RU2785436C2 - Antibodies against a5 representative of 19 family with similarity of sequences and their application method - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: in the present invention, antibodies are presented, which specifically bind to human FAM19A5, as well as nucleic acid encoding an antibody, an expression vector, a cell for antibody expression, a composition for inhibition of FAM19A5 activity, a kit for inhibition of FAM19A5 activity, a method for the production of an antibody, which specifically binds to human FAM19A5 protein, and a method for the treatment of disorders, such as damage to the central nervous system, degenerative brain disease, or neuropathic pain, by administration of an antibody, which specifically binds to human FAM19A5.

EFFECT: obtainment of an antibody against human FAM19A5.

37 cl, 25 dwg, 9 tbl, 12 ex

Description

Cross-references to related applications

This PCT application claims priority advantage of US Provisional Application Nos. 62/787711, filed January 2, 2019, and 62/838190, filed April 24, 2019, each of which is hereby incorporated by reference in its entirety.

The link to the list of sequences is given in electronic form

The contents of the electronically submitted sequence listing in an ASCII text file (Name: 3763_016PC02_SeqListing_ST25.txt; Size: 90,231 bytes; and Creation Date: December 30, 2019) filed with the application are incorporated herein by reference in their entirety.

Government Support Statement

This work was supported by the Innovative Industrial Technology Program (10081300, Development of a Novel Monoclonal Antibody Therapeutic through a Glial Scar Inhibition Mechanism in Ischemic Stroke) funded by the Ministry of Commerce, Industry and Energy (MOTIE, Korea).

The field of technology to which the invention relates

The present disclosure provides antibodies (e.g., deimmunized or affinity matured) that specifically bind to a member of A5 family 19 with sequence similarity (FAM19A5), compositions containing such antibodies, and a method of using such antibodies to prevent or treat disorders or diseases, e.g. caused by damage to the subject's central nervous system.

Prior Art

FAM19A5 is a member of the TAFA protein subfamily, which consists of five highly homologous small proteins. Tang T. Y. et al., Genomics 83(4):727-34 (2004). These proteins contain conserved cysteine residues in fixed positions and are distantly related to macrophage inflammatory protein 1-alpha (MIP-1-alpha), a member of the CC chemokine family. TAFA proteins are predominantly expressed in certain areas of the brain and spinal cord. These proteins are believed to be produced and secreted by adult neural stem cells during neurogenesis.

FAM19A5 is predominantly expressed in the vertebrate brain, and FAM19A5 is believed to be important for the development, differentiation, formation of the entire central nervous system, and may be used to prevent or treat damage and/or diseases of the central nervous system. US Patent Publication No. 2015/0118230.

Although inhibition of FAM19A5 may play an important role in the treatment of the central nervous system, there is still a need to develop antibodies that specifically bind to FAM19A5 and that are able to modulate FAM19A5 activity, especially those antibodies that can be used in humans without side effects.

Summary of Disclosure

Provided herein is an isolated antibody, or an antigen-binding portion thereof, that specifically binds a human A5 family 19 protein with sequence similarity (FAM19A5) ("anti-FAM19A5 antibody") comprising heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3. chains, where CDR1, CDR2 and CDR3 of the heavy chain contain the amino acid sequences indicated in SEQ ID NO: 5, 6 and 7, respectively, each of which optionally contains one, two, three, four or five mutations, while CDR1, CDR2 and The light chain CDR3s comprise the amino acid sequences set forth in SEQ ID NOs: 8, 9, and 10, respectively, wherein at least one of a light chain CDR1, CDR2, and CDR3 containing one, two, three, four, or five mutations, and wherein the antibody has reduced immunogenicity in humans compared to a reference antibody containing the VH indicated in SEQ ID NO: 11 and the VL indicated in SEQ ID NO: 12.

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

In some embodiments, the heavy chain CDR1 of the anti-FAM19A5 antibody comprises the amino acid sequence set forth in SEQ ID NO: 5 with one or two mutations. In some embodiments, the mutations include changing the threonine at amino acid 3 of SEQ ID NO: 5 to an acidic amino acid. In additional embodiments, the mutations include the replacement of serine at amino acid 5 of SEQ ID NO: 5 with an acidic amino acid. In some embodiments, the acidic amino acid includes aspartic acid or glutamic acid.

In some embodiments, the heavy chain CDR2 of the anti-FAM19A5 antibody comprises the amino acid sequence set forth in SEQ ID NO: 6 with one, two, three, four, or five mutations. In some embodiments, the mutations include the replacement of arginine at amino acid 16 of SEQ ID NO: 6 with a basic amino acid. In some embodiments, the basic amino acid includes lysine. In additional embodiments, the mutations include one or more of the following: (a) changing the glycine at amino acid 6 of SEQ ID NO: 6 to an acidic amino acid; (b) replacing serine at amino acid 7 in SEQ ID NO: 6 with an acidic amino acid; (c) replacing serine at amino acid 8 of SEQ ID NO: 6 with an acidic amino acid; (d) replacing the threonine in amino acid 9 of SEQ ID NO: 6 with an acidic amino acid; and (e) replacing the arginine at amino acid 16 of SEQ ID NO: 6 with a basic amino acid. In some embodiments, the acidic amino acid includes aspartic acid or glutamic acid. In some embodiments, the basic amino acid includes lysine.

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

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

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

Also provided herein is an isolated antibody, or an antigen-binding portion thereof, that specifically binds to the human FAM19A5 protein, comprising a heavy chain CDR1, CDR2 and CDR3 and a light chain CDR1, CDR2 and CDR3, wherein: (i) the heavy chain CDR1 comprises the amino acid sequence indicated in SEQ ID NO: 5; (ii) the heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 13; (iii) the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 7; (iv) the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 8; (v) the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 20; and (vi) the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 10.

Also provided herein is an isolated antibody, or an antigen-binding portion thereof, that specifically binds to the human FAM19A5 protein, comprising a heavy chain CDR1, CDR2 and CDR3 and a light chain CDR1, CDR2 and CDR3, wherein: (i) the heavy chain CDR1 comprises the amino acid sequence indicated in SEQ ID NO: 14; (ii) the heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15; (iii) the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 7; (iv) the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 21; (v) the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 22; and (vi) the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 23.

Also provided herein is an isolated antibody, or an antigen-binding portion thereof, that specifically binds to the human FAM19A5 protein, comprising a heavy chain CDR1, CDR2 and CDR3 and a light chain CDR1, CDR2 and CDR3, wherein: (i) the heavy chain CDR1 comprises the amino acid sequence indicated in SEQ ID NO: 14; (ii) the heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15; (iii) the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 7; (iv) the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 21; (v) the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 24; and (vi) the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 23.

Also provided herein is an isolated antibody, or an antigen-binding portion thereof, that specifically binds to the human FAM19A5 protein, comprising a heavy chain CDR1, CDR2 and CDR3 and a light chain CDR1, CDR2 and CDR3, wherein: (i) the heavy chain CDR1 comprises the amino acid sequence indicated in SEQ ID NO: 14; (ii) the heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15; (iii) the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 7; (iv) the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 8; (v) the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 25; and (vi) the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 23.

Also provided herein is an isolated antibody, or an antigen-binding portion thereof, that specifically binds to the human FAM19A5 protein, comprising a heavy chain CDR1, CDR2 and CDR3 and a light chain CDR1, CDR2 and CDR3, wherein: (i) the heavy chain CDR1 comprises the amino acid sequence indicated in SEQ ID NO: 14; (ii) the heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15; (iii) the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 7; (iv) the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 8; (v) the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 24; and (vi) the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 23.

Also provided herein is an isolated antibody, or an antigen-binding portion thereof, that specifically binds to the human FAM19A5 protein, comprising a heavy chain CDR1, CDR2 and CDR3 and a light chain CDR1, CDR2 and CDR3, wherein: (i) the heavy chain CDR1 comprises the amino acid sequence indicated 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.

Also provided herein is an isolated antibody, or an antigen-binding portion thereof, that specifically binds to the human FAM19A5 protein, comprising a heavy chain CDR1, CDR2 and CDR3 and a light chain CDR1, CDR2 and CDR3, wherein: (i) the heavy chain CDR1 comprises the amino acid sequence indicated 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.

In some embodiments, an anti-FAM19A5 antibody disclosed herein includes 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% identical, at least 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% of the specified amino acid sequence in SEQ ID NO: 11 and/or where VL includes an amino acid sequence that is at least about 80% identical, at least about 85% identical, 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% amino acid sequence specified in SEQ ID NO: 12.

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

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

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

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

In some embodiments, the heavy chain CDR3 of the anti-FAM19A5 antibody comprises the amino acid sequence shown in SEQ ID NOS: 18, 128, or 129.

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

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

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

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

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

In some embodiments, an anti-FAM19A5 antibody disclosed herein includes 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% identical, at least 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% of the specified amino acid sequence in SEQ ID NO: 35 and/or where VL includes an amino acid sequence that is at least about 80% identical, at least about 85% identical, 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% amino acid sequence specified in SEQ ID NO: 45.

In some embodiments, the anti-FAM19A5 antibody cross-competes with a reference antibody that includes a heavy chain variable region (VH) and a light chain variable region (VL), where VH includes the amino acid sequence shown in SEQ ID NO: 36 and VL includes the amino acid sequence specified in SEQ ID NO: 46. In other embodiments, the anti-FAM19A5 antibody cross-competes with a reference antibody that includes a heavy chain variable region (VH) and a light chain variable region (VL), where VH includes the amino acid sequence indicated in SEQ ID NO: 37, and VL comprises the amino acid sequence shown in SEQ ID NO: 46. In additional embodiments, the anti-FAM19A5 antibody cross-competes with a reference antibody that includes a heavy chain variable region (VH) and a light chain variable region (VL), where VH includes the amino acid sequence shown in SEQ ID NO: 130 and VL includes the amino acid sequence range as set forth in SEQ ID NO: 46. In some embodiments, the anti-FAM19A5 antibody cross-competes with a reference antibody that includes a heavy chain variable region (VH) and a light chain variable region (VL), where VH comprises the amino acid sequence specified in SEQ ID NO: 131, and VL includes the amino acid sequence shown in SEQ ID NO: 46.

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

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

In some embodiments, the anti-FAM19A5 antibody exhibits one or more of the following properties: (a) binds to soluble human FAM19A5 with a KD of 10 nM or less, as measured by enzyme immunoassay (ELISA); (b) binds to human membrane-bound FAM19A5 with a KD of 10 nM or less as measured by ELISA; (c) reduce, reverse, delay and/or prevent the onset of reactive gliosis; (d) inhibits the excessive proliferation of reactive astrocytes; (e) reduces the expression of chondroitin sulfate proteoglycans, including neurocan and neuron-glial antigen 2 (NG2); (f) increases c-fos and pERK expression in neuronal nuclei; (g) promotes neuronal survival; (h) increases GAP43 expression in neurons; (i) promotes axon regrowth; (j) causes normalization of blood vessels, for example, in a tumor; (k) inhibits tumor growth; (l) enhances the penetration of immune cells into the tumor; (m) enhances the infiltration of neuronal cells into the tumor; (n) enhances the phagocytic activity of the macrophage or microglia; (o) increases the potential of the mitochondrial membrane of a macrophage or microglia; (p) reduces the recruitment of myeloid suppressor cells (MDSC) to the tumor; (q) reduce tumor necrosis and swelling; (r) reduces the permeability of tumor tissue; and (s) increases the rate of blood flow in the tumor.

Also provided herein are nucleic acids encoding the anti-FAM19A5 antibodies disclosed herein, vectors containing the nucleic acid, cells containing the vector, and immunoconjugates containing the anti-FAM19A5 antibodies of the present disclosure. Compositions containing anti-FAM19A5 antibodies, nucleic acids, vectors, cells or immunoconjugates according to the present disclosure, and a carrier are also disclosed herein. This disclosure also provides kits containing anti-FAM19A5 antibodies, nucleic acids, vectors, cells or immunoconjugates in accordance with the present disclosure, as well as instructions for use.

The present document also provides a method for producing an antibody that specifically binds to the human FAM19A5 protein, comprising culturing the cells described herein under suitable conditions and isolating the antibody.

The present disclosure further provides a method for treating a disease or condition in a subject in need thereof, comprising administering an anti-FAM19A5 antibody, nucleic acid, vector, cell, or immunoconjugate as described herein. In some embodiments, the disease or condition includes tumor, fibrosis, glaucoma, mood disorder, retinopathy, age-related macular degeneration, or neuropathic pain. In some embodiments, the disease or condition is a tumor.

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

In some embodiments, the anti-FAM19A5 antibody, nucleic acid, vector, cell, or immunoconjugate of the present disclosure causes normalization of blood vessels. In some embodiments, the normalization of blood vessels is accompanied by changes in the properties of blood vessels, including increased connectivity, increased wall thickness, reduced vessel diameter, more correct direction and distribution of vessels, increased number of vessels, decreased leakage and permeability, increased pericyte coverage, and proximity to blood vessels, increased oxygenation, or combinations thereof.

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

In some embodiments, an anti-FAM19A5 antibody, nucleic acid, vector, cell, or immunoconjugate disclosed herein enhances immune cell infiltration into a tumor. In some embodiments, the immune cells comprise 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, increased infiltration of immune cells into the tumor is accompanied by increased infiltration of neuronal cells into the tumor. In some embodiments, neuronal cells include astrocytes, glial cells, or combinations thereof.

In some embodiments, the anti-FAM19A5 antibody, nucleic acid, vector, cell, or immunoconjugate enhances the phagocytic activity of a macrophage or microglia. In some embodiments, the anti-FAM19A5 antibody, nucleic acid, vector, cell, or immunoconjugate increases the mitochondrial membrane potential of a macrophage or microglia.

In some embodiments, an anti-FAM19A5 antibody, nucleic acid, vector, cell, or immunoconjugate disclosed herein reduces recruitment of myeloid-derived suppressor cells (MDSCs) to a tumor. In some embodiments, the anti-FAM19A5 antibody, nucleic acid, vector, cell, or immunoconjugate reduces necrosis and edema in a tumor. In some embodiments, the anti-FAM19A5 antibody, nucleic acid, vector, cell, or immunoconjugate reduces tumor tissue permeability. In some embodiments, the anti-FAM19A5 antibody, nucleic acid, vector, cell, or immunoconjugate increases the rate of blood flow in the tumor.

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

Brief description of the drawings

In FIG. 1A, 1B and 1C are analysis of the binding of individual scFv clones to the FAM19A5 protein. Absorbance was measured at 405 nm. Clone numbers are indicated on the x-axis. FIG. 1A, 1B and 1C show the analysis of 96 clones from the 4th, 5th or 5th round of biopanning obtained from the first chick, the second chick and the third chick, respectively. For each of the clones shown in FIG. 1A, 1B and 1C, the vertical bars correspond to FAM19A5 protein, negative control protein, hemagglutinin protein and BSA (from left to right). On each of the FIGS. 1A, 1B and 1C, the boxed clones are those 8 clones that were selected for further analysis (see Example 4).

In FIG. 2A and 2B show the approximate size and binding capacity of various anti-FAM19A5 scFvs, respectively. The size of the antibodies is shown by SDS-Page and the binding capacity is measured by ELISA. Antibodies shown include (from left to right): 1-28, 1-85, 2-13, 2-14, 2-20, 2-29, 3-2 and 3-26. In FIG. 2B, for each of the antibodies shown, the band on the left represents antibody binding to the FAM19A5 protein. The lane on the right represents the negative control (measured binding in the presence of blocking buffer alone, ie without recombinant FAM19A5 protein). The column labeled "2nd only" represents another negative control to show the background level of the assay (measured binding in the absence of primary anti-FAM19A5 antibodies).

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

In FIG. 4 shows the amino acid sequences of the F1-F6 epitopes (BSA-conjugated) and their location on the human FAM19A5 polypeptide. The upper amino acid sequence shown is wild type FAM19A5 isoform 2 (no signal peptide). The second amino acid sequence shown is the same sequence, but the cysteine residues have been mutated to serine to reduce non-specific activity during peptide synthesis.

In FIG. 5 shows ELISA results for binding of antibody 3-2 to epitope fragments F1 to F6. The leftmost column ("FAM19A5") is a positive control and shows the binding of anti-FAM19A5 antibody 3-2 to the entire FAM19A5 protein. The "irrelevant protein" and "only blocking" (ie blocking buffer only, ie no FAM19A5 protein) groups are negative controls. For each of the groups, the band on the left corresponds to the isotype control, and the band on the right corresponds to antibody 3-2.

In FIG. 6A, 6B, 6C, 6D, 6E, 6F, 6G, 6H, 6I and 6J are alanine scan analysis results showing specific amino acid residues in the F2 epitope fragment that are important for binding of various variants of antibodies 3-2 to the FAM19A5 protein. In FIG. 6A, 6B, 6C and 6D show the results for the following antibodies generated in HEK293F cells: (A) wild type 3-2 antibody, (B) 1-30 antibody, (C) 1-32 antibody, and (D) 6- 10, respectively. Fig. 6E, 6F, 6G, 6H, 6I and 6J show the results for the following antibodies generated in CHO cells: (E) antibody 1-17, (F) antibody 1-30, (G) antibody 1-32, (H) antibody 4-11, (I) antibody 6-10 and (J) low PI antibody, respectively. As discussed in Example 7, mutant peptides were generated wherein each of the mutant peptides had an alanine substitution at one amino acid residue within the F2 epitope fragment. The binding of antibodies to various mutant peptides was measured using ELISA.

Fig. 7 identifies potential immunogenicity sites in the light chain (VL) variable region (upper three rows) and heavy chain variable region (VH) (lower three rows) of antibody 3-2. VL corresponds to SEQ ID NO: 12 and VH corresponds to SEQ ID NO: 11. Also shown are the human germline sequences used for antibody framework regions 3-2: VL = immunoglobulin lambda, variable 1-51 (IGLV1-51*02) and lambda immunoglobulin connecting 2 (IGLJ2*01); VH = immunoglobulin variable heavy chain 3-64 (IGHV3-64*04) and immunoglobulin heavy chain linking 1 (IGHJ1*01). The human germ lines used were the human germ lines (IgBLAST, NCBI) most homologous to clone 3-2. Shown are promiscuous peptides binding MHC class II with high and moderate immunogenicity potential as determined by the ITOPE™ assay. In particular, peptides that bind MHCII with high immunogenicity potential are the following: (i) peptide No. 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 no. 10: residues 79-87 of SEQ ID NO: 11 (VRLQLNNPG). MHCII binding peptides with moderate immunogenic potential are: (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 no. 11: residues 81-89 of SEQ ID NO: 11 (LQLNNPGAE). The homologous peptides from the T-cell epitope database are as follows: Peptide #5, Peptide #6, and Peptide #9. A total of 11 binding peptides were identified and designated as peptides #1-#11. "P1" for each of the binding peptides indicates the first anchor position.

Fig. 8 identifies potential immunogenicity sites in the light chain variable region (VL) (upper three rows) and heavy chain variable region (VH) (lower three rows) of antibody 2-13. VL corresponds to SEQ ID NO: 45 and VH corresponds to SEQ ID NO: 35. Also shown are the human germline sequences used for antibody framework regions 2-13: VL = immunoglobulin lambda variable 3-27 (IGLV3-27*01) and lambda immunoglobulin connecting 2 (IGLJ2*01); VH = immunoglobulin variable heavy chain 3-64 (IGHV3-64*04) and immunoglobulin heavy chain linking 1 (IGHJ1*01). The human germ lines used were most homologous to clone 2-13 (IgBLAST, NCBI). Shown are promiscuous peptides binding MHC class II with high and moderate immunogenicity potential as determined by the ITOPE™ assay. In particular, MHCII binding peptides with high potential for immunogenicity are: (i) Peptide #1: residues 16-24 of SEQ ID NO: 45 (VKITCSGGS); (ii) Peptide #2: residues 80-88 of SEQ ID NO: 45 (VYFCGTEDI); and (iii) peptide #8: residues 79-87 of SEQ ID NO: 35 (VRLQLNNLR). MHCII binding peptides with moderate immunogenic potential are: (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 no. 5: residues 36-44 of SEQ ID NO: 35 (WVRQTPGKG); (iv) Peptide #6: residues 47-55 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 no. 10: residues 86-94 of SEQ ID NO: 35 (LRAEDTGTY). Homologous peptides from the database of T-cell epitopes are as follows: Peptide #4 and Peptide #5. A total of 10 binding peptides have been identified and are designated peptides #1-#10. "P1" for each of the binding peptides indicates the first anchor position.

In FIG. 9A, 9B and 9C show the binding assay of deimmunized antibodies 3-2. In FIG. 9A shows schematically where various amino acid mutations were made to deimmunize antibody 3-2. In FIG. 9B shows a sequence comparison of the light chain variable region (VL) and the heavy chain variable region (VH) among the following: (i) wild-type 3-2 antibody ("clone 3-2"), (ii) fully deimmunized 3-2 antibody ( "completely deimmunized clone 3-2") and (iii) deimmunized except for one amino acid in heavy chain CDR2) antibody ("deimmunized clone 3-2"). Amino acid residues with high and moderate potential for immunogenicity, as determined by the ITOPE™ assay, are boxed and labeled "1" and "2", respectively. Fig. 9C shows a comparison of the binding of (i) wild type 3-2 antibody, (ii) fully deimmunized 3-2 antibody, and (iii) deimmunized except for one amino acid to the heavy chain CDR2 ("deimmunized clone-3-2") antibody to FAM19A5 protein as measured by ELISA. Each phage presenting a single chain variable fragment (scFv) was added to the wells of a microtiter plate coated with FAM19A5 (■) or anti-HA antibody (□). Background signal was measured in control wells coated with BSA. The wells were tested with HRP-conjugated anti-M13 antibody. Optical density was measured at 405 nm. The results are presented as the mean ± standard deviation obtained from four replicates of the experiments.

On figa and 10B shows the analysis of two different deimmunized antibodies 2-13: (i) completely deimmunized and (ii) deimmunized, except for one amino acid in the CDR2 of the heavy chain) antibodies ("deimmunized clone 2-13"). In FIG. 10A shows a comparison of the light chain variable region (VL) (upper three rows) and the heavy chain variable region (VH) (lower three rows) for two deimmunized antibodies 2-13 to wild-type antibodies 2-13 ("clone 2-13"). . Amino acid residues with high and moderate potential for immunogenicity, as determined by the ITOPE™ assay, are boxed and labeled "1" and "2", respectively. Fig. 10B shows a comparison of the binding of (i) wild-type antibody 2-13, (ii) fully deimmunized antibody 2-13, and (iii) deimmunized except for one amino acid to heavy chain CDR2 ((deimmunized clone 2-13) of anti-FAM19A5 protein antibody as measured by ELISA Each phage presenting a single chain variable fragment (scFv) was added to the wells of a microtiter plate coated with FAM19A5 (■) or anti-HA antibody (□) Background signal was measured in control wells coated with BSA The wells were examined HRP-conjugated anti-M13 antibody Optical density was measured at 405 nm The results are presented as the mean ± standard deviation obtained from four replicates of the experiments.

In FIG. 11 shows a comparison of the binding capacity of different versions of deimmunized clone 2-13. The identity of different antibody variants is shown on the abscissa. Each amino acid residue of CDRL1, CDRL2, CDRL3, CDRH1 and CDRH2 was replaced with glutamic acid and aspartic acid. The reactivity of 70 antibody variants was analyzed by phage immunoassay. Each displayed scFv phage was added to the wells of a microtiter plate coated with FAM19A5 (■) or anti-HA antibody (□). Background signal was measured in control wells coated with BSA. The wells were tested with HRP-conjugated anti-M13 antibody. Optical density was measured at 405 nm. The results are shown as the mean value ± standard deviation obtained from three repetitions of the experiments.

Fig. 12 is a schematic of the methods used to generate deimmunized anti-FAM19A5 antibodies with improved physicochemical properties.

Fig. 13A and 13B show a comparison of the physicochemical properties of several deimmunized 3-2 variants with the wild-type (ie, non-deimmunized) 3-2 ("stock") antibody. Antibody variants shown include: (i) low isoelectric point (“Low_PI”) antibody, (ii) 1-17, (iii) 1-30, (iv) 1-32, (v) 4-11, and (vi) 6-10. In FIG. 13A shows an analysis of antibody binding to the FAM19A5 protein. Results are presented as mean ± standard deviation. In FIG. 13B shows solubility (CamSol score) and hydrophobicity (GRAVY score) data.

Fig. 14A and 14B show sequence alignments of the heavy chain variable regions (FIG. 14A) and the light chain variable regions (FIG. 14B) for different variants of deimmunized antibodies 3-2. The deimmunized variant antibodies shown are related to the 3-2 antibody in that these antibodies are equivalent to the 3-2 antibody, except that they have been deimmunized to reduce immunogenicity when administered to humans. Amino acid residues with high and moderate potential for immunogenicity, as determined by the ITOPE™ assay, are boxed and labeled "1" and "2", respectively. Other amino acid residues of interest are also noted: "3" = differences in human germline proximal sequence; "4" = potential arginine or lysine methylation; "5" = potential oxidation of tryptophan or methionine; "6" = potential deamidation of asparagine; "7" = potential isomerization of aspartate; "8" = rare amino acid insertion; and "9" = free cysteine or non-canonical cysteine pair. Such amino acid residues can lead to product variants during natural cellular processing and degradation.

In FIG. 15 shows sequence alignments of light chain variable regions (top three rows) and heavy chain variable regions (bottom three rows) for various deimmunized and/or affinity matured antibody variants 2-13. These variant antibodies are related to antibody 2-13 in that they are equivalent to antibody 2-13, except that they have been deimmunized and/or affinity matured. Amino acid residues with high and moderate potential for immunogenicity, as determined by the ITOPE™ assay, are boxed and labeled "1" and "2", respectively. Other amino acid residues of interest are also noted: "3" = potential arginine or lysine methylation; "4" = potential oxidation of tryptophan or methionine; "5" = potential deamidation of asparagine; "6" = potential isomerization of aspartate; "7" = rare amino acid insertion; and "8" = free cysteine or non-canonical cysteine pair. Such amino acid residues can lead to product variants during natural cellular processing and degradation.

In FIG. 16A, 16B and 16C are expression levels of two variants (2-13D-37-1.5W-41 and 2-13D-37-3W-16) of the deimmunized antibody 2-13D-37 measured by SDS-PAGE and Western blotting. . Fig. 16A and 16B show the results using SDS-PAGE for culture medium antibodies and purified protein. Fig. 16C shows the results obtained using Western blotting. On each of the FIGS. 16A, 16B and 16C, lanes "1" and "2" correspond to antibodies 2-13D-37-1.5W-41 and 2-13D-37-3W-16, respectively. In lanes "1" and "2", "A" and "B" correspond to the states before centrifugation and after centrifugation, respectively. In FIG. 16B, the left panel shows a decreasing SDS-PAGE and the right panel shows a non-reducing SDS-PAGE.

In FIG. 17A and 17B are analyzes of two variants (2-13D-37-1.5W-41 and 2-13D-37-3W-16) of deimmunized antibody 2-13D-37 that were generated by affinity maturation. In FIG. 17A shows the amino acid sequence alignment of the light chain variable region (upper three boxes) and the heavy chain variable region (lower 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. Amino acid residues with high and moderate potential for immunogenicity, as determined by the ITOPE™ assay, are boxed and labeled "1" and "2", respectively. Other amino acid residues of interest are also noted: "3" = potential oxidation of tryptophan or methionine; "4" = potential deamidation of asparagine; "5" = potential isomerization of aspartate; and "6" = non-canonical cysteine pair. Such amino acid residues can lead to product variants during natural cellular processing and degradation. Fig. 17B shows binding analysis 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 phage presenting a single chain variable fragment (scFv) was added to the wells of a microtiter plate coated with FAM19A5 (■) or anti-HA antibody (□). Background signal was measured in control wells coated with BSA. The wells were tested with HRP-conjugated anti-M13 antibody. Optical density was measured at 405 nm.

In FIG. 18 is a diagram of the general workflow of the HDX-MS analysis used in Example 11.

In FIG. 19 is a table summarizing the percent coverage achieved under various experimental conditions tested for the HDX-MS assay described in Example 11. One or more of the following parameters were adjusted: (i) sample concentration, (ii) quench conditions, (iii) pepsin concentration and/or duration of hydrolysis, and (v) quench hold time (minutes). Depending on the assay, C18 or C8 immobilized pepsin columns were used with either real-time (on) or offline (off) digestion as shown in FIG. 19.

In FIG. 20A and 20B show the results of hydrolysis of the FAM19A5 protein with pepsin using the optimized conditions described in Example 11. FIG. 20A shows the percentage coverage and redundancy of 44 peptides that were identified relative to the mature FAM19A5 protein (SEQ ID NO: 101, ie, SEQ ID NO: 2 minus the signal peptide that corresponds to the first 25 amino acids). Each of the horizontal bands represents a separate peptide. In FIG. 20B shows the amino acid sequences of the 44 peptides, including their start and end sites relative to SEQ ID NO: 101, single ion mass (MHP), and retention time (RT).

Fig. 21 shows the percent coverage and redundancy of 22 peptides that were identified when FAM19A5 protein was digested with pepsin after labeling with deuterium as described in Example 11. Each of the horizontal bars represents a single peptide.

In FIG. 22A, 22B, 22C, 22D and 22E compare deuterium uptake between single (antigen only, "1") and antigen-antibody complex (2-13) ("2") as a function of time. The y-axis shows the maximum absorption of deuterium (if the peptide contained proline, the maximum value was [(number of amino acids - 1) - (number of proline)]). The abscissa shows the duration of labeling with deuterium. Fig. 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). Fig. 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) (bottom left graph), and (vi) ARIIKTKQWC (SEQ ID NO : 113) (lower right graph). Fig. 22C provides data for the following peptides: (i) ARIIKTKQWCDM (SEQ ID NO: 114) (top left graph), (ii) ARIIKTKQWCDML (SEQ ID NO: 115) (top right graph), (iii) ARIIKTKQWCDMLPCL (SEQ ID NO: 116) (middle left graph), (iv) RIIKTKQWCDM (SEQ ID NO: 117) (middle right graph), (v) RIIKTKQWCDML (SEQ ID NO: 118) (lower left graph), and (vi) WCDMLPCL (SEQ ID NO : 119) (lower right graph). Fig. 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). Fig. 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 peptide information (i.e. start and end sites (SEQ ID NO: 101, i.e. SEQ ID NO: 2 minus the signal peptide that corresponds to the first 25 amino acids) and size) are presented in the upper corner each graph.

In FIG. 23A, 23B and 23C show key amino acid residues along the FAM19A5 protein with significant differences in deuterium uptake (i.e. greater than ±05 Da) between single and complex samples. In FIG. 23A is a butterfly diagram analysis of deuterium uptake by a single antigen (antigen only, top graph) and an antigen-antibody complex (2-13) (bottom graph). Fig. 23B is a graph of the difference in deuterium uptake between individual and complex samples. Fig. 23C shows the data as the sum of the deuterium uptake difference less than 15 Da. In FIG. 23A, 23B and 23C each of the lines represents a different duration of deuterium labeling: (i) "1" (033 minutes or 20 seconds), (ii) "2" (10 minutes), (iii) "3". (60 minutes) and (iv) "4" (240 minutes). Also, each dot corresponds to a separate peptide. In FIG. 23B and 23C show start and end sites (SEQ ID NO: 101) of peptides with differences in deuterium uptake of more than ±05 Da between a single antigen (antigen only) and an antigen-peptide complex. The dotted frame in Fig. 23B and 23C show differences in deuterium uptake of less than ±05 Da.

Fig. 24 is a heat map analysis showing regions of the FAM19A5 protein with significant differences in deuterium uptake between single (antigen only) and complex antigen-antibody samples (2-13). Residues circled in dotted red line represent key amino acid residues: (i) CRKGQIAGTTRAR (amino acid residues 38-50 of SEQ ID NO: 101 or amino acid residues 63-75 of SEQ ID NO: 2) and (ii) PACVDARIIKTKQW (amino acid residues 51-64 SEQ ID NO: 101 or amino acid residues 76-85 of SEQ ID NO: 2).

In FIG. 25 shows the 3D structure of the FAM19A5 protein and the location of the key binding epitope for antibody 2-13.

Detailed description of the disclosure

Disclosed herein is an isolated monoclonal antibody, or an antigen-binding portion thereof, that specifically binds to a protein of member A5 of family 19 with sequence similarity (FAM19A5) ("anti-FAM19A5 antibody") and exhibits one or more of the properties disclosed herein. In particular, the anti-FAM19A5 antibody has been deimmunized to reduce immunogenicity in humans.

To facilitate understanding of the disclosure presented herein, a number of terms and phrases are defined. Additional definitions are given in the detailed description.

I. Definitions

In this disclosure, terms in the singular include the plural; for example, "antibody" means one or more antibodies. As such, the terms "one or more" and "at least one" may be used interchangeably herein.

In addition, "and/or" in this context should be understood as a specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and/or" as used herein in a phrase such as "A and/or B" is intended to include "A and B", "A or B", "A" (separately), and " B" (separately). Similarly, the term "and/or" used in a phrase such as "A, B and/or C" is intended to cover each of the following: 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 (separately); B (separately); and C (separately).

It is understood that wherever aspects are described herein in the language of "comprising", similar aspects described in terms of "consisting of" and/or "consisting essentially of" are also contemplated.

Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as generally understood by a person skilled in the art to which this disclosure pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide those skilled in the art with a general vocabulary of many of the terms used in this disclosure.

Units, prefixes, and symbols are designated in their accepted Système International de Unites (SI) form. Numeric ranges include numbers that define the range. Unless otherwise indicated, amino acid sequences are written from left to right in the amino-terminal to carboxy-terminal orientation. The headings provided herein are not intended to limit the various aspects of the disclosure that may be obtained by referring to the description as a whole. Accordingly, the terms defined immediately below are more fully defined with reference to the description as a whole.

The term "about" is used in this document to mean about, approximately, approximately, or in the area. When the term "about" is used in conjunction with a numeric range, it modifies that range by extending the boundaries above and below the specified numeric values. In general, the term "about" can change the numerical value above and below the specified value with a deviation of, for example, 10 percent, up or down (higher or lower).

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

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

(I) Isoform 1 (UniProt: Q7Z5A7-1, transmembrane protein): this isoform was chosen as the canonical sequence.

MAPSPRTGSR QDATALPSMS STFWAFMILA SLLIAYCSQL AAGTCEIVTL DRDSSQPRRT IARQTARCAC RKGQIAGTTR ARPACVDARI IKTKQWCDML PCLEGEGCDL LINRSGWTCT QPGGRIKTTT VS (SEQ ID NO: 1)

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

MQLLKALWAL AGAALCCFLV LVIHAQFLKE GQLAAGTCEI VTLDRDSSQP RRTIARQTAR CACRKGQIAG TTRARPACVD ARIIKTKQWC DMLPCLEGEG CDLLINRSGW TCTQPGGRIK TTTVS (SEQ ID NO: 2)

(III) Isoform 3 (UniProt: Q7Z5A7-3):

MYHHREWPAR IIKTKQWCDM LPCLEGEGCD LLINRSGWTC TQPGGRIKTT TVS (SEQ ID NO: 3)

The term "FAM19A5" includes any variants or isoforms of FAM19A5 that are naturally expressed by cells. Accordingly, the antibodies described herein may cross-react with different isoforms of the same species (eg, different isoforms of human FAM19A5) or cross-react with FAM19A5 from other non-human species (eg, mouse FAM19A5). Alternatively, the antibodies may be specific for human FAM19A5 and may not be cross-reactive with other species. FAM19A5 or any of its variants and isoforms can either be isolated from cells or tissues that naturally express them or produced recombinantly. The polynucleotide encoding human FAM19A5 has GenBank accession number BC039396 and the following sequence:

Table 1A. Human FAM19A5 polynucleotide sequence

Polynucleotide sequence (SEQ ID NO: 4) FAM19A5
(GenBank account number BC039396) ggcggcggag gatggcgcgc gcggggcccg cacgtggagg ccggcgcggg ggcgcgggca gggccggctg ctgagacgcg ctgctgcccc ccgcgcgggc gccgcggctt caatggcgcc atcgcccagg accggcagcc ggcaagatgc gaccgccctg cccagcatgt cctcaacttt ctgggcgttc atgatcctgg ccagcctgct catcgcctac tgcagtcagc tggccgccgg cacctgtgag attgtgacct tggaccggga cagcagccag cctcggagga cgatcgcccg gcagaccgcc cgctgtgcgt gtagaaaggg gcagatcgcc ggcaccacga gagcccggcc cgcctgtgtg gacgcaagaa tcatcaagac caagcagtgg tgtgacatgc ttccgtgtct ggagggggaa ggctgcgact tgttaatcaa ccggtcaggc tggacgtgca cgcagcccgg cgggaggata aagaccacca cggtctcctg acaaacacag cccctgaggg ggccccggga gtggccttgg ctccctggag agcccacgtc tcagccacag ttctccactc gcctcggact tcacccgttc tctgccgccc gcccactccg tttccctgtg gtccgtgaag gacggcctca ggccttggca tcctgagctt cggtctgtcc agccgacccg aggaggccgg actcagacac ataggcgggg ggcggcacct ggcatcagca atacgcagtc tgtgggagcc cggccgcgcc cagcccccgc cgaccgtggc gttggccctg ctgtcctcag aggaggagga ggaggaggca gctccggcag ccacagaagg ctgcagccca gcccgcctga gacacgacgc ctgccccagg ggactgtcag gcacagaagc ggcctcctcc cgtgccccag actgtccgaa ttgcttttat tttcttatac tttcagtata ctccatagac caaagagcaa aatctatctg aacctggacg caccctcact gtcagggtcc ctggggtcgc ttgtgcgggc gggagggcaa tggtggcaga gacatgctgg tggccccggc ggagcggaga gggcggccgt ggtggaggcc tccaccccag gagcaccccg cacaccctcg gaggacgggc ttcggctgcg cggaggccgt ggcacacctg cgggaggcag cgacggcccc cacgcagacg ccgggaacgc aggccgcttt attcctctgt acttagatca acttgaccgt actaaaatcc ctttctgttt taaccagtta aacatgcctc ttctacagct ccatttttga tagttggata atccagtatc tgccaagagc atgttgggtc tcccgtgact gctgcctcat cgatacccca tttagctcca gaaagcaaag aaaactcgag taacacttgt ttgaaagaga tcattaaatg tattttgcaa agcccaaaaa aaaaaaaaaa a

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

The VH and VL regions can be further divided into regions of hypervariability called binding complementarity regions (CDRs), which are interspersed with regions that are more conserved, called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino to carboxy in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with the antigen. Antibody constant regions can mediate immunoglobulin binding to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (C1q) of the classical complement system.

The term "Kabat numbering" and similar terms are known in the art and refer to the numbering system for amino acid residues in the heavy and light chain variable regions of an antibody or antigen-binding portion thereof. In some aspects, the CDR of an antibody can be determined according to the Kabat numbering system (see, for example, 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). Using the Kabat numbering system, CDRs in an antibody heavy chain molecule are typically present at amino acid positions 31 to 35, which may optionally include one or two additional amino acids following 35 (referred to in the Kabat numbering scheme as 35A and 35B) (CDR1), in amino acid positions 50 to 65 (CDR2) and amino acid positions 95 to 102 (CDR3). Using the Kabat numbering system, CDRs in an antibody light chain molecule are typically present at amino acid positions 24 to 34 (CDR1), amino acid positions 50 to 56 (CDR2), and amino acid positions 89 to 97 (CDR3). In a specific embodiment, the CDRs of the antibodies described herein were determined according to the Kabat numbering scheme.

The phrases "numbering amino acid positions as in Kabat", "Kabat position" and their grammatical variants refer to the numbering system used for heavy chain variable domains or light chain variable domains when compiling antibodies in Kabat et al., Sequences of Proteins of Immunological Interest , 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening or insertion in the FW or CDR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insertion (Kabat residue 52a) after residue 52 of H2 and inserted residues (e.g., Kabat residues 82a, 82b and 82c, etc.) after residue 82 of the FW heavy chain. See table 1B.

Table 1B

The loop Kabat AbM Chothia L1 L24-L34 L24-L34 L24-L34 L2 L50-L56 L50-L56 L50-L56 L3 L89-L97 L89-L97 L89-L97 H1 H31-H35B H26-H35B H26-H32…34 (Kabat numbering) H1 H31-H35 H26-H35 H26-H32 (Chothia numbering) H2 H50-H65 H50-H58 H52-H56 H3 H95-H102 H95-H102 H95-H102

Kabat residue numbering can be determined for a given antibody by aligning regions of antibody sequence homology with a "standard" Kabat numbered sequence. Chothia refers instead to the arrangement of structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). Chothia's CDR-H1 loop end when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places inserts in H35A and H35B; if 35A or 35B is missing, the loop ends at 32, if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions are a compromise between Kabat CDRs and Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software.

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 was based on the alignment of over 5000 sequences, structural data and hypervariable loop characteristics and allows easy comparison of variable and CDR regions across species. According to the IMGT numbering scheme, VH-CDR1 is in positions 26 to 35, VH-CDR2 is in positions 51 to 57, VH-CDR3 is in positions 93 to 102, VL-CDR1 is in positions 27 to 32, VL -CDR2 is in positions 50 to 52 and VL-CDR3 is in positions 89 to 97.

For all heavy chain constant region amino acid positions discussed herein, the numbering follows the EU index first described in Edelman et al., 1969, Proc. Natl. Acad. sci. USA 63 (1): 78-85 describing the amino acid sequence of the EU myeloma protein, which is the first human IgG1 sequenced. The EU index from Edelman et al. also outlined in Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda. Thus, the phrases "EU index according to Kabat" or "EU index from Kabat" and "position ... according to the EU index set out in Kabat" and their grammatical variants refer to a residue numbering system based on the human lgG1 EU antibody from Edelman et al. as outlined in Kabat 1991.

The numbering system used for the variable domains (both heavy chain and light chain) and the amino acid sequence of the light chain constant region is set forth in Kabat 1991.

Antibodies can be of any type (eg, IgG, IgE, IgM, IgD, IgA, or IgY), any class (eg, IgD, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (eg, IgG1, IgG2, IgG3, and IgG4 in humans and IgG1, IgG2a, IgG2b and IgG3 in mice) immunoglobulin molecules. Immunoglobulins, such as IgG1, exist as several allotypes that differ from each other by no more than a few amino acids. An antibody described herein may belong to any of the commonly known isotypes, classes, subclasses, or allotypes. In some embodiments, the antibodies described herein are of the IgG1, IgG2, IgG3, or IgG4 subclass, or any hybrid thereof. In some embodiments, the antibodies are of the IgG2, IgG4, or IgG2/IgG4 subclass.

"Antibody" includes, for example, both 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 containing two heavy chain molecules and two light chain molecules; an antibody light chain monomer; an antibody heavy chain monomer; antibody light chain dimer, antibody heavy chain dimer; an antibody light chain-antibody heavy chain pair; intrabody; heteroconjugated antibodies; monovalent antibodies; single chain antibodies; camel antibodies; affi-body; anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies) and single-domain antibodies (sdAbs), which include binding molecules consisting of a single monomeric antibody variable domain that are fully capable of binding an antigen (for example, the VH domain or VL domain). Harmen M. M. and Haard H. J. Appl Microbiol Biotechnol. 77(1): 13-22 (2007)).

