Antibodies against sequence-similar family 19 member A5 and methods of use thereof

Abstract

The present disclosure provides antibodies that specifically bind to human FAM19A5 and compositions comprising such antibodies. In one particular aspect, antibodies are de-immunized to reduce immunogenicity in a human subject. In some embodiments, the anti-FAM19A5 antibodies may utilize such antibodies to modulate FAM19A5 activity, such as to prevent, inhibit, reduce, or reverse the onset of reactive glial hyperplasia and / or reactive astrocyte hyperproliferation. The present disclosure also provides methods of treating a disorder, such as central nervous system injury, degenerative brain disorder, neuropathic pain, or cancer, by administering an antibody that specifically binds to human FAM19A5.

Description

[0001] References to electronically submitted sequence listings

[0002] The contents of the sequence listing submitted electronically as an ASCII text file (name: 3763_011PC02_SeqListing_ST25.txt; size: 34,309 bytes; creation date: May 10, 2019) submitted with this application are incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure provides antibodies that specifically bind (e.g., deimmunize) to sequence similar family 19 member A5 (FAM19A5), compositions comprising such antibodies, and methods of using such antibodies to prevent or treat conditions or diseases caused by damage to the central nervous system of a subject. Background Art

[0004] 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 at fixed positions and are distantly related to macrophage inflammatory protein 1-α (MIP-1-α), a member of the CC chemokine family. TAFA proteins are mainly expressed in specific areas of the brain and spinal cord. It is believed that these proteins are produced and secreted by adult neural stem cells during neurogenesis.

[0005] FAM19A5 is mainly expressed in the brain of vertebrates, and it is believed that FAM19A5 is important for the development, differentiation and formation of a complete central nervous system, and can be used to prevent or treat central nervous system damage and / or diseases. US Patent Publication No. 2015 / 0118230.

[0006] Although inhibition of FAM19A5 can play an important role in treating central nervous system diseases, there is still a need to develop antibodies that specifically bind to FAM19A5 and are able to modulate FAM19A5 activity. Summary of the invention

[0007] The present disclosure provides antibodies or antigen-binding portions thereof that specifically bind to a sequence-similar family 19 member A5 (FAM19A5) protein ("anti-FAM19A5 antibodies"). In some embodiments, the anti-FAM19A5 antibodies comprise heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3, wherein the heavy chain CDR1, CDR2 and CDR3 comprise the amino acid sequences shown in SEQ ID NOs: 5, 6 and 7, respectively, each of which optionally comprises one, two or three mutations, wherein the light chain CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NOs: 8, 9 and 10, respectively, wherein at least one of the light chain CDR1, CDR2 and CDR3 comprises one, two or three mutations, and wherein the antibody has reduced immunogenicity in humans compared to a reference antibody comprising a VH shown in SEQ ID NO: 11 and a VL shown in SEQ ID NO: 12. In some embodiments, the heavy chain CDR3 comprises the amino acid sequence shown in SEQ ID NO: 7 (GSASYITAATIDA). In some embodiments, the heavy chain CDR1 comprises the amino acid sequence (SYQMG) set forth in SEQ ID NO: 5, which optionally has one, two or three mutations. In some embodiments, the heavy chain CDR2 comprises the amino acid sequence (VINKSGSDTS) set forth in SEQ ID NO: 6, which optionally has one, two or three mutations.

[0008] In some embodiments, the light chain CDR3 comprises the amino acid sequence shown in SEQ ID NO: 10 (GNDDYSSDSGYVGV), which optionally has one, two or three mutations.

[0009] In some embodiments, the light chain CDR1 comprises the amino acid sequence (SGGGSSGYGYG) of SEQ ID NO:8, which has one, two or three mutations. In some embodiments, the mutation comprises substitution of glycine at amino acid 4 of SEQ ID NO:8 with an aliphatic amino acid. In certain embodiments, the aliphatic amino acid comprises alanine, valine, leucine or isoleucine.

[0010] In some embodiments, the light chain CDR2 comprises the amino acid sequence (WNDKRPS) shown in SEQ ID NO:9, which has one, two or three mutations. In some embodiments, the mutation comprises that the tryptophan at amino acid 1 of SEQ ID NO:9 is replaced by a basic amino acid. In certain embodiments, the basic amino acid comprises arginine, histidine or lysine. In some embodiments, the mutation comprises that the asparagine at amino acid 2 of SEQ ID NO:9 is replaced by an acidic amino acid. In some embodiments, wherein the acidic amino acid comprises aspartic acid or glutamic acid. In some embodiments, the mutation comprises that the lysine at amino acid 4 of SEQ ID NO:9 is replaced by an acidic amino acid. In certain embodiments, the acidic amino acid comprises aspartic acid or glutamic acid.

[0011] In some embodiments, the anti-FAM19A5 antibodies disclosed herein comprise 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:5; (ii) the heavy chain CDR2 comprises the amino acid sequence shown in SEQ ID NO:6; (iii) the heavy chain CDR3 comprises the amino acid sequence shown in SEQ ID NO:7; (iv) the light chain CDR1 comprises the amino acid sequence shown in SEQ ID NO:13; (v) the light chain CDR2 comprises the amino acid sequence shown in SEQ ID NO:14; and (vi) the light chain CDR3 comprises the amino acid sequence shown in SEQ ID NO:10.

[0012] In some embodiments, the anti-FAM19A5 antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to the amino acid sequence shown in SEQ ID NO:11, and / or wherein VL comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to the amino acid sequence shown in SEQ ID NO:12.

[0013] In some embodiments, the anti-FAM19A5 antibodies of the present disclosure cross-compete with a reference antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH comprises the amino acid sequence set forth in SEQ ID NO: 11, and VL comprises the amino acid sequence set forth in SEQ ID NO: 12. In certain embodiments, the anti-FAM19A5 antibodies cross-compete with a reference antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH comprises the amino acid sequence set forth in SEQ ID NO: 30, and VL comprises the amino acid sequence set forth in SEQ ID NO: 32. In other embodiments, the anti-FAM19A5 antibodies cross-compete with a reference antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH comprises the amino acid sequence set forth in SEQ ID NO: 31, and VL comprises the amino acid sequence set forth in SEQ ID NO: 32. In a further embodiment, the anti-FAM19A5 antibodies of the present disclosure cross-compete with a reference antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), and wherein VH comprises the amino acid sequence shown in SEQ ID NO:31, and VL comprises the amino acid sequence shown in SEQ ID NO:32.

[0014] In some embodiments, the anti-FAM19A5 antibody of the present disclosure is selected from the group consisting of: IgG1, IgG2, IgG3, IgG4, variants thereof, and any combination thereof. In some embodiments, the anti-FAM19A5 antibody is a chimeric antibody, a human antibody, or a humanized antibody.

[0015] In some embodiments, the anti-FAM19A5 antibodies disclosed herein comprise Fab, Fab', F(ab')2, Fv or single-chain Fv (scFv). In some embodiments, the anti-FAM19A5 antibody is scFv. In certain embodiments, the scFv comprises VH and VL, wherein VH comprises the amino acid sequence shown in SEQ ID NO: 17, and VL comprises the amino acid sequence shown in SEQ ID NO: 18.

[0016] In some embodiments, the anti-FAM19A5 antibodies disclosed herein exhibit one or more of the following properties: (a) binding to soluble human FAM19A5 with a KD of 10 nM or less as determined by enzyme-linked immunosorbent assay (ELISA); (b) binding to membrane-bound human FAM19A5 with a KD of 10 nM or less as determined by ELISA; (c) reducing, reversing, delaying and / or preventing the onset of reactive gliosis; (d) inhibiting reactive astrocyte overproliferation; (e) reducing the expression of chondroitin sulfate proteoglycans including neurocan and neuron-glial antigen 2 (NG2); (f) increasing c-fos and pERK expression in neuronal nuclei; (g) promoting neuronal survival; (h) increasing GAP43 expression in neurons; (i) promoting axonal regeneration; (j) inducing normalization of blood vessels, such as in tumors; (k) inhibiting tumor growth; (l) enhancing the infiltration of immune cells into tumors; (m) enhancing the infiltration of neuronal cells into tumors; (n) enhancing the phagocytic activity of macrophages or microglial cells; (o) increasing the mitochondrial membrane potential of macrophages or microglial cells; (p) reducing the recruitment of myeloid-derived suppressor cells (MDSC) to tumors; (q) reducing necrosis and edema in tumors; (r) reducing the tissue permeability of tumors; and (s) increasing the blood flow rate in tumors.

[0017] Also provided herein are nucleic acids encoding the anti-FAM19A5 antibodies disclosed herein; vectors comprising the nucleic acids; cells comprising the vectors; and immunoconjugates comprising the anti-FAM19A5 antibodies disclosed herein. Also disclosed herein are compositions comprising the anti-FAM19A5 antibodies, nucleic acids, vectors, cells or immunoconjugates disclosed herein, and vectors. The disclosure also provides kits comprising the anti-FAM19A5 antibodies, nucleic acids, vectors, cells or immunoconjugates disclosed herein, and instructions for use.

[0018] Also provided herein is a method for producing an antibody that specifically binds to human FAM19A5 protein, which comprises culturing the cell disclosed herein under suitable conditions and isolating the antibody.

[0019] 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 described herein. In some embodiments, the disease or condition comprises a tumor, fibrosis, glaucoma, mood disorders, neurodegenerative diseases (e.g., Alzheimer's disease), stroke or neuropathic pain. In certain embodiments, the disease or condition is a tumor.

[0020] In some embodiments, the tumor comprises melanoma, pancreatic cancer, glioma, breast cancer, lymphoma, lung cancer, kidney cancer, prostate cancer, fibrosarcoma, colon adenocarcinoma, liver cancer, or ovarian cancer. In certain embodiments, the glioma is glioblastoma multiforme (GBM).

[0021] In some embodiments, the anti-FAM19A5 antibodies, nucleic acids, vectors, cells or immunoconjugates of the present disclosure induce normalization of blood vessels. In certain embodiments, normalization of blood vessels is accompanied by changes in vascular properties, including increased connectivity, increased wall thickness, reduced vascular diameter, more regular vascular direction and distribution pattern, increased number of blood vessels, reduced leakage and permeability, increased coverage and proximity of pericytes on blood vessels, increased oxygenation or a combination thereof.

[0022] In some embodiments, the anti-FAM19A5 antibodies, nucleic acids, vectors, cells, or immunoconjugates of the present disclosure inhibit the growth of a tumor.

[0023] In some embodiments, the anti-FAM19A5 antibodies, nucleic acids, vectors, cells or immunoconjugates disclosed herein enhance the infiltration of immune cells into tumors. In certain embodiments, immune cells include macrophages, dendritic cells, T lymphocytes, B lymphocytes, natural killer (NK) cells or combinations thereof. In certain embodiments, immune cells further show hypertrophy. In certain embodiments, the infiltration of immune cells into tumors is enhanced with an increase in the infiltration of neuronal cells into tumors. In certain embodiments, neuronal cells include astrocytes, glial cells or combinations thereof.

[0024] In some embodiments, anti-FAM19A5 antibodies, nucleic acids, vectors, cells or immunoconjugates enhance the phagocytic activity of macrophages or microglial cells. In some embodiments, anti-FAM19A5 antibodies, nucleic acids, vectors, cells or immunoconjugates increase the mitochondrial membrane potential of macrophages or microglial cells.

[0025] In some embodiments, anti-FAM19A5 antibodies, nucleic acids, carriers, cells or immunoconjugates disclosed herein reduce the recruitment of myeloid suppressor cells (MDSC) to tumors. In some embodiments, anti-FAM19A5 antibodies, nucleic acids, carriers, cells or immunoconjugates reduce necrosis and edema in tumors. In some embodiments, anti-FAM19A5 antibodies, nucleic acids, carriers, cells or immunoconjugates reduce the tissue permeability of tumors. In some embodiments, anti-FAM19A5 antibodies, nucleic acids, carriers, cells or immunoconjugates increase the blood flow rate in tumors.

[0026] In some embodiments, the method of treating a disease or disorder further comprises administering an additional therapeutic agent. In certain embodiments, the additional therapeutic agent comprises chemotherapy, immunotherapy, radiation therapy, or a combination thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1A and 1B Provided is the heavy chain variable region of 1-65 antibody ( Figure 1A ) and light chain variable region ( Figure 1B ) were aligned with the following deimmunized 1-65 antibodies: (i) SS01-13; (ii) SS01-13-s5; and (iii) S5-SG (also described herein as S5-2.GKNG antibody or 1-65-S53G antibody). The heavy and light chain CDRs and FRs are in Figure 1A and 1B Indicated in.

[0028] Figure 2 Potential immunogenic sites on the light chain variable region (VL) (top three rows) and heavy chain variable region (VH) (bottom three rows) of the 1-65 antibody were identified. VL corresponds to SEQ ID NO: 12, and VH corresponds to SEQ ID NO: 11. Also shown are the sequences of the human germlines used for the framework regions of the 1-65 antibody: VL = immunoglobulin lambda variable region gene 3-25 (IGLV3-25*02) and immunoglobulin lambda binding region gene 2 (IGLJ2*01); VH = immunoglobulin heavy chain variable region gene 3-23 (IGHV3-23*02) and immunoglobulin heavy chain binding region gene 1 (IGHJ1*01). The human germline used is the most homologous human germline to the 1-65 clone (IgBLAST, NCBI). The sequences as determined by ITOPE TMPromiscuous MHC class II binding peptides identified as having high and moderate immunogenic potential were analyzed. Specifically, MHCII binding peptides with high immunogenic potential are as follows: (i) peptide #2: residues 46-54 of SEQ ID NO: 12 (IYWNDKRPS), (ii) peptide #8: residues 48-56 of SEQ ID NO: 11 (VGVINKSGS), (iii) peptide #10: residues 79-87 of SEQ ID NO: 11 (VRLQLNNLR); and (iv) peptide #13: residues 103-111 of SEQ ID NO: 11 (YITAATIDA). MHCII binding peptides with moderate immunogenicity are as follows: (i) peptide #1: residues 16-24 of SEQ ID NO: 12 (VKITCSGGG); (ii) peptide #3: residues 71-79 of SEQ ID NO: 12 (LTITGVQAE); (iii) peptide #4: residues 18-26 of SEQ ID NO: 11 (LSLVCKASG); (iv) peptide #5: residues 103-111 of SEQ ID NO: 11 (YITAATIDA). NO:11 residues 20-28 (LVCKASGFT); (v) peptide #6: residues 32-40 (YQMGWVRQA) of SEQ ID NO:11; (vi) peptide #7: residues 45-53 (LEWVGVINK) of SEQ ID NO:11; (vii) peptide #9: residues 64-72 (VKGRATISR) of SEQ ID NO:11; (viii) peptide #11: residues 81-87 (LQLNNLR) of SEQ ID NO:11; (ix) peptide #12: residues 86-94 (LRAEDTGTY) of SEQ ID NO:11. Homologous peptides from the T cell epitope database are shown below: peptide #5, peptide #6, peptide #9 and peptide #12. A total of 13 binding peptides were identified, labeled peptides #1 to #13. "P1" of each of the binding peptides indicates the first anchor position.

[0029] Figure 3A and 3B Binding analysis of the deimmunized 1-65 antibody is provided. To deimmunize the antibody, non-identical amino acid residues in the MHC class II binding region of clone 1-65 were replaced with the corresponding amino acids in the human germline sequence. Figure 3AA sequence comparison of the light chain variable region (VL) and heavy chain variable region (VH) of (i) a normal 1-65 antibody ("clone 1-65"), (ii) a completely deimmunized 1-65 antibody ("fully deimmunized clone 1-65"), and (iii) a deimmunized antibody ("deimmunized clone 3-2") except for two amino acids (residue 23 (FR1 region) and residue 54 (HCDR2 region)) of VH (SEQ ID NO: 11). The "deimmunized clone 3-2" antibody is also described herein as the "SS01-13-s5" antibody (see, e.g., Figure 1A and 1B ). With the ITOPE TM The amino acid residues determined to have high and moderate immunogenic potential are indicated within the box by "1" and "2", respectively. Figure 3B Comparison of binding of (i) normal 1-65 antibody, (ii) completely deimmunized 1-65 antibody and (iii) deimmunized ("deimmunized clone 1-65") antibody except at one amino acid with heavy chain CDR2 for FAM19A5 protein as determined by ELISA is shown. Each single chain variable fragment (scFv) of the displayed phage is added to the wells of the microtiter plate coated with FAM19A5 (■) or anti-HA antibody (□). The background signal is measured in the control wells coated with BSA. The wells are probed with HRP-labeled anti-M13 antibody. The absorbance at 405nm is measured. The results are shown as the mean ± SD obtained from quadruplicate experiments.

[0030] Figure 4A , 4B 4C and 4D provide analysis of two different deimmunized 1-65 antibodies: (i) "deimmunized clone 1-65" Figure 3A and 3B The deimmunized antibodies shown are identical; and (ii) "deimmunized clone S5-SG" ("S5-SG") is identical to "deimmunized clone 1-65", except that it has been modified to remove a potential N-glycosylation site in HCDR2 (S to G at amino acid 5 of HCDR2 (i.e., residue 54 of SEQ ID NO: 11), identified by "*"). Figure 4A and 4B The size and expression level of the antibodies measured using SDS-PAGE are shown. Figure 4A In the figure, lanes "1" and "2" correspond to antibodies SS01-13-S5 and S5-SG, respectively. In lanes "1" and "2", "A" and "B" correspond to before and after centrifugation, respectively. Figure 4B The left panel shows reduced SDS-PAGE, while the right panel shows non-reduced SDS-PAGE. Figure 4CSequence comparisons of the light chain variable region (VL) and heavy chain variable region (VH) of the antibodies are provided. Figure 4D Comparison of binding of (i) normal 1-65 antibody ("clone 1-65"), (ii) deimmunized 1-65 antibody (also described herein as SS01-13-S5 antibody), and (iii) deimmunized clone S5-SG antibody to FAM19A5 protein as determined by ELISA is shown. Each single-chain variable fragment (scFv) of the displayed phage was added to a well of a microtiter plate coated with FAM19A5 (■) or anti-HA antibody (□). Background signal was measured in a BSA-coated control well. The wells were probed with an HRP-labeled anti-M13 antibody. The absorbance at 405 nm was measured. The results are shown as the mean ± SD obtained from quadruplicate experiments.

[0031] Figure 5A , 5B and 5C provide antibodies 1-65 ( Figure 5A )、SS01-13-S5( Figure 5B ) and S5-SG( Figure 5C ) and FAM19A5 protein binding SPR test results. Figures 5A to 5C In each figure, the sensorgram is provided at the top, while the quantitative values ​​of the binding affinity of the antibodies (ka, kd, K D and Rmax).

[0032] Fig. 6A , 6B , 6C and 6D provide the ELISA results of the binding of 1-65, SS01-13-S5 and S5-SG antibodies to FAM19A5 protein. Fig. 6A , 6B and 6C show the results for antibodies 1-65, SS01-13-S5, and S5-SG, respectively, as bar graphs at different antibody concentrations. Fig.6D A table including EC50 values ​​for the antibodies is provided.

[0033] Fig. 7A , 7B and 7C provided the BV2 cells with PHRODO TM Comparison of phagocytic uptake of green E. coli bioparticles. BV2 cells were treated with one of the following: (i) PBS, (ii) human FAM19A5-Fc protein alone (0 μg / mL group), or (iii) human FAM19A5-Fc protein combined with different concentrations (3.125 μg / mL, 6.25 μg / mL, 12.5 μg / mL, or 25 μg / mL) of 1-65 ( Fig. 7A )、SS01-13-S5( Figure 7B ) or S5-SG( Figure 7C ) Antibody combinations. Data are shown as mean ± SD. DETAILED DESCRIPTION

[0034] Disclosed herein is an isolated monoclonal antibody or antigen-binding portion thereof that specifically binds to human sequence similar family 19 member A5 (FAM19A5) protein ("anti-FAM19A5 antibody") and exhibits one or more properties disclosed herein. Specifically, the anti-FAM19A5 antibody has been deimmunized to reduce immunogenicity in human subjects.

[0035] To facilitate understanding of the disclosure disclosed herein, various terms and phrases are defined. Additional definitions are set forth throughout the detailed description.

[0036] I. Definitions

[0037] Throughout this disclosure, the term "a" or "an" entity refers to one or more of that entity; for example, "antibody" is understood to represent one or more antibodies. Thus, the terms "a" (or "a"), "one or more" and "at least one" can be used interchangeably herein.

[0038] Furthermore, "and / or" as used herein should be considered as a specific disclosure that each of the two specified features or components has or does not have the other. Thus, the term "and / or" used in phrases such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" used in phrases 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 (alone); B (alone); and C (alone).

[0039] It should be understood that wherever an aspect is described with the language "comprising," other similar aspects described with "consisting of" and / or "consisting essentially of" are also provided.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure relates. For example, 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 Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press provide a general dictionary of many of the terms used in the present disclosure for one of ordinary skill.

[0041] Units, prefixes and symbols are expressed in a form accepted by the International System of Units (SI). Numerical ranges include numbers that define ranges. Unless otherwise indicated, amino acid sequences are written from left to right in the direction of amino to carboxyl. The titles provided herein are not limitations on the various aspects of the disclosure, which can be obtained by reference to the specification as a whole. Therefore, the terms defined immediately below are more fully defined by reference to the entire specification.

[0042] As used herein, the term "about" means approximately, roughly, about, or within the range of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries of the numerical values ​​set forth above or below. In general, the term "about" can modify a numerical value to be greater or less than the stated value by a variable above or below (higher or lower), such as 10%.

[0043] The term "family of sequence similarity 19 member A5" or "FAM19A5" refers to a protein belonging to the TAFA family (also known as the FAM19 family) of five highly homologous proteins that are primarily expressed in the brain and spinal cord. FAM19A5 is also known as TAFA5 or chemokine-like protein TAFA-5.

[0044] In humans, the gene encoding FAM19A5 is located on chromosome 22. There are multiple isoforms of human FAM19A5 (UniProt: Q7Z5A7) that are thought to be produced by alternative splicing: isoform 1 (UniProt: Q7Z5A7-1), consisting of 132 amino acids; isoform 2 (UniProt: Q7Z5A7-2), consisting of 125 amino acids; isoform 3 (UniProt: Q7Z5A7-3), consisting of 53 amino acids. The human FAM19A5 protein is thought to exist in membrane-bound and soluble (secreted) forms. Isoform 1 is thought to be membrane-bound with one transmembrane region. Isoform 2, reported as a secreted protein (soluble) in Tang TY et al., Genomics 83 (4): 727-34 (2004), contains a signal peptide at amino acid positions 1-25. Isoform 1 is thought to be a membrane protein. Below are the amino acid sequences of the three known human FAM19A5 isoforms.

[0045] (I) Isoform 1 (UniProt: Q7Z5A7-1, transmembrane protein): This isoform has been selected as the canonical sequence.

[0046] MAPSPRTGSR QDATALPSMS STFWAFMILA SLLIAYCSQL AAGTCEIVTL DRDSSQPRRTIARQTARCAC RKGQIAGTTR ARPACVDARI IKTKQWCDML PCLEGEGCDL LINRSGWTCT QPGGRIKTTTVS(SEQ ID NO:1)

[0047] (II) Isoform 2 (UniProt: Q7Z5A7-2, soluble protein):

[0048] MQLLKALWAL AGAALCCFLV LVIHAQFLKE GQLAAGTCEI VTLDRDSSQP RRTIARQTARCACRKGQIAG TTRARPACVD ARIIKTKQWC DMLPCLEGEG CDLLINRSGW TCTQPGGRIK TTTVS(SEQID NO:2)

[0049] (III) Isoform 3 (UniProt: Q7Z5A7-3):

[0050] MYHHREWPAR IIKTKQWCDM LPCLEGEGCD LLINRSGWTC TQPGGRIKTT TVS(SEQ ID NO:3)

[0051] The term "FAM19A5" includes any variant or isoform of FAM19A5 naturally expressed by cells. Therefore, the antibodies described herein can cross-react with different isoforms in the same species (e.g., different isoforms of human FAM19A5), or cross-react with FAM19A5 from species other than humans (e.g., mouse FAM19A5). Alternatively, the antibody can be specific to human FAM19A5 without cross-reactivity with other species. FAM19A5 or any of its variants and isoforms can be separated or recombinantly produced from cells or tissues that naturally express them. The polynucleotide encoding human FAM19A5 has GenBank accession number BC039396 and the following sequence:

[0052] Table 1A. Polynucleotide sequence of human FAM19A5

[0053]

[0054]

[0055] The term "antagonist against FAM19A5 protein" refers to all antagonists that inhibit the expression of FAM19A5 protein. Such antagonists can be peptides, nucleic acids or compounds. More specifically, the antagonist can be an antisense oligonucleotide, siRNA, shRNA, miRNA, dsRNA, aptamer, PNA (peptide nucleic acid) targeting FAM19A5 or a vector comprising the same. In some embodiments, the antagonist can be an antibody or an antigen binding portion thereof that specifically binds to the FAM19A5 protein.

