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

Abstract

To provide an antibody that specifically binds to human FAM19A5, and a composition including the antibody.SOLUTION: In a specific aspect, antibodies specifically bind to human FAM19A5 and modulate FAM19A5 activity, for example, inhibit, suppress, reduce, or reverse onset of reactive gliosis and / or excessive proliferation of reactive astrocytes, utilizing such antibodies. The present disclosure also provides methods for treating disorders, such as central nervous system damage, a degenerative brain disorder, or a neuropathic pain, by administering an antibody that specifically binds to human FAM19A5.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to antibodies that specifically bind to Family 19, member A5 (FAM19A5) with sequence similarity, compositions comprising the antibodies, and uses of the antibodies to prevent or treat disorders or diseases such as central nervous system injuries in subjects. [Background technology]

[0002] FAM19A5 is a member of the TAFA subfamily of proteins, which consists of five highly homologous small proteins (Tang TY et al., Genomics 83(4):727-34(2004)). These proteins contain conserved cysteine ​​residues at fixed positions and are related to macrophage inflammatory protein 1-alpha (MIP-1-alpha), a member of the CC-chemokine family. TAFA proteins are preferentially expressed in specific regions of the brain and spinal cord. These proteins are known to be produced and secreted during neural development and by adult neural stem cells.

[0003] FAM19A5 is preferentially expressed in the brain of vertebrates and is believed to be important in the development, differentiation, and formation of the central nervous system, and can be used to prevent and treat damage and / or diseases of the central nervous system (Patent Document 1). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] US Patent Publication No. 2015 / 0118230 Summary of the Invention [Problem to be solved by the invention]

[0005] Inhibition of FAM19A5 may play an important role in the treatment of central nervous system disorders, and there is a need to develop antibodies that can specifically bind to FAM19A5 and modulate FAM19A5 activity. [Means for solving the problem]

[0006] Disclosed are antibodies that specifically bind to human FAM19A5, e.g., monoclonal antibodies or antigen-binding portions thereof (anti-FAM19A5 antibodies), compositions comprising the antibodies or antigen-binding portions thereof, nucleic acids encoding the antibodies or antigen-binding portions thereof, vectors comprising the nucleic acids, or cells comprising the vectors.

[0007] In one specific example (embodiment), the anti-FAM19A5 antibody is capable of competing for the human FAM19A5 epitope with: (1) a heavy chain variable region (VH) comprising SEQ ID NO:5 and a light chain variable region (VL) comprising SEQ ID NO:6; (2) a heavy chain variable region (VH) comprising SEQ ID NO:103 and a light chain variable region (VL) comprising SEQ ID NO:114; (3) a heavy chain variable region (VH) comprising SEQ ID NO:104 and a light chain variable region (VL) comprising SEQ ID NO:115; (4) a heavy chain variable region (VH) comprising SEQ ID NO:105 and a light chain variable region (VL) comprising SEQ ID NO:116; (5) a heavy chain variable region (VH) comprising SEQ ID NO:106 and a light chain variable region (VL) comprising SEQ ID NO:117; (6) a heavy chain variable region (VH) comprising SEQ ID NO:107 and a light chain variable region (VL) comprising SEQ ID NO:108; (7) a heavy chain variable region (VH) comprising SEQ ID NO: 108 and a light chain variable region (VL) comprising SEQ ID NO: 119; (8) a heavy chain variable region (VH) comprising SEQ ID NO: 109 and a light chain variable region (VL) comprising SEQ ID NO: 120; (9) a heavy chain variable region (VH) comprising SEQ ID NO: 110 and a light chain variable region (VL) comprising SEQ ID NO: 121; (10) a heavy chain variable region (VH) comprising SEQ ID NO: 111 and a light chain variable region (VL) comprising SEQ ID NO: 122; (11) a heavy chain variable region (VH) comprising SEQ ID NO: 112 and a light chain variable region (VL) comprising SEQ ID NO: 123; or (12) a heavy chain variable region (VH) comprising SEQ ID NO: 113 and a light chain variable region (VL) comprising SEQ ID NO: 124. It cross-competes with a reference antibody that comprises a light chain variable region (VL) that comprises NO: 124. In one embodiment, the anti-FAM19A5 antibody binds to the same FAM19A5 epitope as the reference antibody.

[0008] In one embodiment, the anti-FAM19A5 antibody binds to at least one FAM19A5 epitope in SEQ ID NO:2 at one or more amino acids corresponding to amino acid residues 99 to 107 of SEQ ID NO:4 (i.e., EGCDLLINR), e.g., amino acid residues 102, 103, 105 and 107 (i.e., DL-IR), amino acid residues 99, 100, 102, 103, 105 and 107 (i.e., EG-DL-IR), amino acid residues 99, 100 and 107 (i.e., EG----R). In one embodiment, the anti-FAM19A5 antibody binds to at least one FAM19A5 epitope identified as EP6, EP7 and / or EP8, where EP6 comprises, consists essentially of, or consists of the amino acids KTKQWCDML (SEQ ID NO:139), EP7 comprises, consists essentially of, or consists of the amino acids GCDLLINR (SEQ ID NO:140), and EP8 comprises, consists essentially of, or consists of the amino acids TCTQPGGR (SEQ ID NO:141).

[0009] In one embodiment, the anti-FAM19A5 antibody comprises a heavy chain CDR1, CDR2 and CDR3, and a light chain CDR1, CDR2 and CDR3, wherein (i) the heavy chain CDR3 comprises SEQ ID NO:9, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:43, SEQ ID NO:49, SEQ ID NO:55, SEQ ID NO:61, SEQ ID NO:67, SEQ ID NO:73, SEQ ID NO:79, SEQ ID NO:85, or SEQ ID NO:91; (ii) the heavy chain CDR1 comprises SEQ ID NO:7, SEQ ID NO:29, SEQ ID NO:35, SEQ ID NO:41, SEQ ID NO:47, SEQ ID NO:53, SEQ ID NO:59, SEQ ID NO:65, SEQ ID NO:71, SEQ ID NO:77, SEQ ID (iii) the heavy chain CDR2 comprises SEQ ID NO:8, SEQ ID NO:30, SEQ ID NO:36, SEQ ID NO:42, SEQ ID NO:48, SEQ ID NO:54, SEQ ID NO:60, SEQ ID NO:66, SEQ ID NO:72, SEQ ID NO:78, SEQ ID NO:84, or SEQ ID NO:90; (iv) the light chain CDR1 comprises SEQ ID NO:10, SEQ ID NO:32, SEQ ID NO:38, SEQ ID NO:44, SEQ ID NO:50, SEQ ID NO:56, SEQ ID NO:62, SEQ ID NO:68, SEQ ID NO:74, SEQ ID NO:80, SEQ ID NO:86, or SEQ ID NO:92; (v) the light chain CDR2 comprises SEQ ID SEQ ID NO:11, SEQ ID NO:33, SEQ ID NO:39, SEQ ID NO:45, SEQ ID NO:51, SEQ ID NO:57, SEQ ID NO:63, SEQ ID NO:69, SEQ ID NO:75, SEQ ID NO:81, SEQ ID NO:87, or SEQ ID NO:93;and / or (vi) the light chain CDR3 comprises SEQ ID NO:12, SEQ ID NO:34, SEQ ID NO:40, SEQ ID NO:46, SEQ ID NO:52, SEQ ID NO:58, SEQ ID NO:64, SEQ ID NO:70, SEQ ID NO:76, SEQ ID NO:82, SEQ ID NO:88, or SEQ ID NO:94;

[0010] In one embodiment, the anti-FAM19A5 antibody comprises: (1) a heavy chain variable region (VH) comprising SEQ ID NO:5 and a light chain variable region (VL) comprising SEQ ID NO:6; (2) a heavy chain variable region (VH) comprising SEQ ID NO:103 and a light chain variable region (VL) comprising SEQ ID NO:114; (3) a heavy chain variable region (VH) comprising SEQ ID NO:104 and a light chain variable region (VL) comprising SEQ ID NO:115; (4) a heavy chain variable region (VH) comprising SEQ ID NO:105 and a light chain variable region (VL) comprising SEQ ID NO:116; (5) a heavy chain variable region (VH) comprising SEQ ID NO:106 and a light chain variable region (VL) comprising SEQ ID NO:117; (6) a heavy chain variable region (VH) comprising SEQ ID NO:107 and a light chain variable region (VL) comprising SEQ ID NO:118; (7) a heavy chain variable region (VH) comprising SEQ ID NO:107 and a light chain variable region (VL) comprising SEQ ID NO:118; (8) a heavy chain variable region (VH) comprising SEQ ID NO:109 and a light chain variable region (VL) comprising SEQ ID NO:120; (9) a heavy chain variable region (VH) comprising SEQ ID NO:110 and a light chain variable region (VL) comprising SEQ ID NO:121; (10) a heavy chain variable region (VH) comprising SEQ ID NO:111 and a light chain variable region (VL) comprising SEQ ID NO:122; (11) a heavy chain variable region (VH) comprising SEQ ID NO:112 and a light chain variable region (VL) comprising SEQ ID NO:123; or (12) a heavy chain variable region (VH) comprising SEQ ID NO:113 and a light chain variable region (VL) comprising SEQ ID NO:124.

[0011] In one embodiment, the anti-FAM19A5 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region 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 NOs:5 and 103-113, and / or the light chain variable region 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 NOs:6 and 114-124.

[0012] In one embodiment, the anti-FAM19A5 antibody is a chimeric antibody, a human antibody or a humanized antibody.

[0013] In one specific example, the anti-FAM19A5 antibody comprises a heavy chain comprising SEQ ID NOs:27 and 145-155 and a light chain comprising SEQ ID NOs:28 and 156-166.

[0014] In one embodiment, an anti-FAM19A5 antibody exhibits any one or more of the following characteristics: (a) K of 10 nM or less as measured by enzyme-linked immunosorbent assay (ELISA). D binds to soluble human FAM19A5; (b) K of 10 nM or less as measured by ELISA D binds to membrane-bound human FAM19A5; (c) reducing, reversing, delaying, and / or preventing the onset of reactive gliosis; (d) inhibiting excessive proliferation of reactive astrocytes; (e) decreasing the expression of chondroitin sulfate proteoglycans, including neurocan and neuron-glial antigen 2 (NG2); (f) increasing the expression of c-fos and pERK in the neuronal nuclei; (g) promoting neuronal survival; (h) increasing the expression of GAP43 in neurons; and (i) Promotes axon regrowth.

[0015] In one embodiment, the present invention provides a FAM19A5 epitope that consists essentially of, or consists of, 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 NOs:2 and 139-141, which epitope can be specifically bound by a reference antibody comprising a heavy chain variable region and a light chain variable region as presented in Tables 4 and 5, respectively.

[0016] In one embodiment, the invention provides nucleic acids encoding the disclosed anti-FAM19A5 antibodies or epitopes.

[0017] In one embodiment, the invention provides a composition comprising the disclosed anti-FAM19A5 antibody and a carrier.

[0018] In one embodiment, the invention provides an anti-FAM19A5 antibody for use in the therapeutic treatment of a disease or condition.

[0019] Examples Example 1 1. An isolated monoclonal antibody, or antigen-binding portion thereof, that specifically binds to human family 19, member A5 (FAM19A5) with sequence similarity and exhibits any one or more of the following properties: (a) K of 10 nM or less as measured by enzyme-linked immunosorbent assay (ELISA). D binds to soluble human FAM19A5; (b) K of 10 nM or less as measured by ELISA D binds to membrane-bound human FAM19A5; (c) reducing, reversing, delaying, and / or preventing the onset of reactive gliosis; (d) inhibiting excessive proliferation of reactive astrocytes; (e) decreasing the expression of chondroitin sulfate proteoglycans, including neurocan and neuron-glial antigen 2 (NG2); (f) increasing the expression of c-fos and pERK in the neuronal nuclei; (g) promoting neuronal survival; (h) increasing the expression of GAP43 in neurons; and (i) Promotes axon regrowth.

[0020] Example 2 An isolated monoclonal antibody or antigen-binding portion thereof, which competes with a reference antibody comprising heavy chain CDR1, CDR2 and CDR3, and light chain CDR1, CDR2 and CDR3 for binding to a human FAM19A5 epitope, wherein the heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO:7, the heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO:8, the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO:9, the light chain CDR1 comprises the amino acid sequence of SEQ ID NO:10, the light chain CDR2 comprises the amino acid sequence of SEQ ID NO:11, and the light chain CDR3 comprises the amino acid sequence of SEQ ID NO:12.

[0021] Example 3 An isolated monoclonal antibody, or an antigen-binding portion thereof, that binds to the same FAM19A5 epitope as a reference antibody comprising heavy chain CDR1, CDR2 and CDR3, and light chain CDR1, CDR2 and CDR3, wherein heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO:7, heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO:8, heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO:9, light chain CDR1 comprises the amino acid sequence of SEQ ID NO:10, light chain CDR2 comprises the amino acid sequence of SEQ ID NO:11, and light chain CDR3 comprises the amino acid sequence of SEQ ID NO:12.

[0022] Example 4 The monoclonal antibody or antigen-binding portion thereof of Example 2 or 3, which binds to at least one FAM19A5 epitope, which is SEQ ID NO:2.

[0023] Example 5 The monoclonal antibody or antigen-binding portion thereof of any one of Examples 2 to 4, which binds only to FAM19A5, and wherein the antibody or antigen-binding portion thereof is SEQ ID NO:2.

[0024] Example 6 The monoclonal antibody or antigen-binding portion thereof of any one of Examples 2-4, wherein the antibody or antigen-binding portion thereof further binds to an additional FAM19A5 epitope.

[0025] Example 7 The monoclonal antibody or antigen-binding portion thereof of any one of specific examples 2 to 4, wherein the antibody or antigen-binding portion thereof further binds to an additional FAM19A5 epitope selected from the group consisting of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, and any combination thereof.

[0026] Example 8 Any one of the preceding specific examples of a monoclonal antibody or antigen-binding portion thereof, wherein the monoclonal antibody or antigen-binding portion thereof comprises heavy chain CDR1, CDR2 and CDR3, and light chain CDR1, CDR2 and CDR3.

[0027] Example 9 The monoclonal antibody or antigen-binding portion thereof of embodiment 8, wherein the heavy chain CDR3 of the monoclonal antibody or antigen-binding portion thereof comprises SEQ ID NO:9.

[0028] Example 10 10. The monoclonal antibody or antigen-binding portion thereof of embodiment 8 or 9, wherein the heavy chain CDR1 of the monoclonal antibody or antigen-binding portion thereof comprises SEQ ID NO:7.

[0029] Example 11 11. The monoclonal antibody or antigen-binding portion thereof of any one of specific examples 8 to 10, wherein the heavy chain CDR2 of the monoclonal antibody or antigen-binding portion thereof comprises SEQ ID NO:8.

[0030] Example 12 12. The monoclonal antibody or antigen-binding portion thereof of any one of specific examples 8 to 11, wherein the light chain CDR1 of the monoclonal antibody or antigen-binding portion thereof comprises SEQ ID NO:10.

[0031] Example 13 13. The monoclonal antibody or antigen-binding portion thereof of any one of specific examples 8 to 12, wherein the light chain CDR2 of the monoclonal antibody or antigen-binding portion thereof comprises SEQ ID NO:11.

[0032] Example 14 14. The monoclonal antibody or antigen-binding portion thereof of any one of specific examples 8 to 13, wherein the light chain CDR3 of the monoclonal antibody or antigen-binding portion thereof comprises SEQ ID NO:12.

[0033] Example 15 A monoclonal antibody or antigen-binding portion thereof according to any one of the preceding embodiments, comprising a heavy chain variable domain comprising SEQ ID NO:5 and a light chain variable domain comprising SEQ ID NO:6.

[0034] Example 16 Any one of the preceding specific examples of a monoclonal antibody or antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region 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:5.

[0035] Example 17 Any one of the preceding embodiments of a monoclonal antibody or antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein the light chain variable region 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:6.

[0036] Example 18 The monoclonal antibody of any one of the preceding embodiments, wherein the antibody is a single domain antibody.

[0037] Example 19 The monoclonal antibody or antigen-binding portion thereof according to any one of the preceding embodiments, wherein the antibody is selected from the group consisting of IgG1, IgG2, IgG3, IgG4 and variants thereof.

[0038] Example 20 20. The monoclonal antibody or antigen-binding portion thereof of embodiment 19, wherein the antibody is an IgG2 antibody, an IgG4 antibody, or a combination thereof.

[0039] Example 21 20. The monoclonal antibody or antigen-binding portion thereof of embodiment 19, wherein the antibody comprises an IgG2 / IgG4 isotype antibody.

[0040] Example 22 2. The monoclonal antibody or antigen-binding portion thereof according to any one of the preceding embodiments, further comprising a constant region that lacks Fc function.

[0041] Example 23 A monoclonal antibody or antigen-binding portion thereof according to any one of the preceding embodiments, which is a chimeric antibody, a human antibody or a humanized antibody.

[0042] Example 24 The monoclonal antibody of any one of embodiments 1-23, wherein the monoclonal antibody comprises a heavy chain comprising SEQ ID NO:27 and a light chain comprising SEQ ID NO:28.

[0043] Example 25 The antigen-binding portion of any one of Examples 1-23, wherein the antigen-binding portion is a Fab, Fab', F(ab')2, Fv, or single chain Fv (scFv).

[0044] Example 26 A bispecific molecule comprising a monoclonal antibody, or antigen-binding portion thereof, according to any one of the preceding embodiments linked to a molecule having a second binding moiety.

[0045] Example 27 A human Family 19, member A5 (FAM19A5) epitope with sequence similarity that consists essentially of or consists of 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:2 and that can be specifically bound by a reference antibody comprising a heavy chain variable region of SEQ ID NO:5 and a light chain variable region of SEQ ID NO:6.

[0046] Example 28 A nucleic acid encoding the monoclonal antibody or antigen-binding portion thereof of any one of Examples 1-25, the bispecific molecule of Example 26, or the epitope of Example 27.

[0047] Example 29 A vector comprising the nucleic acid of Example 28.

[0048] Example 30 The vector of embodiment 29 for use in gene therapy.

[0049] Example 31 A cell transformed with the expression vector of Example 29.

[0050] Example 32 An immunoconjugate comprising a monoclonal antibody or antigen-binding portion thereof according to any one of Examples 1 to 25 or a bispecific molecule of Example 26, bound to a pharmaceutical agent.

[0051] Example 33 A composition comprising the monoclonal antibody or antigen-binding portion thereof of any one of Examples 1 to 25, the bispecific molecule of Example 26, or the immunoconjugate of Example 32, and a carrier.

[0052] Example 34 A kit comprising the monoclonal antibody or antigen-binding portion thereof of any one of Examples 1 to 25, the bispecific molecule of Example 26, or the immunoconjugate of Example 32, and instructions for use.

[0053] Example 35 A method of making an anti-FAM19A5 antibody or antigen-binding portion thereof, comprising the steps of immunizing a non-human animal with the epitope of Example 27 and producing the antibody or antigen-binding portion thereof.

[0054] Example 36 A method for producing an anti-FAM19A5 antibody or antigen-binding portion thereof, comprising culturing the host cell of Example 31 under suitable conditions and isolating the antibody or antigen-binding portion thereof.

[0055] Example 37 A method for reducing, reversing, delaying and / or preventing the onset of reactive gliosis in a subject, comprising administering a monoclonal antibody or antigen-binding portion thereof of Examples 1 to 25, a bispecific molecule of Example 26, or an immunoconjugate of Example 32, such that the onset of gliosis is delayed.

[0056] Example 38 A method of inhibiting excessive proliferation of reactive astrocytes in a subject, comprising administering to the subject a monoclonal antibody or antigen-binding portion thereof of Examples 1-25, a bispecific molecule of Example 26, or an immunoconjugate of Example 32, such that excessive proliferation of reactive astrocytes is inhibited.

[0057] Example 39 A method for reducing expression of neurocan and chondroitin sulfate proteoglycans, including neuron-glial antigen 2 (NG2), in a subject, comprising administering to the subject a monoclonal antibody or antigen-binding portion thereof of Examples 1-25, a bispecific molecule of Example 26, or an immunoconjugate of Example 32, wherein expression of neurocan and chondroitin sulfate proteoglycans, including neuron-glial antigen 2 (NG2), is reduced.

[0058] Example 40 A method for promoting neuronal survival in a subject, comprising administering to the subject a monoclonal antibody or antigen-binding portion thereof of Examples 1-25, a bispecific molecule of Example 26, or an immunoconjugate of Example 32, wherein survival of the neuron is promoted.

[0059] Example 41 A method for promoting axon regrowth in a subject, comprising administering to the subject a monoclonal antibody or antigen-binding portion thereof of Examples 1 to 25, a bispecific molecule of Example 26, or an immunoconjugate of Example 32, wherein axon regrowth is promoted.

[0060] Example 42 A method for treating central nervous system injury in a subject, comprising administering to the subject a monoclonal antibody or antigen-binding portion thereof of Examples 1 to 25, a bispecific molecule of Example 26, or an immunoconjugate of Example 32.

[0061] Example 43 The method of embodiment 42, wherein the central nervous system injury includes traumatic brain injury, cerebrospinal cord injury, stroke, and brain tumor.

[0062] Example 44 A method for treating a cerebrospinal or neurological disorder in a subject, comprising administering to the subject a monoclonal antibody or antigen-binding portion thereof of Examples 1 to 25, a bispecific molecule of Example 26, or an immunoconjugate of Example 32.

[0063] Example 45 The method of embodiment 44, wherein the degenerative brain disorders include Huntington's disease, Parkinson's disease, Alzheimer's disease, multiple sclerosis, and amyotrophic lateral sclerosis (ALS).

[0064] Example 46 A method for treating neuropathic pain in a subject, comprising administering to the subject a monoclonal antibody or antigen-binding portion thereof of Examples 1 to 25, a bispecific molecule of Example 26, or an immunoconjugate of Example 32.

[0065] Example 47 The method of any one of Examples 35-46, wherein the monoclonal antibody, or antigen-binding portion thereof, is administered intravenously, orally, parenterally, intraspinal, intracerebroventricular, pulmonary, subcutaneous, or intraventricularly.

[0066] Example 48 The method of any one of embodiments 35-47, wherein the subject is a human.

[0067] Example 49 Use of a monoclonal antibody or antigen-binding portion thereof according to any one of Examples 1 to 25, a bispecific molecule of Example 26 or an immunoconjugate of Example 32 for the preparation of a medicament for the treatment of central nervous system damage, a degenerative brain disorder or neuropathic pain.

[0068] Example 50 Specific examples of the monoclonal antibody or antigen-binding portion thereof according to any one of claims 1 to 49 for use in therapeutic treatment of a disease or condition.

[0069] Example 51 51. The monoclonal antibody, or antigen-binding portion thereof, for use in accordance with embodiment 50, wherein the disease or condition is a cerebrospinal system injury, a degenerative cerebrospinal or neurological disorder, or neuropathic pain.

[0070] Example 52 A method for diagnosing a subject, comprising contacting a biological sample from the subject with any one of the monoclonal antibodies of Examples 1-49. [Brief description of the drawings]

