Anti-family with sequence similarity 19, member a5 antibodies and method of use thereof

Abstract

To provide antibodies that specifically bind to human FAM19A5, and to provide compositions comprising the antibodies.SOLUTION: In some embodiments, antibodies are de-immunized to reduce immunogenicity in a human subject. In certain embodiments, antibodies have undergone affinity maturation. In some embodiments, the anti-FAM19A5 antibodies can modulate FAM19A5 activity, e.g., inhibit, suppress, reduce, or reverse the onset of reactive gliosis and / or excessive proliferation of reactive astrocytes, utilizing the antibodies. The present disclosure also provides methods for treating disorders, such as central nervous system damage, a degenerative brain disorder, a neuropathic pain, or a cancer, by administering an antibody that specifically binds to human FAM19A5.SELECTED DRAWING: Figure 13A

Description

Detailed Description of the Invention

[0001] [Technical field] CROSS-REFERENCE TO RELATED APPLICATIONS This PCT application claims priority to U.S. Provisional Application Nos. 62 / 787,711, filed January 2, 2019, and 62 / 838,190, filed April 24, 2019, each of which is incorporated by reference in its entirety. References for Electronically Submitted Sequence Listings The contents of the electronically submitted sequence listing in ASCII text file (Name: 3763_016PC02_SeqListing_ST25.txt; Size: 90,231 bytes; and Created on: December 30, 2019) submitted with this application are hereby incorporated by reference in their entirety. Statement of government support This research was supported by a research grant from the Ministry of Trade, Industry and Energy (MOTIE) of the Republic of Korea for the Industrial Technology Innovation Project (10081300, Development of a new therapeutic monoclonal antibody drug using the inhibitory mechanism of glial scar formation for ischemic stroke).

[0002] The present disclosure provides antibodies (e.g., deimmunized or affinity matured antibodies) that specifically bind to family with sequence similarity 19, member A5 (FAM19A5), compositions comprising the antibodies, and methods of using the antibodies to prevent or treat disorders or diseases, such as disorders or diseases resulting from central nervous system injury, in a subject.

[0003] [Background technology] FAM19A5 is a member of the TAFA subfamily of proteins, which is composed of five highly homologous small proteins. See Tang TY et al., Genomics 83(4):727-34 (2004). These proteins contain a conserved cysteine ​​residue at a specific position and are loosely related to macrophage inflammatory protein 1-alpha (MIP-1-alpha), a member of the CC-chemokine family. The TAFA proteins are primarily expressed in specific regions of the brain and spinal cord. These proteins are believed to be produced and secreted by adult neural stem cells during neurogenesis.

[0004] FAM19A5 is primarily expressed in the vertebrate brain and is important for the development, differentiation, and formation of the intact central nervous system, and may be used to prevent or treat central nervous system injuries and / or diseases. See U.S. Patent Publication No. 2015 / 0118230.

[0005] [Summary of the Invention] [Problem to be solved by the invention] Although FAM19A5 inhibition can play an important role in treating central nervous system disorders, there remains a need to develop antibodies that can specifically bind to FAM19A5 and modulate FAM19A5 activity, particularly antibodies that can be used in human subjects without side effects.

[0006] [Means for solving the problem] Provided herein is an isolated antibody ("anti-FAM19A5 antibody"), or antigen-binding portion thereof, that specifically binds to a human family member A5 (FAM19A5) protein with sequence similarity 19, and that comprises heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3, wherein the heavy chain CDR1, CDR2, and CDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 5, 6, and 7, respectively, and each of the sequences optionally comprises 1, 2, 3, 4, or 5 mutations; the light chain CDR1, CDR2, and CDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 8, 9, and 10, respectively, and at least one of the light chain CDR1, CDR2, and CDR3 comprises 1, 2, 3, 4, or 5 mutations; and the antibody has reduced immunogenicity in humans compared to a reference antibody comprising a VH set forth in SEQ ID NO: 11 and a VL set forth in SEQ ID NO: 12.

[0007] In some embodiments, the heavy chain CDR3 of the anti-FAM19A5 antibody disclosed herein comprises the amino acid sequence set forth in SEQ ID NO:7.

[0008] In some embodiments, the heavy chain CDR1 of the anti-FAM19A5 antibody disclosed herein comprises the amino acid sequence set forth in SEQ ID NO: 5 with one or two mutations. In particular embodiments, the mutation comprises a substitution of an acidic amino acid for threonine at amino acid 3 of SEQ ID NO: 5. In other embodiments, the mutation comprises a substitution of an acidic amino acid for serine at amino acid 5 of SEQ ID NO: 5. In particular embodiments, the acidic amino acid comprises aspartic acid or glutamic acid.

[0009] In some embodiments, the heavy chain CDR2 of the anti-FAM19A5 antibody comprises the amino acid sequence set forth in SEQ ID NO:6 with one, two, three, four, or five mutations. In some embodiments, the mutation comprises a substitution of an arginine with a basic amino acid at amino acid 16 of SEQ ID NO:6. In certain embodiments, the basic amino acid comprises lysine. In other embodiments, the mutation comprises any one or more of the following: (a) a substitution of an acidic amino acid for glycine at amino acid 6 of SEQ ID NO:6; (b) a substitution of an acidic amino acid for serine at amino acid 7 of SEQ ID NO:6; (c) a substitution of an acidic amino acid for serine at amino acid 8 of SEQ ID NO:6; (d) a substitution of an acidic amino acid for threonine at amino acid 9 of SEQ ID NO:6; and (e) a substitution of a basic amino acid for arginine at amino acid 16 of SEQ ID NO:6. In certain embodiments, the acidic amino acid comprises aspartic acid or glutamic acid. In certain embodiments, the basic amino acid comprises lysine.

[0010] In some embodiments, the light chain CDR3 of an anti-FAM19A5 antibody disclosed herein comprises the amino acid sequence set forth in SEQ ID NO: 10 with one, two, three, four, or five mutations. In some embodiments, the mutations include one or more of the following: (a) substitution of serine with an acidic amino acid or an aliphatic amino acid at amino acid 6 of SEQ ID NO: 10; (b) substitution of asparagine with an acidic amino acid or a hydroxyl- or sulfur / selenium-containing amino acid at amino acid 7 of SEQ ID NO: 10; (c) substitution of glycine with an acidic amino acid or a hydroxyl- or sulfur / selenium-containing amino acid at amino acid 8 of SEQ ID NO: 10; (d) substitution of glycine with an acidic amino acid or a hydroxyl- or sulfur / selenium-containing amino acid at amino acid 9 of SEQ ID NO: 10; and (e) substitution of isoleucine with a basic amino acid at amino acid 10 of SEQ ID NO: 10. In certain embodiments, the acidic amino acid comprises aspartic acid or glutamic acid. In some embodiments, the hydroxyl- or sulfur / selenium-containing amino acid comprises serine. In other embodiments, the basic amino acid comprises histidine.

[0011] In some embodiments, the light chain CDR1 of an anti-FAM19A5 antibody of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 8 with one, two, three, or four mutations. In particular embodiments, the mutations include one or more of the following: (a) a substitution of tyrosine with an acidic amino acid at amino acid 6 of SEQ ID NO: 8; (b) a substitution of arginine with an acidic amino acid at amino acid 7 of SEQ ID NO: 8; (c) a substitution of glycine with an acidic amino acid at amino acid 8 of SEQ ID NO: 8; and (d) a substitution of serine with an acidic amino acid at amino acid 9 of SEQ ID NO: 8. In some embodiments, the acidic amino acid comprises glutamic acid or glutamine.

[0012] In some embodiments, the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO:9 with one, two, three, or four mutations. In some embodiments, the mutations include one or more of the following: (a) a glutamic acid substitution at amino acid 1 of SEQ ID NO:9 for an acidic amino acid; (b) a serine substitution at amino acid 2 of SEQ ID NO:9 for an acidic amino acid; (c) an asparagine substitution at amino acid 3 of SEQ ID NO:9 for an acidic amino acid, a basic amino acid, or an aliphatic amino acid; and (d) a lysine substitution at amino acid 4 of SEQ ID NO:9 for an acidic amino acid or an aliphatic amino acid. In certain embodiments, the acidic amino acid comprises glutamine, asparagine, aspartic acid, or glutamic acid. In some embodiments, the basic amino acid comprises histidine. In other embodiments, the aliphatic amino acid comprises leucine. In certain embodiments, the mutation includes a serine substitution at amino acid 2 of SEQ ID NO:9 for an acidic amino acid. In some embodiments, the acidic amino acid comprises aspartate, glutamate, asparagine, glutamine, or a combination thereof, hi certain embodiments, the acidic amino acid is asparagine.

[0013] The present specification also provides an isolated antibody or antigen-binding portion thereof that specifically binds to human FAM19A5 protein and comprises heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3, wherein (i) the heavy chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 5; (ii) the heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 13; (iii) the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 7; (iv) the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 8; (v) the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 20; and (vi) the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 10.

[0014] The present specification provides an isolated antibody or antigen-binding portion thereof that specifically binds to human FAM19A5 protein and comprises heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3, wherein (i) the heavy chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 14; (ii) the heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15; (iii) the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 7; (iv) the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 21; (v) the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 22; and (vi) the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 23.

[0015] The present specification provides an isolated antibody or antigen-binding portion thereof that specifically binds to human FAM19A5 protein and comprises heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3, wherein (i) the heavy chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 14; (ii) the heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15; (iii) the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 7; (iv) the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 21; (v) the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 24; and (vi) the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 23.

[0016] The present specification provides an isolated antibody or antigen-binding portion thereof that specifically binds to human FAM19A5 protein and comprises heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3, wherein (i) the heavy chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 14; (ii) the heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15; (iii) the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 7; (iv) the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 8; (v) the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 25; and (vi) the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 23.

[0017] The present specification provides an isolated antibody or antigen-binding portion thereof that specifically binds to human FAM19A5 protein and comprises heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3, wherein (i) the heavy chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 14; (ii) the heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15; (iii) the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 7; (iv) the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 8; (v) the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 24; and (vi) the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 23.

[0018] The present specification provides an isolated antibody or antigen-binding portion thereof that specifically binds to human FAM19A5 protein and comprises heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3, wherein (i) the heavy chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 14; (ii) the heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15; (iii) the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 7; (iv) the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 8; (v) the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 26; and (vi) the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 27.

[0019] The present specification provides an isolated antibody or antigen-binding portion thereof that specifically binds to human FAM19A5 protein and comprises heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3, wherein (i) the heavy chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 14; (ii) the heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15; (iii) the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 7; (iv) the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 8; (v) the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 28; and (vi) the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 29.

[0020] In some embodiments, the anti-FAM19A5 antibodies disclosed herein comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO: 11, and / or the VL comprises an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO: 12.

[0021] In some embodiments, the anti-FAM19A5 antibody cross-competes with a reference antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein (a) the VH comprises the amino acid sequence set forth in SEQ ID NO: 33, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 38; (b) the VH comprises the amino acid sequence set forth in SEQ ID NO: 34, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 39; or (c) the VH comprises the amino acid sequence set forth in SEQ ID NO: 34, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 41. (d) the VH comprises the amino acid sequence set forth in SEQ ID NO: 34 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 40; (e) the VH comprises the amino acid sequence set forth in SEQ ID NO: 34 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 42; (f) the VH comprises the amino acid sequence set forth in SEQ ID NO: 34 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 43; or (g) the VH comprises the amino acid sequence set forth in SEQ ID NO: 34 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 44.

[0022] In some embodiments, the anti-FAM19A5 antibodies disclosed herein bind to the same human FAM19A5 epitope as a reference antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein (a) the VH comprises the amino acid sequence set forth in SEQ ID NO: 33, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 38; (b) the VH comprises the amino acid sequence set forth in SEQ ID NO: 34, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 39; or (c) the VH comprises the amino acid sequence set forth in SEQ ID NO: 34, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 41. (d) the VH comprises the amino acid sequence set forth in SEQ ID NO: 34 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 40; (e) the VH comprises the amino acid sequence set forth in SEQ ID NO: 34 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 42; (f) the VH comprises the amino acid sequence set forth in SEQ ID NO: 34 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 43; or (g) the VH comprises the amino acid sequence set forth in SEQ ID NO: 34 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 44.

[0023] In some embodiments, the human FAM19A5 epitope comprises the amino acid sequence set forth in SEQ ID NO: 90, 91, or 92.

[0024] The present disclosure also provides an isolated antibody ("anti-FAM19A5 antibody"), or antigen-binding portion thereof, that specifically binds to human family, member A5 (FAM19A5) protein with sequence similarity 19, and comprises heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3, wherein the heavy chain CDR1, CDR2, and CDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively, each of which optionally comprises one, two, or three mutations; and the light chain CDR1, CDR2, and CDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 30, 31, and 32, respectively, and at least one of the light chain CDR1, CDR2, and CDR3 comprises one, two, or three mutations; and the antibody has reduced immunogenicity in humans and higher binding affinity for human FAM19A5 protein compared to a reference antibody comprising a VH set forth in SEQ ID NO: 35 and a VL set forth in SEQ ID NO: 45.

[0025] In some embodiments, the heavy chain CDR3 of the anti-FAM19A5 antibody comprises the amino acid sequence set forth in SEQ ID NO: 18, 128, or 129.

[0026] In some embodiments, the heavy chain CDR1 of the anti-FAM19A5 antibody comprises the amino acid sequence set forth in SEQ ID NO: 16. In other embodiments, the heavy chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 19.

[0027] In some embodiments, the heavy chain CDR2 of the anti-FAM19A5 antibody comprises the amino acid sequence set forth in SEQ ID NO:18.

[0028] In some embodiments, the light chain CDR3 of the anti-FAM19A5 antibody comprises the amino acid sequence set forth in SEQ ID NO:32.

[0029] In some embodiments, the light chain CDR2 of the anti-FAM19A5 antibody comprises the amino acid sequence set forth in SEQ ID NO:31.

[0030] In some embodiments, the light chain CDR1 of the anti-FAM19A5 antibody disclosed herein comprises the amino acid sequence set forth in SEQ ID NO: 30, with one mutation. In particular embodiments, the mutation comprises a substitution of a serine with an aliphatic amino acid at amino acid 4 of SEQ ID NO: 30. In some embodiments, the aliphatic amino acid comprises a valine.

[0031] In some embodiments, the anti-FAM19A5 antibodies disclosed herein comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO: 35, and / or the VL comprises an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO: 45.

[0032] In some embodiments, the anti-FAM19A5 antibody cross-competes with a reference antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the amino acid sequence set forth in SEQ ID NO: 36, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 46. In other embodiments, the anti-FAM19A5 antibody cross-competes with a reference antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the amino acid sequence set forth in SEQ ID NO: 37, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 46. In other embodiments, the anti-FAM19A5 antibody cross-competes with a reference antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the amino acid sequence set forth in SEQ ID NO: 130, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 46. In some embodiments, the anti-FAM19A5 antibody cross-competes with a reference antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the amino acid sequence set forth in SEQ ID NO: 131 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 46.

[0033] In some embodiments, the anti-FAM19A5 antibody is selected from the group consisting of IgG1, IgG2, IgG3, IgG4, variants thereof, and any combination thereof. In certain embodiments, the anti-FAM19A5 antibody is a chimeric antibody, a human antibody, or a humanized antibody. In some embodiments, the anti-FAM19A5 antibody comprises a Fab, a Fab', a F(ab')2, an Fv, or a single-chain Fv (scFv).

[0034] In some embodiments, the anti-FAM19A5 antibody disclosed herein is an scFv. In particular embodiments, the scFv comprises a VH and a VL: (a) the VH comprises the amino acid sequence set forth in SEQ ID NO: 33, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 38; (b) the VH comprises the amino acid sequence set forth in SEQ ID NO: 34, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 39; (c) the VH comprises the amino acid sequence set forth in SEQ ID NO: 34, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 41; (d) the VH comprises the amino acid sequence set forth in SEQ ID NO: 34, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 40; (e) the VH comprises the amino acid sequence set forth in SEQ ID NO: 34, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 42; or (f) the VH comprises the amino acid sequence set forth in SEQ ID NO: 34. (g) the VH comprises the amino acid sequence set forth in SEQ ID NO: 34 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 44; (h) the VH comprises the amino acid sequence set forth in SEQ ID NO: 36 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 46; (i) the VH comprises the amino acid sequence set forth in SEQ ID NO: 37 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 46; (j) the VH comprises the amino acid sequence set forth in SEQ ID NO: 130 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 46; or (k) the VH comprises the amino acid sequence set forth in SEQ ID NO: 131 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 46.

[0035] In some embodiments, the anti-FAM19A5 antibody exhibits any one or more of the following properties: (a) binding to soluble human FAM19A5 with a KD of 10 nM or less as measured by enzyme-linked immunosorbent assay (ELISA); (b) binding to membrane-bound human FAM19A5 with a KD of 10 nM or less as measured by ELISA; (c) reducing, reversing, delaying, and / or preventing the onset of reactive gliosis; (d) inhibiting the hyperproliferation of reactive astrocytes; (e) reducing the expression of chondroitin sulfate proteoglycans, including neurocan and neuroglial antigen 2 (NG2); (f) increasing the expression of c-fos and pERK in neuronal nuclei; (g) promoting neuronal survival. (h) the property of increasing the expression of GAP43 in neurons; (i) the property of promoting axonal regrowth; (j) the property of inducing, for example, intratumoral vascular normalization; (k) the property of suppressing tumor growth; (l) the property of increasing immune cell infiltration into tumors; (m) the property of increasing neuronal infiltration into tumors; (n) the property of enhancing the phagocytic activity of macrophages or microglia; (o) the property of increasing the mitochondrial membrane potential of macrophages or microglia; (p) the property of decreasing the recruitment of myeloid-derived suppressor cells (MDSCs) to tumors; (q) the property of decreasing necrosis and edema in tumors; (r) the property of decreasing tumor tissue permeability; and (s) the property of increasing blood flow rate in tumors.

[0036] The present disclosure also provides nucleic acids encoding the anti-FAM19A5 antibodies disclosed herein, vectors containing the nucleic acids, cells containing the vectors, and immunoconjugates containing the anti-FAM19A5 antibodies of the present disclosure. The present disclosure also discloses compositions containing the anti-FAM19A5 antibodies, nucleic acids, vectors, cells, or immunoconjugates of the present disclosure and a carrier. The present disclosure also provides kits containing the anti-FAM19A5 antibodies, nucleic acids, vectors, cells, or immunoconjugates of the present disclosure and instructions for use.

[0037] The present specification also provides a method for producing an antibody that specifically binds to human FAM19A5 protein, the method comprising culturing the cells disclosed herein under appropriate conditions and isolating the antibody.

[0038] The present disclosure also provides a method of treating a disease or condition in a subject in need thereof, comprising administering to the subject an anti-FAM19A5 antibody, nucleic acid, vector, cell, or immunoconjugate described herein. In some embodiments, the disease or condition comprises a tumor, fibrosis, glaucoma, affective disorder, retinal disease, age-related macular degeneration, or neuropathic pain. In certain embodiments, the disease or condition is a tumor.

[0039] In some embodiments, the tumor comprises melanoma, pancreatic cancer, glioma, breast cancer, lymphoma, lung cancer, renal cancer, prostate cancer, fibrosarcoma, colon adenocarcinoma, liver cancer, or ovarian cancer. In certain embodiments, the glioma is glioblastoma multiforme (GBM).

[0040] In some embodiments, the anti-FAM19A5 antibodies, nucleic acids, vectors, cells, or immunoconjugates of the present disclosure induce vascular normalization, which in certain embodiments is accompanied by changes in vascular properties, including increased connectivity, increased wall thickness, decreased vessel diameter, more regular vascular orientation and distribution patterns, increased blood vessel number, decreased leakage and permeability, increased pericyte coverage and proximity on blood vessels, increased oxygen supply, or a combination thereof.

[0041] In some embodiments, the anti-FAM19A5 antibodies, nucleic acids, vectors, cells or immunoconjugates of the present disclosure inhibit tumor growth.

[0042] In some embodiments, the anti-FAM19A5 antibodies, nucleic acids, vectors, cells, or immunoconjugates disclosed herein increase immune cell infiltration into tumors. In certain embodiments, the immune cells include macrophages, dendritic cells, T lymphocytes, B lymphocytes, natural killer (NK) cells, or a combination thereof. In some embodiments, the immune cells further exhibit hypertrophy. In some embodiments, the increased immune cell infiltration into tumors is accompanied by increased neuronal infiltration into tumors. In certain embodiments, the neuronal cells include astrocytes, glial cells, or a combination thereof.

[0043] In some embodiments, the anti-FAM19A5 antibody, nucleic acid, vector, cell, or immunoconjugate enhances the phagocytic activity of macrophages or microglia, hi some embodiments, the anti-FAM19A5 antibody, nucleic acid, vector, cell, or immunoconjugate increases the mitochondrial membrane potential of macrophages or microglia.

[0044] In some embodiments, the anti-FAM19A5 antibodies, nucleic acids, vectors, cells, or immunoconjugates disclosed herein reduce the recruitment of myeloid-derived suppressor cells (MDSCs) to tumors. In some embodiments, the anti-FAM19A5 antibodies, nucleic acids, vectors, cells, or immunoconjugates reduce necrosis and edema in tumors. In some embodiments, the anti-FAM19A5 antibodies, nucleic acids, vectors, cells, or immunoconjugates reduce tumor tissue permeability. In some embodiments, the anti-FAM19A5 antibodies, nucleic acids, vectors, cells, or immunoconjugates increase blood flow rate in tumors.

[0045] In some embodiments, the method of treating the disease or disorder further comprises administering an additional therapeutic agent, hi certain embodiments, the additional therapeutic agent comprises chemotherapy, immunotherapy, radiation therapy, or a combination thereof.

[0046] [Brief description of the drawing] Figures 1A-1C provide analysis of the binding of individual scFv clones to FAM19A5 protein. Absorbance was measured at 405 nm. Clone numbers are provided on the x-axis. Figures 1A-1C show analysis of 96 clones derived from chickens 1, 2, and 3 by the fourth, fifth, or sixth round of biopanning, respectively. For each clone shown in Figures 1A-1C, the vertical bars correspond to FAM19A5 protein, negative control protein, hemagglutinin protein, and BSA (from left to right). In each of Figures 1A, 1B, and 1C, the clones in the boxes represent eight clones selected for further analysis (see Example 4).

[0047] Figures 2A and 2B provide the approximate size and binding capacity of different anti-FAM19A5 scFvs, respectively. Antibody sizes were shown using SDS-Page, and binding capacities were measured using ELISA. The antibodies shown include (from left to right): 1-28, 1-85, 2-13, 2-14, 2-20, 2-29, 3-2, and 3-26. In Figure 2B, for each antibody shown, the left bar represents the binding of the antibody to FAM19A5 protein. The right bar represents the negative control (binding measured in the presence of blocking buffer alone, i.e., in the absence of recombinant FAM19A5 protein). nd The column labeled "only" represents yet another negative control group representing the background level of the assay (binding measured in the absence of the primary anti-FAM19A5 antibody).

[0048] Figures 3A and 3B provide a comparison of the ability of different anti-FAM19A5 antibodies to neutralize FAM19A5 expression in mouse and human glial cells, respectively. Neutralization is shown as the percentage reduction in FAM19A5 expression, expressed as mean fluorescence intensity (MFI). The percentage reduction can be calculated using the following formula: 100% - [((MFI of FAM19A5 + anti-FAM19A5 antibody) / (MFI of FAM19A5 + control antibody)) x 100]. The percentage reduction for each antibody is shown in parentheses.

[0049] Figure 4 provides the amino acid sequences of epitopes F1-F6 (conjugated to BSA) and their locations in the human FAM19A5 polypeptide. The top amino acid sequence shown is wild-type FAM19A5 isoform 2 (no signal peptide). The second amino acid sequence shown is the same sequence, but with cysteine ​​residues mutated to serine during peptide synthesis to reduce nonspecific activity.

[0050] Figure 5 provides ELISA results for the binding of the 3-2 antibody to epitope fragments F1 to F6. The leftmost column ("FAM19A5") represents the positive control group, showing the binding of the 3-2 anti-FAM19A5 antibody to the whole FAM19A5 protein. The "irrelevant protein" and "blocking alone" (i.e., blocking buffer alone, i.e., no FAM19A5 protein) groups represent negative controls. For each group, the left bar corresponds to the isotype control group, and the right bar corresponds to the 3-2 antibody.

[0051] Figures 6A-6J provide alanine scanning analysis results showing specific amino acid residues within the epitope F2 fragment that are important for binding of various 3-2 antibody mutants to the FAM19A5 protein. Figures 6A-6D show the results for (A) wild-type 3-2 antibody, (B) 1-30 antibody, (C) 1-32 antibody, and (D) 6-10 antibody, respectively, produced in HEK293F cells. Figures 6E-6J show the results for (E) 1-17 antibody, (F) 1-30 antibody, (G) 1-32 antibody, (H) 4-11 antibody, (I) 6-10 antibody, and (J) low-PI antibody, respectively, produced in CHO cells. As discussed in Example 7, mutant peptides each containing alanine substitutions at a single amino acid residue within the epitope fragment F2 were generated. Antibody binding to the different mutant peptides was measured using ELISA.

[0052] Figure 7 identifies potential immunogenic sites in the light chain variable region (VL) (top three rows) and heavy chain variable region (VH) (bottom three rows) of the 3-2 antibody. The VL corresponds to SEQ ID NO: 12, and the VH corresponds to SEQ ID NO: 11. The sequences for the human germlines used for the framework regions of the 3-2 antibody are also shown: VL = immunoglobulin lambda variable 1-51 (IGLV1-51*02) and immunoglobulin lambda joining 2 (IGLJ2*01); VH = immunoglobulin heavy chain variable 3-64 (IGHV3-64*04) and immunoglobulin heavy chain joining 1 (IGHJ1*01). The human germlines used were those most homologous to the 3-2 clone (IgBLAST, NCBI). ITOPE TMAs determined by the analysis, promiscuous MHC class II binding peptides with high and intermediate immunogenic potential have been shown to be: (i) peptide #3: residues 85-93 of SEQ ID NO: 12 (IYYCGSWDS); (ii) peptide #9: residues 64-72 of SEQ ID NO: 11 (VRGRATISR); and (iii) peptide #10: residues 79-87 of SEQ ID NO: 11 (VRLQLNNPG). The MHCII-binding peptides with intermediate immunogenic potential are as follows: (i) residues 16-24 of SEQ ID NO: 12 (VKITCSGGG); (ii) peptide #2: residues 48-56 of SEQ ID NO: 12 (IYESNKRPS); (iii) peptide #4: residues 18-26 of SEQ ID NO: 11 (LSLVCKASG); (iv) peptide #5: residues 19-27 of SEQ ID NO: 11 (SLVCKASGF); (v) peptide #6: residues 32-40 of SEQ ID NO: 11 (FNMFWVRQA); (vi) peptide #7: residues 45-53 of SEQ ID NO: 11 (LEYVAQISS); (vii) peptide #8: residues 48-56 of SEQ ID NO: 11 (VAQISSSGS); and (viii) peptide #11: residues 81-89 of SEQ ID NO: 11 (LQLNNPGAE). Homologous peptides from the T cell epitope database are as follows: peptide #5, peptide #6, and peptide #9. A total of 11 binding peptides were identified, which are designated as peptides #1 to #11. For each of the binding peptides, "P1" indicates the first anchor position.

[0053] Figure 8 identifies potential immunogenic sites in the light chain variable region (VL) (top three rows) and heavy chain variable region (VH) (bottom three rows) of the 2-13 antibody. The VL corresponds to SEQ ID NO: 45, and the VH corresponds to SEQ ID NO: 35. The sequences for the human germlines used for the framework regions of the 2-13 antibody are also shown: VL = immunoglobulin lambda variable 3-27 (IGLV3-27*01) and immunoglobulin lambda joining 2 (IGLJ2*01); VH = immunoglobulin heavy chain variable 3-64 (IGHV3-64*04) and immunoglobulin heavy chain joining 1 (IGHJ1*01). The human germlines used were those most homologous to the 2-13 clone (IgBLAST, NCBI). ITOPE TM As determined by the analysis, promiscuous MHC class II binding peptides with high and intermediate immunogenic potential have been shown, specifically, the MHC class II binding peptides with high immunogenic potential are as follows: (i) peptide #1: residues 16-24 of SEQ ID NO:45 (VKITCSGGS); (ii) peptide #2: residues 80-88 of SEQ ID NO:45 (VYFCGTEDI); and (iii) peptide #8: residues 79-87 of SEQ ID NO:35 (VRLQLNNLR). MHCII-binding peptides with intermediate immunogenic potential are as follows: (i) peptide #3: residues 18-26 of SEQ ID NO:35 (LSLVCKASG); (ii) peptide #4: residues 20-28 of SEQ ID NO:35 (LVCKASGFT); (iii) peptide #5: residues 36-44 of SEQ ID NO:35 (WVRQTPGKG); (iv) peptide #6: residues 47-55 of SEQ ID NO:35 (YVAEITNDG); (v) peptide #7: residues 64-72 of SEQ ID NO:35 (VKGRATISR); (vi) peptide #9: residues 81-89 of SEQ ID NO:35 (LQLNNLRAE); and (vii) peptide #10: residues 86-94 of SEQ ID NO:35 (LRAEDTGTY). Homologous peptides from the T cell epitope database are as follows: peptide #4 and peptide #5. A total of 10 binding peptides were identified, designated peptides #1-#10. For each of the above binding peptides, "P1" indicates the first anchor position.

[0054] Figures 9A-9C provide binding analyses of the deimmunized 3-2 antibody. Figure 9A provides a schematic diagram showing the locations where different amino acid mutations were made to deimmunize the 3-2 antibody. Figure 9B provides a comparison of the sequences of the light chain variable region (VL) and heavy chain variable region (VH) among (i) wild-type 3-2 antibody ("clone 3-2"), (ii) a fully deimmunized 3-2 antibody ("fully deimmunized clone 3-2"), and (iii) an antibody deimmunized except for one amino acid in the heavy chain CDR2 ("deimmunized clone 3-2"). ITOPE TM Amino acid residues with high and intermediate immunogenic potential, as determined by the analysis, are boxed and labeled "1" and "2," respectively. Figure 9C compares the binding of (i) wild-type 3-2 antibody, (ii) fully deimmunized 3-2 antibody, and (iii) an antibody deimmunized in the heavy chain CDR2 except for one amino acid ("deimmunized clone-3-2") to the FAM19A5 protein, as measured by ELISA. Each single-chain variable fragment (scFv)-displaying phage was added to wells of a microtiter plate coated with FAM19A5 (filled squares) or anti-HA antibody (□). SU background signal was measured in control wells coated with BSA. Wells were probed with HRP-conjugated anti-M13 antibody. Absorbance was measured at 405 nm. Results are shown as the mean ± SD from four replicate experiments.

