyces cerevisiae (Saccharomyces cerevisiae) or common baker's yeast are most commonly used among lower eukaryotic host microorganisms. However, many other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe (Schizosaccharomyces pombe); kluyveromyces hosts such as kluyveromyces lactis, kluyveromyces fragilis (k.fragilis) (ATCC 12,424), kluyveromyces bulgaricus (k.bulgaricus) (ATCC 16,045), kluyveromyces wilkerii (k.wickramii) (ATCC 24,178), kluyveromyces volvatus (k.walliti) (ATCC 56,500), kluyveromyces drosophilus (k.drosophilarium) (ATCC 36,906), kluyveromyces thermotolerans (k.thermotolerans), and kluyveromyces marxianus (k.xmaranus); yarrowia (EP 402,226); pichia yeast (Pichia pastoris) (EP 183,070); candida (Candida); trichoderma reesei (Trichoderma reesei) (EP 244,234); red bread mold (Neurospora crassa); schwanniomyces (Schwanniomyces), such as Schwanniomyces occidentalis (Schwanniomyces occidentalis); and filamentous fungi, such as Rhodomyces (Neurospora), Penicillium (Penicillium), Tolypocladium (Tolypocladium), and Aspergillus (Aspergillus) hosts, such as Aspergillus nidulans (A.nidulans) and Aspergillus niger (A.niger).

Suitable host cells for expression of the glycosylated anti-Sema 3A antibody are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells, including, for example, various baculovirus strains and variants from hosts such as Spodoptera frugiperda (Lepidoptera larvae), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori (silkworm), as well as corresponding permissive insect host cells. Various viral strains for transfection are publicly available, such as the L-1 variant of Autographa californica (Autographa californica) NPV and the Bm-5 strain of Bombyx mori NPV, and these viruses are particularly useful for transfection of Spodoptera frugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco may also be used as hosts.

The anti-Sema 3A antibodies of the present invention may also be incorporated into a viral vector, i.e., a polynucleotide encoding the anti-Sema 3A antibody or antigen-binding fragment thereof is introduced into a viral vector and then expressed in a patient following infection with the virus.

In another aspect, expression of the anti-Sema 3A antibody is effected in a vertebrate cell. Propagation of vertebrate cells in culture (tissue culture) has become a widely available routine procedure and technique. Examples of useful mammalian host cell lines are the SV40 transformed monkey kidney CV1 (COS-7, ATCC CRL1651), the human embryonic kidney cell line (293 or 293 cells that have been sub-selected 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/-DHFR 1(CHO, Urlaub et al, 1980, Proc.Natl.Acad.Sci.USA 77: 4216; e.g.DG 44), mouse Sauteri cells (sertoli cell) (TM4, Mather,1980, biol.Reprod.23: 243-), monkey kidney cells (ATCC CCL 70), African green monkey kidney cells (Sertoli-76, ATCC CRL-1587), human cervical carcinoma cells (HELA, ATCC 2), canine kidney cells (ATCC 1443 ), bovine liver cells (CCL 38975), bovine liver cells (CCL) and mouse kidney cells (ATCC 38775), HB 8065), mouse mammary tumors (MMT 060562, ATCC CCL51), TR1 cells (Mather et al, 1982, Annals N.Y.Acad.Sci.383:44-68), MRC 5 cells, FS4 cells, and human hepatoma cell lines (Hep G2).

The host cells are transformed with the above-described expression or cloning vectors for anti-Sema 3A antibody production and cultured in conventional nutrient media modified, if appropriate, for inducing promoters, selecting transformants or amplifying genes encoding the desired sequences.

The host cells used to produce the anti-Sema 3A antibodies described herein can be cultured in a variety of media. Any one of a commercially available Medium (e.g., one or more of the media described in Ham's F10(Sigma-Aldrich Co., st. louis, Mo.), minimal essential Medium ((MEM), (Sigma-Aldrich Co.), RPMI-1640(Sigma-Aldrich Co.), and Dubek's Modified Eagle's Medium (DMEM), Sigma-Aldrich Co.)) is suitable for culturing host cells, hi addition, 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, WO 90/103430, and WO 87/00195 may be used as a Medium for culturing host cells, if any one or more of such media is supplemented with insulin (e.g., growth factors and/or other growth factors (e.g., insulin supplementation of insulin) Transferrin or epidermal growth factor), salts (e.g. sodium chloride, calcium, magnesium and phosphate), buffers (e.g. HEPES), nucleotides (e.g. adenosine and thymidine), antibiotics (e.g. gentamicin), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Other supplements may also be included at appropriate concentrations known to those skilled in the art. Culture conditions (e.g., temperature, pH, and the like) are those previously used to select for host cells for expression, and will be apparent to one of ordinary skill in the art.