[0071] [Figure 1A] Binding analysis of individual scFv clones to FAM19A5 protein. Absorbance was measured at 405 nM. Clone numbers are indicated on the x-axis. Analysis of 96 clones from the tertiary bio-panning derived from the first chicken is shown. [Figure 1B] Binding analysis of individual scFv clones to FAM19A5 protein. Absorbance was measured at 405 nM. Clone numbers are indicated on the x-axis. Analysis of 96 clones from the fourth biopanning derived from the second chicken is shown. [Figure 1C] Binding analysis of individual scFv clones to FAM19A5 protein. Absorbance was measured at 405 nM. Clone numbers are indicated on the x-axis. Analysis of 96 clones from the 5th biopanning derived from chicken 3 is shown. [Diagram 2] FIG. 2 shows a schematic diagram for subcloning of anti-FAM19A5 antibody (scFv) into a mammalian expression vector. [Diagram 3] Figure 3 shows the SDS-PAGE results of chimeric anti-FAM19A5-IgG2 / 4 monoclonal antibody (1-65). The left panel shows reduced SDS-PAGE, and the right panel shows non-reduced SDS-PAGE. [Figure 4] FIG. 4 shows that chimeric anti-FAM19A5-IgG2 / 4 monoclonal antibody (1-65) specifically binds to human FAM19A5. [Figure 5A] FIG. 5A shows that the chimeric anti-FAM19A5-IgG2 / 4 monoclonal antibody (1-65) is specific for the FAM19A5 protein but does not bind to other proteins of the FAM19A subfamily. [Figure 5B]FIG. 5B shows immunocytochemical staining (ICC, left panel) and immunohistochemical staining (IHC, right three panels) analysis for FAM19A5 expression. The ICC results show that chimeric anti-FAM19A5-IgG2 / 4 monoclonal antibody (1-65) binds to FAM19A5 protein conjugated with His tag. The IHC results show that FAM19A5 protein is expressed in GFAP protein-expressing cells in the subventricular zone of normal mice (left), in the injured penumbra of mice with traumatic brain injury for 5 days (TBI5D, center), and in the injured penumbra of rats with ischemic brain injury for 7 days (IBI7D, right). [Figure 6] Figure 6 shows that chimeric anti-FAM19A5-IgG2 / 4 monoclonal antibody (1-65) (lower panel) inhibits, reduces, delays, and / or reverses the onset of gliosis in a mouse model of traumatic brain injury (TBI). Normal human control immunoglobulin (HCI) was used as a control (upper panel). Anti-FAM19A5 antibody or control antibody was administered to mice at one of the following concentrations: 0.1 μg, 0.3 μg, 1 μg, 3 μg, 5 μg, or 10 μg. [Figure 7] FIG. 7 shows the neuroprotective effect of chimeric anti-FAM19A5-IgG2 / 4 monoclonal antibody (1-65) (right panel) in a traumatic brain injury mouse model. Normal human control immunoglobulin (HCI) was used as a control (left panel). Animals were administered 5 μg of antibody. The top panel shows GFAP (glial fibrillary acidic protein) expression within the penumbra of the injured area. The bottom panel shows co-expression of GFAP and NeuN (neuronal nuclei) within a magnified section of the injured area in TBI mice. The dotted line indicates the lesion border after exposure to traumatic brain injury. [Figure 8]FIG. 8 shows the functional recovery results of animals with spinal cord injury treated with humanized anti-FAM19A5-IgG2 / 4 antibody (1-65) (circles, group C), rabbit anti-FAM19A5 polyclonal antibody (squares, group B), or vehicle control group (diamonds, group A). ​​Treatment of animals with anti-FAM19A5 antibody improves locomotor activity in both the BBB motility assay (left) and the inclined plane test (right). The table below each figure shows the specific points measured at different days after TBI induction ("dpi"). [Figure 9] Figure 9 shows the alignment of FAM19A5 amino acid sequences of different species (i.e., human, mouse, rat and chicken). Fragments F1-F6 used for epitope mapping analysis are indicated. The signal peptide is underlined. [Figure 10] Figure 10 shows the amino acid sequences of epitopes F1-F6 (conjugated to BSA) and their position on the human FAM19A5 polypeptide. The top amino acid sequence is wild type FAM19A5 isoform 2 (no signal peptide). The bottom amino acid sequence is the same sequence but with cysteine ​​residues mutated to serine during peptide synthesis to reduce non-specific activity. The sizes of the individual epitope fragments are indicated in brackets. [Figure 11] FIG. 11 shows the Western blot results for the binding of monoclonal antibody clone 1-65 to epitope fragments F1 to F6 (lanes 3 to 8, respectively). FAM19A5-CK (lane 1), PSA-CK (lane 2), peptide NDV-BSA (lane 9) and BSA (lane 10) were used as controls. The size of each of the antigens used is indicated on the right side of the blot. The amount of antigen used per well is 300 ng. The primary antibody used for the Western blot was 1-65-scFv-rabbit-Fc-SSS (2 μg / ml) and the secondary antibody used was anti-rabbit IgG (Fc specific)-HRP (1:4000). [Figure 12A]ELISA results for binding of several anti-FAM19A5 antibodies to epitope fragments F1 to F6 are shown. Results are shown for anti-FAM19A5 antibodies 1-65, 2-13, and 3-2. For the 3-2 antibody, two different isotypes are shown: human IgG1 ("h3-2") and mouse IgG1 ("m3-2"). [Figure 12B] ELISA results for binding of several anti-FAM19A5 antibodies to epitope fragments F1-F6 are shown. Results for anti-FAM19A5P2-C12 antibody are shown. For each antibody, the 1st, 2nd, 3rd, 4th, 5th and 6th bars (starting from the left) show binding to epitope fragments F1, F2, F3, F4, F5 and F6, respectively. The rightmost bar (black color) represents the positive control group (i.e., His-tagged FAM19A5 protein). The exact OD value is indicated on top of each bar. [Figure 13A] Figure 1 shows the ELISA results for binding of anti-FAM19A5 antibody 1-65 to eight different FAM19A5 fragment 5 mutant peptides (F5-1 to F5-8). Exact OD values ​​are indicated at the top of each bar. [Figure 13B] Figure 1 shows the ELISA results for binding of anti-FAM19A5P2-C12 to eight different FAM19A5 fragment 5 mutant peptides (F5-1 to F5-8). Exact OD values ​​are indicated at the top of each bar. [Figure 14] Figure 14 shows the amino acid sequence alignment of individual members of the FAM19A family (i.e., FAM19A1-5). Regions of greatest amino acid diversity among the members are boxed and designated EP1-EP8. [Figure 15A] ELISA results for binding of individual anti-FAM19A5 antibodies to FAM19A5 mutations M1-M8 are shown. Results are shown for anti-FAM19A5 antibodies 1-65, 1-28, 2-13 and 3-2. The eight bars for the antibodies correspond to mutations M1-M8 (from left to right). [Figure 15B]ELISA results for binding of individual anti-FAM19A5 antibodies to FAM19A5 mutations M1-M8 are shown. Results are shown for anti-FAM19A5 antibodies 13B4, 13F7 and 15A9. The eight bars for the antibodies correspond to mutations M1-M8 (from left to right). [Figure 15C] ELISA results for binding of individual anti-FAM19A5 antibodies to FAM19A5 mutations M1-M8 are shown. Results are shown for anti-FAM19A5 antibodies P1-A08, P1-F02, P1-G09, P2-A01, P2-A03, P2-C12, P2-F07 and P2-F11. The eight bars for the antibodies correspond to mutations M1-M8 (from left to right). [Figure 16A] FIG. 1 shows a schematic diagram of the two-site sandwich ELISA assay used to assess cross-competition between different anti-FAM19A5 antibodies. [Figure 16B] Figure 1 shows the results of a cross-competition analysis for six different anti-FAM19A5 antibodies: 1-65, P2-A03, P2-F11, 13B4, 2-13 and 3-2. The term "S / N" refers to the signal-to-noise ratio, which is measured as follows: [OD at 10 ng / mL antigen] / [OD at 0 ng / mL antigen]. Grey boxes represent cross-competition (i.e., S / N ratios less than 2). [Figure 17A] 1 shows the results of an OCTET assay for the binding of antibody 1-65 to FAM19A5. Kd values ​​are also displayed for each antibody. Each line corresponds to a distinct concentration of anti-FAM19A5 antibody used in the assay (i.e., 300 nM, 100 nM, 33 nM, 11 nM, 3.3 nM, or 1.1 nM). [Figure 17B] 1 shows the results of an OCTET assay for the binding of antibody 13B4 to FAM19A5. Kd values ​​are also displayed for each antibody. Each line corresponds to a distinct concentration of anti-FAM19A5 antibody used in the assay (i.e., 300 nM, 100 nM, 33 nM, 11 nM, 3.3 nM, or 1.1 nM). [Figure 17C]1 shows the results of an OCTET assay for the binding of antibody 13F7 to FAM19A5. Kd values ​​are also displayed for each antibody. Each line corresponds to a distinct concentration of anti-FAM19A5 antibody used in the assay (i.e., 300 nM, 100 nM, 33 nM, 11 nM, 3.3 nM, or 1.1 nM). [Figure 17D] 1 shows the results of an OCTET assay for the binding of antibody 15A9 to FAM19A5. Kd values ​​are also displayed for each antibody. Each line corresponds to a distinct concentration of anti-FAM19A5 antibody used in the assay (i.e., 300 nM, 100 nM, 33 nM, 11 nM, 3.3 nM, or 1.1 nM). [Figure 18A] ELISA results for binding of several anti-FAM19A5 antibodies to FAM19A5 are shown. Results for the following antibodies are shown (from left to right): 1-65, 13B4, 13F7, 1-28, 2-13, 3-2, P1-A03, P1-A08, P1-D03, P1-F02, P1-G09, P2-A01, P2-A03, P2-C12, P2-F07 and P2-F11. Bar graph showing results for various concentrations of anti-FAM19A5 antibodies. [Figure 18B] ELISA results for binding of several anti-FAM19A5 antibodies to FAM19A5 are shown. Results for the following antibodies are shown: 1-65, 13B4, 13F7, 1-28, 2-13, 3-2, P1-A03, P1-A08, P1-D03, P1-F02, P1-G09, P2-A01, P2-A03, P2-C12, P2-F07 and P2-F11 (from left to right). Kd (nM) for individual anti-FAM19A5 antibodies is shown. [Figure 19A]Representative immunohistochemical staining images of damaged areas of brain tissue from animals treated with different FAM19A5 antibodies (100 μg / mouse) are provided. Representative images for antibody 13B4 at two different concentrations, 100 μg (top row) and 10 μg (bottom row), are shown. In each image, brain tissue sections were stained for GFAP (glial fibrillary acidic protein, green) and nestin (red), known to be derived from reactive astrocytes after brain injury. Dotted lines indicate lesion borders after exposure to traumatic brain injury. [Figure 19B] Representative immunohistochemical staining images of damaged areas of brain tissue from animals treated with different FAM19A5 antibodies (100 μg / mouse) are provided. Six different representative images for antibody 13F7 are shown. In each image, brain tissue sections were stained for GFAP (glial fibrillary acidic protein, green) and nestin (red), known to be derived from reactive astrocytes after brain injury. Dotted lines indicate lesion borders after exposure to traumatic brain injury. [Figure 19C] Representative immunohistochemical staining images of damaged areas of brain tissue from animals treated with different FAM19A5 antibodies (100 μg / mouse) are provided. Three different representative images for antibody 15A9 are shown. In each image, brain tissue sections were stained for GFAP (glial fibrillary acidic protein, green) and nestin (red), known to be derived from reactive astrocytes after brain injury. Dotted lines indicate lesion borders after exposure to traumatic brain injury. [Figure 19D] Representative immunohistochemical staining images of damaged areas of brain tissue from animals treated with different FAM19A5 antibodies (100 μg / mouse) are provided. Three representative images are shown for antibodies P2-A03 (top row) and P2-F11 (bottom row), respectively. In each image, brain tissue sections were stained with GFAP (glial fibrillary acidic protein, green) and nestin (red), which are known to be derived from reactive astrocytes after brain injury. Dotted lines indicate the lesion borders after exposure to traumatic brain injury. [Figure 19E]Representative immunohistochemical staining images of damaged areas of brain tissue from animals treated with different FAM19A5 antibodies (100 μg / mouse) are provided. Three representative images for antibody P1-A03 are shown. In each image, brain tissue sections were stained for GFAP (glial fibrillary acidic protein, green) and nestin (red), which are known to be derived from reactive astrocytes after brain injury. Dotted lines indicate the lesion borders after exposure to traumatic brain injury. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0072] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 418,674, filed November 7, 2016, the entire contents of which are incorporated herein by reference.

[0073] References to electronically submitted sequence listings The entire contents of the sequence listing submitted electronically in an ASCII text file submitted with this application (Filename: 3763.003PC01_ST25.txt, Size: 133,676 bytes, Created: November 6, 2017) are incorporated herein by reference.

[0074] Disclosed is an isolated monoclonal antibody, or antigen-binding portion thereof, that specifically binds to human family 19, member 5 (FAM19A5) with sequence similarity and exhibits one or more of the properties disclosed herein.

[0075] In one embodiment, the monoclonal antibody, or antigen-binding portion thereof, competes with a reference antibody comprising a heavy chain and a light chain for binding to a human FAM19A5 epitope, and (i) the heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO:7; SEQ ID NO:29, SEQ ID NO:35, SEQ ID NO:41, SEQ ID NO:47, SEQ ID NO:53, SEQ ID NO:59, SEQ ID NO:65, SEQ ID NO:71, SEQ ID NO:77, SEQ ID NO:83 or SEQ ID NO:89, and (ii) the heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO:8; SEQ ID NO:30, SEQ ID NO:36, SEQ ID NO:42, SEQ ID NO:48, SEQ ID NO:54, SEQ ID NO:60, SEQ ID NO:66, SEQ ID NO:72, SEQ ID NO:78, SEQ ID NO:89, and and (iii) the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO:9; SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:43, SEQ ID NO:49, SEQ ID NO:55, SEQ ID NO:61, SEQ ID NO:67, SEQ ID NO:73, SEQ ID NO:79, SEQ ID NO:85 or SEQ ID NO:91.In some embodiments, (iv) the light chain CDR1 comprises the amino acid sequence of SEQ ID NO:10; SEQ ID NO:32, SEQ ID NO:38, SEQ ID NO:44, SEQ ID NO:50, SEQ ID NO:56, SEQ ID NO:62, SEQ ID NO:68, SEQ ID NO:74, SEQ ID NO:80, SEQ ID NO:86 or SEQ ID NO:92; (v) the light chain CDR2 comprises the amino acid sequence of SEQ ID NO:11; SEQ ID NO:33, SEQ ID NO:39, SEQ ID NO:45, SEQ ID NO:51, SEQ ID NO:57, SEQ ID NO:63, SEQ ID NO:69, SEQ ID NO:75, SEQ ID NO:81, SEQ ID NO:87 or SEQ ID NO:93; and (vi) the light chain CDR3 comprises the amino acid sequence of SEQ ID NO:12; SEQ ID NO:34, SEQ ID NO:40, SEQ ID NO:46, SEQ ID NO:52, SEQ ID NO:58, SEQ ID NO:64, SEQ ID NO:70, SEQ ID NO:76, SEQ ID NO:82, SEQ ID NO:88 or SEQ ID NO:94.

[0076] In some embodiments, the reference antibody is selected from the group consisting of: (1) a heavy chain variable region (VH) comprising SEQ ID NO:5 and a light chain variable region (VL) comprising SEQ ID NO:6; (2) a heavy chain variable region (VH) comprising SEQ ID NO:103 and a light chain variable region (VL) comprising SEQ ID NO:114; (3) a heavy chain variable region (VH) comprising SEQ ID NO:104 and a light chain variable region (VL) comprising SEQ ID NO:115; (4) a heavy chain variable region (VH) comprising SEQ ID NO:105 and a light chain variable region (VL) comprising SEQ ID NO:116; (5) a heavy chain variable region (VH) comprising SEQ ID NO:106 and a light chain variable region (VL) comprising SEQ ID NO:117; (6) a heavy chain variable region (VH) comprising SEQ ID NO:107 and a light chain variable region (VL) comprising SEQ ID NO:118; (7) a heavy chain variable region (VH) comprising SEQ ID NO:107 and a light chain variable region (VL) comprising SEQ ID NO:118; (8) a heavy chain variable region (VH) comprising SEQ ID NO:109 and a light chain variable region (VL) comprising SEQ ID NO:120; (9) a heavy chain variable region (VH) comprising SEQ ID NO:110 and a light chain variable region (VL) comprising SEQ ID NO:121; (10) a heavy chain variable region (VH) comprising SEQ ID NO:111 and a light chain variable region (VL) comprising SEQ ID NO:122; (11) a heavy chain variable region (VH) comprising SEQ ID NO:112 and a light chain variable region (VL) comprising SEQ ID NO:123; or (12) a heavy chain variable region (VH) comprising SEQ ID NO:113 and a light chain variable region (VL) comprising SEQ ID NO:124.

[0077] In some embodiments, the monoclonal antibody, or antigen binding portion thereof, binds to at least one FAM19A5 epitope, which is SEQ ID NO: 2. In other embodiments, the monoclonal antibody, or antigen binding portion thereof, binds only one FAM19A5 epitope, which is SEQ ID NO: 2. In some embodiments, the monoclonal antibody, or antigen binding portion thereof, further binds to an additional FAM19A5 epitope selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and combinations thereof.

[0078] In some embodiments, the monoclonal antibody or antigen binding portion thereof binds to at least one FAM19A5 epitope identified as EP6, EP7, and / or EP8, where EP6 comprises, consists essentially of, or consists of amino acids KTKQWCDML (SEQ ID NO:139), EP7 comprises, consists essentially of, or consists of amino acids GCDLLINR (SEQ ID NO:140), and EP8 comprises, consists essentially of, or consists of amino acids TCTQPGGR (SEQ ID NO:141). In certain embodiments, the monoclonal antibody or antigen binding portion thereof binds only to EP6, EP7 and / or EP8. In other embodiments, the monoclonal antibody, or antigen-binding portion thereof, further binds to an additional FAM19A5 epitope selected from the group consisting of SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, and any combination thereof.

[0079] To facilitate understanding of the disclosed subject matter, a number of terms and phrases are defined. Additional definitions are presented throughout the detailed description.

[0080] I. Definition Throughout this disclosure, the term "a" or "an" entity means one or more of that entity; for example, "an antibody" is understood to refer to one or more antibodies. Thus, the terms "a" (or "an"), "one or more," and "at least one" are interchangeable.

[0081] It should also be understood that "and / or" is used as a specific disclosure of each of the specified features or components, either together with or without the other. Thus, "A and / or B" is intended to include "A and B", "A or B", "A (alone)", and "B (alone)". Similarly, "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)".

[0082] When an embodiment is used with the word "comprising", it is understood that other similar embodiments described in the "consisting of" and / or "consisting essentially of" aspects are also provided.

[0083] Unless otherwise defined, all technical and scientific terms disclosed herein have the same meaning as commonly understood by one of ordinary skill in the relevant art. See, e.g., Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry and

[0084] Molecular Biology, Revised, 2000, Oxford University Press provides one of ordinary skill in the art with the common dictionary meaning for most of the terms used in this disclosure.

[0085] Units, prefixes, and symbols are denoted in the form accepted by the Systeme International de Unite (SI). Numerical ranges are inclusive of both ends of the numbers that define the range. Unless otherwise noted, amino acid sequences are presented left to right in amino to carboxy orientation. The headings provided are not limitations of the various aspects of the invention, and they may be used as a reference for the specification as a whole. Thus, the terms defined below are more fully defined in the specification as a whole.

[0086] The term "about" means approximately, in the region of, near, or in the vicinity. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the limits above and below the numerical range set forth. In general, the term "about" can modify a numerical range above and below the stated value by, for example, a variance of 10% above or below (higher or lower).

[0087] The term "Family 19 with sequence similarity, member A5" or "FAM19A5" refers to a protein that belongs to the TAFA family of five highly homologous proteins (also referred to as the FAM19 family) and is expressed predominantly in the brain and spinal cord. FAM19A5 is also known as TAFA5 or chemokine-like protein TAFA-5.

[0088] In humans, the gene encoding FAM19A5 is located on chromosome 22. There are multiple human FAM19A5 (UniProt: Q7Z5A7) isoforms, which can be generated 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 exists in membrane-bound and soluble (secreted) forms. Isoform 1 is membrane with one transmembrane domain. Isoform 2 is reported as a secreted protein (soluble) in Tang TY et al., Genomics 83(4): 727-34 (2004) and contains a signal peptide at amino acid positions 1-25. Isoform 1 is considered a membrane protein. Next, the amino acid sequences of the three known human FAM19A5 isoforms are presented. (I) Isoform 1 (UniProt: Q7Z5A7-1, transmembrane protein): This isoform was selected as the official sequence. 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:4) (III) Isoform 3 (UniProt:Q7Z5A7-3): MYHHREWPAR IIKTKQWCDM LPCLEGEGCD LLINRSGWTC TQPGGRIKTT TVS(SEQ ID NO:26)

[0089] The term "FAM19A5" includes any variant or isoform of FAM19A5 that is naturally expressed by a cell. Thus, the antibodies disclosed herein may cross-react with different isoforms in the same species (e.g., different isoforms of human FAM19A5) or with FAM19A5 from another species other than human (e.g., mouse FAM19A5). Also, an antibody may be specific for human FAM19A5 and may not exhibit cross-reactivity with another species. FAM19A5 or any variants and isoforms thereof may be isolated from cells or tissues that naturally express them or recombinantly produced. A polynucleotide encoding human FAM19A5 has GenBank Accession No. BC039396 and the following sequence:

[0090] [Table 1A]

[0091] The terms "antibody" and "antibodies" are terms of the art and are used interchangeably herein to refer to a molecule that has an antigen-binding site that specifically binds to an antigen. As used herein, the term includes whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") or single chains thereof. In one embodiment, "antibody" refers to a glycoprotein or antigen-binding portion thereof that comprises at least two heavy (H) and two light (L) chains interconnected by disulfide bonds. In another embodiment, "antibody" refers to a single-chain antibody that comprises a single variable domain, e.g., a VHH domain. Each heavy chain is composed of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region. In naturally occurring antibodies, the heavy chain constant region is composed of three domains, CH1, CH2, and CH3. In naturally occurring antibodies, each light chain is composed of a light chain variable region (abbreviated as VL) and a light chain constant region. The light chain constant region is composed of one domain, CL.

[0092] The VH and VL regions can be further subdivided into regions of hypervariability, designated complementarity determining regions (CDRs), interspersed with more conserved regions designated framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibody may mediate the binding of the immunoglobulin 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.

[0093] 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 by the Kabat numbering system (see, e.g., Kabat EA & Wu TT (1971) Ann NY Acad Sci 190:382-391 and Kabat EA et al, (1991) Sequences of Proteins of Immunological Interest, 5th Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system, the CDRs in an antibody heavy chain molecule are typically located at amino acid positions 31-35 (which can optionally include one or two additional amino acids following number 35 (referred to as 35A and 35B in the Kabat numbering system)) (CDR1), 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 molecule 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 disclosed herein have been determined according to the Kabat numbering scheme. The phrases "amino acid position numbering as set out in Kabat", "Kabat position" and variations thereof refer to the numbering system used for the heavy or light chain variable domains of an antibody population 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 fewer or additional amino acids corresponding to a shortening of, or insertion into, a FW or CDR of the variable domain.For example, the heavy chain variable domain can include a single amino acid insertion following residue 52 of H2 (residue 52a by Kabat) and can include inserted residues following heavy chain FW residue 82 (e.g., residues 82a, 82b, and 82c, etc., by Kabat). See Table 1B.

[0094] [Table 1B]

[0095] Kabat numbering of residues can be determined for a given antibody by alignment with the "standard" Kabat numbering sequence in the homologous region of the antibody sequence. Chothia proposes the location of the structural loops (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 is variable between H32 and H34 depending on the length of the loop (this is because the Kabat numbering system places the insert at H35A and H35B. If both 35A and 35B are absent, the loop ends at 32. If only 35A is present, the loop ends at 33. If both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent the offset between the Kabat CDRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software.

[0096] IMGT (ImMunoGeneTics) also provides a numbering system for immunoglobulin variable regions, including CDRs. See, for example, Lefranc, MP et al, Dev. Comp. Immunol. 27:55-77 (2003), which is incorporated herein by reference. The IMGT numbering system is based on the alignment of over 5,000 sequences, structural data, and characterization of hypervariable loops, allowing easy 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.

[0097] The numbering for all heavy chain constant region amino acid positions disclosed herein is according to the EU index as originally disclosed in Edelman et al, 1969, Proc. Natl. Acad. Sci. USA 63(1):78-85, which describes the amino acid sequence of the myeloma protein EU, the first sequenced human IgG1. 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). Thus, the phrases "EU index as presented in Kabat" or "Kabat's EU index" and "...positions according to the EU index presented in Kabat" and variations thereof refer to the residue numbering system based on the human IgG1 EU antibody of Edelman et al. as presented in Kabat 1991.

[0098] The numbering system used for the variable domains (both heavy and light chains) and light chain constant region amino acid sequences is that presented in Kabat 1991.

[0099] The antibody can have any type (e.g., IgG, IgE, IgM, IgD, IgA or IgY), any class (e.g., Igd, IgG2, IgG3, IgG4, IgA1 or IgA2), or any subclass (e.g., IgG1, IgG2, IgG3 and IgG4 in humans, and IgG1, IgG2a, IgG2b and IgG3 in mice) of immunoglobulin molecule. Immunoglobulins, such as IgG1, exist in several allotypes, which differ from each other by up to several amino acids. The antibodies disclosed herein may be derived from any of the commonly known isotypes, classes, subclasses or allotypes. In certain embodiments, the antibodies presented herein belong to the IgG1, IgG2, IgG3 or IgG4 subclasses or any hybrids thereof. In certain embodiments, the antibodies belong to the IgG2, IgG4 or IgG2 / IgG4 subclasses.

[0100] Antibodies include, 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 molecules 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 comprised of a single monomeric variable antibody domain (e.g., a VH domain or a VL domain) sufficient for antigen binding (Harmen MM and Haard HJ Appl Microbiol Biotechnol. 77(1):13-22 (2007)).

[0101] As used herein, the term "antigen-binding portion" of an antibody refers to one or more fragments of an antibody (e.g., human FAM19A5) that retain the ability to specifically bind to an antigen. Such a "fragment" is, for example, about 8 to about 1500 amino acids in length, preferably about 8 to about 745 amino acids in length, about 8 to about 300 amino acids, about 8 to about 200 amino acids, or about 10 to about 50 or 100 amino acids in length. It has become clear that the antigen-binding function of an antibody is performed by a fragment of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody, e.g., an anti-FAM19A5 antibody disclosed herein, include: (i) a Fab fragment, a monovalent fragment composed of the VL, VH, CL, and CH1 domains; (ii) an F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment composed of the VH and CH1 domains; (iv) an Fv fragment composed of the VL and VH domains of one arm of an antibody, and a disulfide-linked Fv (sdFv); (v) a dAb fragment (Ward et al, (1989) Nature 341:544-546) composed of a VH domain, and (vi) an isolated complementarity determining region (CDR) or (vii) a combination of two or more isolated CDRs which may optionally be linked by a synthetic linker. Alternatively, the two domains of an Fv fragment, VL and VH, are encoded by separate genes but can be linked using recombinant methods by a synthetic linker that allows pairing of the VL and VH regions into a single protein chain forming a monovalent molecule (single-chain Fv (scFv)) (see 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 included within the "antigen-binding portion" of an antibody. These antibody fragments are obtained by conventional techniques known to those of skill 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.

[0102] As used herein, the terms "variable region" and "variable domain" are used interchangeably. A variable region typically refers to a portion of an antibody, generally a portion of either the light or heavy chain, typically about the amino terminal 110-120 amino acids in the mature heavy chain and about 90-115 amino acids in the mature light chain, which differ extensively in sequence from one antibody to another and which characterize the binding and specificity of a particular antibody for 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).

[0103] The light and heavy chain CDRs are primarily responsible for antibody-antigen interaction and specificity. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and human framework regions (FRs). In certain embodiments, the variable region is a primate (e.g., non-human primate) variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and primate (e.g., non-human primate) framework regions (FRs).

[0104] The term "heavy chain" as provided herein, when used in connection with an antibody, can refer to any of the different types, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ) and mu (μ), based on the amino acid sequence of the constant domain, which give rise to the IgA, IgD, IgE, IgG and IgM types of antibodies, including subclasses of IgG, e.g., IgG1, IgG2, IgG3 and IgG4, respectively.

[0105] The term "light chain" as provided herein, when used in conjunction with an antibody, can refer to any of the different types, e.g., kappa (κ) or 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.

[0106] The terms "VL" and "VL domain" are used interchangeably to refer to the light chain variable region of an antibody.

[0107] The terms "VH" and "VH domain" are used interchangeably and refer to the variable region of an antibody heavy chain.

[0108] As used herein, the terms "constant region" or "constant domain" can be used interchangeably. A constant region is that portion of an antibody, e.g., the carboxy-terminal portion of the light and / or heavy chains, 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 regions of an immunoglobulin molecule generally have a more conserved amino acid sequence than the immunoglobulin variable domains. Yes.

[0109] "Fc region" (fragment crystallizable region), "Fc domain" or "Fc" refers to the C-terminal region of an antibody heavy chain that mediates binding of the immunoglobulin 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 (Clq) of the classical complement system. Thus, the Fc region includes the constant regions of an antibody except for the first constant region immunoglobulin domain (e.g., CH1 or CL). In IgG, IgA and IgD antibody isotypes, the Fc region includes 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 include three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. In the case of IgG, the Fc region includes immunoglobulin domains Cγ2 and Cγ3, and the hinge between Cγ1 and Cγ2. Although the boundaries of the Fc region of an immunoglobulin heavy chain may be variable, the human IgG heavy chain Fc region is generally defined to stretch from an amino acid residue at position C226 or P230 (or an amino acid between these two amino acids) to the carboxy-terminus of the heavy chain, where numbering is according to the EU index as set forth in Kabat. The CH2 domain of the human IgG Fc region stretches from about amino acid 231 to about amino acid 340, and the CH3 domain is located C-terminal to the Cm domain in the Fc region, i.e., stretches from about amino acid 341 to about amino acid 447 of IgG. The Fc region provided herein may be a native sequence Fc or a variant Fc (e.g., a non-naturally occurring Fc), including any allotypic variants. The Fc may also be an Fc-containing protein polypeptide, such as a "binding protein comprising an Fc region", also referred to as an "Fc fusion protein" (e.g., an antibody or immunoconjugate).

[0110] A "native sequence Fc region" or "native sequence Fc" comprises an amino acid sequence identical to the amino acid sequence 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 allotypes of Fes (e.g., Jefferis et al, (2009) mAbs 1:1; Vidarsson G. et al., Front Immunol. 5:520 (published online 2014-10-20)).

[0111] 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, including allelic variants and alternatively spliced ​​forms of these receptors. The FcγR family is composed of three activating receptors (FcγRI, FcγRIII, and FcγRIV in mice; FcγRIA, FcγRIIA, and FcγRIIIA in humans) and one inhibitory (FcγRIIB) receptor. Human IgG1 binds to the majority of human Fc receptors and elicits the most potent Fc effector functions. Human IgG4 is considered equivalent to murine IgG2a in terms of the type of activating Fc receptor bound. On the other hand, human IgG4 elicits minimal Fc effector functions (Vidarsson G. et al., Front Immunol. 5:520 (published online 2014-10-20)).

[0112] The constant region may be engineered, e.g., by recombinant techniques, to eliminate one or more effector functions. An "effector function" refers to the interaction of, or a biochemical event resulting therefrom, an antibody Fc region with an Fc receptor or ligand. Exemplary "effector functions" include Clq 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 receptor; BCR). Such effector functions generally require an Fc region that may be combined with a binding domain (e.g., an antibody variable domain). Thus, the term "constant region lacking an Fc function" includes a constant region that has reduced or no effector functions mediated by the Fc region.

[0113] The effector functions of antibodies can be reduced or avoided by different approaches. The effector functions of antibodies can be reduced or avoided by antibody fragments lacking the Fc region (such as Fab, F(ab')2, single chain Fv (scFv), or sdAbs composed of monomeric VH or VL domains). Also, sugars attached to specific residues in the Fc region can be removed to generate so-called aglycosylated antibodies, which can reduce the effector functions of the antibody while maintaining other valuable attributes of the Fc region (e.g., extended half-life and heterodimerization). Aglycosylated antibodies can be generated, for example, by deleting or altering the residue to which the sugar is attached, by enzymatic sugar removal, by producing the antibody in cells cultured in the presence of glycosylation inhibitors, or by expressing the antibody in cells (e.g., bacterial host cells) that cannot glycosylate proteins (see, for example, US Patent Publication No. 20120100140). Another approach is to utilize Fc regions from IgG subclasses with reduced effector function, for example IgG2 and IgG4 antibodies are characterized by a lower level of Fc effector function than IgG1 and IgG3. The residues closest to the hinge region in the CH2 domain of the Fc portion are responsible for the effector function of the antibody, which has closely overlapping binding sites for Clq (complement) and the IgG-Fc receptor (FcγR) on effector cells of the innate immune system (Vidarsson G. et al., Front Immunol. 5:520 (Published online 2014-10-20)). Thus, antibodies with reduced or unreduced Fc effector function can be made, for example, by generating a chimeric Fc region comprising a CH2 domain from an IgG antibody of the IgG4 isotype and a CH3 domain from an IgG antibody of the IgG1 isotype, or a chimeric Fc region comprising a hinge region from IgG2 and a CH2 region from IgG4 (see, for example, Lau C. et al, J. Immunol. 191:4769-4777 (2013)), or an Fc region with mutations that result in altered Fc effector function, e.g. reduced or unreduced Fc function.Such Fc regions with mutations are known in the art (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); the disclosures of which are incorporated by reference in their entireties).

[0114] "Hinge", "hinge domain", "hinge region" or "antibody hinge region" refers to the domain of the heavy chain constant region that connects the CH1 and CH2 domains, including 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 both heavy chain constant regions. The hinge presented herein 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, e.g., Kabat EA et al, (1991) Sequences of Proteins of Immunological Interest, 5th Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242; Vidarsson G. et al., Front Immunol. 5:520 (published online 2014-10-20)).