[0055] Figures 10A and 10B provide an analysis of two different deimmunized 2-13 antibodies: (i) a fully deimmunized 2-13 antibody, and (ii) an antibody deimmunized except for one amino acid in the heavy chain CDR2 ("deimmunized clone 2-13"). Figure 10A provides a comparison of the light chain variable region (VL) (top three rows) and heavy chain variable region (VH) (bottom three rows) for the two deimmunized 2-13 antibodies versus the wild-type 2-13 antibody ("clone 2-13"). TMAmino acid residues with high and intermediate immunogenic potential, as determined by the analysis, are boxed and labeled "1" and "2," respectively. Figure 10B compares the binding of (i) wild-type 2-13 antibody, (ii) fully deimmunized 2-13 antibody, and (iii) an antibody deimmunized in the heavy chain CDR2 except for one amino acid (deimmunized clone 2-13) to the FAM19A5 protein, as measured by ELISA. Each single-chain variable fragment (scFv)-displaying phage was added to wells of a microtiter plate coated with FAM19A5 (filled squares) or anti-HA antibody (□). Background signals were measured from control wells coated with BSA. Wells were probed with HRP-conjugated anti-M13 antibody. Absorbance was measured at 405 nm. Results are shown as the mean ± SD from four replicate experiments.

[0056] Figure 11 compares the binding ability of different deimmunized clone 2-13 mutants. The identity of the different mutant antibodies is provided along the x-axis. Each amino acid residue in CDRL1, CDRL2, CDRL3, CDRH1, and CDRH2 was replaced with glutamic acid and aspartic acid, respectively. The reactivity of 70 mutant antibodies was analyzed by phage enzyme immunoassay. Each phage-displayed scFv was added to wells of a microtiter plate coated with FAM19A5 (filled squares) or anti-HA antibody (□). Background signals were measured from control wells coated with BSA. Wells were probed with HRP-conjugated anti-M13 antibody. Absorbance was measured at 405 nm. Results are presented as the mean ± SD from four replicate experiments.

[0057] FIG. 12 provides a schematic diagram of the method used to generate deimmunized anti-FAM19A5 antibodies with improved physicochemical properties.

[0058] Figures 13A and 13B provide a comparison of the physicochemical properties of several deimmunized 3-2 variants compared to the wild-type (i.e., non-deimmunized) 3-2 antibody ("original antibody"). The variant antibodies shown include: (i) low isoelectric point antibody ("Low_PI"), (ii) 1-17, (iii) 1-30, (iv) 1-32, (v) 4-11, and (vi) 6-10. Figure 13A shows an analysis of the binding of the antibodies to FAM19A5 protein. Results are presented as mean ± SD. Figure 13B shows solubility (CamSol score) and hydrophobicity (GRAVY score) data.

[0059] Figures 14A and 14B provide sequence alignments of the heavy chain variable region (Figure 14A) and light chain variable region (Figure 14B) for different deimmunized 3-2 antibody variants. The presented deimmunized variant antibodies are related to the 3-2 antibody in that they are identical to the 3-2 antibody except that they have been deimmunized to reduce immunogenicity when administered to human subjects. TM Amino acid residues with high and intermediate immunogenic potential, as determined by analysis, are boxed and labeled "1" and "2," respectively. Other amino acid residues of interest are also labeled: "3" = closest human germline sequence difference; "4" = potential arginine or lysine methylation; "5" = potential tryptophan or methionine oxidation; "6" = potential asparagine deamidation; "7" = potential aspartate isoformation; "8" = rare amino acid insertion; and "9" = free cysteine ​​or non-canonical cysteine ​​pair. Such amino acid residues may appear as product variants during natural cellular processing and degradation reactions.

[0060] Figure 15 provides sequence alignments of the light chain variable region (top three rows) and heavy chain variable region (bottom three rows) for different deimmunized and / or affinity matured 2-13 antibody variants. These variant antibodies are related to the 2-13 antibody in that they are identical to the 2-13 antibody except that they have undergone deimmunization and / or affinity maturation. TMAmino acid residues with high and intermediate immunogenic potential, as determined by analysis, are boxed and labeled "1" and "2," respectively. Other amino acid residues of interest are also labeled: "3" = potential arginine or lysine methylation; "4" = potential tryptophan or methionine oxidation; "5" = potential asparagine deamidation; "6" = potential aspartate isoformation; "7" = rare amino acid insertion; and "8" = free cysteine ​​or non-canonical cysteine ​​pair. Such amino acid residues may appear as product variants during natural cellular processing and degradation reactions.

[0061] Figures 16A-16C show the expression levels of two variants of the deimmunized 2-13D-37 antibody (2-13D-37-1.5W-41 and 2-13D-37-3W-16) measured by SDS-PAGE and Western blotting. Figures 16A and 16B show the results of SDS-PAGE analysis of antibodies from culture medium and purified protein. Figure 16C shows the results of Western blotting. In each of Figures 16A-16C, lanes "1" and "2" correspond to antibodies 2-13D-37-1.5W-41 and 2-13D-37-3W-16, respectively. In lanes "1" and "2," "A" and "B" correspond to the data before and after centrifugation, respectively. In Figure 16B, the left panel shows reduced SDS-PAGE, and the right panel shows non-reduced SDS-PAGE.

[0062] Figures 17A and 17B provide an analysis of two variants of the deimmunized 2-13D-37 antibody (2-13D-37-1.5W-41 and 2-13D-37-3W-16) generated by affinity maturation. Figure 17A shows the amino acid sequence alignment of the light chain variable region (top three boxes) and heavy chain variable region (bottom three boxes) for the following antibodies: deimmunized clone 2-13D-37, deimmunized clone 2-13D-37-1.5W-41, and deimmunized clone 2-13D-37-3W-16. TMAmino acid residues with high and intermediate immunogenic potential, as determined by the analysis, are boxed and labeled "1" and "2," respectively. Other amino acid residues of interest are also labeled: "3" = potential tryptophan or methionine oxidation; "4" = potential asparagine deamidation; "5" = potential aspartate isomerization; and "6" = non-canonical cysteine ​​pairing. Such amino acid residues may appear as product variants during natural cellular processing and degradation reactions. Figure 17B shows the analysis of binding of deimmunized clone 2-13D-37, deimmunized clone 2-13D-37-1.5W-41, and deimmunized clone 2-13D-37-3W-16 to the FAM19A5 protein. Each single-chain variable fragment (scFv)-displaying phage was added to wells of a microtiter plate coated with FAM19A5 (filled squares) or anti-HA antibody (□). Background signals were measured from control wells coated with BSA. Wells were probed with HRP-conjugated anti-M13 antibody. Absorbance was measured at 405 nm. Results are presented as the mean ± SD from quadruplicate experiments.

[0063] FIG. 18 provides a schematic diagram of the overall workflow of the HDX-MS analytical method used in Example 11.

[0064] Figure 19 provides a table summarizing the coverage achieved under different experimental conditions tested for the HDX-MS analytical method described in Example 11. One or more of the following parameters were adjusted: (i) sample concentration, (ii) quenching conditions, (iii) pepsin concentration and / or digestion duration, and (v) quenching hold time (minutes). Depending on the analysis, a C18 or C8 pepsin-immobilized column was used with on-line or off-line digestion, as shown in Figure 19.

[0065] Figures 20A and 20B provide the results of pepsin digestion of the FAM19A5 protein using the optimized conditions described in Example 11. Figure 20A shows the coverage and overlap of 44 peptides identified for the mature FAM19A5 protein (SEQ ID NO: 101, i.e., SEQ ID NO: 2 minus the signal peptide corresponding to the first 25 amino acids). Each horizontal bar represents an individual peptide. Figure 20B provides the amino acid sequences, single ion masses (MHPs), and retention times (RTs) of 44 peptides, including the start and end regions of SEQ ID NO: 101.

[0066] Figure 21 shows the coverage and overlap rates of 22 peptides identified by pepsin digestion of FAM19A5 protein after deuterium labeling, as described in Example 11. Each horizontal bar represents an individual peptide.

[0067] Figures 22A-22E provide a comparison of deuterium uptake rates between single (antigen alone, "1") and antigen-antibody (2-13) complexes ("2") as a function of time. The y-axis represents the maximum deuterium uptake rate (if the peptide contains proline, the maximum value is [(number of amino acids - 1) - (number of prolines)]). The x-axis represents the deuterium labeling period. Figure 22A provides data for the following peptides: (i) FLKEGQL (SEQ ID NO: 102) (top left graph), (ii) FLKEGQLAAGTCE (SEQ ID NO: 103) (top right graph), (iii) LKEGQLAAG (SEQ ID NO: 104) (middle left graph), (iv) LKEGQLAAGTCEI (SEQ ID NO: 105) (middle right graph), (v) LKEGQLAAGTCEIVTL (SEQ ID NO: 106) (bottom left graph), and (vi) AAGTCEI (SEQ ID NO: 107) (bottom right graph). Figure 22B provides data for the following peptides: (i) RDSSQPPRTIARQTARCAC (SEQ ID NO: 108) (top left graph), (ii) QPPRTIARQTA (SEQ ID NO: 109) (top right graph), (iii) ACRKGQIAGTTRARPAC (SEQ ID NO: 110) (middle left graph), (iv) ACRKGQIAGTTRARPACVD (SEQ ID NO: 111) (middle right graph), (v) ACRKGQIAGTTRARPACVDA (SEQ ID NO: 112) (bottom left graph), and (vi) ARIIKTKQWC (SEQ ID NO: 113) (bottom right graph). Figure 22C provides data for the following peptides: (i) ARIIKTKQWCDM (SEQ ID NO: 114) (top left graph), (ii) ARIIKTKQWCDML (SEQ ID NO: 115) (top right graph), (iii) ARIIKTKQWCDMLPCL (SEQ ID NO: 116) (middle left graph), (iv) RIIKTKQWCDM (SEQ ID NO: 117) (middle right graph), (v) RIIKTKQWCDML (SEQ ID NO: 118) (bottom left graph), and (vi) WCDMLPCL (SEQ ID NO: 119) (bottom right graph).Figure 22D provides data for the following peptides: (i) LPCLEGEG (SEQ ID NO: 120) (top left graph), (ii) PCLEGEG (SEQ ID NO: 121) (top right graph), (iii) PCLEGEGCD (SEQ ID NO: 122) (middle left graph), (iv) PCLEGEGCDL (SEQ ID NO: 123) (middle right graph), (v) EGEGCDL (SEQ ID NO: 124) (bottom left graph), and (vi) EGEGCDLL (SEQ ID NO: 125) (bottom right graph). Figure 22E provides data for the following peptides: (i) LLINRSGWTCTQPGGRIKTTT (SEQ ID NO: 126) (left graph) and (ii) LINRSGWTCTQPGGRIKTTT (SEQ ID NO: 127) (right graph). In each of the graphs, additional information about the peptide (i.e., start and end locations (SEQ ID NO: 101, i.e., SEQ ID NO: 2 minus the signal peptide corresponding to the first 25 amino acids) and size) is provided in the top corner of each graph.

[0068] Figures 23A-C show the major amino acid residues along the FAM19A5 protein with significant deuterium uptake differences (i.e., greater than ±0.5 Da) between single and combined samples. Figure 23A provides a butterfly map analysis of the deuterium uptake rates of the single (antigen alone, top graph) and antigen-antibody (2-13) complex (bottom graph). Figure 23B provides a plot of the deuterium uptake difference between the single and combined samples. Figure 23C shows the data as the sum of deuterium uptake differences less than 1.5 Da. In Figures 23A-C, each line represents a different deuterium labeling period: (i) "1" (0.33 minutes or 20 seconds), (ii) "2" (10 minutes), (iii) "3" (60 minutes), and (iv) "4" (240 minutes). Each point corresponds to an individual peptide. In Figures 23B and 23C, the start and end positions of the peptide (of SEQ ID NO: 101) where there is a difference in deuterium absorption rate between the antigen alone and the antigen-peptide complex of more than ±0.5 Da are shown. The dashed boxes in Figures 23B and 23C indicate a difference in deuterium absorption rate of less than ±0.5 Da.

[0069] Figure 24 provides a heat map analysis showing regions of the FAM19A5 protein with significant deuterium uptake differences between the single (antigen alone) and antigen-antibody (2-13) complex samples. Residues surrounded by red dashed lines indicate key amino acid residues: (i) CRKGQIAGTTRAR (amino acid residues 38-50 of SEQ ID NO:101 or amino acid residues 63-75 of SEQ ID NO:2), and (ii) PACVDARIIKTKQW (amino acid residues 51-64 of SEQ ID NO:101 or amino acid residues 76-85 of SEQ ID NO:2).

[0070] FIG. 25 provides the three-dimensional structure of the FAM19A5 protein and the location of the major binding epitope for the 2-13 antibody.

[0071] [Mode for Carrying Out the Invention] Disclosed herein is an isolated monoclonal antibody ("anti-FAM19A5 antibody"), or an antigen-binding portion thereof, that specifically binds to a human family member A5 (FAM19A5) protein with sequence similarity 19 and exhibits any one or more of the properties disclosed herein. Specifically, the anti-FAM19A5 antibody has been deimmunized to reduce immunogenicity in human subjects.

[0072] To facilitate understanding of the disclosure provided herein, a number of terms and phrases are defined. Additional definitions are set forth throughout the detailed description.

[0073] [I.Definition] Throughout this disclosure, the terms "a" or "an" entity are understood to refer to one or more of that entity, for example, "an antibody" refers to one or more antibodies. Thus, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.

[0074] Also, as used herein, "and / or" should be considered as specifically disclosing each one of the two specified features or components together with or alone the other feature or component. Thus, the term "and / or" used herein in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" alone, and "B" alone. Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to include each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B, or C; A and C; A and B; B and C; A alone; B alone; and C alone.

[0075] Whenever an embodiment is described herein using the term "comprising," it is understood that other similar embodiments described in terms of "consisting of" and / or "consisting essentially of" are also provided.

[0076] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press provide those of ordinary skill in the art with many of the terms used in this disclosure.

[0077] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) recognized form. Numerical ranges are inclusive of the numbers limiting the range. Unless otherwise indicated, amino acid sequences are written from left to right in amino to carboxy order. The headings provided herein are not limitations on the various aspects of the disclosure, which may be referenced in their entirety. Accordingly, the terms defined below are more fully defined by reference to the specification as a whole.

[0078] The term "about" is used herein to mean approximately, roughly, to the extent of, or within a range thereof. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values ​​set forth. In general, the term "about" can modify the numerical value above and below the stated value by, for example, a variance of 10% above or below (higher or lower).

[0079] The term "family with sequence similarity 19, member A5" or "FAM19A5" refers to a protein that belongs to the TAFA family of five highly homologous proteins (also known as the FAM19 family) and is expressed primarily in the brain and spinal cord. FAM19A5 is also known as TAFA5 or chemokine-like protein TAFA-5.

[0080] The gene encoding FAM19A5 in humans is located on chromosome 22. There are several human FAM19A5 (UniProt: Q7Z5A7) isoforms, likely produced by alternative splicing: isoform 1 (UniProt: Q7Z5A7-1) consisting of 132 amino acids, isoform 2 (UniProt: Q7Z5A7-2) consisting of 125 amino acids, and isoform 3 (UniProt: Q7Z5A7-3) consisting of 53 amino acids. Human FAM19A5 protein is believed to exist in membrane-bound and soluble (secreted) forms. Isoform 1 is believed to be membrane-bound with a single transmembrane domain. Isoform 2, reported as a secreted (soluble) protein in Tang TY et al., Genomics 83(4):727-34 (2004), contains a signal peptide at amino acid positions 1-25. Isoform 1 is believed to be a membrane protein, predicted based on EST data. Below are the amino acid sequences of the three known human FAM19A5 isoforms.

[0081] (I) Isotype 1 (UniProt: Q7Z5A7-1, transmembrane protein): This isotype was selected as the reference sequence.

[0082] MAPSPRTGSR QDATALPSMS STFWAFMILA SLLIAYCSQL AAGTCEIVTL DRDSSQPRRT IARQTARCAC RKGQIAGTTR ARPACVDARI IKTKQWCDML PCLEGEGCDL LINRSGWTCT QPGGRIKTTT VS (SEQ ID NO: 1) (II) Isoform 2 (UniProt: Q7Z5A7-2, soluble protein): MQLLKALWAL AGAALCCFLV LVIHAQFLKE GQLAAGTCEI VTLDRDSSQP RRTIARQTAR CACRKGQIAG TTRARPACVD ARIIKTKQWC DMLPCLEGEG CDLLINRSGW TCTQPGGRIK TTTVS (SEQ ID NO: 2) (III) Isotype 3 (UniProt: Q7Z5A7-3): MYHHREWPAR IIKTKQWCDM LPCLEGEGCD LLINRSGWTC TQPGGRIKTT TVS (SEQ ID NO: 3) The term "FAM19A5" includes any variant or isoform of FAM19A5 that is naturally expressed by cells. Thus, the antibodies described herein may cross-react with different isoforms within the same species (e.g., different isoforms of human FAM19A5) or with FAM19A5 of species other than human (e.g., murine FAM19A5). Alternatively, the antibody may be specific for human FAM19A5 and may not exhibit any cross-reactivity with other species. FAM19A5 or any variants and isoforms thereof may be isolated from cells or tissues that naturally express them or may be recombinantly produced. A polynucleotide encoding human FAM19A5 has GenBank accession number BC039396 and the following sequence:

[0083] [Table 1A]

[0084] The terms "antibody" and "antibodies" are terms of the art and are used interchangeably herein to refer to a molecule having an antigen-binding site that specifically binds to an antigen. As used herein, the terms include whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") or single chain thereof. In one embodiment, an "antibody" refers to a glycoprotein, or antigen-binding portion thereof, comprising at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds. In yet another embodiment, an "antibody" refers to a single-chain antibody comprising a single variable domain, e.g., a VHH domain. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. In certain naturally occurring antibodies, the heavy chain constant region consists of three domains, CH1, CH2, and CH3. In certain naturally occurring antibodies, each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL.

[0085] The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity-determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The heavy and light chain variable regions contain binding domains that interact with antigens. The antibody constant regions can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0086] The term "Kabat numbering" and similar terms are recognized in the art and refer to a system for numbering amino acid residues in the heavy and light chain variable regions of an antibody or antigen-binding portion thereof. In certain embodiments, the CDRs of an antibody may be determined according to the Kabat numbering system (see, e.g., Kabat EA & Wu, TT (1971) Ann NY Acad Sci 190:382-391 and See Kabat EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242. Using the Kabat numbering system, the CDRs in an antibody heavy chain are typically located at amino acid positions 31-35 (CDR1) (which may optionally include one or two additional amino acids beyond position 35 (referred to as 35A and 35B in the Kabat numbering system), amino acid positions 50-65 (CDR2), and amino acid positions 95-102 (CDR3). Using the Kabat numbering system, the CDRs in an antibody light chain are typically located at amino acid positions 24-34 (CDR1), amino acid positions 50-56 (CDR2), and amino acid positions 89-97 (CDR3). In certain embodiments, the CDRs of the antibodies described herein are determined according to the Kabat numbering system.

[0087] The phrases "amino acid position numbering as in Kabat," "Kabat position," and grammatical variations thereof refer to the numbering system used for the heavy or light chain variable domains of antibody compilations in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering system, the actual linear amino acid sequence may contain additional amino acids corresponding to shortening of, or insertion into, the FW or CDRs of the variable domain. For example, a heavy chain variable domain may contain a single amino acid inserted after residue 52 of H2 (residue 52a according to Kabat) and residues inserted after heavy chain FW residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat). See Table 1B.

[0088] [Table 1B]

[0089] The Kabat numbering of residues for a given antibody can be determined by aligning the regions of homology of the antibody's sequence with a "standard" Kabat numbering sequence. Alternatively, Chothia refers to the location of the structural loop (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). When numbered using the Kabat numbering convention, the end of the Chothia CDR-H1 loop varies between H32 and H34, depending on the length of the loop (this is because the Kabat numbering system places insertions at H35A and H35B; without 35A or 35B, the loop ends at 32; with only 35A, the loop ends at 33; with both 35A and 35B, the loop ends at 34). The AbM hypervariable regions represent the trade-offs between the Kabat CDRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software.

[0090] IMGT (ImMunoGeneTics) also provides a numbering system for immunoglobulin variable regions, including CDRs. See, e.g., Lefranc, MP et al., Dev. Comp. Immunol. 27:55-77 (2003), incorporated herein by reference. The IMGT numbering system is based on alignments of over 5,000 sequences, structural data, and characterization of hypervariable loops, facilitating comparison of variable and CDR regions across all species. According to the IMGT numbering scheme, VH-CDR1 is located at positions 26-35, VH-CDR2 is located at positions 51-57, VH-CDR3 is located at positions 93-102, VL-CDR1 is located at positions 27-32, VL-CDR2 is located at positions 50-52, and VL-CDR3 is located at positions 89-97.

[0091] For all heavy chain constant region amino acid positions discussed in this disclosure, the numbering follows the EU index first described in Edelman et al., 1969, Proc. Natl. Acad. Sci. USA 63(1):78-85, which described the amino acid sequence of myeloma protein EU, the first human IgG1 to be sequenced. The EU index of Edelman et al. is also presented in Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda. Hereby, the phrases "EU index as presented in Kabat" or "Kabat EU index" and "positions according to the EU index presented in Kabat..." and grammatical variations thereof refer to the residue numbering system based on the human IgG1 EU antibody of Edelman et al. as presented in Kabat 1991.

[0092] The numbering system used for the variable domains (both heavy and light chain) and light chain constant region amino acid sequences is that presented in Kabat 1991.

[0093] An antibody can be any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgD, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subtype (e.g., IgG1, IgG2, IgG3, and IgG4 in humans; and IgG1, IgG2a, IgG2b, and IgG3 in mice). Immunoglobulins, such as IgG1, exist in various allotypes that differ from each other in up to a few amino acids. The antibodies disclosed herein can be of any of the commonly known isotypes, classes, subtypes, or allotypes. In certain embodiments, the antibodies described herein are of the IgG1, IgG2, IgG3, or IgG4 subtype, or any hybrid thereof. In certain embodiments, the antibodies are of the IgG2, IgG, or IgG2 / IgG4 subtype.

[0094] "Antibody" includes, for example, naturally occurring and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human and non-human antibodies, fully synthetic antibodies; single-chain antibodies; monospecific antibodies; multispecific antibodies (including bispecific antibodies); tetrameric antibodies comprising two heavy chain and two light chain molecules; antibody light chain monomers; antibody heavy chain monomers; antibody light chain dimers; antibody heavy chain dimers; antibody light chain-antibody heavy chain pairs; intrabodies; heteroconjugate antibodies; monovalent antibodies; single-chain antibodies; camelized antibodies; affibodies; anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), and single domain antibodies (sdAbs), which include binding molecules consisting entirely of a single monomeric variable antibody domain (e.g., a VH domain or a VL domain) capable of antigen binding (Harmen MM and Haard HJ Appl Microbiol Biotechnol.77(1):13-22(2007)).

[0095] As used herein, the term "antigen-binding portion" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., human FAM19A5). Such "fragments" are, for example, about 8 to about 1500 amino acids in length, preferably about 8 to about 745 amino acids in length, and more preferably about 8 to about 300 amino acids, e.g., about 8 to about 200 amino acids or about 10 to about 50 or 100 amino acids in length. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody, for example, an anti-FAM19A5 antibody described herein, include: (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide linkage at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody and disulfide-linked Fvs (sdFv); (v) a dAb fragment consisting of the VH domain (Ward et al., (1989) Nature 341:544-546); and (vi) an isolated complementarity-determining region (CDR) or (vii) a combination of two or more isolated CDRs, optionally joined by a synthetic linker. Alternatively, although the two domains of an Fv fragment, VL and VH, are encoded by separate genes, they can be joined by a synthetic linker that allows them to be produced using recombinant methods as a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as a single-chain Fv (scFv)); see, e.g., Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883. Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

[0096] As used herein, the terms "variable region" and "variable domain" are interchangeable and are commonly used in the art. The variable region typically refers to a portion of an antibody, generally a portion of either the light or heavy chain, typically comprising approximately the amino-terminal 110-120 amino acids in the mature heavy chain and approximately 90-115 amino acids in the mature light chain, which vary extensively in sequence between antibodies and are used in determining the binding and specificity of a particular antibody to a particular antigen. The sequence variability is concentrated in regions called complementarity-determining regions (CDRs), while the more highly conserved regions of the variable domain are called framework regions (FRs).

[0097] Without being limited to a particular mechanism or theory, the CDRs of the light and heavy chains are believed to be primarily responsible for antigen-antibody interaction and specificity. In certain embodiments, the variable regions are human variable regions. In certain embodiments, the variable regions comprise rodent or murine CDRs and human framework regions (FRs). In certain embodiments, the variable regions are primate (e.g., non-human primate) variable regions. In certain embodiments, the variable regions comprise rodent or murine CDRs and primate (e.g., non-human primate) framework regions (FRs).

[0098] As used herein, the term "heavy chain (HC)" when used in reference to an antibody can refer to any of the distinct types, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), which include IgG subtypes, e.g., IgG1, IgG2, IgG3, and IgG4, and which give rise to the IgA, IgD, IgE, IgG, and IgM types of antibodies, respectively, based on the amino acid sequence of the constant domain.

[0099] As used herein, the term "light chain (LC)" when used in connection with an antibody can refer to any distinct type, e.g., kappa (κ) and lambda (λ), based on the amino acid sequence of the constant domain. Light chain amino acid sequences are well known in the art. In certain embodiments, the light chain is a human light chain.

[0100] The terms "VL" and "VL domain" are used interchangeably to refer to the light chain variable region of an antibody.

[0101] The terms "VH" and "VH domain" are used interchangeably to refer to the heavy chain variable region of an antibody.

[0102] As used herein, the terms "constant region" and "constant domain" are interchangeable and have their usual meaning in the art. The constant domain is the portion of an antibody, e.g., the carboxy-terminal portion of the light and / or heavy chain, that is not directly involved in binding the antibody to an antigen, but may exhibit various effector functions, such as interaction with Fc receptors. The constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence than the immunoglobulin variable domain.

[0103] "Fc region" (Fragment crystallizable region) or "Fc domain" or "Fc" refers to the C-terminal region of an antibody heavy chain that mediates immunoglobulin binding to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or the first component (C1q) of the classical complement system. The Fc region thereby includes the constant region of an antibody excluding the first constant region immunoglobulin domain (e.g., CH1 or CL). In IgG, IgA, and IgD antibody isotypes, the Fc region comprises two identical protein fragments derived from the second (CH2) and third (CH3) constant domains of the antibody's two heavy chains; IgM and IgE Fc regions comprise three heavy chain constant domains (CH domains 2-4) on each polypeptide chain. In IgG, the Fc region includes the hinge between immunoglobulin domains Cγ2 and Cγ3 and between Cγ1 and Cγ2. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is generally defined as stretching from the amino acid residue at C226 or P230 (or an amino acid between these two amino acids) to the carboxy-terminus of the heavy chain, where the numbering follows the EU index as in Kabat. The CH2 domain of the human IgG Fc region extends from about amino acid 231 to about amino acid 340, and the CH3 domain extends to the C-terminal end of the Fc region to the Cm domain, i.e., from about amino acid 341 to about amino acid 447 of IgG. As used herein, an Fc region may refer to a native-sequence Fc, including any allogeneic variant, or a variant Fc (e.g., a non-naturally occurring Fc). Fc may also refer to an Fc-containing protein polypeptide, such as an "Fc region-containing binding protein," also referred to as an "Fc fusion protein" (e.g., an antibody or immunoadhesion).

[0104] A "native sequence Fc region" or "native sequence Fc" comprises an amino acid sequence identical to that of an Fc region found in nature. Native sequence human Fc regions include native sequence human IgG1 Fc regions; native sequence human IgG2 Fc regions; native sequence human IgG3 Fc regions; and native sequence human IgG4 Fc regions, as well as naturally occurring variants thereof. Native sequence Fc includes the various allogeneic forms of Fc (see, e.g., Jefferis et al. (2009) mAbs 1:1; Vidarsson G. et al. Front Immunol. 5:520 (published online October 20, 2014)).

[0105] An "Fc receptor" or "FcR" is a receptor that binds to the Fc region of an immunoglobulin. FcRs that bind IgG antibodies include receptors of the FcγR family, as well as allelic variants and alternatively spliced ​​forms of these receptors. The FcγR family consists of three activating receptors (mouse FcγRI, FcγRIII, and FcγRIV; human FcγRIA, FcγRIIA, and FcγRIIIA) and one inhibitory receptor (FcγRIIB). Human IgG1 binds to the majority of human Fc receptors and elicits the strongest Fc effector functions. Human IgG1 can be considered equivalent to murine IgG2a in terms of the type of activating Fc receptor it binds. Conversely, human IgG4 elicits minimal Fc effector functions (see Vidarsson G. et al. Front Immunol. 5:520 (published online October 20, 2014)).

[0106] The constant region can be engineered, e.g., by recombinant techniques, to remove one or more effector functions. "Effector function" refers to the interaction of an antibody Fc region with an Fc receptor or ligand or the biochemical reaction resulting therefrom. Exemplary "effector functions" include C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, FcγR-mediated effector functions such as ADCC and antibody-dependent cell-mediated phagocytosis (ADCP), and down-regulation of cell surface receptors (e.g., B cell receptors; BCRs). Such effector functions generally require that the Fc region be combined with a binding domain (e.g., an antibody variable domain). Thus, the term "constant region without Fc function" includes a constant region in which one or more effector functions mediated by the Fc region are reduced or absent.