The term "antigen-binding portion" of an antibody, as used herein, refers to one or more antibody fragments that retain the ability to specifically bind to an antigen (eg, human FAM19A5). Such "fragments" are, for example, about 8 to about 1500 amino acids in length, respectively, from about 8 to about 745 amino acids in length, respectively, from about 8 to about 300, for example, from about 8 to about 200 amino acids, or about 10 up to about 50 or 100 amino acids in length. It has been shown that the antigen-binding function of an antibody can be accomplished using fragments of a full length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody, such as an anti-FAM19A5 antibody described herein, include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) an F(ab')2 fragment, a divalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region; (iii) an Fd fragment consisting of VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of one antibody arm and disulfide-linked Fvs (sdFv) (v) a dAb fragment (Ward et al., (1989) Nature 341: 544-546) which consists of the domain VH; and (vi) a dedicated complementarity determining region (CDR) or (vii) a combination of two or more dedicated CDRs, which may optionally be joined by a synthetic linker. In addition, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be connected using recombinant methods with a synthetic linker that allows them to be in the form of a single protein chain in which VL and pairs of VH regions form monovalent molecules (known as single chain Fv (scFv); see, for example, Bird et al., (1988) Science 242: 423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single chain antibodies are also intended to be encompassed by the term "antigen-binding portion" of an antibody. These antibody fragments are prepared using conventional methods known to those skilled in the art, and the fragments are tested for suitability in the same manner as intact antibodies. Antigen-binding portions can be obtained by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

As used herein, the terms "variable region" and "variable domain" are used interchangeably and are common in the art. Variable region usually refers to a portion of an antibody, typically a portion of the light or heavy chain, typically around amino-terminal amino acids 110 to 120 in the mature heavy chain and about 90 to 115 amino acids in the mature light chain, which vary greatly in sequence among antibodies and are used for the binding and specificity of a particular antibody for its particular antigen. Sequence variability is concentrated in those regions called complementarity determining regions (CDRs), while the more highly conserved regions in the variable domain are called framework regions (FRs).

Without regard to any particular mechanism or theory, it is believed that the light and heavy chain CDRs are primarily responsible for antibody-antigen interaction and specificity. In some embodiments, the variable region is a human variable region. In some embodiments, the variable region includes rodent or mouse CDRs and human framework regions (FRs). In specific embodiments, the variable region is a primate (eg, non-human primate) variable region. In some embodiments, the variable region comprises rodent or mouse CDRs and framework regions (FRs) of primates (eg, non-human primates).

As used herein, the term "heavy chain" (HC), when used in relation to an antibody, can refer to any single type, such as alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ) based on the constant domain amino acid sequence that gives rise to the IgA, IgD, IgE, IgG, and IgM antibody classes, respectively, including IgG subclasses, eg, IgG1, IgG2, IgG3, and IgG4.

As used herein, the term "light chain" (LC) when used in relation to an antibody can refer to any particular type, such as kappa (κ) or lambda (λ) based on the amino acid sequence of the constant domains. Light chain amino acid sequences are well known in the art. In specific embodiments, the light chain is a human light chain.

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

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

As used herein, the terms "constant region" and "constant domain" are used interchangeably and have the generally accepted meaning in the art. A constant region is a portion of an antibody, such as the carboxyl terminus of the light and/or heavy chain, that is not directly involved in antibody-antigen binding, but may exhibit various effector functions such as interaction with the Fc receptor. The constant region of an immunoglobulin molecule generally has a more conservative amino acid sequence than the variable domain of an immunoglobulin.

"Fc region" (crystallizing fragment region) or "Fc domain" or "Fc" refers to the C-terminal region of an antibody heavy chain that mediates binding of an immunoglobulin to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (eg, effector cells) or with the first component (C1q) of the classical complement system. Thus, the Fc region includes the constant region of the antibody, with the exception of the first immunoglobulin domain of the constant region (eg, CH1 or CL). In the IgG, IgA, and IgD antibody isotypes, the Fc region comprises two identical protein fragments derived from the second (CH2) and third (CH3) constant domains of the two antibody heavy chains; The IgM and IgE Fc regions contain three heavy chain constant domains (CH 2-4 domains) in each polypeptide chain. For IgG, the Fc region includes the Cγ2 and Cγ3 immunoglobulin domains and the hinge between Cγ1 and Cγ2. Although the boundaries of the immunoglobulin heavy chain Fc region may vary, the human IgG heavy chain Fc region is generally defined as extending from the amino acid residue at position C226 or P230 (or the amino acid between those two amino acids) to the carboxyl terminus of the heavy chain, where the numbering corresponds to the index EU, as in Kabat. The CH2 domain of the human IgG Fc region extends from about amino acid 231 to about amino acid 340, while the CH3 domain is located on the C-terminal side of the Cm domain in the Fc region, i.e. it extends from about amino acid 341 to about amino acid 447 in IgG. In this context, the Fc region may be a native sequence Fc, including any allotypic variant or Fc variant (eg, a non-naturally occurring Fc). Fc can also refer to this region alone or in the context of an Fc-containing polypeptide, such as a "binding protein containing an Fc region", also referred to as an "Fc fusion protein" (eg, antibody or immunoadhesion).

A "native sequence Fc region" or "native sequence Fc" includes an amino acid sequence that is identical to the amino acid sequence of a naturally occurring Fc region. Native sequence human Fc regions include 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, as well as natural variants thereof. The native Fc sequence includes various Fc allotypes (see e.g. Jefferis et al., (2009) mAbs 1:1; Vidarsson G. et al., Front Immunol. 5:520 (published online Oct 20, 2014)) .

An "Fc receptor" or "FcR" is a receptor that binds to the Fc region of an immunoglobulin. FcRs that bind to an IgG antibody include the FcγR family of receptors, including allelic variants and alternatively spliced forms of these receptors. The FcγR family consists of three activating (FcγRI, FcγRIII and FcγRIV in mice; FcγRIA, FcγRIIA and FcγRIIIA in humans) and one inhibitory (FcγRIIB) receptor. Human IgG1 binds to most human Fc receptors and elicits the strongest Fc effector functions. It is considered to be equivalent to mouse IgG2a in terms of the types of activating Fc receptors it binds to. In contrast, human IgG4 elicits the least Fc effector functions. Vidarsson G. et al., Front Immunol. 5:520 (published online Oct 20, 2014).

The constant region can be manipulated, for example by recombinant technology, to eliminate one or more effector functions. "Effector function" refers to the interaction of the Fc region of an antibody with an Fc receptor or ligand, or the biochemical event that results from it. Examples of "effector functions" include C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, FcγR-mediated effector functions such as ADCC and antibody-dependent cell-mediated phagocytosis (ADCP), and cell surface receptor downregulation. (eg, B-cell receptor; BCR). Such effector functions typically require that the Fc region be combined with a binding domain (eg, an antibody variable domain). Accordingly, the term "constant region without Fc function" includes constant regions with reduced or absent one or more effector functions mediated by the Fc region.

The effector functions of an antibody can be reduced or avoided by various approaches. Antibody effector functions can be reduced or avoided by using antibody fragments lacking an Fc region (eg, such as Fab, F(ab')2, single chain Fv (scFv), or sdAb consisting of a VH or VL monomeric domain). Alternatively, so-called aglycosylated antibodies can be made by removing sugars that are associated with certain residues in the Fc region to reduce the effector functions of the antibody while maintaining other valuable attributes of the Fc region (eg, prolonged half-life and heterodimerization). Aglycosylated antibodies can be produced, for example, by removing or altering the residue to which the sugar is attached, enzymatically removing sugars, producing the antibody in cells cultured in the presence of a glycosylation inhibitor, or by expressing the antibody in cells incapable of protein glycosylation (e.g., bacterial cells -owners). See, for example, pub. US No. 20120100140. Another approach is to use Fc regions from the IgG subclass that have reduced effector function, for example, IgG2 and IgG4 antibodies have lower levels of Fc effector functions than IgG1 and IgG3. The residues most proximal to the hinge region in the CH2 domain of the Fc portion are responsible for the effector functions of antibodies, as it contains a highly overlapping binding site for C1q (complement) and IgG-Fc (FcγR) receptors on effector cells of the innate immune system. Vidarsson G. et al., Front Immunol. 5:520 (published online Oct 20, 2014). Accordingly, antibodies with or without reduced Fc effector functions can be generated by constructing, for example, a chimeric Fc region that includes a CH2 domain from an IgG isotype IgG antibody and a CH3 domain from an IgG isotype IgG antibody or a chimeric Fc region that includes a hinge a region from IgG2 and a CH2 region from IgG4 (see, for example, Lau C. et al., J. Immunol. 191: 4769-4777 (2013)), or Fc regions with mutations that result in altered Fc effector functions, for example, reduced or absent Fc functions. Such mutated Fc regions are known in the art. See, for example, pub. US No. 20120100140 and the PCT and US applications cited therein and in An et al., mAbs 1: 6, 572-579 (2009); the disclosure of which is incorporated by reference in its entirety.

"Hinge", "hinge domain" or "hinge region" or "antibody hinge region" refers to the domain of the heavy chain constant region that connects the CH1 domain to the CH2 domain and includes the upper, middle and lower parts of the hinge (Roux et al., J Immunol 1998 161: 4083). The hinge provides various levels of flexibility between the binding and effector regions of the antibody, and also provides sites for intermolecular disulfide bonds between the two heavy chain constant regions. In this context, the hinge starts at Glu216 and ends at Gly237 for all IgG isotypes (Roux et al., 1998 J Immunol 161: 4083). The wild-type IgG1, IgG2, IgG3 and IgG4 hinge sequences are known in the art. See, for example, 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 Oct 20, 2014).

The term "CH1 domain" refers to a heavy chain constant region linking a variable domain to a hinge in a heavy chain constant domain. As used herein, the CH1 domain begins at A118 and ends at V215. The term "CH1 domain" includes wild-type CH1 domains as well as their natural variants (eg, allotypes). CH1 domain sequences from IgG1, IgG2, IgG3 and IgG4 (including wild type and allotypes) are known in the art. See, for example, Kabat EA et al., (1991) supra and Vidarsson G. et al., Front Immunol. 5:520 (published online Oct 20, 2014). Examples of CH1 domains include CH1 domains with mutations that modify the biological activity of the antibody, such as half-life, such as those described in pub. US Pat. No. 20120100140 and US Patents and Publications and PCT Publications cited therein.

The term "CH2 domain" refers to a heavy chain constant region linking a hinge to a CH3 domain in the heavy chain constant domain. In this context, the CH2 domain starts at P238 and ends at K340. The term "CH2 domain" includes wild-type CH2 domains as well as their natural variants (eg, allotypes). The CH2 domain sequences of IgG1, IgG2, IgG3 and IgG4 (including wild type and allotypes) are known in the art. See, for example, Kabat EA et al., (1991) supra and Vidarsson G. et al., Front Immunol. 5:520 (published online Oct 20, 2014). Exemplary CH2 domains include CH2 domains with mutations that modify the biological activity of the antibody, such as half-life and/or reduced Fc effector function, such as those described in pub. US No. 20120100140 and US patents and publications, as well as PCT publications cited therein.

The term "CH3 domain" refers to a heavy chain constant region that is C-terminal to the CH2 domain in the heavy chain constant domain. In this context, the CH3 domain starts at G341 and ends at K447. The term "CH3 domain" includes wild-type CH3 domains as well as their natural variants (eg, allotypes). The CH3 domain sequences of IgG1, IgG2, IgG3 and IgG4 (including wild type and allotypes) are known in the art. See, for example, Kabat EA et al., (1991) supra and Vidarsson G. et al., Front Immunol. 5:520 (published online Oct 20, 2014). Examples of CH3 domains include CH3 domains with mutations that modify the biological activity of the antibody, such as half-life, such as those described in pub. US No. 20120100140 and US patents and publications, as well as PCT publications cited therein.

As used herein, the term "isotype" refers to a class of antibodies (eg, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibodies) that are encoded by heavy chain constant region genes.

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

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".

The term "isolated antibody" as used herein is intended to mean an antibody that is substantially free of other antibodies having a different antigen specificity (e.g., an isolated antibody that specifically binds to FAM19A5 is substantially free of antibodies that specifically bind antigens other than FAM19A5). However, an isolated antibody that specifically binds to an FAM19A5 epitope may be cross-reactive with other FAM19A5 proteins from different species.

"Binding affinity" generally refers to the strength of the total amount of non-covalent interactions between one binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). Unless otherwise indicated, as used herein, the term "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of a molecule X for its partner Y can usually be represented by a dissociation constant (KD). Affinity can be measured and/or expressed in a number of ways known in the art, including, but not limited to, equilibrium dissociation constant (KD) and equilibrium association constant (KA). KD is calculated as a quotient of koff/kon and is expressed as a molar concentration (M), while KA is calculated as a quotient of kon/koff. kon refers to the rate constant of association of eg an antibody with an antigen, and koff refers to the dissociation of eg an antibody with an antigen. Kon and koff can be determined by methods known to those of ordinary skill in the art, such as immunoassays (eg, enzyme-linked immunosorbent assay (ELISA)), BIAcore®, or kinetic exclusion assay (KinExA).

As used herein, the terms “specifically binds,” “specifically recognizes,” “specifically binds,” “selectively binds,” and “selectively binds” are analogous terms in the context of antibodies and refer to molecules (e.g., antibodies) that bind to an antigen ( for example, an epitope or an immune complex), as such binding is clear to a person skilled in the art. For example, a molecule that specifically binds to an antigen may bind to other peptides or polypeptides, typically with lower affinity, as determined, for example, by immunoassay, BIAcore®, KinExA 3000 instrument (Sapidyne Instruments, Boise, ID) or other assays known in the art. In a specific embodiment, molecules that specifically bind to an antigen bind to the antigen with a KA that is at least 2 log, 25 log, 3 log, 4 log, or greater than the KA when the molecules bind to another antigen.

Antibodies typically bind specifically to their cognate antigen with high affinity, as reflected by a dissociation constant (KD) of 10 ^-5 to 10 ^-11 M or less. Generally, any KD greater than about 10 ^-4 M is considered to indicate non-specific binding. In this context, an antibody that "specifically binds" to an antigen refers to an antibody that binds to an antigen and substantially identical antigens with high affinity, meaning that the KD is 10 ^-7 M or less, preferably 10 ^-8 M or less, even more preferably 10 ^-9 M or less, and most preferably 10 ^-8 M to 10 ^-10 M or less, as determined by, for example, immunoassay (eg, ELISA) or Surface Plasmon Resonance (SPR) technology in a BIACORE instrument 2000 using a predetermined antigen, but does not bind with high affinity to unrelated antigens.

As used herein, the term "antigen" refers to any natural or synthetic immunogenic substance, such as a protein, peptide, or hapten. The antigen may be FAM19A5 or a fragment thereof.

As used herein, the term "epitope" refers to a localized region of an antigen to which an antibody can specifically bind. An epitope may be, for example, contiguous amino acids of a polypeptide (linear or continuous epitope), or an epitope may, for example, be derived together from two or more non-contiguous regions of a polypeptide or polypeptides (conformational, non-linear, discontinuous or non-contiguous epitope). Epitopes derived from contiguous amino acids are usually, but not always, retained upon exposure to denaturing solvents, while epitopes derived from tertiary folding are usually lost upon exposure to denaturing solvents. An epitope typically comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20 amino acids in a unique spatial conformation. Methods for determining which epitopes are associated with a given antibody (e.g., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays in which overlapping or contiguous peptides from (e.g., from FAM19A5) are tested for reactivity with a given an antibody (eg, an anti-FAM19A5 antibody). Methods for determining the spatial conformation of epitopes include methods in the art and methods described herein, for example, X-ray crystallography, 2D nuclear magnetic resonance, and HDX-MS (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996)).

In some embodiments, the epitope to which the antibody binds can be determined, for example, using NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen/deuterium exchange in combination with mass spectrometry (for example, liquid chromatography with electrospray mass spectrometry), array scanning assays based on oligopeptides and/or mutagenesis mapping (eg site-directed mutagenesis mapping). For X-ray crystallography, crystallization can be performed using any of the methods known in the art (e.g., Giege R et al., (1994) Acta Crystallogr D Biol Crystallogr 50 (Pt 4): 339-350; McPherson A (1990)) Eur J Biochem 189: 1-23; Chayen NE (1997) Structure 5: 1269-1274; McPherson A (1976) J Biol Chem 251: 6300-6303). Antibody:antigen crystals can be studied using well-known X-ray diffraction techniques and can be refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see e.g. Meth Enzymol (1985) volumes 114 & 115, eds Wyckoff HW et al.; U.S. 2004/0014194) and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49 (Pt 1): 37-60; Bricogne G (1997) Meth Enzymol 276A: 361-423, ed Carter CW Roversi P et al. (2000) Acta Crystallogr D Biol Crystallogr 56 (Pt 10): 1316-1323). Mutagenesis mapping studies can be performed using any method known to one of skill in the art. See, for example, Champe M. et al., (1995) J Biol Chem 270: 1388-1394 and Cunningham BC & Wells JA (1989) Science 244: 1081-1085 for descriptions of mutagenesis methods, including alanine scan mutagenesis methods.

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

The term "binds to the same epitope" in relation to two or more antibodies means that the antibodies bind to the same segment of amino acid residues, as determined by this method. Methods for determining whether antibodies with the "same epitope on FAM19A5" bind to the antibodies described herein include, for example, epitope mapping methods such as X-ray analysis of crystals of antigen:antibody complexes, which provides atomic resolution of the epitope and mass hydrogen-deuterium exchange spectrometry (HDX-MS). Other methods control antibody binding to antigen fragments or mutated antigen variants, where loss of binding due to modification of an amino acid residue in the antigen sequence is often considered to be indicative of an epitope component. In addition, computational combinatorial methods for epitope mapping can also be used. These methods rely on the ability of the antibody of interest to affinity isolate specific short peptides from phage display combinatorial peptide libraries. Antibodies having the same VH and VL sequences or the same CDR1, 2 and 3 sequences are expected to bind to the same epitope.

Antibodies that "compete with another antibody for binding to a target" refer to antibodies that inhibit (partially or completely) the binding of another antibody to a target. Do two antibodies compete with each other for binding to the target, i.e. whether and to what extent one antibody inhibits the binding of another antibody to a target can be determined by known competition experiments. In some embodiments, an antibody competes with another antibody and inhibits its binding to a target by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. The level of inhibition or competition may be different depending on which antibody is the "blocking antibody" (ie, cold antibody that is first incubated with the target). Competitive assays can be performed as described, for example, in Ed Harlow and David Lane, Cold Spring Harb Protoc; 2006; doi: 10.1101/pdb.prot4277 or Chapter 11 "Using Antibodies" by Ed Harlow and David Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA 1999. Competing antibodies bind to the same epitope, an overlapping epitope, or with neighboring epitopes (which, for example, is confirmed by steric hindrance).

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

A "bispecific" or "bifunctional antibody" is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites. Bispecific antibodies can be obtained using various methods, including the fusion of hybridomas or the connection of Fab' fragments. See, for example, Songsivilai & Lachmann, Clin. Exp., Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992).

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

The term "recombinant human antibody" as used herein includes all human antibodies that are produced, expressed, generated, or isolated by recombinant methods, such as (a) antibodies isolated from an animal (e.g., mouse) that is transgenic or transchromosomal for immunoglobulin genes a human or a hybridoma derived therefrom, (b) antibodies isolated from a host cell transformed to express the antibody, e.g. from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies obtained, expressed, constructed, or isolated by any other means that involve splicing human immunoglobulin gene sequences with other DNA sequences. Such recombinant human antibodies contain variable and constant regions that use specific human germline immunoglobulin sequences encoded by germline genes, but include subsequent rearrangements and mutations that occur, for example, during antibody maturation. As known in the art (see, for example, Lonberg (2005) Nature Biotech. 23 (9): 1117-1125), the variable region contains an antigen-binding domain that is encoded by various genes that rearrange to form an antibody specific for a foreign antigen. In addition to rearrangement, the variable region can be further modified by multiple single amino acid substitutions (called a somatic mutation or hypermutation) to increase the affinity of the antibody for the foreign antigen. The constant region will change with further antigen response (i.e. isotype switching). Therefore, rearranged and somatically mutated nucleic acid molecules that encode light chain and heavy chain immunoglobulin polypeptides in response to an antigen may not be sequence identical to the original nucleic acid molecules, but will instead be substantially identical or similar (i.e., have at least 80% identity).

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

A "humanized" antibody refers to an antibody in which some, most, or all of the amino acids outside the CDR domains of the non-human antibody have been replaced with corresponding amino acids derived from human immunoglobulins. In one embodiment of the humanized form of the antibody, some, most, or all of the amino acids outside the CDR domains have been replaced with amino acids from human immunoglobulins, while some, most, or all of the amino acids in one or more CDR regions are unchanged. Small 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. The "humanized" antibody retains antigenic specificity similar to the original antibody.

As used herein, the term "deimmunized" or "deimmunization" refers to a process in which an antibody, or antigen-binding portion thereof, is modified to reduce its immunogenicity, for example, in humans. For example, the heavy chain variable region (VH) and light chain variable region (VL) sequences of the parent antibody can be analyzed and a "map" of human T-cell epitopes can be generated from each V region showing the location of the epitopes in relation to the complementarity determining regions (CDRs) and other key residues in the sequence. Individual T cell epitopes from the T cell epitope map are analyzed to identify alternative amino acid substitutions with a low risk of altering the activity of the final antibody. A number of alternative VH and VL sequences have been developed, including combinations of amino acid substitutions, and these sequences are subsequently included in a number of FAM19A5-specific antibodies, or antigen-binding portion thereof, for use in the diagnostic and therapeutic methods disclosed herein, which are then tested for functionality. The complete heavy and light chain genes containing the modified V and human C regions are then cloned into expression vectors and subsequent plasmids are introduced into cell lines to generate the whole antibody. The antibodies are then compared in appropriate biochemical and biological assays and the optimal variant is determined. The antibody can be deimmunized using the methods described herein or by any other methods known in the art. See, for example, WO 98/52976 or WO 00/34317.

A "chimeric antibody" refers to an antibody in which the variable regions are from one species and the constant regions are from another species, such as an antibody in which the variable regions are from a mouse antibody and the constant regions are from a human antibody.

The term "cross-reaction" as used herein refers to the ability of an antibody described herein to bind to another species of FAM19A5. For example, an antibody described herein that binds human FAM19A5 can also bind another species of FAM19A5 (eg, mouse FAM19A5). In this context, cross-reactivity can be measured by determining specific reactivity with purified antigen in binding assays (eg SPR, ELISA) or binding or other functional interaction with cells physiologically expressing FAM19A5. Methods for determining cross-reactivity include standard binding assays as described herein, for example Biacore® surface plasmon resonance (SPR) analysis using a Biacore® 2000 SPR instrument (Biacore AB, Uppsala, Sweden) or flow cytometry methods.

The term "naturally occurring" as used herein in relation to an object refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence present in an organism (including viruses) that can be isolated from a natural source and that has not been deliberately modified by humans in a laboratory is naturally occurring.

"Polypeptide" refers to a chain containing at least two consecutively linked amino acid residues without an upper chain length limit. One or more amino acid residues in a protein may contain a modification such as, but not limited to, glycosylation, phosphorylation, or disulfide bond formation. "Protein" may include one or more polypeptides.

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

As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid", which refers to a circular double stranded loop of DNA into which additional DNA segments can be ligated. Another type of vector is the viral vector, in which additional DNA segments can be ligated into the viral genome. Some vectors exhibit the ability to autonomously replicate in the host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and mammalian episomal vectors). Other vectors (eg, non-episomal mammalian vectors) integrate into the genome of a host cell when introduced into the host cell, and thus replicate along with the host genome. Moreover, some vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. In the present description, the terms "plasmid" and "vector" can be used interchangeably, since the plasmid is the most commonly used form of the vector. However, other forms of expression vectors are also included, such as viral vectors (eg, replication-defective retroviruses, adenoviruses, and adeno-associated viruses) that perform equivalent functions.

The term "recombinant host cell" (or simply "host cell") as used herein is intended to refer to a cell that contains a nucleic acid that is not naturally present in the cell, and may be a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to a specific entity, but also to the progeny of such a cell. Because certain modifications may occur in subsequent generations due to mutations or environmental influences, such progeny may not actually be identical to the parent cell, but are still within the scope of the term "host cell" as used herein.

As used herein, the term "associated" refers to the association of two or more molecules. The bond may be covalent or non-covalent. The connection may also be genetic (ie, recombinantly fused). Such linkages can be achieved using a wide range of techniques recognized in the art, such as chemical conjugation and production of recombinant proteins.

As used herein, the term "administration" refers to the physical administration of a therapeutic agent or a composition containing a therapeutic agent to an object using any of the various delivery methods and systems known to those skilled in the art. Preferred routes of administration of the antibodies described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, or other parenteral routes of administration, such as by injection or infusion. The phrase "parenteral administration" in the context of the present description means methods of administration other than enteral and topical, usually by injection, and includes, without limitation, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injections and infusions, as well as in vivo electroporation. Alternatively, an antibody described herein can be administered by a non-parenteral route, eg, topical, epidermal, or mucosal route, eg, intranasally, orally, vaginally, rectally, sublingually, or topically. The administration may also be performed, for example, once, multiple times, and/or over one or more extended periods.

The terms "treat", "treat" and "treatment", as used herein, refer to any type of intervention or process performed, or administration of an active agent to a subject for the purpose of reversing, alleviating, ameliorating, inhibiting, or slowing down or preventing the progression, development, the severity or recurrence of a symptom, complication, condition, or biochemical parameter associated with the disease. Treatment may refer to an object suffering from a disease or an object that does not have a disease (eg, for prophylaxis).

As used herein, the term "object" includes any non-human human or animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, and the like.

As used herein, the term "onset of gliosis" or "onset of reactive gliosis" includes the onset or initiation of gliosis. Gliosis is a non-specific reactive change in glial cells in the central nervous system (CNS, e.g., brain and/or spinal cord) in response to injury or injury, e.g. trauma, cerebrospinal injury, brain tumor, infection, ischemia, stroke, autoimmune responses and/or neurodegenerative diseases and include the proliferation or hypertrophy of several different types of glial cells, including astrocytes, microglia, and oligodendrocytes. The onset of gliosis can lead to scar formation that prevents axonal regeneration in an injured or damaged part of the CNS. The deleterious effects of the onset of gliosis include irreversible or permanent damage to neurons and/or prevention of repair of surrounding neurons. Accordingly, the terms "delay the onset of gliosis" and "delay the onset of reactive gliosis" include inhibiting, slowing, suppressing or preventing the onset or occurrence of gliosis and its associated deleterious effects on the CNS.

As used herein, the term "excessive proliferation of reactive astrocytes" includes an abnormal increase in the number of astrocytes due to destruction of nearby neurons, such as from CNS injury, trauma, injury, cerebrospinal injury, brain tumor, infection, ischemia, stroke, autoimmune reactions, and /or a neurodegenerative disease. An overgrowth of reactive astrocytes can lead to detrimental effects in the CNS, including scarring that prevents axonal regeneration in the part of the CNS that has been injured or damaged, increased inflammation, production and release of neurotoxic levels of reactive oxygen species, release of potential excitotoxic glutamate, a potential contribution to genesis of seizures, impaired function of the blood-brain barrier, cytotoxic edema during trauma and stroke, the possibility of chronic cytokine activation of astrocytes contributing to chronic pain, and secondary degeneration after CNS injury. Sofroniew, Michael V. (2009) Trends in Neurosciences, 32(12):638-47; McGraw, J. et al. (2001) Journal of Neuroscience Research 63(2):109-15; and Sofroniew, M. V. (2005) The Neuroscientist 11(5): 400-7. Accordingly, the terms "suppression of excessive proliferation of reactive astrocytes" include inhibition, slowing down, suppression, containment or prevention of excessive or abnormal proliferation of reactive astrocytes and associated detrimental effects on the CNS.

As used herein, the term "chondroitin sulfate proteoglycans" includes proteoglycans consisting of a protein core and chondroitin sulfate. Chondroitin sulfate proteoglycans, also known as CSPGs, are extracellular matrix molecules widely expressed in the developing and adult CNS. CSGPs play a key role in the development of the nervous system and the formation of glial scars, and they inhibit axonal regeneration after injury in the CNS. Known CSPGs include aggrecan (CSPG1), versican (CSPG2), neurocan (CSPG3), CSPG4 (or neuroglial antigen 2 (NG2)), CSPG5, SMC3 (CSPG6, chromosome 3 structural maintenance), brevican (CSPG7), and CD44 (CSPG8, differentiation cluster 44), phosphacaneurocan (CSPG3). Rhodes, K. E. and Fawcett, J. W. (2004) Journal of Anatomy. 204(1):33-48. Thus, the term "decreased expression of chondroitin sulfate proteoglycans" includes a decrease, inhibition, decrease in the level of one or more CSGPs, or a decrease in the activity or conversion to an inactive one or more CSGPs. In some embodiments, the term includes a decrease, inhibition, decrease in the level of neurocan, NG2, or both, or a decrease in activity, or turning into an inactive neurocan, NG2, or both.

As used herein, the term "neuron" includes electrically excitable cells that process and transmit information through electrical and chemical signals. Neurons are the main components of the brain and spinal cord of the CNS and ganglia of the peripheral nervous system (PNS) and can connect to each other to form neural networks. A typical neuron consists of a cell body (soma), dendrites, and an axon. The soma (cell body) of a neuron contains the nucleus. The dendrites of a neuron are cellular extensions with many branches where most of the input to the neuron occurs. The axon is a thinner, cable-like protrusion that extends from the soma and carries nerve signals from the soma and certain types of information back to the soma. The term "facilitate the regrowth of neurons" includes the stimulation, promotion, increase or activation of neuronal growth, preferably after injury or damage.

The term "c-fos" as used herein includes the c-fos proto-oncogene, which 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 characterized. See Curran, T, The c-fos proto-oncogene, pp 307-327 (The Oncogene Handbook, Reddy EP et al., (eds.) Elsevier) (1988). Expression of c-fos can be determined by methods known in the art, such as Northern blotting, quantitative PCR, or immunohistochemistry. The term "increases c-fos expression" includes an increase in c-fos mRNA, c-fos protein, or c-fos protein activity.

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

As used herein, the term "GAP43", also known as "growth-associated protein 43", is a neural tissue-specific protein that promotes neurite formation, regeneration, and plasticity. 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 sequence of the human GAP43 polypeptide (UniProt: KB - P17677) and the cDNA sequence encoding the polypeptide are known in the art. Kosik K. S. et al., (1988) Neuron 1(2):127-32; Ng S. C. et al., (1988) Neuron 1(2):133-9. GAP43 expression can be determined by methods known in the art (eg, Northern blotting, quantitative PCR, or immunohistochemistry). The term "increase in GAP43 in neurons" includes an increase or increase in the level of GAP43 mRNA, GAP43 protein, or an increase in GAP43 protein activity.

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

II. Anti-FAM19A5 antibodies

Disclosed herein are antibodies, such as monoclonal antibodies, that are characterized by specific functionalities or properties. For example, antibodies that specifically bind human FAM19A5 are mutated (eg, by substitution or deletion) by removing and/or modifying regions or residues that are highly immunogenic in humans (ie, deimmunized). Accordingly, the antibodies disclosed herein, i. an anti-FAM19A5 antibody has reduced immunogenicity when administered to a human subject compared to a reference antibody (eg, a corresponding antibody that has not been deimmunized, eg, antibody 3-2 or 2-13).

In addition, the antibodies described herein have one or more of the following functional properties:

(a) bind to soluble human FAM19A5 with a KD of 10 nM or less;

(b) bind to human membrane-bound FAM19A5 with a KD of 10 nM or less;

(c) reduce, reverse, delay and/or prevent the onset of reactive gliosis;

(d) suppress excessive proliferation of reactive astrocytes

(e) reduce the expression of chondroitin sulfate proteoglycans, including neurocan and neuron-glial antigen 2 (NG2);

(e) increase c-fos and pERK expression in neuronal nuclei;

(g) ensure the survival of neurons.

(h) increase GAP43 expression in neurons; and

(i) provide axon regrowth.

In some embodiments, the anti-FAM19A5 antibody has been deimmunized such that the antibody is less immunogenic when administered to a human subject compared to a reference antibody (e.g., a corresponding antibody that has not been deimmunized, e.g., antibody 3-2 or 2-13). In some embodiments, the immunogenicity of an 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 antibody that has not been deimmunized, e.g. , antibody 3-2 or 2-13). In some embodiments, the deimmunization process does not change the binding affinity of the antibody.

In some embodiments, an anti-FAM19A5 antibody disclosed herein has undergone affinity maturation such that the antibody binds to the FAM19A5 protein with greater affinity than a reference antibody (e.g., a corresponding antibody that has not undergone affinity maturation, such as antibody 2-13 ). 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 at least about 70%, at least about 80%, at least about 90%, or at least about 100% or more 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 alter the immunogenicity of the antibody.

In some embodiments, an anti-FAM19A5 antibody disclosed herein has been deimmunized and affinity matured such that compared to a reference antibody (e.g., a corresponding antibody that has not been deimmunized or affinity matured, such as a 3-2 antibody or 2-13), the antibody is not only less immunogenic when administered to a human, but also binds to the FAM19A5 protein with higher affinity. In some embodiments, the immunogenicity of an 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 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 about 100% or more compared to the reference antibody.

In some embodiments, the anti-FAM19A5 antibody specifically binds to soluble human FAM19A5 or membrane bound human with high affinity, e.g., a KD of 10 ^-7 M or less, 10 ^-8 M (10 nM) or less, 10 ^-9 M (1 nM) or less, 10 ^-10 M (01 nM) or less, 10 ^-11 M or less, or 10 ^-12 M or less, for example, 10 ^-12 M - 10 ^-7 M, 10 ^-11 M - 10 ^-7 M , 10 ^-10 M - 10 ^-7 M, or 10 ^-9 M - 10 ^-7 M, eg, 10 ^-12 M, 5 X 10 -12 M, ^{10 -11 M} , 5 X 10 ^-11 M, 10 ^{- 10} M, 5 X 10 ^-10 M, 10 ^-9 M, 5 X 10 ^-9 M, 10 ^-8 M, 5 X 10 ^-8 M, 10 ^-7 M, or 5 X 10 ^-7 M. Standard assays for assessments of the ability of an antibody to bind to human FAM19A5 of various species are known in the art, including, for example, ELISA, Western blot, and RIA. Suitable assays are detailed in the Examples. The binding kinetics (eg, binding affinity) of antibodies can also be assessed by standard assays known in the art such as ELISA, Biacore® assay, or KinExA. Assays to evaluate the effects of antibodies on the functional properties of FAM19A5 (eg, ligand binding) are described in more detail below and in the examples.

In some embodiments, the anti-FAM19A5 antibody specifically binds to soluble human FAM19A5 with a KD, e.g., as determined by ELISA, 10 ^-7 M or less, 10 ^-8 M (10 nM) or less, 10 ^-9 M (1 nM) or less, 10 ^-10 M or less, from 10 ^-12 M to 10 -7 M, from 10 ^-11 M to 10 ^-7 M, from 10 ^-10 M to 10 ^-7 M, from 10 ^-9 M to 10 ^-7 M or 10 ^-8 M to 10 ^-7 M. In some embodiments, the anti-FAM19A5 antibody specifically binds to soluble FAM19A5 with a KD of 10 nM or less, eg, 01 to 10 nM, 01 to 5 nM. 01 to 1 nM, 05 to 10 nM, 05 to 5 nM, 05 to 1 nM, 1 to 10 nM, 1 to 5 nM, or 5 to 10 nM. In some embodiments, the anti-FAM19A5 antibody specifically binds to soluble human FAM19A5 with a KD of 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, as determined by ELISA.

In some embodiments, the anti-FAM19A5 antibody specifically binds to a membrane-bound human with a KD, e.g., as determined by ELISA, 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 from 10 ^-8 M to 10 ^-7 M. In some embodiments, the anti-FAM19A5 antibody specifically binds to membrane-bound human FAM19A5 with a KD of 10 nM or less, as determined by ELISA, for example, between 01 and 10 nM, from 01 up to 5 nM, 01 to 1 nM, 05 to 10 nM, 05 to 5 nM, 05 to 1 nM, 1 to 10 nM, 1 to 5 nM, or 5 to 10 nM. In some embodiments, the anti-FAM19A5 antibody specifically binds to membrane-bound human FAM19A5 with a KD of 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, as determined by ELISA.

The anti-FAM19A5 antibody of the present disclosure can delay or inhibit the onset of gliosis, e.g., delay, slow down, or inhibit the onset or onset of a non-specific reactive change in glial cells in the central nervous system (CNS, e.g., brain and/or spinal cord) in response to injury or injury, such as trauma, cerebrospinal injury, brain tumor, infection, ischemia, stroke, autoimmune reactions, and/or neurodegenerative disease.

The anti-AM19A5 antibody of the present invention can retard, inhibit, retard, suppress, inhibit or prevent excessive or abnormal proliferation of reactive astrocytes and its associated detrimental effects on the CNS. For example, an anti-FAM19A5 antibody of the present invention can inhibit or prevent an abnormal increase in astrocytes due to neuronal destruction, such as CNS injury, trauma, injury, cerebrospinal injury, brain tumor, infection, ischemia, stroke, autoimmune reactions, and / or a neurodegenerative disease, inhibit or prevent CNS scarring, inhibit or reduce the release of neurotoxic levels of reactive oxygen species or the release of potentially excitotoxic glutamate, reduce or inhibit convulsions, pain and / or secondary degeneration after CNS injury. The anti-AM19A5 antibody of the present invention may promote, stimulate, increase, or activate neuronal and/or axonal regrowth, preferably after injury or damage to the CNS.

The anti-AM19A5 antibody of the present invention can inhibit the expression of chondroitin sulfate proteoglycans, including core protein and chondroitin sulfate (CSGP) proteoglycans such as aggrecan (CSPG1), versican (CSPG2), neurocan (CSPG3), CSPG4 (or neuron-glial antigen). 2 (NG2)), CSPG5, SMC3 (CSPG6, structural maintenance of chromosomes 3), brevican (CSPG7), CD44 (CSPG8, differentiation cluster 44), and phosphacaneurocan (CSPG3). In some embodiments, the anti-FAM19A5 antibody of the present disclosure inhibits, reduces or reduces the level of Neurocan and/or NG2, or the activity of Neurocan and/or NG2.

The anti-AM19A5 antibody of the present invention can increase the expression of c-fos and pERK in the nucleus of neurons, for example, increase the mRNA, protein and/or protein activity of c-fos and pERK. The anti-AM19A5 antibody of the present invention can also increase or increase the expression level of GAP43 mRNA, GAP43 protein, or increase or increase the activity of GAP43 protein.

In some embodiments, the anti-FAM19A5 antibody of the present disclosure comprises a heavy chain CDR1, CDR2, and CDR3 and a light chain CDR1, CDR2, and CDR3, wherein the heavy chain CDR1, CDR2, and CDR3 comprise a set of amino acid sequences further in SEQ ID NOs: 5, 6, and 7 , respectively, each of which optionally contains one, two, three, four or five mutations, while light chain CDR1, CDR2 and CDR3 contain the amino acid sequences indicated in SEQ ID NOs: 8, 9 and 10, respectively, where at least at least one of the light chain CDR1, CDR2 and CDR3 containing one, two, three, four or five mutations, and where the antibody has reduced immunogenicity in humans compared to a reference antibody containing the VH specified in SEQ ID NO: 11, and VL listed in SEQ ID NO: 12.

In some embodiments, the mutations included in the antibody are substitution, deletion and/or insertion. In one embodiment, the mutation is a substitution, such as a conservative substitution. "Conservative substitution" (also referred to as conservative substitution) as used herein means an amino acid substitution that changes a given amino acid to another amino acid with similar biochemical properties (eg, charge, hydrophobicity, and size). While there are many ways to classify amino acids, they are often divided into six major groups based on their structure and the general chemical characteristics of their R groups.

Table 2. Classes of amino acids

Class Amino acids Aliphatic Glycine, alanine, valine, leucine, isoleucine Hydroxyl or sulfur/selenium containing Serine, cysteine, selenocysteine, threonine, methionine Cyclic Proline aromatic Phenylalanine, tyrosine, tryptophan Main Histidine, lysine, arginine Acids and their amides Aspartate, glutamate, asparagine, glutamine

Conversely, a radical substitution or a radical substitution is an amino acid substitution in which the original amino acid is replaced by a final amino acid with different physicochemical properties. In some embodiments, the amino acid mutation in the FAM19A5 antibody is a radical substitution. In other embodiments, the amino acid mutation in the FAM19A5 antibody is a combination of a conservative substitution and a radical substitution.

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

In some embodiments, the heavy chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 5, optionally with one or two mutations. In some embodiments, the mutations include changing the threonine at amino acid 3 of SEQ ID NO: 5 to an acidic amino acid. In additional embodiments, the mutations include the replacement of serine at amino acid 5 of SEQ ID NO: 5 with an acidic amino acid. In some embodiments, the acidic amino acid includes aspartic acid or glutamic acid.

In some embodiments, the heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 6, optionally with one, two, three, four, or five mutations. In some embodiments, the mutations include the replacement of arginine at amino acid 16 of SEQ ID NO: 6 with a basic amino acid. In some embodiments, the basic amino acid includes lysine. In some embodiments, the mutations include one or more of the following:

(a) replacing the glycine in amino acid 6 of SEQ ID NO: 6 with an acidic amino acid;

(b) replacing serine at amino acid 7 in SEQ ID NO: 6 with an acidic amino acid;

(c) replacing serine at amino acid 8 in SEQ ID NO: 6 with an acidic amino acid;

(d) replacing the threonine in amino acid 9 of SEQ ID NO: 6 with an acidic amino acid; and

(e) replacing arginine at amino acid 16 of SEQ ID NO: 6 with a basic amino acid.

In some embodiments, the acidic amino acid includes aspartic acid or glutamic acid. In some embodiments, the basic amino acid includes lysine.

In some embodiments, the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 10, optionally with one, two, three, four, or five mutations. In some embodiments, the mutations include one or more of the following:

(a) replacing serine at amino acid 6 of SEQ ID NO: 10 with an acidic amino acid or an aliphatic amino acid;

(b) replacing the asparagine at amino acid 7 of SEQ ID NO: 10 with an acidic amino acid or with a hydroxyl or sulfur/selenium containing amino acid;

(c) replacing the glycine in amino acid 8 of SEQ ID NO: 10 with an acidic amino acid or with a hydroxyl or sulfur/selenium containing amino acid;

(d) replacing the glycine in amino acid 9 of SEQ ID NO: 10 with an acidic amino acid or with a hydroxyl or sulfur/selenium containing amino acid; and

(e) replacing the isoleucine at amino acid 10 of SEQ ID NO: 10 with a basic amino acid.

In some embodiments, the acidic amino acid includes aspartic acid or glutamic acid. In some embodiments, the hydroxyl or sulfur/selenium containing amino acid includes serine. In additional embodiments, the basic amino acid includes histidine.

In some embodiments, the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 8 with one, two, three, or four mutations. In some embodiments, the mutations include one or more of the following:

(a) replacing the tyrosine in amino acid 6 of SEQ ID NO: 8 with an acidic amino acid;

(b) replacing arginine at amino acid 7 in SEQ ID NO: 8 with an acidic amino acid;

(c) replacing the glycine in amino acid 8 of SEQ ID NO: 8 with an acidic amino acid; and

(d) replacing the serine at amino acid 9 in SEQ ID NO: 8 with an acidic amino acid.

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

In some embodiments, the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 9 with one, two, three, or four mutations. In some embodiments, the mutations include one or more of the following:

(a) replacing the glutamic acid in amino acid 1 of SEQ ID NO: 9 with an acidic amino acid;

(b) replacing the serine in amino acid 2 of SEQ ID NO: 9 with an acidic amino acid;

(c) replacing asparagine in amino acid 3 of SEQ ID NO: 9 with an acidic amino acid, a basic amino acid, or an aliphatic amino acid; and

(d) replacing the lysine at amino acid 4 of SEQ ID NO: 9 with an acidic amino acid or an aliphatic amino acid.

In some embodiments, the acidic amino acid includes glutamine, asparagine, aspartic acid, or glutamic acid. In some embodiments, the basic amino acid includes histidine. In further embodiments, the aliphatic amino acid comprises leucine.

In some embodiments, the mutations include changing the serine at amino acid 2 of SEQ ID NO: 9 to an acidic amino acid. In some embodiments, the acidic amino acid includes aspartate, glutamate, asparagine, glutamine, or combinations thereof. In further embodiments, the acidic amino acid is asparagine.

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

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

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

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

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

In some embodiments, the anti-FAM19A5 antibody comprises a heavy chain CDR1, CDR2, and CDR3 and a light chain CDR1, CDR2, and CDR3, wherein: (i) the heavy chain 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.