[0056] The terms "antibody" and "antibodies" are terms of the art and are used interchangeably herein and refer to molecules having an antigen binding site that specifically binds to an antigen. The terms used herein include complete antibodies and any antigen binding fragments thereof (i.e., "antigen binding portion") or single chains thereof. In one embodiment, an "antibody" refers to a glycoprotein or antigen binding portion thereof comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. In another embodiment, an "antibody" refers to a single-chain antibody comprising a single variable domain, such as a VHH domain. Each heavy chain consists of a heavy chain variable region (abbreviated herein 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 herein as VL) and a light chain constant region. The light chain constant region consists of one domain CL.

[0057] The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). VH and VL are each composed of three CDRs and four FRs, arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody can mediate the binding of an immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0058] The term "Kabat numbering" and similar terms are well-known in the art and refer to a system for numbering amino acid residues in the heavy chain and light chain variable regions of an antibody or its antigen-binding portion thereof. In some aspects, the CDR of an antibody can be determined according to the Kabat numbering system (see, e.g., Kabat EA & Wu TT (1971) Ann NY Acad Sci 190: 382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system, the CDR in an antibody heavy chain molecule is usually present at amino acids 31 to 35, and one or two additional amino acids (referred to as 35A and 35B in the Kabat numbering scheme) (CDR1) may be optionally included after 35; amino acids 50 to 65 (CDR2); and amino acids 95 to 102 (CDR3). Using the Kabat numbering system, the CDRs within an antibody light chain molecule are typically present at amino acids 24 to 34 (CDR1), amino acids 50 to 56 (CDR2), and amino acids 89 to 97 (CDR3). In specific embodiments, the CDRs of the antibodies described herein have been determined according to the Kabat numbering scheme.

[0059] The phrase "amino acid position numbering as in Kabat", "Kabat position" and grammatical variants thereof refer to the numbering system used for the heavy chain variable domain or light chain variable domain of the antibody compilation 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 correspond to a shortening or insertion of a FW or CDR of the variable domain containing fewer or additional amino acids. For example, a heavy chain variable domain may include a single amino acid insertion fragment after residue 52 of H2 (residue 52a according to Kabat) and an inserted residue after heavy chain FW residue 82 (e.g., residues 82a, 82b, and 82c, etc. according to Kabat). See Table 1B.

[0060] Table 1B

[0061]

[0062] The Kabat numbering of the residues of a given antibody can be determined by comparing the homology region of the sequence of the antibody with the sequence of the "standard" Kabat numbering. In contrast, Chothia refers to the position of the structural loop (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987)). Depending on the length of the loop, when the numbering is specified using the Kabat numbering, the end of the Chothia CDR-H1 loop changes between H32 and H34 (this is because the Kabat numbering scheme places the insertion at H35A and H35B; if both 35A and 35B are absent, the loop ends at 32; if only 35A is present, the loop ends at 33; and if 35A and 35B are present at the same time, the loop ends at 34). The AbM hypervariable region represents a compromise between the Kabat CDR and Chothia structural loops and is used by the AbM antibody modeling software of Oxford Molecular.

[0063] IMGT (ImMunoGeneTics) also provides a numbering system for immunoglobulin variable regions including CDRs. See, for example, Lefranc, MP et al., Dev. Comp. Immunol. 27: 55-77 (2003), which is incorporated herein by reference. The IMGT numbering system is based on the alignment of more than 5,000 sequences, structural data, and characterization of hypervariable loops, and allows easy comparison of variable regions and CDR regions of all species. According to the IMGT numbering scheme, VH-CDR1 is located at positions 26 to 35, VH-CDR2 is located at positions 51 to 57, VH-CDR3 is located at positions 93 to 102, VL-CDR1 is located at positions 27 to 32, VL-CDR2 is located at positions 50 to 52, and VL-CDR3 is located at positions 89 to 97.

[0064] For all heavy chain constant region amino acid positions discussed in this disclosure, numbering is according to the EU index first described in Edelman et al., 1969, Proc. Natl. Acad. Sci. USA 63 (1): 78-85, which describes the amino acid sequence of myeloma protein EU - the first sequenced human IgG1. The EU index of Edelman et al. is also described in Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda. Thus, the phrases "EU index as set forth in Kabat" or "EU index of Kabat" and "positions according to the EU index as set forth in Kabat ..." and grammatical variants thereof refer to the residue numbering system of Edelman et al., as set forth in Kabat 1991, based on the human IgG1 EU antibody.

[0065] The numbering system for variable domains (both heavy and light chains) and light chain constant region amino acid sequences is described in Kabat 1991.

[0066] The antibody can be an immunoglobulin molecule of any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgD, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG1, IgG2, IgG3, and IgG4 in humans, and IgG1, IgG2a, IgG2b, and IgG3 in mice). Immunoglobulins, such as IgG1, exist in several allotypes that differ from each other in a maximum of a few amino acids. The antibodies disclosed herein can be from any commonly known isotype, class, subclass, or allotype. In certain embodiments, the antibodies described herein are of the IgG1, IgG2, IgG3, or IgG4 subclasses, or any hybrid thereof. In certain embodiments, the antibodies are of the human IgG1 subclass, the human IgG2 subclass, the human IgG4 subclass, or the human IgG2 / IgG4 subclass.

[0067] "Antibodies" include, for example, naturally occurring antibodies and non-naturally occurring antibodies; monoclonal antibodies and polyclonal antibodies; chimeric antibodies and humanized antibodies; human antibodies and non-human antibodies; fully synthetic antibodies; single-chain antibodies; monospecific antibodies; multispecific antibodies (including bispecific antibodies); tetrameric antibodies comprising two heavy chain molecules and two light chain molecules; antibody light chain monomers; antibody heavy chain monomers; antibody light chain dimers, antibody heavy chain dimers; antibody light chain-antibody heavy chain pairs; intrabodies; conjugated antibodies; monovalent antibodies; single-chain antibodies; camelized antibodies; affybodies; anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies) and single domain antibodies (sdAbs), which include binding molecules composed of a single monomeric variable antibody domain that has complete antigen binding ability (e.g., VH domain or VL domain). Harmen M.M. and Haard HJ Appl Microbiol Biotechnol. 77(1):13-22 (2007)).

[0068] As used herein, the terms "antigen binding portion" and "antigen binding fragment" of an antibody are interchangeable and refer to one or more fragments of an antibody that retain specific binding ability to an antigen (e.g., human FAM19A5). Such "fragments" are, for example, between about 8 and about 1500 amino acids in length, suitably between about 8 and about 745 amino acids, suitably about 8 to about 300, such as about 8 to about 200 amino acids, or about 10 to about 50 or 100 amino acids. It has been shown that the antigen binding function of an antibody can be performed by a fragment 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 VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody and a disulfide-linked Fv (sdFv); (v) a dAb fragment consisting of a VH domain (Ward et al., (1989) Nature 341:544-546); and (vi) an isolated complementarity determining region (CDR) or (vii) a combination of two or more isolated CDRs that may optionally be linked by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by independent genes, they can be linked using recombinant methods by synthetic linkers that enable them to become one protein chain, wherein the VL region and the VH region pair to form a monovalent molecule (referred to as single-chain Fv (scFv); see, (1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies. Antigen-binding portions can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulins.

[0069] As used herein, the terms "variable region" and "variable domain" are used interchangeably and are common in the art. The variable region generally refers to a portion of an antibody, generally a portion of a light chain or a heavy chain, generally about 110 to 120 amino acids at the amino terminal of a mature heavy chain, about 90 to 115 amino acids of a mature light chain (sequences vary greatly between different antibodies), and is used for the binding and specificity of a particular antibody to its specific antigen. The variability of the sequence is concentrated in those regions called complementary determining regions (CDRs), while the more highly conserved regions in the variable domains are called framework regions (FRs).

[0070] Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light chain and heavy chain are primarily responsible for the interaction and specificity of the antibody with the antigen. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region comprises rodent or mouse CDRs and human framework regions (FRs). In specific embodiments, the variable region is a primate (e.g., non-human primate) variable region. In certain embodiments, the variable region comprises rodent or mouse CDRs and primate (e.g., non-human primate) framework regions (FRs).

[0071] As used herein, the term "heavy chain" (HC) when used in relation to antibodies may refer to any of the different types based on the amino acid sequence of the constant domain, such as alpha (α), delta (δ), epsilon (ε), gamma (γ) and mu (μ), which give rise to antibodies of the IgA, IgD, IgE, IgG and IgM classes, respectively, including subclasses of IgG, such as IgG1, IgG2, IgG3 and IgG4.

[0072] As used herein, the term "light chain" (LC) when used with respect to an antibody can refer to any of the different types of amino acid sequences based on the constant domain, such as kappa (κ) or lambda (λ). Light chain amino acid sequences are well known in the art. In a specific embodiment, the light chain is a human light chain.

[0073] The terms "VL" and "VL domain" are used interchangeably to refer to the light chain variable region of an antibody.

[0074] The terms "VH" and "VH domain" are used interchangeably to refer to the heavy chain variable region of an antibody.

[0075] As used herein, the terms "constant region" and "constant domain" are interchangeable and have their ordinary meaning in the art. The constant region is the portion of an antibody, such as the carboxyl terminal portion of a light chain and / or a heavy chain, which is not directly involved in binding the antibody to an antigen, but may exhibit a variety of effector functions, such as interactions with Fc receptors. Relative to immunoglobulin variable domains, the constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence.

[0076] "Fc region" (fragment crystallizable region) or "Fc domain" or "Fc" refers to the C-terminal region of an antibody heavy chain that mediates the binding of immunoglobulins to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or to the first component (C1q) of the classical complement system. Thus, the Fc region comprises the constant region of an antibody (e.g., CH1 or CL) except for the first constant region immunoglobulin domain. In IgG, IgA, and IgD antibody isotypes, the Fc region comprises two identical protein fragments, the second (CH2) and third (CH3) constant domains from the two heavy chains of the antibody, respectively. IgM and IgE Fc regions comprise three heavy chain constant domains (CH domains 2 to 4) in each polypeptide chain. For IgG, the Fc region comprises immunoglobulin domains Cγ2 and Cγ3 and a hinge between Cγ1 and Cγ2. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the human IgG heavy chain Fc region is generally defined as extending from amino acid residue C226 or P230 (or amino acids between these two amino acids) to the carboxyl terminus of the heavy chain, wherein numbering is according to the EU index as in Kabat. The CH2 domain of the human IgG Fc region extends from about amino acid 231 to about amino acid 340, 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 of IgG to about amino acid 447. As used herein, the Fc region may be a native sequence Fc, including any allotypic variants, or a variant Fc (e.g., a non-naturally occurring Fc). Fc may also refer to this region alone, or in the context of a protein polypeptide comprising Fc, such as a "binding protein comprising an Fc region", also referred to as an "Fc fusion protein" (e.g., an antibody or immunoadhesin).

[0077] A "native sequence Fc region" or "native sequence Fc" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include native sequence human IgG1 Fc regions; native sequence human IgG2 Fc regions; native sequence human IgG3 Fc regions; and native sequence human IgG4 Fc regions and naturally occurring variants thereof. Native sequence Fc includes various allotypes of Fc (see, e.g., Jefferis et al., (2009) mAbs 1:1; Vidarsson G. et al., Front Immunol. 5:520 (published online Oct. 20, 2014)).

[0078] "Fc receptor" or "FcR" is a receptor that binds to the Fc region of an immunoglobulin. FcRs that bind to IgG antibodies include receptors of the FcγR family, including allelic variants and alternative splicing 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 triggers the strongest Fc effector function. It is considered equivalent to mouse IgG2a relative to the type of activating Fc receptor bound to it. In contrast, human IgG4 triggers the least Fc effector function. Vidarsson G. et al., Front Immunol. 5:520 (published online on October 20, 2014).

[0079] 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 an antibody Fc region with an Fc receptor or ligand or the biochemical events resulting therefrom. Exemplary "effector functions" include C1q binding, complement dependent cytotoxicity (CDC), Fc receptor binding, FcγR-mediated effector functions, such as ADCC and antibody-dependent cell-mediated phagocytosis (ADCP), and downregulation of cell surface receptors (e.g., B cell receptor; BCR). Such effector functions generally require the Fc region to be combined with a binding domain (e.g., an antibody variable domain). Therefore, the term "constant region without Fc function" includes a constant region with one or more effector functions reduced or not mediated by the Fc region.

[0080] The effector function of an antibody can be reduced or avoided by different methods. By using an antibody fragment lacking an Fc region (e.g., such as Fab, F(ab')2, single-chain Fv (scFv) or sdAb consisting of a monomeric VH or VL domain), the effector function of an antibody can be reduced or avoided. Alternatively, so-called non-glycosylated antibodies can be produced in the following manner: removing the sugars connected to specific residues in the Fc region to reduce the effector function of the antibody while retaining other valuable properties of the Fc region (e.g., extended half-life and heterodimerization). Antibodies can be produced in cells cultured in the presence of glycosylation inhibitors by, for example, missing or changing the residues to which sugars are attached, enzymatic removal of sugars, or by expressing antibodies in cells (e.g., bacterial host cells) that cannot glycosylate proteins to produce non-glycosylated antibodies. See, for example, U.S. Patent Publication No. 20120100140. Another method is to use an Fc region with reduced effector function from an IgG subclass, for example, IgG2 and IgG4 antibodies are characterized by having a lower level of Fc effector function than IgG1 and IgG3. The residues closest to the hinge region in the CH2 domain of the Fc portion are responsible for the effector function of the antibody because it contains highly overlapping binding sites for C1q (complement) and IgG-Fc receptors (FcγR) on effector cells of the innate immune system. Vidarsson G. et al., Front Immunol.5:520 (published online on October 20, 2014). Therefore, it is possible to prepare antibodies with reduced or no Fc effector function by producing, for example, a chimeric Fc region comprising a CH2 domain of an IgG antibody from an IgG4 isotype and a CH3 domain of an IgG antibody from an IgG1 isotype, or a chimeric Fc region comprising a hinge region from IgG2 and a CH2 region from IgG4 (see, for example, Lau C. et al., J. Immunol.191:4769-4777 (2013)), or an Fc region having a mutation that causes a change in Fc effector function, such as a reduction or absence of Fc function. Such Fc regions with mutations are known in the art. See, e.g., U.S. Publication No. 20120100140 and the U.S. and PCT applications cited therein and An et al., mAbs 1:6, 572-579 (2009); the disclosures of which are incorporated by reference in their entireties.

[0081] "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 different levels of flexibility between the binding region and the effector region of the antibody, and also provides a site for intermolecular disulfide bonding between the two heavy chain constant regions. As used herein, for all IgG isotypes, the hinge starts at Glu216 and ends at Gly237 (Roux et al., 1998 J. Immunol. 161: 4083). The sequences of wild-type IgG1, IgG2, IgG3 and IgG4 hinges are known in the art. See, e.g., Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242; Vidarsson G. et al., Front Immunol. 5:520 (published online Oct. 20, 2014).

[0082] The term "CH1 domain" refers to the heavy chain constant region of the hinge to which the variable domain is connected in the 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, and naturally occurring variants thereof (e.g., allotypes). The CH1 domain sequences of IgG1, IgG2, IgG3, and IgG4 (including wild-type and allotypes) are known in the art. See, e.g., Kabat EA et al., (1991) supra and Vidarsson G. et al., Front Immunol. 5: 520 (published online on October 20, 2014). Exemplary CH1 domains include CH1 domains with mutations that modify the biological activity of the antibody, such as half-life, such as described in U.S. Patents and publications cited therein and PCT publications.

[0083] The term "CH2 domain" refers to the heavy chain constant region that connects the hinge to the CH3 domain in the heavy chain constant domain. As used herein, the CH2 domain begins at P238 and ends at K340. The term "CH2 domain" includes wild-type CH2 domains, as well as naturally occurring variants thereof (e.g., allotypes). The CH2 domain sequences of IgG1, IgG2, IgG3, and IgG4 (including wild-type and allotypes) are known in the art. See, e.g., Kabat EA et al., (1991) supra and Vidarsson G. et al., Front Immunol. 5:520 (published online on October 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 described in U.S. Patent No. 20120100140 and the U.S. Patents and Publications cited therein and PCT Publications.

[0084] The term "CH3 domain" refers to the heavy chain constant region that is the C-terminal end of the CH2 domain in the heavy chain constant domain. As used herein, the CH3 domain starts at G341 and ends at K447. The term "CH3 domain" includes wild-type CH3 domains, and naturally occurring variants thereof (e.g., allotypes). The CH3 domain sequences of IgG1, IgG2, IgG3, and IgG4 (including wild-type and allotypes) are known in the art. See, e.g., Kabat EA et al., (1991) supra and Vidarsson G. et al., Front Immunol. 5: 520 (published online on October 20, 2014). Exemplary CH3 domains include CH3 domains with mutations that modify the biological activity of the antibody, such as half-life, such as described in U.S. Patent No. 20120100140 and the U.S. Patents and Publications cited therein and PCT Publications.

[0085] As used herein, "isotype" refers to the antibody class encoded by the heavy chain constant region genes (eg, IgG1, IgG2, IgG3, IgG4, IgM, IgA1 IgA2, IgD, and IgE antibodies).

[0086] "Allotype" refers to naturally occurring variants within a particular isotype group that differ in a few amino acids (see, e.g., Jefferis et al., (2009) mAbs 1:1). The antibodies described herein can be of any allotype. Allotypes of IgG1, IgG2, IgG3, and IgG4 are known in the art. See, e.g., Kabat EA et al., (1991) supra; Vidarsson G. et al., Front Immunol. 5:520 (published online Oct. 20, 2014); and Lefranc MP, mAbs 1:4, 1-7 (2009).

[0087] The phrases "an antibody that recognizes an antigen" and "an antibody specific for an antigen" are used interchangeably herein with the term "an antibody that specifically binds to an antigen."

[0088] As used herein, "isolated antibody" means an antibody that is substantially free of other antibodies with different antigenic specificities (e.g., an isolated antibody that specifically binds to FAM19A5 is substantially free of antibodies that specifically bind to antigens other than FAM19A5). However, an isolated antibody that specifically binds to an epitope of FAM19A5 may have cross-reactivity with other FAM19A5 proteins from different species.

[0089] "Binding affinity" generally refers to the overall strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed in terms of the dissociation constant (K D Affinity can be measured and / or expressed in a variety of ways known in the art, including but not limited to the equilibrium dissociation constant (K D ) and equilibrium association constant (K A ). D By k 关闭 / k 开启 The quotient is calculated and expressed as molar concentration (M), while K A By k 开启 / k 关闭 Calculate the quotient of k 开启 is, for example, the rate constant for the association of an antibody with an antigen, while k 关闭 Refers to, for example, the dissociation of antibodies and antigens. 开启 and k 关闭 The determination can be made by techniques known to those of ordinary skill in the art, such as immunoassays (e.g., enzyme-linked immunosorbent assay (ELISA)), or equilibrium elimination immunoassay (KinExA).

[0090] As used herein, the terms "specific binding", "specific recognition", "specific binding", "selective binding", and "selective binding" are analogous terms in the context of antibodies and refer to molecules (e.g., antibodies) that bind to an antigen (e.g., an epitope or an immune complex) as understood by those skilled in the art. For example, a molecule that specifically binds to an antigen may bind to other peptides or polypeptides, typically with a lower affinity, which can be determined, for example, by immunoassays, KinExA 3000 instrument (Sapidyne Instruments, Boise, ID) or other assays known in the art. In specific embodiments, a molecule that specifically binds to an antigen has a K of at least 2 log, 2.5 log, 3 log, 4 log or more than the K of the molecule when it binds to another antigen. A Bigger K A Binds to antigen.

[0091] Antibodies usually bind specifically to their cognate antigens with high affinity, as indicated by -5 Up to 10 -11 M or less dissociation constant (K D ) reflects. It is generally believed that any -4 M of K D Indicates non-specific binding. As used herein, an antibody that "specifically binds" to an antigen refers to an antibody that binds to the antigen and substantially identical antigens with high affinity, but does not bind to unrelated antigens with high affinity, wherein the high affinity binding to the antigen and substantially identical antigens means that the binding affinity is 10 when measured by immunoassay (e.g., ELISA) or surface plasmon resonance (SPR) technology using a predetermined antigen with a BIACORE 2000 instrument. -7 M or less, preferably 10 -8 M or less, even more preferably 10 -9 M, and most preferably 10 -8 M and 10 -10 K between M or below D .

[0092] 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.

[0093] As used herein, "epitope" is a term in the art, and refers to a local region of an antigen to which an antibody can specifically bind. An epitope can be, for example, a continuous amino acid (linear or continuous epitope) of a polypeptide, or an epitope can be, for example, simultaneously from two or more non-continuous regions (conformation, non-linearity, discontinuous or non-continuous epitopes) of one or more polypeptides. An epitope formed by continuous amino acids is usually, but not always, retained when exposed to a denaturing solvent, while an epitope formed by a tertiary folding is usually lost when treated with a denaturing solvent. An epitope generally includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 20 amino acids of a unique spatial conformation. The method for determining which epitopes a given antibody binds to (i.e., epitope mapping) is well known in the art, including, for example, immunoblotting and immunoprecipitation assays, wherein a given antibody (e.g., anti-FAM19A5 antibody) is used to test the reactivity of overlapping or continuous peptides from (e.g., from FMAM19A5). Methods for determining the spatial conformation of epitopes include techniques in the art and described herein, for example, X-ray crystallography, two-dimensional nuclear magnetic resonance, and HDX-MS (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996)).

[0094] In certain embodiments, the epitope to which the antibody binds can be determined by, for example, NMR spectroscopy, X-ray diffraction crystallography, ELISA assays, hydrogen / deuterium exchange in combination with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligopeptide scanning assays and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). For X-ray crystallography, any of the methods known in the art can be used to complete crystallization (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 optimized using computer software such as X-PLOR (Yale University, 1992, published 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 those skilled in the art. For a description of mutagenesis techniques, including alanine scanning mutagenesis, see, for example, Champe M et al., (1995) J Biol Chem 270: 1388-1394 and Cunningham BC & Wells JA (1989) Science 244: 1081-1085.

[0095] The term "epitope mapping" refers to the process of identifying molecular determinants for antibody-antigen recognition.

[0096] With respect to two or more antibodies, the term "binding to the same epitope" refers to the binding of an antibody to the same fragment of amino acid residues, as determined by a given method. The technology used to determine whether an antibody binds to the same epitope on FAM19A5 as the antibody described herein includes, for example, an epitope mapping method, such as providing an atomic resolution of an epitope: X-ray analysis of a crystal of an antibody complex, and hydrogen / deuterium exchange mass spectrometry (HDX-MS). Other methods monitor the binding of an antibody to an antigen fragment or a mutational variation of an antigen, wherein it is generally believed that the binding loss caused by the modification of amino acid residues in the antigen sequence is an indication of an epitope component. In addition, a computational combination method for epitope mapping can also be used. These methods rely on the ability of an antibody of interest to affinity separate specific short peptides from a combinatorial phage display peptide library. Antibodies with identical VH and VL or identical CDR1, 2, and 3 sequences are expected to bind to the same epitope.

[0097] "An antibody that competes with another antibody for binding to a target" refers to an antibody that inhibits (partially or completely) the binding of another antibody to a target. Known competition experiments can be used to determine whether two antibodies compete with each other for binding to a target, i.e., whether one antibody inhibits the binding of another antibody to a target and to what extent. In certain embodiments, an antibody competes with another antibody and inhibits the binding of another antibody to a target by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. Depending on which antibody is a "blocking antibody" (i.e., a cold antibody that is first incubated with the target), the level of inhibition or competition may be different. Competition 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 in Chapter 11 of "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 to an adjacent epitope (e.g., as demonstrated by steric hindrance).

[0098] Other competitive binding assays include: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see Stahli et al., Methods in Enzymology 9:242 (1983)); solid phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614 (1986)); solid phase direct labeling assay, solid phase direct labeling sandwich assay (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988); solid phase direct labeling RIA using 1-125 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)).

[0099] "Bispecific" or "bifunctional antibodies" are artificial hybrid antibodies with two different heavy / light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods, including fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992).

[0100] 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. Thus, the term "human monoclonal antibody" refers to an antibody or antibody composition that exhibits a single binding specificity and has variable and optional constant regions derived from human germline immunoglobulin sequences. In one embodiment, human monoclonal antibodies are produced by a hybridoma comprising a B cell obtained from a transgenic non-human animal (e.g., a transgenic mouse) having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.

[0101] As used herein, the term "recombinant human antibody" includes all human antibodies prepared, expressed, produced or isolated by recombinant means, such as (a) antibodies isolated from animals (e.g., mice) transgenic or transchromosomal for human immunoglobulin genes or hybridomas prepared therefrom, (b) antibodies isolated from host cells transformed to express antibodies, such as antibodies isolated from transfectomas, (c) antibodies isolated from recombinant combinatorial human antibody libraries, and (d) antibodies prepared, expressed, produced or isolated by any other method involving splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies include variable regions and constant regions that utilize 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, e.g., Lonberg (2005) Nature Biotech. 23 (9): 1117-1125), variable regions contain antigen binding domains encoded by various genes that rearrange to form antibodies specific for exogenous antigens. In addition to rearrangement, the variable region can be further modified by multiple single amino acid changes (called somatic mutations or hypermutations) to increase the affinity of the antibody for foreign antigens. The constant region will further change in response to the antigen (i.e., isotype switching). Therefore, the rearranged and somatically mutated nucleic acid molecules encoding light and heavy chain immunoglobulin polypeptides in response to the antigen cannot have sequence identity with the original nucleic acid molecules, but will be substantially identical or similar (i.e., at least 80% identical).