[0115] The term "CH1 domain" refers to the heavy chain constant region that connects the hinge and variable domains of the heavy chain constant domain. The CH1 domain presented herein starts at A118 and ends at V215. The term "CH1 domain" includes wild-type CH1 domains as well as naturally occurring variants thereof (e.g., allotypes). IgG1, IgG2, IgG3, and IgG4 CH1 domain sequences (including wild-type and allotypes) 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 2014-10-20)). Exemplary CH1 domains include CH1 domains that have domains that modify the biological activity, e.g., half-life, of an antibody (see, e.g., U.S. Patent Publication No. 20120100140 and the U.S. patents and patent publications and PCT publications cited therein).

[0116] The term "CH2 domain" refers to the heavy chain constant region that connects the hinge with the CH3 domain of the heavy chain constant domain. The CH2 domain presented herein starts at P238 and ends at K340. The term "CH2 domain" includes wild-type CH2 domains as well as naturally occurring variants thereof (e.g., allotypes). CH2 domain sequences (including wild-type and allotypes) of IgG1, IgG2, IgG3 and IgG4 are known in the art (see, for example, Kabat EA et al, (1991) supra and Vidarsson G. et al., Front Immunol. 5:520 (published online 2014-10-20)). Exemplary CH2 domains include CH2 domains having domains that alter the biological activity of an antibody, e.g., half-life and / or reduced Fc effector function (see, e.g., U.S. Patent Publication No. 20120100140 and the U.S. patents and patent publications and PCT publications cited therein).

[0117] The term "CH3 domain" refers to a heavy chain constant region that is C-terminal to the CH2 domain of the heavy chain constant domain. The CH3 domain presented herein begins at G341 and ends at K447. The term "CH3 domain" includes wild-type CH3 domains as well as naturally occurring variants thereof (e.g., allotypes). IgG1, IgG2, IgG3, and IgG4 CH3 domain sequences (including wild-type and allotypes) 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 2014-10-20)). Exemplary CH3 domains include CH3 domains that have domains that modify the biological activity, e.g., half-life, of an antibody (see, e.g., U.S. Patent Publication No. 20120100140 and the U.S. patents and patent publications and PCT publications cited therein).

[0118] "Isotype" as referred to herein refers to the antibody type (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE antibodies) that is encoded by the heavy chain constant region genes.

[0119] "Allotype" refers to naturally occurring variants with some amino acid differences within a particular isotype group (e.g., Jefferis et al, (2009) mAbs 1:1). The antibodies disclosed herein can have any allotype. The allotypes of IgG1, IgG2, IgG3 and IgG4 are known in the art (see, e.g., Kabat EA et al, (1991) supra; Vidarsson G. et al., Front Immunol. 5:520 (published online 2014-10-20); and Lefranc MP, mAbs 1:4, 1-7 (2009)).

[0120] The phrases "an antibody that recognizes an antigen" and "an antibody specific for an antigen" are used interchangeably herein with the term "an antibody that specifically binds to an antigen."

[0121] An "isolated antibody" as referred to herein refers to an antibody that is substantially free of other antibodies having different antigen specificity (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 has cross-reactivity to other FAM19A5 proteins from different species.

[0122] "Binding affinity" generally refers to the strength of the sum of non-covalent 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" disclosed herein refers to the intrinsic binding affinity that reflects 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 measured by the dissociation constant (K D Affinity can be expressed by, but is not limited to, the equilibrium dissociation constant (K D ) and the equilibrium binding constant (K A ) can be measured and / or expressed in a number of ways known in the art. D is k off / k on It is calculated from and expressed as molar concentration (M), K A k on / k off It is calculated from k on is the binding rate constant of the antibody and antigen, and k off is the dissociation of the antibody and antigen. on and k off can be determined by techniques known in the art such as immunoassays (eg, enzyme-linked immunosorbent assay (ELISA)), BIACORE®, or dynamic exclusion assay (KinExA).

[0123] The terms "specifically bind," "specifically recognize," "specific binding," "selective binding," and "selectively bind," as disclosed herein, are similar 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 such binding would be understood by one of skill in the art. For example, a molecule that specifically binds to an antigen may bind to other peptides or polypeptides with lower affinity, as determined by, for example, an immunoassay, a BIACORE®, KinExA3000 instrument (Sapidyne Instruments, Boise, ID), or other assays known in the art. In certain embodiments, a molecule that specifically binds to an antigen has a K A At least 2 logs, 2.5 logs, 3 logs, 4 logs or more K A binds to the antigen.

[0124] Antibodies are typically -5 ~10 -11 The dissociation constant (K D ) specifically binds to its cognate antigen with high affinity, reflected by approximately 10 -4 Any K greater than M D As provided herein, an antibody that "specifically binds" to an antigen refers to an antibody that binds to the antigen and substantially the same antigen with high affinity, but does not bind to unrelated antigens with high affinity, where high affinity is, for example, greater than 10 as determined by immunoassays (e.g., ELISA) or surface plasmon resonance (SPR) techniques on a BIACORE2000 instrument using a defined antigen. -7 M or less, preferably 10 -8 M or less, more preferably 10 -9 M or less, and most preferably 10 -8 M~10 -10 K below M D It means having

[0125] The term "antigen" disclosed herein refers to any natural or synthetic immunogenic substance, such as a protein, peptide, or hapten. The antigen may be FAM19A5 or a fragment thereof.

[0126] "Epitope" as disclosed herein is a term of the art and refers to a localized region of an antigen to which an antibody can specifically bind. For example, an epitope can be contiguous amino acids of a polypeptide (linear or contiguous epitope), or an epitope can be combined together from two or more non-contiguous regions of a polypeptide or multiple polypeptides (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 the manner in which an epitope is bound by a given antibody (i.e., epitope mapping) are known in the art and include, for example, immunoblotting and immunoprecipitation analysis, in which overlapping or adjacent peptides (e.g., from FMAM19A5) are tested for reactivity with a given antibody (e.g., anti-FAM19A5 antibody). Methods for determining the spatial conformation of an epitope include those known in the art and presented herein, such as x-ray crystallography, 2-dimensional nuclear magnetic resonance, and FIDX-MS (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996)).

[0127] In certain embodiments, the epitope to which the antibody binds can be determined, for example, by NMR spectroscopy, x-ray diffraction crystallography studies, 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 accomplished using any method known in the art (see, for example, Giege R et al, (1994) Acta Crystallogr D Biol Crystallogr 50(Pt4):339-350; McPherson A (1990) Eur J Biochem 189:1-23; Chayen E (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 and verified using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, e.g., Meth Enzymol (1985) volumes 114&115, eds Wyckoff HW et al; US2004 / 0014194) and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1):37-60; Bricogne G (1997) Meth Enzymol 276 A: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 in the art.For information regarding mutagenesis techniques, including alanine scanning mutagenesis techniques, see, e.g., Champe M et al., (1995) J Biol Chem 270:1388-1394 and Cunningham BC & Wells JA (1989) Science 244:1081-1085.

[0128] The term "epitope mapping" refers to the process of identifying molecular determinants for antibody-antigen recognition.

[0129] 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 presented herein include, for example, epitope mapping methods, such as x-ray analysis and hydrogen / deuterium exchange mass spectrometry (HDX-MS) on antigen:antibody complex crystals that provide atomic resolution of the epitope. Other methods monitor the binding of the antibody to antigen fragments or mutated variants of the antigen, where loss of binding due to modification of amino acid residues within the antigen sequence is primarily considered as an indication of epitope composition. Also, combinatorial computational methods for epitope mapping may be used. These methods rely on the ability of the antibody of interest to affinity isolate specific short peptides from a combinatorial phage display peptide library. Antibodies with identical VH and VL or identical CDR1, 2 and 3 sequences are expected to bind to the same epitope.

[0130] An antibody that "competes with another antibody for binding to a target" refers to an antibody that inhibits (partially or completely) the binding of the remaining antibody to the target. Whether or not both antibodies compete with each other for binding to a target, i.e., whether and to what extent one antibody inhibits the binding of the remaining antibody to the target, can be determined by known competition experiments. In certain embodiments, an antibody competes with another antibody for binding to a target and inhibits the binding of the other antibody to the target 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). Competitive analyses can be performed, for example, as disclosed in Ed Harlow and David Lane, Cold Spring Harb Protoc; 2006; doi:10.1101 / pdb.prot4277 or in Chapter 11 of "Using Antibodies", Ed Harlow and David Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA 1999. Competitive antibodies bind to the same epitope, overlapping epitopes or adjacent epitopes (e.g., as evidenced by steric hindrance).

[0131] Other competitive binding assays include solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see Stahli et al, Methods in Enzymology 9:242 (1983)); solid phase direct biotin-avidin EIA (Kirkland et al. al, J. Immunol. 137:3614 (1986)); solid phase direct labeling analysis; solid phase direct labeling sandwich analysis (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid phase direct labeling RIA using the I-125 label (Morel et al. al., Mol. Immunol. 25(1):7 (1988)); solid phase direct biotin-avidin EIA (see Cheung et al, Virology 176:546 (1990)); and direct labeled RIA (Moldenhauer et al. al, Scand. J. Immunol. 32:77 (1990)).

[0132] A "bispecific" or "bifunctional" antibody is an artificial hybrid antibody having two different heavy / light chain pairs and 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)).

[0133] A "monoclonal antibody" as referred to herein refers to an antibody or a combination of antibodies where all of the antibodies display a single binding specificity and affinity for a particular epitope. The term "human monoclonal antibody" thus refers to an antibody or antibody composition which displays a single binding specificity and has variable regions and optional constant regions derived from human germline immunoglobulin sequences. In one embodiment, a human monoclonal antibody is produced by a hybridoma comprising a B cell obtained from a transgenic non-human animal, e.g., a transgenic mouse, whose genome comprises a human heavy chain transgene and a human light chain transgene fused to an immortalized cell.

[0134] A "recombinant human antibody" as provided herein includes all human antibodies made, expressed, produced or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or hybridomas made therefrom, (b) antibodies isolated from a host cell that has been transformed to express the antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies made, expressed, produced or isolated by any other means involving splicing of human immunoglobulin gene sequences and other DNA sequences. Such recombinant human antibodies include variable and constant regions, which utilize specific human germline immunoglobulin sequences encoded by germline genes, but which include 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 the antigen-binding domains and are encoded by different genes that rearrange to form antibodies specific to foreign antigens. In addition to rearrangement, the variable regions may be further modified by multiple single amino acid changes to increase the affinity of the antibody for the foreign antigen (somatic mutation or hypermutation). The constant regions will change in additional response to the antigen (i.e., isotype switching). Thus, the rearranged and somatically mutated nucleic acid molecules that encode light and heavy immunoglobulin polypeptides in response to an antigen may not have sequence identity to the parental nucleic acid molecule, but instead will be substantially identical or similar (i.e., have at least 80% identity).

[0135] 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. Also, if the antibody contains constant regions, the constant regions are also derived from human germline immunoglobulin sequences. The antibodies disclosed 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, "human antibodies" disclosed herein do not include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. The terms "human" antibody and "fully human" antibody are used interchangeably.

[0136] 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 replaced with the corresponding amino acids derived from a human immunoglobulin. In one embodiment of a humanized form of an antibody, some, most or all of the amino acids outside the CDR domains are replaced with amino acids from a human immunoglobulin, while some, most or all of the amino acids within one or more CDR regions remain intact. Small additions, deletions, insertions, substitutions or modifications of amino acids are permissible as long as they do not eliminate the ability of the antibody to bind a particular antigen. A "humanized" antibody retains similar antigen specificity as the parent antibody.

[0137] "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, e.g., the variable region is derived from a murine antibody and the constant region is derived from a human antibody.

[0138] The term "cross-react" as disclosed herein refers to the ability of the antibodies presented herein to bind to FAM19A5 from different species. For example, an antibody that binds to human FAM19A5 may also bind to other species of FAM19A5 (e.g., mouse FAM19A5). Cross-reactivity can be measured by detecting specific reactivity with purified antigen in a binding assay (e.g., SPR, ELISA), or by detecting binding or other functional interaction with cells that physiologically express FAM19A5. Methods for determining cross-reactivity include standard binding assays presented herein, such as BIACORE® surface plasmon resonance (SPR) assays using a BIACORE 2000 SPR instrument (Biacore AB, Uppsala, Sweden), or flow cytometry techniques.

[0139] The term "naturally occurring" as disclosed herein, when applied to an object, means that the object 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 viruses) that has not been intentionally modified by man in a laboratory is naturally occurring.

[0140] A "polypeptide" refers to a chain containing at least two constitutively linked amino acid residues, with an upper limit to the length of the chain. One or more amino acid residues in a protein can contain modifications such as, but not limited to, glycosylation, phosphorylation, or disulfide bond formation. A "protein" can include one or more polypeptides.

[0141] The term "nucleic acid molecule" disclosed herein includes DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded and may be cDNA.

[0142] The term "vector" disclosed herein refers to a nucleic acid molecule capable of transporting another nucleic acid 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, in 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. Certain vectors are also 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 of utility in recombinant DNA techniques are primarily in the form of plasmids. As used herein, "plasmid" and "vector" can be used interchangeably, with the plasmid being the most commonly used form of vector. However, other forms of expression vectors, such as viral vectors, which serve equivalent functions (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), are also included.

[0143] The term "recombinant host cell" (or simply, "host cell") disclosed herein refers to a cell that contains a nucleic acid that is not naturally present in the cell, and may be a cell into which a recombinant expression vector has been introduced. It will be understood that such terms refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain variations can occur in successive generations due to mutations 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" disclosed herein.

[0144] The term "linked" as disclosed herein 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 linkages can be achieved using a wide range of art-recognized techniques, such as chemical conjugation and recombinant protein production.

[0145] "Administering" as disclosed herein refers to physically introducing a therapeutic agent or a 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 provided herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, or other parenteral routes of administration, such as by injection or infusion. "Parenteral administration" as disclosed herein means modes of administration other than intestinal and topical administration, generally by injection, and includes, but is not limited to, intravenous, intraperitoneal, intramuscular, intraarterial, intraspinal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intravascular, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, and in vivo electroporation. Antibodies provided herein may also be administered via non-parenteral routes, such as topical, epithelial, or mucosal routes of administration, such as intranasal, oral, vaginal, rectal, sublingual, or topical routes. Administration can also be, for example, once, multiple times, and / or over one or more extended periods of time.

[0146] The terms "treat" and "treatment" disclosed herein refer to any type of intervention or process performed on a subject, or administering an active agent to a subject, for the purpose of reversing, alleviating, ameliorating, inhibiting or slowing the progression, occurrence, severity or recurrence of symptoms, complications, conditions or biochemical indicators associated with a disease, or preventing the recurrence of such symptoms, complications, conditions or biochemical indicators. Treatment can be performed on subjects with a disease or subjects without a disease (e.g., for prophylaxis).

[0147] The term "subject" disclosed herein 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.

[0148] The term "initiation of gliosis" or "initiation of reactive gliosis" disclosed herein includes the beginning or initiation of gliosis. Gliosis is a non-specific reactive change of glial cells in the central nervous system (CNS, e.g., brain and / or spinal cord) in response to injury or damage from, for example, trauma, brain and spinal cord injury, brain tumor, infection, ischemia, stroke, autoimmune response, and / or neurodegenerative disease, and includes proliferation and hypertrophy of several different types of glial cells, including astrocytes, microneurons, and oligodendrocytes. The initiation of gliosis can lead to scar formation and inhibit axon regeneration in parts of the CNS that have been traumatized or injured. The deleterious effects of the initiation of gliosis include irreversible or permanent damage to nerves and / or prevention of the recovery of surrounding neurons. Thus, the terms "delaying the initiation of gliosis" and "delaying the initiation of reactive gliosis" include inhibiting, delaying, suppressing, or preventing the initiation or initiation of gliosis and its associated deleterious effects on the CNS.

[0149] The term "excessive proliferation of reactive astrocytes" as disclosed herein includes an abnormal increase in the number of astrocytes due to, for example, CNS injury, trauma, injury, brain / spinal cord injury, brain tumor, infection, ischemia, stroke, autoimmune response and / or destruction of surrounding neurons due to neurodegenerative disease. Excessive proliferation of reactive astrocytes can lead to deleterious effects in the CNS, including the formation of scars that inhibit axonal regeneration in areas of the CNS that have been traumatized or injured, exacerbated inflammation, production and release of neurotoxic levels of reactive oxygen species, release of potentially excitotoxic glutamate, a potential contribution to seizure genesis, impairment of blood-brain barrier function, cytotoxic edema during trauma and stroke, the potential for chronic cytokine activation of astrocytes that can contribute to chronic pain, and secondary degeneration following CNS injury (see 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 excessive proliferation of reactive astrocytes" includes inhibiting, slowing, suppressing, limiting or preventing the excessive or abnormal proliferation of reactive astrocytes and its associated deleterious effects on the CNS.

[0150] The term "chondroitin sulfate proteoglycans" disclosed herein includes proteoglycans that consist of a protein core and chondroitin sulfate. Chondroitin sulfate proteoglycans, also known as CSPGs, are extracellular matrix molecules that are widely expressed throughout the developing and adult CNS. CSPGs play an important role in neurogenesis and formation of the glial scar and inhibit axon regeneration after injury in the CNS. Known CSPGs include aggrecan (CSPG1), versican (CSPG2), neurocan (CSPG3), CSPG4 (or neuron-glial antigen 2 (NG2)), CSPG5, SMC3 (CSPG6, maintenance of chromosome structure 3), brevican (CSPG7), CD44 (CSPG8, cluster of differentiation 44), and phosphacan-neurocan (CSPG3) (Rhodes, KE and

[0151] (See Fawcett, JW (2004) Journal of Anatom. 204(1):33-48). Thus, the term "reducing expression of chondroitin sulfate proteoglycans" includes lowering, inhibiting, decreasing the level of one or more CSGPs, or reducing or inactivating the activity of one or more CSGPs. In certain embodiments, the term includes lowering, inhibiting, decreasing the level of neurocan, NG2, or both, and reducing or inactivating the activity of neurocan, NG2, or both.

[0152] The term "neuron" as disclosed herein includes electrically excitable cells that process and transmit information through electrical and chemical signals. Neurons are the major components of the brain and spinal cord of the CNS and the neuroglia of the peripheral nervous system (PNS) and can be interconnected to form a neuronal network. A typical neuron consists of a nerve cell body (soma), dendrites and an axon. The soma of a neuron contains the nucleus. The dendrites of a neuron are cell extensions with many branches where most neuronal input originates. The axon is a finer cable-like protective extension from the soma that carries neural signals away from the soma and carries certain types of information back from the soma. The term "promoting regrowth of neurons" preferably includes stimulating, promoting, increasing or activating the growth of neurons after injury or damage.

[0153] The term "c-fos" disclosed herein includes the proto-oncogene c-fos, which is rapidly induced by neurotransmitter stimulation and is present in many species, including mice and humans. The c-fos gene and protein are known and characterized (see Curran, T, The c-fos proto-oncogene, pp 307-327 (The Oncogene Handbook, Reddy EP et al., (eds.) Elsevier) (1988)). Expression of c-fos can be determined by methods known in the art, such as Northern blot, quantitative PCR, or immunohistochemistry. The term "increasing expression of c-fos" includes increasing the levels of c-fos mRNA, c-fos protein, or c-fos protein activity.

[0154] The term "pERK" disclosed herein 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 upstream kinases to form pERK, which then activates downstream targets. ERK is involved in neural and synaptic plasticity, which underlies learning and memory and pain hypersensitivity (see Ji RR et al, Nat Neurosci (1999) 2:1114-1119). The ERK gene, protein, phosphorylation and activation are known and characterized, and expression of ERK and pERK can be determined by methods known in the art, such as Northern blot, quantitative PCR, or immunohistochemistry (see Gao YJ and Ji RR, Open Pain J. (2009) 2:11-17). The term "increasing expression of pERK" includes increasing the levels of ERK mRNA, ERK protein or pERK activity.

[0155] The term "GAP43" disclosed herein, known as "growth associated protein 43", is a neural tissue specific protein that promotes neurite formation, regeneration and plasticity (see Benowitz LI and Routtenberg A. (1997) Trends in Neurosciences 20(2):84-91; Aarts LHet 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 this polypeptide are known in the art (see Kosik KS et al, (1988) Neuron 1(2):127-32; Ng SC et al, (1988) Neuron 1(2):133-9). Expression of GAP43 can be determined by methods known in the art (e.g., Northern blot, quantitative PCR, or immunohistochemistry). The term "increasing GAP43 in neurons" includes enhancing or increasing the levels of GAP43 mRNA, GAP43 protein, or increasing the activity of GAP43 protein.

[0156] The term "therapeutically effective amount" as disclosed herein refers to an amount of a drug, alone or in combination with other therapeutic agents, 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" refers to 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). Thus, a "therapeutically effective" amount is an amount that reduces or provides some relief, alleviation of 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.

[0157] II. Anti-FAM19A5 antibody Disclosed are antibodies, e.g., monoclonal antibodies, characterized by particular functional features or characteristics. For example, the antibodies specifically bind to human FAM19A5, including soluble FAM19A5 and membrane-bound FAM19A5. In addition to specifically binding to soluble and / or membrane-bound human FAM19A5, the antibodies provided herein exhibit any one or more of the following functional characteristics: (a) K of 10 nM or less as measured by enzyme-linked immunosorbent assay (ELISA). D binds to soluble human FAM19A5; (b) K of 10 nM or less as measured by ELISA D binds to membrane-bound human FAM19A5; (c) reducing, reversing, delaying and / or preventing the onset of reactive gliosis; (d) inhibiting excessive proliferation of reactive astrocytes; (e) decreasing the expression of chondroitin sulfate proteoglycans, including neurocan and neuron-glial antigen 2 (NG2); (f) increasing the expression of c-fos and pERK in the neuronal nuclei; (g) promoting neuronal survival; (h) increasing the expression of GAP43 in neurons; and (i) Promotes axon regrowth.

[0158] In some embodiments, the anti-FAM19A5 antibody, or antigen-binding portion thereof, is administered with high affinity, e.g., at 10 -7 M or less, 10 -8 M 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, for example, 10 -12 M, 5X10-12 M, 10 -11 M, 5X10 -11 M, 10 -10 M, 5X10 -10 M, 10 -9 M, 5X10 -9 M, 10 -8 M, 5X10 -8 M, 10 -7 M or 5X10 -7 K of M D The antibody specifically binds to soluble human FAM19A5 or membrane-bound human FAM19A5 at 100° C. Standard assays for evaluating the binding ability of antibodies directed to human FAM19A5 of various species are known in the art and include, for example, ELISA, Western blot, and RIA. Suitable assays are described in detail in the Examples. The binding kinetics (e.g., binding affinity) of the antibody can also be evaluated by standard assays known in the art, such as ELISA, BIACORE assay, or KinExA. Assays for evaluating the effect of the antibody on the functional properties (e.g., ligand binding) of FAM19A5 are described in further detail below and in the Examples.

[0159] In some embodiments, the anti-FAM19A5 antibody, or antigen-binding portion thereof, has a cytoplasmic affinity of 10, as determined, for example, by 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 K of M D In some specific examples, the anti-FAM19A5 antibody or antigen-binding portion thereof binds to soluble human FAM19A5 with a K of 10 nM or less, for example, 0.1 to 10 nM, 0.1 to 5 nM, 0.1 to 1 nM, 0.5 to 10 nM, 0.5 to 5 nM, 0.5 to 1 nM, 1 to 10 nM, 1 to 5 nM, or 5 to 10 nM. DIt binds to soluble FAM19A5 at 100 ng / mL. In some embodiments, the anti-FAM19A5 antibody, or antigen-binding portion thereof, has a K of about 1 pM, 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 8 pM, 9 pM, 10 pM, 20 pM, 30 pM, 40 pM, 50 pM, 60 pM, 70 pM, 80 pM, 90 pM, 100 pM, 200 pM, 300 pM, 400 pM, 500 pM, 600 pM, 700 pM, 800 pM, or 900 pM, or about 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, or 9 nM, or about 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, or 90 nM, as determined by ELISA. D It specifically binds to soluble human FAM19A5 at 100 ng / mL.

[0160] In some embodiments, the anti-FAM19A5 antibody, or antigen-binding portion thereof, has a cytoplasmic affinity of 10, as determined, for example, by 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 K of M D In certain specific examples, the anti-FAM19A5 antibody or antigen-binding portion thereof has 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. DIn some embodiments, the anti-FAM19A5 antibody or antigen-binding portion thereof specifically binds to membrane-bound human FAM19A5 at about 1 pM, 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 8 pM, 9 pM, 10 pM, 20 pM, 30 pM, 40 pM, 50 pM, 60 pM, 70 pM, 80 pM, 90 pM, 100 pM, 200 pM, 300 pM, 350 pM, 400 pM, 500 pM, 600 pM, 70 pM, 80 pM, 90 pM, 100 pM, 200 pM, 350 pM, 400 pM, 500 pM, 500 pM, 600 pM, 700 pM, 800 pM, 900 pM, 10 ... pM, 300 pM, 400 pM, 500 pM, 600 pM, 700 pM, 800 pM or 900 pM, or about 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, or 9 nM, or about 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM or 90 nM D It binds to membrane-bound human FAM19A5 at 100 ng / s.

[0161] The anti-FAM19A5 antibodies, or antigen-binding portions thereof, of the invention can delay or inhibit the onset of gliosis, and can prevent, delay 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) in response to injury or damage due to, for example, trauma, brain / spinal cord injury, brain tumor, infection, ischemia, stroke, autoimmune response and / or neurodegenerative disease.

[0162] The anti-FAM19A5 antibodies or antigen-binding portions thereof of the present invention can inhibit, inhibit, delay, suppress, limit or prevent excessive or abnormal proliferation of reactive astrocytes and their associated deleterious effects on the CNS. For example, the anti-FAM19A5 antibodies or antigen-binding portions thereof of the present invention can inhibit or prevent an abnormal increase in the number of astrocytes due to destruction of neurons due to, for example, CNS injury, trauma, injury, brain and spinal cord injury, brain tumor, infection, ischemia, stroke, autoimmune 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 the release of potentially excitotoxic glutamate, and reduce or inhibit seizures, pain and / or secondary degeneration after CNS injury. The anti-FAM19A5 antibodies or antigen-binding portions thereof of the present invention can preferably promote, stimulate, increase or activate regrowth of neurons and / or axons after CNS injury or damage.

[0163] The anti-FAM19A5 antibodies of the invention, or antigen-binding portions thereof, can inhibit expression of chondroitin sulfate proteoglycans, including proteoglycans consisting of a protein core and chondroitin sulfate (CSGPs), such as aggrecan (CSPG1), versican (CSPG2), neurocan (CSPG3), CSPG4 (or neuron-glial antigen 2 (NG2)), CSPG5, SMC3 (CSPG6, maintenance of chromosome structure 3), brevican (CSPG7), CD44 (CSPG8, cluster of differentiation 44), and phosphacan neurocan (CSPG3). In some embodiments, the anti-FAM19A5 antibodies of the invention, or antigen-binding portions thereof, inhibit, reduce or decrease the levels of neurocan and / or NG2, or the activity of neurocan and / or NG2.

[0164] The anti-FAM19A5 antibody or antigen-binding portion thereof of the present invention can increase the expression of c-fos and pERK in neuronal nuclei, for example, can increase c-fos and pERK mRNA, protein and / or protein activity. The anti-FAM19A5 antibody or antigen-binding portion thereof of the present invention can also increase or enhance the expression levels of GAP43 mPNA, GAP43 protein, or increase or enhance GAP43 protein activity.