When using recombinant techniques, the antibody may be produced intracellularly, in the periplasmic space, or secreted directly into the culture medium. If the antibody is produced intracellularly, the cell can be disrupted to release the protein as a first step. Particulate debris, i.e., host cells or lysed fragments, may be removed, for example, by centrifugation or ultrafiltration. Carter et al, 1992, Bio/Technology10:163-167 describe methods for isolating antibodies secreted into the periplasmic space of E.coli. Briefly, the cell paste was thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) within about 30 minutes. Cell debris can be removed by centrifugation. In the case of secretion of antibodies into the culture medium, the supernatant from such expression systems is typically first concentrated using commercially available protein concentration filters (e.g., Amicon or Millipore Pellicon ultrafiltration units). A protease inhibitor such as PMSF may be included in any of the preceding steps to inhibit proteolysis, and an antibiotic may be included to prevent the growth of adventitious contaminants. Antibodies can be isolated from host cells using a variety of methods.

Antibody compositions prepared from cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being a typical purification technique. The suitability of protein a as an affinity ligand depends on the type and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human gamma 1, gamma 2, or gamma 4 heavy chains (see, e.g., Lindmark et al, 1983J. Immunol. meth.62: 1-13). Protein G has been suggested for use in all mouse isotypes and human gamma 3 (see, e.g., Guss et al, 1986EMBO J.5: 1567-1575). The matrix to which the affinity ligand is attached is most often agarose, but other matrices may be used. Mechanically stable matrices, such as controlled pore glass or poly (styrene divinyl) benzene, allow faster flow rates and shorter processing times than can be achieved using agarose. In the case of antibodies comprising C_H3In the case of a Domain, Bakerbond ABX^TMResins (j.t.baker, phillips burg, n.j.) can be used for purification. Depending on the antibody to be recovered, other protein purification techniques may also be used, such as ion-exchange column fractionation, ethanol precipitation, reverse phase HPLC, chromatography on silica gel, heparin Sepharose^TMChromatography, chromatography on anion or cation exchange resins (e.g., polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation.

After any preliminary purification steps, the mixture comprising the antibody of interest and contaminants can be subjected to low pH hydrophobic interaction chromatography using a elution buffer having a pH between about 2.5-4.5, typically performed at low salt concentrations (e.g., about 0-0.25M salt).

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

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

It will be appreciated that in such anti-Sema 3A antibodies and antibody fragments, the nucleic acid sequences encoding the CDRs remain unchanged (in terms of the amino acids they encode, equivalent versions of the DNA sequences are possible due to codon degeneracy), but surrounding regions (e.g., the FR regions) may be engineered.

Article of manufacture

In another aspect, articles of manufacture comprising materials useful for treating the above-described conditions are included. The article comprises a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The container may be formed from a variety of materials, such as glass or plastic. The container holds a composition effective to treat a condition and may have a sterile infusion port. For example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle. The active agent in the composition is an anti-Sema 3A antibody or antigen-binding fragment thereof. A label on or associated with the container indicates that the composition is used to treat the selected condition. The article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as phosphate buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

The invention is further illustrated in the following examples, which are not intended to limit the scope of the invention.

Examples

Example 1: upregulation of Sema3A in the vitreous of DME and PDR patients

Expression of Sema3A in retinas of samples from human donors with a history of diabetic retinopathy was investigated by immunohistochemistry. The immunostaining protocol was as follows:

1. thaw the slide and allow the sample to air dry at Room Temperature (RT) for 30 min;

2. drawing with a pap pen box and drying;

3. antigen was repaired in 1% SDS for 5min at RT;

4. the slides were washed 3 times in PBS for 5 min;

5. sections were blocked for 30min at RT in 1% BSA/0.3% Triton X100/PBS solution (blocking solution);

6. rabbit anti-Sema 3a (abcam, ab23393) No. 1 antibody was diluted 1:200 in blocking solution. Sections on slides were incubated overnight at RT;

7. wash slides in PBS 3 times for 5 min;

8. antibody 2 was incubated with donkey anti-rabbit Alexa fluor546(invitrogen, A10040) diluted 1:400 in DAPI/0.3% Triton X100/PBS. The sections on the slides were incubated at RT for 3 hours;

9. washing the slide in PBS for 3-5 times for 5 min;

10. coverslipping the sections with Aquamount and air drying;

11. image slice and intensity level at 40x magnification.