[0102] "HuMAb" refers to an antibody with a variable region, wherein the framework region and the CDR region are derived from human germline immunoglobulin sequences. Further, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. The antibodies described herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which the CDR sequences derived from the germline of another mammalian species such as a mouse have been grafted onto human framework sequences. The terms "human" antibody and "fully human" antibody are synonyms.

[0103] "Humanized" antibodies refer to antibodies in which some, most or all of the amino acids outside the CDR domains of non-human antibodies are replaced by corresponding amino acids derived from human immunoglobulins. In one embodiment of a humanized form of an antibody, some, most or all of the amino acids outside the CDR domains have been replaced by 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 to amino acids are permitted as long as they do not eliminate the ability of the antibody to bind to a specific antigen. "Humanized" antibodies retain antigenic specificity similar to that of the original antibody.

[0104] As used herein, the term "deimmunized" or "deimmunization" refers to a process in which an antibody or its antigen-binding portion is modified, for example, in a human subject to reduce its immunogenicity. For example, the heavy chain variable region (VH) and light chain variable region (VL) sequences from the original antibody can be analyzed, and a human T cell epitope "map" can be generated from each V region to show the position of the epitope in the sequence relative to the complementary determining region (CDR) and other key residues. Analyze each T cell epitope from the T cell epitope map to identify alternative amino acid substitutions with a lower risk of changing the activity of the final antibody. Design a series of alternative VH and VL sequences containing amino acid substitution combinations, and then incorporate these sequences into a series of FAM19A5-specific antibodies or their antigen-binding portions for use in the diagnostic and therapeutic methods disclosed herein, and then test the function of the diagnostic and therapeutic methods. Then, the complete heavy and light chain genes containing the modified V and human C regions are cloned into expression vectors and plasmids subsequently introduced into cell lines to produce complete antibodies. The antibodies are then compared with appropriate biochemical and biological assays, and the best variants are identified. Antibodies can be deimmunized by using the methods described herein or by any other method known in the art. See, for example, WO 98 / 52976 or WO 00 / 34317.

[0105] A "chimeric antibody" refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species, such as an antibody in which the variable region is derived from a mouse antibody and the constant region is derived from a human antibody.

[0106] As used herein, the term "cross-reactivity" refers to the ability of an antibody described herein to bind to FAM19A5 from another different species. For example, an antibody described herein that binds to human FAM19A5 may also bind to FAM19A5 of another species (e.g., mouse FAM19A5). As used herein, cross-reactivity can be measured by detecting specific reactions with purified antigens using binding assays (e.g., SPR, ELISA), or binding or functionally interacting with cells that physiologically express FAM19A5. Methods for determining cross-reactivity include standard binding assays as described herein, for example, by using Surface plasmon resonance (SPR) analysis was performed using a Biacore 2000SPR instrument (Biacore AB, Uppsala, Sweden), or flow cytometry techniques.

[0107] The term "naturally occurring" as applied to the objects disclosed herein means 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 source in nature and has not been intentionally modified by man in a laboratory is naturally occurring.

[0108] "Polypeptide" refers to a chain of at least two consecutively linked amino acid residues, with no upper limit to the length of the chain. One or more amino acid residues in a protein may contain modifications, such as but not limited to glycosylation, phosphorylation or disulfide bond formation. A "protein" may contain one or more polypeptides.

[0109] As used herein, the term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, and may be a cDNA.

[0110] As used herein, the term "vector" means a nucleic acid molecule capable of transporting another nucleic acid connected thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop, wherein additional DNA fragments can be connected. Another type of vector is a viral vector, wherein additional DNA fragments can be connected to the viral genome. Some vectors can replicate autonomously in the host cell into which they are introduced (e.g., bacterial vectors and free mammalian vectors with bacterial replication origins). After being introduced into the host cell, other vectors (e.g., non-free mammalian vectors) can be integrated into the genome of the host cell, thereby replicating with the host genome. In addition, some vectors can guide the expression of genes operably connected thereto. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Generally, expression vectors available in recombinant DNA technology are generally in the form of plasmids. In this specification, "plasmid" and "vector" are used interchangeably because plasmids are the most commonly used vector forms. However, other forms of expression vectors are also included, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses) that play equivalent functions.

[0111] As used herein, the term "recombinant host cell" (or simply "host cell") means 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 refer not only to the specific subject cell, but also to the progeny of such cells. Because mutations or environmental influences may cause certain modifications to occur in the progeny, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0112] As used herein, the term "linked" refers to the association of two or more molecules. The link can be covalent or non-covalent. The link can also be genetic (i.e., recombinant fusion). Such links can be achieved using a variety of art-recognized techniques, such as chemical conjugation and recombinant protein production.

[0113] As used herein, "administering" refers to using any of the various methods and delivery systems known to those skilled in the art to physically introduce a therapeutic agent or a composition comprising a therapeutic agent into a subject. Preferred routes of administration of antibodies described herein include, for example, intravenous, intraperitoneal, intramuscular, subcutaneous, spinal or other parenteral routes of administration by injection or infusion. As used herein, the phrase "parenteral administration" refers to administration generally by injection rather than enteral and topical administration, and includes but is not limited to intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, and in vivo electroporation. Alternatively, antibodies described herein can be via non-parenteral routes, such as local, epidermal or mucosal administration routes, such as intranasal, oral, vaginal, rectal, sublingual or topical administration. It can also be administered, for example, once, repeatedly and / or in one or more extended periods.

[0114] As used herein, the terms "treat", "treating" and "treatment" refer to any type of intervention or process performed on a subject, or the administration of an active agent to a subject, to achieve the following goals: reversing, alleviating, ameliorating, inhibiting or slowing down or preventing the progression, development, severity or recurrence of symptoms, complications, pathologies or biochemical signs associated with a disease. The subjects treated can be subjects with the disease or subjects without the disease (e.g., for prevention).

[0115] As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, etc.

[0116] As used herein, the term "onset of gliosis" or "onset of reactive gliosis" includes the onset or initiation of gliosis. Gliosis is a nonspecific reactive change of glial cells in the central nervous system (CNS, such as the brain and / or spinal cord) in response to damage and injury from, for example, trauma, cerebrospinal cord injury, brain tumors, infection, ischemia, stroke, autoimmune reactions and / or neurodegenerative diseases, and includes 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, thereby inhibiting axonal regeneration in parts of the CNS that have been traumatized or injured. The deleterious effects of the onset of gliosis include irreversible or permanent damage to neurons and / or hindering the recovery of peripheral neurons. Thus, the terms "delaying the onset of gliosis" and "delaying the onset of reactive gliosis" include arresting, slowing, inhibiting or preventing the onset or initiation of gliosis and its associated deleterious effects on the CNS.

[0117] As used herein, the term "reactive astrocyte overproliferation" includes, for example, an abnormal increase in the number of astrocytes caused by the destruction of nearby neurons caused by CNS damage, trauma, injury, cerebrospinal cord damage, brain tumors, infection, local ischemia, stroke, autoimmune reactions and / or neurodegenerative diseases. Excessive proliferation of reactive astrocytes can lead to harmful effects on the CNS, including: scar formation, thereby inhibiting axonal regeneration in the traumatized or damaged part of the CNS; exacerbating inflammation; generating and releasing reactive oxygen species at neurotoxic levels; potentially releasing excitotoxic glutamate; potentially leading to epilepsy; impairing blood-brain barrier function; causing cytotoxic edema during trauma and stroke; chronic cytokine activation of astrocytes potentially leading to chronic pain; and causing 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, MV (2005) The Neuroscientist 11(5):400-7. Therefore, the term "inhibiting excessive proliferation of reactive astrocytes" includes preventing, slowing, inhibiting, suppressing or preventing excessive or abnormal proliferation of reactive astrocytes and their related harmful effects on the CNS.

[0118] As used herein, the term "chondroitin sulfate proteoglycan" includes proteoglycans consisting of a protein core and chondroitin sulfate. Chondroitin sulfate proteoglycans, also known as CSPGs, are extracellular matrix molecules that are widely expressed throughout the development and maturation of the CNS. CSGPs play a key role in neural development and glial scar formation, and inhibit axon regeneration after CNS injury. Known CSPGs include aggrecan (CSPG1), versican (CSPG2), neurocan (CSPG3), CSPG4 (or neuron-glial antigen 2 (NG2)), CSPG5, SMC3 (CSPG6, structural maintenance of chromosome 3), short proteoglycan (CSPG7), CD44 (CSPG8, differentiation cluster 44) and phosphatase proteoglycan neurocan (CSPG3). Rhodes, KE and Fawcett, JW (2004) Journal of Anatom.204 (1): 33-48. Thus, the term "reducing the expression of chondroitin sulfate proteoglycans" includes reducing, inhibiting, decreasing the level of one or more CSGPs, or reducing the activity of one or more CSGPs or inactivating them. In certain embodiments, the term includes reducing, inhibiting, decreasing the level of neurocan, NG2, or both, or reducing the activity of neurocan, NG2, or both or inactivating them.

[0119] As used herein, the term "neuron" includes electrically excitable cells that process and transmit information through electrical and chemical signals. Neurons are the main components of the brain and spinal cord of the CNS and the ganglia of the peripheral nervous system (PNS), and can be connected to each other to form a neural network. A typical neuron is composed of a cell body (soma), dendrites and axons. The soma (cell body) of a neuron contains a nucleus. The dendrites of a neuron are cell extensions with many branches, where most of the inputs of a neuron occur at the cell extension. An axon is a thinner, cable-like projection extending from a somatic cell, and transmits nerve signals from a somatic cell and certain types of information back to the somatic cell. The term "promoting the regeneration of neurons" includes stimulating, promoting, increasing or activating the growth of neurons, preferably after injury or damage.

[0120] As used herein, the term "c-fos" includes the proto-oncogene c-fos, which is rapidly induced by stimulating neurotransmitters. c-fos is present in many species, including mice and humans. The c-fos gene and protein are known and characterized. See Curran, T, The c-fos proto-oncogene, pp 307-327 (The Oncogene Handbook, Reddy EP et al., (eds.) Elsevier) (1988). The expression of c-fos can be determined by methods known in the art, such as Northern blot, quantitative PCR or immunohistochemistry. The term "increasing the expression of c-fos" includes increasing the level of c-fos mRNA, c-fos protein or c-fos protein activity.

[0121] As used herein, the term "pERK" includes phosphorylated extracellular signal-regulated kinases. Extracellular signal-regulated kinases or ERKs (including ERK1 and ERK2) are members of the mitogen-activated protein kinase (MAPK) family. ERK is activated by phosphorylation of its upstream kinase to form pERK, which then activates downstream targets. ERK is involved in neural and synaptic plasticity-based learning, as well as memory and pain hypersensitivity. Ji RR et al., Nat Neurosci (1999) 2: 1114-1119. ERK genes, proteins, phosphorylation and activation are known and characterized, and the expression of ERK and pERK can be determined by methods known in the art (e.g., Noser hybridization, quantitative PCR or immunohistochemistry). See Gao YJ and Ji RR, Open Pain J. (2009) 2: 11-17. The term "increasing the expression of pERK" includes increasing the level of ERK mRNA, ERK protein or pERK activity.

[0122] 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 LI and Routtenberg A. (1997) Trends in Neurosciences 20 (2): 84-91; Aarts LH et al., (1998) Advances in Experimental Medicine and Biology 446: 85-106. Human GAP43 is encoded by the GAP43 gene. The human GAP43 polypeptide sequence (UniProt: KB-P17677) and the cDNA sequence encoding the polypeptide are known in the art. Kosik KS et al., (1988) Neuron 1 (2): 127-32; Ng SC et al., (1988) Neuron 1 (2): 133-9. The expression of GAP43 can be determined by methods known in the art (e.g., Noser hybridization, quantitative PCR or immunohistochemistry). The term "increasing GAP43 in neurons" includes enhancing or increasing the level of GAP43 mRNA, GAP43 protein or increasing the activity of GAP43 protein.

[0123] As used herein, the term "therapeutically effective amount" refers to the amount of a drug (alone or in combination with another therapeutic agent) that effectively "treats" a disease or condition (e.g., central nervous system damage) of a subject or reduces its risk, potential, probability or incidence. A "therapeutically effective amount" includes the amount of a drug or therapeutic agent that provides some improvement or benefit to a subject suffering from or in the condition of suffering from a disease or condition (e.g., central nervous system damage, such as brain trauma or other diseases disclosed herein). Therefore, a "therapeutically effective" amount is an amount that reduces the risk, potential, probability or incidence of a disease or condition, or provides a certain degree of relief, mitigation and / or reduces at least one indicator (e.g., the onset of reactive gliosis), and / or alleviates at least one clinical symptom of a disease or condition.

[0124] II. Anti-FAM19A5 Antibody

[0125] Disclosed herein are antibodies characterized by specific functional features or properties, such as monoclonal antibodies. For example, an antibody that specifically binds to human FAM19A5 has been mutated (e.g., substituted or deleted) by removing and / or modifying regions or residues that are highly immunogenic in humans (i.e., deimmunized). Therefore, compared to a reference antibody (e.g., a corresponding antibody that has not been deimmunized, e.g., 1-65 antibody), the antibodies disclosed herein, i.e., anti-FAM19A5 antibodies, have reduced immunogenicity when administered to a human subject.

[0126] Additionally, the antibodies described herein exhibit one or more of the following functional properties:

[0127] (a) With a K of 10 nM or less D Binds to soluble human FAM19A5;

[0128] (b) With a K of 10 nM or less D Binds to membrane-bound human FAM19A5;

[0129] (c) reducing, reversing, delaying and / or preventing the onset of reactive gliosis;

[0130] (d) inhibiting the excessive proliferation of reactive astrocytes;

[0131] (e) decreased expression of chondroitin sulfate proteoglycans including neurocan and neuron-glial antigen 2 (NG2);

[0132] (f) increased expression of c-fos and pERK in neuronal nuclei;

[0133] (g) Promote neuronal survival.

[0134] (h) increasing the expression of GAP43 in neurons; and

[0135] (i) Promote axon regeneration.

[0136] In some embodiments, the anti-FAM19A5 antibody has been deimmunized so that the immunogenicity of the antibody is lower when administered to a human subject compared to a reference antibody (e.g., a corresponding antibody that has not been deimmunized, e.g., antibody 1-65). In some embodiments, the immunogenicity of the antibody has been reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% compared to a reference antibody (e.g., a corresponding antibody that has not been deimmunized, e.g., antibody 1-65). In some embodiments, the deimmunization process does not change the binding affinity of the antibody.

[0137] In some embodiments, the anti-FAM19A5 antibody binds with high affinity (e.g., with 10 -7 M or smaller, 10 -8 M or smaller, 10 -9 M (1nM) or less, 10 -10 M (0.1nM) or less, 10 -11 M or smaller, or 10 -12 M or smaller, e.g. 10 -12 M to 10 -7 M, 10 -11 M to 10 -7 M, 10 -10 M to 10 -7 M, or 10 -9 M to 10 -7 M, for example 10 -12 M, 5×10 -12 M, 10 -11 M, 5×10 -11 M, 10 -10 M, 5×10 -10 M, 10 -9 M, 5×10 -9 M, 10 -8 M, 5×10 -8 M, 10 -7 M or 5×10 -7 M of K D ) specifically binds to soluble human FAM19A5 or membrane-bound human FAM19A5. Standard assays for assessing the binding ability of antibodies to human FAM19A5 of different species are known in the art, including, for example, ELISA, Western blot, and RIA. Suitable assays are described in detail in the Examples. The binding kinetics (e.g., binding affinity) of the antibodies can also be assessed by standard assays known in the art, such as by ELISA, BIACORE TM Analysis or Assays for evaluating the effects of antibodies on the functional properties of FAM19A5 (eg, ligand binding) are described in further detail below and in the Examples.

[0138] In some embodiments, anti-FAM19A5 antibodies are expressed at 10 -7 M or smaller, 10 -8 M (10nM) or less, 10 -9 M (1nM) or less, 10 -10 M or smaller, 10 -12 M to 10 -7 M, 10 -11 M to 10 -7 M, 10 -10 M to 10 - 7 M, 10 -9 M to 10 -7 M or 10 -8 M to 10 -7 M of K DIn some embodiments, the anti-FAM19A5 antibody has a K of 10 nM or less, such as between 0.1 and 10 nM, between 0.1 and 5 nM, between 0.1 and 1 nM, between 0.5 and 10 nM, between 0.5 and 5 nM, between 0.5 and 1 nM, between 1 and 10 nM, between 1 and 5 nM, or between 5 and 10 nM. D Binds to soluble FAM19A5. In some embodiments, the anti-FAM19A5 antibody has a K of about 1 pM, 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 8 pM, 9 pM, 10 pM, 20 pM, 30 pM, 40 pM, 50 pM, 60 pM, 70 pM, 80 pM, 90 pM, 100 pM, 200 pM, 300 pM, 400 pM, 500 pM, 600 pM, 700 pM, 800 pM, 900 pM, or about 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, or 9 nM, or about 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, or 90 nM as determined by ELISA. D Binds specifically to soluble human FAM19A5.

[0139] In some embodiments, anti-FAM19A5 antibodies are expressed at 10 -7 M or smaller, 10 -8 M (10nM) or less, 10 -9 M (1nM) or less, 10 -10 M or smaller, 10 -12 M to 10 -7 M, 10 -11 M to 10 -7 M, 10 -10 M to 10 - 7 M, 10 -9 M to 10 -7 M, or 10 -8 M to 10 -7 M of K D In certain embodiments, the anti-FAM19A5 antibody has a K of 10 nM or less, such as between 0.1 and 10 nM, between 0.1 and 5 nM, between 0.1 and 1 nM, between 0.5 and 10 nM, between 0.5 and 5 nM, between 0.5 and 1 nM, between 1 and 10 nM, between 1 and 5 nM, or between 5 and 10 nM as determined by ELISA. DBinds specifically to membrane-bound human FAM19A5. In some embodiments, the anti-FAM19A5 antibody has a K of about 1 pM, 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 8 pM, 9 pM, 10 pM, 20 pM, 30 pM, 40 pM, 50 pM, 60 pM, 70 pM, 80 pM, 90 pM, 100 pM, 200 pM, 300 pM, 400 pM, 500 pM, 600 pM, 700 pM, 800 pM, or 900 pM, or about 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, or 9 nM, or about 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, or 90 nM as determined by ELISA. D Binds to membrane-bound human FAM19A5.

[0140] The anti-FAM19A5 antibodies of the present disclosure can delay or inhibit the onset of gliosis, for example, delay, slow or inhibit non-specific reactive changes of glial cells in the central nervous system (CNS, e.g., brain and / or spinal cord) in response to injury and damage from, for example, trauma, cerebrospinal cord injury, brain tumor, infection, ischemia, stroke, autoimmune response and / or neurodegenerative disease.

[0141] The anti-FAM19A5 antibodies disclosed herein can delay, prevent, slow down, inhibit, contain or prevent the excessive or abnormal proliferation of reactive astrocytes and their related harmful effects on the CNS. For example, the anti-FAM19A5 antibodies disclosed herein can inhibit or prevent the abnormal increase in the number of astrocytes caused by the destruction of neurons from, for example, CNS damage, trauma, injury, cerebrospinal cord damage, brain tumors, infections, local ischemia, stroke, autoimmune reactions and / or neurodegenerative diseases, inhibit or prevent scar formation in the CNS, inhibit or reduce the release of neurotoxic levels of reactive oxygen species or the release of potential excitotoxic glutamate, reduce or inhibit epileptic seizures, pain and / or secondary degeneration after CNS damage. The anti-FAM19A5 antibodies disclosed herein can promote, stimulate, increase or activate the regeneration of neurons and / or axons, preferably after the CNS is damaged or damaged.

[0142] The anti-FAM19A5 antibodies of the present disclosure can inhibit the expression of chondroitin sulfate proteoglycans, including proteoglycans composed of a protein core and chondroitin sulfate (CSGP), such as aggrecan (CSPG1), versican (CSPG2), neurocan (CSPG3), CSPG4 (or neuron-glial antigen 2 (NG2)), CSPG5, SMC3 (CSPG6, structural maintenance of chromosome 3), brevican (CSPG7), CD44 (CSPG8, cluster of differentiation 44), and phosphatase proteoglycan neurocan (CSPG3). In some embodiments, the anti-FAM19A5 antibodies of the present disclosure inhibit, reduce or decrease the level of neurocan and / or NG2, or the activity of neurocan and / or NG2.

[0143] The anti-FAM19A5 antibodies disclosed herein can increase the expression of c-fos and pERK in neuronal nuclei, for example, increase the mRNA, protein and / or protein activity of c-fos and pERK. The anti-FAM19A5 antibodies disclosed herein can also increase or enhance the expression level of GAP43 mRNA, GAP43 protein, or increase or enhance the activity of GAP43 protein.

[0144] In some embodiments, the anti-FAM19A5 antibodies of the present disclosure comprise heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3, wherein the heavy chain CDR1, CDR2 and CDR3 comprise the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 5, 6 and 7, each of which optionally comprises one, two or three mutations, wherein the light chain CDR1, CDR2 and CDR3 comprise the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 8, 9 and 10, wherein at least one of the light chain CDR1, CDR2 and CDR3 comprises one, two or three mutations, and wherein the antibody has reduced immunogenicity in human subjects compared to a reference antibody comprising the VH shown in SEQ ID NO: 11 and the VL shown in SEQ ID NO: 12.

[0145] In some embodiments, the mutation included in the antibody is a substitution, deletion and / or insertion. In one embodiment, the mutation is a substitution, such as a conservative substitution. As used herein, a "conservative substitution" (also referred to as a conservative substitution) refers to an amino acid substitution in which a given amino acid is changed to another amino acid with similar biochemical properties (e.g., charge, hydrophobicity, and size). Although there are many ways to classify amino acids, they are generally divided into six main groups based on their structure and the general chemical characteristics of their R groups.

[0146] Table 2. Amino acid categories

[0147]

[0148]

[0149] In contrast, free radical replacement or free radical substitution is an amino acid replacement in which the initial amino acid is exchanged for a final amino acid with different physicochemical properties. In certain embodiments, the amino acid mutation at the FAM19A5 antibody is a free radical replacement. In other embodiments, the amino acid mutation at the FAM19A5 antibody is a combination of conservative substitutions and free radical substitutions.

[0150] In some embodiments, the heavy chain CDR3 of the anti-FAM19A5 antibody disclosed herein comprises the amino acid sequence (GSASYITAATIDA) shown in SEQ ID NO:7. In certain embodiments, the heavy chain CDR3 comprises the amino acid sequence shown in SEQ ID NO:7, which has one, two or three mutations. In some embodiments, the heavy chain CDR1 comprises the amino acid sequence (SYQMG) shown in SEQ ID NO:5. In certain embodiments, the heavy chain CDR1 comprises the amino acid sequence shown in SEQ ID NO:5, which has one, two or three mutations. In some embodiments, the heavy chain CDR2 comprises the amino acid sequence (VINKSGSDTS) shown in SEQ ID NO:6. In certain embodiments, the heavy chain CDR2 comprises the amino acid sequence shown in SEQ ID NO:6, which has one, two or three mutations. In certain embodiments, the mutation comprises valine at amino acid 1 of SEQ ID NO:6 being replaced by an aliphatic amino acid. In certain embodiments, the aliphatic amino acid comprises alanine. In some embodiments, the mutation comprises serine at amino acid 5 of SEQ ID NO:6 being replaced by an aliphatic amino acid. In certain embodiments, the aliphatic amino acid comprises glycine.

[0151] In some embodiments, the light chain CDR1 of the anti-FAM19A5 antibody of the present disclosure comprises the amino acid sequence (SGGGSSGYGYG) shown in SEQ ID NO:8. In certain embodiments, the light CDR1 comprises the amino acid sequence shown in SEQ ID NO:8, and the amino acid sequence has one, two or three mutations. In some embodiments, the mutation comprises that the glycine at amino acid 4 of SEQ ID NO:8 is replaced by an aliphatic amino acid. In some embodiments, the aliphatic amino acid comprises alanine, valine, leucine or isoleucine. In certain embodiments, the aliphatic amino acid is alanine.

[0152] In some embodiments, the light chain CDR2 comprises the amino acid sequence (WNDKRPS) shown in SEQ ID NO:9. In certain embodiments, the light chain CDR2 comprises the amino acid sequence shown in SEQ ID NO:9, which has one, two, three or four mutations. In some embodiments, the mutation comprises that the tryptophan at the amino acid 1 of SEQ ID NO:9 is replaced by a basic amino acid. In some embodiments, the basic amino acid comprises arginine, histidine or lysine. In certain embodiments, the basic amino acid is lysine. In some embodiments, the mutation comprises that the asparagine at the amino acid 2 of SEQ ID NO:9 is replaced by an acidic amino acid. In certain embodiments, the acidic amino acid comprises aspartic acid or glutamic acid. In some embodiments, the acidic amino acid is aspartic acid. In some embodiments, the mutation comprises that the aspartic acid at the amino acid 3 of SEQ ID NO:9 is replaced by a hydroxyl or sulfur / selenoic acid-containing amino acid. In certain embodiments, the hydroxyl or sulfur / selenoic acid-containing amino acid comprises serine. In some embodiments, the mutation comprises that the lysine at the amino acid 4 of SEQ ID NO:9 is replaced by an acidic amino acid. In some embodiments, the acidic amino acid comprises aspartic acid or glutamic acid. In some embodiments, the acidic amino acid is glutamic acid.

