Anti-FAM19a5 antibodies and uses thereof

Abstract

To provide antibodies that specifically bind to human FAM19A5, compositions comprising such antibodies, and methods for treating fibrosis and cancer using anti-FAM19A5 antibodies.SOLUTION: Disclosed is an isolated monoclonal antibody that specifically binds to the member A5 (FAM19A5) protein (anti-FAM19A5 antibody) or antigen binding part thereof, where (i) the anti-FAM19A5 antibody binds to at least one epitope comprising an amino acid sequence TLDRDSSQPRRTIARQTARC; (ii) the epitope can specifically bind to a reference antibody comprising a heavy chain variable domain and a light chain variable domain; and (iii) the anti-FAM19A5 antibody binds to a soluble human FAM19A5 or membrane-bound human FAM19A5 with a KD of 0.1 to 1 nM when measured by enzyme-linked immunosorbent assay (ELISA).SELECTED DRAWING: None

Description

Detailed Description of the Invention

[0001] [Technical field] (Reference to electronically submitted sequence listing) The contents of the Sequence Listing submitted electronically in ASCII text file format with this application (Filename: 3763.005PC02_SeqListing_ST25.txt; Size: 166,889 bytes; Created: June 26, 2018) are hereby incorporated by reference in their entirety.

[0002] (Technical field) The present invention provides antibodies that specifically bind to family with sequence homology 19, member A5 (FAM19A5), compositions comprising such antibodies, and methods of using the antibodies to prevent or treat disorders or diseases, such as fibrosis and / or cancer (e.g., brain tumors, e.g., glioblastoma), in a subject.

[0003] [Background technology] FAM19A5 is a member of the TAFA protein subfamily, 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 predominantly expressed in specific regions of the brain and spinal cord. These proteins appear to be produced and secreted by adult neural stem cells during neurogenesis.

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

[0005] In addition to regulating the nervous system, FAM19A5 may also play a role in regulating immune cells. Fibrosis is a common health-related condition that occurs frequently in a variety of pathological processes and is characterized by infiltrating mononuclear immune cells that release cytokines that stimulate fibroblasts to transform connective tissue. Therefore, there is a need to develop antibodies that can specifically bind to FAM19A5 and modulate FAM19A5 activity.

[0006] Summary of the Invention [Means for solving the problems] BRIEF SUMMARY OF THEINVENTION Provided herein is an isolated antibody ("anti-FAM19A5 antibody"), or an antigen-binding portion thereof, that specifically binds to a human family, member A5 ("FAM19A5") protein having sequence homology 19 and cross-competes for binding to a human FAM19A5 epitope with a reference antibody comprising a heavy chain CDR1, CDR2, and CDR3 and a light chain CDR1, CDR2, and CDR3, and wherein (i) the heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO:11, the heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO:12, the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO:13, the light chain CDR1 comprises the amino acid sequence of SEQ ID NO:23, the light chain CDR2 comprises the amino acid sequence of SEQ ID NO:24, and the light chain CDR3 comprises the amino acid sequence of SEQ ID NO:25; or (ii) the heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO:14 and the heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO:26. wherein the heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO:15, and the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO:16, and the light chain CDR1 comprises the amino acid sequence of SEQ ID NO:26, and the light chain CDR2 comprises the amino acid sequence of SEQ ID NO:27, and the light chain CDR3 comprises the amino acid sequence of SEQ ID NO:28; or (iii) wherein the heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO:20, and the heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO:21, and the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO:22, and the light chain CDR1 comprises the amino acid sequence of SEQ ID NO:32, and the light chain CDR2 comprises the amino acid sequence of SEQ ID NO:33, and the light chain CDR3 comprises the amino acid sequence of SEQ ID NO:34.

[0007] Also provided herein are isolated antibodies ("anti-FAM19A5 antibodies"), or antigen-binding portions thereof, that specifically bind to a human family member A5 ("FAM19A5") protein having sequence homology 19 and bind to a FAM19A5 epitope identical to a reference antibody comprising a heavy chain CDR1, CDR2, and CDR3 and a light chain CDR1, CDR2, and CDR3, and that have either (i) a heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO:11, a heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO:12, and a heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO:13, a light chain CDR1 comprises the amino acid sequence of SEQ ID NO:23, a light chain CDR2 comprises the amino acid sequence of SEQ ID NO:24, and a light chain CDR3 comprises the amino acid sequence of SEQ ID NO:25; or (ii) a heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO:14 and a heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO:15. or (iii) heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO:20, heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO:21, heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO:22, light chain CDR1 comprises the amino acid sequence of SEQ ID NO:32, light chain CDR2 comprises the amino acid sequence of SEQ ID NO:33 and light chain CDR3 comprises the amino acid sequence of SEQ ID NO:34.

[0008] In some embodiments, an anti-FAM19A5 antibody of the invention binds to at least one FAM19A5 epitope that is SEQ ID NO: 6. In particular embodiments, an anti-FAM19A5 antibody binds to a FAM19A5 epitope that is SEQ ID NO: 6 at one or more amino acids corresponding to amino acid residues (i) 46-51 (i.e., DSSQPR), (ii) 46, 50, and 52 (i.e., D---PR), or (iii) 46, 47, 48, and 50 (i.e., DSS-P). In some embodiments, an anti-FAM19A5 antibody binds to at least one FAM19A5 epitope that is SEQ ID NO: 9.

[0009] In certain embodiments, the anti-FAM19A5 antibody binds to at least one FAM19A5 epitope identified as EP2, EP4, and / or EP8, where EP2 contains the amino acids DSSQP (SEQ ID NO:66) and EP4 contains the amino acids ARCACRK (SEQ ID NO:67). EP8 contains the amino acids TCTQPGGR (SEQ ID NO:72), and EP9 contains the amino acids TCTQPGGR (SEQ ID NO:73).

[0010] In some embodiments, the anti-FAM19A5 antibody disclosed herein comprises a heavy chain CDR1, CDR2, and CDR3 and a light chain CDR1, CDR2, and CDR3, (i) the heavy chain CDR1 comprises SEQ ID NO:11, SEQ ID NO:14, or SEQ ID NO:20; (ii) the heavy chain CDR2 comprises SEQ ID NO:12, SEQ ID NO:15, or SEQ ID NO:21; (iii) the heavy chain CDR3 comprises SEQ ID NO:13, SEQ ID NO:16, or SEQ ID NO:22; (iv) the light chain CDR1 comprises SEQ ID NO:23, SEQ ID NO:26, or SEQ ID NO:32; (v) the light chain CDR2 comprises SEQ ID NO:24, SEQ ID NO:27, or SEQ ID NO:33; and / or (vi) the light chain CDR3 comprises SEQ ID NO:25, SEQ ID NO:28, or SEQ ID NO:34.

[0011] In some embodiments, the anti-FAM19A5 antibody comprises (i) a heavy chain variable domain comprising SEQ ID NO:35 and a light chain variable domain comprising SEQ ID NO:39; (ii) a heavy chain variable domain comprising SEQ ID NO:36 and a light chain variable domain comprising SEQ ID NO:40; or (iii) a heavy chain variable domain comprising SEQ ID NO:38 and a light chain variable domain comprising SEQ ID NO:42.

[0012] In some embodiments, 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 presented as SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:38, 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 presented as SEQ ID NO:39, SEQ ID NO:40, or SEQ ID NO:42.

[0013] In some embodiments, the anti-FAM19A5 antibody comprises (i) a heavy chain comprising SEQ ID NO:57, SEQ ID NO:58, or SEQ ID NO:60; and (ii) a light chain comprising SEQ ID NO:61, SEQ ID NO:62, or SEQ ID NO:64.

[0014] In some embodiments, an anti-FAM19A5 antibody of the invention is a chimeric antibody, a human antibody, or a humanized antibody. In some embodiments, the anti-FAM19A5 antibody comprises one or more of the following properties: (a) reducing, reversing, slowing, and / or preventing fibrosis; (b) reducing excessive extracellular matrix (ECM) formation; (c) slowing tumor growth or progression; (d) binding to soluble human FAM19A5 with a KD of 10 nM or less as measured by enzyme-linked immunosorbent assay (ELISA); (e) binding to membrane-bound human FAM19A5 with a KD of 10 nM or less as measured by ELISA; (f) reducing, reversing, slowing, and / or preventing the onset of reactive gliosis; (g) inhibiting excessive proliferation of reactive astrocytes; (h) reducing expression of chondroitin sulfate proteoglycans, including neurocan and neuron-glial antigen 2 (NG2); (i) increasing expression of c-fos and pERK in neuronal nuclei; (j) promoting neuronal survival; (k) increasing expression of GAP43 in neurons; and (l) promoting axonal regrowth.

[0015] The present invention also provides a human family, member A5 (FAM19A5) epitope having sequence homology 19 consisting essentially of or consisting 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:5, 6, 9, or 10, said epitope comprising (i) a heavy chain variable domain comprising SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:38, and (ii) a heavy chain variable domain comprising SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, It is capable of specifically binding to a reference antibody comprising a light chain variable domain comprising SEQ ID NO:39, SEQ ID NO:40, or SEQ ID NO:42.

[0016] Also disclosed herein are nucleic acids encoding the anti-FAM19A5 antibodies or FAM19A5 epitopes described herein.

[0017] Also provided are compositions comprising the anti-FAM19A5 antibodies or FAM19A5 epitopes of the invention.

[0018] In some embodiments, the present invention also provides anti-FAM19A5 antibodies for treating brain cancer.

[0019] In another aspect, the present invention provides a method of diagnosing a subject in need of diagnosis, comprising the step of contacting a biological sample from the subject with an anti-FAM19A5 antibody disclosed herein.

[0020] (Specific examples) Example 1. An isolated monoclonal antibody or antigen-binding portion thereof that specifically binds to human family member A5 (FAM19A5) having sequence homology 19 and exhibits one or more of the following properties: (a) reducing, reversing, slowing and / or preventing fibrosis; (b) a reduction in excessive extracellular matrix (ECM) formation; and (c) delaying tumor growth or progression.

[0021] Example 2. The monoclonal antibody or antigen-binding portion thereof of Example 1, further comprising one or more of the following features: (d) binds to soluble human FAM19A5 with a KD of 10 nM or less as measured by enzyme-linked immunosorbent assay (ELISA); (e) binds to membrane-bound human FAM19A5 with a KD of 10 nM or less as measured by ELISA; (f) reducing, reversing, delaying and / or preventing the onset of reactive gliosis; (g) inhibition of excessive proliferation of reactive astrocytes; (h) decreased expression of chondroitin sulfate proteoglycans, including neurocan and neuron-glial antigen 2 (NG2); (i) Increased c-fos and pERK expression in neuronal nuclei; (j) promoting neuronal survival; (k) increased expression of GAP43 in neurons; and (l) Promotion of axonal regrowth.

[0022] Example 3. (i) a reference antibody comprising a heavy chain CDR1, CDR2, and CDR3 and a light chain CDR1, CDR2, and CDR3, wherein heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO:11, heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO:12, heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO:13, light chain CDR1 comprises the amino acid sequence of SEQ ID NO:23, light chain CDR2 comprises the amino acid sequence of SEQ ID NO:24, and light chain CDR3 comprises the amino acid sequence of SEQ ID NO:25; (ii) a reference antibody comprising a heavy chain CDR1, CDR2, and CDR3 and a light chain CDR1, CDR2, and CDR3, wherein heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO:14, heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO:15, heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO:16, light chain CDR1 comprises the amino acid sequence of SEQ ID NO:26, light chain CDR2 comprises the amino acid sequence of SEQ ID NO:27, and light chain CDR3 comprises the amino acid sequence of SEQ ID NO:28; or (iii) a heavy chain CDR1, CDR2, and CDR3 and a light chain CDR1, CDR2, and CDR3, wherein the heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO:20, the heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO:21, and the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: a reference antibody comprising the amino acid sequence of SEQ ID NO:22, wherein the light chain CDR1 comprises the amino acid sequence of SEQ ID NO:32, the light chain CDR2 comprises the amino acid sequence of SEQ ID NO:33, and the light chain CDR3 comprises the amino acid sequence of SEQ ID NO:34. 3. The monoclonal antibody or antigen-binding portion thereof of Example 1 or 2, which cross-competes for binding to the human FAM19A5 epitope with

[0023] Example 4. (i) a reference antibody comprising a heavy chain CDR1, CDR2, and CDR3 and a light chain CDR1, CDR2, and CDR3, wherein heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO:11, heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO:12, heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO:13, light chain CDR1 comprises the amino acid sequence of SEQ ID NO:23, light chain CDR2 comprises the amino acid sequence of SEQ ID NO:24, and light chain CDR3 comprises the amino acid sequence of SEQ ID NO:25; (ii) a reference antibody comprising a heavy chain CDR1, CDR2, and CDR3 and a light chain CDR1, CDR2, and CDR3, wherein heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO:14, heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO:15, heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO:16, light chain CDR1 comprises the amino acid sequence of SEQ ID NO:26, light chain CDR2 comprises the amino acid sequence of SEQ ID NO:27, and light chain CDR3 comprises the amino acid sequence of SEQ ID NO:28; or (iii) a heavy chain CDR1, CDR2, and CDR3 and a light chain CDR1, CDR2, and CDR3, wherein the heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO:20, the heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO:21, and the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: a reference antibody comprising the amino acid sequence of SEQ ID NO:22, wherein the light chain CDR1 comprises the amino acid sequence of SEQ ID NO:32, the light chain CDR2 comprises the amino acid sequence of SEQ ID NO:33, and the light chain CDR3 comprises the amino acid sequence of SEQ ID NO:34. 3. The monoclonal antibody or antigen-binding portion thereof of Example 1 or 2, which binds to the same human FAM19A5 epitope as in

[0024] Example 5. The monoclonal antibody or antigen-binding portion thereof of Example 3 or 4, which binds to at least one FAM19A5 epitope, which is SEQ ID NO:6.

[0025] Example 6. The monoclonal antibody or antigen-binding portion thereof of any one of Examples 2-5, wherein the monoclonal antibody or antigen-binding portion thereof binds only to FAM19A5, which has SEQ ID NO:6.

[0026] Example 7. The monoclonal antibody or antigen-binding portion thereof of any one of Examples 2-5, wherein the monoclonal antibody or antigen-binding portion thereof further binds to an additional FAM19A5 epitope.

[0027] Example 8. Additional FAM19A5 epitopes are SEQ ID NO:5, SEQ ID NO:6, The monoclonal antibody or antigen-binding portion thereof of Example 7, selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and any combination thereof.

[0028] Example 9. The monoclonal antibody or antigen-binding portion thereof of any one of Examples 1-8, comprising a heavy chain CDR1, CDR2, and CDR3 and a light chain CDR1, CDR2, and CDR3.

[0029] Example 10. The monoclonal antibody or antigen-binding portion thereof of Example 9, wherein the heavy chain CDR1 comprises SEQ ID NO:11, SEQ ID NO:14, or SEQ ID NO:20.

[0030] Example 11. The monoclonal antibody or antigen-binding portion thereof of Example 9 or 10, wherein the heavy chain CDR2 comprises SEQ ID NO:12, SEQ ID NO:15, or SEQ ID NO:21.

[0031] Example 12. The monoclonal antibody or antigen-binding portion thereof of any one of Examples 9 to 11, wherein the heavy chain CDR3 comprises SEQ ID NO:13, SEQ ID NO:16, or SEQ ID NO:22.

[0032] Example 13. The monoclonal antibody or antigen-binding portion thereof of any one of Examples 9 to 12, wherein the light chain CDR1 comprises SEQ ID NO:23, SEQ ID NO:26, or SEQ ID NO:32.

[0033] Example 14. The monoclonal antibody or antigen-binding portion thereof of any one of Examples 9 to 13, wherein the light chain CDR2 comprises SEQ ID NO:24, SEQ ID NO:27, or SEQ ID NO:33.

[0034] Example 15. The monoclonal antibody or antigen-binding portion thereof of any one of Examples 9 to 14, wherein the light chain CDR3 comprises SEQ ID NO:25, SEQ ID NO:28, or SEQ ID NO:34.

[0035] Example 16. A monoclonal antibody or antigen-binding portion thereof according to any one of Examples 1 to 15, comprising (i) a heavy chain variable domain comprising SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:38, and (ii) a light chain variable domain comprising SEQ ID NO:39, SEQ ID NO:40, or SEQ ID NO:42.

[0036] Example 17. A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is 17. The monoclonal antibody or antigen-binding portion thereof of any one of Examples 1-16, comprising 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 presented as SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:38.

[0037] Example 18. A heavy chain variable region and a light chain variable region, wherein the light chain variable region is 17. The monoclonal antibody or antigen-binding portion thereof of any one of Examples 1-17, comprising 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:39, SEQ ID NO:40, or SEQ ID NO:42.

[0038] Example 19. The monoclonal antibody of any one of Examples 1 to 18, wherein the monoclonal antibody is a single domain antibody.

[0039] Example 20. The monoclonal antibody or antigen-binding portion thereof of any one of Examples 1 to 19, wherein the monoclonal antibody is selected from the group consisting of IgG1, IgG2, IgG3, IgG4, variants thereof, or combinations thereof.

[0040] Example 21. The monoclonal antibody or antigen-binding portion thereof of Example 20, wherein the monoclonal antibody is a human IgG1 antibody.

[0041] Example 22. The monoclonal antibody or antigen-binding portion thereof of Example 20, wherein the monoclonal antibody comprises a human IgG2 or human IgG4 isotype antibody.

[0042] Example 23. The monoclonal antibody or antigen-binding portion thereof of any one of Examples 1 to 22, further comprising a constant region that lacks Fc function.

[0043] Example 24. A monoclonal antibody or an antigen-binding portion thereof according to any one of Examples 1 to 23, which is a chimeric antibody, a human antibody, or a humanized antibody.

[0044] Example 25. A monoclonal antibody comprising: (i) SEQ ID NO:57; The monoclonal antibody of any one of embodiments 1-24, comprising (ii) a heavy chain comprising SEQ ID NO:58, or SEQ ID NO:60; and (ii) a light chain comprising SEQ ID NO:61, SEQ ID NO:62, or SEQ ID NO:64.

[0045] Example 26. The antigen-binding portion of any one of Examples 1-25, wherein the antigen-binding portion is a Fab, Fab', F(ab')2, Fv, or single chain Fv (scFv).

[0046] Example 27. A bispecific molecule comprising the monoclonal antibody, or antigen-binding portion thereof, of any one of Examples 1-26 linked to a molecule having a second binding moiety.

[0047] Example 28. A human family, member A5 (FAM19A5) epitope having sequence homology 19 essentially consisting of or consisting 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:5, 6, 9, or 10, said epitope comprising (i) a heavy chain variable domain comprising SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:38, and (ii) a heavy chain variable domain comprising SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, An epitope capable of specifically binding to a reference antibody comprising a light chain variable domain comprising SEQ ID NO:39, SEQ ID NO:40, or SEQ ID NO:42.

[0048] Example 29. A nucleic acid encoding the monoclonal antibody or antigen-binding portion thereof of any one of Examples 1-26, the bispecific molecule of Example 27, or the epitope of Example 28.

[0049] Example 30. A vector comprising the nucleic acid of Example 29.

[0050] Example 31. The vector of Example 30 for use in gene therapy.

[0051] Example 32. A cell transformed with the expression vector of Example 30.

[0052] Example 33. An immunoconjugate comprising the monoclonal antibody or antigen-binding portion thereof of any one of Examples 1 to 26 or the bispecific molecule of Example 27, linked to an agent.

[0053] Example 34. A composition comprising (i) the monoclonal antibody or antigen-binding portion thereof of any one of Examples 1-26, or the immunoconjugate of Example 33, and (ii) a carrier.

[0054] Example 35. A kit comprising (i) the monoclonal antibody or antigen-binding portion thereof of any one of Examples 1-26, or the bispecific molecule of Example 27, and (ii) instructions for use.

[0055] Example 36. A method for producing an anti-FAM19A5 antibody, or an antigen-binding portion thereof, comprising immunizing a non-human animal with the epitope of Example 28, and producing the antibody, or an antigen-binding portion thereof.

[0056] Example 37. A method for producing an anti-FAM19A5 antibody or antigen-binding portion thereof, comprising culturing the cell of Example 32 under suitable conditions, and isolating the antibody or antigen-binding portion thereof.

[0057] Example 38. A method for reducing, reversing, and / or preventing fibrosis in a subject in need of treatment comprising administering a monoclonal antibody or antigen-binding portion thereof of any one of Examples 1 to 26, a bispecific molecule of Example 27, or an immunoconjugate of Example 33, such that the onset of fibrosis is delayed.

[0058] Example 39. A method for reducing excess extracellular matrix (ECM) formation in a subject in need of treatment, comprising administering to the subject a monoclonal antibody or antigen-binding portion thereof of any one of Examples 1 to 26, a bispecific molecule of Example 27, or an immunoconjugate of Example 33, wherein the formation of excess ECM is reduced.

[0059] Example 40. A method for preventing, ameliorating, or treating liver cancer, lung cancer, kidney cancer, breast cancer, and / or pancreatic cancer in a subject in need of such treatment, comprising administering to the subject a monoclonal antibody or antigen-binding portion thereof of any one of Examples 1 to 26, a bispecific molecule of Example 27, or an immunoconjugate of Example 33.

[0060] Example 41. The method of Example 40, wherein the liver cancer is hepatocellular carcinoma.

[0061] Example 42. A method for preventing, ameliorating, or treating liver cancer, lung cancer, kidney cancer, breast cancer, and / or pancreatic cancer associated with fibrosis in a subject in need of such treatment, comprising administering to the subject a monoclonal antibody or antigen-binding portion thereof of any one of Examples 1 to 26, a bispecific molecule of Example 27, or an immunoconjugate of Example 33.

[0062] Example 43. The method of Example 38 or 42, wherein the fibrosis comprises liver fibrosis, pulmonary fibrosis, kidney fibrosis, bone marrow fibrosis, pancreatic fibrosis, skin fibrosis, cardiac fibrosis, arterial fibrosis, arthrofibrosis, breast fibrosis, muscle fibrosis, retroperitoneal fibrosis, thyroid fibrosis, lymph node fibrosis, bladder fibrosis, and / or pleural fibrosis.

[0063] Example 44. The method of any one of Examples 38-43, wherein the monoclonal antibody, or antigen-binding portion thereof, is administered via an intravenous, oral, parenteral, intrathecal, intracerebroventricular, pulmonary, subcutaneous, or intraventricular route.

[0064] Example 45. The method of any one of Examples 38-44, wherein the subject is a human.

[0065] Example 46. A method for measuring the level of FAM19A5 in a sample from a subject in need of treatment, comprising contacting the sample obtained from the subject with any one of the monoclonal antibodies or antigen-binding portions thereof of Examples 1 to 26, the bispecific molecule of Example 27, or the immunoconjugate of Example 33.

[0066] Example 47. The method of Example 46, wherein the level of FAM19A5 is increased in the subject sample compared to the reference sample.

[0067] Example 48. A method for diagnosing fibrosis in a subject in need of diagnosis, comprising contacting a sample obtained from the subject with a monoclonal antibody or antigen-binding portion thereof of any one of Examples 1 to 26, a bispecific molecule of Example 27, or an immunoconjugate of Example 33.

[0068] Example 49. The method of Example 48, wherein the fibrosis is hepatic or pulmonary fibrosis.

[0069] Example 50. The method of example 48 or 49, wherein the level of FAM19A5 is increased in the subject sample compared to the level of FAM19A5 in a reference sample from a subject without fibrosis.

[0070] Example 51. A method for diagnosing a tumor with fibrosis in a subject in need of diagnosis, comprising contacting a sample obtained from the subject with a monoclonal antibody or antigen-binding portion thereof of any one of Examples 1 to 26, a bispecific molecule of Example 27, or an immunoconjugate of Example 33.

[0071] Example 52. The method of Example 51, wherein the level of FAM19A5 is increased in the subject sample compared to the level of FAM19A5 in a reference sample from the tumor-free subject.

[0072] Example 53. The method of any one of Examples 46-52, wherein the sample is blood, serum, plasma, cerebrospinal fluid, tissue biopsy, organ biopsy, or a combination thereof.

[0073] Example 54. The method of any one of Examples 46-52, wherein the sample is a fresh tumor sample, a frozen tumor sample, or a formalin-fixed paraffin-embedded sample.

[0074] Example 55. The method of any one of Examples 46-52, wherein the contacting step includes an analyzing step.

[0075] Example 56. The method of Example 54, wherein the analysis comprises Western blot analysis, slot blot analysis, enzyme-linked immunosorbent assay analysis (ELISA), radioimmunoassay analysis (RIA), flow cytometry-based immunofluorescence analysis (FACS), mass spectroscopy, surface plasmon resonance (SPR), or a combination thereof.

[0076] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the amino acid sequences of epitopes F1-F6 (conjugated to BSA) and their locations on the human FAM19A5 polypeptide. The sizes of these different epitope fragments are indicated in brackets.

[0077] FIG. 2 shows ELISA results for binding of anti-FAM19A5 antibodies 1-65, 2-13, and 3-2. In the case of the 3-2 antibody, two different isotypes are shown, namely human IgG1 ("h3-2") and mouse IgG1 ("m3-2"). For each antibody, the first, second, third, fourth, fifth, and sixth bars (starting from the left) represent binding to epitope fragments F1, F2, F3, F4, F5, and F6, respectively. The bar furthest from the right is the positive control (i.e., FAM19A5 protein with a His tag). The exact OD value is indicated on each bar.

[0078] [Figures 3A-3C] ELISA results for binding of anti-FAM19A5 antibodies 3-2 and 1-28 to 13 different FAM19A5 epitope F2 fragment mutant peptides: (i) F2-01-BSA (#1), (ii) F2-02-BSA (#2), (iii) F2-03-BSA (#3), (iv) F2-04-BSA (#4), (v) F2-05-BSA (#5), (vi) F2-06-BSA (#6), (vii) F2-07-BSA (#7), (vii) F2-09-BSA (#8), (vii) F2-100-BSA (#9), (vii) F2-110-BSA (#10), (vii) F2-120-BSA (#11), (vii) F2-130-BSA (#12), (vii) F2-140-BSA (#13), (vii) F2-150-BSA (#14), (vii) F2-160-BSA (#15), (vii) F2-170-BSA (#16), (vii) F2-180-BSA (#17), (vii) F2-190-BSA (#18), (vii) F2-200-BSA (#19), (vii) F2-210-BSA (#20), (vii) F2-220-BSA (#21), (vii) F2-230-BSA (#22), (vii) F2-240-BSA (#23), (vii) F2-250-BSA (#24), (vii) F2-260-BSA (#25), (vii) F2-270-BSA (#26), (vii) F2-280 -BSA (#5), (vi) F2-06-BSA (#6), (vii) F2-07-BSA (#7), (viii) F2-08-BSA (#8), (ix) F2-09-BSA (#9), (x) F2-10-BSA (#10), (xi) F2-11-BSA (#11), (xii) F2-12-BSA (#12), and (xiii) F2-13-BSA (#13). Figure 3A shows the results for the 3-2 antibody, which has a human IgG1 isotype. Figure 3B shows the results for the 3-2 antibody, which has a mouse IgG1 isotype. Figure 3C shows the results for the 1-28 antibody. The exact OD values ​​are indicated above each bar.