[0165] In certain embodiments, the anti-FAM19A5 antibodies or antigen-binding portions thereof of the present invention compete with (or inhibit binding to) an anti-FAM19A5 antibody comprising the CDRs or variable regions disclosed herein in binding to a human FAM19A5 epitope. In certain embodiments, the anti-FAM19A5 antibody, or antigen-binding portion thereof, inhibits binding of a reference antibody comprising a heavy chain CDR1, CDR2, and CDR3, and a light chain CDR1, CDR2, and CDR3, wherein (1) the heavy chain CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively, and the light chain CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively; (2) the heavy chain CDR1, CDR2, and CDR3 comprise SEQ ID NOs:29, 30, and 31, respectively, and the light chain CDR1, CDR2, and CDR3 comprise SEQ ID NOs:32, 33, and 34, respectively; (3) the heavy chain CDR1, CDR2, and CDR3 comprise SEQ ID (4) heavy chain CDR1, CDR2 and CDR3 comprise SEQ ID NOs: 41, 42 and 43, respectively, and light chain CDR1, CDR2 and CDR3 comprise SEQ ID NOs: 44, 45 and 46, respectively; (5) heavy chain CDR1, CDR2 and CDR3 comprise SEQ ID NOs: 47, 48 and 49, respectively, and light chain CDR1, CDR2 and CDR3 comprise SEQ ID NOs: 50, 51 and 52, respectively; (6) heavy chain CDR1, CDR2 and CDR3 comprise SEQ ID NOs: 53, 54 and 55, respectively, and light chain CDR1, CDR2 and CDR3 comprise SEQ ID NOs: 54, 55 and 56, respectively. (7) heavy chain CDR1, CDR2 and CDR3 comprise SEQ ID NOs:59, 60, 61, respectively, and light chain CDR1, CDR2 and CDR3 comprise SEQ ID NOs:62, 63, 64, respectively; (8) heavy chain CDR1, CDR2 and CDR3 comprise SEQ ID NOs:65, 66, 67, respectively, and light chain CDR1, CDR2 and CDR3 comprise SEQ ID NOs:68, 69, 70, respectively;(9) the heavy chain CDR1, CDR2 and CDR3 each comprise SEQ ID NOs: 71, 72, 73, and the light chain CDR1, CDR2 and CDR3 each comprise SEQ ID NOs: 74, 75, 76; (10) the heavy chain CDR1, CDR2 and CDR3 each comprise SEQ ID NOs: 77, 78, 79, and the light chain CDR1, CDR2 and CDR3 each comprise SEQ ID NOs: 80, 81, 82; (11) the heavy chain CDR1, CDR2 and CDR3 each comprise SEQ ID NOs: 83, 84, 85, and the light chain CDR1, CDR2 and CDR3 each comprise SEQ ID NOs: 86, 87, 88; or (12) the heavy chain CDR1, CDR2 and CDR3 each comprise SEQ ID NOs: NOs:89, 90, 91, and light chain CDR1, CDR2 and CDR3 comprise SEQ ID NOs:92, 93, 94, respectively;

[0166] In some embodiments, the reference antibody is selected from the group consisting of: (1) a heavy chain variable region (VH) comprising SEQ ID NO:5 and a light chain variable region (VL) comprising SEQ ID NO:6; (2) a heavy chain variable region (VH) comprising SEQ ID NO:103 and a light chain variable region (VL) comprising SEQ ID NO:114; (3) a heavy chain variable region (VH) comprising SEQ ID NO:104 and a light chain variable region (VL) comprising SEQ ID NO:115; (4) a heavy chain variable region (VH) comprising SEQ ID NO:105 and a light chain variable region (VL) comprising SEQ ID NO:116; (5) a heavy chain variable region (VH) comprising SEQ ID NO:106 and a light chain variable region (VL) comprising SEQ ID NO:117; (6) a heavy chain variable region (VH) comprising SEQ ID NO:107 and a light chain variable region (VL) comprising SEQ ID NO:118; (7) a heavy chain variable region (VH) comprising SEQ ID NO:107 and a light chain variable region (VL) comprising SEQ ID NO:118; (8) a heavy chain variable region (VH) comprising SEQ ID NO:109 and a light chain variable region (VL) comprising SEQ ID NO:120; (9) a heavy chain variable region (VH) comprising SEQ ID NO:110 and a light chain variable region (VL) comprising SEQ ID NO:121; (10) a heavy chain variable region (VH) comprising SEQ ID NO:111 and a light chain variable region (VL) comprising SEQ ID NO:122; (11) a heavy chain variable region (VH) comprising SEQ ID NO:112 and a light chain variable region (VL) comprising SEQ ID NO:123; or (12) a heavy chain variable region (VH) comprising SEQ ID NO:113 and a light chain variable region (VL) comprising SEQ ID NO:124.

[0167] In certain embodiments, the anti-FAM19A5 antibody or antigen-binding portion thereof inhibits the binding of such a reference antibody to human FAM19A5 by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. Competing 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 by competition experiments known in the art, such as RIA and EIA.

[0168] In certain embodiments, the anti-FAM19A5 antibody, or antigen-binding portion thereof, binds to the same FAM19A5 epitope as a reference antibody disclosed herein, including heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3, where (1) the heavy chain CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively, and the light chain CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively; (2) the heavy chain CDR1, CDR2, and CDR3 comprise SEQ ID NOs:29, 30, and 31, respectively, and the light chain CDR1, CDR2, and CDR3 comprise SEQ ID NOs:32, 33, and 34, respectively; (3) the heavy chain CDR1, CDR2, and CDR3 comprise SEQ ID NOs:33, 34, and 35, respectively. (4) heavy chain CDR1, CDR2 and CDR3 comprise SEQ ID NOs: 41, 42 and 43, respectively, and light chain CDR1, CDR2 and CDR3 comprise SEQ ID NOs: 44, 45 and 46, respectively; (5) heavy chain CDR1, CDR2 and CDR3 comprise SEQ ID NOs: 47, 48 and 49, respectively, and light chain CDR1, CDR2 and CDR3 comprise SEQ ID NOs: 50, 51 and 52, respectively; (6) heavy chain CDR1, CDR2 and CDR3 comprise SEQ ID NOs: 53, 54 and 55, respectively, and light chain CDR1, CDR2 and CDR3 comprise SEQ ID NOs: 54, 55 and 56, respectively. (7) heavy chain CDR1, CDR2 and CDR3 comprise SEQ ID NOs:59, 60, 61, respectively, and light chain CDR1, CDR2 and CDR3 comprise SEQ ID NOs:62, 63, 64, respectively; (8) heavy chain CDR1, CDR2 and CDR3 comprise SEQ ID NOs:65, 66, 67, respectively, and light chain CDR1, CDR2 and CDR3 comprise SEQ ID NOs:68, 69, 70, respectively; (9) heavy chain CDR1, CDR2 and CDR3 comprise SEQ ID NOs:71, 72, 73, respectively, and light chain CDR1, CDR2 and CDR3 comprise SEQ ID NOs:74, 75, 76, respectively;(10) the heavy chain CDR1, CDR2 and CDR3 comprise SEQ ID NOs: 77, 78, 79, respectively, and the light chain CDR1, CDR2 and CDR3 comprise SEQ ID NOs: 80, 81, 82, respectively; (11) the heavy chain CDR1, CDR2 and CDR3 comprise SEQ ID NOs: 83, 84, 85, respectively, and the light chain CDR1, CDR2 and CDR3 comprise SEQ ID NOs: 86, 87, 88, respectively; or (12) the heavy chain CDR1, CDR2 and CDR3 comprise SEQ ID NOs: 89, 90, 91, respectively, and the light chain CDR1, CDR2 and CDR3 comprise SEQ ID NOs: 92, 93, 94, respectively;

[0169] In some embodiments, the reference antibody is selected from the group consisting of: (1) a heavy chain variable region (VH) comprising SEQ ID NO:5 and a light chain variable region (VL) comprising SEQ ID NO:6; (2) a heavy chain variable region (VH) comprising SEQ ID NO:103 and a light chain variable region (VL) comprising SEQ ID NO:114; (3) a heavy chain variable region (VH) comprising SEQ ID NO:104 and a light chain variable region (VL) comprising SEQ ID NO:115; (4) a heavy chain variable region (VH) comprising SEQ ID NO:105 and a light chain variable region (VL) comprising SEQ ID NO:116; (5) a heavy chain variable region (VH) comprising SEQ ID NO:106 and a light chain variable region (VL) comprising SEQ ID NO:117; (6) a heavy chain variable region (VH) comprising SEQ ID NO:107 and a light chain variable region (VL) comprising SEQ ID NO:118; (7) a heavy chain variable region (VH) comprising SEQ ID NO:107 and a light chain variable region (VL) comprising SEQ ID NO:118; (8) a heavy chain variable region (VH) comprising SEQ ID NO:109 and a light chain variable region (VL) comprising SEQ ID NO:120; (9) a heavy chain variable region (VH) comprising SEQ ID NO:110 and a light chain variable region (VL) comprising SEQ ID NO:121; (10) a heavy chain variable region (VH) comprising SEQ ID NO:111 and a light chain variable region (VL) comprising SEQ ID NO:122; (11) a heavy chain variable region (VH) comprising SEQ ID NO:112 and a light chain variable region (VL) comprising SEQ ID NO:123; or (12) a heavy chain variable region (VH) comprising SEQ ID NO:113 and a light chain variable region (VL) comprising SEQ ID NO:124.

[0170] 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 that provide atomic resolution of the epitope, hydrogen / deuterium exchange mass spectroscopy (HDX-MS), methods that monitor the binding of antibodies to antigen fragments or mutated variants of the antigen, and computer-assisted methods for epitope mapping, where loss of binding due to modification of amino acid residues within the antigen sequence is primarily viewed as a signature of the epitope component.

[0171] Anti-FAM19A5 antibodies, or antigen-binding portions thereof, of the invention can bind to at least one epitope of mature human FAM19A5, e.g., as determined by binding of the antibody to a fragment of human FAM19A5. In some embodiments, the anti-FAM19A5 antibodies, or antigen-binding portions thereof, bind to at least one epitope having the amino acid sequence CDMLPCLEGEGCDLLINRSG (SEQ ID NO:2, epitope F5, Example 10, amino acid residues 90-109 of SEQ ID NO:4), or a fragment located within the amino acid sequence of SEQ ID NO:2, e.g., an epitope having at least 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:2. In some embodiments, an anti-FAM19A5 antibody or antigen-binding portion thereof binds to amino acid residues 99-107 (i.e., EGCDLLINR) of SEQ ID NO:4. In certain embodiments, an anti-FAM19A5 antibody or antigen-binding portion thereof binds to amino acid residues 99, 100, 102, 103, 105, and 107 (i.e., EG-DL-IR) of SEQ ID NO:4. In other embodiments, an anti-FAM19A5 antibody or antigen-binding portion thereof binds to amino acid residues 102, 103, 105, and 107 (i.e., DL-IR) of SEQ ID NO:4. In some embodiments, an anti-FAM19A5 antibody or antigen-binding portion thereof binds to amino acid residues 99, 100, and 107 (i.e., EG------R) of SEQ ID NO:4. In some embodiments, 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:2. In some embodiments, the anti-FAM19A5 antibody, or antigen-binding portion thereof, binds only to a human FAM19A5 epitope that is SEQ ID NO:2, or to a fragment located within the amino acid sequence of SEQ ID NO:2, such as an epitope having 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:2.

[0172] In some embodiments, the anti-FAM19A5 antibodies, or antigen-binding portions thereof, of the invention bind to SEQ ID NO:2, or a fragment thereof, in its native conformation (i.e., non-denatured). In some embodiments, the anti-FAM19A5 antibodies, or antigen-binding portions thereof, bind to both glycosylated and non-glycosylated human FAM19A5.

[0173] In some embodiments, an anti-FAM19A5 antibody or antigen-binding portion thereof further binds to one or more additional FAM19A5 epitopes. Thus, a particular anti-FAM19A5 antibody or antigen-binding portion thereof binds to the epitope of SEQ ID NO:2 and an additional epitope. In some embodiments, the one or more additional FAM19A5 epitopes are: QFLKEGQLAAGTCEIVTLDR (SEQ ID NO: 13, epitope F1), TLDRDSSQPRRTiARQTARC (SEQ ID NO: 14, epitope F2); TARCACRKGQIAGTTRARPA (SEQ ID NO: 15, epitope F3), ARPACVDARIIKTKQWCDML (SEQ ID NO:16, epitope F4), or NRSGWTCTQPGGRIKTTTVS (SEQ ID NO:17, epitope F6), or a fragment located within the amino acid sequence of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16 or SEQ ID NO:17, or any combination thereof. Fragments located within the amino acid sequence of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16 or SEQ ID NO:17 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 any of SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17. In some embodiments, the one or more additional FAM19A5 epitopes are selected from SEQ ID NO:14, SEQ ID NO:15, fragments located within the amino acid sequence of SEQ ID NO:14, SEQ ID NO:15, such as 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:14 or SEQ ID NO:15, or any combination thereof. In some embodiments, an anti-FAM19A5 antibody or antigen-binding portion thereof of the invention binds to any of the one or more additional epitopes in its native conformation (i.e., non-denatured). In some embodiments, an anti-FAM19A5 antibody or antigen-binding portion thereof binds to glycosylated and non-glycosylated versions of the one or more additional FAM19A5 epitopes.

[0174] In some embodiments, the anti-FAM19A5 antibody or antigen-binding portion thereof binds to at least one FAM19A5 epitope identified as EP6, EP7, or EP8, where EP6 comprises amino acids KTKQWCDML (SEQ ID NO:139), EP7 comprises amino acids GCDLLINR (SEQ ID NO:140), and EP8 comprises amino acids TCTQPGGR (SEQ ID NO:141). 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 EP6, EP7, or EP8. In some embodiments, the anti-FAM19A5 antibody or antigen-binding portion thereof binds only to EP6, EP7, or EP8. In some embodiments, the anti-FAM19A5 antibody or antigen-binding portion thereof binds to EP6, EP7, and EP8. In some embodiments, the anti-FAM19A5 antibody, or antigen-binding portion thereof, binds to EP7 and EP8. In some embodiments, the anti-FAM19A5 antibody, or antigen-binding portion thereof, binds to EP7. In certain embodiments, antibodies or antigen-binding portions thereof are provided 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 even higher affinity than to other molecules of the FAM19A family, as measured, for example, by immunoassay (e.g., ELISA), surface plasmon resonance or dynamic exclusion assay. In certain embodiments, antibodies or antigen-binding portions thereof are provided that bind to FAM19A5 (e.g., human FAM19A5) without cross-reactivity with other molecules of the FAM19A family, as measured, for example, by immunoassay (e.g., ELISA), surface plasmon resonance or dynamic exclusion assay.

[0175] In certain embodiments, the anti-FAM19A5 antibody is not a native or naturally occurring antibody, e.g., the anti-FAM19A5 antibody has different post-translational modifications than naturally occurring antibodies, such as having more, fewer, or different types of post-translational modifications.

[0176] III. Exemplary Anti-FAM19A5 Antibodies Particular antibodies provided herein are antibodies, e.g., monoclonal antibodies, having the CDR and / or variable region sequences of antibodies 1-65, P2-C12, 13B4, 13F7, 15A9, P1-A03, P1-A08, P1-F02, P2-A01, P2-A03, P2-F07, or P2-F1, and antibodies having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or at least 99% identity) to the variable region or CDR sequences thereof. The VH and VL amino acid sequences of the anti-FAM19A5 antibodies of the invention are provided in Tables 2 and 3, respectively.

[0177] [Table 2]

[0178] [Table 3]

[0179] [Table 4]

[0180] [Table 5]

[0181] Thus, disclosed is an isolated anti-FAM19A5 antibody, or antigen-binding portion thereof, comprising a heavy chain and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NOs:5 or 103-113. In other embodiments, the isolated anti-FAM19A5 antibody, or antigen-binding portion thereof, comprises the CDRs of the heavy chain variable region selected from the group consisting of SEQ ID NOs:5 or 103-113.

[0182] Also disclosed is an isolated anti-FAM19A5 antibody, or antigen-binding portion thereof, comprising a heavy chain and a light chain variable region, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NOs: 6 or 114 to 124. In another embodiment, the isolated anti-FAM19A5 antibody, or antigen-binding portion thereof, comprises the CDRs of a light chain variable region selected from the group consisting of SEQ ID NOs: 6 or 114 to 124.

[0183] In certain embodiments, the isolated anti-FAM19A5 antibody or antigen-binding portion thereof comprises a CDR of a heavy chain variable region selected from the group consisting of SEQ ID NOs:5 or 103-113 and a CDR of a light chain variable region selected from the group consisting of SEQ ID NOs:6 or 114-124.

[0184] Also disclosed is an isolated anti-FAM19A5 antibody or antigen-binding portion thereof comprising a heavy chain and a light chain variable region, (i) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:5 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:6; (ii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:103 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:114; (iii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:104 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:115; (iv) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:105 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:116; (v) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:106 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:117; (vi) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:107 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:118; (vii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:108 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:119; (viii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:109 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:120; (ix) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:110 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:121; (x) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:111 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:122; (xi) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:112 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:123; and (xii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:113, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:124.

[0185] An isolated anti-FAM19A5 antibody or antigen-binding portion thereof is disclosed, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region 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 NOs:5 or 103-113.

[0186] Also disclosed is an isolated anti-FAM19A5 antibody or antigen-binding portion thereof comprising a heavy chain variable region and a light chain variable region, wherein the light chain variable region 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 NOs:6 or 114-124.

[0187] Also disclosed is an isolated anti-FAM19A5 antibody or antigen-binding portion thereof comprising a heavy chain and a light chain variable region, wherein the heavy chain variable region 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 NOs:5 or 103-113, and the light chain variable region 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 NOs:6 or 114-124.

[0188] In some embodiments, the invention provides an isolated anti-FAM19A5 antibody, or antigen-binding portion thereof, comprising: (a) heavy and light chain variable region sequences comprising SEQ ID NOs:5 and 6, respectively; (b) heavy and light chain variable region sequences comprising SEQ ID NOs: 103 and 114, respectively; (c) heavy and light chain variable region sequences comprising SEQ ID NOs: 104 and 115, respectively; (d) heavy and light chain variable region sequences comprising SEQ ID NOs:105 and 116, respectively; (e) heavy and light chain variable region sequences comprising SEQ ID NOs: 106 and 117, respectively; (f) heavy and light chain variable region sequences comprising SEQ ID NOs:107 and 118, respectively; (g) heavy and light chain variable region sequences comprising SEQ ID NOs: 108 and 119, respectively; (h) heavy and light chain variable region sequences comprising SEQ ID NOs:109 and 120, respectively; (i) heavy and light chain variable region sequences comprising SEQ ID NOs: 110 and 121, respectively; (j) heavy and light chain variable region sequences comprising SEQ ID NOs:111 and 122, respectively; (k) heavy and light chain variable region sequences comprising SEQ ID NOs: 112 and 123, respectively; or (l) Heavy and light chain variable region sequences comprising SEQ ID NOs: 113 and 124, respectively. Includes.

[0189] In certain embodiments, the anti-FAM19A5 antibody or antigen-binding portion thereof of the present invention comprises: (i) a heavy chain CDR1, CDR2 and CDR3 of 1-65, or a combination thereof, and / or a light chain CDR1, CDR2 and CDR3 of 1-65, or any combination thereof; (ii) a heavy chain CDR1, CDR2 and CDR3 of P2-C12, or a combination thereof, and / or a light chain CDR1, CDR2 and CDR3 of P2-C12, or any combination thereof; (iii) a heavy chain CDR1, CDR2 and CDR3 of 13B4, or a combination thereof. (iv) the heavy chain CDR1, CDR2 and CDR3 of 13F7, or a combination thereof, and / or the light chain CDR1, CDR2 and CDR3 of 13F7, or any combination thereof; (v) the heavy chain CDR1, CDR2 and CDR3 of 15A9, or a combination thereof, and / or the light chain CDR1, CDR2 and CDR3 of 15A9, or any combination thereof; (vi) the heavy chain CDR1, CDR2 and CDR3 of P1-A03, or any combination thereof. (vii) the heavy chain CDR1, CDR2 and CDR3 of P1-A08, or a combination thereof, and / or the light chain CDR1, CDR2 and CDR3 of P1-A08, or any combination thereof; (viii) the heavy chain CDR1, CDR2 and CDR3 of P1-F02, or a combination thereof, and / or the light chain CDR1, CDR2 and CDR3 of P1-F02, or any combination thereof; (ix) the heavy chain CDR1, CDR2 and CDR3 of P2-A01, or any combination thereof; (x) the heavy chain CDR1, CDR2 and CDR3, or a combination thereof, and / or the light chain CDR1, CDR2 and CDR3 of P2-A03, or any combination thereof; (xi) the heavy chain CDR1, CDR2 and CDR3, or a combination thereof, and / or the light chain CDR1, CDR2 and CDR3 of P2-F07, or any combination thereof;or (xii) the heavy chain CDR1, CDR2 and CDR3 of P2-F11, or a combination thereof, and / or the light chain CDR1, CDR2 and CDR3 of P2-F11, or any combination thereof. The amino acid sequences of the VH CDR1, CDR2 and CDR3 for the different anti-FAM19A5 antibodies disclosed herein are provided in Table 2. The amino acid sequences of the VL CDR1, CDR2 and CDR3 for the different anti-FAM19A5 antibodies disclosed herein are provided in Table 3.;

[0190] In some embodiments, an anti-FAM19A5 antibody, or antigen-binding portion thereof, that specifically binds human FAM19A5: (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO:7; and / or (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:8; and / or (c) VH CDR3 comprising the amino acid sequence of SEQ ID NO:9 Includes.

[0191] In certain embodiments, the antibody, or antigen-binding portion thereof, comprises any one, two, or all three of the above VH CDRs.

[0192] In some embodiments, an anti-FAM19A5 antibody, or antigen-binding portion thereof, that specifically binds human FAM19A5: (a) a VL CDR1 comprising the amino acid sequence of SEQ ID NO:10; and / or (b) a VL CDR2 comprising the amino acid sequence of SEQ ID NO:11; and / or (c) VL CDR3 comprising the amino acid sequence of SEQ ID NO:12 Includes.

[0193] In certain embodiments, the antibody, or antigen-binding portion thereof, comprises any one, two, or all three of the VL CDRs.

[0194] In some embodiments, an anti-FAM19A5 antibody, or antigen-binding portion thereof, that specifically binds human FAM19A5: (a) VH CDR1 comprising the amino acid sequence of SEQ ID NO:7; (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:8; (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO:9; (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO:10; (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO:11; and / or (f) VL CDR3 comprising the amino acid sequence of SEQ ID NO:12 Includes.

[0195] In some embodiments, an anti-FAM19A5 antibody, or antigen-binding portion thereof, that specifically binds human FAM19A5: (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO:29; and / or (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:30; and / or (c) VH CDR3 comprising the amino acid sequence of SEQ ID NO:31 Includes.

[0196] In certain embodiments, the antibody, or antigen-binding portion thereof, comprises any one, two, or all three of the above VH CDRs.

[0197] In some embodiments, an anti-FAM19A5 antibody, or antigen-binding portion thereof, that specifically binds human FAM19A5: (a) a VL CDR1 comprising the amino acid sequence of SEQ ID NO:32; and / or (b) a VL CDR2 comprising the amino acid sequence of SEQ ID NO:33; and / or (c) VL CDR3 comprising the amino acid sequence of SEQ ID NO:34 Includes.

[0198] In certain embodiments, the antibody, or antigen-binding portion thereof, comprises any one, two, or all three of the VL CDRs.

[0199] In some embodiments, an anti-FAM19A5 antibody, or antigen-binding portion thereof, that specifically binds human FAM19A5: (a) VH CDR1 comprising the amino acid sequence of SEQ ID NO:29; (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:30; (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO:31; (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO:32; (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO:33; and / or (f) VL CDR3 comprising the amino acid sequence of SEQ ID NO:34 Includes.

[0200] In some embodiments, an anti-FAM19A5 antibody, or antigen-binding portion thereof, that specifically binds human FAM19A5: (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO:35; and / or (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:36; and / or (c) VH CDR3 comprising the amino acid sequence of SEQ ID NO:37 Includes.

[0201] In certain embodiments, the antibody, or antigen-binding portion thereof, comprises any one, two, or all three of the above VH CDRs.

[0202] In some embodiments, an anti-FAM19A5 antibody, or antigen-binding portion thereof, that specifically binds human FAM19A5: (a) a VL CDR1 comprising the amino acid sequence of SEQ ID NO:38; and / or (b) a VL CDR2 comprising the amino acid sequence of SEQ ID NO:39; and / or (c) VL CDR3 comprising the amino acid sequence of SEQ ID NO:40 Includes.

[0203] In certain embodiments, the antibody, or antigen-binding portion thereof, comprises any one, two, or all three of the VL CDRs.

[0204] In some embodiments, an anti-FAM19A5 antibody, or antigen-binding portion thereof, that specifically binds human FAM19A5: (a) VH CDR1 comprising the amino acid sequence of SEQ ID NO:35; (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:36; (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO:37; (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO:38; (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO:39; and / or (f) VL CDR3 comprising the amino acid sequence of SEQ ID NO:40 Includes.

[0205] In some embodiments, an anti-FAM19A5 antibody, or antigen-binding portion thereof, that specifically binds human FAM19A5: (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO:41; and / or (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:42; and / or (c) VH CDR3 comprising the amino acid sequence of SEQ ID NO:43 Includes.

[0206] In certain embodiments, the antibody, or antigen-binding portion thereof, comprises any one, two, or all three of the above VH CDRs.

[0207] In some embodiments, an anti-FAM19A5 antibody, or antigen-binding portion thereof, that specifically binds human FAM19A5: (a) a VL CDR1 comprising the amino acid sequence of SEQ ID NO:44; and / or (b) a VL CDR2 comprising the amino acid sequence of SEQ ID NO:45; and / or (c) VL CDR3 comprising the amino acid sequence of SEQ ID NO:46 Includes.

[0208] In certain embodiments, the antibody, or antigen-binding portion thereof, comprises any one, two, or all three of the VL CDRs.

[0209] In some embodiments, an anti-FAM19A5 antibody, or antigen-binding portion thereof, that specifically binds human FAM19A5: (a) VH CDR1 comprising the amino acid sequence of SEQ ID NO:41; (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:42; (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO:43; (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO:44; (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO:45; and / or (f) VL CDR3 comprising the amino acid sequence of SEQ ID NO:46 Includes.

[0210] In some embodiments, an anti-FAM19A5 antibody, or antigen-binding portion thereof, that specifically binds human FAM19A5: (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO:47; and / or (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:48; and / or (c) VH CDR3 comprising the amino acid sequence of SEQ ID NO:49 Includes.

[0211] In certain embodiments, the antibody, or antigen-binding portion thereof, comprises any one, two, or all three of the above VH CDRs.

[0212] In some embodiments, an anti-FAM19A5 antibody, or antigen-binding portion thereof, that specifically binds human FAM19A5: (a) a VL CDR1 comprising the amino acid sequence of SEQ ID NO:50; and / or (b) a VL CDR2 comprising the amino acid sequence of SEQ ID NO:51; and / or (c) VL CDR3 comprising the amino acid sequence of SEQ ID NO:52 Includes.

[0213] In certain embodiments, the antibody, or antigen-binding portion thereof, comprises any one, two, or all three of the VL CDRs.

[0214] In some embodiments, an anti-FAM19A5 antibody, or antigen-binding portion thereof, that specifically binds human FAM19A5: (a) VH CDR1 comprising the amino acid sequence of SEQ ID NO:47; (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:48; (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO:49; (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO:50; (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO:51; and / or (f) VL CDR3 comprising the amino acid sequence of SEQ ID NO:52 Includes.

[0215] In some embodiments, an anti-FAM19A5 antibody, or antigen-binding portion thereof, that specifically binds human FAM19A5: (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO:53; and / or (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:54; and / or (c) VH CDR3 comprising the amino acid sequence of SEQ ID NO:55 Includes.

[0216] In certain embodiments, the antibody, or antigen binding portion thereof, comprises any one, two, or all three of the above VH CDRs.

[0217] In some embodiments, an anti-FAM19A5 antibody, or antigen-binding portion thereof, that specifically binds human FAM19A5: (a) a VL CDR1 comprising the amino acid sequence of SEQ ID NO:56; and / or (b) a VL CDR2 comprising the amino acid sequence of SEQ ID NO:57; and / or (c) VL CDR3 comprising the amino acid sequence of SEQ ID NO:58 Includes.

[0218] In certain embodiments, the antibody, or antigen-binding portion thereof, comprises any one, two, or all three of the VL CDRs.

[0219] In some embodiments, an anti-FAM19A5 antibody, or antigen-binding portion thereof, that specifically binds human FAM19A5: (a) VH CDR1 comprising the amino acid sequence of SEQ ID NO:53; (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:54; (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO:55; (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO:56; (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO:57; and / or (f) VL CDR3 comprising the amino acid sequence of SEQ ID NO:58 Includes.

[0220] In some embodiments, an anti-FAM19A5 antibody, or antigen-binding portion thereof, that specifically binds human FAM19A5: (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO:59; and / or (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:60; and / or (c) VH CDR3 comprising the amino acid sequence of SEQ ID NO:61 Includes.

[0221] In certain embodiments, the antibody, or antigen-binding portion thereof, comprises any one, two, or all three of the above VH CDRs.

[0222] In some embodiments, an anti-FAM19A5 antibody, or antigen-binding portion thereof, that specifically binds human FAM19A5: (a) a VL CDR1 comprising the amino acid sequence of SEQ ID NO:62; and / or (b) a VL CDR2 comprising the amino acid sequence of SEQ ID NO:63; and / or (c) VL CDR3 comprising the amino acid sequence of SEQ ID NO:64 Includes.

[0223] In certain embodiments, the antibody, or antigen-binding portion thereof, comprises any one, two, or all three of the VL CDRs.

[0224] In some embodiments, an anti-FAM19A5 antibody, or antigen-binding portion thereof, that specifically binds human FAM19A5: (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO:59; (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:60; (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO:61; (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO:62; (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO:63; and / or (f) VL CDR3 comprising the amino acid sequence of SEQ ID NO:64 Includes.