Groups of three sections from each donor were immunostained with Sema 3A. Sema3A labeling was evaluated independently in each of the zones using a 5-point ranking scheme (0 ═ no detection, 1 ═ low intensity, few spots, 2 ═ medium intensity, several spots, 3 ═ bright intensity, extensive staining, and 4 ═ very bright intensity, rich detection) by observers previously trained for this particular task. The observer is unaware of the health of the eye donor. Within the retina, Sema3A is associated with the vascular system walls of the retinal vessels. Expression of Sema3A in retinal vasculature and retinal parenchyma was increased in patients with diabetic macular edema compared to diabetic patients without ocular pathology (fig. 1).

Example 2: potency in cell Permeability assays

Trans-cellular permeability was measured by penetration of FITC-polydextrose into monolayers of Human Retinal Microvascular Endothelial Cells (HRMECs).

Briefly, in vitro endothelial permeability was measured using the Millipore kit ("ex vivo vascular permeability assay" catalog No. ECM642) and Human Retinal Microvascular Endothelial Cells (HRMECs). The assay kit provides a 96-well receiver plate with cell culture inserts. The insert contained 1 μm pores and was coated with type I rat tail collagen. HRMECs were seeded into inserts at a density of 25000 cells/well and cells were grown as monolayers for 3 days. Cells were treated overnight with recombinant VEGF-A, Sema3A and antibody. The high molecular weight FITC-polydextrose solution provided in the kit was added to the insert so that the fluorescent molecules passed through the endothelial cell monolayer. In vitro permeability was determined by measuring fluorescence of the receptor well solutions at 485nM/535nM (excitation and absorption).

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

The anti-Sema 3A antibody of the invention completely inhibited the permeability induced by Sema3A, but not by VEGF-a (fig. 2). Importantly, the permeability of Sema3A is independent of VEGF-A. This effect was demonstrated using a control antibody against TNP due to the specific target of the antibody of the invention for Sema 3A.

Example 3: measurement of cytoskeletal collapse in cellular assays

Cellular activity of the exemplary antibodies of the invention (pure line I) was evaluated by measuring cytoskeletal collapse in Human Retinal Microvascular Endothelial Cells (HRMECs) using the XCELLigence system (e.g., Real Time Cell Analysis Instruments sold by ACEA Biosciences). The system measures cell attachment and confluency via cell impedance. HRMEC endogenously expresses cyclin-1 (Nrp1) and plexin, which are components of the full pan 3-class of semaphorins. By binding to this receptor complex, semaphorins induce the collapse of F-actin fibers in the endothelium. In this functional assay, the addition of recombinant Sema3A protein to confluent layers of human retinal microvascular endothelial cells reduced cellular impedance as measured by the reduction in cellular impedance due to cytoskeletal collapse and subsequent cell contraction.

Briefly, E-Plates View was coated with Attachment Factor. Cells were seeded at a density of 20000 cells/well and then grown as a monolayer in an xcelligene device overnight under their normal growth conditions.

Combinations of Sema3A (or other class 3 semaphores) with and without the anti-Sema 3A antibody of the present invention were added in the presence of 3mM CaCl 2. The cell index was normalized to the time point before addition of the substance. And calculating after stimulating for 5 h.

The anti-Sema 3A antibodies of the invention completely prevented Sema 3A-induced cytoskeletal collapse. The anti-Sema 3A antibody of the invention did not prevent cytoskeletal collapse induced by other test semaphorins (B, C, E and F), confirming the specificity of the antibody of the invention for Sema3A (fig. 3).

Example 4: affinity and cell efficacy

A) Affinity of the amino acid sequence

The running buffer and all dilutions (except those described) used for this experiment were performed in PBS-T-EDTA with 0.01% Tween20 [ 100. mu.L of 100% Tween20 was added to 2LPBS-T-EDTA to give a final Tween20 concentration of 0.01% ]. The GLM sensor chip was normalized and preprocessed according to the manufacturer's recommendations. The sensor chip was activated with an equal mixture of EDC/s-NHS for 300sec in the horizontal direction at a flow rate of 30 μ l/min and immobilized with human Fab binder (10 μ g/ml in 10mM acetate, pH 5.0) for 300sec in the horizontal direction at a flow rate of 30 μ l/min, resulting in about 6739-7414RU of human Fab binder on the surface. The sensor chip was deactivated with 1M ethanolamine HCl in the horizontal direction at a flow rate of 30. mu.l/min for 300 sec. The sensor chip was stabilized horizontally 1 time and vertically 1 time with 18sec of 10mM glycine (pH 2.1) at a flow rate of 100. mu.l/min.