[0079] FIG. 4 shows the amino acid alignment of different members of the FAM19A family (i.e., FAM19A1-5). Regions with the greatest amino acid diversity among the members are boxed and designated EP1-EP8. An IgG antibody ("IgG only") is presented as a control. The y-axis provides the OD value.

[0080] FIG. 5 shows ELISA results for binding of anti-FAM19A5 antibodies 1-65, 1-28, 2-13, and 3-2 to FAM19A5 mutants M1 to M8. For each antibody, eight bars correspond to mutants M1 to M8 (from left to right).

[0081] FIG. 6A shows a diagram of the two-site sandwich ELISA assay used to assess cross-competition between different anti-FAM19A5 antibodies.

[0082] FIG. 6B shows the results of cross-competition analysis for six different anti-FAM19A5 antibodies, namely 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 indicate cross-competition (i.e., S / N ratio of 2 or less).

[0083] 7A and 7B show ELISA results for the binding of several anti-FAM19A5 antibodies to FAM19A5. Results for the following antibodies are displayed: 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). FIG. 7A shows the results for various concentrations of anti-FAM19A5 antibodies in a bar graph. FIG. 7B shows the Kd (nM) of the different anti-FAM19A5 antibodies.

[0084] [Figure 8] Rats with hepatic fibrosis induced by bile duct ligation (BDL) (n=5) ( The serum FAM19A5 protein levels in the “disease model” were compared with healthy rats (the “normal” ) (n=3). FAM19A5 protein levels are expressed as fold change relative to levels observed in normal control animals. "*" indicates a statistically significant difference (p<0.005) compared to normal control animals.

[0085] FIG. 9 shows FAM19A5 protein levels in serum of rats (n=5) with idiopathic pulmonary fibrosis induced by intratracheal injection of 3 mg / kg bleomycin ("Disease model") compared to healthy rats ("Normal") (n=5). FAM19A5 protein levels are expressed as fold change compared to levels observed in normal control animals. "*" indicates a statistically significant difference (p<0.005) compared to normal control animals.

[0086] FIG. 10 shows the fold change in FAM19A5 protein levels in serum from human patients with confirmed liver fibrosis, e.g., cirrhosis (Patient #1-10) and healthy individuals (Normal #1-3). The specific values ​​presented on each bar graph represent the fold change relative to the average concentration of FAM19A5 protein detected in serum from healthy individuals.

[0087] FIG. 11 shows a comparison of H&E (hematoxylin and eosin) staining of left ventricular tissue in myocardial infarction-induced animals treated with control antibody ("NHI") or anti-FAM19A5 antibodies ("1-65", "3-2", and "1-28"). Healthy animals (i.e., non-myocardial infarction-induced, "naive") served as an additional control group.

[0088] Figures 12A and 12B show a comparison of collagen accumulation in left ventricular tissues in myocardial infarction-induced animals. Myocardial infarction-induced animals were administered either a control antibody ("NHI") or one of the anti-FAM19A5 antibodies ("1-65", "3-2", and "1-28"). Healthy animals (i.e., non-infarction-induced, "naive") served as an additional control group. Figure 12A shows collagen accumulation using Masson's trichrome staining. Figure 12B shows collagen accumulation (i.e., fibrotic area) as a ratio of fibrotic area to total left ventricular area. Data are presented as mean ± SD (n = 5-8 per group). "*** / **" on the bars indicates a statistically significant difference compared to the "naive" group (healthy animals) (p < 0.001, p < 0.05, respectively). The "#" on the bar indicates a statistically significant difference compared to the "NHI" group (inducing myocardial infarction + control antibody) (p<0.05).

[0089] FIG. 13 shows immunohistochemical analysis of FAM19A5 protein expression in liver biopsies from three different patients with liver cancer. As shown, each patient has various degrees of fibrosis: (i) stage #0 (left column), (ii) stage #2 (middle column), and (iii) stage #4 (right column). The lower row is a larger enlargement of the boxed area in the upper row. Arrows indicate examples of FAM19A5-positive hepatic stellate cells.

[0090] FIG. 14A shows the weight (g) of animals as a function of time (weeks post-inoculation). Some animals were inoculated with Hep3B cells only and treated with either normal human immunoglobulin (circles, group: “Hep3B+NHI”, n=3) or anti-FAM19A5 antibody (closed box, group: “Hep3B+FAM19A5 Ab”, n=3). Other animals were inoculated with both Hep3B cells and human hepatic stellate cells (HHSteC) and treated with either normal human immunoglobulin (diamonds, group: “Hep3B+HHSteC+NHI”, n=3) or anti-FAM19A5 antibody (open box, group: “Hep3B+HHSteC+FAM19A5 Ab”, n=3). Patients were treated with one of the following treatments: 1) sham anti-inflammatory drug (Ab”, n=3); data are presented as mean ± SD.

[0091] FIG. 14B shows the mean tumor volume observed in the different groups of animals as a function of time (weeks post-inoculation). The groups shown are the same as those described in FIG. 14A. Tumor volume was calculated by the formula: 0.5×length×width. 2 = Tumor volume (mm 3 ). Data are presented as mean ± SD.

[0092] [Figure 14C and Figure 14D] Photographed images and weights (g), respectively, of tumors isolated from the animals described in Figure 14A on day 42 post-inoculation. In Figure 14C, (i) upper left side, tumors from the "Hep3B+NHI" group (n=2); (ii) upper right side, tumors from the "Hep3B+HHSteC+NHI" group (n=2); (iii) lower left side, tumors from the "Hep3B+FAM19A5 Ab" group (n=3); (iv) lower right side, tumors from the "Hep3B+HHSteC+FAM19A5 Ab" group (n=3). Figure 14D shows the average weights (g) of tumors isolated from the different groups.

[0093] FIG. 15A and FIG. 15B show the effect of anti-FAM19A5 antibodies on reactive gliosis after traumatic brain injury. FIG. 15A provides representative immunohistochemical images of damaged areas of brain tissue of animals treated with different anti-FAM19A5 antibodies: (i) 1-65 (2 / 4) (row 1); (ii) 1-28 (row 2); (iii) 2-13 (row 3); and (iv) 3-2 (row 4). Brain tissue sections were stained for GFAP (glial fibrillary acidic protein, green) and nestin (red), which are known to be induced in reactive astrocytes after brain injury. The dotted line (white) indicates the lesion border after exposure to TBI. FIG. 15B provides the average distance of GFAP- and / or nestin-positive astrocytes from the center of the TBI lesion in animals treated with 1-65, 1-28, 2-13, and 3-2 anti-FAM19A5 antibodies.

[0094] FIG. 16A provides a diagram depicting antibody dosing schedule and intracranial injection of glioblastoma cancer cells into mice.

[0095] Figure 16B shows the anti-cancer effect of anti-FAM19A5 antibody in an animal model of glioblastoma evaluated using CLARITY, which evaluates the transparency of tissue samples. Opaque tumor areas in brains harvested from animals treated with either a human IgG control antibody (left) or anti-FAM19A5 antibody (right) are indicated by dotted lines.

[0096] FIG. 17A and FIG. 17B show the anti-cancer effect of anti-FAM19A5 antibody in glioblastoma animal models evaluated using hematoxylin and eosin (H&E) staining. FIG. 17A provides four representative H&E stained images of brain tissue sections from animals treated with either a control human IgG antibody (upper row) or anti-FAM19A5 antibody (lower row). Glioblastomas correspond to the dark areas in this image. FIG. 17B quantifies the data presented in FIG. 17A by providing the data as a percentage of the control group. Black bars correspond to the control group (treated with human IgG antibody). Grey bars correspond to the group treated with anti-FAM19A5 antibody. The numbers on the x-axis correspond to the representative images shown in FIG. 17A.

[0097] 18A-18D show the anti-tumor effect of anti-FAM19A5 antibody in a glioblastoma animal model evaluated using Hoechst nuclear staining. FIG. 18A shows images of four representative brain tissue sections from animals treated with a control human IgG antibody (upper row) or anti-FAM19A5 antibody (lower row). In each image, the light gray area corresponds to the tumor. FIG. 18B provides a table showing values ​​for the number of cells (Hoeschst positive staining) within the tumor ("number of spots") and tumor volume ("volume") measured in the four representative brain tissue sections in FIG. 18A. FIG. 18C (cell number) and 18D (tumor volume) are graphical representations of the data presented in FIG. 18B. In FIG. 18C and FIG. 18D, data are expressed as % of the corresponding values ​​observed in control animals. Numbers on the x-axis correspond to the representative brain tissue sections in FIG. 18A. Black bars correspond to animals treated with a control human IgG antibody. Grey bars correspond to animals treated with anti-FAM19A5 antibody.

[0098] Figures 19A and 19B show the effect of anti-FAM19A5 antibody on vascular normalization in a mouse model of glioblastoma. Figure 19A provides immunohistochemical analysis of CD31 expression (a vascular marker) in similar regions of brains isolated from animals treated with a control human IgG antibody (left image) or anti-FAM19A5 antibody (right image). In Figure 19A, the white dotted line indicates the outer border of the glioblastoma. Figure 19B provides an enlargement of a representative region of Figure 19A.

[0099] [Figures 20A and 20B] The effect of anti-FAM19A5 antibody on macrophage infiltration into tumors in a mouse model of glioblastoma. Figure 20A provides immunohistochemistry images of Ibal expression (macrophage marker) in glioblastomas of mice treated with a control human IgG antibody (upper image) or anti-FAM19A5 antibody (lower image). Figure 20B compares the volume of macrophages observed in the images shown in Figure 20A.

[0100] FIG. 21 shows MRI scan images of the brains of rat glioma models treated with either a control human IgG antibody (upper row) or anti-FAM19A5 antibody (lower row). These images show various characteristics of the tumors, including: (i) T2-morphological analysis; (ii) R2-fluid content; (iii) CBV-cerebral vascular volume; (iv) EnhRate and IAUC30 (initial area under the curve at 30 seconds)-tissue permeability. In these images, tumors are marked with black dots.

[0101] Figure 22 shows the effect of anti-FAM19A5 antibody on survival in a mouse brain cancer model. Black circles indicate animals treated with a human IgG control antibody. White circles indicate animals treated with anti-FAM19A5 antibody.

[0102] [Mode for carrying out the invention] Disclosed herein is an isolated monoclonal antibody, or antigen-binding portion thereof, that specifically binds to human family member A5 having sequence homology 19 and exhibits one or more of the properties disclosed herein, such as reducing, reversing, slowing, and / or preventing fibrosis, reducing excessive extracellular matrix (ECM) formation, slowing tumor growth or progression, binding to soluble human FAM19A5 with a KD of 10 nM or less as measured by enzyme-linked immunosorbent assay (ELISA), binding to membrane-bound human FAM19A5 with a KD of 10 nM or less as measured by ELISA, reducing, reversing, slowing, and / or preventing the onset of reactive gliosis, inhibiting excessive proliferation of reactive astrocytes, reducing expression of chondroitin sulfate proteoglycans including neurocan and neuron-glial antigen 2 (NG2), increasing expression of c-fos and pERK in neuronal nuclei, promoting neuronal survival, increasing expression of GAP43 in neurons, and promoting axonal regrowth.

[0103] To facilitate understanding of the present disclosure, a number of terms and phrases are defined. Additional definitions are provided throughout the detailed description.

[0104] I. Definition Throughout the present invention, the term "a" or "an" entity refers to 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" can be used interchangeably herein.

[0105] Also, "and / or", when used herein, must be interpreted as meaning that each of the two specified features or components is specifically disclosed in conjunction with or alone the remaining one. Thus, the term "and / or" used in a phrase such as "A and / or B" is intended to include "A and B", "A or B", "A" (single), and "B" (single). Similarly, the term "and / or" used in a phrase such as "A, B and / or C" is intended to include the 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 (single); B (single); and C (single), respectively.

[0106] It is understood that when an aspect is presented with "comprising," other similar aspects described in terms of "consisting of" and / or "consisting essentially of" are also provided.

[0107] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. For example, see the literature (the 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 Molecular Biology (Revised, 2000, Oxford University Press) provide one of skill in the art with common dictionary meanings for most of the terms used in the present invention.

[0108] Units, prefixes, and symbols are expressed in the form accepted by the Systeme International de Unites (SI). Numerical ranges include the numbers that limit the range inclusively. Unless otherwise indicated, amino acid sequences are written left to right in the amino to carboxy orientation. The headings provided herein are not limitations of the various aspects of the invention, which may be read in their entirety by reference to the specification. Thus, the terms defined below are more fully defined by reference to the specification in its entirety.

[0109] The term "about" is used herein to mean approximately, roughly, around, or within a certain range. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values ​​set forth. In general, the term "about" can modify a numerical value above or below the stated value, for example, by up to a variance of 10 percent (up or down).

[0110] The term "family with sequence homology 19, member A5" or "FAM19A5" refers to a protein that belongs to the TAFA family of five highly homologous proteins (also known as the FAM19 family) that are primarily expressed in the brain and spinal cord. FAM19A5 is also known as TAFA5 or chemokine-like protein TAFA-5.

[0111] In humans, the gene encoding FAM19A5 is located on chromosome 22. There are three human FAM19A5 (UniProt:Q7Z5A7) isoforms, which appear to be generated by alternative splicing: isoform 1 (UniProt:Q7Z5A7-1), which consists of 132 amino acids; isoform 2 (UniProt:Q7Z5A7-2), which consists of 125 amino acids; and isoform 3 (UniProt:Q7Z5A7-3), which consists of 53 amino acids. Human FAM19A5 protein appears to exist as a membrane-bound and a soluble (secreted) form. Isoform 1 appears to be a membrane protein with one transmembrane domain. Isoform 2 has been reported in the literature (Tang TY et al., Genomics 83(4):727-34 (2004)) as a secreted protein (soluble) and contains a signal peptide at amino acid positions 1-25. Isoform 3 is predicted based on EST data. Below are the amino acid sequences of the three known human FAM19A5 isoforms.

[0112] (I) Isoform 1 (UniProt: Q7Z5A7-1, a transmembrane protein): This isoform was chosen as the standard sequence.

[0113] [ka]

[0114] (II) Isoform 2 (UniProt: Q7Z5A7-2, soluble protein):

[0115] [ka]

[0116] (III) Isoform 3 (UniProt:Q7Z5A7-3):

[0117] [ka]

[0118] The term "FAM19A5" includes any variant or isoform of FAM19A5 that is naturally expressed by cells. Thus, the antibodies disclosed herein may cross-react with different isoforms of the same species (e.g., different isoforms of human FAM19A5) or may cross-react with FAM19A5 of a species other than human (e.g., mouse FAM19A5). Alternatively, the antibodies may be specific for human FAM19A5 and may not exhibit cross-reactivity with other species. FAM19A5, or any variants and isoforms thereof, may be isolated from cells or tissues that naturally express them or produced recombinantly. A polynucleotide encoding human FAM19A5 has GenBank Accession No. BC039396 and has the following sequence:

[0119] [Table 1A]

[0120] The terms "antibody" and "antibodies" are terms of the art and may be used interchangeably herein to refer to a molecule having an antigen-binding site that specifically binds to an antigen. As used herein, the term includes all antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") or single chains thereof. In one embodiment, "antibody" refers to a glycoprotein comprising at least two heavy (H) and two light (L) chains inter-connected by disulfide bonds, or an antigen-binding portion thereof. In another embodiment, "antibody" refers to a single chain antibody comprising a single variable region, e.g., a VHH domain. Each heavy chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. In certain naturally occurring antibodies, the heavy chain constant region comprises three domains, CH1, CH2 and CH3. In certain naturally occurring antibodies, each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one domain, CL.

[0121] The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from 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 can 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 (C1q) of the canonical complement system.

[0122] The term "Kabat numbering" and similar terms are art-recognized and refer to a system for numbering amino acid residues in the heavy and light chain variable regions of an antibody, or an antigen-binding portion thereof. In certain embodiments, the CDRs of an antibody can 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, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system, within an antibody heavy chain molecule, the CDRs are typically located at amino acid positions 31-35 (which may optionally include one or two additional amino acids after 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, within an antibody light chain molecule, the CDRs are typically located at amino acid positions 24-34 (CDR1), amino acid positions 50-56 (CDR2), and amino acid positions 89-97 (CDR3). In certain embodiments, the CDRs of the antibodies described in the present invention have been determined according to the Kabat numbering system.

[0123] The phrases "amino acid position numbering according to Kabat", "Kabat position" and grammatical variations thereof refer to the numbering system used in the literature (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) for the heavy or light chain variable regions of antibody sequences. With this numbering system, the actual linear amino acid sequence can contain fewer or additional amino acids. This corresponds to a shortening of the FW or CDR of the variable region, or an insertion into the FW or CDR. For example, the heavy chain variable region can contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and an inserted residue after heavy chain FW residue 82 (e.g., residues 82a, 82b, and 82c, etc., according to Kabat) (see Table 1b).

[0124] [Table 1B]

[0125] Residual Kabat numbering can be determined for a particular antibody by alignment of the homology regions of the antibody sequence with the "standard" Kabat numbered sequence. Chothia refers instead to the location of the structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The ends of the Chothia CDR-H1 loop, when numbered using the Kabat numbering convention, range from H32 to H34, depending on the length of the loop (this is because the Kabat numbering system places the insertion at H35A and H35B; if neither 35A nor 35B is present, 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 a compromise between the Kabat CDRs and the Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software.

[0126] IVIGT (ImMunoGeneTics) 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 IVIGT 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 IVIGT 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.

[0127] For all heavy chain constant region amino acid positions discussed in this invention, the numbering is according to the EU index first described in the literature (Edelman et al., 1969, Proc. Natl. Acad. Set USA 63(1):78-85), which described the amino acid sequence of the myeloma protein EU, the first human IgG1 to be sequenced. The EU index of Edelman et al. is also presented in the literature (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 grammatical variations thereof refer to the residual numbering system based on the human IgG1 EU antibody of Edelman et al. as presented in Kabat 1991.

[0128] The numbering system used for the variable region (both heavy and light chain) and light chain constant region amino acid sequences is that presented in Kabat 1991.

[0129] 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 as several allotypes, which differ from each other by at most a few amino acids. The antibodies disclosed in the present invention can be derived from any of the commonly known isotypes, classes, subclasses, or allotypes. In certain embodiments, the antibodies disclosed in the present invention are of the IgG1, IgG2, IgG3, or IgG4 subclass, or any hybrid thereof. In certain embodiments, the antibodies are of the human IgG1 subclass or human IgG2 or human IgG4 subclass.

[0130] "Antibody" includes, by way of example, naturally occurring and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human and non-human antibodies, fully synthetic antibodies; single chain antibodies; monospecific antibodies; multispecific antibodies (including bispecific antibodies); tetrameric antibodies comprising two heavy chain and two light chain molecules; antibody light chain monomers; antibody heavy chain monomers; antibody light chain dimers; antibody heavy chain dimers; antibody light chain-antibody heavy chain pairs; intrabodies; heteroconjugate antibodies; monovalent antibodies; camelized antibodies; affibodies; anti-idiotypic (anti-Id) antibodies (including, e.g., anti-anti-Id antibodies), and single domain antibodies (sdAbs), which include binding molecules consisting of a single monomeric variable antibody domain (e.g., a VH domain or a VL domain) sufficient to bind an antigen (see Harmen MM and Haard HJ Appl Microbiol Biotechnol. 77(1): 13-22 (2007)).

[0131] The term "antigen-binding portion" of an antibody as used herein refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., human FAM19A5). Such "fragments" are, for example, about 8 to about 1500 amino acids in length, suitably about 8 to about 745 amino acids in length, more suitably about 8 to about 300, e.g., about 8 to about 200 amino acids, or about 10 to about 50 or 100 amino acids in length. It has been revealed that the antigen-binding function of an antibody can be 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 described in the present invention, include (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, and a disulfide-linked Fvs (sdFv); (v) a dAb fragment consisting of the VH domain (Ward et al. (a119.8, 9) Nature 341:544- 546); and (vi) an isolated complementarity determining region (CDR) or (vii) a combination of two or more isolated CDRs, which can be selectively linked by a synthetic linker. Alternatively, the two domains of the Fv fragment, VL and VH, can be encoded by separate genes but linked using recombinant methods by a synthetic linker, such that the VL and VH domains pair to form a single protein chain that forms a monovalent molecule (referred to as single-chain Fv (scFv)) (see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also encompassed within the term "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 usefully screened 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.

[0132] As used herein, the terms "variable region" and "variable domain" are commonly used interchangeably in the art. 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 a mature heavy chain and about 90-115 amino acids in a mature light chain, which vary widely in sequence from antibody to antibody and are used to determine the binding and specificity of a particular antibody to a particular antigen. Sequence variability is concentrated in regions called complementarity determining regions (CDRs), and the more highly conserved regions of a variable region are called framework regions (FRs).

[0133] The CDRs of the light and heavy chains are believed to be primarily responsible for antigen-antibody interaction and specificity. In certain embodiments, the variable regions are human variable regions. In certain embodiments, the variable regions comprise rodent or murine CDRs and human framework regions (FRs). In certain embodiments, the variable regions are primate (e.g., non-human primate) variable regions. In certain embodiments, the variable regions comprise rodent or murine CDRs and primate (e.g., non-human primate) framework regions (FRs).

[0134] As used herein, the term "heavy chain", when used in reference to an antibody, can refer to any of the different types, such as alpha (α), delta (δ), epsilon (ε), gamma (γ) and mu (μ), which give rise to the IgA, IgD, IgE, IgG and IgM classes of antibodies, based on the amino acid sequence of the constant domains, including subclasses of IgG, such as IgG1, IgG2, IgG3 and IgG4, respectively.

[0135] As used herein, the term "light chain" when used in relation to an antibody can refer to any of the different types, such as kappa (κ) and lambda (λ), based on the amino acid sequence of the constant domain. Light chain amino acid sequences are well known in the art. In a particular embodiment, the light chain is a human light chain.

[0136] The terms "VL" and "VL domain" are used interchangeably to refer to the light chain variable region of an antibody.

[0137] The terms "VH" and "VH domain" are used interchangeably to refer to the variable region of an antibody heavy chain.

[0138] As used herein, the terms "constant region" or "constant domain" are interchangeable and have their usual meaning in the art. A constant domain is a portion of an antibody, e.g., the carboxy-terminal portion of the light and / or heavy chain, that is not directly involved in binding an antibody to an antigen but can exhibit various effector functions, such as interaction with Fc receptors. The constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence than the immunoglobulin variable region.

[0139] "Fc region" (fragment crystallizable region) or "Fc domain" or "Fc" refers to the C-terminal region of an antibody heavy chain which mediates the binding of 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 (C1q) of the canonical complement system. Thus, the Fc region includes the constant regions of an antibody excluding the first constant region immunoglobulin domain (e.g., CH1 or CL). In IgG, IgA, and IgD antibody isotypes, the Fc region 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 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 vary, the human IgG heavy chain Fc region is generally defined as extending 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 of Kabat. The CH2 domain of the human IgG Fc region extends from about amino acid 231 to about amino acid 340, and the CH3 domain is located C-terminal to the Cm domain in the Fc region. It extends from about amino acid 341 to about amino acid 447 of IgG. An Fc region as used herein can be a native sequence Fc, including any allotype variant, or a variant Fc (e.g., a non-naturally occurring Fc). Fc may also refer to an isolated region or to the region associated with a protein polypeptide comprising an Fc, such as an "Fc region-containing binding protein", also referred to as an "Fc fusion protein" (e.g., an antibody or immunoadhesion).

[0140] 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 Fe (e.g., Jefferis et al. (2009) mAbs 1:1; Vidarsson et al. (2010 ... G. et al. Front Immunol. 5:520 (published online October 20, 2014).

[0141] 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 Fcγ family, including allelic variants and alternatively spliced ​​forms of these receptors. The Fcγ family consists of three activating receptors (FcγRI, FcγRIII, and FcγRIV in mice; FcγRIA, FcγRIIA, and FcγRIIIA in humans) and one inhibitory receptor (FcγRIIB). Human IgG1 binds the majority of human Fc receptors and elicits the strongest Fc effector functions. It is considered equivalent to murine IgG2a in terms of the types of activating Fc receptors it binds. Human IgG4, on the other hand, elicits minimal Fc effector functions (see Vidarsson G. et al. Front Immunol. 5:520 (Published online October 20, 2014)).

[0142] The constant region can be engineered, e.g., by recombinant techniques, to eliminate one or more effector functions. "Effector function" refers to the interaction of an antibody Fc region with an Fc receptor or ligand, or the biochemical events resulting therefrom. Exemplary "effector functions" include C1q binding, complement dependent cytotoxicity (CDC), Fc receptor binding, FcγR-mediated effector functions, such as ADCC and antibody-dependent cell-mediated phagocytosis (ADCP), and down-regulation of cell surface receptors (e.g., B cell receptor; BCR). Such effector functions generally require that the Fc region be engineered with a binding domain (e.g., an antibody variable region). Thus, the term "constant region lacking an Fc function" includes a constant region in which one or more effector functions mediated by the Fc region are reduced or eliminated.

[0143] The effector functions of antibodies can be reduced or avoided by different approaches. The effector functions of antibodies can be reduced or avoided by using antibody fragments lacking the Fc region (e.g., Fab, F(ab')2, single chain Fv (scFv), or sdAbs consisting of monomeric VH or VL domains). Alternatively, so-called aglycosylated antibodies can be generated by removing sugars linked to specific residues in the Fc region to reduce the effector functions of antibodies while retaining other valuable attributes of the Fc region (e.g., extended half-life and heterodimerization). Aglycosylated antibodies can be generated, for example, by deleting or modifying the residues to which the sugars are attached and enzymatically removing the sugars, 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, e.g., U.S. Patent Publication No. 20120100140). Another approach uses Fc regions from IgG subclasses with reduced effector functions. For example, IgG2 and IgG4 antibodies are characterized by having lower levels of Fc effector functions than IgG1 and IgG3. The remainder of the CH2 domain of the Fc portion closest to the hinge region is responsible for the effector functions of the antibody and contains highly overlapping binding sites for C1q (complement) and the IgG-Fc receptor (FcγR) on effector cells of the innate immune system (see Vidarsson G. et al. Front Immunol. 5:520 (Published online October 20, 2014)). Thus, antibodies with or without reduced Fc effector function can be produced, 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, e.g., Lau C. et al. J. Immunol. 191:4769-4777 (2013)), or an Fc region with altered Fc effector function, e.g., a mutation that reduces or reduces Fc function. Such Fc regions with mutations are known in the art.See, for example, U.S. Patent Publication No. 20120100140 and the U.S. and PCT applications and references cited therein (An et al. mAbs 1:6, 572- 579 (2009)), the contents of which are incorporated by reference in their entireties.

