Uses of anti-FAM19A1 antagonists for treating central nervous system disorders

Abstract

To provide a composition for treating a disease or disorder associated with an abnormality in CNS function.SOLUTION: The present invention provides a pharmaceutical composition that comprises an anti-FAM19A1 antibody or an antigen binding fragment thereof ("anti-FAM19A1 antibody"), a polynucleotide encoding the anti-FAM19A1 antibody, a vector including the polynucleotide, a cell including the polynucleotide, or any combination of these.SELECTED DRAWING: Figure 6

Description

Detailed description of the invention

[0001] [Technical Field] (Cross-references to related applications) This PCT application claims priority to U.S. Extraordinary Application No. 62 / 984,166, filed on 2 March 2020, which is included herein by reference in its entirety.

[0002] (References for electronically submitted sequence lists) The contents of the sequence listing, electronically submitted with this application as an ASCII text file (name: 3763.017PC01_SeqListing_ST25.txt, size: 32,234 bytes; generated: March 1, 2021), are included in their entirety herein for reference.

[0003] (Statement of government support) This research was supported by the Industry-Academia-Research Collaboration Technology Development Project, funded by the Ministry of Small and Medium Enterprises and Startups of the Republic of Korea in 2017.

[0004] This disclosure provides a family having sequence similarity 19, an antagonist (e.g., an antibody) that specifically binds to member A1 (FAM19A1), a composition comprising such an antagonist, and a method of using such an antagonist to prevent and / or treat central nervous system diseases and / or disorders.

[0005] [Background technology] Diseases and disorders of the central nervous system (CNS) generally include a heterogeneous group of disorders whose causes and etiologies are unknown. Currently, there are no treatments for CNS diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), traumatic brain injury (TBI), neuropathic pain, and glaucoma. In fact, in most cases, available treatments for CNS offer relatively little symptom relief, effectively only temporary relief. Furthermore, with increasing life expectancy and population growth worldwide, the number of individuals suffering from CNS diseases and disorders is expected to increase further (Feigin, VL, et al., Lancet Neurol 16(11):877-897 (2017)). [Summary of the Invention] [Problems the invention aims to solve] Thus, there is still a need for more effective treatment options for CNS-related diseases and disorders.

[0006] [Means for solving the problem] This specification provides therapeutic antagonists that specifically bind to a family having sequence similarity 19, member A1 (FAM19A1) ("FAM19A1 antagonist"). In some embodiments, the FAM19A1 antagonist can treat diseases or disorders in the target population.

[0007] In some forms, the disease or disorder includes central nervous system (CNS) related diseases or disorders. In some forms, the CNS related diseases or disorders are associated with abnormal neural circuits. In some forms, the CNS related diseases or disorders include mood disorders, psychiatric disorders, or all of these. In certain ways, the CNS-related disorders or conditions include anxiety, depression, post-traumatic stress disorder (PTSD), bipolar disorder, attention deficit / hyperactivity disorder (ADHD), autism, schizophrenia, neuropathic pain, glaucoma, intoxication, arachnoid cyst, sclerosis, encephalitis, epilepsy / seizures, fixed syndrome, meningitis, migraine, multiple sclerosis, osteomyelopathy, Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Batten's disease, Tourette's syndrome, traumatic brain injury, spinal cord injury, stroke, tremor (essential or Parkinsonian), dysarthria, intellectual disability, brain tumors, or combinations thereof.

[0008] In some embodiments, the CNS-related disorder or condition is anxiety, depression, PTSD, or a combination thereof. In some embodiments, the FAM19A1 antagonist can improve one or more symptoms associated with anxiety and / or depression (e.g., it can increase the subject's motor activity and / or increase the subject's ability to respond to external stress).

[0009] In certain embodiments, the CNS-related disease or disorder treatable by the present disclosure is glaucoma. In certain embodiments, the FAM19A1 antagonist can reduce, alleviate, or inhibit inflammation associated with glaucoma. In certain embodiments, the FAM19A1 antagonist can improve the electroretinomyelitis in the retina. In certain embodiments, the glaucoma is selected from the group consisting of open-angle glaucoma, closed-angle glaucoma, normal-tension glaucoma ("NTG"), congenital glaucoma, secondary glaucoma, pigmentary glaucoma, pseudoexfoliation glaucoma, traumatic glaucoma, neovascular glaucoma, iris-corneal endothelial syndrome, uveitis glaucoma, and combinations thereof. In some manifestations, the glaucoma is associated with optic nerve damage, retinal ganglion cell (RGC) loss, elevated intraocular pressure (IOP), damaged blood-retinal barrier, and / or increased microglial activity levels within the retina and / or optic nerve of the subject. In some manifestations, the glaucoma is induced by mechanical damage to the optic disc and / or increased inflammatory levels within the retina and / or optic nerve of the subject.

[0010] In certain embodiments, the FAM19A1 antagonist can delay the onset of retinal neuronal degeneration in a subject. In certain embodiments, the FAM19A1 antagonist can reduce retinal ganglion cell loss and / or restore the number of retinal ganglion cells in the subject's retina. In certain embodiments, the retinal ganglion cell loss is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% compared to a baseline (e.g., the relevant value in a subject not receiving the FAM19A1 antagonist or the relevant value in a subject before administration of the FAM19A1 antagonist). In some embodiments, the number of retinal ganglion cells is restored to at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% compared to a baseline (e.g., the relevant value for a subject not receiving the FAM19A1 antagonist or the relevant value for a subject before administration of the FAM19A1 antagonist). In some embodiments, the FAM19A1 antagonist can protect the neural connections of the inner plexiform layer of the subject's retina.

[0011] In some cases, the CNS-related disorders or conditions that can be treated with the FAM19A1 antagonists disclosed herein are neuropathic pain.

[0012] In some embodiments, the FAM19A1 antagonist can increase the threshold or latency to external stimuli in subjects requiring it. In certain embodiments, the external stimulus is a mechanical stimulus. In some embodiments, the external stimulus is a thermal stimulus. In some embodiments, the threshold or latency to the external stimulus increases by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% compared to a baseline (e.g., the relevant value in subjects not receiving the FAM19A1 antagonist or the relevant value in subjects before administration of the FAM19A1 antagonist).

[0013] In certain embodiments, the FAM19A1 antagonist can increase or modulate sensory nerve conduction velocity in subjects where it is needed. In specific embodiments, the sensory nerve conduction velocity increases by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% or more compared to a baseline (e.g., the relevant value in a subject not receiving the FAM19A1 antagonist or the relevant value in a subject before administration of the FAM19A1 antagonist).

[0014] In some cases, the neuropathic pain is central neuropathic pain or peripheral neuropathic pain. In some cases, the neuropathic pain is associated with bodily injury, infection, diabetes, cancer treatment, alcoholism, amputation, muscle weakness of the back, foot, buttocks or face, trigeminal neuralgia, multiple sclerosis, herpes zoster, spinal surgery, or any combination thereof. In some cases, the neuropathic pain includes carpal tunnel syndrome, central pain syndrome, degenerative disc disease, diabetic neuropathy, phantom limb pain, postherpetic neuralgia (herpes zoster), pudendal neuralgia, sciatica, lower back pain, trigeminal neuralgia, or any combination thereof. In certain cases, the neuropathic pain is induced by nerve compression. In some cases, the diabetic neuropathy is diabetic peripheral neuropathy. In some cases, the neuropathic pain is sciatica.

[0015] In certain embodiments, the FAM19A1 antagonist can modulate or improve central nervous system function in the target population. In certain embodiments, the central nervous system function includes limbic system-related function, olfactory system-related function, sensory system-related function, visual system-related function, or a combination thereof.

[0016] In certain embodiments, the FAM19A1 antagonist can reduce the expression levels of FAM19A1 protein and / or FAM19A1 mRNA in brain regions. In certain embodiments, the brain regions include the cerebral cortex, hippocampus, hypothalamus, midbrain, prefrontal cortex, amygdala (e.g., lateral amygdala and basomedial amygdala), piriform cortex, anterior olfactory nucleus, lateral entorhinal cortex, habenula, or a combination thereof.

[0017] In certain embodiments, the FAM19A1 antagonist can reduce the expression levels of FAM19A1 protein and / or FAM19A1 mRNA in the retinal region. In certain embodiments, the retinal region includes the ganglion cell layer (GCL) or the inner reticular layer (INL).

[0018] In certain embodiments, the FAM19A1 antagonist can reduce the expression level of FAM19A1 protein and / or FAM19A1 mRNA in the spinal region. In certain embodiments, the spinal region includes the dorsal horn.

[0019] In some manner, the expression level of the FAM19A1 protein and / or the FAM19A1 mRNA is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% or more compared to a baseline (e.g., the relevant value in a subject not receiving a FAM19A1 antagonist or the relevant value in a subject before administration of a FAM19A1 antagonist).

[0020] In some manner, the FAM19A1 antagonist can regulate, induce, or increase the differentiation of neural stem cells in the target population.

[0021] In some embodiments, the FAM19A1 antagonist can increase neurite growth in differentiated neural stem cells compared to a baseline (e.g., the relevant value in a subject not receiving the FAM19A1 antagonist or the relevant value in a subject before administration of the FAM19A1 antagonist). In some embodiments, the neurite growth increases by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% or more compared to the baseline.

[0022] Furthermore, this specification discloses a method for diagnosing central nervous system (CNS) dysfunction in a subject of interest, comprising contacting a FAM19A1 antagonist with a sample of the subject and measuring the FAM19A1 protein level or FAM19A1 mRNA level in the sample. The present application also provides a method for identifying a subject with central nervous system (CNS) dysfunction, comprising contacting a FAM19A1 antagonist with a sample of the subject and measuring the FAM19A1 protein level or FAM19A1 mRNA level in the sample.

[0023] In some cases, the contact and measurement are performed inside a test tube.

[0024] In some manifestations, the CNS function includes limbic system-related functions, olfactory system-related functions, sensory system-related functions, visual system-related functions, or combinations thereof. In certain manifestations, abnormalities in the CNS function are associated with abnormal neural circuits.

[0025] In some forms, abnormalities in CNS function are associated with CNS-related disorders or conditions. In some forms, CNS-related disorders or conditions include mood disorders, mental disorders, or all of these. In certain forms, CNS-related disorders or conditions include anxiety, depression, post-traumatic stress disorder (PTSD), bipolar disorder, attention deficit / hyperactivity disorder (ADHD), autism, schizophrenia, neuropathic pain, glaucoma, intoxication, arachnoid cysts, sclerosis, encephalitis, epilepsy / seizures, fixed syndromes, meningitis, migraines, multiple sclerosis, osteomyelopathy, Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Batten's disease, Tourette's syndrome, traumatic brain injury, spinal cord injury, stroke, tremor (essential or Parkinsonian), dysarthria, intellectual disability, brain tumors, or combinations thereof. In some forms, the CNS-related disorder or condition is anxiety, depression, PTSD, or a combination thereof. In some forms, the CNS-related disorder or condition is glaucoma, neuropathic pain, or all of these.

[0026] In some manner, the central nervous system dysfunction is associated with increased levels of FAM19A1 protein and / or FAM19A1 mRNA in the sample compared to a baseline (e.g., the corresponding value for a subject without central nervous system dysfunction, e.g., a healthy subject). In some manner, the FAM19A1 protein and / or FAM19A1 mRNA levels are increased by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% or more compared to the baseline.

[0027] In some manner, the abnormalities in central nervous system function are associated with a decrease in FAM19A1 protein and / or FAM19A1 mRNA levels in the sample compared to a baseline (e.g., the corresponding value in a subject without central nervous system function abnormalities, e.g., a healthy subject). In some manner, the FAM19A1 protein and / or FAM19A1 mRNA levels are decreased by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% or more compared to the baseline.

[0028] In some configurations, the FAM19A1 protein level is measured by immunohistochemistry, Western blotting, radioimmunoanalysis, enzyme-linked immunosorbent assay (ELISA), radioimmunodiffusion, immunoprecipitation, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, complement fixation analysis, FACS, protein chips, or a combination thereof. In some configurations, the FAM19A1 mRNA level is measured by reverse transcription polymerase chain reaction (RT-PCR), real-time polymerase chain reaction, Northern blotting, or a combination thereof.

[0029] In some forms, the sample includes tissue, cells, blood, serum, plasma, saliva, urine, cerebrospinal fluid (CSF), or a combination thereof.

[0030] In certain embodiments, the diagnostic or confirmatory methods disclosed herein further include administering a FAM19A1 antagonist to a target when the FAM19A1 protein level and / or FAM19A1 mRNA level is elevated compared to a baseline. In certain embodiments, the diagnostic or confirmatory methods disclosed herein further include administering an agonist against FAM19A1 ("FAM19A1 agonist") when the FAM19A1 protein level and / or FAM19A1 mRNA level is decreased compared to a baseline.

[0031] In some embodiments, the FAM19A1 agonist is the FAM19A1 protein. In some embodiments, the FAM19A1 antagonist is an antisense oligonucleotide, siRNA, shRNA, miRNA, dsRNA, aptamer, PNA, or vector containing the same that specifically targets FAM19A1. In some embodiments, the FAM19A1 antagonist is an anti-FAM19A1 antibody, a polynucleotide coding the anti-FAM19A1 antibody, a vector containing the polynucleotide, a cell containing the polynucleotide, or any combination thereof. In some embodiments, the FAM19A1 antagonist is an anti-FAM19A1 antibody.

[0032] In some cases, the target audience is male.

[0033] This specification states that (a) when measured by ELISA, K D (b) The property of binding to soluble human FAM19A1 with a concentration of 10 nM or less, when measured by ELISA, K D The present invention provides an anti-FAM19A1 antibody or its antigen-binding fragment ("anti-FAM19A1 antibody") that exhibits the property of binding to membrane-bound human FAM19A1 having a concentration of 10 nM, or a property selected from all of (c)(a) and (b).

[0034] In some manner, the anti-FAM19A1 antibody cross-competes with a reference antibody containing heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3, in order to bind to the human FAM19A1 epitope. (i) The heavy chain CDR1 includes the amino acid sequence shown in SEQ ID NO: 10, the heavy chain CDR2 includes the amino acid sequence shown in SEQ ID NO: 11, the heavy chain CDR3 includes the amino acid sequence shown in SEQ ID NO: 12, the light chain CDR1 includes the amino acid sequence shown in SEQ ID NO: 13, the light chain CDR2 includes the amino acid sequence shown in SEQ ID NO: 14, and the light chain CDR3 includes the amino acid sequence shown in SEQ ID NO: 15; (ii) The heavy chain CDR1 includes the amino acid sequence shown in SEQ ID NO: 4, the heavy chain CDR2 includes the amino acid sequence shown in SEQ ID NO: 5, the heavy chain CDR3 includes the amino acid sequence shown in SEQ ID NO: 6, the light chain CDR1 includes the amino acid sequence shown in SEQ ID NO: 7, the light chain CDR2 includes the amino acid sequence shown in SEQ ID NO: 8, and the light chain CDR3 includes the amino acid sequence shown in SEQ ID NO: 9; (iii) The heavy chain CDR1 includes the amino acid sequence shown in SEQ ID NO: 16, the heavy chain CDR2 includes the amino acid sequence shown in SEQ ID NO: 17, the heavy chain CDR3 includes the amino acid sequence shown in SEQ ID NO: 18, the light chain CDR1 includes the amino acid sequence shown in SEQ ID NO: 19, the light chain CDR2 includes the amino acid sequence shown in SEQ ID NO: 20, and the light chain CDR3 includes the amino acid sequence shown in SEQ ID NO: 21; or (iv) The heavy chain CDR1 includes the amino acid sequence shown in SEQ ID NO: 22, the heavy chain CDR2 includes the amino acid sequence shown in SEQ ID NO: 23, the heavy chain CDR3 includes the amino acid sequence shown in SEQ ID NO: 24, the light chain CDR1 includes the amino acid sequence shown in SEQ ID NO: 25, the light chain CDR2 includes the amino acid sequence shown in SEQ ID NO: 26, and the light chain CDR3 includes the amino acid sequence shown in SEQ ID NO: 27.

[0035] In some configurations, the anti-FAM19A1 antibody binds to the same FAM19A1 epitope as the reference antibody, which includes heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3. (i) The heavy chain CDR1 includes the amino acid sequence shown in SEQ ID NO: 10, the heavy chain CDR2 includes the amino acid sequence shown in SEQ ID NO: 11, the heavy chain CDR3 includes the amino acid sequence shown in SEQ ID NO: 12, the light chain CDR1 includes the amino acid sequence shown in SEQ ID NO: 13, the light chain CDR2 includes the amino acid sequence shown in SEQ ID NO: 14, and the light chain CDR3 includes the amino acid sequence shown in SEQ ID NO: 15; (ii) The heavy chain CDR1 includes the amino acid sequence shown in SEQ ID NO: 4, the heavy chain CDR2 includes the amino acid sequence shown in SEQ ID NO: 5, the heavy chain CDR3 includes the amino acid sequence shown in SEQ ID NO: 6, the light chain CDR1 includes the amino acid sequence shown in SEQ ID NO: 7, the light chain CDR2 includes the amino acid sequence shown in SEQ ID NO: 8, and the light chain CDR3 includes the amino acid sequence shown in SEQ ID NO: 9; (iii) The heavy chain CDR1 includes the amino acid sequence shown in SEQ ID NO: 16, the heavy chain CDR2 includes the amino acid sequence shown in SEQ ID NO: 17, the heavy chain CDR3 includes the amino acid sequence shown in SEQ ID NO: 18, the light chain CDR1 includes the amino acid sequence shown in SEQ ID NO: 19, the light chain CDR2 includes the amino acid sequence shown in SEQ ID NO: 20, and the light chain CDR3 includes the amino acid sequence shown in SEQ ID NO: 21; or (iv) The heavy chain CDR1 includes the amino acid sequence shown in SEQ ID NO: 22, the heavy chain CDR2 includes the amino acid sequence shown in SEQ ID NO: 23, the heavy chain CDR3 includes the amino acid sequence shown in SEQ ID NO: 24, the light chain CDR1 includes the amino acid sequence shown in SEQ ID NO: 25, the light chain CDR2 includes the amino acid sequence shown in SEQ ID NO: 26, and the light chain CDR3 includes the amino acid sequence shown in SEQ ID NO: 27.

[0036] In some configurations, the anti-FAM19A1 antibody binds to at least one epitope selected from the group consisting of D112, M117, A119, T120, N122, and combinations thereof.

[0037] In some embodiments, the anti-FAM19A1 antibody comprises heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3, wherein the heavy chain CDR3 comprises the amino acid sequence shown in SEQ ID NOs. 12, 6, 18, or 24. In certain embodiments, the heavy chain CDR1 comprises the amino acid sequence shown in SEQ ID NOs. 10, 4, 16, or 22. In other embodiments, the heavy chain CDR2 comprises the amino acid sequence shown in SEQ ID NOs. 11, 5, 17, or 23. In some embodiments, the light chain CDR1 comprises the amino acid sequence shown in SEQ ID NOs. 13, 7, 19, or 25. In some embodiments, the light chain CDR2 comprises the amino acid sequence shown in SEQ ID NOs. 14, 8, 20, or 26. In certain embodiments, the light chain CDR3 comprises the amino acid sequence shown in SEQ ID NOs. 15, 9, 21, or 27.

[0038] In some configurations, the anti-FAM19A1 antibody comprises heavy chain CDR1, CDR2 and CDR3, and light chain CDR1, CDR2 and CDR3. (i) The heavy chains CDR1, CDR2, and CDR3 each contain the amino acid sequences shown in SEQ ID NOs. 10-12, and the light chains CDR1, CDR2, and CDR3 each contain the amino acid sequences shown in SEQ ID NOs. 13-15; (ii) The heavy chains CDR1, CDR2, and CDR3 each contain the amino acid sequences shown in SEQ ID NOs: 4-6, and the light chains CDR1, CDR2, and CDR3 each contain the amino acid sequences shown in SEQ ID NOs: 7-9; (iii) The heavy chains CDR1, CDR2, and CDR3 each contain the amino acid sequences shown in SEQ ID NOs. 16-18, and the light chains CDR1, CDR2, and CDR3 each contain the amino acid sequences shown in SEQ ID NOs. 19-21; or (iv) The heavy chains CDR1, CDR2, and CDR3 each contain the amino acid sequences shown in SEQ ID NOs. 22-24, and the light chains CDR1, CDR2, and CDR3 each contain the amino acid sequences shown in SEQ ID NOs. 25-27.

[0039] In some embodiments, the anti-FAM19A1 antibody includes a heavy chain variable domain containing the amino acid sequence shown in SEQ ID NOs. 30, 28, 32, or 34 and / or a light chain variable domain containing the amino acid sequence shown in SEQ ID NOs. 31, 29, 33, or 35.

[0040] In some embodiments, the anti-FAM19A1 antibody includes a heavy chain variable domain containing the amino acid sequence shown in SEQ ID NO: 30, and a light chain variable domain containing the amino acid sequence shown in SEQ ID NO: 31. In certain embodiments, the anti-FAM19A1 antibody includes a heavy chain variable domain containing the amino acid sequence shown in SEQ ID NO: 28, and a light chain variable domain containing the amino acid sequence shown in SEQ ID NO: 29. In some embodiments, the anti-FAM19A1 antibody includes a heavy chain variable domain containing the amino acid sequence shown in SEQ ID NO: 32, and a light chain variable domain containing the amino acid sequence shown in SEQ ID NO: 33. In some embodiments, the anti-FAM19A1 antibody includes a heavy chain variable domain containing the amino acid sequence shown in SEQ ID NO: 34, and a light chain variable domain containing the amino acid sequence shown in SEQ ID NO: 35.

[0041] In some embodiments, the anti-FAM19A1 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 NOs. 30, 28, 32, or 34 / or the VL comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence shown in SEQ ID NOs. 31, 29, 33, or 35.

[0042] In some embodiments, the anti-FAM19A1 antibody is a chimeric antibody, a human antibody, or a humanized antibody. In some embodiments, the anti-FAM19A1 antibody comprises Fab, Fab', F(ab')2, Fv, or single-chain Fv(scFv). In some embodiments, the anti-FAM19A1 antibody is selected from the group consisting of IgG1, IgG2, IgG3, IgG4, their variants, and any combination thereof. In some embodiments, the anti-FAM19A1 antibody is an IgG1 antibody. In some embodiments, the anti-FAM19A1 antibody contains a constant region without Fc function.

[0043] In some embodiments, the anti-FAM19A1 antibody is conjugated to an agent to form an immunoconjugate. In certain embodiments, the anti-FAM19A1 antibody is formulated with a pharmaceutically acceptable carrier.

[0044] In some aspects, a FAM19A1 antagonist useful in the methods disclosed herein is the anti-FAM19A1 antibody provided herein.

[0045] In some configurations, the FAM19A1 antagonist is administered intravenously, orally, parenterally, intrathecally, intraventricularly, intrapulmonaryly, intramuscularly, subcutaneously, intravitreously, or intraventricularly. In some configurations, the subject is human.

[0046] This specification also provides nucleic acids comprising a nucleotide sequence coding for the anti-FAM19A1 antibody of this disclosure. Furthermore, this disclosure provides vectors comprising the nucleic acids disclosed herein and one or more promoters operably linked to the nucleic acids. Furthermore, this disclosure provides cells comprising the nucleic acids or vectors described herein. This specification discloses compositions comprising the anti-FAM19A1 antibody and a carrier of this disclosure. This disclosure provides a kit comprising the anti-FAM19A1 antibody and instructions for use of this disclosure.