The inventors tested an exemplary antibody according to the invention (pure line I). The antibody (0.5 μ g/ml) was captured vertically on the human Fab adhesive surface for 300sec at a flow rate of 25 μ L/min, resulting in a capture content of about 180 RU. Baseline was stabilized by injecting PBS-T-EDTA at a flow rate level of 40. mu.l/min for 60 sec. The analyte was injected at a flow rate level of 40 μ L/min onto the captured antibody for 600sec and disassociated for 7200 sec. The analyte concentrations were 0nM, 0.625nM, 1.25nM, 2.5nM, 5nM and 10 nM. The surface was regenerated by injecting 10mM glycine (pH 2.1) at a flow rate of 100. mu.l/min once horizontally and once vertically for 18 seconds. PBS-T-EDTA was injected perpendicularly at a flow rate of 25. mu.l/min for 60 sec.

The intermediate point (interspot) (interaction with the sensor surface) and blank (PBS-T-EDTA with 0.01% Tween20 or 0nM analyte) were subtracted from the raw data. The sensorgram was then fit to 1:1 Langmuir binding in its entirety to provide the association rate (ka), dissociation rate (KD) and affinity(K_D) The value is obtained.

B) Cell efficacy

To determine functional efficacy in cytoskeletal collapse assays, combinations of antibodies at increasing concentrations of the Sema3A concentration response curve were used as ICs₅₀And (4) switching experiments. Gaddum Schild plots were performed to calculate pA2 values (negative log of antibody concentration required to shift the Sema3A concentration response curve by a factor of 2). Efficacy (expressed as pM) was calculated from pA2 values as efficacy (10; -X).

The results are summarized in table 6 below.

Table 6:

example 5: evaluation of immunogenicity of antibody of the present invention

The inventors evaluated the predicted immunogenicity of the exemplary antibody clones I according to the invention. The antibody comprises heavy and light chains comprising the amino acid sequences of SEQ ID NO 14 and SEQ ID NO 15, respectively.

For this purpose, it uses a computer tool for predicting T cell epitopes (EpiMatrix developed by EpiVax).

By screening the sequences of many human antibody isolates, EpiVax has identified several highly conserved HLA ligands thought to have regulatory potential. Experimental evidence suggests that many of these peptides are actually actively tolerogenic in most individuals. These highly conserved, regulatory and promiscuous T cell epitopes are now known as Tregitopes (De Groot et al blood.2008Oct 15; 112(8): 3303-11). In the presence of a large number of Tregitopes, the immunogenic potential of the neoepitopes contained in the humanized antibodies can be effectively controlled.

For the purpose of antibody immunogenicity analysis, EpiVax has developed a Tregitope-adjusted EpiMatrix score and corresponding prediction of anti-therapeutic antibody responses. To calculate the Tregitope-adjusted EpiMatrix score, the Tregitope score was subtracted from the EpiMatrix protein score. Tregitope-adjusted scores have been shown to be well correlated with the observed clinical immune responses in the group of 23 commercial antibodies (De Groot et al Clin Immunol.2009, 5 months; 131(2): 189-.

The results on the EpiMatrix scale are summarized in table 7 below.

Table 7:

the sequence of the antibodies of the invention was scored at the low end of the EpiMatrix scale, indicating that the antibodies of the invention have a strongly limited immunogenic potential. This EpiMatrix scale is well known to the person skilled in the art and can be found, inter alia, in the publication Mufarrege et al Clin immunol.2017, 3 months; 176:31-41 in fig. 2.

Example 6: effect on in vivo apical cell density and avascular zone (oxygen-induced retinopathy OIR model)

The effect of the exemplary antibody of the invention (pure line I) on revascularization of ischemic avascular regions was studied in a mouse model of oxygen-induced retinopathy (OIR). From day 7 after birth to day 12 after birth, a litter of C57Bl/6J mice was exposed to an atmosphere of 75% oxygen. This results in the degeneration of blood vessels and the formation of avascular regions in the central retina. After returning to normal oxygenated conditions, this area becomes ischemic. Pups received a single intravitreal injection of 10 μ g of antibody in 0.5 μ l of solution in each eye under anesthesia with isoflurane on postnatal day 12. On postnatal day 17, animals were sacrificed and eyes were enucleated. The eyes were fixed in formalin (formalin) and retinal plates were prepared in which retinal blood vessels were stained with isolectin B4. The number of tip cells (specialized endothelial cells that initiate neovascularization) was counted along the avascular front end of the entire retina (the boundary between the vascularized peripheral region and the avascular central region of the retina).