[0107] Antibody effector functions can be reduced or avoided by different approaches. Antibody effector functions can be reduced or avoided by using antibody fragments lacking the Fc region (e.g., Fab, F(ab')2, single-chain Fv (scFv), or sdAbs consisting of monomeric VH or VL domains). Alternatively, so-called aglycosylated antibodies can be generated by removing sugars linked to specific residues in the Fc region to reduce antibody effector functions while retaining other valuable properties of the Fc region (e.g., long half-life and heterodimerization). Aglycosylated antibodies can be generated, for example, by deleting or altering the residue to which the sugar is attached, by enzymatically removing the sugar, by producing the antibody in cells cultured in the presence of a glycosylation inhibitor, or by expressing the antibody in cells (e.g., bacterial host cells) that cannot glycosylate proteins. See, e.g., U.S. Patent Publication No. 20120100140. Another approach is to utilize the Fc region of IgG subtypes with reduced effector function; for example, IgG2 and IgG4 antibodies are characterized by lower levels of Fc effector function compared to IgG1 and IgG3. Residues closest to the hinge region in the CH2 domain of the Fc portion are responsible for the effector function of the antibody and contain largely overlapping binding sites for C1q (complement) and IgG-Fc receptors (FcγRs) on effector cells of the innate immune system (Vidarsson G. et al. Front Immunol. 5:520 (Published online October 20, 2014)). Thus, antibodies with reduced or absent Fc effector function can be produced, for example, by generating a chimeric Fc region comprising the CH2 domain of an IgG antibody of the IgG4 isotype and the CH3 domain of an IgG antibody of the IgG1 isotype, or a chimeric Fc region comprising an IgG2 hinge region and an IgG4 CH2 region (see, e.g., Lau C. et al. J. Immunol. 191:4769-4777 (2013)), or an Fc region with mutations that alter Fc effector function, e.g., reduce or eliminate Fc function. Such Fc regions with mutations are known in the art.See, for example, U.S. Patent Publication No. 20120100140 and the U.S. and PCT applications cited therein, the disclosures of which are incorporated herein by reference in their entirety, and An et al., mAbs 1:6, 572-579 (2009).

[0108] "Hinge," "hinge domain," or "hinge region" or "antibody hinge region" refers to the domain of the heavy chain constant region that joins the CH1 domain to the CH2 domain and includes the upper, middle, and lower portions of the hinge (Roux et al., J. Immunol. 1998 161:4083). The hinge provides varying levels of flexibility between the binding and effector regions of the antibody and also provides a site for intermolecular disulfide bonding between the two heavy chain constant regions. As used herein, hinge begins at Glu216 and ends at Gly237 for all IgG isotypes (Roux et al., 1998 J Immunol 161:4083). The sequences of wild-type IgG1, IgG2, IgG3, and IgG4 hinges are known in the art. See, for example, Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242; Vidarsson G. et al., Front Immunol. 5:520 (published online October 20, 2014).

[0109] The term "CH1 domain" refers to the heavy chain constant region that connects the variable domain to the hinge in the heavy chain constant domain. As used herein, the CH1 domain begins at A118 and ends at V215. The term "CH1 domain" includes not only wild-type CH1 domains but also naturally occurring variants thereof (e.g., allogeneic forms). CH1 domain sequences for IgG1, IgG2, IgG3, and IgG4 (including wild-type and allogeneic forms) are known in the art (see, e.g., Kabat EA et al., (1991) supra and Vidarsson G. et al., Front Immunol. 5:520 (published online October 20, 2014)). Exemplary CH1 domains include CH1 domains with mutations that alter the biological activity, e.g., half-life, of antibodies, as described, for example, in U.S. Patent Publication No. 20120100140 and the U.S. patents, publications, and PCT publications cited therein.

[0110] The term "CH2 domain" refers to the heavy chain constant region that connects the hinge to the CH3 domain. As used herein, the CH2 domain begins at P238 and ends at K340. The term "CH2 domain" includes wild-type CH2 domains as well as naturally occurring variants thereof (e.g., allogeneic forms). CH2 domain sequences for IgG1, IgG2, IgG3, and IgG4 (including wild-type and allogeneic forms) are known in the art (see, e.g., Kabat EA et al., (1991) supra and Vidarsson G. et al., Front Immunol. 5:520 (published online October 20, 2014)). Exemplary CH2 domains include CH2 domains with mutations that alter antibody biological activity, e.g., half-life and / or reduced Fc effector function, as described, for example, in U.S. Patent Publication No. 20120100140 and the U.S. patents, publications, and PCT publications cited therein.

[0111] The term "CH3 domain" refers to the heavy chain constant region C-terminal to the CH2 domain in the heavy chain constant domain. As used herein, the CH3 domain begins at G341 and ends at K447. The term "CH3 domain" includes not only wild-type CH3 domains but also naturally occurring variants thereof (e.g., allogeneic variants). CH3 domain sequences for IgG1, IgG2, IgG3, and IgG4 (including wild-type and allogeneic variants) are known in the art (see, e.g., Kabat EA et al., (1991) supra and Vidarsson G. et al., Front Immunol. 5:520 (published online October 20, 2014)). Exemplary CH3 domains include CH3 domains with mutations that alter the biological activity, e.g., half-life, of antibodies, as described, for example, in U.S. Patent Publication No. 20120100140 and the U.S. patents, publications, and PCT publications cited therein.

[0112] As used herein, "isotype" refers to the antibody type (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibodies) encoded by heavy chain constant region genes.

[0113] "Isotype" refers to naturally occurring variants within a particular isotype group that differ by a number of amino acids (see, e.g., Jefferis et al., (2009) mAbs 1:1). The antibodies described herein can have any isotype. Isotypes of IgG1, IgG2, IgG3, and IgG4 are known in the art. See, e.g., Kabat EA et al., (1991); Vidarsson G. et al., Front Immunol. 5:520 (published online October 20, 2014); and Lefranc MP, mAbs 1:4, 1-7 (2009), supra.

[0114] The phrases "antibody that recognizes an antigen" and "antibody specific for an antigen" are used interchangeably herein with the term "antibody that specifically binds to an antigen."

[0115] As used herein, an "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigen specificities (e.g., an isolated antibody that specifically binds to FAM19A5 is substantially free of antibodies that specifically bind to antigens other than FAM19A5). However, an isolated antibody that specifically binds to an epitope of FAM19A5 may have cross-reactivity with other FAM19A5 proteins from different species.

[0116] "Binding affinity" generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity," as used herein, refers to the intrinsic binding affinity, reflecting a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for a partner Y is generally determined by the dissociation constant (K D Affinity can be expressed as the equilibrium dissociation constant (K D ) and the equilibrium binding constant (K A ) can be measured and / or displayed in a number of ways known in the art, including, but not limited to, D is k off / k on It is calculated from the quotient of K A is k on / k off It is calculated from the quotient of k on means, for example, the binding rate constant of an antibody to an antigen, and k off For example, k refers to the dissociation of an antibody against an antigen. on and k off can be determined by techniques known to those skilled in the art such as immunoassays (eg, enzyme-linked immunosorbent assays (ELISAs)), BIACORE®, or kinetic exclusion assays (KinExA).

[0117] As used herein, the terms "specifically bind," "specifically recognize," "specific binding," "selective binding," and "selectively bind" are analogous terms in the context of antibodies and refer to a molecule (e.g., an antibody) that binds to an antigen (e.g., an epitope or immune complex), as understood by those of skill in the art. For example, a molecule that specifically binds to an antigen may bind to other peptides or polypeptides that typically have a lower affinity, as determined by, for example, immunoassays, a BIACORE®, a KinExA 3000 instrument (Sapidyne Instruments, Boise, ID), or other assays known in the art. In certain embodiments, a molecule that specifically binds to an antigen exhibits a K A Compared to A and binds to the antigen.

[0118] Antibodies are typically -5 ~10 -11 The dissociation constant (K D ) specifically bind to their cognate antigens with high affinity, reflected by approximately 10 -4 Any K greater than M D As used herein, an antibody that "specifically binds" to an antigen refers to an antibody that binds to the antigen with high affinity, and to substantially the same antigen, as determined by, for example, immunoassays (e.g., ELISA) or surface plasma resonance (SPR) techniques on a BIACORE® 2000 instrument using the given antigen, with a binding affinity of 10 or more. -7 M or less, preferably 10 -8 M or less, preferably 10 -9 M or less, most preferably 10 -8 M~10 -10 K below M D This means that the antibody has a high affinity to the antigen, but does not bind with high affinity to unrelated antigens.

[0119] As used herein, "antigen" refers to any natural or synthetic immunogenic substance, such as a protein, peptide, or hapten. The antigen can be FAM19A5 or a fragment thereof.

[0120] As used herein, the term "epitope" refers to a localized region of an antigen to which an antibody can specifically bind. An epitope can be, for example, contiguous amino acids of a polypeptide (a linear or contiguous epitope), or an epitope can be, for example, a collection of two or more non-contiguous regions of a polypeptide (a conformational, non-linear, discontinuous, or non-contiguous epitope). Epitopes formed from contiguous amino acids are typically, but not always, maintained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20 amino acids in a unique spatial conformation. Methods for determining which epitope is bound by a given antibody (i.e., epitope mapping) are well known in the art and include, for example, immunoblot and immunoprecipitation analyses in which overlapping or adjacent peptides (e.g., of FAM19A5) are tested for reactivity with a given antibody (e.g., an anti-FAM19A5 antibody). Methods for determining the spatial conformation of epitopes include techniques known in the art and described herein, such as x-ray crystallography, two-dimensional nuclear magnetic resonance, and HDX-MS (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996)).

[0121] In certain embodiments, the epitope to which the antibody binds can be determined by, for example, NMR spectroscopy, X-ray diffraction crystallography, ELISA analysis, hydrogen / deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography-electrospray mass spectrometry), array-based oligopeptide scanning analysis, and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). For X-ray crystallography, crystallization can be achieved using any method known in the art (see, e.g., Giege R et al. (1994) Acta Crystallogr D Biol Crystallogr 50(Pt4):339-350; McPherson A (1990) Eur J Biochem 189:1-23; Chayen NE (1997) Structure 5:1269-1274; McPherson A (1976) J Biol Chem 251:6300-6303). Antibody:antigen crystals can be studied using well-known X-ray diffraction techniques, such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, e.g., Meth. Enzymol (1985) volumes 114 & 115, eds. Wyck offMutagenesis mapping studies can be performed using computer software such as BUSTER (Bricogne G. (1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1):37-60; Bricogne G. (1997) Meth Enzymol 276A:361-423, ed. Carter CW; Roversi P. et al., (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10):1316-1323). Mutagenesis mapping studies can be performed using any method known to those skilled in the art. For a description of mutagenesis techniques, including alanine scanning mutagenesis techniques, see, for example, Champe M. et al. (1995) J Biol Chem 270:1388-1394 and Cunningham BC & Wells JA (1989) Science 244:1081-1085.

[0122] The term "epitope mapping" refers to the process of identifying the molecular determinants for antibody antigen recognition.

[0123] The term "binds to the same epitope" in the context of two or more antibodies means that the antibodies bind to the same segment of amino acid residues, as determined by a given method. Techniques for determining whether an antibody binds to the "same epitope on FAM19A5" as an antibody described herein include epitope mapping methods, such as x-ray analysis of crystals of antigen:antibody complexes, which provide atomic resolution of the epitope, and hydrogen / deuterium exchange mass spectrometry (HDX-MS). Another method is to monitor the binding of an antibody to antigen fragments or mutated variants of the antigen, where loss of binding due to variations in amino acid residues within the antigen sequence is generally considered to be indicative of epitope components. Combinatorial computational methods for epitope mapping are also available. These methods rely on the ability of the antibody of interest to affinity isolate specific short peptides from combinatorial phage display peptide libraries. Antibodies with the same VH and VL or the same CDR1, 2, and 3 sequences are predicted to bind to the same epitope.

[0124] An antibody that "competes with another antibody for binding to a target" refers to an antibody that inhibits (partially or completely) the target binding of the other antibody. Whether two antibodies compete with each other for binding to a target, i.e., whether and to what extent one antibody inhibits the target binding of the other antibody, can be determined using known competition experiments. In certain embodiments, one antibody competes with the other antibody for target binding and inhibits this binding by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. The level of inhibition or competition can vary depending on whether the antibody is a "blocking antibody" (i.e., a cold antibody that is first incubated with the target). Competition assays can be performed, for example, as described in Chapter 11 of "Using Antibodies" by Ed. Harlow and David Lane, Cold Spring Harbor Protoc; 2006; doi:10.11 01 / pdb.prot 4277 or Ed. Harlow and David Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA 1999. Competing antibodies may bind to the same epitope, overlapping epitopes, or adjacent epitopes (e.g., as evidenced by steric hindrance).

[0125] Other competitive binding assays include solid-phase direct or indirect radioimmunoassays (RIA), solid-phase direct or indirect enzyme immunoassays (EIA), and sandwich competitive assays (see Stahl et al., Methods in Enzymology 9:242 (1983)); solid-phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614 (1986)); solid-phase direct label assays, solid-phase direct label sandwich assays (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid-phase direct label RIA using 1-125 labels (see Morel et al., Mol. Immunol. 25(1):7 (1988)); and solid-phase direct biotin-avidin EIA (see Cheung et al., Virology 176:546 (1990); and direct labeling RIA (see Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)).

[0126] A "bispecific" or "bifunctional antibody" is an artificial hybrid antibody in which two different heavy / light chain pairs have two different binding sites. Bispecific antibodies can be produced by a variety of methods, including fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992).

[0127] As used herein, a "monoclonal antibody" refers to an antibody or antibody composition in which all of the antibodies exhibit a single binding specificity and affinity for a particular epitope. Thus, the term "human monoclonal antibody" refers to an antibody or antibody composition that exhibits a single binding specificity and has variable and optional constant regions derived from human germline immunoglobulin sequences. In some embodiments, human monoclonal antibodies are produced by hybridomas containing B cells from a transgenic non-human animal, e.g., a transgenic mouse, whose genome contains human heavy chain transfer genes and light chain transfer genes fused to an immortalized cell.

[0128] As used herein, the term "recombinant human antibody" includes all human antibodies produced, expressed, produced, or isolated by recombinant means, such as (a) antibodies isolated from animals (e.g., mice) transgenic or transchromosomal for human immunoglobulin genes, or hybridomas produced therefrom, (b) antibodies isolated from host cells transformed to express the antibody, e.g., transfectomas, (c) antibodies isolated from recombinant combinatorial human antibody libraries, and (d) antibodies produced, expressed, produced, or isolated by any other means involving splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies use specific human germline immunoglobulin sequences encoded by germline genes, but contain variable and constant regions that contain subsequent rearrangements and mutations that occur, for example, during antibody maturation. As known in the art (see, e.g., Lonberg (2005) Nature Biotech. 23(9):1117-1125), the variable regions contain antigen-binding domains encoded by different genes that are rearranged to form antibodies specific to foreign antigens. In addition to rearrangement, the variable regions can be further modified by multiple single amino acid changes (referred to as somatic mutation or hypermutation) to increase the affinity of the antibody for the foreign antigen. The constant regions will change in response to additional antigens (i.e., isotype changes). Thus, rearranged and somatically mutated nucleic acid molecules encoding light and heavy chain immunoglobulin polypeptides in response to an antigen may not share sequence identity with the original nucleic acid molecule, but instead will be substantially identical or similar (i.e., have at least 80% identity).

[0129] A "human" antibody (HuMAb) refers to an antibody having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Additionally, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The antibodies described herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of other mammalian species, such as a mouse, have been grafted onto human framework sequences. The terms "human" antibody and "fully human" antibody are used interchangeably.

[0130] A "humanized" antibody refers to an antibody in which some, most, or all of the amino acids outside the CDR domains of a non-human antibody have been substituted with the corresponding amino acids derived from a human immunoglobulin. In some embodiments of humanized forms of antibodies, some, most, or all of the amino acids outside the CDR domains are substituted with amino acids from a human immunoglobulin, while some, most, or all of the amino acids within one or more CDR regions remain unchanged. Minor additions, deletions, insertions, substitutions, or modifications of amino acids are permissible as long as they do not eliminate the antibody's ability to bind to a particular antigen. A "humanized" antibody maintains an antigen specificity similar to that of the original antibody.

[0131] As used herein, the term "deimmunized" or "deimmunization" refers to a process in which an antibody or its antigen-binding portion is modified to reduce its immunogenicity, e.g., in a human subject. For example, the heavy chain variable region (VH) and light chain variable region (VL) sequences of an original antibody can be analyzed, and a human T cell epitope "map" can be generated from each V region, indicating the location of epitopes relative to the complementarity-determining regions (CDRs) and other key residues within the sequence. Individual T cell epitopes are analyzed from the T cell epitope map to identify alternative amino acid substitutions that are unlikely to alter the activity of the final antibody. Various alternative VH and VL sequences containing combinations of amino acid substitutions are designed, and these sequences are then incorporated into a wide range of FAM19A5-specific antibodies or their antigen-binding portions for use in the diagnostic and therapeutic methods disclosed herein, and then tested for functionality. The complete heavy and light chain genes, including the modified VH and human C regions, are then cloned into expression vectors and subsequent plasmids that are introduced into cell lines for whole antibody production. The antibodies are then compared using appropriate biochemical and biological assays to identify the optimal variant. The antibody may be deimmunized by the methods described herein or any other method known in the art. See, e.g., WO98 / 52976 or WO00 / 34317.

[0132] "Chimeric antibody" refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species, such as an antibody in which the variable region is derived from a mouse antibody and the constant region is derived from a human antibody.

[0133] As used herein, the term "cross-reactivity" refers to the ability of an antibody described herein to bind to FAM19A5 of a different species. For example, an antibody described herein that binds to human FAM19A5 can also bind to FAM19A5 of other species (e.g., murine FAM19A5). As used herein, cross-reactivity can be measured by detecting specific reactivity with purified antigen in a binding assay (e.g., SPR, ELISA), or by binding to or functionally interacting with cells that physiologically express FAM19A5. Methods for determining cross-reactivity include standard binding assays described herein, such as BIACORE® surface plasma resonance (SPR) analysis using a BIACORE® 2000 SPR instrument (Biacore AB, Uppsala, Sweden), or flow cytometry techniques.

[0134] As used herein, the term "naturally occurring," when applied to a subject, refers to the fact that the subject can be found in nature. For example, a polypeptide or polynucleotide sequence that is isolatable from a natural source and present in an organism (including a virus) that has not been intentionally modified by man in the laboratory is naturally occurring.

[0135] A "polypeptide" refers to a chain comprising at least two consecutively linked amino acid residues, with no upper limit to the length of the chain. One or more amino acid residues within a protein may contain modifications such as, but not limited to, glycosylation, phosphorylation, or disulfide bond formation. A "protein" can include one or more polypeptides.

[0136] As used herein, the term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. A nucleic acid molecule may be single-stranded or double-stranded, and may be cDNA.

[0137] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting additional nucleic acids to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, into which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. As used herein, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), which perform equivalent functions are also included.

[0138] As used herein, the term "recombinant host cell" (or simply "host cell") refers to a cell that contains nucleic acid that is not naturally occurring within a cell, such as a cell into which a recombinant expression vector has been introduced. Such terms should be understood to refer not only to the particular subject cell but also to the progeny of such a cell. While certain variations may occur in successive generations due to mutation or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0139] As used herein, the term "linked" refers to the association of two or more molecules. The linkage may be covalent or non-covalent. The linkage may also be genetic (i.e., recombinant fusion). Such linkage may be achieved using a variety of art-recognized techniques, such as chemical conjugation and recombinant protein production.

[0140] As used herein, "administration" refers to the physical introduction of a therapeutic agent or composition containing a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those of skill in the art. Preferred routes of administration for the antibodies described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, or other parenteral routes of administration, for example, by injection or infusion. As used herein, the phrase "parenteral administration" generally refers to modes of administration other than enteral and topical administration by injection, and includes, but is not limited to, intravenous, intraperitoneal, intramuscular, intraarterial, intraspinal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intravascular, transtracheal, subcutaneous, subcuticular, intraarticular, subcutaneous, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, as well as in vivo electroporation. Alternatively, the antibodies described herein may be administered parenterally, for example, via topical, epidermal, or mucosal routes of administration, for example, intranasal, oral, intravaginal, oral, sublingual, or topical. Administration can also be, for example, once, multiple times, and / or over one or more extended periods of time.

[0141] As used herein, the terms "treat," "treating," and "treatment" refer to any type of intervention or process performed on a subject, or the administration of an active agent to a subject, for the purpose of attempting to reverse, alleviate, ameliorate, inhibit, slow, or prevent the progression, development, severity, or recurrence of disease-related syndromes, complications, symptoms, or biochemical manifestations. Treatment can be performed on subjects with a disease or subjects without a disease (e.g., prophylactically).

[0142] As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, etc.

[0143] As used herein, the terms "onset of gliosis" or "onset of reactive gliosis" include the onset or initiation of gliosis. Gliosis is a nonspecific reactive change in glial cells within the central nervous system (CNS, e.g., brain and / or spinal cord) due to injury or damage from, for example, trauma, cerebrospinal cord injury, brain tumor, infection, ischemia, stroke, reaction, and / or neurodegenerative disease, and includes proliferation or hypertrophy of various different types of glial cells, including astrocytes, microglia, and oligodendrocytes. The onset of gliosis can result in scar formation that inhibits axonal regeneration in parts of the CNS that have been traumatized or damaged. Harmful effects of the onset of gliosis include irreversible or permanent neuronal damage and / or prevention of recovery of surrounding neurons. Thus, the terms "delayed onset of gliosis" and "delayed onset of reactive gliosis" include inhibition, delay, suppression, or prevention of the onset or initiation of gliosis and its associated harmful effects on the CNS.

[0144] As used herein, the term "reactive astrocyte hyperproliferation" includes an abnormal increase in the number of astrocytes due to destruction of surrounding neurons, e.g., from CNS injury, trauma, injury, cerebrospinal cord injury, brain tumor, infection, ischemia, stroke, reaction, and / or neurodegenerative disease. Reactive astrocyte hyperproliferation can have deleterious effects on the CNS, including scar formation that inhibits axonal regeneration in parts of the CNS that have been traumatized or damaged, exacerbated inflammation, production and release of neurotoxic levels of reactive oxygen species, release of potentially excitotoxic glutamate, a potential contribution to seizure development, impaired blood-brain barrier function, cytotoxic edema in trauma and stroke, the potential for chronic cytokine activation of astrocytes contributing to chronic pain, and secondary degeneration following CNS injury. Sofroniew, Michael V. (2009) Trends in Neurosciences, 32(12):638-47; McGraw, J. et al. (2001) Journal of Neuroscience Research 63(2):109-15; and Sofroniew, MV (2005) The Neuroscientist 11(5):400-7. Thus, the term "inhibiting reactive astrocyte hyperproliferation" includes inhibiting, slowing, suppressing, limiting or preventing excessive or abnormal proliferation of reactive astrocytes and the associated deleterious CNS effects.

[0145] As used herein, the term "chondroitin sulfate proteoglycan" includes proteoglycans composed of a protein core and chondroitin sulfate. Chondroitin sulfate proteoglycans, also known as CSPGs, are extracellular matrix molecules widely expressed throughout the developing and adult CNS. CSPGs play important roles in neuronal development and glial scar formation, and they inhibit axonal regeneration after injury in the CNS. Known CSPGs include aggrecan (CSPG1), versican (CSPG2), neurocan (CSPG3), CSPG4 (or neuroglial antigen 2 (NG2)), CSPG5, SMC3 (CSPG6, structural maintenance of chromosome 3), brevican (CSPG7), and CD44 (CSPG8, cluster of differentiation 44), phosphacan-neurocan (CSPG3). Rhodes, K. E. and Fawcett, J. W. (2004) Journal of Anatomy. 204(1):33-48. Accordingly, the term "reducing expression of chondroitin sulfate proteoglycans" includes reducing, inhibiting, or suppressing the level of one or more CSGPs, or reducing the activity of one or more CSGPs, or inactivating one or more CSGPs. In certain embodiments, the term includes reducing, inhibiting, or suppressing the level of neurocan, NG2, or both, or reducing the activity of neurocan, NG2, or both, or inactivating neurocan, NG2, or both.

[0146] As used herein, the term "neuron" includes electrically excitable cells that process and transmit information through electrical and chemical signals. Neurons are the main components of the brain and spinal cord of the CNS and the ganglia of the peripheral nervous system (PNS), and can be interconnected to form a neuronal network. A typical neuron is composed of a soma (cell body), dendrites, and an axon. The soma (cell body) of a neuron contains the nucleus. The dendrites of a neuron are branched cell extensions from which the majority of neuronal input originates. The axon is a relatively fine, cable-shaped protrusion extending from the soma, which transmits neural signals far from the soma and further transmits specific types of information to the soma. The term "promoting neuron regrowth" preferably includes stimulating, promoting, increasing, or activating the growth of neuronal cells after injury or damage.

[0147] As used herein, the term "c-fos" includes the proto-oncogene c-fos, which is rapidly induced by neurotransmitter stimulation. c-fos is present in many species, including mice and humans. The c-fos gene and protein are known and have been characterized. See Curran, T., The c-fos proto-oncogene, pp. 307-327 (The Oncogene Handbook, Reddy EP et al., (eds.) Elsevier) (1988). c-fos expression may be determined by methods known in the art, such as Northern blot, quantitative PCR, or immunohistochemical methods. The term "increased expression of c-fos" includes an increase in the level of c-fos mRNA, c-fos protein, or c-fos protein activity.

[0148] As used herein, the term "pERK" includes phosphorylated extracellular signal-regulated kinase. Extracellular signal-regulated kinase, or ERK, including ERK1 and ERK2, is a member of the mitogen-activated protein kinase (MAPK) family. ERK is activated by phosphorylation by its upstream kinase to form pERK, which then activates downstream targets. ERK is involved in neuronal and synaptic plasticity, which underlies learning, memory, and pain sensitivity. Ji RR et al., Nat Neurosci (1999) 2:1114-1119. The ERK gene, protein, phosphorylation, and activation are known and characterized, and the expression of ERK and pERK can be determined by methods known in the art (e.g., Northern blot, quantitative PCR, or immunohistochemistry). See Gao YJ and Ji RR, Open Pain J. (2009) 2:11-17. The term "increased pERK expression" includes an increase in ERK mRNA, ERK protein or pERK activity levels.

[0149] As used herein, the term "GAP43," also known as "growth-associated protein 43," is a nervous tissue-specific protein that promotes neurite formation, regeneration, and plasticity. See Benowitz, L. I. and Routtenberg, A. (1997) Trends in Neurosciences 20(2):84-91; Aarts, L. H. et al., (1998) Advances in Experimental Medicine and Biology 446:85-106. Human GAP43 is encoded by the GAP43 gene. The human GAP43 polypeptide sequence (UniProt: KB-P17677) and the cDNA sequence encoding the polypeptide are known in the art. Kosik KS et al., (1988) Neuron 1(2):127-32; Ng SC et al., (1988) Neuron 1(2):133-9. GAP43 expression can be determined by methods known in the art (e.g., Northern blot, quantitative PCR, or immunohistochemistry). The term "increased GAP43 in neurons" includes increased or increased levels of GAP43 mRNA, GAP43 protein, or increased activity of GAP43 protein.

[0150] As used herein, the term "therapeutically effective amount" refers to an amount of a drug alone or in combination with another therapeutic agent that is effective to "treat" a disease or disorder in a subject or to reduce the risk, latency, likelihood, or occurrence of a disease or disorder (e.g., central nervous system injury). A "therapeutically effective amount" includes an amount of a drug or therapeutic agent that provides some improvement or benefit to a subject having or at risk of having a disease or disorder (e.g., central nervous system injury such as traumatic brain injury or other diseases disclosed herein). Hereby, a "therapeutically effective amount" is an amount that reduces or provides some relief, alleviation, or reduction in the risk, latency, likelihood, or occurrence of a disease or disorder, and / or reduces at least one indicator (e.g., the onset of reactive gliosis), and / or reduces at least one clinical symptom of a disease or disorder.

[0151] [II. Anti-FAM19A5 antibody] The present specification discloses antibodies, e.g., monoclonal antibodies, characterized by particular functional characteristics or properties. For example, the antibodies that specifically bind to human FAM19A5 have been mutated (e.g., substituted or deleted) by removing and / or modifying regions or residues that are highly immunogenic in humans (i.e., deimmunized). Thus, the antibodies disclosed herein, i.e., anti-FAM19A5 antibodies, have reduced immunogenicity when administered to a human subject compared to a reference antibody (e.g., a non-deimmunized counterpart, e.g., 3-2 or 2-13 antibody).

[0152] The antibodies described herein also exhibit any one or more of the following functional properties: (a)K D the property of binding to soluble human FAM19A5 with an affinity of 10 nM or less; (b)K D the property of binding to membrane-bound human FAM19A5 with an affinity of 10 nM or less; (c) the property of reducing, reversing, delaying and / or preventing the onset of reactive gliosis; (d) properties that inhibit the overgrowth of reactive astrocytes; (e) the ability to reduce the expression of chondroitin sulfate proteoglycans, including neurocan and neuroglial antigen 2 (NG2); (f) the ability to increase the expression of c-fos and pERK in the nuclei of neurons; (g) properties that promote neuronal survival; (h) the property of increasing the expression of GAP43 in neurons; and (i) Properties that promote axonal regrowth.

[0153] In some embodiments, the anti-FAM19A5 antibody has been deimmunized to render the antibody less immunogenic when administered to a human subject compared to a reference antibody (e.g., a non-deimmunized counterpart, e.g., antibody 3-2 or 2-13). In some embodiments, the immunogenicity of the antibody is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% compared to a reference antibody (e.g., a non-deimmunized counterpart, e.g., antibody 3-2 or 2-13). In some embodiments, the deimmunization process does not alter the binding affinity of the antibody.

[0154] In some embodiments, the anti-FAM19A5 antibodies disclosed herein have undergone affinity maturation such that the antibodies bind to FAM19A5 protein with greater affinity than a reference antibody (e.g., a counterpart antibody that has not undergone affinity maturation, e.g., antibody 2-13). In certain embodiments, the binding affinity of the antibody is increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% compared to the reference antibody (e.g., a counterpart antibody that has not undergone affinity maturation, e.g., antibody 2-13). In some embodiments, the affinity maturation process does not alter the immunogenicity of the antibody.

[0155] In some embodiments, the anti-FAM19A5 antibodies disclosed herein have undergone both deimmunization and affinity maturation such that, when administered to a human subject, they are less immunogenic than a reference antibody (e.g., a non-deimmunized counterpart, e.g., antibody 3-2 or 2-13) and bind to the FAM19A5 protein with greater affinity. In some embodiments, the immunogenicity of the antibody is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% compared to the reference antibody. In some embodiments, the binding affinity of the antibody is increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least 100% or more compared to a reference antibody.

