Family with anti-array similarity 19, member A5 antibody, and method of using the same

Abstract

The present invention provides antibodies that can specifically bind to FAM19A5 and regulate FAM19A5 activity, particularly antibodies that can be used in human subjects without side effects. [Solution] An isolated antibody or its antigen-binding portion is provided, wherein the heavy chain CDR1, CDR2 and CDR3 each comprise a specific amino acid sequence, each of which optionally contains one, two or three mutations, the light chain CDR1, CDR2 and CDR3 each comprise a specific amino acid sequence, and at least one of the light chain CDR1, CDR2 and CDR3 comprises one, two or three mutations, and the antibody has reduced immunogenicity in humans and higher binding affinity to human FAM19A5 protein compared to a reference antibody comprising a VH consisting of a specific amino acid sequence and a VL consisting of a specific amino acid sequence.

Description

Detailed Description of the Invention

[0001] [Technical Field] Cross - References to Related Applications This PCT application claims priority to U.S. Provisional Application No. 62 / 787,711, filed on January 2, 2019, and No. 62 / 838,190, filed on April 24, 2019, the entire contents of each of which are incorporated herein by reference. References to Electronically Submitted Sequence Listings The content of the electronically submitted sequence listing of the ASCII text file (name: 3763_016PC02_SeqListing_ST25.txt; size: 90,231 bytes; and generation date: December 30, 2019) submitted with this application is incorporated herein by reference in its entirety. Statement of Government Support This research was conducted with the support of a research grant from the Ministry of Trade, Industry and Energy (MOTIE), Republic of Korea, for the Industrial Technology Innovation Program (10081300, Development of a New Therapeutic Monoclonal Antibody Using the Inhibitory Mechanism of Glial Scar Formation against Ischemic Stroke).

[0002] The present disclosure provides an antibody (e.g., a de - immunized or affinity - matured antibody) that specifically binds to Family with Sequence Similarity 19, Member A5 (FAM19A5), a composition comprising the antibody, and a method of using the antibody to prevent or treat a disorder or disease such as a disorder or disease resulting from central nervous system injury in a subject.

[0003] [Background Art] FAM19A5 is a member of the TAFA subfamily of proteins that consists of five small, highly homologous proteins. See Tang T.Y. et al., Genomics 83(4):727-34(2004). These proteins contain cysteine residues conserved at specific positions and are weakly related to macrophage inflammatory protein 1-alpha (MIP-1-alpha), a member of the CC-chemokine family. The TAFA proteins are mainly expressed in specific regions of the brain and spinal cord. These proteins are thought to be produced and secreted by adult neural stem cells during neurogenesis.

[0004] FAM19A5 is mainly expressed in the vertebrate brain, is important for the development, differentiation, and formation of the complete central nervous system, and can be used for the prevention or treatment of central nervous system damage and / or diseases. See U.S. Patent Publication No. 2015 / 0118230.

[0005] [Summary of the Invention] [Problems to be Solved by the Invention] Although FAM19A5 inhibition can play an important role in treating the central nervous system, there is still a need to develop antibodies that specifically bind to FAM19A5 and regulate FAM19A5 activity, particularly antibodies that can be used in human subjects without side effects.

[0006] [Means for Solving the Problems] This specification provides a separated antibody (“anti - FAM19A5 antibody”) or an antigen - binding portion thereof that specifically binds to the human family with sequence similarity 19, member A5 (FAM19A5) protein and includes heavy - chain CDR1, CDR2, and CDR3 as well as light - chain CDR1, CDR2, and CDR3. The heavy - chain CDR1, CDR2, and CDR3 each include the amino - acid sequences shown in SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively, and each of said sequences optionally includes 1, 2, 3, 4, or 5 mutations. The light - chain CDR1, CDR2, and CDR3 each include the amino - acid sequences shown in SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, respectively, and at least one of the light - chain CDR1, CDR2, and CDR3 includes 1, 2, 3, 4, or 5 mutations. The antibody is a separated antibody or an antigen - binding portion thereof that has reduced immunogenicity in humans compared to a reference antibody including VH shown in SEQ ID NO: 11 and VL shown in SEQ ID NO: 12.

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

[0008] In some embodiments, the heavy - chain CDR1 of the anti - FAM19A5 antibody disclosed herein includes the amino - acid sequence shown in SEQ ID NO: 5 having 1 or 2 mutations. In a specific embodiment, the mutation includes a substitution of threonine with an acidic amino - acid at amino - acid 3 of SEQ ID NO: 5. In other embodiments, the mutation includes a substitution of serine with an acidic amino - acid at amino - acid 5 of SEQ ID NO: 5. In a particular embodiment, the acidic amino - acid includes aspartic acid or glutamic acid.

[0009] In some embodiments, the heavy chain CDR2 of the anti-FAM19A5 antibody comprises the amino acid sequence shown in SEQ ID NO: 6 and has 1, 2, 3, 4 or 5 mutations. In some embodiments, the mutation comprises a substitution of the basic amino acid arginine at amino acid 16 of SEQ ID NO: 6. In certain embodiments, the basic amino acid comprises lysine. In other embodiments, the mutation comprises any one or more of the following: (a) substitution of the acidic amino acid aspartic acid or glutamic acid for glycine at amino acid 6 of SEQ ID NO: 6; (b) substitution of the acidic amino acid aspartic acid or glutamic acid for serine at amino acid 7 of SEQ ID NO: 6; (c) substitution of the acidic amino acid aspartic acid or glutamic acid for serine at amino acid 8 of SEQ ID NO: 6; (d) substitution of the acidic amino acid aspartic acid or glutamic acid for threonine at amino acid 9 of SEQ ID NO: 6; and (e) substitution of the basic amino acid lysine for arginine at amino acid 16 of SEQ ID NO: 6. In certain embodiments, the acidic amino acid comprises aspartic acid or glutamic acid. In certain embodiments, the basic amino acid comprises lysine.

[0010] In some embodiments, the light chain CDR3 of the anti-FAM19A5 antibody disclosed herein comprises the amino acid sequence shown in SEQ ID NO: 10 and has 1, 2, 3, 4 or 5 mutations. In some embodiments, the mutation comprises any one or more of the following: (a) substitution of an acidic amino acid or an aliphatic amino acid for serine at amino acid 6 of SEQ ID NO: 10; (b) substitution of an acidic amino acid or a hydroxyl or sulfur / selenium-containing amino acid for asparagine at amino acid 7 of SEQ ID NO: 10; (c) substitution of an acidic amino acid or a hydroxyl or sulfur / selenium-containing amino acid for glycine at amino acid 8 of SEQ ID NO: 10; (d) substitution of an acidic amino acid or a hydroxyl or sulfur / selenium-containing amino acid for glycine at amino acid 9 of SEQ ID NO: 10; and (e) substitution of the basic amino acid histidine for isoleucine at amino acid 10 of SEQ ID NO: 10. In certain embodiments, the acidic amino acid comprises aspartic acid or glutamic acid. In some embodiments, the hydroxyl or sulfur / selenium-containing amino acid comprises serine. In other embodiments, the basic amino acid comprises histidine.

[0011] In some embodiments, the light chain CDR1 of the anti-FAM19A5 antibody of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 8 and has 1, 2, 3, or 4 mutations. In certain embodiments, the mutation(s) include any one or more of the following: (a) substitution of tyrosine with an acidic amino acid at amino acid 6 of SEQ ID NO: 8; (b) substitution of arginine with an acidic amino acid at amino acid 7 of SEQ ID NO: 8; (c) substitution of glycine with an acidic amino acid at amino acid 8 of SEQ ID NO: 8; and (d) substitution of serine with an acidic amino acid at amino acid 9 of SEQ ID NO: 8. In some embodiments, the acidic amino acid includes glutamic acid or glutamine.

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

[0013] This specification also provides a separated antibody or its antigen-binding portion that specifically binds to human FAM19A5 protein and includes heavy chain CDR1, CDR2, and CDR3 as well as light chain CDR1, CDR2, and CDR3, wherein (i) the heavy chain CDR1 includes the amino acid sequence shown in SEQ ID NO: 5; (ii) the heavy chain CDR2 includes the amino acid sequence shown in SEQ ID NO: 13; (iii) the heavy chain CDR3 includes the amino acid sequence shown in SEQ ID NO: 7; (iv) the light chain CDR1 includes the amino acid sequence shown in SEQ ID NO: 8; (v) the light chain CDR2 includes the amino acid sequence shown in SEQ ID NO: 20; (vi) the light chain CDR3 includes the amino acid sequence shown in SEQ ID NO: 10.

[0014] This specification provides a separated antibody or its antigen-binding portion that specifically binds to human FAM19A5 protein and includes heavy chain CDR1, CDR2, and CDR3 as well as light chain CDR1, CDR2, and CDR3, wherein (i) the heavy chain CDR1 includes the amino acid sequence shown in SEQ ID NO: 14; (ii) the heavy chain CDR2 includes the amino acid sequence shown in SEQ ID NO: 15; (iii) the heavy chain CDR3 includes the amino acid sequence shown in SEQ ID NO: 7; (iv) the light chain CDR1 includes the amino acid sequence shown in SEQ ID NO: 21; (v) the light chain CDR2 includes the amino acid sequence shown in SEQ ID NO: 22; (vi) the light chain CDR3 includes the amino acid sequence shown in SEQ ID NO: 23.

[0015] This specification provides a separated antibody or its antigen-binding portion that specifically binds to the human FAM19A5 protein and contains heavy-chain CDR1, CDR2, and CDR3 as well as light-chain CDR1, CDR2, and CDR3, wherein (i) the heavy-chain CDR1 contains the amino acid sequence shown in SEQ ID NO: 14; (ii) the heavy-chain CDR2 contains the amino acid sequence shown in SEQ ID NO: 15; (iii) the heavy-chain CDR3 contains the amino acid sequence shown in SEQ ID NO: 7; (iv) the light-chain CDR1 contains the amino acid sequence shown in SEQ ID NO: 21; (v) the light-chain CDR2 contains the amino acid sequence shown in SEQ ID NO: 24; (vi) the light-chain CDR3 contains the amino acid sequence shown in SEQ ID NO: 23.

[0016] This specification provides a separated antibody or its antigen-binding portion that specifically binds to the human FAM19A5 protein and contains heavy-chain CDR1, CDR2, and CDR3 as well as light-chain CDR1, CDR2, and CDR3, wherein (i) the heavy-chain CDR1 contains the amino acid sequence shown in SEQ ID NO: 14; (ii) the heavy-chain CDR2 contains the amino acid sequence shown in SEQ ID NO: 15; (iii) the heavy-chain CDR3 contains the amino acid sequence shown in SEQ ID NO: 7; (iv) the light-chain CDR1 contains the amino acid sequence shown in SEQ ID NO: 8; (v) the light-chain CDR2 contains the amino acid sequence shown in SEQ ID NO: 25; (vi) the light-chain CDR3 contains the amino acid sequence shown in SEQ ID NO: 23.

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

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

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

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

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

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

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

[0024] The present disclosure also provides a separated antibody (“anti-FAM19A5 antibody”) or an antigen-binding portion thereof that specifically binds to a human family with sequence similarity 19, member A5 (FAM19A5) protein and comprises heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3, wherein the heavy chain CDR1, CDR2, and CDR3 each comprise the amino acid sequences shown in SEQ ID NO: 16, 17, and 18, respectively, each of which sequences optionally contains 1, 2, or 3 mutations, the light chain CDR1, CDR2, and CDR3 each comprise the amino acid sequences shown in SEQ ID NO: 30, 31, and 32, respectively, and at least one of the light chain CDR1, CDR2, and CDR3 contains 1, 2, or 3 mutations, and the antibody has reduced immunogenicity in humans and higher binding affinity for the human FAM19A5 protein compared to a reference antibody comprising VH shown in SEQ ID NO: 35 and VL shown in SEQ ID NO: 45.

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

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

[0027] In some embodiments, the heavy chain CDR2 of the anti-FAM19A5 antibody comprises the amino acid sequence shown in SEQ ID NO: 17.

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

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

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

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

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

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

[0034] In some embodiments, the anti-FAM19A5 antibody disclosed herein is a scFv. In certain embodiments, the scFv comprises VH and VL: (a) the VH comprises the amino acid sequence shown in SEQ ID NO: 33 and the VL comprises the amino acid sequence shown in SEQ ID NO: 38; (b) the VH comprises the amino acid sequence shown in SEQ ID NO: 34 and the VL comprises the amino acid sequence shown in SEQ ID NO: 39; (c) the VH comprises the amino acid sequence shown in SEQ ID NO: 34 and the VL comprises the amino acid sequence shown in SEQ ID NO: 41; (d) the VH comprises the amino acid sequence shown in SEQ ID NO: 34 and the VL comprises the amino acid sequence shown in SEQ ID NO: 40; (e) the VH comprises the amino acid sequence shown in SEQ ID NO: 34 and the VL comprises the amino acid sequence shown in SEQ ID NO: 42; (f) the VH comprises the amino acid sequence shown in SEQ ID NO: 34 and the VL comprises the amino acid sequence shown in SEQ ID NO: 43; (g) the VH comprises the amino acid sequence shown in SEQ ID NO: 34 and the VL comprises the amino acid sequence shown in SEQ ID NO: 44; (h) the VH comprises the amino acid sequence shown in SEQ ID NO: 36 and the VL comprises the amino acid sequence shown in SEQ ID NO: 46; (i) the VH comprises the amino acid sequence shown in SEQ ID NO: 37 and the VL comprises the amino acid sequence shown in SEQ ID NO: 46; (j) the VH comprises the amino acid sequence shown in SEQ ID NO: 130 and the VL comprises the amino acid sequence shown in SEQ ID NO: 46; or (k) the VH comprises the amino acid sequence shown in SEQ ID NO: 131 and the VL comprises the amino acid sequence shown in SEQ ID NO: 46.

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

[0036] This specification also provides a nucleic acid encoding the anti-FAM19A5 antibody disclosed herein, a vector containing the nucleic acid, a cell containing the vector, and an immunoconjugate containing the anti-FAM19A5 antibody of the present disclosure. This specification also discloses a composition comprising the anti-FAM19A5 antibody, nucleic acid, vector, cell, or immunoconjugate of the present disclosure and a carrier. The present disclosure also provides a kit comprising the anti-FAM19A5 antibody, nucleic acid, vector, cell, or immunoconjugate of the present disclosure and instructions for use.

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

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

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

[0040] In some embodiments, the anti-FAM19A5 antibody, nucleic acid, vector, cell or immunoconjugate of the present disclosure induces normalization of blood vessels. In certain embodiments, the normalization of blood vessels is accompanied by changes in the properties of blood vessels including increased connectivity, increased wall thickness, decreased vessel diameter, more regular vessel orientation and distribution patterns, increased number of blood vessels, decreased leakage and permeability, increased perivascular cell coverage and proximity on blood vessels, increased oxygen supply or a combination thereof.

[0041] In some embodiments, the anti-FAM19A5 antibody, nucleic acid, vector, cell or immunoconjugate of the present disclosure inhibits tumor growth.

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

[0043] In some embodiments, the anti-FAM19A5 antibodies, nucleic acids, vectors, cells or immunoconjugates enhance the phagocytic activity of macrophages or microglia. In some embodiments, the anti-FAM19A5 antibodies, nucleic acids, vectors, cells or immunoconjugates increase the mitochondrial membrane potential of macrophages or microglia.

