Anti-SEMA3A antibodies and uses thereof

Abstract

The present disclosure relates to an isolated antibody or antigen-binding fragment thereof that binds to human Sema3A. The isolated antibody or antigen-binding fragment according to the present disclosure i) binds to human Sema3A of sequence SEQ ID NO: 600 with a dissociation constant (KD) of 50 nM or less, 20 nM or less, 10 nM or less, 1 nM or less, or 0.1 nM or less, ii) cross-reacts with mouse, cynomolgus monkey, rat, pig and / or dog Sema3A, in particular, the isolated antibody or antigen-binding fragment thereof binds to mouse, cynomolgus monkey, rat, pig and / or dog Sema3A with a dissociation constant (KD) of 50 nM or less, 20 nM or less, 10 nM or less, 1 nM or less, or 0.1 nM or less, and iii) binds to human Sema3A of sequence SEQ ID NO: 600 with a binding activity of 60% or more, 70% or more, 80% or more, or 90% or more as measured by surface plasmon resonance (SPR). iv) inhibit the activity of human Sema3A of sequence SEQ ID NO: 600 in an in vitro mesangial cell migration assay with an EC50 of 10 nM or less, 5 nM or less, 2.5 nM or less or 1 nM or less; v) inhibit the activity of human Sema3A of sequence SEQ ID NO: 600 in an in vitro growth cone collapse assay with an EC50 of 50 nM or less, 25 nM or less, 10 nM or less or 5 nM or less; vi) inhibit the activity of human Sema3A of sequence SEQ ID NO: 600 in an in vitro HUVEC repulsion assay with an EC50 of 1 nM or less, or 0.3 nM or less, 0.1 nM or less, 0.07 nM or less, 0.06 nM or less; and / or vii) show enhanced potency against HUVEC repulsion induced by cellular Sema3A of sequence SEQ ID NO: 600.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention provides an isolated antibody or antigen-binding fragment thereof that binds to human semaphorin 3A (Sema3A). The isolated antibody or antigen-binding fragment thereof according to the present invention i) binds to human Sema3A of sequence SEQ ID NO: 600 with a dissociation constant (KD) of 50 nM or less, 20 nM or less, 10 nM or less, 1 nM or less, or 0.1 nM or less, ii) cross-reacts with mouse, cynomolgus monkey, rat, pig and / or dog Sema3A, in particular, the isolated antibody or antigen-binding fragment thereof binds to mouse, cynomolgus monkey, rat, pig and / or dog Sema3A with a dissociation constant (KD) of 50 nM or less, 20 nM or less, 10 nM or less, 1 nM or less, or 0.1 nM or less, iii) binds to human Sema3A of sequence SEQ ID NO: 600 with a binding activity of 60% or more, 70% or more, 80% or more, or 90% or more as measured by surface plasmon resonance (SPR), and iv) cross-reacts with mouse, cynomolgus monkey, rat, pig and / or dog Sema3A. In a vitro mesangial cell migration assay, the sequence of SEQ ID NO: 600 inhibits the activity of human Sema3A with an EC50 of 10 nM or less, 5 nM or less, 2.5 nM or less or 1 nM or less; v) in an in vitro growth cone collapse assay, the sequence of SEQ ID NO: 600 inhibits the activity of human Sema3A with an EC50 of 50 nM or less, 25 nM or less, 10 nM or less or 5 nM or less; and / or vi) in an in vitro HUVEC repulsion assay, the sequence of SEQ ID NO: 600 inhibits the activity of human Sema3A with an EC50 of 1 nM or less, or 0.3 nM or less, 0.1 nM or less, 0.07 nM or less, 0.06 nM or less; and / or vii) shows enhanced potency against HUVEC repulsion induced by cellular Sema3A of the sequence of SEQ ID NO: 600. The invention further provides isolated nucleic acid sequences encoding the antibodies or antigen-binding fragments thereof and vectors containing same, isolated cells expressing the antibodies or antigen-binding fragments thereof, methods of producing the antibodies or antigen-binding fragments thereof, and pharmaceutical compositions and kits comprising the antibodies or antigen-binding fragments thereof.

[0002] The antibodies according to the present invention may be used in the treatment of diseases associated with increased levels or activity of Sema3A, such as Alport syndrome, acute kidney injury (AKI), primary focal segmental glomerulosclerosis (FSGS) or chronic kidney disease (CKD). [Background technology]

[0003] 2. Background of the Invention Semaphorin 3A (Sema3A) is a secreted dimeric protein that functions as a guidance protein. It forms a ternary complex with neuropilin-1 and various plexins, which leads to the activation of various signaling pathways. It is a key regulator of cell migration, adhesion, cytoskeleton stabilization and apoptosis. Sema3A is expressed in podocytes in the adult kidney where it is induced after injury.

[0004] Excessive Sema3A impedes the glomerular filtration barrier and induces ultrastructural changes in the filtration barrier, leading to podocyte foot process effacement and albuminuria. Sema3A is also highly induced after AKI and exacerbates the injury by promoting tubular inflammation, tubular epithelial cell apoptosis, and ultrastructural abnormalities of the filtration barrier. Genetic deletion or pharmacological inhibition of Sema3A in rodents results in reduced renal injury in various animal models of renal disease.

[0005] Furthermore, Sema3A is expressed in retinal neurons and endothelium. It has been shown to increase vascular permeability, promote retinal inflammation and cellular senescence, and inhibit retinal vascular regeneration in rodent models. Sema3A also plays a role in CNS injury. Inhibition of Sema3A promotes the regeneration and / or maintenance of damaged axons, reduces the number of apoptotic cells, and enhances angiogenesis, resulting in significantly better functional recovery.

[0006] WO 20141 / 23186 discloses a chicken-mouse chimeric antibody (derived from clone number 4-2 line) and two humanized IgG1 variants thereof, suggesting their suitability in the treatment of Alzheimer's disease.

[0007] WO 2017 / 074013 discloses anti-Sema3A IgG antibodies A08, C10 and F11 and suggests their suitability in the treatment of cancer (hereinafter referred to as cancer).

[0008] Currently, therapeutic options that inhibit the interaction of Sema3A with its receptor are not available for the treatment of patients with proteinuric kidney diseases, such as Alport syndrome, for which therapeutic Sema3A monoclonal antibodies are believed to be optimal. Thus, there is a significant unmet need for novel therapeutic Sema3A antibodies useful for the treatment of diseases associated with enhanced levels or activity of Sema3A, such as Alport syndrome, acute kidney injury (AKI), primary focal segmental glomerulosclerosis (FSGS) or chronic kidney disease (CKD). Summary of the Invention

[0009] Object of the invention In view of the prior art, it is an object of the present invention to provide a novel therapeutic Sema3A antibody that overcomes the shortcomings of the prior art Sema3A antibody.In particular, it is an object of the present invention to provide a novel Sema3A antibody that is a high affinity binder of human Sema3A and effectively blocks Sema3A activity.Desirable Sema3A antibodies are cross-reactive with Sema3A of multiple species so that preclinical experiments are possible.They are non-immunogenic in human therapy, i.e., they are human or humanized antibodies.Desirable Sema3A antibodies are selective for Sema3A, they do not bind to off-targets, and in particular, they do not cross-react with other semaphorin protein family members.

[0010] Such novel Sema3A antibodies would provide a major advance in the treatment of diseases associated with enhanced levels or activity of Sema3A, such as Alport syndrome, acute kidney injury (AKI), primary focal segmental glomerulosclerosis (FSGS) or chronic kidney disease (CKD).

[0011] Overview of the Invention These and other objects are achieved by the teachings of the present invention, which is based on the discovery of novel antibodies that have specific affinity for Sema3A and can provide therapeutic benefit to a subject.

[0012] Thus, in a first aspect, the present invention relates to an isolated antibody or antigen-binding fragment thereof that binds to human Sema3A, wherein the isolated antibody or antigen-binding fragment thereof i) binds to human Sema3A of sequence SEQ ID NO: 600 with a dissociation constant (KD) of 50 nM or less, 20 nM or less, 10 nM or less, 1 nM or less, or 0.1 nM or less, ii) cross-reacts with mouse, cynomolgus monkey, rat, pig and / or dog Sema3A, in particular, the isolated antibody or antigen-binding fragment thereof binds to mouse, cynomolgus monkey, rat, pig and / or dog Sema3A with a dissociation constant (KD) of 50 nM or less, 20 nM or less, 10 nM or less, 1 nM or less, or 0.1 nM or less, iii) is 60% or more, 70% or more, 80% or more, or 90% or more, as measured by surface plasmon resonance (SPR). 600 with an EC50 of 10 nM or less, 5 nM or less, 2.5 nM or less or 1 nM or less; iv) in an in vitro mesangial cell migration assay, inhibit the activity of human Sema3A of sequence SEQ ID NO: 600 with an EC50 of 10 nM or less, 5 nM or less, 2.5 nM or less or 1 nM or less; v) in an in vitro growth cone collapse assay, inhibit the activity of human Sema3A of sequence SEQ ID NO: 600 with an EC50 of 50 nM or less, 25 nM or less, 10 nM or less or 5 nM or less; vi) in an in vitro HUVEC repulsion assay, inhibit the activity of human Sema3A of sequence SEQ ID NO: 600 with an EC50 of 1 nM or less, or 0.3 nM or less, 0.1 nM or less, 0.07 nM or less, 0.06 nM or less; and / or vii) show enhanced potency against HUVEC repulsion induced by cellular Sema3A of sequence SEQ ID NO: 600.

[0013] The isolated antibody or antigen-binding fragment according to the present invention binds to human Sema3A with high affinity and inhibits its function. Thus, the isolated antibody or antigen-binding fragment according to the present invention can be used in the treatment of diseases associated with enhanced levels or activity of Sema3A, such as the following diseases: i) kidney diseases, in particular acute and chronic kidney diseases, diabetic kidney disease, Alport syndrome, acute and chronic renal failure, polycystic kidney disease (PCKD) and syndrome of insufficient ADH secretion (SIADH); ii) sequelae of kidney failure, in particular pulmonary edema, cardiac failure, uremia, anemia, electrolyte disorders, such as hyperkalemia and hyponatremia, and disorders of bone and carbohydrate metabolism; iii) vascular hyperpermeability, diabetic retinopathy, deterioration of the blood-retinal barrier, macular edema, in particular age-related macular edema, non-proliferative disorders, and disorders of the retina and retina; idiopathic age-related macular oedema and non-proliferative diabetic macular oedema; iv) diseases of the central or peripheral nervous system, in particular neuropathic pain, spinal cord injury, multiple sclerosis, traumatic brain injury, cerebral oedema and neurodegenerative diseases, in particular Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, progressive supranuclear palsy, black matter degeneration, Shy-Drager syndrome, olivopontocerebellar atrophy and spinocerebellar degeneration; v) cancer, in particular intestinal cancer, colorectal cancer, lung cancer, breast cancer, brain cancer, melanoma, renal cell carcinoma, leukemia, lymphoma, T-cell lymphoma, gastric cancer, pancreatic cancer, cervical cancer, endometrial cancer, ovarian cancer, oesophageal cancer, liver cancer, squamous cell carcinoma of the head and neck, skin cancer, urinary tract cancer, prostate cancer, choriocarcinoma, pharyngeal cancer and laryngeal cancer.

[0014] The isolated antibodies or antigen-binding fragments according to the invention may further be used in the diagnosis of Sema3A-associated disorders.

[0015] In another aspect, the present invention relates to an isolated nucleic acid sequence encoding an antibody or antigen-binding fragment according to the invention.

[0016] In another aspect, the present invention relates to a vector comprising a nucleic acid sequence according to the invention.

[0017] In another aspect, the present invention relates to an isolated cell expressing an antibody or antigen-binding fragment according to the invention and / or comprising a nucleic acid according to the invention or a vector according to the invention.

[0018] In another aspect, the present invention relates to a method for producing an isolated antibody or antigen-binding fragment according to the invention, comprising culturing a cell according to the invention and optionally purifying the antibody or antigen-binding fragment thereof.

[0019] In another aspect, the present invention relates to a pharmaceutical composition comprising the isolated antibody or antigen-binding fragment according to the invention or an antibody-conjugate according to the invention.

[0020] In another aspect, the present invention relates to a kit comprising an isolated antibody or antigen-binding fragment according to the invention or a conjugate according to the invention and instructions for use. In one aspect, the present invention provides the following: [Item 1] 1. An isolated antibody or antigen-binding fragment thereof that binds to human Sema3A, comprising: i) binds to human Sema3A of sequence SEQ ID NO: 600 with a dissociation constant (KD) of 50 nM or less, 20 nM or less, 10 nM or less, 1 nM or less, or 0.1 nM or less; ii) cross-reacts with mouse, cynomolgus monkey, rat, pig and / or dog Sema3A, in particular, the isolated antibody or antigen-binding fragment thereof binds to mouse, cynomolgus monkey, rat, pig and / or dog Sema3A with a dissociation constant (KD) of 50 nM or less, 20 nM or less, 10 nM or less, 1 nM or less, or 0.1 nM or less; iii) binds to human Sema3A of sequence SEQ ID NO: 600 with a binding activity of 60% or more, 70% or more, 80% or more, or 90% or more as measured by surface plasmon resonance (SPR); iv) inhibiting the activity of human Sema3A of sequence SEQ ID NO: 600 in an in vitro mesangial cell migration assay with an EC50 of 10 nM or less, 5 nM or less, 2.5 nM or less, or 1 nM or less; v) inhibiting the activity of human Sema3A of sequence SEQ ID NO: 600 in an in vitro growth cone collapse assay with an EC50 of 50 nM or less, 25 nM or less, 10 nM or less, or 5 nM or less; and / or vi) The above isolated antibody or antigen-binding fragment thereof, which inhibits the activity of human Sema3A of sequence SEQ ID NO: 600 in an in vitro HUVEC repulsion assay with an EC50 of 1 nM or less, or 0.3 nM or less, 0.1 nM or less, 0.07 nM or less, or 0.06 nM or less. [Item 2] The isolated antibody or antigen-binding fragment of item 1, wherein the isolated antibody or antigen-binding fragment cross-reacts with mouse, cynomolgus monkey, rat, pig and / or dog Sema3A and in particular has an affinity for mouse, cynomolgus monkey, rat, pig and / or dog Sema3A that differs by less than 100-fold, in particular less than 50-fold, more particularly less than 25-fold, even more particularly less than 10-fold, and most particularly less than 5-fold compared to the affinity for human Sema3A. [Item 3] The isolated antibody or antigen-binding fragment thereof described in item 1 or 2, wherein the isolated antibody or antigen-binding fragment thereof does not significantly cross-react with human Sema3B, Sema3C, Sema3D, Sema3E, Sema3F and / or Sema3G, in particular, the isolated antibody or antigen-binding fragment thereof does not significantly cross-react with human Sema3G. [Item 4] The isolated antibody or antigen-binding fragment of any one of the preceding claims, wherein the isolated antibody or antigen-binding fragment thereof inhibits Sema3A-induced albuminuria and / or proteinuria. [Item 5] The isolated antibody or antigen-binding fragment of any one of the preceding claims, wherein the isolated antibody or antigen-binding fragment thereof inhibits Sema3A-induced fibrosis. [Item 6] An isolated antibody or antigen-binding fragment thereof that competes for binding to Sema3A with the isolated antibody or antigen-binding fragment described in any one of the preceding claims, wherein the isolated antibody or antigen-binding fragment does not compete with the binding to Sema3A of an antibody comprising i) SEQ ID NO: 800 and SEQ ID NO: 804, or ii) SEQ ID NO: 810 and SEQ ID NO: 811. [Item 7] i) a heavy chain variable domain that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 141, and a light chain variable domain that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 145, or ii) a heavy chain variable domain that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO:61, and a light chain variable domain that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO:65. 3. The isolated antibody or antigen-binding fragment of item 1 or 2, comprising: [Item 8] i) a heavy chain antigen-binding region comprising a) an H-CDR3 comprising the sequence RDDYTSRDAFDX (SEQ ID NO: 594), where X is selected from the group consisting of Y and V, in particular X is Y, and b) a light chain antigen-binding region comprising an L-CDR3 comprising the sequence X1AWDDSLNX2X3X4V (SEQ ID NO: 598), where X1 is selected from the group consisting of A and H, X2 is selected from the group consisting of V, D and G, in particular X2 is selected from the group consisting of V and D, X3 is selected from the group consisting of I and Y, and X4 is selected from the group consisting of P and V; or ii) a heavy chain antigen-binding region comprising a) an H-CDR3 comprising the sequence SGYSSSWFDPDFDY (SEQ ID NO: 64); and b) a light chain antigen-binding region comprising an L-CDR3 comprising the sequence X1SYX2GX3NPYVV (SEQ ID NO: 599), wherein X1 is selected from the group consisting of S and Q, X2 is selected from the group consisting of E and A, and X3 is selected from the group consisting of P, I and S, in particular, X3 is selected from the group consisting of P and I. [Item 9] i) H-CDR1 comprising the sequence SYX1MX2 (SEQ ID NO: 588) (wherein X1 is selected from G and A and X2 is selected from H, S and L), in particular the sequence SYAMX (SEQ ID NO: 589) (wherein X is selected from S and L); H-CDR2 comprising the sequence AIGX1GGDTYYADSVX2G (SEQ ID NO: 590) (wherein X1 is selected from T and Y and X2 is selected from K and M), in particular the sequence AIGXGGDTYYADSVKG (SEQ ID NO: 591) (wherein X is selected from T and Y); and the sequence RDDYTSRDAFDX (SEQ ID NO: 594) (wherein X is selected from the group consisting of Y and V, in particular X is Y). and b) a heavy chain antigen-binding region comprising an H-CDR3 comprising the sequence SGSSSNIGSNTVN (SEQ ID NO: 46); an L-CDR2 comprising the sequence YDDLXPS (SEQ ID NO: 596) (wherein X is selected from L and R), in particular an L-CDR2 comprising the sequence YDDLRPS (SEQ ID NO: 127); and a light chain antigen-binding region comprising an L-CDR3 comprising the sequence X1AWDDSLNX2X3X4V (SEQ ID NO: 598) (wherein X1 is selected from the group consisting of A and H, X2 is selected from the group consisting of V, D and G, in particular X2 is selected from the group consisting of V and D, X3 is selected from the group consisting of I and Y, and X4 is selected from the group consisting of P and V); or ii) a heavy chain antigen binding region comprising an H-CDR1 comprising the sequence SYEMN (SEQ ID NO: 62); an H-CDR2 comprising the sequence GISWWNSGX1IX2YADSVKG (SEQ ID NO: 592) (wherein X1 is selected from W and S, and X2 is selected from G and D), in particular an H-CDR2 comprising the sequence GISWWNSGWIXYADSVKG (SEQ ID NO: 593) (wherein X is selected from G and D); and an H-CDR3 comprising the sequence SGYSSSWFDPDFDY (SEQ ID NO: 64); and b) a heavy chain antigen binding region comprising the sequence TGSSSXIGAGYD a light chain antigen-binding region comprising an L-CDR1 comprising VH (SEQ ID NO:595) (wherein X is selected from N and D); an L-CDR2 comprising the sequence GXSNRPS (SEQ ID NO:597) (wherein X is selected from N and A); and an L-CDR3 comprising the sequence X1SYX2GX3NPYVV (SEQ ID NO:599) (wherein X1 is selected from the group consisting of S and Q, X2 is selected from the group consisting of E and A, and X3 is selected from the group consisting of P, I and S, in particular, X3 is selected from the group consisting of P and I). 2. The isolated antibody or antigen-binding fragment of any one of the preceding claims, comprising: [Item 10] In the isolated antibody or antigen-binding fragment according to option i) of item 8, the amino acid residues immediately adjacent to the H-CDR1 at its 5' end are S or Y; or in the isolated antibody or antigen-binding fragment according to option ii) of item 8, the three amino acid residues immediately adjacent to the H-CDR1 at its 5' end are X1FX2, where X1 is selected from T and D and X2 is selected from S and D. [Item 11] i) a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO: 42, H-CDR2 comprising SEQ ID NO: 43 and H-CDR3 comprising SEQ ID NO: 44, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO: 46, L-CDR2 comprising SEQ ID NO: 47 and L-CDR3 comprising SEQ ID NO: 48; or ii) a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO: 62, H-CDR2 comprising SEQ ID NO: 63 and H-CDR3 comprising SEQ ID NO: 64, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO: 66, L-CDR2 comprising SEQ ID NO: 67 and L-CDR3 comprising SEQ ID NO: 68; or iii) a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO: 102, H-CDR2 comprising SEQ ID NO: 103 and H-CDR3 comprising SEQ ID NO: 104, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO: 106, L-CDR2 comprising SEQ ID NO: 107 and L-CDR3 comprising SEQ ID NO: 108; or iv) a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO: 122, H-CDR2 comprising SEQ ID NO: 123 and H-CDR3 comprising SEQ ID NO: 124, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO: 126, L-CDR2 comprising SEQ ID NO: 127 and L-CDR3 comprising SEQ ID NO: 128; or v) a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO: 142, H-CDR2 comprising SEQ ID NO: 143 and H-CDR3 comprising SEQ ID NO: 144, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO: 146, L-CDR2 comprising SEQ ID NO: 147 and L-CDR3 comprising SEQ ID NO: 148; or vi) a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO: 162, H-CDR2 comprising SEQ ID NO: 163 and H-CDR3 comprising SEQ ID NO: 164, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO: 166, L-CDR2 comprising SEQ ID NO: 167 and L-CDR3 comprising SEQ ID NO: 168; or vii) a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO: 182, H-CDR2 comprising SEQ ID NO: 183 and H-CDR3 comprising SEQ ID NO: 184, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO: 186, L-CDR2 comprising SEQ ID NO: 187 and L-CDR3 comprising SEQ ID NO: 188; or viii) a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO: 202, H-CDR2 comprising SEQ ID NO: 203 and H-CDR3 comprising SEQ ID NO: 204, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO: 206, L-CDR2 comprising SEQ ID NO: 207 and L-CDR3 comprising SEQ ID NO: 208; or ix) a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO: 222, H-CDR2 comprising SEQ ID NO: 223 and H-CDR3 comprising SEQ ID NO: 224, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO: 226, L-CDR2 comprising SEQ ID NO: 227 and L-CDR3 comprising SEQ ID NO: 228; or x) a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO: 242, H-CDR2 comprising SEQ ID NO: 243 and H-CDR3 comprising SEQ ID NO: 244, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO: 246, L-CDR2 comprising SEQ ID NO: 247 and L-CDR3 comprising SEQ ID NO: 248; or xi) a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO: 262, H-CDR2 comprising SEQ ID NO: 263 and H-CDR3 comprising SEQ ID NO: 264, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO: 266, L-CDR2 comprising SEQ ID NO: 267 and L-CDR3 comprising SEQ ID NO: 268; or xii) a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO:282, H-CDR2 comprising SEQ ID NO:283 and H-CDR3 comprising SEQ ID NO:284, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO:286, L-CDR2 comprising SEQ ID NO:287 and L-CDR3 comprising SEQ ID NO:288; or xiii) a heavy chain antigen-binding region comprising an H-CDR1 comprising SEQ ID NO: 302, an H-CDR2 comprising SEQ ID NO: 303, and an H-CDR3 comprising SEQ ID NO: 304, and a light chain antigen-binding region comprising an L-CDR1 comprising SEQ ID NO: 306, an L-CDR2 comprising SEQ ID NO: 307, and an L-CDR3 comprising SEQ ID NO: 308; or xiv) a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO: 322, H-CDR2 comprising SEQ ID NO: 323 and H-CDR3 comprising SEQ ID NO: 324, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO: 326, L-CDR2 comprising SEQ ID NO: 327 and L-CDR3 comprising SEQ ID NO: 328; or xv) a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO: 342, H-CDR2 comprising SEQ ID NO: 343 and H-CDR3 comprising SEQ ID NO: 344, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO: 346, L-CDR2 comprising SEQ ID NO: 347 and L-CDR3 comprising SEQ ID NO: 348; or xvi) a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO: 362, H-CDR2 comprising SEQ ID NO: 363 and H-CDR3 comprising SEQ ID NO: 364, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO: 366, L-CDR2 comprising SEQ ID NO: 367 and L-CDR3 comprising SEQ ID NO: 368; or xvii) a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO: 382, ​​H-CDR2 comprising SEQ ID NO: 383, and H-CDR3 comprising SEQ ID NO: 384, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO: 386, L-CDR2 comprising SEQ ID NO: 387, and L-CDR3 comprising SEQ ID NO: 388; or xviii) a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO: 402, H-CDR2 comprising SEQ ID NO: 403 and H-CDR3 comprising SEQ ID NO: 404, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO: 406, L-CDR2 comprising SEQ ID NO: 407 and L-CDR3 comprising SEQ ID NO: 408; or xix) a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO: 422, H-CDR2 comprising SEQ ID NO: 423 and H-CDR3 comprising SEQ ID NO: 424, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO: 426, L-CDR2 comprising SEQ ID NO: 427 and L-CDR3 comprising SEQ ID NO: 428; or xx) a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO: 442, H-CDR2 comprising SEQ ID NO: 443 and H-CDR3 comprising SEQ ID NO: 444, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO: 446, L-CDR2 comprising SEQ ID NO: 447 and L-CDR3 comprising SEQ ID NO: 448; or xxi) a heavy chain antigen-binding region comprising an H-CDR1 comprising SEQ ID NO: 462, an H-CDR2 comprising SEQ ID NO: 463, and an H-CDR3 comprising SEQ ID NO: 464, and a light chain antigen-binding region comprising an L-CDR1 comprising SEQ ID NO: 466, an L-CDR2 comprising SEQ ID NO: 467, and an L-CDR3 comprising SEQ ID NO: 468; or xxii) a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO: 482, H-CDR2 comprising SEQ ID NO: 483 and H-CDR3 comprising SEQ ID NO: 484, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO: 486, L-CDR2 comprising SEQ ID NO: 487 and L-CDR3 comprising SEQ ID NO: 488; or xxiii) a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO: 502, H-CDR2 comprising SEQ ID NO: 503 and H-CDR3 comprising SEQ ID NO: 504, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO: 506, L-CDR2 comprising SEQ ID NO: 507 and L-CDR3 comprising SEQ ID NO: 508; or xxiv) a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO: 522, H-CDR2 comprising SEQ ID NO: 523 and H-CDR3 comprising SEQ ID NO: 524, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO: 526, L-CDR2 comprising SEQ ID NO: 527 and L-CDR3 comprising SEQ ID NO: 528; or xxv) a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO: 542, H-CDR2 comprising SEQ ID NO: 543 and H-CDR3 comprising SEQ ID NO: 544, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO: 546, L-CDR2 comprising SEQ ID NO: 547 and L-CDR3 comprising SEQ ID NO: 548; or xxvi) a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO: 562, H-CDR2 comprising SEQ ID NO: 563 and H-CDR3 comprising SEQ ID NO: 564, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO: 566, L-CDR2 comprising SEQ ID NO: 567 and L-CDR3 comprising SEQ ID NO: 568; 2. The isolated antibody or antigen-binding fragment of any one of the preceding claims, comprising: [Item 12] i) a variable heavy chain domain comprising SEQ ID NO: 41 and a variable light chain domain comprising SEQ ID NO: 45; or ii) a variable heavy chain domain comprising SEQ ID NO: 61 and a variable light chain domain comprising SEQ ID NO: 65; or iii) a variable heavy chain domain comprising SEQ ID NO: 101 and a variable light chain domain comprising SEQ ID NO: 105; or iv) a variable heavy chain domain comprising SEQ ID NO: 121 and a variable light chain domain comprising SEQ ID NO: 125; or v) a variable heavy chain domain comprising SEQ ID NO: 141 and a variable light chain domain comprising SEQ ID NO: 145; or vi) a variable heavy chain domain comprising SEQ ID NO: 161 and a variable light chain domain comprising SEQ ID NO: 165; or vii) a variable heavy chain domain comprising SEQ ID NO: 181 and a variable light chain domain comprising SEQ ID NO: 185; or viii) a variable heavy chain domain comprising SEQ ID NO: 201 and a variable light chain domain comprising SEQ ID NO: 205; or ix) a variable heavy chain domain comprising SEQ ID NO: 221 and a variable light chain domain comprising SEQ ID NO: 225; or x) a variable heavy chain domain comprising SEQ ID NO: 241 and a variable light chain domain comprising SEQ ID NO: 245; or xi) a variable heavy chain domain comprising SEQ ID NO: 261 and a variable light chain domain comprising SEQ ID NO: 265; or xii) a variable heavy chain domain comprising SEQ ID NO: 281 and a variable light chain domain comprising SEQ ID NO: 285; or xiii) a variable heavy chain domain comprising SEQ ID NO: 301 and a variable light chain domain comprising SEQ ID NO: 305; or xiv) a variable heavy chain domain comprising SEQ ID NO: 321 and a variable light chain domain comprising SEQ ID NO: 325; or xv) a variable heavy chain domain comprising SEQ ID NO: 341 and a variable light chain domain comprising SEQ ID NO: 345; or xvi) a variable heavy chain domain comprising SEQ ID NO: 361 and a variable light chain domain comprising SEQ ID NO: 365; or xvii) a variable heavy chain domain comprising SEQ ID NO: 381 and a variable light chain domain comprising SEQ ID NO: 385; or xviii) a variable heavy chain domain comprising SEQ ID NO: 401 and a variable light chain domain comprising SEQ ID NO: 405; or xix) a variable heavy chain domain comprising SEQ ID NO: 421 and a variable light chain domain comprising SEQ ID NO: 425; or xx) a variable heavy chain domain comprising SEQ ID NO: 441 and a variable light chain domain comprising SEQ ID NO: 445; or xxi) a variable heavy chain domain comprising SEQ ID NO: 461 and a variable light chain domain comprising SEQ ID NO: 465; or xxii) a variable heavy chain domain comprising SEQ ID NO: 481 and a variable light chain domain comprising SEQ ID NO: 485; or xxiii) a variable heavy chain domain comprising SEQ ID NO: 501 and a variable light chain domain comprising SEQ ID NO: 505; or xxiv) a variable heavy chain domain comprising SEQ ID NO: 521 and a variable light chain domain comprising SEQ ID NO: 525; or xxv) a variable heavy chain domain comprising SEQ ID NO: 541 and a variable light chain domain comprising SEQ ID NO: 545; or xxvi) a variable heavy chain domain comprising SEQ ID NO: 561 and a variable light chain domain comprising SEQ ID NO: 565 2. The isolated antibody or antigen-binding fragment of any one of the preceding claims, comprising: [Item 13] 10. The isolated antibody of any one of the preceding claims, which is an IgG antibody, in particular an IgG1 or IgG4 antibody. [Item 14] i) a heavy chain comprising SEQ ID NO:57 and a light chain comprising SEQ ID NO:58; or ii) a heavy chain comprising SEQ ID NO: 77 and a light chain comprising SEQ ID NO: 78; or iii) a heavy chain comprising SEQ ID NO: 117 and a light chain comprising SEQ ID NO: 118; or iv) a heavy chain comprising SEQ ID NO: 137 and a light chain comprising SEQ ID NO: 138; or v) a heavy chain comprising SEQ ID NO: 157 and a light chain comprising SEQ ID NO: 158; or vi) a heavy chain comprising SEQ ID NO: 177 and a light chain comprising SEQ ID NO: 178; or vii) a heavy chain comprising SEQ ID NO: 197 and a light chain comprising SEQ ID NO: 198; or viii) a heavy chain comprising SEQ ID NO:217 and a light chain comprising SEQ ID NO:218; or ix) a heavy chain comprising SEQ ID NO: 237 and a light chain comprising SEQ ID NO: 238; or x) a heavy chain comprising SEQ ID NO: 257 and a light chain comprising SEQ ID NO: 258; or xi) a heavy chain comprising SEQ ID NO:277 and a light chain comprising SEQ ID NO:278; or xii) a heavy chain comprising SEQ ID NO:297 and a light chain comprising SEQ ID NO:298; or xiii) a heavy chain comprising SEQ ID NO: 317 and a light chain comprising SEQ ID NO: 318; or xiv) a heavy chain comprising SEQ ID NO: 337 and a light chain comprising SEQ ID NO: 338; or xv) a heavy chain comprising SEQ ID NO: 357 and a light chain comprising SEQ ID NO: 358; or xvi) a heavy chain comprising SEQ ID NO: 377 and a light chain comprising SEQ ID NO: 378; or xvii) a heavy chain comprising SEQ ID NO: 397 and a light chain comprising SEQ ID NO: 398; or xviii) a heavy chain comprising SEQ ID NO: 417 and a light chain comprising SEQ ID NO: 418; or xix) a heavy chain comprising SEQ ID NO: 437 and a light chain comprising SEQ ID NO: 438; or xx) a heavy chain comprising SEQ ID NO: 457 and a light chain comprising SEQ ID NO: 458; or xxi) a heavy chain comprising SEQ ID NO: 477 and a light chain comprising SEQ ID NO: 478; or xxii) a heavy chain comprising SEQ ID NO: 497 and a light chain comprising SEQ ID NO: 498; or xxiii) a heavy chain comprising SEQ ID NO: 517 and a light chain comprising SEQ ID NO: 518; or xxiv) a heavy chain comprising SEQ ID NO: 537 and a light chain comprising SEQ ID NO: 538; or xxv) a heavy chain comprising SEQ ID NO: 557 and a light chain comprising SEQ ID NO: 558; or xxvi) a heavy chain comprising SEQ ID NO: 577 and a light chain comprising SEQ ID NO: 578 2. The isolated antibody of claim 1 , comprising: [Item 15] 2. The antigen-binding fragment of any one of the preceding claims, which is an scFv, Fab, Fab' fragment or F(ab')2 fragment. [Item 16] 2. The isolated antibody or antigen-binding fragment of any one of the preceding claims, which is a monoclonal antibody or antigen-binding fragment thereof. [Item 17] 2. The isolated antibody or antigen-binding fragment of any one of the preceding claims, which is a human antibody, a humanized antibody or a chimeric antibody or an antigen-binding fragment thereof. [Item 18] An isolated antibody or antigen-binding fragment thereof that competes with the isolated antibody or antigen-binding fragment of any preceding claim for binding to human Sema3A. [Item 19] An antibody conjugate comprising the isolated antibody or antigen-binding fragment of any one of the preceding claims. [Item 20] 18. An isolated nucleic acid sequence encoding the antibody or antigen-binding fragment according to any one of items 1 to 17. [Item 21] 20. A vector comprising the nucleic acid sequence of item 19. [Item 22] 21. An isolated cell expressing the antibody or antigen-binding fragment according to any one of items 1 to 17 and / or comprising the nucleic acid according to item 19 or the vector according to item 20. [Item 23] 22. The isolated cell of item 21, wherein the cell is a prokaryotic or eukaryotic cell. [Item 24] A method for producing the isolated antibody or antigen-binding fragment according to any one of items 1 to 17, comprising culturing the cell according to any one of items 21 or 22, and optionally purifying the antibody or antigen-binding fragment thereof. [Item 25] A pharmaceutical composition comprising the isolated antibody or antigen-binding fragment according to any one of items 1 to 17 or the antibody conjugate according to item 18. [Item 26] 25. The isolated antibody or antigen-binding fragment according to any one of items 1 to 17, or the conjugate according to item 18, or the pharmaceutical composition according to item 24 for use as a medicament. [Item 27] 19. The isolated antibody or antigen-binding fragment according to any one of items 1 to 17 or the antibody conjugate according to item 18 for use as a diagnostic agent. [Item 28] i) kidney diseases, in particular acute and chronic kidney diseases, diabetic kidney disease, Alport syndrome, acute and chronic renal failure, polycystic kidney disease (PCKD) and syndrome of insufficient ADH secretion (SIADH); ii) sequelae of renal failure, in particular pulmonary edema, cardiac failure, uremia, anemia, electrolyte disorders such as hyperkalemia and hyponatremia, and disorders of bone and carbohydrate metabolism; iii) vascular hyperpermeability, diabetic retinopathy, deterioration of the blood-retinal barrier, macular edema, in particular age-related macular edema, non-proliferative age-related macular edema and non-proliferative diabetic macular edema; iv) diseases of the central or peripheral nervous system, in particular neuropathic pain, spinal cord injury, multiple sclerosis, traumatic brain injury, cerebral edema and neurodegenerative diseases, in particular Alzheimer's disease. 20. The method of claim 19, further comprising administering to said patient a therapeutically effective amount of at least one of the following: Hymer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, progressive supranuclear palsy, nigra degeneration, Shy-Drager syndrome, olivopontocerebellar atrophy and spinocerebellar degeneration; or v) the isolated antibody or antigen-binding fragment according to any one of items 1 to 17 or the conjugate according to item 18 or the pharmaceutical composition according to item 24 for use in the treatment and / or prevention of cancer, in particular intestinal cancer, colorectal cancer, lung cancer, breast cancer, brain cancer, melanoma, renal cell carcinoma, leukemia, lymphoma, T-cell lymphoma, gastric cancer, pancreatic cancer, cervical cancer, endometrial cancer, ovarian cancer, esophageal cancer, liver cancer, squamous cell carcinoma of the head and neck, skin cancer, cancer of the urinary tract, prostate cancer, choriocarcinoma, pharyngeal cancer and laryngeal cancer. [Item 29] 25. The isolated antibody or antigen-binding fragment according to any one of items 1 to 17, or the conjugate according to item 18, or the pharmaceutical composition according to item 24, for use in combination for simultaneous, separate or sequential administration with one or more additional therapeutically active compounds. [Item 30] A kit comprising the isolated antibody or antigen-binding fragment according to any one of items 1 to 17 or the conjugate according to item 18 together with instructions for use.