[0156] In some embodiments, the anti-FAM19A5 antibody has high affinity, e.g., K D is 10 -7 M or less, 10 -8 M (10nM) or less, 10 -9 M (1nM) or less, 10 -10 M(0.1nM) or less, 10 -11 M or less or 10 -12 M or less, e.g. 10 -12 M~10 -7 M, 10 -11 M~10 -7 M, 10 -10 M~10 -7 M or 10 -9 M~10 -7 M, e.g. 10 -12 M, 5 x 10 -12 M, 10 -11 M, 5 x 10 -11 M, 10 -10 M, 5 x 10 -10 M, 10-9 M, 5 x 10 -9 M, 10 -8 M, 5 x 10 -8 M, 10 -7 M or 5 x 10 -7 The antibody specifically binds to soluble human FAM19A5 or membrane-bound human FAM19A5, which is M. Standard assays for assessing the binding ability of antibodies to human FAM19A5 of various species are known in the art, including, for example, ELISA, Western blot, and RIA. Suitable assays are described in detail in the Examples section. The binding kinetics (e.g., binding affinity) of the antibody can be assessed by standard assays known in the art, such as ELISA, BIACORE® analysis, or KinExA. Assays for assessing the effect of an antibody on the functional properties (e.g., ligand binding) of FAM19A5 are described in further detail below and in the Examples section.

[0157] In some embodiments, the anti-FAM19A5 antibody has a K D is 10 -7 M or less, 10 -8 M (10nM) or less, 10 -9 M (1nM) or less, 10 -10 M or less, 10 -12 M~10 -7 M, 10 -11 M~10 -7 M, 10 -10 M~10 -7 M, 10 -9 M~10 -7 M or 10 -8 M~10 -7 In some embodiments, the anti-FAM19A5 antibody binds to soluble human FAM19A5, which is K D In some embodiments, the anti-FAM19A5 antibody binds to soluble FAM19A5 with a K of 10 nM or less, e.g., 0.1-10 nM, 0.1-5 nM, 0.1-1 nM, 0.5-10 nM, 0.5-5 nM, 0.5-1 nM, 1-10 nM, 1-5 nM, or 5-10 nM, as determined by ELISA. Dis about 1 pM, about 2 pM, about 3 pM, about 4 pM, about 5 pM, about 6 pM, about 7 pM, about 8 pM, about 9 pM, about 10 pM, about 20 pM, about 30 pM, about 40 pM, about 50 pM, about 60 pM, about 70 pM, about 80 pM, about 90 pM, about 100 pM, about 200 pM, about 300 pM, about 400 pM, about 500 pM, about 600 pM, about 700 The antibody specifically binds to soluble human FAM19A5 at a binding affinity of about 100 pM, about 800 pM, or about 900 pM, or about 1 nM, about 2 nM, about 3 nM, about 4 nM, about 5 nM, about 6 nM, about 7 nM, about 8 nM, or about 9 nM, or about 10 nM, about 20 nM, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, or about 90 nM.

[0158] In some embodiments, the anti-FAM19A5 antibody is D , as determined by, for example, ELISA, -7 M or less, 10 -8 M (10nM) or less, 10 -9 M (1nM) or less, 10 -10 M or less, 10 -12 M~10 -7 M, 10 -11 M~10 -7 M, 10 -10 M~10 -7 M, 10 -9 M~10 -7 M or 10 -8 M~10 -7 In certain embodiments, the anti-FAM19A5 antibody binds to membrane-bound human FAM19A5, which has a K D In some embodiments, the anti-FAM19A5 antibody specifically binds to membrane-bound human FAM19A5 having a K of 10 nM or less, e.g., 0.1-10 nM, 0.1-5 nM, 0.1-1 nM, 0.5-10 nM, 0.5-5 nM, 0.5-1 nM, 1-10 nM, 1-5 nM, or 5-10 nM, as determined by ELISA. Dis about 1 pM, about 2 pM, about 3 pM, about 4 pM, about 5 pM, about 6 pM, about 7 pM, about 8 pM, about 9 pM, about 10 pM, about 20 pM, about 30 pM, about 40 pM, about 50 pM, about 60 pM, about 70 pM, about 80 pM, about 90 pM, about 100 pM, about 200 pM, about 300 pM, about 400 pM, about 500 pM, about 600 pM, about 7 00 pM, about 800 pM or about 900 pM, or about 1 nM, about 2 nM, about 3 nM, about 4 nM, about 5 nM, about 6 nM, about 7 nM, about 8 nM or about 9 nM, or about 10 nM, about 20 nM, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM or about 90 nM of membrane-bound human FAM19A5.

[0159] The anti-FAM19A5 antibodies of the present disclosure can delay or inhibit the onset of gliosis, for example, delay, slow, or inhibit the onset or initiation of non-specific reactive changes in glial cells in the central nervous system (CNS, e.g., brain and / or spinal cord) due to injury or damage from trauma, cerebrospinal cord injury, brain tumor, infection, ischemia, stroke, reaction, and / or neurodegenerative disease.

[0160] The anti-FAM19A5 antibodies of the present disclosure can delay, inhibit, slow, suppress, limit, or prevent excessive or abnormal proliferation of reactive astrocytes and the associated deleterious effects on the CNS. For example, the anti-FAM19A5 antibodies of the present disclosure can inhibit or prevent the abnormal increase in astrocyte numbers due to destruction of neurons, e.g., from CNS injury, trauma, injury, cerebrospinal cord injury, brain tumor, infection, ischemia, stroke, reaction, and / or neurodegenerative disease; inhibit or prevent scar formation in the CNS; inhibit or reduce the release of neurotoxic levels of reactive oxygen species or potentially excitotoxic glutamate; and reduce or inhibit seizures, pain, and / or secondary degeneration after CNS injury. The anti-FAM19A5 antibodies of the present disclosure can preferably promote, stimulate, increase, or activate the regrowth of neurons and / or axons after CNS injury or damage.

[0161] Anti-FAM19A5 antibodies of the present disclosure can inhibit the expression of proteoglycans (CSPGs) composed of a protein core and chondroitin sulfate, such as aggrecan (CSPG1), versican (CSPG2), neurocan (CSPG3), CSPG4 (or neuronal glial antigen 2 (NG2)), CSPG5, SMC3 (CSPG6, structural maintenance of chromosome 3), brevican (CSPG7), CD44 (CSPG8, cluster of differentiation 44), and phosphacan neurocan (CSPG3). In some embodiments, anti-FAM19A5 antibodies of the present disclosure inhibit, decrease, or reduce the levels of neurocan and / or NG2, or the activity of neurocan and / or NG2.

[0162] The anti-FAM19A5 antibodies of the present disclosure can increase the expression of c-fos and pERK in the nuclei of neurons, for example, by increasing c-fos and pERK mRNA, protein, and / or protein activity. The anti-FAM19A5 antibodies of the present disclosure can also increase or enhance the expression levels of GAP43 mRNA, GAP43 protein, or GAP43 protein activity.

[0163] In some embodiments, the anti-FAM19A5 antibody of the present disclosure comprises heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3, wherein the heavy chain CDR1, CDR2, and CDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 5, 6, and 7, respectively, each of which optionally contains 1, 2, 3, 4, or 5 mutations; the light chain CDR1, CDR2, and CDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 8, 9, and 10, respectively, wherein at least one of the light chain CDR1, CDR2, and CDR3 comprises 1, 2, 3, 4, or 5 mutations; and the antibody has reduced immunogenicity in humans compared to a reference antibody comprising a VH set forth in SEQ ID NO: 11 and a VL set forth in SEQ ID NO: 12.

[0164] In some embodiments, the mutations contained in the antibody are substitutions, deletions, and / or insertions. In one embodiment, the mutations are substitutions, e.g., conservative substitutions. As used herein, a "conservative substitution" (also referred to as a conservative substitution) refers to an amino acid substitution that changes a given amino acid to another amino acid with similar biochemical properties (e.g., charge, hydrophobicity, and size). There are many ways to classify amino acids, but they are often divided into six major groups based on their structure and the common chemical properties of their R groups.

[0165] [Table 2]

[0166] Conversely, radical substitutions or radical replacements are amino acid replacements that replace an initial amino acid with a final amino acid with different physicochemical properties. In certain embodiments, the amino acid mutations in the FAM19A5 antibody are radical substitutions. In other embodiments, the amino acid mutations in the FAM19A5 antibody are a combination of conservative and radical substitutions.

[0167] In some embodiments, the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 7. In particular embodiments, the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 7 with one, two, or three mutations.

[0168] In some embodiments, the heavy chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 5, optionally with one or two mutations. In certain embodiments, the mutation comprises a substitution of an acidic amino acid for a threonine at amino acid 3 of SEQ ID NO: 5. In other embodiments, the mutation comprises a substitution of an acidic amino acid for a serine at amino acid 5 of SEQ ID NO: 5. In some embodiments, the acidic amino acid comprises aspartic acid or glutamic acid.

[0169] In some embodiments, the heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO:6, optionally with 1, 2, 3, 4, or 5 mutations. In particular embodiments, the mutation comprises a substitution of an arginine with a basic amino acid at amino acid 16 of SEQ ID NO:6. In some embodiments, the basic amino acid comprises a lysine. In some embodiments, the mutation comprises any one or more of the following: (a) substitution of glycine with an acidic amino acid at amino acid 6 of SEQ ID NO:6; (b) substitution of serine with an acidic amino acid at amino acid 7 of SEQ ID NO:6; (c) substitution of serine with an acidic amino acid at amino acid 8 of SEQ ID NO:6; (d) a substitution of threonine with an acidic amino acid at amino acid 9 of SEQ ID NO: 6; and (e) Substitution of arginine with a basic amino acid at amino acid 16 of SEQ ID NO:6.

[0170] In certain embodiments, the acidic amino acid includes aspartic acid or glutamic acid, hi some embodiments, the basic amino acid includes lysine.

[0171] In some embodiments, the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 10, optionally with 1, 2, 3, 4, or 5 mutations. In particular embodiments, the mutations include any one or more of the following: (a) substitution of serine with an acidic amino acid or an aliphatic amino acid at amino acid 6 of SEQ ID NO: 10; (b) substitution of asparagine with an acidic amino acid or a hydroxyl- or sulfur / selenium-containing amino acid at amino acid 7 of SEQ ID NO: 10; (c) substitution of glycine with an acidic amino acid or a hydroxyl- or sulfur / selenium-containing amino acid at amino acid 8 of SEQ ID NO: 10; (d) substitution of glycine with an acidic amino acid or a hydroxyl or sulfur / selenium-containing amino acid at amino acid 9 of SEQ ID NO: 10; and (e) Substitution of an isoleucine with a basic amino acid at amino acid 10 of SEQ ID NO:10.

[0172] In some embodiments, the acidic amino acid includes aspartic acid or glutamic acid. In certain embodiments, the hydroxyl or sulfur / selenium-containing amino acid includes serine. In other embodiments, the basic amino acid includes histidine.

[0173] In some embodiments, the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO:8 with 1, 2, 3, or 4 mutations. In particular embodiments, the mutations include any one or more of the following: (a) substitution of tyrosine with an acidic amino acid at amino acid 6 of SEQ ID NO:8; (b) substitution of arginine with an acidic amino acid at amino acid 7 of SEQ ID NO:8; (c) a substitution of glycine with an acidic amino acid at amino acid 8 of SEQ ID NO:8; and (d) Substitution of serine with an acidic amino acid at amino acid 9 of SEQ ID NO:8.

[0174] In some embodiments, the acidic amino acid comprises glutamic acid or glutamine.

[0175] In some embodiments, the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO:9 with 1, 2, 3, or 4 mutations. In particular embodiments, the mutations include any one or more of the following: (a) substitution of glutamic acid with an acidic amino acid at amino acid 1 of SEQ ID NO: 9; (b) substitution of serine with an acidic amino acid at amino acid 2 of SEQ ID NO:9; (c) a substitution of asparagine at amino acid 3 of SEQ ID NO: 9 with an acidic amino acid, a basic amino acid, or an aliphatic amino acid; and (d) Substitution of lysine at amino acid 4 of SEQ ID NO: 9 with an acidic or aliphatic amino acid.

[0176] In some embodiments, the acidic amino acid includes glutamine, asparagine, aspartic acid, or glutamic acid. In certain embodiments, the basic amino acid includes histidine. In other embodiments, the aliphatic amino acid includes leucine.

[0177] In some embodiments, the mutation comprises a substitution of an acidic amino acid for serine at amino acid 2 of SEQ ID NO: 9. In certain embodiments, the acidic amino acid comprises aspartate, glutamate, asparagine, glutamine, or a combination thereof. In other embodiments, the acidic amino acid is asparagine.

[0178] In some embodiments, the anti-FAM19A5 antibody disclosed herein comprises heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3: (i) the heavy chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 5; (ii) the heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 13; (iii) the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 7; (iv) the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 8; (v) the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 20; and (vi) the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 10.

[0179] In some embodiments, the anti-FAM19A5 antibody comprises heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3: (i) the heavy chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 14; (ii) the heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15; (iii) the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 7; (iv) the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 21; (v) the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 22; and (vi) the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 23.

[0180] In some embodiments, the anti-FAM19A5 antibody of the present disclosure comprises heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3: (i) the heavy chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 14; (ii) the heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15; (iii) the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 7; (iv) the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 21; (v) the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 24; and (vi) the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 23.

[0181] In some embodiments, the anti-FAM19A5 antibody comprises heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3: (i) the heavy chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 14; (ii) the heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15; (iii) the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 7; (iv) the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 8; (v) the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 25; and (vi) the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 23.

[0182] In some embodiments, the anti-FAM19A5 antibody disclosed herein comprises heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3: (i) the heavy chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 14; (ii) the heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15; (iii) the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 7; (iv) the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 8; (v) the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 24; and (vi) the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 23.

[0183] In some embodiments, the anti-FAM19A5 antibody comprises heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3: (i) the heavy chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 14; (ii) the heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15; (iii) the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 7; (iv) the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 8; (v) the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 26; and (vi) the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 27.

[0184] In some embodiments, the anti-FAM19A5 antibody comprises heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3: (i) the heavy chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 14; (ii) the heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15; (iii) the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 7; (iv) the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 8; (v) the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 28; and (vi) the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 29.

[0185] In some embodiments, the anti-FAM19A5 antibodies of the present disclosure are humanized. In other embodiments, the humanized anti-FAM19A5 antibodies comprise framework regions of a human antibody. In certain embodiments, the anti-FAM19A5 antibodies comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7 or more) mutations in the framework regions (i.e., FR1, FR2, FR3, and FR4 of the VH and / or FR1, FR2, FR3, and FR4 of the VL).

[0186] In some embodiments, the anti-FAM19A5 antibody comprises a mutation within FR1 of VH, in particular embodiments, the mutation comprises an amino acid substitution at residue 19 of SEQ ID NO: 11 (e.g., substitution of serine with a basic amino acid, such as arginine) and / or an amino acid substitution at residue 21 (e.g., substitution of valine with a hydroxyl or sulfur / selenium-containing amino acid, such as serine).

[0187] In some embodiments, the anti-FAM19A5 antibodies disclosed herein comprise a mutation within FR2 of VH. In certain embodiments, the mutation comprises an amino acid substitution at residue 49 of SEQ ID NO: 11 (e.g., a substitution of alanine with a hydroxyl or sulfur / selenium-containing amino acid, such as serine).

[0188] In some embodiments, the anti-FAM19A5 antibody of the present disclosure comprises a mutation within FR3 of VH. In particular embodiments, the mutation comprises an amino acid substitution at residue 79 of SEQ ID NO: 11 (e.g., substitution of valine with an aliphatic amino acid, such as leucine), at residue 80 (e.g., substitution of arginine with an aromatic amino acid, such as tyrosine), at residue 83 (e.g., substitution of leucine with a hydroxyl- or sulfur / selenium-containing amino acid, such as methionine), at residue 85 (e.g., substitution of asparagine with a hydroxyl- or sulfur / selenium-containing amino acid, such as serine), at residue 86 (e.g., substitution of proline with an aliphatic amino acid, such as leucine), and / or at residue 87 (e.g., substitution of glycine with a basic amino acid, such as arginine).

[0189] In some embodiments, the anti-FAM19A5 antibody comprises a mutation within FR2 of the VL, hi certain embodiments, the mutation comprises a deletion of amino acid residue 39 of SEQ ID NO:12.

[0190] In some embodiments, the anti-FAM19A5 antibodies disclosed herein comprise a mutation within FR3 of the VL, which may comprise an amino acid substitution at residue 81 of SEQ ID NO: 12 (e.g., an aliphatic amino acid, such as glycine, for aspartic acid) and / or an amino acid substitution at residue 85 (e.g., an acidic amino acid, such as aspartic acid, for isoleucine).

[0191] In some embodiments, an anti-FAM19A5 antibody of the present disclosure comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO: 11, and / or the VL comprises an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO: 12, and the antibody has reduced immunogenicity compared to a reference antibody comprising the VH set forth in SEQ ID NO: 11 and the VL set forth in SEQ ID NO: 12.

[0192] In some embodiments, the anti-FAM19A5 antibodies disclosed herein cross-compete with a reference antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), (a) the VH comprises the amino acid sequence set forth in SEQ ID NO: 33 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 38; (b) the VH comprises the amino acid sequence set forth in SEQ ID NO: 34 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 39; (c) the VH comprises the amino acid sequence set forth in SEQ ID NO: 34 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 41; (d) the VH comprises the amino acid sequence set forth in SEQ ID NO: 34 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 40; (e) the VH comprises the amino acid sequence set forth in SEQ ID NO: 34 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 42; (f) the VH comprises the amino acid sequence set forth in SEQ ID NO: 34 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 43; or (g) the VH comprises the amino acid sequence set forth in SEQ ID NO: 34, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 44.

[0193] In some embodiments, the anti-FAM19A5 antibody binds to the same human FAM19A5 epitope as a reference antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL); (a) the VH comprises the amino acid sequence set forth in SEQ ID NO: 33 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 38; (b) the VH comprises the amino acid sequence set forth in SEQ ID NO: 34 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 39; (c) the VH comprises the amino acid sequence set forth in SEQ ID NO: 34 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 41; (d) the VH comprises the amino acid sequence set forth in SEQ ID NO: 34 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 40; (e) the VH comprises the amino acid sequence set forth in SEQ ID NO: 34 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 42; (f) the VH comprises the amino acid sequence set forth in SEQ ID NO: 34 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 43; or (g) the VH comprises the amino acid sequence set forth in SEQ ID NO: 34, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 44.

[0194] In some embodiments, the anti-FAM19A5 antibody disclosed herein comprises heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3, wherein the heavy chain CDR1, CDR2, and CDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively, and each of the sequences optionally comprises one, two, or three mutations; the light chain CDR1, CDR2, and CDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 30, 31, and 32, respectively, and at least one of the light chain CDR1, CDR2, and CDR3 comprises one, two, or three mutations; and the antibody has reduced immunogenicity in humans and / or higher binding affinity to human FAM19A5 protein compared to a reference antibody comprising the VH set forth in SEQ ID NO: 35 and the VL set forth in SEQ ID NO: 45.

[0195] In some embodiments, the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 18. In particular embodiments, the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 18, optionally with one or two mutations. In some embodiments, the mutations include one or more of the following: (a) a substitution of a hydroxyl- or sulfur / selenium-containing amino acid for threonine at amino acid 2 of SEQ ID NO: 18; and (b) a substitution of an aliphatic amino acid for glutamic acid at amino acid 4 of SEQ ID NO: 18. In particular embodiments, the hydroxyl- or sulfur / selenium-containing amino acid comprises serine. In some embodiments, the aliphatic amino acid comprises valine. In some embodiments, the mutations include one or more of the following: (a) a substitution of an acidic amino acid for threonine at amino acid 2 of SEQ ID NO: 18; and (b) a substitution of an aliphatic amino acid for glutamic acid at amino acid 4 of SEQ ID NO: 18. In particular embodiments, the acidic amino acid comprises asparagine. In certain embodiments, the aliphatic amino acid comprises alanine.

[0196] In some embodiments, the heavy chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 16. In specific embodiments, the heavy chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 16, optionally with one mutation. In some embodiments, the mutation comprises a substitution of threonine with an acidic amino acid at amino acid 3 of SEQ ID NO: 16. In specific embodiments, the acidic amino acid comprises aspartic acid.

[0197] In some embodiments, the heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO:17.

[0198] In some embodiments, the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 32. In certain embodiments, the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 31. In some embodiments, the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 31 with one, two, or three mutations.

[0199] In other embodiments, the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 30, optionally with one mutation. In certain embodiments, the mutation comprises a substitution of an aliphatic amino acid for serine at amino acid 4 of SEQ ID NO: 30. In some embodiments, the aliphatic amino acid comprises valine.

[0200] In some embodiments, the anti-FAM19A5 antibody disclosed herein comprises heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3: (i) the heavy chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 16; (ii) the heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 17; (iii) the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 18; (iv) the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 80; (v) the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 31; and (vi) the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 32.

[0201] In some embodiments, the anti-FAM19A5 antibody disclosed herein comprises heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3: (i) the heavy chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 19; (ii) the heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 17; (iii) the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 18; (iv) the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 80; (v) the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 31; and (vi) the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 32.

[0202] In some embodiments, the anti-FAM19A5 antibody disclosed herein comprises heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3: (i) the heavy chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 19; (ii) the heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 17; (iii) the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 128; (iv) the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 80; (v) the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 31; and (vi) the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 32.

[0203] In some embodiments, the anti-FAM19A5 antibody disclosed herein comprises heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3: (i) the heavy chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 19; (ii) the heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 17; (iii) the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 129; (iv) the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 80; (v) the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 31; and (vi) the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 32.

[0204] In some embodiments, the anti-FAM19A5 antibodies of the present disclosure are humanized. In other embodiments, the humanized anti-FAM19A5 antibodies comprise framework regions of a human antibody. In certain embodiments, the anti-FAM19A5 antibodies comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7 or more) mutations within the framework regions of the antibody (i.e., FR1, FR2, FR3, and FR4 of the VH and / or FR1, FR2, FR3, and FR4 of the VL).

[0205] In some embodiments, the anti-FAM19A5 antibodies disclosed herein comprise a mutation within FR1 of VH. In particular embodiments, the mutation comprises an amino acid substitution at residue 19 of SEQ ID NO: 35 (e.g., a basic amino acid, such as arginine, for serine), at residue 21 (e.g., a hydroxyl- or sulfur / selenium-containing amino acid, such as serine, for valine), and / or at residue 23 (e.g., a hydroxyl- or sulfur / selenium-containing amino acid, such as serine, for lysine).

[0206] In some embodiments, the anti-FAM19A5 antibody comprises a mutation within FR2 of VH, in particular embodiments, the mutation comprises an amino acid substitution at residue 40 of SEQ ID NO: 35 (e.g., substitution of tyrosine with an aliphatic amino acid, such as alanine) and / or an amino acid substitution at residue 49 (e.g., substitution of alanine with a hydroxyl- or sulfur / selenium-containing amino acid, such as serine).

[0207] In some embodiments, the anti-FAM19A5 antibody comprises a mutation within FR3 of VH. In particular embodiments, the mutation comprises an amino acid substitution at residue 79 of SEQ ID NO: 35 (e.g., substitution of valine with an aliphatic amino acid, such as leucine), at residue 80 (e.g., substitution of arginine with an aromatic amino acid, such as tyrosine), at residue 83 (e.g., substitution of leucine with a hydroxyl or sulfur / selenium-containing amino acid, such as methionine), and / or at residue 85 (e.g., substitution of asparagine with a hydroxyl or sulfur / selenium-containing amino acid, such as serine).

[0208] In some embodiments, the anti-FAM19A5 antibody comprises a mutation within FR1 of the VL, hi certain embodiments, the mutation comprises an amino acid substitution at residue 16 of SEQ ID NO: 45 (e.g., a substitution of valine with an aliphatic amino acid, such as alanine).

[0209] In some embodiments, the anti-FAM19A5 antibody comprises a mutation within FR2 of the VL, hi certain embodiments, the mutation comprises a deletion of amino acid residue 34 of SEQ ID NO:45.

[0210] In some embodiments, the anti-FAM19A5 antibodies disclosed herein comprise a mutation within FR3 of the VL, such as an amino acid substitution at residue 76 of SEQ ID NO: 45 (e.g., substitution of an aspartic acid with an acidic amino acid, such as glutamic acid), an amino acid substitution at residue 80 (e.g., substitution of a valine with an acidic amino acid, such as aspartic acid), and / or an amino acid substitution at residue 82 (e.g., substitution of a phenylalanine with an aromatic amino acid, such as tyrosine).

[0211] In some embodiments, the anti-FAM19A5 antibodies disclosed herein comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO: 35, and / or the VH comprises an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO: 45.

[0212] In some embodiments, the anti-FAM19A5 antibody of the present disclosure cross-competes with a reference antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein (i) the VH comprises the amino acid sequence set forth in SEQ ID NO: 36 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 46; (ii) the VH comprises the amino acid sequence set forth in SEQ ID NO: 37 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 46; (iii) the VH comprises the amino acid sequence set forth in SEQ ID NO: 130 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 46; or (iv) the VH comprises the amino acid sequence set forth in SEQ ID NO: 131 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 46.

[0213] In some embodiments, the anti-FAM19A5 antibodies disclosed herein bind to the same human FAM19A5 epitope as a reference antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein (i) the VH comprises the amino acid sequence set forth in SEQ ID NO: 36 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 46; (ii) the VH comprises the amino acid sequence set forth in SEQ ID NO: 37 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 46; (iii) the VH comprises the amino acid sequence set forth in SEQ ID NO: 130 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 46; or (iv) the VH comprises the amino acid sequence set forth in SEQ ID NO: 131 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 46.

[0214] In some embodiments, an anti-FAM19A5 antibody of the present disclosure cross-competes with (or inhibits) binding to the human FAM19A5 epitope with a reference antibody (e.g., the 3-2 or 2-13 antibody).

[0215] In some embodiments, the anti-FAM19A5 antibody inhibits the binding of such a reference antibody (e.g., 3-2 or 2-13 antibody) to human FAM19A5 by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%. Competitive antibodies bind to the same epitope, overlapping epitopes, or adjacent epitopes (e.g., as evidenced by steric hindrance). Whether two antibodies compete with each other for binding to a target can be determined using competition experiments known in the art, such as RIA and EIA.

[0216] Techniques for determining whether two antibodies bind to the same epitope include, for example, epitope mapping methods such as x-ray analysis of crystals of antigen:antibody complexes and hydrogen / deuterium exchange mass spectrometry (HDX-MS), which provide atomic resolution of the epitope; methods that monitor antibody binding to antigen fragments or mutated variants of the antigen, in which loss of binding due to modification of amino acid residues within the antigen sequence is generally considered to be indicative of epitope components; and combinatorial computational methods for epitope mapping.

[0217] Anti-FAM19A5 antibodies useful in the methods disclosed herein can bind to at least one epitope in mature human FAM19A5, e.g., as determined by antibody binding to a fragment of human FAM19A5. In some embodiments, the anti-FAM19A5 antibody binds to at least one epitope having the amino acid sequence TLDRDSSQPRRTIARQTARC (amino acid residues 42-61 of SEQ ID NO:90 or SEQ ID NO:2), or a fragment located within the amino acid sequence of SEQ ID NO:90, e.g., an epitope having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids of SEQ ID NO:90. In some embodiments, the anti-FAM19A5 antibodies disclosed herein bind to one or more amino acids corresponding to amino acid residues 44 to 52 (i.e., DRDSSQPRR) of SEQ ID NO: 2, such as amino acid residues 45, 46, 50, 51, and 52 (i.e., RD---PRR), such as amino acid residues 45, 50, 51, and 52 (i.e., R----PRR), or amino acid residues 45, 50, and 51 (i.e., R----PR).

[0218] In some embodiments, the anti-FAM19A5 antibody binds to at least one epitope having the amino acid sequence TARCACRKGQIAGTTRARPA (SEQ ID NO: 91 or amino acid residues 58-77 of SEQ ID NO: 2), or a fragment located within the amino acid sequence of SEQ ID NO: 91, e.g., an epitope having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids of SEQ ID NO: 91. In some embodiments, the anti-FAM19A5 antibody binds to one or more amino acids corresponding to amino acid residues 63-75 of SEQ ID NO: 2 (i.e., CRKGQIAGTTRAR). In some embodiments, the anti-FAM19A5 antibody binds to one or more epitopes having the amino acid sequence ARPACVDARIIKTKQWCDML (amino acid residues 74-93 of SEQ ID NO:92 or SEQ ID NO:2), or a fragment located within the amino acid sequence of SEQ ID NO:92, e.g., an epitope having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In some embodiments, the anti-FAM19A5 antibody binds to one or more amino acids corresponding to amino acid residues 76-89 of SEQ ID NO:2 (i.e., PACVDARIIKTKQW).

[0219] In some embodiments, the at least one epitope has an amino acid sequence that is at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 90, 91, or 92.

[0220] In some embodiments, the anti-FAM19A5 antibody or antigen-binding portion thereof binds only to the human FAM19A5 epitope of SEQ ID NO: 89, 90, 91, 92, 93, or 94, or a fragment located within the amino acid sequence of SEQ ID NO: 89, 90, 91, 92, 93, or 94, e.g., an epitope having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids of SEQ ID NO: 89, 90, 91, 92, 93, or 94.

[0221] In some embodiments, the anti-FAM19A5 antibodies of the present disclosure bind to SEQ ID NO: 90 or a fragment thereof in its native conformation (i.e., native). In some embodiments, the anti-FAM19A5 antibodies, or antigen-binding portions thereof, bind to both glycosylated and non-glycosylated human FAM19A5.

[0222] In some embodiments, the anti-FAM19A5 antibody binds to one or more additional FAM19A5 epitopes, such as QLAAGTCEIVTLDR (SEQ ID NO: 89, epitope F1), TLDRDSSQPRRTIARQTARC (SEQ ID NO: 90, epitope F2), TARCACRKGQIAGTTRARPA (SEQ ID NO: 91, epitope F3), ARPACVDARIIKTKQW CDML (SEQ ID NO: 92, epitope F4), CDMLPCLEGEGCDLLINRSG (SEQ ID NO: 93, epitope F5), or NRSGWTCTQPGGRIKTTTVS (SEQ ID NO: 94, epitope F6), or a fragment located within the amino acid sequence of SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, or SEQ ID NO: 94, or any combination thereof. Fragments located within the amino acid sequence of SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93 or SEQ ID NO:94 include fragments having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids of SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93 or SEQ ID NO:94. In some embodiments, the one or more additional FAM19A5 epitopes are selected from SEQ ID NOs: 89, 90, 91, 92, 93, or 94, or fragments located within the amino acid sequence of SEQ ID NOs: 89, 90, 91, 92, 93, or 94, e.g., fragments having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids of SEQ ID NOs: 89, 90, 91, 92, 93, or 94, or combinations thereof. In some embodiments, an anti-FAM19A5 antibody, or antigen-binding portion thereof, of the present disclosure binds to a portion of the one or more additional epitopes in their native conformation (i.e., native). In some embodiments, the anti-FAM19A5 antibody, or antigen-binding portion thereof, binds to both the glycosylated and non-glycosylated one or more additional FAM19A5 epitopes.