[0047] This specification provides a method for producing anti-FAM19A1 antibody, comprising culturing the cells disclosed herein under appropriate conditions and isolating the anti-FAM19A1 antibody. [Brief explanation of the drawing]

[0048] Figure 1 shows the ELISA results from each round of biopanning described in Example 2 for binding to the positive poly-scFv-phage antibody pool. The illustrated positive poly-scFv-phage antibody pool includes antibody pools from (i) biopanning round 1 ("1'Fc"), (ii) biopanning round 2 ("2'mFc"), and (iii) biopanning round 3 ("3'Fc"). M13 phage #38 and the entire library were used as control groups. For each illustrated scFv-phage antibody pool, binding to FAM19A1-Fc, FAM19A1-mFc, and non-FAM19A1 protein (ITGA6-Fc) is shown from left to right.

[0049] Figure 2 shows the ELISA results for binding of individual mono-scFv-phage clones isolated from round 3 of biopanning to the FAM19A1 protein. The illustrated clones (from left to right) include 1A1, 1A2, 1A3, 1A4, 1A5, 1A6, 1A7, 1A8, 1A9, 1A10, 1A11, 1A12, 1B1, 1B2, 1B3, 1B4, 1B5, 1B6, 1B7, 1B8, 1B9, 1B10, 1B11, 1B12, 1C1, 1C2, 1C3, 1C4, 1C5, 1C6, 1C7, 1C8, 1C9, 1C10, 1C11, 1C12, 1D1, 1D2, 1D3, 1D4, 1D5, 1D6, 1D7, 1D8, 1D9, 1D10, 1D11, and 1D12. For each antibody clone, the bar on the left indicates binding to FAM19A1-Fc. For each antibody clone, the bar on the right indicates binding to the negative control group (non-FAM19A1-Fc).

[0050] Figure 3 shows BstNI fingerprinting analysis of distinct mono-scFv-phage clones isolated from round 3 of biopanning. The illustrated clones include (from left to right) 1A11, 1C1, M, 2A10, 2C9, 2D12, 2E1, 2G7, 2G8, 2H4, 2H9, 2H11, 2H12, 3A4, 3A5, 3A8, 3A11, 3B6, 3B8, 3B10, 3C5, 3D1, 3D11, 3D12, 3E2, 3E7, 3E12, 3F12, 3G3, 3G4, 3G10, 3G12, 3H2, 3H3, 3H9, 4A2, 4D1, 4E10, 4G8, 4H11, 5A3, 5C1, 5C3, 5C6, 5E11, 5G1, 6E12, and 7G8. The following clones were able to specifically bind to FAM19A1 with high affinity (i.e., they did not bind to the non-FAM19A1 control protein): 1A11, 1C1, 2G7, and 3A8. Antibody clones 2C9, 5A3, and 2E1 either did not specifically bind to FAM19A1, were not monophages, or bound to FAM19A1 with low affinity, respectively.

[0051] Figure 4 shows the ELISA results for binding of all FAM19A1 and non-FAM19A1 proteins to different mono-scFv-phage clones. For each of the clones, binding to the following proteins is shown (from left to right): (i) FAM19A1-MYC / DKK(Origene), (ii) FAM19A1-N-Fc, (iii) FAM19A1-N-mFc, (iv) ITGA6-Fc, (v) CD58-Fc, (vi) hRAGE-Fc, (vii) AITR-Fc, (viii) c-Fc, and (ix) mFc. The three FAM19A1 proteins differ from each other only in the tags used for binding detection.

[0052] Figures 5A, 5B, 5C, and 5D show the distinct characteristics of the anti-FAM19A1 IgG1 antibodies produced as described in Example 3. Figure 5A provides a schematic configuration of the expression vector used to produce the anti-FAM19A1 IgG1 antibody. Figure 5B provides the purity and mobility of the antibody as confirmed by SDS-PAGE analysis. Figure 5C provides production yield data. Figure 5D provides the binding analysis of the antibody to the FAM19A1 protein as measured by ELISA. The table below the graphs provides the Kd values. In Figures 5B, 5C, and 5D, the illustrated anti-FAM19A1 IgG1 antibodies include clones 1A11, 1C1, 2G7, and 3A8.

[0053] Figure 6 shows the ELISA results for binding of different anti-FAM19A1 antibody clones to the FAM19A1 mutant M1-M7. Wild-type FAM19A1 and PBS were used as control groups. For each of the FAM19A1 proteins, the five bars shown correspond to anti-FAM19A1 antibody clones (i) 1A11 ("A1-1A11-Ybio"), (ii) 1C1 ("FAM19A1-1C1"), (iii) F41H5, (iv) D6 ("D6-a-Fam19A1"), and E1 ("E1-a-Fam19A1") (from left to right).

[0054] Figure 7 shows FAM19A1 mRNA expression in different tissues of mice. The illustrated tissues include tissues from different brain regions (i.e., cerebral cortex, cerebellum, midbrain, spinal cord, hippocampus, olfactory bulb, hypothalamus, and pituitary gland) and peripheral tissues (i.e., heart, liver, spleen, stomach, small intestine, testes, kidneys, and lungs). The brain region tissues are indicated by boxes.

[0055] Figures 8A, 8B, 8C, 8D, 8E, 8F, 8G, and 8H show FAM19A1 expression in the FAM19A1 LacZ knock-in (KI) mice generated as described in the Examples. Figure 8A is a schematic diagram of the gene structure of the FAM19A1 LacZ KI mouse. The LacZ gene sequence was inserted directly after the start codon in exon 2 of the FAM19A1 gene. This gene structure was expressed using the native FAM19A1 promoter, and the resulting product was β-galactosidase without any portion of the FAM19A1 protein due to the poly-A tail behind the LacZ sequence. Therefore, the isozygous FAM19A1 LacZ KI mouse was considered a complete knockout of FAM19A1. E1, exon 1; E2, exon 2; E3, exon 3; E4, exon 4; E5, exon 5; lacZ, lacZ gene; neo, amino 3'-glycosyl phosphotransferase gene; pA, poly-A tail. Figure 8B provides genomic DNA PCR results comparing β-galactosidase (343 bp) and FAM19A1 (243 bp) in wild-type FAM19A1 LacZ KI(+ / -) and FAM19A1 LacZ KI(- / -) animals. Figure 8C provides RT-PCR results comparing FAM19A1 expression in all cortical (CTX) and hippocampal (HIP) regions of different animals. Figure 8D provides comparison of endogenous FAM19A1 protein expression in cortical (CTX) and hippocampal (HIP) regions of different animals using FAM19A1-specific antibodies. The exposure time during generation with the ECL solution was 30 minutes for FAM19A1 and 1 minute for β-actin. Figures 8E (cortex) and 8F (hippocampus) provide quantitative analysis of the results shown in Figure 8D. Figure 8G shows all of the FAM19A1 mRNA and protein expression in various regions of the brain of wild-type animals. The exposure time during generation with the ECL solution was 30 minutes for FAM19A1 and 1 minute for β-actin.The illustrated regions include (i) the cortex (CTX), (ii) the hippocampus (HIP), (iii) the olfactory bulb (OB), (iv) the cerebellum (CB), (v) the thalamus + hypothalamus (TH + HYP), (vi) the midbrain (MB), and (vii) the pons (pons, PO). Figure 8H provides a quantitative analysis of the results shown in Figure 8G. In Figures 8E, 8F, and 8H, "au" represents any unit. Data are presented as mean ± standard error of the mean (SEM). One-way ANOVA and Bonferroni post-hoc studies showed **p<0.01 and ***p<0.001 compared to WT.

[0056] Figures 9A, 9B, and 9C show whole-brain X-gal staining in FAM19A1 LacZ KI(- / -) mice at different developmental stages. Figure 9A provides a comparison of β-galactosidase expression in WT and FAM19A1 LacZ KI mice at embryonic day 12.5 (E12.5). Figure 9B shows β-galactosidase expression in FAM19A1 LacZ KI mice at embryonic days 14.5, 16.5, and 18.5. Figure 9C shows β-galactosidase expression in FAM19A1 LacZ KI mice at postnatal days 0.5, 2.5, 7.5, 14.5, and 56.6. In Figures 9A, 9B, and 9C, the scale bar is 2 mm.

[0057] Figures 10A and 10B provide X-gal staining of the brains of embryonic and postnatal FAM19A1 LacZ knock-in (KI) (+ / -) mice. Figure 10A shows the detection of X-gal signals in coronary brain sections at 14.5 days (E14.5) and 18.5 days (E18.5) of embryonic development. Figure 10B shows the detection of X-gal signals in various regions at 0.5 days (P0.5), 7.5 days (P7.5), and 14.5 days (P14.5) postnatally. ACo, anterior cortical amygdala; Amy, amygdala; AO, anterior olfactory nucleus; Au, auditory cortex; BMA; basomedial amygdala, anterior part; CEn, entorhinal cortex; CPf, piriform cortex; FR, fasciculus retroflexus; Hip, hippocampus; LS, lateral septal nucleus; M, motor cortex; MGV, medial geniculate nucleus, ventral part; Op, superior colliculus colliculus) optic nerve layer; PF, pontine flexure; PMCo, amygdala nucleus of the posteromedial cortex; Pn, pontine nucleus; PrL, prelimbic cortex; RMC, red nucleus, magnocellular region; S, somatosensory cortex; V, visual cortex.

[0058] Figures 11A, 11B, and 11C show FAM19A1 expression patterns in the brains of different adult mice. In Figure 11A, X-gal precipitate (red) was detected in some cortical L2-3 CUX1-positive neurons (green) of FAM19A1 LacZ KI mice. In Figure 11B, β-galactosidase (green) was identified in some cortical L5 CTIP2-positive neurons (magenta) of FAM19A1 LacZ KI mice. The heads of the arrows indicate cortical marker cells expressing X-gal or β-galactosidase. Figure 11C shows X-gal staining in coronal brain sections of adult mice as measured by immunohistochemistry. Different panels show different brain regions of related animals. AO, anterior olfactory nucleus; Apir, amygdala-piriform transition area; BLA, basomedial amygdala; BLP, posterior basolateral amygdala; CEn, entorhinal cortex; CPf, piriform cortex; D3V, dorsal third ventricle; FrA, prefrontal association cortex; L2-3, cortical layers 2-3; L5, cortical layer 5; CA1, 2 and 3, fields of the CA1, CA2 and CA3 regions of the hippocampus; LaDL, lateral amygdala; LHb, lateral habenula; LO, lateral orbital cortex; LS, lateral septal nucleus; LV, lateral ventricle; MGN, medial geniculate nucleus; MO, medial orbital cortex; Op, optic nerve layer of the superior colliculus; PMCo, posteromedial cortical amygdala; PrL, anterior limbic cortex; Py, pyramidal cell layer of the hippocampus; RG, splenic granulocortex; VO, ventral orbital cortex.

[0059] Figure 12 shows FAM19A1 expression (shown by X-gal staining) in the brain, spinal cord, and dorsal ganglia of adult mice. Panel AG shows X-gal stained coronal sections from different brain regions of heterozygous FAM19A1 LacZ knock-in (KI) mice. Panels H and I show X-gal stained coronal sections from different brain regions of isozygous FAM19A1 LacZ KI mice. Panel JM shows X-gal stained coronal spinal cord sections of heterozygous FAM19A1 LacZ KI mice. Panel N shows X-gal stained dorsal ganglia of heterozygous FAM19A1 LacZ KI mice. 3V, third ventricle; 7N, facial nerve nucleus; cp, peduncle; DC, dorsal cochlear nucleus; Ecu, lateral cuneate nucleus; ic, internal capsule; IPDL, interpedunclear nucleus, dorsolateral subnucleus; lfp, longitudinal fasciculus of the pons fasciculus); LPO, lateral preoptic area; LRt, lateral reticular nucleus; ml, medial lemniscus; MPOM, medial preoptic nucleus; MVeMC, medial vestibular nucleus, giant cell portion; MVePC, medial vestibular nucleus, parietal portion; Pn, pontine nuclei; Po, posterior thalamic nuclei; Pr, anterior nucleus; py, pyramidal tract; RtTg, pontine reticular tegmental nucleus; Sp5I, spinal trigeminal nucleus, interpoleal portion; Sp5O, spinal trigeminal nucleus, oral portion; SpVe, spinal vestibular nucleus; SuVe, supravestibular nucleus; VMH, ventromedial hypothalamic nucleus; X, nucleus X.

[0060] Figure 13 shows FAM19A1 mRNA expression in the brains of growing and mature wild-type (WT) rats by in situ hybridization using a FAM19A1 mRNA probe. The age of the rat for each illustrated brain section is provided in the lower right corner of each panel: (i) 14.5 days postnatal (E14.5), (ii) 16.5 days postnatal (E16.5), (iii) 18.5 days postnatal (E18.5), (iv) 0.5 days postnatal (P0.5), (v) 7.5 days postnatal (P7.5), (vi) 14.5 days postnatal (P14.5), (vii) 21.5 days postnatal (P21.5), and (viii) different visual states (sagittal, horizontal, and coronal) of the adult brain. Amy, amygdala; AO, anterior olfactory nucleus; Cer, cerebellum; CTX, cortex; Hb, hand rete; Hip, hippocampus; Mes, midbrain; SC, spinal cord; Tel, telencephalon; Th, thalamus.

[0061] Figure 14 provides a table showing the number and percentage (%) of offspring produced from heterozygous conjugation FAM19A1 LacZ KI parents.

[0062] Figures 15A, 15B, 15C, 15D, 15E, 15F, 15G, 15H, 15I, and 15J provide a comparison of morphological differences between wild-type and FAM19A1 LacZ knock-in (KI) mice. Figures 15A and 15B show age-related changes in body weight in male and female mice, respectively. Figure 15C provides whole-mount views of brain tissue specimens from WT and FAM19A1(- / -) adult mice. Figure 15D shows the motor cortex layer in Nissl-stained brain tissue from WT heterozygous FAM19A1 LacZ KI (FAM19A1 + / -) and isozygous FAM19A1 LacZ KI (FAM19A1 - / -) mice. Figures 15E, 15F, and 15G show the total brain length, cerebral cortical length, and brain width of WT (n=9), FAM19A1 + / - (n=8), and FAM19A1 - / - (n=8) adult mice, respectively. Figures 15H, 15I, and 15J show the cortical thickness in the motor, somatosensory, and visual cortices of WT (n=5), FAM19A1 + / - (n=5), and FAM19A1 - / - (n=4) adult mice, respectively. Data are presented as mean ± mean standard error (SEM). One-way or two-way ANOVA and Bonferroni post-hoc studies showed *p<0.05, **p<0.01, and ***p<0.001 for WT and FAM19A1 + / -.

[0063] Figures 16A and 16B provide a comparison of estimated cortical volume (Figure 16A) and total number of neurons (Figure 16B) in the cerebral cortex of wild-type (WT), FAM19A1 + / -, and FAM19A1 - / - adult mice. Data are presented as mean ± mean standard error (SEM).

[0064] Figures 17A, 17B, 17C, 17D, 17E, and 17F provide a comparison of cortical layer thicknesses in wild-type, FAM19A1 + / -, and FAM19A1 - / - adult mice. Figures 17A, 17B, and 17C show the cortical layer thicknesses of the motor, somatosensory, and visual cortices, respectively. Figures 17D, 17E, and 17F show the ratio of the thickness of each cortical layer to the total cortical thickness in the motor, somatosensory, and visual cortices, respectively. The distinct cortical layers are provided on the x-axis. For each of the cortical layers (x-axis), the bars (from left to right) represent wild-type (WT), FAM19A1 + / -, and FAM19A1 - / - adult mice, respectively. Data are presented as mean ± mean standard error (SEM). One-way analysis of variance (ANOVA) showed that *p<0.05, **p<0.001, and ***p<0.001 for WT mice.

[0065] Figures 18A, 18B, 18C, and 18D provide a comparison of neuronal density in the motor cortex layers of wild-type, FAM19A1 + / -, and FAM19A 1- / - adult mice. In Figure 18A, NeuN was used as a marker for neurons. The distinct cortical layers are distinguished as L1, L2-3, L4, L5, and L6. Figures 18B, 18C, and 18D provide quantitative comparisons of neuronal density, volume, and total NeuN-positive cells in each of the cortical layers shown in Figure 18A, respectively. In Figures 18A, 18B, 18C, and 18D, for each of the cortical layers (x-axis), the bars (from left to right) represent wild-type (WT), FAM19A1 + / -, and FAM19A1 - / - adult mice, respectively. Data are presented as mean ± mean standard error (SEM).

[0066] Figures 19A, 19B, 19C, 19D, and 19E provide a comparison of the number of cortical glial cells in the motor cortex of wild-type, FAM19A1 + / -, and FAM19A1 - / - adult mice. In Figure 19A, GFAP (green)-positive astrocytes and Iba1 (red)-positive microglia were detected in the motor cortex. In Figure 19B, Olig2-positive oligodendrocytes (positive cells exemplified by white arrows) were identified in the motor cortex. Figures 19C, 19D, and 19E show the number of GFAP-positive cells, Iba1-positive cells, and Olig2-positive cells in the motor cortex layer, respectively. In Figures 19C, 19D, and 19E, for each of the cortical layers (x-axis), the bars (from left to right) represent wild-type (WT), FAM19A1 + / -, and FAM19A1 - / - adult mice, respectively. The data is presented as mean ± standard error of the mean (SEM).

[0067] Figures 20A, 20B, 20C, 20D, 20E, 20F, 20G, and 20H provide a comparison of hyperactivity in wild-type, FAM19A1 + / -, and FAM19A1 - / - adult mice. Figures 20A and 20B show the total time and total distance traveled in the open arm, measured in the elevated cross maze (EPM) test, respectively. Figures 20C and 20D show the total time and total distance traveled in the center, measured in the open field test (OFT), respectively. Figure 20E provides a simple tracking of animal movement in the OFT arena. Figure 20F shows the percentage of immobile time measured in the tail suspension test (TST). Figures 20G and 20H show spontaneous change and total distance traveled, measured in the Y-maze test, respectively. Data are presented as mean ± mean standard error (SEM). One-way ANOVA and Bonferroni post-hoc studies showed *p<0.05, **p<0.01, and ***p<0.001 for WT or FAM19A1 + / -.

[0068] Figures 21A, 21B, 21C, 21D, 21E, and 21F provide a comparative overview of short-term and long-term memory formation in wild-type, FAM19A1 + / -, and FAM19A1 - / - adult mice. Figures 21A, 21B, and 21C show the total time required for exploration, object preference, and discrimination index measured in the short-term memory novel object recognition (NOR) test, respectively. Figures 21D, 21E, and 21F show the total time required for exploration, object preference, and discrimination index measured in the long-term memory NOR test, respectively. In Figures 21A, 21B, 21D, and 21E, for each animal group, the bar on the left represents the result for familiar objects, and the bar on the right represents the result for new objects. Data are presented as mean ± mean standard error (SEM). One-way ANOVA and Bonferroni post-hoc studies showed *p<0.05, **p<0.01, and ***p<0.001 for WT or FAM19A1 + / -.

[0069] Figures 22A, 22B, 22C, and 22D provide a comparative view of fear responses in wild-type, FAM19A1 + / -, and FAM19A1 - / - adult mice. Figure 22A shows fear conditioning during the acquisition phase of Pavlov's fear conditioning test. Arrows indicate the associated group. Figures 22B and 22C show the results of situational and auditory memory tests conducted 24 hours after the acquisition phase, respectively. Figure 22D shows the results of an innate fear test using 2,5-dihydro-2,4,5-trimethylthiazoline (TMT), a synthesized fox fecal odor. Arrows indicate the associated group. Data are presented as mean ± mean standard error (SEM). Two-way ANOVA and Bonferroni post-hoc trials or Student's t trials showed *p<0.05, **p<0.01, and ***p<0.001 compared to the win condition (WT).

[0070] Figures 23A and 23B provide a comparative view of FAM19A1 and FAM19A5 expression in the brain (verified by β-galactosidase expression) using FAM19A1 LacZ KI and FAM19A5 LacZ KI mice. Figure 23A shows the schematic structure of the FAM19A5 LacZ KI mouse gene. The LacZ gene sequence is inserted by homologous recombination. The resulting product is a fusion of β-galactosidase and FAM19A5. Figure 23B shows the expression of FAM19A1 (left) and FAM19A5 (right) in the brain. The illustrated region includes L2-3 (cortical layers 2 and 3); L5b (cortical layer 5b); CA1 (CA1 region of the hippocampus); CA2 (CA2 region of the hippocampus); CA3 (CA3 region of the hippocampus); DG (dentate gyrus); CC (corpus callosum); CTX (cortex); TH (thalamus); and fi (fimbriae of the hippocampus). Scale bar = 500 μm.

[0071] Figure 24 provides a comparison of neurite growth of differentiated neurons within neural stem cells in adult mice treated with (i) control IgG antibody (left panel), (ii) anti-FAM19A1 antibody (middle panel), or (iii) FAM19A1 protein (right panel).

[0072] Figure 25 shows a comparison of intraocular pressure (IOP) in glaucoma-induced animals treated with human IgG1 ("hIgG"; empty square) or anti-FAM19A1 antibody ("FAM19A1 Ab"; filled square). Healthy, normal animals (i.e., animals not induced with glaucoma) ("naive") were used as the control group. IOP was measured on days 0, 14, and 28 after glaucoma induction. Data are shown as mean ± SD. "***" indicates a statistically significant difference (p<0.001) compared to the untreated control group.

[0073] Figure 26 compares the rhythmic wave intensity in glaucoma-induced animals treated with human IgG1 ("hIgG") or anti-FAM19A1 antibody ("FAM19A1Ab"). Healthy normal animals (i.e., animals without induced glaucoma) ("untreated") were used as the control group. Data are shown as mean ± SD. "***" indicates a statistically significant difference (p<0.001) compared to the untreated control group. "###" indicates a statistically significant difference (p<0.001) compared to the hIgG group.

[0074] Figures 27A and 27B compare the number of retinal ganglion cells (RGCs) observed in the retinal ganglion cell layer of glaucoma-induced animals treated with human IgG1 ("hIgG") or anti-FAM19A1 antibody ("FAM19A1 Ab"). Healthy normal animals (i.e., animals without induced glaucoma) ("untreated") were used as the control group. Figure 27A shows the number of RGC cells as an absolute value. Data are shown as mean ± SD. "***" indicates a statistically significant difference (p<0.001) compared to the untreated control group. "##" indicates a statistically significant difference (p<0.01) compared to the hIgG group. Figure 27B shows fluorescence images (magnification 100×) of the retinal ganglion cell layer of representative animals in each of the above groups.

[0075] Figure 28 shows a comparison of paw retraction thresholds in chronic stenotic injury (CCI)-induced rats treated with saline (empty square) or anti-FAM19A1 antibody (filled square). Normal, healthy animals (i.e., animals without CCI induction) ("untreated") were used as the control group. Paw retraction thresholds were measured at 7, 14, and 21 days after CCI induction. Data are shown as mean ± SD. "#" indicates a statistically significant difference compared to the saline group (p<0.05).

[0076] Figure 29 compares rotarod delay times (the time it takes for an animal to fall off a rotarod-treadmill, as described in the examples) in chronic stenotic injury (CCI)-induced rats treated with saline (empty squares) or anti-FAM19A1 antibody (filled squares). Normal, healthy animals (i.e., animals without CCI induction) ("untreated") were used as the control group. Delay times were measured at 7, 14, and 21 days after CCI induction. Data are shown as mean ± SD. "#" indicates a statistically significant difference (p<0.05) compared to the saline group.

[0077] Figures 30A, 30B, 30C, 30D, 30E, and 30F show the effects of anti-FAM19A1 treatment on neurite growth and dendritic branching in primary mouse hippocampal neurons. Figures 30A and 30B show neurite growth via immunohistochemistry in mouse hippocampal neurons treated with vehicle-control or anti-FAM19A1 antibody, respectively. Figure 30C shows the mean total length (μm) of neurites. Figure 30D shows the number of primary neurites. Figure 30E shows the number of branching points. Figure 30F shows the number of secondary neurites. In Figures 30C to 30F, data are shown as mean ± SD. "*" indicates a statistically significant difference (p<0.05). In Figures 30A and 30B, the scale bar is 20 μm.