[0021] Detailed Description of the Invention The present invention may be understood more readily by reference to the following detailed description of the invention and the examples included therein.

[0022] definition Unless otherwise indicated, all scientific and technical terms used herein have the meanings commonly understood by those skilled in the art to which the present invention belongs. However, the following references may provide a person skilled in the art to which the present invention pertains with a general definition of many of the terms used in the present invention, and such definitions may be consulted and used to the extent that they are consistent with the meanings commonly understood in the art. Such references include, but are not limited to: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker, ed., 1988); Hale & Marham, The Harper Collins Dictionary of Biology (1991); Lackie et al., The Dictionary of Cell & Molecular Biology (3d ed. 1999); and Cellular and Molecular Immunology, Abbas, Lichtman and Pober, eds., 2nd Edition, WB Saunders Company. Reference may be made to additional technical documents available to those skilled in the art, which provide definitions of the terms used herein that have the meanings generally understood in the art. For the purposes of the present invention, the following terms are further defined. Additional terms are defined elsewhere in the description. As used in this specification and the appended claims, the singular forms include plural referents unless the context clearly indicates otherwise. Thus, for example, a reference to a "gene" is a reference to one or more genes, including equivalents thereof known to those skilled in the art, and so forth.

[0023] In the context of the present invention, the words "comprise" or "comprises" mean "including but not limited to." The term is intended to be open-ended, specifying the presence of any indicated features, elements, integers, steps, or components, and is not intended to exclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. Thus, the word "comprise" includes the more restrictive terms "consisting of" and "consisting essentially of." In one embodiment, the word "comprise" used throughout this application, and particularly in the claims, can be replaced by the word "consisting of."

[0024] In this context, the term "about" or "approximately" means within 80%-120%, or within 90%-110%, including within 95%-105%, of a given value or range.

[0025] The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.

[0026] As used herein, "Sema3A" refers to "semaphorin 3A," which is also known as "HH16," "SemD," "COLL1," "SEMA1," "SEMAD," "SEMAL," "coll-1," "Hsema-I," "SEMAIII," "Hsema-III," "collapsin 1," "semaphorin D," "semaphorin III," and "semaphorin L."

[0027] The terms "anti-Sema3A antibody" and "antibody that binds to Sema3A" refer to an antibody that can bind to Sema3A with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent targeting Sema3A. In one embodiment, the degree of binding of the anti-Sema3A antibody to an unrelated non-Sema3A protein is less than about 10%, less than about 5%, or less than about 2% of the binding of the antibody to Sema3A, as measured, for example, by standard ELISA methods. In certain embodiments, the antibody that binds to Sema3A has a binding affinity of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -8 M or less, e.g. 10 -8 M~10 -13 M, for example 10 -9 M~10 -13 M) in binding activity (EC50). In certain embodiments, the anti-Sema3A antibody binds to an epitope of Sema3A that is conserved among Sema3A from various species.

[0028] The term "antibody" as used herein is intended to mean an immunoglobulin molecule. An antibody may comprise four polypeptide chains, typically two heavy (H) chains (about 50-70 kDa) and two light (L) chains (about 25 kDa) linked together by disulfide bonds. In certain embodiments, an antibody is composed of two identical pairs of polypeptide chains. The amino-terminal portion of each chain comprises a "variable" region of about 100-110 or more amino acids that are primarily responsible for antigen recognition. The heavy chain variable region is abbreviated herein as VH, and the light chain variable region is abbreviated herein as VL. The carboxyl-terminal portion of each chain defines a constant region that is primarily responsible for effector function. The heavy chain constant region may comprise three domains, e.g., CH1, CH2, and CH3. The light chain constant region is composed of one domain (CL). The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), which are interspersed between more conserved regions called framework regions (FRs). Each VH and VL typically consists of three CDRs and up to four FRs, which are arranged from the amino terminus to the carboxy terminus, for example in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0029] The term "complementarity determining region" (CDR; e.g., CDR1, CDR2 and CDR3) as used herein refers to the amino acid residues of an antibody variable domain whose presence is necessary for antigen binding. Each variable domain typically has three CDR regions, identified as CDR1, CDR2 and CDR3. Each complementarity determining region may contain amino acid residues from the "complementarity determining regions" defined by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or from the "hypervariable loops" (Chothia and Lesk; J Mol Biol 196:901-917 (1987)). In some cases, a complementarity determining region may contain amino acids from both the CDR regions and the hypervariable loops defined according to Kabat.

[0030] "Framework" or FR residues are those variable domain residues other than the hypervariable region residues.

[0031] The term "constant region" refers to the portion of the antibody molecule that confers effector functions.

[0032] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or constant region is in accordance with the EU numbering system, also referred to as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0033] Immunoglobulins can be assigned to different classes depending on the amino acid sequence of the constant domain of their heavy chains. Heavy chains are classified as mu (μ), delta (Δ), gamma (γ), alpha (α) and epsilon (ε), defining the isotype of the antibody as IgM, IgD, IgG, IgA and IgE, respectively. In a particular embodiment, the antibody according to the invention is an IgG antibody. Some of these may be further classified into subclasses or isotypes, for example IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. In a particular embodiment, the antibody according to the invention is an IgG1, IgG2, IgG3 or IgG4 antibody, more particularly an IgG1 or IgG4 antibody. Different isotypes may have different effector functions. Human light chains are classified into kappa (κ) light chains and lambda (λ) light chains. Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 more amino acids. See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)).

[0034] An "antigen-binding antibody fragment" or "functional fragment" of an antibody / immunoglobulin is defined herein as a fragment of an antibody / immunoglobulin (e.g., the variable region of an IgG) that retains the antigen-binding region. The "antigen-binding region" of an antibody is typically found in one or more hypervariable regions, e.g., the CDR1, 2 and / or 3 regions, of an antibody, although the variable "framework" regions may also play an important role in antigen binding, e.g., by providing a scaffold for the CDRs. Preferably, the "antigen-binding region" comprises at least amino acid residues 4-103 of the variable light chain (VL) and amino acid residues 5-109 of the variable heavy chain (VH), more preferably amino acid residues 3-107 of the VL and amino acid residues 4-111 of the VH, with the complete VL and VH chains (amino acids 1-109 of the VL and 1-113 of the VH; numbering according to WO 97 / 08320).

[0035] Non-limiting examples of "functional fragments" or "antigen-binding antibody fragments" include: Fab, Fab', F(ab')2, Fv fragments, domain antibodies (dAbs), complementarity determining region (CDR) fragments, single chain antibodies (scFv), single chain antibody fragments, diabodies, triabodies, tetrabodies, minibodies, linear antibodies (Zapata et al., Protein Eng., 8(10):1057-1062 (1995)); chelating recombinant antibodies, tribodies or bibodies, intrabodies, nanobodies, small modular immunopharmaceuticals (SMIPs), antigen-binding domain immunoglobulin fusion proteins, camelized antibodies, VHH-containing antibodies or muteins or derivatives thereof, and polypeptides containing at least a portion of an immunoglobulin sufficient to confer specific antigen binding to the polypeptide, such as, for example, a CDR sequence, provided that the antibody retains the desired biological activity; and multispecific antibodies, such as bispecific and trispecific antibodies formed from antibody fragments (CAKs). Borrebaeck, ed. (1995) Antibody Engineering (Breakthroughs in Molecular Biology), Oxford University Press; R. Kontermann & S. Duebel, eds. (2001) Antibody Engineering (Springer Laboratory Manual), Springer Verlag). An antibody other than a "bispecific" or "bifunctional" antibody is understood to have each of its binding sites identical. F(ab')2 or Fab refers to a C H1 Domain and C LThe domains can be engineered to minimize or completely eliminate intermolecular disulfide interactions that occur between them. Papain digestion of an antibody produces two identical antigen-binding fragments (called "Fab" fragments), each with a single antigen-binding site, and a remaining "Fc" fragment, a name that reflects its ability to crystallize easily. Pepsin treatment produces an F(ab')2 fragment, which has two "Fv" fragments. The "Fv" fragment is the smallest antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy-chain variable domain and one light-chain variable domain associated by tight non-covalent bonds. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind to an antigen.

[0036] "Single-chain Fv" or "sFv" or "scFv" antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain.

[0037] Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the Fv to form the desired structure for antigen binding. For a review of Fv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994).

[0038] Fab fragments also contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab fragments differ from Fab' fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteine ​​residues from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residues of the constant domains contain a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteine ​​residues between them.

[0039] The terms "mutein" or "variant" can be used interchangeably and refer to an antibody or antigen-binding fragment that contains at least one amino acid substitution, deletion, or insertion in the variable region or an equivalent of the variable region, provided that the mutein or variant retains the desired binding affinity or biological activity. Variants of antibodies or antigen-binding antibody fragments contemplated in the present invention are molecules that retain the binding activity of the antibody or antigen-binding antibody fragment.

[0040] A "chimeric antibody" or antigen-binding fragment thereof is defined herein as one in which the variable domains are of non-human origin and some or all of the constant domains are of human origin.

[0041] "Humanized antibodies" contain, for example, CDR regions from a non-human species, such as mouse, grafted into a V region from a human sequence with necessary framework back mutations. Thus, in most cases, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from the recipient's hypervariable region are replaced by residues from the hypervariable region of a non-human species (donor antibody), such as mouse, rat, rabbit or non-human primate, with the desired specificity, affinity and capacity. See, for example, U.S. Patent Nos. 5,225,539, 5,585,089, 5,693,761, 5,693,762, 5,859,205, each of which is incorporated herein by reference. In some instances, framework residues of the human immunoglobulin are replaced by corresponding non-human residues (see, e.g., U.S. Pat. Nos. 5,585,089, 5,693,761, and 5,693,762, each of which is incorporated herein by reference). Furthermore, humanized antibodies may include residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance (e.g., to obtain a desired affinity). In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin sequence. The humanized antibody may optionally also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 331:522-25 (1986); Riechmann et al., Nature 332:323-27 (1988); and Presta, Curr. Opin. Struct. Biol. 2:593-96 (1992), each of which is incorporated herein by reference.

[0042] A "human antibody" or "fully human antibody" comprises human-derived CDRs, i.e., CDRs of human origin. A fully human antibody may contain a small number of germline deviations compared to the closest human germline reference determined based on the IMGT database (http: / / www.imgt.org). For example, a fully human antibody according to the invention may contain up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 germline deviations in the CDRs compared to the closest human germline reference. Fully human antibodies can be generated from human-derived B cells by cloning techniques combined with cell enrichment or immortalization steps. However, the majority of fully human antibodies are isolated from immunized mice transgenic for the human IgG locus or from elaborate combinatorial libraries by phage display (Bruggemann M., Osborn MJ, Ma B., Hayre J., Avis S., Lundstrom B. and Buelow R., Human Antibody Production in Transgenic Animals, Arch Immunol Ther Exp(Warsz.) 63(2015), 101-108; Carter PJ, Potent antibody therapeutics by design, Nat Rev Immunol 6(2006), 343-357; Frenzel A., Schirrmann T. and Hust M., Phage display-derived human antibodies in clinical development and therapy, MAbs 8(2016), 1177-1194; Nelson AL, Dhimolea E. and Reichert JM, Development trends for human monoclonal antibodies therapeutics, Nat Rev Drug Discov 9(2010), 767-774).

[0043] Several techniques are available for generating fully human antibodies (see WO 2008 / 112640 A3). Cambridge Antibody Technologies (CAT) and Dyax have obtained antibody cDNA sequences from peripheral B cells isolated from immunized humans and devised phage display libraries to identify human variable region sequences of particular specificity. Briefly, antibody variable region sequences are fused to either Gene III or Gene VIII constructs of M13 bacteriophage. These antibody variable region sequences are expressed as Fab or single chain Fv (scFv) constructs at the tip of the phage carrying the respective sequence. Through rounds of panning processes using different levels of antigen binding conditions (stringency), phages expressing Fab or scFv constructs specific for the antigen of interest can be selected and isolated. The antibody variable region cDNA sequences of the selected phages can then be revealed using standard sequencing methods. These sequences can then be used to reconstitute complete antibodies with the desired isotype using established antibody engineering techniques. Antibodies constructed according to this method are considered to be fully human antibodies (including CDRs). To improve the immunoreactivity (antigen binding affinity and specificity) of the selected antibodies, an in vitro maturation process can be introduced, including combinatorial binding of different heavy and light chains, deletions / additions / mutations in the CDR3 of the heavy and light chains (to mimic VJ and VDJ recombination), and random mutations (to mimic somatic hypermutation). One example of a "fully human" antibody generated by this method is the anti-tumor necrosis factor alpha antibody Humira (adalimumab).

[0044] "Human Engineered™" antibodies are produced by modifying the parent sequences according to the methods described in Studnicka et al., US Pat. No. 5,766,886.

[0045] The antibodies of the invention can be derived from a recombinant antibody gene library. The development of techniques for the generation of repertoires of recombinant human antibody genes and the display of encoded antibody fragments on the surface of filamentous bacteriophage provides a recombinant means for the direct generation and selection of human antibodies, which can be applied to humanized, chimeric, murine or mutein antibodies. Antibodies produced by phage technology are produced in bacteria as antigen-binding fragments (usually Fv or Fab fragments) and therefore lack effector functions. Effector functions can be introduced by one of two methods. The fragments can be engineered into complete antibodies for expression in mammalian cells, or into bispecific antibody fragments with a second binding site that can elicit effector functions. Typically, the heavy chains VH-CH1 and light chains VL-CL of the antibodies are cloned separately by PCR and randomly recombined in a combinatorial phage display library, which can then be selected for binding to a specific antigen. Fab fragments are expressed on the phage surface, i.e., physically linked to the gene encoding the fragment. Thus, selection of Fabs by antigen binding simultaneously selects for Fab-encoding sequences that can then be amplified. Through several rounds of antigen binding and re-amplification, a method called panning, Fabs specific for the antigen are enriched and finally isolated.

[0046] Various methods have been described for the generation of human antibodies from phage display libraries. Such libraries can be constructed on a single master framework into which diverse in vivo generated (i.e., human-derived) CDRs can be recombined, as described in Carlsson and Soderlind Exp. Rev. Mol. Diagn. 1(1), 102-108 (2001); Soderlin et al., Nat. Biotech. 18, 852-856 (2000); and U.S. Pat. No. 6,989,250. Alternatively, such antibody libraries can be based on amino acid sequences encoded by synthetically generated nucleic acids designed in silico. In silico design of antibody sequences can be achieved, for example, by analyzing a database of human sequences and using the data obtained therefrom to devise polypeptide sequences. Methods by which in silico generated sequences may be designed are described, for example, in Knappik et al., J. Mol. Biol. (2000) 296:57; Krebs et al., J. Immunol. Methods. (2001) 254:67; and U.S. Patent No. 6,300,064. For a review of phage display screening, see, for example, Hoet RM et al., Nat Biotechnol 2005;23(3):344-8), well-established hybridoma technology (see, for example, Kohler and Milstein Nature. 1975 Aug 7;256(5517):495-7), or immunization of mice, particularly hMAb mice (e.g., VelocImmune Mouse®).

[0047] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are identical except for possible mutations that may be present in minor amounts, such as naturally occurring mutations. Thus, the term "monoclonal" refers to the property of an antibody that is not a mixture of different antibodies. In contrast to polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. In addition to their specificity, monoclonal antibody preparations are also advantageous in that they are typically free of contamination by other immunoglobulins. The term "monoclonal" should not be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies used may be made by the hybridoma method first described by Kohler et al., Nature, 256:495

[1975] , or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). A "monoclonal antibody" may also be, for example, a recombinant monoclonal antibody, a chimeric monoclonal antibody, a humanized monoclonal antibody, a human monoclonal antibody, a Human Engineered™, or an antibody fragment.

[0048] An "isolated" antibody is one that has been identified and separated from components of the cell in which it is expressed. Contaminating components of the cell are substances that would interfere with diagnostic or therapeutic uses of the antibody, and may include enzymes, hormones and other proteinaceous or nonproteinaceous solutes.

[0049] An "isolated" nucleic acid is one that has been identified and separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0050] As used herein, an antibody that "specifically binds to," "is specific for," or "specifically recognizes" an antigen of interest, e.g., Sema3A, is an antibody that binds to the antigen with sufficient affinity such that the antibody is useful as a therapeutic agent in targeting cells or tissues expressing the antigen. As used herein, the terms "specifically recognize" or "specifically bind to" or "specific for" an epitope on a particular polypeptide or particular polypeptide target refer to, for example, an antibody that "specifically binds to," "is specific for," or "specific for" an epitope on a particular polypeptide target. -4 Less than M or about 10 -5 Less than M or about 10 -6 Less than M or about 10 -7 Less than M or about 10 -8 Less than M or about 10 -9 Less than M or about 10 -10 Less than M or about 10 -11 Less than M or about 10 -12 Monovalent K against antigen less than M D The antibody or antigen-binding fragment thereof may be represented by:

[0051] An antibody is said to "selectively bind", "selective for" or "selectively recognize" an antigen if it can distinguish between such an antigen and one or more reference antigens. In particular, an antibody that "selectively binds" an antigen does not significantly cross-react with proteins other than orthologs and variants (e.g., mutants, splice variants or proteolytic truncated forms) of said antigen target. "Selective binding", "selectively bind", "selectively" or "selectively recognize" in its most general form refers to the ability of an antibody to distinguish between an antigen of interest and unrelated antigens, for example, as determined according to one of the following methods. Such methods include, but are not limited to, surface plasmon resonance (SPR), Western blot, ELISA test, RIA test, ECL test, IRMA test and peptide scan. For example, a standard ELISA assay can be performed. Scoring can be performed by standard color development (e.g., secondary antibody in the presence of horseradish peroxidase and tetramethylbenzidine in the presence of hydrogen peroxide). The reaction in a particular well is scored by the optical density at, for example, 450 nm. A typical background (=negative reaction) may be 0.1 OD. A typical positive reaction may be 1 OD. This means that the positive / negative difference is more than 5-fold (i.e., more than 5-fold), more than 10-fold, more than 50-fold, preferably more than 100-fold. Typically, the determination of binding selectivity is performed using a combination of about 3-5 unrelated antigens (e.g., milk powder, BSA, transferrin, etc.) rather than a single reference antigen.