[0223] In some embodiments, the present disclosure provides antibodies or antigen-binding fragments thereof that bind to FAM19A5 (e.g., human FAM19A5) with 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or higher affinity relative to other proteins in the FAM19A family, as measured, for example, by immunoassay (e.g., ELISA), surface plasma resonance, or dynamic exclusion assay. In certain embodiments, the anti-FAM19A5 antibodies or antigen-binding fragments thereof bind to FAM19A5 (e.g., human FAM19A5) without cross-reactivity with other proteins in the FAM19A family, as measured, for example, by immunoassay.

[0224] In some embodiments, the anti-FAM19A5 antibodies of the present disclosure are not native or naturally occurring antibodies, e.g., in some embodiments, the anti-FAM19A5 antibodies have post-translational modifications that differ from naturally occurring antibodies, such as by having more, fewer, or different types of post-translational modifications.

[0225] The amino acid sequences of the VH and VL CDRs of exemplary antibodies of the disclosure are provided in Tables 3 and 4, respectively. The VH and VL amino acid sequences are provided in Tables 5 and 6, respectively.

[0226] [Table 3]

[0227] [Table 4]

[0228] [Table 5]

[0229] [Table 6]

[0230] In some embodiments, the anti-FAM19A5 antibody of the present disclosure comprises heavy and light chain variable regions, wherein the heavy chain variable region (VH) comprises the heavy chain CDR1, CDR2, and CDR3 of SEQ ID NO: 33, and / or the light chain variable region (VL) comprises the light chain CDR1, CDR2, and CDR3 of SEQ ID NO: 38. In other embodiments, the anti-FAM19A5 antibody comprises a VH and a VL, wherein the VH comprises the amino acid sequence set forth in SEQ ID NO: 33, and / or the VL comprises the amino acid sequence set forth in SEQ ID NO: 38.

[0231] In some embodiments, the anti-FAM19A5 antibody of the present disclosure comprises heavy and light chain variable regions, wherein the heavy chain variable region (VH) comprises the heavy chain CDR1, CDR2, and CDR3 of SEQ ID NO: 34, and / or the light chain variable region (VL) comprises the light chain CDR1, CDR2, and CDR3 of SEQ ID NO: 39. In other embodiments, the anti-FAM19A5 antibody comprises a VH and a VL, wherein the VH comprises the amino acid sequence set forth in SEQ ID NO: 34, and / or the VL comprises the amino acid sequence set forth in SEQ ID NO: 39.

[0232] In some embodiments, the anti-FAM19A5 antibody of the present disclosure comprises heavy and light chain variable regions, wherein the heavy chain variable region (VH) comprises the heavy chain CDR1, CDR2, and CDR3 of SEQ ID NO: 34, and / or the light chain variable region (VL) comprises the light chain CDR1, CDR2, and CDR3 of SEQ ID NO: 41. In other embodiments, the anti-FAM19A5 antibody comprises a VH and a VL, wherein the VH comprises the amino acid sequence set forth in SEQ ID NO: 34, and / or the VL comprises the amino acid sequence set forth in SEQ ID NO: 41.

[0233] In some embodiments, the anti-FAM19A5 antibody of the present disclosure comprises heavy and light chain variable regions, wherein the heavy chain variable region (VH) comprises the heavy chain CDR1, CDR2, and CDR3 of SEQ ID NO: 34, and / or the light chain variable region (VL) comprises the light chain CDR1, CDR2, and CDR3 of SEQ ID NO: 40. In other embodiments, the anti-FAM19A5 antibody comprises a VH and a VL, wherein the VH comprises the amino acid sequence set forth in SEQ ID NO: 34, and / or the VL comprises the amino acid sequence set forth in SEQ ID NO: 40.

[0234] In some embodiments, the anti-FAM19A5 antibody of the present disclosure comprises heavy and light chain variable regions, wherein the heavy chain variable region (VH) comprises the heavy chain CDR1, CDR2, and CDR3 of SEQ ID NO: 34, and / or the light chain variable region (VL) comprises the light chain CDR1, CDR2, and CDR3 of SEQ ID NO: 42. In other embodiments, the anti-FAM19A5 antibody comprises a VH and a VL, wherein the VH comprises the amino acid sequence set forth in SEQ ID NO: 34, and / or the VL comprises the amino acid sequence set forth in SEQ ID NO: 42.

[0235] In some embodiments, the anti-FAM19A5 antibody of the present disclosure comprises heavy and light chain variable regions, wherein the heavy chain variable region (VH) comprises the heavy chain CDR1, CDR2, and CDR3 of SEQ ID NO: 34, and / or the light chain variable region (VL) comprises the light chain CDR1, CDR2, and CDR3 of SEQ ID NO: 43. In other embodiments, the anti-FAM19A5 antibody comprises a VH and a VL, wherein the VH comprises the amino acid sequence set forth in SEQ ID NO: 34, and / or the VL comprises the amino acid sequence set forth in SEQ ID NO: 43.

[0236] In some embodiments, the anti-FAM19A5 antibody of the present disclosure comprises heavy and light chain variable regions, wherein the heavy chain variable region (VH) comprises the heavy chain CDR1, CDR2, and CDR3 of SEQ ID NO: 34, and / or the light chain variable region (VL) comprises the light chain CDR1, CDR2, and CDR3 of SEQ ID NO: 44. In other embodiments, the anti-FAM19A5 antibody comprises a VH and a VL, wherein the VH comprises the amino acid sequence set forth in SEQ ID NO: 34, and / or the VL comprises the amino acid sequence set forth in SEQ ID NO: 44.

[0237] In some embodiments, the anti-FAM19A5 antibody of the present disclosure comprises heavy and light chain variable regions, wherein the heavy chain variable region (VH) comprises the heavy chain CDR1, CDR2, and CDR3 of SEQ ID NO: 36, and / or the light chain variable region (VL) comprises the light chain CDR1, CDR2, and CDR3 of SEQ ID NO: 46. In other embodiments, the anti-FAM19A5 antibody comprises a VH and a VL, wherein the VH comprises the amino acid sequence set forth in SEQ ID NO: 36, and / or the VL comprises the amino acid sequence set forth in SEQ ID NO: 46.

[0238] In some embodiments, the anti-FAM19A5 antibody of the present disclosure comprises heavy and light chain variable regions, wherein the heavy chain variable region (VH) comprises the heavy chain CDR1, CDR2, and CDR3 of SEQ ID NO: 37, and / or the light chain variable region (VL) comprises the light chain CDR1, CDR2, and CDR3 of SEQ ID NO: 46. In other embodiments, the anti-FAM19A5 antibody comprises a VH and a VL, wherein the VH comprises the amino acid sequence set forth in SEQ ID NO: 37, and / or the VL comprises the amino acid sequence set forth in SEQ ID NO: 46.

[0239] In some embodiments, the anti-FAM19A5 antibody of the present disclosure comprises heavy and light chain variable regions, wherein the heavy chain variable region (VH) comprises the heavy chain CDR1, CDR2, and CDR3 of SEQ ID NO: 130, and / or the light chain variable region (VL) comprises the light chain CDR1, CDR2, and CDR3 of SEQ ID NO: 46. In other embodiments, the anti-FAM19A5 antibody comprises a VH and a VL, wherein the VH comprises the amino acid sequence set forth in SEQ ID NO: 37, and / or the VL comprises the amino acid sequence set forth in SEQ ID NO: 46.

[0240] In some embodiments, the anti-FAM19A5 antibody of the present disclosure comprises heavy and light chain variable regions, wherein the heavy chain variable region (VH) comprises the heavy chain CDR1, CDR2, and CDR3 of SEQ ID NO: 131, and / or the light chain variable region (VL) comprises the light chain CDR1, CDR2, and CDR3 of SEQ ID NO: 46. In other embodiments, the anti-FAM19A5 antibody comprises a VH and a VL, wherein the VH comprises the amino acid sequence set forth in SEQ ID NO: 37, and / or the VL comprises the amino acid sequence set forth in SEQ ID NO: 46.

[0241] In some embodiments, the anti-FAM19A5 antibody of the present disclosure comprises a VH and a VL, wherein the VH comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO: 33, 34, 36, 37, 130, or 131.

[0242] In some embodiments, the anti-FAM19A5 antibodies of the present disclosure comprise a VH and a VL, wherein the VL comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO: 38, 39, 40, 41, 42, 43, 44, or 46.

[0243] In some embodiments, the anti-FAM19A5 antibody of the present disclosure comprises a VH and a VL: (a) the VH comprises the amino acid sequence set forth in SEQ ID NO: 33 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 38; (b) the VH comprises the amino acid sequence set forth in SEQ ID NO: 34 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 39; (c) the VH comprises the amino acid sequence set forth in SEQ ID NO: 34 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 41; (d) the VH comprises the amino acid sequence set forth in SEQ ID NO: 34 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 40; (e) the VH comprises the amino acid sequence set forth in SEQ ID NO: 34 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 42; (f) the VH comprises the amino acid sequence set forth in SEQ ID NO: 34 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 43; (g) the VH comprises the amino acid sequence set forth in SEQ ID NO: 34 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 44; (h) the VH comprises the amino acid sequence set forth in SEQ ID NO: 36 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 46; (i) the VH comprises the amino acid sequence set forth in SEQ ID NO: 37 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 46; (j) the VH comprises the amino acid sequence set forth in SEQ ID NO: 130 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 46; or (k) the VH comprises the amino acid sequence set forth in SEQ ID NO: 131, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 46.

[0244] A VH domain described herein, or one or more CDRs thereof, may be linked to a constant domain to form a heavy chain, e.g., a full-length heavy chain. Similarly, a VL domain described herein, or one or more CDRs thereof, may be linked to a constant domain to form a light chain, e.g., a full-length light chain. A full-length heavy chain and a full-length light chain may be combined to generate a full-length antibody.

[0245] Thus, in certain embodiments, the present specification provides antibodies comprising an antibody light chain and a heavy chain, e.g., separate light and heavy chains. With respect to the light chain, in certain embodiments, the light chain of an antibody described herein is a kappa light chain. In other specific embodiments, the light chain of an antibody described herein is a lambda light chain. In yet other specific embodiments, the light chain of an antibody described herein is a human kappa light chain or a human lambda light chain. In certain embodiments, an antibody described herein that specifically binds to a FAM19A5 polypeptide (e.g., human FAM19A5) comprises a light chain comprising any of the VL or VL CDR amino acid sequences described herein, and the constant region of the light chain comprises the amino acid sequence of a human kappa light chain constant region. In certain embodiments, an antibody described herein that specifically binds to a FAM19A5 polypeptide (e.g., human FAM19A5) comprises a light chain comprising a VL or VL CDR amino acid sequence described herein, and the constant region of the light chain comprises the amino acid sequence of a human lambda light chain constant region. Non-limiting examples of human constant region sequences are described in the art. See, e.g., U.S. Patent No. 5,693,780 and Kabat EA et al. (1991), supra.

[0246] With respect to the heavy chain, in some embodiments, the heavy chain of the antibodies described herein can be an alpha (α), delta (δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In other specific embodiments, the heavy chain of the described antibodies can comprise a human alpha (α), delta (δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In some embodiments, an antibody disclosed herein that specifically binds to FAM19A5 (e.g., human FAM19A5) comprises a heavy chain comprising a VH or VH CDR amino acid sequence described herein, and the constant region of the heavy chain comprises the amino acid sequence of a human gamma (γ) heavy chain constant region. In yet other embodiments, an antibody described herein that specifically binds to FAM19A5 (e.g., human FAM19A5) comprises a heavy chain comprising a VH or VH CDR amino acid sequence described herein, and the constant region of the heavy chain comprises the amino acid sequence of a human heavy chain described herein or known in the art. Non-limiting examples of human constant region sequences are described in the art, see, e.g., U.S. Patent No. 5,693,780 and Kabat EA et al., (1991), supra.

[0247] In some embodiments, antibodies described herein that specifically bind to FAM19A5 (e.g., human FAM19A5) comprise a VL domain and a VH domain comprising the VH or VH CDRs and the VL and VL CDRs described herein, wherein the constant region comprises the amino acid sequence of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, or a constant region of a human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule. In other specific embodiments, antibodies described herein that specifically bind to FAM19A5 (e.g., human FAM19A5) comprise a VL domain and a VH domain comprising any amino acid sequence described herein, wherein the constant region comprises the amino acid sequence of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, or any subtype of immunoglobulin molecule (e.g., IgG1, IgG2, IgG3, IgG4, IgAI, and IgA2). In some embodiments, the constant region comprises the amino acid sequence of the constant region of a native human IgG, including subtypes (e.g., IgG1, IgG2, IgG3, or IgG4) and allogeneic subtypes (e.g., Glm, G2m, G3m, and nG4m) and variants thereof. See, e.g., Vidarsson G. et al. Front Immunol. 5:520 (published online October 20, 2014) and Jefferis R. and Lefranc MP, mAbs 1:4, 1-7 (2009). In some embodiments, the constant region comprises the amino acid sequence of the constant region of human IgG1, IgG2, IgG3, or IgG4, or variants thereof.

[0248] In certain embodiments, the anti-FAM19A5 antibodies disclosed herein lack Fc effector functions, such as complement-dependent cytotoxicity (CDC) and / or antibody-dependent cellular phagocytosis (ADCP). Effector functions are mediated by the Fc region, and the residues closest to the hinge region in the CH2 domain of the Fc region contain highly overlapping binding sites for Clq (complement) and IgG-Fc receptors (FcγR) on effector cells of the innate immune system and are therefore responsible for the antibody's effector functions. Furthermore, IgG2 and IgG4 antibodies have lower levels of Fc effector functions than IgG1 and IgG3 antibodies. Antibody effector function can be reduced by (1) using antibody fragments lacking the Fc region (e.g., Fab, F(ab')2, single-chain Fv (scFv), or sdAbs consisting of monomeric VH or VL domains); (2) generating aglycosylated antibodies, for example, by deleting or altering the residue to which the carbohydrate is attached, by enzymatically removing the carbohydrate, by producing the antibody in cells cultured in the presence of a glycosylation inhibitor, or by expressing the antibody in cells that cannot glycosylate proteins (e.g., bacterial host cells; see, e.g., U.S. Patent Publication No. 20120100140); or (3) using Fc regions of IgG subtypes with reduced effector function (e.g., Fc regions of IgG2 and IgG4 antibodies or chimeric Fc regions comprising the CH2 domain of an IgG2 or IgG4 antibody, see, e.g., U.S. Patent Publication No. 20120100140 and Lau C. et al.). al. J. Immunol. 191:4769-4777 (2013); and (4) can be reduced or avoided by different approaches known in the art, including generating Fc regions with mutations that reduce or eliminate Fc function. See, e.g., U.S. Patent Publication No. 20120100140 and the U.S. and PCT applications cited therein, and An et al., mAbs 1:6, 572-579 (2009).

[0249] Thus, in some embodiments, the anti-FAM19A5 antibodies disclosed herein are Fab, Fab', F(ab'), Fv, single-chain Fv (scFv), or sdAbs consisting of monomeric VH or VL domains. Such antibody fragments are well known in the art and are described above.

[0250] In some embodiments, the anti-FAM19A5 antibody is a single-chain Fv. The amino acid sequences of exemplary anti-FAM19A5 scFvs are provided in Table 7 below.

[0251] [Table 7]

[0252] JPEG2025124689000010.jpg172169

[0253] In some embodiments, the anti-FAM19A5 antibodies disclosed herein comprise an Fc region with reduced or absent Fc effector function. In some embodiments, the constant region comprises the amino acid sequence of a human IgG2 or IgG4 Fc region, and in some embodiments, the anti-FAM19A5 antibody has an IgG2 / IgG4 isotype. In some embodiments, the anti-FAM19A5 antibody comprises a chimeric Fc region comprising a CH2 domain of an IgG antibody of the IgG4 isotype and a CH3 domain of an IgG antibody of the IgG1 isotype, or a chimeric Fc region comprising an IgG2 hinge region and an IgG4 CH2 region, or an Fc region with a mutation that results in reduced or absent Fc effector function. Fc regions with reduced or absent Fc effector function include those known in the art. See, e.g., Lau C. et al., J. Immunol. 191:4769-4777 (2013); An et al., mAbs 1:6, 572-579 (2009); and U.S. Patent Publication No. 20120100140 and the U.S. patents and publications and PCT publications cited therein. Additionally, Fc regions with reduced or absent Fc effector function can be readily engineered by one of skill in the art.

[0254] [III. Nucleic acid molecule] Still other aspects described herein relate to one or more nucleic acid molecules encoding any one of the antibodies described herein. The nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is "isolated" or "substantially pure" when purified from other cellular components or other contaminants, such as other cellular nucleic acids (e.g., other chromosomal DNA, e.g., chromosomal DNA linked to naturally isolated DNA) or proteins, by standard techniques, including alkali / SDS treatment, CsCl banding, column chromatography, restriction enzymes, agarose gel electrophoresis, and other techniques well known in the art. See F. Ausubel, et al., ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. The nucleic acids described herein may be, for example, DNA or RNA, and may or may not contain intronic sequences. In certain embodiments, the nucleic acid is a cDNA molecule.

[0255] The nucleic acids described herein can be obtained using standard molecular biology techniques. In the case of antibodies expressed by hybridomas (e.g., hybridomas produced from transgenic mice carrying human immunoglobulin genes, as described further below), cDNAs encoding the light and heavy chains of the antibodies produced by the hybridomas can be obtained by standard PCR amplification or cDNA cloning techniques. In the case of antibodies obtained from an immunoglobulin gene library (e.g., using phage display technology), nucleic acids encoding the antibodies can be recovered from the library.

[0256] Specific nucleic acid molecules described herein encode the VH and VL sequences of various anti-FAM19A5 antibodies of the present disclosure. Exemplary DNA sequences encoding the VH and VL sequences of these antibodies are shown in Tables 8 and 9, respectively.

[0257] [Table 8]

[0258] JPEG2025124689000012.jpg143169

[0259] [Table 9]

[0260] JPEG2025124689000014.jpg57169

[0261] Methods for producing the anti-FAM19A5 antibodies disclosed herein include expressing the heavy and light chains together with a signal peptide in a cell line containing nucleotide sequences encoding the heavy and light chains. Host cells containing these nucleotide sequences are included herein.

[0262] Once the DNA fragments encoding the VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example, to convert the variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these manipulations, the VL- or VH-encoding DNA fragment is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operably linked" as used in this context is intended to mean that the two DNA fragments are joined so that the amino acid sequences encoded by the two DNA fragments remain in-frame.

[0263] The isolated DNA encoding the VH region can be converted into a full-length heavy chain gene by operably linking the VH-encoding DNA to a further DNA molecule encoding a heavy chain constant region (hinge, CH1, CH2, and / or CH3). The sequences of human heavy chain constant region genes are known in the art (see, for example, Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, e.g., an IgG2 and / or IgG4 constant region. In the case of a Fab fragment heavy chain gene, the VH-encoding DNA can be operably linked to a further DNA molecule encoding only the heavy chain CH1 constant region.

[0264] The isolated DNA encoding the VL region can be converted into a full-length light chain gene (as well as a Fab light chain gene) by operatively linking the VL-encoding DNA to another DNA molecule encoding a light chain constant region (CL). The sequences of human light chain constant region genes are known in the art (e.g., Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region.

[0265] To generate scFv antibodies, the VH- and VL-encoding DNA fragments can be operably linked to a flexible linker, such as another fragment encoding the amino acid sequence (Gly4-Ser)3, and the VH and VL sequences can be expressed as a contiguous single-chain protein in which the VL and VH regions are joined by the 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., (1990) Nature 348:552-554). The amino acid sequence of an exemplary anti-FAM19A5 scFv is provided in Table 7.

[0266] In some embodiments, the present disclosure provides a vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody, hi other embodiments, the vector can be used for gene therapy.

[0267] Suitable vectors for the present disclosure include expression vectors, viral vectors, and plasmid vectors. In one embodiment, the vector is a viral vector.

[0268] As used herein, an expression vector refers to any nucleic acid construct that contains the necessary elements for the transcription and translation of an inserted coding sequence, or, in the case of RNA viral vectors, the elements necessary for replication and translation upon introduction into a suitable host cell. Expression vectors can include plasmids, phagemids, viruses, and derivatives thereof.

[0269] An expression vector of the present disclosure can include a polynucleotide encoding an antibody described herein. In one embodiment, the antibody-encoding sequence is operably linked to an expression control sequence. As used herein, two nucleic acid sequences are operably linked when they are covalently linked in a manner that allows each component nucleic acid sequence to maintain its functionality. A coding sequence and a gene expression control sequence are operably linked when they are covalently linked such that expression, transcription, and / or translation of the coding sequence is under the influence or control of the gene expression control sequence. Two DNA sequences are operably linked if induction of a promoter in the 5' gene expression sequence results in transcription of the coding sequence, and the binding properties between the two DNA sequences do not (1) introduce frame-shift mutations, (2) interfere with the ability of the promoter region to direct transcription of the coding sequence, or (3) interfere with the ability of the corresponding RNA transcript to be translated into protein. Thus, a gene expression sequence is operably linked to a coding nucleic acid sequence when the gene expression sequence is capable of affecting transcription of the coding nucleic acid sequence, such that the resulting transcript is translated into the desired antibody.

[0270] Viral vectors include, but are not limited to, nucleic acid sequences from viruses such as retroviruses, e.g., Moloney murine leukemia virus, Harvey murine sarcoma virus, murine mammary tumor virus, and Rous sarcoma virus; lentiviruses; adenoviruses; adeno-associated viruses; SV40-type viruses; polyomaviruses; Epstein-Barr viruses; papillomaviruses; herpes viruses; vaccinia viruses; polioviruses; and RNA viruses such as retroviruses. Other vectors known in the art can be readily used. Certain viral vectors are based on non-cytopathic eukaryotic viruses in which nonessential genes have been replaced with a gene of interest. Non-cytopathic viruses include retroviruses whose life cycle involves reverse transcription of genomic viral RNA into DNA followed by proviral integration into host cell DNA. Retroviruses have been approved for human gene therapy trials. The most useful are replication-defective retroviruses (i.e., capable of directing the synthesis of desired proteins but unable to produce infectious particles). Such genetically modified retroviral expression vectors are generally useful for highly efficient transduction of genes in vivo. Standard protocols for producing replication-defective retroviruses (including the steps of incorporating exogenous genetic material into a plasmid, transfecting a packaging cell line with the plasmid, producing recombinant retrovirus by the packaging cell line, recovering viral particles from tissue culture medium, and infecting target cells with the viral particles) are provided in Kriegler, M., Gene Transfer and Expression, A Laboratory Manual, W.H. Freeman Co., New York (1990) and Murry, E.T., Methods in Molecular Biology, Vol. 7, Humana Press, Inc., Cliffton, NJ (1991).

[0271] In one embodiment, the virus is an adeno-associated virus, a double-stranded DNA virus. Adeno-associated viruses can be engineered to be replication-defective and can infect a wide range of cell types and species. They also have the advantages of heat and lipid solvent stability; high transduction frequencies in cells of various lineages, including hematopoietic cells; and a lack of superinfection inhibition, allowing for a diverse range of transduction sequences. Reportedly, adeno-associated viruses can integrate into human cellular DNA in a site-specific manner, minimizing the potential for insertional mutagenesis and the variability of inserted gene expression profiles following retroviral infection. Furthermore, wild-type adeno-associated virus infections have been tracked in tissue culture for over 100 passages in the absence of selective pressure, indicating that adeno-associated virus genome integration is a relatively stable response. Adeno-associated viruses may also act in an extrachromosomal manner.

[0272] In other embodiments, the vector is derived from a lentivirus. In certain embodiments, the vector is a recombinant lentiviral vector capable of infecting non-dividing cells.

[0273] Lentiviral genomes and proviral DNA generally contain three genes, gag, pol, and env, flanked by two long terminal repeat (LTR) sequences found in retroviruses. The gag gene encodes internal structural (matrix, capsid, and nucleocapsid) proteins; the pol gene encodes RNA-directed DNA polymerase (reverse transcriptase), protease, and integrase; and the env gene encodes viral envelope glycoproteins. The 5' and 3' LTRs facilitate transcription and polyadenylation of virion RNA. The LTRs contain all other cis-acting sequences required for viral replication. Lentiviruses possess additional genes, including vif, vpr, tat, rev, vpu, nef, and vpx (in HIV-1, HIV-2, and / or SIV).

[0274] The 5'LTR is flanked by sequences required for reverse transcription of the genome (tRNA primer binding site) and efficient encapsulation of viral RNA into particles (Psi site). If the sequences required for encapsulation (or packaging of retroviral RNA into infectious virions) are missing from the viral genome, a cis defect prevents encapsulation of genomic RNA.

[0275] However, the resulting mutant remains capable of directing the synthesis of all virion proteins. The present disclosure provides a method for producing recombinant lentivirus capable of infecting non-dividing cells, which involves transfecting suitable host cells with two or more vectors that retain packaging functions, i.e., gag, pol, and env, as well as rev and tat. As disclosed below, vectors lacking a functional tat gene are preferred for certain applications. Thus, for example, one vector can provide nucleic acid encoding viral gag and viral pol, while another vector can provide nucleic acid encoding viral env for producing packaging cells. Introducing a vector delivering a heterologous gene, identified herein as a transfer vector, into packaging cells results in producer cells that release infectious viral particles carrying the foreign gene of interest.

[0276] According to the vector and foreign gene configurations described above, the second vector can provide nucleic acid encoding a viral envelope (env) gene. The env gene can be derived from almost any suitable virus, including retroviruses. In some embodiments, the env protein is an amphoteric envelope protein, allowing for transduction of cells of both human and other species.

[0277] Examples of env genes from retroviruses include, but are not limited to, Moloney Murine Leukemia Virus (MoMuLV or MMLV), Harvey Murine Sarcoma Virus (HaMuSV or HSV), Murine Mammary Tumor Virus (MuMTV or MMTV), Gibbon Ape Leukemia Virus, Human Immunodeficiency Virus (HIV), and Rous Sarcoma Virus. Other env genes, such as vesicular stomatitis virus (VSV) protein G (VSV G), hepatitis virus, and influenza genes, can also be used.

[0278] The vector providing the viral env nucleic acid sequence is operably linked to the regulatory sequences described elsewhere herein.

[0279] In certain embodiments, the vector comprises a lentiviral vector in which the HIV virulence genes env, vif, vpr, vpu, and nef have been deleted without impairing the ability of the vector to transduce non-dividing cells.

[0280] In some embodiments, the vector comprises a lentiviral vector comprising a deletion of the U3 region of the 3'LTR, which may be a complete or partial deletion of the U3 region.

[0281] In some embodiments, a lentiviral vector of the present disclosure comprising a FVIII nucleotide sequence described herein may be transfected with (a) a first nucleotide sequence comprising the gag, pol, or gag and pol genes, and (b) a second nucleotide sequence comprising a heterologous env gene; wherein the lentiviral vector lacks a functional tat gene. In other embodiments, the cells are further transfected with a fourth nucleotide sequence comprising the rev gene. In certain embodiments, the lentiviral vector lacks a functional gene selected from vif, vpr, vpu, vpx, and nef, or a combination thereof.

[0282] In certain embodiments, the lentiviral vector comprises one or more nucleotide sequences encoding a gag protein, a Rev response element, a central polypurine track (cPPT), or any combination thereof.

[0283] Examples of lentiviral vectors are disclosed in WO9931251, WO9712622, WO9817815, WO9817816 and WO9818934, which are incorporated by reference in their entirety.

[0284] Other vectors include plasmid vectors. Plasmid vectors have been described extensively in the art and are well known to those skilled in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. Over the past few years, plasmid vectors have emerged as particularly advantageous for transferring genes to cells in vivo because they cannot replicate or integrate into the host genome. However, these plasmids, which contain a promoter compatible with the host cell, are capable of expressing peptides from genes operably encoded within the plasmid. Some commonly used plasmids available from commercial suppliers include pBR322, pLTCl8, pLTCl9, various pcDNA plasmids, pRC / CMV, various pCMV plasmids, pSV40, and pBlueScript. Additional examples of specific plasmids are pcDNA3.1, catalog number V79020; pcDNA3.1 / hygro, catalog number V87020; pcDNA4 / myc-His, catalog number V86320; and pBudCE4.1, catalog number V53220 (all from Invitrogen, Carlsbad, CA). Other plasmids are well known to those of skill in the art. Additionally, plasmids can be customized using standard molecular biology techniques to remove and / or add specific segments of DNA.

[0285] [VI. Antibody Production]Antibodies or fragments thereof that immunospecifically bind to FAM19A5 (e.g., human FAM19A5) can be produced by any method known in the art for the synthesis of antibodies, such as chemical synthesis or recombinant expression techniques. The methods disclosed herein utilize, unless otherwise indicated, conventional techniques in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and other relevant technical fields. These techniques are fully described, for example, in the references incorporated herein.For example, refer to Maniatis T et al., (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Sambrook J et al., (1989), Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press; Sambrook J et al., (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel FM et al., Current Protocols in Molecular Biology, John Wiley & Sons (1987 and annual updates); Current Protocols in Immunology, John Wiley & Sons (1987 and annual updates) Gait (ed.) (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein (ed.) (1991) Oligonucleotides and Analogues: A Practical Approach, IRL Press; Birren B et al., (eds.) (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press.

[0286] In certain embodiments, the antibodies described herein are antibodies (e.g., recombinant antibodies) that are manufactured, expressed, produced, or isolated by any means involving, for example, synthesis, production using genetic engineering of DNA sequences. In certain embodiments, such antibodies comprise sequences (e.g., DNA sequences or amino acid sequences) that do not naturally occur within the antibody germline repertoire of an animal or mammal (e.g., a human) in vivo. In some embodiments, the anti-FAM19A5 antibodies disclosed herein are deimmunized.