In some embodiments, the anti-FAM19A5 antibody comprises a heavy chain CDR1, CDR2, and CDR3 and a light chain CDR1, CDR2, and CDR3, wherein: (i) the heavy chain 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.

In some embodiments, the anti-FAM19A5 antibody of the present disclosure is humanized. In other embodiments, the humanized anti-FAM19A5 comprises a human antibody framework region. In some embodiments, the anti-FAM19A5 antibody contains one or more (e.g., one, two, three, four, five, six, seven, or more) mutations in the framework region (e.g., FR1, FR2, FR3, and FR4 from VH and/or FR1 , FR2, FR3 and FR4 from VL) antibodies.

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

In some embodiments, an anti-FAM19A5 antibody disclosed herein comprises a mutation in FR2 from VH. In some embodiments, the mutation involves an amino acid substitution at residue 49 (e.g., alanine to hydroxyl or sulfur/selenium containing amino acid, e.g. serine) of SEQ ID NO: 11.

In some embodiments, the anti-FAM19A5 antibody of the present disclosure contains a mutation in FR3 from VH. In some embodiments, the mutation includes an amino acid change at residue 79 (e.g., valine to an aliphatic amino acid, e.g., leucine), residue 80 (e.g., arginine to an aromatic amino acid, e.g., tyrosine), residue 83 (e.g., leucine to hydroxyl or sulfur/selenium containing amino acid, e.g. methionine), residue 85 (e.g. asparagine per hydroxyl or sulfur/selenium containing amino acid e.g. serine), residue 86 (e.g. proline per aliphatic amino acid e.g. leucine) and/or residue 87 (e.g. glycine per basic amino acid, e.g. arginine) of SEQ ID NO: 11.

In some embodiments, the anti-FAM19A5 antibody comprises a mutation in FR2 from VL. In some embodiments, the mutation includes a deletion of amino acid residue 39 of SEQ ID NO: 12.

In some embodiments, an anti-FAM19A5 antibody disclosed herein comprises a mutation in FR3 from VL. In some embodiments, the mutation comprises an amino acid substitution at residue 81 (e.g., aspartic acid with an aliphatic amino acid, e.g., glycine) and/or at residue 85 (e.g., isoleucine with an acidic amino acid, e.g., aspartic acid) of SEQ ID NO: 12.

In some embodiments, an anti-FAM19A5 antibody of the present disclosure includes 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% identical, 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% of the amino acid sequence specified as SEQ ID NO: 11, and/or where VL includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90% identical , at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% of the amino acid sequence specified by SEQ ID NO: 12, where the antibody has reduced immunogenicity compared to the reference antibody, containing a VH listed as SEQ ID NO: 11 and a VL listed as SEQ ID NO: 12.

In some embodiments, an 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), where:

(a) VH includes the amino acid sequence set forth in SEQ ID NO: 33 and VL includes the amino acid sequence set forth in SEQ ID NO: 38;

(b) VH includes the amino acid sequence shown in SEQ ID NO: 34 and VL includes the amino acid sequence shown in SEQ ID NO: 39;

(c) VH includes the amino acid sequence set forth in SEQ ID NO: 34 and VL includes the amino acid sequence set forth in SEQ ID NO: 41;

(d) VH includes the amino acid sequence set forth in SEQ ID NO: 34 and VL includes the amino acid sequence set forth in SEQ ID NO: 40;

(e) VH includes the amino acid sequence set forth in SEQ ID NO: 34 and VL includes the amino acid sequence set forth in SEQ ID NO: 42;

(f) VH includes the amino acid sequence set forth in SEQ ID NO: 34 and VL includes the amino acid sequence set forth in SEQ ID NO: 43; or

(g) VH includes the amino acid sequence shown in SEQ ID NO: 34 and VL includes the amino acid sequence shown in SEQ ID NO: 44.

In some embodiments, the anti-FAM19A5 antibody binds to the same human FAM19A5 epitope as the reference antibody, which includes a heavy chain variable region (VH) and a light chain variable region (VL), where:

(a) VH includes the amino acid sequence set forth in SEQ ID NO: 33 and VL includes the amino acid sequence set forth in SEQ ID NO: 38;

(b) VH includes the amino acid sequence shown in SEQ ID NO: 34 and VL includes the amino acid sequence shown in SEQ ID NO: 39;

(c) VH includes the amino acid sequence set forth in SEQ ID NO: 34 and VL includes the amino acid sequence set forth in SEQ ID NO: 41;

(d) VH includes the amino acid sequence set forth in SEQ ID NO: 34 and VL includes the amino acid sequence set forth in SEQ ID NO: 40;

(e) VH includes the amino acid sequence set forth in SEQ ID NO: 34 and VL includes the amino acid sequence set forth in SEQ ID NO: 42;

(f) VH includes the amino acid sequence set forth in SEQ ID NO: 34 and VL includes the amino acid sequence set forth in SEQ ID NO: 43; or

(g) VH includes the amino acid sequence shown in SEQ ID NO: 34 and VL includes the amino acid sequence shown in SEQ ID NO: 44.

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

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

In some embodiments, the heavy chain CDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 16. In some embodiments, the heavy chain CDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 16, optionally with a single mutation. In some embodiments, the mutation involves replacing the threonine at amino acid 3 of SEQ ID NO: 16 with an acidic amino acid. In some embodiments, the acidic amino acid includes aspartic acid.

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

In some embodiments, the light chain CDR3 includes the amino acid sequence listed in SEQ ID NO: 32. In some embodiments, the light chain CDR2 includes the amino acid sequence listed in SEQ ID NO: 31. In some embodiments, the light chain CDR2 includes the amino acid sequence listed in SEQ ID NO: 31. NO: 31, with one, two or three mutations.

In further embodiments, the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 30, optionally with a single mutation. In some embodiments, the mutation involves the replacement of serine at amino acid 4 of SEQ ID NO: 30 with an aliphatic amino acid. In some embodiments, the aliphatic amino acid includes valine.

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

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

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

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

In some embodiments, the anti-FAM19A5 antibody of the present disclosure is humanized. In other embodiments, the humanized anti-FAM19A5 comprises a human antibody framework region. In some embodiments, the anti-FAM19A5 antibody contains one or more (e.g., one, two, three, four, five, six, seven, or more) mutations in the framework region (e.g., FR1, FR2, FR3, and FR4 from VH and/or FR1 , FR2, FR3 and FR4 from VL) antibodies.

In some embodiments, an anti-FAM19A5 antibody disclosed herein comprises a mutation in FR1 from VH. In some embodiments, the mutation involves an amino acid change at residue 19 (e.g., serine to a basic amino acid, e.g., arginine), residue 21 (e.g., valine to hydroxyl or sulfur/selenium containing amino acid, e.g. serine), and/or residue 23 (e.g., from lysine to hydroxyl or a sulfur/selenium containing amino acid, e.g. serine) of SEQ ID NO: 35.

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

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

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

In some embodiments, the anti-FAM19A5 antibody comprises a mutation in FR2 from VL. In some embodiments, the mutation includes a deletion of amino acid residue 34 of SEQ ID NO: 45.

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

In some embodiments, an anti-FAM19A5 antibody disclosed herein includes 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% identical, at least 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% of the specified amino acid sequence in SEQ ID NO: 35 and/or where VL includes an amino acid sequence that is at least about 80% identical, at least about 85% identical, 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% amino acid sequence specified in SEQ ID NO: 45.

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

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

In some embodiments, an anti-FAM19A5 antibody of the present disclosure competes for binding (or inhibits binding) of a human FAM19A5 epitope to a reference antibody (eg, antibody 3-2 or 2-13).

In some embodiments, the anti-FAM19A5 antibody inhibits the binding of such reference antibody (e.g., antibody 3-2 or 2-13) 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%. Competing antibodies bind to the same epitope, an overlapping epitope, or adjacent epitopes (as evidenced by steric hindrance, for example). Whether two antibodies compete with each other for binding to a target can be determined using competition experiments known in the art such as RIA and EIA.

Techniques for determining whether two antibodies bind to the same epitope include, for example, epitope mapping methods such as X-ray crystal analysis of antigen:antibody complexes, which provides atomic resolution of the epitope, and hydrogen/deuterium exchange mass spectrometry (HDX-MS ), methods for monitoring antibody binding to antigen fragments or mutated antigen variations, where loss of binding due to modification of an amino acid residue in the antigen sequence is often considered a criterion for an epitope component, computational combinatorial methods for epitope mapping.

An anti-AM19A5 antibody that can be used in the methods disclosed herein can bind to at least one epitope of mature human FAM19A5, as determined, for example, by binding antibodies to human FAM19A5 fragments. In some embodiments, the anti-FAM19A5 antibody binds to at least one epitope that has the amino acid sequence TLDRDSSQPRRTIARQTARC (SEQ ID NO: 90 or amino acid residues 42 to 61 of SEQ ID NO: 2), or binds to a fragment located in the amino acid sequence of SEQ ID NO: 90, e.g., an epitope having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, or 20 amino acids of SEQ ID NO: 90. In some embodiments, an anti-FAM19A5 antibody disclosed herein binds to one or more amino acids corresponding to amino acid residues 44 to 52 (ie, DRDSSQPRR) of SEQ ID NO: 2, e.g. amino acid residues 45, 46, 50, 51, and 52 (RD---PRR), e.g. amino acid residues 45, 50, 51 and 52 (i.e. R----PRR), for example, amino acid residues 43, 50 and 51 (i.e. R----PR).

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

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

In some embodiments, the anti-FAM19A5 antibody, or antigen-binding portion thereof, only binds to a human FAM19A5 epitope that is 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, 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 from SEQ ID NO: 89, 90, 91, 92, 93 or 94.

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

In some embodiments, the anti-FAM19A5 antibody binds to one or more additional FAM19A5 epitopes. In some embodiments, one or more additional FAM19A5 epitopes are selected from QLAAGTCEIVTLDR (SEQ ID NO: 89, F1 epitope), TLDRDSSQPRRTIARQTARC (SEQ ID NO: 90, F2 epitope), TARCACRKGQIAGTTRARPA (SEQ ID NO: 91), ARPACVDARIIKTKQW CDML epitope (SEQ ID NO: 92, epitope F4), CDMLPCLEGEGCDLLINRSG (SEQ ID NO: 93, epitope F5) or NRSGWTCTQPGGRIKTTTVS (SEQ ID NO: 94, epitope F6) or a fragment located within the amino acid sequence of SEQ 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. 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 includes 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 acids of any 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, 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 acids of SEQ ID NO: 89, 90, 91, 92, 93 or 94, or any combination thereof. In some embodiments, an anti-FAM19A5 antibody, or an antigen-binding portion thereof, as described herein, binds to any of one or more additional epitopes in their native (ie, undenatured) conformation. In some embodiments, the anti-FAM19A5 antibody, or antigen-binding portion thereof, binds to both glycosylated and non-glycosylated one or more additional FAM19A5 epitopes.

In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that binds to FAM19A5 (e.g., human FAM19A5) with 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% , 65%, 70%, 75%, 80%, 85%, 90%, 95% or higher affinity than the affinity for another protein of the FAM19A family as measured, for example, by immunoassay (e.g. ELISA), surface plasmon resonance or kinetic exclusion analysis. In some embodiments, the anti-FAM19A5 antibody, or antigen-binding fragment thereof, binds to FAM19A5 (eg, human FAM19A5) without cross-reactivity with another FAM19A family protein, as measured, for example, by immunoassay.

In some embodiments, the anti-FAM19A5 antibodies of the present disclosure are not native antibodies or are not naturally occurring antibodies. For example, in some embodiments, anti-FAM19A5 antibodies have post-translational modifications that are different from the antibody modifications that occur naturally, such as having a greater, lesser, or different type of post-translational modification.

The amino acid sequences of the CDRs from the VH and VL of exemplary antibodies of the present invention are shown in Tables 3 and 4, respectively. The amino acid sequences of VH and VL are shown in Tables 5 and 6, respectively.

Table 3. Amino acid sequences of the heavy chain variable region CDRs (identified using IMGT)

Antibody VH-CDR1 VH-CDR2 VH-CDR3 Reference anti-FAM19A5 antibody
("3-2") GFTFSSFNMF (SEQ ID NO: 5) QISSSGSSTNYAPAVRG (SEQ ID NO: 6) SSYDCPYGHCSSGVDSAGEIDA
(SEQ ID NO: 7) 1-7A-IT GFTFSSFNMF (SEQ ID NO: 5) QISSSGSSTNYAPAVKG (SEQ ID NO: 13) SSYDCPYGHCSSGVDSAGEIDA (SEQ ID NO: 7) Low-PI GFDFESFNMF (SEQ ID NO: 14) QISSSEEDENYAPAVKG (SEQ ID NO: 15) SSYDCPYGHCSSGVDSAGEIDA (SEQ ID NO: 7) 1-30 GFDFESFNMF (SEQ ID NO: 14) QISSSEEDENYAPAVKG (SEQ ID NO: 15) SSYDCPYGHCSSGVDSAGEIDA (SEQ ID NO: 7) 1-17 GFDFESFNMF (SEQ ID NO: 14) QISSSEEDENYAPAVKG (SEQ ID NO: 15) SSYDCPYGHCSSGVDSAGEIDA (SEQ ID NO: 7) 1-32 GFDFESFNMF (SEQ ID NO: 14) QISSSEEDENYAPAVKG (SEQ ID NO: 15) SSYDCPYGHCSSGVDSAGEIDA (SEQ ID NO: 7) 4-11 GFDFESFNMF (SEQ ID NO: 14) QISSSEEDENYAPAVKG (SEQ ID NO: 15) SSYDCPYGHCSSGVDSAGEIDA (SEQ ID NO: 7) 6-10 GFDFESFNMF (SEQ ID NO: 14) QISSSEEDENYAPAVKG (SEQ ID NO: 15) SSYDCPYGHCSSGVDSAGEIDA (SEQ ID NO: 7) Reference anti-FAM19A5 antibody
("2-13") GFTFSSHGMF (SEQ ID NO: 16) EITNDGSGTNYGSAVKG (SEQ ID NO: 17) STYECPGGFSCWGDTGQIDA (SEQ ID NO: 18) 2-13D GFTFSSHGMF (SEQ ID NO: 16) EITNDGSGTNYGSAVKG (SEQ ID NO: 17) STYECPGGFSCWGDTGQIDA (SEQ ID NO: 18) 2-13D-37 GFDFSSHGMF (SEQ ID NO: 19) EITNDGSGTNYGSAVKG (SEQ ID NO: 17) STYECPGGFSCWGDTGQIDA (SEQ ID NO: 18) 2-13D-37-1.5W-41 GFDFSSHGMF (SEQ ID NO: 19) EITNDGSGTNYGSAVKG (SEQ ID NO: 17) SSYVCPGGFSCWGDTGQIDA (SEQ ID NO: 128) 2-13D-37-3W-16 GFDFSSHGMF (SEQ ID NO: 19) EITNDGSGTNYGSAVKG (SEQ ID NO: 17) SNYACPGGFSCWGDTGQIDA (SEQ ID NO: 129)

Table 4. Light chain variable region CDR amino acid sequences (identified using IMGT)

Antibody VL-CDR1 VL-CDR2 VL-CDR3 Reference anti-FAM19A5 antibody
("3-2") SGGGSYAGSYYYG (SEQ ID NO: 8) ESNKRPS (SEQ ID NO: 9) GSWDSSNGGI
(SEQ ID NO: 10) 1-7A-IT SGGGSYAGSYYYG (SEQ ID NO: 8) ENNKRPS (SEQ ID NO: 20) GSWDSSNGGI (SEQ ID NO: 10) Low-PI SGGGSEEEQYYYG (SEQ ID NO: 21) EDEERPS (SEQ ID NO: 22) GSWDSEDEDH (SEQ ID NO: 23) 1-30 SGGGSEEEQYYYG (SEQ ID NO: 21) QDEERPS (SEQ ID NO: 24) GSWDSEDEDH (SEQ ID NO: 23) 1-17 SGGGSYAGSYYYG (SEQ ID NO: 8) EDEQRPS (SEQ ID NO: 25) GSWDSEDEDH (SEQ ID NO: 23) 1-32 SGGGSYAGSYYYG (SEQ ID NO: 8) QDEERPS (SEQ ID NO: 24) GSWDSEDEDH (SEQ ID NO: 23) 4-11 SGGGSYAGSYYYG (SEQ ID NO: 8) EDHERPS (SEQ ID NO: 26) GSWDSSDEDH (SEQ ID NO: 27) 6-10 SGGGSYAGSYYYG (SEQ ID NO: 8) QDLLRPS (SEQ ID NO: 28) GSWDSLSSSH (SEQ ID NO: 29) Reference anti-FAM19A5 antibody
("2-13") SGGSYSYG (SEQ ID NO: 30) WDDERPS (SEQ ID NO: 31) GTEDISGTAGV (SEQ ID NO: 32) 2-13D SGGVYSYG (SEQ ID NO: 80) WDDERPS (SEQ ID NO: 31) GTEDISGTAGV (SEQ ID NO: 32) 2-13D-37 SGGVYSYG (SEQ ID NO: 80) WDDERPS (SEQ ID NO: 31) GTEDISGTAGV (SEQ ID NO: 32) 2-13D-37-1.5W-41 SGGVYSYG (SEQ ID NO: 80) WDDERPS (SEQ ID NO: 31) GTEDISGTAGV (SEQ ID NO: 32) 2-13D-37-3W-16 SGGVYSYG (SEQ ID NO: 80) WDDERPS (SEQ ID NO: 31) GTEDISGTAGV (SEQ ID NO: 32)

Table 5. Amino acid sequence of the heavy chain variable region

Antibody VH amino acid sequence (SEQ ID NO) Reference anti-FAM19A5 antibody
("3-2") AVTLDESGGGLQTPGGALSLVCKASGFTFSSFNMFWVRQAPGKGLEYVAQISSSGSSTNYAPAVRGRATISRDNGQSTVRLQLNNPGAEDTGTYYCAKSSYDCPYGHCSSGVDSAGEIDAWGHGTEVIVSS (SEQ ID NO: 11) 1-7A-IT AVTLDESGGGLQTPGGALRLSCKASGFTFSSFNMFWVRQAPGKGLEYVSQISSSGSSTNYAPAVKGRATISRDNGQSTLYLQMNSLRAEDTGTYYCAKSSYDCPYGHCSSGVDSAGEIDAWGHGTEVIVSS (SEQ ID NO: 33) Low-PI AVTLDESGGGLQTPGGALRLSCKASGFDFESFNMFWVRQAPGKGLEYVSQISSSEEDENYAPAVKGRATISRDNGQSTLYLQMNSLRAEDTGTYYCAKSSYDCPYGHCSSGVDSAGEIDAWGHGTEVIVSS (SEQ ID NO: 34) 1-30 AVTLDESGGGLQTPGGALRLSCKASGFDFESFNMFWVRQAPGKGLEYVSQISSSEEDENYAPAVKGRATISRDNGQSTLYLQMNSLRAEDTGTYYCAKSSYDCPYGHCSSGVDSAGEIDAWGHGTEVIVSS (SEQ ID NO: 34) 1-17 AVTLDESGGGLQTPGGALRLSCKASGFDFESFNMFWVRQAPGKGLEYVSQISSSEEDENYAPAVKGRATISRDNGQSTLYLQMNSLRAEDTGTYYCAKSSYDCPYGHCSSGVDSAGEIDAWGHGTEVIVSS (SEQ ID NO: 34) 1-32 AVTLDESGGGLQTPGGALRLSCKASGFDFESFNMFWVRQAPGKGLEYVSQISSSEEDENYAPAVKGRATISRDNGQSTLYLQMNSLRAEDTGTYYCAKSSYDCPYGHCSSGVDSAGEIDAWGHGTEVIVSS (SEQ ID NO: 34) 4-11 AVTLDESGGGLQTPGGALRLSCKASGFDFESFNMFWVRQAPGKGLEYVSQISSSEEDENYAPAVKGRATISRDNGQSTLYLQMNSLRAEDTGTYYCAKSSYDCPYGHCSSGVDSAGEIDAWGHGTEVIVSS (SEQ ID NO: 34) 6-10 AVTLDESGGGLQTPGGALRLSCKASGFDFESFNMFWVRQAPGKGLEYVSQISSSEEDENYAPAVKGRATISRDNGQSTLYLQMNSLRAEDTGTYYCAKSSYDCPYGHCSSGVDSAGEIDAWGHGTEVIVSS (SEQ ID NO: 34) Reference anti-FAM19A5 antibody
("2-13") AVTLDESGGGLQTPGGALSLVCKASGFTFSSHGMFWVRQTPGKGLEYVAEITNDGSGTNYGSAVKGRATISRDNGQSTVRLQLNNLRAEDTGTYFCARSTYECPGGFSCWGDTGQIDAWGHGTEVIVSS (SEQ ID NO: 35) 2-13D AVTLDESGGGLQTPGGALRLSCSASGFTFSSHGMFWVRQAPGKGLEYVSEITNDGSGTNYGSAVKGRATISRDNGQSTLYLQMNSLRAEDTGTYFCARSTYECPGGFSCWGDTGQIDAWGHGTEVIVSS (SEQ ID NO: 36) 2-13D-37 AVTLDESGGGLQTPGGALRLSCSASGFDFSSHGMFWVRQAPGKGLEYVSEITNDGSGTNYGSAVKGRATISRDNGQSTLYLQMNSLRAEDTGTYFCARSTYECPGGFSCWGDTGQIDAWGHGTEVIVSS (SEQ ID NO: 37) 2-13D-37-1.5W-41 AVTLDESGGGLQTPGGALRLSCSASGFDFSSHGMFWVRQAPGKGLEYVSEITNDGSGTNYGSAVKGRATISRDNGQSTLYLQMNSLRAEDTGTYFCARSSYVCPGGFSCWGDTGQIDAWGHGTEVIVSS (SEQ ID NO: 130) 2-13D-37-3W-16 AVTLDESGGGLQTPGGALRLSCSASGFDFSSHGMFWVRQAPGKGLEYVSEITNDGSGTNYGSAVKGRATISRDNGQSTLYLQMNSLRAEDTGTYFCARSNYACPGGFSCWGDTGQIDAWGHGTEVIVSS (SEQ ID NO: 131)

Table 6. Amino acid sequence of the light chain variable region

Antibody VL Amino Acid Sequence (SEQ ID NO) Reference anti-FAM19A5 antibody
("3-2") ALTQPSSVSANPGETVKITCSGGGSYAGSYYYGWYQQKAPGSAPVTLIYESNKRPSDIPSRFSGSTSGSTATLTITGVQADDEAIYYCGSWDSSNGGIFGAGTTLTVL (SEQ ID NO: 12) 1-7A-IT ALTQPSSVSANPGETVKITCSGGGSYAGSYYYGWYQQKPGSAPVTLIYENNKRPSDIPSRFSGSTSGSTATLTITGVQAGDEADYYCGSWDSSNGGIFGAGTTLTVL (SEQ ID NO: 38) Low-PI ALTQPSSVSANPGETVKITCSGGGSEEEQYYYGWYQQKPGSAPVTLIYEDEERPSDIPSRFSGSTSGSTATLTITGVQAGDEADYYCGSWDSEDEDHFGAGTTLTVL (SEQ ID NO: 39) 1-30 ALTQPSSVSANPGETVKITCSGGGSEEEQYYYGWYQQKPGSAPVTLIYQDEERPSDIPSRFSGSTSGSTATLTITGVQAGDEADYYCGSWDSEDEDHFGAGTTLTVL (SEQ ID NO: 40) 1-17 ALTQPSSVSANPGETVKITCSGGGSYAGSYYYGWYQQKPGSAPVTLIYEDEQRPSDIPSRFSGSTSGSTATLTITGVQAGDEADYYCGSWDSEDEDHFGAGTTLTVL (SEQ ID NO: 41) 1-32 ALTQPSSVSANPGETVKITCSGGGSYAGSYYYGWYQQKPGSAPVTLIYQDEERPSDIPSR FSGSTSGSTATLTITGVQAGDEADYYCGSWDSEDEDHFGAGTTLTVL (SEQ ID NO: 42) 4-11 ALTQPSSVSANPGETVKITCSGGGSYAGSYYYGWYQQKPGSAPVTLIYEDHERPSDIPSRFSGSTSGSTATLTITGVQAGDEADYYCGSWDSSDEDHFGAGTTLTVL (SEQ ID NO: 43) 6-10 ALTQPSSVSANPGETVKITCSGGGSYAGSYYYGWYQQKPGSAPVTLIYQDLLRPSDIPSRFSGSTSGSTATLTITGVQAGDEADYYCGSWDSLSSSHFGAGTTLTVL (SEQ ID NO: 44) Reference anti-FAM19A5 antibody
("2-13") ALTQPSSVSANPGETVKITCSGGSYSYGWFQQKSPGSALVTVIYWDDERPSDIPSRFSGALSGSTNTLTITGVQADDEAVYFCGTEDISGTAGVFGAGTTLTVL (SEQ ID NO: 45) 2-13D ALTQPSSVSANPGETAKITCSGGVYSYGWFQQKPGSALVTVIYWDDERPSDIPSRFSGALSGSTNTLTITGVQAEDEADYYCGTEDISGTAGVFGAGTTLTVL (SEQ ID NO: 46) 2-13D-37 ALTQPSSVSANPGETAKITCSGGVYSYGWFQQKPGSALVTVIYWDDERPSDIPSRFSGALSGSTNTLTITGVQAEDEADYYCGTEDISGTAGVFGAGTTLTVL (SEQ ID NO: 46) 2-13D-37-1.5W-41 ALTQPSSVSANPGETAKITCSGGVYSYGWFQQKPGSALVTVIYWDDERPSDIPSRFSGALSGSTNTLTITGVQAEDEADYYCGTEDISGTAGVFGAGTTLTVL (SEQ ID NO: 46) 2-13D-37-3W-16 ALTQPSSVSANPGETAKITCSGGVYSYGWFQQKPGSALVTVIYWDDERPSDIPSRFSGALSGSTNTLTITGVQAEDEADYYCGTEDISGTAGVFGAGTTLTVL (SEQ ID NO: 46)

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

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

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

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

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

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

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

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

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

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

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

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

In some embodiments, the anti-FAM19A5 antibody of the present disclosure comprises VH and VL, where VL includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90% identical, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% of the amino acid sequence specified in SEQ ID NO : 38, 39, 40, 41, 42, 43, 44 or 46.

In some embodiments, the anti-FAM19A5 antibody of the present disclosure contains VH and VL, where:

(a) VH includes the amino acid sequence set forth in SEQ ID NO: 33 and VL includes the amino acid sequence set forth in SEQ ID NO: 38;

(b) VH includes the amino acid sequence shown in SEQ ID NO: 34 and VL includes the amino acid sequence shown in SEQ ID NO: 39;

(c) VH includes the amino acid sequence set forth in SEQ ID NO: 34 and VL includes the amino acid sequence set forth in SEQ ID NO: 41;

(d) VH includes the amino acid sequence set forth in SEQ ID NO: 34 and VL includes the amino acid sequence set forth in SEQ ID NO: 40;

(e) VH includes the amino acid sequence set forth in SEQ ID NO: 34 and VL includes the amino acid sequence set forth in SEQ ID NO: 42;

(f) VH includes the amino acid sequence set forth in SEQ ID NO: 34 and VL includes the amino acid sequence set forth in SEQ ID NO: 43;

(g) VH includes the amino acid sequence set forth in SEQ ID NO: 34 and VL includes the amino acid sequence set forth in SEQ ID NO: 44;

(h) VH includes the amino acid sequence set forth in SEQ ID NO: 36 and VL includes the amino acid sequence set forth in SEQ ID NO: 46;

(i) VH includes the amino acid sequence set forth in SEQ ID NO: 37 and VL includes the amino acid sequence set forth in SEQ ID NO: 46;

(j) VH includes the amino acid sequence set forth in SEQ ID NO: 130 and VL includes the amino acid sequence set forth in SEQ ID NO: 46; or

(k) VH includes the amino acid sequence set out in SEQ ID NO: 131 and VL includes the amino acid sequence set out in SEQ ID NO: 46.

The VH domain, or one or more of its CDRs described herein, may be linked to a constant domain to form a heavy chain, eg, a full length heavy chain. Similarly, a VL domain or one or more of its CDRs described herein can be linked to a constant domain to form a light chain, eg, a full length light chain. The full length heavy chain and full length light chain combine to form a full length antibody.

Accordingly, in specific embodiments, provided herein is an antibody comprising a light chain and a heavy chain of an antibody, eg, separate light chain and heavy chain. With regard to the light chain, in a particular embodiment, the light chain of an antibody described herein is a kappa light chain. In another specific embodiment, the light chain of an antibody described herein is a lambda light chain. In yet another specific embodiment, the light chain of an antibody described herein is a human kappa light chain or a human lambda light chain. In a specific embodiment, an antibody described herein that specifically binds to a FAM19A5 polypeptide (e.g., human FAM19A5) comprises a light chain that contains any of the VL amino acid sequences or CDRs from VL described herein, and wherein the light chain constant region includes the amino acid sequence human kappa light chain constant region. In a specific embodiment, an antibody described herein that specifically binds to a FAM19A5 polypeptide (e.g., human FAM19A5) comprises a light chain that contains the amino acid sequences of VL or CDRs from VL as described herein, and wherein the light chain constant region comprises the amino acid sequence of the constant human lambda light chain region. Non-limiting examples of human constant region sequences have been described in the art, for example, see Patent No. 5693780 and Kabat EA et al. (1991) above.

With respect to the heavy chain, in some embodiments, the heavy chain of an antibody described herein may be an alpha (α), delta (δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In another specific embodiment, the heavy chain of the described antibody may include a human alpha (α), delta (δ), epsilon (ε), gamma (γ) or mu (μ) heavy chain. In one embodiment, an antibody described herein that specifically binds to FAM19A5 (e.g., human FAM19A5) comprises a heavy chain that contains the amino acid sequence of VH or a CDR of VH described herein, and wherein the heavy chain constant region contains the amino acid sequence of constant the gamma (γ) region of the human heavy chain. In another embodiment, an antibody described herein that specifically binds to FAM19A5 (e.g., human FAM19A5) comprises a heavy chain that includes the amino acid sequence of VH or a CDR of the VH described herein, and wherein the heavy chain constant region includes the amino acid sequence of a human heavy chain acid as described herein or known in the art. Non-limiting examples of human constant region sequences have been described in the art, for example, see Patent No. 5693780 and Kabat EA et al. (1991) above.

In some embodiments, an antibody described herein that specifically binds to FAM19A5 (e.g., human FAM19A5) comprises a VL domain and a VH domain containing a VH or CDR from VH and VL and a CDR from VL described herein, and at the same time constant the regions comprise the amino acid sequences of the constant regions of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule or a human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule. In another specific embodiment, an antibody described herein that specifically binds to FAM19A5 (e.g., human FAM19A5) comprises a VL domain and a VH domain containing any of the amino acid sequences described herein, and wherein the constant regions comprise the amino acid sequences of the constant region of an immunoglobulin molecule. IgG, IgE, IgM, IgD, IgA or IgY, any subclass (eg IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) of the immunoglobulin molecule. In some embodiments, the constant regions comprise amino acid sequences of human IgG constant regions that occur naturally, including subclasses (eg, IgG1, IgG2, IgG3, or IgG4) and allotypes (eg, G1m, G2m, G3m, and nG4m) and variants thereof. See, for example, Vidarsson G. et al. Front Immunol. 5:520 (published online Oct 20, 2014) and Jefferis R. and Lefranc MP, mAbs 1:4, 1-7 (2009). In some embodiments, the constant regions comprise the amino acid sequences of human IgG1, IgG2, IgG3, or IgG4 constant regions, or variants thereof.

In some embodiments, the anti-FAM19A5 antibody disclosed herein lacks 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 most proximal to the hinge region in the CH2 domain of the Fc region are responsible for antibody effector functions, since this domain contains a highly overlapping binding site for C1q (complement) and IgG-Fc receptors (FcγR ) on effector cells of the innate immune system. In addition, 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 various approaches known in the art, including (1) the use of antibody fragments lacking an Fc region (eg, such as Fab, F(ab')2, single chain Fv (scFv), or sdAb consisting of a VH or VL monomeric domain); (2) the creation of aglycosylated antibodies, which can be obtained, for example, by removing or changing the residue to which the sugar is attached, by enzymatic removal of sugars, by producing the antibody in cells cultured in the presence of a glycosylation inhibitor, or by expressing the antibody in cells incapable of glycosylation of proteins (for example, bacterial host cells, see, for example, US Pub. No. 20120100140); (3) using Fc regions from the IgG subclass that have reduced effector function (e.g., Fc regions from IgG2 or IgG4 antibodies or a chimeric Fc region containing a CH2 domain from IgG2 or IgG4 antibodies, see, for example, Pub. No. 20120100140 and Lau C. et al., J. Immunol 191: 4769-4777 (2013)); and (4) creating an Fc region with mutations that result in reduced or absent Fc functions. See, for example, pub. US No. 20120100140 and the US and PCT applications cited therein, and An et al., mAbs 1: 6, 572-579 (2009).

Thus, in some embodiments of the invention, the anti-FAM19A5 antibody disclosed herein is a Fab, Fab', F(ab')2, Fv, single chain Fv (scFv), or sdAb consisting of a VH or VL monomeric domain. Such antibody fragments are well known in the art and are described above.

In some embodiments, the anti-FAM19A5 antibody is a single chain Fv. The amino acid sequences of a typical anti-FAM19A5 scFv are shown in Table 7 below.

Table 7. Amino acid sequences of anti-FAM19A5 scFv

ScFv antibody VL Amino Acid Sequence (SEQ ID NO) Reference anti-FAM19A5 antibody
("3-2") ALTQPSSVSANPGETVKITCSGGGSYAGSYYYGWYQQKAPGSAPVTLIYESNKRPSDIPSRFSGSTSGSTATLTITGVQADDEAIYYCGSWDSSNGGIFGAGTTLTVLGQSSRSSGGGGSSGGGGSAVTLDESGGGLQTPGGALSLVCKASGFTFSSFNMFWVRQAPGKGLEYVAQISSSGSSTNYAPAVRGRATISRDNGQSTVRLQLNNPGAEDTGTYYCAKSSYDCPYGHCSSGVDSAGEIDAWGHGTEVIVSS (SEQ ID NO: 47) 1-7A-IT ALTQPSSVSANPGETVKITCSGGGSYAGSYYYGWYQQKPGSAPVTLIYENNKRPSDIPSRFSGSTSGSTATLTITGVQAGDEADYYCGSWDSSNGGIFGAGTTLTVLGQSSRSSGGGGSSGGGGSAVTLDESGGGLQTPGGALRLSCKASGFTFSSFNMFWVRQAPGKGLEYVSQISSSGSSTNYAPAVKGRATISRDNGQSTLYLQMNSLRAEDTGTYYCAKSSYDCPYGHCSSGVDSAGEIDAWGHGTEVIVSS (SEQ ID NO: 48) Low-PI ALTQPSSVSANPGETVKITCSGGGSEEEQYYYGWYQQKPGSAPVTLIYEDEERPSDIPSRFSGSTSGSTATLTITGVQAGDEADYYCGSWDSEDEDHFGAGTTLTVLGQSSRSSGGGGSSGGGGSAVTLDESGGGLQTPGGALRLSCKASGFDFESFNMFWVRQAPGKGLEYVSQISSSEEDENYAPAVKGRATISRDNGQSTLYLQMNSLRAEDTGTYYCAKSSYDCPYGHCSSGVDSAGEIDAWGHGTEVIVSS (SEQ ID NO: 49) 1-30 ALTQPSSVSANPGETVKITCSGGGSEEEQYYYGWYQQKPGSAPVTLIYQDEERPSDIPSRFSGSTSGSTATLTITGVQAGDEADYYCGSWDSEDEDHFGAGTTLTVLGQSSRSSGGGGSSGGGGSAVTLDESGGGLQTPGGALRLSCKASGFDFESFNMFWVRQAPGKGLEYVSQISSSEEDENYAPAVKGRATISRDNGQSTLYLQMNSLRAEDTGTYYCAKSSYDCPYGHCSSGVDSAGEIDAWGHGTEVIVSS (SEQ ID NO: 50) 1-17 ALTQPSSVSANPGETVKITCSGGGSYAGSYYYGWYQQKPGSAPVTLIYEDEQRPSDIPSRFSGSTSGSTATLTITGVQAGDEADYYCGSWDSEDEDHFGAGTTLTVLGQSSRSSGGGGSSGGGGSAVTLDESGGGLQTPGGALRLSCKASGFDFESFNMFWVRQAPGKGLEYVSQISSSEEDENYAPAVKGRATISRDNGQSTLYLQMNSLRAEDTGTYYCAKSSYDCPYGHCSSGVDSAGEIDAWGHGTEVIVSS (SEQ ID NO: 51) 1-32 ALTQPSSVSANPGETVKITCSGGGSYAGSYYYGWYQQKPGSAPVTLIYQDEERPSDIPSR FSGSTSGSTATLTITGVQAGDEADYYCGSWDSEDEDHFGAGTTLTVLGQSSRSSGGGGSSGGGGSAVTLDESGGGLQTPGGALRLSCKASGFDFESFNMFWVRQAPGKGLEYVSQISSSEEDENYAPAVKGRATISRDNGQSTLYLQMNSLRAEDTGTYYCAKSSYDCPYGHCSSGVDSAGEIDAWGHGTEVIVSS (SEQ ID NO: 52) 4-11 ALTQPSSVSANPGETVKITCSGGGSYAGSYYYGWYQQKPGSAPVTLIYEDHERPSDIPSRFSGSTSGSTATLTITGVQAGDEADYYCGSWDSSDEDHFGAGTTLTVLGQSSRSSGGGGSSGGGGSAVTLDESGGGLQTPGGALRLSCKASGFDFESFNMFWVRQAPGKGLEYVSQISSSEEDENYAPAVKGRATISRDNGQSTLYLQMNSLRAEDTGTYYCAKSSYDCPYGHCSSGVDSAGEIDAWGHGTEVIVSS (SEQ ID NO: 53) 6-10 ALTQPSSVSANPGETVKITCSGGGSYAGSYYYGWYQQKPGSAPVTLIYQDLLRPSDIPSRFSGSTSGSTATLTITGVQAGDEADYYCGSWDSLSSSHFGAGTTLTVLGQSSRSSGGGGSSGGGGSAVTLDESGGGLQTPGGALRLSCKASGFDFESFNMFWVRQAPGKGLEYVSQISSSEEDENYAPAVKGRATISRDNGQSTLYLQMNSLRAEDTGTYYCAKSSYDCPYGHCSSGVDSAGEIDAWGHGTEVIVSS (SEQ ID NO: 54) Reference anti-FAM19A5 antibody
("2-13") ALTQPSSVSANPGETVKITCSGGSYSYGWFQQKSPGSALVTVIYWDDERPSDIPSRFSGALSGSTNTLTITGVQADDEAVYFCGTEDISGTAGVFGAGTTLTVLGQSSRSSGGGGSSGGGGSAVTLDESGGGLQTPGGALSLVCKASGFTFSSHGMFWVRQTPGKGLEYVAEITNDGSGTNYGSAVKGRATISRDNGQSTVRLQLNNLRAEDTGTYFCARSTYECPGGFSCWGDTGQIDAWGHGTEVIVSS (SEQ ID NO: 55) 2-13D ALTQPSSVSANPGETAKITCSGGVYSYGWFQQKPGSALVTVIYWDDERPSDIPSRFSGALSGSTNTLTITGVQAEDEADYYCGTEDISGTAGVFGAGTTLTVLGQSSRSSGGGGSSGGGGSAVTLDESGGGLQTPGGALRLSCSASGFTFSSHGMFWVRQAPGKGLEYVSEITNDGSGTNYGSAVKGRATISRDNGQSTLYLQMNSLRAEDTGTYFCARSTYECPGGFSCWGDTGQIDAWGHGTEVIVSS (SEQ ID NO: 56) 2-13D-37 ALTQPSSVSANPGETAKITCSGGVYSYGWFQQKPGSALVTVIYWDDERPSDIPSRFSGALSGSTNTLTITGVQAEDEADYYCGTEDISGTAGVFGAGTTLTVLGQSSRSSGGGGSSGGGGSAVTLDESGGGLQTPGGALRLSCSASGFDFSSHGMFWVRQAPGKGLEYVSEITNDGSGTNYGSAVKGRATISRDNGQSTLYLQMNSLRAEDTGTYFCARSTYECPGGFSCWGDTGQIDAWGHGTEVIVSS (SEQ ID NO: 57) 2-13D-37-1.5W-41 ALTQPSSVSANPGETAKITCSGGVYSYGWFQQKPGSALVTVIYWDDERPSDIPSRFSGALSGSTNTLTITGVQAEDEADYYCGTEDISGTAGVFGAGTTLTVLGQSSRSSGGGGSSGGGGSAVTLDESGGGLQTPGGALRLSCSASGFDFSSHGMFWVRQAPGKGLEYVSEITNDGSGTNYGSAVKGRATISRDNGQSTLYLQMNSLRAEDTGTYFCARSSYVCPGGFSCWGDTGQIDAWGHGTEVIVSS (SEQ ID NO: 132) 2-13D-37-3W-16 ALTQPSSVSANPGETAKITCSGGVYSYGWFQQKPGSALVTVIYWDDERPSDIPSRFSGALSGSTNTLTITGVQAEDEADYYCGTEDISGTAGVFGAGTTLTVLGQSSRSSGGGGSSGGGGSAVTLDESGGGLQTPGGALRLSCSASGFDFSSHGMFWVRQAPGKGLEYVSEITNDGSGTNYGSAVKGRATISRDNGQSTLYLQMNSLRAEDTGTYFCARSNYACPGGFSCWGDTGQIDAWGHGTEVIVSS (SEQ ID NO: 133)

In some embodiments of the invention, the anti-FAM19A5 antibody disclosed herein contains an Fc region with or without reduced Fc effector function. In some embodiments, the constant regions comprise the amino acid sequences of the human IgG2 or IgG4 Fc region, in some embodiments, the anti-FAM19A5 antibody is of the IgG2/IgG4 isotype. In some embodiments, the anti-FAM19A5 antibody comprises a chimeric Fc region that includes a CH2 domain from an IgG antibody of the IgG4 isotype and a CH3 domain from an IgG antibody of the IgG1 isotype, or a chimeric Fc region that includes a hinge region from IgG2 and CH2 regions from IgG4 or Fc- areas with mutations that result in decreased or absent Fc function. Fc regions with or without reduced Fc effector function include regions known in the art. See, for example, Lau C. et al., J. Immunol. K191: 4769-4777 (2013); An et al., MAbs 1: 6, 572-579 (2009); and publ. US No. 20120100140 and US Patents and Publications and PCT Publications cited therein. Also, Fc regions with reduced or no Fc effector function can be easily created by one of skill in the art.

III. Nucleic Acid Molecules

Another aspect described herein refers to one or more nucleic acid molecules that encode any of the antibodies described herein. The nucleic acids may be present in whole cells, in a cell lysate, or in partially purified or substantially pure form. A nucleic acid is "isolated" or "substantially pure" when purified from other cellular components or other contaminants, such as other cellular nucleic acids (eg, other chromosomal DNA, such as chromosomal DNA that is naturally associated with isolated DNA) or proteins standard methods, including treatment with alkali/SDS, separation in CsCl, column chromatography, restriction enzymes, agarose gel electrophoresis, and others well known in the art. See F. Ausubel, et al., Ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. The nucleic acid described herein may be, for example, DNA or RNA, and may or may not contain intron sequences. In some embodiments, the nucleic acid is a cDNA molecule.

Described in this document, the nucleic acids can be obtained using standard methods of molecular biology. For antibodies expressed by hybridomas (e.g., hybridomas derived from transgenic mice carrying human immunoglobulin genes as described below), cDNAs encoding the light and heavy chains of the antibody produced by the hybridoma can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies derived from an immunoglobulin gene library (eg, using phage display techniques), the nucleic acid encoding the antibody can be retrieved from the library.