The apical cells were identified by their specific morphology showing pseudofiliform extensions. For analysis, the number of tip cells was normalized to the length of the avascular front. The size of the avascular zone was determined using confocal microscopy and image analysis software.

The anti-Sema 3A antibody of the invention increased apical cell density in the mouse OIR model (fig. 4). Furthermore, it shows a reduction of the avascular zone. There is a negative correlation between the tip cell density and the size of the avascular zone, indicating a mechanical dependence of the two parameters. The use of a control antibody against TNP confirms that this effect is due to the specific target of the antibody of the invention for Sema 3A.

In summary, the anti-Sema 3A antibody reduced the size of ischemic avascular regions in an animal model of oxygen-induced retinopathy, indicating a beneficial effect on diabetic macular ischemia.

Example 7: anti-Sema 3A and Avastin (Avastin) t in rabbit eye_1/2Comparison of

The results are summarized in table 8 below.

Table 8:

the calculated half-lives in vitreous, retinal and aqueous humor were 3.9, 4.1 and 3.3 days, respectively. These half-lives were similar to those of the clinically used recombinant humanized monoclonal IgG1 antibody avastin (anti-VEGF, bevacizumab, Bakri et al, Opthalmology,2007) reported in the literature, which was also confirmed in internal experiments. The results are expected because the intravitreal clearance of full-length IgG is largely dependent on its molecular size, which is similar to the antibodies and avastin of the present invention. Thus, human PK (including the antibodies of the invention and avastin's ocular half-life) is expected to be similar. Human ocular half-life of avastin is reported to be 9.73 ± 1.48 days (Hutton-Smith, 2016).

Example 8: comparison of binding affinity between antibodies of the invention and Chiome antibodies

For comparison purposes, the present inventors developed a humanized antibody against Sema3A disclosed in WO2014123186 (chip Bioscience) with the following characteristics:

the heavy chain is as shown in SEQ ID NO 11 in WO2014123186, and

the light chain is as shown in SEQ ID No. 12 in WO 2014123186.

The inventors have developed 2 forms of this antibody:

a format on IgG1KO Fc, hereinafter referred to as "Chiome antibody A" and

one formatted on naked IgG1KO-FcRn, hereinafter referred to as "chome antibody B".

High surface density anti-human Fab antibodies (GE Healthcare) were immobilized on GLM chips (BioRad) via direct amine coupling on 6 horizontal channels according to the BioRad manufacturer manual.

Antibodies of the invention (clone I) and chome antibodies were captured on the anti-human Fab antibody surface at minimum surface density on 5 of 6 vertical channels for kinetic binding analysis. Human Sema3A was prepared in PBS-T-EDTA buffer (BioRad) at concentrations of 100, 50, 25, 12.5, 10, 6.25, 5, 2.5, 1.25, 0.625, and 0 nM. PBS-T-EDTA buffer injection was used as a dual reference for kinetic data analysis. Each of the human Sema3A solution and PBS-T-EDTA buffer was injected simultaneously on 6 horizontal channels at a flow rate of 40 μ L/min for 10min, followed by a 2hr dissociation period. The surface was regenerated by injecting 18sec 10mM glycine HCl pH2.1 (GE healthcare) at a flow rate of 100. mu.L/min followed by 60sec PBS-T-EDTA at a flow rate of 25. mu.L/min. Binding sensorgrams were fitted to the 1:1 langmuir model to calculate the on-rate, off-rate and affinity.

The kinetics and affinity data for binding of the antibodies and chome antibodies of the invention to human Sema3A are presented in table 9 below.

Table 9:

sample name	KD to HuSema3A
		Chiome antibody A	56.4nM
Chiome antibody B	55.9nM
		Antibodies of the invention (pure line I)	32.0pM

Conclusion

The results show that the antibodies of the present invention demonstrate superior binding affinity to human Sema3A over the prior art antibodies disclosed by Chiome Bioscience.

Example 9: comparison of binding affinity between the antibodies of the invention and the Samsung scFv

Comparison has been made with scFv fragments as disclosed in WO2017074013 (Samsung).

For comparison purposes, the inventors have developed 3 disclosed fragments having the characteristics disclosed in table 10 below.