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

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

[0046] [Brief Description of the Drawings] Figures 1A - 1C provide an analysis of the binding of individual scFv clones to the FAM19A5 protein. Absorbance was measured at 405 nm. Clone numbers are provided on the X - axis. Figures 1A - 1C respectively show the analysis of 96 clones derived from the first chicken, the second chicken, and the third chicken by 4th, 5th, or 6th - round biopanning. For each clone shown in Figures 1A - 1C, the vertical bars correspond to the FAM19A5 protein, negative - control protein, lectin protein, and BSA (in order from left to right). In each of Figures 1A, 1B, and 1C, the clones in the box are the 8 clones selected for additional analysis (see Example 4).

[0047] Figures 2A and 2B respectively provide the approximate sizes and binding abilities of different anti - FAM19A5 scFvs. The size of the antibody was shown using SDS - Page, and the binding ability was measured using ELISA. The antibodies shown are (from left to right): 1 - 28, 1 - 85, 2 - 13, 2 - 14, 2 - 20, 2 - 29, 3 - 2, and 3 - 26. In Figure 2B, for each antibody shown, the bar on the left represents the binding of the antibody to the FAM19A5 protein. The bar on the right represents the negative - control group (binding measured in the presence of only the blocking buffer, i.e., in the absence of recombinant FAM19A5 protein). The column labeled “2 nd only” represents yet another negative - control group showing the background level of the said analytical method (binding measured in the absence of the primary anti - FAM19A5 antibody).

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

[0049] Figure 4 provides the amino acid sequences of epitopes F1 - F6 (conjugated to BSA) and their positions in the human FAM19A5 polypeptide. The upper amino acid sequence shown is wild - type FAM19A5 isoform 2 (without the signal peptide). The second amino acid sequence shown is the same sequence, but during peptide synthesis, cysteine residues have been mutated to serine to reduce non - specific activity.

[0050] Figure 5 provides ELISA results for the binding of the 3 - 2 antibody to epitope fragments F1 - F6. The left - most column (“FAM19A5”) represents the positive control group and shows the binding of the 3 - 2 anti - FAM19A5 antibody to the whole FAM19A5 protein. The “unrelated protein” and “blocking alone” (i.e., blocking buffer alone, i.e., without FAM19A5 protein) groups represent the negative control groups. The left bar for each group corresponds to the isotype control group, and the right bar corresponds to the 3 - 2 antibody.

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

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

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

[0054] Figures 9A - 9C provide an analysis of the binding of the deimmunized 3 - 2 antibody. Figure 9A provides a schematic diagram showing the positions where different amino acid mutations were made to deimmunize the 3 - 2 antibody. Figure 9B provides a comparison of the sequences of the light - chain variable region (VL) and the heavy - chain variable region (VH) among (i) the wild - type 3 - 2 antibody (“Clone 3 - 2”), (ii) the fully deimmunized 3 - 2 antibody (“Fully deimmunized Clone 3 - 2”), and (iii) the antibody deimmunized except for one amino acid in the heavy - chain CDR2 (“Deimmunized Clone 3 - 2”). ITOPE TM When determined by analysis, amino acid residues with high immunogenic potential and intermediate immunogenic potential are boxed and labeled “1” and “2” respectively. Figure 9C compares the binding of (i) the wild - type 3 - 2 antibody, (ii) the fully deimmunized 3 - 2 antibody, and (iii) the antibody deimmunized by the heavy - chain CDR2 except for one amino acid (“Deimmunized Clone - 3 - 2”) to the FAM19A5 protein by ELISA measurement. Each single - chain variable fragment (scFv) displayed on phage was added to the wells of a microtiter plate coated with FAM19A5 (black - filled squares) or anti - HA antibody (□). The SU background signal was measured in control wells coated with BSA. The wells were probed with an HRP - conjugated anti - M13 antibody. The absorbance was measured at 405 nm. The results are shown as the mean ± SD obtained from an experiment repeated 4 times.

[0055] Figures 10A and 10B provide an analysis of two different deimmunized 2 - 13 antibodies: (i) the fully deimmunized 2 - 13 antibody, and (ii) the antibody deimmunized except for one amino acid in the heavy - chain CDR2 (“Deimmunized Clone 2 - 13”). Figure 10A provides a comparison of the light - chain variable region (VL) (top 3 rows) and the heavy - chain variable region (VH) (bottom 3 rows) of the wild - type 2 - 13 antibody (“Clone 2 - 13”) versus the two deimmunized 2 - 13 antibodies. ITOPE TMWhen determined by analysis, amino acid residues with high immunogenic potential and intermediate immunogenic potential are boxed and labeled "1" and "2", respectively. Figure 10B compares the binding of (i) wild-type 2-13 antibody, (ii) fully deimmunized 2-13 antibody, and (iii) antibody deimmunized by heavy-chain CDR2 except for one amino acid (deimmunized clone 2-13) to FAM19A5 protein by ELISA measurement. Each single-chain variable fragment (scFv) displaying phage was added to wells of a microtiter plate coated with FAM19A5 (black squares) or anti-HA antibody (□). The background signal was measured from control wells coated with BSA. The wells were probed with HRP-conjugated anti-M13 antibody. Absorbance was measured at 405 nm. The results are shown as mean ± SD obtained from an experiment repeated 4 times.

[0056] Figure 11 provides a comparison of the binding ability of different deimmunized clone 2-13 mutants. The identities of the different mutant antibodies are provided along the x-axis. Each amino acid residue of CDRL1, CDRL2, CDRL3, CDRH1, and CDRH2 was substituted with glutamic acid and aspartic acid. The reactivity of 70 mutant antibodies was analyzed by phage enzyme immunoassay. Each scFv displaying phage was added to wells of a microtiter plate coated with FAM19A5 (black squares) or anti-HA antibody (□). The background signal was measured from control wells coated with BSA. The wells were probed with HRP-conjugated anti-M13 antibody. Absorbance was measured at 405 nm. The results are shown as mean ± SD obtained from an experiment repeated 4 times.

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

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

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

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

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

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

[0063] Figure 18 provides a schematic overview of the overall workflow of the HDX-MS analysis method used in Example 11.

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

[0065] Figures 20A and 20B provide the pepsin digestion results of the FAM19A5 protein using the optimized conditions described in Example 11. Figure 20A shows the coverage rate and duplication rate of 44 peptides identified against the mature FAM19A5 protein (SEQ ID NO: 101, i.e., the sequence obtained by subtracting the signal peptide corresponding to the first 25 amino acids from SEQ ID NO: 2). Each of the horizontal bars indicates an individual peptide. Figure 20B provides the amino acid sequences, single ion masses (MHP), and retention times (RT) of 44 peptides including the start and end sites against SEQ ID NO: 101.

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

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

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

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

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

[0071] [Mode for Carrying Out the Invention] This specification discloses an isolated monoclonal antibody (“anti - FAM19A5 antibody”) or an antigen - binding portion thereof that specifically binds to a human family with sequence similarity 19, member A5 (FAM19A5) protein and exhibits any one or more of the characteristics disclosed herein. Specifically, the anti - FAM19A5 antibody is de - immunized to reduce its immunogenicity in a human subject.

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

[0073] [I. Definitions] Throughout this disclosure, the term “a” or “an” entity refers to one or more of that entity; for example, “an antibody” is understood to represent one or more antibodies. Thus, the terms “a” (or “an”), “one or more,” and “at least one” are used interchangeably herein.

[0074] Also, as used herein, "and / or" shall be construed to specifically disclose each one of two specified features or components, either together with another feature or component or alone. Accordingly, the term "and / or" as used in phrases such as "A and / or B" herein is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B and / or C" is intended to include each of the following aspects: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0075] In this specification, if an aspect is described using the term "comprising", it is understood that other similar aspects described in terms of "consisting of" and / or "consisting essentially of" are also provided.

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

[0077] Units, prefixes, and symbols are expressed in the form approved by these International Systems of Units (SI). Numerical ranges include the numbers defining the range. Unless otherwise indicated, amino acid sequences are written from left to right from amino to carboxy. The headings provided in this specification are not limitations on the various aspects of the disclosure, and they may refer to the specification as a whole. Accordingly, the terms defined below are more fully defined by reference to the entire specification.

[0078] As used herein, the term "about" is used to mean approximately, generally, in the order of, or in the region of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the stated numerical values. Generally, the term "about" can vary the numerical values above and below the stated value by, for example, up to or down from (higher or lower) 10%.

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

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

[0081] (I) Isoform 1 (UniProt: Q7Z5A7-1, transmembrane protein): This isoform was selected as the standard sequence.

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

[0083]

Table 1A

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

[0085] The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs) in which more conserved regions called framework regions (FRs) are interspersed. Each of VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant region of the antibody can mediate the binding of immunoglobulins to various cells of the immune system (e.g., effector cells) and host tissues or factors including the first component of the classical complement system (Clq).

[0086] The term "Kabat numbering" and similar terms are recognized in the art and refer to a system for numbering amino acid residues in the heavy and light chain variable regions of an antibody or an antigen-binding portion thereof. In certain embodiments, the CDRs of an antibody can be determined according to the Kabat numbering system (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, U.S. Department of Health and Human Services, NIH Publication No. 91 - 3242 (referenced). Using the Kabat numbering system, the CDRs within the antibody heavy chain molecule are typically at amino acid positions 31 - 35 (CDR1) (optionally including one or two additional amino acids after position 35, referred to as 35A and 35B in the Kabat numbering system), amino acid positions 50 - 65 (CDR2), and amino acid positions 95 - 102 (CDR3). Using the Kabat numbering system, the CDRs within the antibody light chain molecule are typically at amino acid positions 24 - 34 (CDR1), amino acid positions 50 - 56 (CDR2), and amino acid positions 89 - 97 (CDR3). In certain embodiments, the CDRs of the antibodies described herein are determined by the Kabat numbering system.

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

[0088] [Table 1B]

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

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

[0091] For all heavy-chain constant region amino acid positions discussed in this disclosure, the numbering follows the EU index first described in Edelman et al., 1969, Proc. Natl. Acad. Sci. USA 63(1):78-85), which describes 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 Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda. Accordingly, the phrases "EU index 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 residue numbering system based on the human IgG1 EU antibody of Edelman et al. presented in Kabat 1991.

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

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

[0094] "Antibody" includes, for example, naturally occurring and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human and non-human antibodies, fully synthetic antibodies; single-chain antibodies; monospecific antibodies; multispecific antibodies (including bispecific antibodies); tetrameric antibodies comprising two heavy chains 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; intracellular antibodies (intrabodies); heteroconjugate antibodies; monovalent antibodies; single-chain antibodies; camelized antibodies; affybodies; anti-idiotype (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), and single-domain antibodies (sdAbs) comprising a binding molecule consisting of a fully antigen-binding single monomeric variable antibody domain (e.g., a VH domain or a VL domain) (Harmen M.M. and Haard H.J. Appl Microbiol Biotechnol. 77(1):13 - 22(2007)).

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

[0096] As used herein, the terms "variable region" and "variable domain" are interchangeable and are common in the art. The variable region typically refers to a part of an antibody, generally part of the light chain or heavy chain. Typically, there are about 110-120 amino acids at the amino terminus in the mature heavy chain and about 90-115 amino acids in the mature light chain. These vary widely in sequence among antibodies and are used in the binding and specificity of a particular antibody to a particular antigen. The sequence variability is concentrated in regions called complementarity-determining regions (CDRs), while the more highly conserved regions in the variable domain are called framework regions (FRs).

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

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

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

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

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

[0102] The terms "constant region" or "constant domain" as used herein are interchangeable and have their ordinary meaning in the art. The constant domain is the carboxy-terminal portion of the light and / or heavy chains that does not directly participate in the binding of the antibody, e.g., to an antigen, but may exhibit various effector functions such as interaction with Fc receptors. The constant regions of immunoglobulin molecules generally have more conserved amino acid sequences compared to the immunoglobulin variable domains.

[0103] The "Fc region" (fragment crystallizable region) or "Fc domain" or "Fc" refers to the C-terminal region of the heavy chain of an antibody that includes binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or the first component (C1q) of the classical complement system, and mediates the binding of immunoglobulins to host tissues or factors. Thereby, the Fc region includes the constant region of the 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 two heavy chains of the antibody; IgM and IgE Fc regions include three heavy chain constant domains (CH domains 2-4) in their respective polypeptide chains. In the case of IgG, the Fc region includes the hinges between immunoglobulin domains Cγ2 and Cγ3 and between Cγ1 and Cγ2. The boundaries of the Fc region of the immunoglobulin heavy chain can vary, but the human IgG heavy chain Fc region is generally limited to that extending from the amino acid residue at position C226 or P230 (or the amino acids between these two amino acids) to the carboxy terminus of the heavy chain, and the numbering follows the EU index as in Kabat. The CH2 domain of the human IgG Fc region extends from approximately amino acid 231 to approximately amino acid 340, and the CH3 domain is located on the C-terminal side of the Cm domain in the Fc region, i.e., it extends from approximately amino acid 341 to approximately amino acid 447 of IgG. The Fc region used herein can be a native sequence Fc including any allotype variant, or a variant Fc (e.g., non-naturally occurring Fc). Fc can also mean this region in a separated state or in association with an Fc-containing protein polypeptide such as a "binding protein containing an Fc region" also called an "Fc fusion protein" (e.g., an antibody or an immunoadhesion).

[0104] "Native sequence Fc region" or "native sequence Fc" includes an amino acid sequence identical to that of the Fc region discovered in nature. The native sequence human Fc region includes not only the native sequence human IgG1 Fc region; native sequence human IgG2 Fc region; native sequence human IgG3 Fc region; and native sequence human IgG4 Fc region, but also natural occurring variants thereof. Native sequence Fc includes various allotypes of Fc (see, e.g., Jefferis et al. (2009) mAbs 1:1; Vidarsson G. et al. Front Immunol. 5:520 (published online Oct. 20, 2014)).

[0105] "Fc receptor" or "FcR" is a receptor that binds to the Fc region of an immunoglobulin. FcRs that bind IgG antibodies include receptors of the FcγR family and allelic variants of these receptors and forms alternatively spliced in other ways. The FcγR 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 to most human Fc receptors and elicits the strongest Fc effector functions. Human IgG1 can be considered equivalent to murine IgG2a in terms of the types of activating Fc receptors to which it binds. In contrast, human IgG4 elicits minimal Fc effector functions (see Vidarsson G. et al. Front Immunol. 5:520 (published online Oct. 20, 2014)).