[0153] In some embodiments, the light chain CDR3 comprises the amino acid sequence (GNDDYSSDSGYVGV) set forth in SEQ ID NO: 10. In certain embodiments, the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 10 having one, two or three mutations.

[0154] In some embodiments, the anti-FAM19A5 antibodies of the present disclosure are humanized. In other embodiments, humanized anti-FAM19A5 comprises the framework region of human antibodies. In certain embodiments, the anti-FAM19A5 antibodies are included in one or more (e.g., one, two, three, four, five, six, seven or more) mutations in the framework region of the antibody (i.e., FR1, FR2, FR3 and FR4 of VH and / or FR1, FR2, FR3 and FR4 of VL). In certain embodiments, the anti-FAM19A5 antibodies include mutations in the FR1 of VH. In certain embodiments, the mutations are included in amino acid substitutions at residue 21 of SEQ ID NO:11 (e.g., valine is replaced by aliphatic amino acids, such as serine). In certain embodiments, the mutations are included in amino acid substitutions at residue 19 of SEQ ID NO:11 (e.g., serine is replaced by basic amino acids, such as arginine). In other embodiments, the anti-FAM19A5 antibodies include one or more mutations in FR2 of VH. In certain embodiments, the mutation comprises an amino acid substitution at residue 49 of SEQ ID NO: 11 (e.g., glycine is substituted with a hydroxyl or sulfur / seleno amino acid, such as serine). In some embodiments, the anti-FAM19A5 antibodies of the present disclosure comprise one or more mutations (e.g., one, two, three, four, five, six, or seven mutations) within FR3 of VH. In certain embodiments, the mutation comprises an amino acid substitution at residue 79 of SEQ ID NO: 11 (e.g., valine is substituted with a basic amino acid, such as lysine), an amino acid substitution at 80 (e.g., arginine is substituted with an aromatic amino acid, such as tyrosine), an amino acid substitution at 83 (e.g., lysine is substituted with a hydroxyl or sulfur / seleno amino acid, such as methionine), an amino acid substitution at 85 (e.g., asparagine is substituted with a hydroxyl or sulfur / seleno amino acid, such as serine), an amino acid substitution at 92 (e.g., glycine is substituted with an aliphatic amino acid, such as alanine), and / or an amino acid substitution at 93 (e.g., threonine is substituted with an aliphatic amino acid, such as valine).

[0155] In some embodiments, the anti-FAM19A5 antibodies of the present disclosure include mutations (e.g., one or two mutations) within FR1 of VL. In certain embodiments, the mutation includes an amino acid substitution at residue 16 (e.g., valine is substituted with an aliphatic amino acid, such as alanine). In certain embodiments, the mutation includes an amino acid substitution at residue 17 of SEQ ID NO: 12 (e.g., lysine is substituted with a basic amino acid, such as arginine). In some embodiments, the anti-FAM19A5 antibodies disclosed herein include one or more mutations within FR2 of VL. In certain embodiments, the mutation includes a deletion at amino acid residue 37 of SEQ ID NO: 12. In some embodiments, the anti-FAM19A5 antibodies include mutations within FR3 of VL. In certain embodiments, the mutation includes a substitution at amino acid residue 64 of SEQ ID NO: 12 (e.g., lysine is substituted with a hydroxyl or sulfur / seleno amino acid, such as serine). In some embodiments, the mutation includes a substitution at residue 73 of SEQ ID NO: 12 (e.g., threonine is substituted with a hydroxyl or sulfur / seleno amino acid, such as serine).

[0156] In some embodiments, the anti-FAM19A5 antibodies disclosed herein comprise 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:5; (ii) the heavy chain CDR2 comprises the amino acid sequence shown in SEQ ID NO:6; (iii) the heavy chain CDR3 comprises the amino acid sequence shown in SEQ ID NO:7; (iv) the light chain CDR1 comprises the amino acid sequence shown in SEQ ID NO:13; (v) the light chain CDR2 comprises the amino acid sequence shown in SEQ ID NO:14; and (vi) the light chain CDR3 comprises the amino acid sequence shown in SEQ ID NO:10.

[0157] In some embodiments, the anti-FAM19A5 antibodies disclosed herein comprise 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:5; (ii) the heavy chain CDR2 comprises the amino acid sequence shown in SEQ ID NO:27; (iii) the heavy chain CDR3 comprises the amino acid sequence shown in SEQ ID NO:7; (iv) the light chain CDR1 comprises the amino acid sequence shown in SEQ ID NO:13; (v) the light chain CDR2 comprises the amino acid sequence shown in SEQ ID NO:29; and (vi) the light chain CDR3 comprises the amino acid sequence shown in SEQ ID NO:10.

[0158] In some embodiments, the anti-FAM19A5 antibodies disclosed herein comprise 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:5; (ii) the heavy chain CDR2 comprises the amino acid sequence shown in SEQ ID NO:28; (iii) the heavy chain CDR3 comprises the amino acid sequence shown in SEQ ID NO:7; (iv) the light chain CDR1 comprises the amino acid sequence shown in SEQ ID NO:13; (v) the light chain CDR2 comprises the amino acid sequence shown in SEQ ID NO:29; and (vi) the light chain CDR3 comprises the amino acid sequence shown in SEQ ID NO:10.

[0159] In some embodiments, the anti-FAM19A5 antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical amino acids to the amino acid sequence shown in SEQ ID NO: 11, and / or wherein the VL comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence shown in SEQ ID NO: 12, wherein the immunogenicity of the antibody is reduced compared to a reference antibody comprising the VH shown in SEQ ID NO: 11 and the VL shown in SEQ ID NO: 12.

[0160] In some embodiments, the anti-FAM19A5 antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO: 17, and / or wherein the VL comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO: 18, wherein the immunogenicity of the antibody is reduced compared to a reference antibody comprising the VH set forth in SEQ ID NO: 11 and the VL set forth in SEQ ID NO: 12.

[0161] In some embodiments, the anti-FAM19A5 antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO:30, and / or wherein VL comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO:32, wherein the immunogenicity of the antibody is reduced compared to a reference antibody comprising the VH set forth in SEQ ID NO:11 and the VL set forth in SEQ ID NO:12.

[0162] In some embodiments, the anti-FAM19A5 antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence shown in SEQ ID NO:31, and / or wherein the VL comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence shown in SEQ ID NO:32, wherein the immunogenicity of the antibody is reduced compared to a reference antibody comprising the VH shown in SEQ ID NO:11 and the VL shown in SEQ ID NO:12.

[0163] In some embodiments, the anti-FAM19A5 antibodies of the present disclosure cross-compete with a reference antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the amino acid sequence set forth in SEQ ID NO: 11, and wherein the VL comprises the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the anti-FAM19A5 antibodies bind to the same human FAM19A5 epitope as the reference antibody comprising VH and VL, and wherein the VH comprises the amino acid sequence set forth in SEQ ID NO: 11, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the human FAM19A5 epitope comprises the amino acid sequence set forth in SEQ ID NO: 15. In some embodiments, the human FAM19A5 epitope comprises the amino acid GCDLLINR (SEQ ID NO: 16).

[0164] In certain embodiments, the anti-FAM19A5 antibodies of the present disclosure compete with (or inhibit binding to) a reference antibody (eg, 1-65 antibody) for binding to a human FAM19A5 epitope.

[0165] In certain embodiments, anti-FAM19A5 antibodies inhibit the binding of such reference antibodies (e.g., 1-65 antibodies) to human FAM19A5 by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. Competitive antibodies bind to the same epitope, overlapping epitopes, or to adjacent epitopes (e.g., as demonstrated by steric hindrance). Competition experiments known in the art such as RIA and EIA can be used to determine whether two antibodies compete with each other for binding to the target.

[0166] Techniques for determining whether two antibodies bind to the same epitope include, for example, epitope mapping methods, such as X-ray analysis of crystals of the antigen:antibody complex, and hydrogen / deuterium exchange mass spectrometry (HDX-MS), which provide atomic resolution of the epitope; methods for monitoring antibody binding to antigen fragments or mutational variants of the antigen, in which loss of binding resulting from modification of amino acid residues in the antigen sequence is generally taken as an indication of epitope composition; computational combinatorial methods for epitope mapping.

[0167] Anti-FAM19A5 antibodies useful in the methods disclosed herein can bind to at least one epitope of mature human FAM19A5, as determined by, for example, binding the antibody to a fragment of human FAM19A5. In some embodiments, the anti-FAM19A5 antibodies of the present disclosure bind to a fragment located within the amino acid sequence CDMLPCLEGEGCDLLINRSG (amino acids 90 to 109 of SEQ ID NO: 15 or SEQ ID NO: 2), such as an epitope having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids of SEQ ID NO: 15. In certain embodiments, the anti-FAM19A5 antibodies bind to SEQ ID NO: 15 at one or more amino acid residues 99 to 107 (i.e., EGCDLLINR), such as amino acid residues 102, 103, 105 and 107 (i.e., DL-IR).

[0168] In some embodiments, the at least one epitope has an amino acid sequence that is at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 15. In some embodiments, the anti-FAM19A5 antibodies of the present disclosure bind to SEQ ID NO: 15 or a fragment thereof in its native conformation (i.e., undenatured). In some embodiments, the anti-FAM19A5 antibodies bind to glycosylated and unglycosylated human FAM19A5.

[0169] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that binds to FAM19A5 (e.g., human FAM19A5) with an affinity greater than 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more than another protein in the FAM19A family, as measured by, for example, an immunoassay (e.g., ELISA), surface plasmon resonance, or equilibrium elimination immunoassay. In certain embodiments, an anti-FAM19A5 antibody or antigen-binding fragment thereof binds to FAM19A5 (e.g., human FAM19A5) without cross-reactivity with another protein in the FAM19A family, as measured by, for example, an immunoassay.

[0170] In some embodiments, the anti-FAM19A5 antibodies of the present disclosure are not natural antibodies or are not naturally occurring antibodies. For example, in some embodiments, the anti-FAM19A5 antibodies have different post-translational modifications than naturally occurring antibodies, such as by having more, fewer or different types of post-translational modifications.

[0171] Tables 3 and 4 provide the amino acid sequences of the VH and VL CDRs of exemplary antibodies of the present disclosure, respectively. Tables 5 and 6 provide the amino acid sequences of the VH and VL, respectively.

[0172] Table 3. Variable heavy chain CDR amino acid sequences (identified using Kabat)

[0173]

[0174]

[0175] Table 4. Variable light chain CDR amino acid sequences (identified using Kabat)

[0176]

[0177] Table 5. Variable heavy chain amino acid sequences

[0178]

[0179] Table 6. Variable light chain amino acid sequences

[0180]

[0181] In some embodiments, the anti-FAM19A5 antibodies of the present disclosure comprise a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region (VH) comprises the heavy chain CDR1, CDR2 and CDR3 of SEQ ID NO: 17, and / or the light chain variable region (VL) comprises the light chain CDR1, CDR2 and CDR3 of SEQ ID NO: 18. In other embodiments, the anti-FAM19A5 antibody comprises the VH set forth in the amino acid sequence of SEQ ID NO: 17, and / or the VL comprises the amino acid sequence of SEQ ID NO: 18.

[0182] In some embodiments, the anti-FAM19A5 antibodies of the present disclosure comprise a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region (VH) comprises the heavy chain CDR1, CDR2 and CDR3 of SEQ ID NO: 30, and / or the light chain variable region (VL) comprises the light chain CDR1, CDR2 and CDR3 of SEQ ID NO: 32. In other embodiments, the anti-FAM19A5 antibody comprises the VH shown in the amino acid sequence of SEQ ID NO: 30, and / or the VL comprises the amino acid sequence of SEQ ID NO: 32.

[0183] In some embodiments, the anti-FAM19A5 antibodies of the present disclosure comprise a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region (VH) comprises the heavy chain CDR1, CDR2 and CDR3 of SEQ ID NO: 31, and / or the light chain variable region (VL) comprises the light chain CDR1, CDR2 and CDR3 of SEQ ID NO: 32. In other embodiments, the anti-FAM19A5 antibody comprises the VH shown in the amino acid sequence of SEQ ID NO: 31, and / or the VL comprises the amino acid sequence of SEQ ID NO: 32.

[0184] In some embodiments, the anti-FAM19A5 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to the amino acid sequence shown in SEQ ID NO: 17, 30 or 31, and / or the light chain variable region comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to the amino acid sequence shown in SEQ ID NO: 18 or 32, wherein the VH comprises CDR1, CDR2 and CDR3 of SEQ ID NO: 17, 30 or 31, and wherein the VL comprises CDR1, CDR2 and CDR3 of SEQ ID NO: 18 or 32.

[0185] The VH domains described herein or one or more CDRs thereof can be connected to a constant domain to form a heavy chain, such as a full-length heavy chain. Similarly, the VL domains described herein or one or more CDRs thereof can be connected to a constant domain to form a light chain, such as a full-length light chain. The full-length heavy chain and the full-length light chain are combined to form a full-length antibody.

[0186] Therefore, in a specific embodiment, provided herein are antibodies comprising antibody light chains and heavy chains, such as separated light chains and heavy chains. About light chain, in a specific embodiment, the light chain of the antibody described herein is a κ light chain. In another specific embodiment, the light chain of the antibody described herein is a λ light chain. In another specific embodiment, the light chain of the antibody described herein is a human κ light chain or a human λ light chain. In a specific embodiment, the antibody described herein specifically bound to a FAM19A5 polypeptide (e.g., human FAM19A5) comprises a light chain, the light chain comprises any VL or VL CDR amino acid sequence described herein, and wherein the constant region of the light chain comprises the amino acid sequence of a human κ light chain constant region. In a specific embodiment, the antibody described herein specifically bound to a FAM19A5 polypeptide (e.g., human FAM19A5) comprises a light chain, the light chain comprises a VL or VL CDR amino acid sequence described herein, and wherein the constant region of the light chain comprises the amino acid sequence of a human λ light chain constant region. Non-limiting examples of human constant region sequences have been described in the art, see, eg, US Pat. No. 5,693,780 and Kabat EA et al., (1991) supra.

[0187] With regard to the heavy chain, in some embodiments, the heavy chain of the 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 antibody may comprise a human alpha (α), delta (δ), epsilon (ε), gamma (γ) or mu (μ) heavy chain. In one embodiment, the antibody described herein that specifically binds to FAM19A5 (e.g., human FAM19A5) comprises a heavy chain comprising a VH or VHCDR amino acid sequence described herein, and wherein the constant region of the heavy chain comprises the amino acid sequence of a human gamma (γ) heavy chain constant region. In another embodiment, the antibody described herein that specifically binds to FAM19A5 (e.g., human FAM19A5) comprises a heavy chain comprising a VH or VH CDR amino acid sequence disclosed herein, and wherein the constant region of the heavy chain comprises the amino acid of a human heavy chain described herein or known in the art. Non-limiting examples of human constant region sequences have been described in the art, see, eg, US Pat. No. 5,693,780 and Kabat EA et al., (1991) supra.

[0188] In some embodiments, an antibody described herein that specifically binds to FAM19A5 (e.g., human FAM19A5) comprises a VL domain and a VH domain comprising a VH or VH CDR and a VL and VL CDR as described herein, and wherein the constant region comprises an amino acid sequence of a constant region of an IgG, IgE, IgM, IgD, IgA or IgY immunoglobulin molecule or a human IgG, IgE, IgM, IgD, IgA or IgY immunoglobulin molecule. In another specific embodiment, an antibody described herein that specifically binds to FAM19A5 (e.g., human FAM19A5) comprises a VL domain and a VH domain comprising any amino acid sequence described herein, and wherein the constant region comprises an amino acid sequence of a constant region of an IgG, IgE, IgM, IgD, IgA or IgY immunoglobulin molecule, any subclass of an immunoglobulin molecule (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). In some embodiments, the constant region comprises the amino acid sequence of the constant region of a naturally occurring human IgG, including subclasses (e.g., IgG1, IgG2, IgG3, or IgG4) and allotypes (e.g., G1m, G2m, G3m, and nG4m) and variants thereof. See, e.g., Vidarsson G. et al., Front Immunol. 5:520 (published online on October 20, 2014) and Jefferis R. and Lefranc MP, mAbs 1:4, 1-7 (2009). In some embodiments, the constant region comprises the amino acid sequence of the constant region of human IgG1, IgG2, IgG3, or IgG4, or variants thereof.

[0189] In certain embodiments, the anti-FAM19A5 antibodies disclosed herein do not have Fc effector functions, such as complement-dependent cytotoxicity (CDC) and / or antibody-dependent cellular phagocytosis (ADCP). The effector functions are mediated by the Fc region, and the residues closest to the hinge region in the CH2 domain of the Fc region are responsible for the effector functions of the antibody because they contain highly overlapping binding sites for C1q (complement) and IgG-Fc receptors (FcγR) on the effector cells of the innate immune system. Moreover, IgG2 and IgG4 antibodies have lower levels of Fc effector functions than IgG1 and IgG3 antibodies. The effector function of an antibody can be reduced or avoided by various methods known in the art, including (1) using an antibody fragment lacking an Fc region (e.g., such as Fab, F(ab')2, single-chain Fv (scFv), or sdAb composed of a monomeric VH or VL domain); (2) producing aglycosylated antibodies, which can be produced by, for example, deleting or changing the residue to which the sugar is attached, enzymatically removing the sugar, producing the antibody in cells cultured in the presence of a glycosylation inhibitor, or by expressing the antibody in cells that are unable to glycosylate proteins (e.g., bacterial host cells, see, e.g., U.S. Publication No. 20120100140); (3) using an Fc region from the IgG subclass with reduced effector function (e.g., an Fc region from an IgG2 or IgG4 antibody or a chimeric Fc region comprising a CH2 domain from an IgG2 or IgG4 antibody, see, e.g., U.S. Publication No. 20120100140 and Lau et al.); C. et al., J. Immunol. 191:4769-4777 (2013)); and (4) generating an Fc region with mutations resulting in reduced or no Fc function. See, e.g., U.S. Publication No. 20120100140 and the U.S. and PCT applications cited therein and An et al., mAbs 1:6, 572-579 (2009).

[0190] Thus, in some embodiments, the anti-FAM19A5 antibodies disclosed herein are Fab, Fab', F(ab')2, Fv, single-chain Fv (scFv) or sdAb composed of monomeric VH or VL domains. Such antibody fragments are well known in the art and are described above.

[0191] In some embodiments, the anti-FAM19A5 antibody is a single chain Fv. Table 7 below provides the amino acid sequence of an exemplary anti-FAM19A5 scFv.

[0192] Table 7. Amino acid sequences of anti-FAM19A5 scFv

[0193]

[0194]

[0195] In some embodiments, the anti-FAM19A5 antibodies disclosed herein comprise an Fc region with reduced or no Fc effector function. In some embodiments, the constant region comprises the amino acid sequence of the Fc region of human IgG2 or IgG4. In some embodiments, the anti-FAM19A5 antibody is of IgG2 / IgG4 isotype. In some embodiments, the anti-FAM19A5 antibody comprises: a chimeric Fc region comprising a CH2 domain of an IgG antibody from an IgG4 isotype and a CH3 domain of an IgG antibody from an IgG1 isotype; a chimeric Fc region comprising a hinge region from IgG2 and a CH2 region from IgG4; or an Fc region with a mutation that results in reduced or no Fc function. Fc regions with reduced or no Fc effector function include those known in the art. See, e.g., Lau C. et al., J. Immunol. 191: 4769-4777 (2013); An et al., mAbs 1: 6,572-579 (2009); and U.S. Publication No. 20120100140 and the U.S. patents and publications cited therein and PCT publications. One of ordinary skill in the art can also readily prepare an Fc region with reduced or no Fc effector function.

[0196] III. Nucleic Acid Molecules

[0197] Another aspect described herein relates to one or more nucleic acid molecules encoding any of the antibodies described herein. Nucleic acids can exist in intact cells, in cell lysates, or in partially purified or substantially pure forms. Nucleic acids are "isolated" or "become substantially pure" when purified from other cell components or other contaminants, such as other cell nucleic acids (e.g., other chromosomal DNA, such as chromosomal DNA essentially connected to isolated DNA) or proteins by standard techniques, including alkali / SDS treatment, CsCl density gradient centrifugation (CsCl banding), column chromatography, restriction enzymes, agarose gel electrophoresis, and other well-known techniques in the art. See F.Ausubel et al., ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. Nucleic acids described herein can be, for example, DNA or RNA, and may or may not contain intron sequences. In certain embodiments, nucleic acids are cDNA molecules.

[0198] The nucleic acids described herein can be obtained using standard molecular biology techniques. For hybridoma-expressed antibodies (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes, as further described below), cDNAs encoding the light and heavy chains of the antibodies prepared by the hybridomas can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display technology), nucleic acids encoding antibodies can be recovered from the libraries.

[0199] Certain nucleic acid molecules described herein are those encoding the VH and VL sequences of various anti-FAM19A5 antibodies of the present disclosure. Tables 8 and 9 show exemplary DNA sequences encoding the VH and VL sequences of such antibodies, respectively.

[0200] Table 8. Variable heavy chain polynucleotide sequences

[0201]

[0202] Table 9. Variable light chain polynucleotide sequences

[0203]

[0204]

[0205] Methods for preparing the anti-FAM19A5 antibodies disclosed herein may comprise expressing the heavy and light chains in a cell line comprising nucleotide sequences encoding the heavy and light chains, respectively, with signal peptides such as SEQ ID NOs: 21 and 22. Host cells comprising these nucleotide sequences are contemplated herein.

[0206] Once the DNA fragments encoding the VH and VL fragments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, such as converting the variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these manipulations, the DNA fragment encoding VL or VH is operably linked to another DNA fragment encoding another protein (such as an antibody constant region or a flexible linker). As used in this context, the term "operably linked" refers to linking two DNA fragments together so that the amino acid sequences encoded by the two DNA fragments remain in frame.

[0207] By operably linking the DNA encoding VH to another DNA molecule encoding the heavy chain constant region (hinge, CH1, CH2 and / or CH3), the isolated DNA encoding the VH region can be converted into a full-length heavy chain gene. The sequence of the human heavy chain constant region gene is known in the art (see, e.g., Kabat, EA, et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242) and the DNA fragments covering these regions can be obtained by standard PCR amplification. The heavy chain constant region can be IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, such as IgG2 and / or IgG4 constant region. For Fab fragment heavy chain genes, the DNA encoding VH can be operably linked to another DNA molecule encoding only the heavy chain CH1 constant region.

[0208] By operably linking the DNA encoding VL to another DNA molecule encoding the light chain constant region CL, the separated DNA encoding the VL region can be converted into a full-length light chain gene (and a Fab light chain gene). The sequence of human light chain constant region genes is known in the art (see, e.g., Kabat, EA, et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242), and the DNA fragments covering these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region.

[0209] To generate scFv genes, the DNA fragments encoding VH and VL are operably linked to another fragment encoding a flexible linker, such as a fragment encoding an amino acid sequence (Gly4-Ser)3, so that the VH and VL sequences can be expressed as a continuous single-chain protein, wherein the VL and VH regions are connected by a flexible linker (see, e.g., Bird et al., (1988) Science 242:423-426; Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature 348:552-554). The amino acid and nucleotide sequences of exemplary anti-FAM19A5 scFvs are provided in Table 7 (above) and Table 10, respectively.

[0210] Table 10. Nucleotide sequences of anti-FAM19A5 scFv

[0211]

[0212]

[0213] In some embodiments, the present disclosure provides a vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody. In other embodiments, the vector can be used for gene therapy.

[0214] Suitable vectors for use in the present disclosure include expression vectors, viral vectors, and plasmid vectors. In one embodiment, the vector is a viral vector.

[0215] As used herein, an expression vector refers to any nucleic acid construct that contains the necessary elements for transcription and translation of an inserted coding sequence, or in the case of an RNA viral vector, contains the necessary elements for replication and translation when introduced into a suitable host cell. Expression vectors may include plasmids, phagemids, viruses and derivatives thereof.

[0216] The expression vector of the present disclosure can include polynucleotides encoding antibodies or described herein. In one embodiment, the coding sequence of the antibody is operably connected to the expression control sequence. As used herein, when two nucleic acid sequences are covalently connected in a manner that allows each component nucleic acid sequence to retain its functionality, they are operably connected. When the coding sequence and the gene expression control sequence are covalently connected in a manner that places the expression or transcription and / or translation of the coding sequence under the influence or control of the gene expression control sequence, they are referred to as being operably connected. If the induction promoter causes the transcription of the coding sequence in the 5' gene expression sequence, and if the connection essence between the two DNA sequences does not have (1) the introduction of frameshift mutations, (2) the ability of the promoter region to interfere with the transcription of the coding sequence, or (3) the ability of the corresponding RNA transcript to be translated into protein, the two DNA sequences are referred to as being operably connected. Therefore, if the gene expression sequence can achieve the transcription of the coding nucleic acid sequence so that the resulting transcript is translated into the desired antibody, the gene expression sequence will be operably connected to the coding nucleic acid sequence.