Table 10:

high surface density anti-His antibody (GE Healthcare) was immobilized on GLM chips (BioRad) via direct amine coupling on 6 horizontal channels according to the BioRad manufacturer manual. The Samsung ScFv antibody was captured on the anti-His antibody surface at minimum surface density on 5 of the 6 vertical channels for kinetic binding analysis. Human Sema3A was prepared in PBS-T-EDTA buffer (BioRad) at concentrations of 100, 50, 25, 12.5, 10, 6.25, 5, 2.5, 1.25, 0.625, and 0 nM. PBS-T-EDTA buffer injection was used as a dual reference for kinetic data analysis. Each of the human Sema3A solution and PBS-T-EDTA buffer was injected simultaneously on 6 horizontal channels at a flow rate of 40 μ L/min for 10min, followed by a 1hr dissociation period. The surface was regenerated by injecting 18sec 10mM glycine HCl pH2.1 (GE healthcare) at a flow rate of 100. mu.L/min followed by 60sec PBS-T-EDTA at a flow rate of 25. mu.L/min. Binding sensorgrams were fitted to the 1:1 langmuir model to calculate the on-rate, off-rate and affinity.

Binding of the antibodies of the invention to human Sema3A (pure line I) was performed using a similar method, but using goat anti-human igg (invitrogen) to capture the antibodies of the invention. The binding of the antibody of the present invention and the Samsung ScFv to cynomolgus monkey, mouse, rat or rabbit Sema3A was also performed using the same method.

The kinetic and affinity data for the antibodies of the invention and Samsung scFv are listed in table 11 below.

Table 11:

conclusion

The binding affinity of the antibodies of the invention to human, cynomolgus monkey, mouse or rabbit Sema3A is higher than 3 Samsung scfvs as disclosed in WO 2017074013.

Example 10: comparison of the affinity of two antibodies according to the invention

The present inventors have developed two antibodies with CDRs as depicted in SEQ ID NOs 1-6. The difference in Fc region between the two antibodies is:

an antibody comprises a combination of L234A and L235A (antibody A), and

the other antibody comprises the mutation H435A (antibody B),

residues are numbered according to EU index of Kabat.

No statistical difference was shown in the binding affinity of the two antibodies to human Sema3A having the CDRs as depicted in SEQ ID NOs: 1 to 6. Thus, affinity for human Sema3A was maintained in the antibodies of the invention against Sema 3A.

Example 11: efficacy of commercial antibodies against Sema3A

For comparison, the present inventors tested the cellular activity of a commercial antibody targeting Sema 3A. This Antibody is commercialized by Creative Biolabs under the reference "Anti-Human SEMA3A Therapeutic Antibody, Humanized (CAT #: TAB-556 CL)". This commercially available antibody is hereinafter referred to as "Creative Biolabs antibody".

Cellular activity of the Creative Biolabs antibodies was evaluated by measuring cytoskeletal collapse in Human Retinal Microvascular Endothelial Cells (HRMECs) using the same protocol disclosed in example 3. Efficacy was calculated and measured 5h after stimulation.

Under these conditions, the Creative Biolabs antibody did not show any activity on Sema 3A-induced cytoskeletal collapse. The Creative Biolabs antibody did not actually prevent Sema 3A-induced cytoskeletal collapse.

Thus, it was demonstrated that the Creative Biolabs antibody could not prevent Sema 3A-induced cytoskeletal collapse in retinal cells, while under the same conditions, the antibody of the invention could prevent. This confirms the surprising and unexpected effects of the antibodies of the invention.

Sequence listing

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<120> anti-SEMA 3A antibodies and their use for treating ocular or ocular diseases

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<212> PRT

<213> Artificial sequence

<220>

<223> HCDR2

<400> 2

Thr Ile Ile Lys Ser Gly Gly Tyr Ala Tyr Tyr Pro Asp Ser Val Lys

1 5 10 15

Asp

<210> 3

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> HCDR3

<400> 3

Gly Gly Gln Gly Ala Met Asp Tyr

1 5

<210> 4

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> LCDR1

<400> 4

Arg Ala Ser Gln Ser Ile Gly Asp Tyr Leu His

1 5 10

<210> 5

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> LCDR2

<400> 5

Tyr Ala Ser Gln Ser Ile Ser

1 5

<210> 6

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> LCDR3

<400> 6

Gln Gln Gly Tyr Ser Phe Pro Tyr Thr

1 5

<210> 7

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain variable region-variant 1