[0225] In some embodiments, an anti-FAM19A5 antibody, or antigen-binding portion thereof, that specifically binds human FAM19A5: (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO:65; and / or (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:66; and / or (c) VH CDR3 comprising the amino acid sequence of SEQ ID NO:67 Includes.

[0226] In certain embodiments, the antibody, or antigen-binding portion thereof, comprises any one, two, or all three of the above VH CDRs.

[0227] In some embodiments, an anti-FAM19A5 antibody, or antigen-binding portion thereof, that specifically binds human FAM19A5: (a) a VL CDR1 comprising the amino acid sequence of SEQ ID NO:68; and / or (b) a VL CDR2 comprising the amino acid sequence of SEQ ID NO:69; and / or (c) VL CDR3 comprising the amino acid sequence of SEQ ID NO:70 Includes.

[0228] In certain embodiments, the antibody, or antigen-binding portion thereof, comprises any one, two, or all three of the VL CDRs.

[0229] In some embodiments, an anti-FAM19A5 antibody, or antigen-binding portion thereof, that specifically binds human FAM19A5: (a) VH CDR1 comprising the amino acid sequence of SEQ ID NO:65; (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:66; (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO:67; (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO:68; (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO:69; and / or (f) VL CDR3 comprising the amino acid sequence of SEQ ID NO:70 Includes.

[0230] In some embodiments, an anti-FAM19A5 antibody, or antigen-binding portion thereof, that specifically binds human FAM19A5: (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO:71; and / or (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:72; and / or (c) VH CDR3 comprising the amino acid sequence of SEQ ID NO:73 Includes.

[0231] In certain embodiments, the antibody, or antigen-binding portion thereof, comprises any one, two, or all three of the above VH CDRs.

[0232] In some embodiments, an anti-FAM19A5 antibody, or antigen-binding portion thereof, that specifically binds human FAM19A5: (a) a VL CDR1 comprising the amino acid sequence of SEQ ID NO:74; and / or (b) a VL CDR2 comprising the amino acid sequence of SEQ ID NO:75; and / or (c) VL CDR3 comprising the amino acid sequence of SEQ ID NO:76 Includes.

[0233] In certain embodiments, the antibody, or antigen-binding portion thereof, comprises any one, two, or all three of the VL CDRs.

[0234] In some embodiments, an anti-FAM19A5 antibody, or antigen-binding portion thereof, that specifically binds human FAM19A5: (a) VH CDR1 comprising the amino acid sequence of SEQ ID NO:71; (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:72; (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO:73; (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO:74; (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO:75; and / or (f) VL CDR3 comprising the amino acid sequence of SEQ ID NO:76 Includes.

[0235] In some embodiments, an anti-FAM19A5 antibody, or antigen-binding portion thereof, that specifically binds human FAM19A5: (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO:77; and / or (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:78; and / or (c) VH CDR3 comprising the amino acid sequence of SEQ ID NO:79 Includes.

[0236] In certain embodiments, the antibody, or antigen-binding portion thereof, comprises any one, two, or all three of the above VH CDRs.

[0237] In some embodiments, an anti-FAM19A5 antibody, or antigen-binding portion thereof, that specifically binds human FAM19A5: (a) a VL CDR1 comprising the amino acid sequence of SEQ ID NO:80; and / or (b) a VL CDR2 comprising the amino acid sequence of SEQ ID NO:81; and / or (c) VL CDR3 comprising the amino acid sequence of SEQ ID NO:82 Includes.

[0238] In certain embodiments, the antibody, or antigen-binding portion thereof, comprises any one, two, or all three of the VL CDRs.

[0239] In some embodiments, an anti-FAM19A5 antibody, or antigen-binding portion thereof, that specifically binds human FAM19A5: (a) VH CDR1 comprising the amino acid sequence of SEQ ID NO:77; (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:78; (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO:79; (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO:80; (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO:81; and / or (f) VL CDR3 comprising the amino acid sequence of SEQ ID NO:82 Includes.

[0240] In some embodiments, an anti-FAM19A5 antibody, or antigen-binding portion thereof, that specifically binds human FAM19A5: (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO:83; and / or (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:84; and / or (c) VH CDR3 comprising the amino acid sequence of SEQ ID NO:85 Includes.

[0241] In certain embodiments, the antibody, or antigen-binding portion thereof, comprises any one, two, or all three of the above VH CDRs.

[0242] In some embodiments, an anti-FAM19A5 antibody, or antigen-binding portion thereof, that specifically binds human FAM19A5: (a) a VL CDR1 comprising the amino acid sequence of SEQ ID NO:86; and / or (b) a VL CDR2 comprising the amino acid sequence of SEQ ID NO:87; and / or (c) VL CDR3 comprising the amino acid sequence of SEQ ID NO:88 Includes.

[0243] In certain embodiments, the antibody, or antigen-binding portion thereof, comprises any one, two, or all three of the VL CDRs.

[0244] In some embodiments, an anti-FAM19A5 antibody, or antigen-binding portion thereof, that specifically binds human FAM19A5: (a) VH CDR1 comprising the amino acid sequence of SEQ ID NO:83; (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:84; (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO:85; (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO:86; (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO:87; and / or (f) VL CDR3 comprising the amino acid sequence of SEQ ID NO:88 Includes.

[0245] In some embodiments, an anti-FAM19A5 antibody, or antigen-binding portion thereof, that specifically binds human FAM19A5: (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO:89; and / or (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:90; and / or (c) VH CDR3 comprising the amino acid sequence of SEQ ID NO:91 Includes.

[0246] In certain embodiments, the antibody, or antigen-binding portion thereof, comprises any one, two, or all three of the above VH CDRs.

[0247] In some embodiments, an anti-FAM19A5 antibody, or antigen-binding portion thereof, that specifically binds human FAM19A5: (a) a VL CDR1 comprising the amino acid sequence of SEQ ID NO:92; and / or (b) a VL CDR2 comprising the amino acid sequence of SEQ ID NO:93; and / or (c) VL CDR3 comprising the amino acid sequence of SEQ ID NO:94 Includes.

[0248] In certain embodiments, the antibody, or antigen-binding portion thereof, comprises any one, two, or all three of the VL CDRs.

[0249] In some embodiments, an anti-FAM19A5 antibody, or antigen-binding portion thereof, that specifically binds human FAM19A5: (a) VH CDR1 comprising the amino acid sequence of SEQ ID NO:89; (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:90; (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO:91; (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO:92; (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO:93; and / or (f) VL CDR3 comprising the amino acid sequence of SEQ ID NO:94 Includes.

[0250] In certain embodiments, the anti-FAM19A5 antibody, or antigen-binding portion thereof, comprises any one, two, three, four, five or six of the above CDRs.

[0251] A VH domain provided 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 provided 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 form a full-length antibody.

[0252] Thus, in certain embodiments, antibodies are provided herein that include antibody light and heavy chains, for example separated light and heavy chains. In relation to the light chain, in certain embodiments, the light chain of the antibody provided herein is a kappa light chain. In other specific embodiments, the light chain of the antibody provided herein is a lambda light chain. In yet other specific embodiments, the light chain of the antibody provided herein is a human kappa light chain or a human lambda light chain. In certain embodiments, the antibody provided herein that specifically binds to a FAM19A5 polypeptide (e.g., human FAM19A5) includes a light chain that includes any of the VL or VL CDR amino acid sequences provided herein, and the constant region of the light chain includes the amino acid sequence of a human kappa light chain constant region. In certain embodiments, the antibody provided herein that specifically binds to a FAM19A5 polypeptide (e.g., human FAM19A5) includes a light chain that includes any of the VL or VL CDR amino acid sequences provided herein, and the constant region of the light chain includes the amino acid sequence of a human lambda light chain constant region. Non-limiting examples of human constant region sequences are known in the art (see, e.g., U.S. Patent No. 5,693,780 and Kabat EA et al., (1991) supra).

[0253] With respect to the heavy chain, in some embodiments, the heavy chain of the antibodies provided herein can be an alpha (α), delta (δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In other particular embodiments, the heavy chain of the antibodies provided herein can comprise an alpha (α), delta (δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In one embodiment, an antibody provided herein that specifically binds to a FAM19A5 polypeptide (e.g., human FAM19A5) comprises a heavy chain comprising a VH or VH CDR amino acid sequence provided herein, and the constant region of the heavy chain comprises the amino acid sequence of a human gamma (γ) heavy chain constant region. In other embodiments, the antibodies provided herein that specifically bind to a FAM19A5 polypeptide (e.g., human FAM19A5) comprise a heavy chain comprising a VH or VH CDR amino acid sequence provided herein, and the constant region of the heavy chain comprises the amino acids of a human heavy chain provided herein or known in the art. Non-limiting examples of human constant region sequences are known in the art (see, e.g., U.S. Patent No. 5,693,780 and Kabat EA et al, 1991, supra).

[0254] In some embodiments, the antibodies provided herein that specifically bind to a FAM19A5 polypeptide (e.g., human FAM19A5) comprise a VL domain and a VH domain that comprise the VH or VH CDRs and the VL and VL CDRs provided herein, and 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 particular embodiments, the antibodies provided herein that specifically bind to a FAM19A5 polypeptide (e.g., human FAM19A5) comprise a VL domain and a VH domain that comprise any of the amino acid sequences provided herein, and the constant region comprises the amino acid sequence of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, or a constant region of any subclass of immunoglobulin molecule (e.g., IgG1, IgG2, IgG3, IgG4, IgAl, and IgA2). In some embodiments, the constant region comprises the amino acid sequence of the constant region of naturally occurring human IgG, including subclasses (e.g., IgG1, IgG2, IgG3, or IgG4), and allotypes (e.g., G1m, G2m, G3m, and nG4m) and variants thereof (see, e.g., Vidarsson G. et al. Front Immunol. 5:520 (published online 2014-10-20), 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.

[0255] In certain embodiments, the anti-FAM19A5 antibody or antigen-binding portion thereof provided herein does not have 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 in the CH2 domain of the Fc region closest to the hinge region are responsible for the effector functions of the antibody and contain substantially overlapping binding sites for Clq (complement) and IgG-Fc receptor (FcγR) on effector cells of the innate immune system. Also, IgG2 and IgG4 antibodies have lower levels of Fc effector functions than IgG1 and IgG3 antibodies. Antibody effector functions can be reduced or avoided by different approaches known in the art, including: (1) the use of antibody fragments lacking the Fc region (e.g., Fab, F(ab')2, single chain Fv (scFv), or sdAB composed of monomeric VH or VL domains); (2) the generation of aglycosylated antibodies, which can be generated by deleting or altering the residue to which sugar is attached, enzymatically removing sugars, generating the antibody in cells cultured in the presence of glycosylation inhibitors, or expressing the antibody in cells that cannot glycosylate proteins (e.g., bacterial host cells, see U.S. Patent Publication No. 20120100140); (3) the use of Fc regions from IgG subclasses with reduced effector function (e.g., Fc regions from IgG2 or IgG4 antibodies or chimeric Fc regions comprising CH2 domains from IgG2 or IgG4 antibodies, see U.S. Patent Publication No. 20120100140 and Lau C. et al. al, J. Immunol. 191:4769-4777 (2013)); and (4) generating Fc regions with mutations that result in reduced or abolished 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)).

[0256] Thus, in some embodiments, the anti-FAM19A5 antibodies or antigen-binding portions thereof provided herein are Fab, Fab', F(ab')2, Fv, single-chain Fv (scFv), or sdAbs composed of monomeric VH or VL domains. Such antibody fragments are well known in the art and disclosed herein.

[0257] In some embodiments, the anti-FAM19A5 antibody or antigen-binding portion thereof provided herein comprises an Fc region with reduced or no Fc effector function. In some embodiments, the constant region comprises the amino acid sequence of an Fc region of human IgG2 or IgG4. 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 from an IgG antibody of the IgG4 isotype and a CH3 domain from an IgG antibody of the IgG1 isotype, or a chimeric Fc region comprising a hinge region from IgG2 and a CH2 region from IgG4, or an Fc region with mutations that result in reduced or abolished Fc function. Fc regions with reduced or no 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 patent publications and PCT publications cited therein). In addition, Fc regions with reduced or no Fc effector function can be readily generated by one of skill in the art.

[0258] IV. Nucleic acid molecules Other aspects disclosed herein relate to one or more nucleic acid molecules encoding any one of the antibodies or antigen-binding portions thereof provided herein. The nucleic acid 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 it has been purified from other cellular components and other contaminants, such as other cellular nucleic acids (e.g., other chromosomal DNA, e.g., chromosomal DNA linked to the isolated DNA) or proteins by standard techniques, including alkaline / SDS treatment, CsCl banding, column chromatography, restriction enzymes, agarose gel electrophoresis, and other techniques well known in the art (see, e.g., F. Ausubel, et al, ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York). The nucleic acids provided 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.

[0259] The nucleic acids presented herein can be obtained using standard molecular biology techniques. In the case of antibodies expressed by a hybridoma (e.g., a hybridoma generated from a transgenic mouse carrying human immunoglobulin genes, as described in more detail below), cDNA encoding the light and heavy chains of the antibody produced by the hybridoma 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 techniques), nucleic acid encoding the antibody can be recovered from the library.

[0260] Particular nucleic acid molecules provided herein encode the VH and VL sequences of the anti-FAM19A5 antibodies of the invention. Exemplary DNA sequences encoding the VH and VL sequences are provided in Tables 6 and 7, respectively.

[0261] [Table 6]

[0262] [Table 7]

[0263] A method for producing the anti-FAM19A5 antibodies provided herein includes expressing the heavy and light chains in a cell line that contains nucleotide sequences encoding the heavy and light chains, respectively, with signal peptides, e.g., SEQ ID NOs: 18 and 19. Host cells containing those nucleotide sequences are encompassed by the invention.

[0264] Once the DNA fragments encoding the VH and VL segments are obtained, they can be further manipulated by standard recombinant DNA techniques, e.g., to convert the variable region genes into full-length antibody chain genes, into Fab fragment genes, or into scFv genes. In such manipulations, the VL- or VH-encoding DNA fragment is operably linked to another DNA fragment encoding another protein, e.g., an antibody constant region or a flexibility linker. The term "operably linked" means that the two DNA fragments are joined in such a way that the amino acid sequences encoded by the two DNA fragments remain in frame.

[0265] 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 another 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, e.g., Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242) and DNA fragments containing those 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 another DNA molecule encoding only the heavy chain CH1 constant region.

[0266] The isolated DNA encoding the VL region can be converted into a full-length light chain gene (not just a Fab light chain gene) by operably linking the VL-encoding DNA to another DNA molecule encoding the light chain constant region, CL. The sequences of human light chain constant region genes are known in the art (see, e.g., Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region.

[0267] To generate an scFv gene, the VH- and VL-encoding DNA fragments are operably linked to another fragment encoding a flexibility linker, e.g., encoding the amino acid sequence (Gly4-Ser)3, such that the VH and VL sequences can be expressed as a contiguous single-chain protein with the VL and VH domains connected by the flexibility 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).

[0268] In some embodiments, the invention provides a vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antigen-binding portion thereof, hi other embodiments, the vector can be used in gene therapy regimens.

[0269] Suitable vectors for the present invention include expression vectors, viral vectors and plasmid vectors, hi one embodiment, the vector is a viral vector.

[0270] An expression vector as provided herein 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 an RNA viral vector, the necessary elements for replication and translation when introduced into an appropriate host cell. Expression vectors can include plasmids, phagemids, viruses, and their derivatives.

[0271] An expression vector of the invention can include a polynucleotide encoding an antibody or antigen-binding portion thereof provided herein. In one embodiment, the coding sequence for the antibody or antigen-binding portion thereof is operably linked to an expression control sequence. 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 said to be operably linked when they are covalently linked in a manner that allows expression or transcription and / or translation of the coding sequence under the influence and control of the gene expression control sequence. Two DNA sequences are said to be operably linked if the introduction of a promoter 5' to the gene expression sequence results in transcription of the coding sequence, and if the nature of the linkage between the two DNA sequences (1) does not result in the introduction of frame shift mutations, (2) does not interfere with the ability of the promoter region to direct transcription of the coding sequence, or (3) does not interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a gene expression sequence would be operably linked to a coding nucleic acid sequence if the gene expression sequence was capable of effecting transcription of the coding nucleic acid sequence, such that the transcript would be translated into the desired antibody or antigen-binding portion thereof.

[0272] Viral vectors include, but are not limited to, nucleic acid sequences from the following viruses: retroviruses, such as Moloney murine leukemia virus, Harvey murine sarcoma virus, murine mammary tumor virus, and Rous sarcoma virus; lentiviruses; adenoviruses; adeno-associated viruses; SV40-type viruses; polyoma viruses; Epstein-Barr virus; papilloma viruses; herpes viruses; cowpox viruses; polioviruses; and RNA viruses, such as retroviruses. Other vectors known in the art are readily available to those skilled in the art. Certain viral vectors are based on non-cytopathic eukaryotic viruses in which a non-essential gene has 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 the host cell DNA. Retroviruses have been approved for human gene therapy trials. The most useful are retroviruses that are replication-defective (i.e., capable of directing synthesis of desired proteins, but unable to make infectious particles). Such genetically modified retroviral expression vectors have general utility in the highly efficient transduction of genes in vivo. Standard protocols for generating replication-defective retroviruses, including the steps of integrating exogenous genetic material into a plasmid, transfecting a packaging cell line with the plasmid, generating recombinant retrovirus by the packaging cell line, harvesting viral particles from tissue culture medium, and infecting target cells with the viral particles, are presented in Kriegler, M., Gene Transfer and Expression, A Laboratory Manual, W. H. Freeman Co., New York (1990); Murry, E. J., Methods in Molecular Biology, Vol. 7, Humana Press, Inc., Cliffton, NJ (1991).

[0273] In one embodiment, the virus is adeno-associated virus, a double-stranded DNA virus. Adeno-associated virus can be genetically engineered to be replication-defective and can infect a wide range of cell types and species. Adeno-associated virus has the advantages of heat and lipid solvent stability; high transduction frequency in cells of various lineages, including hematopoietic cells; and the lack of superinfection inhibition allows for multiple sequential transductions. Experimental results show that adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the probability of insertional mutagenesis and variability in inserted gene expression that are characteristic of retroviral infection. In addition, wild-type adeno-associated virus infection has been tracked in tissue culture for more than 100 passages in the absence of selection pressure, implying that adeno-associated virus genome integration is a relatively stable event. Adeno-associated virus can also function in an extrachromosomal manner.

[0274] 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.

[0275] Lentiviral genomes and proviral DNA typically contain three genes found in retroviruses, namely gag, pol and env, which are flanked by two long terminal repeat (LTR) sequences. The gag gene encodes the internal structural (matrix, capsid and nucleocapsid) proteins, the pol gene encodes the RNA-directed DNA polymerase (reverse transcriptase), protease and integrase, and the env gene encodes the viral envelope glycoproteins. The 5' and 3' LTRs act to promote transcription and polyadenylation of virion RNA. The LTRs contain all the cis-acting sequences essential for viral replication. Lentiviruses have additional genes including vif, vpr, tat, rev, vpu, nef and vpx.

[0276] Adjacent to the 5'LTR are sequences required for reverse transcription of the genome (tRNA primer binding site) and for efficient encapsidation of viral RNA into particles (Psi site). If the sequences required for encapsidation (or packaging of retroviral RNA into infectious virions) are absent from the viral genome, cis deficiency prevents encapsidation of genomic DNA.

[0277] However, the mutations are maintained so that they are capable of directing the synthesis of all virion proteins. The present invention provides a method for generating recombinant lentivirus capable of infecting non-dividing cells, which method comprises transfecting a suitable host cell with two or more vectors with packaging functions, namely gag, pol and env, or rev and tat. As disclosed below, vectors lacking a functional tat gene are preferred for certain applications. Thus, for example, a first vector can provide nucleic acid encoding viral gag and viral pol, and another vector can provide nucleic acid encoding viral env capable of generating a packaging cell. Introducing a vector providing a heterologous gene, identified as a transfer vector, into the packaging cell provides a producer cell that releases infectious viral particles carrying the foreign gene of interest.

[0278] With the above presented configuration of vector and foreign gene, the second vector can provide a nucleic acid encoding a viral envelope (env) gene. The env gene can be derived from any suitable virus, including retroviruses. In some embodiments, the env protein is a bilateral envelope protein that allows transduction of cells of human and other species.

[0279] Non-limiting examples of retrovirus-derived env genes include 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 (GaLV or GALV), human immunodeficiency virus (HIV), and Rous sarcoma virus (RSV). Other env genes, such as vesicular stomatitis virus (VSV) protein G (VSV G), hepatitis virus, and influenza, can also be used.

[0280] The vector providing the viral env nucleic acid sequence is operably associated with the regulatory sequences presented herein.

[0281] In a particular embodiment, the vector comprises a lentiviral vector in which the HIV virulence genes env, vif, vpr, vpu and nef have been deleted without compromising the ability of the vector to transduce non-dividing cells.

[0282] In some embodiments, the vector comprises a lentiviral vector that comprises a deletion of the U3 region of the 3'LTR. The deletion of the U3 region can be a complete deletion or a partial deletion.

[0283] In some embodiments, the lentiviral vector of the present invention comprising the FVIII nucleotide sequence provided herein can be transfected into a cell with (a) a first nucleotide sequence comprising 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 cell is further transfected with a fourth nucleotide sequence comprising a rev gene. In certain embodiments, the lentiviral vector lacks a functional gene selected from vif, vpr, vpu, vpx, and nef, or a combination thereof.

[0284] In certain embodiments, the lentivirus comprises one or more nucleotide sequences encoding a gag protein, a Rev-responsive element, a central polypurine track (cPPT), or any combination thereof.

[0285] Examples of lentiviral vectors are disclosed in WO9931251, W09712622, W09817815, W09817816, and W09818934, which are incorporated by reference in their entireties.

[0286] Other vectors include plasmid vectors. Plasmid vectors are widely known in the art (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989). In the past few years, plasmid vectors have proven to be particularly useful for delivering genes to cells in vivo, since they do not have the ability to replicate and integrate in the host genome. However, plasmids with promoters compatible with the host cell can express peptides from genes operably encoded within the plasmid. Some commonly used plasmids available from commercial suppliers include pBR322, pUC18, pUC19, various pcDNA plasmids, pRC / CMV, various pCMV plasmids, pSV40, and pBlueScript. Additional examples of specific plasmids include pcDNA3.1, catalog number V79020; pcDNA3.1 / hygro, catalog number V87020; pcDNA4 / myc-His, catalog number V86320; and pBudCE4.1, catalog number V53220, all available from Invitrogen (Carlsbad, Calif.). Other plasmids will be known to those of skill in the art. Additionally, plasmids can be custom designed using standard molecular biology techniques to remove and / or add specific segments of DNA.

[0287] V. Antibody generation 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 by 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 related fields of the art. These techniques are described in detail in the references cited herein (e.g., 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, 2nd 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 annually updated); Current Protocols in Immunology, John Wiley & Sons (1987 and annually updated); Gait (ed.) (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein (ed.) (1991) Oligonucleotides and Analogues: A Practical Approach, IRL Press). Approach, IRL Press; see Birren B et al, (eds.) (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press).

[0288] In certain embodiments, the antibodies provided herein are antibodies that have been created, expressed, generated, or isolated by any means involving, for example, synthesis of DNA sequences, production by genetic engineering (e.g., recombinant antibodies). In certain embodiments, such antibodies contain sequences (e.g., DNA sequences or amino acid sequences) that do not naturally exist within the antibody germline repertoire of an animal or mammal (e.g., human) in vivo.

[0289] In certain embodiments, disclosed are methods for making an antibody or antigen-binding fragment thereof that immunospecifically binds to FAM19A5 (e.g., human FAM19A5), comprising culturing a cell or host cell as provided herein. In certain embodiments, disclosed are methods for making 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 as provided herein (e.g., a cell or host cell comprising a polynucleotide encoding an antibody as provided 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 obtained from the cell or host cell.

[0290] Methods for generating polyclonal antibodies are known in the art (see, e.g., Chapter 11, Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel FM et al, eds., John Wiley and Sons, New York).

[0291] Monoclonal antibodies can be produced using a wide range of techniques known in the art, including hybridoma, recombinant and phage display technologies, or a combination thereof. Monoclonal antibodies are known in the art and can be produced using hybridoma techniques, for example, as taught in Harlow E & Lane D, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed.1988); Hammerling GJ et al., Monoclonal Antibodies and T-Cell Hybridomas 563681 (Elsevier, NY, 1981). The "monoclonal antibodies" presented herein are not limited to antibodies produced by hybridoma techniques. For example, monoclonal antibodies can be produced recombinantly from host cells exogenously expressing the antibodies presented herein or fragments thereof, such as the light chain and / or heavy chain of such antibodies.

[0292] In certain embodiments, a "monoclonal antibody" as provided herein is an antibody produced by a single cell (e.g., a hybridoma or host cell producing a recombinant antibody) that 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 as provided in the Examples disclosed herein. In certain embodiments, a monoclonal antibody may be a chimeric or humanized antibody. In certain embodiments, a monoclonal antibody is a monovalent antibody or a multivalent (e.g., bivalent) antibody. In certain embodiments, a monoclonal antibody is a monospecific or multispecific antibody (e.g., bispecific antibody). A monoclonal antibody as provided herein may be produced by hybridoma methods, e.g., as described in Kohler G & Milstein C (1975) Nature 256:495, or may be isolated from a phage library using techniques as provided herein. Other methods for generating clonal cell lines and the monoclonal antibodies expressed in this manner are also well known in the art (e.g., Chapter 11, Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel FM et al., supra).

[0293] Methods for producing and screening specific antibodies using hybridoma technology are well known in the art. For example, in the hybridoma method, mice or other suitable host animals, such as sheep, goats, rabbits, rats, hamsters, or macaques, are immunized to elicit lymphocytes that produce or can produce antibodies that specifically bind to the protein used for immunization (e.g., human FAM19A5). Lymphocytes may also be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, thereby forming hybridoma cells (see Goding JW (Ed), Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Additionally, RIMMS (repeated immunization multiple sites) technology may be utilized to immunize animals (Kilpatrick KE et al, (1997) Hybridoma 16:381-9, incorporated herein by reference in its entirety).

[0294] In some embodiments, a mouse (or other animal, e.g., chicken, rat, monkey, donkey, pig, sheep, hamster, or dog) may be immunized with an antigen (e.g., FAM19A5, e.g., human FAM19A5), an immune response is detected, antibodies specific to the antigen are detected in the mouse serum, the mouse spleen is harvested, and the spleen cells are isolated. The spleen cells are then fused by known techniques to any suitable myeloma cells, e.g., cells from cell line SP20 available from American Type Culture Collection (ATCC®) (Manassas, VA), thereby forming hybridomas. Hybridomas are selected and cloned by limiting dilution. In certain embodiments, lymph nodes of the immunized mouse are harvested and fused with NSO myeloma cells.

[0295] The hybridoma cells thus produced are preferably seeded and grown in an appropriate culture medium containing one or more substances which 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), then the hybridoma culture medium will typically contain hypoxanthine, aminopterin, and thymidine (HAT medium), which substances will prevent the growth of HGPRT-deficient cells.

[0296] A specific embodiment utilizes myeloma cells that efficiently fuse selected antibody-producing cells and thus support stable high-level antibody production, and are sensitive to media such as HAT medium. Among these myeloma cell lines are murine myeloma cell lines, such as the NSO cell line or 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 demonstrated to produce human monoclonal antibodies (see 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)).

[0297] Culture medium in which the hybridoma cells are grown is evaluated 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).

[0298] After hybridoma cells producing antibodies of the desired specificity, affinity and / or activity are identified, the clones may be subcloned by limiting dilution processes and grown by standard methods (Goding JW (Ed), Monoclonal Antibodies: Principles and Practice, supra). Suitable culture media for this purpose include, for example, D-MEM or RPMI 1640 medium. Hybridoma cells may also be grown in vivo as ascites tumors in animals.

[0299] The monoclonal antibodies secreted by the subclones can be 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.

[0300] The antibodies provided herein include antibody fragments that recognize specific FAM19A5 (e.g., human FAM19A5) and can be generated by any technique known to those skilled in the art. For example, the Fab and F(ab')2 fragments provided herein can be generated by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (to generate Fab fragments) or pepsin (to generate 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 disulfide bonds at the hinge region.

[0301] The antibodies or antigen-binding fragments thereof provided 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 carrying the polynucleotide sequences encoding them. In particular, DNA sequences encoding the VH and VL domains are amplified from animal cDNA libraries (e.g., human or non-human, e.g., murine or chicken cDNA libraries of diseased tissues). The DNA encoding the VH and VL domains are recombined with an scFv linker by PCR and cloned into a phagemid vector. The vector is electroporated into E. coli, which 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 gene can be selected and confirmed using antigen, e.g., labeled antigen or antigen linked or captured to a solid surface or bead.Examples of phage display methods that can be used to generate the antibodies provided 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) Advances in Immunology 1999, 11:111-112, and in J Immunol 2000, 11:111-112. 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; and U.S. Patent Publication Nos. 5,698,426, 5,2 ... including those disclosed in Nos. 3,409, 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.