[0106] The constant region can be manipulated, for example, by recombinant techniques to remove one or more effector functions. "Effector function" refers to the interaction of the antibody Fc region with Fc receptors or ligands or the biochemical reactions resulting therefrom. Exemplary "effector functions" include FcγR-mediated effector functions such as C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, ADCC, and antibody-dependent cell-mediated phagocytosis (ADCP), and downregulation of cell surface receptors (e.g., B cell receptor; BCR). Such effector functions generally require the Fc region to be combined with a binding domain (e.g., the antibody variable domain). Thus, the term "constant region without Fc function" includes constant regions in which one or more effector functions mediated by the Fc region are reduced or absent. The effector functions of an antibody can be reduced or avoided by different approaches. The effector functions of an antibody can be reduced or avoided by different approaches. The effector functions of an antibody can be reduced or absent in antibody fragments lacking the Fc region (e.g., Fab, F(ab’)

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

[0108] "Hinge", "hinge domain", or "hinge region" or "antibody hinge region" refers to the domain of the heavy chain constant region that joins the CH1 domain to the CH2 domain and includes the upper, middle, and lower portions of the hinge (Roux et al., J. Immunol. 1998 161:4083). The hinge provides a level 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 herein starts at Glu216 and ends at Gly237 for all IgG isotypes (Roux et al., 1998 J Immunol 161:4083). The sequences of wild-type IgG1, IgG2, IgG3, and IgG4 hinges are known in the art. For example, see Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; Vidarsson G. et al., Front Immunol. 5:520 (published online October 20, 2014).

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

[0110] The term "CH2 domain" refers to the heavy chain constant region that links the hinge to the CH3 domain in the heavy chain constant domain. The CH2 domain used herein starts at P238 and ends at K340. The term "CH2 domain" includes not only the wild-type CH2 domain but also its naturally occurring variants (e.g., allotypes). The CH2 domain sequences of IgG1, IgG2, IgG3, and IgG4 (including wild-type and allotypes) are known in the art (e.g., Kabat EA et al., (1991) and Vidarsson G. et al., Front Immunol. 5:520 (published online October 20, 2014) supra). Exemplary CH2 domains include, for example, CH2 domains having mutations that alter the biological activity of the antibodies described in US Patent Publication No. 20120100140 and the US patents, published applications, and PCT publications cited therein, such as the half-life and / or reduced Fc effector function.

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

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

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

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

[0115] As used herein, an "isolated antibody" means an antibody 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 can have cross-reactivity with other FAM19A5 proteins of different species.

[0116] "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 reflecting a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for partner Y can generally be expressed by the dissociation constant (K D ). Affinity can be measured and / or expressed in a number of ways known in the art, including, but not limited to, the equilibrium dissociation constant (K D ) and the equilibrium association constant (K A ). The K D is calculated from the quotient of k off / k on and is expressed in molar concentration (M), and K A is calculated from the quotient of k on / k off . k on means, for example, the association rate constant of an antibody for an antigen, and k off means, for example, the dissociation of an antibody from an antigen. k on and k off can be determined by techniques known to those of skill in the art, such as immunoassays (e.g., enzyme-linked immunosorbent assay (ELISA)), BIACORE® or kinetic exclusion analysis (KinExA).

[0117] 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 refer to a molecule (e.g., an antibody) that binds to an antigen (e.g., an epitope or immune complex), as understood by one of skill in the art. For example, a molecule that specifically binds to an antigen may bind to other peptides or polypeptides that typically have a lower affinity, as determined by, for example, immunoassays, a BIACORE®, KinExA 3000 instrument (Sapidyne Instruments, Boise, ID), or other assays known in the art. In certain embodiments, a molecule that specifically binds to an antigen has a K A Compared to A and binds to the antigen.

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

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

[0120] As used herein, "epitope" is a term in the art and refers to a localized region of an antigen to which an antibody can specifically bind. An epitope can be, for example, adjacent amino acids of a polypeptide (linear or contiguous epitope), or an epitope can be, for example, a collection of two or more non-adjacent regions of a polypeptide or polypeptide (conformational, non-linear, discontinuous or non-contiguous epitope). Epitopes formed from adjacent amino acids are typically, but not always, maintained upon exposure to a denaturing solvent, whereas epitopes formed by tertiary folding are typically lost upon treatment with a denaturing solvent. Epitopes typically contain at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 20 amino acids within a unique spatial conformation. Methods for determining which epitope is bound by a given antibody (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays in which overlapping or contiguous peptides (e.g., of FMA19A5) are tested for reactivity with a given antibody (e.g., an anti-FAM19A5 antibody). Methods for determining the spatial conformation of an epitope include techniques in the art and techniques 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, G.E. Morris, Ed. (1996)).

[0121] In certain embodiments, the epitope to which the antibody binds can be determined, for example, by NMR spectroscopy, X-ray crystallographic studies, ELISA assays, hydrogen / deuterium exchange combined with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligopeptide scanning assays, and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). In the case of X-ray crystallography, crystallization can be achieved using any of the methods known in the art (e.g., see Giege R et al., (1994) Acta Crystallogr D Biol Crystallogr 50(Pt4):339-350; McPherson A (1990) Eur J Biochem 189:1-23; Chayen NE (1997) Structure 5:1269-1274; McPherson A (1976) J Biol Chem 251:6300-6303). Antibody:antigen crystals can be studied using well-known X-ray diffraction techniques, such as X-PLOR (distributed by Yale University, 1992, Molecular Simulations, Inc.; e.g., Meth Enzymol (1985) volumes 114 & 115, eds Wyck off H.W. et al.,; see U.S. 2004 / 0014194) and BUSTER (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 It can be refined using computer software such as Crystallogr 56(Pt 10):1316-1323). The mutagenesis mapping study can be carried out using any method known to those skilled in the art. For a description of mutagenesis techniques including alanine scanning mutagenesis techniques, see, for example, Champe M. et al., (1995) J Biol Chem 270:1388-1394 and Cunningham B.C. & Wells J.A. (1989) Science 244:1081-1085.

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

[0123] In the context of two or more antibodies, the term "bind to the same epitope" means that the antibodies bind to the same segment of amino acid residues when determined by a given method. Techniques for determining whether an antibody binds to the "same epitope on FAM19A5" as the antibodies described herein include epitope mapping methods such as x-ray analysis of crystals of the antigen:antibody complex that provide atomic resolution of the epitope and hydrogen / deuterium exchange mass spectrometry (HDX-MS). Other methods involve monitoring the binding of the antibody to antigen fragments or mutated variants of the antigen, where loss of binding due to modification of amino acid residues within the antigen sequence is generally regarded as indicative of the epitope component. Computational combinatorial methods for epitope mapping are also available. These methods rely on the ability of the antibody of interest to affinity purify specific short peptides from a combinatorial phage display peptide library. Antibodies having the same VH and VL or the same CDR1, 2, and 3 sequences are expected to bind to the same epitope.

[0124] An antibody that "competes with another antibody for binding to a target" refers to an antibody that (partially or completely) inhibits the target binding of the other antibody. Whether two antibodies compete with each other for binding to a target, i.e., whether one antibody inhibits the target binding of the other antibody, and the degree of inhibition can be determined using known competition assays. In certain embodiments, one antibody competes in the target binding of the other antibody and inhibits this binding by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. The inhibition or competition level may vary depending on whether the antibody is a "blocking antibody" (i.e., the antibody first cultured with the target). Competition analysis can be performed as described, for example, in Chapter 11 of "Using Antibodies" by Ed. Harlow and David Lane, Cold Spring Harb Protoc; 2006; doi:10.1101 / pdb.prot4277 or Ed. Harlow and David Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA 1999. Competing antibodies bind to the same epitope, overlapping epitopes or adjacent epitopes (e.g., demonstrated by steric hindrance).

[0125] Other competitive binding assays include solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competitive assay (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 labeled assay, solid phase direct labeled sandwich assay (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid phase direct labeled RIA using 1-125 label (see Morel et al., Mol. Immunol. 25(1):7 (1988)); solid phase direct biotin-avidin EIA (see Cheung et al., Virology 176:546 (1990)); and direct labeled RIA (see Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)).

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

[0127] As used herein, the term "monoclonal antibody" refers to an antibody that exhibits a single binding specificity and affinity for a particular epitope, or to an antibody composition in which all antibodies exhibit a single binding specificity and affinity for a particular epitope. Thus, the term "human monoclonal antibody" refers to an antibody or antibody composition that exhibits a single binding specificity and has variable and selected constant regions derived from human germline immunoglobulin sequences. In some embodiments, a human monoclonal antibody is produced, for example, by a hybridoma containing B cells obtained from a transgenic non-human animal, such as a transgenic mouse, having a genome that includes a human heavy chain transgene and a light chain transgene fused to immortalized cells.

[0128] As used herein, the term "recombinant human antibody" includes all human antibodies produced, expressed, generated, or isolated by recombinant means, such as (a) antibodies isolated from or made from hybridomas isolated from transgenic or transchromosomal animals (e.g., mice) to which human immunoglobulin genes have been introduced, (b) antibodies isolated from host cells transformed to express the antibody, such as a transfectoma, (c) antibodies isolated from a recombinant combinatorial human antibody library, and (d) antibodies produced, expressed, generated, or isolated by any other means involving splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies utilize specific human germline immunoglobulin sequences encoded by germline genes, and include variable and constant regions that include subsequent rearrangements and mutations that occur, for example, during antibody maturation. As is known in the art (e.g., Lonberg (2005) Nature (see Biotech.23(9):1117-1125). The variable region contains antigen-binding domains encoded by various genes that are rearranged to form antibodies specific for foreign antigens. In addition to rearrangement, the variable region can be further modified by numerous single amino acid changes (termed somatic mutations or hypermutations) to increase the affinity of the antibody for the foreign antigen. The constant region will vary according to additional reactions to the antigen (i.e., isotype switching). Thus, a rearranged and somatic-mutated nucleic acid molecule encoding a light and heavy chain immunoglobulin polypeptide that reacts to an antigen will not be able to have sequence identity with the original nucleic acid molecule, but instead will be substantially identical or similar (i.e., have at least 80% identity).

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

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

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

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

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

[0134] When applied to an object, the term "naturally occurring" as used herein means the fact that the object can be discovered from nature. For example, a polypeptide or polynucleotide sequence present within an organism (including a virus) that is separable from a natural source and not intentionally modified by a person in the laboratory is naturally occurring.

[0135] "Polypeptide" refers to a chain containing at least two contiguous linked amino acid residues, and there is no upper limit to the length of said chain. One or more amino acid residues within a protein can contain modifications such as glycosylation, phosphorylation, or disulfide bond formation, but are not limited thereto. "Protein" can comprise one or more polypeptides.

[0136] The term "nucleic acid molecule" as used in the present invention is intended to include DNA molecules and RNA molecules. The nucleic acid molecule can be single-stranded or double-stranded and can be cDNA.

[0137] As used herein, the term "vector" is intended to mean a nucleic acid molecule that can transport a further nucleic acid that has been ligated thereto. One type of vector is a "plasmid" which refers to a circular double-stranded DNA loop to which additional DNA fragments can be ligated. Still other types of vectors are viral vectors to which additional DNA fragments can be ligated to the viral genome. Certain vectors are capable of self-replication in the host cells into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian). Other vectors (e.g., non-episomal mammalian vectors) may integrate into the genome of the host cell upon introduction into the host cell and are thereby replicated along with the host genome. Further, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Generally, expression vectors useful in recombinant DNA technology are often in the form of plasmids. As used herein, the terms "plasmid" and "vector" are used interchangeably since the plasmid is the most commonly used form of a vector. However, other forms of expression vectors that perform equivalent functions are also included, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses and adeno-associated viruses).

[0138] As used in the present invention, the term "recombinant host cell" (or simply "host cell") refers to a cell that contains a nucleic acid that does not naturally occur within the cell and can be a cell into which a recombinant expression vector has been introduced. Such terms should be understood to refer not only to the particular subject cell but also to the progeny of such a cell. Although such progeny may not actually be identical to the parent cell due to mutations or environmental influences that may cause certain modifications in the next generation, they are still included within the scope of the term "host cell" as used herein.

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

[0140] As used herein, "administering" refers to physically introducing a therapeutic agent or a composition comprising a therapeutic agent to a subject using any of a variety of methods and delivery systems known to those of skill in the art. Preferred routes of administration for the antibodies described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, intraspinal, or other parenteral routes of administration such as by injection or infusion. As used herein, the phrase "parenteral administration" generally means a mode of administration other than enteral and topical administration by injection and includes, but is not limited to, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intravascular, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation. In contrast, the antibodies described herein can be administered by parenteral routes, for example, local, epidermal or mucosal routes of administration, for example, intranasal, oral, vaginal, workplace, sublingual or topical administration. Administration can also be carried out, for example, once, a plurality of times, and / or over one or more extended periods.

[0141] As used herein, the terms "treat", "treating" and "treatment" refer to any type of intervention or process performed on a subject, or the administration of an active agent to a subject, for the purpose of reversing, alleviating, improving, inhibiting or delaying or preventing the progression, development, severity or recurrence of a disease-related syndrome, complication, symptom or biochemical sign. Treatment can be carried out on a subject with a disease or a subject without a disease (e.g., for prophylaxis).

[0142] As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, for example, mammals and non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, and other non-mammals.

[0143] As used herein, the term "onset of gliosis" or "onset of reactive gliosis" includes the beginning or initiation of gliosis. Gliosis is, for example, a non-specific reactive change of glial cells within the central nervous system (CNS, e.g., brain and / or spinal cord) due to injury or damage from trauma, cerebrospinal injury, brain tumor, infection, ischemia, stroke, reaction, and / or neurodegenerative disease, and includes the proliferation or hypertrophy of various different types of glial cells, including astrocytes, microglia, and oligodendrocytes. The onset of gliosis may result in the formation of a scar that inhibits axonal regeneration in a part of the CNS that has been traumatized or damaged. The detrimental effects of the onset of gliosis include irreversible or permanent neuronal damage and / or interference with the recovery of peripheral neurons. Accordingly, the terms "delaying the onset of gliosis" and "delaying the onset of reactive gliosis" include inhibiting, delaying, suppressing, or preventing the beginning or initiation of gliosis and the associated detrimental effects on the CNS.

[0144] As used herein, the term "reactive astrogliosis" includes, for example, an abnormal increase in the number of astrocytes due to destruction of peripheral neurons from CNS injury, trauma, lesion, spinal cord injury, brain tumor, infection, ischemia, stroke, response and / or neurodegenerative disease. Reactive astrogliosis can lead to the formation of scars that inhibit axonal regeneration in a part of the CNS that has been traumatized or damaged, exacerbation of inflammation, generation and release of reactive oxygen species at neurotoxic levels, release of glutamate, which may be excitotoxic, a potential contribution to seizure occurrence, impairment of blood-brain barrier function, cytotoxic edema in trauma and stroke, the potential for chronic cytokine activation of astrocytes contributing to chronic pain, and secondary degeneration after CNS injury, which can have harmful effects on the CNS. 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, M. V. (2005) The Neuroscientist 11(5):400-7. Accordingly, the term "inhibition of reactive astrogliosis" includes inhibiting, delaying, suppressing, limiting or preventing the excessive or abnormal proliferation of reactive astrocytes and the associated harmful effects on the CNS.

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

[0146] As used herein, the term "neuron" includes electrically excitable cells that process and transmit information by electrical and chemical signals. Neurons are the major components of the brain and spinal cord of the CNS and ganglia of the peripheral nervous system (PNS), and can be interconnected to form neural networks. A typical neuron consists of a soma (cell body), dendrites, and an axon. The soma (cell body) of a neuron contains the nucleus. The dendrites of a neuron are the highly branched cell extensions where most of the neuronal input occurs. The axon is a relatively slender cable-shaped projection extending from the soma, which transmits nerve signals far from the soma and further transmits certain types of information to the soma. The term "promote neuronal regrowth" preferably includes stimulating, promoting, increasing, or activating the growth of neurons after injury or damage.