<400> 7

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Thr Ile Ile Lys Ser Gly Gly Tyr Ala Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Val Arg Gly Gly Gln Gly Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser

115

<210> 8

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain variable region-variant 2

<400> 8

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Phe Ser Ser Tyr

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Thr Ile Ile Lys Ser Gly Gly Tyr Ala Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Val Arg Gly Gly Gln Gly Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser

115

<210> 9

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain variable region-variant 3

<400> 9

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Leu Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Thr Ile Ile Lys Ser Gly Gly Tyr Ala Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Val Lys Gly Gly Gln Gly Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser

115

<210> 10

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain variable region variant 4

<400> 10

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Leu Gln Leu Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Thr Ile Ile Lys Ser Gly Gly Tyr Ala Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Asn

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Val Lys Gly Gly Gln Gly Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser

115

<210> 11

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> light chain variable region-variant a

<400> 11

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Gly Asp Tyr

20 25 30

Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Gly Tyr Ser Phe Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 12

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> light chain variable region-variant b

<400> 12

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Gly Asp Tyr

20 25 30

Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Gly Tyr Ser Phe Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 13

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> light chain variable region-variant c

<400> 13

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Gly Asp Tyr

20 25 30

Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Gly Tyr Ser Phe Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 14

<211> 446

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain-clone I

<400> 14

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Thr Ile Ile Lys Ser Gly Gly Tyr Ala Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Val Arg Gly Gly Gln Gly Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 15

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> light chain-clone I

<400> 15

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Gly Asp Tyr

20 25 30

Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Gly Tyr Ser Phe Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 16

<211> 446

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain-clone II

<400> 16

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Phe Ser Ser Tyr

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Thr Ile Ile Lys Ser Gly Gly Tyr Ala Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Val Arg Gly Gly Gln Gly Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 17

<211> 446

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain-clone III

<400> 17

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Leu Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Thr Ile Ile Lys Ser Gly Gly Tyr Ala Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Val Lys Gly Gly Gln Gly Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 18

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> light chain-clone III

<400> 18

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Gly Asp Tyr

20 25 30

Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Gly Tyr Ser Phe Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 19

<211> 446

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain-clone IV

<400> 19

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Leu Gln Leu Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Thr Ile Ile Lys Ser Gly Gly Tyr Ala Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Asn

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Val Lys Gly Gly Gln Gly Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 20

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> light chain-clone IV

<400> 20

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Gly Asp Tyr

20 25 30

Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Gly Tyr Ser Phe Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 21