[0144] "Hinge", "hinge domain", "hinge region" or "antibody hinge region" are used interchangeably to refer to the domain of the heavy chain constant region that connects the CH1 domain to the CH2 domain and includes the upper, middle, and lower portions of the hinge (see 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 bonds between the two heavy chain constant regions. The hinge used in the present invention begins at Glu216 and ends at Gly237 for all IgG isotypes (see Roux et al 19., 98 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, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242; Vidarsson G. et al. Front Immunol. 5:520 (Published online October 20, 2014)).

[0145] The term "CH1 domain" refers to a heavy chain constant domain that connects the variable region to the hinge. The CH1 domain used in the present invention 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 (including wild-type and allotype) CH1 domain sequences are known in the art. See, for example, (Kabat EA et a (1991) supra and Vidarsson G. et al. Front Immunol. 5:520 (published online October 20, 2014)). Exemplary CH1 domains include CH1 domains with mutations that alter the biological activity, e.g., half-life, of the antibody, as disclosed, for example, in U.S. Patent Publication No. 20120100140 and the U.S. patents and applications and PCT applications cited therein.

[0146] The term "CH2 domain" refers to a heavy chain constant domain, a region of the heavy chain that connects the hinge and the CH3 domain. The CH2 domain used in the present invention 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). IgG1, IgG2, IgG3, and IgG4 CH2 domain sequences (including wild-type and allotypes) 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 October 20, 2014)). Exemplary CH2 domains include CH2 domains with mutations that alter the biological activity of the antibody, e.g., half-life and / or reduced Fc effector function, as disclosed, for example, in U.S. Patent Publication No. 20120100140 and the U.S. patents and applications and PCT applications cited therein.

[0147] The term "CH3 domain" refers to a heavy chain constant domain and a heavy chain constant region that is C-terminal to the CH2 domain. The CH3 domain as used 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 (including wild-type and allotype) CH3 domain sequences 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 October 20, 2014). Exemplary CH3 domains include CH3 domains with mutations that alter the biological activity, e.g., half-life, of the antibody, as disclosed, for example, in U.S. Patent Publication No. 20120100140 and the U.S. patents and applications and PCT applications cited therein.

[0148] As used herein, "isotype" refers to the antibody class (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibodies) encoded by heavy chain constant region genes.

[0149] "Allotype" refers to naturally occurring variants within a particular isotype group that differ in a small number of amino acids (see, e.g., Jefferis et al. (2009) mAbs 1:1). The antibodies described in the present invention can have any allotype. 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 October 20, 2014); and Lefranc MPd, mAbs 1:4, 1- 7 (2009)).

[0150] 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."

[0151] As used herein, an "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to FAM19A5 is substantially free of antibodies that specifically bind to antigens other than FAM19A5). However, an isolated antibody that specifically binds to an epitope of FAM19A5 may have cross-reactivity to other FAM19A5 proteins from different species.

[0152] "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" as used 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 displayed in a number of ways known in the art. D 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 means, for example, the binding rate constant of an antibody to an antigen, and k off k means, for example, the dissociation rate constant of an antibody against an antigen. on and k off can be determined by techniques known to those skilled in the art, such as immunoassays (eg, enzyme-linked immunosorbent (ELISA)), BIACORE®, or equilibrium exclusion assays (KINEXA®).

[0153] As used herein, the terms "specifically bind," "specifically recognize," "specific binding," "selective binding," and "selectively bind" are similar terms in the context of antibodies and are terms that relate to molecules (e.g., antibodies) that bind to an antigen (e.g., 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 generally bind to other peptides or polypeptides with a lower affinity as determined, for example, by immunoassay, BIACORE®, KINEXA® 3000 instrument (Sapidyne Instruments, Boise, India), or other assays known in the art. In certain embodiments, a molecule that specifically binds to an antigen is one that is capable of binding to other antigens with a K A K at least 2 logs, 2.5 logs, 3 logs, 4 logs or more greater than A and binds to the antigen.

[0154] Antibodies are typically 10 -5 ~10 -11 Specific binding to cognate antigens with high affinity, reflected by a dissociation constant of approximately 10 -4 K exceeds M D As used herein, an antibody that "specifically binds" to an antigen refers to an antibody that binds to the antigen with high affinity and substantially the same antigen, for example, in an immunoassay (e.g., ELISA) or BIACORE with a defined antigen. TM 10 as determined by surface plasmon resonance (SPR) technology on a 2000 instrument -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 This means that the antibody has a high affinity for binding to unrelated antigens.

[0155] The term "antigen" as used herein means any natural or synthetic immunogenic substance, such as a protein, peptide or hapten. The antigen may be FAM19A5 or a fragment thereof.

[0156] "Epitope" as used herein is a term of the art and refers to a localized region of an antigen to which an antibody can specifically bind. An epitope can be, for example, contiguous amino acids of a polypeptide (linear or continuous epitope), or an epitope can be, for example, a combination of two or more non-contiguous regions of a polypeptide or polypeptides (conformational, non-linear, discontinuous, or discontinuous epitope). Epitopes formed from contiguous amino acids are typically, but not always, retained when exposed to denaturing solvents, while epitopes formed by tertiary folding are typically lost when treated with denaturing solvents. An epitope typically includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20 amino acids in a unique spatial conformation. Methods for determining which epitopes are bound by a particular antibody (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation analysis, in which overlapping or consecutive peptides (e.g., from FMAM19A5) are tested for reactivity with a particular antibody (e.g., anti-FAM19A5 antibody). Methods for determining the spatial conformation of epitopes include those known in the art and described herein, such as X-ray crystallography, two-dimensional nuclear magnetic resonance, and HDX-MS (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996)).

[0157] In certain embodiments, the epitope bound by the antibody can be determined by, for example, NMR spectroscopy, X-ray diffraction crystallography studies, ELISA analysis, hydrogen / deuterium exchange coupled with mass spectroscopy (e.g., liquid chromatography electrospray mass spectrometry), array-based oligopeptide scanning analysis, and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). In the case of X-ray crystallography, crystallization can be accomplished using any of the methods known in the art (see, e.g., Giege R. et al. (1994) Acta Crystallogr D. Biol Crystallogr. 50(Pt 4): 339-350; McPherson A. (1990) Eur. J. Biochem. 189: 1-23; Chayen NE (1997) Structure 5: 1269- 1274; McPherson A. (1976) J. Biol. Chem. 251: 6300-6303). Antibody:antigen crystals can be studied using well-known X-ray diffraction techniques, such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, e.g., Meth. Enzymol. (1985) Volumes 114 & 115, eds Wyckoff HW et al.,; see U.S. Patent Publication No. 2004 / 0014194), BUSTER (see Bricogne G (1993) Acta. Crystallogr. D. Biol. Crystallogr. 49(Pt1): 37-60; Bricogne G (1997) Meth. Enzymol. 276A: 361-423, ed Carter CW; Roversi P. et al., (2000) Acta. Crystallogr. D. Biol. Crystallogr. 56(Pt 10): 1316-1323). Mutagenesis mapping studies can be accomplished using any method known to those of skill in the art. For details of mutagenesis techniques, including alanine scanning mutagenesis, see, for example, Champe M. et al., (1995) J. Biol. Chem. 270: 1388-1394 and Cunningham BC & Wells JA (1989) Science 244: 1081-1085.

[0158] The term "epitope mapping" refers to the process of identifying molecular determinants for antibody-antigen recognition.

[0159] In the context of two or more antibodies, the term "binds to the same epitope" means that the antibodies bind to the same segment of amino acid residues as determined by a particular method. Techniques for determining whether an antibody binds to the "same epitope on FAM19A5" as an antibody described in the present invention include, for example, epitope mapping methods, such as X-ray analysis of crystals of antigen:antibody complexes that provide atomic resolution of the epitope, and hydrogen / deuterium exchange mass spectrometry (HDX-MS). Other methods monitor the binding of antibodies to antigen fragments or mutated variants of the antigen, where loss of binding due to alterations in amino acid residues within the antigen sequence is primarily taken as an indication of epitope components. Also, computational methods for epitope mapping can be used. These methods rely on the ability of the antibody in question to affinity isolate specific short peptides from recombinant phage display peptide libraries. Antibodies with the same VH and VL or the same CDR1, 2 and 3 sequences are expected to bind to the same epitope.

[0160] 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 other antibody to the target. Whether two antibodies compete with each other for binding to a target, i.e., whether and to what extent one antibody inhibits the binding of the other antibody to the target, can be determined by competition experiments as known in the art. In certain embodiments, an antibody competes with another antibody for binding to a target and inhibits this binding by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. The level of inhibition or competition can vary depending on whether the antibody is a "blocking antibody" (i.e., a cold antibody that has been previously incubated with the target). Competitive assays can be performed, for example, as described in Chapter 11 of Ed Harlow and David Lane, Cold Spring Harb Protoc; 2006; doi: 10.1101 / pdb.prot4277 or in Chapter 11 of "Using Antibodies" by 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., by steric hindrance).

[0161] Other competitive binding assays include solid-phase direct or indirect radioimmunoassays (RIA), solid-phase direct or indirect enzyme immunoassays (EIA), sandwich competition assays (see Stahli et al., Methods in Enzymology 9:242 (1983)); solid-phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614 (1986)); solid-phase direct label assays, solid-phase direct label sandwich assays (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid-phase direct label RIA using 1-125 label (see Morel et al., μΙ. Immunol. 25(1):7 (1988)); solid-phase direct biotin-avidin EIA (Cheung et al, Virology 176:546 (1990)); and direct label RIA (Moldenhauer et al, Scand. J. Immunol. 32:77 (1990)).

[0162] 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)).

[0163] As used herein, the term "monoclonal antibody" refers to an antibody or composition of antibodies in which all of the antibodies exhibit a single binding specificity and affinity for a particular epitope. Thus, the term "human monoclonal antibody" refers to an antibody or composition of antibodies that exhibits a single binding specificity and has variable and optional constant regions derived from human germline immunoglobulin sequences. In one embodiment, human monoclonal antibodies are produced by hybridomas comprising B cells obtained from a transgenic non-human animal, e.g., a transgenic mouse whose genome includes human heavy chain and light chain transgenes fused to an immortalized cell.

[0164] The term "recombinant human antibody" as used herein includes all human antibodies that are produced, expressed, produced or isolated by recombinant means, such as (a) antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal to human immunoglobulin genes, or hybridomas produced therefrom, (b) antibodies isolated from host cells transformed to express the antibody, e.g., from transfectomas, (c) antibodies isolated from recombinant, combinatorial human antibody libraries, and (d) antibodies produced, expressed, produced or isolated by any other means involving splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies contain variable and constant regions that are encoded by germline genes but use specific human germline immunoglobulin sequences including subsequent rearrangements and mutations that occur, e.g., during antibody maturation. As known in the art (see, e.g., Lonberg (2005) Nature Biotech. 23(9): 1117-1125), the variable regions comprise the antigen-binding domains encoded by the divergent genes that are rearranged to form antibodies specific to foreign antigens. In addition to rearrangement, the variable regions can be further modified by multiple single amino acid changes (termed somatic mutation or hypermutation) to increase the affinity of the antibody for the foreign antigen. The constant regions will be altered in response to additional antigens (i.e., isotype switching). Thus, the rearranged and somatically mutated nucleic acid molecules that encode light and heavy chain immunoglobulin polypeptides in response to an antigen will not have sequence identity to the original nucleic acid molecule, but will instead be substantially identical or nearly identical (i.e., have at least 80% identity).

[0165] 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 described herein may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term "human antibody" does not include antibodies in which CDR sequences derived from the germline of other mammalian species, such as mouse, have been grafted onto human framework sequences. The terms "human" antibody and "fully human" antibody are used synonymously.

[0166] The term "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 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 human immunoglobulin, while some, most or all of the amino acids within one or more CDR regions remain intact. Amino acid additions, deletions, insertions, substitutions or modifications are permissible so long as they do not eliminate the ability of the antibody to bind a particular antigen. A "humanized" antibody retains antigen specificity similar to that of the original antibody.

[0167] "Chimeric antibody" refers to an antibody in which the variable region derives from one species and the constant region derives from another species, e.g., the variable region derives from a murine antibody and the constant region derives from a human antibody.

[0168] The term "cross-react" as used herein refers to the ability of the antibodies described herein to bind to FAM19A5 from different species. For example, an antibody described herein that binds to human FAM19A5 can also bind to FAM19A5 from other species (e.g., mouse FAM19A5). Cross-reactivity as used herein can be measured by detecting specific reactivity with purified antigen in a binding assay (e.g., SPR, ELISA), or by detecting binding or functional interaction with cells that physiologically express FAM19A5. Methods for determining cross-reactivity include standard binding assays described herein, such as BIACORE® surface plasmon resonance (SPR) assays using a BIACORE® 2000SPR instrument (Biacore AB, Uppsala, Sweden), or flow cytometry techniques.

[0169] As used herein, the term "naturally occurring" as applied to an entity refers to the fact that the entity can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism that can be isolated from a natural source and has not been intentionally modified by man in the laboratory is naturally occurring.

[0170] "Polypeptide" means a chain containing at least two contiguously linked amino acid residues, with no upper limit on the length of the chain. One or more of the amino acid residues in a protein may contain modifications such as, but not limited to, glycosylation, phosphorylation, or disulfide bond formation. A "protein" may include one or more polypeptides.

[0171] As used herein, the term "nucleic acid molecule" includes DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded and may be cDNA.

[0172] The term "vector" 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 (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors) are capable of autonomous replication in a host cell into which they are introduced. Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell when introduced into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors of utility in recombinant DNA techniques are primarily in the form of plasmids. As used herein, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, other forms of expression vectors, such as viral (eg, replication-coupled retroviruses, adenoviruses and adeno-associated viruses) vectors, are also included, which serve equivalent functions.

[0173] As used herein, the term "recombinant host cell" (or simply "host cell") means a cell that contains nucleic acid that is not naturally occurring in the cell, and may be a cell into which a recombinant expression vector has been introduced. It should be understood that such terms refer not only to the particular relevant cell, but 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" as used herein.

[0174] As used herein, the term "linked" refers to the association of two or more molecules. Linkage can be covalent or non-covalent. Linkage can also be genetic (recombinant fusion). Linkage can also be achieved using a variety of art-recognized techniques, such as chemical linkage and recombinant protein production.

[0175] The term "administering" as used herein means 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 skilled in the art. Different routes of administration for the antibodies described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal or other parenteral routes of administration, such as by injection or infusion. The phrase "parenteral administration" as used herein means other modes of administration than enteral and topical administration, and generally refers to modes of administration by injection, including, but not limited to, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraventricular, intravitreal, epidural, and intrasternal injection and infusion, as well as in vivo electroporation. Alternatively, the antibodies described herein can be administered via a non-parenteral route, such as via a topical, epithelial or mucosal route of administration, such as via an intranasal, oral, vaginal, rectal, sublingual or topical route, and administration can be accomplished, for example, once, multiple times, and / or over one or more extended periods of time.

[0176] As used herein, the terms "treat", "treating" and "treatment" refer to a type of intervention or process performed on a subject, or administering an active agent to a subject, for the purpose of reversing, ameliorating, alleviating, inhibiting or slowing the progression, occurrence, severity or recurrence of symptoms, complications, conditions or biochemical indicators associated with a disease. Treatment can be performed on any subject with a disease or on subjects not having a disease (e.g., for prophylaxis).

[0177] As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, etc.

[0178] The term "onset of gliosis" or "onset of reactive gliosis" as used herein includes the onset or initiation of gliosis. Gliosis is a non-specific reactive change in glial cells of the central nervous system (CNS, e.g., brain and / or spinal cord) that occurs 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, and includes proliferation and hypertrophy of several different types of glial cells, including astrocytes, microglia, and oligodendrocytes. The onset of gliosis can result in scar formation, which inhibits axonal regeneration in parts of the CNS that have been traumatized or damaged. The deleterious effects of the onset of gliosis include irreversible or permanent damage to neurons and / or the inability of surrounding neurons to recover. Thus, the terms "delayed onset of gliosis" and "delayed onset of reactive gliosis" include inhibition, delay, suppression, or prevention of the onset or initiation of gliosis and its associated deleterious effects on the CNS.

[0179] As used herein, the term "excessive proliferation of reactive astrocytes" includes an abnormal increase in the number of astrocytes due to, for example, CNS injury, trauma, injury, cerebrospinal 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 result in deleterious effects in the CNS, including scar formation that inhibits axonal regeneration in traumatized or damaged portions of the CNS, exacerbated inflammation, production and release of neurotoxic levels of reactive oxygen species, release of potentially excitotoxic glutamate, a potential contribution to seizure generation, impairment of blood-brain barrier function, cytotoxic edema during trauma and stroke, potential for late cytokine activation of astrocytes that can contribute to late pain syndromes, 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, arresting, or preventing excessive or abnormal proliferation of reactive astrocytes and the associated deleterious effects on the CNS.

[0180] The term "chondroitin sulfate proteoglycans" as used 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 glial scar formation, and they inhibit axonal 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, structural maintenance of chromosome 3), brevican (CSPG7), and CD44 (CSPG8, cluster of differentiation 44), phosphacan-neurocan (CSPG3) (see Rhodes, KE and Fawcett, JW (2004) Journal of Anatom. 204(l):33-48). Thus, the term "reducing chondroitin sulfate proteoglycan expression" includes reducing, inhibiting, decreasing the level of one or more CSGPs, or reducing the activity of one or more CSGPs or inactivating one or more CSGPs. In certain embodiments, the term includes reducing, inhibiting, decreasing the level of neurocan, NG2, or both, or reducing the activity of neurocan, NG2, or both, or inactivating neurocan, NG2, or both.

[0181] The term "neuron" as used herein includes electrically excitable cells that process and transmit information via electrochemical signals. Neurons are important components of the brain and spinal cord of the CNS, have ganglia in the peripheral nervous system (PNS), and can be connected to each other to form a neuronal network structure. A typical neuron consists of a 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 of the input to the neuron occurs. The axon is a fine cable-like projection that extends from the soma and carries nerve signals away from the soma and also carries certain types of information to the soma. The term "promoting regrowth of neurons" includes stimulating, promoting, increasing, or activating the growth of neurons, preferably after injury or damage.

[0182] As used herein, the term "c-fos" includes the proto-oncogene c-fos, which is rapidly induced by neurotransmitter stimulation. c-fos is present in many species, including mouse and human. The c-fos gene and protein are known and have been characterized (see Curran, T, The c-fos proto-oncogene, pp 307-327 (The Oncogene Handbook, Reddy EP et al., (eds.) Elsevier) (1988)). Expression of c-fos can be determined by methods known in the art, such as Northern blot, quantitative PCR, or immunohistochemistry. The term "increased expression of c-fos" includes an increase in the levels of c-fos mRNA, c-fos protein, or c-fos protein activity.

[0183] As used herein, the term "pERK" includes phosphorylated extracellular signal-regulated kinase. Extracellular signal-regulated kinase or ERK includes ERK1 and ERK2, which are members of the mitogen-activated protein kinase (MAPK) family. ERK is activated by its upstream kinases through phosphorylation to form pERK, which subsequently activates downstream targets. ERK is involved in neural and synaptic plasticity, which is fundamental to learning, 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 (Gao YJ and Ji RR, Open (See Pain J. (2009) 2:11-17.) The term "increased expression of pERK" includes increased levels of ERK mRNA, ERK protein, or pERK activity.

[0184] The term "GAP43" as used herein is also referred to as "growth-associated protein 43" and is a neural tissue-specific protein that promotes axon formation, regeneration, and plasticity (see Benowitz LI and Routtenberg A. (1997) Trends in Neurosciences 20 (2): 84-91; Aarts LH et al., (1998) Advances in Experimental Medicine and Biology 446: 85-106). Human GAP43 is encoded by the GAP43 gene. Human GAP43 is a polypeptide sequence (UniProt:KB-P17677), and cDNA encoding this polypeptide is known in the art (Kosik KS et al., (1988) Neuron 1(2): 127-32; Ng et al., (1999) Neuron 2(2): 23-26). (See, 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 "increased neuronal GAP43" includes an enhancement or increase in the levels of GAP43 mRNA, GAP43 protein, or an increase in the activity of GAP43 protein.

[0185] As used herein, the term "therapeutically effective amount" refers to an amount of a drug, alone or in combination with other therapeutic agents, that is effective to "treat" a disease or disorder in a subject or to reduce the risk, latency, likelihood or occurrence of a disease or disorder (e.g., central nervous system injury). A "therapeutically effective amount" includes an amount of a drug or therapeutic agent that provides some improvement or benefit to a subject having or at risk of having a disease or disorder (e.g., central nervous system injury such as traumatic brain injury or other diseases disclosed herein). 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., gliosis) and / or reduces at least one clinical symptom of a disease or disorder.

[0186] II. Anti-FAM19A5 antibody Disclosed herein are antibodies, e.g., monoclonal antibodies, characterized by special functional features and 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 described herein exhibit one or more of the following functional characteristics: (a) reducing, reversing, slowing and / or preventing fibrosis; (b) reduction in excessive extracellular matrix (ECM) formation; (c) slowing the growth or progression of tumors; (d) binds to soluble human FAM19A5 with a KD of 10 nM or less as measured by enzyme-linked immunosorbent assay (ELISA); (e) binds to membrane-bound human FAM19A5 with a KD of 10 nM or less as measured by ELISA; (f) reducing, reversing, delaying and / or preventing the onset of reactive gliosis; (g) inhibition of excessive proliferation of reactive astrocytes; (h) decreased expression of chondroitin sulfate proteoglycans, including neurocan and neuron-glial antigen 2 (NG2); (i) Increased c-fos and pERK expression in neuronal nuclei; (j) promoting neuronal survival; (k) increased neuronal GAP43 expression; and (l) Promotion of axonal regrowth.

[0187] In some embodiments, the anti-FAM19A5 antibody or antigen-binding portion thereof is administered intracellularly, 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 (1 pM) 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, 5×10 -12 M, 10 -11 M, 5×10 -11 M, 10 -10 M, 5×10 -10 M, 10 -9 M, 5×10 -9 M, 10 -8 M, 5×10 -8 M, 10 -7 M or 5 x 10 -7 K of M DThe antibody specifically binds to soluble human FAM19A5 or membrane-bound human FAM19A5 with high affinity of 0.01 to 0.01. Standard assays for evaluating the binding ability of an antibody to human FAM19A5 of various species are known in the art, including, for example, ELISA, Western blotting, 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 an antibody on the functional properties (e.g., ligand binding) of FAM19A5 are described in more detail below and in the Examples.

[0188] 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, e.g., 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 human FAM19A5 at 600 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.

[0189] 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 embodiments, 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, 400 pM, 500 pM, 600 pM, 70 pM, 80 pM, 90 pM, 100 pM, 200 pM, 300 pM, 400 pM, 500 pM, 600 pM, 700 pM, 800 pM, 90 ... and binds to membrane-bound human FAM19A5 with a KD of 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.

[0190] The anti-FAM19A5 antibodies of the invention, or antigen-binding portions thereof, can reduce, prevent, reverse, delay, or inhibit the onset of reactive gliosis, and can delay, block, or inhibit 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.

[0191] The anti-FAM19A5 antibodies or antigen-binding portions thereof of the present invention can delay, inhibit, block, inhibit, limit, or prevent excessive or abnormal proliferation of reactive astrocytes and the 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, for example, destruction of neurons due to CNS injury, trauma, injury, cerebrospinal cord injury, brain tumor, infection, ischemia, stroke, autoimmune reaction, and / or neurodegenerative disease, inhibit or prevent scarring in the CNS, inhibit or reduce the release of neurotoxic levels of reactive oxygen species or potentially excitotoxic glutamate, and reduce or inhibit secondary degeneration following seizures, pain, and / or 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 following CNS injury or damage.

[0192] 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 sulfates (CSGPs), such as aggrecan (CSPG1), versican (CSPG2), neurocan (CSPG3), CSPG4 (or neuron-glial antigen 2 (NG2)), CSPG5, SMC3 (CSPG6, structural maintenance of chromosome 3), brevican (CSPG7), CD44 (CSPG8, cluster of differentiation 44), phosphacan-neurocan (CSPG3), or any combination thereof. 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 inhibit, reduce, or decrease the activity of neurocan and / or NG2.

[0193] The anti-FAM19A5 antibodies or antigen-binding portions thereof of the present invention can increase the expression of c-fos and pERK in neuronal nuclei, for example, by increasing c-fos and pERK mRNA, protein and / or protein activity, and can increase or enhance the expression levels of GAP43 mRNA, GAP43 protein, or increase or enhance GAP43 protein activity.

[0194] In certain embodiments, an anti-FAM19A5 antibody of the invention, or an antigen-binding portion thereof, cross-competes (or inhibits binding) to a human FAM19A5 epitope with an anti-FAM19A5 antibody comprising a CDR or variable region disclosed in the invention (e.g., 2-13, 3-2, or 1-28).

[0195] 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 to a light chain CDR1, CDR2, and CDR3, wherein (i) the heavy chain CDR1, CDR2, and CDR3 of the reference antibody are each represented by SEQ ID NO: (ii) the heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO:14, the heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO:15, and the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO:16, the light chain CDR1 comprises the amino acid sequence of SEQ ID NO:26, the light chain CDR2 comprises the amino acid sequence of SEQ ID NO:27, and the light chain CDR3 comprises the amino acid sequence of SEQ ID NO:28; (iii) the heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO:20, the heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO:21, and the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO:22, and the light chain CDR1 comprises the amino acid sequence of SEQ ID NO:22, the light chain CDR2 comprises the amino acid sequence of SEQ ID NO:32, the light chain CDR2 comprises the amino acid sequence of SEQ ID NO:33, and the light chain CDR3 comprises the amino acid sequence of SEQ ID NO:34. In some embodiments, the reference antibody comprises (i) a heavy chain variable domain comprising SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:38, and (ii) a light chain variable domain comprising SEQ ID NO:39, SEQ ID NO:40, or SEQ ID NO:42.

[0196] 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., due to 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.

[0197] In certain embodiments, the anti-FAM19A5 antibody, or antigen-binding portion thereof, binds to the same FAM19A5 epitope as a reference antibody disclosed herein comprising heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3, and wherein (i) the heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO:11, the heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO:12, the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO:13, the light chain CDR1 comprises the amino acid sequence of SEQ ID NO:23, the light chain CDR2 comprises the amino acid sequence of SEQ ID NO:24, and the light chain CDR3 comprises the amino acid sequence of SEQ ID NO:25; or (ii) the heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO:14, the heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO:15, the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO:16, and the light chain CDR1 comprises the amino acid sequence of SEQ ID NO: (iii) heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO:20 and heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO:21; and light chain CDR3 comprises the amino acid sequence of SEQ ID NO:22; (iv) heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO:23 and heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO:24; heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 32; light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 33; and light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 34. In some embodiments, the reference antibody comprises the amino acid sequence of SEQ ID NO: 34. In some embodiments, the reference antibody comprises (i) a heavy chain variable domain comprising SEQ ID NO: 35, SEQ ID NO: 36, or SEQ ID NO: 38, and (ii) a light chain variable domain comprising SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 42.