[0052] "Binding affinity" or "affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule and its binding partner. Unless otherwise indicated, "binding affinity" as used herein refers to the inherent binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The dissociation constant "K D" is commonly used to describe the affinity between a molecule (e.g., an antibody) and its binding partner (e.g., an antigen), i.e., how tightly a ligand binds to a particular protein. Ligand-protein affinity is influenced by non-covalent intermolecular interactions between the two molecules. Affinity can be measured by common methods known in the art, including those described herein. In one embodiment, the "K" used in the present invention is D " or "K D The "value" is measured by using a surface plasmon resonance assay using a Biacore T200 instrument (GE Healthcare Biacore, Inc.). Other suitable instruments include BIACORE T100, BIACORE®-2000, BIACORe 4000, BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) or ProteOn XPR36 instrument (Bio-Rad Laboratories, Inc.).

[0053] The term "epitope" as used herein includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains or combinations thereof and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.

[0054] An "antibody that binds to the same epitope" as a reference antibody or an "antibody that competes for binding" to a reference antibody means an antibody that blocks 10%, 20%, 30%, 40%, 50% or more of the binding of the reference antibody to its antigen in a competition assay, and conversely, the reference antibody blocks 10%, 20%, 30%, 40%, 50% or more of the binding of the antibody to its antigen in a competition assay.

[0055] The term "mature antibody" or "mature antigen-binding fragment" (e.g., mature Fab variants or "optimized" variants) includes derivatives of antibodies or antibody fragments that exhibit stronger binding, i.e., binding with increased affinity, to a given antigen, such as the extracellular domain of a target protein. Maturation is the process of identifying a small number of mutations within the six CDRs of an antibody or antibody fragment that result in this increased affinity. The maturation process is a combination of molecular biology methods for introducing mutations into an antibody and for screening to identify improved binders.

[0056] "Percentage of sequence identity" to a reference polynucleotide or polypeptide sequence, respectively, is defined as the percentage of nucleic acid or amino acid residues in a candidate sequence that are identical to the nucleic acid or amino acid residues in the reference polynucleotide or polypeptide sequence, respectively, after aligning the sequences and introducing gaps as necessary to obtain the maximum sequence identity percentage. Conservative substitutions are not considered as part of the sequence identity. Alignment without gaps is preferred. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in various ways that are within the skill of the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the entire length of the sequences to be compared.

[0057] "Sequence homology" indicates the percentage of amino acids that are identical or represent conservative amino acid substitutions.

[0058] An "antagonist" or "blocking" antibody is an antibody that significantly inhibits (partially or completely) the biological activity of the antigen to which it binds. In certain embodiments, an antibody or antigen-binding fragment according to the invention is a Sema3A blocking antibody or antigen-binding fragment thereof.

[0059] The term "antibody conjugate" refers to an antibody conjugated (bound) to one or more molecules, including drugs (in which case the antibody conjugate is referred to as an "antibody drug conjugate" ("ADC")) and large molecular weight molecules, such as peptides or proteins.

[0060] The term "antibody drug conjugate" or "ADC" refers to an antibody conjugated to one or more cytotoxic or cytostatic agents, such as chemotherapeutic agents, drugs, growth inhibitors, toxins (e.g., protein toxins, enzymatically active toxins or fragments thereof derived from bacteria, fungi, plants or animals), or radioisotopes (i.e., radioconjugates). Immunoconjugates have been used for the localized delivery of cytotoxic agents, i.e., drugs that kill cells or inhibit cell growth or proliferation, in the treatment of cancer (e.g., Liu et al., Proc Natl. Acad. Sci. (1996), 93, 8618-8623). Immunoconjugates allow targeted delivery of and intracellular accumulation of drug moieties to tumors, where systemic administration of unconjugated drugs may result in unacceptable levels of toxicity to normal cells and / or tissues. Toxins used in antibody-toxin conjugates include bacterial toxins, such as diphtheria toxin, plant toxins, such as ricin, and small molecule toxins, such as geldanamycin. Toxins may exert their cytotoxic effects by mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition.

[0061] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may also be referred to by their commonly accepted one-letter symbols.

[0062] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures and vectors that are integrated into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0063] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which at least one exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants," "transformed cells," "transfectants," "transfected cells," and "transduced cells," including the primary transformed / transfected / transduced cell and its progeny, regardless of the number of passages. Progeny may not be completely identical in nucleic acid content to the parent cell, and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0064] The term "therapeutically effective amount" as used herein is intended to mean an amount of therapeutic or prophylactic antibody that, when administered in accordance with a desired treatment regimen, is adequate to elicit a desired therapeutic or prophylactic effect or response, including alleviating some or all of such symptoms of a disease or reducing predisposition to a disease.

[0065] The term "pharmaceutical formulation" / "pharmaceutical composition" refers to a preparation (formulation) that is in a form that allows the biological activity of the active ingredient contained therein to be effective and that does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered.

[0066] Antibodies according to the invention In one embodiment, the present invention relates to an isolated antibody or antigen-binding fragment thereof that binds to human Sema3A, wherein the isolated antibody or antigen-binding fragment thereof binds to human Sema3A of sequence SEQ ID NO: 600 with a dissociation constant (KD) of 50 nM or less, 20 nM or less, 10 nM or less, 1 nM or less, or 0.1 nM or less. In particular, the isolated antibody or antigen-binding fragment thereof binds to His-tagged human Sema3A domain of SEQ ID NO: 582 with a dissociation constant (KD) of 50 nM or less, 20 nM or less, 10 nM or less, 1 nM or less, or 0.1 nM or less.

[0067] In another aspect, the present invention relates to an isolated antibody or antigen-binding fragment thereof that binds to human Sema3A, wherein the isolated antibody or antigen-binding fragment thereof cross-reacts with mouse, cynomolgus monkey, rat, pig and / or dog Sema3A, in particular, the isolated antibody or antigen-binding fragment thereof binds to mouse, cynomolgus monkey, rat, pig and / or dog Sema3A with a dissociation constant (KD) of 50 nM or less, 20 nM or less, 10 nM or less, 1 nM or less or 0.1 nM or less.

[0068] In certain such embodiments, the affinity is for mouse Sema3A of SEQ ID NO: 601, cynomolgus Sema3A of SEQ ID NO: 602, rat Sema3A of SEQ ID NO: 603, porcine Sema3A of SEQ ID NO: 604, and dog of SEQ ID NO: 605. In certain embodiments, the affinity is for His-tagged mouse Sema3A domain of SEQ ID NO: 583, His-tagged cynomolgus Sema3A domain of SEQ ID NO: 586, His-tagged rat Sema3A domain of SEQ ID NO: 584, His-tagged porcine Sema3A domain of SEQ ID NO: 587, and His-tagged dog Sema3A domain of SEQ ID NO: 585.

[0069] In another aspect, the present invention relates to an isolated antibody or antigen-binding fragment thereof that binds to human Sema3A, the isolated antibody or antigen-binding fragment thereof binds to human Sema3A with a binding activity of 60% or more, 70% or more, 80% or more, or 90% or more as measured by surface plasmon resonance (SPR). In a particular embodiment, the isolated antibody or antigen-binding fragment thereof binds to human Sema3A of sequence SEQ ID NO: 600 with a binding activity of 60% or more, 70% or more, 80% or more, or 90% or more as measured by surface plasmin resonance (SPR). In a particular embodiment, the isolated antibody or antigen-binding fragment thereof binds to the His-tagged human Sema3A domain of sequence SEQ ID NO: 582 with a binding activity of 60% or more, 70% or more, 80% or more, or 90% or more as measured by surface plasmin resonance (SPR).

[0070] In another aspect, the present invention relates to an isolated antibody or antigen-binding fragment thereof that binds to human Sema3A, wherein the isolated antibody or antigen-binding fragment thereof inhibits the activity of human Sema3A of sequence SEQ ID NO: 600 in an in vitro mesangial cell migration assay with an EC50 of 10 nM or less, 5 nM or less, 2.5 nM or less, or 1 nM or less.

[0071] In particular, the isolated antibody or antigen-binding fragment according to the present invention inhibits the activity of human Sema3A of sequence SEQ ID NO: 600 in an in vitro scratch assay, which is described in more detail in Example 9 using human primary mesangial cells.

[0072] In another aspect, the present invention relates to an isolated antibody or antigen-binding fragment thereof that binds to human Sema3A, wherein the isolated antibody or antigen-binding fragment thereof inhibits the activity of human Sema3A of sequence number 600 in an in vitro growth cone collapse assay with an EC50 of 50 nM or less, 25 nM or less, 10 nM or less, or 5 nM or less.

[0073] In particular, an isolated antibody or antigen-binding fragment according to the invention inhibits Sema3A-induced cytoskeletal collapse in an in vitro growth cone collapse assay using mouse dorsal root ganglion (DRG) neurons, as described in further detail in Example 10. The in vitro growth cone assay described in Example 10 is a modified version of the growth cone assay described in Mikule et al. (PMID: 12077190).

[0074] In another aspect, the present invention relates to an isolated antibody or antigen-binding fragment thereof that binds to human Sema3A, wherein the isolated antibody or antigen-binding fragment thereof inhibits the activity of human Sema3A of sequence SEQ ID NO: 600 in an in vitro HUVEC repulsion assay with an EC50 of 1 nM or less, or 0.3 nM or less, 0.1 nM or less, 0.07 nM or less, or 0.06 nM or less.

[0075] In particular, the isolated antibody or antigen-binding fragment according to the present invention inhibits Sema3A-induced cell repulsion in an in vitro repulsion assay using HEK293 cells expressing Sema3A of sequence number 600 seeded onto a confluent monolayer of human umbilical vein endothelial cells (HUVEC) as described in Example 11.

[0076] In another aspect, the present invention relates to an isolated antibody or antigen-binding fragment thereof that binds to Sema3A of sequence SEQ ID NO: 600, wherein the isolated antibody or antigen-binding fragment thereof shows improved potency in a HUVEC repulsion assay, wherein i) the isolated antibody or antigen-binding fragment thereof is selected from the group consisting of TPP-17755 having SEQ ID NO: 81, 85, 97, 98, or TPP-11489 having SEQ ID NO: 1, 5, 17, 18, or TPP-30788 having SEQ ID NO: 800, 804, 810, 811, and exhibits improved potency in a HUVEC repulsion assay compared to TPP-30789 having SEQ ID NOs: 814, 818, 824, 825, or TPP-30790 having SEQ ID NOs: 828, 832, 838, 839, or TPP-30791 having SEQ ID NOs: 842, 846, 852, 853; and ii) the isolated antibody or antigen-binding fragment thereof exhibits improved potency in a HUVEC repulsion assay compared to TPP-17755 having SEQ ID NOs: 81, 85, 97, 98, or TPP-11489 having SEQ ID NOs: 1, 5, 17, 18, or TPP-30791 having SEQ ID NOs: 800, 804, 810, 811. or TPP-30791 having SEQ ID NOs: 842, 846, 852, 853, or TPP-30788, or TPP-30789 having SEQ ID NOs: 814, 818, 824, 825, or TPP-30790 having SEQ ID NOs: 828, 832, 838, 839, or TPP-30791 having SEQ ID NOs: 842, 846, 852, 853, based on EC50 values, preferably exhibits greater than 400 fold (i.e., greater than 400 fold), preferably greater than 50 fold, preferably greater than 5 fold, preferably greater than 2 fold enhanced potency against cellular Sema3A-induced HUVEC repulsion, based on EC50 values, compared to TPP-30788, or TPP-30789 having SEQ ID NOs: 814, 818, 824, 825, or TPP-30790 having SEQ ID NOs: 828, 832, 838, 839, or TPP-30791 having SEQ ID NOs: 842, 846, 852, 853, and iii) the isolated antibody or antigen-binding fragment thereof is and iv) the isolated antibody or antigen-binding fragment thereof has double-digit picomolar activity against human Sema3A in an in vitro HUVEC repulsion assay, but is not more than TPP-17755, TPP-11489, TPP-30788, TPP-30789, TPP-30790 or TPP-30791 having the above sequence.The potency of the prior art antibodies TPP-30790 or TPP-30791 is in the triple digit picomolar or even nanomolar range, and v) the isolated antibody or antigen-binding fragment thereof inhibits the activity of human Sema3A in an in vitro HUVEC repulsion assay as described in Example 11 with an EC50 of 1 nM or less, or 0.3 nM or less, 0.1 nM or less, 0.07 nM or less, or 0.06 nM or less.

[0077] The isolated antibodies or antigen-binding fragments of the present invention show improved potency in HUVEC repulsion assays compared to TPP-30788 to TPP-30791 (BI clones I to IV), which may be due to binding to a different epitope on human Sema3A.

[0078] In another aspect, the present invention relates to an isolated antibody or its antigen-binding fragment that binds to Sema3A, wherein the isolated antibody or its antigen-binding fragment inhibits the activity of Sema3A in vivo.Because the antibody according to the present invention reduces Sema3A-induced urinary albumin excretion.Therefore, in another aspect, the present invention relates to an isolated antibody or its antigen-binding fragment that binds to Sema3A, wherein the isolated antibody or its antigen-binding fragment shows improved Sema3A inhibitory activity in vivo, wherein i) the antibody shows enhanced reduction in Sema3A-induced urinary albumin excretion compared to TPP-30788 (BI clone I); ii) the antibody shows enhanced reduction in Sema3A-induced urinary albumin excretion compared to TPP-17755 (Samsung); and iii) the antibody shows enhanced reduction in Sema3A-induced urinary albumin excretion compared to TPP-11489 (Chiome), as described in Example 12.

[0079] The isolated antibodies or antigen-binding fragments of the present invention show improved efficacy in an in vivo model of induced urinary albumin excretion compared to TPP-30788 to TPP-30791 (BI clones I to IV), which may be due to binding to a different epitope on human Sema3A.

[0080] Thus, in another aspect, the present invention relates to an isolated antibody or antigen-binding fragment thereof that binds to human Sema3A, wherein the isolated antibody or antigen-binding fragment thereof i) exhibits enhanced stability (e.g., enhanced stress stability when diluted to 25 mg / ml in PBS and incubated at 700 rpm for 2 weeks at 40° C.) compared to TPP-30788 (BI clone I); ii) the enhanced stability exhibits an increased amount of monomeric anti-Sema3A antibody compared to TPP-30788 (BI clone I) as measured by SEC; and iii) enhanced stability exhibits an increased amount of monomeric anti-Sema3A antibody compared to TPP-30788 (BI clone I). The enhanced stability indicates a reduction in the percentage of LC and HC of the anti-Sema3A antibody compared to TPP-30788 (BI clone I) as measured by cGE, demonstrating a reduced rate of degradation as measured by the presence of residual LC and HC; iv) enhanced stability indicates that the amount of monomeric anti-Sema3A antibody is maintained (e.g., after incubation at 40°C, 700 rpm for 2 weeks, Δ% monomer=1); v) enhanced stability indicates that the amount of LC and HC of the anti-Sema3A antibody is maintained (e.g., after incubation at 40°C, 700 rpm for 2 weeks, Δ% LC+HC<1).

[0081] Thus, in another aspect, the present invention relates to TPP-23298 that binds to human Sema3A, wherein the isolated antibody or antigen-binding fragment thereof i) exhibits enhanced stability (e.g., enhanced stress stability when diluted to 25 mg / ml in PBS and incubated at 700 rpm at 40° C. for 2 weeks) compared to TPP-30788 (BI clone I); ii) the enhanced stability exhibits an increased amount of monomeric anti-Sema3A antibody compared to TPP-30788 (BI clone I) as measured by SEC; and iii) the enhanced stability exhibits c) shows a decrease in the percentage of LC and HC of the anti-Sema3A antibody compared to TPP-30788 (BI clone I) as measured by GE, demonstrating a decreased rate of degradation as measured by the presence of residual LC and HC; iv) enhanced stability indicates that the amount of monomeric anti-Sema3A antibody is maintained (e.g., after incubation at 40°C, 700 rpm for 2 weeks, Δ% monomer=1); v) enhanced stability indicates that the amount of LC and HC of the anti-Sema3A antibody is maintained (e.g., after incubation at 40°C, 700 rpm for 2 weeks, Δ% LC+HC<1).

[0082] Thus, in another aspect, the present invention relates to an isolated antibody or antigen-binding fragment thereof that binds to human Sema3A, wherein the isolated antibody or antigen-binding fragment thereof i) exhibits increased solubility, ii) the increased solubility is measured in mg / ml after concentration at 90% recovery, iii) the solubility is increased compared to TPP-30788 (BI clone I), iv) the solubility is increased by no more than 1-fold, no more than 1.5-fold, no more than 2-fold compared to TPP-30788 (BI clone I), and v) the increased solubility indicates that the proportion of monomeric anti-Sema3A antibody is not increased after concentration (e.g., Δ% monomer<1 as measured by SEC).

[0083] Thus, in another aspect, the present invention relates to TPP-23298 that binds to human Sema3A, wherein the isolated antibody or antigen-binding fragment thereof i) exhibits increased solubility, ii) the increased solubility is measured in mg / ml after concentration at 90% recovery, iii) the solubility is increased compared to TPP-30788 (BI clone I), iv) the solubility is increased by no more than 1-fold, no more than 1.5-fold, no more than 2-fold compared to TPP-30788 (BI clone I), and v) the increased solubility indicates that the proportion of monomeric anti-Sema3A antibody is not increased after concentration (e.g., Δ% monomer<1 as measured by SEC).

[0084] Thus, in another aspect, the present invention relates to an isolated antibody or antigen-binding fragment thereof that binds to human Sema3A, wherein the isolated antibody or antigen-binding fragment thereof i) exhibits increased viscosity compared to water or PBS, ii) exhibits reduced viscosity in PBS compared to TPP-30788 (BI clone I), and iii) wherein the viscosity is measured by Viscosizer and exhibits a cP value of 5.1 (150 mg / ml).

[0085] Thus, in another aspect, the present invention relates to TPP-23298 that binds to human Sema3A, wherein the isolated antibody or antigen-binding fragment thereof i) exhibits increased viscosity compared to water or PBS, ii) exhibits reduced viscosity in PBS compared to TPP-30788 (BI clone I), and iii) wherein the viscosity is measured by Viscosizer and exhibits a cP value of 5.1 (150 mg / ml).

[0086] In particular, the isolated antibodies or antigen-binding fragments according to the present invention exhibit much higher solubility and stability than TPP-30788, are more resistant to heat stress, and have lower viscosity in PBS buffer, as described in Example 17.

[0087] In particular, TPP-23298 exhibits much higher solubility and stability than TPP-30788, is more resistant to heat stress, and has a lower viscosity in PBS buffer, as described in Example 17.

[0088] In another aspect, the present invention relates to an isolated antibody or antigen-binding fragment thereof that binds to human Sema3A, which can be produced in high titer in mammalian cells, i) wherein high titer is 200 mg / L or less, as described in Example 16.

[0089] In another aspect, the present invention relates to an isolated antibody or antigen-binding fragment thereof that binds to human Sema3A, wherein the antibody shows higher binding selectivity for active Sema3A (TPP-13211) compared to truncated Sema3A TPP-19068, and i) wherein the antibody shows higher binding selectivity for active Sema3A (TPP-13211) compared to the binding selectivity of TPP-30788 to TPP-30791 for active Sema3A, as described in Example 8.

[0090] In another aspect, the present invention relates to TPP-23298 which binds to human Sema3A, wherein the antibody shows higher binding selectivity for active Sema3A (TPP-13211) compared to truncated Sema3A TPP-19068, and i) wherein the antibody shows higher binding selectivity for active Sema3A (TPP-13211) compared to the binding selectivity of TPP-30788 to TPP-30791 for active Sema3A, as described in Example 8.

[0091] In another aspect, the present invention relates to an isolated antibody or antigen-binding fragment thereof that binds to human Sema3A, wherein the antibody binds to a different epitope on Sema3A compared to TPP-30788, i) wherein epitope binding is measured in an SPR assay as described in Example 5a. Any antibody that binds to the same epitope and competes with the binding of the isolated antibody or antigen-binding fragment according to the present invention is included in the present invention.

[0092] In another aspect, the present invention relates to TPP-23298 that binds to human Sema3A, wherein the antibody binds to a different epitope on Sema3A compared to TPP-30788, i) wherein epitope binding is measured in an SPR assay as described in Example 5a. Any antibody that binds to the same epitope and competes with the binding of the isolated antibody or antigen-binding fragment according to the present invention is included in the present invention.

[0093] In another aspect, the present invention relates to an isolated antibody or antigen-binding fragment thereof that competes for binding to Sema3A with an isolated antibody or antigen-binding fragment thereof described in any one of the preceding claims, wherein the isolated antibody or antigen-binding fragment thereof does not compete with the binding to Sema3A of an antibody having SEQ ID NO: 800, SEQ ID NO: 804, SEQ ID NO: 810 or SEQ ID NO: 811.

[0094] An isolated antibody or antigen-binding fragment according to the invention can exhibit any combination of the above characteristics.

[0095] The isolated antibody or antigen-binding fragment according to the present invention is a Sema3A blocking antibody or antigen-binding fragment thereof. In certain embodiments, the antibody specifically and more particularly selectively binds to the Sema3A domain of Semaphorin 3A and prevents the interaction of its receptor Neuropilin 1.

[0096] In a specific embodiment, the isolated antibody or antigen-binding fragment thereof according to the present invention cross-reacts with mouse, cynomolgus monkey, rat, pig and / or dog Sema3A and in particular has an affinity for mouse, cynomolgus monkey, rat, pig and / or dog Sema3A that differs by less than 100-fold, in particular less than 50-fold, more particularly less than 25-fold, even more particularly less than 10-fold, and most particularly less than 5-fold compared to the affinity for human Sema3A.

[0097] In a specific embodiment, the isolated antibody or antigen-binding fragment thereof according to the present invention does not significantly cross-react with human Sema3B, Sema3C, Sema3D, Sema3E, Sema3F and / or Sema3G. In particular, the isolated antibody or antigen-binding fragment thereof does not significantly cross-react with human Sema3G.

[0098] In a specific embodiment, the isolated antibody or antigen-binding fragment thereof according to the invention inhibits Sema3A-induced albuminuria and / or proteinuria.

[0099] In certain embodiments, the isolated antibody or antigen-binding fragment thereof according to the invention inhibits Sema3A-induced fibrosis.

[0100] In certain embodiments, an isolated antibody or antigen-binding fragment according to the invention comprises a heavy chain variable domain that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO:141 and a light chain variable domain that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO:145.

[0101] In certain other embodiments, an isolated antibody or antigen-binding fragment according to the invention comprises a heavy chain variable domain that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO:61 and a light chain variable domain that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO:65.

[0102] In a specific embodiment, an isolated antibody or antigen-binding fragment according to the invention comprises a heavy chain antigen-binding region comprising an H-CDR3 comprising the sequence RDDYTSRDAFDX (SEQ ID NO:594), where X is selected from the group consisting of Y and V, and in particular, X is Y.

[0103] In a specific embodiment, an isolated antibody or antigen-binding fragment according to the invention comprises a light chain antigen-binding region comprising an L-CDR3 comprising the sequence X1AWDDSLNX2X3X4V (SEQ ID NO:598), where X1 is selected from the group consisting of A and H, X2 is selected from the group consisting of V, D and G, in particular, X2 is selected from the group consisting of V and D, X3 is selected from the group consisting of I and Y, and X4 is selected from the group consisting of P and V.

[0104] In a specific embodiment, an isolated antibody or antigen-binding fragment according to the invention comprises a heavy chain antigen-binding region comprising an H-CDR3 as defined above, and a light chain antigen-binding region comprising an L-CDR3 as defined above.

[0105] In a specific embodiment, an isolated antibody or antigen-binding fragment according to the invention comprises a heavy chain antigen-binding region comprising an H-CDR3 comprising the sequence SGYSSSWFDPDFDY (SEQ ID NO:64).

[0106] In a specific embodiment, an isolated antibody or antigen-binding fragment according to the invention comprises a light chain antigen-binding region comprising an L-CDR3 comprising the sequence X1SYX2GX3NPYVV (SEQ ID NO:599), where X1 is selected from the group consisting of S and Q, X2 is selected from the group consisting of E and A, and in particular, X3 is selected from the group consisting of P, I and S.

[0107] In a specific embodiment, an isolated antibody or antigen-binding fragment according to the invention comprises a heavy chain antigen-binding region comprising an H-CDR3 as defined above, and a light chain antigen-binding region comprising an L-CDR3 as defined above.

[0108] In a particular embodiment, an isolated antibody or antigen-binding fragment according to the invention comprises a heavy chain antigen-binding region comprising an H-CDR1 comprising the sequence SYX1MX2 (SEQ ID NO:588), where X1 is selected from G and A, and X2 is selected from H, S and L. In particular, the heavy chain antigen-binding region comprises an H-CDR1 comprising the sequence SYAMX (SEQ ID NO:589), where X is selected from S and L.

[0109] In a particular embodiment, an isolated antibody or antigen-binding fragment according to the invention comprises a heavy chain antigen-binding region comprising an H-CDR2 comprising the sequence AIGX1GGDTYYADSVX2G (SEQ ID NO:590), where X1 is selected from T and Y, and X2 is selected from K and M. In particular, the heavy chain antigen-binding region comprises an H-CDR2 comprising the sequence AIGXGGDTYYADSVKG (SEQ ID NO:591), where X is selected from T and Y.

[0110] In a particular embodiment, an isolated antibody or antigen-binding fragment according to the invention comprises a heavy chain antigen-binding region comprising an H-CDR3 comprising the sequence RDDYTSRDAFDX (SEQ ID NO:594), where X is selected from the group consisting of Y and V. In particular, X is Y.

[0111] In a specific embodiment, an isolated antibody or antigen-binding fragment according to the invention comprises a heavy chain antigen-binding region comprising H-CDR1, H-CDR2 and H-CDR3 as defined above.

[0112] In a specific embodiment, an isolated antibody or antigen-binding fragment according to the invention comprises a light chain antigen-binding region comprising an L-CDR1 comprising the sequence SGSSSNIGSNTVN (SEQ ID NO:46).

[0113] In a particular embodiment, an isolated antibody or antigen-binding fragment according to the invention comprises a light chain antigen-binding region comprising an L-CDR2 comprising the sequence YDDLXPS (SEQ ID NO:596), where X is selected from L and R. In particular, the light chain antigen-binding region comprises an L-CDR2 comprising the sequence YDDLRPS (SEQ ID NO:127).

[0114] In a specific embodiment, an isolated antibody or antigen-binding fragment according to the invention comprises a light chain antigen-binding region comprising an L-CDR3 comprising the sequence X1AWDDSLNX2X3X4V (SEQ ID NO:598), where X1 is selected from the group consisting of A and H, X2 is selected from the group consisting of V, D and G, in particular, X2 is selected from the group consisting of V and D, X3 is selected from the group consisting of I and Y, and X4 is selected from the group consisting of P and V.