[0287] As described in the Examples section (e.g., Example 2), the anti-FAM19A5 antibodies were first generated by immunizing chickens with a synthetic FAM19A5 peptide. Therefore, to minimize the risk of immunogenicity when administered to human subjects, the anti-FAM19A5 antibodies (e.g., 3-2 and 2-13) were modified to more closely resemble the immunogenic sequence of a human antibody. In some embodiments, the deimmunized anti-FAM19A5 antibodies disclosed herein have similar binding affinity to human FAM19A5 compared to their non-deimmunized counterparts. In some embodiments, the anti-FAM19A5 antibodies disclosed herein also underwent affinity maturation. Methods for deimmunizing antibodies are disclosed herein and known in the art.

[0288] In certain embodiments, the description provides a method for producing an antibody or antigen-binding fragment thereof that immunospecifically binds to FAM19A5 (e.g., human FAM19A5), comprising culturing a cell or host cell described herein. In certain embodiments, the description provides a method for producing an antibody or antigen-binding fragment thereof that immunospecifically binds to FAM19A5 (e.g., human FAM19A5), comprising expressing (e.g., recombinantly expressing) the antibody or antigen-binding fragment thereof using a cell or host cell described herein (e.g., a cell or host cell comprising a polynucleotide encoding an antibody described herein). In certain embodiments, the cell is an isolated cell. In certain embodiments, an exogenous polynucleotide has been introduced into the cell. In certain embodiments, the method further comprises purifying the antibody or antigen-binding fragment thereof from the cell or host cell.

[0289] Methods for producing polyclonal antibodies are known in the art (see, for example, Chapter 11 in: Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel FM et al., eds., John Wiley and Sons, New York).

[0290] Monoclonal antibodies can be produced using a variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma technology, including those techniques known in the art and taught, for example, in Harlow E & Lane D, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling GJ et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563681 (Elsevier, NY, 1981). As used herein, the term "monoclonal antibody" is not limited to antibodies produced by hybridoma technology. For example, monoclonal antibodies can be recombinantly produced from host cells that exogenously express the antibodies or fragments thereof described herein, e.g., the light and / or heavy chains of those antibodies.

[0291] In certain embodiments, a "monoclonal antibody," as used herein, refers to an antibody produced by a single cell (e.g., a recombinant antibody-producing hybridoma or host cell), which immunospecifically binds to FAM19A5 (e.g., human FAM19A5), as determined, for example, by ELISA or other antigen-binding or competitive binding assays known in the art or described in the Examples section provided herein. In certain embodiments, the monoclonal antibody may be a chimeric or humanized antibody. In certain embodiments, the monoclonal antibody is a monovalent or polyvalent (e.g., bivalent) antibody. In certain embodiments, the monoclonal antibody is a monospecific or multispecific antibody (e.g., bispecific antibody). The monoclonal antibodies described herein may be made by hybridoma methods, e.g., as described in Kohler G & Milstein C (1975) Nature 256:495, or may be isolated from phage libraries, e.g., using the techniques described herein. Other methods for producing clonal cell lines and the monoclonal antibodies expressed thereby are well known in the art (see, e.g., Chapter 11 in: Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel FM et al., supra).

[0292] Methods for producing and screening for specific antibodies using hybridoma technology are conventional and well known in the art. For example, in the hybridoma method, a mouse or other suitable host animal, such as a sheep, goat, rabbit, rat, hamster, or macaque, is immunized to induce lymphocytes that produce or are capable of producing antibodies that specifically bind to the protein used for immunization (e.g., human FAM19A5). Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells (Goding JW (Ed.), Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Additionally, animals can be immunized using RIMMS (repeated immunization multiple site) technology (Kilpatrick KE et al., (1997) Hybridoma 16:381-9, incorporated by reference in its entirety).

[0293] In some embodiments, a mouse (or other animal, such as a chicken, rat, monkey, donkey, pig, sheep, hamster, or dog) can be immunized with an antigen (e.g., FAM19A5, such as human FAM19A5), and upon detection of an immune response, e.g., upon detection of antibodies specific to the antigen in the mouse serum, the mouse spleen is harvested and splenocytes are isolated. The splenocytes are then fused to any suitable myeloma cells, e.g., cells of cell line SP20 available from the American Type Culture Collection (ATCC®) (Manassas, VA), by well-known techniques to form hybridomas. The hybridomas are selected and cloned by limiting dilution. In certain embodiments, lymph nodes from the immunized mouse are harvested and fused with NSO myeloma cells.

[0294] The hybridoma cells thus produced are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the hybridoma culture medium will typically contain hypoxanthine, aminopterin, and thymidine (HAT medium), substances that prevent the growth of HGPRT-deficient cells.

[0295] A specific embodiment uses myeloma cells that fuse efficiently, are conducive to stable, high-level production of antibody by selected antibody-producing cells, and are sensitive to a medium such as HAT medium. These myeloma cell lines include the NSO cell line, or murine myeloma cell lines such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, CA, USA, and SP-2 or X63-Ag8.653 cells available from the American Type Culture Collection, Rockville, MD, USA. Human myeloma and mouse-human heteromyeloma cell lines have also been described for human monoclonal antibody production (Kozbor D (1984) J Immunol 133:3001-5; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

[0296] The culture medium in which the hybridoma cells grow is analyzed for production of monoclonal antibodies directed against FAM19A5 (e.g., human FAM19A5). The binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by methods known in the art, such as immunoprecipitation or in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0297] After hybridoma cells producing antibodies of the desired specificity, affinity, and / or activity are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods (see Goding JW (Ed.), Monoclonal Antibodies: Principles and Practice, supra). Suitable culture media for such purposes include, for example, D-MEM or RPMI 1640 medium. Hybridoma cells can also be grown in vivo as multiple tumors in animals.

[0298] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0299] The antibodies described herein recognize a specific FAM19A5 (e.g., human FAM19A5) and include antibody fragments that can be produced by any technique known to those skilled in the art. For example, the Fab and F(ab')2 fragments described herein can be produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments). The Fab fragment corresponds to one of the two identical arms of an antibody molecule and contains an intact light chain paired with the VH and CH1 domains of the heavy chain. The F(ab')2 fragment contains the two antigen-binding arms of an antibody molecule linked by a disulfide bond at the hinge region.

[0300] The antibodies or antigen-binding fragments thereof described herein may also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles that carry the polynucleotide sequences encoding them. In particular, DNA sequences encoding VH and VL domains are amplified from animal cDNA libraries (e.g., human cDNA libraries or non-human cDNA libraries, such as murine or chicken cDNA libraries of infected tissues). The DNA encoding the VH and VL domains is recombined with an scFv linker by PCR and cloned into a phagemid vector. The vector is electroporated in E. coli, and the E. coli is infected with helper phage. The phages used in these methods are typically filamentous phages, including fd and M13, and the VH and VL domains are generally recombinantly fused to phage gene III or gene VIII. Phage expressing an antigen binding domain that binds to a particular antigen can be selected or identified using antigen, eg, labeled antigen or antigen bound or captured to a solid surface or bead.Examples of phage display methods that can be used to produce the antibodies described herein are described in Brinkman U et al., (1995) J Immunol Methods 182:41-50; Ames RS et al., (1995) J Immunol Methods 184:177-186; Kettleborough CA et al., (1994) Eur J Immunol 24:952-958; Persic L et al., (1997) Gene 187:9-18; Burton DR & Barbas CF (1994) Advan Immunol 57:191-280; PCT Application No. PCT / GB91 / 001134; International Publication Nos. WO90 / 02809, WO91 / 10737, WO92 / 01047, WO92 / 18619, WO93 / 11236, WO95 / 15982, WO95 / 20401, and WO97 / 13844; U.S. Patent Nos. 5,698,426 and 5,223,409 , 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743 and 5,969,108.

[0301] As described in the above references, after phage selection, the antibody-coding region of the phage can be isolated and used to generate whole antibodies or any other desired antigen-binding fragment, including human antibodies, and can be expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, as described below. Techniques for recombinantly producing antibody fragments, such as Fab, Fab', and F(ab')2 fragments, can also be employed using methods known in the art, as disclosed in PCT Publication No. WO 92 / 22324; Mullinax R L et al., (1992) BioTechniques 12(6):864-9; Sawai H et al., (1995) Am J Reprod Immunol 34:26-34; and Better M et al., (1988) Science 240:1041-1043.

[0302] In one embodiment, to generate a whole antibody, PCR primers containing the VH or VL nucleotide sequence, restriction sites, and flanking sequences to protect the restriction sites can be used to amplify the VH or VL sequence from a template, e.g., an scFv clone. Using cloning techniques known to those skilled in the art, the PCR-amplified VH domain can be cloned into a vector expressing a VH constant region, and the PCR-amplified VL domain can be cloned into a vector expressing a VL constant region, e.g., a human kappa or lambda constant region. The VH and VL domains can be cloned into a single vector expressing the necessary constant regions. The heavy chain conversion vector and light chain conversion vector can then be co-transfected into a cell line using techniques known to those skilled in the art to generate a stable or transient cell line expressing a full-length antibody, e.g., an IgG.

[0303] A chimeric antibody is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules. For example, a chimeric antibody can contain the variable region of a non-human animal (e.g., mouse, rat, or chicken) monoclonal antibody fused to the constant region of a human antibody. Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison SL (1985) Science 229:1202-7; Oi VT & Morrison SL (1986) BioTechniques 4:214-221; Gillies SD et al., (1989) J Immunol Methods 125:191-202; and U.S. Patent Nos. 5,807,715, 4,816,567, 4,816,397, and 6,331,415.

[0304] A humanized antibody can bind to a predetermined antigen and comprises a framework region having substantially the amino acid sequence of a human immunoglobulin and CDRs having substantially the amino acid sequence of a non-human immunoglobulin (e.g., a murine or chicken immunoglobulin). In certain embodiments, the humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. The antibody may also comprise the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. The humanized antibody may be selected from any type of immunoglobulin, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including IgG1, IgG2, IgG3, and IgG4.Humanized antibodies can be produced using a variety of techniques known in the art, including, but not limited to, CDR-grafting (European Patent No. EP 239400; International Publication No. WO 91 / 09967; and U.S. Patent Nos. 5,225,539, 5,530,101, and 5,585,089), veneering, or resurfacing (European Patent Nos. EP 592106 and EP 519596; Padlan EA (1991) Mol Immunol 28(4 / 5):489-498; Studnicka GM et al., (1994) Prot Engineering 7(6):805-814; and Roguska MA et al., (1994) PNAS 91:969-973), chain shuffling (U.S. Patent No. 5,565,332) and, for example, U.S. Patent No. 6,407,213, U.S. Patent No. 5,766,886, International Publication No. WO93 / 17105; Tan P et al., (2002) J Immunol 169:1119-25; Caldas C et al., (2000) Protein Eng. 13(5):353-60; Morea V et al., (2000) Methods 20(3):267-79; Baca M et al., (1997) J Biol Chem 272(16):10678-84; Roguska MA et al., (1996) Protein Eng 9(10):895-904; Couto JR et al. al., (1995) Cancer Res. 55(23Supp):5973s-5977s; Couto JR et al., (1995) Cancer Res 55(8):1717-22; Sandhu JS (1994) Gene 150(2):409-10 and Pedersen JT et al., (1994) J Mol Biol 235(3):959-73. See also U.S. Application Publication No. US2005 / 0042664A1 (February 24, 2005).

[0305] Methods for producing multispecific (e.g., bispecific) antibodies have been described (see, e.g., U.S. Patent Nos. 7,951,917; 7,183,076; 8,227,577; 5,837,242; 5,989,830; 5,869,620; 6,132,992; and 8,586,713).

[0306] Single domain antibodies, e.g., antibodies lacking light chains, can be produced by methods well known in the art (see Riechmann L & Muyldermans S (1999) J Immunol 231:25-38; Nuttall SD et al., (2000) Curr Pharm Biotechnol 1(3):253-263; Muyldermans S, (2001) J Biotechnol 74(4):277-302; U.S. Patent No. 6,005,079; and International Publication Nos. WO94 / 04678, WO94 / 25591, and WO01 / 44301).

[0307] Antibodies that immunospecifically bind to the FAM19A5 antigen can also be used to generate anti-genotypic antibodies that "mimicking" the antigen, using techniques well known to those skilled in the art (see, e.g., Greenspan NS & Bona CA (1989) FASEB J 7(5):437-444; and Nissinoff A (1991) J Immunol 147(8):2429-2438).

[0308] In certain embodiments, an antibody described herein that binds to the same epitope of FAM19A5 (e.g., human FAM19A5) as an anti-FAM19A5 antibody described herein is a human antibody or antigen-binding fragment thereof. In certain embodiments, an antibody described herein that competitively blocks (e.g., in a dose-dependent manner) the binding of an antibody described herein to FAM19A5 (e.g., human FAM19A5) is a human antibody or antigen-binding fragment thereof.

[0309] Human antibodies can be produced using any method known in the art. For example, transgenic mice that are unable to express functional endogenous immunoglobulins but can express human immunoglobulin genes can be used. In particular, human heavy and light chain immunoglobulin gene complexes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, in addition to the human heavy and light chain genes, human variable regions, constant regions, and diversity regions can be introduced into mouse embryonic stem cells. The mouse heavy and light chain immunoglobulin genes can be rendered non-functional by homologous recombination, either separately or simultaneously with the introduction of human immunoglobulin loci. In particular, homozygous deletion of the JH region renders endogenous antibody production impossible. The modified embryonic stem cells are expanded and microinjected into blastocysts to generate chimeric mice. The chimeric mice are then bred to generate homozygous offspring that express human antibodies. The transgenic mice are immunized in the normal manner with a selected antigen, such as all or part of an antigen (e.g., FAM19A5). Monoclonal antibodies directed against the antigen can be obtained from the immunized transgenic mice using conventional hybridoma technology. The human immunoglobulin transfer genes harbored by the transgenic mice rearrange during B cell differentiation and subsequently undergo class switching and somatic mutation. This technology therefore allows for the production of therapeutically useful IgG, IgA, IgM, and IgE antibodies. For an overview of this technology for human antibody production, see Lonberg N & Huszar D (1995) Int Rev Immunol 13:65-93. For a detailed discussion of the technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., International Publication Nos. WO 98 / 24893, WO 96 / 34096, and WO 96 / 33735; and U.S. Patent Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, and 5,939,598. An example of a mouse capable of producing human antibodies is the XENOMOUSE.TM (Abgenix, Inc.; U.S. Patent Nos. 6,075,181 and 6,150,184), HUAB-MOUSE TM (Mederex, Inc. / Gen Pharm; U.S. Patent Nos. 5,545,806 and 5,569,825), TRANS CHROMO MOUSE TM (Kirin) and KM MOUSE TM (Medarex / Kirin).

[0310] Human antibodies that specifically bind to FAM19A5 (e.g., human FAM19A5) can be produced by various methods known in the art, including the phage display methods described above, using antibody libraries derived from human immunoglobulin sequences (see also U.S. Patent Nos. 4,444,887, 4,716,111, and 5,885,793; and International Publication Nos. WO98 / 46645, WO98 / 50433, WO98 / 24893, WO98 / 16654, WO96 / 34096, WO96 / 33735, and WO91 / 10741).

[0311] In some embodiments, human antibodies can be produced using mouse-human hybridomas. For example, human peripheral blood lymphocytes transformed with Epstein-Barr virus (EBV) can be fused with mouse myeloma cells to produce mouse-human hybridomas that secrete human monoclonal antibodies, and these mouse-human hybridomas can be screened to determine those that secrete human monoclonal antibodies that immunospecifically bind to a target antigen (e.g., FAM19A5, such as human FAM19A5). These methods are known and described in the art (see, e.g., Shinmoto H et al., (2004) Cytotechnology 46:19-23; Naganawa Y et al., (2005) Human Antibodies 14:27-31).

[0312] V. Methods for Engineering Antibodies As discussed above, anti-FAM19A5 antibodies having the VH and VL sequences disclosed herein can be used to generate new anti-FAM19A5 antibodies by modifying the VH and / or VL sequences or the constant regions attached thereto. Thus, in yet another embodiment described herein, the structural features of the anti-FAM19A5 antibodies described herein can be used to generate structurally related anti-FAM19A5 antibodies that retain at least one functional property of the antibodies described herein, such as binding to human FAM19A5. For example, the starting material for the engineering method is a VH and / or VL sequence provided herein or one or more CDR regions thereof. To generate the engineered antibody, it is not necessary to actually produce (i.e., express as a protein) an antibody having one or more VH and / or VL sequences or one or more CDR regions thereof provided herein. Instead, the information contained in the sequence can be used as a starting material to generate "second-generation" sequences derived from the original sequence, and then the "second-generation" sequences can be produced and expressed as proteins.

[0313] Therefore, in this specification, (a) providing (i) a heavy chain variable region sequence comprising the CDR1, CDR2 and / or CDR3 sequence set forth in Table 3, or the CDR1, CDR2 and / or CDR3 of a heavy chain variable region set forth in Table 5; and (ii) a light chain variable region sequence comprising the CDR1, CDR2 and / or CDR3 sequence set forth in Table 4, or the CDR1, CDR2 and / or CDR3 of a heavy chain variable region set forth in Table 6; (b) modifying at least one amino acid residue within the heavy chain variable region sequence and / or the light chain variable region sequence to generate at least one modified antibody sequence; (c) A method for producing an anti-FAM19A5 antibody is provided, which comprises expressing the modified antibody sequence as a protein.

[0314] The altered antibody sequence can be prepared and expressed using standard molecular biology techniques.

[0315] In some embodiments, the antibody encoded by the modified antibody sequence retains one, some, or all of the functional properties of the anti-FAM19A5 antibodies described herein, including: (1) reduced immunogenicity in human subjects; (2) For example, when measured by Biacore, K D binds to soluble human FAM19A5 at a binding affinity of 10 nM or less (e.g., 0.01 nM to 10 nM); (3) For example, when measured by ELISA, K D binds to membrane-bound human FAM19A5 at a binding affinity of 10 nM or less (e.g., 0.01 nM to 1 nM); (4) binds to membrane-bound human FAM19A5 with an EC50 of 1 nM or less (e.g., 0.01 nM to 1 nM), e.g., as measured by ELISA; (5) reducing, reversing, delaying, and / or preventing the onset of reactive gliosis; (6) suppression of reactive astrocyte hyperproliferation; (7) decreased expression of chondroitin sulfate proteoglycans, including neurocan and neuroglial antigen 2 (NG2); (8) increased expression of c-fos and pERK in neuronal nuclei; (9) Promoting neuronal survival; (10) Increased expression of GAP43 in neurons; (11) promoting axonal regrowth; and (12) competes with the anti-FAM19A5 antibodies disclosed herein in either one or both directions for binding to human FAM19A5.

[0316] The modified antibodies may exhibit one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or all of the functional properties set forth above in (1) to (12). The functional properties of the modified antibodies can be assessed using standard assays available in the art and / or described herein (e.g., ELISA, FACS), as shown in the Examples section.

[0317] In certain embodiments of the antibody engineering methods described herein, mutations can be introduced randomly or selectively along all or part of an anti-FAM19A5 antibody coding sequence, and the resulting modified anti-FAM19A5 antibodies can be screened for binding activity and / or other functional properties as described herein. Mutational methods are described in the art. For example, PCT Publication WO 02 / 092780 to Short describes methods for generating and screening antibody mutations using saturation mutagenesis, synthetic ligation assembly, or a combination thereof. In contrast, PCT Publication WO 03 / 074679 to Lazar et al. describes methods that utilize computational screening methods to optimize the physicochemical properties of antibodies.

[0318] VI. Cells and Vectors In certain aspects, the description provides cells (e.g., host cells) that express (e.g., recombinantly) antibodies (or antigen-binding fragments thereof) described herein that specifically bind to FAM19A5 (e.g., human FAM19A5) and related polynucleotides and expression vectors. The description provides vectors (e.g., expression vectors) comprising a polynucleotide that includes a nucleotide sequence encoding an anti-FAM19A5 antibody or fragment for recombinant expression in a host cell, e.g., a mammalian cell. The description also provides host cells comprising such vectors for recombinantly expressing anti-FAM19A5 antibodies (e.g., human or humanized antibodies). In certain aspects, the description provides methods for producing the antibodies described herein, comprising expressing the antibodies from a host cell.

[0319] Recombination of an antibody described herein (e.g., a full-length antibody, antibody heavy and / or light chain, or single-chain antibody described herein) that specifically binds to FAM19A5 (e.g., human FAM19A5) involves the creation of an expression vector containing a polynucleotide encoding the antibody. Once a polynucleotide encoding an antibody molecule described herein, antibody heavy and / or light chain, or fragment thereof (e.g., heavy and / or light chain variable domain) is obtained, vectors for the production of the antibody molecule can be produced by recombinant DNA technology using techniques well known in the art. Accordingly, methods for producing proteins by expressing polynucleotides containing nucleotide sequences encoding antibodies or antibody fragments (e.g., light or heavy chains) are described herein. Methods well known to those skilled in the art can be used to construct expression vectors containing antibody or antibody fragment (e.g., light or heavy chain) coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Also provided are replicable vectors containing nucleotide sequences encoding the antibody molecules described herein, the antibody heavy or light chains, the antibody heavy or light chain variable domains or fragments thereof, or the heavy or light chain CDRs operably linked to a promoter. Such vectors can include, for example, nucleotide sequences encoding the constant regions of the antibody molecules (see, e.g., International Publication Nos. WO 86 / 05807 and WO 89 / 01036; and U.S. Patent No. 5,122,464), and the antibody variable domains can be cloned into the vector for expression of the entire heavy chain, the entire light chain, or both the entire heavy and light chains.

[0320] The expression vector can be transferred to a cell (e.g., a host cell) by conventional techniques, and the resulting cells can then be cultured by conventional techniques to produce an antibody described herein (e.g., an antibody comprising any one or more of the VH and / or VL, or VH and / or VL CDRs, of an anti-FAM19A5 antibody of the present disclosure) or fragment thereof. Accordingly, the present disclosure provides a host cell containing a polynucleotide encoding an antibody or fragment thereof described herein, or a heavy or light chain thereof, or a fragment thereof, or a single-chain antibody described herein, operably linked to a promoter for expression of the sequence in the host cell. In certain embodiments, vectors encoding both the heavy and light chains separately for expression of a double-chain antibody can be coexpressed in a host cell for expression of a whole immunoglobulin molecule, as described in detail below. In certain embodiments, the host cell contains a vector comprising a polynucleotide encoding both the heavy and light chains, or fragments thereof, of an antibody described herein. In certain embodiments, the host cell contains two different vectors: a first vector containing a polynucleotide encoding the heavy chain or heavy chain variable region of an antibody described herein or a fragment thereof, and a second vector containing a polynucleotide encoding the light chain or light chain variable region of an antibody described herein or a fragment thereof. In other embodiments, a first host cell contains a first vector containing a polynucleotide encoding the heavy chain or heavy chain variable region of an antibody described herein or a fragment thereof, and a second host cell contains a second vector containing a polynucleotide encoding the light chain or light chain variable region of an antibody described herein. In certain embodiments, the heavy chain / heavy chain variable region expressed by the first cell associates with the light chain / light chain variable region of the second cell to form an anti-FAM19A5 antibody or antigen-binding fragment thereof described herein. In certain embodiments, the present specification provides a population of host cells comprising the first host cell and the second host cell.

[0321] In certain embodiments, the present specification provides a vector collection comprising a first vector comprising a polynucleotide encoding the light chain / light chain variable region of an anti-FAM19A5 antibody described herein and a second vector comprising a polynucleotide encoding the heavy chain / heavy chain variable region of an anti-FAM19A5 antibody described herein.

[0322] A variety of host-expression vector systems can be used to express the antibody molecules described herein. Such host-expression systems represent vehicles in which coding sequences of interest can be produced and subsequently purified, as well as cells that, when transformed or transfected with the appropriate nucleotide coding sequences, are capable of expressing the antibody molecules described herein in situ. These include microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing antibody coding sequences; and plant cell systems (e.g., Chlamydomonas sp.) infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences. reinhardtii); or mammalian cell systems (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK293, NSO, PER.C6, VERO, CRL7030, HsS78Bst, HeLa, and NIH3T3, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, and BMT10 cells) harboring a recombinant expression construct containing a promoter derived from the genome of a mammalian cell (e.g., a metallothionein promoter) or a promoter derived from a mammalian virus (e.g., an adenovirus late promoter; a cowpox virus 7.5K promoter). In certain embodiments, cells for expressing the antibodies or antigen-binding fragments thereof described herein are CHO cells, e.g., CHO GS SYSTEM TM(Lonza) CHO cells. In certain embodiments, cells for expressing the antibodies described herein are human cells, e.g., human cell lines. In certain embodiments, the mammalian expression vector is POPTIVEC™ or pcDNA3.3. In certain embodiments, bacterial cells such as Escherichia coli or eukaryotic cells (e.g., mammalian cells), particularly for the expression of whole recombinant antibody molecules, are used to express recombinant antibody molecules. For example, mammalian cells such as Chinese hamster ovary (CHO) cells are effective expression systems for antibodies in conjunction with vectors such as the human cytomegalovirus major intermediate-early gene promoter element (Foecking MK & Hofstetter H (1986) Gene 45:101-5; and Cockett MI et al., (1990) Biotechnology 8(7):662-7). In certain embodiments, the antibodies described herein are produced in CHO cells or NSO cells. In certain embodiments, expression of nucleotide sequences encoding antibodies described herein that immunospecifically bind to FAM19A5 (e.g., human FAM19A5) is regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

[0323] In bacterial systems, numerous expression vectors may be advantageously selected depending on the intended use of the antibody molecule being expressed. For example, when producing large quantities of the antibody for preparation of a pharmaceutical composition of the antibody molecule, vectors that direct the expression of high levels of a fusion protein product that is easily purified may be preferred. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruether U & Mueller-Hill B (1983) EMBO J 2:1791-1794), in which the antibody-coding sequence is individually ligated into the vector with the lac Z coding region to produce the fusion protein; pIN vector (Inouye S & Inouye M (1985) Nuc Acids Res 13:3101-3109; Van Heeke G & Schuster SM (1989) J Biol Chem 24:5503-5509); and the like. For example, pGEX vectors may be used to express foreign polypeptides as fusion proteins with glutathione 5-transferase (GST). Generally, the fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to matrix glutathione agarose beads followed by elution in the presence of free glutathione. The pGEX vectors may be engineered to contain thrombin or factor Xa protease cleavage sites, allowing the cloned target gene product to be released from the GST moiety.

[0324] In an insect system, for example, Autographa californica nuclear polyhedron virus (AcNPV) can be used as a vector to express foreign genes. The virus is grown in Spodoptera frugiperda cells. The antibody coding sequence can be cloned individually into non-essential regions (e.g., the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (e.g., the polyhedrin promoter).

[0325] Numerous viral expression systems can be used in mammalian host cells. When adenovirus is used as an expression vector, the antibody coding sequence of interest can be ligated to an adenovirus transcription / translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (e.g., region E1 or E3) will generate recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts (see, e.g., Logan J & Shenk T (1984) PNAS 81(12):3655-9). Specific initiation signals may also be required for efficient translation of the inserted antibody coding sequence. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to allow translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see, eg, Bitter G et al., (1987) Methods Enzymol. 153:516-544).

[0326] Additionally, a host cell strain can be selected that modulates the expression of the inserted sequence or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for the function of the protein. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. An appropriate cell line or host system can be selected to ensure the correct modification and processing of the expressed foreign protein. To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, Hela, MDCK, HEK293, NIH3T3, W138, BT483, Hs578T, HTB2, BT20, and T47D, NSO (a murine myeloma cell line that does not produce any immunoglobulin chains in vivo), CRL7030, COS (e.g., COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, BMT10, and HsS78Bst cells. In certain embodiments, the anti-FAM19A5 antibodies described herein are produced in mammalian cells, such as CHO cells.

[0327] In certain embodiments, the antibodies or antigen-binding portions thereof described herein have reduced or no fucose content. Such antibodies can be produced using techniques known to those of skill in the art. For example, the antibodies can be expressed in cells that have insufficient or no fucosylation capacity. In certain embodiments, a cell line in which two alleles of 1,6-fucosyltransferase have been knocked out can be used to produce antibodies or antigen-binding portions thereof with reduced fucose content. The POTELLIGENT® system (Lonza) is an example of such a system that can be used to produce antibodies or antigen-binding portions thereof with reduced fucose content.

[0328] Stable expression cells can be engineered for long-term, high-yield production of recombinant proteins. For example, cell lines can be engineered that stably express the anti-FAM19A5 antibodies or antigen-binding portions thereof described herein. In certain embodiments, the cells provided herein stably express the light chain / light chain variable domains and heavy chain / heavy chain variable domains that assemble to form the antibodies or antigen-binding portions thereof described herein.

[0329] In certain embodiments, instead of using expression vectors containing viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequence, transcription terminator, polyadenylation site, etc.) and a selectable marker. After introduction of the foreign DNA / polynucleotide, engineered cells can be grown in an enriched medium for 1-2 days and then switched to a selective medium. The selectable marker in the recombinant plasmid confers resistance to selection, forming foci that can be cloned and expanded into cell lines by allowing cells to stably integrate the plasmid into their chromosomes and grow. This method can be advantageously used to engineer cell lines that express the anti-FAM19A5 antibodies or antibody-binding portions thereof described herein. Such engineered cell lines may be particularly useful for screening and evaluating compositions that interact directly or indirectly with antibody molecules.

[0330] A number of selection systems can be used, including, but not limited to, the herpes simplex virus thymidine kinase (Wigler M et al., (1977) Cell 11(1):223-32), hypoxanthine guanine phosphoribosyltransferase (Szybalska EH & Szybalski W (1962) PNAS 48(12):2026-2034), and adenine phosphoribosyltransferase (Lowy I et al., (1980) Cell 22(3):817-23) genes, which can be used in tk-, hgprt-, or aprt- cells, respectively. Additionally, antimetabolite resistance can be used as the basis for selection against genes such as dhfr, which confers methotrexate resistance (Wigler M et al., (1980) PNAS 77(6):3567-70; O'Hare K et al., (1981) PNAS 78:1527-31); gpt, which confers mycophenolic acid resistance (Mulligan RC & Berg P (1981) PNAS 78(4):2072-6); neo, which confers resistance to the aminoglycoside G-418 (Wu GY & Wu CH (1991) Biotherapy 3:87-95; Tolstoshev P (1993) Ann Rev Pharmacol Toxicol 32:573-596; Mulligan RC (1993) Science 260:926-932; and Morgan RA & Anderson WF (1993) Ann Rev Biochem 62:191-217; Nabel GJ & Feigner PL (1993) Trends Biotechnol 11(5):211-5); and hygro, which confers hygromycin resistance (Santerre RF et al., (1984) Gene 30(1-3):147-56).Conventional methods known in the art of recombinant DNA technology may generally be applied to select the desired recombinant clone, and such methods are described, for example, in Ausubel FM et al., (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler M, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and Chapters 12 and 13, Dracopoli NC et al., (eds.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colbere-Garapin F et al., (1981) J Mol Biol 150:1-14, the entire contents of which are incorporated herein by reference.