[0217] Viral vectors include, but are not limited to, nucleic acid sequences from the following viruses: retroviruses, such as Moloney murine leukemia virus, Harvey murine sarcoma virus, mouse mammary tumor virus, and Rous sarcoma virus; lentivirus; adenovirus; adeno-associated virus; SV40 virus; polyomavirus; Epstein-Barr virus; papillomavirus; herpes virus; vaccinia virus; poliovirus; and RNA viruses, such as retroviruses. Other vectors well known in the art can be readily adopted. Certain viral vectors are based on non-cellular eukaryotic viruses, in which non-essential genes have been replaced by genes of interest. Non-cellular pathogenic viruses include retroviruses, whose life cycle includes reverse transcription of genomic viral RNA into DNA, followed by integration of the provirus into host cell DNA. Retroviruses have been approved for human gene therapy trials. The most useful are those replication-defective retroviruses (i.e., capable of directing the synthesis of desired proteins, but unable to make infectious particles). The general use of such genetically altered retroviral expression vectors is the efficient transduction of genes in vivo. Standard protocols for producing replication-defective retroviruses (comprising incorporating foreign genetic material into plasmids, transfecting packaging cell lines with the plasmids, producing recombinant retroviruses by the packaging cell lines, collecting viral particles from tissue culture medium, and infecting target cells with the viral particles) are provided in Kriegler, M., Gene Transfer and Expression, A Laboratory Manual, WH Freeman Co., New York (1990) and Murry, EJ, Methods in Molecular Biology, Vol. 7, Humana Press, Inc., Cliffton, NJ (1991).

[0218] In one embodiment, the virus is an adeno-associated virus, a double-stranded DNA virus. Adeno-associated viruses can be transformed into replication-defective types and can infect a range of cell types and species. It also has other advantages, such as thermal and lipid solvent stability; high transduction frequencies of various lineage cells including hematopoietic cells; and no repeated infection inhibition, so multiple series of transductions can be performed. It is reported that adeno-associated viruses can be integrated into human cell DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and the variability of the expression characteristics of inserted genes infected by retroviruses. In addition, in the absence of selection pressure, wild-type adeno-associated virus infection is passaged more than 100 times in tissue culture, which means that adeno-associated virus genome integration is a relatively stable event. Adeno-associated viruses can also work in an extrachromosomal manner.

[0219] In other embodiments, the vector is derived from a lentivirus. In certain embodiments, the vector is a recombinant lentiviral vector capable of infecting non-dividing cells.

[0220] The lentiviral genome and proviral DNA typically have three genes found in retroviruses: gag, pol, and env, which are flanked by two long terminal repeat (LTR) sequences. The gag gene encodes the internal structural (matrix, capsid, and nucleocapsid) proteins; the pol gene encodes the RNA-directed DNA polymerase (reverse transcriptase), protease, and integrase; and the env gene encodes the viral envelope glycoprotein. The 5' and 3' LTRs serve to facilitate transcription and polyadenylation of the virion RNA. The LTRs contain all other cis-acting sequences required for viral replication. Lentiviruses have additional genes, including vif, vpr, tat, rev, vpu, nef, and vpx (in HIV-1, HIV-2, and / or SIV).

[0221] Adjacent to the 5' LTR are sequences necessary for reverse transcription of the genome (tRNA primer binding site) and for efficient encapsidation of viral RNA into particles (Psi site). If sequences required for encapsidation (or packaging of retroviral RNA into infectious virions) are missing from the viral genome, the cis defect prevents encapsidation of the genomic RNA.

[0222] However, the resulting mutants are still able to direct the synthesis of all virion proteins. The present disclosure provides a method for producing a recombinant lentivirus capable of infecting non-dividing cells, the method comprising transfecting a suitable host cell with two or more vectors carrying packaging functions, i.e., gag, pol and env, as well as rev and tat. As will be disclosed below, vectors lacking a functional tat gene are required for certain applications. Thus, for example, a first vector can provide nucleic acids encoding viral gag and viral pol, while another vector can provide nucleic acids encoding viral env to produce packaging cells. A vector providing a heterologous gene (identified herein as a transfer vector) is introduced into the packaging cell to produce a production cell that releases infectious viral particles carrying an exogenous gene of interest.

[0223] According to the above configuration of the vector and the foreign gene, the second vector can provide a nucleic acid encoding a viral envelope (env) gene. The env gene can be derived from almost any suitable virus, including retroviruses. In some embodiments, the env protein is an amphiphilic envelope protein that allows transduction of cells of humans and other species.

[0224] Examples of env genes from retroviral sources include, but are not limited to, Moloney murine leukemia virus (MoMuLV or MMLV), Harvey murine sarcoma virus (HaMuSV or HSV), mouse mammary tumor virus (MuMTV or MMTV), gibbon ape leukemia virus (GaLV or GALV), human immunodeficiency virus (HIV), and Rous sarcoma virus (RSV). Other env genes, such as vesicular stomatitis virus (VSV) protein G (VSV G), hepatitis virus genes, and influenza virus genes, may also be used.

[0225] The vector providing the viral env nucleic acid sequence is operably associated with the regulatory sequences described elsewhere herein.

[0226] In certain embodiments, the vector comprises a lentiviral vector in which the HIV virulence genes env, vif, vpr, vpu, and nef are deleted without compromising the ability of the vector to transduce non-dividing cells.

[0227] In some embodiments, the vector comprises a lentiviral vector comprising a deletion of the U3 region of the 3' LTR. The deletion of the U3 region may be a complete deletion or a partial deletion.

[0228] In some embodiments, the lentiviral vector of the present disclosure comprising the FVIII nucleotide sequence described herein can be transfected in cells with (a) a first nucleotide sequence comprising gag, pol, or gag and pol genes and (b) a second nucleotide sequence comprising a heterologous env gene; wherein the lentiviral vector lacks a functional tat gene. In other embodiments, the cell is further transfected with a fourth nucleotide sequence comprising a rev gene. In certain embodiments, the lentiviral vector lacks a functional gene selected from vif, vpr, vpu, vpx, and nef, or a combination thereof.

[0229] In certain embodiments, the lentiviral vector comprises one or more nucleotide sequences encoding a gag protein, a Rev response element, a central polypurine tract (cPPT), or any combination thereof.

[0230] Examples of lentiviral vectors are disclosed in WO9931251, W09712622, W09817815, W09817816 and WO9818934, which are incorporated herein by reference in their entirety.

[0231] Other vectors include plasmid vectors. Plasmid vectors have been widely described in the art and are well known to those skilled in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. In recent years, it has been found that, due to being unable to replicate in the host genome and be integrated into the host genome, plasmid vectors are particularly advantageous for delivering genes to cells in vivo. However, these plasmids with promoters compatible with host cells can express peptides from genes operably encoded in the plasmid. Some conventional plasmids can be obtained from commercial suppliers, including 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; and pcDNA3.1 / hygro, catalog number V87020; pcDNA4 / myc-His, catalog number V86320; and pBudCE4.1, catalog number V53220, all from Invitrogen (Carlsbad, California). Other plasmids are well known to those of ordinary skill in the art. In addition, plasmids can be custom designed using standard molecular biology techniques to remove and / or add specific segments of DNA.

[0232] IV. Antibody Production

[0233] Antibodies or fragments thereof that immunospecifically bind to FAM19A5 (e.g., human FAM19A5) can be produced by any method known in the art for antibody synthesis, such as by chemical synthesis or by recombinant expression technology. Unless otherwise indicated, the methods described herein employ 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 technical scope of the art. For example, these techniques are described in the references cited herein and are fully described 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 (1987and annual updates); Current Protocols in Immunology, John Wiley&Sons (1987and 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.

[0234] In specific embodiments, the antibodies described herein are antibodies (e.g., recombinant antibodies) prepared, expressed, produced, or isolated by any method involving, for example, synthesis, genetic engineering of DNA sequences. In certain embodiments, such antibodies comprise sequences (e.g., DNA sequences or amino acid sequences) that are not naturally present in the antibody germline library of an animal or mammal (e.g., human) in vivo. In some embodiments, the anti-FAM19A5 antibodies disclosed herein have been deimmunized.

[0235] As described in the examples (e.g., Example 2), anti-FAM19A5 antibodies were initially produced by immunizing chickens with synthetic FAM19A5 peptides. Therefore, in order to minimize the immunogenicity risk when applied to human subjects, anti-FAM19A5 antibodies (e.g., 1-65) have been modified to be more similar to the immunogenic sequence of human antibodies. In certain embodiments, the anti-FAM19A5 antibodies of deimmunization disclosed herein have similar binding affinity to human FAM19A5 compared to their corresponding corresponding antibodies that have not yet been deimmunized. Methods for deimmunizing antibodies are disclosed herein and are also known in the art.

[0236] In one aspect, provided herein is a method for preparing an antibody or its antigen binding fragment that immunospecifically binds to FAM19A5 (e.g., human FAM19A5), the method comprising culturing cells or host cells as described herein. In one aspect, provided herein is a method for preparing an antibody or its antigen binding fragment that immunospecifically binds to FAM19A5 (e.g., human FAM19A5), the method comprising using cells or host cells as described herein (e.g., cells or host cells comprising polynucleotides encoding antibodies as described herein) to express (e.g., recombinantly express) antibodies or their antigen binding fragments. In a particular embodiment, the cell is a separated cell. In a particular embodiment, an exogenous polynucleotide has been introduced into the cell. In a particular embodiment, the method further comprises a step of purifying the antibody or its antigen binding fragment obtained from the cell or host cell.

[0237] Methods for producing polyclonal antibodies are known in the art (see, e.g., Chapter 11 in: Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel FM et al., eds., John Wiley and Sons, New York).

[0238] Monoclonal antibodies can be prepared using a variety of techniques known in the art, including the use of hybridomas, recombinant and phage display techniques or combinations thereof. For example, monoclonal antibodies can be produced using hybridoma technology, including those known in the art and, for example, Harlow E & Lane D, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al.,: Monoclonal Antibodies and T-Cell Hybridomas 563 681 (Elsevier, NY, 1981). As used herein, the term "monoclonal antibody" is not limited to antibodies produced by hybridoma technology. For example, monoclonal antibodies can be produced by recombinant host cells that exogenously express antibodies described herein or fragments thereof, such as light chains and / or heavy chains of such antibodies.

[0239] In specific embodiments, as used herein, "monoclonal antibody" is an antibody produced by a single cell (e.g., a hybridoma or host cell producing a recombinant antibody), wherein the antibody is immunospecifically bound to FAM19A5 (e.g., human FAM19A5), as determined, for example, by ELISA, or other antigen binding or competitive binding 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 certain embodiments, the monoclonal antibody is a monovalent antibody or a multivalent (e.g., bivalent) antibody. In specific embodiments, the monoclonal antibody is a monospecific or multispecific antibody (e.g., a bispecific antibody). The monoclonal antibodies described herein can be prepared, for example, by the hybridoma method as described in Kohler G & Milstein C (1975) Nature 256: 495, or can be separated from a phage library, for example, using the techniques described herein. Other methods for preparing clonal cell lines and monoclonal antibodies expressed thereby are well known in the art (see, e.g., Chapter 11 in: Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel FM et al., supra).

[0240] The method of using hybridoma technology to produce and screen specific antibodies is conventional and well known in the art. For example, in the hybridoma method, mice or other suitable host animals, such as sheep, goats, rabbits, rats, hamsters or macaques are immunized to induce or produce lymphocytes that will specifically bind to the protein (e.g., human FAM19A5) used for immunization. Alternatively, lymphocytes can be immunized in vitro. Then, lymphocytes are fused with myeloma cells using suitable fusion agents such as polyethylene glycol to form hybridoma cells (Goding JW (Ed), Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)). In addition, animals can be immunized using multi-site repeated immunization (RIMMS) technology (Kilpatrick KE et al., (1997) Hybridoma 16:381-9, which is incorporated herein by reference in its entirety).

[0241] 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, such as antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is collected and splenocytes are isolated. The splenocytes are then fused to any suitable myeloma cells, such as those available from the American Type Culture Collection, by well-known techniques. Cells obtained from the cell line SP20 of immunized mice (Manassas, Virginia) are used to form hybridomas. Hybridomas are selected and cloned by limiting dilution. In certain embodiments, lymph nodes of immunized mice are harvested and fused with NSO myeloma cells.

[0242] The hybridoma cells thus prepared are seeded and grown in an appropriate culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas will typically include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.

[0243] Specific embodiments employ myeloma cells that fuse efficiently, support stable high-level production of antibodies by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these myeloma cell lines are murine myeloma lines, such as the NSO cell line, or those derived from MOPC-21 and MPC-11 mouse tumors (available from the Salk Institute Cell Distribution Center in San Diego, California, USA), and SP-2 or X63-Ag8.653 cells (available from the American Type Culture Collection in Rockville, Maryland, USA). Human myeloma and mouse human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor D (1984) J Immunol 133: 3001-5; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

[0244] The culture medium in which the hybridoma cells are grown is assayed for the production of monoclonal antibodies against FAM19A5 (e.g., human FAM19A5). The binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by methods known in the art, such as immunoprecipitation, or by in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0245] After hybridoma cells producing antibodies with the desired specificity, affinity and / or activity are identified, clones can be subcloned by 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 in animals as ascites tumors.

[0246] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0247] The antibodies described herein include antibody fragments that recognize specific FAM19A5 (e.g., human FAM19A5) and can be produced by any technique known to those skilled in the art. For example, Fab and F(ab')2 fragments described herein can be produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (producing Fab fragments) or pepsin (producing F(ab')2 fragments). The Fab fragment corresponds to one of the 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 connected by a disulfide bond in the hinge region.

[0248] Further, various phage display methods known in the art can also be used to produce antibodies or antigen-binding fragments thereof described herein. In phage display methods, functional antibody domains are displayed on the surface of phage particles with polynucleotide sequences encoding them. In particular, DNA sequences encoding VH and VL domains are amplified from animal cDNA libraries (e.g., human or non-human, such as mouse or chicken cDNA libraries of diseased tissues). By PCR, the DNA encoding VH and VL domains is recombined with scFv linkers and cloned into phagemid vectors. The vector is electroporated in E. coli, and E. coli is infected with helper phage. The phages used in these methods are generally filamentous phages including fd and M13, and the VH and VL domains are generally recombinantly fused to phage gene III or gene VIII. Antigens can be used, such as labeled antigens or antigens bound or captured to a solid surface or beads, to select or identify phages expressing antigen-binding domains that bind to specific antigens. Examples of phage display methods that can be used to prepare the antibodies described herein include those described in: Brinkman U et al., (1995) J Immunol Methods 182:41-50; Ames RS et al., (1995) J Immunol Methods 184:177-186; Kettleborough CA et al., (1994) Eur J Immunol 24:952-958; Persic L et al., (1997) Gene 187:9-18; Burton DR & Barbas CF (1994) Advan Immunol 57:191-280; PCT Application No. PCT / GB91 / 001134; International Publication Nos. 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 U.S. Patents Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743, and 5,969,108.

[0249] As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to produce complete antibodies, including human antibodies or any other desired antigen-binding fragments, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast and bacteria, for example, as described below. Recombinant techniques for producing antibody fragments such as Fab, Fab' and F(ab')2 fragments can also be employed 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.

[0250] On the one hand, to produce complete antibodies, PCR primers including VH or VL nucleotide sequences, restriction sites, and flanking sequences protecting the restriction sites can be used to amplify VH or VL sequences from templates such as scFv clones. Using cloning techniques known to those skilled in the art, the PCR-amplified VH domain can be cloned into a vector expressing a VH constant region, and the PCR-amplified VL domain can be cloned into a vector expressing a VL constant region, such as a human kappa or lambda constant region. The VH and VL domains can also be cloned into a vector expressing an essential constant region. Then, using techniques known to those skilled in the art, the heavy chain conversion vector and the light chain conversion vector are co-transfected into a cell line to produce a stable or transient cell line expressing a full-length antibody such as an IgG.

[0251] Chimeric antibodies are molecules in which different parts of the antibody are derived from different immunoglobulin molecules. For example, a chimeric antibody may contain a variable region of a non-human animal (e.g., mouse, rat, or chicken) monoclonal antibody fused to a human antibody constant region. Methods for producing chimeric antibodies are known in the art. See, for example, Morrison SL (1985) Science 229: 1202-7; Oi VT & Morrison SL (1986) BioTechniques 4: 214-221; Gillies SD et al., (1989) J Immunol Methods 125: 191-202; and U.S. Pat. Nos. 5,807,715, 4,816,567, 4,816,397, and 6,331,415.

[0252] Humanized antibodies can be combined with a predetermined antigen, and comprise a framework region having substantially the amino acid sequence of a human immunoglobulin and a CDR having substantially the amino acid sequence of a non-human immunoglobulin (e.g., mouse or chicken immunoglobulin). In a specific embodiment, the humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), which is typically a constant region of a human immunoglobulin. The antibody can 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 produced using a variety of techniques known in the art, including but not limited to CDR grafting (European Patent No. EP 239400; International Publication No. WO 91 / 09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (European Patent Nos. EP 592106 and EP 519596; Padlan EA (1991) Mol Immunol 28(4 / 5):489-498; Studnicka GM et al., (1994) Prot Engineering 7(6):805-814; and Roguska MA et al., (1994) PNAS 91:969-973), chain shuffling (U.S. Pat. No. 5,565,332), and the techniques disclosed in, for example, U.S. Pat. No. 6,407,213, U.S. Pat. No. 5,766,886, 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. Application Publication No. US2005 / 0042664 Al (Feb. 24, 2005), which is incorporated herein by reference in its entirety.

[0253] Methods for making multispecific (eg, bispecific antibodies) have been described, see, e.g., U.S. Pat. Nos. 7,951,917; 7,183,076; 8,227,577; 5,837,242; 5,989,830; 5,869,620; 6,132,992 and 8,586,713.

[0254] Single domain antibodies, e.g., antibodies lacking light chains, can be produced by methods well known in the art, see Riechmann L & Muyldermans S (1999) J Immunol 231: 25-38; Nuttall SD et al., (2000) Curr Pharm Biotechnol 1(3): 253-263; Muyldermans S, (2001) J Biotechnol 74(4): 277-302; U.S. Pat. No. 6,005,079; and International Publication Nos. WO 94 / 04678, WO 94 / 25591, and WO 01 / 44301.

[0255] Further, antibodies that immunospecifically bind to the FAM19A5 antigen can be used in turn to generate anti-idiotypic antibodies that "mimic" the antigen using techniques well known to those skilled in the art (see, e.g., Greenspan NS & Bona CA (1989) FASEB J 7(5): 437-444; and Nissinoff A (1991) J Immunol 147(8): 2429-2438).

[0256] In a specific embodiment, the antibody described herein that is combined with the same epitope of FAM19A5 (e.g., people FAM19A5) as the anti-FAM19A5 antibody described herein is a human antibody or its antigen-binding fragment. In a specific embodiment, the antibody described herein that competitively blocks (e.g., in a dose-dependent manner) antibodies described herein (e.g., 1-65) combined with FAM19A5 (e.g., people FAM19A5) is a human antibody or its antigen-binding fragment.

[0257] Any method known in the art can be used to produce human antibodies. For example, transgenic mice that cannot express functional endogenous immunoglobulins but can express human immunoglobulin genes can be used. In particular, human heavy chain and light chain immunoglobulin gene complexes can be introduced randomly or introduced into mouse embryonic stem cells by homologous recombination. Alternatively, in addition to human heavy chain and light chain genes, human variable regions, constant regions and diversity regions can also be introduced into mouse embryonic stem cells. Mouse heavy chain and light chain immunoglobulin genes can be made non-functional by homologous recombination alone or simultaneously with the introduction of human immunoglobulin loci. In particular, homozygous deletions in the JH region prevent endogenous antibody production. Modified embryonic stem cells are amplified and microinjected into blastocysts to produce chimeric mice. Then, chimeric mice are bred to produce homozygous offspring expressing human antibodies. Transgenic mice are immunized in a normal manner with selected antigens, such as all or part of the antigens (e.g., FAM19A5). Monoclonal antibodies against antigens can be obtained from immunized transgenic mice using conventional hybridoma technology. The human immunoglobulin transgene carried by transgenic mice is rearranged during the B cell differentiation process, followed by class switching and somatic mutation. Therefore, using such technology, useful IgG, IgA, IgM and IgE antibodies for treatment can be produced. For an overview 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 producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., International Publication Nos. WO 98 / 24893, WO 96 / 34096, and WO 96 / 33735; and U.S. Pat. Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, and 5,939,598. Examples of mice capable of producing human antibodies include the XENOMOUSE TM (Abgenix; U.S. Patent Nos. 6,075,181 and 6,150,184), HUAB-MOUSE TM (Mederex Corporation / Gen Pharm; U.S. Patents 5,545,806 and 5,569,825), TRANS CHROMO MOUSE TM (Kirin) and KM MOUSE TM (Medarex / Kirin).

[0258] Human antibodies that specifically bind to FAM19A5 (e.g., human FAM19A5) can be prepared using antibody libraries derived from human immunoglobulin sequences by various methods known in the art, including the above-mentioned phage display method. See also U.S. Patents Nos. 4,444,887, 4,716,111, and 5,885,793; and International Publications Nos. WO 98 / 46645, WO 98 / 50433, WO 98 / 24893, WO 98 / 16654, WO 96 / 34096, WO 96 / 33735, and WO 91 / 10741.

[0259] In certain embodiments, human antibodies can be produced using mouse-human hybridomas. For example, human peripheral blood lymphocytes transformed with Epstein-Barr virus (EBV) can be fused with mouse myeloma cells to produce mouse-human hybridomas that secrete human monoclonal antibodies, and these mouse-human hybridomas can be screened to determine the hybridomas that secrete human monoclonal antibodies that are immunospecifically bound to target antigens (e.g., 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.

[0260] V. Methods of Engineering Antibodies

[0261] As described above, the anti-FAM19A5 antibodies with VH and VL sequences disclosed herein can be used to produce new anti-FAM19A5 antibodies by modifying VH and / or VL sequences or constant regions connected thereto. Therefore, in another aspect described herein, the structural features of the anti-FAM19A5 antibodies described herein are used to produce structurally related anti-FAM19A5 antibodies, which retain at least one functional property of the antibodies described herein, such as binding to human FAM19A5. For example, the starting material for the engineering method is a VH and / or VL sequence or one or more CDR regions thereof provided herein. In order to produce engineered antibodies, it is not necessary to actually prepare (i.e., express in protein form) antibodies with one or more VH and / or VL sequences or one or more CDR regions thereof provided herein. On the contrary, the information contained in the sequence is used as a starting material to produce a "second generation" sequence derived from the original sequence, and then the "second generation" sequence is prepared and expressed in protein.

[0262] Therefore, provided herein is a method for preparing an anti-FAM19A5 antibody, comprising:

[0263] (a) providing: (i) a heavy chain variable region sequence comprising a CDR1, CDR2 and / or CDR3 sequence shown in Table 3, or a CDR1, CDR2 and / or CDR3 of a heavy chain variable region shown in Table 5; and (ii) a light chain variable region sequence comprising a CDR1, CDR2 and / or CDR3 sequence shown in Table 4, or a CDR1, CDR2 and / or CDR3 of a heavy chain variable region shown in Table 6;

[0264] (b) changing at least one amino acid residue within the heavy chain variable region sequence and / or the light chain variable region sequence to generate at least one altered antibody sequence; and

[0265] (c) Antibody sequences with altered protein expression.

[0266] Standard molecular biology techniques can be used to prepare and express altered antibody sequences.

[0267] In some embodiments, the antibody encoded by the altered antibody sequence is an antibody that retains one, some or all of the functional properties of the anti-FAM19A5 antibodies described herein, including:

[0268] (1) Reduced immunogenicity in human subjects;

[0269] (2) As determined by Biacore, with a K of 10 nM or less D (e.g., 0.01 nM to 10 nM) binds to soluble human FAM19A5;

[0270] (3) As measured by ELISA, with a K of 10 nM or less D (e.g., 0.01 nM to 1 nM) binds to membrane-bound human FAM19A5;

[0271] (4) binds to membrane-bound human FAM19A5 with an EC50 of 1 nM or less (e.g., 0.01 nM to 1 nM) as determined by ELISA;

[0272] (5) reduce, reverse, delay and / or prevent the onset of reactive gliosis;

[0273] (6) inhibiting the excessive proliferation of reactive astrocytes;

[0274] (7) reduce the expression of chondroitin sulfate proteoglycans including neurocan and neuron-glial antigen 2 (NG2);

[0275] (8) Increased expression of c-fos and pERK in neuronal nuclei;

[0276] (9) Promote neuronal survival;

[0277] (10) Increase the expression of GAP43 in neurons;

[0278] (11) promoting axon regeneration; and

[0279] (12) Using the anti-FAM19A5 antibodies disclosed herein to compete for binding with human FAM19A5 in one or two directions.