[0115] In a particular embodiment, an isolated antibody or antigen-binding fragment according to the invention comprises a light chain antigen-binding region comprising L-CDR1, L-CDR2 and L-CDR3 as defined above.

[0116] In a specific embodiment, an isolated antibody or antigen-binding fragment according to the invention comprises a heavy chain antigen-binding region comprising H-CDR1, H-CDR2 and H-CDR3 as defined above, and a light chain antigen-binding region comprising L-CDR1, L-CDR2 and L-CDR3 as defined above.

[0117] In certain such embodiments, the amino acid residue immediately adjacent to the 5' end of the H-CDR1 (corresponding to residue 30 of the reference VH domain of SEQ ID NO:121) is S or Y.

[0118] In certain other embodiments, an isolated antibody or antigen-binding fragment according to the invention comprises a heavy chain antigen-binding region comprising an H-CDR1 comprising the sequence SYEMN (SEQ ID NO:62).

[0119] In a particular embodiment, an isolated antibody or antigen-binding fragment according to the invention comprises a heavy chain antigen-binding region comprising an H-CDR2 comprising the sequence GISWWNSGX1IX2YADSVKG (SEQ ID NO:592), where X1 is selected from W and S, and X2 is selected from G and D. In particular, the heavy chain antigen-binding region comprises an H-CDR2 comprising the sequence GISWWNSGWIXYADSVKG (SEQ ID NO:593), where X is selected from G and D.

[0120] In a specific embodiment, an isolated antibody or antigen-binding fragment according to the invention comprises a heavy chain antigen-binding region comprising an H-CDR3 comprising the sequence SGYSSSWFDPDFDY (SEQ ID NO:64).

[0121] In a specific embodiment, an isolated antibody or antigen-binding fragment according to the invention comprises a heavy chain antigen-binding region comprising H-CDR1, H-CDR2 and H-CDR3 as defined above.

[0122] In a specific embodiment, an isolated antibody or antigen-binding fragment according to the invention comprises a light chain antigen-binding region comprising an L-CDR1 comprising the sequence TGSSSXIGAGYDVH (SEQ ID NO:595), where X is selected from N and D.

[0123] In a specific embodiment, an isolated antibody or antigen-binding fragment according to the invention comprises a light chain antigen-binding region comprising an L-CDR2 comprising the sequence GXSNRPS (SEQ ID NO:597), where X is selected from N and A.

[0124] In a particular embodiment, an isolated antibody or antigen-binding fragment according to the invention comprises a light chain antigen-binding region comprising an L-CDR3 comprising the sequence X1SYX2GX3NPYVV (SEQ ID NO:599), where X1 is selected from the group consisting of S and Q, X2 is selected from the group consisting of E and A, and X3 is selected from the group consisting of P, I and S. In particular, X3 is selected from the group consisting of P and I.

[0125] In a particular embodiment, an isolated antibody or antigen-binding fragment according to the invention comprises a light chain antigen-binding region comprising L-CDR1, L-CDR2 and L-CDR3 as defined above.

[0126] In a specific embodiment, an isolated antibody or antigen-binding fragment according to the invention comprises a heavy chain antigen-binding region comprising H-CDR1, H-CDR2 and H-CDR3 as defined above, and a light chain antigen-binding region comprising L-CDR1, L-CDR2 and L-CDR3 as defined above.

[0127] In certain such embodiments, the three amino acid residues immediately adjacent to the 5' end of H-CDR1 (corresponding to residues 28-30 of the reference VH domain of SEQ ID NO: 101) are X1FX2, where X1 is selected from T and D and X2 is selected from S and D.

[0128] In certain embodiments, an isolated antibody or antigen-binding fragment according to the invention comprises: i) a heavy chain antigen-binding region comprising an H-CDR3 comprising SEQ ID NO: 44 and a light chain antigen-binding region comprising an L-CDR3 comprising SEQ ID NO: 48; or ii) a heavy chain antigen-binding region comprising an H-CDR3 comprising SEQ ID NO:64 and a light chain antigen-binding region comprising an L-CDR3 comprising SEQ ID NO:68; or

[0129] iii) a heavy chain antigen-binding region comprising an H-CDR3 comprising SEQ ID NO: 104 and a light chain antigen-binding region comprising an L-CDR3 comprising SEQ ID NO: 108; or iv) a heavy chain antigen-binding region comprising an H-CDR3 comprising SEQ ID NO: 124 and a light chain antigen-binding region comprising an L-CDR3 comprising SEQ ID NO: 128; or v) a heavy chain antigen-binding region comprising an H-CDR3 comprising SEQ ID NO: 144 and a light chain antigen-binding region comprising an L-CDR3 comprising SEQ ID NO: 148; or vi) a heavy chain antigen-binding region comprising an H-CDR3 comprising SEQ ID NO: 164 and a light chain antigen-binding region comprising an L-CDR3 comprising SEQ ID NO: 168; or vii) a heavy chain antigen-binding region comprising an H-CDR3 comprising SEQ ID NO: 184 and a light chain antigen-binding region comprising an L-CDR3 comprising SEQ ID NO: 188; or viii) a heavy chain antigen-binding region comprising an H-CDR3 comprising SEQ ID NO: 204 and a light chain antigen-binding region comprising an L-CDR3 comprising SEQ ID NO: 208; or ix) a heavy chain antigen-binding region comprising an H-CDR3 comprising SEQ ID NO: 224 and a light chain antigen-binding region comprising an L-CDR3 comprising SEQ ID NO: 228; or x) a heavy chain antigen-binding region comprising an H-CDR3 comprising SEQ ID NO: 244 and a light chain antigen-binding region comprising an L-CDR3 comprising SEQ ID NO: 248; or xi) a heavy chain antigen-binding region comprising an H-CDR3 comprising SEQ ID NO:264 and a light chain antigen-binding region comprising an L-CDR3 comprising SEQ ID NO:268; or xii) a heavy chain antigen-binding region comprising an H-CDR3 comprising SEQ ID NO:284 and a light chain antigen-binding region comprising an L-CDR3 comprising SEQ ID NO:288; or xiii) a heavy chain antigen-binding region comprising an H-CDR3 comprising SEQ ID NO: 304 and a light chain antigen-binding region comprising an L-CDR3 comprising SEQ ID NO: 308; or xiv) a heavy chain antigen-binding region comprising an H-CDR3 comprising SEQ ID NO: 324 and a light chain antigen-binding region comprising an L-CDR3 comprising SEQ ID NO: 328; or xv) a heavy chain antigen-binding region comprising an H-CDR3 comprising SEQ ID NO: 344 and a light chain antigen-binding region comprising an L-CDR3 comprising SEQ ID NO: 348; or xvi) a heavy chain antigen-binding region comprising an H-CDR3 comprising SEQ ID NO: 364 and a light chain antigen-binding region comprising an L-CDR3 comprising SEQ ID NO: 368; or xvii) a heavy chain antigen-binding region comprising an H-CDR3 comprising SEQ ID NO: 384 and a light chain antigen-binding region comprising an L-CDR3 comprising SEQ ID NO: 388; or xviii) a heavy chain antigen-binding region comprising an H-CDR3 comprising SEQ ID NO: 404 and a light chain antigen-binding region comprising an L-CDR3 comprising SEQ ID NO: 408; or xix) a heavy chain antigen-binding region comprising an H-CDR3 comprising SEQ ID NO: 424 and a light chain antigen-binding region comprising an L-CDR3 comprising SEQ ID NO: 428; or xx) a heavy chain antigen-binding region comprising an H-CDR3 comprising SEQ ID NO: 444 and a light chain antigen-binding region comprising an L-CDR3 comprising SEQ ID NO: 448; or xxi) a heavy chain antigen-binding region comprising an H-CDR3 comprising SEQ ID NO: 464 and a light chain antigen-binding region comprising an L-CDR3 comprising SEQ ID NO: 468; or xxii) a heavy chain antigen-binding region comprising an H-CDR3 comprising SEQ ID NO: 484 and a light chain antigen-binding region comprising an L-CDR3 comprising SEQ ID NO: 488; or xxiii) a heavy chain antigen-binding region comprising an H-CDR3 comprising SEQ ID NO: 504 and a light chain antigen-binding region comprising an L-CDR3 comprising SEQ ID NO: 508; or xxiv) a heavy chain antigen-binding region comprising an H-CDR3 comprising SEQ ID NO: 524 and a light chain antigen-binding region comprising an L-CDR3 comprising SEQ ID NO: 528; or xxv) a heavy chain antigen-binding region comprising an H-CDR3 comprising SEQ ID NO: 544 and a light chain antigen-binding region comprising an L-CDR3 comprising SEQ ID NO: 548; or xxvi) A heavy chain antigen-binding region comprising an H-CDR3 comprising SEQ ID NO:564 and a light chain antigen-binding region comprising an L-CDR3 comprising SEQ ID NO:568.

[0130] In certain embodiments, an isolated antibody or antigen-binding fragment according to the invention comprises a heavy chain antigen-binding region comprising an H-CDR1 comprising any one of SEQ ID NOs: 42, 62, 102, 122, 142, 162, 182, 202, 222, 242, 262, 282, 302, 322, 342, 362, 382, ​​402, 422, 442, 462, 482, 502, 522, 542 and 562.

[0131] In certain embodiments, an isolated antibody or antigen-binding fragment according to the invention comprises a heavy chain antigen-binding region comprising an H-CDR2 comprising any one of SEQ ID NOs: 43, 63, 103, 123, 143, 163, 183, 203, 223, 243, 263, 283, 303, 323, 343, 363, 383, 403, 423, 443, 463, 483, 503, 523, 543 and 563.

[0132] In certain embodiments, an isolated antibody or antigen-binding fragment according to the invention comprises a light chain antigen-binding region comprising an L-CDR1 comprising any one of SEQ ID NOs: 46, 66, 106, 126, 146, 166, 186, 206, 226, 246, 266, 286, 306, 326, 346, 366, 386, 406, 426, 446, 466, 486, 506, 526, 546 and 566.

[0133] In certain embodiments, an isolated antibody or antigen-binding fragment according to the invention comprises a light chain antigen-binding region comprising an L-CDR2 comprising any one of SEQ ID NOs: 47, 67, 107, 127, 147, 167, 187, 207, 227, 247, 267, 287, 307, 327, 347, 367, 387, 407, 427, 447, 467, 487, 507, 527, 547 and 567.

[0134] In certain embodiments, an isolated antibody or antigen-binding fragment according to the invention comprises: i) a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO: 42, H-CDR2 comprising SEQ ID NO: 43 and H-CDR3 comprising SEQ ID NO: 44, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO: 46, L-CDR2 comprising SEQ ID NO: 47 and L-CDR3 comprising SEQ ID NO: 48; or ii) a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO: 62, H-CDR2 comprising SEQ ID NO: 63 and H-CDR3 comprising SEQ ID NO: 64, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO: 66, L-CDR2 comprising SEQ ID NO: 67 and L-CDR3 comprising SEQ ID NO: 68; or iii) a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO: 102, H-CDR2 comprising SEQ ID NO: 103 and H-CDR3 comprising SEQ ID NO: 104, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO: 106, L-CDR2 comprising SEQ ID NO: 107 and L-CDR3 comprising SEQ ID NO: 108; or iv) a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO: 122, H-CDR2 comprising SEQ ID NO: 123 and H-CDR3 comprising SEQ ID NO: 124, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO: 126, L-CDR2 comprising SEQ ID NO: 127 and L-CDR3 comprising SEQ ID NO: 128; or v) a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO: 142, H-CDR2 comprising SEQ ID NO: 143 and H-CDR3 comprising SEQ ID NO: 144, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO: 146, L-CDR2 comprising SEQ ID NO: 147 and L-CDR3 comprising SEQ ID NO: 148; or vi) a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO: 162, H-CDR2 comprising SEQ ID NO: 163 and H-CDR3 comprising SEQ ID NO: 164, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO: 166, L-CDR2 comprising SEQ ID NO: 167 and L-CDR3 comprising SEQ ID NO: 168; or vii) a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO: 182, H-CDR2 comprising SEQ ID NO: 183 and H-CDR3 comprising SEQ ID NO: 184, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO: 186, L-CDR2 comprising SEQ ID NO: 187 and L-CDR3 comprising SEQ ID NO: 188; or viii) a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO: 202, H-CDR2 comprising SEQ ID NO: 203 and H-CDR3 comprising SEQ ID NO: 204, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO: 206, L-CDR2 comprising SEQ ID NO: 207 and L-CDR3 comprising SEQ ID NO: 208; or ix) a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO: 222, H-CDR2 comprising SEQ ID NO: 223 and H-CDR3 comprising SEQ ID NO: 224, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO: 226, L-CDR2 comprising SEQ ID NO: 227 and L-CDR3 comprising SEQ ID NO: 228; or x) a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO: 242, H-CDR2 comprising SEQ ID NO: 243 and H-CDR3 comprising SEQ ID NO: 244, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO: 246, L-CDR2 comprising SEQ ID NO: 247 and L-CDR3 comprising SEQ ID NO: 248; or xi) a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO: 262, H-CDR2 comprising SEQ ID NO: 263 and H-CDR3 comprising SEQ ID NO: 264, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO: 266, L-CDR2 comprising SEQ ID NO: 267 and L-CDR3 comprising SEQ ID NO: 268; or xii) a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO:282, H-CDR2 comprising SEQ ID NO:283 and H-CDR3 comprising SEQ ID NO:284, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO:286, L-CDR2 comprising SEQ ID NO:287 and L-CDR3 comprising SEQ ID NO:288; or xiii) a heavy chain antigen-binding region comprising an H-CDR1 comprising SEQ ID NO: 302, an H-CDR2 comprising SEQ ID NO: 303, and an H-CDR3 comprising SEQ ID NO: 304, and a light chain antigen-binding region comprising an L-CDR1 comprising SEQ ID NO: 306, an L-CDR2 comprising SEQ ID NO: 307, and an L-CDR3 comprising SEQ ID NO: 308; or xiv) a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO: 322, H-CDR2 comprising SEQ ID NO: 323 and H-CDR3 comprising SEQ ID NO: 324, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO: 326, L-CDR2 comprising SEQ ID NO: 327 and L-CDR3 comprising SEQ ID NO: 328; or xv) a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO: 342, H-CDR2 comprising SEQ ID NO: 343 and H-CDR3 comprising SEQ ID NO: 344, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO: 346, L-CDR2 comprising SEQ ID NO: 347 and L-CDR3 comprising SEQ ID NO: 348; or xvi) a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO: 362, H-CDR2 comprising SEQ ID NO: 363 and H-CDR3 comprising SEQ ID NO: 364, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO: 366, L-CDR2 comprising SEQ ID NO: 367 and L-CDR3 comprising SEQ ID NO: 368; or xvii) a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO: 382, ​​H-CDR2 comprising SEQ ID NO: 383, and H-CDR3 comprising SEQ ID NO: 384, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO: 386, L-CDR2 comprising SEQ ID NO: 387, and L-CDR3 comprising SEQ ID NO: 388; or xviii) a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO: 402, H-CDR2 comprising SEQ ID NO: 403 and H-CDR3 comprising SEQ ID NO: 404, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO: 406, L-CDR2 comprising SEQ ID NO: 407 and L-CDR3 comprising SEQ ID NO: 408; or xix) a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO: 422, H-CDR2 comprising SEQ ID NO: 423 and H-CDR3 comprising SEQ ID NO: 424, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO: 426, L-CDR2 comprising SEQ ID NO: 427 and L-CDR3 comprising SEQ ID NO: 428; or xx) a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO: 442, H-CDR2 comprising SEQ ID NO: 443 and H-CDR3 comprising SEQ ID NO: 444, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO: 446, L-CDR2 comprising SEQ ID NO: 447 and L-CDR3 comprising SEQ ID NO: 448; or xxi) a heavy chain antigen-binding region comprising an H-CDR1 comprising SEQ ID NO: 462, an H-CDR2 comprising SEQ ID NO: 463, and an H-CDR3 comprising SEQ ID NO: 464, and a light chain antigen-binding region comprising an L-CDR1 comprising SEQ ID NO: 466, an L-CDR2 comprising SEQ ID NO: 467, and an L-CDR3 comprising SEQ ID NO: 468; or xxii) a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO: 482, H-CDR2 comprising SEQ ID NO: 483 and H-CDR3 comprising SEQ ID NO: 484, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO: 486, L-CDR2 comprising SEQ ID NO: 487 and L-CDR3 comprising SEQ ID NO: 488; or xxiii) a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO: 502, H-CDR2 comprising SEQ ID NO: 503 and H-CDR3 comprising SEQ ID NO: 504, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO: 506, L-CDR2 comprising SEQ ID NO: 507 and L-CDR3 comprising SEQ ID NO: 508; or xxiv) a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO: 522, H-CDR2 comprising SEQ ID NO: 523 and H-CDR3 comprising SEQ ID NO: 524, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO: 526, L-CDR2 comprising SEQ ID NO: 527 and L-CDR3 comprising SEQ ID NO: 528; or xxv) a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO: 542, H-CDR2 comprising SEQ ID NO: 543 and H-CDR3 comprising SEQ ID NO: 544, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO: 546, L-CDR2 comprising SEQ ID NO: 547 and L-CDR3 comprising SEQ ID NO: 548; or xxvi) A heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO: 562, H-CDR2 comprising SEQ ID NO: 563 and H-CDR3 comprising SEQ ID NO: 564, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO: 566, L-CDR2 comprising SEQ ID NO: 567 and L-CDR3 comprising SEQ ID NO: 568.

[0135] In certain embodiments, an isolated antibody or antigen-binding fragment according to the invention comprises: i) a variable heavy chain domain comprising SEQ ID NO: 41 and a variable light chain domain comprising SEQ ID NO: 45; or ii) a variable heavy chain domain comprising SEQ ID NO: 61 and a variable light chain domain comprising SEQ ID NO: 65; or iii) a variable heavy chain domain comprising SEQ ID NO: 101 and a variable light chain domain comprising SEQ ID NO: 105; or iv) a variable heavy chain domain comprising SEQ ID NO: 121 and a variable light chain domain comprising SEQ ID NO: 125; or v) a variable heavy chain domain comprising SEQ ID NO: 141 and a variable light chain domain comprising SEQ ID NO: 145; or vi) a variable heavy chain domain comprising SEQ ID NO: 161 and a variable light chain domain comprising SEQ ID NO: 165; or vii) a variable heavy chain domain comprising SEQ ID NO: 181 and a variable light chain domain comprising SEQ ID NO: 185; or viii) a variable heavy chain domain comprising SEQ ID NO: 201 and a variable light chain domain comprising SEQ ID NO: 205; or ix) a variable heavy chain domain comprising SEQ ID NO: 221 and a variable light chain domain comprising SEQ ID NO: 225; or x) a variable heavy chain domain comprising SEQ ID NO: 241 and a variable light chain domain comprising SEQ ID NO: 245; or xi) a variable heavy chain domain comprising SEQ ID NO: 261 and a variable light chain domain comprising SEQ ID NO: 265; or xii) a variable heavy chain domain comprising SEQ ID NO: 281 and a variable light chain domain comprising SEQ ID NO: 285; or xiii) a variable heavy chain domain comprising SEQ ID NO: 301 and a variable light chain domain comprising SEQ ID NO: 305; or xiv) a variable heavy chain domain comprising SEQ ID NO: 321 and a variable light chain domain comprising SEQ ID NO: 325; or xv) a variable heavy chain domain comprising SEQ ID NO: 341 and a variable light chain domain comprising SEQ ID NO: 345; or xvi) a variable heavy chain domain comprising SEQ ID NO: 361 and a variable light chain domain comprising SEQ ID NO: 365; or xvii) a variable heavy chain domain comprising SEQ ID NO: 381 and a variable light chain domain comprising SEQ ID NO: 385; or xviii) a variable heavy chain domain comprising SEQ ID NO: 401 and a variable light chain domain comprising SEQ ID NO: 405; or xix) a variable heavy chain domain comprising SEQ ID NO: 421 and a variable light chain domain comprising SEQ ID NO: 425; or xx) a variable heavy chain domain comprising SEQ ID NO: 441 and a variable light chain domain comprising SEQ ID NO: 445; or xxi) a variable heavy chain domain comprising SEQ ID NO: 461 and a variable light chain domain comprising SEQ ID NO: 465; or xxii) a variable heavy chain domain comprising SEQ ID NO: 481 and a variable light chain domain comprising SEQ ID NO: 485; or xxiii) a variable heavy chain domain comprising SEQ ID NO: 501 and a variable light chain domain comprising SEQ ID NO: 505; or xxiv) a variable heavy chain domain comprising SEQ ID NO: 521 and a variable light chain domain comprising SEQ ID NO: 525; or xxv) a variable heavy chain domain comprising SEQ ID NO: 541 and a variable light chain domain comprising SEQ ID NO: 545; or xxvi) A variable heavy domain comprising SEQ ID NO: 561 and a variable light domain comprising SEQ ID NO: 565.

[0136] In certain embodiments, the isolated antibody according to the invention is an IgG antibody. In certain such embodiments, the isolated antibody according to the invention is an IgG1, IgG2, IgG3 or IgG4 antibody. Most particularly, the isolated antibody according to the invention is an IgG1 or IgG4 antibody.

[0137] In certain embodiments, an isolated antibody according to the invention comprises: i) a heavy chain comprising SEQ ID NO:57 and a light chain comprising SEQ ID NO:58; or ii) a heavy chain comprising SEQ ID NO: 77 and a light chain comprising SEQ ID NO: 78; or iii) a heavy chain comprising SEQ ID NO: 117 and a light chain comprising SEQ ID NO: 118; or iv) a heavy chain comprising SEQ ID NO: 137 and a light chain comprising SEQ ID NO: 138; or v) a heavy chain comprising SEQ ID NO: 157 and a light chain comprising SEQ ID NO: 158; or vi) a heavy chain comprising SEQ ID NO: 177 and a light chain comprising SEQ ID NO: 178; or vii) a heavy chain comprising SEQ ID NO: 197 and a light chain comprising SEQ ID NO: 198; or viii) a heavy chain comprising SEQ ID NO:217 and a light chain comprising SEQ ID NO:218; or ix) a heavy chain comprising SEQ ID NO: 237 and a light chain comprising SEQ ID NO: 238; or x) a heavy chain comprising SEQ ID NO: 257 and a light chain comprising SEQ ID NO: 258; or xi) a heavy chain comprising SEQ ID NO:277 and a light chain comprising SEQ ID NO:278; or xii) a heavy chain comprising SEQ ID NO:297 and a light chain comprising SEQ ID NO:298; or xiii) a heavy chain comprising SEQ ID NO: 317 and a light chain comprising SEQ ID NO: 318; or xiv) a heavy chain comprising SEQ ID NO: 337 and a light chain comprising SEQ ID NO: 338; or xv) a heavy chain comprising SEQ ID NO: 357 and a light chain comprising SEQ ID NO: 358; or xvi) a heavy chain comprising SEQ ID NO: 377 and a light chain comprising SEQ ID NO: 378; or xvii) a heavy chain comprising SEQ ID NO: 397 and a light chain comprising SEQ ID NO: 398; or xviii) a heavy chain comprising SEQ ID NO: 417 and a light chain comprising SEQ ID NO: 418; or xix) a heavy chain comprising SEQ ID NO: 437 and a light chain comprising SEQ ID NO: 438; or xx) a heavy chain comprising SEQ ID NO: 457 and a light chain comprising SEQ ID NO: 458; or xxi) a heavy chain comprising SEQ ID NO: 477 and a light chain comprising SEQ ID NO: 478; or xxii) a heavy chain comprising SEQ ID NO: 497 and a light chain comprising SEQ ID NO: 498; or xxiii) a heavy chain comprising SEQ ID NO: 517 and a light chain comprising SEQ ID NO: 518; or xxiv) a heavy chain comprising SEQ ID NO: 537 and a light chain comprising SEQ ID NO: 538; or xxv) a heavy chain comprising SEQ ID NO: 557 and a light chain comprising SEQ ID NO: 558; or xxvi) A heavy chain comprising SEQ ID NO: 577 and a light chain comprising SEQ ID NO: 578.

[0138] In certain embodiments, an antigen-binding fragment according to the invention is an scFv, Fab, Fab' fragment or F(ab')2 fragment.

[0139] In certain embodiments, the isolated antibody or antigen-binding fragment according to the invention is a monoclonal antibody or antigen-binding fragment thereof.

[0140] In certain embodiments, the isolated antibody or antigen-binding fragment according to the invention is a human, humanized or chimeric antibody or antigen-binding fragment thereof, more particularly, a fully human antibody or antigen-binding fragment thereof.

[0141] In certain embodiments, the isolated antibody or antigen-binding fragment according to the invention is a monospecific antibody. In certain other embodiments, the isolated antibody or antigen-binding fragment according to the invention is a multispecific antibody, e.g., a bispecific, trispecific, or tetraspecific antibody, that binds to Sema3A and at least one additional antigen.

[0142] In another aspect, the present invention relates to an isolated antibody or antigen-binding fragment thereof that competes with the isolated antibody or antigen-binding fragment according to the present invention for binding to human Sema3A.

[0143] In another aspect, the present invention relates to an antibody conjugate comprising the isolated antibody or antigen-binding fragment according to the present invention. For example, the antibody can be conjugated to a cytotoxic agent, an immunotoxin, a toxophore, or a radioisotope. Also provided is an anti-Sema3A antibody conjugated to a detectable marker. Preferred markers are radiolabels, enzymes, chromophores, or fluorophores. The antibody can also be conjugated to a high molecular weight molecule, such as a peptide or protein, such as an interleukin.

[0144] The ADCs according to the present invention comprise an anti-Sema3A antibody conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitors, toxins (e.g., protein toxins, enzymatically active toxins or fragments thereof derived from bacteria, fungi, plants or animals), or radioactive isotopes.

[0145] In one embodiment, an ADC according to the invention comprises an anti-Sema3A antibody as described herein conjugated to one or more drugs, including, but not limited to, a maytansinoid (see U.S. Pat. Nos. 5,208,020, 5,416,064, and European Patent No. EP 0425235); an auristatin, such as monomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483, 5,780,588, and 7,498,298); a dolastatin; a calicheamicin or a derivative thereof; an anthracycline, such as daunomycin or doxorubicin; methotrexate; vindesine; a taxane, such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; a trichothecene; and CC1065.

[0146] In another embodiment, an ADC according to the invention comprises an anti-Sema3A antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, such as, but not limited to, diphtheria A chain, non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alphasarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana protein (P API, P APII and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and a trichothecene.

[0147] In another embodiment, the ACD according to the present invention comprises an anti-Sema3A antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes are available for the production of radioconjugates. Specific examples include 227Th, 225Ac, 211At, 131I, 125I, 90Y, 186Re, 188Re, 153Sm, 212Bi, 32P, 212Pb, and Lu radioisotopes. When a radioconjugate is used for detection, it can comprise a radioactive atom for scintigraphy studies, such as Tc99m, or a spin label for nuclear magnetic resonance (NMR) imaging, such as iodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0148] Conjugates of antibodies and cytotoxic agents can be prepared using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene).

[0149] The linker may be a "cleavable linker" that facilitates release of the cytotoxic drug inside the cell, such as an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker (Chari et al., Cancer Res. 52:127-131 (1992)).