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

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

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

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

[0151] [II. Anti-FAM19A5 Antibody] This specification discloses antibodies characterized by specific functional features or characteristics, such as monoclonal antibodies. For example, the antibody that specifically binds to human FAM19A5 has been mutated (i.e., deimmunized) by removing and / or modifying regions or residues that are highly immunogenic in humans, such as by substitution or deletion. Thus, the antibodies disclosed herein, i.e., anti-FAM19A5 antibodies, have reduced immunogenicity when administered to humans compared to a reference antibody (e.g., a corresponding non-deimmunized antibody, such as antibody 3-2 or 2-13).

[0152] In addition, the antibodies described herein exhibit any one or more of the following functional characteristics: (a) The property of binding to soluble human FAM19A5 with a K D of 10 nM or less; (b) The property of binding to membrane-bound human FAM19A5 with a K D of 10 nM or less; (c) The property of reducing, reversing, delaying and / or preventing the onset of reactive gliosis; (d) The property of suppressing the overgrowth of reactive astrocytes; (e) The property of reducing the expression of chondroitin sulfate proteoglycans including neurocan and glial antigen 2 (NG2); (f) The property of increasing the expression of nuclear c-fos and pERK in neurons; (g) The property of promoting the survival of neurons; (h) The property of increasing the expression of GAP43 in neurons; and (i) The property of promoting axonal regrowth.

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

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

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

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

[0157] In some embodiments, the anti-FAM19A5 antibody has a K D of 10 -7 M or less, 10 -8 M (10 nM) or less, 10 -9 M (1 nM) or less, 10 -10 M or less, 10 -12 M to 10 -7 M, 10 -11 M to 10 -7 M, 10 -10 M to 10 -7 M, 10 -9 M to 10 -7 M, or 10 -8 M to 10 -7 M and binds to soluble human FAM19A5. In some embodiments, the anti-FAM19A5 antibody has a K D of 10 nM or less, for example, 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 and binds to soluble FAM19A5. In some embodiments, the anti-FAM19A5 antibody has a K DSpecifically binds to soluble human FAM19A5 at about 1 pM, about 2 pM, about 3 pM, about 4 pM, about 5 pM, about 6 pM, about 7 pM, about 8 pM, about 9 pM, about 10 pM, about 20 pM, about 30 pM, about 40 pM, about 50 pM, about 60 pM, about 70 pM, about 80 pM, about 90 pM, about 100 pM, about 200 pM, about 300 pM, about 400 pM, about 500 pM, about 600 pM, about 700 pM, about 800 pM or about 900 pM, or about 1 nM, about 2 nM, about 3 nM, about 4 nM, about 5 nM, about 6 nM, about 7 nM, about 8 nM or about 9 nM or about 10 nM, about 20 nM, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM or about 90 nM.

[0158] In some embodiments, the anti-FAM19A5 antibody has a K D which, when determined by, for example, ELISA, is 10 -7 M or less, 10 -8 M (10 nM) or less, 10 -9 M (1 nM) or less, 10 -10 M or less, 10 -12 M to 10 -7 M, 10 -11 M to 10 -7 M, 10 -10 M to 10 -7 M, 10 -9 M to 10 -7 M or 10 -8 M to 10 -7 M and binds to membrane-bound human FAM19A5. In certain embodiments, the anti-FAM19A5 antibody has a K D which is 10 nM or less when determined by ELISA, for example, 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 and specifically binds to membrane-bound human FAM19A5. In some embodiments, the anti-FAM19A5 antibody has a K DIt binds to membrane-bound human FAM19A5 at about 1 pM, about 2 pM, about 3 pM, about 4 pM, about 5 pM, about 6 pM, about 7 pM, about 8 pM, about 9 pM, about 10 pM, about 20 pM, about 30 pM, about 40 pM, about 50 pM, about 60 pM, about 70 pM, about 80 pM, about 90 pM, about 100 pM, about 200 pM, about 300 pM, about 400 pM, about 500 pM, about 600 pM, about 700 pM, about 800 pM or about 900 pM, or about 1 nM, about 2 nM, about 3 nM, about 4 nM, about 5 nM, about 6 nM, about 7 nM, about 8 nM or about 9 nM, or about 10 nM, about 20 nM, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM or about 90 nM.

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

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

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

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

[0163] In some embodiments, the anti-FAM19A5 antibodies of the present disclosure include heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3, wherein the heavy chain CDR1, CDR2, and CDR3 each include the amino acid sequences shown in SEQ ID NOs: 5, 6, and 7, respectively, and each of the sequences may include 1, 2, 3, 4, or 5 mutations in some cases. The light chain CDR1, CDR2, and CDR3 each include the amino acid sequences shown in SEQ ID NOs: 8, 9, and 10, respectively, and at least one of the light chain CDR1, CDR2, and CDR3 includes 1, 2, 3, 4, or 5 mutations. The antibody has reduced immunogenicity in humans compared to a reference antibody including VH shown in SEQ ID NO: 11 and VL shown in SEQ ID NO: 12.

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

[0165]

Table 2

[0166] Conversely, a radical substitution or radical mutation is an amino acid substitution that exchanges the initial amino acid for a final amino acid having different physicochemical properties. In certain embodiments, in the FAM19A5 antibody, the amino acid mutation is a radical substitution. In other embodiments, in the FAM19A5 antibody, the amino acid mutation is a combination of conservative substitution and radical substitution.

[0167] In some embodiments, the heavy chain CDR3 includes the amino acid sequence shown in SEQ ID NO: 7. In certain embodiments, the heavy chain CDR3 includes the amino acid sequence shown in SEQ ID NO: 7 having one, two, or three mutations.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0191] In some embodiments, the anti-FAM19A5 antibody of the present disclosure includes a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH contains 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% the same as the amino acid sequence shown in SEQ ID NO: 11, and / or the VL contains 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% the same as the amino acid sequence shown in SEQ ID NO: 12. The antibody has reduced immunogenicity compared to a reference antibody that includes the VH shown in SEQ ID NO: 11 and the VL shown in SEQ ID NO: 12.

[0192] In some embodiments, the anti-FAM19A5 antibody disclosed herein cross-competes with a reference antibody that includes a heavy chain variable region (VH) and a light chain variable region (VL), (a) the VH contains the amino acid sequence shown in SEQ ID NO: 33 and the VL contains the amino acid sequence shown in SEQ ID NO: 38; (b) the VH contains the amino acid sequence shown in SEQ ID NO: 34 and the VL contains the amino acid sequence shown in SEQ ID NO: 39; (c) the VH contains the amino acid sequence shown in SEQ ID NO: 34 and the VL contains the amino acid sequence shown in SEQ ID NO: 41; (d) the VH contains the amino acid sequence shown in SEQ ID NO: 34 and the VL contains the amino acid sequence shown in SEQ ID NO: 40; (e) Does the VH contain the amino acid sequence shown in SEQ ID NO: 34 and does the VL contain the amino acid sequence shown in SEQ ID NO: 42? (f) Does the VH contain the amino acid sequence shown in SEQ ID NO: 34 and does the VL contain the amino acid sequence shown in SEQ ID NO: 43? Or (g) The VH contains the amino acid sequence shown in SEQ ID NO: 34 and the VL contains the amino acid sequence shown in SEQ ID NO: 44.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0216] Techniques for determining whether two antibodies bind to the same epitope include, for example, epitope mapping methods such as x-ray analysis of crystals of antigen:antibody complexes that provide atomic resolution of the epitope and hydrogen / deuterium exchange mass spectrometry (HDX-MS), methods for monitoring the binding of an antibody to an antigen fragment or a mutated variant of an antigen in which loss of binding due to modification of an amino acid residue within the antigen sequence is generally regarded as an indication of an epitope component, and combinatorial computational methods for epitope mapping.

[0217] Anti-FAM19A5 antibodies useful in the methods disclosed herein can bind to at least one epitope of mature human FAM19A5, as determined, for example, by the binding of an antibody to a fragment of human FAM19A5. In some embodiments, 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: 90 or SEQ ID NO: 2), or to a fragment located within the amino acid sequence of SEQ ID NO: 90, 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: 90. In some embodiments, the anti-FAM19A5 antibodies disclosed herein bind to one or more amino acids corresponding to amino acid residues 44-52 of SEQ ID NO: 2 (i.e., DRDSSQPRR), such as amino acid residues 45, 46, 50, 51 and 52 (i.e., RD---PRR), such as amino acid residues 45, 50, 51 and 52 (i.e., R----PRR), amino acid residues 45, 50 and 51 (i.e., R----PR).

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

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

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

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

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

[0223] In some embodiments, the present specification provides an antibody or an antigen-binding fragment thereof that binds to FAM19A5 (e.g., human FAM19A5) with an affinity that is 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or higher compared to other proteins within the FAM19A family when measured by, for example, immunoassay (e.g., ELISA), surface plasmon resonance or size exclusion chromatography. In certain embodiments, the anti-FAM19A5 antibody or antigen-binding fragment thereof binds to FAM19A5 (e.g., human FAM19A5) without cross-reactivity with other proteins within the FAM19A family when measured by, for example, immunoassay.

[0224] In some embodiments, the anti-FAM19A5 antibody of the present disclosure is not a natural antibody or a naturally occurring antibody. For example, in some embodiments, the anti-FAM19A5 antibody has post-translational modifications that are more, fewer or different types than those of a naturally occurring antibody, resulting in post-translational modifications that are different from those of a naturally occurring antibody.

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

[0226] [Table 3]

[0227] [Table 4]

[0228] [Table 5]

[0229] [Table 6]

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

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

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

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

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

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

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

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

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

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

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

[0241] In some embodiments, the anti-FAM19A5 antibody of the present disclosure includes VH and VL, wherein the VH includes 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% the same as the amino acid sequences shown in SEQ ID NOs: 33, 34, 36, 37, 130, or 131.

[0242] In some embodiments, the anti-FAM19A5 antibody of the present disclosure includes VH and VL, wherein the VL includes 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% the same as the amino acid sequences shown in SEQ ID NOs: 38, 39, 40, 41, 42, 43, 44, or 46.

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

[0244] The VH domain or one or more CDRs thereof described herein can be linked to a constant domain for forming a heavy chain, such as a full length heavy chain. Similarly, the VL domain or one or more CDRs thereof described herein can be linked to a constant domain for forming a light chain, such as a full length light chain. The full length heavy chain and the full length light chain combine to generate a full length antibody.

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

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

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

[0248] In certain embodiments, the anti-FAM19A5 antibodies disclosed herein do not have Fc effector functions, such as complement-dependent cytotoxicity (CDC) and / or antibody-dependent cellular phagocytosis (ADCP). Effector functions are mediated by the Fc region, and the residues in the CH2 domain of the Fc region closest to the hinge region contain overlapping binding sites for Clq (complement) and IgG-Fc receptors (FcγR) on effector cells of the innate immune system, thus responsible for the effector functions of the antibody. Also, IgG2 and IgG4 antibodies have lower levels of Fc effector functions than IgG1 and IgG3 antibodies. The effector functions of an antibody can be reduced or avoided by different approaches known in the art, including (1) using antibody fragments lacking the Fc region (e.g., Fab, F(ab’) 2 , single-chain Fv (scFv), or sdAb consisting of monomeric VH or VL domains); (2) generating non-glycosylated antibodies by, for example, deleting or modifying residues to which sugars are attached, enzymatically removing sugars, generating antibodies in cells cultured in the presence of glycosylation inhibitors, or expressing antibodies in cells that cannot glycosylate proteins (e.g., bacterial host cells, see, for example, U.S. Patent Publication No. 20120100140); (3) using Fc regions of IgG subtypes with reduced effector functions (e.g., Fc regions of IgG2 and IgG4 antibodies or chimeric Fc regions containing the CH2 domain of IgG2 or IgG4 antibodies, 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 reduce or abolish Fc function. See, for example, U.S. Patent Publication No. 20120100140 and the U.S. and PCT applications cited therein and An et al, mAbs 1:6,572-579 (2009).

[0249] Thus, in some embodiments, the anti-FAM19A5 antibodies disclosed herein are Fab, Fab’, F(ab’) 2It is an sdAb consisting of Fv, single-chain Fv (scFv), or monomeric VH or VL domains. Such antibody fragments are well known in the art and are described above.

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

[0251] [Table 7]

[0252] JPEG0007687703000009.jpg172169

[0253] In some embodiments, the anti-FAM19A5 antibody disclosed herein comprises an Fc region with reduced or absent Fc effector function. In some embodiments, the constant region comprises the amino acid sequence of the Fc region of human IgG2 or IgG4, and in some embodiments, the anti-FAM19A5 antibody has an IgG2 / IgG4 isotype. In some embodiments, the anti-FAM19A5 antibody comprises a chimeric Fc region comprising the CH2 domain of an IgG antibody of the IgG4 isotype and the CH3 domain of an IgG antibody of the IgG1 isotype, or a chimeric Fc region comprising the hinge region of IgG2 and the CH2 region of IgG4, or an Fc region having a mutation with reduced or absent Fc effector function. Fc regions with reduced or absent Fc effector function include those known in the art. See, for example, 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 published publications and PCT published publications cited therein. Also, Fc regions with reduced or absent Fc effector function can be readily made by those skilled in the art.

[0254] [III. Nucleic Acid Molecules] Still other aspects described herein relate to one or more nucleic acid molecules encoding any one of the antibodies described herein. The nucleic acids can be present in whole cells, cell lysates, or in a partially purified or substantially pure form. Nucleic acids are “isolated” or “substantially purified” when purified by standard techniques including, but not limited to, alkaline / SDS treatment, CsCl banding, column chromatography, restriction enzymes, agarose gel electrophoresis, and other techniques well known in the art, from other cellular components or other contaminants, such as other cellular nucleic acids (e.g., other chromosomal DNA, e.g., chromosomal DNA ligated to naturally isolated DNA) or proteins. See 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 can contain or lack intron sequences. In certain embodiments, the nucleic acids are cDNA molecules.

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

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

[0257]

Table 8

[0258] JPEG0007687703000011.jpg143169

[0259]

Table 9

[0260] JPEG0007687703000013.jpg57169

[0261] The method for producing an anti-FAM19A5 antibody disclosed in this specification can include expressing the heavy and light chains together with a signal peptide in a cell line containing nucleotide sequences encoding the heavy and light chains. Host cells containing these nucleotide sequences are included herein.

[0262] When DNA fragments encoding VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example, the variable region genes can be converted into full-length antibody chain genes, Fab fragment genes or scFv genes. In these manipulations, the VL or VH-coding DNA fragments are operably linked to other DNA fragments encoding further other proteins such as antibody constant regions or flexible linkers. The term "operably linked" used in connection with this is intended to mean that two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in-frame.

[0263] The isolated DNA encoding the VH region can be converted into a full-length heavy chain gene by operably linking the VH-coding DNA to other DNA molecules encoding heavy chain constant regions (hinge, CH1, CH2 and / or CH3). The sequences of human heavy chain constant region genes are known in the art (e.g., Kabat, EA, el al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91 - 3242 (reference), 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, such as an IgG2 and / or IgG4 constant region. In the case of the Fab fragment heavy chain gene, the VH - coding DNA can be operably linked to another DNA molecule that encodes only the heavy chain CH1 constant region.