[0280] The 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 listed in (1) to (12) above. The functional properties of the altered antibody may be assessed using standard assays available in the art and / or described herein, such as those listed in the Examples (e.g., ELISA, FACS).

[0281] In certain embodiments of the engineered antibody methods described herein, mutations can be introduced randomly or selectively along all or part of the anti-FAM19A5 antibody coding sequence, and the resulting modified anti-FAM19A5 antibodies can be screened for binding activity and / or other functional properties, as described herein. Mutation methods have been described in the art. For example, Short's PCT Publication WO 02 / 092780 describes methods for generating and screening antibody mutations using saturation mutagenesis, synthetic ligation assembly, or a combination thereof. Alternatively, Lazar et al.'s PCT Publication WO 03 / 074679 describes methods for optimizing the physicochemical properties of antibodies using computational screening methods.

[0282] VI. Cells and Vectors

[0283] In certain aspects, cells (e.g., host cells) expressing (e.g., recombinantly) antibodies (or antigen-binding fragments thereof) described herein that specifically bind to FAM19A5 (e.g., human FAM19A5) are provided herein, as well as related polynucleotides and expression vectors. Provided herein are vectors (e.g., expression vectors) comprising polynucleotides comprising nucleotide sequences encoding anti-FAM19A5 antibodies or fragments for recombinant expression in host cells such as mammalian cells. Also provided herein are host cells comprising such vectors for recombinant expression of anti-FAM19A5 antibodies (e.g., human or humanized antibodies) described herein. In specific aspects, provided herein are methods for producing antibodies described herein, comprising expressing such antibodies from host cells.

[0284] The recombinant expression of an antibody described herein (e.g., a full-length antibody, a heavy chain and / or a light chain of an antibody, or a single-chain antibody described herein) that specifically binds to FAM19A5 (e.g., human FAM19A5) involves the construction of an expression vector containing a polynucleotide encoding the antibody. Once a polynucleotide encoding an antibody molecule, a heavy chain and / or a light chain of an antibody, or a fragment thereof (e.g., a heavy chain and / or a light chain variable domain) as described herein has been obtained, a vector for producing an antibody molecule can be produced by recombinant DNA technology using techniques well known in the art. Therefore, a method for preparing a protein by expressing a polynucleotide containing an antibody or an antibody fragment (e.g., a light chain or a heavy chain) encoding nucleotide sequence is described herein. An expression vector containing an antibody or an antibody fragment (e.g., a light chain or a heavy chain) encoding sequence and suitable transcription and translation control signals can be constructed using methods well known to those skilled in the art. These methods include, for example, in vitro recombinant DNA technology, synthetic techniques, and in vivo genetic recombination. Also provided is a carrier comprising a nucleotide sequence encoding an antibody molecule described herein, a heavy chain or light chain of an antibody, a heavy chain or light chain variable domain of an antibody or its fragment, or a heavy chain or light chain CDR that is operably connected to a promoter. Such carriers can, for example, include a nucleotide sequence encoding a constant region of an antibody molecule (see, for example, International Publication No. WO 86 / 05807 and No. WO 89 / 01036; and U.S. Patent No. 5,122,464), and the variable domains of an antibody can be cloned into such carriers to express the entire heavy, entire light chain, or entire heavy and light chains.

[0285] The expression vector can be transferred to a cell (e.g., a host cell) by conventional techniques, and then the resulting cell can be cultured by conventional techniques to produce an antibody as described herein (e.g., an antibody comprising one or more of the VH and / or VL of an anti-FAM19A5 antibody disclosed herein, or its VH and / or VL CDR) or its fragment. Therefore, a host cell is provided herein, which contains a polynucleotide operably connected to a promoter to express such a sequence in a host cell, and the polynucleotide encodes an antibody as described herein or its fragment, or its heavy chain or light chain or its fragment, or a single-chain antibody as described herein. In certain embodiments, in order to express a double-chain antibody, a vector encoding a heavy chain and a light chain, respectively, can be co-expressed in a host cell to express the entire immunoglobulin molecule, as described below. In certain embodiments, the host cell contains a vector comprising a polynucleotide encoding both the heavy chain and the light chain of an antibody as described herein or its fragment. In a specific embodiment, the host cell contains two different vectors, the first vector comprising a polynucleotide encoding the heavy chain or heavy chain variable region of an antibody as described herein or its fragment, and the second vector comprising a polynucleotide encoding the light chain or light chain variable region of an antibody as described herein or its fragment. In other embodiments, the first host cell comprises a first vector comprising a polynucleotide encoding a heavy chain or heavy chain variable region of an antibody described herein or a fragment thereof, and the second host cell comprises a second vector comprising a polynucleotide encoding a light chain or light chain variable region of an antibody described herein. In a specific embodiment, the heavy chain / heavy chain variable region expressed by the first cell associated with the light chain / light chain variable region of the second cell is used to form an anti-FAM19A5 antibody or antigen-binding fragment thereof described herein. In certain embodiments, a host cell population comprising such a first host cell and such a second host cell is provided herein.

[0286] In a specific embodiment, provided herein is a set of vectors comprising a first vector comprising a polynucleotide encoding the light chain / light chain variable region of an anti-FAM19A5 antibody described herein; and a second vector comprising a polynucleotide encoding the heavy chain / heavy chain variable region of an anti-FAM19A5 antibody described herein.

[0287] A variety of host-expression vector systems can be used to express the antibody molecules described herein. Such host-expression systems represent vectors by which the coding sequence of interest can be produced and subsequently purified, and also represent cells that can in situ express the antibody molecules described herein when transformed or transfected with the appropriate nucleotide coding sequence. These include, but are not limited to, microorganisms such as bacteria (e.g., Escherichia coli and Bacillus subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Pichia pastoris) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) 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., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK293, 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), the mammalian cell system comprises a recombinant expression construct containing a promoter derived from a mammalian cell genome (e.g., a metallothionein promoter) or a mammalian virus (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter). In a specific embodiment, the cell used to express the antibody or antigen-binding fragment thereof described herein is a CHO cell, such as from a CHO GS SYSTEM TM In certain embodiments, the cells used to express the antibodies described herein are human cells, e.g., human cell lines. In specific embodiments, the mammalian expression vector is a POPTIVEC TMOr pcDNA3.3. In a specific embodiment, bacterial cells such as Escherichia coli, or eukaryotic cells (e.g., mammalian cells), particularly cells for expressing the entire recombinant antibody molecule, are used to express the recombinant antibody molecule. For example, mammalian cells (such as Chinese hamster ovary (CHO) cells) together with vectors (such as major immediate early gene promoter elements from human cytomegalovirus) are effective expression systems for antibodies (Foecking MK & Hofstetter H (1986) Gene 45: 101-5; and Cockett MI et al., (1990) Biotechnology 8 (7): 662-7). In certain embodiments, the antibodies described herein are produced by CHO cells or NSO cells. In a specific embodiment, the expression of the nucleotide sequence encoding the antibodies described herein that immunospecifically bind to FAM19A5 (e.g., human FAM19A5) is regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

[0288] In bacterial systems, depending on the intended use of the antibody molecule being expressed, many expression vectors can be advantageously selected. For example, when such antibodies are to be produced in large quantities, in order to generate a pharmaceutical composition of the antibody molecule, it may be necessary to instruct a vector that is easy to purify a high-level fusion protein product to express. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruether U & Mueller-Hill B (1983) EMBO J 2: 1791-1794), in which the antibody coding sequence can be individually connected to the vector in frame with the lac Z coding region to produce a fusion protein; pIN vectors (Inouye S & Inouye M (1985) Nuc Acids Res 13: 3101-3109; Van Heeke G & Schuster SM (1989) J Biol Chem 24: 5503-5509) etc. For example, pGEX vectors can also be used to express exogenous polypeptides as fusion proteins with glutathione 5-transferase (GST). Typically, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to the matrix glutathione agarose beads followed by elution in the presence of free glutathione. pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

[0289] In insect systems, the Autographa californica nuclear polyhedrosis virus (AcNPV), for example, can be used as a vector for expressing foreign genes. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence can be cloned individually into a non-essential region of the virus (e.g., the polyhedrin gene) and placed under the control of an AcNPV promoter (e.g., the polyhedrin promoter).

[0290] In mammalian host cells, a variety of virus-based expression systems can be used. In the case of using adenovirus as an expression vector, the antibody coding sequence of interest can be connected to an adenovirus transcription / translation control complex, such as a late promoter and a triplet leader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion of the non-essential region of the viral genome (e.g., region E1 or E3) will produce a recombinant virus that can survive and express antibody molecules in an infected host (see, e.g., Logan J & Shenk T (1984) PNAS 81 (12): 3655-9). In order to effectively translate the inserted antibody coding sequence, a specific start signal may also be required. These signals include an ATG start codon and adjacent sequences. Further, the start codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translation control signals and start codons can be of various sources--natural and synthetic. The efficiency of expression can be increased by including appropriate transcription enhancer elements, transcription terminators, and the like (see, for example, Bitter G et al., (1987) Methods Enzymol. 153: 516-544).

[0291] In addition, a host cell strain can be selected that regulates the expression of the inserted sequence, or modifies and processes the gene product in a desired ad hoc manner. Such modifications (e.g., glycosylation) and processing (e.g., lysis) of the protein product may be important for the function of the protein. Different host cells have characteristics and specific mechanisms for post-translational processing and modification of proteins and gene products. Suitable cell lines or host systems can be selected to ensure the correct modification and processing of the exogenous protein expressed. For this reason, eukaryotic host cells can be used that have cellular machinery for appropriate primary transcript processing, 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 (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7030, COS (e.g., COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC 1, BSC40, YB / 20, BMT10 and HsS78Bst cells. In certain embodiments, the anti-FAM19A5 antibodies described herein are produced in mammalian cells such as CHO cells.

[0292] In specific embodiments, the antibodies or antigen-binding portions thereof described herein have reduced or no fucose content. Such antibodies can be produced using techniques known to those skilled in the art. For example, the antibodies can be expressed in cells lacking or lacking the ability to fucosylate. In a specific example, a cell line in which both alleles of 1,6-fucosyltransferase are knocked out can be used to produce antibodies or antigen-binding portions thereof having reduced fucose content. The CAR-T® system (Lonza) is an example of a system that can be used to generate antibodies, or antigen-binding portions thereof, with reduced fucose content.

[0293] In order to produce recombinant proteins in a long-term and high-yield manner, stable expression cells can be generated. For example, cell lines that stably express the anti-FAM19A5 antibodies and antigen-binding portions thereof described herein can be engineered. In a specific embodiment, the cells provided herein stably express light chains / light chain variable domains and heavy chains / heavy chain variable domains, which combine to form antibodies or antigen-binding portions thereof described herein.

[0294] In some aspects, host cells can be transformed using DNA controlled by suitable expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and selectable markers, rather than using expression vectors containing viral origins of replication. After introducing exogenous DNA / polynucleotides, engineered cells can be allowed to grow in enriched medium for 1 to 2 days and then converted to selective medium. The selectable marker in the recombinant plasmid causes resistance to selection, and allows cells to stably integrate the plasmid into its chromosome and grow to form a focus, which can then be cloned and expanded to a cell line. This method can be advantageously used for engineering cell lines expressing anti-FAM19A5 antibodies described herein or their antibody binding portions. Such engineered cell lines are particularly useful when screening and evaluating compositions that interact directly or indirectly with antibody molecules.

[0295] A number of selection systems can be used, including but not limited to herpes simplex virus thymidine kinase (Wigler M et al., (1977) Cell 11(1):223-32), hypoxanthine guanine phosphoribosyltransferase (Szybalska EH & Szybalski W (1962) PNAS 48(12):2026-2034), and adenine phosphoribosyltransferase (Lowy I et al., (1980) Cell 22(3):817-23) genes can be used in tk, hgprt or aprt cells, respectively. In addition, antimetabolite resistance can be used as the basis for selection for the following gene: 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 the aminoglycoside G-418 (Wu GY & Wu CH (1991) Biotherapy 3:87-95; Tolstoshev P (1993) Ann Rev Pharmacol Toxicol 32:573-596; Mulligan RC (1993) Science 260:926-932; and Morgan RA & Anderson WF (1993) Ann Rev Biochem 62:191-217; Nabel GJ & Feigner PL (1993) Trends Biotechnol 11(5):211-5); and hygro, which confers resistance to hygromycin (Santerre RF et al., (1984) Gene 30(1-3):147-56).Methods generally known in the field of recombinant DNA technology can be routinely used to select desired recombinant clones, and such methods are described, for example, in Ausubel FM et al., (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler M, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli NC et al., (eds.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colbere-Garapin F et al., (1981) J Mol Biol 150: 1-14, which are incorporated herein by reference in their entirety.

[0296] The expression level of antibody molecules can be increased by vector amplification (for review, see Bebbington CR & Hentschel CCG, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, New York, 1987)). When the marker in the vector system expressing the antibody is amplifiable, the increase in the level of inhibitors present in the host cell culture will increase the copy number of the marker gene. Since the amplified region is associated with the antibody gene, the production of the antibody will also increase (Crouse GF et al., (1983) Mol Cell Biol 3: 257-66).

[0297] Host cells can be co-transfected with two or more expression vectors as described herein, wherein the first vector encodes a polypeptide from a heavy chain source, and the second vector encodes a polypeptide from a light chain source. The two vectors can contain the same selectable marker, which enables the expression of heavy chain polypeptides and light chain polypeptides to be equal. Host cells can be co-transfected with two or more expression vectors of different amounts. For example, host cells can be transfected with the first expression vector and the second expression vector in any ratio: 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.

[0298] Alternatively, a single vector can be used, which encodes and can express heavy chain polypeptides and light chain polypeptides simultaneously. In this case, the light chain should be placed before the heavy chain to avoid excessive non-toxic heavy chain (Proudfoot NJ (1986) Nature 322: 562-565; and Kohler G (1980) PNAS 77: 2197-2199). The coding sequence of heavy chain and light chain can include cDNA or genomic DNA. The expression vector can be a monocistronic or polycistronic. Polycistronic nucleic acid constructs can encode 2, 3, 4, 5, 6, 7, 8, 9, 10 or more genes / nucleotide sequences, or genes / nucleotide sequences in the range of 2 to 5, 5 to 10 or 10 to 20. For example, a bicistronic nucleic acid construct can include a promoter, a first gene (e.g., a heavy chain of an antibody described herein) and a second gene and (e.g., a light chain of an antibody described herein) in the following order. In such expression vectors, transcription of both genes can be driven by the promoter, while translation of mRNA from the first gene can occur by a cap-dependent scanning mechanism, and translation of mRNA from the second gene can occur by a cap-dependent mechanism, such as through an IRES.

[0299] Once the antibody molecules described herein are produced by recombinant expression, they can be purified by any method known in the art for purifying immunoglobulin molecules, such as by chromatography (e.g., ion exchange; affinity, particularly by affinity for a specific antigen after protein A; and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for purifying proteins. Further, the antibodies described herein can be fused to heterologous polypeptide sequences described herein or known in the art to facilitate purification.

[0300] In a specific embodiment, an antibody or its antigen-binding portion described herein is separated or purified. Generally, an isolated antibody is an antibody that is substantially free of other antibodies with different antigenic specificity. For example, in a specific embodiment, the preparation of the antibody described herein is substantially free of cellular material and / or chemical precursors. The statement "substantially free of cellular material" includes preparations of such antibodies, i.e., wherein the antibody is separated from the cellular components of the cells from which the antibody is separated or recombined to produce the antibody. Therefore, antibodies substantially free of cellular material include variants of heterologous proteins (also referred to herein as "contaminating proteins") and / or antibodies with less than about 30%, 20%, 10%, 5%, 2%, 1%, 0.5% or 0.1% (by dry weight), such as different post-translational modified forms of antibodies (or antibody binding portions) or other different versions of antibody preparations. When the antibody is recombinantly produced, it is also generally substantially free of culture medium, i.e., culture medium accounts for less than about 20%, 10%, 2%, 1%, 0.5% or 0.1% of the volume of the protein preparation. When an antibody is produced by chemical synthesis, it is generally substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals involved in protein synthesis. Thus, preparations of such antibodies have less than about 30%, 20%, 10% or 5% (by dry weight) of chemical precursors or compounds other than the antibody of interest. In specific embodiments, the antibodies described herein are isolated or purified.

[0301] VII. Assay

[0302] The binding of antibodies described herein to FAM19A5 can be tested by, for example, standard ELISA. In brief, a microtiter plate is coated with 1 to 2 μg / ml of purified FAM19A5 in PBS, and then blocked with 5% bovine serum albumin in PBS. A dilution of the antibody (e.g., a dilution of the plasma from FAM19A5 immune mice) is added to each well and incubated at 37°C for 1 to 2 hours. The plate is washed with PBS / Tween and then incubated at 37°C for 1 hour with a second reagent (e.g., for human antibodies, goat anti-human IgG Fc specific polyclonal reagent) conjugated to horseradish peroxidase (HRP). After washing, the plate is developed with ABTS substrate (Moss, product: ABTS-1000) and analyzed at OD 415-495 with a spectrophotometer. Then, the combination of the cell line expressing human FAM19A5 is further screened by flow cytometry, but the serum from immune mice combined with the control cell line not expressing FAM19A5 is not combined. In brief, the combination of anti-FAM19A5 antibodies is assessed by incubating the CHO cells expressing FAM19A5 with anti-FAM19A5 antibodies at a dilution of 1:20. The cells are washed and the combination is detected with anti-human IgG Ab labeled with PE. Flow cytometric analysis is performed using a FACS tank flow cytometer (Becton Dickinson, San Jose, California). Preferably, the mouse producing the highest titer is used for fusion.

[0303] The ELISA assay as described above can be used to screen antibodies, and thus hybridomas producing antibodies that show positive reactivity with the FAM19A5 immunogen can be screened. Then, hybridomas producing antibodies that preferably bind to FAM19A5 with high affinity can be subcloned and further characterized. Then, a clone (by ELISA) that retains the reactivity of the parental cells can be selected from each hybridoma to make a cell bank and perform antibody purification.

[0304] To purify anti-FAM19A5 antibodies, the selected hybridomas can be grown in two-liter spinner bottles for monoclonal antibody purification. The supernatant can be filtered and concentrated, and then affinity chromatography analysis is performed with agarose protein A (Pharmacia, Piscataway, New Jersey). The 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 a 1.43 extinction coefficient. The monoclonal antibody can be aliquoted and stored at -80°C.

[0305] To determine whether the selected anti-FAM19A5 monoclonal antibodies bind to unique epitopes, each antibody can be biotinylated using commercially available reagents (Pierce, Rockford, IL). Biotinylated MAb binding can be detected using a streptavidin-labeled probe. Competition studies using unlabeled and biotinylated monoclonal antibodies can be performed using FAM19A5-coated ELISA plates as described above.

[0306] To determine the isotype of the purified antibody, an isotype ELISA can be performed using reagents specific for antibodies of a particular isotype. For example, to determine the isotype of a human monoclonal antibody, the wells of a microtiter plate can be coated with 1 μg / ml anti-human immunoglobulin overnight at 4°C. After blocking with 1% BSA, the plate is reacted with 1 μg / ml or less of the test monoclonal antibody or purified isotype control at ambient temperature for one to two hours. The wells can then be reacted with a probe conjugated to alkaline phosphatase specific for human IgG1 or human IgM. The plate is developed and analyzed as described above.

[0307] As described in the examples, in order to test the binding of monoclonal antibodies to living cells expressing FAM19A5, flow cytometry can be used. In brief, cell lines expressing membrane-bound FAM19A5 (grown under standard growth conditions) were mixed with various concentrations of monoclonal antibodies in PBS containing 0.1% BSA at 4 ° C for 1 hour. After washing, cells were reacted with fluorescein-labeled anti-IgG antibodies under the same conditions as primary antibody staining. Samples can be analyzed by FACScan instruments using light and side scatter properties to gate single cells, and determine the binding of labeled antibodies. In addition to or in place of flow cytometry assays, alternative assays using fluorescence microscopy can be used. Cells can be stained exactly as described above and examined by fluorescence microscopy. This method visualizes single cells, but depending on the density of the antigen, sensitivity may be reduced.

[0308] The reactivity of anti-FAM19A5 antibodies to FAM19A5 antigens can be further tested by Western blotting. In brief, 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 probed with the monoclonal antibodies to be tested. IgG binding can be detected using anti-IgG alkaline phosphatase and developed with BCIP / NBT substrate sheets (Sigma Chem. Co., St. Louis, Missouri).

[0309] Methods for analyzing the binding affinity, cross-reactivity, and binding kinetics of various anti-FAM19A5 antibodies include standard detection methods known in the art, for example, using BIACORE TM BIACORE 2000SPR instrument (Biacore AB, Uppsala, Sweden) TM Surface plasmon resonance (SPR) analysis.

[0310] In one embodiment, the antibody specifically binds to a soluble form of human FAM19A5. In one embodiment, the antibody specifically binds to a membrane-bound form of human FAM19A5. The antibody can specifically bind to a specific epitope of FAM19A5 (e.g., a fragment of SEQ ID NO: 15 or SEQ ID NO: 15). In certain embodiments, the antibody preferably specifically binds to human FAM19A5 with high affinity and does not cross-react with other members of the FAM19 subfamily of proteins.

[0311] VIII. Bispecific Molecules

[0312] The antibodies described herein can be used to form bispecific molecules. Anti-FAM19A5 antibodies or antigen-binding portions thereof can be derivatized or connected to another functional molecule, such as another peptide or protein (e.g., another antibody or receptor ligand), to produce a bispecific molecule that binds to at least two different binding sites or target molecules. Cytokines such as IL-6, CNTF, LIF, EGF, and TGFα are considered to be the cause of gliosis and / or reactive astrogliosis (Balasingam et al., J. Neurosci. 14 (2): 846-56 (1994); Winter et al., Proc. Natl. Acad. Sci. USA 20; 92 (13): 5865-9 (1995)), which activates protein signal transduction and transcription activator 3 (STAT3), and 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 reduction of STAT3 leads to a reduction in the upregulation of glial fibrillary acid protein (GFAP), a failure of astrocyte hypertrophy, an increase in the spread of inflammation, an increase in the size of lesions, and a partial reduction in motor recovery after CNS injury. Herrmann JE et al., J. Neurosci. 28(28):7231-7243 (2008). Thus, for example, an anti-FAM19A5 antibody can be linked to an antibody or scFv for combined therapy that specifically binds to any protein involved in inhibiting the onset of glial cell proliferation and / or reactive astrogliosis, such as an antibody that binds to IL-6, CNTF, LIF, EGF, or TGFα.

[0313] Moreover, the anti-FAM19A5 antibody can be linked to an antibody or scFv that treats a disease or condition in a subject, including central nervous system injury (e.g., traumatic brain injury, cerebrospinal cord injury, stroke, or brain tumor), cerebrospinal system damage, brain degenerative disorders (e.g., Huntington's disease, Parkinson's disease, Alzheimer's disease, multiple sclerosis, ALS), cerebrospinal or neurodegenerative disorders, or neuropathic pain (see diseases or conditions in Section XII below). For example, an anti-FAM19A5 antibody can be linked to an antibody or scFv, such as natalizumab, for treating multiple sclerosis. Alemtuzumab

[0314] In fact, the antibodies described herein can be derivatized or connected to more than one other functional molecule to produce multispecific molecules that bind to more than two different binding sites and / or target molecules; such multispecific molecules are also intended to be covered by the term "bispecific molecule" as used herein. In order to produce bispecific molecules as described herein, the antibodies described herein can be functionally connected (e.g., by chemical coupling, genetic fusion, non-covalent binding or other means) to one or more other binding molecules (such as another antibody, its antibody binding portion, peptide or binding mimetic), thereby producing bispecific molecules. In one embodiment, the bispecific molecule binds to FAM19A5 and VEGF. In another embodiment, the bispecific molecule binds to FAM19A5 and EGF.

[0315] Thus, 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 embodiments where the bispecific molecules described herein are multispecific, the molecules may further comprise a third binding specificity.

[0316] In one embodiment, the bispecific molecules described herein comprise at least one antibody or antibody binding portion thereof as a binding specificity, including, for example, Fab, Fab', F(ab')2, Fv or single-chain Fv (scFv). The antibody may also be a light chain or heavy chain dimer or any minimal fragment thereof, such as an Fv or single-chain construct, as described by Ladner et al. in U.S. Pat. No. 4,946,778, the contents of which are expressly incorporated by reference.

[0317] 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.

[0318] The bispecific molecules described herein can be prepared by conjugating the component binding specificities using methods known in the art. For example, each binding specificity of the bispecific molecule can be generated separately and then conjugated to each other. When the binding specificity is a protein or peptide, a variety of coupling agents or cross-linking agents can be used for covalent binding. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetylthioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) (see, e.g., Karpovsky et al., (1984) J. Exp. Med. 160:1686; Liu, MA et al., (1985) Proc. Natl. Acad. Sci. USA 82:8648). Other methods 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, IL).

[0319] When the binding specificities are antibodies, they can be conjugated via sulfhydryl bonds in the C-terminal hinge regions of the two heavy chains. In a particularly preferred embodiment, the hinge region is modified to contain an odd number of sulfhydryl residues, preferably one, prior to conjugation.