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> epitope

<400> 21

Asp Ser Thr Lys Asp Leu Pro Asp Asp Val Ile Thr Phe

1 5 10

<210> 22

<211> 751

<212> PRT

<213> Intelligent people

<400> 22

Asn Tyr Gln Asn Gly Lys Asn Asn Val Pro Arg Leu Lys Leu Ser Tyr

1 5 10 15

Lys Glu Met Leu Glu Ser Asn Asn Val Ile Thr Phe Asn Gly Leu Ala

20 25 30

Asn Ser Ser Ser Tyr His Thr Phe Leu Leu Asp Glu Glu Arg Ser Arg

35 40 45

Leu Tyr Val Gly Ala Lys Asp His Ile Phe Ser Phe Asp Leu Val Asn

50 55 60

Ile Lys Asp Phe Gln Lys Ile Val Trp Pro Val Ser Tyr Thr Arg Arg

65 70 75 80

Asp Glu Cys Lys Trp Ala Gly Lys Asp Ile Leu Lys Glu Cys Ala Asn

85 90 95

Phe Ile Lys Val Leu Lys Ala Tyr Asn Gln Thr His Leu Tyr Ala Cys

100 105 110

Gly Thr Gly Ala Phe His Pro Ile Cys Thr Tyr Ile Glu Ile Gly His

115 120 125

His Pro Glu Asp Asn Ile Phe Lys Leu Glu Asn Ser His Phe Glu Asn

130 135 140

Gly Arg Gly Lys Ser Pro Tyr Asp Pro Lys Leu Leu Thr Ala Ser Leu

145 150 155 160

Leu Ile Asp Gly Glu Leu Tyr Ser Gly Thr Ala Ala Asp Phe Met Gly

165 170 175

Arg Asp Phe Ala Ile Phe Arg Thr Leu Gly His His His Pro Ile Arg

180 185 190

Thr Glu Gln His Asp Ser Arg Trp Leu Asn Asp Pro Lys Phe Ile Ser

195 200 205

Ala His Leu Ile Ser Glu Ser Asp Asn Pro Glu Asp Asp Lys Val Tyr

210 215 220

Phe Phe Phe Arg Glu Asn Ala Ile Asp Gly Glu His Ser Gly Lys Ala

225 230 235 240

Thr His Ala Arg Ile Gly Gln Ile Cys Lys Asn Asp Phe Gly Gly His

245 250 255

Arg Ser Leu Val Asn Lys Trp Thr Thr Phe Leu Lys Ala Arg Leu Ile

260 265 270

Cys Ser Val Pro Gly Pro Asn Gly Ile Asp Thr His Phe Asp Glu Leu

275 280 285

Gln Asp Val Phe Leu Met Asn Phe Lys Asp Pro Lys Asn Pro Val Val

290 295 300

Tyr Gly Val Phe Thr Thr Ser Ser Asn Ile Phe Lys Gly Ser Ala Val

305 310 315 320

Cys Met Tyr Ser Met Ser Asp Val Arg Arg Val Phe Leu Gly Pro Tyr

325 330 335

Ala His Arg Asp Gly Pro Asn Tyr Gln Trp Val Pro Tyr Gln Gly Arg

340 345 350

Val Pro Tyr Pro Arg Pro Gly Thr Cys Pro Ser Lys Thr Phe Gly Gly

355 360 365

Phe Asp Ser Thr Lys Asp Leu Pro Asp Asp Val Ile Thr Phe Ala Arg

370 375 380

Ser His Pro Ala Met Tyr Asn Pro Val Phe Pro Met Asn Asn Arg Pro

385 390 395 400

Ile Val Ile Lys Thr Asp Val Asn Tyr Gln Phe Thr Gln Ile Val Val

405 410 415

Asp Arg Val Asp Ala Glu Asp Gly Gln Tyr Asp Val Met Phe Ile Gly

420 425 430

Thr Asp Val Gly Thr Val Leu Lys Val Val Ser Ile Pro Lys Glu Thr

435 440 445

Trp Tyr Asp Leu Glu Glu Val Leu Leu Glu Glu Met Thr Val Phe Arg

450 455 460

Glu Pro Thr Ala Ile Ser Ala Met Glu Leu Ser Thr Lys Gln Gln Gln

465 470 475 480

Leu Tyr Ile Gly Ser Thr Ala Gly Val Ala Gln Leu Pro Leu His Arg

485 490 495

Cys Asp Ile Tyr Gly Lys Ala Cys Ala Glu Cys Cys Leu Ala Arg Asp

500 505 510

Pro Tyr Cys Ala Trp Asp Gly Ser Ala Cys Ser Arg Tyr Phe Pro Thr

515 520 525

Ala Lys Arg Arg Thr Arg Arg Gln Asp Ile Arg Asn Gly Asp Pro Leu

530 535 540

Thr His Cys Ser Asp Leu His His Asp Asn His His Gly His Ser Pro

545 550 555 560

Glu Glu Arg Ile Ile Tyr Gly Val Glu Asn Ser Ser Thr Phe Leu Glu

565 570 575

Cys Ser Pro Lys Ser Gln Arg Ala Leu Val Tyr Trp Gln Phe Gln Arg

580 585 590

Arg Asn Glu Glu Arg Lys Glu Glu Ile Arg Val Asp Asp His Ile Ile

595 600 605

Arg Thr Asp Gln Gly Leu Leu Leu Arg Ser Leu Gln Gln Lys Asp Ser

610 615 620

Gly Asn Tyr Leu Cys His Ala Val Glu His Gly Phe Ile Gln Thr Leu

625 630 635 640

Leu Lys Val Thr Leu Glu Val Ile Asp Thr Glu His Leu Glu Glu Leu

645 650 655

Leu His Lys Asp Asp Asp Gly Asp Gly Ser Lys Thr Lys Glu Met Ser

660 665 670

Asn Ser Met Thr Pro Ser Gln Lys Val Trp Tyr Arg Asp Phe Met Gln

675 680 685

Leu Ile Asn His Pro Asn Leu Asn Thr Met Asp Glu Phe Cys Glu Gln

690 695 700

Val Trp Lys Arg Asp Arg Lys Gln Arg Arg Gln Arg Pro Gly His Thr

705 710 715 720

Pro Gly Asn Ser Asn Lys Trp Lys His Leu Gln Glu Asn Lys Lys Gly

725 730 735

Arg Asn Arg Arg Thr His Glu Phe Glu Arg Ala Pro Arg Ser Val

740 745 750