Certain nucleic acid molecules described herein encode the VH and VL sequences of various anti-FAM19A5 antibodies of the present invention. Exemplary DNA sequences encoding the VH and VL sequences of such antibodies are shown in Tables 8 and 9, respectively.

Table 8. Heavy chain variable region polynucleotide sequence

Antibody Heavy chain variable region polynucleotide sequence (SEQ ID NO) Reference anti-FAM19A5 antibody
("3-2") GCCGTGACGTTGGACGAGTCCGGGGGCGGCCTCCAGACGCCCGGAGGAGCGCTCAGCCTCGTCTGCAAGGCCTCCGGGTTCACCTTCAGCAGCTTCAACATGTTCTGGGTGCGACAGGCGCCCGGCAAGGGGCTGGAATACGTCGCTCAAATTAGCAGCAGTGGTAGTAGCACAAACTACGCACCCGCGGTGAGGGGCCGTGCCACCATCTCGAGGGACAACGGGCAGAGCACAGTGAGGCTGCAGCTGAACAACCCCGGGGCTGAAGACACCGGCACCTACTACTGCGCCAAAAGTAGTTATGACTGTCCTTACGGTCATTGTAGTAGTGGTGTTGATAGTGCTGGTGAGATCGACGCATGGGGCCACGGGACCGAAGTCATCGTCTCCTCC (SEQ ID NO: 58) 1-7A-IT GCCGTGACGTTGGATGAATCCGGGGGCGGCCTCCAGACGCCCGGAGGAGCGCTCCGCCTCAGCTGCAAGGCCTCTGGGTTCACCTTCAGCAGCTTCAACATGTTCTGGGTGCGACAGGCGCCCGGCAAGGGGCTGGAATACGTCTCGCAGATTAGCAGCAGTGGTAGTAGCACAAACTACGCACCCGCGGTGAAAGGCCGTGCCACCATCTCGAGGGACAACGGGCAGAGCACACTGTATCTGCAGATGAACAGCCTGCGCGCTGAAGACACCGGCACCTACTACTGCGCCAAAAGTAGTTATGACTGTCCTTACGGTCATTGTAGTAGTGGTGTTGATAGTGCTGGTGAGATCGACGCATGGGGCCACGGGACCGAAGTCATCGTCTCCTCC (SEQ ID NO: 59) Low-PI GCCGTGACGTTGGATGAATCCGGGGGCGGCCTCCAGACGCCCGGAGGAGCGCTCCGCCTCAGCTGCAAGGCCTCTGGGTTCACCTTCAGCAGCTTCAACATGTTCTGGGTGCGACAGGCGCCCGGCAAGGGGCTGGAATACGTCTCGCAGATTAGCAGCAGTGGTAGTAGCACAAACTACGCACCCGCGGTGAAAGGCCGTGCCACCATCTCGAGGGACAACGGGCAGAGCACACTGTATCTGCAGATGAACAGCCTGCGCGCTGAAGACACCGGCACCTACTACTGCGCCAAAAGTAGTTATGACTGTCCTTACGGTCATTGTAGTAGTGGTGTTGATAGTGCTGGTGAGATCGACGCATGGGGCCACGGGACCGAAGTCATCGTCTCCTCC (SEQ ID NO: 60) 1-30 GCCGTGACGTTGGATGAATCCGGGGGCGGCCTCCAGACGCCCGGAGGAGCGCTCCGCCTCAGCTGCAAGGCCTCTGGCTTTGATTTTGAAAGCTTCAACATGTTCTGGGTGCGACAGGCGCCCGGCAAGGGGCTGGAATACGTCTCGCAGATTAGCAGCAGTGAAGAAGATGAAAACTACGCACCCGCGGTGAAAGGCCGTGCCACCATCTCGAGGGACAACGGGCAGAGCACACTGTATCTGCAGATGAACAGCCTGCGCGCTGAAGACACCGGCACCTACTACTGCGCCAAAAGTAGTTATGACTGTCCTTACGGTCATTGTAGTAGTGGTGTTGATAGTGCTGGTGAGATCGACGCATGGGGCCACGGGACCGAAGTCATCGTCTCCTCC (SEQ ID NO: 61) 1-17 GCCGTGACGTTGGATGAATCCGGGGGCGGCCTCCAGACGCCCGGAGGAGCGCTCCGCCTCAGCTGCAAGGCCTCTGGCTTTGATTTTGAAAGCTTCAACATGTTCTGGGTGCGACAGGCGCCCGGCAAGGGGCTGGAATACGTCTCGCAGATTAGCAGCAGTGAAGAAGATGAAAACTACGCACCCGCGGTGAAAGGCCGTGCCACCATCTCGAGGGACAACGGGCAGAGCACACTGTATCTGCAGATGAACAGCCTGCGCGCTGAAGACACCGGCACCTACTACTGCGCCAAAAGTAGTTATGACTGTCCTTACGGTCATTGTAGTAGTGGTGTTGATAGTGCTGGTGAGATCGACGCATGGGGCCACGGGACCGAAGTCATCGTCTCCTCC (SEQ ID NO: 62) 1-32 GCCGTGACGTTGGATGAATCCGGGGGCGGCCTCCAGACGCCCGGAGGAGCGCTCCGCCTCAGCTGCAAGGCCTCTGGCTTTGATTTTGAAAGCTTCAACATGTTCTGGGTGCGACAGGCGCCCGGCAAGGGGCTGGAATACGTCTCGCAGATTAGCAGCAGTGAAGAAGATGAAAACTACGCACCCGCGGTGAAAGGCCGTGCCACCATCTCGAGGGACAACGGGCAGAGCACACTGTATCTGCAGATGAACAGCCTGCGCGCTGAAGACACCGGCACCTACTACTGCGCCAAAAGTAGTTATGACTGTCCTTACGGTCATTGTAGTAGTGGTGTTGATAGTGCTGGTGAGATCGACGCATGGGGCCACGGGACCGAAGTCATCGTCTCCTCC (SEQ ID NO: 63) 4-11 GCCGTGACGTTGGATGAATCCGGGGGCGGCCTCCAGACGCCCGGAGGAGCGCTCCGCCTCAGCTGCAAGGCCTCTGGCTTTGATTTTGAAAGCTTCAACATGTTCTGGGTGCGACAGGCGCCCGGCAAGGGGCTGGAATACGTCTCGCAGATTAGCAGCAGTGAAGAAGATGAAAACTACGCACCCGCGGTGAAAGGCCGTGCCACCATCTCGAGGGACAACGGGCAGAGCACACTGTATCTGCAGATGAACAGCCTGCGCGCTGAAGACACCGGCACCTACTACTGCGCCAAAAGTAGTTATGACTGTCCTTACGGTCATTGTAGTAGTGGTGTTGATAGTGCTGGTGAGATCGACGCATGGGGCCACGGGACCGAAGTCATCGTCTCCTCC (SEQ ID NO: 64) 6-10 GCCGTGACGTTGGATGAATCCGGGGGCGGCCTCCAGACGCCCGGAGGAGCGCTCCGCCTCAGCTGCAAGGCCTCTGGCTTTGATTTTGAAAGCTTCAACATGTTCTGGGTGCGACAGGCGCCCGGCAAGGGGCTGGAATACGTCTCGCAGATTAGCAGCAGTGAAGAAGATGAAAACTACGCACCCGCGGTGAAAGGCCGTGCCACCATCTCGAGGGACAACGGGCAGAGCACACTGTATCTGCAGATGAACAGCCTGCGCGCTGAAGACACCGGCACCTACTACTGCGCCAAAAGTAGTTATGACTGTCCTTACGGTCATTGTAGTAGTGGTGTTGATAGTGCTGGTGAGATCGACGCATGGGGCCACGGGACCGAAGTCATCGTCTCCTCC (SEQ ID NO: 65) Reference anti-FAM19A5 antibody
("2-13") GCCGTGACGTTGGACGAGTCCGGGGGCGGCCTCCAGACGCCCGGAGGAGCGCTCAGCCTCGTCTGCAAGGCCTCCGGGTTCACCTTCAGCAGCCATGGCATGTTCTGGGTGCGACAGACGCCCGGCAAGGGGTTGGAATATGTCGCTGAAATTACCAATGATGGTAGTGGCACAAACTACGGGTCGGCGGTGAAGGGCCGTGCCACCATCTCGAGGGACAACGGGCAGAGCACAGTGAGGCTGCAGCTGAACAACCTCAGGGCTGAGGACACCGGCACCTACTTCTGCGCCAGATCTACTTATGAATGTCCTGGTGGTTTTAGTTGTTGGGGTGATACTGGTCAAATAGACGCATGGGGCCACGGGACCGAAGTCATCGTCTCCTCC (SEQ ID NO: 66) 2-13D GCCGTGACGTTGGACGAGTCCGGGGGCGGCCTCCAGACGCCCGGAGGAGCGCTCCGCCTTAGCTGCAGCGCCTCCGGGTTCACCTTCAGCAGCCATGGCATGTTCTGGGTGCGACAGGCGCCCGGCAAGGGGTTGGAATATGTCTCGGAGATTACCAATGATGGTAGTGGCACAAACTACGGGTCGGCGGTGAAGGGCCGTGCCACCATCTCGAGGGACAACGGGCAGAGCACACTGTATCTGCAGATGAACAGCCTCAGGGCTGAGGACACCGGCACCTACTTCTGCGCCAGATCTACTTATGAATGTCCTGGTGGTTTTAGTTGTTGGGGTGATACTGGTCAAATAGACGCATGGGGCCACGGGACCGAAGTCATCGTCTCCTCC (SEQ ID NO: 67) 2-13D-37 GCCGTGACGTTGGACGAGTCCGGGGGCGGCCTCCAGACGCCCGGAGGAGCGCTCCGCCTTAGCTGCAGCGCCTCCGGGTTCGATTTCAGCAGCCATGGCATGTTCTGGGTGCGACAGGCGCCCGGCAAGGGGTTGGAATATGTCTCGGAGATTACCAATGATGGTAGTGGCACAAACTACGGGTCGGCGGTGAAGGGCCGTGCCACCATCTCGAGGGACAACGGGCAGAGCACACTGTATCTGCAGATGAACAGCCTCAGGGCTGAGGACACCGGCACCTACTTCTGCGCCAGATCTACTTATGAATGTCCTGGTGGTTTTAGTTGTTGGGGTGATACTGGTCAAATAGACGCATGGGGCCACGGGACCGAAGTCATCGTCTCCTCC (SEQ ID NO: 68) 2-13D-37-1.5W-41 GCCGTGACGTTGGACGAGTCCGGGGGCGGCCTCCAGACGCCCGGAGGAGCGCTCCGCCTTAGCTGCAGCGCCTCCGGGTTCGATTTCAGCAGCCATGGCATGTTCTGGGTGCGACAGGCGCCCGGCAAGGGGTTGGAATATGTCTCGGAGATTACCAATGATGGTAGTGGCACAAACTACGGGTCGGCGGTGAAGGGCCGTGCCACCATCTCGAGGGACAACGGGCAGAGCACACTGTATCTGCAGATGAACAGCCTCAGGGCTGAGGACACCGGCACCTACTTCTGCGCCAGATCTTCTTATGTTTGTCCTGGTGGTTTTAGTTGTTGGGGTGATACTGGTCAAATAGACGCATGGGGCCACGGGACCGAAGTCATCGTCTCCTCC (SEQ ID NO: 134) 2-13D-37-3W-16 GCCGTGACGTTGGACGAGTCCGGGGGCGGCCTCCAGACGCCCGGAGGAGCGCTCCGCCTTAGCTGCAGCGCCTCCGGGTTCGATTTCAGCAGCCATGGCATGTTCTGGGTGCGACAGGCGCCCGGCAAGGGGTTGGAATATGTCTCGGAGATTACCAATGATGGTAGTGGCACAAACTACGGGTCGGCGGTGAAGGGCCGTGCCACCATCTCGAGGGACAACGGGCAGAGCACACTGTATCTGCAGATGAACAGCCTCAGGGCTGAGGACACCGGCACCTACTTCTGCGCCAGATCTAATTATGCTTGTCCTGGTGGTTTTAGTTGTTGGGGTGATACTGGTCAAATAGACGCATGGGGCCACGGGACCGAAGTCATCGTCTCCTCC (SEQ ID NO: 135)

Table 9 Variable Light Chain Polynucleotide Sequence

Antibody Light chain variable region polynucleotide sequence (SEQ ID NO) Reference anti-FAM19A5 antibody
("3-2") GCCCTGACTCAGCCGTCCTCGGTGTCAGCAAACCCAGGAGAAACCGTCAAGATCACCTGCTCCGGGGGTGGCAGCTATGCTGGAAGTTACTATTATGGCTGGTACCAGCAGAAGGCACCTGGCAGTGCCCCTGTCACTCTGATCTATGAAAGCAACAAGAGACCCTCGGACATCCCTTCACGATTCTCCGGTTCCACATCTGGCTCCACAGCCACACTAACCATCACTGGGGTCCAAGCCGATGACGAGGCTATCTATTACTGTGGGAGCTGGGACAGTAGCAATGGTGGTATATTTGGGGCCGGGACAACCCTGACCGTCCTA (SEQ ID NO: 69) 1-7A-IT GCCCTGACTCAGCCGTCCTCGGTGTCAGCAAACCCAGGAGAAACCGTCAAGATCACCTGCTCCGGGGGTGGCAGCTATGCTGGAAGTTACTATTATGGCTGGTATCAGCAGAAGCCTGGCAGTGCCCCTGTCACTCTGATCTATGAAAACAACAAGAGACCCTCGGACATCCCTTCACGATTCTCCGGTTCCACATCTGGCTCCACAGCCACACTAACCATCACTGGGGTCCAAGCCGGCGACGAGGCTGATTATTACTGTGGGAGCTGGGACAGTAGCAATGGTGGTATATTTGGGGCCGGGACAACCCTGACCGTCCTA (SEQ ID NO: 70) Low-PI GCCCTGACTCAGCCGTCCTCGGTGTCAGCAAACCCAGGAGAAACCGTCAAGATCACCTGCTCCGGGGGTGGCAGCTATGCTGGAAGTTACTATTATGGCTGGTATCAGCAGAAGCCTGGCAGTGCCCCTGTCACTCTGATCTATGAAAACAACAAGAGACCCTCGGACATCCCTTCACGATTCTCCGGTTCCACATCTGGCTCCACAGCCACACTAACCATCACTGGGGTCCAAGCCGGCGACGAGGCTGATTATTACTGTGGGAGCTGGGACAGTAGCAATGGTGGTATATTTGGGGCCGGGACAACCCTGACCGTCCTA (SEQ ID NO: 71) 1-30 GCCCTGACTCAGCCGTCCTCGGTGTCAGCAAACCCAGGAGAAACCGTCAAGATCACCTGCTCCGGGGGTGGCAGCGAAGAAGAACAGTACTATTATGGCTGGTATCAGCAGAAGCCTGGCAGTGCCCCTGTCACTCTGATCTATCAGGATGAAGAAAGACCCTCGGACATCCCTTCACGATTCTCCGGTTCCACATCTGGCTCCACAGCCACACTAACCATCACTGGGGTCCAAGCCGGCGACGAGGCTGATTATTACTGTGGGAGCTGGGACAGTGAAGATGAAGATCATTTTGGGGCCGGGACAACCCTGACCGTCCTA (SEQ ID NO: 72) 1-17 GCCCTGACTCAGCCGTCCTCGGTGTCAGCAAACCCAGGAGAAACCGTCAAGATCACCTGCTCCGGGGGTGGCAGCTATGCTGGAAGTTACTATTATGGCTGGTATCAGCAGAAGCCTGGCAGTGCCCCTGTCACTCTGATCTATGAAGATGAACAGAGACCCTCGGACATCCCTTCACGATTCTCCGGTTCCACATCTGGCTCCACAGCCACACTAACCATCACTGGGGTCCAAGCCGGCGACGAGGCTGATTATTACTGTGGGAGCTGGGACAGTGAAGATGAAGATCATTTTGGGGCCGGGACAACCCTGACCGTCCTA (SEQ ID NO: 73) 1-32 GCCCTGACTCAGCCGTCCTCGGTGTCAGCAAACCCAGGAGAAACCGTCAAGATCACCTGCTCCGGGGGTGGCAGCTATGCTGGAAGTTACTATTATGGCTGGTATCAGCAGAAGCCTGGCAGTGCCCCTGTCACTCTGATCTATCAGGATGAAGAAAGACCCTCGGACATCCCTTCACGATTCTCCGGTTCCACATCTGGCTCCACAGCCACACTAACCATCACTGGGGTCCAAGCCGGCGACGAGGCTGATTATTACTGTGGGAGCTGGGACAGTGAAGATGAAGATCATTTTGGGGCCGGGACAACCCTGACCGTCCTA (SEQ ID NO: 74) 4-11 GCCCTGACTCAGCCGTCCTCGGTGTCAGCAAACCCAGGAGAAACCGTCAAGATCACCTGCTCCGGGGGTGGCAGCTATGCTGGAAGTTACTATTATGGCTGGTATCAGCAGAAGCCTGGCAGTGCCCCTGTCACTCTGATCTATGAAGACCACGAGAGACCCTCGGACATCCCTTCACGATTCTCCGGTTCCACATCTGGCTCCACAGCCACACTAACCATCACTGGGGTCCAAGCCGGCGACGAGGCTGATTATTACTGTGGGAGCTGGGACAGTAGCGATGAAGATCATTTTGGGGCCGGGACAACCCTGACCGTCCTA (SEQ ID NO: 75) 6-10 GCCCTGACTCAGCCGTCCTCGGTGTCAGCAAACCCAGGAGAAACCGTCAAGATCACCTGCTCCGGGGGTGGCAGCTATGCTGGAAGTTACTATTATGGCTGGTATCAGCAGAAGCCTGGCAGTGCCCCTGTCACTCTGATCTATCAGGATCTGCTGAGACCCTCGGACATCCCTTCACGATTCTCCGGTTCCACATCTGGCTCCACAGCCACACTAACCATCACTGGGGTCCAAGCCGGCGACGAGGCTGATTATTACTGTGGGAGCTGGGACAGTCTGAGCAGCAGCCATTTTGGGGCCGGGACAACCCTGACCGTCCTA (SEQ ID NO: 76) Reference anti-FAM19A5 antibody
("2-13") GCCCTGACTCAGCCGTCCTCGGTGTCAGCAAACCCAGGAGAAACCGTCAAGATAACCTGCTCCGGGGGTAGCTATAGCTATGGCTGGTTCCAGCAGAAGTCTCCTGGCAGTGCCCTTGTCACTGTGATCTACTGGGATGATGAGAGACCCTCGGACATCCCTTCACGATTCTCCGGTGCCCTATCCGGCTCCACAAACACATTAACCATCACTGGGGTCCAAGCCGACGACGAGGCTGTCTATTTCTGTGGGACTGAAGACATCAGCGGCACTGCTGGTGTATTTGGGGCCGGGACAACCCTGACCGTCCTG (SEQ ID NO: 77) 2-13D GCCCTGACTCAGCCGTCCTCGGTGTCAGCAAACCCAGGAGAAACCGCGAAGATAACCTGCTCCGGGGGTGTGTATAGCTATGGCTGGTTCCAGCAGAAGCCTGGCAGTGCCCTTGTCACTGTGATCTACTGGGATGATGAGAGACCCTCGGACATCCCTTCACGATTCTCCGGTGCCCTATCCGGCTCCACAAACACATTAACCATCACTGGGGTCCAAGCCGAAGACGAGGCTGATTATTATTGTGGGACTGAAGACATCAGCGGCACTGCTGGTGTATTTGGGGCCGGGACAACCCTGACCGTCCTG (SEQ ID NO: 78) 2-13D-37 GCCCTGACTCAGCCGTCCTCGGTGTCAGCAAACCCAGGAGAAACCGCGAAGATAACCTGCTCCGGGGGTGTGTATAGCTATGGCTGGTTCCAGCAGAAGCCTGGCAGTGCCCTTGTCACTGTGATCTACTGGGATGATGAGAGACCCTCGGACATCCCTTCACGATTCTCCGGTGCCCTATCCGGCTCCACAAACACATTAACCATCACTGGGGTCCAAGCCGAAGACGAGGCTGATTATTATTGTGGGACTGAAGACATCAGCGGCACTGCTGGTGTATTTGGGGCCGGGACAACCCTGACCGTCCTG (SEQ ID NO: 79) 2-13D-37-1.5W-41 GCCCTGACTCAGCCGTCCTCGGTGTCAGCAAACCCAGGAGAAACCGCGAAGATAACCTGCTCCGGGGGTGTGTATAGCTATGGCTGGTTCCAGCAGAAGCCTGGCAGTGCCCTTGTCACTGTGATCTACTGGGATGATGAGAGACCCTCGGACATCCCTTCACGATTCTCCGGTGCCCTATCCGGCTCCACAAACACATTAACCATCACTGGGGTCCAAGCCGAAGACGAGGCTGATTATTATTGTGGGACTGAAGACATCAGCGGCACTGCTGGTGTATTTGGGGCCGGGACAACCCTGACCGTCCTG (SEQ ID NO: 136) 2-13D-37-3W-16 GCCCTGACTCAGCCGTCCTCGGTGTCAGCAAACCCAGGAGAAACCGCGAAGATAACCTGCTCCGGGGGTGTGTATAGCTATGGCTGGTTCCAGCAGAAGCCTGGCAGTGCCCTTGTCACTGTGATCTACTGGGATGATGAGAGACCCTCGGACATCCCTTCACGATTCTCCGGTGCCCTATCCGGCTCCACAAACACATTAACCATCACTGGGGTCCAAGCCGAAGACGAGGCTGATTATTATTGTGGGACTGAAGACATCAGCGGCACTGCTGGTGTATTTGGGGCCGGGACAACCCTGACCGTCCTG (SEQ ID NO: 137)

The method disclosed herein for producing an anti-FAM19A5 antibody may include expressing the heavy chain and light chains in a cell line containing nucleotide sequences encoding the heavy and light chains using a signal peptide. Host cells containing these nucleotide sequences are included herein.

Once the DNA fragments encoding the VH and VL segments have been obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, e.g., to convert variable region genes to full-length antibody chain genes, Fab fragment genes, or scFv genes. In these manipulations, a DNA fragment encoding a VL or VH is operably linked to another DNA fragment encoding a different protein, such as an antibody constant region or a flexible linker. The term "operably linked" as used herein is intended to mean that two DNA fragments are connected in such a way that the amino acid sequences encoded by the two DNA fragments remain in reading frame.

The isolated DNA encoding the VH region can be converted into a full length heavy chain gene by operably linking the DNA encoding the VH to another DNA molecule encoding the heavy chain constant regions (hinge, CH1, CH2 and/or CH3). Human heavy chain constant region gene sequences are known in the art (see, e.g., Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91 -3242) and DNA fragments covering these regions can be obtained using standard PCR amplification. The heavy chain constant region may be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, e.g., an IgG2 and/or IgG 4 constant region. linked to another DNA molecule encoding only the CH1 constant region of the heavy chain.

An isolated DNA encoding a VL region can be converted into a full-length light chain gene (as well as a Fab light chain gene) by operably linking the DNA encoding VL to another DNA molecule encoding a light chain constant region, CL. Human light chain constant region gene sequences are known in the art (see, e.g., Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91 -3242) and DNA fragments covering these regions can be obtained using standard PCR amplification. The light chain constant region may be a kappa or lambda constant region.

To create the scFv gene, DNA fragments encoding VH and VL are operably linked to another fragment encoding a flexible linker, for example encoding the amino acid sequence (Gly4-Ser) 3, so that the VH and VL sequences can be expressed. as a continuous single chain protein, with the VL and VH regions connected 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). Amino acid sequences of exemplary anti-FAM19A5 scFvs are shown in Tables 7.

In some embodiments, the present invention provides a vector containing an isolated nucleic acid molecule containing a nucleotide sequence encoding an antibody. In other embodiments, the vectors can be used for gene therapy.

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

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

Expression vectors as described herein may include polynucleotides encoding an antibody or as described herein. In one embodiment, the antibody coding sequences are operably linked to an expression control sequence. In this context, two nucleic acid sequences are operably linked when they are covalently linked in such a way as to allow each component nucleic acid sequence to retain its functionality. A coding sequence and a gene expression control sequence are considered to be operably linked if they are covalently linked in such a way as to allow expression or transcription and/or translation of the coding sequence under the influence or control of the gene expression control sequence. Two DNA sequences are considered operably linked if induction of a promoter in the 5' gene expression sequence results in transcription of the coding sequence and if the nature of the linkage between the two DNA sequences (1) does not introduce a frameshift mutation, (2) does not interfere with the ability of the promoter region to direct transcription of the coding sequence, or (3) does not interfere with the ability of the corresponding RNA transcript to be translated into protein. Thus, a gene expression sequence would be operably linked to a nucleic acid coding sequence if the gene expression sequence were capable of influencing the transcription of that nucleic acid coding sequence such that the resulting transcript was translated into the antibody of interest.

Viral vectors include, but are not limited to, nucleic acid sequences from the following viruses: a retrovirus such as Moloney mouse leukemia virus, Harvey mouse sarcoma virus, mouse mammary tumor virus, and Rous sarcoma virus; lentivirus; adenovirus; adeno-associated virus; viruses of the SV40 type; polyomaviruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; poliomyelitis virus; and an RNA virus such as a retrovirus. Other vectors well known in the art can be used without problems. Some viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes are replaced by a gene of interest. Non-cytopathic viruses include retroviruses whose life cycle involves reverse transcription of viral genomic RNA into DNA followed by proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy clinical trials. The most useful are those retroviruses that are replication-defective (ie, able to direct the synthesis of the desired proteins, but unable to create an infectious particle). Such genetically modified retroviral expression vectors are of general use for highly efficient in vivo gene transduction. Standard protocols for the production of replication-deficient retroviruses (including the steps of incorporating exogenous genetic material into a plasmid, transfecting a packaging cell line with a plasmid, generating recombinant retroviruses with a packaging cell line, harvesting viral particles from tissue culture media, and infecting target cells with viral particles) are presented in Kriegler. , M., Gene Transfer and Expression, A Laboratory Manual, W. H. Freeman Co., New York (1990) and Murry, E. J., Methods in Molecular Biology, Vol. 7, Humana Press, Inc., Cliffton, N.J. (1991).

In one embodiment, the virus is an adeno-associated virus, a double-stranded DNA virus. Adeno-associated virus can be engineered to be replication defective and is capable of infecting a wide range of cell types and species. In addition, this virus has advantages such as resistance to heat and lipid solvents; high transduction frequencies in various cell lines, including hematopoietic cells; and no suppression of superinfection, allowing for multiple series of transductions. Adeno-associated virus reportedly can be integrated into human cellular DNA in a site-specific manner; thus minimizing the likelihood of insertion mutagenesis and the variability in expression of the inserted genes characteristic of retroviral infection. In addition, wild-type adeno-associated virus infections were monitored in tissue culture for more than 100 passages in the absence of selective pressure, confirming that adeno-associated virus genomic integration is a relatively stable event. Adeno-associated virus can also act extrachromasomally.

In other embodiments, the vector is derived from a lentivirus. In some embodiments, the vector is a recombinant lentivirus vector capable of infecting non-dividing cells.

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

Next to the 5' LTR are the sequences required for reverse transcription of the genome (tRNA primer binding site) and for efficient encapsidation of viral RNA into particles (Psi site). If the sequences required for encapsidation (or packaging of retroviral RNA into infectious virions) are not present in the viral genome, the cis defect prevents encapsidation of the genomic RNA.

However, the resulting mutant remains capable of controlling the synthesis of all virion proteins. The description provides a method for obtaining a recombinant lentivirus capable of infecting a non-dividing cell, comprising transfection of a suitable host cell with two or more vectors that carry packaging functions, namely gag, pol and env, as well as rev and tat. As will be disclosed herein below, vectors lacking a functional tat gene are desirable for certain applications. Thus, for example, a first vector may provide a nucleic acid encoding a viral gag and a viral pol, and another vector may provide a nucleic acid encoding a viral env to obtain a packaging cell. Introduction of a vector providing a heterologous gene, herein referred to as a transfer vector, into this packaging cell results in a producer cell that releases infectious viral particles carrying the foreign gene of interest.

According to the above configuration of vectors and foreign genes, the second vector may provide a nucleic acid encoding a viral envelope (env) gene. The env gene can be derived from virtually any suitable virus, including retroviruses. In some embodiments, the env protein is an amphotropic envelope protein that allows transduction of human and non-human cells.

Examples of env genes derived from retroviruses include, but are not limited to: Moloney mouse leukemia virus (MoMuLV or MMLV), Harvey mouse sarcoma virus (HaMuSV or HSV), mouse mammary tumor virus (MuMTV or MMTV), gibbon leukemia virus (GaLV or GALV), human immunodeficiency virus (HIV), and Rous sarcoma virus (RSV). Other env genes can also be used, such as the vesicular stomatitis virus (VSV) G protein (VSV G), hepatitis and influenza virus genes.

The vector providing the env viral nucleic acid sequence is operably linked to the control sequences described elsewhere herein.

In some embodiments, the vector includes a lentiviral vector in which the HIV env, vif, vpr, vpu, and nef virulence genes have been deleted without compromising the ability of the vector to transduce non-dividing cells.

In some embodiments, the vector includes a lentiviral vector that contains a deletion of the 3'-LTR U3 region. The removal of the U3 area can be complete or partial.

In some embodiments, the lentiviral vector of the disclosure comprising the FVIII nucleotide sequence described herein can be transfected into a cell with (a) a first nucleotide sequence containing the gag, pol or gag and pol genes, and (b) a second nucleotide sequence containing a heterologous env gene; where the functional tat gene is absent in the lentiviral vector. In other embodiments, the cell is further transfected with a fourth nucleotide sequence containing the rev gene. In some embodiments, the lentiviral vector lacks functional genes selected from vif, vpr, vpu, vpx, and nef, or combinations thereof.

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

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

Other vectors include plasmid vectors. Plasmid vectors are described in detail in this field and are well known to specialists in this field. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. In the last few years, plasmid vectors have been found to be particularly useful for delivering genes to cells in vivo due to their inability to replicate in the host genome and integrate into it. However, these plasmids having a promoter compatible with the host cell can express a peptide from a gene operably encoded in the plasmid. Some commonly used plasmids available from commercial vendors include pBR322, pUC18, pUC19, various pcDNA plasmids, pRC/CMV, various pCMV plasmids, pSV40 and pBlueScript. Additional examples of specific plasmids include pcDNA3.1, catalog number V79020; pcDNA3.1/hygro, catalog number V87020; pcDNA4/myc-His, catalog number V86320; and pBudCE4.1, catalog number V53220, all from Invitrogen (Carlsbad, CA). Other plasmids are well known to those of ordinary skill in the art. In addition, plasmids can be specifically designed using standard molecular biology techniques to remove and/or add specific DNA fragments.

IV. Obtaining antibodies

Antibodies or fragments thereof that immunospecifically bind to FAM19A5 (eg, human FAM19A5) can be made by any method known in the art for antibody synthesis, eg, chemical synthesis or recombinant expression methods. The methods described herein use, unless otherwise indicated, conventional techniques in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields within the skill of this areas. These methods are described, for example, in the references cited in this document and fully explained in the literature. See, for example, Maniatis T et al., (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Sambrook J et al., (1989), Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press; Sambrook J et al., (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel FM et al., Current Protocols in Molecular Biology, John Wiley & Sons (1987 and annual updates); Current Protocols in Immunology, John Wiley & Sons (1987 and annual updates) Gait (ed.) (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein (ed.) (1991) Oligonucleotides and Analogues: A Practical Approach, IRL Press; Birren B et al., (eds.) (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press.

In a specific embodiment, an antibody described herein is an antibody (eg, a recombinant antibody) obtained, expressed, generated, or isolated by any means, which includes the creation, for example, through synthesis, genetic engineering of DNA sequences. In some embodiments, such an antibody contains sequences (eg, DNA sequences or amino acid sequences) that do not naturally exist in the germline repertoire of an animal or mammalian (eg, human) antibody in vivo. In some embodiments of the invention, the anti-FAM19A5 antibodies disclosed herein have been deimmunized.

As described in the examples (eg, in example 2), anti-FAM19A5 antibodies were originally obtained by immunizing chickens with synthetic FAM19A5 peptide. Therefore, to minimize the risk of immunogenicity when administered to humans, anti-FAM19A5 antibodies (eg, 3-2 and 2-13) have been modified to more closely resemble human antibody immunogenic sequences. In some embodiments, deimmunized anti-FAM19A5 antibodies disclosed herein have similar binding affinity for human FAM19A5 compared to their corresponding antibody counterparts that have not been deimmunized. In some embodiments, the anti-FAM19A5 antibodies disclosed herein have also undergone affinity maturation. Methods for deimmunizing an antibody are disclosed herein and are also known in the art.

In a specific aspect, provided herein is a method for producing an antibody, or antigen-binding fragment thereof, that immunospecifically binds to FAM19A5 (eg, human FAM19A5), comprising culturing a host cell or cell as described herein. In a specific aspect, provided herein is a method for producing an antibody, or antigen-binding fragment thereof, that immunospecifically binds to FAM19A5 (e.g., human FAM19A5), comprising expression (e.g., recombinant expression) of the antibody or antigen-binding fragment thereof using a cell or host cell described in this document (for example, cells or host cells containing polynucleotides encoding the antibody described in this document). In a specific embodiment, the cell is an isolated cell. In a specific embodiment, exogenous polynucleotides have been introduced into the cell. In a specific embodiment, the method further comprises the step of purifying the antibody, or an antigen-binding fragment thereof, derived from a host cell or cell.

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

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

In specific embodiments, a "monoclonal antibody" as used herein is an antibody produced by a single cell (e.g., a hybridoma or a host cell producing a recombinant antibody), wherein the antibody immunospecifically binds to FAM19A5 (e.g., human FAM19A5) as defined., for example, using ELISA or other antigen binding or competitive binding assay known in the art or in the Examples provided herein. In specific embodiments, the monoclonal antibody may be a chimeric antibody or a humanized antibody. In some embodiments, the monoclonal antibody is a monovalent antibody or a polyvalent (eg, bivalent) antibody. In specific embodiments, the monoclonal antibody is a monospecific or multispecific antibody (eg, a bispecific antibody). The monoclonal antibodies described herein may, for example, be prepared by the hybridoma method as described in Kohler G & Milstein C (1975) Nature 256:495, or may be isolated, for example, from phage libraries using, for example, the methods described in this document. Other methods for obtaining clonal cell lines and the monoclonal antibodies they express are well known in the art (see, for example, Chapter 11 in: Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel FM et al, supra).

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

In some embodiments, mice (or other animals such as chickens, rats, monkeys, donkeys, pigs, sheep, hamsters, or dogs) can be immunized with an antigen (e.g., FAM19A5, such as human FAM19A5) and once an immune response is detected, for example, antibodies specific to the antigen are detected in mouse serum, mouse spleen is harvested and splenocytes are isolated. The splenocytes are then fused using well known methods with any suitable myeloma cells, such as those from the SP20 cell line available from the American Type Culture Collection (ATCC®) (Manassas, VA) to form hybridomas. Hybridomas are selected and cloned by limited dilution. In some embodiments, lymph nodes from immunized mice are harvested and fused with NSO myeloma cells.

The hybridoma cells thus obtained are seeded and grown in a suitable culture medium, which preferably contains one or more substances that inhibit the growth or survival of the unfused original myeloma cells. For example, if the original myeloma cells lack the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), then culture media for hybridomas will typically include hypoxanthine, aminopterine, and thymidine ("HAT media"), substances that prevent the growth of HGPRT-deficient cells. .

In specific embodiments, myeloma cells are used that fuse efficiently, maintain stable antibody production at high levels by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these myeloma cell lines are mouse myeloma cell lines such as the NSO cell line or lines derived from mouse tumors MOPC-21 and MPC-11 available from the Salk Institute Cell Distribution Center, San Diego, CA, USA, and SP cells. -2 or X63-Ag8.653 are available from the American Type Culture Collection, Rockville, MD, USA. Human myeloma and murine and 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).

The culture medium in which the hybridoma cells grow is examined for the production of monoclonal antibodies directed against FAM19A5 (eg, human FAM19A5). The binding specificity of monoclonal antibodies produced by hybridoma cells is determined by methods known in the art, for example, by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme immunoassay (ELISA).

After identifying hybridoma cells producing antibodies of the desired specificity, affinity and/or activity, clones can be subcloned using limiting dilution procedures and grown by standard methods (Goding JW (Ed), Monoclonal Antibodies: Principles and Practice, supra). Suitable culture media for this purpose include, for example, D-MEM or RPMI 1640 medium. In addition, hybridoma cells can be grown in vivo as ascitic tumors in animals.

The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascitic fluid or serum by conventional immunoglobulin purification procedures such as, for example, protein A-sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.

Antibodies described herein include antibody fragments that recognize specific FAM19A5 (eg, human FAM19A5) and can be generated by any method known to those skilled in the art. For example, the Fab and F(ab')2 fragments described herein can be prepared by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments). The Fab fragment corresponds to one of two identical arms of the antibody molecule and contains a complete light chain paired with the VH and CH1 domains of the heavy chain. The F(ab')2 fragment contains two antigen-binding arms of the antibody molecule linked by disulfide bonds in the hinge region.

In addition, the antibodies described herein, or antigen-binding fragments thereof, can also be obtained using various phage display methods known in the art. In phage display methods, functional domains of antibodies are displayed on the surface of phage particles that carry their encoding polynucleotide sequences. In particular, DNA sequences encoding the VH and VL domains are amplified from animal cDNA libraries (eg, human or non-human cDNA libraries, such as diseased mouse or chicken cDNA libraries). The DNA encoding the VH and VL domains are recombined with the scFv linker by PCR and cloned into a phagemid vector. The vector is electroporated into E. coli and the E. coli is infected with a helper phage. The phage used in these methods is typically filamentous phage, including fd and M13, and the VH and VL domains are typically recombinantly fused to either the phage III gene or the VIII gene. A phage expressing an antigen-binding domain that binds to a particular antigen can be selected or identified by the antigen, for example using a labeled antigen or an antigen bound or captured on a solid surface or bead. Examples of phage display methods that can be used to produce the antibodies described herein include those disclosed in Brinkman U et al., (1995) J Immunol Methods 182: 41-50; Ames RS et al., (1995) J Immunol Methods 184: 177-186; Kettleborough CA et al., (1994) Eur J Immunol 24: 952-958; Persic L et al., (1997) Gene 187: 9-18; Burton DR & Barbas CF (1994) Advan Immunol 57: 191-280; PCT Application No. PCT/GB91/001134; International Publications Nos. WO 90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO 93/1 1236, WO 95/15982, WO 95/20401 and WO 97/13844; and Pat. U.S. Nos. 5698426, 5223409, 5403484, 5580717, 5427908, 5750753, 5821047, 5571698, 5427908, 5516637, 5780225, 5658727, 5733743 and 5969108.

As described in the references above, once the phage has been selected, antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen-binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast and bacteria, for example, as described below. Methods for recombinant production of antibody fragments such as Fab, Fab' and F(ab')2 fragments can also be used using methods known in the art such as those disclosed in PCT Publication No. WO 92/22324; 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.

In one aspect, to generate complete antibodies, PCR primers, including VH or VL nucleotide sequences, a restriction site, and a flanking sequence to protect the restriction site, can be used to amplify VH or VL sequences from a template, for example scFv clones. Using cloning techniques known to those skilled in the art, PCR amplified VH domains can be cloned into vectors expressing the VH constant region, and PCR amplified VL domains can be cloned into vectors expressing a VL constant region, such as human kappa or lambda constant regions. The VH and VL domains can also be cloned into a single vector expressing the required constant regions. Heavy chain conversion vectors and light chain conversion vectors are then co-transfected into cell lines to generate stable or transient cell lines that express full-length antibodies, eg, IgG, using methods known to those skilled in the art.

A chimeric antibody is a molecule in which different parts of the antibody come from different immunoglobulin molecules. For example, a chimeric antibody may comprise the variable region of a non-human (eg, mouse, rat, or chicken) monoclonal antibody fused to a constant region of a human antibody. Methods for making 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 Pat. US Nos. 5807715, 4816567, 4816397 and 6331415.

The humanized antibody is capable of binding to a predetermined antigen and includes a framework region having the substantially amino acid sequence of a human immunoglobulin and CDRs having the substantially amino acid sequence of a non-human immunoglobulin (eg, mouse or chicken immunoglobulin). In specific embodiments, the humanized antibody also includes at least a portion of an immunoglobulin (Fc) constant region, typically a human immunoglobulin. The antibody may also include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. The humanized antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgG1, IgG2, IgG3 and IgG4. Humanized antibodies can be obtained using various methods known in the art, including, but not limited to, CDR transplantation (European Patent No. EP 239400; International Publication No. WO 91/09967; and US Pat. No. 5225539, 5530101 and 5585089) , veneer or repositioning (EP 592106 and EP 519596; Padlan EA (1991) Mol Immunol 28 (4/5): 489-498; Studnicka GM et al. (1994) Prot Engineering 7 (6): 805-814 ; and Roguska MA et al., (1994) PNAS 91: 969-973), chain shuffling (US Pat. US No. 5565332) and methods disclosed, for example, in US Pat. USA No. 6407213, USA. Pat. US No. 5766886, International Publication No. WO 93/17105; Tan P et al., (2002) J Immunol 169: 1119-25; Caldas C et al., (2000) Protein Eng. 13(5): 353-60; Morea V et al., (2000) Methods 20(3): 267-79; Baca M et al., (1997) J Biol Chem 272(16): 10678-84; Roguska MA et al., (1996) Protein Eng 9(10): 895 904; Couto JR et al., (1995) Cancer Res. 55 (23 Supp): 5973s-5977s; Couto JR et al., (1995) Cancer Res 55(8): 1717-22; Sandhu JS (1994) Gene 150(2): 409-10 and Pedersen JT et al. (1994) J Mol Biol 235(3): 959-73. See also U.S. Publication No. US 2005/0042664 A1 (Feb. 24, 2005), which is incorporated herein by reference in its entirety.

Methods for creating multispecific (eg, bispecific antibodies) have been described, see, for example, US Pat. US No. 7951917; 7183076; 8227577; 5837242; 5989830; 5869620; 6132992 and 8586713.

Single domain antibodies, such as antibodies lacking light chains, can be prepared 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; Patent No. 6005079; and International Publication Nos. WO 94/04678, WO 94/25591 and WO 01/44301.

In addition, antibodies that immunospecifically bind to the FAM19A5 antigen can in turn be used to generate anti-idiotypic antibodies that "mimic" the antigen using methods well known to those skilled in the art. (See, for example, Greenspan NS & Bona CA (1989) FASEB J 7(5): 437-444; and Nissinoff A (1991) J Immunol 147(8): 2429-2438).

In specific embodiments, an antibody described herein that binds to the same FAM19A5 epitope (eg, human FAM19A5) as an anti-FAM19A5 antibody described herein is a human antibody or an antigen-binding fragment thereof. In specific embodiments, an antibody described herein that competitively blocks (eg, in a dose-dependent manner) antibodies described herein from binding to FAM19A5 (eg, human FAM19A5) is a human antibody or an antigen-binding fragment thereof.

Human antibodies can be obtained using any method known in this field. For example, transgenic mice that are unable to express functional endogenous immunoglobulins but can express human immunoglobulin genes can be used. In particular, human immunoglobulin heavy and light chain gene complexes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, constant region and diversity region can be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse immunoglobulin heavy and light chain genes can be rendered non-functional alone or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, a homozygous deletion of the JH region prevents endogenous antibody production. Modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring that express human antibodies. Transgenic mice are immunized in the usual way with the selected antigen, for example, the entire antigen or part of the antigen (eg, FAM19A5). Monoclonal antibodies directed against the antigen can be obtained from immunized transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes contained in transgenic mice are rearranged during B cell differentiation and then undergo class switching and somatic mutations. Thus, the use of such a method is possible for the production of therapeutically useful antibodies IgG, IgA, IgM and IgE. For a review of this technology for producing human antibodies, see Lonberg N & Huszar D (1995) Int Rev Immunol 13: 65-93. For a detailed discussion of this technology for the production of human antibodies and human monoclonal antibodies and protocols for the production of such antibodies, see, for example, International Publications No. WO 98/24893, WO 96/34096 and WO 96/33735; and Pat. US Pat. Mouse™ (Mederex, Inc./Gen Pharm; US Pat. Nos. 5545806 and 5569825), Trans Chromo Mouse™ (Kirin), and KM Mouse™ (Medarex/Kirin).