[0150] ACDs according to the present invention include those prepared using cross-linking reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate), which are commercially available from Pierce Biotechnology, Inc., Rockford, Ill., USA.

[0151] The amino acid and nucleic acid sequences of a preferred antibody according to the invention and three prior art antibodies are listed in Tables 1 and 1A.

[0152] Peptide variants The antibody or antigen-binding fragment of the present invention is not limited to the specific peptide sequence provided herein.Rather, the present invention also encompasses variants of these polypeptides.With reference to this disclosure and the techniques and references available in the past, those skilled in the art can produce, test and use functional variants of the antibodies disclosed herein, and it is understood that these variants that have the ability to bind to Sema3A are included within the scope of the present invention.

[0153] Variants can include, for example, antibodies with at least one complementarity determining region (CDR) (hypervariable) and / or framework (FR) (variable) domain / position that is altered compared to the disclosed peptide sequences.

[0154] By modifying one or more amino acid residues in the CDR or FR regions, one skilled in the art can routinely generate mutated or diversified antibody sequences, which can be screened, for example, against the antigen for new or improved properties.

[0155] Another preferred embodiment of the present invention is an antibody or antigen-binding fragment thereof, in which the VH and VL sequences are selected as shown in Table 1 and Table 1A. Those skilled in the art can use the data in Table 1 and Table 1A to design peptide variants that are within the scope of the present invention. The variants are preferably constructed by changing amino acids in one or more CDR regions, and the variants may also have one or more modified framework regions. For example, the peptide FR domains may be altered, in which there is a deviation in residues compared to the germline sequence.

[0156] Alternatively, one skilled in the art could perform the same analysis by comparing the amino acid sequences disclosed herein with known sequences of the same class of such antibodies, for example using the procedures described in Knappik A. et al., JMB 2000, 296:57-86.

[0157] Furthermore, variants can be obtained by using an antibody as a starting point for further optimization, diversifying one or more amino acid residues in the antibody, preferably in one or more CDRs, and screening the resulting collection of antibody variants for variants with improved properties. Particularly preferred is diversifying one or more amino acid residues in CDR3 of VL and / or VH. Diversification can be performed, for example, by synthesizing a collection of DNA molecules using trinucleotide mutagenesis (TRIM) technology (Virnekas B. et al., Nucl. Acids Res. 1994, 22:5600). Antibodies or antigen-binding fragments thereof include molecules with modifications / mutations, including, but not limited to, modifications that result in a change in half-life (e.g., modification of the Fc portion or attachment of an additional molecule such as PEG), modifications that result in a change in binding affinity, or modifications that result in a change in ADCC or CDC activity.

[0158] Conservative amino acid variants Polypeptide variants that retain the overall molecular structure of the antibody peptide sequences described herein may be produced. Some rational substitutions will be recognized by those skilled in the art, taking into account the properties of individual amino acids. Amino acid substitutions, i.e. "conservative substitutions", may be made, for example, on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphipathic nature of the residues involved.

[0159] For example, (a) nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. (b) polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. (c) positively charged (basic) amino acids include arginine, lysine, and histidine. (d) negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Substitutions may typically be made within groups (a)-(d). Glycine and proline may also be substituted for one another based on their ability to disrupt α-helices. Similarly, certain amino acids such as alanine, cysteine, leucine, methionine, glutamic acid, glutamine, histidine and lysine are more commonly found in α-helices, while valine, isoleucine, phenylalanine, tyrosine, tryptophan and threonine are more commonly found in β-pleated sheets. Glycine, serine, aspartic acid, asparagine and proline are found in order of frequency. Some preferred substitutions can be made within the following groups: (i) S and T; (ii) P and G; and (iii) A, V, L and I. With the known genetic code and recombinant and synthetic DNA techniques, skilled scientists can readily construct DNAs that code for conservative amino acid variants.

[0160] Glycosylation variants If the antibody comprises an Fc region, the carbohydrate attached thereto can be modified. Natural antibodies produced by mammalian cells typically contain branched biantennary oligosaccharides that are generally attached by an N-linkage to Asn297 (using Kabat EU numbering) of the CH2 domain of the Fc region. See, e.g., Wright et al., Trends Biotechnol. 15:26-32 (1997).

[0161] In certain embodiments, the antibodies provided herein are modified to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by modifying the expression system (e.g., host cell) and / or by modifying the amino acid sequence such that one or more glycosylation sites are created or removed.

[0162] In one embodiment of the invention, aglycosyl antibodies or antibody derivatives with reduced effector function are produced by expression in prokaryotic hosts. Suitable prokaryotic hosts include, but are not limited to, E. coli, Bacillus subtilis, Salmonella typhimurium, and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus.

[0163] In one embodiment, an antibody variant with reduced effector function is provided, characterized by a modification at a conserved N-linked site in the CH2 domain of the Fc portion of the antibody. In one embodiment of the present invention, the modification comprises a mutation at the heavy chain glycosylation site to prevent glycosylation at that site. Thus, in a preferred embodiment of the present invention, an aglycosyl antibody or antibody derivative is produced by mutation at the heavy chain glycosylation site, i.e., mutation at N297 using Kabat EU numbering, and expressed in a suitable host cell.

[0164] In another embodiment of the present invention, the aglycosyl antibody or antibody derivative has reduced effector function, wherein the modification at the conserved N-linked site in the CH2 domain of the Fc portion of the antibody or antibody derivative comprises removing the CH2 domain glycan, i.e., deglycosylation. These aglycosyl antibodies can be produced by conventional methods and then deglycosylated enzymatically. Methods for the enzymatic deglycosylation of antibodies are well known in the art (e.g., Winkelhake & Nicolson (1976), J Biol Chem. 251 (4): 1074-80).

[0165] In another embodiment of the invention, deglycosylation can be achieved using the glycosylation inhibitor tunicamycin (Nose & Wigzell (1983), Proc Natl Acad Sci USA, 80(21):6632-6). That is, the modification prevents glycosylation at the conserved N-linked site in the CH2 domain of the Fc portion of the antibody.

[0166] In one embodiment, antibody variants are provided that have carbohydrate structures that lack fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be 1%-80%, 1%-65%, 5%-65%, or 20%-40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all glycan structures (e.g., complex, hybrid and high mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry, for example as described in WO 2008 / 077546. Asn297 refers to an asparagine residue located at about position 297 (Eu numbering of Fc region residues) in the Fc region. However, Asn297 can be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function.

[0167] Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include Okazaki et al. J Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004).

[0168] Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells, which are deficient in protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); and WO 2004 / 056312) and knockout cell lines, such as the α-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006)).

[0169] Further provided are antibody variants with bisected oligosaccharides, for example, where the bisected oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003 / 011878, U.S. Patent No. 6,602,684 and US 2005 / 0123546.

[0170] Also provided is an antibody variant that has at least one galactose residue in the oligosaccharide attached to the Fc region. Such an antibody variant can have improved CDC function. Such an antibody variant is described, for example, in WO 1997 / 30087, WO 1998 / 58964 and WO 1999 / 22764.

[0171] FC domain variants In certain embodiments, one or more amino acid modifications (e.g., substitutions) can be introduced into the Fc region of an antibody provided herein (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region), thereby generating an Fc region variant.

[0172] In certain embodiments, the present invention contemplates antibody variants that retain some, but not necessarily all, effector functions, making them desirable candidates for applications where the half-life of the antibody in vivo is important, but certain effector functions (e.g., complement and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / depleted CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to confirm that the antibody lacks FcγR binding (and thus potentially lacks ADCC activity) but retains FcRn binding ability. In some embodiments, modifications are made to the Fc region that result in altered (i.e., improved or decreased) C1q binding and / or complement-dependent cytotoxicity (CDC).

[0173] In certain embodiments, the present invention contemplates antibody variants with increased or decreased half-life. Antibodies with increased half-life and improved binding to neonatal Fc receptor (FcRn), which results in transfer of maternal IgG to the fetus (Guyer et al., J Immunol. 117:587 (1976) and Kim et al., J Immunol. 24:249 (1994)), have been described in US2005 / 0014934 (Hinton et al.). These antibodies comprise an Fc region having one or more substitutions therein that improve binding of the Fc region to FcRn.

[0174] Antibody production The antibody of the present invention can be derived from a recombinant antibody library based on amino acid sequences isolated from the antibodies of a large number of healthy volunteers. For example, using n-CoDeR® technology, fully human CDRs are recombined into new antibody molecules (Carlson & Soderlind, Expert Rev Mol Diagn. 2001 May; 1(1): 102-8). Alternatively, it is possible to isolate Sema3A-specific antibodies using an antibody library, for example as a fully human antibody phage display library as described in Hoet RM et al., Nat Biotechnol 2005; 23(3): 344-8. Antibodies or antibody fragments isolated from a human antibody library are considered herein as human antibodies or human antibody fragments.

[0175] Additionally, human antibodies can be produced by administering immunogens to transgenic animals that have been modified to produce intact human antibodies or intact antibodies containing human variable regions in response to antigenic challenge. Such animals typically contain all or part of the human immunoglobulin loci that replace endogenous immunoglobulin loci or that are present extrachromosomally or randomly integrated into the animal's chromosomes. For example, immunization of genetically engineered mice can be performed, particularly hMAb mice (e.g., VelocImmune Mouse® or XENOMOUSE®).

[0176] Additionally, antibodies can be produced using hybridoma technology (see, e.g., Kohler and Milstein Nature. 1975 Aug. 7;256(5517):495-7), which can provide, for example, mouse, rat or rabbit antibodies which can be converted into chimeric or humanized antibodies. Humanized antibodies and methods for their production are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and described in further detail in: Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Natl Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osboum et al., Methods 36:61-68 (2005), and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing a "guided selection" approach for FR shuffling).

[0177] An example of the generation of antibodies using a recombinant antibody library is provided.

[0178] DNA molecules according to the invention The present invention also relates to isolated nucleic acid sequences encoding the antibodies or antigen-binding fragments according to the invention. The isolated nucleic acid sequences encoding the antibodies or antigen-binding fragments according to the invention can be produced, for example, by the techniques described in Sambrook et al., 1989 and Ausubel et al., 1989, or by chemical synthesis, for example, the techniques described in Oligonucleotide Synthesis (1984, Gait, ed., IRL Press, Oxford). The DNA sequences and respective SEQ ID NOs used for the expressed antibodies are shown in Tables 1 and 1A. These sequences are optimized for mammalian expression in certain cases. The DNA molecules of the present invention are not limited to the sequences disclosed herein, but also include variants thereof. The DNA variants included in the present invention can be described by reference to their physical properties in hybridization. The skilled artisan will recognize that DNA can be used to identify its complement, and that since DNA is double-stranded, it is possible to identify its equivalents or homologs using nucleic acid hybridization techniques. It will also be recognized that hybridization can occur with less than 100% complementarity. However, with appropriate selection of conditions, hybridization techniques can be used to distinguish DNA sequences based on their structural relatedness to a particular probe. For guidance regarding such conditions, see Sambrook et al., 1989 (supra) and Ausubel et al., 1995 (Ausubel, FM, Brent, R., Kingston, RE, Moore, DD, Sedman, JG, Smith, JA, & Struhl, K., eds. (1995). Current Protocols in Molecular Biology. New York: John Wiley and Sons).

[0179] The structural similarity between two polynucleotide sequences can be expressed as a function of the "stringency" of the conditions under which the two sequences hybridize to each other.The term "stringency" as used herein refers to the degree to which the conditions prevent hybridization.Stringent conditions strongly prevent hybridization, and under such conditions, only the most structurally related molecules hybridize to each other.Conversely, non-stringent conditions promote the hybridization of molecules that show a lower degree of structural relatedness.Therefore, the stringency of hybridization directly correlates with the structural relationship of two nucleic acid sequences.

[0180] The stringency of hybridization is a function of many factors, including total DNA concentration, ionic strength, temperature, probe size, and the presence of substances that disrupt hydrogen bonds. Factors that promote hybridization include high DNA concentration, high ionic strength, low temperature, longer probe size, and the absence of substances that disrupt hydrogen bonds. Hybridization is typically carried out in two stages: a "binding" stage and a "washing" stage.

[0181] Functionally equivalent DNA variants Yet another class of DNA variants within the scope of the invention can be described with reference to the products they encode. These functionally equivalent polynucleotides are characterized in that they encode the same peptide sequence because of the degeneracy of the genetic code.

[0182] It will be appreciated that variants of the DNA molecules provided herein can be constructed in several different ways. For example, they can be constructed as fully synthetic DNA. Methods for efficiently synthesizing oligonucleotides are widely available. See Ausubel et al., section 2.11, Supplement 21 (1993). Overlapping oligonucleotides can be synthesized and combined by methods first reported in Khorana et al., J. Mol. Biol. 72:209 217 (1971). See also Ausubel et al., supra, Section 8.2. Synthetic DNA is preferably designed to contain convenient restriction sites engineered at the 5' and 3' ends of the gene to facilitate cloning into an appropriate vector.

[0183] As indicated, the method for making mutants is to start with one of the DNAs disclosed herein and then perform site-directed mutagenesis. See Ausubel et al., supra, chapter 8, Supplement 37 (1997). In a typical method, the target DNA is cloned into a single-stranded DNA bacteriophage vehicle. The single-stranded DNA is isolated and hybridized to an oligonucleotide containing the desired nucleotide alteration. The complementary strand is synthesized and the double-stranded phage is introduced into a host. Some of the resulting progeny will contain the desired mutation, which can be confirmed using DNA sequencing. Also, various methods are available to increase the probability that the progeny phage will be the desired mutation. These methods are well known to those skilled in the art, and kits for making such mutants are commercially available.

[0184] Recombinant DNA constructs and expression The present invention further provides a recombinant DNA construct comprising one or more of the nucleotide sequences according to the invention. The recombinant construct of the present invention may be used in combination with a vector, such as a plasmid, phagemid, phage or viral vector, into which a DNA molecule encoding an antibody or antigen-binding fragment thereof or variant thereof of the present invention is inserted.

[0185] Thus, in one aspect, the present invention relates to a vector comprising a nucleic acid sequence according to the invention.

[0186] The antibody, antigen-binding portion or variant thereof provided herein can be produced by recombinant expression in a host cell of the nucleic acid sequence encoding the light chain and heavy chain or a portion thereof. To recombinantly express the antibody, antigen-binding portion or variant thereof, one or more recombinant expression vectors containing DNA fragments encoding the light chain and / or heavy chain or a portion thereof are transfected into a host cell to express the light chain and heavy chain in the host cell. Standard recombinant DNA methods are used to prepare and / or obtain the nucleic acids encoding the heavy chain and light chain, incorporate these nucleic acids into recombinant expression vectors, and introduce the vectors into the host cell. Such methods are described, for example, in Sambrook, Fritsch and Maniatis (eds.), Molecular Cloning; A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, (1989), Ausubel, FM et al. (eds.), Current Protocols in Molecular Biology, Greene Publishing Associates, (1989), and U.S. Patent No. 4,816,397 to Boss et al.

[0187] Also, the nucleic acid sequences encoding the variable regions of the heavy and / or light chains can be converted to nucleic acid sequences encoding, for example, full-length antibody chains, Fab fragments, or scFvs. The DNA fragments encoding the VL or VH can be operably linked to another DNA fragment encoding, for example, an antibody constant region or a flexible linker (so that the amino acid sequences encoded by the two DNA fragments are in frame). The sequences of human heavy and light chain constant regions are known in the art (see, for example, Kabat, EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification.

[0188] To generate a polynucleotide sequence encoding an scFv, the nucleic acid encoding the VH and VL can be operably linked to another fragment encoding a flexible linker such that the VH and VL sequences are expressed as a continuous single-chain protein containing the VL and VH regions linked by a flexible linker (see, e.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., Nature (1990) 348:552-554).

[0189] Standard recombinant DNA expression methods can be used to express antibodies, their antigen-binding fragments or variants thereof (see, for example, Goeddel; Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). For example, the DNA encoding the desired polypeptide can be inserted into an expression vector, which can then be transfected into a suitable host cell. Suitable host cells are prokaryotic and eukaryotic cells. Examples of prokaryotic host cells include, for example, bacteria, and examples of eukaryotic host cells include yeast, insects and insect cells, plants and plant cells, transgenic animals or mammalian cells. Introduction of the recombinant construct into the host cell can be performed using standard techniques, such as calcium phosphate transfection, DEAE-dextran-mediated transfection, electroporation, transduction or phage infection.

[0190] In some embodiments, the DNA encoding the heavy and light chains is inserted into separate vectors. In other embodiments, the DNA encoding the heavy and light chains is inserted into the same vector. It is understood that the design of the expression vector, including the selection of regulatory sequences, will be influenced by factors such as the choice of host cell, the expression level of the desired protein, and whether expression is constitutive or inducible.

[0191] Therefore, in another aspect, the present invention relates to an isolated cell expressing an antibody or antigen-binding fragment according to the invention and / or comprising a nucleic acid according to the invention or a vector according to the invention.

[0192] The isolated cell can be virtually any cell for which an expression vector is available. The isolated cell can be, for example, a higher eukaryotic host cell, such as a mammalian cell, a lower eukaryotic host cell, such as a yeast cell, or a prokaryotic cell, such as a bacterial cell.

[0193] In another aspect, the invention relates to a method for producing an isolated antibody or antigen-binding fragment according to the invention, comprising culturing a cell according to the invention. In a particular embodiment, the cell according to the invention is cultured under suitable conditions for antibody expression and the antibody or antigen-binding fragment thereof is recovered. In a particular embodiment, the antibody or antigen-binding fragment thereof is purified, in particular to at least 95% homogeneity by weight.

[0194] Bacterial expression Expression vectors useful for bacterial use are constructed by inserting a DNA sequence encoding a desired protein with appropriate translation initiation and termination signals in functional reading phase in the presence of a functional promoter. The vector contains one or more phenotypic selection markers and an origin of replication to ensure maintenance of the vector and, if necessary, provide amplification within the host. Suitable prokaryotic hosts for transformation include, but are not limited to, E. coli, Bacillus subtilis, Salmonella typhimurium, and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus.

[0195] Bacterial vectors can be, for example, bacteriophage, plasmid or phagemid based. These vectors can contain a selection marker and a bacterial origin of replication derived from a commercially available plasmid that typically contains elements of the well-known cloning vector pBR322 (ATCC 37017). After transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is derepressed / induced by appropriate means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time. Typically, cells are harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract is retained for further purification.

[0196] In bacterial systems, a number of expression vectors may be advantageously selected depending on the use intended for the expressed protein, e.g., when producing large quantities of such proteins for the generation of antibodies or screening peptide libraries, vectors which direct the expression of high levels of fusion protein products that are easily purified, for example, may be desirable.

[0197] Thus, one embodiment of the invention is an expression vector comprising a nucleic acid sequence encoding the novel antibody of the invention.

[0198] The antibodies or antigen-binding fragments thereof or variants thereof of the present invention include naturally purified products, products of chemical synthetic methods, and products produced by recombinant techniques from prokaryotic hosts (e.g., including E. coli, Bacillus subtilis, Salmonella typhimurium, and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus).

[0199] Mammalian Expression Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and / or enhancers from cytomegalovirus (CMV) (e.g., CMV promoter / enhancer), Simian Virus 40 (SV40) (e.g., SV40 promoter / enhancer), adenovirus (e.g., adenovirus major late promoter (AdMLP)) and polyoma. Antibody expression can be constitutive or regulated (e.g., inducible by addition or removal of a small molecule inducer such as tetracycline in combination with the Tet system). For further description of viral regulatory elements and sequences thereof, see, for example, US 5,168,062 by Stinski, US 4,510,245 by Bell et al. and US 4,968,615 by Schaffner et al. The recombinant expression vector may also contain an origin of replication and a selection marker (see, e.g., US 4,399,216, 4,634,665 and US 5,179,017). Suitable selection markers include genes that confer resistance to drugs, such as G418, puromycin, hygromycin, blasticidin, zeocin / bleomycin or methotrexate, in a host cell into which the vector has been introduced, or selection markers that utilize auxotrophy, such as glutamine synthetase (Bebbington et al., Biotechnology (NY). 1992 Feb; 10(2): 169-75). For example, the dihydrofolate reductase (DHFR) gene confers resistance to methotrexate, the neo gene confers resistance to G418, the bsd gene from Aspergillus terreus confers resistance to blasticidin, puromycin N-acetyltransferase confers resistance to puromycin, the Sh ble gene product confers resistance to zeocin, and resistance to hygromycin is conferred by the E. coli hygromycin resistance gene (hyg or hph). Also, selection markers such as DHFR or glutamine synthetase are useful in amplification techniques in combination with MTX and MSX.

[0200] Transfection of the expression vector into the host cell can be carried out using standard techniques, such as electroporation, nucleofection, calcium phosphate precipitation, lipofection, polycation-based transfection, such as polyethyleneimine (PEI)-based transfection, and DEAE-dextran transfection.

[0201] Suitable mammalian host cells for expressing the antibodies, antigen-binding fragments thereof or variants thereof provided herein include Chinese Hamster Ovary (CHO cells), such as CHO-K1, CHO-S, CHO-K1SV [DHFR markers, e.g., as described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220 and Urlaub et al., Cell. 1983 Jun;33(2):405-12, used with those described in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol. 159:601-621; and dhfr-CHO cells, as described in Fan et al., Biotechnol Bioeng. 2012 Apr;109(4):1007-15], NS0 myeloma cells, COS cells, HEK293 cells, HKB11 cells, BHK21 cells, CAP cells, EB66 cells and SP2 cells.

[0202] Also, in expression systems such as HEK293, HEK293T, HEK293-EBNA, HEK293E, HEK293-6E, HEK293-Freestyle, HKB11, Expi293F, 293EBNALT75, CHO Freestyle, CHO-S, CHO-K1, CHO-K1SV, CHOEBNALT85, CHOS-XE, CHO-3E7 or CAP-T cells, expression can be transient or semi-stable (e.g., Durocher et al., Nucleic Acids Res. 2002 Jan 15;30(2):E9).

[0203] In some embodiments, the expression vector is designed so that the expressed protein is secreted into the medium that the host cells are grown in. The antibody, antigen-binding fragment thereof, or variant thereof can be recovered from the medium using standard protein purification methods.

[0204] purification The antibody or antigen-binding fragment or variant thereof of the present invention can be recovered and purified from recombinant cell culture by well-known methods, including, but not limited to, ammonium sulfate or ethanol precipitation, acid extraction, protein A chromatography, protein G chromatography, anion or cation exchange chromatography, phospho-cellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. High performance liquid chromatography ("HPLC") can also be used for purification. See, e.g., Colligan, Current Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY, NY, (1997-2001), e.g., Chapters 1, 4, 6, 8, 9, 10 (each of which is incorporated herein by reference in its entirety).

[0205] The antibody or antigen-binding fragment or variant thereof of the present invention includes naturally purified products, products of chemical synthesis methods, and products produced by recombinant techniques from eukaryotic hosts, including, for example, yeast, higher plants, insects, and mammalian cells. Depending on the host used in the recombinant production method, the antibody of the present invention may be glycosylated or aglycosylated. Such methods are described in many standard laboratory manuals, for example, Sambrook (supra), Sections 17.37-17.42; Ausubel (supra), Chapters 10, 12, 13, 16, 18, and 20.

[0206] In preferred embodiments, the antibody is purified to: (1) greater than 95% by weight of the antibody, and in more preferred embodiments, greater than 99% by weight of the antibody, as measured, for example, by the Lowry method, UV-Vis spectroscopy, or SDS-capillary gel electrophoresis (e.g., on a Caliper LabChip GXII, GX90, or Biorad Bioanalyzer instrument); (2) sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence; or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain. An isolated naturally occurring antibody includes an antibody in situ within a recombinant cell, since at least one component of the antibody's natural environment will not be present. Ordinarily, however, an isolated antibody will be prepared by at least one purification step.

[0207] Treatment method The method of treatment includes administering a therapeutically effective amount of the antibody or its antigen-binding fragment or variant contemplated by the present invention to a subject in need of treatment. A "therapeutically effective" amount is defined herein as an amount of the antibody or its antigen-binding fragment, alone or in combination with other substances, as a single dose or according to a multiple dose regimen, sufficient to reduce Sema3A activity in a subject, resulting in the alleviation of a harmful condition, but toxicologically acceptable. The subject may be a human or non-human animal (e.g., rabbit, rat, mouse, dog, monkey or other lower primate).

[0208] Thus, in one aspect, the present invention relates to an isolated antibody or antigen-binding fragment according to the invention, or a conjugate comprising the isolated antibody or antigen-binding fragment according to the invention, or a pharmaceutical composition comprising the isolated antibody or antigen-binding fragment according to the invention, for use as a medicament.

[0209] The isolated antibodies or antigen-binding fragments according to the invention may be used as therapeutic or diagnostic tools in a variety of Sema3A-related disorders.

[0210] Thus, in another aspect, the present invention relates to an isolated antibody or antigen-binding fragment according to the present invention, or a conjugate comprising the isolated antibody or antigen-binding fragment according to the present invention, or a pharmaceutical composition comprising the isolated antibody or antigen-binding fragment according to the present invention, for use in the treatment and / or prevention of kidney diseases, in particular acute and chronic kidney diseases, diabetic kidney disease, Alport syndrome and acute and chronic kidney failure. The general term "kidney disease" or "kidney disease" refers to a class of pathological conditions in which the kidneys are unable to filter and remove waste products from the blood. There are two main forms of kidney disease: acute kidney disease (acute kidney injury, AKI) and chronic kidney disease (CKD). The isolated antibody or antigen-binding fragment according to the present invention, or a conjugate or pharmaceutical composition comprising it, may further be used in the treatment and / or prevention of sequelae of acute kidney injury resulting from multiple insults such as ischemia-reperfusion injury, administration of radiocontrast agents, cardiopulmonary bypass surgery, shock and sepsis.In the context of the present invention, the terms renal failure and renal insufficiency include both acute and chronic manifestations of renal failure, as well as underlying or associated renal diseases, such as renal hypoperfusion, intradialytic hypotension, obstructive uropathy, glomerulopathy, IgA nephropathy, glomerulonephritis, acute glomerulonephritis, glomerulosclerosis, tubulointerstitial diseases, nephropathy diseases, such as primary renal diseases and congenital renal diseases, nephritis, Alport syndrome, renal inflammation, immune-mediated renal diseases, such as renal transplant rejection, immune complex-induced renal disease, toxic substance-induced nephropathy, contrast-induced nephropathy; minimal change glomerulonephritis (lipoid); membranous glomerulonephritis; focal segmental glomerulosclerosis (FSGS); hemolytic uremic syndrome (HUS) ), amyloidosis, Goodpasture's syndrome, Wegener's granulomatosis, Schönlein-Henoch purpura, diabetic and non-diabetic nephropathy, pyelonephritis, renal cysts, nephrosclerosis, hypertensive nephrosclerosis and nephrotic syndrome; which may be characterized in diagnosis by, for example, an abnormal decrease in creatinine and / or water excretion, an abnormal increase in blood concentrations of urea, nitrogen, potassium and / or creatinine, changes in the activity of renal enzymes such as glutamyl synthetase, changes in urine osmolality or urine volume, increased microalbuminuria, macroalbuminuria, lesions of glomeruli and arterioles, renal tubular dilatation, hyperphosphatemia and / or the need for dialysis. The present invention also relates to an isolated antibody or antigen-binding fragment according to the invention or a conjugate or pharmaceutical composition comprising same for use in the treatment and / or prevention of sequelae of renal failure, in particular pulmonary edema, cardiac failure, uremia, anemia, electrolyte disorders (e.g. hyperkalemia, hyponatremia), and disorders of bone and carbohydrate metabolism. The compounds according to the invention are also suitable for the treatment and / or prevention of polycystic kidney disease (PCKD) and the syndrome of inappropriate ADH secretion (SIADH).