[0078] Figure 31 shows the analgesic effect of anti-FAM19A1 monoclonal antibody (A1-1C1) in CCI-induced mechanical allodynia. The analgesic effect is expressed as the number of leg withdrawal responses from 10 filament applications to each hind leg at 10-second intervals ("Leaning Response Frequency (%)"). CCI-induced animals treated with human IgG control antibody (filled circle) and normal healthy animals (i.e., animals not affected by CCI) (untreated, empty circle) were used as the control group. Leg withdrawal responses were measured on days 6, 10, 13, 17, and 20 after CCI induction. Data are shown as mean ± SD. "*" indicates a statistically significant difference (p<0.01). Arrows indicate the time of administration of anti-FAM19A1 antibody (i.e., days 7 and 14 after CCI induction).

[0079] Figure 32 shows the analgesic effect of anti-FAM19A1 monoclonal antibody (A1-1C1) in CCI-induced thermal hyperalgesia. The analgesic effect is shown as the withdrawal delay time (the time it took for the animal to withdraw its foot in response to the thermal stimulus). CCI-induced animals treated with human IgG control antibody (filled circle) and normal healthy animals (i.e., animals that have not been treated with CCI) (untreated, empty circle) were used as the control group. Foot withdrawal response was measured on days 6, 10, 13, 17, and 20 after CCI induction. Data are shown as mean ± SD. Arrows indicate the time of administration of anti-FAM19A1 antibody (i.e., days 7 and 14 after CCI induction).

[0080] [Modes for carrying out the invention] This specification discloses antagonists (e.g., monoclonal antibodies) that specifically bind to a family member A1 (FAM19A1) having sequence similarity 19 and exhibit one or more of the properties disclosed herein.

[0081] Numerous terms and phrases are defined to facilitate understanding of the disclosures provided herein. Additional definitions are provided throughout the detailed descriptions.

[0082] I. Definition Throughout this disclosure, the terms "one" or "a" are understood to mean one or more entities; for example, "one antibody" is understood to mean one or more antibodies. Thus, the terms "one" (or "a"), "one or more" and "at least one" may be used interchangeably in this specification.

[0083] Furthermore, as used herein, “and / or” should be considered as if each of two specific features or components were a specific disclosure, either together with the other features or components, or on its own. Accordingly, in this specification, the term “and / or” as used in phrases such as “A and / or B” is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Similarly, the term “and / or” as used in phrases such as “A, B and / or C” is intended to include each of the following modes: 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).

[0084] In this specification, when each mode is described with the term “contains,” it is understood that other similar modes described in terms of “consisting of” and / or “essentially consisting of” are also provided.

[0085] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those typically understood by an ordinary person skilled in the art relating to this disclosure. For example, *Concise Dictionary of Biomedicine and Molecular Biology*, Juo, Pei-Show, 2nd ed., 2002, CRC Press; *Dictionary of Cell and Molecular Biology*, 3rd ed., 1999, Academic Press; and *Oxford Dictionary of Biochemistry and Molecular Biology*, Revised, 2000, Oxford University Press provide a majority of the terms used herein to a person skilled in the art.

[0086] Units, prefixes, and symbols are shown in the forms recognized by these SI (Systems International de Unites). Numerical ranges include the digits that limit their range. Unless otherwise indicated, amino acid sequences are written from left to right in the amino-carboxyl direction. The titles provided herein are not limitations on the various aspects of the disclosure, and may refer to the specification as a whole. Thus, each term defined directly below is more fully defined by referring to the specification as a whole.

[0087] In this specification, the term "about" is used to mean almost, generally, approximately, or within that range. When the term "about" is used with a numerical range, it extends the boundaries above and below the stated number, thereby changing the applicable range. Generally, the term "about" can change the numbers above and below the stated value by, for example, 10% above or below (further up or further down).

[0088] The term "family with sequence similarity 19, member A1" or "FAM19A1" refers to proteins belonging to the TAFA family (also known as the FAM19 family) of five highly homologous proteins. These proteins contain conserved cysteine ​​residues and are at least distantly related to MIP-1alpha, a type of CC-chemokine family. FAM19A1 is mainly expressed in the central nervous system (brain and spinal cord). See examples. For example, FAM19A1 is also known as TAFA1 or chemokine-like protein TAFA-1.

[0089] In humans, the gene coding for FAM19A1 is located on chromosome 3. There are two possible isoforms of human FAM19A1 (UniProt:Q7Z5A9): isoform 1 (UniProt:Q7Z5A9-1), consisting of 133 amino acids, and isoform 2 (UniProt:A0A087X2J7), consisting of 52 amino acids, which is predicted based on EST data. The amino acid sequences of these two known human FAM19A1 isoforms are provided in Table 1 below.

[0090] [Table 1] The term "FAM19A1" includes any variant or isotype of FAM19A1 spontaneously expressed by cells. Therefore, the antagonists (e.g., antibodies) described herein may cross-react with different isotypes within the same species (e.g., different isotypes of human FAM19A1) or with non-human species FAM19A1 (e.g., mouse FAM19A1). In contrast, the antibodies may be specific to human FAM19A1 and may not exhibit any cross-reaction with other species. FAM19A1 or any variants and isotypes thereof may be isolated from cells or tissues that spontaneously express them, or produced by recombination. The polynucleotide encoding human FAM19A1 has GenBank accession number NM_213609.3 and the following sequence:

[0091] [Table 2] The term "antagonist against FAM19A1 protein" refers to all antagonists that suppress the expression of the FAM19A1 protein. Such antagonists may be peptides, nucleic acids, or compounds. In some forms, a FAM19A1 antagonist may be an antisense oligonucleotide, siRNA, shRNA, miRNA, dsRNA, aptamer, PNA (peptide nucleic acid), or vector containing the same that targets FAM19A1. In some forms, a FAM19A1 antagonist may include an antibody or its antigen-binding fragment that specifically binds to the FAM19A1 protein.

[0092] The term "FAM19A1 protein agonist" or "FAM19A1 agonist" refers to all agonists that promote the expression of the FAM19A1 protein and / or share the same biological function as the FAM19A1 protein and increase FAM19A1 activity. In some forms, the FAM19A1 agonist is the FAM19A1 protein itself.

[0093] The terms “antibody” and “each antibody” are industry terms, interchangeable herein, and refer to molecules having an antigen-binding site that specifically binds to an antigen. As used herein, the terms include the whole antibody and any antigen-binding fragment thereof (i.e., “antigen-binding fragment”) or a single chain thereof. In some aspects, “antibody” refers to a glycoprotein or an antigen-binding fragment thereof comprising at least two heavy (H) chains and two light (L) chains linked to each other by disulfide bonds. In some aspects, “antibody” refers to a single-chain antibody comprising a single variable domain, e.g., a VHH domain. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. In certain spontaneously occurring antibodies, the heavy chain constant region consists of three domains CH1, CH2, and CH3. In certain spontaneously 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.

[0094] The VH and VL regions can be further subdivided into highly variable regions called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each of the VH and VL regions consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: 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 the antibody can mediate the binding of immunoglobulins to various cells of the immune system (e.g., effector cells) and to host tissues or factors, including the first component (Clq) of the classical complement system.

[0095] The term "Kabat numbering" and similar terms are recognized in this industry and refer to a system for numbering amino acid residues in the heavy and light chain variable regions of antibodies or their antigen-binding fragments. In certain ways, the CDR of an antibody may be determined by the Kabat numbering system (see, for example, Kabat EA & Wu TT (1971) Ann NY Acad Sci 190:382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USD Department of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system, each CDR within the antibody heavy chain molecule is typically located at amino acid positions 31 to 35 (CDR1) (which may contain one or two additional amino acids after position 35 (referred to as 35A and 35B in the Kabat numbering system)), amino acid positions 50 to 65 (CDR2), and amino acid positions 95 to 102 (CDR3). Using the Kabat numbering system, each CDR within the antibody light chain molecule is typically located at amino acid positions 24 to 34 (CDR1), amino acid positions 50 to 56 (CDR2), and amino acid positions 89 to 97 (CDR3). In some cases, the CDRs of the antibodies described herein were determined using the Kabat numbering system.

[0096] The phrases "Kabat-like amino acid position numbering," "Kabat position," and their grammatical variations refer to the numbering system used for heavy chain variable domains or light chain variable domains in antibody compilations, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to shortenings or insertions of the FW or CDR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insertion from H2 residue 52 onwards (residue 52a according to Kabat), and residues inserted from heavy chain FW residue 82 onwards (e.g., residues 82a, 82b, and 82c according to Kabat). See Table 3.

[0097] [Table 3] The Kabat numbering of residues for a given antibody can be determined by alignment in regions of homology between the sequence with the “standard” Kabat numbering and the sequence of the antibody. Alternatively, Chothia refers to the position of the structural loop (Chothia and Lesk, J.Mol.Biol.196:901-917(1987)). When the numbering is expressed using the Kabat numbering convention, the end of the Chothia CDR-H1 loop varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering system places insertions at H35A and H35B; if 35A or 35B is absent, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable region represents a compromise between Kabat CDR and Chothia structural loops and is used by Oxford Molecular's AbM antibody modeling software.

[0098] IMGT (ImMunoGeneTics) also provides a numbering system for immunoglobulin variable regions, including CDRs. For example, see Lefranc, MP et al., Dev. Comp. Immunol. 27:55-77 (2003), which is included as reference herein. The IMGT numbering system is based on the characterization of more than 5,000 sequence alignments, structural data, and hypervariable loops, facilitating the comparison of variable and CDR regions across all species. According to the IMGT numbering system, VH-CDR1 is located at positions 26 to 35, VH-CDR2 at positions 51 to 57, VH-CDR3 at positions 93 to 102, VL-CDR1 at positions 27 to 32, VL-CDR2 at positions 50 to 52, and VL-CDR3 at positions 89 to 97.

[0099] For all heavy chain constant region amino acid positions discussed in this disclosure, the numbering follows the EU index first described in Edelman et al., 1969, Proc. Natl. Acad. Sci. USA 63(1):78-85), which described the amino acid sequence of myeloma protein EU, the first sequenced human IgG1. Furthermore, the EU index by Edelman et al. is presented in Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda. Accordingly, the phrases "the EU index presented in Kabat," "Kabat's EU index," and "position according to the EU index presented in Kabat…" and their grammatical variations refer to the residue numbering system based on the human IgG1 EU antibody presented by Edelman et al. in Kabat 1991.

[0100] The numbering system used for the amino acid sequences of the variable domain (all of the heavy and light chains) and the constant region of the light chain is the one presented in Kabat 1991.

[0101] Antibodies may have any type of immunoglobulin molecule (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any subtype (e.g., IgD, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subtype (e.g., IgG1, IgG2, IgG3, and IgG4 in humans; and IgG1, IgG2a, IgG2b, and IgG3 in mice). Rabbit globulin, for example, IgG1, exists in many allotypes that differ from each other by at most a few amino acids. Antibodies disclosed herein may consist of any of the commonly known isotypes, subtypes, subtypes, and allotypes. In certain embodiments, the antibodies disclosed herein are IgG1, IgG2, IgG3, or IgG4 subtypes, or any hybrids thereof. In certain embodiments, the antibodies have human IgG1 subtype, human IgG2, or human IgG4 subtype.

[0102] "Antibodies" include, for example, spontaneously occurring and non-spontaneous antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human and non-human antibodies, totally synthetic antibodies; single-chain antibodies; single-specific antibodies; multispecific antibodies (including bispecific antibodies); tetramer antibodies containing two heavy chains and two light chain molecules; antibody light chain monomers; antibody heavy chain monomers; antibody light chain dimers; antibody heavy chain dimers; antibody light chain-antibody heavy chain pairs; intrabody antibodies; heteroconjugate antibodies; monovalent antibodies; camelized antibodies; affybody antibodies; anti-idiotype (anti-Id) antibodies (e.g., anti-anti-Id antibodies); and single-domain antibodies (sdAb) containing a binding molecule consisting of a single monomer variable antibody domain (e.g., VH domain or VL domain) capable of complete antigen binding (Harmen MMand Hard HJAppl Microbiol Biotechnol.77(1):13-22(2007)).

[0103] The terms “antigen-binding portion” and “antigen-binding fragment” of an antibody refer to one or more fragments of an antibody that possess the ability to specifically bind to an antigen (e.g., human FAM19A1). Such “fragments” may be, for example, about 8 to 1500 amino acids long, preferably about 8 to 745 amino acids long, preferably about 8 to 300 amino acids, for example, about 8 to 200 amino acids, or about 10 to 50 or 100 amino acids long. It has become clear that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Each example of a binding fragment included in the term “antigen-binding portion” of an antibody, for example, the anti-FAM19A1 antibody described herein, includes (i) a Fab fragment which is a monovalent fragment consisting of VL, VH, CL, and CH1 domains; (ii) an F(ab')2 fragment which is a bivalent fragment containing two Fab fragments linked by a disulfide linkage at a hinge region; (iii) an Fd fragment consisting of VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of the antibody and a disulfide-linked Fvs(sdFv); (v) a dAb fragment consisting of a VH domain (Ward et al., Nature 341:544-546 (1989)); and (vi) a separated complementarity-determining region (CDR), or (vii) a combination of two or more separated CDRs which may be joined by a synthetic linker. Furthermore, the two domains of the Fv fragment, VL and VH, are coded by separate genes, but these can be joined by a synthetic linker that can be created using recombination methods to form a single protein chain (known as single-chain Fv (scFv)) in which the VL and VH regions pair up to form a monovalent molecule; see, for example, Bird et al., Science 242:423-426 (1988); and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988). Such single-chain antibodies are also included in the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using prior art known to those skilled in the art, and each fragment is screened for usefulness in the same manner as intact antibodies.The antigen-binding portion can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulin.

[0104] The terms “variable region” and “variable domain” as used herein are interchangeable and are universal in the industry. The variable region typically refers to a portion of an antibody, generally a portion of the light chain or heavy chain, typically containing about 110 to 120 amino acids at the amino-terminus of the mature heavy chain, and about 90 to 115 amino acids in the mature light chain, which vary widely in sequence among antibodies and are used for the binding and specificity of a particular antibody to a particular antigen. The sequence variability is concentrated in a region called the complementarity-determining region (CDR), while a more highly conserved region within the variable domain is called the framework region (FR).

[0105] While not wishing to be limited to a specific mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for antigen-antibody interaction and specificity. In certain aspects, the variable region is a human variable region. In certain aspects, the variable region includes a rodent or murid CDR and a human framework region (FR). In certain aspects, the variable region is a primate (e.g., non-human primate) variable region. In certain aspects, the variable region includes a rodent or murid CDR and a primate (e.g., non-human primate) framework region (FR).

[0106] As used herein, the term “heavy chain (HC)” may, when used in relation to an antibody, refer to any distinct types of IgG subtypes, e.g., IgG1, IgG2, IgG3, and IgG4, based on the amino acid sequence of the constant domain, that give rise to the IgA, IgD, IgE, IgG, and IgM types of the respective antibodies, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ).

[0107] As used herein, the term “light chain (LC),” when used in relation to an antibody, may refer to any distinct type based on the amino acid sequence of the constant domain, such as kappa (κ) or lambda (λ). Light chain amino acid sequences are well known in the art. In certain manner, the light chain is a human light chain.

[0108] The terms "VL" and "VL domain" are used interchangeably to refer to the variable region of the antibody light chain.

[0109] The terms "VH" and "VH domain" are used interchangeably to refer to the variable region of the antibody's heavy chain.

[0110] The terms “constant region” or “constant domain” as used herein are interchangeable and have the common meaning in the industry. The constant domain is the carboxyl-terminal portion of the light and / or heavy chain that does not directly participate in the binding of the antibody to the antigen, for example, but can exhibit a variety of effector functions, such as interaction with the Fc receptor. The constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence than the immunoglobulin variable domain.

[0111] The "Fc region" (fragment-crystallizable region), "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 includes the constant region of the antibody excluding the first constant region immunoglobulin domain (e.g., CH1 or CL). In IgG, IgA, and IgD antibody isotypes, the Fc region contains two identical protein fragments derived from the second (CH2) and third (CH3) constant domains of the two heavy chains of the antibody; the IgM and IgE Fc regions contain three heavy chain constant domains (CH domains 2-4) in their respective polypeptide chains. In the case of IgG, the Fc region includes the immunoglobulin domains Cγ2 and Cγ3, and the hinge between Cγ1 and Cγ2. While the boundaries of the Fc region of immunoglobulin heavy chains can vary, the human IgG heavy chain Fc region is generally limited to the extension from the amino acid residue at position C226 or P230 (or the amino acid between these two amino acids) to the carboxyl terminus of the heavy chain, although the numbering follows the EU index, as in Kabat. The CH2 domain of the human IgG Fc region extends from approximately amino acid 231 to approximately amino acid 340, and the CH3 domain is located on the C-terminal side of the Cm domain in the Fc region, i.e., extending from approximately amino acid 341 to approximately amino acid 447 of IgG. The Fc region as used herein may be a native sequence Fc containing any allogeneic variant, or a variant Fc (e.g., non-spontaneous Fc). Furthermore, Fc may refer to the region being isolated or to an Fc-containing protein polypeptide such as an "Fc-fusion protein" (e.g., an antibody or immunoadhesion).

[0112] A "natural sequence Fc region" or "natural sequence Fc" contains an amino acid sequence identical to that of a naturally occurring Fc region. Natural sequence human Fc regions include not only the natural sequence human IgG1 Fc region, the natural sequence human IgG2 Fc region, the natural sequence human IgG3 Fc region, and the natural sequence human IgG4 Fc region, but also their naturally occurring variants. Natural sequence Fc includes a diverse range of allogeneic types of each Fc (see, for example, Jefferis et al., mAbs 1:1 (2009); Vidarsson G. et al. Front Immunol. 5:520 (2014)).

[0113] An "Fc receptor" or "FcR" is a receptor that binds to the Fc region of immunoglobulins. FcRs that bind to IgG antibodies include the Fcγ family of receptors, which includes allelic variants and other spliced ​​forms of these receptors. The Fcγ family consists of three activating receptors (FcγRI, FcγRIII, and FcγRIV in mice; FcγRIA, FcγRIIA, and FcγRIIIA in humans) and one inhibitory receptor (FcγRIIB). Human IgG1 binds to most human Fc receptors and elicits the strongest Fc effector function. Human IgG1 can be considered equivalent to rat IgG2a, depending on the type of activating Fc receptor it binds to. Conversely, human IgG4 elicits the least Fc effector function (Vidarsson G. et al. Front Immunol. 5:520 (published online October 20, 2014)).

[0114] The constant region may be manipulated, for example, by recombinant technology, to remove one or more effector functions. “Effector function” refers to the interaction between the antibody Fc region and an Fc receptor or ligand, or the biochemical reaction therefrom. Exemplary “effector functions” include FcγR-mediated effector functions such as C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, ADCC, and antibody-dependent cell-mediated phagocytosis (ADCP), and downward regulation of cell surface receptors (e.g., B cell receptors; BCRs). 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 in which one or more effector functions mediated by the Fc region are reduced or absent.

[0115] The effector function of an antibody can be reduced or avoided by different approaches. This effector function can be reduced or avoided by using antibody fragments lacking the Fc region (e.g., Fab, F(ab')2, single-chain Fv(scFv), or sdAb consisting of monomeric VH or VL domains). Alternatively, so-called aglycosylated antibodies can be produced by removing sugars linked to specific residues in the Fc region, thereby reducing the antibody's effector function, while retaining other valuable properties of the Fc region (e.g., long half-life and heterodimerization). Aglycosylated antibodies can be produced, for example, by deleting or altering the sugar-bound residue, by enzymatically removing the sugar, by producing antibodies in cells cultured in the presence of a glycosylation inhibitor, or by expressing antibodies in cells incapable of protein glycosylation (e.g., bacterial host cells). See, for example, U.S. Patent Publication No. 20120100140. Another approach involves using the Fc region of IgG subtypes with reduced effector function; for example, IgG2 and IgG4 antibodies are characterized by having an even lower level of Fc effector function than IgG1 and IgG3. The residue closest to the hinge region in the CH2 domain of the Fc portion is responsible for the antibody's effector function because it contains a binding site that overlaps significantly with C1q (complement) and the IgG-Fc receptor (FcγR) on effector cells of the innate immune system (Vidarsson G. et al. Front Immunol. 5:520 (2014)). Therefore, antibodies with reduced or absent Fc effector function can be produced, for example, by creating a chimeric Fc region containing the CH2 domain of an IgG4 isotype IgG antibody and the CH3 domain of an IgG1 isotype IgG antibody, or a chimeric Fc region containing the hinge region of IgG2 and the CH2 region of IgG4 (see, e.g., Lau C. et al. J.Immunol. 191:4769-4777 (2013)), or by generating an Fc region with mutations that alter Fc effector function, for example, by reducing or eliminating Fc function. Such Fc regions with mutations are publicly known in the art.For example, refer to U.S. Patent Publication No. 20120100140 and the U.S. applications cited therein, each PCT application, and An et al., mAbs 1:6, 572-579 (2009).

[0116] The terms "hinge," "hinge domain," "hinge region," or "antibody hinge region" refer to the domain of the heavy chain constant region, including the upper, middle, and lower parts of the hinge, formed by the conjugation of the CH1 domain with the CH2 domain (Roux et al., J.Immunol. 161:4083 (1998)). The hinge provides a change in the level of flexibility between antibody binding and the effector region, and also provides a site for intermolecular disulfide bonding between the two heavy chain constant regions. The hinge used herein for all IgG isotypes begins at Glu216 and ends at Gly237 (Roux et al., J Immunol 161:4083 (1998)). The sequences of the wild-type IgG1, IgG2, IgG3, and IgG4 hinges are known in the art. For example, see Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USD Department of Health and Human Services, NIH Publication No. 91-3242; Vidarsson G. et al., Front Immunol. 5:520 (published online October 20, 2014).

[0117] The term "CH1 domain" refers to the heavy chain constant region that hinges a variable domain to a heavy chain constant domain. As used herein, the CH1 domain begins at A118 and ends at V215. The term "CH1 domain" includes not only the wild-type CH1 domain but also its naturally occurring variants (e.g., allogeneic variants). The CH1 domain sequences of IgG1, IgG2, IgG3, and IgG4 (including wild-type and allogeneic variants) are publicly known in the art (e.g., Kabat EA et al., (1991) and Vidarsson G. et al., Front Immunol. 5:520 (see online publication October 20, 2014)). Exemplary CH1 domains include, for example, CH1 domains with mutations that alter the half-life of antibodies described in U.S. Patent Publication 20120100140 and the U.S. patents, publications, and PCT publications cited herein.

[0118] The term "CH2 domain" refers to the heavy chain constant region that hinges to the CH3 domain in the heavy chain constant domain. As used herein, the CH2 domain begins at P238 and terminates at K340. The term "CH2 domain" includes not only the wild-type CH2 domain but also its naturally occurring variants (e.g., allogeneic variants). The CH2 domain sequences of IgG1, IgG2, IgG3, and IgG4 (including wild-type and allogeneic variants) are publicly known in the art (e.g., Kabat EA et al., (1991) and Vidarsson G. et al., Front Immunol. 5:520 (see online publication October 20, 2014)). Exemplary CH2 domains include, for example, CH2 domains having mutations that alter the biological activity of antibodies described in U.S. Patent Publication 20120100140 and the U.S. patents, publications, and PCT publications cited herein, such as half-life and / or reduced Fc effector function.