[0320] Alternatively, both binding specificities can be encoded in the same vector and expressed and assembled in the same host cell. This approach is particularly useful when the bispecific molecule is a mAb x mAb, mAb x Fab, mAb x (scFv) 2, Fab x F(ab') 2, or ligand x Fab fusion protein. The bispecific antibody may comprise an antibody comprising an scFv at the C-terminus of each heavy chain. The bispecific molecules described herein may be single-chain molecules comprising a single-chain antibody and a binding determinant, or single-chain bispecific molecules comprising two binding determinants. The bispecific molecule may comprise at least two single-chain molecules. Methods for making bispecific molecules are described, e.g., in U.S. Pat. No. 5,260,203; U.S. Pat. No. 5,455,030; U.S. Pat. No. 4,881,175; U.S. Pat. No. 5,132,405; U.S. Pat. No. 5,091,513; U.S. Pat. No. 5,476,786; U.S. Pat. No. 5,013,653; U.S. Pat. No. 5,258,498; and U.S. Pat. No. 5,482,858.

[0321] The binding of a bispecific molecule to its specific target can be confirmed using methods recognized in the art, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS assay, bioassay (e.g., growth inhibition) or Western blot assay. Each of these assays typically detects the presence of a protein-antibody complex of particular interest by employing a labeled reagent (e.g., antibody) specific for the complex of interest.

[0322] IX. Diagnosis

[0323] In one embodiment, the moiety linked to the anti-FAM19A5 antibody is selected from the group consisting of: a binding moiety, a labeling moiety, and a biologically active moiety.

[0324] The antibodies described herein can be used for diagnostic purposes, including sample testing and in vivo imaging, and for this purpose, the antibodies (or their binding portions) can be conjugated to suitable detectable agents to form immunoconjugates. For diagnostic purposes, suitable agents are detectable labels, including radioisotopes for whole body imaging, and radioisotopes, enzymes, fluorescent labels and other suitable antibody labels for sample testing.

[0325] The detectable label may be any of a variety of types currently used in the field of in vitro diagnostics, including particulate labels, including metal sols such as colloidal gold; isotopes such as I provided by peptide chelators of the type N2S2, N3S or N4; 125 or Tc 99; chromophores, including fluorescent labels, luminescent labels, phosphorescent labels, etc.; and enzyme labels that convert a given substrate into a detectable label, and polynucleotide tags that are displayed after amplification such as by polymerase chain reaction. Suitable enzyme labels include horseradish peroxidase, alkaline phosphatase, etc. For example, the label can be the enzyme alkaline phosphatase, which is detected by measuring the presence and formation of chemiluminescence after conversion of the following substances: 1,2 dioxetane substrates such as adamantyl methoxyphosphoryloxyphenyl dioxetane (AMPPD), 3-(4-(methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo{3.3.1.1 3,7}dec}-4-yl)phenyl disodium phosphate (CSPD), and CDP and 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 method is determined by the selected label. In the case where the label is in particulate form and accumulates at an appropriate level, the appearance of the label or its reaction product can be seen by the naked eye, or can be seen using instruments such as spectrophotometers, luminometers, fluorometers, etc., all according to standard practice.

[0326] The antibodies described herein can also be conjugated to form immunoconjugates, such as antibody-drug conjugates (ADCs) with therapeutic agents. Suitable therapeutic agents include agents that regulate gliosis and / or reactive astrogliosis and / or treat brain degenerative disorders, central nervous system damage, or neuropathic pain. Therapeutic agents for treating brain degenerative disorders include drugs for treating Huntington's disease, Parkinson's disease, Alzheimer's disease, multiple sclerosis, and amyotrophic lateral sclerosis (ALS). This includes drugs that are commonly used to treat such brain degenerative disorders, such as those disclosed in Section XII below.

[0327] Immunoconjugates can be prepared by methods known in the art. Preferably, the conjugation method results in substantially (or almost) non-immunogenic bonds, such as peptide- (i.e., amide-), sulfide-, (sterically hindered), disulfide-, hydrazone- and ether bonds. These bonds are almost non-immunogenic and show reasonable stability in serum (see, e.g., Senter, PD, Curr. Opin. Chem. Biol. 13 (2009) 235-244; WO 2009 / 059278; WO 95 / 17886).

[0328] Depending on the biochemical properties of the part and the antibody, different conjugation strategies can be used. In the case where the part is naturally occurring or a recombinant of 50 to 500 amino acids, there are standard procedures for describing the chemical methods for synthesizing protein conjugates in textbooks, and technicians can easily follow the standard procedures (see, for example, Hackenberger, CPR, and Schwarzer, D., Angew. Chem. Int. Ed. Engl. 47 (2008) 10030-10074). In one embodiment, the reaction of the maleimido moiety with the cysteine ​​residue in the antibody or part is used. In the case of, for example, using the Fab or Fab' fragment of an antibody, this is a particularly suitable coupling chemical method. Alternatively, in one embodiment, coupling with the C-terminus of the antibody or part is performed. The C-terminal modification of the protein, such as the Fab fragment, can be carried out, for example, according to Sunbul, M. and Yin, J., Org. Biomol. Chem. 7 (2009) 3361-3371.

[0329] Typically, site-specific reactions and covalent couplings are based on converting natural amino acids into amino acids whose reactivity is orthogonal to the reactivity of other functional groups present. For example, specific cysteines in rare sequence contexts can be enzymatically converted in aldehydes (see Frese, MA, and Dierks, T., ChemBioChem. 10 (2009) 425-427). Desired amino acid modifications can also be obtained by exploiting the specific enzymatic reactivity of certain enzymes with natural amino acids in a given sequence context (see, for example, Taki, M. et al., Prot. Eng. Des. Sel. 17 (2004) 119-126; Gautier, A. et al., Chem. Biol. 15 (2008) 128-136; and Protease-catalyzed formation of C—N bonds is used by Bordusa, F., Highlights in Bioorganic Chemistry (2004) 389-403).

[0330] Site-specific reactions and covalent couplings can also be achieved by selective reaction of the terminal amino acid with an appropriate modifier. The reactivity of N-terminal cysteine ​​with benzonitrile (see H. et al., Angew. Chem. Int. Ed. Engl. 48 (2009) 9658-9662) can be used to achieve site-specific covalent coupling. Native chemical ligation can also rely on C-terminal cysteine ​​residues (Taylor, E. Vogel; Imperiali, B, Nucleic Acids and Molecular Biology (2009), 22 (Protein Engineering), 65-96).

[0331] EP 1 074 563 describes a conjugation method based on the faster reaction of a cysteine ​​in a stretch of negatively charged amino acids with a cysteine ​​located in a stretch of positively charged amino acids.

[0332] The moiety may also be a synthetic peptide or a peptide mimetic. In the case of chemically synthesized polypeptides, amino acids with orthogonal chemical reactivity may be incorporated during such synthesis (see, e.g., de Graaf, AJ et al., Bioconjug. Chem. 20 (2009) 1281-1295). Since a wide variety of orthogonal functional groups are at risk and can be introduced into synthetic peptides, the conjugation of such peptides to linkers is a standard chemical method.

[0333] To obtain a single labeled polypeptide, the 1:1 stoichiometric conjugate can be separated from other conjugation byproducts by chromatography. This process can be facilitated by the use of dye-labeled binding pair members and charged linkers. By using such labeled and highly negatively charged binding pair members, the single conjugated polypeptide can be easily separated from non-labeled polypeptides and polypeptides with more than one linker, since differences in charge and molecular weight can be used for separation. Fluorescent dyes can be used to purify the complex from unbound components such as labeled monovalent binders.

[0334] X. Pharmaceutical Compositions

[0335] Provided herein are compositions comprising an antibody or antigen-binding portion thereof described herein (Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA) of the desired purity in a physiologically acceptable carrier, excipient, or stabilizer. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzylchloridonium, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium metal complexes (e.g., zinc protein complexes); and / or nonionic surfactants such as or polyethylene glycol (PEG).

[0336] In a specific embodiment, the pharmaceutical composition comprises an antibody or antigen binding portion thereof, a bispecific molecule or an immunoconjugate as described herein, and optionally one or more additional prophylactic or therapeutic agents in a pharmaceutically acceptable carrier. In a specific embodiment, the pharmaceutical composition comprises an effective amount of an antibody or antigen binding portion thereof as described herein, and optionally one or more additional prophylactic or therapeutic agents in a pharmaceutically acceptable carrier. In some embodiments, the antibody is the only active ingredient included in the pharmaceutical composition. The pharmaceutical compositions described herein can be used to enhance, induce or activate FAM19A5 activity and treat conditions such as central nervous system damage, brain degenerative disorders or neuropathic pain.

[0337] The pharmaceutically acceptable carrier used in the parenteral preparation includes aqueous excipients, non-aqueous excipients, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifiers, masking agents or chelating agents and other pharmaceutically acceptable substances. Examples of aqueous excipients include sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, dextrose and lactated Ringer's injection. Non-aqueous parenteral excipients include fixed oils, cottonseed oil, corn oil, sesame oil and peanut oil of plant origin. Antibacterial agents of antibacterial or antifungal concentrations can be added to the parenteral preparations packaged in multidose containers, and the containers include phenol or cresol, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl parabens, thimerosal, benzalkonium chloride and benzethonium chloride. Isotonic agents include sodium chloride and glucose. Buffers include phosphates and citrates. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcellulose, hydroxypropyl methylcellulose and polyvinyl pyrrolidone. Emulsifiers include polysorbate 80 ( 80). Metal ion sequestering agents or chelating agents include EDTA. Pharmaceutical carriers also include ethanol, polyethylene glycol and propylene glycol for water-miscible excipients; and sodium hydroxide, hydrochloric acid, citric acid or lactic acid for adjusting pH.

[0338] The pharmaceutical composition can be formulated for any route of administration of the subject. Specific examples of routes of administration include intranasal, oral, parenteral, intrathecal, intraventricular, pulmonary, subcutaneous or intraventricular. Parenteral administration is also contemplated herein, characterized by subcutaneous, intramuscular or intravenous injection. Injections can be prepared in conventional forms, can be liquid solutions or suspensions, solid forms suitable for dissolving or suspending in liquids before injection, or emulsions. Injections, solutions and emulsions also contain one or more adjuvants. Suitable adjuvants are, for example, water, saline, dextrose, glycerol or ethanol. In addition, if desired, the pharmaceutical composition to be administered may also contain a small amount of non-toxic auxiliary substances, such as wetting or emulsifying agents, pH buffers, stabilizers, solubility enhancers and other such agents, such as, for example, sodium acetate, sorbitan laurate, triethanolamine oleate and cyclodextrins.

[0339] Preparations for parenteral administration of antibodies include sterile solutions for injection; sterile dry soluble products, such as lyophilized powders that can be combined with solvents before use, including subcutaneous injection tablets; sterile suspensions for injection; sterile dry insoluble products that can be combined with excipients before use; and sterile emulsions. The solution can be aqueous or non-aqueous.

[0340] If administered intravenously, suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents such as glucose, polyethylene glycol and polypropylene glycol and mixtures thereof.

[0341] For local and systemic administration, a topical mixture containing the antibody is prepared as described. The resulting mixture can be a solution, suspension, emulsion, etc., and can be formulated into a cream, gel, ointment, emulsion, solution, elixir, lotion, suspension, tincture, paste, foam, aerosol, rinse, spray, suppository, bandage, skin patch or any other formulation suitable for topical administration.

[0342] The antibodies or antigen-binding portions thereof described herein can be formulated for topical application, such as by inhaled aerosols (see, e.g., U.S. Pat. Nos. 4,044,126, 4,414,209, and 4,364,923, which describe aerosols for delivering steroids useful for treating inflammatory diseases, particularly asthma). These formulations for administration to the respiratory tract can be in the form of an aerosol or solution for a nebulizer, or in the form of a fine powder for insufflation, either alone or in combination with an inert carrier such as lactose. In this case, in one embodiment, the diameter of the particles of the formulation will be less than 50 microns, and in one embodiment less than 10 microns.

[0343] The antibodies or antigen-binding portions thereof described herein can be formulated for topical or external application in the form of gels, creams and lotions, such as topical application to the skin and mucous membranes (such as in the eye), and application to the eye, or application to the cisterns or spine. Topical administration is contemplated for transdermal delivery and also for ocular or mucosal administration, or for inhalation therapy. Nasal solutions of the antibodies can also be administered alone or in combination with other pharmaceutically acceptable excipients.

[0344] Transdermal patches including iontophoresis and electrophoresis devices are well known to those skilled in the art and can be used to administer antibodies. For example, such patches are disclosed in U.S. Pat. Nos. 6,267,983, 6,261,595, 6,256,533, 6,167,301, 6,024,975, 6,010715, 5,985,317, 5,983,134, 5,948,433, and 5,860,957.

[0345] In certain embodiments, the pharmaceutical composition comprising an antibody or antigen-binding portion thereof described herein is a lyophilized powder that can be reconstituted so as to be administered in the form of a solution, emulsion, and other mixtures. It can also be reconstituted and formulated as a solid or gel. The lyophilized powder is prepared by dissolving the antibody or antigen-binding portion thereof described herein or a pharmaceutically acceptable derivative thereof in a suitable solvent. In some embodiments, the lyophilized powder is sterile. The solvent may contain an adjuvant that improves the stability or other pharmacological components of the powder or the reconstituted solution prepared from the powder. The adjuvants that can be used include, but are not limited to, glucose, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose, or other suitable agents. In one embodiment, the solvent may also contain a buffer at about neutral pH, such as citrate, sodium phosphate or potassium phosphate, or other such buffers known to those skilled in the art. The solution is then sterile filtered and then freeze-dried under standard conditions known to those skilled in the art to provide the desired formulation. In one embodiment, the resulting solution is dispensed into vials for freeze-drying. Each vial will contain a single dose or multiple doses of the compound. The lyophilized powder can be stored under appropriate conditions, such as at about 4°C to room temperature.

[0346] This lyophilized powder is reconstituted using water for injection to provide a formulation for parenteral administration. For reconstitution, the lyophilized powder is added to sterile water or other suitable carrier. The exact amount depends on the compound selected. Such an amount can be determined empirically.

[0347] Antibodies or antigen-binding portions thereof, bispecific molecules or immunoconjugates described herein and other compositions provided herein can also be formulated to target specific tissues, receptors or other areas of the body of the subject to be treated. Many such targeting methods are well known to those skilled in the art. All such targeting methods for use in the compositions of the present invention are contemplated herein. For non-limiting examples of targeting methods, see, e.g., U.S. Pat. Nos. 6,316,652, 6,274,552, 6,271,359, 6,253,872, 6,139,865, 6,131,570, 6,120,751, 6,071,495, 6,060,082, 6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, 5,900,252, 5,840,674, 5,759,542, and 5,709,874. In specific embodiments, the antibodies or antigen-binding portions thereof described herein are targeted for treating central nervous system damage, brain degenerative disorders, or neuropathic pain.

[0348] Compositions for in vivo administration may be sterile. This can be easily achieved, for example, by filtration through sterile filtration membranes.

[0349] XI. Kit

[0350] Provided herein are kits comprising one or more antibodies or antigen binding portions thereof, bispecific molecules or immunoconjugates thereof as described herein. In specific embodiments, provided herein are kits or kits comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions described herein, such as one or more antibodies or antigen binding portions thereof provided herein, optionally with instructions for use. In some embodiments, the kits contain the pharmaceutical compositions described herein and any prophylactic or therapeutic agents, such as those described herein.

[0351] XII. Therapeutic Uses and Methods

[0352] Also provided herein are methods of reducing CNS injury or damage in a subject (eg, a human) in need thereof, the method comprising administering to the subject an anti-FAM19A5 antibody, bispecific molecule or immunoconjugate described herein, or a composition thereof.

[0353] In other aspects, methods for preventing, slowing, inhibiting, curbing, mitigating, reversing or preventing the onset or initiation of gliosis and its related harmful effects on the CNS of a subject are proposed herein, the method comprising administering to a subject an anti-FAM19A5 antibody disclosed herein. In some embodiments, methods for preventing, slowing, inhibiting, curbing, mitigating, reversing or preventing excessive or abnormal proliferation of reactive astrocytes and its related harmful effects on the CNS of a subject are proposed herein, the method comprising administering to a subject an anti-FAM19A5 antibody disclosed herein. In some embodiments, methods for reducing, inhibiting or reducing the expression of chondroitin sulfate proteoglycans (including neurocan, NG2 or both levels) in a subject, or reducing the activity of neurocan, NG2 or both in a subject or inactivating them are proposed herein, the method comprising administering to a subject an anti-FAM19A5 antibody described herein. In some embodiments, methods for stimulating, promoting, increasing or activating neuronal growth in a subject are proposed herein, preferably after the subject is injured or damaged, the method comprising administering to a subject an anti-FAM19A5 antibody as described herein. In other embodiments, methods for increasing c-fos mRNA, c-fos protein or c-fos protein activity levels, and increasing ERK mRNA, ERK protein or pERK activity levels in subjects in need thereof are presented herein, preferably in the nucleus of neurons, the method comprising administering to the subject an anti-FAM19A5 antibody disclosed herein. In certain embodiments, methods for enhancing or increasing the level of GAP43 mRNA, GAP43 protein, or increasing the activity of GAP43 protein in subjects in need thereof, preferably in neurons, the method comprising administering to the subject an anti-FAM19A5 antibody as disclosed herein. In some embodiments, methods for enhancing or promoting neuronal survival and / or promoting axonal regeneration in subjects in need thereof are presented herein, the method comprising administering to the subject an anti-FAM19A5 antibody disclosed herein. In some embodiments, the subject is a human, preferably a human whose neurons are damaged or impaired due to, for example, CNS damage, trauma, injury, cerebrospinal cord damage, brain tumor, infection, ischemia, stroke, autoimmune reaction and / or neurodegenerative disease.

[0354] In some aspects, the present invention also proposes a method for treating a disease, disorder or condition in a subject in need thereof, the method comprising administering to the subject an anti-FAM19A5 antibody disclosed herein. In some embodiments, the disease, disorder or condition comprises central nervous system damage, cerebrospinal system damage, brain degenerative disease, cerebrospinal or neurodegenerative disease or neuropathic pain. In some embodiments, central nervous system damage is traumatic brain injury, cerebrospinal damage, stroke, brain tumor or a combination thereof. In some embodiments, brain degenerative disease is Huntington's disease, Parkinson's disease, Alzheimer's disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS) or a combination thereof. Therefore, in certain embodiments, disclosed herein is a method for treating traumatic brain injury, cerebrospinal damage, stroke, brain tumor or a combination thereof in a subject in need thereof, the method comprising administering to the subject an anti-FAM19A5 antibody disclosed herein or a composition thereof. 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, the method comprising administering to the subject an anti-FAM19A5 antibody disclosed herein or a composition thereof. In some embodiments, the subject is a human.

[0355] In some embodiments, an anti-FAM19A5 antibody can be administered in combination with one or more additional agents for the treatment of central nervous system damage (e.g., traumatic brain injury, cerebrospinal cord damage, stroke, or brain tumor), cerebrospinal system damage, brain degenerative disorders (e.g., Huntington's disease, Parkinson's disease, Alzheimer's disease, multiple sclerosis, ALS), cerebrospinal or neurodegenerative disorders, or neuropathic pain.

[0356] In some embodiments, the disease, disorder or condition comprises a tumor, fibrosis, glaucoma or a mood disorder. In certain embodiments, the disease, disorder or condition comprises a tumor. In some embodiments, the tumor comprises melanoma, pancreatic cancer, glioma (e.g., glioblastoma multiforme (GBM)), breast cancer, lymphoma, lung cancer, kidney cancer, prostate cancer, fibrosarcoma, colon adenocarcinoma, liver cancer or ovarian cancer.

[0357] In some embodiments, the anti-FAM19A5 antibodies of the present disclosure induce normalization of blood vessels, for example, within a tumor. In some embodiments, normalization of blood vessels is accompanied by changes in vascular properties, including increased connectivity, increased wall thickness, decreased vessel diameter, more regular vessel orientation and distribution pattern, increased number of vessels, reduced leakage and permeability, increased coverage and proximity of pericytes on blood vessels, increased oxygenation, or a combination thereof.

[0358] In some embodiments, the anti-FAM19A5 antibodies of the present disclosure inhibit the growth of tumors. In some embodiments, compared to a reference (e.g., the growth of a tumor in a subject not receiving an anti-FAM19A5 antibody), the growth of the tumor is inhibited by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.

[0359] In some embodiments, anti-FAM19A5 antibodies enhance the infiltration of immune cells into tumors. In some embodiments, compared with a reference (e.g., a cancer subject who has not received an anti-FAM19A5 antibody), the infiltration of immune cells into tumors is enhanced / increased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. In certain embodiments, immune cells include macrophages, dendritic cells, T lymphocytes, B lymphocytes, natural killer (NK) cells or a combination thereof. In some embodiments, immune cells show hypertrophy. In some embodiments, the infiltration of immune cells into tumors is accompanied by an increase in the infiltration of neuronal cells into tumors. In certain embodiments, neuronal cells include astrocytes, glial cells or a combination thereof.

[0360] In some embodiments, the anti-FAM19A5 antibodies of the present disclosure enhance the phagocytic activity of macrophages or microglial cells. In some embodiments, anti-FAM19A5 antibodies increase the mitochondrial membrane potential of macrophages or microglial cells. In certain embodiments, compared with a reference (e.g., a cancer subject not receiving an anti-FAM19A5 antibody), phagocytic activity or mitochondrial membrane potential improves or increases by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.

[0361] In some embodiments, the anti-FAM19A5 antibodies of the present disclosure reduce necrosis and edema in tumors. In other embodiments, anti-FAM19A5 antibodies reduce the tissue permeability of tumors. In some embodiments, compared with a reference (e.g., a cancer subject not receiving an anti-FAM19A5 antibody), the necrosis and edema or tissue permeability of a tumor is reduced by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.

[0362] In some embodiments, anti-FAM19A5 antibodies increase blood flow velocity in tumors. In certain embodiments, blood flow velocity is increased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% compared to a reference (e.g., a cancer subject not receiving an anti-FAM19A5 antibody).

[0363] In some embodiments, the method for treating a tumor includes administering an additional therapeutic agent. In certain embodiments, the additional therapeutic agent includes chemotherapy, immunotherapy, radiotherapy, or a combination thereof. In some embodiments, immunotherapy includes monoclonal antibodies, chimeric antigen receptor (CAR) therapy, T cell therapy, NK cell therapy, dendritic cell (DC) therapy, adoptive cell transfer (ACT), immune checkpoint regulators, cytokines, cancer vaccines, adjuvants, oncolytic viruses, or a combination thereof. In some embodiments, chemotherapy includes temozolomide, gemcitabine, paclitaxel, carboplatin, cisplatin, elotuzumab, lenalidomide, dexamethasone, oxaliplatin, or a combination thereof.

[0364] In some embodiments, a therapeutically effective amount of an anti-FAM19A5 antibody of the present disclosure or a composition thereof is administered. When treating a subject (eg, a human), the therapeutically effective amount of an anti-FAM19A5 antibody disclosed herein depends on factors such as age, sex, severity of the disease, and the like.

[0365] In some embodiments, an anti-FAM19A5 antibody of the present disclosure, or a composition thereof, is administered intravenously, orally, parenterally, transthecally, intrathecally, intracerebroventricularly, intrapulmonaryly, subcutaneously, intradermally, intramuscularly, or intraventricularly.

[0366] The following examples are intended to be illustrative rather than limiting.

[0367] Examples

[0368] Example 1: Expression and purification of human FAM19A5 protein

[0369] Recombinant human FAM19A5 protein was produced and purified as described below, and the purified protein was used in antibody screening assays based on binding affinity analysis. First, the LPS-hT plasmid expressing the FAM19A5 gene was converted into bacteria, and the overexpression of the protein was induced. After production, the FAM19A5 protein was purified using Ni-NTA affinity chromatography (Qiagen, Valencia, California, USA). The FAM19A5 protein with His tag was removed from the nickel column using imidazoles that gradually increased the concentration. Protein expression in the solution was measured using Coomassie Brilliant Blue R-250 dye. Only a solution containing FAM19A5 imidazole was used to concentrate the FAM19A5 protein using PBS. When the concentration was complete, the purity and concentration of the FAM19A5 protein were measured using Western blotting assay. Subsequently, the concentrated protein was used to screen for FAM19A5 specific antibodies.

[0370] Example 2: Generation of antibody library FAM19A5

[0371] 1. Immunization

[0372] FAM19A5 protein was used as an antigen for immunizing chickens. 50 μg of synthetic peptide KLH conjugate was mixed in 750 μl of phosphate buffered saline (PBS) and incubated at 37 ° C for 30 minutes. Afterwards, the toxin was removed in 2% squalene endotoxin MPL (monophosphorylated lipid A species), and the mycobacteria (mycobacteria) containing the cell wall components of TDW and CWS of oil-in-water emulsified adjuvant (RIBI+MPL+TDM+CWS adjuvant, Sigma, St. Louis, Missouri, USA) were emulsified and then subcutaneously injected into three chickens or four rabbits. Chickens and rabbits were immunized three times and four times respectively, with an interval of about 2 to 3 weeks between each immunization. The titer of the antibodies obtained from the immunized animals was determined by immunoblotting using the lysate of HEK293T cells overexpressing FAM19A5 protein.