[0198] Techniques for determining whether two antibodies bind to the same epitope include, for example, epitope mapping methods, such as X-ray analysis of antigen-antibody complex crystals and hydrogen / deuterium exchange mass spectrometry (HDX-MS), which provide atomic resolution of the epitope, methods monitoring binding of antibodies to antigen fragments or mutated variants of the antigen (wherein binding losses due to alterations of amino acid residues within the antigen sequence are considered primarily indicative of epitope components), and computer-assisted methods for epitope mapping.

[0199] The anti-FAM19A5 antibodies, or antigen-binding portions thereof, of the invention can bind to at least one epitope of mature human FAM19A5, for example, as determined by binding of the antibody to a fragment of human FAM19A5. In some embodiments, the anti-FAM19A5 antibody, or antigen-binding portion thereof, has the sequence TLDRDSSQPRRTIARQTARC (SEQ ID NO: 1). or a fragment located within the amino acid sequence of SEQ ID NO:6, such as an epitope having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids of SEQ ID NO:6. In some embodiments, the anti-FAM19A5 antibody or antigen-binding portion thereof binds at one or more amino acids corresponding to amino acid residues 46-51 (i.e., DSSQPR) of SEQ ID NO:2, such as amino acid residues 46, 50, and 52 (i.e., D---PR), such as amino acid residues 46, 47, 48, and 50 (i.e., DSS-P). In some embodiments, the anti-FAM19A5 antibody, or antigen binding portion thereof, binds to a fragment located within the amino acid sequence of SEQ ID NO:9, e.g., an epitope having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids of SEQ ID NO:9.

[0200] 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:6. 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:9.

[0201] In some embodiments, the anti-FAM19A5 antibody, or antigen-binding portion thereof, binds only to the human FAM19A5 epitope of SEQ ID NO:5, 6, 7, 8, 9, or 10, or to a fragment located within the amino acid sequence of SEQ ID NO:5, 6, 7, 8, 9, or 10, such as an epitope having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids of SEQ ID NO:5, 6, 7, 8, 9, or 10.

[0202] In some embodiments, an anti-FAM19A5 antibody, or antigen-binding portion thereof, of the invention binds to SEQ ID NO:6 or a fragment thereof in a native conformation (i.e., native) state. In some embodiments, an anti-FAM19A5 antibody, or antigen-binding portion thereof, of the invention binds to SEQ ID NO:9 or a fragment thereof in a native conformation (i.e., native) state. In some embodiments, an anti-FAM19A5 antibody, or antigen-binding portion thereof, binds to both glycosylated and non-glycosylated human FAM19A5.

[0203] 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:6 and an additional epitope, or the epitope of SEQ ID NO:9 and an additional epitope. Other anti-FAM19A5 antibodies or antigen-binding portions thereof can bind to the epitope of SEQ ID NO:5, the epitope of SEQ ID NO:9 and an additional epitope. In some embodiments, an anti-FAM19A5 antibody or antigen-binding portion thereof binds to the epitope of SEQ ID NO:6, the epitope of SEQ ID NO:10 and an additional epitope.

[0204] In some embodiments, the one or more additional FAM19A5 epitopes are selected from QLAAGTCEIVTLDR (SEQ ID NO:5, epitope F1), TLDRDSSQPRRTIARQTARC (SEQ ID NO:6, epitope F2), TARCACRKGQIAGTTRARPA (SEQ ID NO:7, epitope F3), ARPACVDARIIKTKQWCDML (SEQ ID NO:8, epitope F4), CDMLPCLEGEGCDLLINRSG (SEQ ID NO:9, epitope F5), or NRSGWTCTQPGGRIKTTTVS (SEQ ID NO:10, epitope F6), or a fragment located within the amino acid sequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10, or any combination thereof. Fragments located within the amino acid sequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10 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:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10. In some embodiments, the one or more additional FAM19A5 epitopes are selected from SEQ ID NO:5, 6, 7, 8, 9, or 10, or fragments located within the amino acid sequence of SEQ ID NO:5, 6, 7, 8, 9, or 10, 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:5, 6, 7, 8, 9, or 10, or any combination thereof. In some embodiments, the anti-FAM19A5 antibody or antigen-binding portion thereof of the invention binds to the one or more additional epitopes in a native conformation (i.e., native) state. In some embodiments, the anti-FAM19A5 antibody or antigen-binding portion thereof binds to both glycosylated and non-glycosylated forms of the one or more additional FAM19A5 epitopes.

[0205] In some embodiments, an anti-FAM19A5 antibody or antigen-binding portion thereof binds to at least one FAM19A5 epitope identified as EP2, EP4, and / or EP8, where EP2 comprises, consists essentially of, or consists of amino acids DSSQP (SEQ ID NO:66), EP4 comprises, consists essentially of, or consists of amino acids ARCACRK (SEQ ID NO:68), and EP8 comprises, consists essentially of, or consists of amino acids TCTQPGGR (SEQ ID NO:72). 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 EP2, EP4, or EP8. In some embodiments, an anti-FAM19A5 antibody or antigen-binding portion thereof binds only to EP2. In some embodiments, an anti-FAM19A5 antibody or antigen-binding portion thereof binds to EP4 and EP8. In some embodiments, the anti-FAM19A5 antibody, or antigen-binding portion thereof, binds to one or more FAM19A5 epitopes selected from the group consisting of SEQ ID NO:65[[EP1-IVTLD]], SEQ ID NO:67[[EP3-RTIAR]], SEQ ID NO:69[[EP5-ARPA]], SEQ ID NO:70[[EP6-KTKQWCDML]], and SEQ ID NO:71[[EP7-GCDLLINR]], and any combination thereof.

[0206] In certain embodiments, provided herein are antibodies or antigen-binding portions thereof that bind to FAM19A5 (e.g., human FAM19A5) with 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more higher affinity than other proteins of the FAM19A family, as measured, for example, by immunoassay (e.g., ELISA), surface plasmon resonance, or equilibrium binding exclusion methods. In certain embodiments, provided herein are antibodies or antigen-binding portions thereof that bind to FAM19A5 (e.g., human FAM19A5) without cross-reactivity with other proteins of the FAM19A family, as measured, for example, by immunoassay.

[0207] In certain embodiments, the anti-FAM19A5 antibody is not a native or naturally occurring antibody, e.g., the anti-FAM19A5 antibody has post-translational modifications that differ from those of naturally occurring antibodies, e.g., by having more, fewer, or different types of post-translational modifications.

[0208] III. Exemplary Anti-FAM19A5 Antibodies Particular antibodies that can be used in the methods disclosed herein include antibodies having the CDRs and / or variable regions of antibodies 2-13, 3-2, 1-65 and 1-28 isolated in Example 1, including monoclonal antibodies as well as antibodies having at least 80% identity (e.g., at least 85%, at least 90%, at least 95% or at least 99% identity) to these variable region or CDR sequences. The VH amino acid sequences of 2-13, 3-2, 1-65 and 1-28 are provided as SEQ ID NOs:35-38. The VH amino acid sequences of 2-13, 3-2, 1-65 and 1-28 are provided as SEQ ID NOs:39-42.

[0209] [Table 2]

[0210] [Table 3]

[0211] [Table 4]

[0212] [Table 5]

[0213] Thus, provided herein 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 NO:35, 36, or 38. 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 NO:35, 36, or 38.

[0214] Also provided 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 NO: 39, 40, or 42. In other embodiments, 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 NO: 39, 40, or 42.

[0215] In certain embodiments, the isolated anti-FAM19A5 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region CDR selected from the group consisting of SEQ ID NO:35, 36, or 38, and a light chain variable region CDR selected from the group consisting of SEQ ID NO:39, 40, or 42.

[0216] Also provided is an isolated anti-FAM19A5 antibody, or antigen-binding portion thereof, comprising heavy and light chain variable regions, wherein (i) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:35 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:39; (ii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:36 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:40; or (iii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:38 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:42.

[0217] Provided herein 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 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 as SEQ ID NO:35, 36 or 38.

[0218] Also provided herein 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 as SEQ ID NO:39, 40 or 42.

[0219] Also provided herein 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 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 provided as SEQ ID NOs:35, 36 or 38, and 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 provided as SEQ ID NOs:39, 40 or 42.

[0220] 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:35 and 39, respectively; (b) heavy and light chain variable region sequences comprising SEQ ID NOs: 36 and 40, respectively; or (c) comprising heavy and light chain variable region sequences comprising SEQ ID NOs:38 and 42, respectively.

[0221] In certain embodiments, an anti-FAM19A5 antibody or antigen-binding portion thereof of the invention comprises: (i) 2-13 heavy chain CDR1, CDR2 and CDR3, or a combination thereof, and / or 2-13 light chain CDR1, CDR2 and CDR3, or any combination thereof; (ii) 3-2 heavy chain CDR1, CDR2 and CDR3, or a combination thereof, and / or 3-2 light chain CDR1, CDR2 and CDR3, or any combination thereof; or (iii) 1-28 heavy chain CDR1, CDR2 and CDR3, or a combination thereof, and / or 1-28 light chain CDR1, CDR2 and CDR3, or any combination thereof.

[0222] The amino acid sequences of VH CDR1, CDR2 and CDR3 for 2-13 are presented as SEQ ID NOs:11, 12 and 13, respectively. The amino acid sequences of VL CDR1, CDR2 and CDR3 for 2-13 are presented as SEQ ID NOs:23, 24 and 25, respectively. The amino acid sequences of VH CDR1, CDR2 and CDR3 for 3-2 are presented as SEQ ID NOs:14, 15 and 16, respectively. The amino acid sequences of VL CDR1, CDR2 and CDR3 for 3-2 are presented as SEQ ID NOs:26, 27 and 28, respectively. The amino acid sequences of VH CDR1, CDR2 and CDR3 for 1-28 are presented as SEQ ID NOs:20, 21 and 22, respectively. The amino acid sequences of VL CDR1, CDR2 and CDR3 for 1-28 are presented as SEQ ID NOs:32, 33 and 34, respectively. The amino acid sequences of VH CDR1, CDR2 and CDR3 for 1-65 are shown in SEQ ID NO: The amino acid sequences of the VL CDR1, CDR2 and CDR3 for 1-65 are presented as SEQ ID NOs: 29, 30 and 31, respectively.

[0223] In some embodiments, an anti-FAM19A5 antibody, or antigen-binding portion thereof, of the invention that specifically binds to human FAM19A5 is: (a) VH CDR1 comprising the amino acid sequence of SEQ ID NO:11; (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:12; and / or (c) VH CDR3 comprising the amino acid sequence of SEQ ID NO:13 Includes.

[0224] In certain embodiments, the antibody, or antigen binding portion thereof, comprises one, two, or all three of said VH CDRs.

[0225] In some embodiments, an anti-FAM19A5 antibody, or antigen-binding portion thereof, that specifically binds to human FAM19A5 is: (a) VL CDR1 comprising the amino acid sequence of SEQ ID NO:23; (b) a VL CDR2 comprising the amino acid sequence of SEQ ID NO:24; and / or (c) VL CDR3 comprising the amino acid sequence of SEQ ID NO:25 Includes.

[0226] In certain embodiments, the antibody, or antigen binding portion thereof, comprises one, two, or all three of said VL CDRs.

[0227] In some embodiments, an anti-FAM19A5 antibody, or antigen-binding portion thereof, that specifically binds to human FAM19A5 is: (a) VH CDR1 comprising the amino acid sequence of SEQ ID NO:11; (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:12; (c) VH CDR3 comprising the amino acid sequence of SEQ ID NO:13 (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO:23; (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO:24; and / or (f) VL CDR3 comprising the amino acid sequence of SEQ ID NO:25 Includes.

[0228] In some embodiments, an anti-FAM19A5 antibody, or antigen-binding portion thereof, of the invention that specifically binds to human FAM19A5 is: (a) VH CDR1 comprising the amino acid sequence of SEQ ID NO:14; (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:15; and / or (c) VH CDR3 comprising the amino acid sequence of SEQ ID NO:16 Includes.

[0229] In certain embodiments, the antibody, or antigen binding portion thereof, comprises one, two, or all three of said VH CDRs.

[0230] In some embodiments, an anti-FAM19A5 antibody, or antigen-binding portion thereof, that specifically binds to human FAM19A5 is: (a) a VL CDR1 comprising the amino acid sequence of SEQ ID NO:26; (b) a VL CDR2 comprising the amino acid sequence of SEQ ID NO:27; and / or (c) VL CDR3 comprising the amino acid sequence of SEQ ID NO:28 Includes.

[0231] In certain embodiments, the antibody, or antigen binding portion thereof, comprises one, two, or all three of said VL CDRs.

[0232] In some embodiments, an anti-FAM19A5 antibody, or antigen-binding portion thereof, that specifically binds to human FAM19A5 is: (a) VH CDR1 comprising the amino acid sequence of SEQ ID NO:14; (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:15; (c) VH CDR3 comprising the amino acid sequence of SEQ ID NO:16 (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO:26; (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO:27; and / or (f) VL CDR3 comprising the amino acid sequence of SEQ ID NO:28 Includes.

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

[0234] In some embodiments, an anti-FAM19A5 antibody, or antigen-binding portion thereof, that specifically binds to human FAM19A5 is: (a) a VL CDR1 comprising the amino acid sequence of SEQ ID NO:29; (b) a VL CDR2 comprising the amino acid sequence of SEQ ID NO:30; and / or (c) VL CDR3 comprising the amino acid sequence of SEQ ID NO:31 Includes.

[0235] In certain embodiments, the antibody, or antigen binding portion thereof, comprises one, two, or all three of said VL CDRs.

[0236] In some embodiments, an anti-FAM19A5 antibody, or antigen-binding portion thereof, of the invention that specifically binds to human FAM19A5 is: (a) VH CDR1 comprising the amino acid sequence of SEQ ID NO:20; (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:21; and / or (c) VH CDR3 comprising the amino acid sequence of SEQ ID NO:22 Includes.

[0237] In certain embodiments, the antibody, or antigen binding portion thereof, comprises one, two, or all three of said VH CDRs.

[0238] In some embodiments, an anti-FAM19A5 antibody, or antigen-binding portion thereof, that specifically binds to human FAM19A5 is: (a) a VL CDR1 comprising the amino acid sequence of SEQ ID NO:32; (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.

[0239] In certain embodiments, the antibody, or antigen binding portion thereof, comprises one, two, or all three of said VL CDRs.

[0240] In some embodiments, an anti-FAM19A5 antibody, or antigen-binding portion thereof, that specifically binds to human FAM19A5 is: (a) VH CDR1 comprising the amino acid sequence of SEQ ID NO:20; (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:21; (c) VH CDR3 comprising the amino acid sequence of SEQ ID NO:22 (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.

[0241] In certain embodiments, the anti-FAM19A5 antibody, or antigen-binding portion thereof, comprises one, two, three, four, five, or six of said CDRs.

[0242] A VH domain as described herein, or one or more CDRs thereof, can be linked to a constant domain to form a heavy chain, e.g., a full-length heavy chain. Similarly, a VL domain as described herein, or one or more CDRs thereof, can 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 can be combined to form a full-length antibody.

[0243] Thus, in certain embodiments, the present invention provides antibodies comprising antibody light and heavy chains, for example separated light and heavy chains. With respect to the light chain, in certain embodiments, the light chain of the antibody described herein is a kappa light chain. In other specific embodiments, the light chain of the antibody described herein is a lambda light chain. In yet other specific embodiments, the light chain of the antibody described herein is a human kappa light chain or a human lambda light chain. In certain embodiments, the antibody described herein that specifically binds to a FAM19A5 polypeptide (e.g., human FAM19A5) comprises a light chain comprising any of the VL or VL CDR amino acid sequences described herein, wherein the constant region of the light chain comprises the amino acid sequence of a human kappa light chain constant region. In certain embodiments, the antibody described herein that specifically binds to a FAM19A5 polypeptide (e.g., human FAM19A5) comprises a light chain comprising any of the VL or VL CDR amino acid sequences described herein, wherein the constant region of the light chain comprises the amino acid sequence of a human lambda light chain constant region. Non-limiting examples of human constant region sequences are described in the art, see, eg, US Pat. No. 5,693,780 and Kabat EA et al, (1991) supra.

[0244] With respect to the heavy chain, in some embodiments, the heavy chain of the antibody described 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 antibody described herein can comprise a human alpha (α), delta (δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In one embodiment, an antibody described herein that specifically binds to FAM19A5 (e.g., human FAM19A5) comprises a heavy chain comprising a VH or VH CDR amino acid sequence described herein, wherein the constant region of the heavy chain comprises the amino acid sequence of a human gamma (γ) heavy chain constant region. In another embodiment, an antibody described herein that specifically binds to FAM19A5 (e.g., human FAM19A5) comprises a heavy chain comprising a VH or VH CDR amino acid sequence described herein, wherein the constant region of the heavy chain comprises an amino acid sequence of a human heavy chain described 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).

[0245] In some embodiments, an antibody described herein that specifically binds to FAM19A5 (e.g., human FAM19A5) comprises a VL domain and a VH domain comprising a VH or VH CDR and a VL or VL CDR described herein, where the constant region comprises an amino acid sequence of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, or a constant region of a human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule. In other specific embodiments, an antibody described herein that specifically binds to FAM19A5 (e.g., human FAM19A5) comprises a VL domain and a VH domain comprising any amino acid sequence described herein, wherein the constant region comprises the amino acid sequence of a constant region of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, any subclass of immunoglobulin molecule (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). In some embodiments, the constant region comprises the amino acid sequence of a constant region of a 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 October 20, 2014) and Jefferis R. and Lefranc MP, mAbs 1:4, 1-7 (2009). In some embodiments, the constant region comprises the amino acid sequence of the constant region of human IgG1, IgG2, IgG3, or IgG4, or a variant thereof.

[0246] In a particular embodiment, the anti-FAM19A5 antibody or antigen-binding portion thereof described in the present invention does not have Fc effector functions, such as complement-dependent cytotoxicity (CDC) and / or antibody-dependent cellular phagocytosis (ADCP). The effector functions are mediated by the Fc region, and the residues of the CH2 domain of the Fc region closest to the hinge region are responsible for the effector functions of the antibody, which contain highly overlapping binding sites for C1q (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. The effector functions of antibodies can be reduced or avoided by different approaches known in the art, such as: (1) the use of antibody fragments lacking the Fc region (e.g., Fab, F(ab')2, single chain Fv (scFv), or sdAbs consisting of monomeric VH or VL domains); (2) the generation of aglycosylated antibodies, which can be generated, for example, by deleting or altering the residues to which sugars are attached, enzymatically removing sugars, generating the antibody in cells cultured in the presence of glycosylation inhibitors, or expressing the antibody in a cell that cannot glycosylate proteins (e.g., a bacterial host cell, 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 a CH2 domain from an IgG2 or IgG4 antibody) (see, for example, U.S. Patent Publication No. 20120100140 and Lau C. et al. J. Immunol. 191:4769- 4777 (2013); and (4) generating Fc regions with mutations that result in reduced or no 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)).

[0247] Thus, in some embodiments, the anti-FAM19A5 antibodies or antigen-binding portions thereof described in the present invention are Fab, Fab', F(ab')2, Fv, single-chain Fv (scFv), or sdAbs consisting of monomeric VH or VL domains. Such antibody fragments are well known in the art and described above.

[0248] In some embodiments, the anti-FAM19A5 antibody or antigen-binding portion thereof described herein comprises an Fc region with or without reduced 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 is of the 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 with or without reduced Fc function. Fc regions with or without reduced 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). Additionally, Fc regions with or without reduced Fc effector function can be readily engineered by one of skill in the art.

[0249] IV. Nucleic acid molecules Another embodiment described herein relates to one or more nucleic acid molecules encoding any one of the antibodies or antigen-binding portions thereof described herein. The nucleic acid can 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 away from other cellular components or other contaminants, such as other cellular nucleic acids (e.g., other chromosomal DNA, e.g., chromosomal DNA linked to naturally isolated DNA) or proteins, by standard techniques, including alkaline / SDS treatment, CsCl banding, column chromatography, restriction enzymes, agarose gel electrophoresis, and other techniques known in the art (F. Ausubel, et al., ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York). The nucleic acids described herein can be, for example, DNA or RNA and may or may not contain intron sequences. In a particular embodiment, the nucleic acid is a cDNA molecule.

[0250] The nucleic acids described in the present invention can be obtained by standard molecular biology techniques. In the case of antibodies expressed by a hybridoma (e.g., a hybridoma produced from a transgenic mouse carrying human immunoglobulin genes, as described in more detail below), cDNAs 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 acids encoding the antibody can be recovered from the library.

[0251] Particular nucleic acid molecules described in the present invention are those encoding the VH and VL sequences of the 2-13, 3-2, 1-65 and 1-28 monoclonal antibodies. Exemplary DNA sequences encoding the VH sequences of 2-13, 3-2, 1-65 and 1-28 are provided as SEQ ID NOs: 43, 44, 45 and 46, respectively. Exemplary DNA sequences encoding the VL sequences of 2-13, 3-2, 1-65 and 1-28 are provided as SEQ ID NOs: 47, 48, 49 and 50.

[0252] [Table 6]

[0253] [Table 7]

[0254] The method for producing the anti-FAM19A5 antibodies described in the present invention can include expressing the heavy and light chains in a cell line that contains nucleotide sequences encoding the heavy and light chains with signal peptides, such as SEQ ID NOs: 43 and 47, SEQ ID NOs: 44 and 48, SEQ ID NOs: 46 and 50, respectively. Host cells containing these nucleotide sequences are included in the present invention.

[0255] After the DNA fragments encoding the VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example to convert the variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these manipulations, the VL- or VH-encoding DNA fragment is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. As used in this context, the term "operably linked" means that the two DNA fragments are ligated such that the amino acid sequences encoded by the two DNA fragments remain in-frame.

[0256] 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). 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, US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, e.g., an IgG2 and / or IgG4 constant region. In the case of a Fab fragment heavy chain gene, the VH-encoding DNA can be operably linked to another DNA molecule encoding only the heavy chain CH1 constant region.

[0257] The isolated DNA encoding the VL region can be converted into a full-length 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 ed.). (See, US Department of Health and Human Services, NIH Publication No. 91-3242, Edition), 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.

[0258] To generate an scFv gene, the VH- and VL-encoding DNA fragments are operably linked to another fragment encoding a flexible 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 linked by the flexible linker (see, e.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature 348:552-554).

[0259] In some embodiments, the invention provides vectors comprising an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antigen-binding portion thereof, hi other embodiments, the vectors can be used in gene therapy.

[0260] Vectors suitable for the present invention include expression vectors, viral vectors, and plasmid vectors, hi one embodiment, the vector is a viral vector.

[0261] As used herein, an expression vector refers to any nucleic acid construct that, when introduced into an appropriate host cell, contains the necessary elements for transcription and translation, or, in the case of RNA viral vectors, replication and translation, of an inserted coding sequence. Expression vectors can include plasmids, phagemids, viruses, and derivatives thereof.

[0262] An expression vector of the invention can include a polynucleotide encoding an antibody or antigen-binding portion thereof described herein. In one embodiment, the coding sequence for the antibody or antigen-binding portion thereof is operably linked to an expression control sequence. As used herein, two nucleic acid sequences are operably linked when they are covalently linked in a manner that permits each component nucleic acid sequence to retain its functionality. A coding sequence and a gene expression control sequence are said to be operably linked when they are covalently linked in such a manner that expression or transcription and / or translation of the coding sequence is under the influence or control of the gene expression control sequence. Two DNA sequences are said to be operably linked if induction of the promoter at the 5' gene expression sequence results in transcription of the coding sequence, and if the nature of the linkage of the two DNA sequences does not (1) result in the introduction of a frameshift mutation, (2) interfere with the ability of the promoter region to direct transcription of the coding sequence, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a gene expression sequence is operably linked to a coding nucleic acid sequence if the gene expression sequence is capable of effecting transcription of the coding nucleic acid sequence, such that the resulting transcript can be translated into the desired antibody or antigen-binding portion thereof.

[0263] Viral vectors include, but are not limited to, 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 viruses; papilloma viruses; herpes viruses; vaccinia viruses; polio viruses; and nucleic acid sequences from RNA viruses, such as retroviruses. Other vectors known in the art can also be readily used. Certain viral vectors are based on non-cytopathic eukaryotic viruses in which a non-essential gene has been replaced with the gene of interest. Non-cytopathic viruses include retroviruses, whose life cycle involves reverse transcription of genomic viral RNA into DNA and subsequent proviral integration into host cell DNA. Retroviruses have been approved for human gene therapy trials. Retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins but unable to manufacture infectious particles) are the most useful. Such genetically engineered retroviral expression vectors have general utility for high-efficiency transduction of genes in vivo. Standard protocols for generating replication-deficient retroviruses (including the steps of integrating foreign 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 provided in the literature (Kriegler, M., Gene Transfer and Expression, A Laboratory Manual, WH Freeman Co., New York (1990) and Murry, EJ, Methods in Molecular Biology, Vol. 7, Humana Press, Inc., Cliffton, NJ (1991).

[0264] In one embodiment, the virus is an adeno-associated virus, which is a double-stranded DNA virus. Adeno-associated viruses can be engineered to be replication-deficient and can infect a wide range of cell types and species. Adeno-associated viruses also have advantages such as heat and lipid solvent safety; high transformation frequency in cells of various lineages, including hematopoietic cells; and lack of superinfection inhibition, which allows for many rounds of transformation. Adeno-associated viruses can reportedly integrate into human cellular DNA in a site-specific manner, thereby minimizing the probability of insertional mutagenesis and the variability of inserted gene expression characteristics of retroviral infection. Wild-type adeno-associated virus infections have also been tracked in tissue cultures for over 100 passages in the absence of selective pressure, suggesting that adeno-associated virus genome integration is a relatively stable event. Adeno-associated viruses can also function in an extrachromosomal manner.

[0265] 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.

[0266] Lentiviral genomes and proviral DNA typically contain three genes found in retroviruses, gag, pol, and env, 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-dependent DNA polymerase (reverse transcriptase), protease, and integrase, and the env gene encodes the viral envelope glycoprotein. The 5' and 3' LTRs act to facilitate transcription and polyadenylation of virion RNA. The LTRs contain all other cis-acting sequences necessary for viral replication. Lentiviruses contain additional genes including vif, vpr, tat, rev, vpu, nef, and vpx (in HIV-1, HIV-2, and / or SIV).

[0267] Adjacent to the 5'LTR are sequences necessary for reverse transcription of the genome (tRNA primer binding site), and the Psi site is effective for encapsidation of viral RNA into particles. If sequences essential for encapsidation (or packaging of retroviral RNA into infectious virions) are missing from the viral genome, this cis deletion prevents encapsidation of genomic DNA.