Human antibodies that specifically bind to FAM19A5 (eg, human FAM19A5) can be generated by various methods known in the art, including the phage display methods described above, using antibody libraries derived from human immunoglobulin sequences. See also Pat. US Nos. 4,444,887, 4,716,111 and 5,885,793; and International Publication Nos. WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735 and WO 91/10741.

In some embodiments, human antibodies can be generated 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 secreting human monoclonal antibodies, and these mouse-human hybridomas can be screened to determine which secrete human monoclonal antibodies that immunospecifically bind to the target antigen (eg FAM19A5, such as human FAM19A5)). Such methods are known and described in the art, see, for example, Shinmoto H et al., (2004) Cytotechnology 46: 19-23; Naganawa Y et al., (2005) Human Antibodies 14: 27-31.

V. Methods for constructing antibodies

As discussed above, an anti-FAM19A5 antibody having the VH and VL sequences described herein can be used to generate a novel anti-FAM19A5 antibody by modifying the VH and/or VL sequences or constant regions attached thereto. Thus, in another aspect described herein, the structural features of an anti-FAM19A5 antibody described herein are used to generate structurally related anti-FAM19A5 antibodies that retain at least one functional property of the antibodies described herein. , such as binding to human FAM19A5. For example, the starting material for the design method are the VH and/or VL sequences provided herein, or one or more of their CDR regions. It is not necessary to actually prepare (ie, 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, to generate an engineered antibody. Rather, the information contained in the sequence(s) is used as input to create a "second generation" sequence(s) derived from the original sequence(s), and then the "second generation" sequence(s) is prepared and expressed as squirrel.

Accordingly, provided herein are methods for producing an anti-FAM19A5 antibody, comprising:

(a) providing: (i) a heavy chain variable region sequence comprising a CDR1, CDR2 and/or CDR3 sequence as specified in Table 3, or a heavy chain variable region CDR1, CDR2 and/or CDR3 sequence as specified in Table 5; and (ii) a light chain variable region sequence comprising a CDR1, CDR2 and/or CDR3 sequence as set forth in Table 4, or a heavy chain variable region CDR1, CDR2 and/or CDR3 sequence as set forth in Table 6;

(b) modifying at least one amino acid residue in the heavy chain variable region sequence and/or the light chain variable region sequence to create at least one altered antibody sequence; and

(c) expressing the altered antibody sequence as a protein.

Standard molecular biology techniques can be used to generate and express an altered antibody sequence.

In some embodiments, the antibody encoded by the altered antibody sequence(s) is an antibody that retains one, some, or all of the functional properties of the anti-FAM19A5 antibodies described herein, which include:

(1) reduced immunogenicity in humans;

(2) binding to soluble human FAM19A5, eg, a KD of 10 nM or less (eg, 001 to 10 nM), eg, as measured by Biacore;

(3) binding to membrane-bound human FAM19A5, eg, a KD of 10 nM or less (eg, 001 to 1 nM), eg, as measured by ELISA;

(4) binding to membrane-bound human FAM19A5, eg EC50 1 nM or less (eg 001 to 1 nM), eg as measured by ELISA;

(5) reduce, reverse, delay and/or prevent the onset of reactive gliosis;

(6) suppression of excessive proliferation of reactive astrocytes;

(7) decreased expression of chondroitin sulfate proteoglycans, including neurocan and neuron-glial antigen 2 (NG2);

(8) increased expression of c-fos and pERK in neuronal nuclei;

(9) stimulation of neuronal survival;

(10) increased expression of GAP43 in neurons;

(11) stimulation of axon regrowth; and

(12) competition in either or both directions for binding to human FAM19A5 with the anti-FAM19A5 antibody disclosed herein.

An altered antibody may exhibit one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven, or all of the functional properties , indicated above in (1) - (12). The functional properties of altered antibodies can be assessed using standard assays available in the art and/or described herein, such as those set forth in the examples (eg, ELISA, FACS).

In some embodiments of the antibody construction methods described herein, mutations can be introduced randomly or selectively along all or part of the coding sequence of an anti-FAM19A5 antibody, and the resulting modified anti-FAM19A5 antibodies can be tested for binding activity and/or other functional properties. as described in this document. Mutation methods are described in the art. For example, PCT Publication WO 02/092780 by Short describes methods for generating and screening for antibody mutations using saturation mutagenesis, synthetic ligation assembly, or a combination thereof. Alternatively, PCT publication WO 03/074679 Lazar et al. describes how to use computer screening methods to optimize the physicochemical properties of antibodies.

VI. Cells and vectors

In certain aspects, provided herein are cells (e.g., host cells) expressing (e.g., recombinantly) the antibodies described herein (or an antigen-binding fragment thereof) that specifically bind to FAM19A5 (e.g., human FAM19A5) and related polynucleotides, and expression vectors. Provided herein are vectors (eg, expression vectors) containing polynucleotides containing nucleotide sequences encoding anti-FAM19A5 antibodies or a fragment for recombinant expression in host cells, eg, mammalian cells. Also provided herein are host cells containing such vectors for recombinant expression of the anti-FAM19A5 antibodies described herein (eg, a human or humanized antibody). In a specific aspect, provided herein are methods for producing an antibody described herein, comprising expressing such an antibody from a host cell.

Recombinant expression of an antibody described herein (e.g., a full length antibody, an antibody heavy and/or light chain, or a single chain antibody described herein) that specifically binds to FAM19A5 (e.g., human FAM19A5) involves generating an expression vector comprising a polynucleotide encoding an antibody. Once a polynucleotide encoding an antibody molecule, an antibody heavy and/or light chain, or fragment thereof (e.g., heavy and/or light chain variable domains) described herein has been obtained, a vector for producing the antibody molecule can be obtained using recombinant DNA technology. using methods well known in the art. Thus, this document describes methods for producing a protein by expressing a polynucleotide containing a nucleotide sequence encoding an antibody or antibody fragment (eg, a light chain or a heavy chain). Methods well known to those skilled in the art can be used to construct expression vectors containing the coding sequences for an antibody or antibody fragment (eg, light chain or heavy chain) and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA methods, synthetic methods, and in vivo genetic recombination. Also provided are replicable vectors containing a nucleotide sequence encoding an antibody molecule as described herein, an antibody heavy or light chain, an antibody heavy or light chain variable domain or a fragment thereof, or a heavy or light chain CDR operably linked to a promoter. Such vectors may, for example, include a nucleotide sequence encoding the constant region of an antibody molecule (see, for example, International Publications Nos. WO 86/05807 and WO 89/01036; and US Pat. No. 5,122,464) and the variable domains of the antibody can be cloned into such a vector for expressing the entire heavy chain, the entire light chain, or both entire heavy and light chains.

The expression vector can be transferred into a cell (e.g., a host cell) by conventional methods, and the resulting cells can then be cultured by conventional methods to produce an antibody described herein (e.g., an antibody containing a VH and/or VL, or one or more VH and/or VL CDR of an anti-FAM19A5 antibody of the present disclosure) or a fragment thereof. Thus, provided herein are host cells comprising a polynucleotide encoding an antibody described herein, or fragments thereof, or a heavy or light chain thereof, or a fragment thereof, or a single chain antibody described herein, operably linked to a promoter. to express such sequences in the host cell. In some embodiments for the expression of double chain antibodies, vectors encoding both the heavy and light chains individually can be co-expressed in a host cell to express the entire immunoglobulin molecule, as detailed below. In some embodiments, the host cell contains a vector containing a polynucleotide encoding both the heavy chain and the light chain of the antibody described herein, or a fragment thereof. In specific embodiments, the host cell comprises two different vectors, a first vector containing a polynucleotide encoding the heavy chain or heavy chain variable region of an antibody described herein, or a fragment thereof, and a second vector containing a polynucleotide encoding a light chain or light chain variable region. an antibody described herein, or a fragment thereof. In other embodiments, the first host cell contains a first vector containing a polynucleotide encoding the heavy chain or variable region of the heavy chain of the antibody described herein, or a fragment thereof, and the second host cell contains a second vector containing a polynucleotide encoding a light chain or variable region. the light chain region of an antibody described herein. In specific embodiments, the heavy chain/heavy chain variable region expressed by the first cell is associated with the light chain/light chain variable region of the second cell to form the anti-FAM19A5 antibody described herein, or an antigen-binding fragment thereof. In certain embodiments, provided herein is a population of host cells including such a first host cell and such a second host cell.

In a specific embodiment, provided herein is a population of vectors comprising a first vector containing a polynucleotide encoding a light chain/light chain variable region of an anti-FAM19A5 antibody described herein and a second vector containing a polynucleotide encoding a heavy chain/heavy chain variable region. anti-FAM19A5 antibody described herein.

Various host-expression vector systems can be used to express the antibody molecules described herein. Such host-expression systems are vehicles by which coding sequences of interest can be generated and subsequently purified, but are also cells that, when transformed or transfected with the appropriate nucleotide coding sequences, can express the antibody molecule described herein in situ. These include, but are not limited to, microorganisms such as bacteria (eg, E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing antibody coding sequences; yeast (eg Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus (eg, baculovirus) expression vectors containing antibody coding sequences; 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. plasmid Ti) containing sequences encoding antibodies; or mammalian cell systems (e.g. COS (e.g. COS1 or COS), CHO, BHK, MDCK, HEK 293, NSO, PER.C6, VERO, CRL7030, HsS78Bst, HeLa and NIH 3T3, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB/20 and BMT10 cells) carrying recombinant expression constructs containing promoters derived from the genome of mammalian cells (eg, metallothionein promoter) or from mammalian viruses (eg, adenovirus late promoter; 7.5K vaccinia virus promoter). In a specific embodiment, the cells for expressing the antibodies described herein, or an antigen-binding fragment thereof, are CHO cells, such as CHO cells from the CHO GS System™ (Lonza). In a specific embodiment, the cells for expressing the antibodies described herein are human cells, eg, human cell lines. In a specific embodiment, the mammalian expression vector is pOptiVEC™ or pcDNA3.3. In a specific embodiment, bacterial cells such as Escherichia coli or eukaryotic cells (eg mammalian cells), especially for expression of the entire recombinant antibody molecule, are used to express the recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary (CHO) cells, in combination with a vector such as the human cytomegalovirus immediate early gene promoter major element, provide an efficient antibody expression system (Foecking MK & Hofstetter H (1986) Gene 45 : 101-5 and Cockett MI et al. (1990) Biotechnology 8(7): 662-7). In some embodiments, the antibodies described herein are produced by CHO cells or NSO cells. In a specific embodiment, expression of nucleotide sequences encoding antibodies described herein that immunospecifically binds to FAM19A5 (eg, human FAM19A5) is regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

In bacterial systems, a number of expression vectors can advantageously be selected depending on the application intended for the antibody molecule to be expressed. For example, when a large amount of such an antibody is to be produced, vectors that drive the expression of fusion products at a high level that are easy to purify may be desirable to create pharmaceutical compositions of the antibody molecule. Such vectors include, but are not limited to, the E. coli pUR278 expression vector (Ruether U & Mueller-Hill B (1983) EMBO J 2: 1791-1794), wherein the antibody coding sequence can be ligated individually into the vector in frame with the coding region. lac Z so that a fusion protein is formed; 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. For example, pGEX vectors can also be used to express foreign polypeptides as glutathione-5-transferase (GST) fusion proteins. Typically, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to glutathione-agarose matrix beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include protease thrombin or factor Xa cleavage sites so that the cloned product of the target gene can be released from the GST group.

In the insect system, the Autographa californica nuclear polyhedrosis virus (AcNPV), for example, can be used as a vector for the expression of foreign genes. The virus grows in the cells of Spodoptera frugiperda. An antibody coding sequence can be cloned individually into non-essential regions (eg, the polyhedrin gene) of the virus and placed under the control of an AcNPV promoter (eg, the polyhedrin promoter).

A number of viral expression systems can be used in mammalian host cells. In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest can be ligated to an adenovirus transcription/translation control complex, eg, a late promoter and a three-component 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 (eg, the E1 or E3 region) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts (see, for example, Logan J & Shenk T (1984).) PNAS 81 ( 12): 3655-9). Inserted antibody coding sequences may also require specific initiation signals for efficient translation. These signals include the initiation codon ATG and neighboring sequences. In addition, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of various origins, both natural and synthetic. Expression efficiency can be increased by including appropriate transcription enhancer elements, transcription terminators, etc. (see, for example, Bitter G et al., (1987) Methods Enzymol. 153: 516-544).

In addition, a host cell strain may be selected that modulates the expression of the inserted sequences or modifies and processes the gene product in the specific manner desired. Such modifications (eg, glycosylation) and processing (eg, cleavage) of protein products may be important for protein function. Various host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be selected to ensure correct modification or processing of the expressed foreign protein. To this end, eukaryotic host cells can be used, which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, Hela, MDCK, HEK 293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT20 and T47D, NSO (mouse myeloma cell line that does not produce endogenously any immunoglobulin chains), CRL7030, COS (eg COS 1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC 1, BSC40, YB/20 , BMT10 and HsS78Bst. In some embodiments, the anti-FAM19A5 antibodies described herein are produced in mammalian cells, such as CHO cells.

In a specific embodiment, the antibodies described herein, or antigen-binding portions thereof, are fucose-reduced or free of fucose. Such antibodies can be obtained using methods known to those skilled in the art. For example, antibodies can be expressed in cells that are deficient or lack the ability to fucosylate. In a specific example, cell lines with a knockout of both 16-fucosyltransferase alleles can be used to generate antibodies or their antigen-binding portions with a reduced fucose content. The Potelligent® System (Lonza) is an example of such a system that can be used to generate reduced fucose antibodies or antigen-binding portions thereof.

For long-term production of recombinant proteins in high yield, stable expressing cells can be created. For example, cell lines can be constructed that stably express the anti-FAM19A5 antibody described herein and its antigen-binding portion. In specific embodiments, the cell provided herein stably expresses a light chain/light chain variable domain and a heavy chain/heavy chain variable domain that bind to form the antibody described herein, or an antigen-binding portion thereof.

In certain aspects, instead of using expression vectors that contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.) and a selectable marker . After the introduction of foreign DNA/polynucleotide engineered cells can be allowed to grow for 1-2 days in enriched medium, and then transferred to selective medium. The selectable marker in the recombinant plasmid confers resistance to selection and allows cells to stably integrate the plasmid into their chromosomes and grow into foci, which in turn can be cloned and propagated into cell lines. This method can be successfully used to construct cell lines that express the anti-FAM19A5 antibody described herein, or its binding portion of the antibody. Such engineered cell lines may be particularly useful in screening and evaluating compositions that interact directly or indirectly with an antibody molecule.

A number of selection systems can be used, including, but not limited to, herpes simplex virus thymidine kinase genes (Wigler M. et al., (1977) Cell 11 (1): 223-32), hypoxanthine guanine phosphoribosyltransferase (Szybalska EH & Szybalski W. (1962) PNAS 48 (12): 2026-2034) and adenine phosphoribosyl transferase (Lowy I et al., (1980) Cell 22 (3): 817-23) can be used in tk, hgprt or aprt cells, respectively. In addition, antimetabolite resistance can be used as a selection basis for the following genes: 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). Methods widely known in the field of recombinant DNA technology can be routinely used to select the desired recombinant clone, and such methods are described, for example, in Ausubel FM et al., (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler M, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and chapters 12 and 13 of Dracopoli NC et al. (eds.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colbere-Garapin F et al., (1981) J Mol Biol 150: 1-14, which are incorporated herein by reference in their entirety.

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

The host cell can be co-transfected with two or more expression vectors described herein, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors may contain identical selectable markers that provide equal expression of the heavy and light chain polypeptides. Host cells can be co-transfected with varying amounts of two or more expression vectors. For example, host cells can be transfected with any of the following ratios of first expression vector and second expression vector: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8 , 1:9, 1:10, 1:12, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45 or 1:50.

Alternatively, a single vector that encodes and is capable of expressing both heavy and light chain polypeptides can be used. In such situations, the light chain must be placed before the heavy chain to avoid excess toxic free heavy chain (Proudfoot NJ (1986) Nature 322: 562-565; and Kohler G (1980) PNAS 77: 2197-2199). The heavy and light chain coding sequences may include cDNA or genomic DNA. The expression vector may be monocistronic or multicistronic. A multicistronic nucleic acid construct may encode 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, or in the range of 2-5, 5-10, or 10-20 gene/nucleotide sequences. For example, a bicistronic nucleic acid construct may comprise, in the following order, a promoter, a first gene (eg, the heavy chain of the antibody described herein), and a second gene (eg, the light chain of the antibody described herein). In such an expression vector, transcription of both genes may be promoter driven, while translation of mRNA from the first gene may be by a cap-dependent scanning mechanism, and translation of mRNA from the second gene may be by a cap-independent scanning mechanism, such as by IRES.

Once an antibody molecule described herein has been produced by recombinant expression, it can be purified by any method known in the art for purifying an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity chromatography, especially affinity chromatography). chromatography to a specific antigen after protein A and size exclusion chromatography), centrifugation, differential solubility, or any other standard protein purification method. In addition, the antibodies described herein may be fused to heterologous polypeptide sequences as described herein or otherwise known in the art to facilitate purification.

In specific embodiments, the antibody or antigen-binding portion described herein is isolated or purified. Typically, an isolated antibody is an antibody that is substantially free of other antibodies with antigenic specificity different from that of the isolated antibody. For example, in a specific embodiment, the antibody preparation described herein is substantially free of cellular material and/or chemical precursors. The phrase "substantially free of cellular material" includes antibody preparations in which the antibody is separated from the cellular components of the cells from which it is isolated or recombinantly produced. Thus, an antibody that is substantially free of cellular material includes antibody preparations containing less than about 30%, 20%, 10%, 5%, 2%, 1%, 05%, or 01% (by dry weight) of the heterologous protein ( also referred to herein as a "contaminating protein") and/or variants of the antibody, for example, various post-translationally modified forms of the antibody or other various versions of the antibody (or antibody-binding portions). When an antibody is produced recombinantly, it also usually contains little or no culture medium, i. e. the culture medium is less than about 20%, 10%, 2%, 1%, 05%, or 01% of the volume of the protein preparation. When an antibody is produced by chemical synthesis, it is usually substantially free of chemical precursors or other chemicals, i.e. it is separated from chemical precursors or other chemicals that are involved in protein synthesis. Accordingly, such antibody preparations contain less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or compounds other than the antibody of interest. In a specific embodiment, the antibodies described herein are isolated or purified.

VII. Quantitative analyzes

Antibodies described herein can be tested for binding to FAM19A5, for example, using a 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 dilutions (eg, plasma dilutions from mice immunized with FAM19A5) are added to each well and incubated for 1-2 hours at 37°C. The plates were washed with PBS/Tween and then incubated with a secondary reagent (eg, for human antibodies, goat anti-human IgG Fc polyclonal reagent) conjugated with horseradish peroxidase (HRP) for 1 hour at 37°C. After washing, the plates were developed with ABTS substrate (Moss Inc, product: ABTS-1000) and analyzed on a spectrophotometer at OD 415-495. Sera from immunized mice were then further screened by flow cytometry for binding to a cell line expressing human FAM19A5, but not to a control cell line that did not express FAM19A5. Briefly, binding of anti-FAM19A5 antibodies was assessed by incubating CHO cells expressing FAM19A5 with anti-FAM19A5 antibody at a 1:20 dilution. Cells were washed and binding to PE-labeled anti-human IgG antibodies was detected. Flow cytometry analyzes were performed using FACS flow cytometry (Becton Dickinson, San Jose, CA). Preferably, the mice with the highest titers were used for fusion.

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

To purify anti-FAM19A5 antibodies, selected hybridomas can be grown in 2 liter spinner flasks for monoclonal antibody purification. Supernatants can be filtered and concentrated prior to protein A-sepharose affinity chromatography (Pharmacia, Piscataway, NJ). Eluted IgG can be checked by gel electrophoresis and high performance liquid chromatography to ensure purity. The buffer solution can be replaced with PBS, and the concentration can be determined by OD 280 using an extinction coefficient of 143. Monoclonal antibodies can be divided into aliquots and stored at -80°C.

To determine if selected monoclonal anti-FAM19A5 antibodies bind to unique epitopes, each antibody can be biotinylated using commercially available reagents (Pierce, Rockford, IL). Binding of biotinylated MAbs can be detected using a streptavidin-labeled probe. Competitive studies using unlabeled monoclonal antibodies and biotinylated monoclonal antibodies can be performed using ELISA plates coated with FAM19A5 as described above.

To determine the isotype of purified antibodies, isotype ELISA can be performed using reagents specific for antibodies of a certain isotype. For example, to determine the isotype of a human monoclonal antibody, the wells of microtiter plates can be coated with 1 μg/ml of human immunoglobulin overnight at 4°C. After blocking with 1% BSA in the plates, the reaction was started with 1 μg/ml or less of the tested monoclonal antibodies or purified isotype controls at ambient temperature for one to two hours. Alkaline phosphatase conjugated to either human IgG1 or human IgM can then be initiated in the wells. The plates were developed and analyzed as described above.

To test the binding of monoclonal antibodies to live cells expressing FAM19A5, flow cytometry can be used as described in the examples. Briefly, cell lines expressing membrane-bound FAM19A5 (grown under standard growth conditions) were mixed with various concentrations of monoclonal antibodies in PBS containing 01% BSA at 4°C for 1 hour. After washing, the cells were reacted with fluorescein-labeled anti-IgG antibody under the same conditions as when stained with the primary antibody. Samples can be analyzed with the FACScan instrument using light and side scatter properties to analyze single cells and determine the binding of labeled antibodies. An alternative analysis using fluorescence microscopy can be used (in addition to or instead of) flow cytometry analysis. Cells can be stained exactly as described above and examined using fluorescence microscopy. This method allows visualization of individual cells, but may have reduced sensitivity depending on the density of the antigen.

Anti-FAM19A5 antibodies can be further tested for reactivity with the FAM19A5 antigen by Western blotting. Briefly, cell extracts from cells expressing FAM19A5 can be prepared and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. After electrophoresis, the separated antigens are transferred to nitrocellulose membranes, blocked with 20% mouse serum, and examined with test monoclonal antibodies. IgG binding can be determined with anti-IgG alkaline phosphatase and visualized with BCIP/NBT substrate tablets (Sigma Chem. Co., St. Louis, MO).

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

In one embodiment, the antibody specifically binds to a soluble form of human FAM19A5. In one embodiment, the antibody specifically binds to a membrane bound form of human FAM19A5. An antibody can specifically bind to a specific FAM19A5 epitope (eg SEQ ID NO: 90 or a fragment in SEQ ID NO: 90). In some embodiments, the antibody specifically binds human FAM19A5, preferably with high affinity, and does not cross-react with other members of the FAM19 protein subfamily.

VIII. Bispecific molecules

Antibodies described herein can be used to form bispecific molecules. The anti-AM19A5 antibody, or antigen-binding portions thereof, may be derivatized or linked to another functional molecule, such as another peptide or protein (eg, another antibody or ligand for a receptor) to create a bispecific molecule that binds to at least two different binding site or target molecule. Cytokines such as IL-6, CNTF, LIF, EGF and TGFα have been implicated as triggers for the onset of gliosis and/or reactive astrogliosis (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)) by activating the protein signal transducer and transcription activator 3 (STAT3), which then regulates many aspects of reactive astrogliosis after CNS injury. . Herrmann J. E. et al., J. Neurosci. 28(28): 7231-7243 (2008). For example, the absence or decrease of STAT3 results in attenuated glial fibrillary acidic protein (GFAP) activation, lack of astrocyte hypertrophy and increased inflammation, increased lesion volume, and partially impaired motor recovery after CNS injury. Herrmann J. E. et al., J. Neurosci. 28(28): 7231-7243 (2008). Thus, for example, an anti-FAM19A5 antibody can be coupled to an antibody or scFv that specifically binds to any protein that is involved in inhibiting the onset of gliosis and/or overproliferation of reactive astrogliosis for combination therapy, such as an anti-IL-6 antibody, CNTF , LIF, EGF or TGFα.

In addition, an anti-FAM19A5 antibody may be linked to an antibody or scFv that treats a disease or disorder, including central nervous system injury (eg, traumatic brain injury, cerebrospinal injury, stroke, or brain tumor), cerebrospinal systemic injury, degenerative disease brain (eg, Huntington's disease, Parkinson's disease, Alzheimer's disease, multiple sclerosis, ALS), degenerative cerebrospinal or nerve disorder, or neuropathic pain in the subject (see Diseases or Disorders in Section XII below). For example, an anti-FAM19A5 antibody can be linked to an antibody or scFv, eg, natalizumab (TYSABRI®), alemtuzumab (LEMTRADA®), which are used to treat multiple sclerosis.

An antibody described herein may in fact be derivatized or linked to more than one other functional molecule to create multispecific molecules that bind to more than two different binding sites and/or target molecules; such multispecific molecules are also encompassed by the term "bispecific molecule" as used herein. To create the bispecific molecule described herein, an antibody described herein may be operably linked (e.g., by chemical linkage, genetic fusion, non-covalent association, or otherwise) to one or more other binding molecules, such as another antibody that binds it. part of an antibody, peptide, or binding mimetic so that a bispecific molecule is obtained. In one embodiment, the bispecific molecule binds FAM19A5 and VEGF. In another embodiment, the bispecific molecule binds to FAM19A5 and EGF.

Accordingly, provided herein are bispecific molecules comprising at least one first binding specificity for FAM19A5 and a second binding specificity for a second target epitope. In the embodiment described herein, in which the bispecific molecule is multispecific, the molecule may further include a third binding specificity.

In one embodiment, the bispecific molecules described herein comprise, as binding specificity, at least one antibody or antibody binding portion thereof, including, for example, Fab, Fab', F(ab')2, Fv, or single chain Fv ( scFv). The antibody can also be a light chain or heavy chain dimer or any minimal fragment thereof, such as an Fv or a single chain construct, as described in Ladner et al., US Pat. US No. 4946778, the contents of which are specifically incorporated by reference.

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

The bispecific molecules described herein can be prepared by conjugating the constituent binding specificities using methods known in the art. For example, each binding specificity of a bispecific molecule can be obtained separately and then conjugated to each other. When the binding specificities are proteins or peptides, various binding or crosslinking agents can be used for covalent conjugation. Examples of crosslinkers include protein A, carbodiimide, N-succinimidyl-S-acetylthioacetate (SATA), 55'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2- pyridyldithio) propionate (SPDP) and sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohaxane-1-carboxylate (sulfo-SMCC) (see e.g. Karpovsky et al., (1984) J. Exp. Med., 160: 1686 Liu, MA et al., (1985) Proc. Natl. Acad. Sci. USA 82:8648). Other methods 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 conjugating agents are SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, Illinois).

When the binding specificities are antibodies, they can be conjugated by sulfhydryl linkage of the C-terminal hinge regions of the two heavy chains. In a particularly preferred embodiment, the hinge region is modified to contain an odd number of sulfhydryl residues, preferably one, prior to conjugation.

Alternatively, both binding specificities can be encoded in the same vector, expressed and assembled in the same host cell. This method is particularly useful when the bispecific molecule is mAb x mAb, mAb x Fab, mAb x (scFv) 2 , Fab x F(ab') 2 or ligand x Fab fusion protein. The bispecific antibody may include an antibody containing an scFv at the C-terminus of each heavy chain. The bispecific molecule described herein can be a single chain molecule containing one single chain antibody and a binding determinant, or a single chain bispecific molecule containing two binding determinants. Bispecific molecules may include at least two single chain molecules. Methods for obtaining bispecific molecules are described, for example, in US Pat. US No. 5260203; Pat. US No. 5455030; Pat. US No. 4881175; Pat. US No. 5,132,405; Pat. US No. 5091513; Pat. US No. 5476786; Pat. US No. 5,013,653; Pat. US #5,258,498; and pat. USA No. 5482858.

Binding of bispecific molecules to their specific targets can be confirmed using art-recognized methods such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS assay, bioassay (eg, growth inhibition), or Western blot assay. Each of these assays typically detects the presence of protein-antibody complexes of particular interest using a labeled reagent (eg, antibody) specific for the complex of interest.

IX. Diagnostics

In one embodiment, the fragment attached to the FAM19A5 antibody is selected from the group consisting of a binding fragment, a labeling fragment, and a biologically active fragment.

The antibodies described herein can be used for diagnostic purposes, including sample analysis and in vivo imaging, and for this purpose, the antibody (or binding portion thereof) can be conjugated with a suitable detectable agent to form an immunoconjugate. For diagnostic purposes, suitable agents are detectable labels, which include radioisotopes for whole body imaging and radioisotopes, enzymes, fluorescent labels, and other suitable antibody labels for testing samples.

Detectable labels can be any of the various types currently used in the field of in vitro diagnostics, including particulate labels, including metal sols such as colloidal gold, isotopes such as I125 or Tc99, provided, for example, with a peptide chelating agent N2S2, N3S. or N4 type, chromophores, including fluorescent markers, luminescent markers, phosphorescent markers, and the like, as well as enzyme labels that convert a given substrate into a detectable marker, and polynucleotide labels that are detected after amplification, such as polymerase chain reaction. Suitable enzyme labels include horseradish peroxidase, alkaline phosphatase, and the like. For example, the label may be an alkaline phosphatase enzyme, detectable by measuring the presence or formation of chemiluminescence after conversion of 12-dioxetane substrates such as adamantylmethoxyphosphoryloxyphenyldioxetane (AMPPD), disodium 3-(4-(methoxyspiro{l, 2-dioxetane-32'-(5 '-chloro)tricyclo {3.3.1.1 37}decan}-4-yl)phenyl phosphate (CSPD) as well as CDP and CDP-star® or other luminescent substrates well known to those skilled in the art, such as chelates of suitable lanthanides such as terbium(III) and europium(III) The detection medium is determined by the selected label The appearance of the label or its reaction products can be achieved with the naked eye if the label is particulate and accumulates at appropriate levels, or with tools such as a spectrophotometer, luminometer, fluorimeter, etc. in accordance with standard practice.

The antibodies described herein can also be conjugated to a therapeutic agent to form an immunoconjugate, such as an antibody-drug conjugate (ADC). Suitable therapeutic agents include agents that modulate the onset of gliosis and/or reactive astrogliosis and/or treat degenerative diseases of the brain, central nervous system injury, or neuropathic pain. Therapeutic agents for the treatment of degenerative diseases of the brain include drugs for the treatment of 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 diseases, such as those described in Section XII below.

Immunoconjugates can be obtained by methods known in this field. Preferably, conjugation methods result in bonds that are essentially (or nearly) non-immunogenic, such as peptide (ie, amide), sulfide (hindered), disulfide, hydrazone, and ether bonds. These bonds are almost non-immunogenic and show reasonable stability in serum (see, for example, Senter, P.D., Curr. Opin. Chem. Biol. 13 (2009) 235-244; WO 2009/059278; WO 95/17886).

Depending on the biochemical nature of the fragment and the antibody, different conjugation strategies can be used. In case the fragment is natural or recombinant and contains 50 to 500 amino acids, textbooks have standard procedures describing the chemistry of the synthesis of protein conjugates that can be easily followed by the skilled person (see, for example, Hackenberger, CPR, and Schwarzer, D. , Angew Chem Int Ed Engl 47 (2008) 10030-10074). In one embodiment, the reaction of a maleimido fragment with a cysteine residue in the antibody or fragment is used. This particularly suitable binding chemistry is used in the case of, for example, a Fab or Fab' fragment of an antibody. Alternatively, in one embodiment, binding is made to the C-terminal end of the antibody or fragment. C-terminal modification of a protein, such as a Fab fragment, for example, can be performed as described (Sunbul, M. and Yin, J., Org. Biomol. Chem. 7 (2009) 3361-3371).

In general, site-specific reaction and covalent bonding are based on the conversion of a naturally occurring amino acid to an amino acid with a reactivity that is orthogonal to that of the other functional groups present. For example, a particular cysteine in the context of a rare sequence can be enzymatically converted to an aldehyde (see Frese, M.A., and Dierks, T., ChemBioChem. 10 (2009) 425-427). It is also possible to obtain the desired amino acid modification using the specific enzymatic reactivity of certain enzymes with a natural amino acid in a given sequence context (see, for example, Taki, M. et al., Prot. Eng. Des. Sel. 17 (2004) 119-126; Gautier, A. et al., Chem Biol 15 (2008) 128-136, and protease-catalyzed C-N bonding is used by Bordusa, F., Highlights in Bioorganic Chemistry (2004) 389-403).

Site-specific reaction and covalent bonding can also be achieved by selectively reacting terminal amino acids with appropriate modifying reagents. The reactivity of N-terminal cysteine with benzonitriles (see Ren, H. et al., Angew. Chem. Int. Ed. Engl. 48 (2009) 9658-9662) can be used to achieve site-specific covalent binding. Native chemical ligation may also depend on C-terminal cysteine residues (Taylor, E. Vogel; Imperiali, B, Nucleic Acids and Molecular Biology (2009), 22 (Protein Engineering), 65-96).

EP 1074 563 describes a conjugation method which is based on a faster reaction of a cysteine in a negatively charged amino acid segment with a cysteine located in a positively charged amino acid segment.

The fragment can also be a synthetic peptide or a peptide mimic. In the case of chemical synthesis of a polypeptide, amino acids with orthogonal chemical reactivity may be included during such synthesis (see, for example, de Graaf, A.J. et al., Bioconjug. Chem. 20 (2009) 1281-1295). Because there is a wide variety of orthogonal functionalities that can be introduced into a synthetic peptide, conjugation of such a peptide to a linker is standard chemistry.

To obtain a mono-labeled polypeptide, the 1:1 stoichiometry conjugate can be separated by chromatography from other conjugation by-products. This procedure can be facilitated by using a dye-labeled binding pair member and a charged linker. Using this kind of labeled and highly negatively charged member of the binding pair, monoconjugated polypeptides are easily separated from unlabeled polypeptides and polypeptides that carry more than one linker because differences in charge and molecular weight can be used for separation. A fluorescent dye can be used to purify the complex of unbound components such as a labeled monovalent binder.

X. Pharmaceutical compositions

Provided herein are compositions comprising an antibody as described herein, or an antigen-binding portion thereof, having the desired purity in a physiologically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, Pennsylvania). Acceptable carriers, excipients or stabilizers are non-toxic to recipients at the dosages and concentrations used and include buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight polypeptides (less than about 10 residues); 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 dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (eg Zn-protein complexes); and/or non-ionic surfactants such as TWEEN®, PLURONICS® or polyethylene glycol (PEG).

In a particular embodiment, the pharmaceutical compositions comprise the antibody or antigen-binding portion thereof, the bispecific molecule or immunoconjugate described herein, and optionally one or more additional prophylactic or therapeutic agents in a pharmaceutically acceptable carrier. In a specific embodiment, the pharmaceutical compositions comprise an effective amount of an antibody or antigen-binding portion thereof as described herein and optionally one or more additional prophylactic therapeutic agents in a pharmaceutically acceptable carrier. 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 a condition such as central nervous system injury, brain degenerative disease, or neuropathic pain.

Pharmaceutically acceptable carriers for use in parenteral formulations include aqueous carriers, non-aqueous carriers, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, binding or chelating agents, and other pharmaceutically acceptable substances. Examples of aqueous vehicles include sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, dextrose injection, and Ringer's lactate injection. Non-aqueous parenteral carriers include vegetable fatty oils, cottonseed oil, corn oil, sesame oil and peanut oil. Antimicrobial agents at bacteriostatic or fungistatic concentrations may be added to parenteral preparations packaged in multi-dose containers, which include phenols or cresols, mercury, benzyl alcohol, chlorobutanol, esters of methyl and propyl p-hydroxybenzoic acid, thimerosal, benzalkonium chloride and chloride benzethonium. Isotonic agents include sodium chloride and dextrose. Buffers include phosphate and citrate. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspension and dispersing agents include sodium carboxymethylcellulose, hydroxypropylmethylcellulose, and polyvinylpyrrolidone. Emulsifying agents include polysorbate 80 (TWEEN® 80). Metal ion binding or chelating agents include EDTA. Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol, and propylene glycol for water-miscible carriers; and sodium hydroxide, hydrochloric acid, citric acid or lactic acid to adjust the pH.

The pharmaceutical composition may be formulated for any route of administration to the subject. Specific examples of routes of administration include intranasal, oral, parenteral, intrathecal, intracerebroventricular, pulmonary, subcutaneous, or intraventricular. This document also discusses parenteral administration, characterized by subcutaneous, intramuscular or intravenous injection. Injectable preparations may be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for dissolution or suspension in a liquid prior to injection, or as emulsions. Injectable formulations, solutions and emulsions also contain one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol or ethanol. In addition, if desired, the pharmaceutical compositions administered may also contain small amounts of non-toxic excipients such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers and other such agents, such as, for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate. and cyclodextrins.

Formulations for parenteral administration of antibodies include sterile solutions ready for injection, sterile dry soluble products such as lyophilized powders ready to be mixed with a solvent immediately before use, including tablets for subcutaneous injection, sterile suspensions ready for injection, sterile dry insoluble products, ready to be mixed with a carrier immediately before use and sterile emulsions. Solutions may be aqueous or non-aqueous.

For intravenous administration, suitable carriers include saline or phosphate buffered saline (PBS), as well as solutions containing thickening and solubilizing agents such as glucose, polyethylene glycol and polypropylene glycol, and mixtures thereof.

Topical mixtures containing the antibody are prepared as described for topical and systemic administration. The resulting mixture may be a solution, suspension, emulsions, and the like. and may be formulated as creams, gels, ointments, emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes, foams, aerosols, drizzles, sprays, suppositories, dressings, skin patches, or any other formulation suitable for topical application.

An antibody or antigen-binding portion thereof described herein may be formulated as an aerosol for topical use such as inhalation (see, for example, US Pat. inflammatory diseases such as asthma). These respiratory formulations may be in the form of an aerosol or solution for nebulization, or as a fine powder for insufflation, alone or in combination with an inert carrier such as lactose. In this case, the particles of the composition in one embodiment will have a diameter of less than 50 microns, in one embodiment less than 10 microns.

The antibody or antigen-binding portion thereof described herein can be formulated for topical or topical application, for example, for topical application to the skin and mucous membranes, for example, in the eyes, in the form of gels, creams and lotions, and also for use in the eye. or for intracisternal or intraspinal use. Topical administration is contemplated for transdermal delivery, as well as for ocular or mucosal administration, or for inhalation therapy. It is also possible to administer nasal solutions of the antibody alone or in combination with other pharmaceutically acceptable excipients.

Transdermal patches, including iontophoretic and electrophoretic devices, are well known to those skilled in the art and can be used to administer an antibody. For example, such patches are disclosed in US Pat. U.S. Nos. 6267983, 6261595, 6256533, 6167301, 6024975, 6010715, 5985317, 5983134, 5948433 and 5860957.

In some embodiments, the pharmaceutical composition containing the antibody or antigen-binding portion described herein is a lyophilized powder that can be reconstituted for administration as solutions, emulsions, and other mixtures. It can also be reconstituted and prepared as solids or gels. The lyophilized powder is prepared by dissolving the antibody, or 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 may contain a stability excipient or other pharmacological component of the powder or reconstituted solution prepared from the powder. Excipients that may be used include, but are not limited to, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose, or other suitable agent. The solvent may also contain a buffer, such as citrate, sodium or potassium phosphate, or other such buffer known to those skilled in the art, in one embodiment at about neutral pH. Subsequent sterile filtration of the solution, followed by lyophilization under standard conditions known to those skilled in the art, provides the desired composition. In one embodiment, the resulting solution will be dispensed into lyophilization vials. Each vial will contain one or more doses of the compound. The lyophilized powder can be stored under appropriate conditions, for example at about 4° C. to room temperature.

Reconstitution of this lyophilized powder with water for injection provides a formulation for parenteral administration. For reconstitution, the lyophilized powder is added to sterile water or other suitable vehicle. The exact amount depends on the selected compound. This amount can be determined empirically.

The antibodies or antigen-binding portions thereof, the bispecific molecule or immunoconjugate described herein, and other compositions provided herein can also be formulated to target a specific tissue, receptor, or other area of the subject's body to be treated. Many such targeting methods are well known to those skilled in the art. All such targeting methods are contemplated herein for use in the present compositions. For non-limiting examples of targeting methods, see, for example, US Pat. США № 6316652, 6274552, 6271359, 6253872, 6139865, 6131570, 6120751, 6071495, 6060082, 6048736, 6039975, 6004534, 5985307, 5972366, 5900252, 5840674, 5759542 и 5709874. В конкретном воплощении антитело или его антигенсвязывающая часть, описанные в данном document, are aimed at the treatment of damage to the central nervous system, degenerative disease of the brain or neuropathic pain.

Compositions used for in vivo administration may be sterile. This is easily achieved by filtration, for example through sterile filter membranes.

XI. Sets

Provided herein are kits containing one or more of the antibodies described herein, or antigen-binding portions, bispecific molecules, or immunoconjugates thereof. In a specific embodiment, provided herein is a pharmaceutical package or kit comprising one or more containers filled with one or more ingredients of the pharmaceutical compositions described herein, such as one or more of the antibodies provided herein, or an antigen-binding portion thereof, optionally instructions for use. In some embodiments, the kits contain the pharmaceutical composition described herein and any prophylactic or therapeutic agent, such as those described herein.

XII. Therapeutic Uses and Methods

Also provided herein are methods of mitigating CNS injury or injury in a subject (eg, human) in need thereof, comprising administering to the subject an anti-FAM19A5 antibody, bispecific molecule, or immunoconjugate described herein, or a composition thereof.

In other aspects provided herein, methods of inhibiting, slowing, suppressing, containing, reducing, reversing or preventing the onset or initiation of gliosis and associated detrimental effects on the CNS in a subject are provided, comprising administering to the subject an anti-FAM19A5 antibody described herein. document. In some embodiments, provided herein are methods of inhibiting, slowing, suppressing, containing, reducing, reversing, or preventing excessive or abnormal proliferation of reactive astrocytes and associated detrimental CNS effects in a subject, comprising administering to the subject an anti-FAM19A5 antibody of the present disclosure. In some embodiments, provided herein are methods of reducing, inhibiting, or reducing the expression of chondroitin sulfate proteoglycans (including the level of Neurocan, NG2, or both), or reducing the activity of, or turning into an inactive Neurocan, NG2, or both in an entity, comprising administering an anti-FAM19A5 antibody to the entity, described in this document. In some embodiments, provided herein are methods of stimulating, promoting, increasing, or activating neuronal growth, preferably after trauma or injury in a subject, comprising administering an anti-FAM19A5 antibody to the subject, as described herein. In other embodiments, provided herein are methods for increasing the activity level of c-fos mRNA, c-fos protein, or c-fos protein, as well as increasing the activity level of ERK mRNA, ERK protein, or pERK, preferably in the nucleus of neurons in an object in need thereof, comprising administering to the subject an anti-FAM19A5 antibody of the present disclosure. In certain embodiments, provided herein are methods for enhancing or increasing the level of GAP43 mRNA, GAP43 protein, or increasing GAP43 protein activity, preferably in neurons, in a subject in need thereof, comprising administering to the subject an anti-FAM19A5 antibody of the present disclosure. In certain embodiments, provided herein are methods of increasing or promoting neuronal survival and/or promoting axon regrowth in a subject in need thereof, comprising administering to the subject an anti-FAM19A5 antibody disclosed herein. In some embodiments, the subject is a human, preferably a human, having a neuronal lesion or injury, e.g., as a result of CNS injury, trauma, injury, cerebrospinal injury, brain tumor, infection, ischemia, stroke, autoimmune reactions, and/or neurodegenerative disease.