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

[0265] To generate the scFv antibody, the VH- and VL-coding DNA fragments are operably linked to another fragment encoding a flexible linker, such as the amino acid sequence (Gly4-Ser)3, such that the VH and VL sequences can be expressed as an adjacent single-chain protein in which the VL and VH regions are joined by a flexible linker (see, for example, Bird et al., (1988) Science 242:423-426; Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature 348:552-554). The amino acid sequence of an exemplary anti-FAM19A5 scFv is provided in Table 7.

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

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

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

[0269] The expression vector of the present disclosure can include a polynucleotide encoding the antibody described herein. In one embodiment, the sequence encoding the antibody is operably linked to an expression regulatory 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 maintain its functionality. A coding sequence and a gene expression regulatory sequence are operably linked when they are covalently linked such that the expression or transcription and / or translation of the coding sequence is under the influence or control of the gene expression regulatory sequence. Induction of a promoter in a 5' gene expression sequence causes transcription of the coding sequence, and the binding characteristics between the two DNA sequences are such that (1) they do not cause the introduction of a frame-shift mutation, (2) they do not interfere with the ability of the promoter region to direct transcription of the coding sequence, or (3) they do not interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, when a gene expression sequence can affect the transcription of a coding nucleic acid sequence, the gene expression sequence is operably linked to the coding nucleic acid sequence such that the transcript produced is translated into the desired antibody.

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

[0271] In one embodiment, the virus is an adeno-associated virus which is a double-stranded DNA virus. The adeno-associated virus may be engineered to have replication defects and can infect a wide range of cell types and species. It also has advantages such as heat and lipid solvent stability; high transduction frequency in various cell lines including hematopoietic cells; lack of superinfection inhibition and the ability to perform various transduction series. According to reports, since adeno-associated virus can integrate into human cellular DNA in a site-specific manner, it can minimize the induction potential of insertional mutations and the variability of the inserted gene expression characteristics of retroviral infections. Also, wild-type adeno-associated virus infection has been tracked in tissue culture during more than 100 passages without selective pressure, which means that adeno-associated virus genome integration is a relatively stable reaction. The adeno-associated virus may act in an episomal manner.

[0272] In other embodiments, the vector is derived from a lentivirus. In a specific embodiment, the vector is a vector of a recombinant lentivirus that can infect non-dividing cells.

[0273] The lentivirus genome and proviral DNA generally have gag, pol, and env, which are three genes flanked by two long terminal repeat (LTR) sequences found in retroviruses. The gag gene encodes internal structure (matrix, capsid, and nucleocapsid) proteins; the pol gene encodes RNA-directed DNA polymerase (reverse transcriptase), protease, and integrase; the env gene encodes viral envelope glycoproteins. The 5' and 3' LTRs play a role in promoting the transcription and polyadenylation of virion RNA. The LTR contains all other cis-acting sequences necessary for virus replication. Lentiviruses have additional genes including vif, vpr, tat, rev, vpu, nef, and vpx (in HIV-1, HIV-2, and / or SIV).

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

[0275] However, as a result, the resulting mutants remain in a state where they can direct the synthesis of all virion proteins. The present disclosure provides a method for producing a recombinant lentivirus capable of infecting non-dividing cells, including transfecting a suitable host cell with two or more vectors having a packaging function, namely, not only gag, pol, and env, but also rev and tat. As disclosed below, vectors lacking a functional tat gene are suitable for certain applications. Thus, for example, a first vector can provide nucleic acids encoding viral gag and viral pol, and another vector can provide nucleic acids encoding viral env for producing packaging cells. As used herein, introducing into a packaging cell a vector that provides a heterologous gene identified as a transfer vector results in a producer cell that releases infectious virus particles carrying the foreign gene of interest.

[0276] According to the vector and foreign gene constructs described above, the second vector can provide nucleic acids encoding the viral envelope (env) gene. The env gene can be derived from substantially all suitable viruses, including retroviruses. In some embodiments, the env protein is a amphotropic envelope protein that enables transduction of cells of humans and other species.

[0277] 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 Ape Leukemia Virus, human immunodeficiency virus (HIV), and Rous sarcoma virus. Other env genes such as the Vesicular stomatitis virus (VSV) protein G (VSV G), hepatitis virus and influenza virus genes may also be used.

[0278] Vectors providing viral env nucleic acid sequences are operably linked to regulatory sequences described elsewhere herein.

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

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

[0281] In some embodiments, the lentiviral vectors of the present disclosure comprising the FVIII nucleotide sequences described herein may be transfected using (a) a first nucleotide sequence comprising the gag, pol or gag and pol genes, and (b) a second nucleotide sequence comprising a heterologous env gene; wherein the lentiviral vector lacks a functional tat gene. In other embodiments, the cell is further transfected using 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 combinations thereof.

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

[0283] Examples of lentiviral vectors are disclosed in W09931251, W09712622, W09817815, W09817816, and W09818934, the entire contents of which are incorporated herein by reference.

[0284] Other vectors include plasmid vectors. Plasmid vectors are widely described in the art and are well known to those skilled in the art. See, for example, Sambrook el al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. In recent years, plasmid vectors have been shown to be particularly advantageous for delivering genes to cells in vivo because they replicate within the host genome and cannot integrate into the host genome. However, these plasmids having a promoter compatible with the host cell can express peptides from genes operably encoded within the plasmid. Some commonly available and generally used plasmids from commercial suppliers include pBR322, pLTCl8, pLTCl9, various pcDNA plasmids, pRC / CMV, various pCMV plasmids, pSV40, and pBlueScript. Additional examples of specific plasmids are pcDNA3.1, catalog number V79020; pcDNA3.1 / hygro, catalog number V87020; pcDNA4 / myc-His, catalog number V86320; and pBudCE4.l, catalog number V53220 (all from Invitrogen (Carlsbad, CA)). Other plasmids are well known to those skilled in the art. Furthermore, plasmids can be custom designed using standard molecular biology techniques to remove and / or add specific fragments of DNA.

[0285] [VI. Antibody Production] Antibodies or fragments thereof that immunospecifically bind to FAM19A5 (e.g., human FAM19A5) can be generated by any method known in the art for antibody synthesis, such as chemical synthesis or recombinant expression techniques. The methods disclosed herein utilize, unless otherwise indicated, conventional techniques in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related technical fields in the art. These techniques are fully described, for example, in the references incorporated herein by reference. 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 Please refer to & Sons (1987 and annual updates), Gait (ed.) (1984), Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein (ed.) (1991), Oligonucleotides and Analogues: A Practical Approach, IRL Press; Birren B et al., (eds.) (1999), Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press.

[0286] In certain embodiments, the antibodies described herein are antibodies (e.g., recombinant antibodies) produced, expressed, generated, or isolated by any means involving synthesis, generation using genetic manipulation 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 a living animal or mammal (e.g., human). In some embodiments, the anti-FAM19A5 antibodies disclosed herein are deimmunized.

[0287] As described in the Examples section (e.g., Example 2), the anti-FAM19A5 antibodies were first generated by immunizing chickens with synthetic FAM19A5 peptides. Thus, to minimize the risk of immunogenicity upon administration to humans, the anti-FAM19A5 antibodies (e.g., 3-2 and 2-13) were engineered to be more similar to the immunogenic sequences of human antibodies. In some embodiments, the deimmunized anti-FAM19A5 antibodies disclosed herein have similar binding affinities for human FAM19A5 as their corresponding non-deimmunized counterparts. In some embodiments, the anti-FAM19A5 antibodies disclosed herein have also undergone affinity maturation. Methods for deimmunizing antibodies are disclosed herein and are known in the art.

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

[0289] Methods for producing 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).

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

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

[0292] 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 induce lymphocytes that produce or are capable of producing antibodies that specifically bind to the protein used for immunization (e.g., human FAM19A5). Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusogen such as polyethylene glycol to form hybridoma cells (Goding JW (Ed), Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Additionally, animals can be immunized using the RIMMS (repeated immunization multiple site) technique (Kilpatrick KE et al., (1997) Hybridoma 16:381-9, which is incorporated by reference in its entirety).

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

[0294] The hybridoma cells thus produced are preferably inoculated and grown in a suitable culture medium containing one or more substances that inhibit the growth or survival of the unfused parental myeloma cells. For example, when the parental myeloma cells lack the enzyme hypoxanthine-guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for hybridomas will typically contain hypoxanthine, aminopterin and thymidine (HAT medium), which are substances that interfere with the growth of HGPRT-deficient cells.

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

[0296] The culture medium in which the hybridoma cells grow is analyzed for 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 (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0297] After identifying hybridoma cells that produce antibodies of 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 above). Culture media suitable for such purposes include, for example, D-MEM or RPMI 1640 medium. In addition, the hybridoma cells can be grown in vivo as multiple tumors in an animal.

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

[0299] The antibodies described herein recognize a specific FAM19A5 (e.g., human FAM19A5) and include antibody fragments that can be generated by any technique known to those skilled in the art. For example, the Fab and F(ab’) 2 fragments described herein can be generated by proteolytic cleavage of the immunoglobulin molecule using an enzyme such as papain (for generating Fab fragments) or pepsin (for generating F(ab’) 2 fragments). The Fab fragment corresponds to one of the two identical arms of the antibody molecule and contains the complete 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 the antibody molecule linked by a disulfide bond in the hinge region.

[0300] In addition, the antibodies or antigen-binding fragments thereof described in this specification may be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles that carry the polynucleotide sequences encoding them. In particular, the DNA sequences encoding the VH and VL domains are amplified from an animal cDNA library (e.g., a non-human cDNA library such as a human cDNA library or a murine or avian cDNA library of infected tissues). The DNA encoding the VH and VL domains is recombined with an scFv linker by PCR and cloned into a phagemid vector. The vector is electroporated into E. coli, and the E. coli is infected with helper phage. The phages used in these methods are typically filamentous phages including fd and M13, and the VH and VL domains are generally recombinantly fused to phage gene III or gene VIII. Phages expressing antigen-binding domains that bind to a particular antigen can be selected or identified using an antigen, e.g., a labeled antigen or an antigen bound or captured on a solid surface or beads. Examples of phage display methods available for producing the antibodies described in this specification are Brinkman U et al., (1995) J Immunol Methods 182:41-50; Ames RS et al., (1995) J Immunol Methods 184:177-186; Kettleborough CA et al., (1994) Eur J Immunol 24:952-958; Persic L et al., (1997) Gene 187:9-18; Burton DR & Barbas CF (1994) Advan Immunol 57:191-280; PCT application number PCT / GB91 / 001134; international publication numbers WO90 / 02809, WO91 / 10737, WO92 / 01047, WO92 / 18619, WO93 / 11236, WO95 / 15982, WO95 / 20401 and WO97 / 13844; US Patent Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743 and 5,969,108, including those disclosed therein.

[0301] As described in the foregoing references, after phage selection, the antibody-coding region of the phage can be isolated and used to generate whole antibodies or any other desired antigen-binding fragment, including human antibodies, and can be expressed in any desired host, including, for example, mammalian cells, insect cells, plant cells, yeast and bacteria, as described below. PCT publication number 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, techniques for recombinantly producing antibody fragments such as Fab, Fab’ and F(ab’) 2 fragments can also be employed.

[0302] In one aspect, the VH or VL nucleotide sequence can be amplified from a template, e.g., an scFv clone, using a PCR primer that includes a VH or VL nucleotide sequence, a restriction site, and a flank sequence for protecting the restriction site to generate a full antibody. Using cloning techniques known to those skilled in the art, the PCR-amplified VH domain can be cloned into a vector that expresses the VH constant region, and the PCR-amplified VL domain can be cloned into a vector that expresses the VL constant region, e.g., a human kappa or lambda constant region. The VH and VL domains can be cloned into one vector that expresses the required constant region. Subsequently, the heavy chain conversion vector and the light chain conversion vector are co-transfected into a cell line using techniques known to those skilled in the art to generate a stable or transient cell line that expresses a full-length antibody, e.g., IgG.

[0303] A chimeric antibody is a molecule in which different parts of the antibody are derived from different immunoglobulin molecules. For example, a chimeric antibody can include the variable region of a monoclonal antibody from a non-human animal (e.g., mouse, rat, or chicken) 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.

[0304] A humanized antibody can bind to a given antigen and comprises a framework region having substantially the amino acid sequence of a human immunoglobulin and CDRs having substantially the amino acid sequence of a non-human immunoglobulin (e.g., murine or avian immunoglobulin). In certain embodiments, the humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin. The antibody can also comprise the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. The humanized antibody can be selected from all types of immunoglobulins including IgM, IgG, IgD, IgA, and IgE and any isotype including IgG1, IgG2, IgG3, and IgG4. The humanized antibody can be produced using, but is not limited to, various techniques known in the art including: CDR-grafting (European Patent No. EP239400; International Publication No. WO91 / 09967; and U.S. Patent Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (European Patent Nos. EP592106 and EP519596; 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 (U.S. Patent No. 5,565,332), chain shuffling (U.S. Patent No. 5,565,332), and, for example, U.S. Patent No. 6,407,213, U.S. Patent No. 5,766,886, International Publication No. WO93 / 17105; Tan P et al., (2002) J Immunol 169:1119-25; Caldas C et al., (2000) Protein Eng. 13(5):353-60; Morea V et al., (2000) Methods 20(3):267-79; Baca M et al., (1997) J Biol Chem 272(16):10678-84; Roguska MA et al., (1996) Protein Eng 9(10):895-904; Couto JR et al., (1995) Cancer Res. 55(23Supp):5973s-5977s; Couto JR et al., (1995) Cancer Res 55(8):1717-22; Sandhu JS(1994) Gene 150(2):409-10 and Pedersen JT et al., (1994) J Mol Biol 235(3):959-73. See also U.S. Patent Application Publication No. US2005 / 0042664A1 (February 24, 2005).

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

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

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

[0308] In certain embodiments, 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 binding of the antibodies described herein to FAM19A5 (e.g., human FAM19A5) are human antibodies or antigen-binding fragments thereof.

[0309] Human antibodies can be produced using any method known in the art. For example, transgenic mice that can express human immunoglobulin genes but not functional endogenous immunoglobulins can be used. In particular, the human heavy and light chain immunoglobulin gene complexes can be introduced into mouse embryonic stem cells either randomly or by homologous recombination. Alternatively, in addition to the human heavy and light chain genes, human variable, constant, and diversity regions can be introduced into mouse embryonic stem cells. The mouse heavy and light chain immunoglobulin genes can be rendered non-functional either separately or simultaneously with the introduction of the human immunoglobulin gene loci by homologous recombination. In particular, homozygous deletion of the JH region renders endogenous antibody production impossible. The modified embryonic stem cells are expanded and microinjected into blastocysts to generate chimeric mice. The chimeric mice are bred to generate homozygous progeny that express human antibodies. The transgenic mice are immunized in a normal manner with a selected antigen, e.g., all or part of an antigen (e.g., FAM19A5). Monoclonal antibodies directed against the antigen can be obtained from the immunized transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes carried by the transgenic mice are rearranged during B cell differentiation and subsequently undergo class switching and somatic hypermutation. Thus, using such techniques, therapeutically useful IgG, IgA, IgM, and IgE antibodies can be produced. See Lonberg N & Huszar D (1995) Int Rev Immunol 13:65-93 for an overview of the technology for human antibody production. For a detailed discussion of the technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, for example, International Publication Nos. WO98 / 24893, WO96 / 34096, and WO96 / 33735; and U.S. Patent Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, and 5,939,598. Examples of mice that can produce human antibodies include XENOMOUSETM (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 (See Medarex / Kirin).