[0268] However, the resulting mutations are still capable of directing the synthesis of all virion proteins. The present invention provides a method for producing recombinant lentiviruses capable of infecting non-dividing cells, which involves transfecting a suitable host cell with two or more vectors having packaging functions, i.e., gag, pol, and env, as well as rev and tat. As disclosed below, vectors lacking a functional tat gene are preferred for certain applications. Thus, for example, a first vector can provide nucleic acid encoding viral gag and viral pol, and another vector can provide nucleic acid encoding viral env, thereby 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.

[0269] Depending on the above configuration of the vector and the foreign gene, the second vector can provide a nucleic acid encoding a viral envelope (env) gene. The env gene can be derived from almost any suitable virus, including retroviruses. In some embodiments, the env protein is an amphotropic envelope protein that allows transformation of cells of human and other species.

[0270] Examples of retrovirus-derived env genes include, but are not limited to, Moloney murine leukemia virus (MoMuLV or MMLV), Harvey murine sarcoma virus (HaMuSV or HSV), murine mammary tumor virus (MuMTV or MMTV), gibbon monkey 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 (VSVG), hepatitis virus, and influenza, can also be used.

[0271] The vector providing the viral env nucleic acid sequence is operably associated with the regulatory sequences described elsewhere in this invention.

[0272] In particular embodiments, 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 transform non-dividing cells.

[0273] 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.

[0274] In some embodiments, the lentiviral vector of the present invention comprising the FVIII nucleotide sequence described in the present invention can be transfected in 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. The lentiviral vector lacks a functional tat gene. In other embodiments, the cell is further transfected with a fourth nucleotide sequence comprising the rev gene. In certain embodiments, the lentiviral vector lacks a functional gene selected from vif, vpr, vpu, vpx, and nef, or any combination thereof.

[0275] In certain embodiments, the lentiviral vector comprises one or more nucleotide sequences encoding a gag protein, a Rev response element, a central polypurine track (cPPT), or any combination thereof.

[0276] Examples of lentiviral vectors are disclosed in WO9931251, WO9712622, WO9817815, WO9817816 and WO9818934, which are incorporated by reference in their entirety.

[0277] Other vectors include plasmid vectors. Plasmid vectors have been described extensively in the art and are known to those of skill in the art. See, for example, (Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989). Over the past few years, plasmid vectors have proven to be particularly useful for delivering genes to cells in vivo because they cannot replicate or integrate into the host genome. However, such plasmids having a promoter 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 of which are products of Invitrogen (Carlsbad, Calif.). Other plasmids will be familiar 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.

[0278] 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 described in the present invention employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields within the skill of the art. These techniques are described in the references and in detail in the following documents:See, for example, (Maniatis T. et al., (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Sambrook J et al., (1989), Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press; Sambrook J. et al., (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel FM. et al., Current Protocols in Molecular Biology, John Wiley & Sons (1987 and annual updates); Current Protocols in Immunology, John Wiley & Sons (1987 and annual updates) Gait (ed.) (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein (ed.) (1991) Oligonucleotides and Analogues: A Practical Approach, IRL Press; Birren B. et al., (eds.) (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press).

[0279] In certain embodiments, the antibodies described herein are antibodies (e.g., recombinant antibodies) that are manufactured, expressed, produced, or isolated by any means involving, for example, synthesis, recombinant production of DNA sequences. In certain embodiments, such antibodies contain sequences (e.g., DNA sequences or amino acid sequences) that do not naturally occur within the antibody germline repertoire of an animal or mammal (e.g., human) in vivo.

[0280] In certain embodiments, provided herein is a method for producing an antibody or antigen-binding fragment thereof that immunospecifically binds to FAM19A5 (e.g., human FAM19A5), comprising culturing a cell or host cell described herein. In certain embodiments, provided herein is a method for producing an antibody or antigen-binding fragment thereof that immunospecifically binds to FAM19A5 (e.g., human FAM19A5), comprising expressing (e.g., recombinantly expressing) the antibody or antigen-binding fragment thereof using a cell or host cell described herein (e.g., a cell or host cell comprising a polynucleotide encoding an antibody described herein). In certain embodiments, the cell is an isolated cell. In certain embodiments, an exogenous polynucleotide has been introduced into the cell. In certain embodiments, the method further comprises purifying the antibody or antigen-binding fragment thereof obtained from the cell or host cell.

[0281] Methods for generating polyclonal antibodies are known in the art (see, e.g., Chapter 11 in: Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel FM et al., eds., John Wiley and Sons, New York).

[0282] Monoclonal antibodies can be produced using a wide variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced by hybridoma techniques, including those known in the art. These include, for example, those disclosed in Harlow E & Lane D, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling GJ et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563 681 (Elsevier, NY, 1981). The term "monoclonal antibody" as used herein is not limited to antibodies produced by hybridoma technology. For example, monoclonal antibodies can be produced recombinantly from host cells exogenously expressing an antibody described herein or a fragment thereof, e.g., the light chain and / or the heavy chain of such an antibody.

[0283] In certain embodiments, a "monoclonal antibody" as used herein is an antibody produced by a single cell (e.g., a recombinant antibody-producing hybridoma or a host cell), which immunospecifically binds to FAM19A5 (e.g., human FAM19A5), e.g., by ELISA or other antigen-binding or competitive binding assays known in the art or as determined in the Examples provided herein. In certain embodiments, a monoclonal antibody can be a chimeric or humanized antibody. In certain embodiments, a monoclonal antibody is a monovalent antibody or a polyvalent (e.g., bivalent) antibody. In certain embodiments, a monoclonal antibody is a monospecific or multispecific antibody (e.g., bispecific antibody). The monoclonal antibodies described herein can be produced, e.g., by the hybridoma method described in Kohler G & Milstein C (1975) Nature 256: 495, or can be isolated from a phage library, e.g., using the techniques described herein. Other methods for producing clonal cell lines and the monoclonal antibodies expressed thereby are also well known in the art (see, e.g., Chapter 11 in: Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel FM et al., supra).

[0284] Methods for producing and screening specific antibodies using hybridoma technology are conventional and well known in the art. For example, in the hybridoma method, a mouse or other suitable host animal, such as a sheep, goat, rabbit, rat, hamster, or macaque, is immunized to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the protein used for immunization (e.g., human FAM19A5). Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, thereby forming hybridoma cells (see Goding JW (Ed), Monoclonal Antibodies: Principles and Practice, pp. 59- 103 (Academic Press, 1986)). Additionally, RFMMS (repeated immunization multiple site) technology can be used to immunize animals (Kilpatrick KE et al., (1997) Hybridoma 16:381-9, incorporated by reference in its entirety).

[0285] In some embodiments, a mouse (or other animal, such as a chicken, rat, monkey, donkey, pig, sheep, hamster, or dog) can be immunized with an antigen (e.g., FAM19A5, e.g., human FAM19A5), and once an immune response is detected, e.g., 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 well-known techniques with any suitable myeloma cells, e.g., cells from cell line SP20 available from the American Type Culture Collection (ATCC) (Manassas, Virginia), to form 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.

[0286] The hybridoma cells thus produced are preferably seeded and grown in an appropriate culture medium containing one or more substances that inhibit the growth or survival of the unfused, parent myeloma cells. For example, if the parent myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or FIPRT), then the hybridoma culture medium will typically contain hypoxanthine, aminopterin, and thymidine (HAT medium), which substances will prevent the growth of the HGPRT-deficient cells.

[0287] A specific embodiment uses myeloma cells that fuse efficiently and support stable high-level production of antibodies by selected antibody-producing cells, and are sensitive to media such as HAT medium. Such myeloma cell lines are in particular mouse 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, California, USA) and SP-2 or X63-Ag8.653 cells available from the American Type Culture Collection (Rockville, Maryland, USA). Human myeloma and mouse-human xenogeneic myeloma cell lines have also been proposed for the production of 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)).

[0288] The culture medium in which the hybridoma cells are grown is analyzed for the 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 assay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0289] After hybridoma cells are identified that produce antibodies with the desired specificity, affinity, and / or activity, the clones can be subcloned by limiting dilution procedures and grown by standard methods (see Goding JW (Ed), Monoclonal Antibodies: Principles and Practice, supra). Suitable culture media for this process include, for example, D-MEM or RPMI 1640 medium. Hybridoma cells can also be grown in vivo as ascites tumors in animals.

[0290] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0291] The antibodies described in the present invention include antibody fragments that recognize a 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 described in the present invention 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 a disulfide bond at the hinge region.

[0292] The antibodies or antigen-binding fragments thereof described herein can 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 an animal cDNA library (e.g., a human or non-human library such as a mouse or chicken cDNA library of invaded tissue). The DNA encoding the VH and VL domains are recombined with an scFv linker by PCR and cloned into a phagemid vector. This vector is electroporated into E. coli, and the E. coli is infected with helper phage. The phages used in the methods are typically filamentous phages including fd and M13, and the VH and VL domains are generally recombinantly fused to either one of the phage gene III or gene VIII. Phage expressing an antigen-binding domain that binds to a particular antigen can be selected or identified with the antigen, for example, using labeled antigen or antigen bound or captured to a solid surface or bead. Examples of phage display methods that can be used to generate the antibodies described in the present invention 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. al., (1997) Gene 187: 9-18; Burton DR & Barbas CF (1994) Advan Immunol 57: 191-280;; PCT Application No. PCT / GB91 / 001134; International Patent Publication Nos. WO90 / 02809, WO91 / 10737, WO92 / 01047, WO92 / 18619, WO93 / 11236, WO95 / 15982, WO95 / 20401, and WO97 / 13844; and U.S. Patent Nos. 5,698,426, 5,222, 5,313, 5,411, 5,512, 5,522, 5,698, 5,712, 5,72 ... 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.

[0293] After phage selection, as described in the above references, the antibody coding region is isolated from the phage and used to generate whole antibodies, including human antibodies, or any other desired antigen-binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, as described below. Additionally, techniques for recombinantly producing antibody fragments, such as Fab, Fab', and F(ab')2 fragments, can be used using methods known in the art, such as those described 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.

[0294] 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 of skill 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 single vector expressing the necessary constant regions. The heavy chain storage vector and the light chain storage vector are then co-transfected into a cell line, using techniques known to those of skill in the art, thereby generating a stable or transient cell line expressing a full-length antibody, e.g., an IgG.

[0295] A chimeric antibody is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules. For example, a chimeric antibody can contain the variable region of a non-human animal (e.g., mouse, rat, or chicken) monoclonal antibody fused to the constant region of a human antibody. Methods for producing chimeric antibodies are known in the art. See, for example, Morrison SL (1985) Science 229: 1202-7; Oi VT & Morrison SL (1986) BioTechniques 4: 214- 221; Gillies SD et al., (1989) J Immunol Methods 125: 191-202; and U.S. Patent Nos. 5,807,715, 4,816,567, 4,816,397, and 6,331,415.

[0296] A humanized antibody can bind to a specific 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., mouse or chicken immunoglobulin). In certain embodiments, the humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically a constant region (Fc) 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 class of immunoglobulin, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including IgG1, IgG2, IgG3, and IgG4.Humanized antibodies can be produced using a variety of techniques known in the art, including, but not limited to, CDR-grafting (European Patent No. EP 239400; International Patent Publication No. WO 91 / 09967; and U.S. Patent Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (European Patent Nos. EP 592106 and EP 519596; Padlan EA (1991) Mol Immunol 28(4 / 5): 489-498; Studnicka GM et al., (1994) Prot Engineering 7(6): 805-814; and Roguska MA et al., (1994) PNAS 91: 969-973), chain remodeling (SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3), and / or other techniques. shuffling (U.S. Pat. No. 5,565,332), and see, for example, U.S. Pat. No. 6,407,213, U.S. Pat. No. 5,766,886, International Patent Publication No. WO93 / 17105; Tan P et al., (2002) J Immunol 169: 1119-25; Caldas C et al., (2000) Protein Eng.13(5): 353-60; Morea V et al., (2000) Methods 20(3): 267- 79; Baca M et al., (1997) J Biol Chem 272(16): 10678-84; Roguska MA et al., (1996) Protein Eng 9(10): 895 904; Couto JR et al., (1995) Cancer Res. 55 (23 Supp): 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): No. 959-73, also see U.S. Patent Application Publication No. US 2005 / 0042664 A1 (February 24, 2005), which is incorporated herein by reference in its entirety.

[0297] Methods for producing multispecific antibodies (e.g., bispecific antibodies) have been described (see, e.g., U.S. Patent Nos. 7,951,917; 7,183,076; 8,227,577; 5,837,242; 5,989,830; 5,869,620; 6,132,992 and 8,586,713).

[0298] Single domain antibodies, e.g., antibodies lacking light chains, can be produced by methods well known in the art (see Riechmann L & Muyldermans S (1999) J Immunol 231: 25-38; Nuttall SD et al., (2000) Curr Pharm Biotechnol 1(3): 253-263; Muyldermans S, (2001) J Biotechnol 74(4): 277-302; U.S. Patent No. 6,005,079; and International Patent Publication Nos. WO94 / 04678, WO94 / 25591 and WO01 / 44301).

[0299] Additionally, antibodies that immunospecifically bind to the FAM19A5 antigen can be used to generate anti-idiotypic antibodies that "mimic" the antigen using techniques well known to those of skill in the art (see, e.g., Greenspan NS & Bona CA (1989) FASEB J 7(5): 437-444; and Nissinoff A (1991) J Immunol 147(8): 2429-2438).

[0300] In certain embodiments, the antibodies described herein that bind to the same epitope of FAM19A5 (e.g., human FAM19A5) as the anti-FAM19A5 antibodies described herein are human antibodies or antigen-binding fragments thereof. In certain embodiments, the antibodies described herein that competitively block (e.g., in a dose-dependent manner) the antibodies described herein (e.g., 2-13 and 3-2) from binding to FAM19A5 are human antibodies or antigen-binding fragments thereof.

[0301] Human antibodies can be produced by any method known in the art. For example, transgenic mice can be used that are incapable of expressing functional endogenous immunoglobulins, but do express human immunoglobulin genes. In particular, human light and heavy chain immunoglobulin gene complexes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, human variable regions, constant regions, and diversity regions can be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes can be rendered non-functional by homologous recombination, either simultaneously with or separately from the introduction of the human immunoglobulin loci. In particular, homozygous deletion of the JH region prevents the production of endogenous antibodies. The modified embryonic stem cells are expanded and microinjected, thereby generating chimeric mice. The chimeric mice are then bred to generate homozygous offspring that express human antibodies. The transgenic mice are immunized in the normal manner with a selected antigen, for example, all or part of an antigen (e.g., FAM19A5). Monoclonal antibodies directed against the antigen can be obtained from the immunized transgenic mice using conventional hybridoma techniques. The human immunoglobulin transgenes harbored by the transgenic mice undergo rearrangement during B cell differentiation and subsequently undergo type reversal and somatic mutation. Thus, using such techniques, therapeutically useful IgG, IgA, IgM and IgE antibodies can be produced. For a general description of the techniques for producing human antibodies, see Lonberg N & Huszar D (1995) Int Rev Immunol 13:65-93. For a detailed discussion of technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., International Patent 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 5m939,598.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).

[0302] Human antibodies that specifically bind to FAM19A5 (e.g., human FAM19A5) can be produced by a variety of methods known in the art, including the above-mentioned phage display method using an antibody library derived from human immunoglobulin sequences (see also U.S. Patent Nos. 4,444,887, 4,716,111 and 5,885,793; International Patent Publication Nos. WO98 / 46645, WO98 / 50433, WO98 / 24893, WO98 / 16654, WO96 / 34096, WO96 / 33735, and WO91 / 10741).

[0303] 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 generate mouse-human hybridomas that secrete human monoclonal antibodies, and these mouse-human hybridomas can be screened to determine those that secrete human monoclonal antibodies that immunospecifically bind to a target antigen (e.g., FAM19A5, e.g., human FAM19A5). Such methods are known and described in the art, see, e.g., Shinmoto H et al., (2004) Cytotechnology 46: 19-23; Naganawa Y et al., (2005) Human Antibodies 14: 27-31.

[0304] VI. Antibody manipulation method As discussed above, anti-FAM19A5 antibodies or antigen-binding portions thereof having the VH and VL sequences disclosed 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 (etc.) attached thereto. Thus, in another embodiment described herein, the structural features of the anti-FAM19A5 antibodies described herein, such as 2-13 and 3-2, can be used to generate structurally related anti-FAM19A5 antibodies that retain at least one functional property of the antibodies described herein, such as binding to human FAM19A5. For example, the starting material for this engineering method is the VH and / or VL sequences provided herein, or one or more CDR regions thereof. It is not necessary to actually produce (i.e., express as a protein) one or more of the VH and / or VL sequences provided herein, or one or more CDR regions thereof, to generate an engineered antibody. Rather, the information contained in the sequence(s) is used as the starting material to generate a "second generation" sequence(s) derived from the original sequence(s), and the "second generation" sequence(s) is then manufactured and expressed as a protein.

[0305] Thus, there is provided herein a method for producing an anti-FAM19A5 antibody, or an antigen-binding portion thereof, comprising the steps of: (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 light chain variable region set out in Table 5; (b) generating at least one altered antibody or antigen-binding subsequence by altering at least one amino acid residue within the heavy chain variable region sequence and / or the light chain variable region sequence; and (c) expressing the altered antibody or antigen-binding subsequence as a protein.

[0306] Standard molecular biology techniques can be used to prepare and express the altered antibody or antigen-binding subsequence.

[0307] In some embodiments, the antibody or antigen-binding portion thereof encoded by the altered antibody or antigen-binding subsequence (s) retains one, some, or all of the functional properties of the anti-FAM19A5 antibodies described herein, including the following: (1) binds to soluble human FAM19A5 with a KD of, e.g., 10 nM or less (e.g., 0.01 nM to 10 nM), e.g., as measured by Biacore; (2) binds to membrane-bound human FAM19A5 with a KD of, e.g., 1 nM or less (e.g., 0.01 nM to 1 nM), e.g., as measured by Biacore; (3) binds to membrane-bound human FAM19A5 with an EC50 of, e.g., 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) suppression of excessive proliferation of reactive astrocytes; (6) decreased expression of chondroitin sulfate proteoglycans, including neurocan and neuron-glial antigen 2 (NG2); (7) increased expression of c-fos and pERK in neuronal nuclei; (8) promoting neuronal survival; (9) increased expression of GAP43 in neurons; (10) promoting axonal regrowth; and (11) Competing with 2-13, 3-2, or 1-28 for binding to human FAM19A5 in one or both directions.

[0308] The modified antibodies or antigen-binding portions thereof can 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, or all of the functional properties listed in (1)-(11) above. The functional properties of the modified antibodies or antigen-binding portions thereof can be assessed using standard assays (e.g., ELISA, FACS) available in the art and / or described herein, such as those provided in the Examples.

[0309] In certain embodiments of the antibody engineering methods described herein, mutations can be introduced randomly or selectively along all or part of the anti-FAM19A5 antibody coding sequence, and the resulting altered anti-FAM19A5 antibodies can be screened for binding activity and / or other functional properties as described herein. Mutational methods are described in the art. For example, PCT Patent Publication No. WO 02 / 092780 by Short describes methods for generating and screening antibody mutations using saturation mutagenesis, synthetic ligation assembly, or a combination thereof. Alternatively, Lazar et al. PCT Patent Publication No. WO 03 / 074679 by al. describes methods of using computational screening methods to optimize the physicochemical properties of antibodies.

[0310] VII. Cells and Vectors In certain aspects, provided herein are cells (e.g., host cells) and related polynucleotides and expression vectors that express (e.g., recombinantly) an antibody (or antigen-binding fragment thereof) described herein that specifically binds to FAM19A5 (e.g., human FAM19A5). Provided herein 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 provided herein are host cells that contain such vectors that recombinantly express an anti-FAM19A5 antibody (e.g., a human or humanized antibody) described herein. In certain aspects, provided herein are methods for producing an antibody described herein, comprising expressing such an antibody from a host cell.

[0311] Recombinant expression of an antibody described herein (e.g., a full-length antibody, a heavy and / or light chain of the antibody, or a single-chain antibody described herein) that specifically binds to FAM19A5 (e.g., human FAM19A5) involves the construction of an expression vector containing a polynucleotide encoding the antibody. After a polynucleotide encoding an antibody molecule described 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 can be generated by recombinant DNA technology using techniques well known in the art. Thus, described herein are methods for producing proteins by expression of a polynucleotide comprising an antibody or antibody fragment (e.g., light or heavy chain) encoding nucleotide sequence. Expression vectors containing an antibody or antibody fragment (e.g., light or heavy chain) coding sequence and appropriate transcriptional and translational control signals can be constructed using methods well known to those of skill in the art. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Also provided are replicable vectors comprising nucleotide sequences encoding the antibody molecules described herein, the antibody heavy or light chains, the antibody heavy or light chain variable domains or fragments thereof, or the heavy or light chain CDRs, operably linked to a promoter. Such vectors can, for example, comprise nucleotide sequences encoding the constant regions of an antibody molecule (see, e.g., International Patent Publications WO 86 / 05807 and WO 89 / 01036; and U.S. Patent No. 5,122,464), and the variable domains of an antibody can be cloned into such vectors for expression of the entire heavy chain, the entire light chain, or the entire heavy and light chains.

[0312] The expression vector can be delivered to a cell (e.g., a host cell) by conventional techniques, and the resulting cells can be cultured by conventional techniques to produce an antibody described herein (e.g., an antibody comprising one or more of VH and / or VL, or VH and / or VL CDR of 2-13, 3-2, or 1-28) or fragment thereof. Thus, provided herein is a host cell comprising a polynucleotide encoding an antibody or fragment thereof described herein, or a heavy or light chain thereof, or a fragment thereof, or a single chain antibody described herein, operably linked to a promoter for expression of 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 comprises a vector comprising a polynucleotide encoding the heavy and light chains, or fragments thereof, of an antibody described herein. In certain embodiments, the host cell comprises two different vectors, a first vector comprising a polynucleotide encoding a heavy chain or heavy chain variable region of an antibody described herein, or a fragment thereof, and a second vector comprising a polynucleotide encoding a light chain or light chain variable region of an antibody described herein, or a fragment thereof. In other embodiments, a first host cell comprises a first vector comprising a polynucleotide encoding a heavy chain or heavy chain variable region of an antibody described herein, or a fragment thereof, and a second host cell comprises a polynucleotide encoding a light chain or light chain variable region of an antibody described herein. In certain embodiments, the heavy chain / light chain variable region expressed by the first cell associates with the light chain / light chain variable region of the second cell to form an anti-FAM19A5 antibody or antigen-binding fragment thereof described herein. In certain embodiments, a population of host cells comprising such a first host cell and such a second host cell is provided herein.

[0313] In a particular embodiment, the present invention provides a population of vectors comprising a first vector comprising a polynucleotide encoding the light chain / light chain variable region of an anti-FAM19A5 antibody described in the present invention, and a second vector comprising a polynucleotide encoding the heavy chain / heavy chain variable region of an anti-FAM19A5 antibody described in the present invention.

[0314] A variety of host expression systems can be used to express the antibody molecules described in the present invention. Such host expression systems not only represent vehicles in which a coding sequence of interest can be produced and subsequently purified, but also represent cells which, when transformed or transfected with the appropriate nucleotide coding sequence, are capable of expressing the antibody molecules described in the present invention in situ. These include, but are not limited to, microorganisms such as bacteria (e.g., Escherichia coli and Bacillus subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing the antibody coding sequence; yeast (e.g., Saccharomyces pichia) transformed with recombinant yeast expression vectors containing the antibody coding sequence; insect cell systems infected with recombinant virus (e.g., baculovirus) expression vectors containing the antibody coding sequence; and cells infected with recombinant virus (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) expression vectors containing the antibody coding sequence or transfected with recombinant plasmid expression vectors (e.g., Ti plasmid). or mammalian cell systems (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK293, NSO, PER.C6, VERO, CRL7030, HsS78Bst, HeLa, and NIH3T3, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, and BMT10 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., the metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). In a particular embodiment, the cell for expressing the antibody or antigen-binding fragment thereof described in the present invention is a CHO cell, e.g., a CHO cell from CHO GS SYSTEM (Lonza). In a particular embodiment, the cell for expressing the antibody described in the present invention is a human cell, e.g., a human cell line. In a particular embodiment, the mammalian expression vector is a POPTIVEC TMor pcDNA3.3. In certain embodiments, bacterial cells such as E. coli, or eukaryotic cells (e.g., mammalian cells) are used for expression of a recombinant antibody molecule, particularly for expression of the whole recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary (CHO) cells provide effective expression systems for antibodies with vectors such as the major intermediate early gene promoter element from human cytomegalovirus (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 described herein are produced by CHO cells or NSO cells. In certain embodiments, expression of a nucleotide sequence encoding an antibody described herein that immunospecifically binds to FAM19A5 (e.g., human FAM19A5) is regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

[0315] In bacterial systems, a number of expression vectors may be advantageously selected depending on the use intended for the antibody molecule being expressed. For example, when large quantities of an antibody are to be produced for the production of a pharmaceutical composition of the antibody molecule, vectors which direct the expression of high levels of fusion protein products that are easily purified may be preferred. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruether U & Mueller-Hill B (1983) EMBO J 2: 1791-1794) (antibody coding sequences can be ligated individually into the vector in frame with the lac Z coding region to generate 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. For example, pGEX vectors can 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.

[0316] In an insect system, for example, Autographa californica nuclear polyhedrosis virus (AcNPV) can be used as a vector to express foreign genes. The virus is grown in Spodoptera frugiperda cells. The antibody coding sequence can be cloned individually into non-essential regions (e.g., the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (e.g., the polyhedrin promoter).

[0317] In mammalian host cells, a number of virus-based expression systems can be used. When adenovirus is used as an expression vector, the antibody coding sequence of interest can be ligated into an adenovirus transcription / translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into non-essential regions of the viral genome (e.g., regions E1 or E3) can result in viable recombinant virus capable of expressing the antibody molecule in infected hosts (see, e.g., Logan J & Shenk T (1984) PNAS 81(12): 3655-9). Specific initiation signals may also be required for efficient translation of the inserted antibody coding sequence. These signals include the ATG initiation codon and adjacent sequences. Also, the initiation codon must 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).

[0318] In addition, a host cell strain can be selected that modulates the expression of the inserted sequences or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for the function of the protein. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. An appropriate cell line or host system can be selected to ensure correct modification and processing of the expressed foreign protein. To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, Hela, MDCK, HEK293, NIH3T3, W138, BT483, Hs578T, HTB2, BT20, and T47D, NSO (a murine myeloma cell line that does not 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 a specific embodiment, the anti-FAM19A5 antibodies described herein are produced in mammalian cells, such as CHO cells.