In some aspects, the present document also provides methods for treating a disease, disorder, or condition in a subject in need thereof, comprising administering to the subject an anti-FAM19A5 antibody according to the present disclosure. In some embodiments, the disease, disorder, or condition includes damage to the central nervous system, damage to the spinal system, degenerative brain disease, degenerative cerebrospinal or nerve disorder, or neuropathic pain. In some embodiments, the central nervous system injury is a traumatic brain injury, cerebrospinal injury, stroke, brain tumor, or a combination thereof. In some embodiments, the degenerative brain disease is Huntington's disease, Parkinson's disease, Alzheimer's disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS), or a combination thereof. Thus, in certain embodiments, disclosed herein is a method of treating a traumatic brain injury, cerebrospinal injury, stroke, brain tumor, or a combination thereof in a subject in need thereof, comprising administering to the subject the disclosed anti-FAM19A5 antibody disclosed herein, or their composition. In some embodiments, disclosed herein is a method of treating Huntington's disease, Parkinson's disease, Alzheimer's disease, multiple sclerosis, ALS in a subject in need thereof, comprising administering to the subject an anti-FAM19A5 antibody disclosed herein, or a composition thereof. In some embodiments, the object is a human.

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

In some embodiments, the disease, disorder, or condition includes a tumor, fibrosis, glaucoma, retinopathy, age-related macular degeneration, or a mood disorder. In some 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, colon adenocarcinoma, liver cancer, or ovarian cancer. .

In some embodiments, the anti-FAM19A5 antibody of the present disclosure causes normalization of blood vessels, for example, within a tumor. In some embodiments, the normalization of blood vessels is accompanied by changes in the properties of blood vessels, including increased connectivity, increased wall thickness, reduced vessel diameter, more correct direction and distribution of vessels, increased number of vessels, decreased leakage and permeability, increased pericyte coverage, and proximity to blood vessels, increased oxygenation, or combinations thereof.

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

In some embodiments, the anti-FAM19A5 antibody enhances immune cell entry into the tumor. In some embodiments, immune cell infiltration into the tumor is enhanced/increased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% by compared to a standard (for example, a subject with a malignant tumor who did not receive an anti-FAM19A5 antibody). In some embodiments, the immune cells comprise macrophages, dendritic cells, T lymphocytes, B lymphocytes, natural killer (NK) cells, or combinations thereof. In some embodiments, immune cells exhibit hypertrophy. In some embodiments, the infiltration of immune cells into the tumor is accompanied by increased infiltration of neuronal cells into the tumor. In some embodiments, neuronal cells include astrocytes, glial cells, or combinations thereof.

In some embodiments, the anti-FAM19A5 antibody of the present disclosure enhances the phagocytic activity of a macrophage or microglia. In some embodiments, the anti-FAM19A5 antibody increases the mitochondrial membrane potential of a macrophage or microglia. In some embodiments, phagocytic activity or mitochondrial membrane potential is increased or increased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to a standard (eg, a subject with a malignant tumor who did not receive an anti-FAM19A5 antibody).

In some embodiments, the anti-FAM19A5 antibody of the present disclosure reduces necrosis and edema in a tumor. In other embodiments, the anti-FAM19A5 antibody reduces tumor tissue permeability. In some embodiments, necrosis and edema or tumor tissue permeability is reduced by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to a standard (eg, a subject with a malignant tumor who did not receive an anti-FAM19A5 antibody).

In some embodiments, the anti-FAM19A5 antibody increases the rate of blood flow in the tumor. In some embodiments, the blood flow rate is increased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% compared to the standard (for example , a cancer subject who did not receive an anti-FAM19A5 antibody).

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

In some embodiments, a therapeutically effective amount of an anti-FAM19A5 antibody of the present disclosure or a composition thereof is administered. When an object (eg, human) is treated, the therapeutically effective amount of the anti-FAM19A5 antibody described herein depends on factors such as age, sex, disease severity.

In some embodiments, the anti-FAM19A5 antibody of the present disclosure, or composition thereof, is administered intravenously, orally, parenterally, transthecally, intrathecally, intracerebroventricularly, pulmonary, subcutaneously, intradermally, intramuscularly, or intraventricularly.

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

EXAMPLES

Example 1 Expression and Purification of Human FAM19A5 Protein

Recombinant human FAM19A5 protein was generated and purified as described below, and the purified protein was used in a binding affinity based antibody screening assay. First, the LPS-hT plasmid expressing the FAM19A5 gene was transformed into bacteria and overexpression of the protein was induced. Once obtained, the FAM19A5 protein was purified by Ni-NTA affinity chromatography (Qiagen, Valencia, CA, USA). Using a gradually increasing concentration of imidazole, the His-tagged FAM19A5 protein was removed from the Ni column. Protein expression in solution was measured using Coomassie Brilliant Blue R-250. The imidazole-containing solution that captured only FAM19A5 was used to concentrate the FAM19A5 protein with PBS. When the concentration was completed, both the purity and the concentration of the FAM19A5 protein were measured by Western blotting. The concentrated protein was subsequently used to screen for antibodies specific to FAM19A5.

Example 2 Preparation of Anti-FAM19a5 Antibody Libraries

1. Immunization

The FAM19A5 protein was used as an antigen to immunize white leghorn chickens. 50 μg of the KLH synthetic peptide conjugate was mixed with 750 μl of phosphate-buffered saline (PBS) and incubated at 37°C for 30 minutes. The toxin was then removed in 2% squalene-endotoxin MPL (monophosphorylated lipid A species) and mycobacteria (mycobacteria) cell wall components TDW and CWS containing water-in-oil emulsion adjuvant (RIBI + MPL + TDM + CWS. adjuvant, Sigma, St. Louis, Missouri, USA) in an emulsified form, which was then injected subcutaneously into chickens. Chickens were immunized a total of four times with an interval of approximately 2-3 weeks between immunizations. The titer of antibodies obtained from immunized animals was measured by immunoblotting using lysates of HEK293T cells that overexpressed the FAM19A5 protein.

2. Obtaining a Library of Single Chain Variable Fragments (scFv) from Immunized Chicken

Using the TRI reagent (Invitrogen, Carlsbad, CA, USA), RNA was extracted from the spleen, bone marrow, and bursa of Fabricius of the immunized chickens described above. Oligo-dT primers and Superscript™ III First Strand Synthesis System (Invitrogen) were used to synthesize first strand cDNA. For cDNA derived from animal immune systems, the Expand High Fidelity PCR System (Roche Molecular Systems, Indiana, USA) was used to generate a library of single chain variable regions. In each reaction, 1 µl of cDNA, 60 pmol of each primer, 10 µl of 10x reaction buffer, 8 µl of 25 mM dNTP (Promega, Madison, WI, USA) and 05 µl of Taq DNA polymerase were mixed with water. The final volume was 100 μl. The PCR reaction was performed using the following conditions: 30 cycles (i) 15 seconds at 94°C, (ii) 30 seconds at 56°C and (iii) 90 seconds at 72°C, followed by a final extension of 10 minutes at 72 °C PCR products containing a fragment of about 350 bp were loaded onto a 15% agarose gel and after electrophoresis, a QIAGEN Gel II extraction kit (QIAGEN, Valencia, CA, USA) was used to purify the nucleotide fragment. The purified PCR product was quantified by reading at OD 260 nm. (1 OD unit = 50 µg/mL).

The VH and VL of the first product from the second PCR were randomly linked by overlapping primer PCR (overlapping PCR). Each PCR reaction was mixed with 100 ng of purified VL and VH product, 60 pmol of each primer, 10 µl of 10x reaction buffer, 8 µl of 25 mM dNTP, 05 µl of Taq DNA polymerase and water in a final volume of 100 µl. PCR was performed under the following conditions: 25 cycles (i) 15 seconds at 94°C, (ii) 30 seconds at 56°C and (iii) 2 minutes at 72°C followed by a final extension of 10 minutes at 72°C. PCR products containing a single chain variable region fragment of about 700 bp were loaded onto a 15% agarose gel and after electrophoresis, the QIAGEN II Gel Extraction Kit (QIAGEN) was used to purify the nucleotide fragment. The purified PCR product was quantified by reading at OD 260 nm. (1 unit OD = 50/mL).

3. Library, ligation and transformation

The scFv fragment of the PCR product and the vector pComb3X - SS (Scripps Research Institute, California, USA) were digested with the restriction enzyme Sfi. 10 μg of the purified overlapping PCT product was mixed with 360 units of Sif I (μg DNA for 16 units, Roche Molecular Systems, Pleasanton, CA, USA), 20 μl of 10x reaction buffer and water to a final volume of 200 μl. 20 μg of pComb3X-SS vector was mixed with 120 units of Sfi I (μg of DNA for 6 units), 20 μl of 10x reaction buffer and water to a final volume of 200 μl. The mixture was hydrolyzed at 50°C for 8 hours. Thereafter, the digested product containing the scFv fragment (approximately 700 bp) and vector (approximately 3400 bp) was loaded onto a 1% agarose gel and purified using the QIAGEN Gel Extraction Kit II (QIAGEN, Valencia, CA, USA). 1400 ng of Sfi I-restricted vector pComb3X and 700 ng of digested scFv fragments were mixed with 5x ligation buffer, 10 μl of T4 DNA ligase (Invitrogen, Carlsbad, CA, USA) and water to a final volume of 200 μl. The mixture was incubated at 16°C for 16 hours for ligation.

After precipitation with ethanol, the DNA precipitate was dissolved in 15 µl of water. To create a library, a ligation sample was transformed into E. coli strain ER2738 (New England Biolabs Inc., Hitchin, North Hertfordshire, SG4 OTY, England, UK) by electroporation using a vibrator gene (Gene pulser: Bio-Rad Laboratories, Hercules, CA , USA). Cells were mixed in 5 ml Super Broth (SB) and incubated with shaking at 250 rpm for one hour at 37°C. Then 3 μl of 100 mg/ml kanamycin was added to 10 ml of SB medium. For library size determination, 01 μl, 1 μl and 10 μl of the culture sample were plated onto Luria Broth (LB) agar plates containing 50 μg/ml kanamycin. After stirring for 1 hour, 45 μl of 100 mg/ml kanamycin was added to the LB culture and stirred for an additional 1 hour. Then, 2 ml of VCM13 helper phage in water (>1011 CFU/ml) was added to the LB medium along with pre-warmed LB (183 ml) containing 925 µl of 100 mg/ml kanamycin. This mixture was stirred at 250 rpm at 37° C. for an additional 2 hours. Then 280 μl (50 mg/ml) kanamycin was added to the culture and stirred overnight at 37°C. The next day, the bacteria pellet was centrifuged using a high speed centrifuge (Beckman, JA-10 rotor) at 3000 g, 4°C. Thereafter, the bacterial pellet was used for phagemid DNA extraction, and the supernatant was transferred into sterile centrifuge vials. A further 8 grams of polyethylene glycol-8000 (PEG-8000, Sigma) and 6 grams of sodium chloride (NaCl, Merck) were added to the supernatant and then kept for 30 minutes on ice. After that, the supernatant was centrifuged for 15 minutes at 15000 g, 4°C. The supernatant was then discarded and the phage pellet suspended in Tris buffered saline (TBS) containing 1% BSA-recovery.

Example 3 Library panning (biopanning) on immobilized antigen

Biopanning was performed using magnetic beads (Dynabeads M-270 Epoxy, Invitrogen). At room temperature, approximately 1 x 10 ⁷ beads were coated with 5 μg of recombinant FAM19A5 protein by swirling the beads and protein together for 20 hours at room temperature. After coating, the beads were washed 4 times with phosphate buffered saline (PBS) and blocked for one hour in PBS containing 3% BSA at room temperature. The coated beads were then cultured for two hours at room temperature with the phage-presented scFv described above. To remove any phage that did not bind to the antigen coated beads, the beads were washed with 0.05% Tween20/PBS. Bound phages were then eluted with 50 µl of 01 M glycine/hydrogen chloride (01 M glycine-HCl, pH 22) and neutralized with 3 µl of 2 M Tris with hydrogen chloride (Tris-HCl, pH 91). These phage-containing supernatants were used to infect E. coli ER2738 cells and the helper phage VCSM13 was used for amplification and overnight rescue. Also, input (input) and production (output) titers of phage from phage-infected cultures were determined by blotting phage-infected cultures on LB agar plates containing 50 μg/ml kanamycin. The next day, PEG-8000 and NaCl were used to precipitate phages, which were subsequently used for biopanning. Biopanning was performed up to five times in total, repeating the process described above. At each amplification, phages were screened and selected for high affinity for the FAM19A5 protein.

Example 4 Clone selection by phage ELISA.

For analysis of clones selected by biopanning, individual clones were randomly selected from the phage-presented scFv and confirmed by ELISA that the clones bind to the recombinant FAM19A5 protein. Recombinant FAM19A5 protein was diluted in 01 M NaHCO3 buffer and 100 ng/well of protein was used to coat 96-well microtiter plates at 4° C. for 16 hours. The next day, the plates were blocked with 3% BSA/PBS at 37°C for 1 hour. The phage supernatant was then mixed with 6% BSA/PBS and cultured for 2 hours at 37°C. The plates containing the supernatant were then washed with 0.05% Tween-20/PBS. HRP-conjugated M13 antibody (a-M13-HRP, Pierce Chemical Co, Rockford, IL, USA) was diluted to 1/5000. 50 μl of the diluted antibody was added to the plates and incubated for 1 hour at 37°C. After incubation and washing, 005 M citrate buffer solution, 1 μg/ml 22'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS, Amresco, Solon, Ohio, USA) and 0.1% H2O2 for color development. The optical density for each well was measured at 405 nm.

Of the 96 clones that were initially identified, 8 scFv clones with unique heavy chain CDR3 (HCDR3) sequences and strong binding to the FAM19A5 protein were selected for further analysis. FIG. 1A-1C.

EXAMPLE 5 PRODUCTION OF ANTI-FAM19A5-IGG2/4 ANTIBODIES

The anti-FAM19A5 ScFv was subcloned into a mammalian expression vector. In the FAM19A5 scFv gene sequence, the human Cκ gene was fused to the light chain variable domain, and the isotypic human IgG2/4 immunoglobulin CH1, CH2 and CH3 genes were fused to the heavy chain variable region. An antibody having each light chain and each heavy chain was synthesized by adding restriction sites (Genscript, USA). The synthesized gene was inserted into a mammalian cell expression vector having a modified restriction site to facilitate cloning. First, the light chain gene was inserted into the vector using restriction enzymes Hind III and Xba I (New England Biolabs, UK), and then the heavy chain gene was added to the vector using restriction enzymes NheΙ and BamHΙ (New England Biolabs, UK).

Mammalian cell transfection and overexpression injection system was used to express and purify the anti-FAM19A5 antibody-IgG2/4. Approximately 2 μg/ml mammalian expression vector was mixed with 4 μg polyethyleneimine (PEI, Polysciences, Warrington, PA, USA) in 150 mM sodium chloride (NaCl, Merck), corresponding to 1/10 cell culture volume. The mixture was left for 15 minutes at room temperature. The mixture was added to HEK293F cells (2×10 ⁶ cells/ml, Invitrogen) which were then incubated in Freestyle™ 293 expression culture medium containing 100 U/ml penicillin and streptomycin (Invitrogen) at 7% CO2 and 37°C and stirred at 135 rpm for six days. Protein A bead affinity gel chromatography (RepliGen, Waltham, MA, USA) was used to purify expressed anti-FAM19A5 IgG2/4 antibodies from cell culture supernatant. Protein A chromatography was performed using 4~12% Bis-Tris gradient gel electrophoresis. Protein size and yield were confirmed by Coomassie brilliant blue staining. The binding capacity of the antibodies was measured using an ELISA assay.

As shown in FIG. 2A, the different antibodies tested (ie 1-28, 1-85, 2-13, 2-14, 2-20, 2-29, 3-2 and 3-26) were comparable in size. With the exception of antibody 1-85, the antibodies tested were able to bind to the FAM19A5 protein to varying degrees. Fig. 2b.

EXAMPLE 6. ANTI-FAM19A5 ANTIBODY NEUTRALIZATION ASSAY

The following methods were used to further evaluate the functional properties of antibodies:

1. Preparation of recombinant fusion protein FAM19A5 with rabbit Fc

To construct FAM19A5 expression vectors, the genes encoding human FAM19A5 were chemically synthesized (Genscript, Piscataway, NJ, USA). The gene was subcloned into a modified mammalian expression vector encoding the human IgG1 hinge region and rabbit IgG CH2-CH3 domains in the 3' region, as previously reported. See Han, J., et al., Exp Mol Med. 48(11):e271 (2016).

An expression vector encoding a rabbit Fc-FAM19A5 fusion was transfected into HEK293F cells (Invitrogen, Carlsbad, CA, USA) using a 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). Rabbit-FAM19A5 fusion protein was purified from culture supernatants of transiently transfected HEK293F cells using a protein A-sepharose column (Repligen, Waltham, MA, USA) according to the manufacturer's instructions.

2. Preparation of recombinant anti-FAM19A5 scFv fusion protein with human Cκ

The genes of selected clones were subcloned into a modified pCEP4 vector encoding a Cκ domain (human immunoglobulin light chain κ constant domain) in the 5' region as previously reported. See Lee, Y., et al., Exp Mol Med. 46:e114 (2014). Expression vectors encoding a human anti-FAM19A5 scFv fusion were transfected into HEK293F cells (Invitrogen) as described above. The scFv-hCκ fusion protein was purified from culture supernatants of transiently transfected HEK293F cells using a protein A-sepharose column (Repligen, Waltham, MA, USA) according to the manufacturer's instructions.

3. Neutralizing efficacy of anti-FAM19A5 antibodies on glial cells

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

As shown in FIG. 3A, all antibodies tested (i.e. 1-28, 1-85, 2-13, 2-14, 2-20, 2-29, 3-2, and 3-26) were able to inhibit FAM19A5 interaction with primary glial cells. mouse cells to varying degrees. In a human glioblastoma cell line, antibody 2-20 could not neutralize the interaction of FAM19A5 with the cell line. FIG. 3b. Other antibodies were able to neutralize FAM19A5 activity, albeit to varying degrees. Antibodies 1-28, 2-13 and 3-2 showed the highest neutralizing efficacy in the human glioblastoma cell line.

EXAMPLE 7. EPITOPE MAPPING ANALYSIS USING FAM19A5 F1-F6 EPITOPE FRAGMENTS

Because of its ability to neutralize FAM19A5 expression on both mouse and human glial cells, antibody 3-2 was chosen for epitope mapping analysis. Overlapping peptide fragments (F1-F6, see Fig. 4) of the human FAM19A5 protein were synthesized and conjugated to BSA. Binding of various anti-FAM19A5 antibodies to BSA-conjugated F1-F6 peptide fragments was determined by ELISA analysis. Briefly, FAM19A5 fragment F1-F6 (diluted to 1 µg/mL in 50 mM carbonate buffer (Biosesang) or to 20 µg/mL for high concentration assay) was used to coat wells of 96-well immunoplates (Thermo Scientific) (100 µL/well ) overnight at 4°C and then washed twice with 1X PBS. The plates were then blocked with blocking buffer (100 μl/well) for 1 hour at room temperature. During the 1 hour incubation, the corresponding anti-FAM19A5 antibodies were diluted to 1 μg/ml (or 20 μg/ml for high concentration assay) in dilution buffer. After washing the plates (2 times with 1x PBS), diluted anti-FAM19A5 antibodies were added to the appropriate wells and the plates were incubated at room temperature for 1 hour. The plates were then washed a total of five times with wash buffer. Then an ODP substrate (prepared by dissolving one tablet of ODP (O-phenylenediamine dihydrochloride, Thermo) in 9 ml of sterilized deionized water and 1 ml of 10-fold stable peroxide-stable buffer (Thermo)) was added to each well, and color change reactions were allowed to run for 10 minutes. This reaction was stopped by adding 100 µl of 2N to the wells. H2SO4 (Daejung). The absorbance value of each well was determined at 492 nm using a 96-well microplate reader (Molecular Device).

As shown in FIG. 5, antibody 3-2 binds strongly to a fragment of the F2 epitope with minimal binding to other fragments.

Scanning alanine analysis was then performed to identify specific amino acid residues within the F2 epitope fragment that antibody 3-2 binds to. As shown in FIG. 6A, when amino acid residues R4, D5, P9, R10, or R11 were changed to alanine, the ability of antibody 3-2 to bind to the F2 epitope fragment was significantly reduced. A similar analysis was performed for several deimmunized variants of the 3-2 antibody (see Example 8 for details on the deimmunization process). As shown in FIG. 6B, 6C, 6D, 6F, 6G, 6I and 6J amino acid residues R4, P9, R10 and R11 are important for the binding of antibodies 1-30, 1-32 and 6-10 to FAM19A5. For antibodies 1-17 and 4-11, the amino acid residues R4, P9 and R10 are important (see Fig. 6E and 6H).

EXAMPLE 8 ANTI-FAM19A5 ANTIBODY DEIMMUNIATION

1. Assessment of immunogenicity in silico

To reduce the risk of immunogenicity when administered to humans, an in silico assay was performed to identify specific regions of high immunogenicity within anti-FAM19A5 antibodies 3-2 and 2-13.

To identify promiscuous MHC class II binding peptides, iTopeTM (Abzena plc., UK) analysis was performed for overlapping 9-mer peptides spanning the entire sequence. Possible T cell epitopes were predicted by interaction analysis with 34 different MHC class II alleles. As shown in FIG. 7 and 8, respectively, clone 3-2 contained a total of 11 binding peptides, and clone 2-13 contained a total of 10 promiscuous, non-germline bound MHC class II peptides.

In addition, MHC class II promiscuous binding peptides were analyzed using TCEDTM (Abzena plc., UK), which is a database of CD4+ T cell epitopes generated by T cell stimulation assay against over 10,000 peptides. Clone 2-13 showed that the two promiscuous MHC class II binding peptides have high homology to known T cell epitopes. Fig. 8 (highlighted in green). Clone 3-2 had three promiscuous class II MHC binding peptides with high homology to known T cell epitopes. Fig. 7 (highlighted in green).

2. Creation of a composite library of antibodies

The antibody composite library was designed to avoid CD4+ T cell epitopes associated with immunogenicity. To select the most homologous human germline for library construction, the framework of clone 2-13 and clone 3-2 was analyzed in the IgBLAST database (NCBI). For clone 2-13, the human germline genes IGLV1-51*02, IGLJ2*01, IGHV3-64*04 and IGHJ1*01 were selected (FIGS. 9A and 9B). For clone 2-13, the human germline genes IGLV3-27*01, IGLJ2*01, IGHV3-64*04 and IGHJ1*01 were selected (FIG. 10).

The library was designed to replace certain non-identical amino acid residues in the MHC class II binding regions of clone 2-13 and clone 3-2 with the corresponding amino acid in the most homologous human germline sequence. To create a site-directed mutagenesis library, as previously described, sequential polymerase chain reaction (PCR) extension by overlapping oligonucleotides encoding degenerate codons covering both human and chicken amino acids was performed. Baek DS, Kim YS. Biochem Biophys Res Commun. 463(3):414-20 (2015). The PCR product was subcloned into a phagemid vector and transformed into Escherichia coli strain ER2738 (New England BioLabs, Ipswich, Massachusetts, USA) to produce phage as previously described. Han, J., et al., Exp Mol Med. 48(11):e271 (2016).

3. Biopanning and selection of clones

To isolate positive clones, biopanning and phage ELISA were performed as previously described (Barbas CF., Phage display: a laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 2001). Phage clones with reactivity against FAM19A5 were selected and their nucleotide sequences determined by Sanger sequencing. Finally, scFv clones with the fewest promiscuous MHC class II binding epitopes with retained affinity for FAM19A5 were selected.

As shown in FIG. 9B, 7 promiscuous class II MHC binding epitopes in clone 3-2 were deleted in deimmunized clone 3-2. The deleted epitopes correspond to binding peptides #2, 3, 4, 5, 9, 10 and 11 as shown in FIG. 7. Promiscuous epitopes binding MHC class II in CDRH1 could not be removed (ie binding peptide #6 in FIG. 7). In addition, the promiscuous MHC class II binding epitope in HFR2 could not be removed because the p3 residue located in CDRH2 plays a critical role in binding activity (ie binding peptide #8 in FIG. 7). As shown in FIG. 9C, complete deimmunization of antibody 3-2 by mutating all of the above residues resulted in antibody 3-2 being unable to bind to the FAM19A5 protein. However, when the Q50 VH amino acid residue was not mutated, the deimmunized 3-2 antibody was able to bind to the FAM19A5 protein, as was the wild-type 3-2 antibody.

As shown in FIG. 10A, 7 promiscuous class II MHC binding epitopes in clone 2-13 were deleted in deimmunized clone 2-13. The deleted epitopes correspond to binding peptides #1, 2, 3, 4, 5, 8 and 9 as shown in FIG. 8. The promiscuous MHC class II binding epitope in HFR2 could not be removed because the p4 residue located in CDRH2 plays a critical role in binding activity (ie, binding peptide #6 in FIG. 8). As with antibody 3-2, complete deimmunization of antibody 2-13 also negatively affects binding to the FAM19A5 protein. Fig. 10b.

The above results suggest that it is possible to reduce the immunogenicity of anti-FAM19A5 antibodies without adversely affecting their ability to bind to the FAM19A5 protein.

EXAMPLE 9 AFFINITY MATURATION OF A DEIMMUNE ANTIBODY 2-13

Electrostatic interaction is important in the association of antibody and antigen. Lee J.Y., et al., Nat Commun. 7:13354 (2016). Each amino acid in the 5 CDRs, except for the CDRH3 of deimmunized clone 2-13, was replaced with glutamic acid and aspartic acid to introduce artificially negatively charged R groups. A total of 70 genes encoding point mutated scFvs were chemically synthesized (Integrated DNA Technologies, Coralville, Iowa, USA) and subcloned into a phagemid vector to construct phage display mutant scFvs as previously described. Yoon, A., et al., PLoS One. 11(1):e0146907 (2016).

Charge variant scFv mutants presented by phage were rescued and subjected to phage immunoassay as previously described (Barbas CF. Phage display: a laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 2001). As shown in FIG. 11, all clones exhibited FAM19A5 binding activity to varying degrees. Two clones (36.H1-3TE and 37.H1-3TD) showed higher binding affinity than deimmunized clone 2-13. Comparison of the VH and VL sequences of (i) wild-type antibody 2-13, (ii) deimmunized clone 2-13 antibody, and (iii) deimmunized affinity matured antibody 2-13 (2-13D-37) are shown in FIG. fifteen.

EXAMPLE 10 AFFINITY MATURATION OF DEIMMUNE ANTIBODY 2-13D-37

To create CDR-H3 site-directed mutagenesis libraries, the gene encoding the deimmunized clone 2-13D-37 scFv was used as a template for PCR. Three consecutive residues in CDR-H3 were randomized with oligonucleotides encoding degenerate NNK codons (N = A, T, G or C, K = G or T) as previously described (Lee Y, Kim H, Chung J. Exp Mol Med 2014;46:e114). Randomized codons were introduced into CDR-H3 by PCR. The amplified scFv fragments by overlapping PCR were subcloned into a phagemid vector and transformed into ER2738 (New England BioLabs) to generate phage as described above. A total of eight randomized CDR-H3 libraries were created.

Biopanning and phage ELISA were performed to isolate positive clones as previously described (Barbas CF. Phage display: a laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 2001; see also Example 8). Phage clones with higher reactivity against FAM19A5 were selected and their nucleotide sequences determined by Sanger sequencing. The size and expression level of the candidate clones (ie 2-13D-37-1.5W-41 and 2-13D-37-3W-16) measured by SDS-PAGE and Western blotting are shown in FIG. 16B-16C. Sequence comparison of the candidate clones (ie 2-13D-37-1.5W-41 and 2-13D-37-3W-16) with deimmunized clone 2-13D-37 is shown in FIG. 17A. Compared to the 2-13D-37 antibody, these deimmunized affinity matured antibodies exhibited greater binding to the FAM19A5 protein as measured by ELISA (FIG. 17B).

EXAMPLE 11. IMPROVEMENT OF PHYSICO-CHEMICAL PROPERTIES OF DEIMMUNE ANTIBODY 3-2

To improve the physicochemical properties of the deimmunized anti-FAM19A5 antibodies, a phage display library was created using randomized sequences from light chain CDR1, CDR2 and CDR3 and from heavy chain CDR1 and CDR2 of antibody 3-2. To create CDR libraries for site-directed mutagenesis, the gene encoding the deimmunized clone 3-2 scFv was used as a template for PCR. Four or five consecutive residues in the CDR were randomized with oligonucleotides encoding degenerate NNK codons (N=A, T, G or C, K=G or T) as previously described. Lee, Y., et al., J. Exp Mol Med. 46:e114 (2014). Randomized codons were introduced into five CDRs except CDRH3 by PCR. The amplified scFv fragments by overlapping PCR were subcloned into a phagemid vector and transformed into ER2738 (New England BioLabs) to generate phage as described above. Fig. 12.

Biopanning and phage ELISA were performed to isolate positive clones as previously described (Barbas CF. Phage display: a laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 2001). Phage clones with reactivity against FAM19A5 were selected and their nucleotide sequences determined by Sanger sequencing. The intrinsic solubility score and the GRAVY score of individual clones were calculated as described previously. Sormanni, P., et al., Sci Rep. 7(1):8200 (2017). Clones with improved solubility were selected showing similar reactivity against FAM19A5: (i) low isoelectric point ("Low_PI") antibody, (ii) 1-17, (iii) 1-30, (iv) 1-32, (v) ) 4-11 and (vi) 6-10. The results are shown in FIG. 13A and 13B. Comparison of the VH and VL sequences of various deimmunized variants of antibodies 3-2 to wild-type antibodies 3-2 is shown in FIG. 14A and 14B, respectively.

EXAMPLE 12 HDX-MX ANTIBODY EPITOPE MAPPING 2-13

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) was used as described below to probe the human FAM19A5 binding epitope for anti-FAM19A5 antibody clone 2-13. See FIG. eighteen.

1. Optimization of conditions for maximum coverage of FAM19A5.

Prior to the epitope mapping experiments, experiments without deuteration were performed to generate a list of common peptides for the FAM19A5 protein. Offline and offline pepsin digestion was used to hydrolyze the FAM19A5 protein. Houde, D., et al., Methods Mol Biol 988: 269-89 (2013). In non-autonomous digestion, an immobilized pepsin column (~20° C.) was used and reaction efficiency was controlled by adjusting the flow rate. Offline, the FAM19A5 protein was manually digested with pepsin at 4°C for 5 minutes, and then the mixture was loaded onto a liquid chromatography column for analysis. To determine the maximum protein coverage condition, one or more of the following parameters were adjusted: potassium phosphate, sodium phosphate, quench dwell time, reducing agent (TCEP), urea concentration, and pepsin concentration. Waters PLGS software was used to analyze the raw mass spectrometry (MS) data obtained to determine if maximum protein coverage had been obtained. To confirm the reproducibility of the experiments, each of the experimental conditions was repeated at least twice, and the peptides that were obtained were confirmed. In FIG. 19 shows the various experimental conditions used and the percent coverage achieved.

Maximum coverage was observed under the following conditions: 1M TCEP, 2M urea (pH 266) and pepsin at a concentration of 1:2. As shown in FIG. 20A and 20B, using these conditions, 44 peptide fragments were identified that covered approximately 97% of the FAM19A5 protein.

2. FAM19A5 epitope mapping

To obtain maximum (100%) binding (KD = 1 nM), even at 15-fold dilution of the antibody-protein complex in labeling buffer, samples of the antigen-antibody complex were incubated for at least 3 hours before labeling with hydrogen/deuterium. After labeling, the samples were brought to the same volume using an equilibrium buffer.

To start the labeling reaction, 25 µl of prepared samples (antigen only, antibody only, or antigen-antibody complex) diluted 1:15 in labeling buffer were mixed with D2O labeling solution (18 µM). Reactions were run for different time periods: 0 min (ie non-deuterated), 20 seconds, 10 minutes, 60 minutes and 240 minutes. For the non-deuterated reaction, the prepared sample was instead mixed with the equilibration buffer. At the end of each labeling period, the reaction was stopped with quenching buffer. Then the samples were shaken, immediately frozen in liquid nitrogen and stored at -80°C until analysis.

Prior to analysis with a mass spectrometer, the stored frozen samples were allowed to melt and then digested with pepsin for about 5 minutes on ice (i.e. the offline method described above). Relative deuterium levels obtained were plotted against exchange time using DynamX 3.0™ software (Waters). The final coverage and sequence redundancy data is shown in FIG. 21.

Comparison of deuterium uptake data (single antigen/antibody versus antigen-antibody complex) for each of the identified peptides (see FIG. 21) is shown in FIG. 22A-22E and 23A-23C. As shown, significant deuterium uptake was observed for the following peptide sequences: (i) ACRKGQIAGTTRARPAC (residues 37-53 of SEQ ID NO: 101), (ii) ACRKGQIAGTTRARPACVD (residues 37-55 of SEQ ID NO: 101), (iii) ACRKGQIAGTTRARPACVDA (residues 37-56 SEQ ID NO: 101), (iv) ARIIKTKQWC (residues 56-65 SEQ ID NO: 101), (v) ARIIKTKQWCDM (residues 56-67 SEQ ID NO: 101), (vi) ARIIKTKQWCDML (residues 56- 68 SEQ ID NO: 101), (vii) ARIIKTKQWCDMLPCL (residues 56-71 of SEQ ID NO: 101), (viii) RIIKTKQWCDM (residues 57-67 of SEQ ID NO: 101), and (ix) RIIKTKQWCDML (residues 57-68 SEQ ID NO: 101).

Using the above results, a heat map was constructed to identify regions in the FAM19A5 protein with the most significant difference in deuterium uptake between a single antigen/antibody sample and an antigen-antibody complex. As shown in FIG. 24 amino acid residues 38-50 (CRKGQIAGTTRAR) and 51-64 (PACVDARIIKTKQW) of SEQ ID NO: 101 were identified as important binding residues for antibody 2-13. In FIG. 25 shows the location of these residues in the three-dimensional structure of the FAM19A5 protein.

It should be understood that the "Detailed Description" section, and not the "Summary" and "Abstract" sections, is intended to be used to interpret the claims. The Summary and Abstract sections may set forth one or more, but not all, of the exemplary embodiments of the present disclosure contemplated by the author(s), and thus are not intended to limit the present disclosure and the appended claims in any way.

The present disclosure has been described above in terms of functional building blocks illustrating the implementation of certain functions and their relationships. The boundaries of these functional building blocks have been arbitrarily defined herein for convenience of description. Alternative boundaries may be defined provided that the specified functions and their interrelationships are properly performed.

The foregoing description of specific embodiments will so fully convey the general nature of the disclosure that others may, by applying knowledge within skill in the art, easily modify and/or adapt such specific embodiments for various applications without undue experimentation without deviating from the general concept of the present disclosure. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments based on the intent and guidance provided herein. It should be understood that the phraseology or terminology in this document is intended to be descriptive and not restrictive, so that the terminology or phraseology of the present description should be interpreted by the skilled artisan in light of the intent and guidance.

The breadth and scope of the present disclosure should not be limited by any of the exemplary embodiments described above, but should only be determined in accordance with the following claims and their equivalents.

All publications, patents, patent applications, Internet sites, and database access numbers/sequences (including both polynucleotide and polypeptide sequences) cited herein are incorporated herein by reference in their entirety for all purposes to the same extent. , as if each individual publication, patent, patent application, website, or database access number/sequence were specifically and individually designated for inclusion by reference.

--->

SEQUENCE LIST

<110> NEWRECL SCIENCE CO., LTD.

SEOUL NATIONAL UNIVERSITY ArandDB FOUNDATION

<120> ANTIBODY AGAINST FAMILY 19 REPRESENTATIVE A5 WITH LIKENESS

SEQUENCES AND METHOD OF THEIR APPLICATION

<130> 3763.016PC02/C-K/DKC

<150> US 62/787,711

<151> 2019-01-02

<150> US 62/838,190

<151> 2019-04-24

<160> 137

<170>PatentIn version 3.5

<210> 1

<211> 132

<212> PRT

<213> Homo sapiens

<400> 1

Met Ala Pro Ser Pro Arg Thr Gly Ser Arg Gln Asp Ala Thr Ala Leu

1 5 10 15

Pro Ser Met Ser Ser Thr Phe Trp Ala Phe Met Ile Leu Ala Ser Leu

20 25 30

Leu Ile Ala Tyr Cys Ser Gln Leu Ala Ala Gly Thr Cys Glu Ile Val

35 40 45

Thr Leu Asp Arg Asp Ser Ser Gln Pro Arg Arg Thr Ile Ala Arg Gln

50 55 60

Thr Ala Arg Cys Ala Cys Arg Lys Gly Gln Ile Ala Gly Thr Thr Arg

65 70 75 80

Ala Arg Pro Ala Cys Val Asp Ala Arg Ile Ile Lys Thr Lys Gln Trp

85 90 95

Cys Asp Met Leu Pro Cys Leu Glu Gly Glu Gly Cys Asp Leu Leu Ile

100 105 110

Asn Arg Ser Gly Trp Thr Cys Thr Gln Pro Gly Gly Arg Ile Lys Thr

115 120 125

Thr Thr Val Ser

130

<210> 2

<211> 125

<212> PRT

<213> Homo sapiens

<400> 2

Met Gln Leu Leu Lys Ala Leu Trp Ala Leu Ala Gly Ala Ala Leu Cys

1 5 10 15

Cys Phe Leu Val Leu Val Ile His Ala Gln Phe Leu Lys Glu Gly Gln

20 25 30

Leu Ala Ala Gly Thr Cys Glu Ile Val Thr Leu Asp Arg Asp Ser Ser

35 40 45

Gln Pro Arg Arg Thr Ile Ala Arg Gln Thr Ala Arg Cys Ala Cys Arg

50 55 60

Lys Gly Gln Ile Ala Gly Thr Thr Arg Ala Arg Pro Ala Cys Val Asp

65 70 75 80

Ala Arg Ile Ile Lys Thr Lys Gln Trp Cys Asp Met Leu Pro Cys Leu

85 90 95

Glu Gly Glu Gly Cys Asp Leu Leu Ile Asn Arg Ser Gly Trp Thr Cys

100 105 110

Thr Gln Pro Gly Gly Arg Ile Lys Thr Thr Thr Val Ser

115 120 125

<210> 3

<211> 53

<212> PRT

<213> Homo sapiens

<400> 3

Met Tyr His His Arg Glu Trp Pro Ala Arg Ile Ile Lys Thr Lys Gln

1 5 10 15

Trp Cys Asp Met Leu Pro Cys Leu Glu Gly Glu Gly Cys Asp Leu Leu

20 25 30

Ile Asn Arg Ser Gly Trp Thr Cys Thr Gln Pro Gly Gly Arg Ile Lys

35 40 45

Thr Thr Thr Val Ser

fifty

<210> 4

<211> 1501

<212> DNA

<213> Homo sapiens

<400> 4

ggcggcggag gatggcgcgc gcggggcccg cacgtggagg ccggcgcggg ggcgcgggca 60

gggccggctg ctgagacgcg ctgctgcccc ccgcgcgggc gccgcggctt caatggcgcc 120

atcgcccagg accggcagcc ggcaagatgc gaccgccctg cccagcatgt cctcaacttt 180

ctgggcgttc atgatcctgg ccagcctgct catcgcctac tgcagtcagc tggccgccgg 240

cacctgtgag attgtgacct tggaccggga cagcagccag cctcggagga cgatcgcccg 300

gcagaccgcc cgctgtgcgt gtagaaaggg gcagatcgcc ggcaccacga gagcccggcc 360

cgcctgtgtg gacgcaagaa tcatcaagac caagcagtgg tgtgacatgc ttccgtgtct 420

ggagggggaa ggctgcgact tgttaatcaa ccggtcaggc tggacgtgca cgcagcccgg 480

cgggaggata aagaccacca cggtctcctg acaaacacag cccctgaggg ggccccggga 540

gtggccttgg ctccctggag agccccgtc tcagccacag ttctccactc gcctcggact 600

tcacccgttc tctgccgccc gcccactccg tttccctgtg gtccgtgaag gacggcctca 660

ggccttggca tcctgagctt cggtctgtcc agccgacccg aggaggccgg actcagacac 720

atacgcgggg ggcggcacct ggcatcagca atacgcagtc tgtgggagcc cggccgcgcc 780

cagcccccgc cgaccgtggc gttggccctg ctgtcctcag aggaggagga ggaggaggca 840

gctccggcag ccacagaagg ctgcagccca gcccgcctga gacacgacgc ctgccccagg 900

ggactgtcag gcacagaagc ggcctcctcc cgtgccccag actgtccgaa ttgcttttat 960

tttcttatac tttcagtata ctccatagac caaaagagcaa aatctatctg aacctggacg 1020

caccctcact gtcagggtcc ctggggtcgc ttgtgcgggc gggagggcaa tggtggcaga 1080

gacatgctgg tggccccggc ggagcggaga gggcggccgt ggtggaggcc tccaccccag 1140

gagcaccccg cacaccctcg gaggacgggc ttcggctgcg cggaggccgt ggcacacctg 1200

cgggaggcag cgacggcccc cacgcagacg ccgggaacgc aggccgcttt attcctctgt 1260

acttagatca acttgaccgt actaaaatcc ctttctgttt taaccagtta aacatgcctc 1320

ttctacagct ccatttttga tagttggata atccagtatc tgccaagagc atgttgggtc 1380

tcccgtgact gctgcctcat cgatacccca tttagctcca gaaagcaaag aaaactcgag 1440

taacacttgt ttgaaagaga tcattaaatg tattttgcaa agcccaaaaa aaaaaaaaaa 1500

a 1501

<210> 5

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy Chain CDR1 - 3-2; 1-7A-IT Abs

<400> 5

Gly Phe Thr Phe Ser Ser Phe Asn Met Phe

1 5 10

<210> 6

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy Chain CDR2 - 3-2 Ab

<400> 6

Gln Ile Ser Ser Ser Gly Ser Ser Thr Asn Tyr Ala Pro Ala Val Arg

1 5 10 15

gly

<210> 7

<211> 22

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy Chain CDR3 - 3-2; 1-7A-IT; Low-PI 1-30; 1-17; 1-32; 4-11;

6-10 Abs

<400> 7

Ser Ser Tyr Asp Cys Pro Tyr Gly His Cys Ser Ser Gly Val Asp Ser

1 5 10 15

Ala Gly Glu Ile Asp Ala

twenty

<210> 8

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> Light Chain CDR1 - 3-2; 1-7A-IT; 1-17; 1-32; 4-11; 6-10 Abs

<400> 8

Ser Gly Gly Gly Ser Tyr Ala Gly Ser Tyr Tyr Tyr Gly

1 5 10

<210> 9

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light Chain CDR2 - 3-2 Ab

<400> 9

Glu Ser Asn Lys Arg Pro Ser

fifteen

<210> 10

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Light Chain CDR3 - 3-2; 1-7A-IT Abs

<400> 10

Gly Ser Trp Asp Ser Ser Asn Gly Gly Ile

1 5 10

<210> 11

<211> 131

<212> PRT

<213> Artificial Sequence

<220>

<223> VH - 3-2 Ab

<400> 11

Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly

1 5 10 15

Ala Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

Asn Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val

35 40 45

Ala Gln Ile Ser Ser Ser Gly Ser Ser Thr Asn Tyr Ala Pro Ala Val

50 55 60

Arg Gly Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Val Arg

65 70 75 80

Leu Gln Leu Asn Asn Pro Gly Ala Glu Asp Thr Gly Thr Tyr Tyr Cys

85 90 95

Ala Lys Ser Ser Tyr Asp Cys Pro Tyr Gly His Cys Ser Ser Gly Val

100 105 110

Asp Ser Ala Gly Glu Ile Asp Ala Trp Gly His Gly Thr Glu Val Ile

115 120 125

Val Ser Ser

130

<210> 12

<211> 108

<212> PRT

<213> Artificial Sequence

<220>

<223> VL - 3-2 Ab

<400> 12

Ala Leu Thr Gln Pro Ser Ser Val Ser Ala Asn Pro Gly Glu Thr Val

1 5 10 15

Lys Ile Thr Cys Ser Gly Gly Gly Ser Tyr Ala Gly Ser Tyr Tyr Tyr

20 25 30

Gly Trp Tyr Gln Gln Lys Ala Pro Gly Ser Ala Pro Val Thr Leu Ile

35 40 45

Tyr Glu Ser Asn Lys Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Thr Ser Gly Ser Thr Ala Thr Leu Thr Ile Thr Gly Val Gln Ala