[0119] The term "CH3 domain" refers to the heavy chain constant region that is C-terminus relative to the CH2 domain. As used herein, the CH3 domain begins at G341 and terminates at K447. The term "CH3 domain" includes not only the wild-type CH3 domain but also its naturally occurring variants (e.g., allogeneic variants). The CH3 domain sequences of IgG1, IgG2, IgG3, and IgG4 (including wild-type and allogeneic variants) are publicly known in the art (e.g., Kabat EA et al., (1991) and Vidarsson G. et al., Front Immunol. 5:520 (online publication October 20, 2014)). Exemplary CH3 domains include, for example, CH3 domains with mutations that alter the half-life of antibodies described in U.S. Patent Publication 20120100140 and the U.S. patents, publications, and PCT publications cited herein.

[0120] As used herein, "isotype" refers to the type of antibody coded by a heavy chain constant region gene (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibodies).

[0121] A "homogeneic variant" refers to a spontaneously occurring variant within a specific isotype group that differs in several amino acids (see, e.g., Jefferis et al., (2009) mAbs 1:1). The antibodies described herein may have any allogeneic variant. Allogeneic variants of IgG1, IgG2, IgG3, and IgG4 are publicly known in the art. See, for example, Kabat EA et al., (1991); Vidarsson G. et al., Front Immunol. 5:520 (published online October 20, 2014); and Lefranc MP, mAbs 1:4, 1-7 (2009).

[0122] The phrases "antibody that recognizes an antigen" and "antibody that is specific to an antigen" are used interchangeably in this specification with the term "antibody that specifically binds to an antigen."

[0123] As used herein, an "isolated antibody" means an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to FAM19A1 is substantially free of antibodies that specifically bind to antigens other than FAM19A1). However, an isolated antibody that specifically binds to an epitope of FAM19A1 can have cross-reactivity with other FAM19A1 proteins of different species.

[0124] "Binding affinity" generally means the overall strength of the interaction between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless presented in a different form, as used herein, "binding affinity" means the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for partner Y can generally be expressed by the dissociation constant (K D ). Affinity can be measured and / or expressed in a number of ways known in the art, including, but not limited to, the equilibrium dissociation constant (K D ) and the equilibrium association constant (K A ). The K D is calculated from the ratio of k off / k on and is expressed as molar concentration (M), and the K A is calculated from the ratio of k on / k off . k on means, for example, the binding rate constant of an antibody to an antigen, and k off means, for example, the dissociation of an antibody from an antigen. k on and k off can be determined by techniques known to those of skill in the art, such as immunoassays (e.g., enzyme-linked immunosorbent assay (ELISA)), BIACORE TM or kinetic exclusion assay (KINEXA®).

[0125] As used herein, the terms “specifically binding,” “specifically recognized,” “specific binding,” “selective binding,” and “selectively binding” are analogous terms in relation to antibodies and refer to molecules (e.g., antibodies) that bind to antigens (e.g., epitopes or immune complexes), and such binding is understood by those skilled in the art. For example, molecules that specifically bind to antigens are used in immunoassays, biorecommended immunoassays, etc. TM When determined by the KINEXA® 3000 instrument (Sapidyne Instruments, Boise, ID) or other analytical methods known to the industry, it is generally possible to bind to other peptides or polypeptides with even lower affinity. In some forms, molecules that specifically bind to an antigen may, when this molecule binds to another antigen, A At least 2logs, 2.5logs, 3logs, 4logs or more, and even larger K A It then binds to the antigen.

[0126] Antibodies are typically 10 -5 M to 10 -11 Dissociation constants (K) less than or equal to M D It specifically binds to these cognate antigens with a high affinity, as reflected by ). -4 Any K greater than M D This is generally considered to mean nonspecific binding. As used herein, an antibody that "specifically binds" to an antigen refers to an antibody that binds to the antigen and substantially the same antigen with high affinity, for example, a BIACORE using the aforementioned antigen. TM When determined by immunoassay (e.g., ELISA) or surface plasmon resonance (SPR) techniques using 2000 instruments, 10 -7 M or less, preferably 10 -8 M or less, far more preferably 10 -9 M or less, most preferably 10 -8 M to 10 -10 K below M D This means that the antibody possesses the necessary properties, but it does not bind to unrelated antigens with high affinity.

[0127] As used herein, “antigen” refers to any natural or synthetic immunogenic substance, such as a protein, peptide, or hapten. The antigen may be FAM19A1 or a fragment thereof.

[0128] As used herein, “epitope” is an industry term referring to a local region of an antigen to which an antibody can specifically bind. An epitope may be, for example, an adjacent amino acid of a polypeptide (linear or adjacent epitope), or an epitope may be, for example, a polypeptide or a combination of two or more non-adjacent regions of each polypeptide (stereomorphic, nonlinear, discontinuous, or non-adjacent epitope). Epitopes formed from adjacent amino acids are typically maintained when exposed to a denaturing solvent, although this is not always the case, while epitopes formed by tertiary folding are typically lost when treated with a denaturing solvent. An epitope typically contains at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or twenty amino acids within a specific spatial stereomorphism. Methods for determining which epitopes are bound by a given antibody (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation analysis for testing the reactivity of superimposed or adjacent peptides (e.g., FAM19A5) with a given antibody (e.g., anti-FAM19A1 antibody). Methods for determining the spatial stereomorphism of epitopes include art in the art and art described herein, e.g., X-ray crystallography, two-dimensional nuclear magnetic resonance and HDX-MS (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GEMorris, Ed. (1996)).

[0129] In a particular manner, the epitopes to which the antibodies bind can be determined, for example, by NMR spectroscopy, X-ray diffraction crystallography studies, ELISA analysis, hydrogen / deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography-electron atomization mass spectrometry), array-based oligo-peptide scanning analysis, and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). In the case of X-ray crystallography, crystallization can be achieved using any method known to the art (see, for example, Giege R et al., Acta Crystallogr D Biol Crystallogr 50(Pt 4):339-350; McPherson A Eur J Biochem 189:1-23(1990); Chayen NE Structure 5:1269-1274(1997); McPherson AJ Biol Chem 251:6300-6303(1976)). Antibody: Antigen crystals can be studied using well-known X-ray diffraction techniques and their structure can be refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; e.g., Meth Enzymol (1985) volumes 114 & 115, eds Wyckoff H. Wet al.,; see US2004 / 0014194) and BUSTER (Bricogne G. Acta Crystallogr D Biol Crystallogr 49(Pt1):37-60(1993); Bricogne G. Meth Enzymol 276A:361-423(1997), ed Carter CW; Roversi P. et al., Acta Crystallogr D Biol Crystallogr 56(Pt 10):1316-1323(2000)). Mutagenesis mapping studies can be carried out using any method known to those skilled in the art.For descriptions of mutagenesis techniques, including alanine scanning mutagenesis, refer to, for example, Champe M. et al., J Biol Chem 270 (1995):1388-1394 and Cunningham BC & Wells JA Science 244:1081-1085 (1989).

[0130] The term "epitope mapping" refers to the process of identifying molecular determinants of antibody-antigen recognition.

[0131] In relation to two or more antibodies, the term “binding to the same epitope” means that each antibody binds to the same segment of amino acid residues, as determined by a predetermined method. Techniques for determining whether each antibody binds to the “same epitope on FAM19A1” as the antibodies described herein include epitope mapping methods, e.g., X-ray analysis of the antigen:antibody complex crystals and hydrogen / deuterium exchange mass spectrometry (HDX-MS) that provide atomic resolution of the epitopes. Other methods involve monitoring the binding of antibodies to antigen fragments or mutant variants of antigens, where binding loss due to deformation of amino acid residues in the antigen sequence is generally considered an indication of the epitope component. Computerized combination methods for epitope mapping can also be used. These methods depend on the ability of the antibody of interest to affinity-separate specific short peptides from a combination phage display peptide library. Antibodies having the same VH and VL or the same CDR1, 2, and 3 sequences are expected to bind to the same epitopes.

[0132] An antibody that “competes with other antibodies for binding to a target” refers to an antibody that (partially or completely) inhibits the target binding of the other antibody. Whether two antibodies compete with each other for binding to a target, that is, whether one antibody inhibits the target binding of another antibody and to what extent, can be determined using known competition experiments. In a particular manner, one antibody competes with the other antibody for target binding, inhibiting this binding by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. The level of inhibition or competition may differ depending on whether the antibody is a “blocking antibody” (i.e., a cold antibody cultured before the target). Competitive analysis can be performed as described in Chapter 11 of "Using Antibodies" by Ed Harlow and David Lane, Cold Spring Harbor Protoc; 2006; doi:10.1101 / pdb.prot4277 or Ed Harlow and David Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA 1999. Competitive antibodies bind to the same epitope, superimposed epitope, or adjacent epitope (e.g., one demonstrated by steric hindrance).

[0133] Other competitive coupling analyses include solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competitive analysis (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 analysis, solid-phase direct labeling sandwich analysis (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid-phase direct labeling RIA using 1-125 labeling (see Morel et al., Mol.Immunol. 25(1):7 (1988)); solid-phase direct biotin-avidin EIA (see Cheung et al., Virology This includes 176:546 (1990) and directly labeled RIA (see Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)).

[0134] A "dual-specific" or "dual-functional" antibody is an artificial hybrid antibody having two distinct heavy / light chain pairs and two distinct binding sites. Dual-specific antibodies can be produced by a variety of methods, including hybridoma fusion or Fab' fragment linking. See, for example, Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992).

[0135] As used herein, "monoclonal antibody" refers to an antibody that exhibits single-binding specificity and affinity to a specific epitope, or an antibody composition in which all antibodies exhibit single-binding specificity and affinity to a specific epitope. Therefore, the term "human monoclonal antibody" refers to an antibody or antibody composition that exhibits single-binding specificity and has variable and selective constant regions derived from human germline immunoglobulin sequences. In some forms, human monoclonal antibodies are produced, for example, by a transgenic non-human animal having a genome containing human heavy chain and light chain transgenes fused to immortalized cells, such as a hybridoma containing B cells obtained from a transgenic mouse.

[0136] As used herein, the term “recombinant human antibody” includes all human antibodies manufactured, expressed, produced or isolated by recombinant means, such as (a) antibodies isolated from or produced from transchromosomal animals (e.g., mice) that have been transchromosomal against human immunoglobulin genes, (b) antibodies isolated from, for example, transfectomas, from host cells transformed to express antibodies, (c) antibodies isolated from recombinant, combined human antibody libraries, and (d) antibodies manufactured, expressed, produced or isolated by any other means involving splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies use specific human germline immunoglobulin sequences coded by germline genes, but include variable and constant regions, such as subsequent rearrangements and mutations that occur during antibody maturation. As is well known in the industry (see, for example, Lonberg Nature Biotech. 23(9):1117-1125 (2005)), the variable region contains antigen-binding domains coded by a variety of genes rearranged to form antibodies specific to foreign antigens. In addition to rearrangement, the variable region can be further modified by numerous single-amino acid changes (referred to as somatic mutations or hypermutations) to increase the affinity of antibodies to foreign antigens. The constant region can be altered by additional reactions to the antigen (i.e., isotypic changes). Therefore, rearranged and somatically mutated nucleic acid molecules that code light-chain and heavy-chain immunoglobulin polypeptides in response to an antigen may not have sequence identity with the original nucleic acid molecule, but may be substantially identical or similar (i.e., have at least 80% identity).

[0137] A “human” antibody (HuMAb) refers to an antibody having a variable region in which the framework and CDR region are entirely derived from a human germline immunoglobulin sequence. Furthermore, if the antibody contains a constant region, the constant region is also derived from a human germline immunoglobulin sequence. Antibodies described herein may contain amino acid residues not coded by a human germline immunoglobulin sequence (e.g., mutations introduced by random or site-directed mutagenesis in vitro, or by somatic mutation in vivo). However, as used herein, the term “human antibody” does not include antibodies in which a CDR sequence derived from the germline of another mammalian species, such as mouse, has been transplanted into a human framework sequence. The terms “human” antibody and “completely human” antibody are used synonymously.

[0138] A "humanized" antibody is an antibody in which some, almost all, or all of the amino acids outside the CDR domain of a non-human antibody are replaced with corresponding amino acids derived from human immunoglobulins. In some forms, some, almost all, or all of the amino acids outside the CDR domain are replaced with amino acids from human immunoglobulins, while some, almost all, or all of the amino acids inside one or more CDR regions remain unchanged. Small additions, deletions, insertions, substitutions, or modifications of amino acids are acceptable as long as they do not eliminate the antibody's ability to bind to a particular antigen. Humanized antibodies maintain antigen specificity similar to that of the proto-antibody.

[0139] A "chimeric antibody" refers to an antibody in which the variable region originates from one species and the constant region originates from another species, such as an antibody in which the variable region originates from a mouse antibody and the constant region originates from a human antibody.

[0140] As used herein, the term "cross-reactivity" refers to the ability of an antibody described herein to bind to a different species of FAM19A1. For example, an antibody described herein that binds to human FAM19A15 may also bind to FAM19A1 of another species (e.g., mouse FAM19A1). As used herein, cross-reactivity may be measured by detecting specific reactivity with an antigen purified by binding analysis (e.g., SPR, ELISA), or by binding to or, if not, functionally interacting with cells that physiologically express FAM19A1. Methods for determining cross-reactivity include standard binding analyses described herein, e.g., BIACORE TM BIACORE using 2000 SPR equipment (Biacore AB, Uppsala, Sweden) TM This includes surface plasmon resonance (SPR) analysis or flow cytometry techniques.

[0141] When the term “spontaneous occurrence” is applied to a subject herein, it means the fact that the subject can be found in nature. For example, polypeptides or polynucleotide sequences present in organisms (including viruses) that can be isolated from a natural source and are not intentionally altered by humans in a laboratory are spontaneously occurring.

[0142] A "polypeptide" refers to a chain containing at least two consecutively linked amino acid residues, and there is no upper limit to the length of the chain. One or more amino acid residues within a protein may, but are not limited to, undergo deformations such as glycosylation, phosphorylation, or disulfide bond formation. A "protein" may contain one or more polypeptides.

[0143] As used herein, the term "nucleic acid molecule" includes DNA molecules and RNA molecules. Nucleic acid molecules may be single-stranded or double-stranded, and may be cDNA.

[0144] As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting other ligated nucleic acids. One type of vector is a “plasmid,” which refers to a circular double-stranded DNA loop to which additional DNA segments can be ligated. Another type of vector is a viral vector, to which additional DNA segments can be ligated to a viral genome. Certain vectors can self-replicate in the host cell into which they are introduced (e.g., bacterial vectors and episomal mammalian vectors with a bacterial replication origin). Other vectors (e.g., non-episomal mammalian vectors), when introduced into a host cell, can be integrated into the host cell's genome, thereby being replicated by the host genome. Furthermore, certain vectors can direct the expression of genes to which they are operatively ligated. Such vectors are referred herein as “recombinant expression vectors” (or simply “expression vectors”). Generally, expression vectors that are useful in recombinant DNA technology are often in the form of plasmids. In this specification, “plasmid” and “vector” may be used interchangeably, as plasmids are the most commonly used form of vectors. However, this also includes other forms of expression vectors that perform equivalent functions, such as viral vectors (e.g., replication-deficient retroviruses, adenoviruses, and adeno-associated viruses).

[0145] As used herein, the term “recombinant host cell” (or simply “host cell”) refers to a cell that contains nucleic acids not naturally present within it, and may be a cell into which a recombinant expression vector has been introduced. These terms should be understood to refer not only to specific target cells but also to the offspring of such cells. Because mutations and environmental influences can cause specific deformations in the next generation, such offspring may not be identical to the parent cells, but they are still included within the scope of the term “host cell” as used herein.

[0146] As used herein, the term "conjugated" refers to the association of two or more molecules. Such conjugations may be covalent or non-covalent. Furthermore, such conjugations may be gene conjugations (i.e., fusions by recombination). Such conjugations may be achieved using a variety of techniques recognized in the industry, such as chemical conjugation and recombinant protein production.

[0147] As used herein, the term “therapeutic dose” refers to the amount of a drug, alone or in combination with other therapeutic agents, that is effective in “treating” a CNS-related disorder or disability in a subject, or in reducing the risk, potential, likelihood, or incidence of a CNS-related disorder or disability. “Therapeutic dose” includes the amount of a drug or therapeutic agent that provides some improvement or practical benefit to a subject who has or is at risk of having a CNS-related disorder or disability. Accordingly, “therapeutic dose” is the amount that reduces the risk, potential, likelihood, or incidence of a CNS-related disorder, or provides relief and mitigation, or reduces at least one indicator, or reduces at least one clinical symptom of a CNS-related disorder or disability. Non-limiting examples of CNS-related disorders or disabilities are provided in other parts of this disclosure.

[0148] As used herein, the terms “to treat” and “treatment” mean any type of intervention or process performed on a subject with the aim of reversing, alleviating, improving, inhibiting, delaying or preventing the progression, development, severity or recurrence of disease-related symptoms, complications, symptoms or biochemical signs, or the administration of an activating agent to a subject. Treatment may be performed on a subject with the disease or on a subject without the disease (e.g., for preventative purposes).

[0149] As used herein, the “central nervous system” (CNS) refers to the nervous system, including the brain and spinal cord. The CNS may further include the retina and optic nerve (cranial nerve II), the olfactory nerve (cranial nerve I), and the olfactory epithelium. The brain is the primary control module of the CNS and can be broadly divided into four lobes: (1) the temporal lobe (processes sensory input and assigns it to emotional meaning; establishes long-term memory and is important in some language recognition); (2) the occipital lobe (the visual processing area of ​​the brain, housing the visual cortex); (3) the parietal lobe (integration of sensory information, including touch, spatial awareness, and navigation; language recognition); and (4) the frontal lobe (contains most of the dopamine-sensitive nerve cells and is therefore involved in concentration, reward, short-term memory, motivation, and conception).

[0150] The brain can be further divided into other regions: (1) basal ganglia (involved in voluntary movement, procedural learning, and control of decision-making regarding which motor activity to perform; diseases affecting this region include Parkinson's disease and Huntington's disease); (2) cerebellum (involved in precise motor control, language, and concentration; damage to this region results in motor control disorders known as ataxia); (3) Broca's area (involved in language processing; damage to this region can result in language disorders); (4) corpus callosum (an extensive band of nerve fibers connecting the left and right hemispheres; children with dyslexia are known to have an even smaller corpus callosum); (5) medulla oblongata (involved in involuntary functions such as vomiting, breathing, sneezing, and maintaining accurate blood pressure); (6) hypothalamus (secretes a variety of neurohormones that affect thermoregulation, thirst, and hunger); (7) thalamus (receives sensory and motor input and transmits information to the rest of the cerebral cortex); and (8) amygdala (located in the temporal lobe and involved in decision-making, memory, and emotional responses).

[0151] As used herein, the term “spinal marrow” refers to a long, thin, tubular structure composed of nerve tissue extending from the medulla oblongata of the brainstem to the lumbar region of the spine. Non-limiting examples of spinal marrow functions include (1) connecting most of the peripheral nervous system to the brain and being responsible for signal transmission between the brain and peripheral tissues; and (2) acting as a small regulatory center responsible for several simple reflexes, such as the retraction reflex.

[0152] As used herein, the term “nerve cell” includes a nerve cell and any part or each part thereof (e.g., the cell body, axon, or dendrite). The term “nerve cell” means a nervous system cell that includes a central cell body or somatic cell and two types of extensions or projections: dendrites, which generally transmit most nerve signals to the cell body, and axons, which generally transmit most nerve signals from the cell body to target nerve cells or effector cells such as muscle cells.

[0153] As used herein, the term "neurite" refers to any projection from the cell body of a nerve cell (e.g., an axon or dendrite).

[0154] As used herein, “administration” means the physical introduction of a therapeutic agent or a composition containing a therapeutic agent into a subject using any of the various methods and delivery systems known to those skilled in the art. Preferred routes of administration for antibodies described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, intravitreal, or other parenteral administration routes, such as by injection or infusion. As used herein, “parenteral administration” generally means, but is not limited to, methods of administration other than intestinal and local administration by injection, including intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, intratracheal, pulmonary, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, intravitreous, epidural, and intrasternal injections and infusions, as well as intra vivo electroporation. In contrast, the antibodies described herein may be administered via parenteral routes, such as topical, cutaneous, or mucosal routes, such as intranasal, oral, vaginal, rectal, sublingual, or topical administration. Furthermore, administration may be performed, for example, once, multiple times, and / or over one or more extended periods.

[0155] As used herein, the term “diagnosis” means a method that can be used to determine or predict whether a patient has a given disease or condition and to identify a subject suitable for treatment. A person skilled in the art can make a diagnosis based on one or more diagnostic markers (e.g., FAM19A1), where the presence, absence, amount, or change in amount of the diagnostic markers indicates the presence, severity, or absence of a condition. In some manner, increased FAM19A1 expression in a biological sample of a subject indicates a CNS-related disease or disorder. The term “diagnosis” does not mean the ability to determine with 100% accuracy whether a particular disease or disorder is present or absent, nor does it mean that a given process or result is more likely to occur than it is unlikely to occur. Instead, a person skilled in the art will understand that the term “diagnosis” means a high probability that a particular disease or disorder is present in a subject. In some manner, the term “diagnosis” includes one or more diagnostic methods to identify a subject with a CNS-related disease or disorder. Non-limiting examples of CNS-related diseases or disorders are provided in other parts of this disclosure.

[0156] A composition for diagnosing CNS dysfunction comprises a formulation for measuring the protein level of FAM19A1 or the nucleic acid (e.g., mRNA) coding for FAM19A1 in a sample of the subject of interest (e.g., a subject suspected of having CNS dysfunction). Such a formulation comprises an oligonucleotide, primer, or nucleic acid probe having a sequence complementary to FAM19A1 mRNA, and an antibody or antigen-binding fragment thereof that specifically binds to the FAM19A1 protein.

[0157] 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-human primates, and non-mammals such as sheep, dogs, cattle, chickens, amphibians, and reptiles. As described herein (e.g., in the Examples), the beneficial effects of the FAM19A1 antagonists disclosed herein are not sex-dependent. Therefore, in certain aspects, the subjects who can benefit from the FAM19A1 antagonists disclosed herein (e.g., improvement of CNS function or treatment of CNS-related diseases or disorders) are male subjects. In certain aspects, the term "male" refers to an individual having X and Y chromosomes. In certain aspects, the subjects who can benefit from the FAM19A1 antagonists disclosed herein (e.g., improvement of CNS function or treatment of CNS-related diseases or disorders) are female subjects. In certain aspects, the term "female" refers to an individual having two X chromosomes. In certain aspects, the subjects who can obtain practical benefits from the FAM19A1 antagonists disclosed herein (e.g., improvement of CNS function or treatment of CNS-related diseases or disorders) include both male and female subjects.

[0158] 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 central nervous system (CNS) and the ganglia of the peripheral nervous system (PNS), and can be interconnected to form neural networks. A typical neuron consists of a somatic cell (cell body), dendrites, and axon. The somatic cell (cell body) of a neuron contains a nucleus. The dendrites of a neuron are branched cell extensions where most of the neuron's input occurs. The axon is a relatively fine, cable-like projection extending from the somatic cell that transmits nerve signals far from the somatic cell and retransmits specific types of information back to the somatic cell. The term “promoting neuronal regrowth” preferably includes stimulating, promoting, increasing, or activating the growth of neurons after injury or damage.

[0159] The term "glaucoma" refers to a group of eye-related diseases and / or disorders characterized by progressive loss of retinal ganglion cells and optic nerve atrophy. Glaucoma may be associated with optic nerve damage, loss of retinal ganglion cells ("RGCs"), elevated intraocular pressure ("IOP"), damaged blood-retinal barrier, and / or increased microglial activity levels within the retina and / or optic nerve of the subject. Glaucoma may be asymptomatic or present with one of the following eye-related symptoms: burning or stinging sensation, laceration, dryness, fatigue, visual field clouding / dim vision, tunnel vision, day blindness, night blindness, halo around lighting, and / or blindness. Lee et al., Arch Ophthalmol 116:861-866 (1998). The term "glaucoma" includes all types of glaucoma, regardless of the cause and any or all of the symptoms.