[0319] In certain embodiments, the antibodies or antigen-binding portions thereof described herein have reduced or no fucose content. Such antibodies can be produced using techniques known to those of skill in the art. For example, the antibodies can be expressed in cells that are deficient or lack fucosylation capability. In certain instances, antibodies or antigen-binding portions thereof with reduced fucose content can be produced using a cell line in which alleles of 1,6-fucosyltransferase have been knocked out. The POTELLIGENT® system (Lonza) is an example of such a system that can be used to produce antibodies or antigen-binding portions thereof with reduced fucose content.

[0320] For high yield production of recombinant protein, suitable expression cells can be generated. For example, cell lines can be engineered that stably express the anti-FAM19A5 antibody or antigen-binding portion thereof described in the present invention. In a particular embodiment, the cells provided herein 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 described in the present invention.

[0321] In certain embodiments, other 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 incubation medium for 1-2 days and then transferred to selective medium. The selectable marker on the recombinant plasmid confers resistance to selection, and the cells are able to stably integrate the plasmid into their chromosomes and grow to form cores, which can then be cloned and expanded into cell lines. This method can be advantageously used to engineer cell lines expressing the anti-FAM19A5 antibodies or antigen-binding portions thereof described in the present invention. Such engineered cell lines can be useful in screening and evaluation of compositions that interact directly or indirectly with the antibody molecule.

[0322] Many systems can be used, including but not limited to herpes simplex virus thymidine kinase (Wigler M et al., (1977) Cell 11(1): 223-32), hypoxanthine guanine phosphoribosyltransferase (Szybalska EH & Szybalski W (1962) PNAS 48(12): 2026-2034), and adenine phosphoribosyltransferase (Lowy I et al., (1980) Cell 22(3): 817-23) genes, which can be used in tk-, hgprt-, or aprt- cells, respectively. Also, antimetabolite resistance can be used as the basis for selection against the following genes: (Wigler M et al., (1980) PNAS 77(6): 3567-70; O'Hare K et al., (1981) PNAS 78: 1527-31); gpt, which confers resistance to mycophenolic acid (Mulligan RC & Berg P (1981) PNAS 78(4): 2072-6); neo, which confers resistance to the aminoglycoside G-418 (Wu GY & Wu CH (1991) Biotherapy 3: 87-95; Tolstoshev P (1993) Ann Rev Pharmacol Toxicol 32: 573-596; Mulligan RC (1993) Science 260: 926-932; and Morgan RA & Anderson WF (1993) Ann Rev Biochem 62: 191-217; Nabel GJ & Feigner PL (1993) Trends Biotechnol 11(5): 211-5; and hygro, which confers resistance to hygromycin (Santerre RF e.g., (1984) Gene 30(1-3): 147-56). Methods commonly known in the recombinant DNA art are generally applied to select the desired recombinant clones, and such methods are described, for example, in Ausubel FM et al., (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler M, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli NC et al., (eds.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colbere-Garapin F et al., (1981) JMolBiol 150: 1-14, which are incorporated herein by reference in their entireties.

[0323] 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 in the antibody expression vector system is amplifiable, increasing the level of inhibitor present in the host cell culture will increase the copy number of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Crouse GF et al., (1983) Mol Cell Biol 3: 257-66).

[0324] A host cell can be co-transformed with two or more expression vectors described herein, where the first vector encodes a heavy chain derived polypeptide and the second vector encodes a light chain derived polypeptide. The two vectors can contain the same selectable marker, which allows for the same expression of heavy and light chain polypeptides. A host cell can be co-transformed with different amounts of two or more expression vectors. For example, a host cell can be transformed with one of the following ratios of the first expression vector to the second expression vector: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:12, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50. Alternatively, a single vector capable of encoding and expressing both heavy and light chain polypeptides can be used. In such a situation, the light chain must precede the heavy chain to avoid excess 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 monocistronic nucleic acid construct can encode 2, 3, 4, 5, 6, 7, 8, 9, 10 or more genes / nucleotide sequences, or in the range of 2-5, 5-10 or 10-20 genes / nucleotide sequences. For example, a bicistronic nucleic acid construct can comprise, in the following order: a promoter, a first gene (e.g., a heavy chain of an antibody described in the present invention), and a second gene (e.g., a light chain of an antibody described in the present invention). In such an expression vector, transcription of the two genes can be 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, e.g., by an IRES.

[0325] Once an antibody molecule described in the present invention has been produced by recombinant expression, it can 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 protein purification. The antibodies described in the present invention can also be fused to heterologous polypeptide sequences described in the present invention or known in the art to facilitate purification.

[0326] In certain embodiments, the antibodies described herein, or antigen-binding portions thereof, are isolated or purified. Generally, an isolated antibody is one that is substantially free of other antibodies having distinct antigenic specificities other than the antibody that was isolated. For example, in certain embodiments, preparations of the antibodies described herein are substantially free of cellular material and / or chemical precursors. "Substantially free of cellular material" is meant to include preparations of the antibody 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 antibody that have about 30%, 20%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (by dry weight) of heterologous proteins (also referred to herein as "contaminating proteins") and / or 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 the antibody is produced recombinantly, it is generally substantially free of culture medium, i.e., culture medium represents less than about 20%, 10%, 2%, 1%, 0.5%, or 0.1% of the volume of the protein preparation. When the antibody is produced by chemical synthesis, it is generally substantially free of chemical precursors or other chemicals, i.e., separated from chemical precursors or other chemicals associated with the synthesis of the protein. Thus, such preparations of the antibody have less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or compounds other than the antibody of interest. In certain embodiments, the antibodies described herein are isolated or purified.

[0327] VIII.Analysis The antibodies described in this invention can be tested for binding to FAM19A5, for example, by standard ELISA. Briefly, microtiter plates are coated with purified FAM19A5 at 1-2 μg / mL in PBS, then blocked with 5% bovine serum albumin in PBS. Dilutions of antibody (e.g., dilutions of plasma from FAM19A5-immunized mice) are added to each well and incubated for 1-2 hours at 37°C. After washing the plate with PBS / Tween, it is incubated with a secondary reagent conjugated with horseradish peroxidase (HRP) (e.g., goat-anti-human IgG Fc specific polyclonal reagent for human antibodies) for 1 hour at 37°C. After washing, the plate is developed with ABTS substrate (Moss Inc.: 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 but not to control cell lines not expressing FAM19A5. Briefly, binding of anti-FAM19A5 antibodies is assessed by incubating FAM19A5-expressing CHO cells with anti-FAM19A5 antibodies at a 1:20 dilution. Cells are washed and binding is detected with PE-labeled anti-human IgG Ab. Flow cytometry is performed using a FACS flow cytometry system (Becton Dickinson, San Jose, Calif.). Preferably, mice that generate the highest titers are used for fusions.

[0328] The ELISA assay described above can be used to screen antibodies and thus hybridomas that produce antibodies that show positive reactivity with the FAM19A5 immunogen. Hybridomas that produce antibodies that bind to FAM19A5, preferably with high affinity, can then be subcloned and further characterized. One clone that retains the reactivity of the parent cell can then be selected from each hybridoma for cell bank formation and antibody purification (by ELISA).

[0329] To purify anti-FAM19A5 antibodies, selected hybridomas can be grown in 2-liter spinner flasks for purification of monoclonal antibodies. After the supernatant is filtered and concentrated, protein A-Sepharose affinity chromatography (Pharmacia, Piscataway, NJ) can be performed. To ensure purity, the eluted IgG can be checked by gel electrophoresis and high performance liquid chromatography. The buffer solution can be exchanged into PBS, and the concentration can be determined by OD280 with an extinction coefficient of 1.43. The monoclonal antibodies can be aliquoted and stored at -80°C.

[0330] To determine whether the selected anti-FAM19A5 monoclonal antibodies bind to unique epitopes, each antibody can be biotinylated using commercially available reagents (Pierce, Rockford, Illinois). 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.

[0331] 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, the 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 less than 1 μg / mL of the test monoclonal antibody or purified isotype control for 1-2 hours at ambient temperature. The wells can then be reacted with either a human IgG1 or human IgM specific alkaline phosphatase conjugated probe. The plate is developed and analyzed as described above.

[0332] To test the binding of monoclonal antibodies to living cells expressing FAM19A5, flow cytometry can be used as described in the Examples. Briefly, cell lines expressing membrane-bound FAM19A5 (grown under standard growth conditions) are mixed with various concentrations of monoclonal antibodies in PBS containing 0.1% BSA for 1 hour at 4°C. After washing, cells are reacted with fluorescein-labeled anti-IgG antibodies under the same conditions as the primary antibody staining. Samples can be analyzed by a FACScan instrument using forward and side scatter with gating properties for single cells, and binding of the labeled antibody is determined. Other analyses using fluorescence microscopy in addition to or instead of flow cytometry can also be used. Cells can be stained exactly as described above and examined by fluorescence microscopy. This method allows visualization of individual cells, but sensitivity can be reduced depending on the density of the antigen.

[0333] Anti-FAM19A5 antibodies can be further tested for reactivity with FAM19A5 antigen by Western blotting. Briefly, cell extracts from cells expressing FAM19A5 can be prepared and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. After electrophoresis, the separated antigens can 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 by BCIP / NBT substrate purification (Sigma Chem. Co., St. Louis, MO).

[0334] Methods for analyzing the binding affinity, cross-reactivity, and binding kinetics of various anti-FAM19A5 antibodies can be performed using standard assays known in the art, such as BIACORE. TM BIACORE 2000 SPR instrument (Biacore AB, Uppsala, Sweden) TM Including surface plasmon resonance (SPR) analysis.

[0335] 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 can specifically bind to a particular epitope of FAM19A5 (e.g., SEQ ID NO:6 or a fragment within SEQ ID NO:6). 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 protein subfamily.

[0336] IX. Bispecific molecules The antibodies described herein can be used to form bispecific molecules. The 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 TGFα have been implicated as triggers for the initiation of gliosis and / or reactive astrogliosis by activating the protein signal transducer and activator of transcription 3 (STAT3) (see Balasingam et al., J. Neurosci. 14(2):846-56 (1994); Winter et al., Proc. Natl. Acad. Sci. USA20; 92(13):5865- 9 (1995)), which regulate many forms of reactive astrogliosis after CNS injury (see Herrmann JE eg, J. Neurosci. 28(28): 7231-7243 (2008)). For example, the absence or reduction of STAT3 leads to attenuated upregulation of glial fibrillary acidic protein (GFAP), impaired astroglial hypertrophy, and increased inflammatory spread, increased lesion volume, and partially impaired motor recovery after CNS injury (see Herrmann JE et al., J. Neurosci. 28(28): 7231- 7243 (2008)). Thus, for example, anti-FAM19A5 antibodies can be combined for combination therapy with antibodies or scFvs that specifically bind to any protein involved in the initiation of gliosis and / or inhibiting excessive proliferation of reactive astrocytes, such as antibodies against IL-6CNTF, LIF, EGF, or TGFα.

[0337] The anti-FAM19A5 antibody can 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 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 (see Diseases or Disorders in Chapter XIII below). For example, the anti-FAM19A5 antibody can be conjugated to an antibody or scFv that treats multiple sclerosis, such as natalizumab (Tysabri), or alemtuzumab (Lemtrada).

[0338] The antibodies described herein can in fact be derivatized or conjugated with two or more other functional molecules, thus generating multispecific molecules that bind to three or more different binding sites and / or target molecules. Such multispecific molecules are also included in the term "bispecific molecule" as used herein. To generate the bispecific molecules described herein, the antibodies described herein can be functionally conjugated (e.g., by chemical coupling, genetic fusion, non-covalent association, or other methods) with one or more other binding molecules, such as other antibodies, antigen-binding portions thereof, proteins, 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.

[0339] Thus, provided herein is a bispecific molecule comprising at least one first binding specificity for FAM19A5 and a second binding specificity for a second target epitope. In one embodiment described herein where the bispecific molecule is multispecific, the molecule can further comprise a third binding specificity.

[0340] In one embodiment, the bispecific molecules described herein comprise as binding specificities at least one antibody or an antigen-binding portion thereof, including, for example, a Fab, Fab', F(ab')2, Fv, or single chain Fv (scFv). The antibody can also be a light or heavy chain dimer, or a minimal fragment thereof, such as an Fv or single chain construct, as described in U.S. Patent No. 4,946,778 to Ladner et al., the contents of which are expressly incorporated herein by reference.

[0341] Although human monoclonal antibodies are preferred, other antibodies that can be used in the bispecific molecules described in this invention are murine, chimeric and humanized monoclonal antibodies.

[0342] Bispecific molecules described herein can be prepared by conjugating the constituent binding specificities using methods known in the art. For example, each binding specificity of the bispecific molecule can be generated separately and then conjugated to one another. When the binding specificities are proteins or peptides, a variety of coupling or cross-linking agents can be used for covalent conjugation. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) (see, e.g., Karpovsky et al., (1984)). J. Exp. Med. 160: 1686; Liu, MA et al., (1985) Proc. Natl. Acad. Sci. USA 82:8648. Other methods are described in Paulus (1985) Be bring Ins. Mitt. No. 78, 118-132; Brennan et al., (1985) Science 229:81-83), and Glennie et al., (1987) J. Immunol. 139: 2367-2375. Suitable conjugating agents are SATA and sulfo-SMCC, both of which can be purchased from Pierce Chemical Co. (Rockford, Ill.).

[0343] When the binding specificity is an antibody, the conjugation can be via sulfhydryl bonding of 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 sulfhydryl residue.

[0344] Alternatively, both binding specificities can be encoded in the same vector and expressed and assembled in the same host cell. This method allows for bispecific molecules to be constructed as mAb x mAb, mAb x mAb, or mAb x mAb. This is particularly useful for mAb x Fab, mAb x (scFv)2, Fab x F(ab')2 or ligand x Fab fusion proteins. Bispecific antibodies can include antibodies that contain scFv at the C-terminus of each heavy chain. Bispecific molecules described herein can be single chain molecules containing one single chain antibody and binding determinant, or single chain bispecific molecules containing two binding determinants. Bispecific molecules can include at least two single chain molecules. Methods for producing bispecific molecules are described, for example, in U.S. Pat. Nos. 5,260,203; 5,455,030; 4,881,175; 5,132,405; 5,091,513; 5,476,786; 5,013,653; 5,258,498; and 5,482,858.

[0345] Binding of the bispecific molecule to a specific target can be determined using art-recognized methods such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassays (e.g., growth inhibition), or Western blot analysis. Each of these assays generally detects the presence of a protein-antibody complex of particular interest using a labeled reagent (e.g., an antibody) specific for the complex of interest.

[0346] 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.

[0347] The antibodies described in the present invention can be used for diagnostic purposes, including sample testing and in vivo imaging, and for this purpose the antibodies (or binding moieties thereof) can be conjugated to a suitable detectable agent to form an immunoconjugate. Suitable agents for diagnostic purposes are detectable labels, including radioisotopes for whole body imaging, and radioisotopes, enzymes, fluorescent labels and other suitable antibody tags for sample testing.

[0348] The detectable label can be any of a wide variety of types currently used in the field of in vitro diagnostics. These include particulate labels including metal sols such as colloidal gold, isotopes such as I125 or Tc99 presented with peptide chelators of the N2S2, N3S or N4 type, chromophores including fluorescent markers, luminescent markers, phosphorescent markers, etc., as well as enzyme labels that convert a given substrate into a detectable marker, and polynucleotide tags that appear after amplification, such as by polymerase chain reaction. Suitable enzyme labels include horseradish peroxidase, alkaline phosphatase, etc. For example, the label can be the enzyme alkaline phosphatase. This is detected by measuring the presence or formation of chemiluminescence following conversion of 1,2-dioxetane substrates such as adamantyl methoxyphosphoryloxyphenyl dioxetane (AMPPD), disodium 3-(4-(methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo{3.3.1.13,7}decan}-4-yl)phenyl phosphate (CSPD), as well as CDP and CDP-STAR®, or other luminescent substrates well known to those skilled in the art, e.g., appropriate lanthanum family chelates such as terbium(III) and europium(III). The means of detection will be determined by the label chosen. The appearance of the label or its reaction products can be seen with the naked eye or, if the label is particulate and has accumulated at appropriate levels, can be seen using instruments such as spectrophotometers, luminometers, fluorometers, and the like, all following standard practices.

[0349] The antibodies described herein can also be conjugated with a therapeutic agent, thereby forming an immunoconjugate such as an antibody-drug conjugate (ADC). Suitable therapeutic agents include agents that modulate the onset of gliosis and / or reactive astrogliosis and / or agents that treat degenerative brain disorders, central nervous system injuries, or neuropathic pain. Therapeutic agents for treating degenerative brain disorders include drugs for treating Huntington's disease, Parkinson's disease, Alzheimer's disease, multiple sclerosis, and amyotrophic lateral sclerosis (ALS). This includes drugs commonly used to treat such degenerative brain disorders, such as those disclosed in Section XII below.

[0350] Immunoconjugates can be prepared by methods known in the art. Preferably, the conjugation method results in a bond that is substantially (or mostly) non-immunogenic, such as peptide-(i.e., amide-), sulfide-, (sterically hindered), disulfide-, hydrazone-, and ether bonds. These bonds are largely non-immunogenic and exhibit adequate safety in serum (see, e.g., Senter, PD, Curr. Opin. Chem. Biol. 13 (2009) 235-244; WO 2009 / 059278; WO 95 / 17886).

[0351] Depending on the biochemical properties of the moiety and the antibody, different conjugation strategies can be used. When the moiety is a naturally occurring or recombinant moiety of 50-500 amino acids, standard procedures found in textbooks describing the synthetic chemistry of protein conjugates are followed, which can be easily followed 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, for example, Fab or Fab' fragments of an antibody are used. Alternatively, in one embodiment, coupling to the C-terminal end of the antibody or moiety is performed. C-terminal modifications of proteins, such as Fab fragments, can be performed, for example, as described in Sunbul, M. and Yin, J., Org. Biomol. Chem. 7 (2009) 3361-3371.

[0352] 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 the remaining existing functional groups. For example, specific cysteines in rare sequence segments can be enzymatically converted into aldehydes (see MA, and Dierks, T., ChemBioChem. 10 (2009) 425-427). Also, the specific enzymatic reactivity of natural amino acids and specific enzymes at a given sequence segment can be used to obtain the desired amino acid modification (see, for example, Taki, M. et al., Prot. Eng. Des. Sel. 17 (2004) 119-126; Gautier, A. eg, Chem. Biol. 15 (2008) 128-136; and Protease-catalyzed formation of C-N bonds is used by Bordusa, F., Highlights in Bioorganic Chemistry (2004) 389-403).

[0353] Alternatively, site-specific reactions and covalent couplings can be achieved by selective reaction of the terminal amino acid with an appropriate modification reagent. Site-specific covalent couplings can be achieved using the reactivity of N-terminal cysteines with benzonitrile (see Ren, H. eg, Angew. Chem. Int. Ed. Engl. 48 (2009) 9658-9662). Native chemical ligation can also be based on C-terminal cysteine ​​residues (see Taylor, E. Vogel; Imperiali, B, Nucleic Acids and Molecular Biology (2009), 22 (Protein Engineering), 65-96).

[0354] EP1074563 describes a conjugation method based on the rapid reaction of cysteines within a negatively charged amino acid stretch with cysteines located in a positively charged amino acid stretch.

[0355] The moiety may also be a synthetic peptide or peptidomimetic. If the polypeptide is chemically synthesized, amino acids with orthogonal chemical reactivity can 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 great variety of orthogonal functional groups can be successfully introduced into the synthetic peptides.

[0356] 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 the use of dye-labeled binding pair members and charged linkers. By using this type of labeled, highly negatively charged binding pair member, the single conjugated polypeptide is easily separated from unlabeled polypeptides and polypeptides with two or more linkers, because the 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.

[0357] XI. Pharmaceutical Compositions Provided herein are compositions comprising the antibodies or antigen-binding portions thereof described herein having the desired degree of purity in a physiologically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, Pennsylvania). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers, such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin. , gelatin, or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0358] In certain embodiments, the pharmaceutical composition comprises an antibody or antigen-binding portion thereof, bispecific molecule, or immunoconjugate described herein, and optionally one or more additional prophylactic or therapeutic agents in a pharma- ceutically acceptable carrier. In certain embodiments, the pharmaceutical composition comprises an effective amount of an antibody or antigen-binding portion thereof described herein, and optionally one or more additional prophylactic or therapeutic agents in a pharma- ceutically acceptable carrier. In some embodiments, the antibody is the only active ingredient contained in the pharmaceutical composition. The pharmaceutical compositions described herein can enhance, induce, or activate FAM19A5 activity and be useful for treating conditions such as central nervous system injury, degenerative brain disorders, or neuropathic pain.

[0359] Pharmaceutically acceptable carriers used in parenteral formulations include aqueous vehicles, non-aqueous vehicles, antimicrobial 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 vehicles include sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, dextrose and lactate Ringer's injection. Non-aqueous parenteral vehicles include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, and peanut oil. Antimicrobial agents in bacteriostatic or fungistatic concentrations can be added to multi-dose packaged parenteral formulations, and these include phenol or cresol, mercurials, 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 citric acid. Antioxidants include sodium bisulfate. Topical anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcellulose, hydroxypropylmethylcellulose, 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.

[0360] The pharmaceutical composition can be formulated to suit any route of administration to a subject. Specific examples of routes of administration include intranasal, oral, parenteral, intrathecal, intraventricular, pulmonary, subcutaneous, or intraventricular routes. Parenteral administration, characterized by subcutaneous, intramuscular, or intravenous injection, is also contemplated by the present invention. Injectables can be prepared in conventional forms, liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or emulsions. Injectables, solutions, and emulsions also contain one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol, or ethyl alcohol. In addition, if necessary, the pharmaceutical composition to be administered can also contain small amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, pH buffers, stabilizers, solubility enhancers, and other such agents, such as sodium acetate, sorbitan monolaurate, triethyl alcoholamine oleate, and cyclodextrins.

[0361] Formulations for parenteral administration of antibodies include sterile, ready-to-inject solutions, sterile, dry soluble products which can be combined with a solvent immediately prior to use, including tablets for subcutaneous injection, such as lyophilized powders, sterile, ready-to-inject suspensions, sterile, dry insoluble products which can be combined with a vehicle immediately prior to use, and sterile emulsions. The solutions can be aqueous or non-aqueous.

[0362] If administered intravenously, suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing viscosity enhancing and solubilizing agents, such as glucose, polyethylene glycol, and polypropylene glycol, and mixtures thereof.

[0363] Topical mixtures containing the antibody are prepared as described for local and systemic administration. The resulting mixture may be a solution, suspension, emulsion, etc., and may be formulated as a cream, gel, ointment, emulsion, solution, elixir, lotion, suspension, tincture, paste, foam, aerosol, irrigate, spray, suppository, bandage, skin patch, or any other form suitable for topical administration.

[0364] The antibodies or antigen-binding portions thereof described herein can be formulated as aerosols for local administration, e.g., by inhalation (see, e.g., 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). The formulations for administration to the respiratory tract can be in the form of an aerosol or solution for nebulizers, or as ultrafine powders for inhalants, used alone or combined with an inert 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.

[0365] The antibodies or antigen-binding portions thereof described herein can be formulated into gels, creams and lotions for local or topical application, e.g., topical application to the skin and mucous membranes, e.g., to the eyes, and can be formulated for application to the eye or for intracapsular or intrathecal application. Topical administration is contemplated for transdermal delivery, and is also contemplated for ocular or mucosal administration, or for inhalation therapy. Nasal solutions of the antibodies can also be administered, either alone or in combination with other pharma- ceutically acceptable excipients. Transdermal patches, including iontophoretic and electrophoretic devices, are known to those skilled in the art and can be used to administer the antibodies. For example, such patches are described 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.

[0366] In certain embodiments, the pharmaceutical composition comprising the antibody or antigen-binding portion thereof described herein is a lyophilized powder, which can be reconstituted for administration as a solution, emulsion, and other mixture. The lyophilized powder can also be reconstituted and formulated as a solid or gel. The lyophilized powder is prepared by dissolving the antibody or antigen-binding portion thereof described herein, or a pharma-ceutically acceptable derivative thereof, in a suitable solvent. In some embodiments, the lyophilized powder is sterile. The solvent can contain excipients that improve the safety or other pharmacological components of the powder or reconstituted solutions prepared from the powder. Excipients that can be used include, but are not limited to, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable formulations. The solvent can also contain a weakly neutral pH buffer, such as citrate, sodium or potassium phosphate, or other buffers known to those of skill in the art. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those of skill in the art provides the desired formulation. In one embodiment, the resulting solution is apportioned into lyophilization vials, each containing 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.

[0367] Reconstitution of this lyophilized powder with water for injection provides a formulation for use in parenteral administration. For reconstitution, the lyophilized powder is added to sterile water or other appropriate carrier. The exact amount depends on the compound selected. Such amounts can be determined empirically.

[0368] Additionally, the antibodies or antigen-binding portions thereof, bispecific molecules, or immunoconjugates described herein and other compositions provided herein can be formulated to target specific tissues, receptors, or other body regions of the subject being treated. Many such targeting methods are well known to those of skill in the art. All such targeting methods are contemplated for use in the present compositions. Non-limiting examples of targeting methods are described, for example, in 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, 6,131,570, 6,131,570, 6,120,751, 6,131,570 ... Nos. 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 anti-FAM19A5 antibodies or antigen-binding portions thereof described herein are targeted to treat central nervous system injuries, degenerative brain disorders, or neuropathic pain.

[0369] Compositions to be used for in vivo administration can be sterilized, for example, by filtration using sterile filtration membranes.

[0370] XII. Kit Provided herein are kits comprising one or more antibodies, or antigen-binding portions thereof, bispecific molecules, or immunoconjugates thereof described herein. In certain embodiments, provided herein are pharmaceutical packs or kits comprising one or more containers filled with one or more of the components of the pharmaceutical compositions described herein, such as one or more antibodies, or antigen-binding portions thereof, provided herein, and optionally instructions for use. In some embodiments, the kits comprise the pharmaceutical compositions described herein and any prophylactic or therapeutic agents, such as those described herein.

[0371] XIII. Therapeutic Uses and Methods In one aspect, the present invention provides a method for alleviating damage or injury to the CNS of a subject, comprising administering to a subject in need of treatment an anti-FAM19A5 antibody or antigen-binding portion thereof, bispecific molecule, or immunoconjugate, or composition thereof, as described herein.