65 70 75 80

Asp Asp Glu Ala Ile Tyr Tyr Cys Gly Ser Trp Asp Ser Ser Asn Gly

85 90 95

Gly Ile Phe Gly Ala Gly Thr Thr Leu Thr Val Leu

100 105

<210> 13

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy Chain CDR2 - 1-7A-IT Ab

<400> 13

Gln Ile Ser Ser Ser Gly Ser Ser Thr Asn Tyr Ala Pro Ala Val Lys

1 5 10 15

gly

<210> 14

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy Chain CDR1 - Low-PI; 1-30; 1-17; 1-32; 4-11; 6-10 Abs

<400> 14

Gly Phe Asp Phe Glu Ser Phe Asn Met Phe

1 5 10

<210> 15

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy Chain CDR2 - Low-PI; 1-30; 1-17; 1-32; 4-11; 6-10 Abs

<400> 15

Gln Ile Ser Ser Ser Glu Glu Asp Glu Asn Tyr Ala Pro Ala Val Lys

1 5 10 15

gly

<210> 16

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy Chain CDR1 - 2-13; 2-13D Abs

<400> 16

Gly Phe Thr Phe Ser His Gly Met Phe

1 5 10

<210> 17

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy Chain CDR2 - 2-13; 2-13D; 2-13D-37 Abs

<400> 17

Glu Ile Thr Asn Asp Gly Ser Gly Thr Asn Tyr Gly Ser Ala Val Lys

1 5 10 15

gly

<210> 18

<211> 20

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy Chain CDR3 - 2-13; 2-13D; 2-13D-37 Abs

<400> 18

Ser Thr Tyr Glu Cys Pro Gly Gly Phe Ser Cys Trp Gly Asp Thr Gly

1 5 10 15

Gln Ile Asp Ala

twenty

<210> 19

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy Chain CDR1 - 2-13D-37 Ab

<400> 19

Gly Phe Asp Phe Ser Ser His Gly Met Phe

1 5 10

<210> 20

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light Chain CDR2 - 1-7A-IT

<400> 20

Glu Asn Asn Lys Arg Pro Ser

fifteen

<210> 21

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> Light Chain CDR1 - Low-PI; 1-30 Abs

<400> 21

Ser Gly Gly Gly Ser Glu Glu Glu Gln Tyr Tyr Tyr Gly

1 5 10

<210> 22

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light Chain CDR2 - Low-PI Ab

<400> 22

Glu Asp Glu Glu Arg Pro Ser

fifteen

<210> 23

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Light Chain CDR3 - Low-PI; 1-30; 1-17; 1-32 Abs

<400> 23

Gly Ser Trp Asp Ser Glu Asp Glu Asp His

1 5 10

<210> 24

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light Chain CDR2 - 1-30; 1-32 Abs

<400> 24

Gln Asp Glu Glu Arg Pro Ser

fifteen

<210> 25

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light Chain CDR2 - 1-17 Ab

<400> 25

Glu Asp Glu Gln Arg Pro Ser

fifteen

<210> 26

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light Chain CDR2 - 4-11 Ab

<400> 26

Glu Asp His Glu Arg Pro Ser

fifteen

<210> 27

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Light Chain CDR3 - 4-11 Ab

<400> 27

Gly Ser Trp Asp Ser Ser Asp Glu Asp His

1 5 10

<210> 28

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light Chain CDR2 - 6-10 Ab

<400> 28

Gln Asp Leu Leu Arg Pro Ser

fifteen

<210> 29

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Light Chain CDR3 - 6-10 Ab

<400> 29

Gly Ser Trp Asp Ser Leu Ser Ser Ser His

1 5 10

<210> 30

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Light Chain CDR1 - 2-13 Ab

<400> 30

Ser Gly Gly Ser Tyr Ser Tyr Gly

fifteen

<210> 31

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light Chain CDR2 - 2-13; 2-13D; 2-13D-37 Abs

<400> 31

Trp Asp Asp Glu Arg Pro Ser

fifteen

<210> 32

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Light Chain CDR3 - 2-13; 2-13D; 2-13D-37 Abs

<400> 32

Gly Thr Glu Asp Ile Ser Gly Thr Ala Gly Val

1 5 10

<210> 33

<211> 131

<212> PRT

<213> Artificial Sequence

<220>

<223> VH - 1-7A-IT Ab

<400> 33

Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly

1 5 10 15

Ala Leu Arg Leu Ser Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

Asn Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val

35 40 45

Ser Gln Ile Ser Ser Ser Gly Ser Ser Thr Asn Tyr Ala Pro Ala Val

50 55 60

Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Gly Thr Tyr Tyr Cys

85 90 95

Ala Lys Ser Ser Tyr Asp Cys Pro Tyr Gly His Cys Ser Ser Gly Val

100 105 110

Asp Ser Ala Gly Glu Ile Asp Ala Trp Gly His Gly Thr Glu Val Ile

115 120 125

Val Ser Ser

130

<210> 34

<211> 131

<212> PRT

<213> Artificial Sequence

<220>

<223> VH - Low-PI; 1-30; 1-17; 1-32; 4-11; 6-10 Abs

<400> 34

Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly

1 5 10 15

Ala Leu Arg Leu Ser Cys Lys Ala Ser Gly Phe Asp Phe Glu Ser Phe

20 25 30

Asn Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val

35 40 45

Ser Gln Ile Ser Ser Ser Glu Glu Asp Glu Asn Tyr Ala Pro Ala Val

50 55 60

Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Gly Thr Tyr Tyr Cys

85 90 95

Ala Lys Ser Ser Tyr Asp Cys Pro Tyr Gly His Cys Ser Ser Gly Val

100 105 110

Asp Ser Ala Gly Glu Ile Asp Ala Trp Gly His Gly Thr Glu Val Ile

115 120 125

Val Ser Ser

130

<210> 35

<211> 129

<212> PRT

<213> Artificial Sequence

<220>

<223> VH - 2-13 Ab

<400> 35

Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly

1 5 10 15

Ala Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser His

20 25 30

Gly Met Phe Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Tyr Val

35 40 45

Ala Glu Ile Thr Asn Asp Gly Ser Gly Thr Asn Tyr Gly Ser Ala Val

50 55 60

Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Val Arg

65 70 75 80

Leu Gln Leu Asn Asn Leu Arg Ala Glu Asp Thr Gly Thr Tyr Phe Cys

85 90 95

Ala Arg Ser Thr Tyr Glu Cys Pro Gly Gly Phe Ser Cys Trp Gly Asp

100 105 110

Thr Gly Gln Ile Asp Ala Trp Gly His Gly Thr Glu Val Ile Val Ser

115 120 125

Ser

<210> 36

<211> 129

<212> PRT

<213> Artificial Sequence

<220>

<223> VH - 2-13D Ab

<400> 36

Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly

1 5 10 15

Ala Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Thr Phe Ser Ser His

20 25 30

Gly Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val

35 40 45

Ser Glu Ile Thr Asn Asp Gly Ser Gly Thr Asn Tyr Gly Ser Ala Val

50 55 60

Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Gly Thr Tyr Phe Cys

85 90 95

Ala Arg Ser Thr Tyr Glu Cys Pro Gly Gly Phe Ser Cys Trp Gly Asp

100 105 110

Thr Gly Gln Ile Asp Ala Trp Gly His Gly Thr Glu Val Ile Val Ser

115 120 125

Ser

<210> 37

<211> 129

<212> PRT

<213> Artificial Sequence

<220>

<223> VH - 2-13D-37 Ab

<400> 37

Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly

1 5 10 15

Ala Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Asp Phe Ser Ser His

20 25 30

Gly Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val

35 40 45

Ser Glu Ile Thr Asn Asp Gly Ser Gly Thr Asn Tyr Gly Ser Ala Val

50 55 60

Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Gly Thr Tyr Phe Cys

85 90 95

Ala Arg Ser Thr Tyr Glu Cys Pro Gly Gly Phe Ser Cys Trp Gly Asp

100 105 110

Thr Gly Gln Ile Asp Ala Trp Gly His Gly Thr Glu Val Ile Val Ser

115 120 125

Ser

<210> 38

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> VL - 1-7A-IT Ab

<400> 38

Ala Leu Thr Gln Pro Ser Ser Val Ser Ala Asn Pro Gly Glu Thr Val

1 5 10 15

Lys Ile Thr Cys Ser Gly Gly Gly Ser Tyr Ala Gly Ser Tyr Tyr Tyr

20 25 30

Gly Trp Tyr Gln Gln Lys Pro Gly Ser Ala Pro Val Thr Leu Ile Tyr

35 40 45

Glu Asn Asn Lys Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly Ser

50 55 60

Thr Ser Gly Ser Thr Ala Thr Leu Thr Ile Thr Gly Val Gln Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gly Ser Trp Asp Ser Ser Asn Gly Gly

85 90 95

Ile Phe Gly Ala Gly Thr Thr Leu Thr Val Leu

100 105

<210> 39

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> VL - Low-PI Ab

<400> 39

Ala Leu Thr Gln Pro Ser Ser Val Ser Ala Asn Pro Gly Glu Thr Val

1 5 10 15

Lys Ile Thr Cys Ser Gly Gly Gly Ser Glu Glu Glu Gln Tyr Tyr Tyr

20 25 30

Gly Trp Tyr Gln Gln Lys Pro Gly Ser Ala Pro Val Thr Leu Ile Tyr

35 40 45

Glu Asp Glu Glu Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly Ser

50 55 60

Thr Ser Gly Ser Thr Ala Thr Leu Thr Ile Thr Gly Val Gln Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gly Ser Trp Asp Ser Glu Asp Glu Asp

85 90 95

His Phe Gly Ala Gly Thr Thr Leu Thr Val Leu

100 105

<210> 40

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> VL - 1-30 Ab

<400> 40

Ala Leu Thr Gln Pro Ser Ser Val Ser Ala Asn Pro Gly Glu Thr Val

1 5 10 15

Lys Ile Thr Cys Ser Gly Gly Gly Ser Glu Glu Glu Gln Tyr Tyr Tyr

20 25 30

Gly Trp Tyr Gln Gln Lys Pro Gly Ser Ala Pro Val Thr Leu Ile Tyr

35 40 45

Gln Asp Glu Glu Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly Ser

50 55 60

Thr Ser Gly Ser Thr Ala Thr Leu Thr Ile Thr Gly Val Gln Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gly Ser Trp Asp Ser Glu Asp Glu Asp

85 90 95

His Phe Gly Ala Gly Thr Thr Leu Thr Val Leu

100 105

<210> 41

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> VL - 1-17 Ab

<400> 41

Ala Leu Thr Gln Pro Ser Ser Val Ser Ala Asn Pro Gly Glu Thr Val

1 5 10 15

Lys Ile Thr Cys Ser Gly Gly Gly Ser Tyr Ala Gly Ser Tyr Tyr Tyr

20 25 30

Gly Trp Tyr Gln Gln Lys Pro Gly Ser Ala Pro Val Thr Leu Ile Tyr

35 40 45

Glu Asp Glu Gln Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly Ser

50 55 60

Thr Ser Gly Ser Thr Ala Thr Leu Thr Ile Thr Gly Val Gln Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gly Ser Trp Asp Ser Glu Asp Glu Asp

85 90 95

His Phe Gly Ala Gly Thr Thr Leu Thr Val Leu

100 105

<210> 42

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> VL - 1-32 Ab

<400> 42

Ala Leu Thr Gln Pro Ser Ser Val Ser Ala Asn Pro Gly Glu Thr Val

1 5 10 15

Lys Ile Thr Cys Ser Gly Gly Gly Ser Tyr Ala Gly Ser Tyr Tyr Tyr

20 25 30

Gly Trp Tyr Gln Gln Lys Pro Gly Ser Ala Pro Val Thr Leu Ile Tyr

35 40 45

Gln Asp Glu Glu Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly Ser

50 55 60

Thr Ser Gly Ser Thr Ala Thr Leu Thr Ile Thr Gly Val Gln Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gly Ser Trp Asp Ser Glu Asp Glu Asp

85 90 95

His Phe Gly Ala Gly Thr Thr Leu Thr Val Leu

100 105

<210> 43

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> VL - 4-11 Ab

<400> 43

Ala Leu Thr Gln Pro Ser Ser Val Ser Ala Asn Pro Gly Glu Thr Val

1 5 10 15

Lys Ile Thr Cys Ser Gly Gly Gly Ser Tyr Ala Gly Ser Tyr Tyr Tyr

20 25 30

Gly Trp Tyr Gln Gln Lys Pro Gly Ser Ala Pro Val Thr Leu Ile Tyr

35 40 45

Glu Asp His Glu Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly Ser

50 55 60

Thr Ser Gly Ser Thr Ala Thr Leu Thr Ile Thr Gly Val Gln Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gly Ser Trp Asp Ser Ser Asp Glu Asp

85 90 95

His Phe Gly Ala Gly Thr Thr Leu Thr Val Leu

100 105

<210> 44

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> VL - 6-10 Ab

<400> 44

Ala Leu Thr Gln Pro Ser Ser Val Ser Ala Asn Pro Gly Glu Thr Val

1 5 10 15

Lys Ile Thr Cys Ser Gly Gly Gly Ser Tyr Ala Gly Ser Tyr Tyr Tyr

20 25 30

Gly Trp Tyr Gln Gln Lys Pro Gly Ser Ala Pro Val Thr Leu Ile Tyr

35 40 45

Gln Asp Leu Leu Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly Ser

50 55 60

Thr Ser Gly Ser Thr Ala Thr Leu Thr Ile Thr Gly Val Gln Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gly Ser Trp Asp Ser Leu Ser Ser Ser

85 90 95

His Phe Gly Ala Gly Thr Thr Leu Thr Val Leu

100 105

<210> 45

<211> 104

<212> PRT

<213> Artificial Sequence

<220>

<223> VL - 2-13 Ab

<400> 45

Ala Leu Thr Gln Pro Ser Ser Val Ser Ala Asn Pro Gly Glu Thr Val

1 5 10 15

Lys Ile Thr Cys Ser Gly Gly Ser Tyr Ser Tyr Gly Trp Phe Gln Gln

20 25 30

Lys Ser Pro Gly Ser Ala Leu Val Thr Val Ile Tyr Trp Asp Asp Glu

35 40 45

Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly Ala Leu Ser Gly Ser

50 55 60

Thr Asn Thr Leu Thr Ile Thr Gly Val Gln Ala Asp Asp Glu Ala Val

65 70 75 80

Tyr Phe Cys Gly Thr Glu Asp Ile Ser Gly Thr Ala Gly Val Phe Gly

85 90 95

Ala Gly Thr Thr Leu Thr Val Leu

100

<210> 46

<211> 103

<212> PRT

<213> Artificial Sequence

<220>

<223> VL - 2-13D; 2-13D-37 Abs

<400> 46

Ala Leu Thr Gln Pro Ser Ser Val Ser Ala Asn Pro Gly Glu Thr Ala

1 5 10 15

Lys Ile Thr Cys Ser Gly Val Tyr Ser Tyr Gly Trp Phe Gln Gln

20 25 30

Lys Pro Gly Ser Ala Leu Val Thr Val Ile Tyr Trp Asp Asp Glu Arg

35 40 45

Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly Ala Leu Ser Gly Ser Thr

50 55 60

Asn Thr Leu Thr Ile Thr Gly Val Gln Ala Glu Asp Glu Ala Asp Tyr

65 70 75 80

Tyr Cys Gly Thr Glu Asp Ile Ser Gly Thr Ala Gly Val Phe Gly Ala

85 90 95

Gly Thr Thr Leu Thr Val Leu

100

<210> 47

<211> 257

<212> PRT

<213> Artificial Sequence

<220>

<223> scFv - 3-2 Ab

<400> 47

Ala Leu Thr Gln Pro Ser Ser Val Ser Ala Asn Pro Gly Glu Thr Val

1 5 10 15

Lys Ile Thr Cys Ser Gly Gly Gly Ser Tyr Ala Gly Ser Tyr Tyr Tyr

20 25 30

Gly Trp Tyr Gln Gln Lys Ala Pro Gly Ser Ala Pro Val Thr Leu Ile

35 40 45

Tyr Glu Ser Asn Lys Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Thr Ser Gly Ser Thr Ala Thr Leu Thr Ile Thr Gly Val Gln Ala

65 70 75 80

Asp Asp Glu Ala Ile Tyr Tyr Cys Gly Ser Trp Asp Ser Ser Asn Gly

85 90 95

Gly Ile Phe Gly Ala Gly Thr Thr Leu Thr Val Leu Gly Gln Ser Ser

100 105 110

Arg Ser Ser Gly Gly Gly Gly Ser Ser Gly Gly Gly Gly Ser Ala Val

115 120 125

Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly Ala Leu

130 135 140

Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser Phe Asn Met

145 150 155 160

Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val Ala Gln

165 170 175

Ile Ser Ser Ser Gly Ser Ser Thr Asn Tyr Ala Pro Ala Val Arg Gly

180 185 190

Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Val Arg Leu Gln

195 200 205

Leu Asn Asn Pro Gly Ala Glu Asp Thr Gly Thr Tyr Tyr Cys Ala Lys

210 215 220

Ser Ser Tyr Asp Cys Pro Tyr Gly His Cys Ser Ser Gly Val Asp Ser

225 230 235 240

Ala Gly Glu Ile Asp Ala Trp Gly His Gly Thr Glu Val Ile Val Ser

245 250 255

Ser

<210> 48

<211> 256

<212> PRT

<213> Artificial Sequence

<220>

<223> scFV - 1-7A-IT Ab

<400> 48

Ala Leu Thr Gln Pro Ser Ser Val Ser Ala Asn Pro Gly Glu Thr Val

1 5 10 15

Lys Ile Thr Cys Ser Gly Gly Gly Ser Tyr Ala Gly Ser Tyr Tyr Tyr

20 25 30

Gly Trp Tyr Gln Gln Lys Pro Gly Ser Ala Pro Val Thr Leu Ile Tyr

35 40 45

Glu Asn Asn Lys Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly Ser

50 55 60

Thr Ser Gly Ser Thr Ala Thr Leu Thr Ile Thr Gly Val Gln Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gly Ser Trp Asp Ser Ser Asn Gly Gly

85 90 95

Ile Phe Gly Ala Gly Thr Thr Leu Thr Val Leu Gly Gln Ser Ser Arg

100 105 110

Ser Ser Gly Gly Gly Gly Ser Ser Gly Gly Gly Gly Ser Ala Val Thr

115 120 125

Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly Ala Leu Arg

130 135 140

Leu Ser Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser Phe Asn Met Phe

145 150 155 160

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val Ser Gln Ile

165 170 175

Ser Ser Ser Gly Ser Ser Thr Asn Tyr Ala Pro Ala Val Lys Gly Arg

180 185 190

Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Leu Tyr Leu Gln Met

195 200 205

Asn Ser Leu Arg Ala Glu Asp Thr Gly Thr Tyr Tyr Cys Ala Lys Ser

210 215 220

Ser Tyr Asp Cys Pro Tyr Gly His Cys Ser Ser Gly Val Asp Ser Ala

225 230 235 240

Gly Glu Ile Asp Ala Trp Gly His Gly Thr Glu Val Ile Val Ser Ser

245 250 255

<210> 49

<211> 256

<212> PRT

<213> Artificial Sequence

<220>

<223> scFv - Low-PI Ab

<400> 49

Ala Leu Thr Gln Pro Ser Ser Val Ser Ala Asn Pro Gly Glu Thr Val

1 5 10 15

Lys Ile Thr Cys Ser Gly Gly Gly Ser Glu Glu Glu Gln Tyr Tyr Tyr

20 25 30

Gly Trp Tyr Gln Gln Lys Pro Gly Ser Ala Pro Val Thr Leu Ile Tyr

35 40 45

Glu Asp Glu Glu Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly Ser

50 55 60

Thr Ser Gly Ser Thr Ala Thr Leu Thr Ile Thr Gly Val Gln Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gly Ser Trp Asp Ser Glu Asp Glu Asp

85 90 95

His Phe Gly Ala Gly Thr Thr Leu Thr Val Leu Gly Gln Ser Ser Arg

100 105 110

Ser Ser Gly Gly Gly Gly Ser Ser Gly Gly Gly Gly Ser Ala Val Thr

115 120 125

Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly Ala Leu Arg

130 135 140

Leu Ser Cys Lys Ala Ser Gly Phe Asp Phe Glu Ser Phe Asn Met Phe

145 150 155 160

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val Ser Gln Ile

165 170 175

Ser Ser Ser Glu Glu Asp Glu Asn Tyr Ala Pro Ala Val Lys Gly Arg

180 185 190

Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Leu Tyr Leu Gln Met

195 200 205

Asn Ser Leu Arg Ala Glu Asp Thr Gly Thr Tyr Tyr Cys Ala Lys Ser

210 215 220

Ser Tyr Asp Cys Pro Tyr Gly His Cys Ser Ser Gly Val Asp Ser Ala

225 230 235 240

Gly Glu Ile Asp Ala Trp Gly His Gly Thr Glu Val Ile Val Ser Ser

245 250 255

<210> 50

<211> 256

<212> PRT

<213> Artificial Sequence

<220>

<223> scFv - 1-30 Ab

<400> 50

Ala Leu Thr Gln Pro Ser Ser Val Ser Ala Asn Pro Gly Glu Thr Val

1 5 10 15

Lys Ile Thr Cys Ser Gly Gly Gly Ser Glu Glu Glu Gln Tyr Tyr Tyr

20 25 30

Gly Trp Tyr Gln Gln Lys Pro Gly Ser Ala Pro Val Thr Leu Ile Tyr

35 40 45

Gln Asp Glu Glu Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly Ser

50 55 60

Thr Ser Gly Ser Thr Ala Thr Leu Thr Ile Thr Gly Val Gln Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gly Ser Trp Asp Ser Glu Asp Glu Asp

85 90 95

His Phe Gly Ala Gly Thr Thr Leu Thr Val Leu Gly Gln Ser Ser Arg

100 105 110

Ser Ser Gly Gly Gly Gly Ser Ser Gly Gly Gly Gly Ser Ala Val Thr

115 120 125

Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly Ala Leu Arg

130 135 140

Leu Ser Cys Lys Ala Ser Gly Phe Asp Phe Glu Ser Phe Asn Met Phe

145 150 155 160

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val Ser Gln Ile

165 170 175

Ser Ser Ser Glu Glu Asp Glu Asn Tyr Ala Pro Ala Val Lys Gly Arg

180 185 190

Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Leu Tyr Leu Gln Met

195 200 205

Asn Ser Leu Arg Ala Glu Asp Thr Gly Thr Tyr Tyr Cys Ala Lys Ser

210 215 220

Ser Tyr Asp Cys Pro Tyr Gly His Cys Ser Ser Gly Val Asp Ser Ala

225 230 235 240

Gly Glu Ile Asp Ala Trp Gly His Gly Thr Glu Val Ile Val Ser Ser

245 250 255

<210> 51

<211> 256

<212> PRT

<213> Artificial Sequence

<220>

<223> scFv - 1-17 Ab

<400> 51

Ala Leu Thr Gln Pro Ser Ser Val Ser Ala Asn Pro Gly Glu Thr Val

1 5 10 15

Lys Ile Thr Cys Ser Gly Gly Gly Ser Tyr Ala Gly Ser Tyr Tyr Tyr

20 25 30

Gly Trp Tyr Gln Gln Lys Pro Gly Ser Ala Pro Val Thr Leu Ile Tyr

35 40 45

Glu Asp Glu Gln Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly Ser

50 55 60

Thr Ser Gly Ser Thr Ala Thr Leu Thr Ile Thr Gly Val Gln Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gly Ser Trp Asp Ser Glu Asp Glu Asp

85 90 95

His Phe Gly Ala Gly Thr Thr Leu Thr Val Leu Gly Gln Ser Ser Arg

100 105 110

Ser Ser Gly Gly Gly Gly Ser Ser Gly Gly Gly Gly Ser Ala Val Thr

115 120 125

Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly Ala Leu Arg

130 135 140

Leu Ser Cys Lys Ala Ser Gly Phe Asp Phe Glu Ser Phe Asn Met Phe

145 150 155 160

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val Ser Gln Ile

165 170 175

Ser Ser Ser Glu Glu Asp Glu Asn Tyr Ala Pro Ala Val Lys Gly Arg

180 185 190

Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Leu Tyr Leu Gln Met

195 200 205

Asn Ser Leu Arg Ala Glu Asp Thr Gly Thr Tyr Tyr Cys Ala Lys Ser

210 215 220

Ser Tyr Asp Cys Pro Tyr Gly His Cys Ser Ser Gly Val Asp Ser Ala

225 230 235 240

Gly Glu Ile Asp Ala Trp Gly His Gly Thr Glu Val Ile Val Ser Ser

245 250 255

<210> 52

<211> 256

<212> PRT

<213> Artificial Sequence

<220>

<223> scFv - 1-32 Ab

<400> 52

Ala Leu Thr Gln Pro Ser Ser Val Ser Ala Asn Pro Gly Glu Thr Val

1 5 10 15

Lys Ile Thr Cys Ser Gly Gly Gly Ser Tyr Ala Gly Ser Tyr Tyr Tyr

20 25 30

Gly Trp Tyr Gln Gln Lys Pro Gly Ser Ala Pro Val Thr Leu Ile Tyr

35 40 45

Gln Asp Glu Glu Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly Ser

50 55 60

Thr Ser Gly Ser Thr Ala Thr Leu Thr Ile Thr Gly Val Gln Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gly Ser Trp Asp Ser Glu Asp Glu Asp

85 90 95

His Phe Gly Ala Gly Thr Thr Leu Thr Val Leu Gly Gln Ser Ser Arg

100 105 110

Ser Ser Gly Gly Gly Gly Ser Ser Gly Gly Gly Gly Ser Ala Val Thr

115 120 125

Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly Ala Leu Arg

130 135 140

Leu Ser Cys Lys Ala Ser Gly Phe Asp Phe Glu Ser Phe Asn Met Phe

145 150 155 160

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val Ser Gln Ile

165 170 175

Ser Ser Ser Glu Glu Asp Glu Asn Tyr Ala Pro Ala Val Lys Gly Arg

180 185 190

Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Leu Tyr Leu Gln Met

195 200 205

Asn Ser Leu Arg Ala Glu Asp Thr Gly Thr Tyr Tyr Cys Ala Lys Ser

210 215 220

Ser Tyr Asp Cys Pro Tyr Gly His Cys Ser Ser Gly Val Asp Ser Ala

225 230 235 240

Gly Glu Ile Asp Ala Trp Gly His Gly Thr Glu Val Ile Val Ser Ser

245 250 255

<210> 53

<211> 256

<212> PRT

<213> Artificial Sequence

<220>

<223> scFv - 4-11 Ab

<400> 53

Ala Leu Thr Gln Pro Ser Ser Val Ser Ala Asn Pro Gly Glu Thr Val

1 5 10 15

Lys Ile Thr Cys Ser Gly Gly Gly Ser Tyr Ala Gly Ser Tyr Tyr Tyr

20 25 30

Gly Trp Tyr Gln Gln Lys Pro Gly Ser Ala Pro Val Thr Leu Ile Tyr

35 40 45

Glu Asp His Glu Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly Ser

50 55 60

Thr Ser Gly Ser Thr Ala Thr Leu Thr Ile Thr Gly Val Gln Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gly Ser Trp Asp Ser Ser Asp Glu Asp

85 90 95

His Phe Gly Ala Gly Thr Thr Leu Thr Val Leu Gly Gln Ser Ser Arg

100 105 110

Ser Ser Gly Gly Gly Gly Ser Ser Gly Gly Gly Gly Ser Ala Val Thr

115 120 125

Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly Ala Leu Arg

130 135 140

Leu Ser Cys Lys Ala Ser Gly Phe Asp Phe Glu Ser Phe Asn Met Phe

145 150 155 160

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val Ser Gln Ile

165 170 175

Ser Ser Ser Glu Glu Asp Glu Asn Tyr Ala Pro Ala Val Lys Gly Arg

180 185 190

Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Leu Tyr Leu Gln Met

195 200 205

Asn Ser Leu Arg Ala Glu Asp Thr Gly Thr Tyr Tyr Cys Ala Lys Ser

210 215 220

Ser Tyr Asp Cys Pro Tyr Gly His Cys Ser Ser Gly Val Asp Ser Ala

225 230 235 240

Gly Glu Ile Asp Ala Trp Gly His Gly Thr Glu Val Ile Val Ser Ser

245 250 255

<210> 54

<211> 256

<212> PRT

<213> Artificial Sequence

<220>

<223> scFv - 6-10 Ab

<400> 54

Ala Leu Thr Gln Pro Ser Ser Val Ser Ala Asn Pro Gly Glu Thr Val

1 5 10 15

Lys Ile Thr Cys Ser Gly Gly Gly Ser Tyr Ala Gly Ser Tyr Tyr Tyr

20 25 30

Gly Trp Tyr Gln Gln Lys Pro Gly Ser Ala Pro Val Thr Leu Ile Tyr

35 40 45

Gln Asp Leu Leu Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly Ser

50 55 60

Thr Ser Gly Ser Thr Ala Thr Leu Thr Ile Thr Gly Val Gln Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gly Ser Trp Asp Ser Leu Ser Ser Ser