[0160] The term “glaucoma” includes, but is not limited to, the following types of glaucoma: open-angle glaucoma, closed-angle glaucoma, normal-tension glaucoma ("NTG"), congenital glaucoma, secondary glaucoma, pigmentary glaucoma, pseudoexfoliation glaucoma, traumatic glaucoma, neovascular glaucoma, iris-corneal endothelial syndrome, and uveitis glaucoma. Some of the aforementioned risk factors include elevated intraocular pressure, genetic predisposition (e.g., family history of glaucoma, certain races), age, diabetes, hypertension, and / or physical injury to the eye.

[0161] The term "inflammation" refers to a complex response of the innate immune system in vascularized tissue, involving the accumulation and activation of immune cells (e.g., microglia) and plasma proteins at the site of injury and / or damage. In healthy individuals, the blood-retinal barrier provides a robust barrier that prevents substances from flowing freely from the blood to the retina and vice versa. However, in glaucoma patients, this barrier is damaged. Such vascular dysregulation restricts blood flow to the optic nerve head of the retina, allowing the production of various inflammatory mediators (e.g., TNF-α, IL-6, IL-9, IL-10, and nitric oxide) that can freely move to the optic nerve head.

[0162] As used herein, the term "optic nerve" refers to the paired nerve that transmits visual information from the retina to the brain. In humans, the optic nerve originates from the optic stalk within 7 weeks of development and is composed of retinal ganglion cell axons and glial cells. The optic nerve extends from the optic disk to the optic chiasm and connects to the lateral geniculate nucleus, pretectal nucleus, and superior colliculus as the visual pathway. Selhorst, JB, et al., Semin Neurol 29(1):29-35 (2009).

[0163] The term "optic nerve injury" refers to any change in the normal structure or function of the optic nerve. Changes in the normal structure or function of the optic nerve can result from any disease, disorder, or injury, including glaucoma. Changes in the normal function of the optic nerve include all changes in the optic nerve's ability to function properly in transmitting visual information from the retina to the brain. Functional changes can be described, for example, as visual field loss, central visual acuity impairment, or abnormal color vision. Examples of structural changes include loss of nerve fibers in the retina, abnormal cupping of the optic nerve, and / or loss of cells from the retinal ganglion cell layer of the retina. As used herein, "optic nerve injury" may include optic nerve injury to one or all of the optic nerves in question.

[0164] Retinal ganglion cells (RGCs) refer to a specific type of nerve cell located near the innermost layer of the retina (i.e., the ganglion cell layer). These cells play a crucial role in transmitting visual information collected from photoreceptors to the brain. Sanes et al., Annu Rev Neurosci 38:221-46 (2015). While RGCs can vary considerably in size, connectivity, and responsiveness to visual stimuli, they all share the defining characteristic of possessing long axons that extend to the brain.

[0165] The term "intraocular pressure" (IOP) refers to the pressure maintained inside the eye. The anterior chamber of the eye is surrounded by the cornea, iris, pupil, and lens, and is filled with aqueous humor, which supplies oxygen and nutrients to the cornea and lens. This aqueous humor provides the pressure necessary to help maintain the shape of the eye. If the normal secretion of aqueous humor is disrupted, it will affect intraocular pressure.

[0166] The term "microglia or microglial cells" refers to a type of glial cell present in the retina. These cells behave like macrophages, continuously exploring the surrounding microenvironment for external antigens and / or damage. Upon such recognition, microglia are activated and rapidly respond to foreign antigens and / or damage by limiting damage through phagocytosis of any potentially harmful debris, secreting inflammatory mediators, and generating an effective immune response through interaction with other immune cells. (Kettenmann et al., Physiol Rev 91(2):461-553 (2011)). Activated microglia are distinguished from resting microglia by increased surface expression of Iba-1. Microglia are also involved in programmed cell death in the developing retina, and nerve growth factor (NGF) released by microglia can induce retinal neuronal death. Ashwell et al., Visual Neuroscience 2(5):437-448 (1989).

[0167] The term "neuropathic pain" refers to pain resulting from injury, damage, and / or improper function affecting any part of the central nervous system (CNS) and / or peripheral nervous system. The term "neuropathic pain" includes all types of neuropathic pain, regardless of the cause of the pain or any or all of the symptoms.

[0168] Neuropathic pain includes central neuropathic pain and peripheral neuropathic pain. As used herein, the term "central neuropathic pain" refers to pain resulting from a disorder, congenital defect, or injury to the central nervous system (i.e., the brain or spinal cord). As used herein, the term "peripheral neuropathic pain" refers to pain resulting from injury to or infection of peripheral sensory nerves.

[0169] Symptoms of neuropathic pain can include not only persistent / chronic pain and spontaneous pain, but also allodynia (e.g., a pain response to stimuli that are normally painless), hyperalgesia (e.g., a marked response to painful stimuli that generally cause only mild discomfort, such as being pricked with a pin), hyperesthesia (e.g., excessive physical hypersensitivity to stimuli, especially skin stimuli), or hyperalgesia (e.g., short-term discomfort can develop into prolonged, severe pain). In some forms, symptoms can persist for a long time and, if a primary cause was present, may persist after that cause has been resolved. Available in Merck Manual, Neuropathic Pain, merckmanuals.com / professional / neurologic-disorders / pain / neuropathic-pain; Campbell JNand Meyer RANeuron 52(1):77-92(2006).

[0170] As used herein, “mononeurosis” is a peripheral neuropathy characterized by loss of motor or sensory function in an area resulting from damage or destruction of a single peripheral nerve or group of nerves. Mononeuropathy most frequently occurs as a result of injury or trauma to a local site, which, for example, causes prolonged pressure / compression on a single nerve. However, certain systemic disorders (e.g., polyconjunctival mononeuritis) can also cause mononeuropathy. In some forms, injury or trauma to the local site can cause destruction of part of the nerve’s myelin sheath (outer covering) or nerve cells (axons), delaying or preventing the conduction of stimuli through the nerve. In some forms, mononeuropathy can affect any part of the body. Examples of mononeuropathic pain include, but are not limited to, sciatic nerve dysfunction, common nasal nerve dysfunction, lumbar nerve dysfunction, ulnar nerve dysfunction, cranial mononeuropathy VI, cranial mononeuropathy VII, cranial mononeuropathy III (compression type), cranial mononeuropathy III (diabetic type), axillary nerve dysfunction, carpal tunnel syndrome, femoral nerve dysfunction, ossicular nerve dysfunction, facial nerve palsy (Bell's palsy), thoracic outlet syndrome, carpal tunnel syndrome, and abducens nerve 6 palsy. See also the Peripheral Neuropathy Fact Sheet from the National Institute of Neurological Disorders and Stroke, available at Finnerup NB et al., Pain 157(8):1599-1606(2016); ninds.nih.gov / disorders / peripheralneuropathy / detail_peripheralneuropathy.htm.

[0171] As used herein, “polyneuropathy” refers to peripheral neuropathy characterized by loss of motor or sensory function in an area resulting from damage or destruction of numerous peripheral nerves. Polyneuropathic pain includes, but is not limited to, post-polio syndrome, post-mastectomy syndrome, diabetic neuropathy, alcoholic neuropathy, amyloidosis, toxins, AIDS, hypothyroidism, uremia, vitamin deficiency, chemotherapy-induced pain, 2',3'-dideoxycytidine (ddC) treatment, Guillain-Barré syndrome, or Fabry disease. Finnerup NBet al., Pain 157(8):1599-1606(2016); National Institute of Neurological Disorders available at ninds.nih.gov / disorders / peripheralneuropathy / detail_peripheralneuropathy.htm You can refer to the Stroke, Peripheral Neuropathy Fact Sheet.

[0172] The term "disease-related neuropathic pain" refers to neuropathic pain that is associated with, caused by, or resulting from a disease or disorder (e.g., those disclosed herein).

[0173] [II. Methods of Disclosure] Methods of treating a disease or disorder This specification discloses FAM19A1 antagonists (e.g., antibodies) that can be used for therapeutic purposes (e.g., treatment of disease or disorder). As described herein, FAM19A1 is expressed in many areas of the CNS (e.g., neural circuits), and abnormal FAM19A1 expression is thought to result in impaired normal CNS function. As demonstrated throughout this application (e.g., examples), the applicant has found that the FAM19A1 antagonists disclosed herein can be used to improve one or more CNS functions. The applicant has also confirmed that the beneficial effects of the FAM19A1 antagonists disclosed herein are independent of the sex of the subject. Therefore, in some embodiments, the FAM19A1 antagonists disclosed herein can be used to treat male subjects (e.g., male subjects with impaired CNS function). In some embodiments, said subjects are not female subjects. In some embodiments, the FAM19A1 antagonists disclosed herein can be used to treat female subjects (e.g., female subjects with impaired CNS function). In certain embodiments, the FAM19A1 antagonists disclosed herein can be used to treat all male and female subjects (e.g., male and female subjects with impaired CNS function).

[0174] In certain aspects, the FAM19A1 antagonists disclosed herein can be used to treat CNS-related diseases or disorders. In certain aspects, the CNS-related diseases or disorders that can be treated with the foregoing are associated with abnormal neural circuits. As used herein, “neural circuit” refers to a group of nerve cells that are interconnected by synapses when activated and perform specific functions. Neural circuits can be interconnected to form large brain networks. Neural circuits are essential for the proper transmission of information from the brain to nerve cells. Furthermore, each different neural circuit is generally associated with a different function. Abnormalities in one or more neural circuits can lead to impairment of CNS function, as observed in a variety of CNS-related diseases or disorders.

[0175] In certain aspects, CNS-related disorders or conditions treatable with the FAM19A1 antagonists disclosed herein include mood disorders, psychiatric disorders, or all of these. In certain aspects, CNS-related disorders or conditions treatable with the disclosures include anxiety, depression, post-traumatic stress disorder (PTSD), bipolar disorder, attention deficit / hyperactivity disorder (ADHD), autism, schizophrenia, neuropathic pain, glaucoma, intoxication, arachnoid cysts, sclerosis, encephalitis, epilepsy / seizures, fixed syndromes, meningitis, migraines, multiple sclerosis, osteomyelopathy, Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Batten's disease, Tourette's syndrome, traumatic brain injury, spinal cord injury, stroke, tremor (essential or Parkinsonian), dysarthria, intellectual disability, brain tumors, or combinations thereof.

[0176] In certain aspects, CNS-related disorders or conditions treatable with the FAM19A1 antagonists disclosed herein are mood disorders. As used herein, “mood disorder” refers to all types of mental disorders affecting an individual’s emotional state. As used herein, “mood” refers to a person’s internal emotional state. In certain aspects, mood disorders treatable with respect to this disclosure may be characterized by prevailing mood and affect associated with behavioral, physiological, cognitive, neurochemical, and psychomotor dysfunction, and by prolonged feelings of helplessness. Mood disorders may be associated with persistent mood elevation (mania), persistent depressed mood, or alternating moods between mania and depression. Mood disorders may actually be genetic or may be triggered by secondary factors (e.g., disease, drugs, medications). Examples of mood disorders include, but are not limited to, major depressive disorder (MDD), bipolar disorder (BD), minor depressive disorder, persistent depressive disorder (low mood), seasonal affective disorder (SAD), psychotic depression, postpartum depression, recurrent short-term depressive disorder, premenstrual dysphoric disorder (PMDD), situational depression, atypical depression, anxiety disorders, and cyclothymic disorders.

[0177] As used herein, the term “major depressive disorder” or “MDD” refers to a mood disorder characterized by the occurrence of two or more major depressive symptoms. Symptoms of MDD may include fatigue, feelings of worthlessness, guilt, difficulty concentrating, indecisiveness, insomnia or hypersomnia, marked loss of interest or pleasure in virtually all activities, restlessness, frequent thoughts of death or suicide, and marked weight loss or gain (a change of 5%). Diagnostic criteria for MDD, along with those for other mood disorders, can be found, for example, in the Diagnostic and Statistical Manual of Mental Disorders, fourth edition, DSM-VI®-TR, American Psychiatric Association (DSM IV), and are useful for assessment.

[0178] The term "bipolar disorder" refers to a mood disorder characterized by alternating periods of extreme mood swings. Individuals with bipolar disorder typically experience mood cycles of excessive joy or anger (mania), followed by sadness and despair (depression), with periods of normal mood in between. The diagnosis of bipolar disorder is described, for example, in DSM-IV. The categories of bipolar disorder include, but are not limited to, bipolar disorder I (mania with or without major depression) and bipolar disorder II (hypomania with major depression). As used herein, the terms "mania" or "manic" refer to a disturbed mental state of excessive excitement. The term "hypomania" refers to a less severe and less extreme manifestation of manic symptoms.

[0179] As is evident from the foregoing disclosure, the FAM19A1 antagonists disclosed herein can be used to treat all types of mood disorders.

[0180] In some embodiments, the damage treatable with the FAM19A1 antagonists disclosed herein is related to the visual system. Accordingly, in some embodiments, this specification provides a method for treating glaucoma in a subject of interest, comprising administering a FAM19A1 antagonist to said subject. In some embodiments, FAM19A1 antagonists useful in this disclosure are antisense oligonucleotides, siRNAs, shRNAs, miRNAs, dsRNAs, aptamers, PNAs, or vectors containing the same that specifically target FAM19A1. In some embodiments, the FAM19A1 antagonist is an anti-FAM19A1 antibody, a polynucleotide coding the anti-FAM19A1 antibody, or a vector containing the polynucleotide. In some embodiments, the FAM19A1 antagonist binds to the FAM19A1 protein and reduces FAM19A1 activity. In other embodiments, the reduced FAM19A1 activity reduces, alleviates, or inhibits inflammation associated with glaucoma.

[0181] In certain configurations, FAM19A1 antagonists (e.g., anti-FAM19A1 antibodies) reduce retinal ganglion cell (RGC) loss and / or restore the number of retinal ganglion cells in the retina of subjects (e.g., glaucoma patients). In specific configurations, retinal ganglion cell loss is reduced by at least approximately 5%, at least approximately 10%, at least approximately 20%, at least approximately 30%, at least approximately 40%, at least approximately 50%, at least approximately 60%, at least approximately 70%, at least approximately 80%, or at least approximately 90% or more compared to a baseline (e.g., the relevant value in a subject not receiving a FAM19A1 antagonist or the relevant value in a subject before administration of a FAM19A1 antagonist). In some conditions, the number of retinal ganglion cells is restored by at least approximately 5%, at least approximately 10%, at least approximately 20%, at least approximately 30%, at least approximately 40%, at least approximately 50%, at least approximately 60%, at least approximately 70%, at least approximately 80%, or at least approximately 90% compared to the baseline (e.g., the relevant value for subjects not receiving FAM19A1 antagonists or the relevant value for subjects before administration of FAM19A1 antagonists).

[0182] In some embodiments, the FAM19A1 antagonists disclosed herein (e.g., anti-FAM19A1 antibodies) delay the onset of retinal neuronal degeneration in subjects (e.g., glaucoma patients). In some embodiments, FAM19A1 antagonists protect the neural connections of the inner retinal plexiform layer in subjects (e.g., glaucoma patients). In some embodiments, FAM19A1 antagonists suppress inflammation around the optic disc of the retina by regulating the activation of microglial cells. In some embodiments, the FAM19A1 antagonists disclosed herein (e.g., anti-FAM19A1 antibodies) have an immediate effect, for example, reducing the high intraocular pressure observed in glaucoma subjects. However, in some embodiments, FAM19A1 antagonists increase and / or improve the electroretinomycete in subjects (e.g., glaucoma patients).

[0183] In some forms, glaucoma includes open-angle glaucoma, closed-angle glaucoma, normal-tension glaucoma ("NTG"), congenital glaucoma, secondary glaucoma, pigmentary glaucoma, pseudoexfoliation glaucoma, traumatic glaucoma, neovascular glaucoma, iris-corneal endothelial syndrome, uveitis glaucoma, or combinations thereof. In certain forms, glaucoma is associated with optic nerve damage, retinal ganglion cell ("RGC") loss, elevated intraocular pressure ("IOP"), damaged blood-retinal barrier, and / or increased microglial activity levels in the retina and / or optic nerve of the subject.

[0184] In some embodiments, a method for treating glaucoma further includes administering one or more additional formulations for the treatment of glaucoma. In certain embodiments, the additional formulation is a prostaglandin analog such as XALATAN®, LUMIGAN®, or TRAVATAN Z®. In some embodiments, the additional formulation is an alpha-agonist such as ALPHAGAN® P and IOPIDINE®. In other embodiments, the additional formulation is a carbonic anhydrase inhibitor such as TRUSOPT® and AZOPT®. Such additional formulations may be administered before, concurrently with, or after the administration of a FAM19A1 antagonist.

[0185] In some manifestations, CNS dysfunction is associated with the sensory system, particularly touch. Therefore, in some manifestations, the present disclosure provides a method for treating, preventing, or reducing neuropathic pain in subjects in need, comprising administering a FAM19A1 antagonist to said subjects.

[0186] In some forms, neuropathic pain is central neuropathic pain, i.e., pain caused by injury or damage affecting any part of the CNS, including the central somatic sensory nervous system (e.g., brain injury and spinal cord injury), or pain associated with or resulting from diseases or disorders such as stroke, multiple sclerosis, or lateral medullary infarction. In some forms, central neuropathic pain can be spontaneous or induced by stimulation. In some forms, central neuropathic pain can include dynamic mechanical allodynia and cold allodynia. Symptoms of central neuropathic pain include sensations such as burning, stinging, stabbing, squeezing, suffocating cold, paresthesia and paresthesia, with tingling, pinching, cold, and pressure being common. The distribution of central neuropathic pain includes, for example, areas ranging from small to large in the periorbital region, areas including half of the body in the case of stroke or the lower body in the case of spinal cord injury, or areas including one side of the face and the opposite side of the torso or limbs. Central neuropathic pain due to spinal cord injury includes "at-level" pain, which is pain perceived as a segmental pattern at the site of injury, and "below-level" pain, which is pain felt below the site of injury. In some manner, the method reduces, reverses, alleviates, reduces, inhibits, delays, or prevents central neuropathic pain, pain-related symptoms, the underlying cause of pain, or a combination thereof.

[0187] In some forms, neuropathic pain is peripheral neuropathic pain, pain resulting from injury or damage to any part of the peripheral nervous system (e.g., motor nerves, sensory nerves, autonomic nerves, or a combination thereof), or pain resulting from or associated with disease or disorder. Injury or damage to motor nerves is associated with symptoms such as muscle weakness (e.g., weakness of the muscles of the back, legs, buttocks, or face), distressing spasms and fasciculation (uncontrolled muscle contractions seen subcutaneously), muscle atrophy (severe contractions of muscle size), and decreased reflexes. Injury or damage to sensory nerves can induce a variety of symptoms, including skin pain and hypersensitivity of pain receptors, and can manifest as allodynia (e.g., severe pain from stimuli that are generally painless).

[0188] In some embodiments, the method comprises administering a FAM19A1 antagonist (e.g., an anti-FAM19A1 antibody) to a subject in need of treatment for one or more types of neuropathic pain. In some embodiments, neuropathic pain that can be treated by the methods disclosed herein includes, but is not limited to, trigeminal neuralgia (TN) (e.g., pain in the facial or oral trigeminal nerve region), atypical trigeminal neuralgia (ATN), occipital neuralgia, postherpetic neuralgia (e.g., pain unilaterally distributed within one or more spinal dermatomes or ophthalmic divisions of the trigeminal nerve), peripheral nerve injury pain (e.g., pain in the nerve distribution area of ​​a lesioned nerve, generally distal to trauma, surgery, or compression), glossopharyngeal neuralgia (e.g., irritation of the ninth cranial nerve that induces severe pain in the throat, tongue, and behind the ear), sciatica, low back pain, and atypical facial pain. In some forms, the neuralgia is caused by or associated with chemical irritation, inflammation, trauma (including surgery), nerve compression by adjacent tissue (e.g., tumor), or infection. In some forms, the neuropathic pain is afferent blockade pain syndrome, including, but not limited to, brain or spinal cord injury, post-stroke pain, phantom limb pain, lower limb paralysis, brachial plexus avulsion injury, and lumbar nerve root compression. In some forms, the neuropathic pain is a complex regional pain syndrome (CRPS), including, but not limited to, CRPS1 and CRPS2. In some forms, CRPS-related symptoms may include severe pain, changes in nails, bones, and skin; and increased touch hypersensitivity in the affected limb. In some forms, the neuropathic pain is a neuropathy (e.g., central or peripheral). Non-limiting examples of neuropathic pain include, for example, mononeuropathy and polyneuropathy.

[0189] In some forms, neuropathic pain is caused by or associated with, for example, (1) traumatic injury or injury including nerve compression (e.g., nerve crush, nerve stretching, nerve entrapment or incomplete nerve dissection); (2) spinal cord injury (e.g., unilateral spinal cord amputation); (3) injury or injury to peripheral nerves (e.g., motor nerves, sensory nerves, autonomic nerves or combination thereof); (4) limb amputation; contusions; inflammation (e.g., spinal cord inflammation); or surgical procedures; and (5) physical injury including, for example, repetitive stress involving repetitive, inconvenient and / or forced activities requiring prolonged movement of a specific group of joints (e.g., ulnar nerve neuropathy and carpal tunnel syndrome). In some forms, the methods described above treat neuropathic pain caused by or associated with exposure to toxic substances in particular.

[0190] In some cases, neuropathic pain can be caused by: (1) ischemic conditions (e.g., stroke or cardiac arrest); (2) multiple sclerosis; (3) metabolic and / or endocrine disorders or conditions (e.g., diabetes, metabolic disorders, and acromegaly, a condition characterized by abnormal enlargement of parts of the skeleton, including joints, which are induced by excessive growth hormone production and cause nerve compression and pain); (4) small vessel diseases that lead to reduced oxygen supply to peripheral nerves, resulting in nerve tissue damage (e.g., vasculitis, i.e., vasculitis); (5) autoimmune diseases (e.g., Sjögren's syndrome, lupus, rheumatoid arthritis, and acute inflammatory demyelinating neuropathy, also known as Guillain-Barré syndrome); (6) kidney disorders; (7) cancer or tumors (e.g., neoplastic tumors, neuromas, paraneoplastic syndromes). (1) Neuropathic pain caused by or associated with one or more diseases or disorders, including (syndrome) and toxicity from chemotherapy and radiation during cancer treatment; (2) infections (e.g., varicella-zoster virus, Epstein-Barr virus, West Nile virus, giant cell virus and herpes simplex virus, viruses such as AIDS, Lyme disease, diphtheria and leprosy, etc.); (3) inflammatory disorders; (4) peripheral neuropathy (e.g., neuroma); (5) congenital or de novo genetic disorders (e.g., Charcot-Marie-Tooth disorder); (6) mononeuropathy; (7) polyneuropathy or combination thereof. In some forms, the neuropathic pain is caused by or associated with diabetes mellitus (type 1 or type 2). In some forms, the neuropathic pain is diabetic peripheral neuropathy.

[0191] In some forms, neuropathic pain is caused by or associated with exposure to toxic substances, such as tick-borne infections, varicella-zoster virus, Epstein-Barr virus, West Nile virus, giant cell virus, herpes simplex virus, AIDS, or toxic substances including drugs, alcohol, heavy metals (e.g., lead, arsenic, mercury), or industrial substances (e.g., solvents, fumes from adhesives, and nitrous oxide).