[0310] Human antibodies that specifically bind to FAM19A5 (e.g., human FAM19A5) can be made by various methods known in the art, including the phage display method described above, 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; and International Publication Nos. WO98 / 46645, WO98 / 50433, WO98 / 24893, WO98 / 16654, WO96 / 34096, WO96 / 33735 and WO91 / 10741.

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

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

[0313] Accordingly, provided herein is (a) (i) a heavy chain variable region sequence comprising the CDR1, CDR2, and / or CDR3 sequences shown in Table 3 or the CDR1, CDR2, and / or CDR3 of the heavy chain variable region shown in Table 5; and (ii) a light chain variable region sequence comprising the CDR1, CDR2, and / or CDR3 sequences shown in Table 4 or the CDR1, CDR2, and / or CDR3 of the heavy chain variable region shown in Table 6; (b) modifying at least one amino acid residue within the heavy chain variable region sequence and / or the light chain variable region sequence to generate at least one engineered antibody sequence; (c) expressing the engineered antibody sequence as a protein, a method for producing an anti-FAM19A5 antibody is provided.

[0314] The engineered antibody sequences can be produced and expressed using standard molecular biology techniques.

[0315] In some embodiments, the antibody encoded by the modified antibody sequence is an antibody that possesses one, some, or all of the functional properties of the anti-FAM19A5 antibodies described herein, including: (1) Reduced immunogenicity in a human subject; (2) Binding to soluble human FAM19A5 with a K D of 10 nM or less (e.g., 0.01 nM to 10 nM), as measured by, for example, Biacore; (3) Binding to membrane-bound human FAM19A5 with a K D of 10 nM or less (e.g., 0.01 nM to 1 nM), as measured by, for example, ELISA; (4) Binding to membrane-bound human FAM19A5 with an EC50 of 1 nM or less (e.g., 0.01 nM to 1 nM), as measured by, for example, ELISA; (5) Reducing, reversing, delaying, and / or preventing the onset of reactive gliosis; (6) Inhibiting the over-proliferation of reactive astrocytes; (7) Reducing the expression of chondroitin sulfate proteoglycans including neurocan and neuronal-glial antigen 2 (NG2); (8) Increasing the expression of c-fos and pERK in the nuclei of neurons; (9) Promoting the survival of neurons; (10) Increasing the expression of GAP43 in neurons; (11) Promoting axonal regrowth; and (12) Competing with the anti-FAM19A5 antibodies disclosed herein in either one or both directions with respect to binding to human FAM19A5.

[0316] The modified antibody 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, eleven, or all of the functional properties shown in (1)-(12) above. The functional properties of the modified antibody are available in the art and / or can be evaluated using standard analytical methods described herein (e.g., ELISA, FACS), as shown in the Examples section.

[0317] In certain embodiments of the methods of manipulating the antibodies described herein, mutations may be introduced randomly or selectively along all or part of the anti-FAM19A5 antibody coding sequence, and the resulting mutated anti-FAM19A5 antibodies can be screened against the binding activities and / or other functional properties described herein. Mutation methods are described in the art. For example, Short's PCT publication WO02 / 092780 describes methods for generating and screening antibody mutations using saturation mutagenesis, synthetic ligation assembly, or combinations thereof. In contrast, PCT publication WO03 / 074679 by Lazar et al. describes methods that utilize computer screening methods to optimize the physicochemical properties of antibodies.

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

[0319] Recombination of the antibodies described herein (e.g., full-length antibodies, heavy and / or light chains of antibodies or single-chain antibodies) that specifically bind to FAM19A5 (e.g., human FAM19A5) involves the preparation of an expression vector containing a polynucleotide encoding said antibody. Once a polynucleotide encoding an antibody molecule, a heavy and / or light chain of an antibody or a fragment thereof (e.g., heavy and / or light chain variable domains) described herein is obtained, a vector for the production of said antibody molecule can be manufactured by recombinant DNA techniques using techniques well known in the art. Thereby, a method for producing a protein by expressing a polynucleotide containing a nucleotide sequence encoding an antibody or an antibody fragment (e.g., a light or heavy chain) is described herein. An expression vector containing an antibody or antibody fragment (e.g., light or heavy chain) coding sequence and appropriate transcriptional and translational control signals can be prepared using methods well known to those skilled in the art. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques and in vivo gene recombination. Also provided is a replicable vector containing a nucleotide sequence encoding an antibody molecule, a heavy or light chain of an antibody, a heavy or light chain variable domain or a fragment thereof, or a heavy or light chain CDR operably linked to a promoter described herein. Such a vector can, for example, contain a nucleotide sequence encoding the constant region of said antibody molecule (see, e.g., International Publication Nos. WO86 / 05807 and WO89 / 01036; and U.S. Patent No. 5,122,464), and the variable domain of said antibody can be cloned into said vector for expression of the entire heavy chain, the entire light chain, or both the entire heavy and light chains.

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

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

[0322] A variety of host-expression vector systems can be used to express the antibody molecules described herein. The host-expression system corresponds to a vehicle that can generate and then purify the coding sequence of interest, but also corresponds to a cell that can express the antibody molecules described herein in situ upon transformation or transfection with an appropriate nucleotide coding sequence. These include microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing 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 expression vectors (e.g., baculovirus) containing the antibody coding sequence; plant cell systems (e.g., green algae such as Chlamydomonas reinhardtii) infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the antibody coding sequence; 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, B-W, L-M, BSCl, BSCl, BSC40, YB / 20 and BMT10 cells) carrying a recombinant expression construct containing a promoter derived from the genome of a mammalian cell (e.g., metallothionein promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter), including but not limited to. In certain embodiments, the cells for expressing the antibodies or antigen-binding fragments thereof described herein are CHO cells, such as the CHO GS SYSTEM TM(Lonza)'s CHO cells. In certain embodiments, the cells for expressing the antibodies described herein are human cells, such as human cell lines. In certain embodiments, the mammalian expression vector is pOPTIvectorTM or pcDNA3.3. In certain embodiments, in particular, bacterial cells such as Escherichia coli or eukaryotic cells (e.g., mammalian cells) for the expression of the whole recombinant antibody molecule are used for the expression of the recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary (CHO) cells are an effective expression system for antibodies in combination with vectors such as the major intermediate early gene promoter element of human cytomegalovirus (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, the expression of the nucleotide sequence encoding the antibodies 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.

[0323] In a bacterial system, a number of expression vectors may be advantageously selected depending on the intended use for the antibody molecule to be expressed. For example, when attempting to produce the antibody in large quantities for making a pharmaceutical composition of the antibody molecule, a vector that directs the expression of a high level of fusion protein product that is easily purified would be preferred. Such vectors include the E. coli expression vector pUR278 in which the antibody coding sequence is ligated individually to the vector in a configuration having the lac Z coding region to produce a fusion protein (Ruether U & Mueller-Hill B (1983) EMBO J 2:1791-1794); 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); etc. are included, but not limited thereto. For example, a pGEX vector may be used to express a foreign polypeptide as a fusion protein with glutathione 5-transferase (GST). Generally, the fusion protein is soluble and can be easily purified from lysed cells by eluting in the presence of free glutathione after adsorption and binding to matrix glutathione agarose beads. The pGEX vector is configured to include a thrombin or factor Xa protease cleavage site, and the cloned target gene product can be released from the GST moiety.

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

[0325] A number of viral expression systems can be used in mammalian host cells. When using an adenovirus as an expression vector, an antibody-coding sequence of interest can be ligated to an adenovirus transcription / translation control complex, such as a late promoter and a tripartite leader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (e.g., region E1 or E3) will generate a recombinant virus that is viable and capable of expressing the antibody molecule in the infected host (see, for example, 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 start codon and adjacent sequences. Also, the start codon must be in frame with the reading frame of the desired coding sequence so that translation of the entire insert is possible. These exogenous translation control signals and start codons can have various origins, both natural and synthetic. Expression efficiency can be increased by including appropriate transcriptional enhancer elements, transcriptional terminator factors, etc. (see, for example, Bitter G et al., (1987) Methods Enzymol. 153:516-544).

[0326] In addition, host cell strains capable of regulating the expression of the inserted array or modifying and processing gene products in a desired specific manner can be selected. 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 the post-translational processing and modification of proteins and gene products. An appropriate cell line or host system can be selected to enable proper modification and processing of the expressed foreign protein. For this purpose, eukaryotic host cells possessing cellular mechanisms for proper processing of primary transcripts, glycosylation, and phosphorylation of gene products can be used. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, Hela, MDCK, HEK293, NIH3T3, W138, BT483, Hs578T, HTB2, BT20, and T47D, NSO (a murine myeloma cell line that does not produce any immunoglobulin chains in vivo), CRL7030, COS (e.g., COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, B-W, L-M, BSC1, BSC40, YB / 20, BMT10, and HsS78Bst cells. In certain embodiments, the anti-FAM19A5 antibodies described herein are produced in mammalian cells such as CHO cells.

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

[0328] Stable expression cells can be created to produce recombinant proteins with high yields over a long period. For example, cell lines that stably express the anti-FAM19A5 antibody or antigen-binding portion thereof described herein can be manipulated. In certain embodiments, the cells provided herein stably express the light chain / light chain variable domain and the heavy chain / heavy chain variable domain that associate to form the antibody or antigen-binding portion thereof described herein.

[0329] In certain embodiments, instead of using an expression vector containing a viral replication origin, host cells can be transformed with DNA and a selection marker controlled by appropriate expression control elements (e.g., promoter, enhancer, sequence, transcription terminator, polyadenylation site, etc.). After introduction of the foreign DNA / polynucleotide, the manipulated cells can be grown in a concentrated medium for 1-2 days and then changed to a selective medium. In the recombinant plasmid, the selection marker confers resistance to selection, and the cells can form foci that can be cloned and expanded into cell lines by stably integrating the plasmid into their chromosomes and growing. This method can be advantageously used to manipulate cell lines that express the anti-FAM19A5 antibody or antibody-binding portion thereof described herein. Cell lines thus manipulated can be particularly useful for screening and evaluating compositions that interact directly or indirectly with the antibody molecule.

[0330] A number of selectable systems can be used, but are not limited to, those containing the herpes simplex virus thymidine kinase (Wigler M et al., (1977) Cell 11(1):223-32), hypoxanthine-guanine phosphoribosyl transferase (Szybalska EH & Szybalski W (1962) PNAS 48(12):2026-2034), and adenine phosphoribosyl transferase (Lowy I et al., (1980) Cell 22(3):817-23) genes, each of which can be used in tk-, hgprt-, or aprt- cells. Also, antimetabolite resistance can be used as the basis for selection for genes such as dhfr, which confers resistance to methotrexate (Wigler M et al., (1980) PNAS 77(6):3567-70; O’Hare K et al., (1981) PNAS 78:1527-31); gpt, which confers resistance to mycophenolic acid (Mulligan RC & Berg P (1981) PNAS 78(4):2072-6); neo, which confers resistance to 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 et al., (1984) Gene 30(1-3):147-56). Conventional methods known in the field of recombinant DNA technology may generally be 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), the entire of which is hereby incorporated by reference; Kriegler M, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and Chapters 12 and 13, Dracopoli NC et al., (eds.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colbere-Garapin F et al., (1981) J Mol Biol 150:1-14.

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

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

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

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

[0335] In certain embodiments, the antibodies or antigen-binding portions thereof described herein are isolated or purified. Generally, an isolated antibody is one in which no other antibodies having antigen specificity different from that of the isolated antibody are substantially present. For example, in certain embodiments, the formulations of the antibodies described herein are substantially free of cellular material and / or chemical precursors. The term "substantially free of cellular material" includes formulations of antibodies that are isolated from cells or produced recombinantly and separated from the cellular components of the cells. Thus, an antibody that is substantially free of cellular material is one that has less than about 30%, 20%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (by dry weight) of heterologous proteins (also referred to herein as "contaminating proteins") and / or variants of the antibody, e.g., different post-translationally modified forms of the antibody or other different variant forms of the antibody (or antibody-binding portion). When the antibody is produced recombinantly, the antibody also generally is substantially free of culture medium, i.e., the culture medium accounts for less than about 20%, 10%, 2%, 1%, 0.5%, or 0.1% of the volume of the protein formulation. When the antibody is produced by chemical synthesis, the antibody generally is substantially free of chemical precursors or other chemicals, i.e., separated from the chemical precursors or other chemicals involved in the synthesis of the protein. Thus, such antibody formulations have less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or compounds other than the antibody of interest. In certain embodiments, the antibodies described herein are isolated or purified.

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

[0337] The above - described ELISA assay can be used to screen antibodies and hybridomas that produce antibodies showing positive reactivity with the FAM19A5 immunogen in relation thereto. Next, preferably, hybridomas that produce antibodies binding to FAM19A5 with high affinity can be subcloned and further characterized. Next, one clone can be made from each of the hybridomas that retain the reactivity of the parental cells (by ELISA) to create a cell bank and selected for antibody purification.

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

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

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

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

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

[0343] Methods for analyzing the binding affinity, cross-reactivity, and binding kinetics of various anti-FAM19A5 antibodies include standard analytical methods known in the art, such as BIACORE TM BIACORE using a 2000 SPR instrument (Biacore AB, Uppsala, Sweden) TM including surface plasmon resonance (SPR) analysis.

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

[0345] [VIII. Bispecific Molecules] The antibodies described herein can be used to form bispecific molecules. The anti-FAM19A5 antibody or antigen-binding portion thereof can be derivatized or linked to another functional molecule, such as another peptide or protein (e.g., another antibody or ligand for a receptor), to generate a bispecific molecule that binds to at least two different binding sites or target molecules. Cytokines such as IL-6, CNTF, LIF, EGF, and TGFα have been implicated as promoters of gliosis and / or reactive astrocytosis by activating signal transducer and activator of transcription 3 (STAT3), a transcriptional protein signaling factor and activator that modulates the profuse aspects of reactive astrocytosis after CNS injury (Balasingam et al., J. Neurosci. 14(2):846-56 (1994); Winter et al., Proc. Natl. Acad. Sci. U.S.A. 20;92(13):5865-9 (1995)). See Herrmann J.E. et al., J. Neurosci. 28(28):7231-7243 (2008). For example, in the absence or reduction of STAT3, it manifests as attenuated upregulation of glial fibrillary acidic protein (GFAP), failure of astrocyte hypertrophy, increased spread of inflammation, increased lesion volume, and partial attenuation of motor function recovery after CNS injury. See Herrmann J.E. et al., J. Neurosci. 28(28):7231-7243 (2008). Thereby, for example, the anti-FAM19A5 antibody can be linked to an antibody or scFv that specifically binds to any protein involved in inhibiting the development of gliosis and / or the overproliferation of reactive astrocytosis for combination therapy, such as an antibody against IL-6, CNTF, LIF, EGF, or TGFα.