[0302] After phage selection, as described in the above references, the antibody coding region can be isolated from the phage and used to produce whole antibodies, including human antibodies, or any other desired antigen-binding fragment, which 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, are also known in the art, such as those disclosed in PCT Publication No. WO92 / 22324; Mullinax RL 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.

[0303] In one embodiment, to generate a whole antibody, PCR primers containing the VH or VL nucleotide sequence, a restriction site, and flanking sequences to protect the restriction site 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. Alternatively, the VH and VL domains can be cloned into a vector expressing the essential constant regions. Stable or transient cell lines expressing full-length antibodies, e.g., IgG, can then be generated by co-transfecting the heavy chain conversion vector and the light chain conversion vector into a cell line using techniques known to those skilled in the art.

[0304] A chimeric antibody is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules. For example, a chimeric antibody may 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, for example, Morrison SL (1985) Science 229:1202-7; Oi VT & Morrison SL (1986) Bio-Techniques 4:214-221; Gillies SD et al, (1989) J Immunol Methods 125:191-202; and U.S. Patent Application Nos. 5,807,715, 4,816,567, 4,816,397, and 6,331,415).

[0305] A humanized antibody is capable of binding to a defined antigen and comprises a framework region having substantially the amino acid sequence of a human immunoglobulin and a CDR having substantially the amino acid sequence of a non-human immunoglobulin (e.g., 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, non-limiting examples of which include CDR-grafting (European Patent No. EP 239400; International Publication No. WO 91 / 09967; and U.S. Pat. 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. al, (1994) PNAS91:969-973), chain shuffling (US Pat. No. 5,565,332), and US Pat. No. 6,407,213, US Pat. 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):895904;Couto JR et 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 (2005-2-24), which is incorporated herein by reference in its entirety.

[0306] Methods for making multispecific (e.g., bispecific) antibodies have been published (see, e.g., U.S. Pat. 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).

[0307] Single domain antibodies, e.g., antibodies lacking light chains, can be produced by methods 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; Muyl-dermans 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).

[0308] In addition, antibodies that immunospecifically bind to the FAM19A5 antigen can be used to generate anti-idiotypic antibodies that "mimic" the antigen, using techniques known to those of skill in the art (see, e.g., Greenspan NS & Bona CA (1989) 77(5):437-444; and Nissinoff A (1991) 7 Immunol 147(8):2429-2438).

[0309] In certain embodiments, an antibody presented herein that binds to the same epitope of FAM19A5 (e.g., human FAM19A5) as an anti-FAM19A5 antibody presented herein is a human antibody or an antigen-binding fragment thereof. In certain embodiments, an antibody presented herein that competitively blocks (e.g., in a dose-dependent manner) an antibody presented herein (e.g., 1-65) from binding to FAM19A5 (e.g., human FAM19A5) is a human antibody or an antigen-binding fragment thereof.

[0310] Human antibodies can be produced using any method known in the art. For example, transgenic mice that are incapable of expressing functional endogenous immunoglobulins, but are capable of expressing 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. Also, human variable, constant and diversity regions can be introduced into mouse embryonic stem cells in addition to human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes can be rendered non-functional simultaneously with or separately from the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents the production of endogenous antibodies. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous progeny that express human antibodies. The transgenic mice are immunized in the normal manner with a selected antigen, for example, all or part of the antigen (e.g., FAM19A5). Monoclonal antibodies directed against antigens are obtained from immunized transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation and then undergo type reversal and somatic mutation. Thus, using such technology, it is possible to generate therapeutically useful IgG, IgA, IgM and IgE antibodies. For a general overview of this technology for generating human antibodies, see Lonberg N & Huszar D (1995) Int Rev Immunol 13:65-93. For details regarding this 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).

[0311] Human antibodies that specifically bind to FAM19A5 (e.g., human FAM19A5) can be made by a variety of 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.

[0312] 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 whether they secrete human monoclonal antibodies that immunospecifically bind to a target antigen (e.g., FAM19A5, such as human FAM19A5). Such methods are known 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).

[0313] VI. Antibody Genetic Engineering Methods As mentioned above, anti-FAM19A5 antibodies or antigen-binding portions thereof having the VH and VL sequences presented herein can be used to generate new anti-FAM19A5 antibodies or antigen-binding portions thereof by modifying the VH and / or VL sequences or the constant regions attached thereto. Thus, in other embodiments, the structural features of the anti-FAM19A5 antibodies presented herein are used to generate structurally related anti-FAM19A5 antibodies that retain at least one functional property of the antibodies presented herein, such as binding to human FAM19A5. For example, the starting material for the genetic engineering method is the VH and / or VL sequences presented herein, or one or more CDR regions thereof. It is not necessary to actually make (i.e., express as a protein) an antibody having any one or more of the VH and / or VL sequences presented herein, or one or more CDR regions thereof, to generate a genetically engineered antibody. Rather, the information contained in the sequence is used as starting material to generate "second generation" sequences derived from the parent sequence, and the "second generation" sequences are then made and expressed as proteins.

[0314] Thus, provided herein is a method for making an anti-FAM19A5 antibody, or an antigen-binding portion thereof, comprising: (a) providing (i) a heavy chain variable region sequence comprising the CDR1, CDR2 and / or CDR3 sequences set out in Table 2 or the CDR1, CDR2 and / or CDR3 of a heavy chain variable region set out in Table 4; and (ii) a light chain variable region sequence comprising the CDR1, CDR2 and / or CDR3 sequences set out in Table 3 or the CDR1, CDR2 and / or CDR3 of a heavy chain variable region set out in Table 5; (b) altering 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 altered antibody or antigen-binding subsequence; and (c) expressing the altered antibody or antigen-binding subsequence as a protein. Includes.

[0315] Standard molecular biology techniques can be used to create and express the altered antibody or antigen-binding subsequence.

[0316] In some embodiments, the antibody or antigen-binding portion thereof encoded by the altered antibody or antigen-binding subsequence retains one, some or all of the functional properties of the anti-FAM19A5 antibodies presented herein, including the following: (1) A K of 10 nM or less (e.g., 0.01 nM to 10 nM) when measured, for example, by Biacore D binds to soluble human FAM19A5; (2) A K of 1 nM or less (e.g., 0.01 nM to 1 nM) as measured, for example, by ELISA D t bound to membrane-bound human FAM19A5; (3) 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; (4) reducing, reversing, delaying, and / or preventing the onset of reactive gliosis; (5) Inhibiting excessive proliferation of reactive astrocytes; (6) decreased expression of chondroitin sulfate proteoglycans, including neurocan and neuron-glial antigen 2 (NG2); (7) increased c-fos and pERK expression in neuronal nuclei; (8) Promoting neuronal survival; (9) increased expression of GAP43 in neurons; (10) promoting axon regrowth; and (11) Compete in one or both directions with the anti-FAM19A5 antibodies presented herein for binding to human FAM19A5.

[0317] The modified antibodies or antigen-binding portions thereof 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 out in (1)-(11) above. The functional properties of the modified antibodies or antigen-binding portions thereof may be assessed by standard assays available in the art such as those set out in the Examples (e.g., ELISA, FACS) and / or as set out herein.

[0318] In certain embodiments of the methods for genetically engineering antibodies provided herein, mutations can be introduced randomly or selectively along all or part of the anti-FAM19A5 antibody coding sequence, and the resulting modified anti-FAM19A5 antibodies can be screened for binding activity and / or other functional properties provided herein. Mutational methods are known in the art (e.g., PCT Publication WO02 / 092780 by Short discloses methods for generating and screening antibody mutations using saturation mutagenesis, synthetic ligation assembly, or a combination thereof). Alternatively, PCT Publication WO03 / 074679 by Lazar et al. discloses methods using computational screening methods to optimize physicochemical properties of antibodies.

[0319] VII. Cells and Vectors In certain aspects, disclosed are cells (e.g., host cells) and related polynucleotides and expression vectors that express (e.g., in a recombinant manner) the antibodies (or antigen-binding portions thereof) presented herein that specifically bind to FAM19A5 (e.g., human FAM19A5). Disclosed are vectors (e.g., expression vectors) that contain 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. Also disclosed are host cells that contain such vectors for recombinantly expressing the anti-FAM19A5 antibodies (e.g., human or humanized antibodies) presented herein. In certain aspects, disclosed are methods of producing the antibodies presented herein, including expressing such antibodies from a host cell.

[0320] Recombinant expression of an antibody presented herein (e.g., a full-length antibody, a heavy and / or light chain of the antibody, or a single-chain antibody presented herein) that specifically binds to FAM19A5 (e.g., human FAM19A5) involves the construction of an expression vector containing a polynucleotide encoding the antibody. Once a polynucleotide encoding an antibody molecule presented herein, a heavy and / or light chain of the antibody, or a fragment thereof (e.g., a heavy and / or light chain variable domain) is obtained, a vector for the production of the antibody molecule can be generated by recombinant DNA technology using techniques known in the art. Thus, disclosed herein are methods for making a protein by expressing a polynucleotide containing an antibody or antibody fragment (e.g., light or heavy chain) encoding nucleotide sequence. Methods known in the art can be utilized to construct an expression vector containing an antibody or antibody fragment (e.g., light or heavy chain) coding sequence 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 disclosed are replicable vectors comprising nucleotide sequences encoding the antibody molecules presented 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, for example, comprise nucleotide sequences encoding the constant regions of the antibody molecules (see, for example, International Publication Nos. WO86 / 05807 and WO89 / 01036; and U.S. Patent No. 5,122,464), and the variable domains of the antibodies can be cloned into such vectors for expression of the entire heavy chain, the entire light chain, or both the entire heavy and light chains.

[0321] The expression vector can be transferred to a cell (e.g., a host cell) by conventional techniques, and the resulting cell can then be cultured by conventional techniques to produce an antibody (e.g., an antibody comprising any one or more of the VH and / or VL, or VH and / or VLCDR, of the anti-FAM19A5 antibody of the invention) or a fragment thereof as presented herein. Thus, disclosed herein are host cells containing a polynucleotide encoding an antibody or fragment thereof as presented herein, or a heavy or light chain thereof, or a fragment thereof, or a single chain antibody as presented herein, operably linked to a promoter for expression of such sequences in the host cell. In certain embodiments, for expression of a double-chain antibody, vectors encoding both the heavy and light chains separately can be co-expressed in a host cell for expression of an entire immunoglobulin molecule, as described in detail below. In certain embodiments, a host cell contains a vector comprising a polynucleotide encoding both the heavy and light chains of an antibody or fragment thereof as presented herein. In certain embodiments, the host cell contains two different vectors, a first vector comprising a polynucleotide encoding the heavy chain or heavy chain variable region of the antibody or fragment thereof presented herein, and a second vector comprising a polynucleotide encoding the light chain or light chain variable region of the antibody or fragment thereof presented herein. In other embodiments, the first host cell comprises a first vector comprising a polynucleotide encoding the heavy chain or heavy chain variable region of the antibody or fragment thereof presented herein, and the second host cell comprises a second vector comprising a polynucleotide encoding the light chain or light chain variable region of the antibody or fragment thereof presented 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 the anti-FAM19A5 antibody or antigen-binding fragment thereof presented herein. In certain embodiments, a host cell population comprising such a first host cell and such a second host cell is disclosed.

[0322] In a particular embodiment, a vector population is disclosed comprising a first vector comprising a polynucleotide encoding the light chain / light chain variable region of an anti-FAM19A5 antibody presented herein, and a second vector comprising a polynucleotide encoding the heavy chain / heavy chain variable region of an anti-FAM19A5 antibody presented herein.

[0323] A variety of host-expression vector systems may be utilized to express the antibody molecules presented herein. Such host-expression systems include not only vehicles in which a coding sequence of interest may be produced and then purified, but also cells which, when transformed or transfected with the appropriate nucleotide coding sequence, are capable of expressing the antibody molecules presented herein in situ. Such host-expression systems include microorganisms such as bacteria (e.g., E. coli and Bacillus subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA containing the antibody coding sequence; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing the antibody coding sequence; plant cell systems (e.g., Chlamydomonas reinhardtii) 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 the antibody coding sequence. reinhardtii); or mammalian cell systems harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., adenovirus late promoter; cowpox virus 7.5K promoter) (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). In a particular embodiment, the cells for expressing the antibodies or antigen-binding fragments thereof provided herein are CHO cells, e.g., CHO cells from the CHO GS SYSTEM™ (Lonza). In certain embodiments, the cells for expressing the antibodies provided 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 E. coli or eukaryotic cells (e.g., mammalian cells) are used for the expression of recombinant antibody molecules, particularly for the expression of whole recombinant antibody molecules. For example, mammalian cells such as Chinese hamster ovary (CHO) cells, together with vectors such as the major intermediate early gene promoter element from human cytomegalovirus, provide effective expression systems for antibodies (see 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 provided herein are produced by CHO cells or NSO cells. In certain embodiments, expression of nucleotide sequences encoding the antibodies provided herein that immunospecifically bind to FAM19A5 (e.g., human FAM19A5) is regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

[0324] In bacterial systems, a number of expression vectors may be advantageously selected depending on the antibody molecule to be expressed. For example, if large quantities of an antibody are to be produced for the production of a pharmaceutical composition of the antibody molecule, a vector directing the expression of high levels of a fusion protein product that is easily purified may be preferred. Non-limiting examples of such vectors include the E. coli expression vector UR278 (Ruether U & Mueller-Hill B (1983) EMBO J2:1791-1794) in which antibody coding sequences may be individually ligated in frame with the lac Z coding region to the vector to produce a fusion protein; pIN vectors (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. Also, pGEX vectors may be used to express foreign polypeptides as fusion proteins with glutathione 5-transferase (GST). In general, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to substrate glutathione agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to contain thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

[0325] In an insect system, for example, Autographa californica nuclear polyhedrosis virus (AcNPV) can be used as a vector to express foreign genes. The virus grows 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).

[0326] In mammalian host cells, a number of virus-based expression systems can be utilized. 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 in a non-essential region of the viral genome (e.g., region E1 or E3) should result in a recombinant virus that is viable and capable of expressing the antibody molecule in an infected host (see, e.g., Logan J & Shenk T (1984) PNAS 81(12):3655-9). Specific initiation signals are also required for efficient translation of the inserted antibody coding sequence. These signals include the ATG initiation codon and adjacent sequences. The initiation codon must also be in phase with the reading frame of the desired coding sequence to ensure 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 can be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see, e.g., Bitter G et al, (1987) Methods Enzymol. 153:516-544).

[0327] Additionally, a host cell strain may be selected which modulates the expression of the inserted sequences, or which modulates and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems may be selected which can ensure correct modification and processing of the expressed foreign protein. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Non-limiting examples of such mammalian host cells include CHO, VERO, BHK, Hela, MDCK, HEK293, NIH3T3, W138, BT483, Hs578T, HTB2, BT20 and T47D, NSO (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), 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 provided herein are produced in mammalian cells, such as CHO cells.

[0328] In certain embodiments, the antibodies or antigen-binding portions thereof provided herein have reduced or no fucose content. Such antibodies can be generated using techniques known to those skilled in the art. For example, the antibodies can be expressed in cells that have a defective or lacking fucosylation ability. In certain instances, cell lines in which two alleles of 1,6-fucosyltransferase have been knocked out can be used to generate antibodies or antigen-binding portions thereof with reduced fucose content. One example of such a system that can be used to generate antibodies or antigen-binding portions thereof with reduced fucose content is the POTELLIGENT® system (Lonza).

[0329] To obtain long-term high yields of recombinant protein, stable expression cells can be generated. For example, cell lines can be engineered that stably express the anti-FAM19A5 antibody or antigen-binding portion thereof provided herein. In certain embodiments, the cells provided herein stably express the light chain / light chain variable domain and the heavy chain / heavy chain variable domain that assemble to form the antibody or antigen-binding portion thereof provided herein.

[0330] In certain embodiments, rather than using an expression vector containing a viral origin of replication, a host cell can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.) and a selectable marker. After introduction of the foreign DNA / polynucleotide, the engineered cells can be grown in enriched medium for 1-2 days and then switched to selective medium. The selectable marker on the recombinant plasmid confers resistance to the selection, allowing the cells to stably integrate the plasmid into their chromosomes and grow to form foci, which can in turn be cloned and expanded into cell lines. This method can be advantageously used to engineer cell lines that express the anti-FAM19A5 antibodies or antibody binding portions thereof presented herein. Such engineered cell lines can be particularly useful for screening and evaluation of compositions that interact directly or indirectly with the antibody molecule.

[0331] A number of selection systems can be used, and by way of non-limiting example, the herpes simplex virus thymidine kinase (Wigler M et al, (1977) Cell 11(1):223-32), hypoxanthine guanine phosphoribosyltransferase (Szybalska EH & Szybalski W (1962) PNAS48(12):2026-2034), and adenine phosphoribosyltransferase (Lowy I et al, (1980) Cell 22(3):817-23) genes can be utilized in tk-, hgprt-, or aprt- cells, respectively. Antimetabolite resistance can also be used as the basis of selection for the following genes: dhfr (confers resistance to methotrexate) (Wigler M et al, (1980) PNAS 77(6):3567-70; O'Hare K et al, (1981) PNAS 78:1527-31); gpt (confers resistance to mycophenolic acid) (Mulligan RC & Berg P (1981) PNAS 78(4):2072-6); neo (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 (confers resistance to hygromycin) (Santerre RF et al, (1984) Gene 30(1-3):147-56).Known methods in the field of recombinant DNA technology can be routinely applied to select the desired recombinant clones, such as those described in, for example, 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); Chapters 12, 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, which are incorporated herein by reference in their entireties.

[0332] The expression level of an antibody molecule can be increased by vector amplification (see Bebbington CR & Hentschel CCG, Use of Gene Amplification-Based Vectors for the Expression of Cloned Genes in Mammalian Cells in DNA Cloning, Vol. 3 (Academic Press, New York, 1987)). If the marker of the antibody-expressing vector system is amplifiable, increasing the level of inhibitor present in the host cell culture should increase the copy number of the marker gene. Since the amplified region will be associated with the antibody gene, production of the antibody should also increase (see Crouse GF et al., (1983) Mol Cell Biol 3:257-66).

[0333] A host cell can be co-transfected with two or more expression vectors as provided herein, i.e., a first vector encoding a heavy chain derived polypeptide and a second vector encoding a light chain derived polypeptide. Both vectors can contain the same selectable marker that allows equal expression of heavy and light chain polypeptides. A host cell can be co-transfected with different amounts of two or more expression vectors. For example, a host cell can be transfected with any one of the following ratios of the first expression vector and 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.

[0334] Alternatively, a single vector can be used that can both encode and express heavy and light chain polypeptides. The light chain must precede the heavy chain to avoid an overdose of non-toxic heavy chain (Proudfoot NJ (1986) Nature 322:562-565; and Kohler G (1980) PNAS 77:2197-2199). The coding sequences for the heavy and light chains 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. For example, a bicistronic nucleic acid construct can comprise, in order, a promoter, a first gene (e.g., a heavy chain of an antibody presented herein) and a second gene (e.g., a light chain of an antibody presented herein). In such an expression vector, transcription of both genes is driven by a promoter, translation of mRNA from the first gene can be driven by a cap-dependent scanning mechanism, and translation of mRNA from the second gene can be driven by a cap-independent mechanism, such as an IRES.

[0335] Once the antibody molecules presented herein have been produced by recombinant expression, they may be purified by any method known in the art for the purification of immunoglobulin molecules, such as, for example, chromatography (e.g., ion exchange, affinity, particularly affinity for specific antigens after Protein A, and sizing column chromatography), centrifugation, differential solubility, or any other standard technique for the purification of proteins. The antibodies presented herein may also be fused to heterologous polypeptide sequences disclosed herein or other methods known in the art to facilitate purification may be applied.

[0336] In certain embodiments, the antibodies or antigen-binding portions thereof provided herein are isolated or purified. Generally, an isolated antibody is one that is substantially free of other antibodies having antigen specificities other than the antibody from which it is isolated. For example, in certain embodiments, preparations of the antibodies provided herein are substantially free of cellular material and / or chemical precursors. The phrase "substantially free of cellular material" includes preparations of the antibodies in which the antibody is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, antibodies that are substantially free of cellular material include preparations of the antibodies that have less than about 30%, 20%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (by dry weight) of heterologous proteins (or contaminating proteins) and / or variants of the antibody, such as different post-translationally modified forms of the antibody or other different versions of the antibody (or antibody-binding portion). When an antibody is recombinantly produced, it is generally substantially free of culture medium, i.e., culture medium represents less than about 20%, less than 10%, 2%, 1%, 0.5%, or less than 0.1% of the volume of the protein preparation. When an antibody is produced by chemical synthesis, it is generally substantially free of chemical precursors and other chemicals, i.e., separated from chemical precursors and other chemicals involved in the synthesis of the protein. Thus, such preparations of an antibody have less than about 30%, 20%, 10%, or 5% (by dry weight) of other chemical precursors or compounds other than the antibody of interest. In certain embodiments, the antibodies provided herein are isolated or purified.

[0337] VIII.Analysis The antibodies presented herein can be tested for binding to FAM19A5, for example, by standard ELISA. 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., dilutions of plasma from FAM19A5-immunized mice) are added to each well and incubated at 37° C. for 1-2 hours. The plates are washed with PBS / Tween, followed by incubation 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, the plates are developed with ABTS substrate (Moss Inc, product: ABTS-1000) and analyzed by spectrophotometer at OD 415-495. Sera from immunized mice are then further screened by flow cytometry for binding to cell lines expressing human FAM19A5, while control cell lines not expressing FAM19A5 are not bound. Anti-FAM19A5 antibody binding is assessed by co-incubating FAM19A5-expressing CHO cells with anti-FAM19A5 antibody at a dilution of 1:20. Cells are washed, and binding is detected with PE-labeled anti-human IgG Ab. Flow cytometry analysis is performed by a FACScan flow cytometry machine (Becton Dickinson, San Jose, CA). Preferably, the mice that develop the highest titers will be used for fusion.

[0338] The ELISA assay described above can be used to screen for antibodies that exhibit positive reactivity with the FAM19A5 immunogen and for hybridomas that produce the antibodies. Hybridomas that produce antibodies that bind with the preferred high affinity to FAM19A5 are then subcloned and further characterized. One clone from each hybridoma that retains the reactivity of the parent cell can then be selected for cell banking and antibody purification (by ELISA).

[0339] To purify anti-FAM19A5 antibodies, selected hybridomas can be grown in 2 L spinner flasks for monoclonal antibody purification. Supernatants can be filtered and concentrated prior to protein A-Sepharose affinity chromatography (Pharmacia, Piscataway, NJ). Eluted IgG can be checked by gel electrophoresis and high performance liquid chromatography to ensure purity. Buffer solution can be exchanged into PBS and concentration can be determined by OD280 using a 1.43 extinction coefficient. Monoclonal antibodies can be aliquoted and stored at -80°C.

[0340] 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.

[0341] 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 of anti-human immunoglobulin overnight at 4° C. After blocking with 1% BSA, the plate is reacted with 1 μg / mL or less of the test monoclonal antibody or purified isotype control at ambient temperature for 1-2 hours. 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.

[0342] To test the binding of monoclonal antibodies to living cells expressing FAM19A5, flow cytometry can be used as described in the examples. 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 the primary antibody staining. The samples can be analyzed by a FACScan instrument using light and side scatter properties to gate on single cells and the binding of the labeled antibodies is measured. Other analyses using fluorescent microscopy are possible in addition to or instead of flow cytometry analysis. Cells can be stained exactly as described above and examined by fluorescent microscopy. This method allows visualization of individual cells but may have reduced sensitivity depending on the density of the antigen.

[0343] Anti-FAM19A5 antibodies can be further tested for reactivity with the FAM19A5 antigen by Western blotting. Cell extracts from cells expressing FAM19A5 can be made and sodium dodecyl sulfate polyacrylamide gel electrophoresis can be performed. After electrophoresis, the separated antigens will be 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 tablets (Sigma Chem. Co., St. Louis, MO).

[0344] Methods for analyzing the binding affinity, cross-reactivity and binding kinetics of various anti-FAM19A5 antibodies include standard analyses known in the art, such as BIACORE™ Surface Plasmon Resonance (SPR) analysis using a BIACORE™ 2000 SPR instrument (Biacore AB, Uppsala, Sweden).

[0345] In one embodiment, the antibody specifically binds to a soluble form of human FAM19A5. In one embodiment, the antibody specifically binds to a membrane-bound form of human FAM19A5. The antibody may specifically bind to a particular epitope of FAM19A5 (e.g., SEQ ID NO:2 or a fragment within SEQ ID NO:2). In certain embodiments, the antibody specifically binds to human FAM19A5, preferably with high affinity, and does not cross-react with other members of the FAM19 subfamily of proteins.

[0346] IX. Bispecific molecules The antibodies provided herein can be used to form bispecific molecules. Anti-FAM19A5 antibodies or antigen-binding portions thereof can be derivatized or conjugated to other functional molecules, such as other peptides or proteins (e.g., other antibodies or receptor ligands), thereby generating bispecific molecules that bind to at least two different binding sites or target molecules. Cytokines such as IL-6, CNTF, LIF, EGF and TGFa have been implicated as triggers for the initiation of gliosis and / or reactive gliosis by activating the protein signal transducer or activator of transcription 3 (STAT3) (see Balasingam et al, J. Neurosci. 14(2):846-56 (1994); Winter et al, Proc. Natl. Acad. Set USA 20;92(13):5865-9 (1995)), which in turn regulates many aspects of reactive astrocytosis following CNS injury (see Herrmann JE et al, J. Neurosci. 28(28):7231-7243 (2008)). For example, absence or reduction of STAT3 leads to weakened upregulation of glial fibrillary acidic protein (GFAP), failure of astrocytic hypertrophy, and increased spread of inflammation, increased lesion volume, and partially impaired motor recovery after CNS injury (see Herrmann JE et al, J. Neurosci. 28(28):7231-7243 (2008)). Thus, anti-FAM19A5 antibodies can be conjugated to antibodies or scFvs that specifically bind to any protein involved in the initiation of gliosis and / or suppressing the excessive proliferation of reactive astrocytosis for combination therapy, for example antibodies against IL-6, CNTF, LIF, EGF, or TGFa.

[0347] The anti-FAM19A5 antibody may also be conjugated 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 cord 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 and neurological disorders, or neuropathic pain (see the diseases or disorders described in Chapter XII below). For example, the anti-FAM19A5 antibody may be conjugated to an antibody or scFv that treats multiple sclerosis, such as natalizumab (TYSABRI®), alemtuzumab (LEMTRADA®).

[0348] The antibodies provided herein may be derivatized or conjugated to two or more other functional molecules to generate multispecific molecules that actually bind to three or more different binding sites and / or target molecules. Such multispecific molecules are also included in the "bispecific molecule". To generate the bispecific molecules provided herein, the antibodies provided herein are functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association, or other methods) to one or more other binding molecules, such as other antibodies, antibody binding portions thereof, peptides, or binding mimetics, thereby resulting in a bispecific molecule. In one embodiment, the bispecific molecule binds to FAM19A5 and VEGF. In another embodiment, the bispecific molecule binds to FAM19A5 and EGF.

[0349] Thus, disclosed herein are bispecific molecules comprising at least one first binding specificity for FAM19A5 and a second binding specificity for a second target epitope. In one embodiment where the bispecific molecule is multispecific, the molecule can further comprise a third binding specificity.

[0350] In one embodiment, the bispecific molecules provided herein comprise as binding specificities at least one antibody or an antibody binding portion thereof, including, for example, a Fab, Fab', F(ab')2, Fv, or single chain Fv (scFv). The antibody may also be a light or heavy chain dimer, or any minimal fragment thereof, such as the Fv or single chain constructs described in Ladner et al., U.S. Patent No. 4,946,778, the contents of which are incorporated herein by reference.

[0351] Although human monoclonal antibodies are preferred, other antibodies that can be used in the bispecific molecules presented herein are murine, chimeric and humanized monoclonal antibodies.

[0352] The bispecific molecules presented herein can be produced by conjugating the constituent binding specificities using methods known in the art. For example, each binding specificity of the bispecific molecule can be produced separately and then conjugated to each other. When the binding specificities are proteins or peptides, a variety of coupling or cross-linking agents can be used for covalent conjugation. Exemplary cross-linkers 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) (see, e.g., Karpovsky et al, (1984) J. Exp. Med. 160:1686; Liu, MA et al, (1985) Proc. Natl. Acad. Set 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 conjugation agents are SATA and sulfo-SMCC, both of which are commercially available from Pierce Chemical Co. (Rockford, IL).

[0353] When the binding specificities are antibodies, they can be conjugated through sulfhydryl bonds in the C-terminal hinge regions of the two heavy chains, in a particularly preferred embodiment, the hinge region is modified prior to conjugation to contain an odd number of sulfhydryl residues, preferably one residue.