[0192] In some forms, neuropathic pain is caused by or associated with bodily injury, infection, diabetes, cancer treatment, alcoholism, amputation, multiple sclerosis, herpes zoster, spinal surgery, sciatica (pain caused by the sciatic nerve), lower back pain, trigeminal neuralgia (e.g., pain in the trigeminal nerve area of ​​the face or oral cavity), painful polyneuropathy (e.g., foot pain may extend to include the leg, thigh and hand), or a combination thereof. In some forms, neuropathic pain is trigeminal neuralgia. In some forms, neuropathic pain is associated with weakness of the muscles of the back, leg, buttocks, or face. In some forms, neuropathic pain is induced by compression of a nerve, e.g., a nerve located in the muscles of the leg, foot, buttocks, or face. In some forms, neuropathic pain includes sciatic nerve injury. In some forms, the said neuropathic pain is sciatica.

[0193] In certain embodiments, the methods of the present disclosure can reverse, alleviate, reduce, inhibit, delay, or prevent one or more symptoms associated with neuropathic pain. Accordingly, in one embodiment, the present disclosure provides a method comprising administering an antagonist to FAM19A1 to a subject as a method for improving hyperalgesia in the subject in need. As used herein, the term “hyperalgesia” refers to an increased or marked response to a painful stimulus (e.g., puncture or hot plate). In certain embodiments, the hyperalgesia relates to a mechanical stimulus such as puncture (mechanical hyperalgesia). In other embodiments, the hyperalgesia relates to a thermal stimulus such as a hot plate (thermal hyperalgesia). In certain embodiments, the subject in need has chronic stenotic injury (e.g., sciatica). In certain embodiments, the subject in need has diabetic peripheral neuropathy.

[0194] In certain embodiments, administration of a FAM19A1 antagonist to a subject requiring it results in the subject having a higher threshold for mechanical stimulation compared to a reference control group (e.g., a subject with neuropathic pain who has not received a FAM19A1 antagonist). As used herein, “threshold for mechanical stimulation” refers to the amount of pressure (of mechanical stimulation) before the subject responds to the stimulation (e.g., by pulling away). Thus, subjects with a higher threshold can tolerate a much larger amount of mechanical stimulation than subjects with a lower threshold. In certain embodiments, the methods disclosed herein can increase the threshold for mechanical stimulation of a subject by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, or at least about 200% compared to a reference control group (e.g., the threshold of a subject before administration of a FAM19A1 antagonist).

[0195] In some embodiments, administration of a FAM19A1 antagonist to a subject requiring such an antagonist increases the subject's delay time to thermal stimuli (e.g., a hot plate) compared to a reference control group (e.g., a subject with neuropathic pain who has not received FAM19A15 antagonists). In some embodiments, the methods disclosed herein can increase the subject's delay time to thermal stimuli by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, or at least about 200% compared to a reference control group (e.g., the subject's threshold before administration of a FAM19A1 antagonist).

[0196] In other parts, this disclosure provides methods for improving sensory nerve conduction velocity in subjects of interest. The term “sensory nerve conduction velocity (SNCV)” refers to the speed at which electrical signals travel through peripheral nerves. Healthy nerves transmit electrical signals faster and more strongly than damaged nerves. See Chouhan S., J Clin Diagn Res 10(1):CC01-3(2016). Therefore, tests that aid in measuring SNCV (e.g., sensory nerve conduction velocity tests) may be useful in confirming potential nerve damage and / or dysfunction in subjects. In certain aspects, the methods disclosed herein can increase the SNCV of a neuropathic pain subject by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, or at least about 200% compared to a reference control group (e.g., the subject's threshold before administration of a FAM19A1 antagonist).

[0197] In some cases, methods for treating neuropathic pain may further include administering additional formulations for treating neuropathic pain. Non-limiting examples of formulations for treating neuropathic pain include anticonvulsants such as venlafaxine (EFFEXOR®), carbamazepine (CARBATROL®, FDA-approved TEGRETOL® for the relief of trigeminal neuralgia), gabapentin (NEURONTIN®, FDA-approved GRALISE® for the management of postherpetic neuralgia (PHN), which is pain that persists for 1 to 3 months after the healing of herpes zoster), and pregabalin (LYRICA®, approved for PHN, painful diabetic neuropathic pain, and fibromyalgia); sodium channel blockers such as lidocaine; adrenergic drugs such as clonidine, phentolamine, phenoxybenzamine, reserpine, and dexmedetomidine; opioids such as morphine; and antidepressants such as amitriptyline, imipramine, and duloxetine.

[0198] The dosage and administration of the aforementioned one or more additional therapeutic agents are publicly known to the industry, for example, as indicated by the respective product labels of the drugs.

[0199] In some cases, the subject of treatment is a non-human animal such as a rat or mouse. In some cases, the subject of treatment is a human.

[0200] [Methods for regulating or improving central nervous system (CNS) function] Without being bound by any particular theory, in certain aspects, the FAM19A1 antagonists disclosed herein can treat diseases or disorders by reducing and / or inhibiting FAM19A1 activity. In certain aspects, the reduced and / or inhibited FAM19A1 activity can improve one or more functions of the central nervous system. Accordingly, in certain aspects, this specification discloses a method comprising administering a FAM19A1 antagonist to a subject as a method for modulating or improving one or more functions of the central nervous system in a subject of interest. In certain aspects, FAM19A1 antagonists useful in this disclosure are antisense oligonucleotides, siRNAs, shRNAs, miRNAs, dsRNAs, aptamers, PNAs, or vectors containing the same that specifically target FAM19A1. In certain aspects, FAM19A1 antagonists include anti-FAM19A1 antibodies, polynucleotides coding anti-FAM19A15 antibodies, or vectors containing the same polynucleotides.

[0201] As mentioned above, although there is some overlap in functions, most tissues within the CNS are involved in specific functions that can be classified into different groups. Therefore, in some forms, central nervous system functions include limbic system-related functions, olfactory system-related functions, sensory system-related functions, visual system-related functions, or combinations thereof.

[0202] As used herein, the term “limbic system-related functions” refers to activities associated with the limbic system. The term “limbic system” refers to the part of the brain that processes three primary functions: emotion, memory, and arousal (or stimulation). In some forms, the limbic system includes the following brain regions: olfactory bulb, hippocampus, hypothalamus, amygdala, anterior thalamic nucleus, fornix, fornix trabecula, mammillary body, septum pellucida, habenular commissure, cingulate gyrus, parahippocampal gyrus, entorhinal cortex, reticular retina, and limbic midbrain regions.

[0203] As used herein, the term "olfactory system-related functions" refers to the olfactory system and related activities, which are a part of the sensory system used for odor detection (olfaction).

[0204] As used herein, the term “sensory system-related functions” refers to activities associated with the sensory system. The sensory system includes sensory nerve cells (including sensory receptor cells), neural pathways, and parts of the brain involved in sensory perception. In some forms, sensory system-related functions include hearing, touch, taste, balance, or a combination thereof.

[0205] As used herein, the term “visual system-related functions” refers to vision and related activities. The term “visual system” refers to the part of the CNS that not only processes visual details (e.g., perceiving and interpreting information from visible light) but also enables the formation of various non-image light response functions (e.g., pupillary light reflex (PLR) and circadian photoentrainment). The visual system performs a number of complex tasks, including light reception and the formation of monocular representations; the accumulation of nuclear binocular perception from a pair of two-dimensional projections; the identification and classification of visual individuals; the evaluation of distance to and between objects; and the guidance of body movements corresponding to visible objects.

[0206] In connection with this disclosure, it has been shown that FAM19A1 is expressed in specific brain regions associated with the CNS functions disclosed herein. See, for example, Example 8. Without being bound by any particular mechanism or theory, abnormal expression of FAM19A1 may cause defects in CNS function.

[0207] In certain embodiments, FAM19A1 antagonists disclosed herein (e.g., anti-FAM19A1 antibodies) can modulate or improve central nervous system function by reducing FAM19A1 protein expression and / or FAM19A1 mRNA expression in brain regions (e.g., regions associated with CNS function as disclosed herein). In certain embodiments, brain regions include the cerebral cortex, hippocampus, hypothalamus, midbrain, prefrontal cortex, amygdala (e.g., lateral amygdala and basomedial amygdala), piriform cortex, anterior olfactory nucleus, lateral entorhinal cortex, reticular retina, or combinations thereof.

[0208] In certain configurations, FAM19A1 antagonists (e.g., anti-FAM19A1 antibodies) reduce FAM19A1 protein expression and / or FAM19A1 mRNA expression in the retinal region. In specific configurations, the retinal region includes the ganglion cell layer (GCL) or the internal plexiform layer (INL).

[0209] In certain configurations, FAM19A1 antagonists (e.g., anti-FAM19A1 antibodies) reduce FAM19A1 protein expression and / or FAM19A1 mRNA expression in the spinal region. In specific configurations, the spinal region includes the dorsal horn.

[0210] In some embodiments, after administration of a FAM19A1 antagonist disclosed herein (e.g., an anti-FAM19A1 antibody), FAM19A1 protein expression and / or FAM19A1 mRNA expression are reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% or more compared to a baseline (e.g., the relevant value in a subject not receiving the FAM19A1 antagonist or the relevant value in a subject before administration of the FAM19A1 antagonist). In some embodiments, the reduced FAM19A1 protein expression and / or FAM19A1 mRNA expression is associated with improved CNS function.

[0211] Methods for regulating, inducing, or increasing the differentiation of nerve cells Neural stem cells (NSCs) possess the ability to divide continuously (i.e., self-regenerative capacity) and differentiate into neurons, astrocytes, and oligodendrocytes of the central nervous system. Differentiation into neurons mainly occurs during the embryonic stage, while differentiation into glial cells occurs after birth. See Bayer et al., J Comp Neurol 307:499-516 (1991); Miller and Gauthier, Neuron 54:357-369 (2007).

[0212] Maintaining normal CNS function requires a numerical balance between neurons and glial cells (e.g., astrocytes). While astrocytes have specific beneficial functions (e.g., structurally supporting neurons, secreting nerve growth factors, and helping to maintain the blood-brain barrier), excessive astrocyte formation can interfere with neuronal regeneration and cause inflammation-mediated damage to CNS tissue. See Myer et al., Brain 129:2761-2772 (2006); Chen and Swanson, J Cereb Blood Flow Metab 23:137-149 (2003); Cunningham et al., Brain 128:1931-1942 (2005); Faden, Curr Opin Neurol 15:707-712 (2002); or see U.S. Publication No. 2015 / 0118230, the full text of which is included herein as reference.

[0213] Gliosis is a phenomenon that frequently occurs in various pathological processes of the central nervous system and is induced by the hyperproliferation and activation of astrocytes due to nerve cell damage. When the central nervous system is damaged, normal astrocytes become hypertrophic reactive astrocytes that increase the production of an intermediate filament protein called glial cell fibrous acidic protein (GFAP). Various glial cells, including reactive astrocytes, hyperproliferate after the injury, forming a solid cell layer called a gliocarp, which is a product of the healing process. Such gliosis is observed in various pathological phenomena of the central nervous system, including degenerative brain diseases such as Huntington's disease, Parkinson's disease, and Alzheimer's disease, spinal cord injury, stroke, and brain tumors. Faideau et al.,Hum Mol Genet 19(15):3053-67(2010);Chen et al.,Curr Drug Targets 5:149-157(2005);Rodriguez et al.,Cell Death Differ 16:378-385(2009);Robel et al.,J Neurosci 31(35):12471-12482(2011);Talbott et al.,Exp Neurol 192:11-24(2005);Shimada et al.,J Neurosci 32(33):7926-40(2012);Sofroniew and Vinters,Acta Neuropathol 119:7-35 (2010).

[0214] Therefore, without being constrained by any particular mechanism or theory, CNS function can be improved, for example, by promoting and / or regulating the differentiation of neurons from neural stem cells. Accordingly, this disclosure provides a method comprising administering a FAM19A1 antagonist (e.g., an anti-FAM19A1 antibody) to a subject as a method for regulating, inducing or increasing the differentiation and / or maturation of neurons in a subject of interest. In some embodiments, the FAM19A1 antagonist increases neurite growth in differentiated neural stem cells (i.e., neurons) compared to a baseline (e.g., the relevant value in a subject not receiving the FAM19A1 antagonist or the relevant value in a subject before administration of the FAM19A1 antagonist). In certain embodiments, neurite growth increases by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% or more compared to the baseline.

[0215] Methods for diagnosing central nervous system (CNS) dysfunction This specification discloses a method for diagnosing CNS dysfunction in a subject of interest, which includes contacting a sample of the subject with a FAM19A1 antagonist (e.g., an anti-FAM19A1 antibody) and measuring the FAM19A1 protein level or FAM19A1 mRNA level in the sample. Furthermore, this specification discloses a method for identifying a subject with central nervous system dysfunction, which includes contacting a sample of the subject with a FAM19A1 antagonist and measuring the FAM19A1 protein level or FAM19A1 mRNA level in the sample.

[0216] The term "central nervous system dysfunction" refers to the inability (or reduced ability) to perform one or more functions related to the CNS. In some forms, central nervous system dysfunction includes limbic system-related functions, olfactory system-related functions, sensory system-related functions, visual system-related functions, or combinations thereof.

[0217] Beyond what is described herein, as otherwise provided, many diseases or disorders affecting the CNS are, to some extent, associated with impaired CNS function (for example, the primary symptom associated with glaucoma is visual impairment). Therefore, in certain manner, the methods disclosed herein can be used to diagnose and / or identify subjects with diseases or disorders associated with the CNS. Non-limiting examples of such diseases or disorders include glaucoma, neuropathic pain, intoxication, arachnoid cyst, attention deficit / hyperactivity disorder (ADHD), autism, bipolar disorder, sclerosis, depression, encephalitis, epilepsy / seizures, fixation syndrome, meningitis, migraine, multiple sclerosis, osteomyelopathy, Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Batten's disease, Tourette's syndrome, traumatic brain injury, post-traumatic stress disorder (PTSD), spinal cord injury, stroke, tremor (essential or Parkinsonian), dysarthria, schizophrenia, intellectual disability, and brain tumors. In some forms, central nervous system dysfunction is associated with glaucoma or neuropathic pain.

[0218] In some embodiments, a method for diagnosing and / or confirming a subject with a central nervous system dysfunction includes administering a FAM19A1 antagonist to the subject before measurement to induce contact between the FAM19A1 antagonist and FAM19A1 in vivo. In some embodiments, the contact and measurement are performed in a test tube.

[0219] In some manner, CNS dysfunction is associated with elevated FAM19A1 protein and / or FAM19A1 mRNA levels in a sample compared to a baseline (e.g., the corresponding value in a sample from a subject without CNS dysfunction, e.g., a healthy subject). In certain manner, the FAM19A1 protein and / or FAM19A1 mRNA levels are increased by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% or more compared to the baseline.

[0220] In some manner, samples from subjects with central nervous system dysfunction have increased FAM19A1 protein levels and / or FAM19A1 mRNA levels by at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, at least 15 times, at least 20 times, at least 25 times, or at least 30 times compared to the reference value (e.g., the corresponding value in samples from subjects without central nervous system dysfunction, e.g., healthy subjects).

[0221] In some manner, CNS dysfunction is associated with decreased levels of FAM19A1 protein and / or FAM19A1 mRNA in a sample compared to a baseline (e.g., the corresponding value in a sample from a subject without CNS dysfunction, e.g., a healthy subject). In certain manner, the FAM19A1 protein and / or FAM19A1 mRNA levels are decreased by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% or more compared to the baseline.

[0222] In some manner, samples from subjects with central nervous system dysfunction exhibit a decrease of at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 15, at least 20, at least 25, or at least 30 times in FAM19A1 protein levels and / or FAM19A1 mRNA levels compared to a reference (e.g., the corresponding value in samples from subjects without central nervous system dysfunction, e.g., healthy subjects).

[0223] In some cases, FAM19A1 protein levels are measured by immunohistochemistry, Western blotting, radioimmunoanalysis, enzyme-linked immunosorbent assay (ELISA), radioimmunodiffusion, immunoprecipitation, Octalony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, complement fixation analysis, FACS, protein chips, or a combination thereof. In some cases, FAM19A1 mRNA levels are measured by reverse transcription polymerase chain reaction (RT-PCR), real-time polymerase chain reaction, Northern blotting, or a combination thereof. In some cases, FAM19A1 protein levels are measured by analytical methods using FAM19A1 antagonists disclosed herein (e.g., anti-FAM19A1 antibodies).

[0224] In some cases, the sample (for example, a sample used to measure FAM19A1 protein levels and / or FAM19A1 mRNA levels) may include tissue, cells, blood, serum, plasma, saliva, urine, cerebrospinal fluid (CSF), or a combination thereof.

[0225] In some manner, a method for diagnosing and / or confirming a subject with central nervous system dysfunction further includes administering a FAM19A1 agonist to the subject when the FAM19A1 protein level and / or FAM19A1 mRNA level increases compared to a standard (e.g., the corresponding value in a sample of a subject without central nervous system dysfunction, e.g., a healthy subject).

[0226] In some manner, a method for diagnosing and / or confirming a subject with central nervous system dysfunction further includes administering a FAM19A1 agonist to the subject when the FAM19A1 protein level and / or FAM19A1 mRNA level decreases compared to a standard (e.g., the corresponding value in a sample of a subject without central nervous system dysfunction, e.g., a healthy subject).

[0227] In some embodiments, the FAM19A1 agonist comprises the FAM19A1 protein. In some embodiments, the FAM19A1 antagonist comprises an anti-FAM19A1 antibody, a polynucleotide coding the anti-FAM19A1 antibody, a vector containing the polynucleotide, a cell containing the polynucleotide, or any combination thereof. In some embodiments, the FAM19A1 antagonist comprises an antisense oligonucleotide, siRNA, shRNA, miRNA, dsRNA, aptamer, PNA, or a vector containing the same that specifically targets FAM19A1. In certain embodiments, the FAM19A1 antagonist is an anti-FAM19A1 antibody.

[0228] [III. FAM19A1 antagonists] In conjunction with this method, one or more FAM19A1 antagonists may be used. In some embodiments, the FAM19A1 antagonist is an antisense oligonucleotide, siRNA, shRNA, miRNA, dsRNA, aptamer, PNA (peptide nucleic acid), or a vector containing the same, which specifically targets FAM19A1. In some embodiments, the FAM19A1 antagonist is an anti-FAM19A1 antibody, a polynucleotide coding the anti-FAM19A1 antibody, or a vector containing the polynucleotide.

[0229] Antibodies useful in the methods disclosed herein include monoclonal antibodies characterized by specific functional features or properties. For example, such antibodies specifically bind to human FAM19A1, including soluble FAM19A1 and membrane-bound FAM19A1. In addition to specific binding to soluble and / or membrane-bound human FAM19A1, the antibodies described herein also (a) K D (b) K D It binds to membrane-bound human FAM19A1 with a concentration of 10 nM or less; or both of (a) and (b).

[0230] In some aspects, the anti-FAM19A1 antibodies disclosed herein are soluble human FAM19A1 or have high affinity, for example, K D10 -7 M or less, 10 -8 M (10nM) or less, 10 -9 M (1nM) or less, 10 -10 M(0.1nM) or less, 10 -11 M or less or 10 -12 M or less, for example, 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 It specifically binds to soluble human FAM19A1 or membrane-bound human FAM19A1, which is M. Standard analytical methods for evaluating the binding ability of antibodies to various species of human FAM19A1 are known in the art, including, for example, ELISA, Western blotting, and RIA. Appropriate analytical methods are described in detail in the Examples section. Furthermore, the binding kinetics (e.g., binding affinity) of the antibody are evaluated using ELISA, BIACORE TM It can also be evaluated by analysis or by standard analytical methods known in the industry, such as KINEXA®.

[0231] In some aspects, the anti-FAM19A1 antibodies disclosed herein, when determined by, for example, ELISA, are K D 10 -7 M or less, 10 -8 M (10nM) or less, 10 -9 M (1nM) or less, 10 -10 M or less, 10 -12 M to 10 -7 M, 10 -11 M to 10-7 M, 10 -10 M to 10 -7 M, 10 -9 M to 10 -7 M or 10 -8 M to 10 -7 It binds to soluble human FAM19A1 which is M. In some embodiments, the anti-FAM19A1 antibody has a K D of 10 nM or less, for example, 0.1 nM to 10 nM, 0.1 nM to 5 nM, 0.1 nM to 1 nM, 0.5 nM to 10 nM, 0.5 nM to 5 nM, 0.5 nM to 1 nM, 1 nM to 10 nM, 1 nM to 5 nM or 5 nM to 10 nM and binds to soluble FAM19A1. In certain embodiments, the anti-FAM19A1 antibody, when determined by ELISA, has a K D 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 and specifically binds to soluble human FAM19A1.

[0232] In some embodiments, the anti-FAM19A1 antibody has a K D of, for example, when determined by ELISA, 10 -7 M or less, 10 -8 M (10 nM) or less, 10 -9 M (1 nM) or less, 10 -10 M or less, 10 -12 M to 10 -7 M, 10 -11 M to 10 -7 M, 10 -10 M to 10 -7 M, 10 -9 M to 10 -7 M or 10 -8 M to 10 -7It binds to membrane-bound human FAM19A1 which is M. In certain embodiments, the anti-FAM19A1 antibody has a K D of 10 nM or less, such as 0.1 nM to 10 nM, 0.1 nM to 5 nM, 0.1 nM to 1 nM, 0.5 nM to 10 nM, 0.5 nM to 5 nM, 0.5 nM to 1 nM, 1 nM to 10 nM, 1 nM to 5 nM or 5 nM to 10 nM and specifically binds to membrane-bound human FAM19A1. In some embodiments, the anti-FAM19A1 antibody or its antigen-binding portion has a K D 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 and binds to membrane-bound human FAM19A1.

[0233] In addition to the above, FAM19A1 antagonists (e.g., anti-FAM19A1 antibodies) exhibit one or more of the following functional properties: (1) Promote the differentiation of nerve cells; (2) Increase neurite outgrowth in differentiated nerve cells; ((3) Reduce, reverse and / or prevent one or more symptoms associated with glaucoma; (4) Improve retinal potential (e.g., evidenced by increased rhythmic-like small waves); (5) Reduce and / or restore the loss of retinal ganglion cells (e.g., observed in glaucoma subjects); (6) Reduce, reverse and / or prevent one or more symptoms associated with neuropathic pain; (7) Increase the delay time and / or threshold to external stimuli; and (8) Increase and / or regulate the sensory nerve conduction velocity.

[0234] Other functional properties of the anti-FAM19A1 antibody disclosed herein are provided throughout this application.

[0235] In certain aspects, the anti-FAM19A1 antibodies disclosed herein cross-compete with anti-FAM19A1 antibodies containing the CDRs or variable regions (e.g., 1C1, 1A11, 2G7, and 3A8) disclosed herein for binding to the human FAM19A1 epitope.

[0236] In certain manner, the anti-FAM19A1 antibody of this disclosure inhibits the binding of a reference antibody comprising heavy chain CDR1, CDR2 and CDR3, and light chain CDR1, CDR2 and CDR3, wherein (i) the heavy chain CDR1, CDR2 and CDR3 of the reference antibody each contain the amino acid sequence shown in SEQ ID NOs. 10-12, and the light chain CDR1, CDR2 and CDR3 of the reference antibody each contain the amino acid sequence shown in SEQ ID NOs. 13-15; (ii) the heavy chain CDR1, CDR2 and CDR3 of the reference antibody each contain the amino acid sequence shown in SEQ ID NOs. 4-6, and the light chain CDR1, CDR3 of the reference antibody (iii) DR2 and CDR3 each contain the amino acid sequences shown in SEQ ID NOs: 7-9; (iii) the heavy chains CDR1, CDR2 and CDR3 of the reference antibody each contain the amino acid sequences shown in SEQ ID NOs: 16-18, and the light chains CDR1, CDR2 and CDR3 of the reference antibody each contain the amino acid sequences shown in SEQ ID NOs: 19-21; or (iv) the heavy chains CDR1, CDR2 and CDR3 of the reference antibody each contain the amino acid sequences shown in SEQ ID NOs: 22-24, and the light chains CDR1, CDR2 and CDR3 of the reference antibody each contain the amino acid sequences shown in SEQ ID NOs: 25-27.