[0373] 2. Preparation of single-chain variable fragment (scFv) libraries from immunized chickens and rabbits

[0374] RNA was extracted from the spleen, bone marrow, and synovial bursa of the immunized chickens described above using TRI reagent (Invitrogen, Carlsbad, CA, USA). TM The first strand cDNA was synthesized using the PCR amplification system (Invitrogen) of the PCR amplification kit. For cDNA obtained from the animal immune system, the single-stranded variable region library was generated using the Expand HighFidelity PCR System (Roche Molecular Systems, Indiana, USA). In each reaction, 1 μL cDNA, 60 pmol of each primer, 10 μL 10× reaction buffer, 8 μL 2.5 mM dNTP (Promega, Madison, Wisconsin, USA) and 0.5 μL Taq DNA polymerase were mixed with water. The final volume was 100 μL, and the PCR reaction was performed using the following conditions: (i) 15 seconds at 94°C, (ii) 30 seconds at 56°C and (iii) 90 seconds at 72°C for 30 cycles, and finally extended at 72°C for 10 minutes. The PCR product containing a fragment of about 350 bp in length was loaded onto a 1.5% agarose gel, and after electrophoresis, the nucleotide fragment was purified using a QIAGEN Gel II extraction kit (QIAGEN, Valencia, CA, USA). The purified PCR product was quantified by reading at OD 260 nm. (1 unit OD = 50 μg / ml).

[0375] The two VH and VL first products from the second PCR are randomly connected by overlap extension PCR (overlap extension PCR). Each PCR reaction is mixed with 100ng purified VL and VH products, 60pmol of each primer, 10μL 10× reaction buffer solution, 8μL 2.5mM dNTP, 0.5μL Taq DNA polymerase and water, and the final volume is 100μL. PCR is carried out under the following conditions: (i) 15 seconds at 94°C, (ii) 30 seconds at 56°C and (iii) 25 cycles at 72°C for 2 minutes, and finally extended at 72°C for 10 minutes. The PCR product containing the single-chain variable region fragment of about 700bp in length is loaded onto 1.5% agarose gel, and after electrophoresis, the nucleotide fragment is purified using QIAGEN II gel extraction kit (QIAGEN). The PCR product of quantitative purification is read at OD 260nm. (1 unit OD=50 / ml).

[0376] 3. Library, ligation and transformation

[0377] The scFv fragment of the PCR product and the vector pComb3X-SS (The Scripps Research Institute, California, USA) were digested with SfI I restriction enzyme. 10 μg of the purified overlapping PCT product was mixed with 360 units of Sif I (per 16 units μg of DNA, Roche Molecular Systems, Pleasanton, California, USA), 20 μL of 10× reaction buffer and water in a final volume of 200 μL. 20 μg of pComb3X-SS vector was mixed with 120 units of Sfi I (per 6 units μg of DNA), 20 μL of 10× reaction buffer and water in a final volume of 200 μL. The mixture was digested at 50°C for 8 hours. Afterwards, the digestion product containing the scFv fragment (about 700 bp) and the vector (about 3400 bp) was loaded on a 1% agarose gel and purified using a gel extraction kit IIQIAGEN (QIAGEN, Valencia, California, USA). 1400 ng of Sfi I restricted pComb3X vector and 700 ng of digested scFv fragment were mixed with 5× ligase buffer, 10 μL 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.

[0378] After ethanol precipitation, the DNA precipitate was dissolved in 15 μL water. In order to produce the library, the ligation sample was transformed into the Escherichia coli strain ER2738 (New England Biolabs Inc., Hitchin, Hertfordshine, SG4 OTY, England, UK) via electroporation using a vibrator gene (Gene Pulser: Bio-Rad Laboratories, Hercules, California, USA). The cells were mixed in 5ml Super Broth (SB) culture medium and incubated for one hour at 37°C with 250rpm stirring. Then, 3 μL 100mg / mL kanamycin was added to 10mL SB culture medium. In order to determine the library size, 0.1 μL, 1 μL and 10 μL of culture samples were applied to Luria Broth (LB) agar plates containing 50 μg / ml kanamycin. After stirring for 1 hour, 4.5 μL 100 mg / mL kanamycin was added to the LB culture and stirred for another 1 hour. Then, 2 ml of VCM13 helper phage (>10 11 cfu / ml) was added to the LB culture medium with 92.5 μL 100mg / mL kanamycin (183mL) of preheating. This mixture was stirred for another 2 hours at 37°C with 250rpm. Then, 280 μL (50mg / mL) kanamycin was added to the culture, and stirred overnight at 37°C. The next day, a high-speed centrifuge (Beckman, JA-10 rotor) was used to centrifuge bacterial precipitation at 4°C with 3,000g. Afterwards, bacterial precipitation was used to extract phagemid DNA, and the supernatant was transferred to a sterile centrifuge bottle at the same time. Then, 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 in ice for 30 minutes. Afterwards, the supernatant was centrifuged for 15 minutes at 4°C with 15,000g. Then, the supernatant was discarded and the phage pellet was resuspended in Tris-buffered saline (TBS) containing 1% BSA.

[0379] Example 3: Library panning of immobilized antigens (biopanning)

[0380] Biopanning was performed using magnetic beads (Dynabeads M-270 Epoxy, Invitrogen). Approximately 1×10 7The beads were washed with phosphate buffered saline (PBS) for 20 hours. Once the coating was completed, the beads were washed 4 times with phosphate buffered saline (PBS) and blocked for one hour at room temperature in PBS containing 3% BSA. Then, the coated beads were incubated at room temperature for two hours with the scFv displayed by the phages described above. In order to remove any phages that were not bound to the antigen-coated beads, the beads were washed with 0.05% Tween20 / PBS. Then, the bound phages were eluted with 50 μL 0.1M glycine / hydrogen chloride (0.1M glycine-HCl, pH 2.2) and neutralized with 3 μL 2M Tris and hydrogen chloride (tris-HCl, pH 9.1). This phage-containing supernatant was used to infect E. coli ER2738 cells, and VCSM13 was used to assist phage amplification and rescue overnight. In addition, by blotting the culture infected by phage on the LB agar plate containing 50 μ g / ml kanamycin, the input (input) and output (output) of the phage titer from the culture infected by phage are determined. The next day, PEG-8000 and NaCl are used to precipitate the phage, which is then used for bio-panning. By repeating the above process, a total of five bio-pannings are carried out. Each amplification is for the high affinity to the FAM19A5 protein, screening and selecting phage.

[0381] Example 4: Clone selection by phage ELISA

[0382] To analyze the clones selected from the bio-panning, individual clones were randomly selected from the scFv displayed by phage, and ELISA was used to confirm that the clones were bound to the FAM19A5 recombinant protein. The FAM19A5 recombinant protein was diluted in 0.1M NaHCO3 buffer and coated with 100ng / well of protein at 4°C for 16 hours in a 96-well microtiter plate. The next day, the plate was blocked with 3% BSA / PBS at 37°C for 1 hour. Then, the phage supernatant was mixed with 6% BSA / PBS and cultured at 37°C for 2 hours. Then, the plate containing the supernatant was washed with 0.05% Tween-20 / PBS. The HRP-conjugated M13 antibody (a-M13-HRP, Pierce Chemical Co, Rockford, Illinois, USA) was diluted to 1 / 5000. 50μl of the diluted antibody was added to the plate and incubated at 37°C for 1 hour. After incubation and washing, 0.05 M citrate buffer, 1 μg / ml 2,2'-azidobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS, Amresco, Solon, Ohio, USA) and 0.1% H2O2 were added to the plate for color development. The absorbance of each well was measured at 405 nm.

[0383] Analyzed 24 clones produced by the chicken of immunity that is combined with FAM19A5 recombinant protein and demonstrates high absorbance.From these 24 clones, 13 scFv clones with unique sequences were obtained.For the clone (data not shown) produced from the rabbit of immunity, 174 clones were initially identified, wherein 164 clones were sequenced.From these clones, 22 final unique ScFv sequences were obtained.

[0384] Example 5: Generation of deimmunized anti-FAM19A5 antibodies

[0385] 1. Deimmunization of anti-FAM19A5 antibody (clone 1-65)

[0386] To reduce the risk of immunogenicity when administered to human subjects, in silico analysis (EpiScreen Immunogenicity Analysis, Antitope Ltd., UK) was performed to identify specific regions within the anti-FAM19A5 antibody (e.g., 1-65) that were highly immunogenic. To identify promiscuous MHC class II binding peptides, iTope was performed on overlapping 9-mer peptides spanning the entire sequence. TM (Abzena plc., UK). Potential T cell epitopes were predicted by analysis of interactions with 34 different MHC class II alleles. Clone 1-65 contained a total of 13 non-germline promiscuous MHC class II binding peptides ( Figure 2 ). Figure 1A and 1B Sequence alignments of the heavy chain variable region and light chain variable region of the 1-65 antibody and SS01-13 (ie, the deimmunized 1-65 antibody) are provided, respectively.

[0387] In addition, through TCED TM (Abzena plc., UK) analyzed promiscuous MHC class II binding peptides, a CD4+ T cell epitope database constructed by T cell stimulation assays against more than 10,000 peptides. Clone 1-65 showed that four promiscuous MHC class II binding peptides had high homology to known T cell epitopes ( Figure 2 ).

[0388] 2. Construction of composite antibody library

[0389] Design composite antibody library to avoid CD4+T cell epitopes associated with immunogenicity. In order to select the most homologous human germline for library construction, the framework of clone 1-65 was analyzed to the IgBLAST database (NCBI). Human germline IGLV3-25*02, IGLJ2*01, IGHV3-23*02 and IGHJ1*01 genes (Fig. 1) were selected for clone 1-65. The library was constructed to replace some non-identical amino acid residues of clone 1-65 MHC class II binding region with the corresponding amino acids in the most homologous human germline sequence. In particular, p1, p4, p6, p7 and p9 residues in the peptide are essential for interaction with the binding minor groove of MHC class II alleles (James EA, Moustakas AK, Bui J, Nouv R, Papadopoulos GK, Kwok WW. J Immunol. 2009; 183 (5): 3249-58).

[0390] Sequence overlap extension polymerase chain reaction (PCR) of oligonucleotides encoding degenerate codons to cover both human and chicken amino acids was performed to generate a site-directed mutagenesis library as previously described (Baek DS, Kim YS. Biochem Biophys Res Commun. 2015; 463(3): 414-20). PCR products were subcloned into phagemid vectors and transformed into Escherichia coli ER2738 strain (New England BioLabs, Ipswich, MA, USA) for phage preparation as previously described (Han J, Lee JH, Park S, Yoon S, Yoon A, Hwang DB, et al., Exp Mol Med. 2016; 48(11): e271).

[0391] 3. Biopanning and clone selection

[0392] To isolate positive clones, biopanning and phage enzyme immunoassay were performed as previously described (Barbas CF. Phage display: a laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 2001). Phage clones reactive to FAM19A5 were selected and their nucleotide sequences were determined by Sanger sequencing. Finally, scFv clones with minimal promiscuous MHC class II binding epitopes that retained affinity for FAM19A5 were selected.

[0393] A total of 6 promiscuous MHC class II binding epitopes in clone 1-65 were removed in the deimmunized clone 1-65 (i.e., SS01-13-S5 antibody) Figure 3A ). It is not possible to remove the promiscuous MHC class II binding epitopes in CDR-H1, CDR-H2 and CDR-H3 (i.e. Figure 3A Furthermore, since the p6 residue in binding protein #4 and the p4 residue in binding protein #5 located in HFR1 play a key role in the binding activity, the promiscuous MHC class II binding epitopes in HFR1 cannot be removed. Figure 3B A comparison of the binding of the deimmunized clone 1-65 antibody to the wild-type 1-65 antibody to the FAM19A5 protein is provided.

[0394] Example 6: Identification of potential N-glycosylation sites in CDR-H2 of deimmunized anti-FAM19A5 antibody (clone 1-65)

[0395] Point Removal

[0396] In order to construct a site-directed mutagenesis library, the gene encoding clone 1-65scFv was used as a template for PCR. S53 in CDR-H2 was randomly assigned with oligonucleotides encoding NNK degenerate codons (N=A, T, G or C, K=G or T), as previously described (Lee HK, Jin J, Kim SI, Kang MJ, Yi EC, Kim JE, et al., Biochem Biophys Res Commun. 2017; 493 (1): 325-31). As described above, the scFv fragment amplified by overlap extension PCR was subcloned into a phagemid vector and transformed into ER2738 (New England BioLabs) for phage preparation.

[0397] From the transformed titration plates, randomly selected mutant scFvs displaying phage were rescued and subjected to phage enzyme immunoassay as previously described (Barbas CF. Phage display: a laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 2001). Phage clones displaying similar reactivity to FAM19A5 were selected and their nucleotide sequences were determined by sanger sequencing. S53 can be replaced by threonine, glycine and leucine. These results indicate that potential N-glycosylation sites in CDR-H2 of clone 1-65 can be removed by replacing serine with leucine or glycine.

[0398] Using oligonucleotides, a deimmunized clone 1-65 (ie, SS01-13-S5 antibody) in which serine (S53G) (ie, S5-SG antibody) was replaced with glycine was constructed by overlap extension PCR ( Figure 4C ). After subcloning into a phagemid vector, the S5-SG antibody displaying phage was rescued and subjected to phage enzyme immunoassay as described above. Figure 4A and 4B The expression level and size of the antibody are provided in the. S5-SG antibody ("deimmunized clone 1-65-S53G") exhibited a lower affinity for FAM19A5 protein than deimmunized clone 1-65 (ie, SS01-13-S5 antibody) ( Figure 4D ).

[0399] Example 7: Binding affinity analysis

[0400] To further evaluate the binding affinity of the various anti-FAM19A5 antibodies disclosed herein to the FAM19A5 protein, both SPR and ELISA assays were used.

[0401] For SPR assay, the following protocol was used. FAM19A5 protein was diluted (2 μg / mL) in a fixed buffer (10 mM sodium acetate, pH 4.5). The CM5 sensor chip was activated for 420 seconds with a mixture of ethyl (dimethylaminopropyl) carbodiimide (EDC) (400 mM) and NHS (100 mM). Then, 2 μg / mL FAM19A5 protein (in a fixed buffer, pH 4.5) was injected into the Fc2 sample channel for 420 seconds at a flow rate of 10 μL / min to reach a level of about 50RU. Afterwards, the chip was deactivated for 420 seconds at a flow rate of 10 μL / min with 1 M ethanolamine hydrochloride. A reference surface Fc1 channel was also prepared, and similar activation and deactivation were performed. Anti-FAM19A5 antibodies were diluted to different concentrations (90nM, 30nM, 10nM, 3.3nM, 1.1nM, 0nM) in running buffer (HBS-EP; 1×HEPES with 0.005% Tween-20, pH 7.4). The diluted anti-FAM19A5 antibodies were injected into the Fc1-Fc2 channel at a flow rate of 30 μL / min for a 60-second association phase. Subsequently, a 300-second dissociation phase was performed via a single-cycle kinetic mode. After each analysis cycle, the sensor chip surface was regenerated for 60 seconds at a flow rate of 30 μL / min with 10mM glycine HCl (pH 1.5). The data were analyzed using a 1:1 fit using BIAevaluation software.

[0402] For ELISA analysis, the following protocol was used. Anti-rabbit Fc antibody (diluted to a concentration of 1 μg / mL in 50 mM carbonate buffer, pH 9.6) was used to coat 96-well plates (100 μL / well) overnight at 4°C. Next, the plates were washed with wash buffer (PBST: 0.05% Tween-20 / PBS) for a total of five times. Then, the plates were blocked with blocking buffer (1% BSA / PBST) (150 μL / well) for 1 hour at 37°C and washed again with wash buffer. Then, 50 ng / mL FAM19A5-rabbit Fc (also referred to as FAM19A5-rFc) antigen diluted in blocking buffer was added to each of the wells. The plates were incubated for another hour at 37°C. After incubation, the plates were washed again with wash buffer for a total of five times. The anti-FAM19A5 antibody of different concentrations (2055pM, 685pM, 228.3pM, 76.1pM, 25.4pM, 8.5pM, 2.8pM, 0pM) of dilution in blocking buffer was added to each of the wells, and the plate was incubated at 37°C for 2 hours. After incubation, the plate was washed (washed five times in total in washing buffer), and then the diluted anti-human κ light chain HRP (1:10000 in blocking buffer) was added to the wells (100 μL / well). The plate was incubated at 37°C for 1 hour. Then, the plate was washed again and treated with TMB solution (100 μL / well). Matrix. This reaction was terminated using 100 μL sulfuric acid (2N H2SO4), and the degree of color change was detected by absorption at 450nm using a 96-well microplate reader (Molecular Device).

[0403] The results of the measurement are Figures 5A to 5C and 6A to 6D. Figures 5A to 5C As shown in Figure 2, the SPR results showed that among the three anti-FAM19A5 antibodies tested, the 1-65 antibody bound to the FAM19A5 protein with the greatest binding affinity. D was 0.345 nM, while the K D The results were 2.201 nM and 1.797 nM respectively. Similar results were observed using ELISA assay, as shown in FIG6 .

[0404] Example 8: Evaluation of the effect of anti-FAM19A5 antibodies on the phagocytic ability of immune cells

[0405] In order to evaluate whether the anti-FAM19A5 antibodies disclosed herein have an effect on the phagocytic capacity of certain immune cells, BV cells (an immortalized mouse microglial cell line) were used. Briefly, BV2 cells were seeded into 96-well plates (5×10 3 / 100 μL / well) and incubated at 37°C in 5% CO2 for 6 hours. Then, cells were treated with human FAM19A5-Fc protein (0.25 μM, batch #170815) alone or in combination with anti-FAM19A5 antibodies 1-65, SS01-13-S5 or S5-SG at different concentrations (3.125 μg / mL, 6.25 μg / mL, 12.5 μg / mL or 25 μg / mL). Cells from the control group were treated with PBS alone. The treated cells were incubated for another 16 hours at 37°C in 5% CO2. After incubation, PHRODO TM Green E. coli bioparticles (Thermofisher, P35366) were added to the cells (15 μg / 100 μL / well). (Sartorius), and the phagocytic uptake of bioparticles was observed by measuring the green fluorescence intensity every 1 hour.

[0406] like Figures 7A to 7C As shown, treatment of BV2 cells with human FAM19A5-Fc protein inhibited the phagocytic ability of BV2 cells. However, treatment of BV2 cells with a combination of human FAM19A5-Fc protein and any of the tested anti-FAM19A5 antibodies restored phagocytosis in a concentration-dependent manner. Compared to the control group (i.e., PBS alone), the amount of phagocytosis observed with antibodies 1-65, SS01-13-S5, and S5-SG was as follows: 86%, 96%, and 89% approximately 10 hours after treatment with bioparticles. These results indicate that, like the 1-65 antibody, deimmunized SS01-13-S5 and S5-SG are able to effectively block FAM19A5-Fc protein-mediated inhibition of BV2 cells.

[0407] Example 9: Use of anti-FAM19A5 antibodies in brain injury animal models

[0408] In order to evaluate the in vivo function of the anti-FAM19A5 antibodies disclosed herein, a traumatic brain injury (TBI) mouse model will be used. Briefly, different anti-FAM19A5 antibodies are administered to mice. Then, the animals are sacrificed after TBI induction (TBI5D). The effects of anti-FAM19A5 antibodies, such as the onset of reactive gliosis in the penumbra after traumatic brain injury, will be measured.

[0409] Example 10: Anti-cancer effect of anti-FAM19A5 antibody in an inducible mouse melanoma model

[0410] In order to evaluate the anti-cancer effect of the anti-FAM19A5 antibody disclosed herein, an inducible mouse melanoma model will be used. In brief, melanoma will be induced by hybridizing male Tyr::CreER, Braf+ / +, Ptenlox / lox mice (Jackson Lab, USA) with female Tyr::CreER, BrafCA / CA, Ptenlox / lox to produce Tyr::CreER, BrafCA / +, Ptenlox / lox mice. See Dankort D. et al., Nat Genet 41(5):544-52(2009). Melanoma will begin to be spontaneously induced about 7 weeks after birth. Mice will be classified according to their tumor volume and the number of melanomas.

[0411] After separation, animals will receive anti-FAM19A5 antibody or human IgG control antibody. Melanoma volume analysis will then be performed in animals at various time points after administration.

[0412] Example 11: Use of anti-FAM19A5 antibodies in animal models of neuropathic pain

[0413] In order to evaluate the in vivo function of the anti-FAM19A5 antibodies disclosed herein, a mouse model of chronic constriction injury (CCI) will be used. In brief, different anti-FAM19A5 antibodies are administered to mice. Then, about 1 week after administration, peripheral nerve injury will be induced in the animal by ligation of the scrotal nerve. At various time points after injury, the animals' mechanical tactile allodynia (response to external body stimuli) and thermal hyperalgesia (response to increased temperature) will be assessed. Mechanical tactile allodynia will be assessed by applying Von Frey monofilament (0.16 g) to the injured foot multiple times and observing the frequency of the animal's response to pain. Thermal hyperalgesia will be assessed using the Hargreaves test, in which a radial heat stimulus (intensity: 30) is applied to the injured foot and the paw withdrawal latency is determined.

[0414] It should be understood that the "Detailed Description" section is intended to be used to interpret the claims, rather than the "Summary" and "Abstract" sections. The "Summary" and "Abstract" sections may set forth one or more but not all exemplary embodiments of the present disclosure contemplated by the inventors, and therefore, are not intended to limit the present disclosure and the appended claims in any way.

[0415] The present disclosure has been described above with the aid of functional building blocks that illustrate the implementation of specific functions and their relationships. For ease of description, the boundaries of these functional building blocks have been arbitrarily defined in this article. Alternative boundaries may also be defined as long as the specific functions and their relationships are appropriately performed.

[0416] The foregoing description of specific embodiments will reveal the general nature of the present disclosure very fully, so that others can easily modify and / or adapt various applications of such specific embodiments without undue experimentation by applying knowledge within the technical scope of the art, without departing from the general concepts of the present disclosure. Therefore, based on the teachings and guidance given herein, such modifications and adaptations are intended to be within the meaning and range of equivalents of the disclosed embodiments. It should be understood that the words or terms in this article are for the purpose of illustration and not limitation, so that the terms or words of this specification will be interpreted by the technician according to the teachings and guidance.

[0417] The breadth and scope of the present disclosure should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

[0418] All publications, patents, patent applications, internet sites, and accession numbers / database sequences (including both polynucleotide and polypeptide sequences) cited herein are incorporated by reference in their entirety for all purposes as if each individual publication, patent, patent application, internet site, or accession number / database sequence was specifically and individually indicated to be incorporated by reference.

[0419] This PCT application claims priority to U.S. Provisional Application Nos. 62 / 669,648, filed on May 10, 2018, and 62 / 838,187, filed on April 24, 2019, each of which is incorporated herein by reference in its entirety.

Claims

1. An isolated antibody or antigen-binding portion thereof that specifically binds to a human family of sequence similarity 19, member A5 (FAM19A5) protein ("anti-FAM19A5 antibody"), the isolated antibody or antigen-binding portion thereof comprising 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 of SEQ ID NOs: 5, 6 and 7, respectively, each of which optionally comprises one, two or three mutations, wherein the light chain CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NOs: 8, 9 and 10, respectively, wherein at least one of the light chain CDR1, CDR2 and CDR3 comprises one, two or three mutations, and wherein the antibody has reduced immunogenicity in humans compared to a reference antibody comprising a VH as set forth in SEQ ID NO: 11 and a VL as set forth in SEQ ID NO:

12.

2. The antibody according to claim 1, wherein the heavy chain CDR3 comprises the amino acid sequence shown in SEQ ID NO:7 (GSASYITAATIDA).

3. The antibody according to claim 1 or 2, wherein the heavy chain CDR1 comprises the amino acid sequence shown in SEQ ID NO:5 (SYQMG), and the amino acid sequence optionally has one, two or three mutations.

4. The antibody according to any one of claims 1 to 3, wherein the heavy chain CDR2 comprises the amino acid sequence shown in SEQ ID NO: 6 (VINKSGSDTS), which optionally has one, two or three mutations.

5. The antibody of claim 4, wherein the mutation comprises substitution of valine at amino acid 1 of SEQ ID NO: 6 with an aliphatic amino acid.

6. The antibody according to claim 4 or 5, wherein the mutation comprises substitution of the serine at amino acid 5 of SEQ ID NO: 6 with an aliphatic amino acid.

7. The antibody according to claim 5 or 6, wherein the aliphatic amino acid comprises alanine or glycine.

8. The antibody according to any one of claims 1 to 7, wherein the light chain CDR3 comprises the amino acid sequence shown in SEQ ID NO: 10 (GNDDYSSDSGYVGV), which optionally has one, two or three mutations.

9. The antibody according to any one of claims 1 to 8, wherein the light chain CDR1 comprises the amino acid sequence shown in SEQ ID NO: 8 (SGGGSSGYGYG), and the amino acid sequence has one, two or three mutations.

10. The antibody of claim 9, wherein the mutation comprises substitution of glycine at amino acid 4 of SEQ ID NO: 8 with an aliphatic amino acid.

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