[0331] The expression level of an antibody molecule can be increased by vector amplification (for a review, see Bebbington CR & Hentschel CCG, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, New York, 1987)). When a marker in an antibody expression vector system is amplifiable, increasing the level of inhibitor present in the host cell culture will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, antibody production will also increase (Crouse GF et al., (1983) Mol Cell Biol 3:257-66).

[0332] The host cells can be co-transfected with two or more expression vectors described herein, a first vector encoding a heavy chain-derived polypeptide and a second vector encoding a light chain-derived polypeptide. The two vectors can contain the same selectable marker that allows for identical expression of the heavy and light chain polypeptides. The host cells can be co-transfected with different amounts of the two or more expression vectors. For example, the host cells can be transfected with any one of the following ratios of the first expression vector to the second expression vector: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:12, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50.

[0333] Alternatively, a single vector can be used that can encode and express both heavy and light chain polypeptides. In this situation, the light chain must precede the heavy chain to prevent excessive toxic free heavy chain (Proudfoot NJ (1986) Nature 322:562-565; and Kohler G (1980) PNAS 77:2197-2199). The heavy and light chain encoding sequences can comprise cDNA or genomic DNA. The expression vector can be monocistronic or multicistronic. A multicistronic nucleic acid construct can encode 2, 3, 4, 5, 6, 7, 8, 9, 10 or more genes / nucleotide sequences, or in the range of 2-5, 5-10, or 10-20 genes / nucleotide sequences. For example, a bicistronic nucleic acid construct can include, in order, a promoter, a first gene (e.g., the heavy chain of an antibody described herein), and a second gene (e.g., the light chain of an antibody described herein). In such an expression vector, transcription of the two genes is driven by the promoter, whereas translation of mRNA from the first gene may be driven by a cap-dependent scanning mechanism and translation of mRNA from the second gene may be driven by a cap-independent mechanism, e.g., an IRES.

[0334] Once the antibody molecules described herein are produced by recombinant expression, they may be purified by any method known in the art for the purification of immunoglobulin molecules, such as chromatography (e.g., ion exchange, affinity, particularly for protein A and specific antigens, and sizing column chromatography), centrifugation, differential solubility, or other standard techniques for protein purification. The antibodies described herein may also be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.

[0335] In certain embodiments, the antibodies or antigen-binding portions thereof described herein are isolated or purified. Generally, an isolated antibody is one that is substantially free of other antibodies whose antigenic specificity differs from that of the isolated antibody. For example, in certain embodiments, preparations of the antibodies described herein are substantially free of cellular material and / or chemical precursors. The term "substantially free of cellular material" includes preparations of antibodies that are isolated from cells or recombinantly produced and separated from cellular components of the cells. Thus, antibodies that are substantially free of cellular material include preparations of antibodies that have less than about 30%, 20%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (by dry weight) of heterologous proteins (also referred to herein as "contaminating proteins") and / or antibody variants, e.g., different post-translationally modified forms of the antibody or other different variant forms of the antibody (or antibody-binding portion). When the antibody is recombinantly produced, the antibody is also generally substantially free of culture medium, i.e., culture medium represents less than about 20%, 10%, 2%, 1%, 0.5%, or 0.1% of the volume of the protein preparation. When the antibody is produced by chemical synthesis, the antibody is generally substantially free of chemical precursors or other chemicals, i.e., separated from chemical precursors or other chemicals involved in protein synthesis. Thus, such antibody preparations have less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or compounds other than the antibody of interest. In certain embodiments, the antibodies described herein are isolated or purified.

[0336] [VII. Analysis method] Antibodies described herein can be tested for binding to FAM19A5, for example, by standard ELISA. Briefly, microtiter plates are coated with purified FAM19A5 at 1-2 μg / ml in PBS, followed by blocking with 5% bovine serum albumin in PBS. Antibody dilutions (e.g., plasma dilutions from FAM19A5-immunized mice) are added to each well and incubated for 1-2 hours at 37°C. Plates are washed with PBS / Tween and then incubated with a secondary reagent conjugated to horseradish peroxidase (HRP) (e.g., for human antibodies, goat anti-human IgG Fc-specific polyclonal reagent) for 1 hour at 37°C. After washing, plates are developed with ABTS substrate (Moss Inc., product: ABTS-1000) and analyzed by spectrophotometer at OD 415-495. Next, sera from mice immunized against the antibody that bind to the cell line expressing human FAM19A5 but not to the control cell line that does not express FAM19A5 are further screened by flow cytometry. Briefly, binding of the anti-FAM19A5 antibody is assessed by incubating FAM19A5-expressing CHO cells with the anti-FAM19A5 antibody at a 1:20 dilution ratio. The cells are washed, and binding is detected with a PE-labeled anti-human IgG Ab. Flow cytometry is performed using a FACS can flow cytometry system (Becton Dickinson, San Jose, CA). Preferably, mice exhibiting the highest titers will be used for fusion.

[0337] The ELISA assay described above can be used to screen for hybridomas producing antibodies and, relatedly, antibodies that show positive reactivity with the FAM19A5 immunogen. Hybridomas producing antibodies that bind to FAM19A5, preferably with high affinity, can then be subcloned and further characterized. One clone from each hybridoma that retains the reactivity of the parental cells (by ELISA) can then be cell banked and selected for antibody purification.

[0338] To purify anti-FAM19A5 antibodies, selected hybridomas can be grown in 2L spinner-flasks for monoclonal antibody purification. The supernatant can be filtered and concentrated before affinity chromatography using protein A-Sepharose (Pharmacia, Piscataway, NJ). To ensure purity, the eluted IgG can be checked by gel electrophoresis and high-performance liquid chromatography. The buffer can be exchanged into PBS, and the concentration can be determined by OD 280 using a 1.43 extinction coefficient. The monoclonal antibody can be aliquoted and stored at -80°C.

[0339] To determine whether the selected anti-FAM19A5 monoclonal antibodies bind to unique epitopes, each antibody can be biotinylated using commercially available reagents (Pierce, Rockford, IL). Biotinylated MAb binding can be detected with a streptavidin-labeled probe. Competition studies using unlabeled and biotinylated monoclonal antibodies can be performed using FAM19A5-coated ELISA plates as described above.

[0340] To determine the isotype of purified antibodies, an isotype ELISA can be performed using reagents specific for antibodies of a particular isotype. For example, to determine the isotype of a human monoclonal antibody, wells of a microtiter plate can be coated with 1 μg / ml anti-human immunoglobulin overnight at 4°C. After blocking with 1% BSA, the plate is reacted with up to 1 μg / ml of the test monoclonal antibody or purified isotype control for 1-2 hours at ambient temperature. The wells can then be reacted with human IgG1- or human IgM-specific alkaline phosphatase-conjugated probes. The plate is developed and analyzed as described above.

[0341] To test the binding of monoclonal antibodies to live cells expressing FAM19A5, flow cytometry can be used, as described in the Examples section. Briefly, cell lines expressing membrane-bound FAM19A5 (grown under standard growth conditions) are mixed with various concentrations of monoclonal antibodies in PBS containing 0.1% BSA for 1 hour at 4°C. After washing, the cells are reacted with fluorescein-labeled anti-IgG antibodies under the same conditions as for primary antibody staining. Samples can be analyzed using a FACScan instrument using light and side scatter characteristics to gate on single cells and determine the binding of the labeled antibody. Other analytical methods using fluorescence microscopy can be used in addition to or instead of flow cytometry. Cells can be precisely stained as described above and examined by fluorescence microscopy. This method allows visualization of individual cells, but sensitivity may be reduced depending on the density of the antigen.

[0342] Anti-FAM19A5 antibodies can be further tested for reactivity with the FAM19A5 antigen by Western blotting. Briefly, cell extracts can be prepared from cells expressing FAM19A5 and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. After electrophoresis, the separated antigens are transferred to a nitrocellulose membrane, blocked with 20% mouse serum, and probed with the monoclonal antibody to be tested. IgG binding can be detected using anti-IgG alkaline phosphatase and developed with BCIP / NBT substrate purification (Sigma Chem. Co., St. Louis, MO).

[0343] Methods for analyzing the binding affinities, cross-reactivities, and binding kinetics of various anti-FAM19A5 antibodies can be performed using standard assays known in the art, such as BIACORE™. TM BIACORE 2000 SPR instrument (Biacore AB, Uppsala, Sweden) TM Surface plasmon resonance (SPR) analysis is included.

[0344] In one embodiment, the antibody specifically binds to the soluble form of human FAM19A5. In another embodiment, the antibody specifically binds to the membrane-bound form of human FAM19A5. The antibody can specifically bind to a specific epitope of FAM19A5 (e.g., SEQ ID NO: 90 or a fragment within SEQ ID NO: 90). In certain embodiments, the antibody preferably specifically binds to human FAM19A5 with high affinity and does not cross-react with other members of the FAM19 subfamily of proteins.

[0345] [VIII. Bispecific molecules] The antibodies described herein can be used to form bispecific molecules. Anti-FAM19A5 antibodies or their antigen-binding portions can be derivatized or linked to additional functional molecules, such as other peptides or proteins (e.g., other antibodies or ligands for receptors), to generate bispecific molecules that bind to at least two different binding sites or target molecules. Cytokines such as IL-6, CNTF, LIF, EGF, and TGFα have since been implicated as promoters of gliosis and / or reactive astrogliosis by activating the protein signal transducer and activator of transcription 3 (STAT3), which regulates many aspects of reactive astrogliosis after CNS injury (Balasingam et al., J. Neurosci. 14(2):846-56 (1994); Winter et al., Proc. Natl. Acad. Sci. USA 20;92(13):5865-9 (1995)). See Herrmann JE et al., J. Neurosci. 28(28):7231-7243(2008). For example, the absence or reduction of STAT3 manifests as impaired upregulation of glial fibrillary acidic protein (GFAP), failure of astrocytic hypertrophy and increased spread of inflammation, increased lesion volume, and partial impairment of motor function recovery after CNS injury. See Herrmann JE et al., J. Neurosci. 28(28):7231-7243(2008). Thus, for example, an anti-FAM19A5 antibody can be linked to an antibody or scFv that specifically binds to any protein involved in the pathogenesis of gliosis and / or inhibiting the hyperproliferation of reactive astrogliosis for combination therapy, such as antibodies against IL-6, CNTF, LIF, EGF, or TGFα.

[0346] The anti-FAM19A5 antibody can also be linked to an antibody or scFv that treats a disease or disorder in a subject, including central nervous system injury (e.g., traumatic brain injury, cerebrospinal injury, stroke, or brain tumor), cerebrospinal system injury, degenerative brain disorder (e.g., Huntington's disease, Parkinson's disease, Alzheimer's disease, multiple sclerosis, ALS), degenerative cerebrospinal or neuropathy, or neuropathic pain (see Diseases or Disorders in Section XII below). For example, the anti-FAM19A5 antibody can be linked to an antibody or scFv that treats multiple sclerosis, such as natalizumab (TYSABRI®) or alemtuzumab (LEMTRADA®).

[0347] The antibodies described herein can actually be derivatized or linked to more than one other functional molecule to generate multispecific molecules that bind to more than two different binding sites and / or target molecules; such multispecific molecules are also intended to be encompassed by the term "bispecific molecule" as used herein. To generate the bispecific molecules described herein, the antibodies described herein can be operatively linked (e.g., by chemical conjugation, genetic fusion, non-covalent association, etc.) to one or more other binding molecules, such as other antibodies, antibody-binding portions thereof, peptides, or binding mimetics, to generate the bispecific molecule. In one embodiment, the bispecific molecule binds to FAM19A5 and VEGF. In yet another embodiment, the bispecific molecule binds to FAM19A5 and EGF.

[0348] Accordingly, the present disclosure provides bispecific molecules comprising at least one first binding specificity for FAM19A5 and a second binding specificity for a second target epitope. In embodiments described herein in which the bispecific molecule is multispecific, the molecule can further comprise a third binding specificity.

[0349] In one embodiment, the bispecific molecules described herein comprise as a binding specificity at least one antibody or antibody-binding portion thereof, including, for example, Fab, Fab', F(ab'), Fv, or single-chain Fv (scFv). The antibody may also be a light or heavy chain dimer, or any minimal fragment thereof, such as an Fv or single-chain construct, as described in U.S. Patent No. 4,946,778 to Ladner et al., the contents of which are expressly incorporated by reference.

[0350] Although human monoclonal antibodies are preferred, other antibodies that can be used in the bispecific molecules described herein are murine, chimeric, and humanized monoclonal antibodies.

[0351] The bispecific molecules described herein can be prepared by conjugating the constituent binding specificities using methods known in the art. For example, each binding specificity of the bispecific molecule can be generated separately and then conjugated to one another. When the binding specificities are proteins or peptides, a variety of coupling or cross-linking agents can be used for covalent conjugation. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) (e.g., Karpovsky et al., (1984) J. Exp. Med. 160:1686; Liu, MA et al., (1985) Proc. Natl. Acad. Sci. USA 82:8648). Other methods include those described by Paulus (1985) Behring Ins. Mitt. No. 78, 118-132; Brennan et al. (1985) Science 229:81-83; and Glennie et al. (1987) J. Immunol. 139:2367-2375. Preferred conjugating agents are SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, IL).

[0352] When the binding specificities are antibodies, they can be joined by sulfhydryl bonds in the C-terminal hinge regions of the two heavy chains. In a particularly preferred embodiment, the hinge region is modified to contain an odd number of sulfhydryl residues, preferably one, prior to joining.

[0353] Alternatively, two binding specificities can be encoded in the same vector and expressed and combined in the same host cell. This method is particularly useful when the bispecific molecule is a mAb x mAb, mAb x Fab, mAb x (scFv)2, Fab x F(ab')2, or ligand x Fab fusion protein. Bispecific antibodies can include antibodies comprising an scFv at the C-terminus of each heavy chain. The bispecific molecules described herein can be single-chain molecules comprising one single-chain antibody and a binding determinant, or single-chain bispecific molecules comprising two binding determinants. Bispecific molecules can comprise at least two single-chain molecules. Methods for producing bispecific molecules are described, for example, in U.S. Patent No. 5,260,203; U.S. Patent No. 5,455,030; U.S. Patent No. 4,881,175; U.S. Patent No. 5,132,405; U.S. Patent No. 5,091,513; U.S. Patent No. 5,476,786; U.S. Patent No. 5,013,653; U.S. Patent No. 5,258,498; and U.S. Patent No. 5,482,858.

[0354] Binding of the bispecific molecule to its specific target can be confirmed using art-recognized methods such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), or Western blot analysis. Each of these assays generally detects the presence of the protein-antibody complex of particular interest by employing a labeled reagent (e.g., an antibody) specific for the complex.

[0355] IX. Diagnosis In one embodiment, the moiety attached to the anti-FAM19A5 antibody is selected from the group consisting of a binding moiety, a labeling moiety, and a biologically active moiety.

[0356] The antibodies described herein can be used for diagnostic purposes, including sample testing and in vivo imaging, and for this purpose the antibodies (or binding portions thereof) can be conjugated to a suitable detectable agent to form an immunoconjugate. Suitable agents for diagnostic purposes are detectable labels, including radioisotopes for whole-body imaging and radioisotopes for sample testing, enzymes, fluorescent labels, and other suitable antibody tags.

[0357] The detectable label can be a particulate label including a metal sol such as colloidal gold, e.g., I presented with a peptide chelator of the N2S2, N3S, or N4 type. 125 or Tc 99 The label may be any of a variety of types currently used in the field of in vitro diagnostics, including isotopes such as , chromophores including fluorescent markers, luminescent markers, phosphorescent markers, etc., as well as enzyme labels that convert a given substrate into a detectable marker and polynucleotide tags that are identified after amplification, for example, by polymerase chain reaction. Suitable enzyme labels include horseradish peroxidase, alkaline phosphatase, etc. For example, the label may be the enzyme alkaline phosphatase, which is detected by measuring the presence or formation of chemiluminescence following conversion of 1,2-dioxetane substrates such as adamantyl methoxyphosphoryloxyphenyl dioxetane (AMPPD), disodium 3-(4-(methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo{3.3.1.13,7}decan}-4-yl)phenyl phosphate (CSPD), as well as other luminescent substrates such as CDP and CDP-star®, or other luminescent substrates well known to those of skill in the art, e.g., appropriate lanthanum chelates such as terbium(III) and europium(III). The means of detection will depend on the label selected. The appearance of the label or its reaction products, if the label is particulate and accumulates to an appropriate level, can be obtained by eye or by instruments such as spectrophotometers, luminometers, fluorometers, etc., using standard practices.

[0358] The antibodies described herein can also be conjugated to a therapeutic agent to form an immunoconjugate, such as an antibody-drug conjugate (ADC). Suitable therapeutic agents include agents that modulate the pathogenesis of gliosis and / or reactive astrogliosis and / or treat degenerative brain disorders, central nervous system injuries, or neuropathic pain. Therapeutic agents for treating degenerative brain disorders include drugs for treating Huntington's disease, Parkinson's disease, Alzheimer's disease, multiple sclerosis, and amyotrophic lateral sclerosis (ALS). This includes drugs commonly used to treat such degenerative brain disorders, such as those disclosed in Section XII below.

[0359] Immunoconjugates can be prepared by methods known in the art. Preferably, the conjugation method results in substantially (or mostly) non-immunogenic linkages, such as peptide (i.e., amide), sulfide, (sterically hindered), disulfide, hydrazone, and ether linkages. These linkages are largely non-immunogenic and exhibit considerable stability in serum (see, e.g., Senter, PD, Curr. Opin. Chem. Biol. 13 (2009) 235-244; WO 2009 / 059278; WO 95 / 17886).

[0360] Depending on the biochemical attributes of the moiety and antibody, different conjugation strategies can be used. Whether the moiety is naturally occurring or recombinant, consisting of 50 to 500 amino acids, standard procedures describing chemical methods for synthesizing protein conjugates are available in textbooks and can be easily followed by those skilled in the art (e.g., Hackenberger, CPR, and Schwarzer, D., Angew. Chem. Int. Ed. Engl. 47 (2008) 10030-10074). In one embodiment, the reaction of a cysteine ​​residue in the antibody or moiety with a maleimide moiety is utilized. This is a particularly suitable coupling chemistry when, for example, an antibody Fab or Fab' fragment is used. Alternatively, in one embodiment, coupling is performed at the C-terminus of the antibody or moiety. C-terminal modifications of proteins, e.g., Fab fragments, can be performed, for example, as described in Sunbul, M. and Yin, J., Org. Biomol. Chem. 7 (2009) 3361-3371.

[0361] Site-specific reactions and covalent bonds are generally based on converting natural amino acids into amino acids with reactivities orthogonal to those of other functional groups present. For example, specific cysteines in rare sequence contexts can be enzymatically converted to aldehydes (see Frese, MA and Dierks, T., ChemBioChem. 10 (2009) 425-427). The specific enzymatic reactivity of specific enzymes with natural amino acids in a given sequence context can also be used to obtain desired amino acid modifications (see, e.g., Taki, M. et al., Prot. Eng. Des. Sel. 17 (2004) 119-126; Gautier, A. et al., Chem. Biol. 15 (2008) 128-136; and protease-catalyzed formation of CN bonds is used by Bordusa, F., Highlights in Bioorganic Chemistry (2004) 389-403).

[0362] Site-specific reactions and covalent bonds can also be achieved by selective reaction of the terminal amino acid with an appropriate modification reagent. The reactivity of benzonitrile with N-terminal cysteine ​​(see Ren, H. et al., Angew. Chem. Int. Ed. Engl. 48 (2009) 9658-9662) can be used to obtain site-specific covalent bonds. Native chemical ligation can also rely on C-terminal cysteine ​​residues (Taylor, E. Vogel; Imperiali, B., Nucleic Acids and Molecular Biology (2009), 22 (Protein Engineering), 65-96).

[0363] EP 1 074 563 describes a conjugation method based on the faster reaction of cysteines located within a series of positively charged amino acids with cysteines located within a series of negatively charged amino acids.

[0364] The moiety may be a synthetic peptide or peptidomimetic. If the polypeptide is chemically synthesized, amino acids with orthogonal chemical reactivity may be introduced during such synthesis (see, for example, de Graaf, AJ et al., Bioconjug. Chem. 20 (2009) 1281-1295). Since a wide variety of orthogonal functional groups are labile and can be introduced into synthetic peptides, conjugating the peptide to a linker is a standard chemical method.

[0365] Conjugates with a 1:1 stoichiometry can be separated from other conjugation by-products by chromatography to yield a single-labeled polypeptide. This process can be facilitated by using dye-labeled binding pair members and charged linkers. By using these types of labels and highly negatively charged binding pair members, single-conjugated polypeptides are easily separated from unlabeled polypeptides and polypeptides carrying more than one linker, since charge and molecular weight differences can be utilized for separation. The fluorescent dyes, like labeled monovalent binding agents, can be useful for purifying complexes from unbound components.

[0366] X. Pharmaceutical Compositions The present specification provides compositions comprising the antibodies or antigen-binding portions thereof described herein, with the desired degree of purity, in a physiologically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (fewer than about 10 residues) polypeptides; serum albumin, gelatin, or immunoglobulins. hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEENTM®, PLURONICS®, or polyethylene glycol (PEG).

[0367] In certain embodiments, the pharmaceutical composition comprises an antibody or antigen-binding portion thereof, bispecific molecule, or immunoconjugate described herein, and optionally one or more additional prophylactic or therapeutic agents, in a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition comprises an effective amount of an antibody or antigen-binding portion thereof described herein, and optionally one or more additional prophylactic or therapeutic agents, in a pharmaceutically acceptable carrier. In some embodiments, the antibody is the only active ingredient contained in the pharmaceutical composition. The pharmaceutical compositions described herein enhance, induce, or activate FAM19A5 activity and may be useful for treating conditions such as central nervous system injury, degenerative brain disorders, or neuropathic pain.

[0368] Pharmaceutically acceptable carriers used in parenteral formulations include aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonicity agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifiers, sequestering or chelating agents, and other pharmaceutically acceptable substances. Examples of aqueous vehicles include sodium chloride injection, intravenous infusion injection, isotonic dextrose injection, sterile water injection, and dextrose and lactate intravenous infusion injection. Non-aqueous parenteral vehicles include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, and peanut oil. Antimicrobial agents in bacteriostatic or fungistatic concentrations may be added to parenteral formulations packaged in multidose containers, including phenol or cresol, mercury-containing substances, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoates, thimerosal, benzalkonium chloride, and benzethonium chloride. Isotonicity agents include sodium chloride and dextrose. Buffering agents include phosphate and citrate. Antioxidants include sodium bisulfate. Topical anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcellulose, hydroxypropyl methylcellulose, and polyvinylpyrrolidone. Emulsifying agents include Polysorbate 80 (TWEEN® 80). Sequestering or chelating agents for metal ions include EDTA. Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol, and propylene glycol for water-miscible vehicles; and sodium hydroxide, hydrochloric acid, citric acid, or lactic acid for pH adjustment.

[0369] Pharmaceutical compositions can be formulated for any route of administration to a subject. Specific examples of routes of administration include intranasal, oral, parenteral, intrathecal, intraventricular, pulmonary, subcutaneous, or intraventricular. Parenteral administration, characterized by subcutaneous, intramuscular, or intravenous injection, is also contemplated herein. Injectables can be prepared in conventional forms, such as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Injectables, solutions, and emulsions also contain one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol, or ethanol. If necessary, the pharmaceutical composition to be administered can also contain minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffers, stabilizers, solubility enhancers, and formulation agents, such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, and cyclodextrins.

[0370] Formulations of antibodies for parenteral administration include sterile, dry soluble products such as lyophilized powders that can be mixed extemporaneously with a solvent immediately before use, including tablets for subcutaneous injection, sterile, dry suspensions that can be mixed extemporaneously with a vehicle immediately before use, and sterile emulsions. The solutions can be aqueous or non-aqueous.

[0371] For intravenous administration, suitable carriers include physiological saline or phosphate buffered saline (PBS) and solutions containing viscosity enhancing and solubilizing agents such as glucose, polyethylene glycol, polypropylene glycol, and mixtures thereof.

[0372] Topical mixtures containing antibodies are prepared as described for local and systemic administration. The resulting mixture may be a solution, suspension, emulsion, etc., and may be formulated as a cream, gel, ointment, emulsion, solution, elixir, lotion, suspension, tincture, paste, foam, aerosol, irrigation, spray, suppository, bandage, skin patch, or any other dosage form suitable for topical administration.

[0373] The antibodies or antigen-binding portions thereof described herein can be formulated as aerosols for topical administration, e.g., by inhalation (see, e.g., U.S. Pat. Nos. 4,044,126, 4,414,209, and 4,364,923, which describe steroid-delivering aerosols useful for treating inflammatory diseases, particularly asthma). These dosage forms for administration to the respiratory tract can be in the form of a nebulizer aerosol or solution, or a fine powder for inhalation, alone or in combination with an inert carrier such as lactose. In such cases, the particles of the dosage form have diameters of less than 50 microns in one embodiment, and in one embodiment, less than 10 microns.

[0374] The antibodies or antigen-binding portions thereof described herein can be formulated for topical or local use, such as topical application to the skin and mucous membranes, including the eye, in the form of gels, creams, and lotions, and for application to the eye or intracisternal or intrathecal applications. Topical administration is also contemplated for transdermal delivery, as well as for administration to the eye or mucous membranes or for inhalation therapy. Nasal solutions of the antibodies can be administered alone or in combination with other pharmaceutically acceptable excipients.

[0375] Transdermal patches, including iontophoretic and electrophoretic devices, are well known to those of skill in the art and can be used to administer antibodies. For example, such patches are disclosed in U.S. Patent Nos. 6,267,983, 6,261,595, 6,256,533, 6,167,301, 6,024,975, 6,010,715, 5,985,317, 5,983,134, 5,948,433, and 5,860,957.

[0376] In certain embodiments, pharmaceutical compositions containing antibodies or antigen-binding portions thereof described herein are lyophilized powders that can be reconstituted for administration as solutions, emulsions, and other mixtures. The lyophilized powders can also be reconstituted and formulated as solids or gels. The lyophilized powders are prepared by dissolving the antibodies or antigen-binding portions thereof described herein, or pharmaceutically acceptable derivatives thereof, in a suitable solvent. In some embodiments, the lyophilized powders are sterile. The solvents can contain excipients that improve the stability or other pharmacological components of the powder or reconstituted solutions prepared from the powders. Excipients that can be used include, but are not limited to, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable formulations. The solvents can also contain buffers such as citrate, sodium phosphate, or potassium phosphate, or other well-known buffers with a near-neutral pH, in one embodiment. The solution is then sterile filtered and lyophilized under standard conditions known to those skilled in the art to provide the desired dosage form. In one embodiment, the resulting solution can be apportioned into lyophilization vials, each containing a single dose or multiple doses of the compound. The lyophilized powder can be stored under appropriate conditions, such as at about 4°C to room temperature.

[0377] Such lyophilized powders are reconstituted with water for injection to provide a dosage form for use in parenteral administration. For reconstitution, the lyophilized powder is added to sterile water or other suitable carrier, the exact amount of which will vary depending on the compound selected. Such amounts can be determined empirically.

[0378] The antibodies or antigen-binding portions thereof, bispecific molecules or immunoconjugates described herein, and other compositions provided herein, may also be formulated to target specific tissues, receptors, or other body regions of the subject to be treated. Many such targeting methods are well known to those of skill in the art. Any such targeting method is contemplated herein for use with the present compositions. For non-limiting examples of targeting methods, see, e.g., U.S. Patent Nos. 6,316,652, 6,274,552, 6,271,359, 6,253,872, 6,139,865, 6,131,570, 6,120,751, 6,071,495, 6,060,082, 6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, 5,900,252, 5,840,674, 5,759,542, and 5,709,874. In certain embodiments, the antibodies, or antigen-binding portions thereof, described herein can be targeted to treat central nervous system injuries, degenerative brain disorders, or neuropathic pain.

[0379] Compositions to be used for in vivo administration can be sterile, which is readily accomplished, for example, by filtration through sterile filtration membranes.

[0380] [VIII. Kit] The present specification provides kits containing one or more antibodies or antigen-binding portions thereof described herein. In certain embodiments, the present specification provides pharmaceutical packs or kits comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions described herein, such as one or more antibodies or antigen-binding portions thereof provided herein, and optionally, instructions for use. In some embodiments, the kits contain the pharmaceutical compositions described herein and any prophylactic or therapeutic agents as described herein.

[0381] XII. Therapeutic Uses and Methods The present specification also provides a method for reducing injury or damage to the CNS in a subject (e.g., a human) in need thereof, comprising administering to the subject an anti-FAM19A5 antibody, bispecific molecule, or immunoconjugate, or composition thereof, described herein.

[0382] In other aspects, the present specification provides methods for inhibiting, delaying, suppressing, limiting, reducing, reversing, or preventing the onset or onset of gliosis and associated deleterious CNS effects in a subject, comprising administering an anti-FAM19A5 antibody disclosed herein to the subject. In some embodiments, the present specification provides methods for inhibiting, delaying, suppressing, limiting, reducing, reversing, or preventing excessive or abnormal proliferation of reactive astrocytes and associated deleterious CNS effects in a subject, comprising administering an anti-FAM19A5 antibody disclosed herein to the subject. In some embodiments, the present specification provides methods for decreasing, inhibiting, or reducing the expression of chondroitin sulfate proteoglycans (including levels of neurocan, NG2, or both), or decreasing or inactivating the activity of neurocan, NG2, or both, in a subject, comprising administering an anti-FAM19A5 antibody disclosed herein to the subject. In some embodiments, the present disclosure provides a method for stimulating, promoting, increasing, or activating neuronal growth in a subject, preferably after injury or damage, comprising administering to the subject an anti-FAM19A5 antibody described herein. In other embodiments, the present disclosure provides a method for increasing the level of c-fos mRNA, c-fos protein, or c-fos protein activity in a subject in need thereof, preferably increasing the level of ERK mRNA, ERK protein, or pERK activity in the nucleus of neuronal cells, comprising administering to the subject an anti-FAM19A5 antibody disclosed herein. In certain embodiments, the present disclosure provides a method for increasing or enhancing the level of GAP43 mRNA or GAP43 protein in a subject in need thereof, preferably increasing the activity of GAP43 protein in neuronal cells, comprising administering to the subject an anti-FAM19A5 antibody disclosed herein. In certain embodiments, the present specification provides a method for enhancing or promoting neuronal survival and / or promoting axonal regrowth in a subject in need thereof, comprising administering to the subject an anti-FAM19A5 antibody disclosed herein.In some embodiments, the subject is a human, preferably a human who has suffered neural damage or injury, e.g., from CNS injury, trauma, injury, cerebrospinal cord injury, brain tumor, infection, ischemia, stroke, reaction, and / or neurodegenerative disease.