[0237] In some configurations, the reference antibody includes (a) heavy chain and light chain variable regions containing the amino acid sequences shown in SEQ ID NOs. 30 and 31, respectively; (b) heavy chain and light chain variable regions containing the amino acid sequences shown in SEQ ID NOs. 28 and 29, respectively; (c) heavy chain and light chain variable regions containing the amino acid sequences shown in SEQ ID NOs. 32 and 33, respectively; or (d) heavy chain and light chain variable regions containing the amino acid sequences shown in SEQ ID NOs. 34 and 35, respectively.

[0238] In certain embodiments, the anti-FAM19A1 antibodies disclosed herein inhibit the binding of such reference antibodies to human FAM19A1 by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. Competitive antibodies bind to the same epitope, superimposed epitope, or adjacent epitope (e.g., demonstrated by steric hindrance). Whether two antibodies compete with each other for binding to a target can be determined using commercially known competitive experiments such as RIA and EIA.

[0239] In some embodiments, the anti-FAM19A1 antibody binds to the same FAM19A1 epitope as the reference antibody disclosed herein, which includes heavy chain CDR1, CDR2 and CDR3, and light chain CDR1, CDR2 and CDR3, wherein (i) the heavy chain CDR1 includes the amino acid sequence shown in SEQ ID NO: 10, the heavy chain CDR2 includes the amino acid sequence shown in SEQ ID NO: 11, the heavy chain CDR3 includes the amino acid sequence shown in SEQ ID NO: 12, the light chain CDR1 includes the amino acid sequence shown in SEQ ID NO: 13, the light chain CDR2 includes the amino acid sequence shown in SEQ ID NO: 14, and the light chain CDR3 includes the amino acid sequence shown in SEQ ID NO: 15; (ii) the heavy chain CDR1 includes the amino acid sequence shown in SEQ ID NO: 4, the heavy chain CDR2 includes the amino acid sequence shown in SEQ ID NO: 5, the heavy chain CDR3 includes the amino acid sequence shown in SEQ ID NO: 6, the light chain CDR1 includes the amino acid sequence shown in SEQ ID NO: 7, and the light chain CDR2 includes the amino acid sequence shown in SEQ ID NO: 8 (iii) The heavy chain CDR1 includes the amino acid sequence shown in SEQ ID NO: 16, the heavy chain CDR2 includes the amino acid sequence shown in SEQ ID NO: 17, the heavy chain CDR3 includes the amino acid sequence shown in SEQ ID NO: 18, the light chain CDR1 includes the amino acid sequence shown in SEQ ID NO: 19, the light chain CDR2 includes the amino acid sequence shown in SEQ ID NO: 20, the light chain CDR3 includes the amino acid sequence shown in SEQ ID NO: 21; or (iv) The heavy chain CDR1 includes the amino acid sequence shown in SEQ ID NO: 22, the heavy chain CDR2 includes the amino acid sequence shown in SEQ ID NO: 23, the heavy chain CDR3 includes the amino acid sequence shown in SEQ ID NO: 24, the light chain CDR1 includes the amino acid sequence shown in SEQ ID NO: 25, the light chain CDR2 includes the amino acid sequence shown in SEQ ID NO: 26, and the light chain CDR3 includes the amino acid sequence shown in SEQ ID NO: 27. In a particular manner, the reference antibody includes (i) a heavy chain variable domain containing the amino acid sequence shown in SEQ ID NOs. 30, 28, 32, or 34, and (ii) a light chain variable domain containing the amino acid sequence shown in SEQ ID NOs. 31, 29, 33, or 35.

[0240] In some embodiments, the reference antibody comprises: (a) heavy and light chain variable regions comprising the amino acid sequences set forth in SEQ ID NOs: 30 and 31, respectively; (b) heavy and light chain variable regions comprising the amino acid sequences set forth in SEQ ID NOs: 28 and 29, respectively; (c) heavy and light chain variable regions comprising the amino acid sequences set forth in SEQ ID NOs: 32 and 33, respectively; or (d) heavy and light chain variable regions comprising the amino acid sequences set forth in SEQ ID NOs: 34 and 35, respectively.

[0241] Techniques for determining whether two antibodies bind to the same epitope include, for example, epitope mapping methods such as x-ray analysis of crystals of antigen:antibody complexes that provide atomic resolution of the epitope and hydrogen / deuterium exchange mass spectrometry (HDX-MS), methods of monitoring the binding of an antibody to an antigen fragment or a mutated variant of an antigen, generally viewing loss of binding due to modification of an amino acid residue within the antigen sequence as an indication of the epitope component, and computational combinatorial methods for epitope mapping.

[0242] The anti-FAM19A1 antibodies of the present disclosure can bind to at least one epitope of mature human FAM19A1, as determined, for example, by the binding of an antibody to a fragment of human FAM19A1. In some embodiments, the anti-FAM19A1 antibody binds to at least one epitope selected from the group consisting of D112, M117, A119, T120, N122, and combinations thereof.

[0243] In some embodiments, the present specification provides anti-FAM19A1 antibodies that bind to FAM19A1 with an affinity that is 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or even higher compared to other proteins within the FAM19A family when measured by, for example, immunoassay (e.g., ELISA), surface plasmon resonance or binding equilibrium exclusion methods. In certain embodiments, the anti-FAM19A1 antibodies disclosed herein do not have cross-reactivity with other proteins within the FAM19A family when measured by, for example, immunoassay (e.g., ELISA), surface plasmon resonance or binding equilibrium exclusion methods.

[0244] In some embodiments, the anti-FAM19A1 antibody is not a natural antibody or a spontaneously occurring antibody. For example, in certain embodiments, the anti-FAM19A1 antibodies of the present disclosure have post-translational modifications that are more, less or different types of post-translational modifications compared to spontaneously occurring antibodies, thereby differing from spontaneously occurring antibodies.

[0245] [IV. Exemplary anti-FAM19A1 antibodies] Certain antibodies that can be used in the methods disclosed herein are antibodies having the sequences of the CDRs and / or variable regions disclosed herein, for example, monoclonal antibodies as well as antibodies having at least 80% identity (e.g., at least 85%, at least 90%, at least 95% or at least 99% identity) to these variable region or CDR sequences. The VH and VL amino acid sequences of the different anti-FAM19A1 antibodies of the present disclosure are provided in Tables 6 and 7, respectively. [Table 4] [Table 5] [Table 6]

[0246] [Table 7] In certain embodiments, the anti-FAM19A1 antibody of this disclosure comprises a heavy chain and a light chain variable region, the heavy chain variable region comprising the amino acid sequence shown in SEQ ID NOs. 30, 28, 32, or 34. In certain embodiments, the anti-FAM19A1 antibody disclosed herein comprises a heavy chain variable region CDR selected from the group consisting of SEQ ID NOs. 30, 28, 32, and 34.

[0247] In some embodiments, the anti-FAM19A1 antibody disclosed herein comprises a heavy chain and a light chain variable region, the light chain variable region comprising the amino acid sequence shown in SEQ ID NOs. 31, 29, 33, or 35. In some embodiments, the anti-FAM19A1 antibody disclosed herein comprises a CDR of the light chain variable region selected from the group consisting of SEQ ID NOs. 31, 29, 33, and 35.

[0248] In some forms, the anti-FAM19A1 antibody includes a heavy chain variable region CDR selected from the group consisting of SEQ ID NOs: 30, 28, 32, and 34, and a light chain variable region CDR selected from the group consisting of SEQ ID NOs: 31, 29, 33, and 35.

[0249] In some embodiments, the anti-FAM19A1 antibody comprises a heavy chain and a light chain variable region, wherein (i) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 30, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 31; (ii) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 28, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 29; (iii) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 32, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 33; (iv) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 34, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 35.

[0250] In some embodiments, the anti-FAM19A1 antibody comprises a heavy chain and a light chain variable region, the heavy chain variable region comprising an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence shown in SEQ ID NOs. 30, 28, 32, or 34.

[0251] In some embodiments, the anti-FAM19A1 antibody comprises heavy chain and light chain variable regions, the light chain variable region comprising an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence shown in SEQ ID NOs. 31, 29, 33, or 35.

[0252] In some embodiments, the anti-FAM19A1 antibody comprises a heavy chain and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence shown in SEQ ID NOs. 30, 28, 32, or 34, and the light chain variable region comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence shown in SEQ ID NOs. 31, 29, 33, or 35.

[0253] In some cases, anti-FAM19A1 antibodies: (a) Heavy chain and light chain variable regions containing the amino acid sequences shown in SEQ ID NOs. 30 and 31, respectively; (b) Heavy chain and light chain variable regions containing the amino acid sequences shown in SEQ ID NOs. 28 and 29, respectively; (c) Heavy chain and light chain variable regions containing the amino acid sequences shown in SEQ ID NOs. 32 and 33, respectively; or (d) a heavy chain and a light chain variable region comprising the amino acid sequences shown in SEQ ID NO: 34 and SEQ ID NO: 35, respectively;

[0254] In some embodiments, the anti-FAM19A1 antibody of the present disclosure comprises: (i) the heavy chain CDR1, CDR2, and CDR3 of 1C1 or combinations thereof and / or the light chain CDR1, CDR2, and CDR3 of 1C1 or any combination thereof; (ii) the heavy chain CDR1, CDR2, and CDR3 of 1A11 or combinations thereof and / or the light chain CDR1, CDR2, and CDR3 of 1A11 or any combination thereof; (iii) the heavy chain CDR1, CDR2, and CDR3 of 2G7 or combinations thereof and / or the light chain CDR1, CDR2, and CDR3 of 2G7 or any combination thereof; or (iv) the heavy chain CDR1, CDR2, and CDR3 of 3A8 or combinations thereof and / or the light chain CDR1, CDR2, and CDR3 of 3A8 or any combination thereof. The amino acid sequences of VH CDR1, CDR2, and CDR3 for the anti-FAM19A1 antibodies different from each other disclosed herein are provided in Table 4. The amino acid sequences of VL CDR1, CDR2, and CDR3 for the anti-FAM19A1 antibodies different from each other disclosed herein are provided in Table 5.

[0255] In some embodiments, an anti-FAM19A1 antibody that specifically binds to human FAM19A1 is: (a) VH CDR1 comprising the amino acid sequence shown in SEQ ID NO: 10; (b) VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 11; and / or (c) VH CDR3 comprising the amino acid sequence shown in SEQ ID NO: 12.

[0256] In some embodiments, the antibody comprises all of one, two, or three of the aforementioned VH CDRs.

[0257] In some embodiments, an anti-FAM19A1 antibody that specifically binds to human FAM19A1 is: (a) VL CDR1 comprising the amino acid sequence shown in SEQ ID NO: 13; (b) VL CDR2 containing the amino acid sequence shown in SEQ ID NO: 14; and / or (c) Contains VL CDR3 with the amino acid sequence shown in SEQ ID NO: 15;

[0258] In some configurations, the antibody contains one, two, or all three of the VL CDRs.

[0259] In some cases, anti-FAM19A1 antibodies that specifically bind to human FAM19A1 are: (a) VH CDR1 containing the amino acid sequence shown in SEQ ID NO: 10; (b) VH CDR2 containing the amino acid sequence shown in SEQ ID NO: 11; (c) VH CDR3 containing the amino acid sequence shown in SEQ ID NO. 12; (d) VL CDR1 containing the amino acid sequence shown in SEQ ID NO: 13; (e) VL CDR2 containing the amino acid sequence shown in SEQ ID NO: 14; and / or (f) Contains VL CDR3 with the amino acid sequence shown in SEQ ID NO: 15;

[0260] In some cases, anti-FAM19A1 antibodies that specifically bind to human FAM19A1 are: (a) VH CDR1 containing the amino acid sequence shown in SEQ ID NO: 4; (b) VH CDR2 containing the amino acid sequence shown in Sequence ID No. 5; and / or (c) Contains VH CDR3 with the amino acid sequence shown in SEQ ID NO: 6;

[0261] In some configurations, the antibody contains one, two, or all three of the aforementioned VH CDRs.

[0262] In some cases, anti-FAM19A1 antibodies that specifically bind to human FAM19A1 are: (a) VL CDR1 containing the amino acid sequence shown in SEQ ID NO: 7; (b) VL CDR2 containing the amino acid sequence shown in SEQ ID NO: 8; and / or (c) Contains VL CDR3 with the amino acid sequence shown in SEQ ID NO: 9;

[0263] In some configurations, the antibody contains one, two, or all three of the VL CDRs.

[0264] In some cases, anti-FAM19A1 antibodies that specifically bind to human FAM19A1 are: (a) VH CDR1 containing the amino acid sequence shown in SEQ ID NO: 4; (b) VH CDR2 containing the amino acid sequence shown in SEQ ID NO: 5; (c) VH CDR3 containing the amino acid sequence shown in SEQ ID NO. 6; (d) VL CDR1 containing the amino acid sequence shown in SEQ ID NO: 7; (e) VL CDR2 containing the amino acid sequence shown in SEQ ID NO: 8; and / or (f) Contains VL CDR3 with the amino acid sequence shown in SEQ ID NO: 9;

[0265] In some cases, anti-FAM19A1 antibodies that specifically bind to human FAM19A1 are: (a) VH CDR1 containing the amino acid sequence shown in SEQ ID NO: 16; (b) VH CDR2 containing the amino acid sequence shown in SEQ ID NO: 17; and / or (c) Contains VH CDR3 with the amino acid sequence shown in SEQ ID NO: 18;

[0266] In some configurations, the antibody contains one, two, or all three of the aforementioned VH CDRs.

[0267] In some cases, anti-FAM19A1 antibodies that specifically bind to human FAM19A1 are: (a) VL CDR1 containing the amino acid sequence shown in SEQ ID NO: 19; (b) VL CDR2 containing the amino acid sequence shown in SEQ ID NO. 20; and / or (c) Contains VL CDR3 with the amino acid sequence shown in SEQ ID NO: 21;

[0268] In some configurations, the antibody contains one, two, or all three of the VL CDRs.

[0269] In some cases, anti-FAM19A1 antibodies that specifically bind to human FAM19A1 are: (a) VH CDR1 containing the amino acid sequence shown in SEQ ID NO: 16; (b) VH CDR2 containing the amino acid sequence shown in SEQ ID NO: 17; (c) VH CDR3 containing the amino acid sequence shown in SEQ ID NO. 18; (d) VL CDR1 containing the amino acid sequence shown in SEQ ID NO: 19; (e) VL CDR2 containing the amino acid sequence shown in SEQ ID NO: 20; and / or (f) Contains VL CDR3 with the amino acid sequence shown in SEQ ID NO: 21;

[0270] In some cases, anti-FAM19A1 antibodies that specifically bind to human FAM19A1 are: (a) VH CDR1 containing the amino acid sequence shown in SEQ ID NO: 22; (b) VH CDR2 containing the amino acid sequence shown in SEQ ID NO: 23; and / or (c) Contains VH CDR3 with the amino acid sequence shown in SEQ ID NO: 24;

[0271] In some configurations, the antibody contains one, two, or all three of the aforementioned VH CDRs.

[0272] In some cases, anti-FAM19A1 antibodies that specifically bind to human FAM19A1 are: (a) VL CDR1 containing the amino acid sequence shown in SEQ ID NO: 25; (b) VL CDR2 containing the amino acid sequence shown in SEQ ID NO: 26; and / or (c) VL CDR3 containing the amino acid sequence shown in sequence 27;

[0273] In some configurations, the antibody contains one, two, or all three of the VL CDRs.

[0274] In some cases, anti-FAM19A1 antibodies that specifically bind to human FAM19A1 are: (a) VH CDR1 containing the amino acid sequence shown in SEQ ID NO: 22; (b) VH CDR2 containing the amino acid sequence shown in SEQ ID NO: 23; (c) VH CDR3 containing the amino acid sequence shown in SEQ ID NO. 24; (d) VL CDR1 containing the amino acid sequence shown in SEQ ID NO: 25; (e) VL CDR2 containing the amino acid sequence shown in SEQ ID NO. 26; and / or (f) Contains VL CDR3 with the amino acid sequence shown in SEQ ID NO: 27;

[0275] The VH domains or one or more CDRs thereof described herein may be linked to a heavy chain, for example, a constant domain for forming a full-length heavy chain. Similarly, the VL domains or one or more CDRs thereof described herein may be linked to a light chain, for example, a constant domain for forming a full-length light chain. The full-length heavy chain and full-length light chain are combined to produce a full-length antibody.

[0276] Accordingly, in certain embodiments, the anti-FAM19A1 antibody comprises an antibody light chain and a heavy chain, for example, separate light and heavy chains. In relation to the light chain, in certain embodiments, the light chain of the antibody described herein is a kappa light chain. In certain embodiments, the light chain of the antibody described herein is a lambda light chain. In certain embodiments, the light chain of the antibody described herein is a human kappa light chain or a human lambda light chain. In certain embodiments, the antibody described herein that specifically binds to a FAM19A1 polypeptide (e.g., human FAM19A1) comprises a light chain comprising any VL or VL CDR amino acid sequence described herein, wherein the constant region of the light chain comprises the amino acid sequence of the human kappa light chain constant region. In certain embodiments, antibodies described herein that specifically bind to the FAM19A1 polypeptide (e.g., human FAM19A1) include a light chain comprising a VL or VL CDR amino acid sequence described herein, wherein the constant region of the light chain comprises the amino acid sequence of the human lambda light chain constant region. Non-restrictive examples of human constant region sequences are described in the art. See, for example, U.S. Patent No. 5,693,780 and Kabat EA et al., (1991) above.

[0277] In relation 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 some embodiments, the heavy chain of the antibody described herein may include a human alpha (α), delta (δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In some embodiments, the antibody described herein that specifically binds to FAM19A1 (e.g., human FAM19A1) comprises a heavy chain containing a VH or VH CDR amino acid sequence described herein, wherein the constant region of the heavy chain contains the amino acid sequence of the human gamma (γ) heavy chain constant region. In some embodiments, the antibody described herein that specifically binds to FAM19A1 (e.g., human FAM19A1) comprises a heavy chain containing a VH or VH CDR amino acid sequence disclosed herein, wherein the constant region of the heavy chain contains amino acids of a human heavy chain described herein or known in the art. Non-restrictive examples of human constant region sequences are described in the industry. See, for example, U.S. Patent No. 5,693,780 and Kabat EA et al., (1991) above.

[0278] In certain embodiments, the antibodies described herein include a VH or VH CDR as described herein, and a VL domain and a VH domain including a VL and a VL CDR, wherein the constant region includes the amino acid sequence of the constant region of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule or a human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule. In certain embodiments, the antibodies described herein that specifically bind to FAM19A1 (e.g., human FAM19A1) include a VL domain and a VH domain containing any amino acid sequence described herein, wherein the constant region includes the amino acid sequence of the constant region of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule and any subtype of immunoglobulin molecule (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). In some embodiments, the constant region includes the amino acid sequence of the constant region of native human IgG, including subtypes (e.g., IgG1, IgG2, IgG3, or IgG4) and allogeneic types (e.g., G1m, G2m, G3m, and nG4m) and their variants. See, for example, Vidarsson G. et al. Front Immunol. 5:520 (published online October 20, 2014) and Jefferis R. and Lefranc MP, mAbs 1:4, 1-7 (2009). In some embodiments, the constant region includes the amino acid sequence of the constant region of human IgG1, IgG2, IgG3, or IgG4 or their variants.

[0279] In some aspects, the anti-FAM19A1 antibodies disclosed herein lack Fc effector function, such as complement-dependent cell damage (CDC) and / or antibody-dependent phagocytosis (ADCP). Effector function is mediated by the Fc region, and the residue in the CH2 domain of the Fc region closest to the hinge region contains a binding site that greatly overlaps with Clq (complement) and the IgG-Fc receptor (FcγR) on effector cells of the innate immune system, thus contributing to the antibody's effector function. Furthermore, IgG2 and IgG4 antibodies have a lower level of Fc effector function than IgG1 and IgG3 antibodies. The effector function of antibodies can be achieved by (1) using antibody fragments lacking an Fc region (e.g., Fab, F(ab')2, single-chain Fv(scFv), or sdAb consisting of monomeric VH or VL domains); (2) generating glycation-free antibodies by, for example, deleting or modifying sugar-attached residues, enzymatically removing sugar, generating antibodies in cells cultured in the presence of glycosylation inhibitors, or expressing antibodies in cells incapable of protein glycosylation (e.g., bacterial host cells, see, e.g., U.S. Patent Publication No. 20120100140); or (3) using Fc regions of IgG subtypes with reduced effector function (e.g., Fc regions of IgG2 and IgG4 antibodies, or chimeric Fc regions containing the CH2 domain of IgG2 and IgG4 antibodies, see, e.g., U.S. Patent Publication No. 20120100140 and Lau C. et al.). See al. J. Immunol. 191:4769-4777 (2013); and (4) it can be reduced or avoided by different approaches known to the art, including reducing Fc function or generating Fc regions having mutations that eliminate Fc function. See, for example, U.S. Patent Publication No. 20120100140 and the U.S. and PCT applications cited therein, and An et al., mAbs 1:6, 572-579 (2009).

[0280] Accordingly, in some manner, the antigen-binding fragments disclosed herein are Fab, Fab', F(ab')2, Fv, single-chain Fv(scFv), or sdAb consisting of monomeric VH or VL domains. Such antibody fragments are well known in the art and are described above.

[0281] In some embodiments, the anti-FAM19A1 antibodies disclosed herein include Fc regions with reduced or absent Fc effector function. In some embodiments, the constant region includes the amino acid sequence of a human IgG2 or IgG4 Fc region, and in some embodiments, the anti-FAM19A1 antibody has an IgG2 / IgG4 isotype. In some embodiments, the anti-FAM19A1 antibody includes a chimeric Fc region containing the CH2 domain of an IgG4 isotype IgG antibody and the CH3 domain of an IgG1 isotype IgG antibody, or a chimeric Fc region containing the hinge region of IgG2 and the CH2 region of IgG4, or an Fc region having a mutation with reduced or absent Fc effector function. Fc regions with reduced or absent Fc effector function include those known in the art. For example, one can refer to Lau C. et al, J.Immunol. 191:4769-4777 (2013); An et al, mAbs 1:6, 572-579 (2009); and U.S. Patent Publication No. 20120100140 and the U.S. patents, publications, and PCT publications cited therein. Furthermore, Fc regions with reduced or absent Fc effector functionality can be easily created by those skilled in the art.

[0282] The anti-FAM19A1 antibodies described herein can be used for diagnostic purposes, including sample testing and in vivo imaging, for which the antibodies can be conjugated to a suitable detectable formulation to form an immunoconjugate. Suitable formulations for diagnostic purposes include radioisotopes for whole-body imaging and detectable labels including radioisotopes, enzymes, fluorescent labels, and other suitable antibody tags for sample testing.