[0211] The isolated antibody or antigen-binding fragment according to the invention or a conjugate or pharmaceutical composition comprising same may also be used for the treatment and / or prevention of vascular hyperpermeability, diabetic retinopathy, deterioration of the blood-retinal barrier and resulting macular edema, preferably age-related macular edema, non-proliferative age-related macular edema and non-proliferative diabetic macular edema.

[0212] Furthermore, the isolated antibody or antigen-binding fragment according to the invention or a conjugate or pharmaceutical composition comprising same is suitable for the prevention or treatment of diseases of the central or peripheral nervous system, such as neuropathic pain, spinal cord injury, multiple sclerosis, traumatic brain injury, cerebral edema and neurodegenerative diseases, wherein the neurodegenerative diseases are Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, progressive supranuclear palsy, black matter degeneration, Shy-Drager syndrome, olivopontocerebellar atrophy and spinocerebellar degeneration.

[0213] Furthermore, the isolated antibody or antigen-binding fragment according to the invention or a conjugate or pharmaceutical composition comprising same may be useful for the treatment and / or prevention of cancer, where the cancer is intestinal cancer, colorectal cancer, lung cancer, breast cancer, brain cancer, melanoma, renal cell carcinoma, leukemia, lymphoma, T-cell lymphoma, gastric cancer, pancreatic cancer, cervical cancer, endometrial cancer, ovarian cancer, esophageal cancer, liver cancer, squamous cell carcinoma of the head and neck, skin cancer, cancer of the urinary tract, prostate cancer, choriocarcinoma, pharyngeal cancer and laryngeal cancer.

[0214] The above disorders are well characterized in humans, but have similar etiologies in other animals, including mammals, and may be treated by administering a pharmaceutical composition according to the present invention.

[0215] The antibodies or antigen-binding fragments or variants thereof according to the invention can be co-administered with known pharmaceuticals, and in some cases the antibodies or antigen-binding fragments thereof themselves can be modified, for example, the antibodies or antigen-binding fragments or variants thereof can be conjugated to a drug or another peptide or protein, potentially further enhancing efficacy.

[0216] The antibodies or antigen-binding fragments or variants thereof of the present invention can be administered as the sole agent or in combination with one or more additional therapeutic substances where such combination does not cause unacceptable adverse effects.

[0217] Thus, in another aspect, the present invention relates to an isolated antibody or antigen-binding fragment according to the invention or a conjugate according to the invention or a pharmaceutical composition according to the invention for use in combination for simultaneous, separate or sequential administration with one or more additional therapeutically active compounds.

[0218] Non-limiting examples of therapeutically active compounds that may be used in combination with an antibody or antigen-binding fragment according to the invention are as follows:

[0219] - antihypertensive agents, for example preferably from the group of calcium antagonists, angiotensin II antagonists, ACE inhibitors, NEP inhibitors, vasopeptidase inhibitors, endothelin antagonists, renin inhibitors, alpha-blockers, beta-blockers, mineralocorticoid receptor antagonists and diuretics; - antidiabetic agents (hypoglycemic or antihyperglycemic agents), such as, preferably, insulin and derivatives, sulfonylureas, biguanides, thiazolidinediones, acarbose, DPP4 inhibitors, GLP-1 analogues or SGLT inhibitors (gliflozins); - compounds that inhibit signal transduction cascades, in particular tyrosine and / or serine / threonine kinase inhibitors, such as nintedanib, dasatinib, nilotinib, bosutinib, regorafenib, sorafenib, sunitinib, cediranib, axitinib, telatinib, imatinib, brivanib, pazopanib, vatalanib, gefitinib, erlotinib, lapatinib, canertinib, lestaurtinib, pelitinib, semaxanib or tandutinib; - anti-inflammatory drugs, such as non-steroidal anti-inflammatory drugs (NSAIDs), such as acetylsalicylic acid (aspirin), ibuprofen and naproxen, glucocorticoids, such as, preferably, prednisone, prednisolone, methylprednisolone, triamcinolone, dexamethasone, beclomethasone, betamethasone, flunisolide, budesonide or fluticasone, or 5-aminosalicylic acid derivatives, leukotriene antagonists, TNF-alpha inhibitors and chemokine receptor antagonists, such as CCR1, 2 and / or 5 inhibitors, NF-κB inhibitors and Nerf2 activators; - Antifibrotic drugs, such as TGF-beta antagonists or microRNA-21 inhibitors; - organic nitrates and NO donors, such as sodium nitroprusside, nitroglycerin, isosorbide mononitrate, isosorbide dinitrate, molsidomine or SIN-1 and inhalable NO; - compounds that inhibit the degradation of cyclic guanosine monophosphate (cGMP), such as inhibitors of phosphodiesterase (PDE) 1, 2, 5 and / or 9, in particular PDE-5 inhibitors, such as sildenafil, vardenafil, tadalafil, udenafil, dasantafil, avanafil, mirodenafil, lodenafil, CTP-499 or PF-00489791; - calcium sensitizers, for example and preferably levosimendan; - antithrombotic agents, in particular those selected from the group consisting of platelet aggregation inhibitors, anticoagulants and fibrinolytic substances; - substances that stimulate NO- and heme-dependent and NO- and heme-independent cGMP synthesis, such as, preferably, soluble guanylate cyclase (sGC) modulators, such as, preferably, riociguat, cinaciguat, vericiguat or BAY1101042; - fat metabolism modifying agents, such as, preferably, those selected from thyroid receptor agonists, cholesterol synthesis inhibitors, such as, preferably, HMG-CoA-reductase or squalene synthesis inhibitors, ACAT inhibitors, CETP inhibitors, MTP inhibitors, PPAR-alpha, PPAR-gamma and / or PPAR-delta agonists, cholesterol absorption inhibitors, lipase inhibitors, polymeric bile acid adsorbents, bile acid reabsorption inhibitors and lipoprotein(a) antagonists.

[0220] In a particular embodiment, an isolated antibody or antigen-binding fragment according to the invention is administered in combination with a platelet aggregation inhibitor, particularly aspirin, clopidogrel, ticlopidine, or dipyridamole.

[0221] In a specific embodiment, an isolated antibody or antigen-binding fragment according to the invention is administered in combination with a thrombin inhibitor, particularly ximelagatran, dabigatran, melagatran, bivalirudin or enoxaparin.

[0222] In a specific embodiment, the isolated antibody or antigen-binding fragment according to the invention is administered in combination with a GPIIb / IIIa antagonist, in particular tirofiban or abciximab.

[0223] In a specific embodiment, an isolated antibody or antigen-binding fragment according to the invention is administered in combination with a factor Xa inhibitor, particularly one selected from rivaroxaban, apixaban, otamixaban, fidexaban, razaxaban, fondaparinux, idraparinux, DU-176b, PMD-3112, YM-150, KFA-1982, EMD-503982, MCM-17, MLN-1021, DX9065a, DPC906, JTV803, SSR-126512, and SSR-128428.

[0224] In a specific embodiment, an isolated antibody or antigen-binding fragment according to the invention is administered in combination with heparin or a low molecular weight (LMW) heparin derivative.

[0225] In a specific embodiment, the isolated antibody or antigen-binding fragment according to the invention is administered in combination with a Vitamin K antagonist, particularly one selected from a coumarin.

[0226] The antihypertensive agent is in particular selected from the group consisting of calcium antagonists, angiotensin AII antagonists, ACE inhibitors, NEP inhibitors, vasopeptidase inhibitors, endothelin antagonists, renin inhibitors, alpha-blockers, beta-blockers, mineralocorticoid receptor antagonists and diuretics.

[0227] In a specific embodiment, an isolated antibody or antigen-binding fragment according to the invention is administered in combination with a calcium antagonist, particularly one selected from nifedipine, amlodipine, verapamil, and diltiazem.

[0228] In a specific embodiment, the isolated antibody or antigen-binding fragment according to the invention is administered in combination with an angiotensin AII receptor antagonist, particularly one selected from the group consisting of losartan, candesartan, valsartan, telmisartan, irbesartan, olmesartan, eprosartan, embrusartan and azilsartan.

[0229] In certain embodiments, an isolated antibody or antigen-binding fragment according to the invention is administered in combination with an ACE inhibitor, particularly one selected from the group consisting of enalapril, captopril, lisinopril, ramipril, delapril, fosinopril, quinopril, perindopril, benazepril and trandopril.

[0230] In certain embodiments, the isolated antibody or antigen-binding fragment according to the invention is administered in combination with an endothelin antagonist, particularly one selected from the group consisting of bosentan, darusentan, ambrisentan, tezosentan, sitaxsentan, avosentan, macitentan and atrasentan.

[0231] In a particular embodiment, the isolated antibody or antigen-binding fragment according to the invention is administered in combination with a renin inhibitor, particularly one selected from the group consisting of aliskiren, SPP-600 and SPP-800.

[0232] In a particular embodiment, the isolated antibody or antigen-binding fragment according to the invention is administered in combination with a mineralocorticoid receptor antagonist, in particular one selected from the group consisting of finerenone, spironolactone, canrenone, potassium canrenoate, eplerenone, esaxerenone (CS-3150) or aparalenone (MT-3995), CS-3150 and MT-3995.

[0233] In certain embodiments, an isolated antibody or antigen-binding fragment according to the invention is administered in combination with a diuretic, particularly one selected from the group consisting of furosemide, bumetanide, piretanide, torsemide, bendroflumethiazide, chlorothiazide, hydrochlorothiazide, xipamide, indapamide, hydroflumethiazide, methyclothiazide, polythiazide, trichloromethiazide, chlorothalidone, metolazone, quinethazone, acetazolamide, dichlorophenamide, methazolamide, glycerin, isosorbide, mannitol, amiloride, and triamterene.

[0234] The fat metabolism modifying agent is in particular selected from the group consisting of CETP inhibitors, thyroid receptor agonists, cholesterol synthesis inhibitors, such as HMG-CoA reductase inhibitors or squalene synthesis inhibitors, ACAT inhibitors, MTP inhibitors, PPAR-alpha, PPAR-gamma and / or PPAR-delta agonists, cholesterol absorption inhibitors, polymeric bile acid adsorbents, bile acid reabsorption inhibitors, lipase inhibitors and lipoprotein(a) antagonists.

[0235] In a specific embodiment, the isolated antibody or antigen-binding fragment according to the invention is administered in combination with a Nerf2 activator, particularly one selected from bardoxolone methyl.

[0236] In certain embodiments, the isolated antibody or antigen-binding fragment according to the invention is administered in combination with a thyroid receptor agonist, particularly one selected from the group consisting of D-thyroxine, 3,5,3'-triiodothyronine (T3), CGS 23425 and axityrom (CGS 26214).

[0237] In a specific embodiment, an isolated antibody or antigen-binding fragment according to the invention is administered in combination with an HMG-CoA reductase inhibitor from the statin class, particularly one selected from the group consisting of lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin, rosuvastatin and pitavastatin.

[0238] In a particular embodiment, the isolated antibody or antigen-binding fragment according to the invention is administered in combination with a PPAR-gamma modulator, particularly one selected from pioglitazone and rosiglitazone.

[0239] In a particular embodiment, the isolated antibody or antigen-binding fragment according to the invention is administered in combination with a PPAR-delta modulator, particularly one selected from the group consisting of ASP1128, GW 501516 and BAY 68-5042.

[0240] In a particular embodiment, an isolated antibody or antigen-binding fragment according to the invention is administered in combination with a cholesterol absorption inhibitor, particularly one selected from the group consisting of ezetimibe, tiqueside and pamaqueside.

[0241] In a particular embodiment, the isolated antibody or antigen-binding fragment according to the invention is administered in combination with a lipase inhibitor, particularly one selected from orlistat.

[0242] In certain embodiments, an isolated antibody or antigen-binding fragment according to the invention is administered in combination with a polymeric bile acid adsorbent, particularly one selected from the group consisting of cholestyramine, colestipol, colesolvam, CholestaGel, and colestimide.

[0243] In certain embodiments, an isolated antibody or antigen-binding fragment according to the invention is administered in combination with a bile acid reabsorption inhibitor, in particular an ASBT (IBAT) inhibitor, such as one selected from the group consisting of AZD-7806, S-8921, AK-105, BARI-1741, SC-435 and SC-635.

[0244] In a specific embodiment, the isolated antibody or antigen-binding fragment according to the invention is administered in combination with a lipoprotein(a) antagonist, particularly one selected from the group consisting of gemcabene calcium (CI-1027) and nicotinic acid.

[0245] In a particular embodiment, the isolated antibody or antigen-binding fragment according to the invention is administered in combination with a TGF-beta antagonist, particularly one selected from pirfenidone and fresolimumab.

[0246] In a particular embodiment, the isolated antibody or antigen-binding fragment according to the invention is administered in combination with an anti-microRNA-21 oligonucleotide, particularly one selected from rademirucene.

[0247] In a specific embodiment, the isolated antibody or antigen-binding fragment according to the invention is administered in combination with a HIF-PH inhibitor, particularly one selected from molidastat and roxadustat.

[0248] In a specific embodiment, the isolated antibody or antigen-binding fragment according to the invention is administered in combination with a CCR2 antagonist, particularly one selected from CCX-140.

[0249] In a particular embodiment, the isolated antibody or antigen-binding fragment according to the invention is administered in combination with a TNF-alpha antagonist, particularly one selected from adalimumab.

[0250] In a specific embodiment, the isolated antibody or antigen-binding fragment according to the invention is administered in combination with a galectin-3 inhibitor, particularly one selected from GCS-100.

[0251] In a particular embodiment, the isolated antibody or antigen-binding fragment according to the invention is administered in combination with a hepatocyte growth factor mimetic, particularly one selected from refanarin.

[0252] In a specific embodiment, the isolated antibody or antigen-binding fragment according to the invention is administered in combination with a p53 modulator, particularly one selected from QPI-1002.

[0253] In a specific embodiment, the isolated antibody or antigen-binding fragment according to the invention is administered in combination with a NOX1 / 4 inhibitor, particularly one selected from GKT-137831.

[0254] In a particular embodiment, an isolated antibody or antigen-binding fragment according to the invention is administered in combination with a medication that affects vitamin D metabolism, particularly one selected from cholecalciferol and paracalcitol.

[0255] In a specific embodiment, the isolated antibody or antigen-binding fragment according to the invention is administered in combination with a cytostatic agent, particularly one selected from cyclophosphamide.

[0256] In certain embodiments, the isolated antibody or antigen-binding fragment according to the invention is administered in combination with an anti-VEGF therapy, particularly one selected from the group consisting of ranibizumab, bevacizumab, and aflibercept.

[0257] In certain embodiments, the isolated antibody or antigen-binding fragment according to the invention is administered in combination with an immunosuppressant, particularly one selected from cyclosporines.

[0258] In certain embodiments, the isolated antibody or antigen-binding fragment according to the invention is administered in combination with a phosphate binding agent, particularly one selected from sevelamer and lanthanum carbonate.

[0259] In a specific embodiment, an isolated antibody or antigen-binding fragment according to the invention is administered in combination with a calcimimetic for the treatment of hyperparathyroidism.

[0260] In a specific embodiment, an isolated antibody or antigen-binding fragment according to the invention is administered in combination with a substance for treating iron deficiency, particularly one selected from iron products.

[0261] In a particular embodiment, an isolated antibody or antigen-binding fragment according to the invention is administered in combination with a hyperuricemia therapeutic, particularly one selected from allopurinol and rasburicase.

[0262] In a specific embodiment, an isolated antibody or antigen-binding fragment according to the invention is administered in combination with a glycoprotein hormone, particularly one selected from erythropoietin, for the treatment of anemia.

[0263] In certain embodiments, the isolated antibody or antigen-binding fragment according to the invention is administered in combination with an immunotherapeutic biologic, particularly one selected from the group consisting of abatacept, rituximab, eculizumab and belimumab.

[0264] In certain embodiments, the isolated antibody or antigen-binding fragment according to the invention is administered in combination with a Jak inhibitor, particularly one selected from the group consisting of ruxolitinib, tofacitinib, baricitinib, CYT387, GSK2586184, lestaurtinib, pacritinib (SB1518) and TG101348.

[0265] In a specific embodiment, an isolated antibody or antigen-binding fragment according to the invention is administered in combination with a prostacyclin analog for the treatment of microthrombi.

[0266] In certain embodiments, the isolated antibody or antigen-binding fragment according to the invention is administered in combination with alkaline therapy, particularly selected from sodium bicarbonate.

[0267] In a particular embodiment, the isolated antibody or antigen-binding fragment according to the invention is administered in combination with an mTOR inhibitor, particularly one selected from everolimus and rapamycin.

[0268] In a particular embodiment, the isolated antibody or antigen-binding fragment according to the invention is administered in combination with an NHE3 inhibitor, particularly one selected from AZD1722.

[0269] In a specific embodiment, the isolated antibody or antigen-binding fragment according to the invention is administered in combination with an eNOS modulator, particularly one selected from sapropterin.

[0270] In a specific embodiment, the isolated antibody or antigen-binding fragment according to the invention is administered in combination with a CTGF inhibitor, particularly one selected from FG-3019.

[0271] In certain embodiments, an isolated antibody or antigen-binding fragment according to the invention is administered in combination with one or more additional therapeutic agents selected from the group consisting of diuretics, angiotensin AII antagonists, ACE inhibitors, beta receptor blockers, mineralocorticoid receptor antagonists, antidiabetic agents, organic nitrates and NO donors, activators and stimulators of soluble guanylate cyclase (sGC), and positive inotropic agents.

[0272] In certain embodiments, an isolated antibody or antigen-binding fragment according to the invention is administered in combination with one or more additional therapeutic agents selected from the group consisting of diuretics, angiotensin AII antagonists, ACE inhibitors, beta receptor blockers, mineralocorticoid receptor antagonists, antidiabetic agents, organic nitrates and NO donors, activators and stimulators of soluble guanylate cyclase (sGC), positive inotropic agents, anti-inflammatory agents, immunosuppressants, phosphate binders, and antibodies that modulate vitamin D metabolism.

[0273] Combination (combined) therapy includes administration of a single pharmaceutical dosage formulation comprising an antibody or antigen-binding fragment or variant thereof according to the invention and one or more additional therapeutic agents, and administration of an antibody or antigen-binding fragment according to the invention and each additional therapeutic agent in its own separate pharmaceutical dosage formulation. For example, an antibody or antigen-binding fragment or variant thereof according to the invention and a therapeutic agent can be administered to a patient together in a single liquid composition, or each agent can be administered in a separate dosage formulation.

[0274] When separate dosage formulations are used, the antibody or antigen-binding fragment or variant thereof according to the invention and the one or more additional therapeutic agents can be administered at substantially the same time (e.g., simultaneously) or at separate, staggered times (e.g., sequentially).

[0275] The antibodies or antigen-binding fragments or variants thereof according to the invention may be used in combination with surgical interventions, including but not limited to: - Major cardiovascular surgery, such as coronary artery bypass graft (CABG), heart valve repair or replacement, pacemaker or implantable cardioverter defibrillator (ICD) insertion, maze surgery, aneurysm repair, carotid artery surgery / endarterectomy and thrombectomy; - Major non-cardiac surgery, e.g. thoracic, orthopedic, urological surgery.

[0276] Diagnostic methods Additionally, antibodies or antigen-binding fragments according to the invention, either by themselves or as compositions, may be used in research and diagnostics, or as analytical reference standards, and the like.

[0277] The anti-Sema3A antibody or antigen-binding fragment thereof can be used to detect the presence of Sema3A. Thus, in another aspect, the present invention relates to an isolated antibody or antigen-binding fragment according to the present invention or an antibody conjugate according to the present invention for use as a diagnostic agent.

[0278] Pharmaceutical Compositions and Administration In another aspect, the present invention relates to a pharmaceutical composition comprising an isolated antibody or antigen-binding fragment according to the invention or an antibody conjugate according to the invention. Pharmaceutical compositions for use according to the invention to treat any of the above disorders may be formulated in any conventional manner using one or more physiologically acceptable carriers, excipients or adjuvants. Further details regarding formulation and administration techniques may be found in the latest edition of Remington's Pharmaceutical Sciences (Ed. Maack Publishing Co, Easton, Pa.).

[0279] The antibody or antigen-binding fragment according to the invention may be administered by any suitable means, which may vary depending on the type of disorder being treated. Possible routes of administration include oral, parenteral and topical administration. Methods of parenteral delivery include intra-arterial, intramuscular, subcutaneous, intramedullary, intrathecal, intracerebroventricular, intravenous, intraperitoneal or intranasal administration. The antibody or antigen-binding fragment according to the invention may also be administered by pulse infusion, for example with decreasing doses of antibody. Preferably, administration is by injection, most preferably intravenous or subcutaneous injection, depending in part on whether administration is brief or chronic. The dosage depends on various factors, such as, for example, clinical symptoms, weight of the individual, whether other drugs are administered, etc. Those skilled in the art will recognize that the route of administration will vary depending on the disorder or condition being treated.

[0280] The pharmaceutical composition according to the invention comprises the antibody or antigen-binding fragment according to the invention, alone or together with at least one other substance, such as a stabilizing compound. The antibody or antigen-binding fragment according to the invention may be administered in any sterile biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose and water. In certain embodiments, the pharmaceutical composition according to the invention may comprise one or more additional pharma- ceutical active compounds, in particular one or more additional pharma- ceutical active compounds suitable for treating Sema3A-associated disorders. Any of these substances may be administered to a patient as a pharmaceutical composition mixed with an excipient or pharma- ceutical acceptable carrier, in combination with other substances or drugs, or alone. In certain embodiments, the pharma- ceutical acceptable carrier is pharma- ceutical inert.

[0281] Pharmaceutical compositions for oral administration can be formulated using pharma- ceutically acceptable carriers well known in the art in amounts suitable for oral administration. Such carriers allow the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.

[0282] Pharmaceutical preparations for oral use can be obtained by combining active compounds with solid excipients, optionally grinding the resulting mixture, adding suitable auxiliary agents as necessary, and then processing the granulated mixture to obtain tablets or dragee cores.Suitable excipients include carbohydrate or protein fillers, such as sugars, such as lactose, sucrose, mannitol or sorbitol; starches from corn, wheat, rice, potato or other plants; celluloses, such as methylcellulose, hydroxypropylmethylcellulose or sodium carboxymethylcellulose; and gums, such as gum arabic and gum tragacanth; and proteins, such as gelatin and collagen.Optionally, disintegrants or solubilizers, such as cross-linked polyvinylpyrrolidone, agar, alginic acid or its salts, such as sodium alginate, can be added.

[0283] Dragee cores may be provided with suitable coatings such as gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol and / or titanium dioxide, lacquer solutions and concentrated sugar solutions which may also contain suitable organic solvents or solvent mixtures.Dyes or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.

[0284] Orally usable pharmaceutical preparations include push-fit capsules made of gelatin and sealed soft capsules made of gelatin and a coating (e.g., glycerol or sorbitol). Push-fit capsules may contain the active ingredient mixed with a filler or binder, such as lactose or starch, a lubricant, such as talc or magnesium stearate, and, optionally, a stabilizer. In soft capsules, the active compound may be dissolved or suspended in a suitable liquid, such as fatty oils, liquid paraffin, or liquid polyethylene glycol, with or without stabilizers.

[0285] Pharmaceutical preparations for parenteral administration include aqueous solutions of active compounds. For injection, the pharmaceutical compositions of the present invention can be formulated in aqueous solutions, preferably in physiologically compatible buffers, such as Hank's solution, Ringer's solution or physiologically buffered saline. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Alternatively, suspensions of active compounds can be prepared as suitable oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. If desired, the suspension can also contain substances that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions or suitable stabilizers.

[0286] For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation, and such penetrants are generally known in the art.

[0287] The pharmaceutical compositions of the present invention may be manufactured in a manner known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, pulverizing, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0288] Pharmaceutical compositions can be provided as salts and can be formed with acids such as, but not limited to, hydrochloric acid, sulfuric acid, acetic acid, lactic acid, tartaric acid, malic acid, succinic acid, and the like. Salts tend to be more soluble in aqueous or other protic solvents than the corresponding free base form. In other cases, a preferred formulation can be a lyophilized powder in 1 mM to 50 mM histidine or phosphate or Tris, 0.1% to 2% sucrose, and / or 2% to 7% mannitol, at a pH range of 4.5 to 7.5, optionally containing additional substances such as polysorbates, that are combined with a buffer prior to use.

[0289] After preparation of pharmaceutical compositions containing the compounds of the present invention formulated in an acceptable carrier, they can be placed in an appropriate container and labeled for treatment of an indicated condition. In the case of administration of an anti-Sema3A antibody or antigen-binding fragment thereof, such labeling would include the amount, frequency and method of administration.

[0290] Therapeutically Effective Amount Pharmaceutical compositions suitable for use according to the present invention include compositions containing an effective amount of the active ingredients to achieve the intended purpose, e.g., treatment of a particular pathology characterized by enhanced levels or activity of Sema3A.

[0291] Determining an effective amount is well within the capabilities of a person skilled in the art. Determining a therapeutically effective amount of the novel antibody or antigen-binding fragment thereof or variant thereof of the present invention depends largely on the characteristics of the individual patient, the route of administration, and the nature of the disorder being treated. General guidelines can be found, for example, in publications from the International Conference on Harmonization and in REMINGTON'S PHARMACEUTICAL SCIENCES, chapters 27 and 28, pp. 484-528 (18th ed., edited by Alfonso R. Gennaro, Easton, Pa.: Mack Pub. Co., 1990). More specifically, determining a therapeutically effective amount depends on factors such as toxicity and efficacy of the drug. Toxicity can be determined using methods well known in the art and found in the above references. Efficacy is determined using the same guidelines along with the methods described in the Examples below.

[0292] For any compound, the therapeutically effective amount can be estimated first in cell culture assays or animal models (usually mice, rabbits, dogs, pigs or monkeys). Animal models are also used to obtain a desirable concentration range and route of administration. Such information can then be used to determine useful doses and routes of administration in humans.

[0293] A therapeutically effective amount refers to that amount of antibody or antigen-binding fragment thereof that ameliorates the symptoms or condition. Therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical methods in cell cultures or experimental animals, e.g., ED 50 (the dose that is therapeutically effective in 50% of the population) and LD 50 (the dose lethal to 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, which is the ED 50 / LD 50The therapeutic index may be expressed as a ratio of ED 1 to ED 2 . Pharmaceutical compositions that exhibit large therapeutic indices are preferred. Data obtained from cell culture assays and animal studies are used in formulating a range of dosages for human use. The dosage of such compounds is preferably within the range of ED 1 to ED 2 with little or no toxicity. 50 The dosage varies within this range depending on the dosage form used, sensitivity of the patient, and the route of administration.

[0294] The exact dosage is chosen by the individual physician in view of the patient to be treated. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Additional factors that may be considered include the severity of the patient's condition, age, weight and sex; diet; time and frequency of administration, drug combinations, reaction sensitivities and tolerance / response to treatment. Long-acting pharmaceutical compositions may be administered, for example, every 3-4 days, every week, once every two weeks or once every three weeks, depending on the half-life and clearance rate of the particular formulation.

[0295] Typical dosages may vary from 0.1 to 100,000 micrograms, up to a total dose of about 10 g, depending on the route of administration. Guidance regarding specific dosages and delivery methods is provided in the literature. See U.S. Patent Nos. 4,657,760, 5,206,344, or 5,225,212.