[0383] In some embodiments, the present disclosure also provides a method for treating a disease, disorder, or condition in a subject in need thereof, comprising administering to the subject an anti-FAM19A5 antibody of the present disclosure. In some embodiments, the disease, disorder, or condition comprises a central nervous system injury, a cerebrospinal system injury, a degenerative brain disorder, a degenerative cerebrospinal or neurological disorder, or neuropathic pain. In some embodiments, the central nervous system injury is a traumatic brain injury, a cerebrospinal injury, a stroke, a brain tumor, or a combination thereof. In some embodiments, the degenerative brain disorder is Huntington's disease, Parkinson's disease, Alzheimer's disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS), or a combination thereof. Thus, in certain embodiments, the present specification discloses a method for treating traumatic brain injury, cerebrospinal cord injury, stroke, brain tumor, or a combination thereof in a subject in need thereof, comprising administering to the subject an anti-FAM19A5 antibody or composition thereof disclosed herein. In some embodiments, the present specification discloses a method for treating Huntington's disease, Parkinson's disease, Alzheimer's disease, multiple sclerosis, or ALS in a subject in need thereof, comprising administering to the subject an anti-FAM19A5 antibody or composition thereof disclosed herein. In some embodiments, the subject is a human.

[0384] In some embodiments, the anti-FAM19A5 antibody may be administered in combination with one or more additional agents for treating central nervous system injury (e.g., traumatic brain injury, cerebrospinal injury, stroke, or brain tumor), cerebrospinal system injury, degenerative brain disorder (e.g., Huntington's disease, Parkinson's disease, Alzheimer's disease, multiple sclerosis, ALS), degenerative cerebrospinal or neuropathy, or neuropathic pain.

[0385] In some embodiments, the disease, disorder, or condition comprises a tumor, fibrosis, glaucoma, retinopathy, age-related macular degeneration, or mood disorder. In certain embodiments, the disease, disorder, or condition comprises a tumor. In some embodiments, the tumor comprises melanoma, pancreatic cancer, glioma (e.g., glioblastoma multiforme (GBM)), breast cancer, lymphoma, lung cancer, kidney cancer, prostate cancer, fibrosarcoma, colon adenocarcinoma, liver cancer, or ovarian cancer.

[0386] In some embodiments, the anti-FAM19A5 antibodies of the present disclosure induce vascular normalization, for example, within tumors, which in some embodiments involves changes in vascular properties, including increased connectivity, increased wall thickness, decreased vessel diameter, more regular vascular orientation and distribution patterns, increased blood vessel number, decreased leakage and permeability, increased pericyte coverage and proximity on blood vessels, increased oxygen supply, or a combination thereof.

[0387] In some embodiments, the anti-FAM19A5 antibodies of the present disclosure inhibit tumor growth by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to a baseline (e.g., tumor growth in a subject not administered the anti-FAM19A5 antibody).

[0388] In some embodiments, the anti-FAM19A5 antibody increases tumor infiltration of immune cells. In some embodiments, the tumor infiltration of immune cells is increased / increased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to a baseline (e.g., a cancer subject not administered the anti-FAM19A5 antibody). In certain embodiments, the immune cells include macrophages, dendritic cells, T lymphocytes, B lymphocytes, natural killer (NK) cells, or a combination thereof. In some embodiments, the immune cells exhibit hypertrophy. In some embodiments, the tumor infiltration of immune cells is accompanied by increased tumor infiltration of neural cells. In certain embodiments, the neural cells include astrocytes, glial cells, or a combination thereof.

[0389] In some embodiments, the anti-FAM19A5 antibodies of the present disclosure enhance the phagocytic activity of macrophages or microglia. In some embodiments, the anti-FAM19A5 antibodies increase the mitochondrial membrane potential of macrophages or microglia. In certain embodiments, the phagocytic activity or mitochondrial membrane potential is enhanced or increased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to a baseline (e.g., a cancer subject not administered an anti-FAM19A5 antibody).

[0390] In some embodiments, the anti-FAM19A5 antibodies of the present disclosure reduce necrosis and edema in tumors. In other embodiments, the anti-FAM19A5 antibodies reduce tumor tissue permeability. In some embodiments, the tumor necrosis and edema or tissue permeability is reduced by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to a baseline (e.g., a cancer subject not administered an anti-FAM19A5 antibody).

[0391] In some embodiments, the anti-FAM19A5 antibody increases blood flow rate in tumors, and in certain embodiments, the blood flow rate is increased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to a baseline (e.g., a cancer subject not administered the anti-FAM19A5 antibody).

[0392] In some embodiments, the tumor treatment method includes administering an additional therapeutic agent. In certain embodiments, the additional therapeutic agent includes chemotherapy, immunotherapy, radiation therapy, or a combination thereof. In some embodiments, the immunotherapy includes a monoclonal antibody, chimeric antigen receptor (CAR) therapy, T-cell therapy, NK-cell therapy, dendritic cell (DC) therapy, adoptive cell transfer (ACT), an immune checkpoint modulator, a cytokine, a cancer vaccine, an adjuvant, an oncolytic virus, or a combination thereof. In some embodiments, the chemotherapy includes temozolomide, gemcitabine, paclitaxel, carboplatin, cisplatin, elotuzumab, lenalidomide, dexamethasone, oxaliplatin, or a combination thereof.

[0393] In some embodiments, a therapeutically effective amount of an anti-FAM19A5 antibody or composition thereof disclosed herein is administered. When treating a subject (e.g., a human), the therapeutically effective amount of an anti-FAM19A5 antibody disclosed herein will vary depending on factors such as age, sex, and disease severity.

[0394] In some embodiments, the anti-FAM19A5 antibodies or compositions thereof of the present disclosure are administered intravenously, orally, parenterally, intrathecally, intrathecally, intracerebroventricularly, pulmonary, subcutaneously, intravascularly, intramuscularly, or intraventricularly.

[0395] The following examples are provided by way of illustration and not limitation.

[0396] [Example] Example 1: Expression and purification of human FAM19A5 protein Recombinant human FAM19A5 protein was produced and purified as described below, and the purified protein was used in antibody screening assays based on binding affinity analysis. First, the LPS-hT plasmid expressing the FAM19A5 gene was transformed into bacteria to induce protein overexpression. After production, the FAM19A5 protein was purified using Ni-NTA affinity chromatography (Qiagen, Valencia, CA, USA). The His-tagged FAM19A5 protein was removed from the Ni-column using increasingly higher concentrations of imidazole. Protein expression in the solution was measured using Coomassie Brilliant Blue R-250 dye. The FAM19A5 imidazole-containing solution was isolated and the FAM19A5 protein was concentrated using PBS. Once concentrated, the purity and concentration of the FAM19A5 protein were measured using Western blot analysis. The concentrated protein was then used to screen for FAM19A5-specific antibodies.

[0397] Example 2: Preparation of anti-FAM19A5 antibody library 1.Immunization FAM19A5 protein was used as an antigen for immunization of White Leghorn chickens. 50 μg of synthetic peptide-KLH conjugate was mixed with 750 μL of phosphate-buffered saline (PBS) and incubated at 37°C for 30 minutes. The mixture was then emulsified with 2% squalene-resistant toxin-resistant MPL (monophosphoryl lipid A species) from TDW and CWS cell wall components containing a water-in-oil emulsifying adjuvant (RIBI+MPL+TDM+CWS adjuvant, Sigma, St. Louis, MO, USA) and toxins removed from mycobacteria. The mixture was then subcutaneously injected into the chickens. Chickens were immunized a total of four times, approximately 2–3 weeks apart. Antibody titers from immunized animals were measured by immunoblotting using lysates from HEK293T cells overexpressing FAM19A5 protein.

[0398] 2. Preparation of single-chain variable fragment (scFv) libraries from immunized chickens RNA was extracted from the spleen, bone marrow, and bursa of Fabricius of the immunized chickens using TRI Reagent (Invitrogen, Carlsbad, CA USA). TM First-strand cDNA was synthesized using the Invitrogen III First-Strand Synthesis System. For cDNA obtained from the immune systems of the animals, single-strand variable region libraries were prepared using the Expand High Fidelity PCR System (Roche Molecular Systems, IN, USA). For each reaction, 1 μL of cDNA, 60 pmol of each primer, 10 μL of 10× reaction buffer, 8 μL of 2.5 mM dNTPs (Promega, Madison, WI, USA), and 0.5 μL of Taq DNA polymerase were mixed with water. The final volume was 100 μL. PCR reactions were performed using the following conditions: 30 cycles of (i) 94°C for 15 seconds, (ii) 56°C for 30 seconds, and (iii) 72°C for 90 seconds, followed by a final extension at 72°C for 10 minutes. PCR products containing approximately 350 bp fragments were loaded onto a 1.5% agarose gel, and the nucleotide fragments were purified after electrophoresis using a QIAGEN Gel II extraction kit (QIAGEN, Valencia, CA, USA). The purified PCR products were quantified by reading at OD 260 nm (1 unit OD = 50 μg / ml).

[0399] In the second PCR, the two VH and VL first products were randomly linked by overlap extension PCR. Each PCR reaction product was mixed with 100 ng of purified VL and VH products, 60 pmol of each primer, 10 μL of 10× reaction buffer, 8 μL of 2.5 mM dNTPs, 0.5 μL of Taq DNA polymerase, and water in a final volume of 100 μL. The PCR reaction was performed using the following conditions: (i) 94°C for 15 seconds, (ii) 56°C for 30 seconds, and (iii) 72°C for 2 minutes, followed by 25 cycles and a final extension at 72°C for 10 minutes. The PCR product, containing a single-stranded variable region fragment approximately 700 bp long, was loaded onto a 1.5% agarose gel, and the nucleotide fragment was purified after electrophoresis using a QIAGEN II gel extraction kit (QIAGEN). The purified PCR products were quantified by reading at OD 260 nm (1 unit OD = 50 μg / ml).

[0400] 3. Library, Ligation, and Transformation The scFv fragment of the PCR product and the vector pComb3X-SS (The Scripps Research Institute, CA, USA) were digested with SfiI restriction enzyme. 10 μg of the purified overlapping PCR product was mixed with 360 units of SifI (16 units per μg of DNA, Roche Molecular Systems, Pleasanton, CA, USA), 20 μL of 10× reaction buffer, and water to a final volume of 200 μL. 20 μg of the pComb3X-SS vector was mixed with 120 units of SfiI (6 units per μg of DNA), 20 μL of 10× reaction buffer, and water to a final volume of 200 μL. The mixture was digested at 50°C for 8 hours. The digested product, containing the scFv fragment (approximately 700 bp) and vector (approximately 3400 bp), was then loaded onto a 1% agarose gel and purified using QIAGEN Gel Extraction II (QIAGEN, Valencia, CA, USA). 1400 ng of SfiI-restricted pComb3X vector and 700 ng of the digested scFv fragment were mixed with 5x ligase buffer, 10 μL of T4 DNA ligase (Invitrogen, Carlsbad, CA, USA), and water to a final volume of 200 μL. The mixture was incubated at 16°C for 16 hours to allow for ligation.

[0401] After ethanol precipitation, the DNA pellet was dissolved in 15 μL of water. To generate the library, the ligated samples were transformed into E. coli strain ER2738 (New England Biolabs Inc., Hitchin, Hertfordshire, SG4 0TY, England, UK) by electroporation using a gene pulser (Bio-Rad Laboratories, Hercules, CA, USA). The cells were mixed in 5 ml of Super Broth (SB) medium and cultured at 37°C for 1 hour with agitation at 250 rpm. Next, 3 μL of 100 mg / mL kanamycin was added to 10 mL of SB medium. To determine the library size, 0.1 μL, 1 μL, and 10 μL of the culture samples were plated on Luria Broth (LB) agar plates containing 50 μg / mL kanamycin. After stirring for 1 hour, 4.5 μL of 100 mg / mL kanamycin was added to the LB culture and stirred for another hour. Next, 2 ml of VCM13 helper phage (>10 11 The culture (cfu / ml) was added to LB medium along with 183 mL of prewarmed LB containing 92.5 μL of 100 mg / mL kanamycin. This mixture was stirred at 250 rpm at 37°C for an additional 2 hours. Next, 280 μL (50 mg / mL) kanamycin was added to the culture and stirred overnight at 37°C. The next day, the bacterial pellet was centrifuged at 3,000 g at 4°C using a high-speed centrifuge (Beckman, JA-10 rotor). Phagemid DNA was then extracted using the bacterial pellet, while the supernatant was transferred to a sterile centrifuge bottle. Next, 8 g of polyethylene glycol-8000 (PEG-8000, Sigma) and 6 g of sodium chloride (NaCl, Merck) were added to the supernatant, which was then stored on ice for 30 minutes. The supernatant was then centrifuged at 15,000 g at 4°C for 15 minutes. The supernatant was then discarded and the phage pellet was resuspended in Tris-buffered saline (TBS) containing 1% BSA.

[0402] Example 3: Library panning (biopanning) on ​​immobilized antigens Biopanning was performed using magnetic beads (Dynabeads M-270 Epoxy, Invitrogen). Approximately 1 × 10 7 Each bead was coated with 5 μg of recombinant FAM19A5 protein by rotating at room temperature for 20 hours. After coating, the beads were washed four times with phosphate-buffered saline (PBS) and blocked with 3% BSA in PBS at room temperature for 1 hour. The coated beads were then incubated with the phage-displayed scFvs described above for 2 hours at room temperature. To remove phages unbound to the antigen-coated beads, the beads were washed with 0.05% Tween 20 / PBS. The bound phages were then eluted with 50 μL of 0.1 M glycine-HCl (0.1 M glycine-HCl, pH 2.2) and neutralized with 3 μL of 2 M Tris-HCl (pH 9.1) with HCl. The phage-containing supernatant was used to infect E. coli ER2738 cells, which were then amplified and rescued overnight using VCSM13 helper phage. The phage-infected culture was also blotted on LB agar plates containing 50 μg / ml kanamycin to determine the phage input and output titers. The next day, the phages were precipitated using PEG-8000 and NaCl and then used for biopanning. The above process was repeated for a total of five rounds of biopanning. After each round of amplification, phages were screened and selected for high affinity to the FAM19A5 protein.

[0403] Example 4: Clone selection by phage ELISA To analyze clones selected from biopanning, individual clones were randomly selected from the phage-displayed scFv and their binding to FAM19A5 recombinant protein was confirmed by ELISA. The FAM19A5 recombinant protein was diluted in 0.1 M NaHCO3 buffer and coated onto a 96-well microtiter plate at 100 ng / well for 16 hours at 4°C. The following day, the plate was blocked with 3% BSA / PBS at 37°C for 1 hour. The phage supernatant was then mixed with 6% BSA / PBS and incubated at 37°C for 2 hours. The plate containing the supernatant was then washed with 0.05% Tween-20 / PBS. HRP-conjugated M13 antibody (α-M13-HRP, Pierce Chemical Co., Rockford, IL, USA) was diluted 1 / 5000. 50 μl of the diluted antibody was added to the plate and incubated at 37°C for 1 hour. After incubation and washing, 0.05 M citrate buffer, 1 μg / ml 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS, Amresco, Solon, OH, USA), and 0.1% H2O2 were added to the plate for color development. The absorbance of each well was measured at 405 nm.

[0404] From the initially identified 96 clones, eight scFv clones with unique heavy chain CDR3 (HCDR3) sequences and high binding to the FAM19A5 protein were selected for further analysis (see Figures 1A-1C).

[0405] Example 5: Production of anti-FAM19A5-IGG2 / 4 antibody The anti-FAM19A5 scFv was subcloned into a mammalian expression vector. In the FAM19A5 scFv gene sequence, a human Cκ gene was linked to the light chain variable domain, and human immunoglobulin isotype IgG2 / 4 (CH1, CH2, and CH3) genes were linked to the heavy chain variable region. Restriction sites (Genscript, USA) were added to synthesize antibodies with each light chain and each heavy chain. The synthesized genes were inserted into a mammalian cell expression vector with modified restriction sites to facilitate cloning. First, the light chain gene was inserted into the vector using HindIII and XbaI restriction enzymes (New England Biolabs, UK), and then the heavy chain gene was added to the vector using NheI and BamHI restriction enzymes (New England Biolabs, UK).

[0406] To express and purify the anti-FAM19A5-IgG2 / 4 antibody, a mammalian cell transfection and overexpression system was used. Approximately 2 μg / ml of mammalian expression vector was mixed with 4 μg of polyethyleneimine (PEI, Polysciences, Warrington, PA, USA) in 150 mM sodium chloride (NaCl, Merck) in a volume equivalent to 1 / 10 of the cell culture volume. The mixture was left at room temperature for 15 minutes. The mixture was then transfected into HEK293F cells (2 × 10 6 After adding the cells (cells / ml, Invitrogen), the cells were incubated in FREESTYLE containing 100 U / ml penicillin and streptomycin (Invitrogen) at 37°C with 7% CO2 and 135 rpm. TM The cells were cultured in 293 expression medium for 6 days. The expressed anti-FAM19A5 IgG2 / 4 antibody was purified from the cell culture supernatant using Protein A bead (RepliGen, Waltham, MA, USA) affinity gel chromatography. Protein A chromatography was performed using 4-12% Bis-Tris gradient gel electrophoresis. Protein size and yield were confirmed by Coomassie Brilliant Blue staining. Antibody binding activity was measured using ELISA analysis.

[0407] As shown in Figure 2A, the different antibodies tested (i.e., 1-28, 1-85, 2-13, 2-14, 2-20, 2-29, 3-2, and 3-26) were similar in size. In addition to the 1-85 antibody, the antibodies tested were able to bind to the FAM19A5 protein at various levels. See Figure 2B.

[0408] Example 6: Analysis of the neutralizing ability of anti-FAM19A5 antibodies To further evaluate the functional properties of the antibodies, the following methods were used.

[0409] 1. Production of Recombinant FAM19A5 Rabbit Fc Fusion Protein To create a FAM19A5 expression vector, a gene encoding human FAM19A5 was chemically synthesized (Genscript, Picataway, NJ, USA). The gene was subcloned into a modified mammalian expression vector encoding the hinge region of human IgG1 and the CH2-CH3 domains of rabbit IgG in the 3' region, as previously reported. See Han, J., et al., Exp Mol Med. 48(11):e271(2016).

[0410] An expression vector encoding the FAM19A5 rabbit Fc fusion was transfected into HEK293F cells (Invitrogen, Carlsbad, CA, USA) using 25-kDa linear polyethyleneimine (Polyscience, Warrington, PA, USA) as previously described (see Boussif, O., et al., Proc Natl Acad Sci U S A. 92(16):7297-301 (1995)). The FAM19A5 rabbit fusion protein was purified from the culture supernatant of transiently transfected HEK293F cells using a protein A Sepharose column (Repligen, Waltham, MA, USA) according to the manufacturer's instructions.

[0411] 2. Production of Recombinant Anti-FAM19A5 scFv Human Cκ Fusion Protein The gene of the selected clone was subcloned into a modified pCEP4 vector encoding a Cκ domain (human immunoglobulin kappa light chain constant domain) in the 5' region, as previously reported (see Lee, Y., et al., Exp Mol Med. 46:e114 (2014)). The expression vector encoding the anti-FAM19A5 scFv-human Cκ fusion protein was transfected into HEK293F cells (Invitrogen) as described above. The scFv-hCκ fusion protein was purified from the culture supernatant of transiently transfected HEK293F cells using a Protein A Sepharose column (Repligen, Waltham, MA, USA) according to the manufacturer's instructions.

[0412] 3. Neutralizing Efficacy of Anti-FAM19A5 Antibodies on Glial Cells To assess the neutralizing efficacy of the antibodies, flow cytometry was performed as previously described (see Kim, M., et al., PLoS One. 7(4):e35100 (2012)). Mouse and human glial cells were plated at a final density of 3 x 10 per well. 5 Cells were seeded in a V-bottom 96-well plate (Corning Inc., Corning, Ny, USA). Cells were treated with 1 μM recombinant FAM19A5 rabbit Fc and 5 μM anti-FAM19A5 scFv human Cκ fusion protein in flow cytometry buffer (1% (w / v) BSA in PBS containing 0.05% (w / v) sodium azide) for 1 h at 37°C. After washing with flow cytometry buffer, cells were incubated with Alexa Fluor 488-conjugated anti-rabbit IgG (Fc-specific) antibody (Jackson Immuno Research Inc., PA, USA) for 1 h at 37°C in the dark. After further washing with the same buffer, cells were resuspended in 300 μL PBS and analyzed using a FACSCANTO microscope equipped with a 488-nm laser. TM The data were analyzed by flow cytometry using a BD Bioscience II instrument (San Jose, CA, USA). Data were analyzed with FlowJo software (TreeStar, Ashland, OR, USA).

[0413] As shown in Figure 3A, all antibodies tested (i.e., 1-28, 1-85, 2-13, 2-14, 2-20, 2-29, 3-2, and 3-26) were able to inhibit FAM19A5 interaction with mouse primary glial cells to varying degrees. In human glioblastoma cell lines, the 2-20 antibody was unable to neutralize FAM19A5 interaction with the cell lines (see Figure 3B). The other antibodies were able to neutralize FAM19A5 activity to varying degrees. The 1-28, 2-13, and 3-2 antibodies showed the greatest neutralizing efficacy in human glioblastoma cell lines.

[0414] Example 7: Epitope mapping analysis using FAM19A5 epitope fragments F1-F6 The 3-2 antibody was selected for epitope mapping analysis due to its ability to neutralize FAM19A5 expression in both mouse and human glial cells. Overlapping peptide fragments of the human FAM19A5 protein (F1–F6, see Figure 4) were synthesized and conjugated to BSA. The binding of different anti-FAM19A5 antibodies to the BSA-conjugated peptide fragments F1–F6 was determined by ELISA analysis. Briefly, FAM19A5 fragments F1–F6 (diluted to 1 μg / mL in 50 mM carbonate buffer (Biosesang) or 20 μg / mL for high-resolution analysis) were used to coat the wells of a 96-well immunoplate (Thermo Scientific) overnight at 4°C (100 μL / well), followed by washing twice with 1X PBS. The plate was then blocked with blocking buffer (100 μL / well) at room temperature for 1 hour. During the 1-hour incubation, the relevant anti-FAM19A5 antibody was diluted to 1 μg / mL (or 20 μg / mL for high-concentration assays) in dilution buffer. After the plate was washed (2x with 1x PBS), the diluted anti-FAM19A5 antibody was added to the appropriate wells and the plate was incubated at room temperature for 1 hour. The plate was then washed a total of five times with wash buffer. Next, ODP substrate (prepared by dissolving 1 aliquot of ODP purified (O-phenylenediamine dihydrochloride, Thermo) in 9 mL of sterile deionized water and 1 mL of 10x hydrogen peroxide stable buffer (Thermo)) was added to each well, and the color reaction was allowed to occur for 10 minutes. The reaction was terminated by adding 100 mL of 2N H2SO4 (Daejung) to the wells. The absorbance of each well was detected at 492 nm using a 96-well microplate reader (Molecular Devices).

[0415] As shown in Figure 5, the 3-2 antibody bound strongly to the epitope fragment F2 and minimally to the other fragments.

[0416] Next, we performed alanine scanning analysis to identify the specific amino acid residues in epitope fragment F2 to which the 3-2 antibody binds. As shown in Figure 6A, when amino acid residues R4, D5, P9, R10, or R11 were mutated to alanine, the ability of the 3-2 antibody to bind to epitope fragment F2 was significantly reduced. Similar analyses were performed on multiple deimmunized variants of the 3-2 antibody (see Example 8 for details on the deimmunization process). As shown in Figures 6B, 6C, 6D, 6F, 6G, 6I, and 6J, amino acid residues R4, P9, R10, and R11 were important for binding of antibodies 1-30, 1-32, and 6-10 to FAM19A5. In the case of antibodies 1-17 and 4-11, amino acid residues R4, P9, and R10 were important (see Figures 6E and 6H).

[0417] Example 8: Deimmunization of anti-FAM19A5 antibody 1. In silico immunogenicity assessment To reduce the risk of immunogenicity upon administration to human subjects, in silico analysis was performed to identify specific regions of high immunogenicity within the 3-2 and 2-13 anti-FAM19A5 antibodies.

[0418] iTope for overlapping 9-mer peptides across the entire sequence to identify promiscuous MHC class II peptides TM (Abzena plc., UK) analysis was performed. Potential T cell epitopes were predicted by analyzing interactions with 34 different MHC class II alleles. As shown in Figures 7 and 8, respectively, clone 3-2 contained a total of 11 binding peptides, and clone 2-13 contained a total of 10 non-germline promiscuous MHC class II binding peptides.

[0419] Furthermore, promiscuous MHC class II-binding peptides were identified using the CD4+ T cell epitope database TCED, which was ...

Claims

1. 1. An isolated antibody ("anti-FAM19A5 antibody"), or an antigen-binding portion thereof, that specifically binds to a human family member A5 (FAM19A5) protein with sequence similarity 19, and that comprises a heavy chain CDR1, CDR2, and CDR3 and a light chain CDR1, CDR2, and CDR3, the anti-FAM19A5 antibody binds to amino acids corresponding to amino acid residues 45, 46, 50, 51 and 52 (RD---PRR), amino acid residues 45, 50, 51 and 52 (R---PRR), or amino acid residues 45, 50 and 51 (R----PR) of SEQ ID NO:2; (a) (i) heavy chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO:5, (ii) heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO:6, (iii) heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:7, (iv) light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO:8, (v) light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO:9, and (vi) light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:10; (b) (i) heavy chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO:5, (ii) heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO:13, (iii) heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:7, (iv) light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO:8, (v) light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO:20, and (vi) light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:10; (c) (i) heavy chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 14, (ii) heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15, (iii) heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 7, (iv) light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 21, (v) light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 22, and (vi) light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 23; (d) (i) heavy chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 14, (ii) heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15, (iii) heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 7, (iv) light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 21, (v) light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 24, and (vi) light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 23; (e) (i) heavy chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 14, (ii) heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15, (iii) heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 7, (iv) light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 8, (v) light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 25, and (vi) light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 23; (f) (i) heavy chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 14, (ii) heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15, (iii) heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 7, (iv) light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 8, (v) light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 24, and (vi) light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 23; (g) (i) the heavy chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 14, (ii) the heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15, (iii) the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 7, (iv) the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 8, (v) the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 26, and (vi) the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 27, or (h) An antibody, wherein (i) the heavy chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 14, (ii) the heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15, (iii) the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 7, (iv) the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 8, (v) the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 28, and (vi) the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:

29.

2. 2. The antibody of claim 1, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO: 11, and / or the VL comprises an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO:

12.

3. the antibody cross-competes with a reference antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL); (a) the VH comprises the amino acid sequence set forth in SEQ ID NO: 11, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 12; (b) the VH comprises the amino acid sequence set forth in SEQ ID NO: 33 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 38; (c) the VH comprises the amino acid sequence set forth in SEQ ID NO: 34, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 39; (d) the VH comprises the amino acid sequence set forth in SEQ ID NO: 34, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 41; (e) the VH comprises the amino acid sequence set forth in SEQ ID NO: 34, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 40; (f) the VH comprises the amino acid sequence set forth in SEQ ID NO: 34, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 42; (g) the VH comprises the amino acid sequence set forth in SEQ ID NO: 34 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 43; or (h) The antibody of claim 1, wherein the VH comprises the amino acid sequence set forth in SEQ ID NO: 34 and the VL comprises the amino acid sequence set forth in SEQ ID NO:

44.

4. The antibody of claim 1 , wherein the antibody is selected from the group consisting of IgG1, IgG2, IgG3, IgG4, variants thereof, and any combination thereof.

5. The antibody of claim 1, which is a chimeric antibody, a human antibody, or a humanized antibody.

6. Fab, Fab', F(ab') ２ 10. The antibody of claim 1, comprising a single-chain Fv (scFv), Fv, or single-chain Fv (scFv).

7. The scFv comprises a VH and a VL: (a) the VH comprises the amino acid sequence set forth in SEQ ID NO: 11, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 12; (b) the VH comprises the amino acid sequence set forth in SEQ ID NO: 33 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 38; (c) the VH comprises the amino acid sequence set forth in SEQ ID NO: 34, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 39; (d) the VH comprises the amino acid sequence set forth in SEQ ID NO: 34, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 41; (e) the VH comprises the amino acid sequence set forth in SEQ ID NO: 34, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 40; (f) the VH comprises the amino acid sequence set forth in SEQ ID NO: 34, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 42; (g) the VH comprises the amino acid sequence set forth in SEQ ID NO: 34, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 43; or (h) The antibody of claim 6, wherein the VH comprises the amino acid sequence set forth in SEQ ID NO: 34 and the VL comprises the amino acid sequence set forth in SEQ ID NO:

44.

8. A nucleic acid encoding the antibody of claim 1.

9. A vector comprising the nucleic acid of claim 8.

10. A cell comprising the vector of claim 9.

11. An immunoconjugate comprising the antibody of claim 1 linked to an agent.

12. A composition comprising the antibody of claim 1, the nucleic acid of claim 8, the vector of claim 9, the cell of claim 10 or the immunoconjugate of claim 11, and a carrier.

13. A kit comprising the antibody of claim 1, the nucleic acid of claim 8, the vector of claim 9, the cell of claim 10 or the immunoconjugate of claim 11 and instructions for use.

14. A method for producing an antibody that specifically binds to human FAM19A5 protein, comprising culturing the cells of claim 10 under appropriate conditions and isolating the antibody.

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