[0354] Alternatively, both binding specificities can be encoded in the same vector and expressed and assembled 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. The bispecific antibody can include an antibody that includes an scFv at the C-terminus of each heavy chain. The bispecific molecules provided herein can be single chain molecules that include a single chain antibody and a binding determinant, or single chain bispecific molecules that include two binding determinants. The bispecific molecule can include at least two single chain molecules. Methods for making 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.

[0355] Binding of the bispecific molecule to its specific target can be determined using methods known in the art, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassays (e.g., growth inhibition), or Western blot analysis, each of which generally detects particular protein-antibody complexes of interest by utilizing a labeled reagent (e.g., an antibody) specific for the complex of interest.

[0356] X. 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.

[0357] The antibodies presented herein can be used for diagnostic purposes, including sample testing and in vivo imaging, for which the antibodies (or binding moieties thereof) can be conjugated to a suitable detectable agent to form an immunoconjugate. For diagnostic purposes, suitable agents are detectable labels including radioisotopes, and for whole body imaging, suitable agents are radioisotopes, enzymes, fluorescent labels and other suitable antibody tags for sample testing.

[0358] The detectable label may be any of a variety of types currently used in the field of in vitro diagnostics, including particulate labels including metal sols such as colloidal gold, e.g., I presented with peptide chelators of the N2S2, N3S or N4 types. 125 or Tc 99 Suitable enzyme labels include isotopes such as , chromophores including fluorescent markers, luminescent markers, phosphorescent markers, etc., or enzyme labels that convert a given substrate into a detectable marker, and polynucleotide tags that are revealed after amplification, such as by polymerase chain reaction. Suitable enzyme labels include horseradish peroxidase, alkaline phosphatase, and the like. For example, the label can be the enzyme alkaline phosphatase, which is detected by measuring the presence or formation of chemiluminescence following conversion of a 1,2-dioxetane substrate, such as adamantyl methoxyphosphoryloxyphenyl dioxetane (AMPPD), disodium 3-(4-(methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo{3.3.1.1 3,7}decan}-4-yl)phenyl phosphate (CSPD), as well as CDP and CDP-star or other luminescent substrates known to those skilled in the art, such as suitable lanthanum group element chelates, such as terbium(III) and europium(III). The means of detection will be determined by the label selected. The appearance of the label or its reaction products can be confirmed with the naked eye, if the label is particulate and accumulates at appropriate levels, or with instruments such as spectrophotometers, luminometers, fluorimeters, and the like, all following standard practice.

[0359] The antibodies provided herein may also be conjugated to a therapeutic agent to form an immunoconjugate, such as an antibody-drug conjugate (ADC). Suitable therapeutic agents include preparations that modulate the onset of gliosis and / or reactive astrocytosis and / or treat degenerative brain disorders, central nervous system injuries, or neuropathic pain. Therapeutic agents for treating degenerative brain disorders include drugs that treat Huntington's disease, Parkinson's disease, Alzheimer's disease, multiple sclerosis, and amyotrophic lateral sclerosis (ALS). Therapeutic agents include drugs commonly used to treat such degenerative brain disorders, such as those disclosed in Chapter XII.

[0360] Immunoconjugates can be produced by methods known in the art. Preferably, the conjugation method results in substantially (or almost) non-immunogenic bonds, such as peptide-(i.e., amide-), sulfide-, disulfide-, hydrazone- and ether bonds. These bonds are mostly non-immunogenic and exhibit suitable stability in serum (see, for example, Senter, PD, Curr. Opin. Chem. Biol. 13 (2009) 235-244; WO2009 / 059278; WO95 / 17886).

[0361] Depending on the biochemical properties of the moiety and the antibody, different conjugation strategies can be used. When the moiety is naturally occurring or recombinant of 50-500 amino acids, standard procedures for the synthesis of protein conjugates are described in the literature and can be easily reproduced by the skilled artisan (see, for example, Hackenberger, CPR, and Schwarzer, D., Angew. Chem. Int. Ed. Engl. 47 (2008) 10030-10074). In one embodiment, reaction of a maleimide moiety with a cysteine ​​residue in the antibody or moiety is used. This is a particularly suitable coupling chemistry when Fab or Fab'-fragments of the antibody are used. Also, in one embodiment, coupling is performed with the C-terminus of the antibody or moiety. C-terminal modifications of proteins, such as Fab-fragments, can be performed, for example, as described in the literature (Sunbul, M. and Yin, J., Org. Biomol. Chem. 7 (2009) 3361-3371).

[0362] In general, site-specific reactions and covalent couplings are based on modifying natural amino acids into amino acids with reactivity orthogonal to the reactivity of other functional groups. For example, specific cysteines in rare sequence contexts can be enzymatically converted into aldehydes (see Frese, MA, and Dierks, T., ChemBioChem. 10 (2009) 425-427). It is also possible to obtain desired amino acid modifications by exploiting the specific enzymatic reactivity of natural amino acids with specific enzymes in a given sequence context (see, for example, 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).

[0363] Site-specific reactions and covalent coupling can also be achieved by selective reaction of the terminal amino acid with an appropriate modification reagent. The reactivity of N-terminal cysteines with benzonitrile can be used to achieve site-specific covalent coupling (see Ren, H. et al, Angew. Chem. Int. Ed. Engl. 48 (2009) 9658-9662). Spontaneous chemical ligation can also rely on C-terminal cysteine ​​residues (see Taylor, E. Vogel; Imperiali, B, Nucleic Acids and Molecular Biology (2009), 22 (Protein Engineering), 65-96).

[0364] EP1074563 discloses a conjugation method based on the rapid reaction of cysteines located in a stretch of positively charged amino acids with cysteines in a stretch of negatively charged amino acids.

[0365] The moiety may also be a synthetic peptide or peptidomimetic. If the polypeptide is chemically synthesized, amino acids with orthogonal chemical reactivity may be integrated during such synthesis (see, for example, de Graaf, AJ et al, Bioconjug. Chem. 20 (2009) 1281-1295). The conjugation of such peptides to linkers is standard chemistry, since a wide variety of orthogonal functional groups are relevant and can be introduced into synthetic peptides.

[0366] To obtain a single labeled polypeptide, the 1:1 stoichiometric conjugate can be separated from other conjugation by-products by chromatography. This process can be facilitated by using dye-labeled binding pair members and charged linkers. By using labeled and highly negatively charged binding pair members, single conjugated polypeptides are easily separated from unlabeled polypeptides and polypeptides with two or more linkers, since charge and molecular weight differences can be used for separation. Fluorescent dyes can be useful for purifying the complex from unbound components, such as labeled monovalent binders.

[0367] XI. Pharmaceutical Compositions Disclosed herein are compositions comprising the antibodies or antigen-binding portions thereof provided herein with a 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 buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium 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 (less than about 10 residues) polypeptides; proteins, such as serum alcohols. Examples of suitable surface active agents include: bufin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; carbohydrates, including monosaccharides, disaccharides, and 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 TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0368] In certain embodiments, the pharmaceutical composition comprises an antibody or antigen-binding portion thereof, bispecific molecule or immunoconjugate as provided herein, and optionally one or more additional prophylactic or therapeutic agents in a pharma- ceutically acceptable carrier. In certain embodiments, the pharmaceutical composition comprises an effective amount of an antibody or antigen-binding portion thereof as provided herein, and optionally one or more additional prophylactic or therapeutic agents in a pharma- ceutically acceptable carrier. In some embodiments, the antibody is the only active ingredient contained in the pharmaceutical composition. The pharmaceutical compositions provided herein can be useful for enhancing, inducing or activating FAM19A5 activity and treating conditions such as central nervous system injury, degenerative brain disorders or neuropathic pain.

[0369] Pharmaceutically acceptable carriers used in parenteral formulations include aqueous vehicles, non-aqueous vehicles, antibacterial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents, and other pharma- ceutically acceptable substances. Examples of aqueous parenteral vehicles include sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, dextrose and lactated Ringer's injection. Non-aqueous parenteral vehicles include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, and peanut oil. Antibacterial agents are added to parenteral formulations packaged in multi-volume containers in bacteriostatic or fungistatic concentrations, and these include phenol or cresol, mercury-containing substances, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride, and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. Buffers include phosphates and citrates. Antioxidants include sodium bisulfate. Local 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 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.

[0370] The pharmaceutical composition can be formulated for any route of administration to a subject. Specific examples of routes of administration include intranasal, oral, parenteral, intraspinal, intraventricular, pulmonary, subcutaneous or intraventricular routes. Parenteral administration, characterized by subcutaneous, intramuscular or intravenous injection, is also contemplated. Injectables can be prepared in conventional forms, as liquid solutions or suspensions, solid forms suitable for solution or suspension 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. The pharmaceutical composition to be administered may also contain small amounts of non-toxic auxiliary substances, such as wetting agents, emulsifying agents, pH buffers, stabilizers, solubility enhancers, and other formulations, for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, and cyclodextrins.

[0371] Preparations for parenteral administration of antibodies include sterile solutions ready for injection immediately prior to use, sterile dry soluble products, such as lyophilized powders that can be combined with a solvent immediately prior to use, such as tablets for subcutaneous injection, sterile suspensions ready for injection immediately prior to use, sterile dry insoluble products that can be combined with a vehicle immediately prior to use, and sterile emulsions. The solutions may be aqueous or non-aqueous.

[0372] If administered intravenously, suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing viscosity enhancing and softening agents, such as glucose, polyethylene glycol and polypropylene glycol, and mixtures thereof.

[0373] Topical mixtures containing the antibodies are prepared as described for local and systemic administration. The resulting mixture may be a solution, suspension, emulsion, etc., and may be made into creams, gels, ointments, emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes, foams, aerosols, irrigations, sprays, suppositories, bandages, skin patches, or any other formulation suitable for topical administration.

[0374] The antibodies or antigen-binding portions thereof provided herein can be formulated as aerosols for topical application, for example, by inhalation (see, for example, U.S. Patent Nos. 4,044,126, 4,414,209, and 4,364,923, which describe aerosols for the delivery of steroids useful in the treatment of inflammatory diseases, particularly asthma). These formulations for administration to the respiratory tract can be in the form of an aerosol or solution for a nebulizer, or a microfine powder for inhalation, either alone or in combination with an inactive carrier such as lactose. In such cases, the particles of the formulation have diameters of less than 50 microns in one embodiment, and less than 10 microns in one embodiment.

[0375] The antibodies or antigen-binding portions thereof presented herein can be formulated for local or topical application, e.g., to the skin and mucous membranes, e.g., for topical application to the eyes, in the form of gels, creams and lotions, and for application to the eye or intrathecal or intraspinal application. Topical administration is contemplated for transdermal delivery and administration to the eye or mucous membranes, or for inhalation therapeutic therapy. Nasal solutions of the antibodies alone or in combination with other pharma- ceutically acceptable excipients can again be administered.

[0376] Transdermal patches, including iontophoretic and electrophoretic devices, are known in the art and can be used to administer an antibody. 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.

[0377] In certain embodiments, pharmaceutical compositions comprising the antibodies or antigen-binding portions thereof presented herein are lyophilized powders and can be reconstituted for administration as solutions, emulsions, and other mixtures. Pharmaceutical compositions can also be reconstituted and formulated as solids or gels. Lyophilized powders are made by dissolving the antibodies or antigen-binding portions thereof presented herein, or pharma-ceutically acceptable derivatives thereof, in a suitable solvent. In some embodiments, the lyophilized powders are sterile powders. The solvent can contain excipients that improve the stability of the powder or reconstituted solutions made from the powder or other pharmaceutical ingredients. Non-limiting examples of excipients that can be used include dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable formulations. The solvent can also contain a neutral pH buffer, such as citrate, sodium or potassium phosphate, or other buffers known in the art. Subsequent sterile filtration of the solvent followed by lyophilization under standard conditions known in the art can provide the desired formulation. In one embodiment, the resulting solution will be apportioned into lyophilization vials. Each vial will have a single or multiple doses of the compound. The lyophilized powder can be stored under appropriate conditions, for example, at about 4° C. to room temperature.

[0378] The lyophilized powder can be reconstituted with water for injection to obtain a formulation for parenteral administration. For reconstitution, the lyophilized powder is added to sterile water or other suitable carrier. The exact amount depends on the compound selected. Such amount can be empirically determined.

[0379] The antibodies or antigen-binding portions thereof, bispecific molecules, or immunoconjugates presented herein and other compositions presented herein can also be formulated to target to the specific tissue, receptor, or other area of ​​the subject's body to be treated. Many targeting methods are known in the art. Such targeting methods are contemplated for use in the compositions of the present invention. For non-limiting examples of targeting methods, see 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, presented herein are targeted to treat central nervous system injuries, degenerative brain disorders, or neuropathic pain.

[0380] Compositions available for in vivo administration can be sterilized, which is readily accomplished, for example, by filtration through sterile filtration membranes.

[0381] XII. Kit Disclosed are kits comprising one or more of the antibodies or antigen-binding portions thereof, bispecific molecules, or immunoconjugates thereof presented herein. In certain embodiments, disclosed are pharmaceutical packs or kits comprising one or more containers filled with any one or more of the components of the pharmaceutical compositions presented herein, such as one or more of the antibodies or antigen-binding portions thereof presented herein, and optional instructions for use. In some embodiments, the kits contain the pharmaceutical compositions presented herein and any of the prophylactic or therapeutic agents presented herein.

[0382] XIII. Therapeutic Uses and Methods In one aspect, a method for alleviating injury or damage to the CNS of a subject is disclosed, the method comprising administering to the subject an anti-FAM19A5 antibody or antigen-binding portion thereof, bispecific molecule or immunoconjugate, or composition thereof, presented herein.

[0383] In one embodiment, a method is disclosed for inhibiting, delaying, suppressing, limiting, or preventing the onset or onset of gliosis in a subject and its associated deleterious effects on the CNS, comprising administering to the subject an anti-FAM19A5 antibody or antigen-binding portion thereof, bispecific molecule, or immunoconjugate, or composition thereof, as provided herein. In one embodiment, a method is disclosed for inhibiting, delaying, suppressing, limiting, or preventing the excessive or abnormal proliferation of reactive astrocytes in a subject and its associated deleterious effects on the CNS, comprising administering to the subject an anti-FAM19A5 antibody or antigen-binding portion thereof, bispecific molecule, or immunoconjugate, or composition thereof, as provided herein.

[0384] In one embodiment, a method is disclosed for decreasing, inhibiting, decreasing the level of neurocan, NG2, or both, or decreasing or inactivating the activity of neurocan, NG2, or both, in a subject, comprising administering to the subject an anti-FAM19A5 antibody or antigen-binding portion thereof, bispecific molecule, or immunoconjugate, or composition thereof, as provided herein. In one embodiment, a method is disclosed for stimulating, promoting, increasing, or activating neuronal growth in a subject after injury or damage, comprising administering to the subject an anti-FAM19A5 antibody or antigen-binding portion thereof, bispecific molecule, or immunoconjugate, or composition thereof, as provided herein. In one embodiment, a method is disclosed for increasing the level of c-fos mRNA, c-fos protein, or c-fos protein activity, and increasing ERK mRNA, ERK protein, or pERK activity, preferably in the nucleus of a neuron, in a subject, comprising administering to the subject an anti-FAM19A5 antibody or antigen-binding portion thereof, bispecific molecule, or immunoconjugate, or composition thereof, as provided herein. In one embodiment, a method is disclosed for enhancing or increasing the level of GAP43 mRNA, GAP43 protein, or increasing the activity of GAP43 protein in a subject in need thereof, preferably in a neuron, comprising administering an anti-FAM19A5 antibody or antigen-binding portion thereof, bispecific molecule or immunoconjugate, or composition thereof, as provided herein. In one embodiment, a method is disclosed for enhancing or promoting neuronal survival and / or promoting axonal regrowth in a subject, comprising administering an anti-FAM19A5 antibody or antigen-binding portion thereof, bispecific molecule or immunoconjugate, or composition thereof, as provided herein. In one embodiment, the subject is a human, e.g., a human having neuronal injury or damage from CNS injury, trauma, injury, brain / spinal cord injury, brain tumor, infection, ischemia, stroke, autoimmune response, and / or neurodegenerative disease.

[0385] In some embodiments, a method is disclosed for treating a disease or disorder, including central nervous system injury, cerebrospinal system injury, degenerative brain injury, degenerative cerebrospinal or neurological disorder, or neuropathic pain, in a subject, comprising administering to the subject an anti-FAM19A5 antibody or antigen-binding portion thereof, bispecific molecule, or immunoconjugate, or composition thereof, as provided herein. In one embodiment, the central nervous system injury is traumatic brain injury, cerebrospinal injury, stroke, brain tumor, or a combination thereof. In one embodiment, the degenerative brain disorder is Huntington's disease, Parkinson's disease, Alzheimer's disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS), or a combination thereof. In one embodiment, a method is disclosed for treating a traumatic brain injury, cerebrospinal injury, stroke, brain tumor, or a combination thereof, in a subject, comprising administering to the subject an anti-FAM19A5 antibody or antigen-binding portion thereof, bispecific molecule, or immunoconjugate, or composition thereof, as provided herein. In one embodiment, a method of treating Huntington's disease, Parkinson's disease, Alzheimer's disease, multiple sclerosis, ALS in a subject is disclosed, the method comprising administering to the subject an anti-FAM19A5 antibody or antigen-binding portion thereof, bispecific molecule or immunoconjugate provided herein, or a composition thereof. In a preferred embodiment, the subject is a human.

[0386] In some embodiments, a therapeutically effective amount of the anti-FAM19A5 antibody or antigen-binding portion thereof, bispecific molecule or immunoconjugate presented herein is administered. When treating a subject (e.g., a human), the therapeutically effective amount of the anti-FAM19A5 antibody or antigen-binding portion thereof, bispecific molecule or immunoconjugate presented herein can be determined by a person skilled in the art (e.g., a physician) according to factors such as age, sex, and severity of the disease. Typically, the anti-FAM19A5 antibody or antigen-binding portion thereof, bispecific molecule or immunoconjugate presented herein is administered at a dose of 0.01 μg to 1000 mg per day. The anti-FAM19A5 antibody or antigen-binding portion thereof, bispecific molecule or immunoconjugate presented herein can be administered once, twice, or more times per day depending on the symptoms and severity of the disease or disorder.

[0387] In some embodiments, the anti-FAM19A5 antibodies or antigen-binding portions thereof, bispecific molecules or immunoconjugates or compositions thereof presented herein are administered intravenously, orally, parenterally, intraspinal, intracerebroventricular, pulmonary, subcutaneous or intraventricular routes.

[0388] In some embodiments, an anti-FAM19A5 antibody or antigen-binding molecule thereof, or a composition thereof, may be administered with one or more additional agents for treating central nervous system injury (e.g., traumatic brain injury, cerebrospinal cord 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 neurological disorder, or neuropathic pain. For example, non-limiting exemplary agents for treating Huntington's disease include tetrabenazine (XENAZINE®), antipsychotic drugs such as haloperidol (HALDOL®), chlorpromazine, risperidone (RISPERDAL®), and quetiapine (SEROQUEL®).

[0389] Non-limiting exemplary formulations for treating Parkinson's disease include levodopa (with or without carbidopa) (LODOSYN®), dopamine agonists such as pramipexole (MIRAPEX®), ropinirole (REQUIP®), and rotigotine (NEUPRO®), and apomorphine (APOKYN®), selegiline (ELDEPRYL®, ZELAPAR®), rasagiline (AZILECT®), entacapone (COMTAN®), benztropine (COGENTIN®), trihexyphenidyl, and amantadine.

[0390] Non-limiting exemplary formulations for treating Alzheimer's disease include donepezil (ARICEPT®), galantamine (RAZADYNE®), and rivastigmine (EXELON®).

[0391] Non-limiting exemplary formulations for treating multiple sclerosis include glatiramer acetate (COPAXONE®), dimethyl fumarate (TECFIDERA®), fingolimod (GILENYA®), teriflunomide (AUBAGIO®), natalizumab (TYSABRI®), alemtuzumab (LEMTRADA®), and mitoxantrone (NOVANTRONE®).

[0392] A non-limiting exemplary formulation for treating ALS includes riluzole (RILUTEK®).

[0393] The dosage and administration of the one or more additional therapeutic agents is known in the art and is described, for example, in the product labeling of the respective agent.

[0394] Working Example The following examples are illustrative and not limiting.

[0395] Example 1: Expression and purification of human FAM19A5 protein Recombinant human FAM19A5 protein was produced and purified as follows, and the purified protein was used for binding affinity analysis and antibody screening analysis. First, LPS-hT plasmid expressing FAM19A5 gene was transformed into bacteria to induce protein overexpression. The produced FAM19A5 protein was purified using Ni-NTA affinity chromatography (Qiagen, Valencia, CA, USA). The His-tagged FAM19A5 protein was removed from the Ni-column by increasing the concentration of imidazole. Protein expression in the solution was measured using Coomassie Brilliant Blue R-250 dye. Only the FAM19A5 imidazole-containing solution was taken, and the FAM19A5 protein was concentrated using PBS. When the concentration was completed, the purity and concentration of FAM19A5 protein were measured using Western blot analysis. The concentrated protein was then used to screen FAM19A5-specific antibodies.

[0396] Example 2: Generation of antibody library FAM19A5 1.Immunization The cell wall components of TDW and CWS were emulsified with a water-in-oil emulsion adjuvant (RIBI+MPL+TDM+CWS adjuvant, Sigma, St. Louis, Mo, USA) and then subcutaneously injected into three chickens or four rabbits. The chickens and rabbits were immunized three and four times, respectively, approximately at 2-3 week intervals. The titers of antibodies obtained from the immunized animals were measured by immunoblotting using cell lysates of HEK293T cells overexpressing FAM19A5 protein.

[0397] 2. Generation of single-chain variable fragment (scFv) libraries from immunized chickens and rabbits RNA was extracted from the spleen, bone marrow and synovial sac of the immunized chickens described above using TRI Reagent (Invitrogen, Carlsbad, CA, USA). First-strand cDNA was synthesized using Oligo-dT primer and SUPERSCRIPT™ III First-Strand Synthesis System (Invitrogen). Single-strand variable region libraries were generated from cDNA obtained from the animal's immune system using the Expand High Fidelity PCR System (Roche Molecular Systems, IN, USA). In each reaction, 1 μL cDNA, 60 pmol of each primer, 10 μL 10x reaction buffer solution, 8 μL 2.5 mM dNTP (Promega, Madison, WI, USA) and 0.5 μL Taq DNA polymerase were mixed with water. The final volume was 100 μL. The PCR reaction was carried out under the following conditions: (i) 94°C for 15 s, (ii) 56°C for 30 s, and (iii) 72°C for 90 s, followed by 30 cycles of a final extension at 72°C for 10 min. The PCR products containing fragments with a length of approximately 350 bp were loaded onto a 1.5% agarose gel and electrophoresed, after which the nucleotide fragments were purified using a QIAGEN Gel II extraction kit (QIAGEN, Valencia, CA, USA). The purified PCR products were quantified by reading at OD260nm (1 unit OD=50 μg / ml).

[0398] The two VH and VL primary products from the secondary PCR were randomly linked by overlap extension PCR. Each PCR reaction was mixed with 100 ng of purified VL and VH products, 60 pmol of each primer, 10 μL 10x reaction buffer, 8 μL 2.5 mM dNTPs, 0.5 μL Taq DNA polymerase, and water to a final volume of 100 μL, and PCR was performed under 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 of final extension at 72°C for 10 minutes. The PCR products containing single-stranded variable region fragments with a length of approximately 700 bp were loaded onto a 1.5% agarose gel and electrophoresed, and then the nucleotide fragments were purified using a QIAGEN Gel II extraction kit (QIAGEN). The purified PCR products were quantified by reading at OD 260 nm (1 unit OD=50 μg / ml).

[0399] 3. Libraries, Ligation and Transformation The scFv fragment of the PCR product and the vector pComb3X-SS (The Scripps Research Institute CA, USA) were digested with Sfi I restriction enzyme. 10 μg of the purified overlapping PCR product was mixed with 360 units of Sif I (16 units per μg DNA, Roche Molecular Systems, Pleasanton, CA, USA), 20 μL 10x reaction buffer, and water to a final volume of 200 μL. 20 μg of the pComb3X-SS vector was mixed with 120 units of Sfi I (6 units per μg DNA), 20 μL 10x reaction buffer solution, and water to a final volume of 200 μL. The mixture was digested at 50°C for 8 hours. The digested product, including the scFv fragment (approximately 700 bp) and the vector (approximately 3400 bp), was then loaded onto a 1% agarose gel and purified using the Gel Extraction Kit II QIAGEN (QIAGEN, Valencia, CA, USA).

[0400] 1400 ng of Sfi I-restricted pComb3X vector and 700 ng of digested scFv fragment were mixed with 5x ligase buffer, 10 μL T4 DNA ligase (Invitrogen, Carlsbad, CA, USA) and water to a final volume of 200 μL. The mixture was incubated for 16 hours at 16°C to carry out ligation.

[0401] After precipitation with ethanol, the DNA pellet was dissolved in 15 μL of water. To generate the library, the ligation samples were transformed into E. coli strain ER2738 (New England Biolabs Inc, Hitchin, Hertfordshine, SG4OTY, England, UK) by electroporation using a vibrator gene (Gene Pulser: Bio-Rad Laboratories, Hercules, CA, USA). The cells were mixed in 5 ml SuperBroth (SB) medium and incubated at 37°C for 1 h with agitation at 250 rpm. Then, 3 μL of 100 mg / mL kanamycin was added to 100 mL of SB medium. To determine the library size, 0.1 μL, 1 μL, and 10 μL of the culture sample were smeared on Luria Broth (LB) agar plates containing 50 μg / mL kanamycin. After 1 h of agitation, 4.5 μL of 100 mg / mL kanamycin was added to the LB culture and further agitated for 1 h. Next, 2 mL of VCM13 helper phage solution (>10 11cfu / ml) was added to LB medium along with pre-warmed LB (183 mL) containing 92.5 μL of 100 mg / mL kanamycin. This mixture was further stirred at 250 rpm for 2 h at 37 °C. Then, 280 μL of kanamycin (50 mg / mL) was added to the culture and stirred overnight at 37 °C. The next day, the bacterial pellet was centrifuged at 3,000 g and 4 °C using a high-speed centrifuge. Then, the bacterial pellet was used to extract phagemid DNA, and 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 and kept on ice for 30 min. Then, the supernatant was centrifuged at 15,000 g and 4 °C for 15 min. Then, the supernatant was discarded and the phage pellet (Tris-reproduced containing 1% BSA) was suspended in buffered saline (TBS).

[0402] Example 3: Library panning against immobilized antigens (biopanning) Biopanning was performed using magnetic beads (Dynabeads M-270 epoxy, Invitrogen). The beads and protein were mixed together by rotation at room temperature for 20 hours, and approximately 1 × 10 7Beads were coated with 5 μg of recombinant FAM19A5 protein. Once coating was complete, the beads were washed four times with phosphate-buffered saline (PBS) and blocked for 1 h at room temperature with PBS containing 3% BSA. The coated beads were then incubated with the phage-displayed scFvs described above for 2 h at room temperature. To remove phages that were not bound to the antigen-coated beads, the beads were washed with 0.05% Tween 20 / PBS. Bound phages were then eluted with 50 μL of 0.1 M glycine / hydrochloric acid (0.1 M glycine-HCl, pH 2.2) and neutralized with 3 μL of 2 M Tris with hydrogen chloride (tris-HCl, pH 9.1). The phage-containing supernatant was used to infect E. coli ER2738 cells and rescued by overnight amplification with VCSM13 helper phage. Phage titer input and output were also determined from the phage-infected cultures by blotting the phage-infected cultures onto LB agar plates containing 50 μg / mL kanamycin. The next day, phages were precipitated with PEG-8000 and NaCl and then used for biopanning. Biopanning was performed up to a total of five times by repeating the above process. After each amplification, phages were screened and selected for high affinity to FAM19A5 protein.

[0403] Example 4: Selection of clones by phage ELISA To analyze the clones selected from biopanning, individual clones were randomly selected from the phage-displayed scFvs and ELISA was used to confirm whether the clones bound to FAM19A5 recombinant protein. FAM19A5 recombinant protein was diluted in 0.1M NaHC03 buffer and 100ng / well protein was used to coat a 96-well microtiter plate for 16 hours at 4°C. The next 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 (a-M13-HRP, Pierce Chemical Co, Rockford, IL, USA) was diluted 1 / 5000. 50μL 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 solution, 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] As shown in Figures 1A-1C, 24 clones generated from immunized chickens that bound to FAM19A5 recombinant protein and showed high absorbance were analyzed. From these 24 clones, 13 scFv clones with unique sequences were obtained. In the case of clones generated from immunized rabbits (data not shown), 174 clones were identified for the first time, and among these, 164 clones were sequenced. From these clones, 22 unique scFv sequences were finally obtained.