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

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

[0348] Accordingly, the present specification provides a bispecific molecule comprising at least one first binding specific entity for FAM19A5 and a second binding specific entity for a second target epitope. In embodiments described herein where the bispecific molecule is multispecific, the molecule can further comprise a third binding specific entity.

[0349] In one embodiment, the bispecific molecules described herein have, as binding specific entities, for example, Fab, Fab', F(ab') 2comprises at least one antibody or an antibody binding portion thereof that includes an Fv or single-chain Fv (scFv). The antibody may also be a light or heavy chain dimer, or any minimal fragment thereof such as an Fv or single-chain construct, as described in U.S. Patent No. 4,946,778 to Ladner et al., the contents of which are hereby incorporated by reference.

[0350] Human monoclonal antibodies are preferred, but other antibodies useful in the bispecific molecules described herein are murine, chimeric, and humanized monoclonal antibodies.

[0351] The bispecific molecules described herein can be produced by conjugating the constituent binding specificities using methods known in the art. For example, each binding specificity of the bispecific molecule can be generated separately and then conjugated to each other. When the binding specificities are proteins or peptides, various binders 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 sulfo-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) (e.g., Karpovsky et al., (1984) J. Exp. Med. 160:1686; Liu, MA et al., (1985) Proc. Natl. Acad. Sci. USA 82:8648). Other methods include those described in Paulus (1985) Behring Ins. Mitt. No. 78, 118-132; Brennan et al., (1985) Science 229:81-83) and Glennie et al., (1987) J. Immunol. 139:2367-2375). Preferred binders are SATA and sulfo-SMCC, both of which are available from Pierce Chemical Co. (Rockford, IL).

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

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

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

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

[0356] The antibodies described herein can be used for diagnostic purposes including sample testing and in vivo imaging, and for this purpose the antibody (or its binding portion) 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.

[0357] The detectable label includes particulate labels including metal sols such as colloidal gold, for example, I presented with a peptide chelating agent of the N2S2, N3S or N4 type 125 or Tc 99Not only chromophores including isotopes, fluorescent markers, luminescent markers, phosphorescent markers, etc., but also enzyme labels that convert a predetermined substrate into a detectable marker and polynucleotide tags that are confirmed after amplification by, for example, polymerase chain reaction may be any of various types currently used in the field of in vitro diagnosis. Suitable enzyme labels include horseradish peroxidase, alkaline phosphatase, etc. For example, the label may be not only adamantylmethoxyphosphoryloxyphenyldioxetane (AMPPD), disodium 3-(4-(methoxyspiro{1,2-dioxetane-3,2’-(5’-chloro)tricyclo{3.3.1.13,7}decane}-4-yl)phenyl phosphate (CSPD), but also 1,2-dioxetane substrates such as CDP and CDP-star (registered trademark) or other chemiluminescent substrates well known to those skilled in the art, for example, an enzyme alkaline phosphatase whose presence or formation of chemiluminescence is detected by measuring after conversion of a suitable lanthanide chelate such as terbium(III) and europium(III). The detection means is determined by the selected label. The appearance of the label or its reaction product, when the label is a microparticle and accumulates at an appropriate level, uses the naked eye or equipment such as a spectrophotometer, luminometer, fluorometer, etc., all of which can be obtained by standard practice.

[0358] The antibodies described herein can also be conjugated to therapeutic agents to form immunoconjugates such as antibody-drug conjugates (ADCs). Suitable therapeutic agents include formulations that modulate the onset of gliosis and / or reactive astrocytosis and / or treat degenerative brain disorders, central nervous system injuries or neuropathic pain. Therapeutic agents for treating degenerative brain disorders include drugs 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, for example, the drugs disclosed in Section XII below.

[0359] Immunoconjugates can be produced by methods known in the art. Preferably, as a result of the conjugation method, bonds that are substantially (or almost) non-immunogenic appear, such as peptide- (i.e., amide-), sulfide-, (steric hindrance), disulfide-, hydrazone- and ether bonds. These bonds are mostly non-immunogenic and show considerable stability in serum (see, for example, Senter, P.D., Curr. Opin. Chem. Biol. 13 (2009) 235-244; WO2009 / 059278; WO95 / 17886).

[0360] Conjugation strategies can be used that vary depending on the biochemical properties of the moiety and the antibody. When the moiety is natural or recombinant of 50 to 500 amino acids, standard procedures describing chemical methods for the synthesis of protein conjugates are available in textbooks and can be easily followed by those skilled in the art (for example, Hackenberger, C.P.R., and Schwarzer, D., Angew. Chem. Int. Ed. Engl. 47 (2008) 10030-10074). In one embodiment, the reaction of cysteine residues in the antibody or moiety with maleimide moieties is utilized. This is a particularly suitable coupling chemistry, for example, when using Fab or Fab’ fragments of an antibody. In contrast, in one embodiment, coupling is performed at the C-terminus of the antibody or moiety. C-terminal modification of a protein, such as a Fab-fragment, can be performed as described, for example, in Sunbul, M. and Yin, J., Org. Biomol. Chem. 7 (2009) 3361-3371.

[0361] Generally, site-specific reactions and covalent bonds are based on converting natural amino acids into amino acids with reactivity orthogonal to the reactivity of other functional groups present. For example, a specific cysteine within a rare sequence context can be enzymatically converted to an aldehyde (see Frese, M.A. and Dierks, T., ChemBioChem. 10 (2009) 425-427). Desired amino acid modifications can also be obtained using the specific enzymatic reactivity of a natural amino acid and a specific enzyme within a given sequence context (e.g., see Taki, M. et al., Prot. Eng. Des. Sel. 17 (2004) 119-126; Gautier, A. et al., Chem. Biol. 15 (2008) 128-136; and Bordusa, F., Highlights in Bioorganic Chemistry (2004) 389-403, where protease-catalyzed formation of C-N bonds is used).

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

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

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

[0365] Conjugates having a 1:1 stoichiometric ratio to obtain a single-labeled polypeptide can be separated chromatographically from other conjugation by-products. This process can be facilitated by using a dye-labeled binding partner member and a charged linker. By using such types of labels and highly negatively charged binding partner members, the charge and molecular weight differences can be utilized for separation, so that the singly conjugated polypeptide is easily separated from the unlabeled polypeptide and the polypeptide having more than one linker. The fluorescent dye can be useful for purifying the complex from the unbound components, like the labeled monovalent binder.

[0366] [X. Pharmaceutical Compositions] This specification provides a composition comprising an antibody or antigen-binding portion thereof described herein, having a desired degree of purity in a physiologically acceptable carrier, excipient, or stabilizer (Remington’s Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA). Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dosages and concentrations employed, and include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium 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 immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; 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 nonionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

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

[0368] Pharmaceutically acceptable carriers used in parenteral formulations include aqueous vehicles, non-aqueous vehicles, antimicrobial agents, tonicity agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents for metal ions, and other pharmaceutically acceptable substances. Examples of aqueous vehicles include sodium chloride injection, infusion injection, isotonic dextrose injection, sterile water for injection, and dextrose and lactate infusion injection. Non-aqueous parenteral vehicles include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, and peanut oil. Antimicrobial agents at bacteriostatic or fungistatic concentrations can be added to parenteral formulations packaged in multi-dose containers containing phenol or cresol, mercury-containing substances, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoate esters, thimerosal, benzalkonium chloride, and benzethonium chloride. Tonicity agents include sodium chloride and dextrose. Buffers include phosphates and citrates. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose, and polyvinyl pyrrolidone. Emulsifying agents include Polysorbate 80 (TWEEN® 80). Sequestering or chelating agents for metal ions include EDTA. Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol, and propylene glycol for water-miscible vehicles; and sodium hydroxide, hydrochloric acid, citric acid, or lactic acid for pH adjustment.

[0369] The pharmaceutical composition can be formulated for any route of administration to a subject. Specific examples of routes of administration include intranasal, oral, parenteral, intrathecal, intracerebroventricular, pulmonary, subcutaneous, or intraventricular. In this specification, parenteral administration characterized by subcutaneous, intramuscular, or intravenous injection is also contemplated. Injectables are in conventional forms and can be manufactured as liquid solutions or suspensions, in solid forms suitable for solution or suspension in a liquid before injection, or as emulsions. Injectables, solutions, and emulsions also contain one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol, or ethanol. Also, 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 formulations such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, and cyclodextrin.

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

[0371] In the case of intravenous administration, suitable carriers include solutions containing thickening and solubilizing agents such as physiological saline or phosphate-buffered saline (PBS) and glucose, polyethylene glycol, polypropylene glycol, and mixtures thereof.

[0372] Topical mixtures containing antibodies are manufactured as described for topical and systemic administration. The resulting mixtures can be solutions, suspensions, emulsions, etc., and can be formulated into creams, gels, ointments, emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes, foams, aerosols, irrigation fluids, sprays, suppositories, dressings, skin patches, or any other dosage form suitable for topical administration.

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

[0374] The antibodies or antigen-binding portions thereof described herein can be formulated for topical or local use, such as topical application to the skin and mucosa as in the eye, in the form of gels, creams, and lotions, and for application to the eye or intracisternal or intraspinal application. Topical administration is contemplated for transdermal delivery and for eye, mucosal, or inhalation therapy. The nasal solution of the antibody can be administered alone or in combination with other pharmaceutically acceptable excipients.

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

[0376] In certain embodiments, a pharmaceutical composition comprising an antibody or an antigen-binding portion thereof described herein is a lyophilized powder that can be reconstituted for administration as a solution, emulsion, and other mixtures. The lyophilized powder can also be reconstituted and formulated as a solid or a gel. The lyophilized powder is formed by dissolving the antibody or an antigen-binding portion thereof described herein, or a pharmaceutically acceptable derivative thereof, in a suitable solvent. In some embodiments, the lyophilized powder is sterile. The solvent can contain excipients that improve the stability of the powder or the reconstituted solution produced from the powder or other pharmacological components. 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 buffering agent such as citrate, sodium phosphate, or potassium phosphate, or other well-known buffering agents having a substantially neutral pH in one embodiment. The solution is then sterile filtered and lyophilized under standard conditions known to those skilled in the art to provide the desired dosage form. In one embodiment, the resulting solution can be dispensed into vials for lyophilization. Each vial can contain a single dose or multiple doses of the compound. The lyophilized powder can be stored under suitable conditions such as about 4°C to room temperature.

[0377] Such a lyophilized powder is reconstituted with water for injection to provide a dosage form for parenteral administration. For reconstitution, the lyophilized powder is added to sterile water or other suitable carriers. The exact amount varies depending on the compound selected. Such amounts can be determined empirically.

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

[0379] Compositions for in vivo administration may be sterile. This can be readily accomplished, for example, by filtration through sterile filtration membranes.

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

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

[0382] In other aspects, the present specification presents a method for inhibiting, delaying, suppressing, limiting, reducing, reversing or preventing the onset or initiation of gliosis and the associated harmful effects on the CNS in a subject, the method comprising administering to the subject an anti-FAM19A5 antibody disclosed herein. In some embodiments, the present specification presents a method for inhibiting, delaying, suppressing, limiting, reducing, reversing or preventing excessive or abnormal proliferation of reactive astrocytes and the associated harmful effects on the CNS in a subject, the method comprising administering to the subject an anti-FAM19A5 antibody of the present disclosure. In some embodiments, the present specification presents a method for decreasing, inhibiting or reducing the expression of chondroitin sulfate proteoglycan (including levels of neurocan, NG2, or both), or for decreasing or inactivating the activity of neurocan, NG2 or both, in a subject, the method comprising administering to the subject an anti-FAM19A5 antibody described herein. In some embodiments, the present specification presents a method for stimulating, promoting, increasing or activating the growth of neurons in a subject, preferably after injury or damage, the method comprising administering to the subject an anti-FAM19A5 antibody described herein. In other embodiments, the present specification presents a method for increasing the level of c-fos mRNA, c-fos protein or c-fos protein activity in a subject in need thereof, preferably for increasing the level of ERK mRNA, ERK protein or pERK activity in the nucleus of neurons, the method comprising administering to the subject an anti-FAM19A5 antibody of the present disclosure. In certain embodiments, the present specification presents a method for increasing or augmenting the level of GAP43 mRNA, GAP43 protein, or preferably for increasing the activity of GAP43 protein in neurons in a subject in need thereof, the method comprising administering to the subject an anti-FAM19A5 antibody disclosed herein. In certain embodiments, the present specification presents a method for enhancing or promoting the survival of neurons and / or for promoting axonal regrowth in a subject in need thereof, the method comprising administering to the subject an anti-FAM19A5 antibody disclosed herein.In some embodiments, the subject is a human, preferably a human in which nerve cells are damaged or injured from, for example, CNS injury, trauma, wound, spinal cord injury, brain tumor, infection, ischemia, stroke, reaction and / or neurodegenerative disease.

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

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

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

[0386] In some embodiments, the anti-FAM19A5 antibody of the present disclosure induces normalization of blood vessels, for example, within a tumor. In some embodiments, the normalization of the blood vessels involves changes in the properties of the blood vessels including increased connectivity, increased wall thickness, decreased blood vessel diameter, more regular blood vessel orientation and distribution patterns, increased number of blood vessels, decreased leakage and permeability, increased perivascular cell coverage and proximity on the blood vessels, increased oxygen supply or combinations thereof.

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

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

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

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

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

[0392] In some embodiments, the tumor treatment method includes administering an additional therapeutic agent. In certain embodiments, the additional therapeutic agent includes chemotherapy, immunotherapy, radiotherapy, or a combination thereof. In some embodiments, the immunotherapy includes monoclonal antibodies, chimeric antigen receptor (CAR) therapy, T-cell therapy, NK-cell therapy, dendritic cell (DC) therapy, adoptive cell transfer (ACT), immune checkpoint modulator, cytokines, cancer vaccines, adjuvants, oncolytic viruses, or a combination thereof. In some embodiments, the chemotherapy includes temozolomide, gemcitabine, paclitaxel, carboplatin, cisplatin, erlotinib, lenalidomide, dexamethasone, oxaliplatin, or a combination thereof.

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

[0394] In some embodiments, the anti-FAM19A5 antibody or composition thereof of the present disclosure is administered intravenously, orally, parenterally, intradurally, intrathecally, intraventricularly, by inhalation, subcutaneously, intravascularly, intramuscularly, or intraventricularly.

[0395] The examples described below are provided as non-limiting illustrations.

[0396] [Examples] [Example 1 Expression and Purification of Human FAM19A5 Protein] The recombinant human FAM19A5 protein was produced and purified as described below, and the purified protein was used for antibody screening analysis based on binding affinity analysis. First, the LPS-hT plasmid expressing the FAM19A5 gene was transformed into bacteria to induce protein overexpression. After production, the FAM19A5 protein was purified using Ni-NTA affinity chromatography (Qiagen, Valencia, CA, USA). The His-tagged FAM19A5 protein was removed from the Ni-column using gradually increasing concentrations of imidazole. Protein expression in the said 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. Once the concentration was complete, the purity and concentration of the FAM19A5 protein were measured using Western blot analysis. Then, the concentrated protein was used to screen for FAM19A5-specific antibodies.