[0372] In one embodiment, the present invention provides a method for inhibiting, delaying, suppressing, preventing, reducing, reversing, or preventing the onset or onset of gliosis in a subject and the 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 disclosed in the present invention. In one embodiment, the present invention provides a method for inhibiting, delaying, suppressing, preventing, reducing, reversing, or preventing the excessive or abnormal proliferation of reactive astrocytes in a subject and the 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 disclosed in the present invention. In one embodiment, the present invention provides a method for decreasing, inhibiting or reducing expression of chondroitin sulfate proteoglycans (including levels of neurocan, NG2, or both) in a subject, or for decreasing or inactivating activity of neurocan, NG2, or both, comprising administering to the subject an anti-FAM19A5 antibody, or antigen-binding portion thereof, bispecific molecule, or immunoconjugate, or composition thereof, disclosed herein. In one embodiment, the present invention provides a method for stimulating, promoting, increasing, or activating neuronal growth in a subject, preferably following injury or damage, comprising administering to the subject an anti-FAM19A5 antibody, or antigen-binding portion thereof, bispecific molecule, or immunoconjugate, or composition thereof, disclosed herein. In one embodiment, the present invention provides a method for increasing the level of c-fos mRNA, c-fos protein, or c-fos protein activity, preferably in a neuronal nucleus, of a subject in need of treatment, comprising administering to the subject an anti-FAM19A5 antibody or antigen-binding portion thereof, bispecific molecule, or immunoconjugate, or a composition thereof disclosed herein. In one embodiment, the present invention provides a method for enhancing or increasing the level of GAP43 mRNA, GAP43 protein, or increasing GAP43 protein activity, preferably in a neuronal nucleus, of a subject in need of treatment, comprising administering to the subject an anti-FAM19A5 antibody or antigen-binding portion thereof, bispecific molecule, or immunoconjugate, or a composition thereof disclosed herein.In one embodiment, the present invention provides a method for enhancing or promoting neuronal survival and / or promoting axonal regrowth in a subject in need of treatment, comprising administering to the subject an anti-FAM19A5 antibody or antigen-binding portion thereof, bispecific molecule, or immunoconjugate, or composition thereof, as disclosed herein. In one embodiment, the subject is a human, preferably a human having neuronal injury or damage, e.g., due to CNS injury, trauma, injury, cerebrospinal cord injury, brain tumor, infection, ischemia, stroke, autoimmune response, and / or neurodegenerative disease.

[0373] In some embodiments, the present invention provides a method 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 in need of such treatment, comprising administering to the subject an anti-FAM19A5 antibody or antigen-binding portion thereof, bispecific molecule, or immunoconjugate disclosed in the present invention, or a composition thereof.

[0374] In one embodiment, the central nervous system injury is traumatic brain injury, cerebrospinal injury, stroke, brain tumor (e.g., glioma or glioblastoma), 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. Thus, in one embodiment, disclosed herein is a method for treating traumatic brain injury, cerebrospinal injury, stroke, brain tumor (e.g., glioma or glioblastoma), or a combination thereof in a subject in need of treatment, comprising administering to the subject an anti-FAM19A5 antibody or antigen-binding portion thereof, bispecific molecule, or immunoconjugate thereof disclosed herein, or a composition thereof. In one embodiment, the present invention discloses a method for treating Huntington's disease, Parkinson's disease, Alzheimer's disease, multiple sclerosis, ALS in a subject in need of such treatment, comprising administering to the subject an anti-FAM19A5 antibody or antigen-binding portion thereof, bispecific molecule, or immunoconjugate, or composition thereof, disclosed herein. In some embodiments, the subject is a human.

[0375] In some embodiments, the methods disclosed herein can be used to treat brain tumors, the methods comprising administering an anti-FAM19A5 antibody (such as those disclosed herein) to a subject in need of treatment. In certain embodiments, the brain tumor is a glioma or glioblastoma.

[0376] In some embodiments, administration of an anti-FAM19A5 antibody reduces tumor (e.g., glioblastoma) growth compared to a baseline (e.g., a corresponding value in a subject who did not receive the anti-FAM19A5 antibody). In certain embodiments, tumor growth (e.g., tumor volume or weight) is reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or up to 100% compared to a baseline (e.g., a corresponding value in a subject who did not receive the anti-FAM19A5 antibody).

[0377] In some embodiments, administration of anti-FAM19A5 antibodies promotes vascular normalization in tumors (e.g., glioblastomas). In certain embodiments, vascular normalization includes (i) an increase in the number of blood vessels, (ii) an improvement in vascular connectivity, (iii) a decrease in vascular permeability, (iv) an increase in blood flow velocity, or (v) any combination thereof. In certain embodiments, the methods disclosed herein increase the number of blood vessels expanded in tumors (along with increased thickness and improved connectivity) by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% or more.

[0378] In some embodiments, administration of an anti-FAM19A5 antibody increases immune cell infiltration into a tumor (e.g., a glioblastoma). In some embodiments, immune cell infiltration into a tumor is increased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% or more compared to a baseline (e.g., the number of immune cells present in a tumor not exposed to an anti-FAM19A5 antibody). In certain embodiments, immune cells include macrophages, dendritic cells, microglia, and T lymphocytes. In additional embodiments, the methods disclosed herein increase the cell volume of immune cells present in a tumor. In certain embodiments, the cell volume of the immune cells is increased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% or more compared to a baseline (e.g., the volume of immune cells present in a tumor not exposed to an anti-FAM19A5 antibody).

[0379] In some embodiments, administration of an anti-FAM19A5 antibody (such as those disclosed herein) increases survival of a subject with a tumor (e.g., a brain tumor). In certain embodiments, survival is increased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% or more compared to a baseline (e.g., a corresponding value for subjects who did not receive the anti-FAM19A5 antibody). In some embodiments, survival is increased by at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, 3 years, 4 years, or 5 years or more.

[0380] In some embodiments, the brain tumor (e.g., glioblastoma) that can be treated with the methods of the present invention can be metastatic, unresectable, or resistant to a prior cancer therapy. In certain embodiments, the prior cancer therapy comprises chemotherapy. In some embodiments, the chemotherapy comprises a platinum-based therapy. In some embodiments, the platinum-based therapy comprises a platinum-based anti-neoplastic agent selected from the group consisting of cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin, tetranitrate, phenanthriplatin, picoplatin, satraplatin, and any combination thereof. In certain embodiments, the platinum-based therapy comprises cisplatin. In additional embodiments, the platinum-based therapy comprises carboplatin. In some embodiments, the prior cancer therapy comprises immunotherapy (e.g., an anti-PD-1 antibody).

[0381] In some embodiments, the anti-FAM19A5 antibodies useful in the methods of the invention (for treating brain tumors, e.g., glioblastoma) can be provided in combination with other compounds, drugs, and / or preparations used to treat cancer. Such compounds, drugs, and / or preparations can include, for example, chemotherapy drugs, small molecule drugs, or antibodies that stimulate an immune response against a given cancer. In some embodiments, the methods described herein can be used in combination with standard cancer treatments (e.g., surgery, radiation, and chemotherapy). In other embodiments, the methods described herein can be used as a maintenance therapy, e.g., a therapy to prevent tumor development or recurrence.

[0382] In some embodiments, anti-FAM19A5 antibodies useful in the present invention can be combined with two or more immuno-oncology agents, e.g., a combinatorial approach that targets multiple elements in the immune pathway, e.g., one or more of the following: therapies that enhance tumor antigen presentation (e.g., dendritic cell vaccines, GM-CSF-secreting cellular vaccines, CpG oligonucleotides, imiquimod); therapies that inhibit negative immune regulation by inhibiting the CTLA-4 and / or PD1 / PD-L1 / PD-L2 pathways and / or depleting or blocking Treg or other immune suppressor cells (e.g., myeloma-derived suppressor cells); therapies that stimulate positive immune regulation by agonists that stimulate the CD-137, OX-40, and / or CD40 or GITR pathways and / or stimulate T cell effector function. therapies that systemically increase the frequency of anti-tumor T cells; therapies that deplete or inhibit Tregs, e.g., tumor Tregs, using CD25 antagonists (e.g., daclizumab) or by ex vivo anti-CD25 bead depletion; therapies that affect the function of tumor suppressor myeloma cells; therapies that enhance the immunogenicity of tumor cells (e.g., anthracyclines); adoptive T cell or NK cell transfer (CAR-T therapy) including genetically modified cells, e.g., cells transformed with chimeric antigen receptors; therapies that inhibit metabolic enzymes such as indoleamine dioxygenase (IDO), dioxygenase, arginase, or nitric oxide synthase; therapies that reverse / prevent T cell anergy or exhaustion; therapies that trigger innate immune activation and / or inflammation at the tumor site; administration of immunostimulatory cytokines; or blockade of immunosuppressive cytokines. In some examples, additional anti-cancer agents that may be combined with anti-FAM19A5 antibodies useful in the present invention may include, but are not limited to, one or more of an anti-CTLA-4 antibody, an anti-PD-1 antibody, an anti-PD-Ll antibody, an anti-OX40 (also known as CD134, TNFRSF4, ACT35 and / or TXGP1L) antibody, an anti-CD137 antibody, an anti-LAG-3 antibody, an anti-GITR antibody, or any combination thereof.

[0383] In certain embodiments, the anti-FAM19A5 antibodies disclosed herein can treat, reduce, reverse, prevent, ameliorate, control, or inhibit fibrosis in a subject in need of treatment, comprising administering an anti-FAM19A5 antibody or an antigen-binding portion thereof to the subject. In one embodiment, the fibrosis is positive (i.e., does not cause symptoms). In other embodiments, the fibrosis is associated with a pathological condition, which may or may not be recurrent. In some embodiments, the fibrosis is selected from the group consisting of liver fibrosis, pulmonary fibrosis, kidney fibrosis, bone marrow fibrosis, pancreatic fibrosis, skin fibrosis, cardiac fibrosis, arterial fibrosis, arthrofibrosis, breast fibrosis, muscle fibrosis, retroperitoneal fibrosis, thyroid fibrosis, lymph node fibrosis, bladder fibrosis, and pleural fibrosis. In one embodiment, the fibrosis is liver fibrosis or pulmonary fibrosis. In other embodiments, the fibrosis is associated with a tumor resulting from a cancer, such as liver cancer, lung cancer, kidney cancer, breast cancer, brain cancer, and / or pancreatic cancer. In one embodiment, the method attenuates, reverses, alleviates, ameliorate, inhibits, or slows or prevents fibrosis, a symptom associated with fibrosis, an underlying cause of fibrosis, or a combination thereof.

[0384] In another embodiment, the anti-FAM19A5 antibodies disclosed herein can treat, reduce, reverse, prevent, ameliorate, control, or inhibit fibrosis associated with a disorder or disease in a subject in need of such treatment, comprising administering to said subject an anti-FAM19A5 antibody, or an antigen-binding portion thereof. The term "fibrosis associated with a disease or disorder" refers to fibrosis associated with a disease or disorder, or fibrosis that is caused by or results from a disease or disorder. This term includes fibrosis resulting from or caused by a disease or disorder disclosed herein, or fibrosis associated with that disease or disorder.

[0385] In yet another embodiment, the anti-FAM19A5 antibody disclosed herein can treat, reduce, reverse, prevent, delay, ameliorate, control, or inhibit liver cancer, lung cancer, kidney cancer, breast cancer, brain cancer (e.g., glioma or glioblastoma), and / or pancreatic cancer in a subject in need of such treatment, comprising administering an anti-FAM19A5 antibody or an antigen-binding portion thereof to the subject. In some embodiments, the liver cancer, lung cancer, kidney cancer, breast cancer, brain cancer, and / or pancreatic cancer is associated with or caused by fibrosis. In some embodiments, the brain cancer is glioma or glioblastoma.

[0386] In some embodiments, a therapeutically effective amount of an anti-FAM19A5 antibody or antigen-binding portion thereof, bispecific molecule, or immunoconjugate disclosed in the present invention is administered. When treating a subject (e.g., a human), the therapeutically effective amount of an anti-FAM19A5 antibody or antigen-binding portion thereof, bispecific molecule, or immunoconjugate disclosed in the present invention depends on factors such as age, sex, and severity of the disease. Typically, an anti-FAM19A5 antibody or antigen-binding portion thereof, bispecific molecule, or immunoconjugate disclosed in the present invention is administered in a dose of 0.01 μg to 1000 mg per day.

[0387] In some embodiments, the anti-FAM19A5 antibody or antigen-binding portion thereof, bispecific molecule or immunoconjugate disclosed herein, or composition thereof, is administered intravenously, orally, parenterally, intratendinously, intrathecally, intracerebroventricularly, pulmonary, subcutaneously, or intraventricularly.

[0388] In some embodiments, an anti-FAM19A5 antibody or antigen-binding portion thereof, or a composition thereof, can be administered in combination with one or more additional agents for treating fibrosis (e.g., pirfenidone (ESBRJET®) or nintedanib (OFEV) for idiopathic pulmonary fibrosis). The dosage and administration of the one or more additional therapeutic agents are known in the art and are described, for example, in the product labeling of each agent.

[0389] In some embodiments, an anti-FAM19A5 antibody or antigen-binding portion thereof, or composition thereof, can be administered in combination with one or more additional agents for treating central nervous system injury (e.g., traumatic brain injury, cerebrospinal injury, stroke, or brain tumor (e.g., glioma or glioblastoma)), cerebrospinal system injury, degenerative brain disorder (e.g., Huntington's disease, Parkinson's disease, Alzheimer's disease, multiple sclerosis, ALS), degenerative cerebrospinal or neurological disorders, or neuropathic pain. For example, non-limiting exemplary agents for treating Huntington's disease include tetrabenazine (XENAZINE®), antipsychotics such as haloperidol (HALDOL®), chlorpromazine, risperidone (RISPERDAL®), and quetiapine (SEROQUEL®).

[0390] Non-limiting exemplary formulations for the treatment of Parkinson's disease include levodopa (with or without carbidopa), dopamine agonists such as pramipexole (MIRAPEX®), ropinirole (REQUIP®), and rotigotine (NEUPRO®), and apomorphine (APOKYN®), selegiline (ELDEPRYL® and ZELAPAR®), rasagiline (AZILECT®), entacafone (COMTAN®), benztropine (COGENTIN®), trihexyphenidyl, and amantadine.

[0391] Non-limiting exemplary agents for the treatment of Alzheimer's disease include donepezil (ARICEPT®), galantamine (RAZADYNE®), and rivastigmine (EXELON®).

[0392] Non-limiting exemplary formulations for the treatment of multiple sclerosis include glatiramer acetate (COPAXONE®), dimethyl fumarate (TECFIDERA®), fingolimod (GILENYA®), teriflunomide (AUBAGIO®), natalizumab (TYSABRI®), alemtuzumab (LEMTRADA®), and mitoxangorone.

[0393] A non-limiting exemplary formulation for the treatment of ALS includes riluzole (RILUTEK®).

[0394] 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 each agent.

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

[0396] (Example) In the examples that follow, reference is made to the following experimental methods and details.

[0397] Example 1: Expression and purification of human FAM19A5 protein Recombinant human FAM19A5 protein was produced and purified, and the purified protein was used for antibody screening assay based on binding affinity analysis. First, LPS-hT plasmid containing FAM19A5 gene was transformed into bacteria to induce protein overexpression. After production, FAM19A5 protein was purified using Ni-NTA affinity chromatography (Qiagen, Valencia, California, USA). The His-tagged FAM19A5 protein was removed from the Ni column by gradually 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 by Western blotting analysis. The concentrated protein was then used to screen FAM19A5-specific antibodies.

[0398] Example 2: Generation of the FAM19A5 antibody library 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, Missouri, USA) and then subcutaneously injected into three chickens. The chickens were immunized a total of three times at intervals of approximately 2-3 weeks. The titers of antibodies obtained from the immunized chickens were measured by immunoblotting using cell lysates of HEK293T cells overexpressing FAM19A5 protein.

[0399] Single-chain variable fragment (scFv) libraries were prepared from immunized chickens using TRI Reagent (Invitrogen, Carlsbad, California, USA), and RNA was extracted from the spleen, bone marrow, and bursa of immunized chickens as described above.

[0400] Oligo-dT primers and SUPERSCRIPT TMFirst-strand cDNA was synthesized using the III First-Strand Synthesis System (Invitrogen). A single-stranded variable region library was generated using the Extended High-Fidelity PCR System (Roche Molecular Systems, Indiana, USA) for cDNA obtained from chicken immune system. For each reaction, 1 μL of cDNA, 60 pmol of each primer, 10 μL of 10× reaction buffer solution, 8 μL of 2.5 mM dNTPs (Promega, Madison, Wisconsin, USA), and 0.5 μL of Taq DNA polymerase were mixed with water. The final volume was 100 μL. PCR reactions were performed under the following conditions: (i) 94°C for 15 s, (ii) 56°C for 30 s, and (iii) 72°C for 90 s for 30 cycles, followed by a final extension at 72°C for 10 min. The PCR product containing a fragment having a length of about 350 bp was loaded onto a 1.5% agarose gel, and after electrophoresis, the nucleotide fragment was purified using a QIAGEN Gel II extraction kit (QIAGEN, Valencia, California, USA). The purified PCR product was quantified by reading at OD 260 nm (1 unit OD = 50 μg / ml).

[0401] 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 of 10× reaction buffer, 8 μL of 2.5 mM dNTPs, 0.5 μL of Taq DNA polymerase, and water to a final volume of 100 μL. PCR was performed under the following conditions: (i) 94° C. for 15 s, (ii) 56° C. for 30 s, and (iii) 72° C. for 2 min for 25 cycles, followed by a final extension at 72° C. for 10 min. The PCR products containing single-stranded variable region fragments with a length of about 700 bp were loaded onto a 1.5% agarose gel, and after electrophoresis, the nucleotide fragments were purified using a QIAGEN Gel II extraction kit (QIAGEN). The purified PCR products were quantified by reading at OD260 nm (1 unit OD=50 μg / ml).

[0402] The scFv fragment of the PCR product and the vector pComb3X-SS (The Scripps Research Institute, California, USA) were digested with Sfi I restriction enzyme. 10 μg of the purified overlap PCR product was mixed with 360 units of Sif I (1 μg DNA per 16 units, Roche Molecular Systems, Pleasanton, California, USA), 20 μL of 10× reaction buffer, and water to a final volume of 200 μL. 20 μg of the pComb3X-SS vector was mixed with 120 units of Sfi I (1 μg DNA per 6 units), 20 μL of 10× reaction buffer solution, and water to a final volume of 200 μL. The mixture was digested at 50° C. for 8 hours. The enzymatically cleaved products, including the scFv fragment (approximately 700 bp) and vector (approximately 3400 bp), were then loaded onto 1% agarose and purified using a Gel Extraction Kit II QIAGEN (QIAGEN, Valencia, California, USA). 1400 ng of the Sfi I restricted pComb3X vector and 700 ng of the enzymatically cleaved scFv fragment were mixed with 5x ligase buffer, T4 DNA ligase (Invitrogen, Carlsbad, California, USA) and water to a final volume of 200 μL. The mixture was incubated at 16°C for 16 hours to accomplish the ligation.

[0403] After ethanol precipitation, the DNA pellet was dissolved in 15 μL of water. To generate the library, the ligation samples were electroporated into Escherichia coli strain ER2738 (New Jersey, USA) using a gene pulser (Bio-Rad Laboratories, Hercules, California, USA). The cells were transformed into 1000 ng / ml PBS (England Biolabs Inc., Hitchin, Hertfordshine, SG4OTY, England, UK). The cells were mixed with 5 mL Superbroth (SB) medium and incubated at 37°C for 1 h with agitation at 250 rpm. 3 μL of 100 mg / mL kanamycin was then added to 10 mL of SB medium. To determine library size, 0.1 μL, 1 μL, and 10 μL culture samples were smeared onto 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 agitated for an additional 1 h. 2 mL of VCM13 helper phage in water (>10 11 cfu / ml) was added to LB medium along with 92.5 μL of pre-warmed LB (183 mL) containing 100 mg / mL kanamycin. The mixture was stirred at 250 rpm at 37 °C for an additional 2 h. 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 at 4 °C in a high-speed centrifuge (Beckman, JA-10 rotor). The bacterial pellet was then used to extract phagemid DNA, and the supernatant was transferred into a sterile centrifuge bottle. Then, 8 g of polyethylene glycol-8000 (PEG-8000, Sigma) and 6 g of sodium chloride (NaCl) were added to the supernatant, which was then kept on ice for 30 min. The supernatant was then centrifuged at 15,000 g for 15 min at 4 °C. The supernatant was then discarded and the phage pellet was suspended in tributyltin-buffered saline (TBS).

[0404] Example 3: Library panning on immobilized antigen (bio-panning) Biopanning was performed using magnetic beads (Dynabeads M-270 Epoxy, Invitrogen). The beads and protein were stirred together at room temperature for 20 hours to obtain approximately 1 × 10 7Beads were coated with 5 μg of recombinant FAM19A5 protein. After coating was complete, the beads were washed four times with phosphate-buffered saline (PBS) and blocked for 1 h at room temperature in 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 / hydrogen chloride (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 using VCSM13 helper phage. In addition, the input and output from the phage-infected cultures were determined by blotting the phage-infected cultures on LB agar plates containing 50 μg / mL kanamycin. The next day, the phages were precipitated using PEG-8000 and NaCl and then used for biopanning. The above process was repeated for a total of five rounds of biopanning. After each amplification, the phages were screened to select those with high affinity for the FAM19A5 protein.

[0405] 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 selected clones bound to FAM19A5 recombinant protein. FAM19A5 recombinant protein was diluted in 0.1M NaHCO3 buffer and 100ng of protein per well was used to coat 96-well microtiter plates at 4°C for 16 hours. The next day, the plates were 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 plates containing the supernatant were then washed with 0.05% Tween 20 / PBS. HRP-conjugated M13 antibody (a-M13-HRP, Pierce Chemical) was used to coat 96-well microtiter plates at 4°C for 16 hours. Co, Rockford, Illinois, USA) was diluted 1 / 5000. 50 μL of the diluted antibody was added to the plate and incubated at 37°C for 1 h. After incubation and washing, 0.05 M citrate buffer, 1 μg / mL 2,2'-azino-bis(3-ethylbenzothiazolyl-6-sulfonic acid) (ABTS, Amresco, Solon, Ohio, USA), and 0.1% H2O2 were added to the plate for color development. The absorbance of each well was measured at 405 nm.

[0406] Then, 24 clones that bound to the FAM19A5 recombinant protein and showed high absorbance were analyzed, and 13 scFv clones with unique sequences were obtained from these. After further selection, clones 2-13, 1-28, and 3-2 were obtained.

[0407] Example 5: Generation of anti-FAM19A5-IgG2 / 4 antibody Anti-FAM19A5 scFv was subcloned into a mammalian expression vector. In the FAM19A5 scFv gene sequence, human Cκ gene was linked to the light chain variable region, and human immunoglobulin isotype IgG2 / 4 of CH1, 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). To facilitate cloning, the synthesized genes were inserted into a mammalian cell expression vector with modified restriction sites. First, the light chain gene was inserted into the vector using HindIII and XbaI (New England Biolabs, UK) restriction enzymes, and then the heavy chain gene was added to the vector using NheI and BamHI (New England Biolabs, UK) restriction enzymes.

[0408] A mammalian cell transformation and overexpression injection system was used to express and purify anti-FAM19A5-IgG2 / 4 antibody. 4 μg of polyethyleneimine (PEI, Polysciences, Warrington, Pennsylvania, USA) and 2 μg / mL of mammalian expression vector were mixed in 150 mM sodium chloride (NaCl, Merck) corresponding 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 (2 × 10 6 After addition of 100 U / mL penicillin and streptomycin (Invitrogen) to the cells, the cells were incubated in FREESTYLE containing 100 U / mL penicillin and streptomycin (Invitrogen) at 37°C with 7% CO2 and stirring at 135 rpm for 6 days. TM The cells were incubated in 293 expression medium. Protein A bead (RepliGen, Waltham, Massachusetts, USA) affinity gel chromatography was used to purify the expressed anti-FAM19A5 IgG2 / 4 antibody from cell culture supernatant. Protein A chromatography was run on a 4-12% Bis-Tris gradient gel electrophoresis. Protein size and yield were confirmed by Coomassie brilliant blue staining.

[0409] Example 6: Epitope mapping analysis using FAM19A5 epitope fragments F1-F6 Overlapping peptide fragments (F1-F6, see FIG. 1) 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 determined by ELISA analysis. Briefly, FAM19A5 fragments F1-F6 (diluted in 50 mM carbonate buffer (Biosesang) at 1 μg / mL or 20 μg / mL for concentration analysis) were used to coat (100 μL / well) wells of a 96-well immunoplate (Thermo Scientific) 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 antibodies were diluted in dilution buffer to 1 μg / mL (or 20 μg / mL for concentration analysis). After washing the plate (twice with 1x PBS), diluted anti-FAM19A5 antibody was added to the appropriate wells and the plate was incubated at room temperature for 1 hour. The plate was then washed a total of five times with washing buffer. ODP substrate (prepared by dissolving one ODP tablet (O-phenylenediamine dihydrochloride, Thermo) in 9 mL of sterile deionized water and 1 mL of 10x stable peroxide stable buffer (Thermo)) was then added to each well and the color change reaction was allowed to occur for 10 minutes. The reaction was terminated by adding 100 μL of 2N H2S04 (Daejung) to the wells. The absorbance value of each well was detected at 492 nm using a 96-well microplate reader (Molecular Device).

[0410] As shown in Figure 2, anti-FAM19A5 antibody 3-2 bound strongly to epitope fragment F2, with minimal binding to the remaining fragments. This was also the case for anti-FAM19A5 antibody 1-28 (data not shown). Meanwhile, 1-65 antibody bound strongly only to epitope fragment F5. Interestingly, it appeared that anti-FAM19A5 antibody 2-13 did not bind to any of the F1-F6 epitope fragments.

[0411] Next, to confirm the specific amino acid residues in the epitope fragment F2 to which 3-2 and 1-28 bound, different amino acid residues in the F2 fragment were substituted with alanine or valine as shown in Table 9 (below). The mutated residues are underlined in bold. The binding affinity of the indicated anti-FAM19A5 antibodies was measured using ELISA as described above.

[0412] [Table 8]

[0413] As shown in Figures 3A and 3B, the 3-2 antibody bound strongly to mutant F2 peptide fragments #1, 2, 4-6 and 9-13, with reduced binding to fragments #3, 7 and 8. The latter three mutant F2 peptide fragments contain alanine substitutions at amino acid residues D5, P9 and R11 (numbered according to SEQ ID NO:6 in Table 9 above), respectively, suggesting that the 3-2 antibody binds to FAM19A5 primarily at these amino acid residues.

[0414] The 1-28 antibody bound strongly to mutant F2 peptide fragments #1, 2, 6 and 8-13, whereas the 1-28 antibody showed much reduced binding to peptide fragments #3, 4, 5 and 7 (see FIG. 3C). This data suggests that the anti-FAM19A5 antibody 1-28 binds to FAM19A5 primarily at amino acid residues D5, S6, S7 and P9 (numbered according to SEQ ID NO:6 in Table 9 above) within the epitope F2 fragment.

[0415] Example 7: Epitope mapping analysis using FAM19A5 mutants M1-M8 To further characterize the binding epitope of the anti-FAM19A5 antibody disclosed in the present invention, the amino acid sequences of different FAM19 family members (i.e., FAM19A1-5) were aligned as shown in Figure 4. Based on this alignment, eight regions (M1-M8) were identified in which the amino acid sequence of the FAM19A5 protein was most significantly different from the remaining members of the FAM19A family (i.e., FAM19A1-4). The amino acid sequences of these regions were replaced with the consensus sequences of the corresponding regions of the FAM19A1-4 protein (see Table 10, where the mutated amino acid residues are underlined in bold).