[0405] Example 5: Generation of anti-FAM19A5-IGG2 / 4 antibodies 1. Subcloning the Anti-FAM19A5scFv into a Mammalian Expression Vector In the FAM19A5 scFv gene sequence, the human Cκ gene was linked to the light chain variable domain, and the human immunoglobulin isotype IgG2 / 4 of CH2, CH2, and CH3 genes was linked to the heavy chain variable region. Antibodies with each light chain and each heavy chain were synthesized by adding restriction sites (Genscript, USA). The synthesized genes were inserted into a mammalian cell expression vector with modified restriction sites to facilitate cloning. First, the light chain genes were inserted into the vector using Hind III and Xba I (New England Biolabs, UK) restriction enzymes, and then the heavy chain genes were added to the vector using Nhel and BamHI (New England Biolabs, UK) restriction enzymes (Figure 2).

[0406] 2. Purification of Anti-FAM19A5 Antibody A mammalian cell transfection and overexpression injection system was used to express and purify anti-FAM19A5-IgG2 / 4 antibody. 2 μg / mL of mammalian expression vector and 4 μg of polyethyleneimine (PEI, Polysciences, Warrington, PA, USA) were mixed in 150 mM sodium chloride (NaCl, Merck), which corresponds to 1 / 10 of the cell culture volume. The mixture was left at room temperature for 15 min. The mixture was then transferred to HEK293F cells (2x10 6 The cells were added to 1000 ng / ml of IgG1000 ng / ml (Invitrogen) and then incubated in FREESTYLE™ 293 expression culture medium containing 100 U / mL penicillin and streptomycin (Invitrogen) at 7% CO2 and 37°C for 6 days under stirring at 135 rpm. Protein A beads (RepliGen, Waltham, MA, USA) affinity gel chromatography was used to purify the expressed anti-FAM19A5 IgG2 / 4 antibody from the cell culture supernatant. Protein A chromatography purified antibody was subjected to 4-12% Bis-Tris gradient gel electrophoresis. Protein size and yield were confirmed by Coomassie Brilliant Blue staining (Figure 3).

[0407] Example 6: Verification of anti-FAM19A5-IGG2 / 4 antibody specificity HEK293T cells stably transfected with genes encoding the FAM19A family of proteins were lysed in a buffer containing 50 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 100 mM mercaptoethanol, and bromophenol blue. Total cell extracts were resolved on SDS-polyacrylamide gels (PAGE) and transferred to nitrocellulose blotting membranes in a Bio-Rad Trans-Blot electrophoresis apparatus (Hercules, CA, USA). Blots were blocked with Tris-buffered saline containing 0.3% Tween 20 and 5% nonfat milk and incubated with anti-FAM19A5 antibody for 3 h at room temperature. They were then washed three times with TBS / 0.3% Tween 20. The blots were then incubated with a secondary antibody conjugated to horseradish peroxidase (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) to detect antibody binding. The blots were washed three times and exposed to x-ray film for 0.5 or 10 min after application of GE healthcare ECL reagent (Buckingham-shire, UK), and the immunoreactive bands were visualized. The results confirmed that anti-FAM19A5 antibody 1-65 specifically bound to the FAM19A5 protein conjugated with the Flag tag, but not to other FAM19A subfamily proteins (Figure 5A). For immunocytochemistry analysis, HEK293 cells expressing FAM19A-family proteins were fixed with 4% PFA. The cells were then blocked with 3% BSA and 0.1% Triton X-100 in PBS for 30 min at room temperature. The primary antibody (anti-FAM19A5 antibody 1-65) was applied overnight at 4°C. After several washes with PBS, appropriate secondary antibodies were applied for 30 min. Cells were then washed and mounted under a fluorescence or confocal microscope (LSM700; Zeiss, Goettingen, Germany). Nuclei were stained with Hoechst 33342. Immunocytochemical analysis of His-tagged FAM19A5 expressed in HEK293T showed that anti-FAM19A5 antibody 1-65 specifically bound to FAM19A5 protein conjugated with His-tag (Figure 5B, left panel).

[0408] Example 7: Epitope mapping analysis using FAM19A5 epitope fragments F1-F6 Overlapping peptide fragments (F1-F6, see FIG. 9) of human FAM19A5 protein were synthesized and conjugated to BSA. The binding of different anti-FAM19A5 antibodies to the BSA-conjugated peptide fragments F1-F6 was confirmed by Western blot analysis or ELISA analysis. For Western blot analysis, the BSA-conjugated FAM19A5 fragments F1-F6 were separated by SDS polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane by standard procedures. The membrane was incubated with an anti-FAM19A5 antibody (e.g., 1-65, 2 μg / ml, 1-65-scFv-rabbitFc-SSS), and the antigen-antibody complex was detected with an appropriate secondary antibody conjugated with horseradish peroxidase (anti-rabbit IgG (Fc specific)-HRP, 1:4000 dilution). For ELISA analysis, the following protocol was used. FAM19A5 fragments F1-F6 (diluted to 1 μg / mL in 50 mM carbonate buffer (Biosesang) or 20 μg / mL for high concentration analysis) were used to coat wells of a 96-well immunoplate (Thermo Scientific) (100 μL / well) overnight at 4° C., followed by washing twice with 1× PBS. The plate was then blocked with blocking buffer (100 μL / well) for 1 h at room temperature. During the 1 h incubation, the relevant anti-FAM19A5 antibody was diluted in dilution buffer to 1 μg / mL (or 20 μg / mL for high concentration analysis). After washing the plate (2× using 1× PBS), the diluted anti-FAM19A5 antibody was added to the appropriate wells and the plate was incubated for 1 h at room temperature. The plate was then washed a total of 5 times with washing buffer. Then, ODP substrate (prepared by dissolving one ODP tablet (O-phenylenediamine dihydrochloride, Thermo) in 9 mL sterile deionized water and 1 mL 10x Peroxide Stable Buffer (Theromo)) was added to each well and allowed to stand for 10 min for the color change reaction to occur. The reaction was terminated by adding 100 μL of 2N H2SO4 (Daejung) to the wells. The absorbance of each well was detected at 492 nm using a 96-well microplate reader (Molecular Device).

[0409] As shown in Figures 11 and 12A, anti-FAM19A5 antibody 1-65 bound strongly to fragment F5 as determined by Western blot and ELISA analysis. Anti-FAM19A5 antibody P2-C12 also bound strongly to epitope fragment F5, but did not bind significantly to other fragments (F1-F4 and F6) (see Figure 12B). On the other hand, anti-FAM19A5 antibody 3-2 did not bind to fragment F5. Instead, the 3-2 antibody bound strongly to epitope fragment F2 and minimally to other fragments (see Figure 12A). This result was the same for anti-FAM19A5 1-28 antibody (data not shown). However, unlike the other antibodies, it was confirmed that anti-FAM19A5 antibody 2-13 did not bind to any of the epitope fragments (see Figure 12A).

[0410] Next, to confirm the specific amino acid residues in the epitope fragment F5 to which the 1-65 and P2-C12 antibodies bind, different amino acid residues in the F5 fragment were substituted with alanine as shown in Table 9 below. The mutated residues are shown in bold and underlined. The binding affinity of the anti-FAM19A5 antibody was measured using ELISA analysis as described above.

[0411] [Table 9]

[0412] Double underline: Cysteine ​​is reactive during peptide synthesis and was replaced with serine to reduce reactivity. Serine is replaced with serine because its structure is closest to cysteine. Such replacements are shown with double underlines.

[0413] As shown in FIG. 13A, the 1-65 antibody could bind to mutant peptides #1, 5, 6, and 7 with similar affinity. However, when amino acid residues D13, L14, I16, and R18 of fragment F5 were mutated to alanine (numbered according to SEQ ID NO:125 in Table 9 above), the 1-65 antibody could no longer bind to this peptide fragment, suggesting that those amino acid residues were important binding sites for the 1-65 antibody. Meanwhile, the P2-C12 antibody showed high binding affinity to mutant peptides #2, 3, 4, 5, and 7, but did not bind to mutant peptides #1, 6, and 8 (see FIG. 13B). As shown in Table 9 (above), mutant peptides #1, 6, and 8 contained alanine substitutions at amino acid residues G11, E10, and R18 (numbered according to SEQ ID NO:125), suggesting that those amino acid residues were important binding sites for the P2-C12 antibody.

[0414] Example 8: Epitope mapping analysis using FAM19A5 mutants M1-M8 To further characterize the binding epitopes of the anti-FAM19A5 antibodies disclosed herein, the amino acid sequences for different FAM19 family members (i.e., FAM19A1-5) were aligned as shown in Figure 14. Based on this alignment, eight regions were identified (M1-M8) in which the amino acid sequence of the FAM19A5 protein is most significantly different from other members of the FAM19A5 family (i.e., FAM19A1-4). The amino acid sequences of these regions were replaced with the consensus sequences of the corresponding regions for the FAM19A1-4 proteins (see Table 10 - the mutated amino acid residues are shown in bold and underlined).

[0415] Mutant FAM19A5 expressing phages were prepared as follows: To prepare phage culture medium, 55.6 mL of 2 M glucose (D-(+)-glucose, Sigma), 5 mL of 1 M MgCl2 (Magnesium chloride, Junsei), and 1 mL of 34 mg / mL chloramphenicol (Sigma) were added to 2x YT medium. Colonies obtained by monophage ELISA were selected and placed in 5 mL of prepared medium (2x YT-GMC) and cultured at 37°C in a shaking incubator (VS-8480, Vision) for 16 hours. 100 μL of each culture was individually transferred to 10 mL 2x YT-GMC and cultured in an incubator at 37°C until the detection value at OD600nm reached 0.5. When the detection value at OD600nm reached 0.5, 5 mL of each culture was taken as a sample and infected. After sample preparation, 50 μL of M1 helper phage was added to each sample, followed by incubation at 37°C for 30 min without shaking and then 30 min with shaking. Separate cultures were centrifuged at 3,850 rpm for 15 min in a Microcentrifuge (Microl2, Hanil). The supernatants of the centrifuged cultures were removed and kept aside and replaced with 5 mL of 2x YT medium containing 1 mL of 1 M IPTG (AG Scientific), 5 mL of 1 M MgCl2, 1 mL of 70 μg / μL kanamycin (Biopure), and 1 mL of chloramphenicol. The resulting pellets were thoroughly dispersed in the freshly added medium and then incubated at 30°C for 16 h with agitation.

[0416] [Table 10]

[0417] With reference to Figure 15A and the data described in Example 7, anti-FAM19A5 antibody 1-65 failed to bind to FAM19A5 mutants M6, M7, and M8, which contain amino acid mutations at sites corresponding to regions within the epitope fragment F5 of FAM19A5 (see Table 10 above).

[0418] Epitope analysis for additional anti-FAM19A5 antibodies is provided in Figures 15B and 15C. Anti-FAM19A5 antibodies 13B4, 13F7, and 15A9 all failed to bind to FAM19A5 mutant M8 and had reduced binding to mutant M7. Also, 13B4 antibody failed to bind to FAM19A5 mutant M6 (see Figure 15B). Similarly, antibodies P1-A08, P1-F02, P2-A01, P2-A03, P2-F07, and P2-F11 all failed to bind to FAM19A5 mutant M8, and most antibodies also had reduced or no binding to mutant M7. Unlike these antibodies, P2-C12 antibody bound strongly to FAM19A5 mutant M8. Instead, the P2-C12 antibody failed to bind to the FAM19A5 mutant M6, a result consistent with previous data demonstrating that the epitope fragment F5 contains the critical FAM19A5 binding site of the P2-C12 antibody.

[0419] Example 9: Reciprocal competition analysis We next assessed whether different anti-FAM19A5 antibodies with similar binding epitopes compete with each other using a two-site sandwich ELISA, as follows (see FIG. 16A).

[0420] First, the incubated anti-FAM19A5 antibody was diluted in 1x PBS at a concentration of 10 μg / mL. The diluted anti-FAM19A5 antibody (10 μg / m in 1x PBS, "capture antibody") was used to coat a 96-well plate (100 μL / well) at 37°C for approximately 1 hour. After incubation, the plate was washed a total of 5 times with washing buffer (0.01% Tween-20 / PBS; also known as 0.01% PBST), blocked with blocking solution (5% BSA / PBS; also known as 5% PBSA, 250 μL / well) at 37°C for 1 hour, and then washed again. Next, 100 μg / mL FAM19A5 antigen (diluted in PBS containing 5% BSA, 0.01% Tween-20; also known as 5% PBSAT; "dilution buffer") was added to each well, and the 96-well plate was incubated at 37°C for 2 hours. After incubation, the plates were washed a total of 5 times using 0.01% PBST. After the final wash, the incubated biotinylated anti-FAM19A5 antibody ("detection antibody", diluted to 1 μg / mL in 5% PBSAT) was added to the relevant wells (100 μL volume) and the plates were further incubated for 1 h at 37°C. Afterwards, the plates were washed again with 0.01% PBST (total of 5 washes). Next, 10 μL of diluted (1 / 2000 in 5% PBSAT) streptavidin-HRP (1 mg / mL, Sigma, USA) was added to the wells and the plates were incubated for 30 min at room temperature. Next, the plates were washed and treated with 100 μL of TMB substrate (then 3,3 plates were washed and treated with 100 μL, Thermo Fisher Scientific). After a further incubation of 30 min at room temperature, TMB substrate was added to induce the color change reaction. The reaction was terminated using 50 μL of sulfuric acid (2N H2SO4) and the extent of color change was detected at 450 nm with a reference wavelength of 620 nm using a 96-well microplate reader (Molecular Device).

[0421] As shown in Figure 16B, the anti-FAM19A5 antibodies 1-65, P2-A03, P2-F11, and 13B4 all competed with each other. This result was consistent with the previous epitope analysis, and indicated that these antibodies bind to FAM19A5 at similar epitopes (i.e., EP6, EP7, and / or EP8) (see Figures 15A-C).

[0422] Example 10: Binding affinity analysis To determine whether there are differences in the binding affinity of different anti-FAM19A5 antibodies to FAM19A5, OCTET® test and ELISA analysis were used. For OCTET® test, the following protocol was used: Ni-NTA biosensor was hydrated with UPLC grade water in Octet QK3. The His-tag of FAM19A5 protein was immobilized on Ni-NTA biosensor (at 2nM level). The incubated anti-FAM19A5 antibody was diluted to concentrations of 300nM, 100nM, 33nM, 11nM, 3.3nM, 1.1nM and 0nM. The binding kinetics of different anti-FAM19A5 antibodies to FAM19A5 immobilized on biosensor was measured. The data was analyzed using Data Analysis Software (version 9.0) program.

[0423] As shown in Figures 17A-17D, anti-FAM19A5 antibodies 1-65 and 13F7 had similar binding affinities (Kd = 0.23 nM and 0.25 nM, respectively) and bound more strongly to FAM19A5 than antibodies 13B4 (Kd = 3.6 nM) and 15A9 (Kd = 5.95 nM). The binding affinities of different anti-FAM19A5 antibodies measured by ELISA are presented in Figures 18A and 18B.

[0424] Example 11: Use of FAM19A5 antibody in animal models of brain injury To evaluate the in vivo function of the anti-FAM19A5 antibodies presented herein, a traumatic brain injury (TBI) mouse model was used. C57BL / 6 adult male mice (8-9 weeks old) were anesthetized with sodium pentobarbital (50 mg / kg). A pre-cooled iron bar was placed on the calvaria for 1 min to induce cryogenic TBI. The skin of the mice was then sutured and housed in the same manner as normal mice (see Moon et al, Neuro Report 22:304-308 (2011)). Approximately 1 day after TBI induction, animals were intravenously administered different anti-FAM19A5 antibodies at various concentrations (0.1, 0.3, 1, 3, 5, 10 or 100 μg / mouse) diluted in phosphate-buffered saline (PBS). Normal human control immunoglobulin (HCI) served as the control group.

[0425] Then, on day 5 after TBI induction (TBI5D), animals were killed by perfusion with 4% paraformaldehyde (PFA) in PBS and brain tissue was harvested. The harvested brain tissue was further fixed in 4% PFA solution for 24 h. Brain tissue was then cryoprotected in 30% sucrose, serially sectioned (40 μm) on a cryostat, and stored in 50% glycerol / 50% PBS at -20 °C until use.

[0426] To stain reactive astrocytes associated with gliosis (nestin positive and GFAP expressing), brain tissue sections were blocked with 3% bovine serum albumin (BSA) and 0.1% Triton X-100 in PBS for 30 min. Primary antibodies were then incubated with the sections overnight at 4°C. Primary antibodies used in this study were mouse anti-nestin (Millipore, Billerica, MA, USA) and rabbit anti-GFAP (Dako, Carpinteria, CA, USA). After several washes with PBS, appropriate secondary antibodies were applied for 30 min. Nuclei were labeled with Hoechst 33342 (Invitrogen, Carlsbad, CA, USA). Sections were then washed and mounted for observation under a fluorescent or confocal microscope (Leica, Wetzlar, Germany).

[0427] To quantify the effect, ROIs (regions of interest) in GFAP-negative regions were first identified in each image, and the area (μm 2 The mean distance from the lesion center to the penumbra (A, A) was calculated using LAS AF light software (Leica Microsystem CMS GmbH, Mannheim, Germany). The outer length of the border of the lesion center bordering the penumbra (μm, B, B) was also measured using the same software. The result of A / B represents the average distance from the lesion center (A / B, μm).

[0428] As shown in Figure 6, unlike the control HCI antibody, anti-FAM19A5 antibody 1-65 significantly reduced, reversed and / or prevented the onset of reactive gliosis in the posterior penumbra of traumatic brain injury, as evidenced by less GFAP-positive staining near the lesion border (shown by the dotted line). Similar results were observed with anti-FAM19A5 antibodies 13B4, 13F7, 15A9, P2-A03, P2-F11 and P1-A03 (see Figures 19A-E). And as shown in Figure 7, immunostaining for GFAP and NeuN in the lesion epicenter and penumbra indicates that anti-FAM19A5 antibody can also promote neuronal survival in the penumbra surrounding the injured area.

[0429] Example 12: Efficacy of anti-FAM19A5 for the treatment of spinal cord injury To test whether anti-FAM19A5 antibody can improve the functional motor activity of injured animals, a spinal cord injury (SCI) animal model was used. Adult male Sprague Dawley rats (DaeHan BioLink Co., Ltd, Korea) were anesthetized with chloral hydrate (500 mg / kg) and the thoracic spinal cord was exposed 8 or 10 times. To simulate the human spinal cord condition, a NYU impactor (Routes, Sciteck Korea Inc.), designed to numerically calculate injury strength, was used. A 10 g weight was dropped at a height of 25 mm onto the thoracic spinal cord number 9, which was exposed by laminectomy without dural damage. The data calculated from the NYU impactor confirmed that the injury was uniformly transmitted within the error range, and the wound was sutured. After applying povidone-iodine to the wound site, the rats were housed 2 per cage, and the bladder was massaged 3 times a day for 7 weeks to promote urination. Each animal then received one of the following antibodies by intravenous route: (i) anti-FAM19A5 antibody 1-65 (60 μg), (ii) anti-FAM19A5 polyclonal Ab (60 μg), or (iii) normal rat IgG (60 μg) in PBS. Motility function of SCI animals was assessed using BBB locomotor scores. BBB scores range from 0 for no hind paw movement to 21 for normal movement. Animals were left to roam freely in an open space and observed by two blinded observers. Animals with a BBB score >1 on the first day after SCI were excluded from the study. Scores were monitored for 40 days. Data were quantified as the mean ± SEM of the two hind paw scores, compiled, and organized graphically. Figure 8 (left panel) shows that a single administration of FAM19A5 antibody 1-65 (C, circles) significantly improved locomotor activity from 21 days post injury (dpi) compared to vehicle-treated rats (A, diamonds). Treatment with anti-FAM19A5 polyclonal Ab (B, squares) also improved locomotor activity from 35 dpi compared to vehicle-treated rats (A, diamonds). Inclined plane testing was also performed weekly for up to 6 weeks (right panel of Figure 8). In the inclined plane test, rats were positioned on an adjustable inclined plane.The maximum tilt angle at which each animal maintained a stable position for at least 5 seconds was assessed by two observers blinded to the animal groups, and the average angle was recorded. The tilt test also showed improved functional activity in animals treated with FAM19A5 antibody 1-65 (C, circles) or anti-FAM19A5 polyclonal Ab (B, squares) compared to the control group (A, diamonds).

[0430] The portions of the Detailed Description of the Invention other than the Summary and Abstract are intended to be used to interpret the claims. The Summary and Abstract may present one or more, but not all, exemplary embodiments of the present disclosure as contemplated by the inventors, and therefore do not limit the scope of the present disclosure and the appended claims in any way.

[0431] The present disclosure has been described above with functional components illustrating the performance of specified functions and their relationships. The boundaries of these functional components have been arbitrarily defined herein for convenience of description. Other boundaries may be defined to the extent that the specified functions and their relationships are appropriately performed.

[0432] The foregoing description of specific embodiments should fully express the general nature of the present disclosure, and others can easily modify and / or adapt such specific embodiments to various applications by applying knowledge in the art without departing from the general concept of the present disclosure and without undue experimentation. Such adjustments and modifications are therefore intended to be within the meaning and range of equivalents of the disclosed embodiments based on the teaching and guidance presented herein. It should be understood that the words or terms presented herein are used for purposes of description and not limitation, and therefore the words and terms herein should be interpreted by those skilled in the art in light of the teaching and guidance.

[0433] The scope of the present disclosure should not be limited by any of the above-described exemplary embodiments, but should be defined only by the following claims and their equivalents.

[0434] All publications cited herein, particularly patent applications, internet sites and accession numbers / database sequences (including polynucleotide and polypeptide sequences), are hereby incorporated by reference in their entirety for all purposes only to the extent that each individual publication, patent, patent application, internet site or accession number / database sequence is specifically and individually indicated to be incorporated by reference herein.

Claims

1. An anti-FAM19A5 antibody, which is a monoclonal antibody or an antigen-binding portion thereof that specifically binds to human family 19, member A5 (FAM19A5), characterized in that the anti-FAM19A5 antibody binds to at least one epitope having the amino acid sequence of SEQ ID NO: 2 or a fragment thereof, The anti-FAM19A5 antibody is heavy chain CDR1, CDR2 and CDR3, and light chain CDR1, CDR2 and CDR3; (2) heavy chain CDR1, CDR2 and CDR3 comprise SEQ ID NO: 29, SEQ ID NO: 30 and SEQ ID NO: 31, respectively; and light chain CDR1, CDR2 and CDR3 comprise SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34, respectively; (3) the heavy chain CDR1, CDR2 and CDR3 comprise SEQ ID NO: 35, SEQ ID NO: 36 and SEQ ID NO: 37, respectively; and the light chain CDR1, CDR2 and CDR3 comprise SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40, respectively; (4) the heavy chain CDR1, CDR2 and CDR3 comprise SEQ ID NO: 41, SEQ ID NO: 42 and SEQ ID NO: 43, respectively; and the light chain CDR1, CDR2 and CDR3 comprise SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46, respectively; (5) the heavy chain CDR1, CDR2 and CDR3 comprise SEQ ID NO: 47, SEQ ID NO: 48 and SEQ ID NO: 49, respectively; and the light chain CDR1, CDR2 and CDR3 comprise SEQ ID NO: 50, SEQ ID NO: 51 and SEQ ID NO: 52, respectively; (6) the heavy chain CDR1, CDR2 and CDR3 comprise SEQ ID NO: 53, SEQ ID NO: 54 and SEQ ID NO: 55, respectively; and the light chain CDR1, CDR2 and CDR3 comprise SEQ ID NO: 56, SEQ ID NO: 57 and SEQ ID NO: 58, respectively; (7) the heavy chain CDR1, CDR2 and CDR3 comprise SEQ ID NO: 59, SEQ ID NO: 60 and SEQ ID NO: 61, respectively; and the light chain CDR1, CDR2 and CDR3 comprise SEQ ID NO: 62, SEQ ID NO: 63 and SEQ ID NO: 64, respectively; (8) the heavy chain CDR1, CDR2 and CDR3 comprise SEQ ID NO: 65, SEQ ID NO: 66 and SEQ ID NO: 67, respectively; and the light chain CDR1, CDR2 and CDR3 comprise SEQ ID NO: 68, SEQ ID NO: 69 and SEQ ID NO: 70, respectively; (9) the heavy chain CDR1, CDR2 and CDR3 comprise SEQ ID NO: 71, SEQ ID NO: 72 and SEQ ID NO: 73, respectively; and the light chain CDR1, CDR2 and CDR3 comprise SEQ ID NO: 74, SEQ ID NO: 75 and SEQ ID NO: 76, respectively; (10) the heavy chain CDR1, CDR2 and CDR3 comprise SEQ ID NO: 77, SEQ ID NO: 78 and SEQ ID NO: 79, respectively; and the light chain CDR1, CDR2 and CDR3 comprise SEQ ID NO: 80, SEQ ID NO: 81 and SEQ ID NO: 82, respectively; (11) the heavy chain CDR1, CDR2 and CDR3 comprise SEQ ID NO: 83, SEQ ID NO: 84 and SEQ ID NO: 85, respectively, and the light chain CDR1, CDR2 and CDR3 comprise SEQ ID NO: 86, SEQ ID NO: 87 and SEQ ID NO: 88, respectively; or (12) The anti-FAM19A5 antibody, wherein the heavy chain CDR1, CDR2 and CDR3 comprise SEQ ID NO: 89, SEQ ID NO: 90 and SEQ ID NO: 91, respectively, and the light chain CDR1, CDR2 and CDR3 comprise SEQ ID NO: 92, SEQ ID NO: 93 and SEQ ID NO: 94, respectively.

2. (2) a heavy chain variable region (VH) comprising SEQ ID NO: 103 and a light chain variable region (VL) comprising SEQ ID NO: 114; (3) a heavy chain variable region (VH) comprising SEQ ID NO: 104 and a light chain variable region (VL) comprising SEQ ID NO: 115; (4) a heavy chain variable region (VH) comprising SEQ ID NO: 105 and a light chain variable region (VL) comprising SEQ ID NO: 116; (5) a heavy chain variable region (VH) comprising SEQ ID NO: 106 and a light chain variable region (VL) comprising SEQ ID NO: 117; (6) a heavy chain variable region (VH) comprising SEQ ID NO: 107 and a light chain variable region (VL) comprising SEQ ID NO: 118; (7) a heavy chain variable region (VH) comprising SEQ ID NO: 108 and a light chain variable region (VL) comprising SEQ ID NO: 119; (8) a heavy chain variable region (VH) comprising SEQ ID NO: 109 and a light chain variable region (VL) comprising SEQ ID NO: 120; (9) a heavy chain variable region (VH) comprising SEQ ID NO: 110 and a light chain variable region (VL) comprising SEQ ID NO: 121; (10) a heavy chain variable region (VH) comprising SEQ ID NO: 111 and a light chain variable region (VL) comprising SEQ ID NO: 122; (11) a heavy chain variable region (VH) comprising SEQ ID NO: 112 and a light chain variable region (VL) comprising SEQ ID NO: 123; or (12) A heavy chain variable region (VH) comprising SEQ ID NO: 113 and a light chain variable region (VL) comprising SEQ ID NO:

124. The anti-FAM19A5 antibody of claim 1, comprising:

3. 3. The anti-FAM19A5 antibody according to claim 1 or 2, characterized in that it exhibits one or more of the following properties: (a) binds to soluble human FAM19A5 with a KD of 10 nM or less, as measured by enzyme-linked immunosorbent assay (ELISA); (b) binds 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 excessive proliferation of reactive astrocytes; (e) reducing the expression of chondroitin sulfate proteoglycans, including neurocan and neuron-glial antigen 2 (NG2); (f) increased expression of c-fos and pERK in neuronal nuclei; (g) promoting neuronal survival; (h) increasing the expression of GAP43 in neurons; and (i) Promotes axon regrowth.

4. A nucleic acid encoding the anti-FAM19A5 antibody of claim 1 or 2.

5. A composition comprising an anti-FAM19A5 antibody described in claim 1 or 2, a bispecific molecule comprising the anti-FAM19A5 antibody and the anti-FAM19A5 antibody bound to a molecule having a second binding moiety, or an immunoconjugate comprising the anti-FAM19A5 antibody or the bispecific molecule, and a carrier.

6. 3. An anti-FAM19A5 antibody according to claim 1 or 2 for use in the therapeutic therapy of a disease or condition.

7. The anti-FAM19A5 antibody of claim 6, wherein the disease or condition comprises traumatic brain injury, cerebrospinal cord injury, stroke or brain tumor.

8. 3. An anti-FAM19A5 antibody according to claim 1 or 2 for use in a method for diagnosing a subject by contacting said antibody with a physiological sample from said subject.

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