[0296] kit In another aspect, the invention relates to a kit comprising an isolated antibody or antigen-binding fragment according to the invention or a conjugate according to the invention and instructions for use. In certain embodiments, the kit comprises one or more containers filled with one or more of the components of the composition of the invention. Such containers may bear a notice in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceutical or biological products. The notice indicates approval by the governmental agency of the manufacture, use or sale of the product for administration to humans. [Brief description of the drawings]

[0297] [Figure 1] Figure 1A: Effect of Sema3A inhibition by TPP-15370 (gray bars), TPP-11489 (striped bars) and TPP-17755 (square bars) on Sema3A-induced albumin excretion in mice. Mean values ​​± SD (n=10) are shown. ***, ****: p<0.001, p<0.0001 vs. isotype control. Dunnett's post-hoc test.

[0298] Figure 1B: Effect of Sema3A inhibition by TPP-23298 (gray bars), TPP-11489 (dotted bars) and TPP-17755 (striped bars) on Sema3A-induced albumin excretion in mice. Mean values ​​± SD (n=10) are shown. *** , **** : p<0.001, p<0.0001 vs. isotype control. Dunnett's post-hoc test. [Diagram 2] FIG. 2A: Sema3A-induced albuminuria in mice following treatment with TPP-15370 (open circles) and TPP-23298 (closed circles) compared to TPP-11489 (closed triangles). Comparisons were made in two separate experiments. Mean values ​​are shown. (n=10). ns=not statistically significant vs. TPP-15370; *=p<0.05 vs. TPP-23298. Unpaired T-test.

[0299] FIG. 2B: Sema3A-induced albuminuria in mice following treatment with TPP-15370 (open circles) and TPP-23298 (closed circles) compared to TPP-17755 (closed squares). Comparisons were made in two separate experiments. Mean values ​​are shown. (n=10). ns=not statistically significant versus TPP-15370; * = p<0.05 vs. TPP-23298. Independent T-test.

[0300] FIG. 2C: Sema3A-induced albuminuria in mice following treatment with TPP-15370 (open circles) and TPP-23298 (closed circles) compared to TPP-30788 (closed diamonds). Comparisons were made in two separate experiments. Mean values ​​are shown. (n=10). ns=not statistically significant versus TPP-15370; * = p<0.05 vs. TPP-23298. Independent T-test. [Diagram 3] Effect of Sema3A inhibition by TPP-23374 (dotted bars), TPP-23298 (gray bars) and TPP-15370 (striped bars) on A: serum creatinine levels, B: serum urea levels and C: urinary albumin excretion after I / R injury in mice. Mean values ​​± SD are shown (n=8-10). *, **, ****: p<0.05, p<0.01, p<0.0001 vs. isotype control. Dunnett's post-hoc test. [Figure 4] Effect of Sema3A inhibition by TPP-15370 (gray bars), TPP-11489 (striped bars) and TPP-17755 (square bars) on A: serum creatinine levels, B: serum urea levels and C: urinary albumin excretion after I / R injury in mice. Mean values ​​± SD are shown (n=8-10). *, **, ****: p<0.05, p<0.01, p<0.0001 vs. isotype control. Dunnett's post-hoc test. [Diagram 5] Effect of Sema3A inhibition by TPP-23298 (gray bars), TPP-11489 (striped bars) and TPP-17755 (square bars) on A: serum creatinine levels, B: serum urea levels and C: urinary albumin excretion after I / R injury in mice. Mean values ​​± SD (n=10-12) are shown. *, **, ****: p<0.05, p<0.01, p<0.0001 vs. isotype control. Dunnett's post-hoc test. [Figure 6]Effect of Sema3A inhibition by TPP-15374 (gray bars), TPP-11489 (striped bars) on A: serum creatinine levels, B: serum urea levels, and C: urinary albumin excretion after I / R injury in mice. Mean values ​​± SD are shown (n=10–12). *, **, ***, ****: p<0.05, p<0.01, p<0.001 p<0.0001 vs. isotype control. Dunnett's post-hoc test. [Figure 7] Effect of Sema3A inhibition by TPP-15370 (gray bars) and TPP-11489 (striped bars) on proteinuria in Alport mice. Mean values ​​± SD are shown (n = 8-10). ****: p < 0.0001 vs. isotype control. Dunnett's post-hoc test. [Figure 8] Effect of Sema3A inhibition by TPP-15370 (gray bars), TPP-11489 (striped bars) on A: serum creatinine levels, B: serum urea levels and fibrosis, C: myofibroblasts, and D: collagen deposition in Alport mice. Mean values ​​± SD are shown (n=8-10). *, ***, ****: p<0.05, p<0.001, p<0.0001 vs. isotype control. Dunnett's post-hoc test. [Figure 9] Effect of Sema3A inhibition by TPP-23298 in a single-dose prophylactic setting in a porcine unilateral renal IRI model during 105 min of ischemia. TPP-23298 (A; black dots) or control IgG (open circles) (10 mg / kg) was administered 30 min before balloon inflation in the left renal artery. Values ​​from SHAM animals are shown as diamonds. Time course of plasma creatinine concentrations for individual animals (A) and time course of mean change in creatinine plasma concentration relative to baseline values ​​at the start of the experiment (0 h) (B). Mean values ​​of creatinine clearance over a 24-27 h interval. Creatinine clearance by side of the left (injured) and right (non-injured) kidney as well as kidneys from sham (sham) animals (C). Total creatinine clearance (D); mean ± SEM, p values ​​in (B) from t test, * / *** in (C) and (D): p<0.05 / 0.001, one-way ANOVA versus matched controls followed by Dunnett's multiple comparisons. [Figure 10] Schematic of a sandwich-based epitope binning experiment using SPR (see also Example 5A). [Figure 11] HRA image analysis steps: 11A) Fluorescence microscopy image of DAPI / CM cells. 11B) Identification of cells in selected regions. 11C) Calculation of cell-free region size (grey area). [Figure 12] Shown is the % inhibition of Sema3A in a HUVEC repulsion assay at an antibody concentration of 80 pM (see Example 11). Each bar represents one antibody in the following order from left to right: TPP-23298 (black bar), TPP-30788, TPP-TPP-30789, TPP-30790 and TPP-30791.

[0301] Working Example Example 1: Sema3A sequence and material preparation [Table 1]

[0302] Sema3A domains were produced by mammalian cell culture using transiently transfected HEK293-6E cells (National Research Council Canada). All constructs are under the control of the CMV promoter and the sequences contain a C-terminal FXa cleavage site followed by a 6xhis tag. Cell culture was performed using F17 medium (Life Technologies) supplemented with 0.1% Pluronic® F68 (Life Technologies) and 4 mM Glutamax (Life Technologies). 24 hours after transfection, 1% FCS ultra-low IgG (Life Technologies) and 0.5 mM valproic acid (Sigma Aldrich) were added. Cell supernatants were sterile filtered and then purified or concentrated by cross-flow filtration prior to purification.

[0303] The Sema3A domain was purified using a two-step purification consisting of affinity chromatography and size-exclusion chromatography. Briefly, a Ni-Cell Chromatograph connected to an Akta Avant system (GE Healthcare) was used. 2+ A -NTA column (GE Healthcare) was loaded with cell culture supernatant. The column was equilibrated with 4 CV of 50 mM NaH2PO4, 300 mM NaCl (pH 8) and then washed with 10 CV of running buffer until baseline was reached. Elution was performed using 6 CV of running buffer containing 250 mM imidazole (pH 8.0). Fractions of the elution peak were collected, concentrated using a Vivaflow 200 Hydrosart membrane (cut-off 10 kDa, Sartorius) and subjected to size exclusion chromatography using a Superdex 200 column (GE Healthcare) connected to an Akta Pure25 system. The column was equilibrated and run in DPBS (pH 7.4). Fractions of the domain elution peak were collected and concentrated using a Vivaflow 200 Hydrosart membrane (cut-off 10 kDa, Sartorius). The quality of the final protein was assessed for purity and monodispersity by analytical size-exclusion chromatography (Superdex 200) and by SDS-PAGE. The Sema domain was aliquoted, flash frozen in liquid nitrogen, and stored at -80°C until further use.

[0304] Example 2: Generation of antibodies from the BioInvent antibody library A fully human antibody phage display library (BioInvent n-CoDeR Fab lambda library) was used to isolate human monoclonal antibodies of the present invention by selection against recombinant human Sema3A (TPP-13211, R&D Systems) using the following protocol. Briefly, immunotubes (Nunc) were coated with 100 μg of target molecule (huSema3A) or irrelevant Fc-containing off-target in 1 ml of PBS (phosphate buffered saline) for 1 hour at room temperature (RT) with rotation. Target and depletion antigen-coated immunotubes as well as empty immunotubes were washed 4 times with PBS+0.05% Tween20 (PBST) and then blocked with 3 ml of 3% milk powder in PBST solution for 1 hour at room temperature with rotation. An aliquot of the phage library was thawed and blocked in a solution of 3% milk powder in PBST for 1 hour at room temperature with rotation. The uncoated depletion immunotube was washed three times in 4 ml of PBS, after which the blocked phage library was added and incubated at room temperature for 30 minutes with rotation. This step was repeated for the non-target antigen-coated depletion immunotube. The huSema3A-coated immunotube was washed three times in 4 ml of PBS, after which the depleted library was added and incubated at room temperature for 90 minutes with rotation. After stringent washing (four times with 4 ml of PBST and one time with 4 ml of PBS), Fab-expressing phages that specifically bound to the coated target were eluted using 500 μl of 100 nM TEA, incubated at room temperature for 10 minutes, and then neutralized by the addition of 500 μl of Tris-HCl (pH 7.5). 500 μl of the eluted phages was used to infect Escherichia coli strain HB101. Phages were then amplified in E. coli strain HB101 using M13KO7 helper phage (Invitrogen™). In two subsequent rounds of selection, the target concentration was reduced to 25 μg / ml.In an initial qualitative assessment, 88 randomly selected Fab-expressing phage clones from each selection round were expressed in single wells and tested for binding to huSema3A compared to irrelevant off-targets. The clone pool from round 3 in this example was found to contain a 60% positive hit rate and was selected for further screening.

[0305] In the next step, global removal of gene III fusions in this pool allowed expression of soluble Fabs, and 2208 single clones were selected for expression in E. coli Top10 strain and evaluation of Fab-containing supernatants in huSema3A binding ELISA. The VH and VL sequences of all 2208 clones were also determined using NGS methods. 154 distinct clones positive for binding to huSema3A were identified. These positive binding Fab fragments were tested in a confirmatory binding ELISA and were also evaluated for binding to mouse Sema3A-Fc (TPP-13357, R&D Systems) and in a specificity test using an additional off-target molecule, mouse Sema3F (R&D Systems). Based on this analysis, 48 ​​human / mouse cross-reactive Sema3A binding Fabs were prioritized. These Fab fragments were then purified from 25 ml expression cultures using Capture Select CH1 matrix (Life Technologies), eluted using 12.5 mM citric acid, pH 2.5, and finally buffer exchanged into PBS using Zeba™ Spin desalting plates (ThermoFisher). All 48 purified Fab fragments were kinetically ranked to examine binding to both human and mouse Sema3A by surface plasmon resonance (SPR), reformatted to full-length human IgG1, and retested for binding in SPR (see Example 4).

[0306] Example 3: Sequence optimization, germlining and affinity maturation of lead antibodies TPP-15370 and TPP-15374 The IgG1 antibodies TPP-15370 and TPP-15374 were subjected to lead optimization procedures with the objectives of (i) optimizing their affinity, (ii) increasing functional efficiency, (iii) reducing the risk of sequence-based immunogenicity, and (iv) improving compatibility with downstream development processes.

[0307] Affinity maturation was performed by an initial single mutation gathering round, followed by recombination of the most affinity- and potency-increasing amino acid substitutions in a germlined, sequence-optimized antibody backbone.

[0308] For mutation collection, NNK (N=A or G or C or T, K=G or T) randomization at the following individual amino acid positions was performed by site-directed mutagenesis using synthetic oligonucleotides containing NNK for codon diversification. For TPP-15370, the following regions were analyzed for their effect on affinity: GFTFSSYGMH (residues 26-35 of VH SEQ ID NO:41), WVSAIGTGGDTYYADSVMG (residues 47-65 of VH SEQ ID NO:41), ARRDDYTSRDAFDV (residues 96-109 of VH SEQ ID NO:41), SGSSSNIGSNTVNWY (residues 23-37 of VL SEQ ID NO:45), LLIYYDDLLPS (residues 47-57 of VL SEQ ID NO:45) and AAWDDSLNGYVV (residues 90-101 of VL SEQ ID NO:45).

[0309] For TPP-15374, the following regions were analyzed for their effect on affinity: GFTFSSYEMN (residues 26-35 of VH SEQ ID NO:61), WVSGISWNSGSIGYADSVKG (residues 47-66 of VH SEQ ID NO:61), ARSGYSSSWFDPDFDY (residues 97-112 of SEQ ID NO:61), TGSSSNIGAGYDVHWY (residues 23-38 of VL SEQ ID NO:65), LLIYGNSNRPS (residues 48-58 of VL SEQ ID NO:65) and SSYAGSNPYV (residues 91-101 of VL SEQ ID NO:65).

[0310] The resulting single NNK library was sequenced and identified approximately 1000 single amino acid substitution mutants of TPP15370 and TPP-15374, respectively, which were expressed by transient transfection of mammalian cells and the resulting expression supernatants were normalized for antibody concentration to be screened in surface plasmon resonance and competitive ELISA.

[0311] In the germline and sequence optimization process of TPP-15370 and TPP-15374, the closest germline family for the light and heavy chains was selected and probed for potential CMC-related residues. The deviations from the closest human germline in the CDR and FW regions and the potential CMC-related residues in the CDR regions were adjusted by site-directed mutagenesis and tested in functional and biophysical assays (non-specific binding, temperature stability in DSC). The resulting single revertants and the following combinatorial IgG variants were expressed by transient transfection of mammalian cells and the resulting expression supernatants were normalized for antibody concentration to be screened in binding assays (SPR, competitive ELISA) and functional assays. This resulted in the germlined and sequence-optimized molecules TPP-21565 for TPP-15370 and TPP-18533 for TPP-15374. TPP-21565 contains the reversions L55R and R80Q in the light chain and G33A, H35S, M64K and V109Y in the heavy chain compared to TPP-15370. TPP-18533 contains the reversions A10V, T13A, S78T, R81Q, S82A in the light chain compared to TPP-15374.

[0312] As the final recombination library of TPP-21565, eight single substitution mutants that were shown to show improved affinity and functional efficiency in the screening process of the NNK library were selected. The light chain mutations A90H, G98D, G98V, Y99I and V100P and the heavy chain mutations S30Y, S35L and T53Y were recombined in one recombination library (contiguous amino acid nomenclature; the reference is TPP-21565 defined by SEQ ID NO: 121-VH and SEQ ID NO: 125-VL).

[0313] As the final recombination library of TPP-18533, 11 single substitution mutants that were shown to show improved affinity and functional efficiency in the screening process of NNK library were selected.Light chain mutations N28D, N53A, S91K, S91Q, A94E, S96I and S96P and heavy chain mutations T28D, S30D, S57W and G59Y were recombined in one recombination library (contiguous amino acid nomenclature; reference is TPP-18533 defined by SEQ ID NO:101-VH and SEQ ID NO:105-VL).

[0314] For TPP-18533, oligonucleotides were generated to introduce the selected mutation or the corresponding wild-type amino acid at each selected position. Library construction was performed using successive rounds of overlap-extension PCR. The final PCR products were ligated into a mammalian IgG4(S228P) expression vector, and the mutants were sequenced using massively parallel sequencing technology. For TPP-21565, the recombinant mutants were engineered as separate clones and cloned into an IgG4(S228P)-containing expression plasmid.

[0315] Over 1000 unique combinatorial amino acid substitution mutants of TPP-18533 and over 100 unique combinatorial amino acid substitution mutants of TPP-21565 were generated in this manner, expressed by transient transfection of mammalian cells, and the resulting expression supernatants were normalized for antibody concentration screened at various numbers in SPR, competitive ELISA, and functional assays. Based on the results in these assays, mutants were classified as either "improved" or "non-improved."

[0316] Tables 1 and 1A list preferred antibody candidates according to the present invention, selected in the combinatorial library screening process as being, inter alia, the most potent in binding to Sema3A and in antagonizing Sema3A-dependent biological activity, as well as the respective amino acid and nucleic acid sequences of the antibodies according to the present invention.

[0317] Example 4: Determining affinity and species cross-reactivity using surface plasmon resonance To evaluate the binding kinetics and affinity of anti-Sema3A antibodies and their species cross-reactivity profile, binding assays were performed using surface plasmon resonance (SPR). Binding assays were performed at 25°C on a Biacore T200 instrument or a Biacore 8K+ instrument (Cytiva) using the assay buffer HBS P+, 300 mM NaCl, 0.75 mM CaCl2, 2.5 mM MgCl2, 1 mg / ml BSA, 0.05% NaN3. Antibodies were captured by anti-human Fc IgG ("Human Antibody Capture Kit", order number BR100839, Cytiva) or, in the case of Fc-tagged analytes, by anti-human Fab IgG ("Human Fab Capture Kit", order number 28958325, Cytiva) [covalently amine-coupled to a series S CM5 sensor chip (Cytiva)]. Amine coupling was performed using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and ethanolamine HCl (pH 8.5) ("Amine Coupling Kit" BR-1000-50, Cytiva) according to the manufacturer's instructions. For phage display hits, Fc-tagged human and mouse Sema3A were used as analytes in the concentration range of 1.56-200 nM. Monovalent Sema3A domains from human, mouse, cynomolgus monkey, rat, dog and pig were used as analytes in a concentration series of 0.024-3.125 nM in multi-cycle kinetic mode or at 100 nM for binding analysis only. The sensor surface was regenerated with glycine (pH 2.0) after each antigen injection. The resulting sensorgrams were double referenced (subtraction of the reference flow cell signal and buffer injection) and fitted to a 1:1 Langmuir binding model to derive kinetic data using the Biacore T200 evaluation software. The results are shown in Tables 3, 4 and 4a. [Table 2] TIFF0007659654000003.tif139161

[0318] The majority of the phage display hits bind to human and mouse dimeric Sema3A in the lower nanomolar range. [Table 3]

[0319] All derivative antibodies of TPP-15370 and TPP-15374 have significantly increased affinity for the Sema3A domain in the lower picomolar range compared to their parent antibodies and most prior art antibodies. [Table 4]

[0320] In contrast to full-length 3H4 IgG1 (TPP-30792), which showed no binding to Sema3A molecules in SPR, TPP-31357, a Fab variant of TPP-30792, shows binding to human Sema3A, albeit with lower affinity than TPP-23298.

[0321] Example 5: Measurement of binding activity using surface plasmon resonance To evaluate the binding activity of anti-Sema3A antibodies, binding assays were performed using surface plasmon resonance (SPR). Binding assays were performed at 25°C on a Biacore T200 instrument (Cytiva) using assay buffer HBS P+, 300 mM NaCl, 0.75 mM CaCl2, 2.5 mM MgCl2, 1 mg / ml BSA, 0.05% NaN3. Antibodies were captured by anti-human Fc IgG ("Human Antibody Capture Kit", order number BR100839, Cytiva) that was covalently amine-coupled to a series S CM5 sensor chip (Cytiva). Amine coupling was performed using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and ethanolamine HCl (pH 8.5) ("Amine Coupling Kit" BR-1000-50, Cytiva) according to the manufacturer's instructions. Monovalent Sema3A domains from human, mouse, cynomolgus monkey, rat, dog and pig were used as analytes at a series of concentrations from 0.024 to 3.125 nM in multi-cycle kinetics mode. The sensor surface was regenerated with glycine (pH 2.0) after each antigen injection. The resulting sensorgrams were double-referenced (subtraction of reference flow cell signal and buffer injection) and fitted to a 1:1 Langmuir binding model using the Biacore T200 evaluation software to obtain the experimentally fitted R Max To calculate the binding activity, we first calculated the theoretical R Max must be calculated according to the following formula 1:

number

[0322] The experimentally determined R according to Eq. Max , the theoretically calculated R Max Binding activity was determined by dividing by .

number

[0323] The binding activity calculated in the SPR experiment is a measure of the activity of the surface-bound ligand. As can be seen from Table 5, TPP-15370, TPP-15374, TPP-23298 and TPP30788-30791 can bind to all tested Sema3A domains with approximately 100% activity. This means that all binding regions can bind completely. Prior art antibody TPP-17755 only reaches an activity level of 50-60% depending on the species. Prior art antibody TPP-11489 shows even lower levels, below 50%, except for mouse and pig, which show higher levels. Surprisingly, to reach such activity levels, the ligand level of TPP-11489 needs to be more than 10 times higher than the other antibodies, which generally shows a much lower binding activity compared to TPP-15370, TPP-15374 and TPP-23298.

[0324] Example 6: Competitive ELISA For screening in competitive ELISA, human Sema3a (TPP-13211) was coated onto 384-well plates (Greiner bio-one, 781077) at a concentration of 0.5 μg / ml in coating buffer (carbonate-based, pH 9.6, Candor 121125) overnight at 10° C. After washing the plates three times with 50 μl PBS 0.05% Tween, the plates were blocked with 50 μl Smart Block® (Candor 113500) for 1 h at 20° C. and washed again three times as described.

[0325] Then, 20 μl of premixed antibody solution was added to the plate and incubated at 10° C. for 18 hours. To obtain the premixed antibody solution, one biotinylated parent antibody, either TPP-15370 or TPP-15374, was mixed with an antibody (recombinant variant) that does not contain a biotin tag and contains one or more amino acid mutations in the CDR region in a ratio of 1:1, 1:5, or 5:1 per well. As an additional control, an isotype control antibody that does not show binding to human Sema3A was also used as a competing antibody. The total concentration of the added antibody solution was 0.25 μg / ml. During the incubation period, the antibodies competitively bound to the plate because they compete for the same epitope on the human Sema3A protein.

[0326] After washing three times with 50 μl PBS 0.05% Tween, 20 μl of streptavidin-HRP solution (R&D Systems, DY998, 1:200 in PBS 0.05% Tween 10% SmartBlock) was then added and incubated for 1 h at 20° C., followed by washing three times with 50 μl PBS 0.05% Tween and adding 20 μl of Amplex Red solution (Invitrogen A12222; 1:1000 in NaP buffer 50 mM, pH 7.6, containing 1:10000 of 30% H2O2). After a final incubation of 20 min at 20° C., the signal was measured using an emission wavelength of 595 nm and an excitation of 530 nm. Due to the biotinylation of the parent antibodies TPP-15370 and TPP-15374, only the binding of these mutants could be detected. Thus, competition with an antibody variant that exhibits superior binding will show a lower binding signal compared to competition with, for example, a non-biotinylated form of the parent antibody itself.

[0327] Overall, 103 recombinant variants of TPP-15370 and 1136 recombinant variants of TPP-15374 were measured. For analysis and to allow correction of plate-to-plate variability, the raw ELISA values ​​were normalized to those of competition with the isotype control antibody TPP-9809.

[0328] Table 6 lists the competitive ELISA values ​​of selected recombinant variants of TPP-15370 and TPP-15374 relative to the isotype control antibody TPP-9809 measured at a ratio of 1:5 or 1:1, respectively. [Table 6]

[0329] Example 5a: Epitope binning using surface plasmon resonance (SPR) By utilizing the classical sandwich method, an epitope binning experiment was performed to determine the epitope bins of anti-Sema3A antibodies using SPR. In this experiment, one antibody is immobilized on an SPR chip, Sema3A is injected, and binding is monitored (Figure 10A). After successful binding of Sema3A to the first antibody, a second antibody is injected onto the complex of immobilized mAb bound to Sema3A, and further binding is monitored (Figures 10B and 10C). If the second antibody competes with the first antibody for binding to Sema3A, no additional binding signal is detected after injection of the second antibody, indicating that the two antibodies bind to the same or closely adjacent Sema3A epitopes (Figure 10C). If the second antibody does not compete with the first antibody for binding to Sema3A, an additional binding signal is detected after injection of the second antibody, indicating that the two antibodies bind to different Sema3A epitopes (Figure 10B).

[0330] Experiments were performed at 25°C on a Biacore T200 instrument (Cytiva) using assay buffer HBS P+, 300 mM NaCl, 0.75 mM CaCl2, 2.5 mM MgCl2, 1 mg / ml BSA, 0.05% NaN3. Antibodies were covalently amine-coupled to a series S CM5 sensor chip (Cytiva). Amine coupling was performed using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and ethanolamine HCl (pH 8.5) ("Amine Coupling Kit" BR-1000-50, Cytiva) according to the manufacturer's instructions. Human monovalent Sema3A domain was used as the first analyte at a concentration of 200 nM, followed by a second injection of a competing antibody. This setup was performed for all possible combinations. The sensor surface was regenerated with glycine (pH 2.0) after each antigen injection. Table 6a shows the binning results. [Table 7]

[0331] Binning experiments strongly indicated a distinct epitope for TPP-23298 compared to TPP-30788 (BI clone I), implying that both antibodies target independent / different epitopes on Sema3A, while TPP-23298 may have overlapping or adjacent epitopes with TPP-17755 (Samsung).

[0332] Example 7: Assessment of off-target binding To assess the specificity of the anti-Sema3A mAb (TPP-15370, parent mAb), off-target screening was performed using Retrogenix technology. In the primary screen, 5484 expression vectors encoding both ZsGreen1 and full-length human cell membrane proteins or cell surface-associated human secreted proteins were arrayed in duplicate across 16 microarray slides. Human HEK293 cells were used for reverse transfection / expression.

[0333] Test antibodies were added to each slide after cell fixation to a final concentration of 20 μg / ml. Detection of binding was performed using the same AF647 anti-hIgG Fc detection antibody used in prescreening. For each of the 16 slide sets, two replicate slides were screened. Hits were classified as "strong, moderate, weak, or very weak" depending on the intensity of the duplicate spots.

[0334] After screening for binding against fixed HEK293 cells expressing 5484 human cell membrane proteins and human secreted proteins, Retrogenix technology did not identify any specific off-target interactions for the test antibody TPP-15370. Binding to Sema3A (its primary target) was observed. These data indicate that TPP-15370 is highly specific for its primary target.

[0335] Example 8: Selectivity of anti-Sema3A mAb Semaphorin proteins can be subdivided into five classes (classes 3-7) found in vertebrates. To evaluate the selectivity profile of the parent anti-Sema3A mAbs, TPP-15370 and TPP-15374, in the semaphorin 3 class (Sema3A-G), ELISA assays were performed using Sema3A, Sema3B, Sema3C, Sema3D, Sema3E and Sema3F molecules from R&D Systems. Neither antibody showed binding to Sema3B, Sema3C, Sema3D, Sema3E or Sema3F.

[0336] Since Sema3G has recently been confirmed to be renal protective (PMID:27180624), it was important to test whether the antibody does not bind to Sema3G. To evaluate the binding selectivity for Sema3A versus Sema3G, 1 nM recombinant human Sema3A-Fc chimera (R&D Systems) or recombinant human GST-Sema3G (Abnova) was coated onto Maxisorb plates and incubated with antibodies in a dose-response curve from 0.00015 to 10 μg / ml, and antibody binding was quantified using HRP-conjugated anti-human antiserum and a chemiluminescent substrate. [Table 8]

[0337] As shown in Table 7, all tested antibodies of the present invention and prior art antibodies do not bind to the renal protective Sema3G.