[0397] 〔Example 2 Production of anti-FAM19A5 antibody library〕 1. Immunization The FAM19A5 protein was used as an antigen for immunization of white leghorn chickens. 50 μg of the synthetic peptide KLH conjugate was mixed with 750 μL of phosphate-buffered saline (PBS) and cultured at 37 °C for 30 minutes. Then, an oil-in-water emulsifying adjuvant (RIBI + MPL + TDM + CWS adjuvant, Sigma, St. Louis, Mo, USA), 2% squalene of the cell wall components of TDW and CWS, detoxified MPL (monophosphoryl lipid A species), and mycobacteria were used to remove the toxin and emulsify it, and then it was subcutaneously injected into the chickens. During immunization, the chickens were immunized a total of 4 times at approximately 2 - 3 week intervals. The titer of the antibody obtained from the immunized animals was measured by immunoblotting using the lysate of HEK293T cells overexpressing the FAM19A5 protein.

[0398] 2. Production of single-chain variable fragment (scFv) library from immunized chickens RNA was extracted from the spleens, bone marrows, and bursa of Fabricius of the above-described immunized chickens using TRI reagent (Invitrogen, Carlsbad, CA, USA). First-strand cDNA was synthesized using oligo-dT primers and SUPERSCRIPT TM III First-Strand Synthesis System (Invitrogen). For cDNA obtained from the immune systems of the animals, a single-chain variable region library was produced using the Expand High Fidelity PCR system (Roche Molecular Systems, IN, USA). In each reaction, 1 μL of cDNA, 60 pmol of each primer, 10 μL of 10× reaction buffer, 8 μL of 2.5 mM dNTP (Promega, Madison, WI, USA), and 0.5 μL of Taq DNA polymerase were mixed with water. The final volume was 100 μL. The PCR reaction was carried out using the following conditions: (i) 15 seconds at 94°C, (ii) 30 seconds at 56°C, (iii) 90 seconds at 72°C for 30 cycles, and finally extended at 72°C for 10 minutes. The PCR product containing a fragment approximately 350 bp in length was loaded onto a 1.5% agarose gel, and the nucleotide fragment was purified using the QIAGEN Gel II extraction kit (QIAGEN, Valencia, CA, USA) after electrophoresis. The purified PCR product was quantified by reading at OD260nm (1 unit OD = 50 μg / ml).

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

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

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

[0402] Example 3 Library panning (biopanning) on immobilized antigen Biotin panning was performed using magnetic beads (Dynabeads M-270 Epoxy, Invitrogen). Approximately 1×10 7 beads were coated with the protein by stirring in the presence of 5 μg of recombinant FAM19A5 protein at room temperature for 20 hours with rotation. After coating was completed, the beads were washed four times with phosphate-buffered saline (PBS) and blocked with PBS containing 3% BSA for 1 hour at room temperature. Next, the coated beads were incubated with the above phage-displayed scFv at room temperature for 2 hours. To remove phage not bound to the antigen-coated beads, the beads were washed with 0.05% Tween20 / PBS. Next, the bound phage was eluted with 50 μL of 0.1 M glycine / hydrochloric acid (0.1 M Glycine-HCl, pH 2.2) and neutralized with 3 μL of 2 M tris (tris-HCl, pH 9.1) together with hydrochloric acid. E. coli ER2738 cells were infected with this phage-containing supernatant and amplified and rescued overnight using VCSM13 helper phage. Also, the phage-infected culture was blotted on an LB agar plate containing 50 μg / ml kanamycin, and the input and output by phage titer were determined from the phage-infected culture. The next day, the phage was precipitated using PEG-8000 and NaCl and subsequently used for biotin panning. The above process was repeated up to a total of five times for biotin panning. Phage were screened and selected for high affinity for the FAM19A5 protein by each amplification.

[0403] Example 4 Clone Selection by Phage ELISA To analyze the clones selected from biotin panning, individual clones were randomly selected from the phage-display scFv, and it was confirmed by ELISA whether the clones bound to the FAM19A5 recombinant protein. The FAM19A5 recombinant protein was dissolved in 0.1 M NaHCO 3Diluted in buffer, a 96-well microtiter plate was coated with 100 ng / well of protein at 4 °C for 16 hours. The next day, the plate was blocked with 3% BSA / PBS at 37 °C for 1 hour. Then, the phage supernatant was mixed with 6% BSA / PBS and cultured at 37 °C for 2 hours. Next, the plate containing the supernatant was washed with 0.05% Tween-20 / PBS. The HRP-conjugated M13 antibody (a-M13-HRP, Pierce Chemical Co, Rockford, IL, USA) was diluted 1 / 5000. 50 μl of the diluted antibody was added to the plate and cultured at 37 °C for 1 hour. After culturing and washing, 0.05 M citrate buffer, 1 μg / ml of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS, Amresco, Solon, OH, USA) and 0.1% H 2 O 2 was added. The absorbance for each well was measured at 405 nm.

[0404] From the initially identified 96 clones, 8 scFv clones with unique heavy chain CDR3 (HCDR3) sequences and high binding to the FAM19A5 protein were selected for further analysis. Refer to FIGS. 1A to 1C.

[0405] 〔Example 5 Production of anti-FAM19A5-IgG2 / 4 antibody〕 The anti-FAM19A5 scFv was subcloned into a mammalian expression vector. In the FAM19A5 scFv gene sequence, the human Cκ gene was ligated to the light chain variable domain, and the human immunoglobulin isotype IgG2 / 4 of the CH1, CH2, and CH3 genes was ligated to the heavy chain variable region. Restriction sites (Genscript, USA) were added to synthesize antibodies with each light chain and each heavy chain. The synthesized gene was inserted into a mammalian cell expression vector with modified restriction sites to facilitate cloning. First, the light chain gene was inserted into the vector using Hind III and Xba I (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.

[0406] To express and purify the anti-FAM19A5-IgG2 / 4 antibody, a mammalian cell transfection and overexpression injection system was used. Approximately 2 μg / ml of the mammalian expression vector was mixed with 4 μg of polyethyleneimine (PEI, Polysciences, Warrington, PA, USA) 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 minutes. After adding the mixture to HEK293F cells (2×10 6 cells / ml, Invitrogen), it was cultured for 6 days in FREESTYLE 2 293 expression culture medium containing 100 U / ml of penicillin and streptomycin (Invitrogen) under stirring conditions of 7% CO TM and 37 °C at 135 rpm. To purify the expressed anti-FAM19A5 IgG2 / 4 antibody from the cell culture supernatant, protein A beads (RepliGen, Waltham, MA, USA) affinity gel chromatography was used. Protein A chromatography was performed on a 4 - 12% Bis-Tris gradient gel electrophoresis. The size and yield of the protein were confirmed by Coomassie Brilliant Blue staining. The binding ability of the antibody was measured using ELISA analysis.

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

[0408] 〔Example 6 Neutralizing Ability Analysis of Anti-FAM19A5 Antibodies〕 To further evaluate the functional characteristics of the antibodies, the following method was used.

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

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

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

[0412] 3. Neutralizing efficacy of anti-FAM19A5 antibody against glial cells To evaluate the neutralizing efficacy of the antibody, it was confirmed by flow cytometry as described above. See Kim, M., et al., PLoS One. 7(4): e35100 (2012). Mouse and human glial cells were seeded into a v-bottom 96-well plate (Corning Inc., Corning, Ny, USA) at a final density of 3×10 5 cells per well. The cells were treated with 1 μM recombinant FAM19A5 rabbit Fc and 5 μM anti-FAM19A5 scFv human Cκ fusion protein in flow cytometry buffer [1% (w / v) BSA in PBS containing 0.05% (w / v) sodium azide] at 37 °C for 1 hour. After washing with flow cytometry buffer, the cells were cultured with Alexa Fluor 488-conjugated anti-rabbit IgG (Fc specific) antibody (Jackson Immuno Research Inc., PA, USA) at 37 °C for 1 hour in the dark. After further washing with the same buffer, the cells were resuspended in 300 μL PBS and analyzed by flow cytometry using a FACSCANTO TM II instrument (BD Bioscience, San Jose, CA, USA). The data were analyzed using FlowJo software (TreeStar, Ashland, OR, USA).

[0413] As shown in FIG. 3A, all of the antibodies tested (i.e., 1-28, 1-85, 2-13, 2-14, 2-20, 2-29, 3-2 and 3-26) were able to suppress the interaction of FAM...

Claims

1. A separated antibody ("anti-FAM19A5 antibody") or its antigen-binding portion that specifically binds to the human family, member A5 (FAM19A5) protein having an array similarity of 19, and that includes heavy-chain CDR1, CDR2, and CDR3 as well as light-chain CDR1, CDR2, and CDR3, wherein (i) the VH of the antibody or its antigen-binding portion includes the amino acid sequence shown in SEQ ID NO: 37, and the VL includes the amino acid sequence shown in SEQ ID NO: 46, (ii) the VH of the antibody or its antigen-binding portion includes the amino acid sequence shown in SEQ ID NO: 130, and the VL includes the amino acid sequence shown in SEQ ID NO: 46, or, (iii) the VH of the antibody or its antigen-binding portion includes the amino acid sequence shown in SEQ ID NO: 131, and the VL includes the amino acid sequence shown in SEQ ID NO: 46, the antibody or its antigen-binding portion.

2. The antibody or its antigen-binding portion according to claim 1, showing any one or more of the following characteristics: (a) The characteristic of binding to soluble human FAM19A5 with a KD of 10 nM or less when measured by enzyme-linked immunosorbent assay (ELISA); (b) The characteristic of binding to membrane-bound human FAM19A5 with a KD of 10 nM or less when measured by ELISA; (c) The characteristic of reducing, reversing, delaying, or preventing the onset of reactive gliosis; (d) The characteristic of suppressing the overgrowth of reactive astrocytes; (e) The characteristic of reducing the expression of chondroitin sulfate proteoglycans including neurocan and glial antigen 2 (NG2); (f) The characteristic of increasing the expression of nuclear c-fos and pERK in nerve cells; (g) The characteristic of promoting the survival of nerve cells; (h) The characteristic of increasing the expression of GAP43 in nerve cells; (i) The characteristic of promoting axon regrowth; (j) The characteristic of inducing, for example, the normalization of blood vessels in tumors; (k) The characteristic of suppressing tumor growth; (l) The characteristic of increasing the infiltration of immune cells into tumors; (m) The characteristic of increasing the infiltration of nerve cells into tumors; (n) The characteristic of enhancing the phagocytic activity of macrophages or microglia; (o) The characteristic of increasing the mitochondrial membrane potential of macrophages or microglia; (p) The characteristic of reducing the mobilization of myeloid-derived suppressor cells (MDSC) against tumors; (q) The characteristic of reducing necrosis and edema in tumors; (r) The characteristic of reducing the tissue permeability of tumors; and (s) The characteristic of increasing the blood flow rate in tumors.

3. A nucleic acid encoding the antibody or its antigen-binding portion of claim 1.

4. A vector comprising the nucleic acid of claim 3.

5. A cell comprising the vector of claim 4.

6. An immunoconjugate comprising the antibody of claim 1 or an antigen-binding portion thereof linked to an agent.

7. A composition comprising the antibody of claim 1 or an antigen-binding portion thereof, the nucleic acid of claim 3, the vector of claim 4, the cell of claim 5, the immunoconjugate of claim 6, and a carrier.

8. A kit comprising the antibody of claim 1 or an antigen-binding portion thereof, the nucleic acid of claim 3, the vector of claim 4, the cell of claim 5, the immunoconjugate of claim 6, and instructions for use.

9. A method for producing an antibody or an antigen-binding portion thereof that specifically binds to a human FAM19A5 protein, the method comprising culturing the cell of claim 5 under appropriate conditions to isolate the antibody.

10. The composition according to claim 7, wherein the composition is a pharmaceutical composition for treating symptoms in a subject in need thereof.

11. The composition according to claim 10, wherein the symptoms include tumor, fibrosis, glaucoma, mood disorder, retinal disease, age-related macular degeneration, or neuropathic pain.

12. The composition according to claim 11, wherein the disease or symptom is a tumor.

13. The composition according to claim 11 or 12, wherein the tumor includes melanoma, pancreatic cancer, glioma, breast cancer, lymphoma, lung cancer, kidney cancer, prostate cancer, fibrosarcoma, colorectal adenocarcinoma, liver cancer, or ovarian cancer.

14. The composition according to claim 13, wherein the glioma is glioblastoma multiforme.

15. The composition according to any one of claims 10 to 14, wherein the antibody or an antigen-binding portion thereof, the nucleic acid, the vector, the cell, or the immunoconjugate induces normalization of blood vessels.

16. The composition according to claim 15, wherein the normalization of blood vessels is accompanied by changes in the properties of blood vessels including increased connectivity, increased wall thickness, decreased blood vessel diameter, more regular blood vessel direction and distribution pattern, increased number of blood vessels, decreased leakage and permeability, increased perivascular cell coverage and proximity on blood vessels, increased oxygen supply, or a combination thereof.

17. The composition according to any one of claims 11 to 16, wherein the antibody or an antigen-binding portion thereof, the nucleic acid, the vector, the cell, or the immunoconjugate inhibits tumor growth.

18. The composition according to any one of claims 11 to 17, wherein the antibody or an antigen-binding portion thereof, the nucleic acid, the vector, the cell, or the immunoconjugate increases infiltration of immune cells into tumors.

19. The composition according to claim 18, wherein the immune cells include macrophages, dendritic cells, T lymphocytes, B lymphocytes, natural killer (NK) cells, or a combination thereof.

20. The composition according to claim 18 or 19, wherein the immune cells further exhibit hypertrophy.

21. The composition according to any one of claims 18 to 20, wherein the increased infiltration of the immune cells into the tumor is accompanied by an increased infiltration of nerve cells into the tumor.

22. The composition according to claim 21, wherein the nerve cells include astrocytes, glial cells, or a combination thereof.

23. The composition according to any one of claims 10 to 22, wherein the antibody or its antigen-binding portion, the nucleic acid, the vector, the cell, or the immunoconjugate enhances the phagocytic activity of macrophages or microglia.

24. The composition according to any one of claims 10 to 23, wherein the antibody or its antigen-binding portion, the nucleic acid, the vector, the cell, or the immunoconjugate increases the mitochondrial membrane potential of macrophages or microglia.

25. The composition according to any one of claims 11 to 23, wherein the antibody or its antigen-binding portion, the nucleic acid, the vector, the cell, or the immunoconjugate reduces the recruitment of myeloid-derived suppressor cells (MDSC) to the tumor.

26. The composition according to any one of claims 11 to 25, wherein the antibody or its antigen-binding portion, the nucleic acid, the vector, the cell, or the immunoconjugate reduces necrosis and edema in the tumor.

27. The composition according to any one of claims 11 to 26, wherein the antibody or its antigen-binding portion, the nucleic acid, the vector, the cell, or the immunoconjugate reduces the tissue permeability of the tumor.

28. The composition according to any one of claims 11 to 27, wherein the antibody or its antigen-binding portion, the nucleic acid, the vector, the cell, or the immunoconjugate increases the blood flow rate in the tumor.

29. The composition according to any one of claims 10 to 28, comprising administering an additional therapeutic agent.

30. The composition according to claim 29, wherein the additional therapeutic agent includes chemotherapy, immunotherapy, radiotherapy, or a combination thereof.

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