85 90 95

His Phe Gly Ala Gly Thr Thr Leu Thr Val Leu Gly Gln Ser Ser Arg

100 105 110

Ser Ser Gly Gly Gly Gly Ser Ser Gly Gly Gly Gly Ser Ala Val Thr

115 120 125

Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly Ala Leu Arg

130 135 140

Leu Ser Cys Lys Ala Ser Gly Phe Asp Phe Glu Ser Phe Asn Met Phe

145 150 155 160

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val Ser Gln Ile

165 170 175

Ser Ser Ser Glu Glu Asp Glu Asn Tyr Ala Pro Ala Val Lys Gly Arg

180 185 190

Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Leu Tyr Leu Gln Met

195 200 205

Asn Ser Leu Arg Ala Glu Asp Thr Gly Thr Tyr Tyr Cys Ala Lys Ser

210 215 220

Ser Tyr Asp Cys Pro Tyr Gly His Cys Ser Ser Gly Val Asp Ser Ala

225 230 235 240

Gly Glu Ile Asp Ala Trp Gly His Gly Thr Glu Val Ile Val Ser Ser

245 250 255

<210> 55

<211> 251

<212> PRT

<213> Artificial Sequence

<220>

<223> scFv - 2-13 Ab

<400> 55

Ala Leu Thr Gln Pro Ser Ser Val Ser Ala Asn Pro Gly Glu Thr Val

1 5 10 15

Lys Ile Thr Cys Ser Gly Gly Ser Tyr Ser Tyr Gly Trp Phe Gln Gln

20 25 30

Lys Ser Pro Gly Ser Ala Leu Val Thr Val Ile Tyr Trp Asp Asp Glu

35 40 45

Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly Ala Leu Ser Gly Ser

50 55 60

Thr Asn Thr Leu Thr Ile Thr Gly Val Gln Ala Asp Asp Glu Ala Val

65 70 75 80

Tyr Phe Cys Gly Thr Glu Asp Ile Ser Gly Thr Ala Gly Val Phe Gly

85 90 95

Ala Gly Thr Thr Leu Thr Val Leu Gly Gln Ser Ser Arg Ser Ser Gly

100 105 110

Gly Gly Gly Ser Ser Gly Gly Gly Gly Ser Ala Val Thr Leu Asp Glu

115 120 125

Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly Ala Leu Ser Leu Val Cys

130 135 140

Lys Ala Ser Gly Phe Thr Phe Ser Ser His Gly Met Phe Trp Val Arg

145 150 155 160

Gln Thr Pro Gly Lys Gly Leu Glu Tyr Val Ala Glu Ile Thr Asn Asp

165 170 175

Gly Ser Gly Thr Asn Tyr Gly Ser Ala Val Lys Gly Arg Ala Thr Ile

180 185 190

Ser Arg Asp Asn Gly Gln Ser Thr Val Arg Leu Gln Leu Asn Asn Leu

195 200 205

Arg Ala Glu Asp Thr Gly Thr Tyr Phe Cys Ala Arg Ser Thr Tyr Glu

210 215 220

Cys Pro Gly Gly Phe Ser Cys Trp Gly Asp Thr Gly Gln Ile Asp Ala

225 230 235 240

Trp Gly His Gly Thr Glu Val Ile Val Ser Ser

245 250

<210> 56

<211> 250

<212> PRT

<213> Artificial Sequence

<220>

<223> scFv - 2-13D Ab

<400> 56

Ala Leu Thr Gln Pro Ser Ser Val Ser Ala Asn Pro Gly Glu Thr Ala

1 5 10 15

Lys Ile Thr Cys Ser Gly Val Tyr Ser Tyr Gly Trp Phe Gln Gln

20 25 30

Lys Pro Gly Ser Ala Leu Val Thr Val Ile Tyr Trp Asp Asp Glu Arg

35 40 45

Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly Ala Leu Ser Gly Ser Thr

50 55 60

Asn Thr Leu Thr Ile Thr Gly Val Gln Ala Glu Asp Glu Ala Asp Tyr

65 70 75 80

Tyr Cys Gly Thr Glu Asp Ile Ser Gly Thr Ala Gly Val Phe Gly Ala

85 90 95

Gly Thr Thr Leu Thr Val Leu Gly Gln Ser Ser Arg Ser Ser Gly Gly

100 105 110

Gly Gly Ser Ser Gly Gly Gly Gly Ser Ala Val Thr Leu Asp Glu Ser

115 120 125

Gly Gly Gly Leu Gln Thr Pro Gly Gly Ala Leu Arg Leu Ser Cys Ser

130 135 140

Ala Ser Gly Phe Thr Phe Ser Ser His Gly Met Phe Trp Val Arg Gln

145 150 155 160

Ala Pro Gly Lys Gly Leu Glu Tyr Val Ser Glu Ile Thr Asn Asp Gly

165 170 175

Ser Gly Thr Asn Tyr Gly Ser Ala Val Lys Gly Arg Ala Thr Ile Ser

180 185 190

Arg Asp Asn Gly Gln Ser Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg

195 200 205

Ala Glu Asp Thr Gly Thr Tyr Phe Cys Ala Arg Ser Thr Tyr Glu Cys

210 215 220

Pro Gly Gly Phe Ser Cys Trp Gly Asp Thr Gly Gln Ile Asp Ala Trp

225 230 235 240

Gly His Gly Thr Glu Val Ile Val Ser Ser

245 250

<210> 57

<211> 250

<212> PRT

<213> Artificial Sequence

<220>

<223> scFv - 2-13D-37Ab

<400> 57

Ala Leu Thr Gln Pro Ser Ser Val Ser Ala Asn Pro Gly Glu Thr Ala

1 5 10 15

Lys Ile Thr Cys Ser Gly Val Tyr Ser Tyr Gly Trp Phe Gln Gln

20 25 30

Lys Pro Gly Ser Ala Leu Val Thr Val Ile Tyr Trp Asp Asp Glu Arg

35 40 45

Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly Ala Leu Ser Gly Ser Thr

50 55 60

Asn Thr Leu Thr Ile Thr Gly Val Gln Ala Glu Asp Glu Ala Asp Tyr

65 70 75 80

Tyr Cys Gly Thr Glu Asp Ile Ser Gly Thr Ala Gly Val Phe Gly Ala

85 90 95

Gly Thr Thr Leu Thr Val Leu Gly Gln Ser Ser Arg Ser Ser Gly Gly

100 105 110

Gly Gly Ser Ser Gly Gly Gly Gly Ser Ala Val Thr Leu Asp Glu Ser

115 120 125

Gly Gly Gly Leu Gln Thr Pro Gly Gly Ala Leu Arg Leu Ser Cys Ser

130 135 140

Ala Ser Gly Phe Asp Phe Ser Ser His Gly Met Phe Trp Val Arg Gln

145 150 155 160

Ala Pro Gly Lys Gly Leu Glu Tyr Val Ser Glu Ile Thr Asn Asp Gly

165 170 175

Ser Gly Thr Asn Tyr Gly Ser Ala Val Lys Gly Arg Ala Thr Ile Ser

180 185 190

Arg Asp Asn Gly Gln Ser Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg

195 200 205

Ala Glu Asp Thr Gly Thr Tyr Phe Cys Ala Arg Ser Thr Tyr Glu Cys

210 215 220

Pro Gly Gly Phe Ser Cys Trp Gly Asp Thr Gly Gln Ile Asp Ala Trp

225 230 235 240

Gly His Gly Thr Glu Val Ile Val Ser Ser

245 250

<210> 58

<211> 393

<212> DNA

<213> Artificial Sequence

<220>

<223> VH -3-2 Ab

<400> 58

60

gtctgcaagg cctccgggtt caccttcagc agcttcaaca tgttctgggt gcgacaggcg 120

cccggcaagg ggctggaata cgtcgctcaa attagcagca gtggtagtag cacaaactac 180

gcacccgcgg tgaggggccg tgccaccatc tcgagggaca acgggcagag cacagtgagg 240

ctgcagctga acaaccccgg ggctgaagac accggcacct actactgcgc caaaagtagt 300

ttgtagtagt ggtgttgata gtgctggtga gatcgacgca 360

tggggccacg ggaccgaagt catcgtctcc tcc 393

<210> 59

<211> 393

<212> DNA

<213> Artificial Sequence

<220>

<223> VH - 1-7A-IT Ab

<400> 59

gccgtgacgt tggatgaatc cggggggcggc ctccagacgc ccggaggagc gctccgcctc 60

agctgcaagg cctctgggtt caccttcagc agcttcaaca tgttctgggt gcgacaggcg 120

cccggcaagg ggctggaata cgtctcgcag attagcagca gtggtagtag cacaaactac 180

gcacccgcgg tgaaaggccg tgccaccatc tcgagggaca acgggcagag cacactgtat 240

ctgcagatga acagcctgcg cgctgaagac accggcacct actactgcgc caaaagtagt 300

ttgtagtagt ggtgttgata gtgctggtga gatcgacgca 360

tggggccacg ggaccgaagt catcgtctcc tcc 393

<210> 60

<211> 393

<212> DNA

<213> Artificial Sequence

<220>

<223> VH - Low-PI Ab

<400> 60

gccgtgacgt tggatgaatc cggggggcggc ctccagacgc ccggaggagc gctccgcctc 60

agctgcaagg cctctgggtt caccttcagc agcttcaaca tgttctgggt gcgacaggcg 120

cccggcaagg ggctggaata cgtctcgcag attagcagca gtggtagtag cacaaactac 180

gcacccgcgg tgaaaggccg tgccaccatc tcgagggaca acgggcagag cacactgtat 240

ctgcagatga acagcctgcg cgctgaagac accggcacct actactgcgc caaaagtagt 300

ttgtagtagt ggtgttgata gtgctggtga gatcgacgca 360

tggggccacg ggaccgaagt catcgtctcc tcc 393

<210> 61

<211> 393

<212> DNA

<213> Artificial Sequence

<220>

<223> VH - 1-30 Ab

<400> 61

gccgtgacgt tggatgaatc cggggggcggc ctccagacgc ccggaggagc gctccgcctc 60

agctgcaagg cctctggctt tgattttgaa agcttcaaca tgttctgggt gcgacaggcg 120

cccggcaagg ggctggaata cgtctcgcag attagcagca gtgaagaaga tgaaaactac 180

gcacccgcgg tgaaaggccg tgccaccatc tcgagggaca acgggcagag cacactgtat 240

ctgcagatga acagcctgcg cgctgaagac accggcacct actactgcgc caaaagtagt 300

ttgtagtagt ggtgttgata gtgctggtga gatcgacgca 360

tggggccacg ggaccgaagt catcgtctcc tcc 393

<210> 62

<211> 393

<212> DNA

<213> Artificial Sequence

<220>

<223> VH - 1-17 Ab

<400> 62

gccgtgacgt tggatgaatc cggggggcggc ctccagacgc ccggaggagc gctccgcctc 60

agctgcaagg cctctggctt tgattttgaa agcttcaaca tgttctgggt gcgacaggcg 120

cccggcaagg ggctggaata cgtctcgcag attagcagca gtgaagaaga tgaaaactac 180

gcacccgcgg tgaaaggccg tgccaccatc tcgagggaca acgggcagag cacactgtat 240

ctgcagatga acagcctgcg cgctgaagac accggcacct actactgcgc caaaagtagt 300

ttgtagtagt ggtgttgata gtgctggtga gatcgacgca 360

tggggccacg ggaccgaagt catcgtctcc tcc 393

<210> 63

<211> 393

<212> DNA

<213> Artificial Sequence

<220>

<223> VH - 1-32 Ab

<400> 63

gccgtgacgt tggatgaatc cggggggcggc ctccagacgc ccggaggagc gctccgcctc 60

agctgcaagg cctctggctt tgattttgaa agcttcaaca tgttctgggt gcgacaggcg 120

cccggcaagg ggctggaata cgtctcgcag attagcagca gtgaagaaga tgaaaactac 180

gcacccgcgg tgaaaggccg tgccaccatc tcgagggaca acgggcagag cacactgtat 240

ctgcagatga acagcctgcg cgctgaagac accggcacct actactgcgc caaaagtagt 300

ttgtagtagt ggtgttgata gtgctggtga gatcgacgca 360

tggggccacg ggaccgaagt catcgtctcc tcc 393

<210> 64

<211> 393

<212> DNA

<213> Artificial Sequence

<220>

<223> VH - 4-11 Ab

<400> 64

gccgtgacgt tggatgaatc cggggggcggc ctccagacgc ccggaggagc gctccgcctc 60

agctgcaagg cctctggctt tgattttgaa agcttcaaca tgttctgggt gcgacaggcg 120

cccggcaagg ggctggaata cgtctcgcag attagcagca gtgaagaaga tgaaaactac 180

gcacccgcgg tgaaaggccg tgccaccatc tcgagggaca acgggcagag cacactgtat 240

ctgcagatga acagcctgcg cgctgaagac accggcacct actactgcgc caaaagtagt 300

ttgtagtagt ggtgttgata gtgctggtga gatcgacgca 360

tggggccacg ggaccgaagt catcgtctcc tcc 393

<210> 65

<211> 393

<212> DNA

<213> Artificial Sequence

<220>

<223> VH - 6-10 Ab

<400> 65

gccgtgacgt tggatgaatc cggggggcggc ctccagacgc ccggaggagc gctccgcctc 60

agctgcaagg cctctggctt tgattttgaa agcttcaaca tgttctgggt gcgacaggcg 120

cccggcaagg ggctggaata cgtctcgcag attagcagca gtgaagaaga tgaaaactac 180

gcacccgcgg tgaaaggccg tgccaccatc tcgagggaca acgggcagag cacactgtat 240

ctgcagatga acagcctgcg cgctgaagac accggcacct actactgcgc caaaagtagt 300

ttgtagtagt ggtgttgata gtgctggtga gatcgacgca 360

tggggccacg ggaccgaagt catcgtctcc tcc 393

<210> 66

<211> 387

<212> DNA

<213> Artificial Sequence

<220>

<223> VH - 2-13 Ab

<400> 66

60

gtctgcaagg cctccgggtt caccttcagc agccatggca tgttctgggt gcgacagacg 120

cccggcaagg ggttggaata tgtcgctgaa attaccaatg atggtagtgg cacaaactac 180

gggtcggcgg tgaagggccg tgccaccatc tcgagggaca acgggcagag cacagtgagg 240

ctgcagctga acaacctcag ggctgaggac accggcacct acttctgcgc cagatctact 300

tatgaatgtc ctggtggttt tagttgttgg ggtgatactg gtcaaataga cgcatggggc 360

cacgggaccg aagtcatcgt ctcctcc 387

<210> 67

<211> 387

<212> DNA

<213> Artificial Sequence

<220>

<223> VH - 2-13D Ab

<400> 67

gccgtgacgt tggacgagtc cggggggcggc ctccagacgc ccggaggagc gctccgcctt

agctgcagcg cctccgggtt caccttcagc agccatggca tgttctgggt gcgacaggcg 120

cccggcaagg ggttggaata tgtctcggag attaccaatg atggtagtgg cacaaactac 180

gggtcggcgg tgaagggccg tgccaccatc tcgagggaca acgggcagag cacactgtat 240

ctgcagatga acagcctcag ggctgaggac accggcacct acttctgcgc cagatctact 300

tatgaatgtc ctggtggttt tagttgttgg ggtgatactg gtcaaataga cgcatggggc 360

cacgggaccg aagtcatcgt ctcctcc 387

<210> 68

<211> 387

<212> DNA

<213> Artificial Sequence

<220>

<223> VH - 2-13D-37 Ab

<400> 68

gccgtgacgt tggacgagtc cggggggcggc ctccagacgc ccggaggagc gctccgcctt

agctgcagcg cctccgggtt cgatttcagc agccatggca tgttctgggt gcgacaggcg 120

cccggcaagg ggttggaata tgtctcggag attaccaatg atggtagtgg cacaaactac 180

gggtcggcgg tgaagggccg tgccaccatc tcgagggaca acgggcagag cacactgtat 240

ctgcagatga acagcctcag ggctgaggac accggcacct acttctgcgc cagatctact 300

tatgaatgtc ctggtggttt tagttgttgg ggtgatactg gtcaaataga cgcatggggc 360

cacgggaccg aagtcatcgt ctcctcc 387

<210> 69

<211> 324

<212> DNA

<213> Artificial Sequence

<220>

<223> VL - 3-2 Ab

<400> 69

gccctgactc agccgtcctc ggtgtcagca aacccaggag aaaccgtcaa gatcacctgc 60

tccggggggtg gcagctatgc tggaagttac tattatggct ggtaccagca gaaggcacct 120

ggcagtgccc ctgtcactct gatctatgaa agcaacaaga gaccctcggga catcccttca 180

cgattctccg gttccacatc tggctccaca gccacactaa ccatcactgg ggtccaagcc 240

gatgacgagg ctatctatta ctgtgggagc tgggacagta gcaatggtgg tatatttgggg 300

gccgggacaa ccctgaccgt ccta 324

<210> 70

<211> 321

<212> DNA

<213> Artificial Sequence

<220>

<223> VL - 1-7A-IT Ab

<400> 70

gccctgactc agccgtcctc ggtgtcagca aacccaggag aaaccgtcaa gatcacctgc 60

tccggggggtg gcagctatgc tggaagttac tattatggct ggtatcagca gaagcctggc 120

agtgcccctg tcactctgat ctatgaaaac aacaagagac ccctcggacat cccttcacga 180

ttctccggtt ccacatctgg ctccacagcc acactaacca tcactggggt ccaagccggc 240

gacgaggctg attattactg tgggagctgg gacagtagca atggtggtat atttggggcc 300

gggacaaccc tgaccgtcct a 321

<210> 71

<211> 321

<212> DNA

<213> Artificial Sequence

<220>

<223> VL - Low-PI Ab

<400> 71

gccctgactc agccgtcctc ggtgtcagca aacccaggag aaaccgtcaa gatcacctgc 60

tccggggggtg gcagctatgc tggaagttac tattatggct ggtatcagca gaagcctggc 120

agtgcccctg tcactctgat ctatgaaaac aacaagagac ccctcggacat cccttcacga 180

ttctccggtt ccacatctgg ctccacagcc acactaacca tcactggggt ccaagccggc 240

gacgaggctg attattactg tgggagctgg gacagtagca atggtggtat atttggggcc 300

gggacaaccc tgaccgtcct a 321

<210> 72

<211> 321

<212> DNA

<213> Artificial Sequence

<220>

<223> VL - 1-30 Ab

<400> 72

gccctgactc agccgtcctc ggtgtcagca aacccaggag aaaccgtcaa gatcacctgc 60

tccggggggtg gcagcgaaga agaacagtac tattatggct ggtatcagca gaagcctggc 120

agtgcccctg tcactctgat ctatcaggat gaagaaagac ccctcggacat cccttcacga 180

ttctccggtt ccacatctgg ctccacagcc acactaacca tcactggggt ccaagccggc 240

gacgaggctg attattactg tgggagctgg gacagtgaag atgaagatca ttttggggcc 300

gggacaaccc tgaccgtcct a 321

<210> 73

<211> 321

<212> DNA

<213> Artificial Sequence

<220>

<223> VL 1-17 Ab

<400> 73

gccctgactc agccgtcctc ggtgtcagca aacccaggag aaaccgtcaa gatcacctgc 60

tccggggggtg gcagctatgc tggaagttac tattatggct ggtatcagca gaagcctggc 120

agtgcccctg tcactctgat ctatgaagat gaacagagac ccctcggacat cccttcacga 180

ttctccggtt ccacatctgg ctccacagcc acactaacca tcactggggt ccaagccggc 240

gacgaggctg attattactg tgggagctgg gacagtgaag atgaagatca ttttggggcc 300

gggacaaccc tgaccgtcct a 321

<210> 74

<211> 321

<212> DNA

<213> Artificial Sequence

<220>

<223> VL 1-32 Ab

<400> 74

gccctgactc agccgtcctc ggtgtcagca aacccaggag aaaccgtcaa gatcacctgc 60

tccggggggtg gcagctatgc tggaagttac tattatggct ggtatcagca gaagcctggc 120

agtgcccctg tcactctgat ctatcaggat gaagaaagac ccctcggacat cccttcacga 180

ttctccggtt ccacatctgg ctccacagcc acactaacca tcactggggt ccaagccggc 240

gacgaggctg attattactg tgggagctgg gacagtgaag atgaagatca ttttggggcc 300

gggacaaccc tgaccgtcct a 321

<210> 75

<211> 321

<212> DNA

<213> Artificial Sequence

<220>

<223> VL 4-11 Ab

<400> 75

gccctgactc agccgtcctc ggtgtcagca aacccaggag aaaccgtcaa gatcacctgc 60

tccggggggtg gcagctatgc tggaagttac tattatggct ggtatcagca gaagcctggc 120

agtgcccctg tcactctgat ctatgaagac cacgagagac cctcggacat cccttcacga 180

ttctccggtt ccacatctgg ctccacagcc acactaacca tcactggggt ccaagccggc 240

gacgaggctg attattactg tgggagctgg gacagtagcg atgaagatca ttttggggcc 300

gggacaaccc tgaccgtcct a 321

<210> 76

<211> 321

<212> DNA

<213> Artificial Sequence

<220>

<223> VL 6-10 Ab

<400> 76

gccctgactc agccgtcctc ggtgtcagca aacccaggag aaaccgtcaa gatcacctgc 60

tccggggggtg gcagctatgc tggaagttac tattatggct ggtatcagca gaagcctggc 120

agtgcccctg tcactctgat ctatcaggat ctgctgagac ccctcggacat cccttcacga 180

ttctccggtt ccacatctgg ctccacagcc acactaacca tcactggggt ccaagccggc 240

gacgaggctg attattactg tgggagctgg gacagtctga gcagcagcca ttttggggcc 300

gggacaaccc tgaccgtcct a 321

<210> 77

<211> 312

<212> DNA

<213> Artificial Sequence

<220>

<223> VL 2-13 Ab

<400> 77

gccctgactc agccgtcctc ggtgtcagca aacccaggag aaaccgtcaa gataacctgc 60

tccggggggta gctatagcta tggctggttc cagcagaagt ctcctggcag tgcccttgtc 120

actgtgatct actgggatga tgagagaccc tcggacatcc cttcacgatt ctccggtgcc 180

ctatccggct ccacaaacac attaaccatc actggggtcc aagccgacga cgaggctgtc 240

tatttctgtg ggactgaaga catcagcggc actgctggtg tatttggggc cgggacaacc 300

ctgaccgtcc tg 312

<210> 78

<211> 309

<212> DNA

<213> Artificial Sequence

<220>

<223> VL 2-13D Ab

<400> 78

gccctgactc agccgtcctc ggtgtcagca aacccaggag aaaccgcgaa gataacctgc 60

tccggggggtg tgtatagcta tggctggttc cagcagaagc ctggcagtgc ccttgtcact 120

gtgatctact gggatgatga gagaccctcg gacatccctt cacgattctc cggtgcccta 180

tccggctcca caaacacatt aaccatcact ggggtccaag ccgaagacga ggctgattat 240

tattgtggga ctgaagacat cagcggcact gctggtgtat ttggggccgg gacaaccctg 300

accgtcctg 309

<210> 79

<211> 309

<212> DNA

<213> Artificial Sequence

<220>

<223> VL 2-13D-37 Ab

<400> 79

gccctgactc agccgtcctc ggtgtcagca aacccaggag aaaccgcgaa gataacctgc 60

tccggggggtg tgtatagcta tggctggttc cagcagaagc ctggcagtgc ccttgtcact 120

gtgatctact gggatgatga gagaccctcg gacatccctt cacgattctc cggtgcccta 180

tccggctcca caaacacatt aaccatcact ggggtccaag ccgaagacga ggctgattat 240

tattgtggga ctgaagacat cagcggcact gctggtgtat ttggggccgg gacaaccctg 300

accgtcctg 309

<210> 80

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Light Chain CDR1-2-13D; 2-13D-37 Abs

<400> 80

Ser Gly Gly Val Tyr Ser Tyr Gly

fifteen

<210> 81

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223>EP1 Epitope

<400> 81

Ile Val Thr Leu Asp

fifteen

<210> 82

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223>EP2 Epitope

<400> 82

Asp Ser Ser Gln Pro

fifteen

<210> 83

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223>EP3 Epitope

<400> 83

Arg Thr Ile Ala Arg

fifteen

<210> 84

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223>EP4 Epitope

<400> 84

Ala Arg Cys Ala Cys Arg Lys

fifteen

<210> 85

<211> 4

<212> PRT

<213> Artificial Sequence

<220>

<223> EP5 Epitope

<400> 85

Ala Arg Pro Ala

one

<210> 86

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223>EP6 Epitope

<400> 86

Lys Thr Lys Gln Trp Cys Asp Met Leu

fifteen

<210> 87

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223>EP7 Epitope

<400> 87

Gly Cys Asp Leu Leu Ile Asn Arg

fifteen

<210> 88

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> EP8 Epitope

<400> 88

Thr Cys Thr Gln Pro Gly Gly Arg

fifteen

<210> 89

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> Epitope F1

<400> 89

Gln Leu Ala Ala Gly Thr Cys Glu Ile Val Thr Leu Asp Arg

1 5 10

<210> 90

<211> 20

<212> PRT

<213> Artificial Sequence

<220>

<223> Epitope F2

<400> 90

Thr Leu Asp Arg Asp Ser Ser Gln Pro Arg Arg Thr Ile Ala Arg Gln

1 5 10 15

Thr Ala Arg Cys

twenty

<210> 91

<211> 20

<212> PRT

<213> Artificial Sequence

<220>

<223> Epitope F3

<400> 91

Thr Ala Arg Cys Ala Cys Arg Lys Gly Gln Ile Ala Gly Thr Thr Arg

1 5 10 15

Ala Arg Pro Ala

twenty

<210> 92

<211> 20

<212> PRT

<213> Artificial Sequence

<220>

<223> Epitope F4

<400> 92

Ala Arg Pro Ala Cys Val Asp Ala Arg Ile Ile Lys Thr Lys Gln Trp

1 5 10 15

Cys Asp Met Leu

twenty

<210> 93

<211> 20

<212> PRT

<213> Artificial Sequence

<220>

<223> Epitope F5

<400> 93

Cys Asp Met Leu Pro Cys Leu Glu Gly Glu Gly Cys Asp Leu Leu Ile

1 5 10 15

Asn Arg Ser Gly

twenty

<210> 94

<211> 20

<212> PRT

<213> Artificial Sequence

<220>

<223> Epitope F6

<400> 94

Asn Arg Ser Gly Trp Thr Cys Thr Gln Pro Gly Gly Arg Ile Lys Thr

1 5 10 15

Thr Thr Val Ser

twenty

<210> 95

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> Linker

<400> 95

Ser Gly Gly Gly Gly Ser Ser Gly Gly Gly Gly Ser

1 5 10

<210> 96

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> 2-13 binding epitope 1

<400> 96

Cys Arg Lys Gly Gln Ile Ala Gly Thr Thr Arg Ala Arg

1 5 10

<210> 97

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> 2-13 binding epitope 2

<400> 97

Pro Ala Cys Val Asp Ala Arg Ile Ile Lys Thr Lys Gln Trp

1 5 10

<210> 98

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> binding epitope

<220>

<221> BINDING

<222> (1)..(2)

<223> Antibody specifically binds to respective residues

<220>

<221> BINDING

<222>(6)..(8)

<223> Antibody specifically binds to respective residues

<400> 98

Arg Asp Ser Ser Gln Pro Arg Arg

fifteen

<210> 99

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> binding epitope

<220>

<221> BINDING

<222> (1)..(1)

<223> Antibody Specifically binds to respective residues

<220>

<221> BINDING

<222>(6)..(8)

<223> Antibody Specifically binds to respective residues

<400> 99

Arg Asp Ser Ser Gln Pro Arg Arg

fifteen

<210> 100

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> binding epitope

<220>

<221> BINDING

<222> (1)..(1)

<223> Antibody Specifically binds to respective residues

<220>

<221> BINDING

<222> (7)..(8)

<223> Antibody Specifically binds to respective residues

<400> 100

Arg Asp Ser Ser Gln Pro Arg Arg

fifteen

<210> 101

<211> 100

<212> PRT

<213> Artificial Sequence

<220>

<223> Mature Human FAM19A5 Isoform 2

<400> 101

Gln Phe Leu Lys Glu Gly Gln Leu Ala Ala Gly Thr Cys Glu Ile Val

1 5 10 15

Thr Leu Asp Arg Asp Ser Ser Gln Pro Arg Arg Thr Ile Ala Arg Gln

20 25 30

Thr Ala Arg Cys Ala Cys Arg Lys Gly Gln Ile Ala Gly Thr Thr Arg

35 40 45

Ala Arg Pro Ala Cys Val Asp Ala Arg Ile Ile Lys Thr Lys Gln Trp

50 55 60

Cys Asp Met Leu Pro Cys Leu Glu Gly Glu Gly Cys Asp Leu Leu Ile

65 70 75 80

Asn Arg Ser Gly Trp Thr Cys Thr Gln Pro Gly Gly Arg Ile Lys Thr

85 90 95

Thr Thr Val Ser

100

<210> 102

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide Sequence #1 (residues 2-8 of SEQ ID NO: 101)

<400> 102

Phe Leu Lys Glu Gly Gln Leu

fifteen

<210> 103

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide Sequence #2 (residues 2-14 of SEQ ID NO: 101)

<400> 103

Phe Leu Lys Glu Gly Gln Leu Ala Ala Gly Thr Cys Glu

1 5 10

<210> 104

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide Sequence #3 (residues 3-11 of SEQ ID NO: 101)

<400> 104

Leu Lys Glu Gly Gln Leu Ala Ala Gly

fifteen

<210> 105

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide Sequence #4 (residues 3-15 of SEQ ID NO: 101)

<400> 105

Leu Lys Glu Gly Gln Leu Ala Ala Gly Thr Cys Glu Ile

1 5 10

<210> 106

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide Sequence #5 (residues 3-18 of SEQ ID NO: 101)

<400> 106

Leu Lys Glu Gly Gln Leu Ala Ala Gly Thr Cys Glu Ile Val Thr Leu

1 5 10 15

<210> 107

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide Sequence #6 (residues 9-15 of SEQ ID NO: 101)

<400> 107

Ala Ala Gly Thr Cys Glu Ile

fifteen

<210> 108

<211> 19

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide Sequence #7 (residues 20-38 of SEQ ID NO: 101)

<400> 108

Arg Asp Ser Ser Gln Pro Pro Arg Thr Ile Ala Arg Gln Thr Ala Arg

1 5 10 15

Cys Ala Cys

<210> 109

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide Sequence #8 (residues 24-34 of SEQ ID NO: 101)

<400> 109

Gln Pro Pro Arg Thr Ile Ala Arg Gln Thr Ala

1 5 10

<210> 110

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide Sequence #9 (residues 37-53 of SEQ ID NO: 101)

<400> 110

Ala Cys Arg Lys Gly Gln Ile Ala Gly Thr Thr Arg Ala Arg Pro Ala

1 5 10 15

Cys

<210> 111

<211> 19

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide Sequence #10 (residues 37-55 of SEQ ID NO: 101)

<400> 111

Ala Cys Arg Lys Gly Gln Ile Ala Gly Thr Thr Arg Ala Arg Pro Ala

1 5 10 15

Cys Val Asp

<210> 112

<211> 20

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide Sequence #12 (residues 56-65 of SEQ ID NO: 101)

<400> 112

Ala Cys Arg Lys Gly Gln Ile Ala Gly Thr Thr Arg Ala Arg Pro Ala

1 5 10 15

Cys Val Asp Ala

twenty

<210> 113

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide Sequence #12 (residues 56-65 of SEQ ID NO: 101)

<400> 113

Ala Arg Ile Ile Lys Thr Lys Gln Trp Cys

1 5 10

<210> 114

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide Sequence #13 (residues 56-67 of SEQ ID NO: 101)

<400> 114

Ala Arg Ile Ile Lys Thr Lys Gln Trp Cys Asp Met

1 5 10

<210> 115

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide Sequence #14 (residues 56-68 of SEQ ID NO: 101)

<400> 115

Ala Arg Ile Ile Lys Thr Lys Gln Trp Cys Asp Met Leu

1 5 10

<210> 116

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide Sequence #15 (residues 56-71 of SEQ ID NO: 101)

<400> 116

Ala Arg Ile Ile Lys Thr Lys Gln Trp Cys Asp Met Leu Pro Cys Leu

1 5 10 15

<210> 117

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide Sequence #16 (residues 57-67 of SEQ ID NO: 101)

<400> 117

Arg Ile Ile Lys Thr Lys Gln Trp Cys Asp Met

1 5 10

<210> 118

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide Sequence #17 (residues 57-68 of SEQ ID NO: 101)

<400> 118

Arg Ile Ile Lys Thr Lys Gln Trp Cys Asp Met Leu

1 5 10

<210> 119

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide Sequence #18 (residues 64-71 of SEQ ID NO: 101)

<400> 119

Trp Cys Asp Met Leu Pro Cys Leu

fifteen

<210> 120

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide Sequence #19 (residues 68-75 of SEQ ID NO: 101)

<400> 120

Leu Pro Cys Leu Glu Gly Glu Gly

fifteen

<210> 121

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide Sequence #20 (residues 69-75 of SEQ ID NO: 101)

<400> 121

Pro Cys Leu Glu Gly Glu Gly

fifteen

<210> 122

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide Sequence #21 (residues 69-77 of SEQ ID NO: 101)

<400> 122

Pro Cys Leu Glu Gly Glu Gly Cys Asp

fifteen

<210> 123

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide Sequence #22 (residues 69-78 of SEQ ID NO: 101)

<400> 123

Pro Cys Leu Glu Gly Glu Gly Cys Asp Leu

1 5 10

<210> 124

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide Sequence #23 (residues 72-78 of SEQ ID NO: 101)

<400> 124

Glu Gly Glu Gly Cys Asp Leu

fifteen

<210> 125

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide Sequence #24 (residues 72-79 of SEQ ID NO: 101)

<400> 125

Glu Gly Glu Gly Cys Asp Leu Leu

fifteen

<210> 126

<211> 21

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide Sequence #25 (residues 78-98 of SEQ ID NO: 101)

<400> 126

Leu Leu Ile Asn Arg Ser Gly Trp Thr Cys Thr Gln Pro Gly Gly Arg

1 5 10 15

Ile Lys Thr Thr Thr

twenty

<210> 127

<211> 20

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide Sequence #26 (residues 79-98 of SEQ ID NO: 101)

<400> 127

Leu Ile Asn Arg Ser Gly Trp Thr Cys Thr Gln Pro Gly Gly Arg Ile

1 5 10 15

Lys Thr Thr Thr

twenty

<210> 128

<211> 20

<212> PRT

<213> Artificial Sequence

<220>

<223> 2-13D-37-1.5W-41 Ab VH-CDR3

<400> 128

Ser Ser Tyr Val Cys Pro Gly Gly Phe Ser Cys Trp Gly Asp Thr Gly

1 5 10 15

Gln Ile Asp Ala

twenty

<210> 129

<211> 20

<212> PRT

<213> Artificial Sequence

<220>

<223> 2-13D-37-3W-16 Ab VH-CDR3

<400> 129

Ser Asn Tyr Ala Cys Pro Gly Gly Phe Ser Cys Trp Gly Asp Thr Gly

1 5 10 15

Gln Ile Asp Ala

twenty

<210> 130

<211> 129

<212> PRT

<213> Artificial Sequence

<220>

<223> 2-13D-37-1.5W-41 Ab VH

<400> 130

Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly

1 5 10 15

Ala Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Asp Phe Ser Ser His

20 25 30

Gly Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val

35 40 45

Ser Glu Ile Thr Asn Asp Gly Ser Gly Thr Asn Tyr Gly Ser Ala Val

50 55 60

Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Gly Thr Tyr Phe Cys

85 90 95

Ala Arg Ser Ser Tyr Val Cys Pro Gly Gly Phe Ser Cys Trp Gly Asp

100 105 110

Thr Gly Gln Ile Asp Ala Trp Gly His Gly Thr Glu Val Ile Val Ser

115 120 125

Ser

<210> 131

<211> 129

<212> PRT

<213> Artificial Sequence

<220>

<223> 2-13D-37-3W-16 Ab VH

<400> 131

Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly

1 5 10 15

Ala Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Asp Phe Ser Ser His

20 25 30

Gly Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val

35 40 45

Ser Glu Ile Thr Asn Asp Gly Ser Gly Thr Asn Tyr Gly Ser Ala Val

50 55 60

Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Gly Thr Tyr Phe Cys

85 90 95

Ala Arg Ser Asn Tyr Ala Cys Pro Gly Gly Phe Ser Cys Trp Gly Asp

100 105 110

Thr Gly Gln Ile Asp Ala Trp Gly His Gly Thr Glu Val Ile Val Ser

115 120 125

Ser

<210> 132

<211> 250

<212> PRT

<213> Artificial Sequence

<220>

<223> scFv - 2-13D-37-1.5W-41 Ab

<400> 132

Ala Leu Thr Gln Pro Ser Ser Val Ser Ala Asn Pro Gly Glu Thr Ala

1 5 10 15

Lys Ile Thr Cys Ser Gly Val Tyr Ser Tyr Gly Trp Phe Gln Gln

20 25 30

Lys Pro Gly Ser Ala Leu Val Thr Val Ile Tyr Trp Asp Asp Glu Arg

35 40 45

Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly Ala Leu Ser Gly Ser Thr

50 55 60

Asn Thr Leu Thr Ile Thr Gly Val Gln Ala Glu Asp Glu Ala Asp Tyr

65 70 75 80

Tyr Cys Gly Thr Glu Asp Ile Ser Gly Thr Ala Gly Val Phe Gly Ala

85 90 95

Gly Thr Thr Leu Thr Val Leu Gly Gln Ser Ser Arg Ser Ser Gly Gly

100 105 110

Gly Gly Ser Ser Gly Gly Gly Gly Ser Ala Val Thr Leu Asp Glu Ser

115 120 125

Gly Gly Gly Leu Gln Thr Pro Gly Gly Ala Leu Arg Leu Ser Cys Ser

130 135 140

Ala Ser Gly Phe Asp Phe Ser Ser His Gly Met Phe Trp Val Arg Gln

145 150 155 160

Ala Pro Gly Lys Gly Leu Glu Tyr Val Ser Glu Ile Thr Asn Asp Gly

165 170 175

Ser Gly Thr Asn Tyr Gly Ser Ala Val Lys Gly Arg Ala Thr Ile Ser

180 185 190

Arg Asp Asn Gly Gln Ser Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg

195 200 205

Ala Glu Asp Thr Gly Thr Tyr Phe Cys Ala Arg Ser Ser Tyr Val Cys

210 215 220

Pro Gly Gly Phe Ser Cys Trp Gly Asp Thr Gly Gln Ile Asp Ala Trp

225 230 235 240

Gly His Gly Thr Glu Val Ile Val Ser Ser

245 250

<210> 133

<211> 250

<212> PRT

<213> Artificial Sequence

<220>

<223> scFv - 2-13D-37-3W-16 Ab

<400> 133

Ala Leu Thr Gln Pro Ser Ser Val Ser Ala Asn Pro Gly Glu Thr Ala

1 5 10 15

Lys Ile Thr Cys Ser Gly Val Tyr Ser Tyr Gly Trp Phe Gln Gln

20 25 30

Lys Pro Gly Ser Ala Leu Val Thr Val Ile Tyr Trp Asp Asp Glu Arg

35 40 45

Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly Ala Leu Ser Gly Ser Thr

50 55 60

Asn Thr Leu Thr Ile Thr Gly Val Gln Ala Glu Asp Glu Ala Asp Tyr

65 70 75 80

Tyr Cys Gly Thr Glu Asp Ile Ser Gly Thr Ala Gly Val Phe Gly Ala

85 90 95

Gly Thr Thr Leu Thr Val Leu Gly Gln Ser Ser Arg Ser Ser Gly Gly

100 105 110

Gly Gly Ser Ser Gly Gly Gly Gly Ser Ala Val Thr Leu Asp Glu Ser

115 120 125

Gly Gly Gly Leu Gln Thr Pro Gly Gly Ala Leu Arg Leu Ser Cys Ser

130 135 140

Ala Ser Gly Phe Asp Phe Ser Ser His Gly Met Phe Trp Val Arg Gln

145 150 155 160

Ala Pro Gly Lys Gly Leu Glu Tyr Val Ser Glu Ile Thr Asn Asp Gly

165 170 175

Ser Gly Thr Asn Tyr Gly Ser Ala Val Lys Gly Arg Ala Thr Ile Ser

180 185 190

Arg Asp Asn Gly Gln Ser Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg

195 200 205

Ala Glu Asp Thr Gly Thr Tyr Phe Cys Ala Arg Ser Asn Tyr Ala Cys

210 215 220

Pro Gly Gly Phe Ser Cys Trp Gly Asp Thr Gly Gln Ile Asp Ala Trp

225 230 235 240

Gly His Gly Thr Glu Val Ile Val Ser Ser

245 250

<210> 134

<211> 387

<212> DNA

<213> Artificial Sequence

<220>

<223> 2-13D-37-1.5W-41 Ab VH

<400> 134

gccgtgacgt tggacgagtc cggggggcggc ctccagacgc ccggaggagc gctccgcctt

agctgcagcg cctccgggtt cgatttcagc agccatggca tgttctgggt gcgacaggcg 120

cccggcaagg ggttggaata tgtctcggag attaccaatg atggtagtgg cacaaactac 180

gggtcggcgg tgaagggccg tgccaccatc tcgagggaca acgggcagag cacactgtat 240

ctgcagatga acagcctcag ggctgaggac accggcacct acttctgcgc cagatcttct 300

tatgtttgtc ctggtggttt tagttgttgg ggtgatactg gtcaaataga cgcatggggc 360

cacgggaccg aagtcatcgt ctcctcc 387

<210> 135

<211> 387

<212> DNA

<213> Artificial Sequence

<220>

<223> 2-13D-37-3W-16 Ab VH

<400> 135

gccgtgacgt tggacgagtc cggggggcggc ctccagacgc ccggaggagc gctccgcctt

agctgcagcg cctccgggtt cgatttcagc agccatggca tgttctgggt gcgacaggcg 120

cccggcaagg ggttggaata tgtctcggag attaccaatg atggtagtgg cacaaactac 180

gggtcggcgg tgaagggccg tgccaccatc tcgagggaca acgggcagag cacactgtat 240

ctgcagatga acagcctcag ggctgaggac accggcacct acttctgcgc cagatctaat 300

tatgcttgtc ctggtggttt tagttgttgg ggtgatactg gtcaaataga cgcatggggc 360

cacgggaccg aagtcatcgt ctcctcc 387

<210> 136

<211> 309

<212> DNA

<213> Artificial Sequence

<220>

<223> 2-13D-37-1.5W-41 Ab VL

<400> 136

gccctgactc agccgtcctc ggtgtcagca aacccaggag aaaccgcgaa gataacctgc 60

tccggggggtg tgtatagcta tggctggttc cagcagaagc ctggcagtgc ccttgtcact 120

gtgatctact gggatgatga gagaccctcg gacatccctt cacgattctc cggtgcccta 180

tccggctcca caaacacatt aaccatcact ggggtccaag ccgaagacga ggctgattat 240

tattgtggga ctgaagacat cagcggcact gctggtgtat ttggggccgg gacaaccctg 300

accgtcctg 309

<210> 137

<211> 309

<212> DNA

<213> Artificial Sequence

<220>

<223> 2-13D-37-3W-16 Ab VL

<400> 137

gccctgactc agccgtcctc ggtgtcagca aacccaggag aaaccgcgaa gataacctgc 60

tccggggggtg tgtatagcta tggctggttc cagcagaagc ctggcagtgc ccttgtcact 120

gtgatctact gggatgatga gagaccctcg gacatccctt cacgattctc cggtgcccta 180

tccggctcca caaacacatt aaccatcact ggggtccaag ccgaagacga ggctgattat 240

tattgtggga ctgaagacat cagcggcact gctggtgtat ttggggccgg gacaaccctg 300

accgtcctg 309

<---

Claims (91)

1. An isolated antibody, or an antigen-binding portion thereof, that specifically binds a protein of a human A5 family member with sequence similarity 19 (FAM19A5) ("anti-FAM19A5 antibody"), comprising heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3, where VH-CDR1 is selected from SEQ ID NO: 5, 14, 16 or 19; VH-CDR2 is selected from SEQ ID NO: 13, 15 or 17; VH-CDR3 is selected from SEQ ID NO: 7, 18, 128 or 129; and VL-CDR1 is selected from SEQ ID NO: 8, 21 or 80; VL-CDR2 is selected from SEQ ID NO: 20, 22, 24, 25, 26, 28 or 31; VL-CDR3 is selected from SEQ ID NO: 10, 23, 27, 29, or 32, and wherein the antibody has reduced immunogenicity in humans compared to a reference antibody containing the VH set forth in SEQ ID NO: 11 and the VL set forth in SEQ ID NO: 12. 2. An isolated antibody, or antigen-binding portion thereof, that specifically binds to the human FAM19A5 protein, comprising a heavy chain CDR1, CDR2 and CDR3 and a light chain CDR1, CDR2 and CDR3, wherein: (i) the heavy chain CDR1 comprises the amino acid sequence shown in SEQ ID NO: 5; (ii) the heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 13; (iii) the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 7; (iv) the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 8; (v) the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 20; and (vi) the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 10. 3. An isolated antibody, or antigen-binding portion thereof, that specifically binds to the human FAM19A5 protein, comprising 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 set forth in SEQ ID NO: 15; (iii) the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 7; (iv) the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 21; (v) the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 22; and (vi) the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 23. 4. An isolated antibody or antigen-binding portion thereof that specifically binds to the human FAM19A5 protein, comprising a heavy chain CDR1, CDR2 and CDR3 and a 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 set forth in SEQ ID NO: 15; (iii) the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 7; (iv) the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 21; (v) the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 24; and (vi) the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 23. 5. An isolated antibody, or antigen-binding portion thereof, that specifically binds to the human FAM19A5 protein, comprising a heavy chain CDR1, CDR2 and CDR3 and a 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 set forth in SEQ ID NO: 15; (iii) the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 7; (iv) the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 8; (v) the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 25; and (vi) the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 23. 6. An isolated antibody or antigen-binding portion thereof that specifically binds to the human FAM19A5 protein, comprising a heavy chain CDR1, CDR2 and CDR3 and a 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 set forth in SEQ ID NO: 15; (iii) the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 7; (iv) the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 8; (v) the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 24; and (vi) the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 23. 7. An isolated antibody or antigen-binding portion thereof that specifically binds to the human FAM19A5 protein, comprising a heavy chain CDR1, CDR2 and CDR3 and a 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 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. 8. An isolated antibody, or antigen-binding portion thereof, that specifically binds to the human FAM19A5 protein, comprising a heavy chain CDR1, CDR2, and CDR3 and a 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 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. 9. An antibody according to any one of paragraphs. 1-8, containing a heavy chain variable region (VH) and a light chain variable region (VL), where VH includes an amino acid sequence that is at least about 80% identical, at least about 85%, at least about 90% identical , at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% of the specified amino acid sequence in SEQ ID NO: 11, and /or where VL includes an amino acid sequence that is at least about 80% identical, 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% of the amino acid sequence specified in SEQ ID NO: 12. 10. An antibody according to any one of paragraphs. 1-9, characterized in that the specified antibody cross-competes with a reference antibody, which includes the variable region of the heavy chain (VH) and the variable region of the light chain (VL), and: (a) VH includes the amino acid sequence set forth in SEQ ID NO: 33 and VL includes the amino acid sequence set forth in SEQ ID NO: 38; (b) VH includes the amino acid sequence shown in SEQ ID NO: 34 and VL includes the amino acid sequence shown in SEQ ID NO: 39; (c) VH includes the amino acid sequence set forth in SEQ ID NO: 34 and VL includes the amino acid sequence set forth in SEQ ID NO: 41; (d) VH includes the amino acid sequence set forth in SEQ ID NO: 34 and VL includes the amino acid sequence set forth in SEQ ID NO: 40; (e) VH includes the amino acid sequence set forth in SEQ ID NO: 34 and VL includes the amino acid sequence set forth in SEQ ID NO: 42; (f) VH includes the amino acid sequence set forth in SEQ ID NO: 34 and VL includes the amino acid sequence set forth in SEQ ID NO: 43; or (g) VH includes the amino acid sequence shown in SEQ ID NO: 34 and VL includes the amino acid sequence shown in SEQ ID NO: 44. 11. An antibody according to any one of paragraphs. 1-10, characterized in that said antibody binds to the same human FAM19A5 epitope as the control antibody, which includes the heavy chain variable region (VH) and the light chain variable region (VL), wherein: (a) VH includes the amino acid sequence set forth in SEQ ID NO: 33 and VL includes the amino acid sequence set forth in SEQ ID NO: 38; (b) VH includes the amino acid sequence shown in SEQ ID NO: 34 and VL includes the amino acid sequence shown in SEQ ID NO: 39; (c) VH includes the amino acid sequence set forth in SEQ ID NO: 34 and VL includes the amino acid sequence set forth in SEQ ID NO: 41; (d) VH includes the amino acid sequence set forth in SEQ ID NO: 34 and VL includes the amino acid sequence set forth in SEQ ID NO: 40; (e) VH includes the amino acid sequence set forth in SEQ ID NO: 34 and VL includes the amino acid sequence set forth in SEQ ID NO: 42; (f) VH includes the amino acid sequence set forth in SEQ ID NO: 34 and VL includes the amino acid sequence set forth in SEQ ID NO: 43; or (g) VH includes the amino acid sequence shown in SEQ ID NO: 34 and VL includes the amino acid sequence shown in SEQ ID NO: 44. 12. An antibody according to claim 11, wherein the human FAM19A5 epitope comprises the amino acid sequence indicated in SEQ ID NO: 90, 91, or 92. 13. An isolated antibody, or an antigen-binding portion thereof, that specifically binds a human A5 family member protein with sequence similarity 19 (FAM19A5) ("anti-FAM19A5 antibody"), comprising heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3, where the heavy chain CDR1 contains an amino acid sequence selected from SEQ ID NO: 16 or 19, the heavy chain CDR2 contains the amino acid sequence of SEQ ID NO: 17 and the heavy chain CDR3 contains an amino acid sequence selected from SEQ ID NO: 18, 128 or 129, where the light chain CDR1 contains an amino acid sequence selected from SEQ ID NO: 30 and SEQ ID NO: 30, in which the amino acid serine at position 4 of SEQ ID NO: 30 is replaced by valine, the light chain CDR2 contains the amino acid sequence of SEQ ID NO: 31 and The light chain CDR3 contains the amino acid sequence of SEQ ID NO: 32, and wherein the antibody has reduced human immunogenicity and/or higher binding affinity for the human FAM19A5 protein compared to a reference antibody containing the VH shown in SEQ ID NO: 35 and the VL shown in SEQ ID NO: 45. 14. An antibody according to claim 13, containing a heavy chain variable region (VH) and a light chain variable region (VL), where VH includes an amino acid sequence that is at least about 80% identical, at least about 85%, at least 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% of the specified amino acid sequence in SEQ ID NO :35, and/or where VL includes an amino acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97%, at least about 98%, at least about 99%, or about 100% of the amino acid sequence set forth in SEQ ID NO: 45. 15. An antibody according to any one of paragraphs. 13, 14, characterized in that the specified antibody cross-competes with a reference antibody that includes the variable region of the heavy chain (VH) and the variable region of the light chain (VL), where VH includes the amino acid sequence indicated in SEQ ID NO: 36, and VL includes the amino acid sequence shown in SEQ ID NO: 46. 16. An antibody according to any one of paragraphs. 13, 14, characterized in that the specified antibody cross-competes with a reference antibody that includes the variable region of the heavy chain (VH) and the variable region of the light chain (VL), where VH includes the amino acid sequence indicated in SEQ ID NO: 37, and VL includes the amino acid sequence shown in SEQ ID NO: 46. 17. An antibody according to any one of paragraphs. 13, 14, characterized in that the specified antibody cross-competes with a reference antibody that includes a heavy chain variable region (VH) and a light chain variable region (VL), where VH includes the amino acid sequence indicated in SEQ ID NO: 130, and VL includes the amino acid sequence shown in SEQ ID NO: 46. 18. An antibody according to any one of paragraphs. 13, 14, characterized in that the specified antibody cross-competes with a reference antibody that includes the variable region of the heavy chain (VH) and the variable region of the light chain (VL), where VH includes the amino acid sequence indicated in SEQ ID NO: 131, and VL includes the amino acid sequence shown in SEQ ID NO: 46. 19. An antibody according to any one of paragraphs. 1-18, characterized in that the antibody is selected from the group consisting of IgG1, IgG2, IgG3, IgG4, its variant and any combination thereof. 20. An antibody according to any one of paragraphs. 1-19, which is a chimeric antibody, a human antibody, or a humanized antibody. 21. An antibody according to any one of paragraphs. 1-20 containing Fab, Fab', F(ab')2, Fv or single chain Fv (scFv). 22. The antibody of claim 21, which is scFv. 23. An antibody according to claim 22, characterized in that scFv includes VH and VL, and: (a) VH includes the amino acid sequence set forth in SEQ ID NO: 33 and VL includes the amino acid sequence set forth in SEQ ID NO: 38; (b) VH includes the amino acid sequence shown in SEQ ID NO: 34 and VL includes the amino acid sequence shown in SEQ ID NO: 39; (c) VH includes the amino acid sequence set forth in SEQ ID NO: 34 and VL includes the amino acid sequence set forth in SEQ ID NO: 41; (d) VH includes the amino acid sequence set forth in SEQ ID NO: 34 and VL includes the amino acid sequence set forth in SEQ ID NO: 40; (e) VH includes the amino acid sequence set forth in SEQ ID NO: 34 and VL includes the amino acid sequence set forth in SEQ ID NO: 42; (f) VH includes the amino acid sequence set forth in SEQ ID NO: 34 and VL includes the amino acid sequence set forth in SEQ ID NO: 43; (g) VH includes the amino acid sequence set forth in SEQ ID NO: 34 and VL includes the amino acid sequence set forth in SEQ ID NO: 44; (h) VH includes the amino acid sequence set forth in SEQ ID NO: 36 and VL includes the amino acid sequence set forth in SEQ ID NO: 46; (i) VH includes the amino acid sequence set forth in SEQ ID NO: 37 and VL includes the amino acid sequence set forth in SEQ ID NO: 46; (j) VH includes the amino acid sequence set forth in SEQ ID NO: 130 and VL includes the amino acid sequence set forth in SEQ ID NO: 46; or (k) VH includes the amino acid sequence set out in SEQ ID NO: 131 and VL includes the amino acid sequence set out in SEQ ID NO: 46. 24. An antibody according to any one of paragraphs. 1-23 which exhibits one or more of the following properties: (a) binding to soluble human FAM19A5 with a KD of 10 nM or less as measured by enzyme immunoassay (ELISA); (b) binding to membrane-bound human FAM19A5 with a KD of 10 nM or less as measured by ELISA; (c) reducing, reversing, delaying and/or preventing the onset of reactive gliosis; (d) suppression of excessive proliferation of reactive astrocytes; (e) decreased expression of chondroitin sulfate proteoglycans, including neurocan and neuron-glial antigen 2 (NG2); (e) increased expression of c-fos and pERK in neuronal nuclei; (g) ensuring the survival of neurons; (h) increase in GAP43 expression in neurons; (i) ensuring the regrowth of the axon; (j) induction of normalization of blood vessels, for example, in a tumor; (k) inhibition of tumor growth; (l) increased infiltration of immune cells into the tumor; (m) increased infiltration of neuronal cells into the tumor; (n) enhancing the phagocytic activity of the macrophage or microglia; (o) increasing the potential of the mitochondrial membrane of the macrophage or microglia; (p) reducing the recruitment of myeloid suppressor cells (MDSC) to the tumor; (q) reduction of tumor necrosis and edema; (r) reducing the permeability of the tumor tissue; and (s) increase in tumor blood flow velocity. 25. Nucleic acid encoding an antibody according to any one of paragraphs. 1-24. 26. An expression vector containing the nucleic acid according to claim 25. 27. A cell for expressing an antibody according to any one of claims 1 to 24, containing a vector according to claim 26. 28. Composition for inhibiting the activity of FAM19A5, comprising an antibody according to any one of paragraphs. 1-24, a nucleic acid according to claim 25, a vector according to claim 26 or a cell according to claim 27, and a pharmaceutically acceptable carrier. 29. A kit for inhibiting FAM19A5 activity, comprising an antibody according to any one of paragraphs. 1-24, a nucleic acid according to claim 25, a vector according to claim 26, a cell according to claim 27, and instructions for use. 30. A method for producing an antibody that specifically binds to the human FAM19A5 protein, comprising culturing a cell according to claim 27 under suitable conditions and isolating the antibody. 31. A method of treating a disease or condition in an object in need thereof, comprising administering to the object an antibody according to any one of paragraphs. 1-24, the nucleic acid of claim 25, the vector of claim 26, the cell of claim 27, wherein the disease or condition is selected from central nervous system injury, degenerative brain disease, and neuropathic pain. 32. The method of claim 31, wherein the antibody, nucleic acid, vector, cell, or immunoconjugate induce normalization of blood vessels. 33. The method according to claim 32, characterized in that the normalization of blood vessels is accompanied by changes in the properties of blood vessels, including increased connectivity, increased wall thickness, reduced vessel diameter, more regular direction and distribution of vessels, increased number of vessels, reduced leakage and permeability, increased pericyte coverage and proximity to blood vessels, increased oxygenation, or combinations thereof. 34. The method according to any one of paragraphs. 31-33, characterized in that the antibody, nucleic acid, vector, cell or immunoconjugate enhance the phagocytic activity of a macrophage or microglia. 35. The method according to any one of paragraphs. 31-34, characterized in that the antibody, nucleic acid, vector, cell or immunoconjugate increase the potential of the mitochondrial membrane of a macrophage or microglia. 36. The method according to any one of paragraphs. 31-35, which includes the introduction of an additional therapeutic agent. 37. The method according to p. 36, characterized in that the additional therapeutic agent includes chemotherapy, immunotherapy, radiation therapy, or combinations thereof.

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