[0416] Mutant FAM19A5 expressing phages were prepared as follows. To prepare a medium for phage culture, 55.6 mL of 2 M glucose (D-(+)-glucose, Sigma), 5 mL of 1 M MgC12 (magnesium chloride, Junsei), and 1 mL of 34 mg / mL chloramphenicol (Sigma) were added to 2x YT medium. Colonies obtained through monophage ELISA were selected and placed in 5 mL of the 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 at 37°C in an incubator until the OD600nm detection value reached 0.5. When the OD600nm detection value reached 0.5, 5 mL of each culture was obtained as an infected sample. After sample preparation, 50 μL of M1 helper phage was added to each sample and incubated at 37°C for 30 min without shaking and another 30 min with shaking. The individual cultures were centrifuged at 3,850 rpm for 15 min in a microcentrifuge (Micro12, 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 pellet was completely dispersed in the freshly added medium and incubated at 30°C for 16 h with stirring.

[0417] [Table 9]

[0418] As shown in Figure 5, anti-FAM19A5 antibodies 1-28 and 3-2 were unable to bind to the FAM19A5 mutant M2. As shown in Table 10 (above), the M2 mutant has a substitution at amino acid residues 21-25 (i.e., epitope EP2), which corresponds to a region within the epitope fragment of FAM19A5. This data is consistent with the data in Examples 1-6 and confirms the importance of epitope fragment F2 in the binding of the 1-28 and 3-2 antibodies to FAM19A5.

[0419] It was found that the 2-13 antibody binds to FAM19A5 with an epitope completely different from that of the 1-28, 3-2 and 1-65 antibodies. As shown in FIG. 5, the anti-FAM19A5 antibody had intermediate to high binding to the FAM19A5 mutants M1-M3 and M5-M7, but was unable to bind to the mutants M4 and M8. This suggests the importance of epitopes EP4 and EP8 in the 2-13 antibody binding to FAM19A5. Considering that the 2-13 antibody was unable to bind to any of the FAM19A5 epitope fragments in Example 7 (see FIG. 2), this data also suggests that the 2-13 antibody has a conformational epitope as opposed to a linear epitope.

[0420] Example 8: Cross-competition analysis As described below, a two-site sandwich ELISA was used to assess whether different anti-FAM19A5 antibodies with similar binding epitopes cross-compete with each other (see FIG. 6A).

[0421] First, anti-FAM19A5 antibody was diluted in 1x PBS to a concentration of 10 μg / mL. The diluted anti-FAM19A5 antibody (10 μg / mL 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) and blocked with blocking solution (5% BSA / PBS; also known as 5% PBSA, 250 μL / well) for 1 hour at 37°C, and then washed again. Then, 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 with 0.01% PBST. After the final wash, 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 incubated for a further 1 hour at 37°C. The plates were then washed a further time with 0.01% PBST (5 washes total). Then, 100 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 minutes at room temperature. The plates were then washed and treated with 100 μL of TMB substrate (Thermo Fisher Scientific). After a further 30 minutes of incubation at room temperature, the TMB substrate was added to induce the color change reaction. The reaction was stopped using 50 μL of sulfuric acid (2N H2S04), and the color change extent was detected by absorbance at 450 nm with a reference wavelength of 620 nm using a 96-well microplate reader (Molecular Device).

[0422] As shown in Figure 6B, anti-FAM19A5 antibodies 1-65, P2-A03, P2-F11 and 13B4 all cross-competed with each other. Similar to the 1-65 antibody, the P2-A03, P2-F11 and 13B4 antibodies all bind to FAM19A5 at epitopes M6, M7 and / or M8 (i.e., within the F5 and / or F6 epitope fragments) (data not shown). Meanwhile, antibodies 2-13 and 3-2 did not cross-compete with the remaining anti-FAM19A5 antibodies. This confirms the epitope mapping analysis described above, which showed that these antibodies bind primarily at the F2 epitope fragment.

[0423] Example 9: Binding affinity analysis To determine the binding affinity of different anti-FAM19A5 antibodies to FAM19A5, ELISA analysis was used as described above (see, e.g., Example 6). As shown in Figures 7A and 7B, the 1-28, 3-2 and 1-65 antibodies bound to FAM19A5 with similar binding affinities (Kd = 0.17 nM, 0.12 nM and 0.10 nM, respectively). The 2-13 antibody bound with a Kd value of about 2.77 nM (see Figure 7B).

[0424] Example 10: Serum analysis in liver fibrosis To evaluate the in vivo function of the anti-FAM19A5 antibody disclosed in the present invention, a rat model of liver fibrosis (i.e., bile duct ligation (BLD)-induced liver fibrosis Sprague-Dawley (SD) rat model) was used to measure the level of FAM19A5 protein in liver fibrosis (see Kountouras et al., Br J Exp Path 65:305-311 (1984)). Briefly, animals were anesthetized with ketamine and xylazine. A 1.5 cm midline gland incision was performed to locate the common bile duct, and double ligation was performed with 3-0 silk ligature. Then, whole blood was collected from the animals on the 21st day after induction of liver fibrosis, and serum was separated from the whole blood.

[0425] FAM19A5 protein levels in serum were measured by FAM19A5 sandwich ELISA assay. 96-well ELISA plates were coated overnight with 1-65 human IgG1 anti-FAM19A5 antibody at 1 μg / mL in a volume of 100 μL. The plates were then washed with PBS and then blocked with 1% BSA in PBS for 1 hour at room temperature. The plates were then washed again with PBS and approximately 100 μL of standards and samples were distributed into the appropriate wells. The plates were then incubated at room temperature for 2 hours. The plates were then washed with 0.05% PBS-Tween 20 (i.e., wash buffer) and approximately 100 μL of detection antibody (horseradish peroxidase (HRP)) conjugated to goat a-rabbit IgG antibody (Santa Cruz, Cat # SC-2030, diluted 1:5000 in 1% BSA in PBS) was added to the wells. The plates were incubated at room temperature for an additional hour. After an additional wash with wash buffer, approximately 100 μL of O-phenylenediamine dihydrochloride (OPD) substrate was added to each well and the plates were developed at room temperature. After approximately 10 minutes, the reaction was stopped by adding approximately 100 μL of 1N H2SO4 (sulfuric acid) to the wells. The plate was then read using a 96-well microplate reader (Molecular The absorbance was measured at a wavelength of 492 nm using a spectrophotometer (Versa Max).

[0426] As shown in FIG. 8, FAM19A5 protein levels were more than three-fold higher in the serum of BDL-induced hepatic fibrosis SD rats ("disease model") compared to normal (ie, non-BDL-induced) rats.

[0427] Example 11: Serum analysis in pulmonary fibrosis To assess whether FAM19A5 protein levels were also increased in pulmonary fibrosis, idiopathic pulmonary fibrosis was induced in 7-week-old male SD rats by intratracheal injection of 3 mg / kg bleomycin. Whole blood was collected from the animals 24 days after bleomycin administration, and serum was separated from the whole blood. Serum FAM19A5 protein levels were measured by FAM19A5 sandwich ELISA assay as described above in Example 10. As shown in FIG. 9, FAM19A5 protein levels were more than 1.5-fold higher in serum from SD rats with pulmonary fibrosis induction ("disease model") compared to normal (i.e., no idiopathic pulmonary fibrosis) rats.

[0428] Example 12: Serum analysis of human liver fibrosis patients Upon hepatocyte injury, serum AST and / or ALT levels can increase, and such increases are primarily thought to indicate liver injury (Gowda et al., Pan Afr Medjy. M (2009); (See Giannini et al., Arch Intern Med 163:218-224 (2003)). However, when damage is severe (e.g., cirrhosis), AST and ALT levels can be shown as normal. Thus, to assess whether FAM19A5 expression is a better indicator of liver damage, FAM19A5 protein levels were measured in serum from human patients with confirmed cirrhosis using a FAM19A5 sandwich ELISA assay.

[0429] 2-13 Anti-FAM19A5 human IgG1 antibody was reconstituted in 50 mM carbonate buffer (Bio-World, Cat. C2070-9.6, Lot. C70-9.616Z11C) at a concentration of 1 μg / μL. The anti-FAM19A5 antibody was then diluted in PBS at a ratio of 1:1000 and then used to coat a 96-well ELISA plate overnight. The next day, the plate was washed with PBS and then blocked with 1% BSA in PBS at room temperature for about 1 hour. The plate was then washed again with PBS and approximately 100 μL of standards and samples were distributed into the appropriate wells. The plate was incubated at room temperature for 2 hours. The plate was then washed with 0.05% PBS-Tween 20 (i.e., wash buffer). Then, 3-2 anti-FAM19A5 mouse IgG2a antibody (reconstituted at a concentration of 1 μg / μL) was diluted in 1% BSA in PBS at a ratio of 1:2000. Approximately 1 μL of this diluted antibody was added to each well, and the plate was incubated at room temperature for another 2 hours. After further washing with washing buffer, approximately 100 μL of horseradish peroxidase (HRP) conjugated to donkey α-mouse IgG antibody (Jackson Lab, Cat. 715-035-151, Lot. 122401) (diluted 1:5000 in 1% BSA in PBS) was added to the well. The plate was then incubated at room temperature for 1 hour. After incubation, the plate was washed with washing buffer and incubated with 1-STEP TM Approximately 1 μL of Ultra TMB-ELISA substrate solution (Pierce, Cat. 34028, Lot. QJ20436540) was added to each well. After 10 minutes of incubation, approximately 100 μL of 1N H2SO4 (sulfuric acid) was added to the wells to terminate the reaction. The absorbance was measured at a wavelength of 450 nm using a 96-well microplate reader (Molecular Device, Versa Max).

[0430] As shown in Figure 10, patients with cirrhosis had FAM19A5 protein levels approximately 2-6 times higher than those observed in the serum of normal healthy individuals. These data are consistent with the animal studies mentioned above, and suggest that serum concentrations of FAM19A5 protein may be an ideal diagnostic marker for cirrhosis.

[0431] (Example 13: Analysis of effect on myocardial infarction) Ischemic cell death that occurs during myocardial infarction leads to a repair response in which damaged tissue is replaced by a fibrotic scar, followed by remodeling of the surrounding tissue (e.g., thinning of the left ventricular wall and ventricular dilation), ultimately impairing cardiac function (Talman et al., Cell Tissue Res 365(3): 563-581 (2016); see Firth et al., Cardiovasc Drugs Ther 4:1363-1374 (1990)). heart To evaluate the effect of anti-FAM19A5 antibody on myocardial infarction, a mouse ischemia-reperfusion model was used. Briefly, Balb / cAnNCrljOri mice (6 weeks old, male) (Orient Bio Inc., Seoul, Korea) were purchased and housed under specific pathogen-free (SPF) conditions for approximately 1 week. The mice were then deeply anesthetized with Zoletil 50 (VIRBAC, France) and xylazine (Rompun®, Bayer AG, Germany). After anesthesia, a thoracotomy was performed on the mice to expose the coronary artery, and the artery was ligated using a 7-0 suture and a PE20 tube to induce myocardial infarction. After air intubation, the endotracheal tube was connected to a ventilator to maintain forced breathing of the mice. The ischemic state was maintained for 40 minutes, and the PE20 tube was removed to allow blood to be reperfused. Anti-FAM19A5 antibodies (1-65, 3-2, or 1-28 antibodies) were administered intravenously to mice on day 3 after induction of myocardial infarction, for a total of three weekly injections. Naive (i.e., healthy) mice and NHI mice served as control groups.

[0432] On the 21st day after induction of myocardial infarction, the mice were sacrificed and the hearts were harvested. The cardiac tissues were fixed in 10% formalin solution. The fixed cardiac tissues were then sliced ​​transversely at 2 mm thickness near the apex, placed in paraffin blocks, and further sliced ​​into 5 μm sections, mounted on glass tissue slides, and analyzed. Hematoxylin and eosin (H&E) staining was performed on the cardiac tissue samples to evaluate the effect of each anti-FAM19A5 antibody (i.e., 1-65, 3-2, and 1-28 antibodies) on myocardial infarction-related tissue remodeling.

[0433] As expected, cardiac tissue from the NHI control group showed significant left ventricular dilatation and thinning of the left ventricular wall (see Figure 11 ("NHI")). In contrast, cardiac tissue from mice treated with anti-FAM19A5 antibody was similar to cardiac tissue from naive ("healthy") animals (i.e., reduced left ventricular dilatation and preserved left ventricular wall tissue).

[0434] To confirm this effect, Masson's trichrome staining, which stains collagen, was performed on cardiac tissue sections. As previously mentioned, collagen is a major component of the extracellular matrix (ECM), so Masson's trichrome staining of increased collagen accumulation in tissue samples may be an accurate indicator of fibrosis. The stained slides were photographed using an optical microscope (Olympus BX53, Japan), and the ratio of fibrotic area to total left ventricular area was calculated using an image analyzer.

[0435] As shown in Figure 12A, mice receiving anti-FAM19A5 antibodies (1-65, 3-2, and 1-28) had much less collagen accumulation than the NHI control group. In the anti-FAM19A5 antibody treatment group, approximately 10-15% of the total left ventricular area was composed of fibrous tissue (see Figure 12B). In contrast, in the NHI control group mice, the fibrous area accounted for approximately 20% of the total left ventricular area. Among the different FAM19A5 antibodies, the 1-65 antibody appeared to have the greatest effect.

[0436] Taken together, the above results demonstrate that administration of FAM19A5 antibody immediately after myocardial infarction can significantly ameliorate reorganization and fibrosis formation.

[0437] Example 14: Analysis of human liver cancer patients Liver fibrosis and cirrhosis are major risk factors for liver cancer. In human liver cancer patients, fibrosis similar to cirrhosis frequently occurs around cancerous tissues. Therefore, to evaluate FAM19A5 expression in liver cancer, we measured FAM19A5 protein expression in human liver biopsies using immunohistochemistry. Briefly, liver samples were obtained from liver cancer patients with various degrees of fibrosis (i.e., stage #0 to stage #4). Tissue samples were immunostained with anti-FAM19A5 antibody and then counterstained with hematoxylin and eosin (H&E).

[0438] As shown in Figure 13, there was a significant increase in FAM19A5 protein expression in liver tissues from stage #2 and stage #4 cancer patients compared to stage #0 patients. The increased FAM19A5 expression was mainly localized near the scar formation area and in hepatic stellate cells. The increase in FAM19A5 expression was associated with disease progression, and stage #4 liver tissues expressed much higher levels of FAM19A5 compared to stage #2 liver tissues. Taken together, this data demonstrates that FAM19A5 also plays an important role in human liver cancer.

[0439] Example 15: Analysis of efficacy of anti-FAM19A5 antibody administration in a liver cancer xenograft model To evaluate the antitumor efficacy of anti-FAM19A5 antibody against human hepatoma, a xenograft mouse model of hepatoma was used. Briefly, nude mice were purchased and housed under special pathogen-free (SPF) conditions. After an adaptation period of approximately one week, Hep3B cells and / or human hepatic stellate cells (HHSteCs) were subcutaneously injected into the animals. For this purpose, the cells were first treated with trypsin to prepare a single cell suspension. The cells were then washed and cultured at approximately 5 × 10 6 Hep3B and / or 0.4 x 10 6The HHSteC cells were resuspended in 100 μL of DMEM medium. The cells were then subcutaneously injected into the right flank of nude mice using an insulin syringe. The animals were observed for tumor formation approximately 3 weeks after injection.

[0440] At the time of tumor formation (3 weeks after injection), animals were injected intravenously with either normal human immunoglobulin (NHI, control group) or 3-2 human IgG1 anti-FAM19A5 antibody (2.5 mg / kg). Animals received antibody once weekly for a total of 3 weeks. Body weight and tumor size were assessed weekly. Animals were sacrificed 42 days after inoculation and tumors were further analyzed.

[0441] As shown in Figure 14A, animals in all different groups have similar body weights throughout the experimental period. As shown in Figures 14B-14D, administration of anti-FAM19A5 antibody has minimal anti-tumor effect on animals inoculated with Hep3B cells alone (i.e., "Hep3B+NHI" and "Hep3B+FAM19A5 Ab"). However, in animals inoculated with Hep3B cells and HHSteC, administration of anti-FAM19A5 antibody resulted in a significant reduction in tumor size / weight (i.e., "Hep3B+HHSteC+NHI" v. "Hep3B+HHSteC+FAM19A5 Ab"). These data suggest that the interaction of anti-FAM19A5 antibody with hepatic stellate cells in the tumor microenvironment can treat liver cancer.

[0442] Example 16: Effect of anti-FAM19A5 antibody administration after traumatic brain injury A traumatic brain injury (TBI) model was used to evaluate the effect of the anti-FAM19A5 antibody disclosed in the present invention on traumatic brain injury. Briefly, C57BL / 6 adult male mice (8-9 weeks old) were deeply anesthetized with isoflurane. Cryogenic TBI was performed by placing an iron bar (pre-cooled using liquid nitrogen) on the calvaria for 1 minute. The skin of the mice was then sutured and confined in the same manner as normal mice (see Moon et al., Neuro Report 22: 304-308 (2011)). Approximately 1 day after TBI induction, the animals were intravenously administered 100 μg of different anti-FAM19A5 antibodies (1-65 (2 / 4); 1-28; 2-13; and 3-2) diluted in phosphate-buffered saline (PBS).

[0443] On day 5 after TBI induction (TBI5D), animals were sacrificed and perfused with 4% paraformaldehyde (PFA) in PBS, and brain tissue was harvested. Harvested brain tissue was fixed in 4% PFA solution for an additional 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.

[0444] To stain reactive astrocytes (positive for nestin and GFAP expression) associated with gliosis, 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, Mesa, 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 and observed under a fluorescence or confocal microscope (Leica, Wetzlar, Germany).

[0445] To quantify the effect, we first identified ROIs (regions of interest) in GFAP-negative regions in each image, then calculated the area (μm 2 , A) were calculated using LAS AF lite software (Leica Microsystem CMS GmbH, Mannheim, Germany). The lateral length of the border of the lesion depth bordering the penumbra (μm, B) was also measured using the same software. The value of A / B indicates the average length from the lesion depth (A / B, μm).

[0446] As shown in Figures 15A and 15B, the 1-28, 2-13, and 3-2 anti-FAM19A5 antibodies similarly reduced, reversed, and / or prevented the onset of reactive gliosis in the reflecting zone after traumatic brain injury. The overall effect was similar to that of the 1-65 anti-FAM19A5 antibody, which has previously been shown to significantly attenuate the generation of reactive astrocytes after traumatic brain injury (see U.S. Patent No. 9,579,398). Despite binding to a different epitope of the FAM19A5 protein than the 1-65 antibody, the 1-28, 2-13, and 3-2 antibodies were effective when administered after traumatic brain injury.

[0447] Example 17: Evaluation of the anticancer effect of FAM19A5 antibody in mouse glioblastoma cell line GL-261 cancer cells To induce the glioblastoma model, fix the heads of 4-week-old C57BL / 6 mice in a brain fixation apparatus and inoculate them with GL-261 cells (1 x 10 5 Glioblastoma was induced by intracranial injection of 1000x1000 cells / 5μL (intracranial injection site: 0.2mm anterior to the bregma, 2.2mm lateral to the midline, 3.5mm deep from the skull surface to the center of the caudate putamen) (Figure 16A). Human IgG (negative control group) and anti-FAM19A5 antibody were intravenously injected on the 7th day after GL-261 inoculation in each animal. Each mouse was administered either human IgG control antibody or anti-FAM19A5 antibody at a dose of 2.5mg / kg once a week for three doses (Figure 16A).

[0448] Mortality began to occur 23 days after GL-261 inoculation, and all surviving mice were sacrificed and their brains were removed and fixed in phosphate-buffered saline containing 4% paraformaldehyde. Three analytical methods were used to measure tumor size in brain tissue: 1. Tissue CLARITY (Figure 16B), 2. Hematoxylin and Eosin (H&E) staining (Figures 17A and 17B), and 3. Hoechst nuclear staining (Figures 18A-D).

[0449] (1) To ensure tissue transparency, the analyzed tissue was first immersed in a hydrogel monomer solution (A4P0) and stored at 4°C for one day to allow the hydrogel to permeate the entire tissue. The temperature was then raised to 37°C to induce polymerization and form a network structure. Then, lipids were removed from the tissue using electrophoretic tissue clearing (ETC). The prepared tissue was placed in a light box. The relatively opaque tissue surface was considered as the tumor surface, and the approximate tumor mass was measured using a scale bar.

[0450] (2) For hematoxylin and eosin staining (H&E staining), the tissues were fixed in phosphate-buffered saline containing 4% paraformaldehyde for 24 hours, and then suspended in phosphate-buffered saline containing 30% sucrose for 24 hours. Then, the tissues were placed in brain tissue molds, frozen on dry ice in OCT compound containing 30% sucrose, and the brain tissues were thinly sectioned at 30 μm using a cryostat microtome (Leica).

[0451] After completion of H&E staining, images of the prepared specimens were acquired via a slide scanner (Carl Zeiss / AxioScanZ), and tumor border surfaces were arbitrarily identified using a ZEN microscope and imaging software (Zeiss) to obtain tumor surface area values.

[0452] (3) For Hoechst staining, the thin-sectioned tissues were treated with Hoechst in phosphate-buffered saline (1:2000) for 30 min. Fluorescent images of the prepared specimens were acquired using a confocal microscope (Leica), and tumor borders were determined using IMARIS 3D software (Bitplane), and tumor volumes and intratumoral cell numbers were obtained.

[0453] As shown in Figures 16B, 17A, 17B, and 18A-18D, measurements of the tumor size of each mouse showed that the size of glioblastomas was significantly reduced in the anti-FAM19A5 antibody-treated group compared to the human IgG-treated group in all three experimental methods.

[0454] Example 18: Promotion of vascular normalization after FAM19A5 antibody treatment in a glioblastoma mouse model The brains obtained by sacrificing glioblastoma-induced mouse models (see Example 17) were fixed in phosphate-buffered saline (PBS) containing 4% paraformaldehyde for 24 hours, and then further infiltrated with PBS containing 30% sucrose for 24 hours. The brains were then placed in brain tissue molds and frozen on dry ice with OCT compound containing 30% sugar solution. The frozen brains were then thin-sectioned at 30 μm using a cryostat microtome. Immunohistochemistry was performed to determine the expression pattern of CD31 protein (a marker for vascular endothelial cells) from the glioblastoma-induced mouse brains. Fluorescent images of the prepared specimens were obtained using a confocal microscope (Leica).

[0455] When the same surface area was examined (Fig. 19A, bordered by white dots), the number and connectivity of blood vessels was very poor in the control group (Fig. 19A, left column), whereas the number and connectivity of blood vessels was relatively high in the anti-FAM19A5 antibody-treated group (Fig. 19A, right column). In this regard, please refer to Fig. 19B, which shows a larger enlargement of a representative region of Fig. 19A.

[0456] The results suggested that anti-FAM19A5 antibody treatment could promote vascular normalization and facilitate the treatment of glioblastoma.

[0457] Example 19: Differential distribution of immune cells after FAM19A5 antibody treatment in a mouse model of glioblastoma Brains isolated from glioblastoma-induced mouse models (see Example 17) were fixed in phosphate-buffered saline (PBS) containing 4% paraformaldehyde for 24 hours, and then further permeabilized in PBS containing 30% sucrose for 24 hours. The brains were then placed in brain tissue molds and frozen on dry ice with OCT compound containing 30% sugar solution. The frozen brains were then thin-sectioned at 30 μm using a cryostat microtome. Immunohistochemistry was performed for GFAP (astrocyte marker) and Iba-1 (macrophage marker) to observe the distribution of astrocytes and macrophages in glioblastoma.

[0458] While no astrocytes were observed in glioblastomas in the control group, a relatively large number of astrocytes were maintained in the tumors in the FAM19A5 antibody-treated group. Tumor astrocytes are expected to have a positive effect on surrounding neurons or the extracellular environment of neurons. Furthermore, analysis of the level of macrophage infiltration into glioblastomas showed that macrophage infiltration was dramatically increased in the anti-FAM19A5 antibody-treated group compared to the negative control human IgG-treated group (Figures 20A and 20B).

[0459] These results suggested that anti-FAM19A5 antibody treatment could promote macrophage infiltration into glioblastoma, facilitating the treatment of glioblastoma.

[0460] Example 20: Evaluation of the anti-cancer efficacy of...

Claims

1. 1. An isolated monoclonal antibody, or antigen-binding portion thereof, that specifically binds to a human family member A5 ("FAM19A5") protein having sequence homology 19 (anti-FAM19A5 antibody), (i) the anti-FAM19A5 antibody binds to at least one epitope having the amino acid sequence of TLDRDSSQPRRTIARQTARC (amino acid residues 42-61 of SEQ ID NO:6 or SEQ ID NO:2); (ii) the epitope is capable of specifically binding to a reference antibody comprising (i) a heavy chain variable domain comprising SEQ ID NO: 36, and (ii) a light chain variable domain comprising SEQ ID NO: 40; and (iii) the anti-FAM19A5 antibody has a K of 0.1 to 1 nM as measured by enzyme-linked immunosorbent assay (ELISA). Ｄ binds to soluble human FAM19A5 or membrane-bound human FAM19A5 at An isolated monoclonal antibody or an antigen-binding portion thereof (anti-FAM19A5 antibody).

2. The anti-FAM19A5 antibody of claim 1, which binds to a FAM19A5 epitope having one or more amino acids corresponding to amino acid residues (i) 46 to 51 (i.e., DSSQPR), (ii) 46, 50, and 52 (i.e., D---P-R), or (iii) 46, 47, 48, and 50 (i.e., DSS-P), which is SEQ ID NO:

6.

3. The anti-FAM19A5 antibody according to claim 1 or 2, which is a chimeric antibody, a human antibody, or a humanized antibody.

4. The anti-FAM19A5 antibody of any one of claims 1 to 3, comprising one or more of the following characteristics: (a) reducing, reversing, slowing and / or preventing fibrosis; (b) reduction in excessive extracellular matrix (ECM) formation; (c) slowing the growth or progression of tumors; (d) reducing, reversing, delaying and / or preventing the onset of reactive gliosis; or (e) Suppression of excessive proliferation of reactive astrocytes.

5. A nucleic acid encoding the anti-FAM19A5 antibody according to any one of claims 1 to 4.

6. A vector comprising the nucleic acid of claim 5.

7. A vector according to claim 6 for use in gene therapy.

8. A cell transformed with the vector according to claim 6.

9. A composition comprising the anti-FAM19A5 antibody according to any one of claims 1 to 4 and a carrier.

10. A kit comprising the anti-FAM19A5 antibody according to any one of claims 1 to 4 and instructions for use.

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