[0338] Sema3A is a secreted protein that contains two furin cleavage sites and exists in active or inactive truncated forms. In the in vivo situation, Sema3A exists side by side in both forms. To test whether anti-Sema3A antibodies can distinguish between inactive and active forms, and to test how the antibodies function in binding to active Sema3A (similar to full-length Sema3A (TPP-13211)) compared to the inactive form that contains only Sema3A domain (similar to truncated Sema3A TPP-19068), ELISA assay was performed. As a reading, the ELISA signal of the test antibody against active Sema3A is divided by the ELISA signal of the test antibody against inactive Sema3A. [Table 9]

[0339] The binding analysis shown in Table 7a clearly demonstrated that the antibody of the present invention (TPP-23298) exhibited enhanced binding to active Sema3A than TPP-30788 to TPP-30791 (BI clones), possibly because they target different epitopes that exhibit higher selectivity for active Sema3A.

[0340] Example 9: In vitro efficacy in mesangial cell migration assay Confluent monolayers of human primary mesangial cells were generated by seeding cells in serum-containing medium in Imagelock plates for 24 hours. After switching to serum-free medium, a scratch was created using the WoundMaker tool, and cells were then treated with 1 nM recombinant human Sema3A-Fc chimera (R&D Systems) in the absence or presence of inhibitory antibodies. Cells were imaged in an Incucyte, and the extent of wound closure was assessed after 24 hours using the Incucyte Integrated Cell Migration Analysis software module. [Table 10] TIFF0007659654000016.tif227164

[0341] The present inventors have identified antibodies with potencies in the triple digit picomolar range in a human mesangial cell migration assay, which are significantly more potent than prior art antibodies, as shown in Table 8.

[0342] Example 10: In vitro efficacy in the growth cone collapse assay To determine the potency of antibodies against Sema3A-induced cytoskeletal collapse, a growth cone collapse assay was used similar to that described (PMID:12077190) with some modifications. Briefly, mouse dorsal root ganglion (DRG) neurons were isolated from E13 C57Bl / 6J mouse embryos and cultured on poly-L-lysine and laminin coated 96-well plates containing Neurobasal medium + 100ng / mL NGF + B-27 + 10% FCS. After 20 hours, cells were treated with 10nM recombinant human Sema3A-Fc chimera (RnD Systems) for 1 hour in the absence or presence of inhibitory antibodies, followed by PFA fixation and staining with Alexa488-phalloidin. The extent of growth cone collapse was assessed using immunofluorescence microscopy by actin growth cone area / shape / texture for more than 100 growth cones per well. [Table 11] TIFF0007659654000018.tif164164

[0343] The identified antibodies also exhibited potency in the single digit nanomolar range in the mouse growth cone collapse assay, again significantly more potent than the prior art antibodies tested (2-3 digit nanomolar potency), as shown in Table 9.

[0344] Example 11: In vitro efficacy in HUVEC repulsion assay Recombinant human Sema3A-Fc chimera (R&D Systems) is not identical to Sema3A in human biological fluids because it contains some mutant amino acids and an extra protein fragment at the carboxy terminus. Furthermore, the assays (human mesangial cell migration assay and mouse growth cone collapse assay) use uniformly distributed Sema3A. This is in contrast to the gradient distribution described for Sema3A in tissues. We hypothesized that these differences may result in different potencies of antibodies against recombinant and endogenous proteins. Therefore, we adopted an assay using a gradient of human wild-type Sema3A as an agonist (PMID:17569671). Briefly, in this HUVEC repulsion assay, human embryonic kidney 293 cells (HEK293) expressing human Sema3A of sequence SEQ ID NO: 600 are seeded on a confluent monolayer of human umbilical vein endothelial cells (HUVEC) in EGM-2 medium in the absence or presence of inhibitory antibodies, cultured for 72 hours, fixed, stained with DAPI, and assessed by immunofluorescence microscopy for the degree of cell repulsion (measurement of cell-free area).Therefore, the substrate human Sema3A is present in excess.

[0345] Based on the immunofluorescence microscopy images of DAPI / CM stained cells (CM = HCS CellMask™ Stain stains the whole cell to define the total cell area), data analysis is performed as follows: Identify cells based on DAPI / CM signal (Figure 11B). Define and select the cell area for analysis. Calculate cell-free area in this area (Figure 11C). Calculate inhibition percentage based on "cell-free area" induced by Sema3A in antibody-treated wells compared to isotype-treated wells. Plot inhibition percentage against antibody concentration and calculate EC50 value for each antibody.

[0346] In detail, the following steps are carried out for data analysis.

[0347] 1. Four fields are imaged per well, representing 80% of the well area. The stitched together set of fields is used to detect cells by DAPI / CM fluorescence.

[0348] 2. Calculate "cell area" based on DAPI / CM area.

[0349] 3. Calculate the "cell-free area" based on "total area" minus "cell area".

[0350] 4. % inhibition is calculated based on the "cell-free area" induced by Sema3A in antibody-treated vs. isotype-treated wells.

[0351] 5. Determine EC50 values ​​using non-linear regression (variable slope model = 4 parameter dose response curve) using the software GraphPad Prism. [Table 12] TIFF0007659654000020.tif99166

[0352] The difference in potency with the prior art antibodies in the human mesangial cell migration assay and mouse growth cone collapse assay described above is even more pronounced in this HUVEC repulsion assay using a gradient of native wild-type Sema3A (a mixture of processed inactive Sema3A and undigested active Sema3A), as shown in Tables 10 and 10a. The improved potency in the HUVEC repulsion assay compared to TPP-17755, TPP-11489, TPP-30788, TPP-30789, TPP-30790 or TPP-30791 is quantified by measuring the picomolar activity as indicated by the corresponding EC50 value. TPP-23298 exhibits double-digit picomolar activity, whereas the potencies of the prior art antibodies TPP-17755, TPP-11489, TPP-30788, TPP-30789, TPP-30790 or TPP-30791 are in the triple-digit picomolar or even nanomolar range.

[0353] As an alternative illustration of the results, improved potency in HUVEC repulsion assay is quantified by measuring the cell-free area at a specified concentration of 80 pM of each antibody. TPP-23298 shows a higher Sema3A inhibition percentage than TPP-30788, TPP-30789, TPP-30790 or TPP-30791 (Figure 12).

[0354] Analysis of the data from both assays shown in Tables 10 and 10a indicates that TPP-23298 shows the highest potency against cell Sema3A-induced HUVEC repulsion. BI antibodies TPP-30788, TPP-30798, TPP-30790 and TPP-30791 showed slightly higher EC50 values ​​(2-5 fold). Samsung antibody TPP-17755 has significantly lower potency (50 fold) than TPP-23298. Chiome antibody TPP-11489 only showed inhibitory activity at the highest tested concentration, resulting in a predicted EC50 value that is more than 400 fold higher than the antibody according to the invention. As shown in Tables 10 and 10a, this indicates that under conditions similar to the natural environment in the absence of added exogenous recombinant Semaphorin 3A, the antibody according to the invention inhibits Sema3A-induced cell repulsion with the strongest activity.

[0355] Example 12: In vivo assay to detect renal protective effects: Inhibition of Sema3A-induced albuminuria in mice Sema3A inhibitors reduce urinary albumin excretion induced by systemic injection of recombinant Sema3A. The beneficial effects of the compounds on reducing albuminuria were examined in a Sema3A-induced albuminuria model as follows.

[0356] Male C57Bl / 6 mice (8–10 weeks old) purchased from Taconic were injected intravenously with anti-Sema3A antibody. Thirty minutes after antibody application, albuminuria was induced by intravenous injection of human recombinant Sema3A (1.0 mg / kg, R&D Systems). Animals were placed in metabolic cages and urine was collected for 4 h. Urinary creatinine was measured by a clinical biochemistry analyzer (Pentra400). For evaluation of urinary albumin, a mouse-specific albumin ELISA (Abcam) was used according to the manufacturer's protocol. Both urinary creatinine and albumin were used to calculate the urinary albumin-to-creatine ratio (ACR). Differences between groups were analyzed by one-way ANOVA with Dunnett's correction for multiple comparisons. Statistical significance was defined as p<0.05. All statistical analyses were performed using GraphPad Prism8.

[0357] Tables 11-15a show dose-response experiments with TPP-15370, TPP-15374, TPP-11489, TPP-17755, TPP-30788 and TPP-23298 in the Sema3A-induced albuminuria model in mice. The effects of TPP-15370, TPP-23298 on albuminuria reduction compared to TPP-11489 and / or TPP-17755 and / or TPP-30788 are shown in Figures 1-2.

[0358] The antibodies according to the invention reduce Sema3A-induced urinary albumin excretion. [Table 13] TIFF0007659654000022.tif140163TIFF0007659654000023.tif140164

[0359] Example 13: In vivo assay to detect renal protective effects: Acute ischemia / reperfusion injury (I / RI) model in mice Unilaterally nephrectomized mice may benefit from treatment with Sema3A inhibitors after ischemia-reperfusion injury. The beneficial effects of Sema3A antibodies on renal function were examined in a renal ischemia-reperfusion injury model in mice as follows.

[0360] Laboratory-bred male C57Bl / 6J mice aged 6–8 weeks were obtained from CharlesRiver. Mice were maintained under standard laboratory conditions with a 12-h light-dark cycle with regular food and drinking water available ad libitum. A total of 8–10 mice were used in each control and experimental group for the ischemia-reperfusion injury model.

[0361] Animals were anesthetized with continuous inhaled isoflurane. Right nephrectomy was performed through a right flank incision 7 days prior to ischemic treatment of the contralateral kidney. Antibodies and appropriate isotype controls were administered intravenously to mice 1 hour prior to the onset of renal ischemia. Mice were anesthetized and the left flank incision was made. The left renal pedicle was incised to expose the renal vessels. Atraumatic vascular clamps were used to stop blood flow (arterial and venous) for 25 minutes (mice) of ischemia. Reperfusion was established by removing the clamps. The abdominal wall (muscle layer and skin) was closed with 5.0 polypropylene sutures. Temgesic® (buprenorphine, 0.025 mg / kg subcutaneously) was applied as analgesic.

[0362] Urine from each animal was collected overnight in metabolic cages and then sacrificed at 24 hours after ischemia. Urinary creatinine was measured on a clinical biochemistry analyzer (Pentra400). For evaluation of urinary albumin, a mouse-specific albumin kit (Hitachi) was used in the Pentra analyzer. Both urinary creatinine and albumin were used to measure albuminuria (albumin / creatinine ratio). At the time of sacrifice, blood samples were taken under terminal anesthesia. Serum was isolated after centrifugation of the blood samples. Both serum creatinine and serum urea were measured on a clinical biochemistry analyzer (Pentra400). Differences between groups were analyzed by one-way ANOVA with Dunnett's correction for multiple comparisons. Statistical significance was defined as p<0.05. All statistical analyses were performed using GraphPad Prism8.

[0363] Tables 16-20 show dose-response studies with TPP-15370, TPP-15374, TPP-11489, TPP-17755 and TPP-23298 in acute renal ischemia / reperfusion injury models in mice. Figure 3 shows efficacy of TPP-23374, TPP-23298 and TPP-15370 following treatment at 15 mg / kg in the I / RI model. The effect of treatment with TPP-15370, TPP-23298 and TPP-15374 compared to TPP-11489 and / or TPP-17755 is shown in Figures 4-6.

[0364] The antibody reduced ischemia / reperfusion-induced renal injury by reducing serum creatinine and serum urea (surrogates of glomerular filtration rate) and urinary albumin excretion. [Table 14] TIFF0007659654000025.tif196163TIFF0007659654000026.tif103162

[0365] Example 14: In vivo assay to detect renal protective effects: Alport syndrome model (Col4α3-deficient mice) The phenotype of Alport mice is similar to that of Alport patients, including characteristic thickening and fragmentation of the glomerular basement membrane and strong proteinuria. Alport mice may benefit from treatment with Sema3A inhibitors due to enhanced Sema3A expression in the kidneys of these mice. The beneficial effect of Sema3A blocking antibodies on renal function was examined in the Alport mouse model as follows. Knockout Col4α3 (129-Col4a3) was introduced into the mouse model by breeding heterozygous animals in the breeding facility at Bayer AG, Wuppertal, Germany. <tm1dec>A colony of Col4α3 / J) mice (Jackson Laboratory, USA) was established. Male and female homozygous and wild-type Col4α3 mice aged 4 to 5 weeks were obtained from the animal care facility at Bayer AG and used in this study.

[0366] Homozygous mice (HOM) were randomized into groups according to their age and sex (n=10 per group). Mice were administered isotype control, TPP-15370 and TPP-23298 once a week. TPP-11489 was administered every other week. Urine was collected from each animal in a metabolic cage once a week, starting before the start of treatment. Urinary creatinine and total protein were measured by a clinical biochemistry analyzer (Pentra400). Both urinary creatinine and albumin were used to measure proteinuria (protein / creatinine ratio). Upon sacrifice on the 21st or 28th day after the start of treatment, blood samples were taken under terminal anesthesia. After centrifugation of the blood samples, serum was isolated. Both serum creatinine and serum urea were measured by a clinical biochemistry analyzer (Pentra400).

[0367] Kidneys were harvested and divided into two parts. One part was snap frozen in liquid nitrogen for mRNA analysis. The other part was stored in Davidson fixative for preparation of tissue sections. Total RNA was isolated from the harvested kidney parts. Kidney tissue was homogenized, RNA was obtained and transcribed into cDNA. TaqMan real-time PCR was used to analyze renal mRNA expression of profibrotic markers in kidney tissue. For evaluation of fibrosis at the protein level, paraffin tissue sections were stained with alpha smooth muscle actin (αSMA) and Sirius Red / Fast Green collagen stains using standard methods.

[0368] Quantitative measurements of alpha smooth muscle actin (αSMA)-positive and Sirius red (collagen)-positive areas within the kidney were obtained by computer image analysis using an AxioScan Z1 (Zeiss) microscope and Zen software.

[0369] All data are expressed as mean ± SD. Differences between groups were analyzed by one-way ANOVA with Dunnett's correction for multiple comparisons. Statistical significance was defined as p < 0.05. All statistical analyses were performed using GraphPad Prism8.

[0370] Tables 21A-21C and 22A-22C show the effects on proteinuria, renal function and renal fibrosis obtained after treatment with TPP-15370 and TPP-23298 in the Alport model. The effects on proteinuria, renal function and renal fibrosis after treatment with TPP-15370 compared to TPP-11489 are shown in Figures 7 and 8.

[0371] Antibodies according to the invention halted the progression of kidney disease in a mouse model of Alport syndrome, reduced urinary protein excretion, decreased creatinine and serum urea (surrogates for glomerular filtration rate), and reduced fibrosis as quantified by myofibroblast staining and collagen deposition. [Table 15] TIFF0007659654000028.tif220162TIFF0007659654000029.tif202163

[0372] Example 15: In vivo assay to detect renal protective effects: Unilateral renal IRI model in pigs TPP-23298 was tested in a minimally invasive unilateral renal artery balloon catheter occlusion model in adult minipigs with follow-up after approximately 24 hours of reperfusion. Female Göttingen minipigs (Ellegaard, Denmark) weighing in the range 14-17 kg were used for the experiments. Animals were randomly assigned to experimental groups.

[0373] In a blinded controlled study, TPP-23298 was administered to six animals and compared to six matched IgG-treated controls. Animals that underwent all treatment procedures without renal artery occlusion and received only the phosphate-buffered saline vehicle served as a sham-treated reference group.

[0374] TPP-23298 was administered by slow intravenous injection in a weight-adjusted dose in a final volume of 1 ml / kg phosphate-buffered saline as a bolus prior to the initiation of renal artery occlusion (prophylactic setting).

[0375] For the first day of intervention, the pigs were anesthetized with a combination of propofol and fentanyl and ventilated with an oral tracheal tube under muscle relaxation with pancuronium. Volume was replaced continuously by continuous infusion of lactated Ringer's solution. Before the start of surgery, antibiotics and thrombosis prophylaxis were given by intramuscular administration of enrofloxacin and intravenous administration of heparin, respectively. Blood pressure and heart rate were monitored using a non-invasive veterinary device with front leg cuffs.

[0376] All the following interventions were performed under strict aseptic conditions. For drug administration, a catheter was passed subcutaneously through the dorsal neck skin into the jugular vein. A sheath was placed and fixed in the femoral artery (preferably the left one), through which a hockey stick catheter with a balloon catheter inside was advanced upstream into the abdominal aorta and its tip was inserted into the opening of the left or right renal artery. The balloon catheter was then ejected and the balloon was inflated to block the blood flow to the kidney. The correct position of the balloon was controlled by Doppler ultrasound using a commercially available ultrasound diagnostic device. Plasma samples were collected at baseline and 2 hours after the beginning of ischemia.

[0377] Renal ischemia was removed at precisely defined time points (ranging from 90 to 120 min) after the onset of occlusion by deflating the balloon and withdrawing the catheter and sheath. After suturing the vessels and closing the wound, the animals were allowed to awaken again from anesthesia and extubated after spontaneous breathing had begun.

[0378] Approximately 22-23 hours after renal artery occlusion, animals were reanesthetized with a combination of Ketacet / Dormicum and pancuronium and artificially ventilated as described. Blood pressure and heart rate were monitored invasively via a carotid catheter. Volume replacement was performed with intravenous administration of lactated Ringer's at a flow rate of 10 ml / kg / h. Through a small incision in the lower abdomen, both ureters were dissected above the bladder wall and catheterized to collect urine separately for volume measurements and urinalysis. Once all parameters were stable (this was typically 24 hours after occlusion), recordings and sample collection were started. Blood samples were collected at baseline and at each time point over a 3-hour period (24-27 hour intervals). In parallel, urine was collected at three intervals of 1 hour.

[0379] Urine volumetric flow rate (V U ) and urinary creatinine concentration ([Crea] U ) and then measure the standard CL Crea =V U * [Crea] U / [Crea] Pl (Here, [Crea] Pl represents the plasma creatinine concentration), and creatinine clearance (CL Crea ) was calculated separately for each side. The CL of the left and right kidney of each animal was Crea The total CL is calculated by adding Crea was calculated.

[0380] The results are shown in Figure 9. TPP-23298 significantly prevented the ischemia / reperfusion-induced deterioration of creatinine clearance in this experimental setting after 105 minutes of unilateral renal artery occlusion when administered prophylactically 30 minutes prior to occlusion.

[0381] Example 16: Expression titers of anti-Sema3A antibodies in mammalian cell culture HEK293-6E cells were transfected with pTT5 plasmids encoding the Fab fragment of TPP-30792 (TPP-31357) or the heavy and light chains of anti-Sema3A antibodies. Two days prior to transfection, HEK293-6E cells were split to a density of 5×10?5 cells / mL in FreeStyle™ F17 Expression Medium (Gibco, A1383501) containing 0.1% Pluronic F68 (Gibco, 24040032) and 4 mM GlutaMax (Gibco, 35050061) in shake flasks, constituting 90% of the desired expression level. HEK293-6E cells were cultured at 37°C, 5% CO2 with shaking at 75 rpm.

[0382] For transfection, the DNA is mixed with polyethyleneimine (Polysciences, 29366) in FreeStyle™ F17 expression medium (Gibco, A1383501) containing 4 mM GlutaMax (Gibco, 35050061), which constitutes 10% of the final expression amount. The solution is incubated for 10 minutes and added to the shake flask. 24 hours after transfection, 1% (v / v) ultra-low IgG FBS (Gibco, 16250078) and 0.05% (v / v) 1N valproic acid (Sigma, P4543) are added to the shake flask.

[0383] Cell viability and density are monitored daily from 4 days after transfection, and when viability is determined to be 70%, the supernatant is harvested by centrifugation and sterile filtration. To determine production titer, 100 μL of harvested supernatant is loaded onto a 0.1 mL Poros A affinity column (Thermo Scientific, 2100100) via an HPLC system (Agilent, 1100 HPLC system) using 50 mM sodium phosphate (Sigma, S0751, S9763), 150 mM NaCl (Sigma, S6546), 5% 2-propanol (sigma, 34863) (pH 7.2) as the running buffer. Protein is then eluted using 12 mM HCl (Sigma, H9892), 150 mM NaCl, 5% 2-propanol (pH 2). A calibration curve from 5 μg / mL to 150 μg / mL is set up using proteins of known size and also applied to a Poros A column via an HPLC system. Taking into account the size and extinction coefficient of the protein in the supernatant, the exact titer can be calculated using the standard curve. Expression in CHO is similar to HEK cells, except that the plasmid pTT22AKT was used for TPP-30792. [Table 16]

[0384] The antibodies of the present invention, and all prior art antibodies except TPP-30792, can be produced in high titers in mammalian cells, as shown in Table 23. TPP-30792 could not be expressed in significant amounts in either HEK or CHO cells. A total of 125 μg of TPP-30792 could be purified from 4.5 liters of HEK293 cell culture. Similarly, the Fab fragment of TPP30792 (TPP-31357) yielded only 200 μg of purified Fab from 5 liters of HEK293 cell culture.

[0385] Example 17: Analysis of stability and solubility of CMC parameters of anti-Sema3A antibodies It is known that high-concentration protein solutions, above 50 mg / ml, usually exhibit higher viscosity compared to lower-concentration protein solutions. Increased viscosity adversely affects the deliverability of protein solutions, especially in the case of low application volumes, which can increase injection time and pain at the injection site. In addition, high viscosity affects industrial large-scale protein production. Therefore, reducing the viscosity of high-concentration protein solutions while maintaining stability over long storage periods is particularly important in in vivo therapeutic situations.

[0386] Proteins in concentrated solutions often exhibit lower stability than those in dilute solutions because, at higher concentrations, proteins tend to aggregate and can reversibly self-associate. Aggregation can adversely affect the structural integrity and therefore the amount of functional and bioavailable protein in an in vivo therapeutic context. This makes delivery by injection even more difficult.

[0387] Protein solubility is another important quality criterion: increasing the solubility of the isolated protein allows the preparation of highly concentrated solutions necessary for in vivo therapeutic situations.

[0388] Therefore, obtaining highly concentrated protein solutions with low viscosity and high stability and solubility is beneficial for the therapeutic applicability of therapeutic molecules.

[0389] To evaluate the stability, solubility and viscosity of CMC (Chemistry, Manufacturing and Control) parameters of anti-Sema3A antibodies for potential therapeutic applications, antibodies TPP-23289 and TPP-30788 (BI clone I) were diluted to 25 mg / ml in PBS and incubated at 700 rpm and 40°C for 2 weeks. Although antibodies are typically stored frozen at 4-10°C for short-term storage or below -18°C or above -81°C for long-term storage, exposure of mammalian antibodies to temperatures above 40°C (the average body temperature of mammals is 36°C-39°C) mimics heat stress conditions. In this heat stress condition, accelerated protein stability / stress stability is tested. Stability analysis was evaluated by size exclusion chromatography using a Superdex 200 column (Cytiva) connected to an Akta system (Cytiva) in PBS buffer, and by capillary gel electrophoresis using a Caliper system (Perkin Elmer). Profile changes were calculated as a percentage (%) of the non-stressed starting material. Solubility was determined by concentrating the anti-Sema3A antibody using an Amicon spin filter (Millipore) with a cutoff of 30 kDa in PBS buffer. Solubility was measured at 90% recovery from the concentrator, and protein concentration was measured using absorbance at UV 280 nm. [Table 17]

[0390] As mentioned above, stability, solubility and viscosity are important CMC parameters for therapeutic molecules. The structural integrity after heat stress conditions such as exposure to 40°C or concentration steps is analyzed by SEC and / or cGE to examine the impact of stress on structural integrity. Less than 1% change after stress compared to the start indicates a stable molecule, while deviations of more than 1% indicate instability in the molecule. TPP-23289 shows much higher solubility in PBS compared to TPP-30788, i.e., more than twice as high, which is very beneficial, for example, to allow low application volumes. Furthermore, TPP-23298 is more stable than TPP-30788, more resistant to heat stress, and less viscous in PBS buffer.

[0391] array [Table 18] TIFF0007659654000033.tif247162TIFF0007659654000034.tif246163TIFF0007659654000035.tif246163TIFF0007659654000036.t if247163TIFF0007659654000037.tif246164TIFF0007659654000038.tif247162TIFF0007659654000039.tif245161TIFF00076596540 00040.tif245162TIFF0007659654000041.tif246161TIFF0007659654000042.tif246162TIFF0007659654000043.tif245161TIFF000 7659654000044.tif244161TIFF0007659654000045.tif245161TIFF0007659654000046.tif245161TIFF0007659654000047.tif125160 [Table 19] TIFF0007659654000049.tif246165TIFF0007659654000050.tif249165TIFF0007659654000051.tif250164TIFF0007659654000052.tif248164TIFF0007659654000053.tif249164TIFF0007659654000054.tif246163TIFF0007659654000055.tif246164TIFF0007659654000056.tif246164TIFF0007659654000057.tif250164TIFF0007659654000058.tif249164TIFF0007659654000059.tif249164TIFF0007659654000060.tif250164TIFF0007659654000061.tif246164TIFF0007659654000062.tif249163TIFF0007659654000063.tif250164TIFF0007659654000064.tif247164TIFF0007659654000065.tif250164TIFF0007659654000066.tif248164TIFF0007659654000067.tif247164TIFF0007659654000068.tif247163TIFF0007659654000069.tif250164TIFF0007659654000070.tif247163TIFF0007659654000071.tif249164TIFF0007659654000072.tif249164TIFF0007659654000073.tif247164TIFF0007659654000074.tif249165TIFF0007659654000075.tif249163TIFF0007659654000076.tif248164TIFF0007659654000077.tif247164TIFF0007659654000078.tif246164TIFF0007659654000079.tif250164TIFF0007659654000080.tif245164TIFF0007659654000081.tif249164TIFF0007659654000082.tif248164TIFF0007659654000083.tif248164TIFF0007659654000084.tif247165TIFF0007659654000085.tif250164TIFF0007659654000086.tif231164.

Claims

1. An isolated antibody or antigen-binding fragment thereof that binds to human Sema3A, comprising a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO: 142, H-CDR2 comprising SEQ ID NO: 143, and H-CDR3 comprising SEQ ID NO: 144, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO: 146, L-CDR2 comprising SEQ ID NO: 147, and L-CDR3 comprising SEQ ID NO:

148.

2. 2. The isolated antibody or antigen-binding fragment of claim 1, comprising a variable heavy chain domain that is at least 98% identical to SEQ ID NO:141, and a variable light chain domain that is at least 98% identical to SEQ ID NO:

145.

3. An isolated antibody or antigen-binding fragment of claim 1, comprising a variable heavy chain domain comprising SEQ ID NO:141 and a variable light chain domain comprising SEQ ID NO:

145.

4. The isolated antibody described in claim 1, which is an IgG1 or IgG4 antibody.

5. An isolated antibody as described in claim 1, comprising a heavy chain comprising sequence number 157 and a light chain comprising sequence number 158.

6. The antigen-binding fragment of claim 1, which is an scFv, Fab, Fab' fragment or F(ab')2 fragment.

7. An antibody conjugate comprising an isolated antibody or antigen-binding fragment according to any one of claims 1 to 6.

8. A pharmaceutical composition comprising the isolated antibody or antigen-binding fragment of any one of claims 1 to 6 or the antibody conjugate of claim 7.

9. A kit comprising the isolated antibody or antigen-binding fragment of any one of claims 1 to 6, the conjugate of claim 7, or the pharmaceutical composition of claim 8, together with instructions for use.

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