[0283] The detectable label is a particulate label containing a metal sol such as colloidal gold, for example, a peptide chelating agent of type N2S2, N3S, or N4 is provided. 125 or Tc 99 This includes not only chromophores containing isotopes, fluorescent markers, luminescent markers, phosphorescent markers, etc., but also enzymatic labels that convert a given substrate into a detectable marker, and polynucleotide tags that can be identified after amplification by, for example, a polymerization enzyme chain reaction, and can be any of the diverse types used in the field of in vitro diagnostics. Suitable enzymatic labels include horseradish peroxidase and alkaline phosphatase. For example, the label may be not only adamantylmethoxyphosphoryloxyphenyl dioxetane (AMPPD) or disodium 3-(4-(methoxyspiro1,2-dioxetane-3,2'-(5'-chloro)tricyclo3.3.1.1 3,7-decane-4-yl)phenyl phosphate (CSPD), but also an alkaline phosphatase enzyme detected by measuring the presence or formation of chemiluminescence after chelation of a 1,2-dioxetane substrate such as CDP and CDP-star®, or other luminescent substrates well known to those skilled in the art, such as terbium(III) and europium(III). The detection means is determined by the selected label. The appearance of the label or its reaction product can be obtained by standard procedures using the naked eye or by instruments such as a spectrophotometer, illuminometer, or fluorometer, provided that the label is a fine particle but accumulates at an appropriate level.

[0284] Immunoconjugates can be prepared by methods known to the art. Preferably, the conjugation method results in substantially (or almost) non-immunogenic linkages, such as peptide-(i.e., amide-), sulfide-, (sterically hindered), disulfide-, hydrazon-, and ether linkages. These linkages are almost non-immunogenic and exhibit considerable stability in serum (see, e.g., Senter, PD, Curr. Opin. Chem. Biol. 13(2009) 235-244; WO2009 / 059278; WO95 / 17886).

[0285] Depending on the biochemical attributes of the moiety and antibody, different conjugation strategies can be used. If the moiety is naturally occurring or a recombinant of 50 to 500 amino acids, standard procedures describing chemical methods for synthesizing the protein conjugate are described in textbooks and can be easily followed by those skilled in the art (e.g., Hackenberger, CPR, and Schwarzer, D., An gew. Chem. Int. Ed. Engl. 47 (2008) 10030-10074). In some embodiments, a reaction is used between a cysteine ​​residue in the antibody or moiety and the maleinimid moiety. This is a particularly suitable coupling chemical method when, for example, the Fab or Fab'-fragment of the antibody is used. In contrast, in some embodiments, coupling is performed at the C-terminus of the antibody or moiety. Protein modifications, such as C-terminal deformation of Fab fragments, can be carried out as described, for example, in Sunbul, M. and Yin, J., Org. Biomol. Chem. 7 (2009) 3361-3371.

[0286] Generally, site-directed reactions and covalent bonding are based on converting native amino acids into amino acids whose reactivity is orthogonal to the reactivity of other functional groups present. For example, certain cysteines in rare sequence contexts can be enzymatically converted to aldehydes (see Frese, MA and Dierks, T., ChemBioChem. 10 (2009) 425-427). Desired amino acid deformations can also be obtained by utilizing the specific enzymatic reactivity of native amino acids and specific enzymes within a given sequence context (see, for example, Taki, M. et al., Prot.Eng.Des.Sel.17(2004)119-126; Gautier, A. et al., Chem.Biol.15(2008)128-136; and Protease-catalyzed formation of CN bonds is used by Bordusa, F., Highlights in Bioorganic Chemistry(2004)389-403).

[0287] Furthermore, site-directed reactions and covalent bonding can be achieved by appropriate deformatives and selective reactions of terminal amino acids. Site-directed covalent bonding can be obtained using the reactivity of benzonitrile with N-terminal cysteine ​​(see Ren, H. et al., Angew. Chem. Int. Ed. Engl. 48 (2009) 9658-9662). In addition, natural chemical ligation can depend on the C-terminal cysteine ​​residue (Taylor, E. Vogel; Imperiali, B, Nucleic Acids and Molecular Biology (2009), 22 (Protein Engineering), 65-96).

[0288] EP 1 074 563 describes a conjugation method based on a faster reaction between cysteine ​​located within a series of positively charged amino acids and cysteine ​​located within a series of negatively charged amino acids.

[0289] The aforementioned moiety may be a synthetic peptide or a peptide mimetic. When polypeptides are chemically synthesized, orthogonal, chemically reactive amino acids can be introduced during such synthesis (see, for example, de Graaf, AJet al., Bioconjug. Chem. 20(2009) 1281-1295). A wide variety of orthogonal functional groups can be introduced into synthetic peptides, albeit in an unstable manner, so linking such peptides to a linker is a standard chemical method.

[0290] To obtain a single-labeled polypeptide, conjugates having a 1:1 stoichiometric ratio can be separated from other conjugation byproducts by chromatography. This process can be facilitated by using dye-labeled binding pair members and charged linkers. By using such types of labeling and strongly negatively charged binding pair members, the difference in charge and molecular weight can be utilized for separation, so that the single-conjugated polypeptide can be easily separated from unlabeled polypeptides and polypeptides having more than one linker. The fluorescent dye may be useful for purifying the complex from unbound components, as with labeled monovalent binders.

[0291] [V. Nucleic acid molecules] Other embodiments described herein relate to one or more nucleic acid molecules coding for any one of the antibodies and their antigen-binding fragments described herein. Such nucleic acids may exist in whole cells, cell lysates, or in partially purified or substantially pure forms. Nucleic acids are “separated” or “substantially pure” when purified from other cellular components or other contaminants, e.g., other cellular nucleic acids (e.g., other chromosomal DNA, e.g., chromosomal DNA linked to naturally isolated DNA) or proteins by standard techniques including alkali / SDS treatment, CsCl banding, column chromatography, restriction enzymes, agarose gel electrophoresis and other techniques well known in the art. See F. Ausubel, el al., ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. The nucleic acids described herein may be, for example, DNA or RNA, and may or may not contain intron sequences. In certain embodiments, such nucleic acids are cDNA molecules.

[0292] The nucleic acids described herein can be obtained using standard molecular biology techniques. In the case of antibodies expressed by hybridomas (e.g., hybridomas produced from genetically modified mice possessing human immunoglobulin genes, as further described below), the cDNAs coding the light and heavy chains of the antibodies produced by the hybridoma can be obtained by standard PCR amplification or cDNA cloning techniques. In the case of antibodies obtained from an immunoglobulin gene library (e.g., using phage display techniques), the nucleic acids coding the antibodies can be recovered from the library.

[0293] Certain nucleic acid molecules code for the VH and VL sequences of the various anti-FAM19A1 antibodies described herein. Exemplary DNA sequences coding for the VH sequences of such antibodies are shown in SEQ ID NOs: 38, 36, 40, and 42. See Table 8. Exemplary DNA sequences coding for the VL sequences of such antibodies are shown in SEQ ID NOs: 39, 37, 41, and 43. See Table 9. [Table 8]

[0294] [Table 9] For example, a method for producing an anti-FAM19A1 antibody disclosed herein may include expressing the associated heavy and light chains of the antibody together with a signal peptide in a cell line containing nucleotide sequences coding the heavy and light chains. Host cells containing these nucleotide sequences are included herein.

[0295] Once DNA fragments coding the VH and VL segments are obtained, these DNA fragments can be further manipulated using standard recombinant DNA techniques to convert, for example, variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these operations, the VL or VH-coding DNA fragment is operatively ligated to another DNA fragment coding another protein, such as an antibody constant region or a flexible linker. The term "operatively ligated" as used in this context is intended to mean that the two DNA fragments are joined together, and the amino acid sequences coded by the two DNA fragments remain within the frame.

[0296] The isolated DNA coding the VH region can be converted into a full-length heavy chain gene by operatively ligating the VH-coding DNA to another DNA molecule coding the heavy chain constant region (hinge, CH1, CH2, and / or CH3). The sequences of human heavy chain constant region genes are publicly known in the art (see, for example, Kabat, EA, el al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region may be the IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, for example, the IgG2 and / or IgG4 constant region. In the case of Fab fragment heavy chain genes, the VH-coding DNA can be operatively ligated to another DNA molecule coding only the heavy chain CH1 constant region.

[0297] The isolated DNA coding the VL region can be converted into a full-length light chain gene (and not just a Fab light chain gene) by operatively ligating the VL-coding DNA to another DNA molecule coding the light chain constant region (CL). The sequences of human light chain constant region genes are publicly known in the art (e.g., Kabat, EA, et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USD Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. The light chain constant region may be a kappa or lambda constant region.

[0298] To generate scFv antibodies, the VH and VL coding DNA fragments are operatively linked to a flexible linker, for example, another fragment coding the amino acid sequence (Gly4-Ser)3, and the VH and VL sequences can be expressed as adjacent single-chain proteins with the VL and VH regions joined by the flexible linker (see, for example, Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); McCafferty et al., Nature 348:552-554 (1990)).

[0299] In some embodiments, the vectors disclosed herein include isolated nucleic acid molecules comprising a nucleotide sequence coding an antibody or an antigen-binding fragment thereof.

[0300] Appropriate vectors for this disclosure include expression vectors, viral vectors, and plasmid vectors. In some aspects, the vector is a viral vector.

[0301] As used herein, "expression vector" refers to any nucleic acid structure containing the elements necessary for the transcription and translation of an inserted coding sequence, or, in the case of an RNA viral vector, the elements necessary for replication and translation upon introduction into a suitable host cell. Expression vectors may include plasmids, phagemids, viruses, and derivatives thereof.

[0302] [VI. Antibody Production] The anti-FAM19A1 antibodies disclosed herein may be produced by any method known in the art for antibody synthesis, such as chemical synthesis or recombinant expression techniques. Unless otherwise stated, the methods described herein utilize prior art in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields of the art. These techniques are described, for example, in the references cited herein and are fully explained therein.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. al.,Current Protocols in Molecular Biology,John Wiley & Sons(1987 and annual updates);Current Protocols in Immunology,John Wiley & Sons(1987 and annual updates)Gait(ed.)(1984)Oligonucleotide Synthesis:A Practical Approach,IRL Press;Eckstein(ed.)(1991)Oligonucleotides and Analogues:A Practical Approach,IRL Press;Birren B et You can refer to al., (eds.) (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press.

[0303] In some aspects, the antibodies described herein are antibodies (e.g., recombinant antibodies) manufactured, expressed, produced, or isolated by any means, such as synthesis or production through genetic engineering of DNA sequences. In certain aspects, such antibodies contain sequences (e.g., DNA sequences or amino acid sequences) that do not naturally exist in the antibody germline repertoire of living animals or mammals (e.g., humans).

[0304] In certain embodiments, this specification provides a method for producing an antibody or antigen-binding fragment thereof that immunospecifically binds to FAM19A1 (e.g., human FAM19A1), comprising culturing cells or host cells described herein. In certain embodiments, this specification provides a method for producing an antibody or antigen-binding fragment thereof that immunospecifically binds to FAM19A1 (e.g., human FAM19A1), comprising the step of expressing the antibody or antigen-binding fragment thereof using cells or host cells described herein (e.g., cells or host cells containing polynucleotides encoding the antibody described herein). In certain embodiments, the cells are isolated cells. In certain embodiments, exogenous polynucleotides are introduced into the cells. In certain embodiments, the method further comprises the step of purifying the antibody or antigen-binding fragment thereof obtained from the cells or host cells.

[0305] Methods for producing polyclonal antibodies are publicly known in the industry (see, for example, Chapter 11 in: Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel FM et al., eds., John Wiley and Sons, New York).

[0306] Monoclonal antibodies can be produced using a variety of technologies known to the art, including the use of hybridoma, recombination, and phage display technologies or combinations thereof. For example, monoclonal antibodies can be produced using hybridoma technologies, including technologies known to the art and taught in, for example, Harlow E & Lane D, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling GJ et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563 681 (Elsevier, NY, 1981). The term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technologies. For example, monoclonal antibodies can be produced by recombination from antibodies or fragments thereof described herein, for example, from host cells exogenously expressing the light and / or heavy chains of such antibodies.

[0307] In certain aspects, the “monoclonal antibody” as used herein is an antibody produced by a single cell (e.g., a hybridoma or host cell producing recombinant antibodies) that immunospecifically binds to FAM19A1 (e.g., human FAM19A1) as determined, for example, by ELISA or other antigen-binding or competitive binding analysis methods known in the art or described in the Examples section provided herein. In certain aspects, a monoclonal antibody may be a chimeric antibody or a humanized antibody. In certain aspects, a monoclonal antibody may be a monovalent or polyvalent (e.g., bivalent) antibody. In certain aspects, a monoclonal antibody may be a monospecific or multispecific (e.g., bispecific) antibody. Monoclonal antibodies described herein can be produced, for example, by a hybridoma method as described in Kohler G & Milstein C (1975) Nature 256:495, or they can be isolated from a phage library, for example, using the techniques described herein. Other methods for producing clonal cell lines and the monoclonal antibodies expressed thereby are well known in the industry (see, for example, Chapter 11 above in: Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel FM et al.).

[0308] Methods for producing and screening specific antibodies using hybridoma technology are commonplace and well-known in the field. For example, hybridoma methods immunize mice or other suitable host animals such as sheep, goats, rabbits, rats, hamsters, or macaque monkeys to produce antibodies that specifically bind to the protein used for immunization (e.g., human FAM19A1), or to derive lymphocytes capable of producing such antibodies. In contrast, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusion agent such as polyethylene glycol to form hybridoma cells (Goding JW (Ed), Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Furthermore, animals can be immunized using RIMMS (Repeated Immunization Multiple Site) technology (Kilpatrick KE et al., (1997) Hybridoma 16:381-9, the full text is included for reference).

[0309] In certain configurations, mice (or other animals such as chickens, rats, monkeys, donkeys, pigs, sheep, hamsters, or dogs) can be immunized with an antigen (e.g., FAM19A1, such as human FAM19A1). Once an immune response is detected, for example, if antigen-specific antibodies are detected in mouse serum, the mouse spleen is harvested and splenic cells are isolated. These splenic cells are then fused with any suitable myeloma cells, for example, cells from the SP20 cell line available from the ATCC (American Type Culture Collection) (Manassas, VA), using well-known techniques to form hybridomas. The hybridomas are then sorted and cloned with limited dilution. In certain configurations, lymph nodes from immunized mice are harvested and fused with NSO myeloma cells.

[0310] The hybridoma cells thus produced are inoculated and grown in a suitable culture medium containing one or more substances that preferably inhibit the growth or survival of unfused parent myeloma cells. For example, if the parent myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the hybridoma culture medium may contain hypoxanthine, aminopterin, and thymidine (HAT medium), which are substances that typically inhibit the growth of HGPRT-deficient cells.

[0311] Specific methods support stable, high-level antibody production by efficiently fused and selected antibody-producing cells, using myeloma cells sensitive to media such as HAT medium. These myeloma cell lines include mouse myeloma cell lines such as NSO cell lines or MOPC-21 and MPC-11 mouse tumor cells available from Salk Institute Cell Distribution Center, San Diego, CA, USA, and SP-2 or X63-Ag8.653 cells available from American Type Culture Collection, Rockville, MD, USA. Human myeloma and mouse-human xenomyeloma cell lines have also been described for human monoclonal antibody production (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)).

[0312] The culture medium in which hybridoma cells grow is analyzed for the production of monoclonal antibodies directed against FAM19A1 (e.g., human FAM19A1). The binding specificity of the monoclonal antibodies produced by hybridoma cells is determined by methods known in the art, such as immunoprecipitation or in vitro binding analysis, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0313] After identifying hybridoma cells that produce antibodies with desired specificity, affinity, and / or activity, the clones can be subcloned with limited dilution procedures and grown by standard methods (see Goding JW (Ed), Monoclonal Antibodies: Principles and Practice above). Suitable culture media for such purposes include, for example, D-MEM or RPMI 1640 medium. Hybridoma cells can also grow in vivo as ascites tumors in animals.

[0314] Monoclonal antibodies secreted by subclones are appropriately separated from culture media, ascites fluid, or serum by conventional immunoglobulin purification procedures such as protein A-Sepharose chromatography, hydroxyl apatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0315] The antibodies described herein include antibody fragments that recognize a specific FAM19A1 (e.g., human FAM19A1) and can be produced by any technique known to those skilled in the art. For example, the Fab and F(ab')2 fragments described herein can be produced by proteolytic cleavage of an immunoglobulin molecule using an enzyme such as papain (for producing the Fab fragment) or pepsin (for producing the F(ab')2 fragment). The Fab fragment corresponds to one of two identical arms of the antibody molecule and includes a complete light chain paired with the VH and CH1 domains of the heavy chain. The F(ab')2 fragment includes two antigen-binding arms of the antibody molecule linked by a disulfide bond at a hinge region.

[0316] Furthermore, the antibodies or antigen-binding fragments described herein can also be generated using a variety of phage display methods known to the art. In phage display methods, the functional antibody domain is displayed on the surface of a phage particle possessing the polynucleotide sequence coding it. In particular, the DNA sequences coding the VH and VL domains are amplified from an animal cDNA library (e.g., a human cDNA library or a non-human cDNA library such as a mouse or chicken cDNA library from infected tissue). The DNA coding the VH and VL domains is recombined with an scFv linker by PCR and cloned into a phagemide vector. The vector is electroperforated with E. coli, and the E. coli is infected with a helper phage. The phages used in these methods are typically filamentous phages containing fd and M13, and the VH and VL domains are generally fused by recombination to phage gene III or gene VIII. Phages expressing antigen-binding domains that bind to specific antigens can be screened or identified using antigens, such as labeled antigens, or antigens bound to or captured on solid surfaces or beads.Examples of phage display methods that can be used for the production of antibodies described herein include: Brinkman U et al., (1995) J Immunol Methods 182:41-50; Ames RS et al., (1995) J Immunol Methods 184:177-186; Kettleborough CA et al., (1994) Eur J Immunol 24:952-958; Persic L et al., (1997) Gene 187:9-18; Burton DR & Barbas CF (1994) Advan Immunol 57:191-280; PCT application number PCT / GB91 / 001134; International publication numbers WO90 / 02809, WO91 / 10737, WO92 / 01047, WO92 / 18619, WO93 / 11236, WO95 / 15982, WO95 / 20401 and WO97 / 13844; U.S. Patent No. 5,698,426 This includes what is disclosed in 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.

[0317] As described in the aforementioned references, after phage selection, the antibody coding region of the phage can be isolated and used to produce a whole antibody or any other desired antigen-binding fragment containing a human antibody, which can be expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, as described below. Techniques for recombinant production of antibody fragments such as Fab, Fab', and F(ab')2 fragments can also be employed using methods known to the art, as disclosed in PCT publication number WO92 / 22324; Mullinax RL et al., (1992) BioTechniques 12(6):864-9; Sawai H et al., (1995) Am J Reprod Immunol 34:26-34; and Better M et al., (1988) Science 240:1041-1043.

[0318] In one manner, to generate a full antibody, the VH or VL sequence can be amplified from a template, for example, an scFv clone, using PCR primers containing a VH or VL nucleotide sequence, a restriction site, and a side sequence to protect the restriction site. The PCR-amplified VH domain can be cloned into a vector expressing the VH constant region using cloning techniques known to those skilled in the art, and the PCR-amplified VL domain can be cloned into a vector expressing the VL constant region, for example, a human kappa or lambda constant region. The VH and VL domains can also be cloned into a single vector expressing the required constant region. Next, the heavy chain conversion vector and the light chain conversion vector are simultaneously transfused into a cell line using techniques known to those skilled in the art to generate a stable or transient cell line expressing a full-length antibody, for example, IgG.

[0319] A chimeric antibody is a molecule in which each distinct portion of the antibody originates from a different immunoglobulin molecule. For example, a chimeric antibody may include a variable region of a non-human animal (e.g., mouse, rat, or chicken) monoclonal antibody fused to the constant region of a human antibody. Various methods for producing chimeric antibodies are known in the art. See, for example, Morrison SL (1985) Science 229:1202-7; Oi VT & Morrison SL (1986) BioTechniques 4:214-221; Gillies SD et al., (1989) J Immunol Methods 125:191-202; and U.S. Patent Nos. 5,807,715, 4,816,567, 4,816,397 and 6,331,415.

[0320] A humanized antibody comprises a framework region that can bind to a given antigen and substantially has the amino acid sequence of a human immunoglobulin, and a CDR that substantially has the amino acid sequence of a non-human immunoglobulin (e.g., mouse or chicken immunoglobulin). In certain embodiments, the humanized antibody comprises an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin. The antibody may also contain the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. The humanized antibody may be selected from all types of immunoglobulins, including IgM, IgG, IgD, IgA, and IgE, and any isotypes, including IgG1, IgG2, IgG3, and IgG4.Humanized antibodies can be produced using a variety of techniques known to the art, including, but are not limited to, the following: CDR grafting (European Patent No. EP239400; International Publication No. WO91 / 09967; and US Patent Nos. 5,225,539, 5,530,101 and 5,585,089), veneering, or resurfacing (European Patent Nos. EP592106 and EP519596; Padlan EA (1991) Mol Immunol 28(4 / 5):489-498; Studnicka GM et al., (1994) Prot Engineering 7(6):805-814; and Roguska MA et al., (1994) PNAS 91:969-973), chain shuffling (US Patent No. 5,565,332), and, for example, US Patent No. 6,407,213, US Patent No. 5,766,886, International Publication No. WO93 / 17105; Tan P et al., (2002) J Immunol 169:1119-25; Caldas C et al., (2000) Protein Eng. 13(5):353-60; Morea V et al., (2000) Methods 20(3):267-79; Baca M et al., (1997) J Biol Chem 272(16):10678-84; Roguska MA et al., (1996) Protein Eng 9(10):895-904; Couto JR et The technology disclosed in 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. Patent Application Publication No. US2005 / 0042664 A1 (February 24, 2005), the full text of which is included herein by reference.

[0321] Methods for producing multiple specific antibodies (e.g., bispecific antibodies) are described (see, for example, U.S. Patent Nos. 7,951,917; 7,183,076; 8,227,577; 5,837,242; 5,989,830; 5,869,620; 6,132,992 and 8,586,713).

[0322] Single-domain antibodies, such as antibodies lacking a light chain, can be produced by methods well known in the art. See Riechmann L & Muyldermans S(1999)J Immunol 231:25-38; Nuttall SD et al.,(2000)Curr Pharm Biotechnol 1(3):253-263; Muyldermans S,(2001)J Biotechnol 74(4):277-302; U.S. Patent No. 6,005,079; and International Publication Nos. WO94 / 04678, WO94 / 25591 and WO01 / 44301.

[0323] Furthermore, antibodies that immunospecifically bind to the FAM19A1 antigen can also be used to generate anti-genotype antibodies that "mimic" the antigen using techniques well known to those skilled in the art (see, for example, Greenspan NS & Bona CA (1989) FASEB J 7(5):437-444; and Nissinoff A (1991) J Immunol 147(8):2429-2438).

[0324] In a particular manner, an antibody described herein that binds to the same FAM19A1 (e.g., human FAM19A1) epitope as the anti-FAM19A1 antibody described herein is a human antibody or its antigen-binding fragment. In a particular manner, an antibody described herein that competitively blocks (e.g., in a dose-dependent manner) the binding of the antibody described herein to FAM19A1 (e.g., human FAM19A1) is a human antibody or its antigen-binding fragment.

[0325] Human antibodies can be produced using any method known to the art. For example, genetically modified mice can be used in which functional endogenous immunoglobulins cannot be expressed but human immunoglobulin genes can be expressed. In particular, the human heavy-chain and light-chain immunoglobulin gene complex can be introduced into mouse embryonic stem cells randomly or by homologous recombination. In contrast, in addition to the human heavy-chain and light-chain genes, human variable regions, constant regions, and diversity regions can also be introduced into mouse embryonic stem cells. The mouse heavy-chain and light-chain immunoglobulin genes can become non-functional by homologous recombination, either separately or simultaneously with the introduction of human immunoglobulin loci. In particular, homozygous deletion of the JH region makes endogenous antibody production impossible. Chimeric mice are generated by expanding the modified embryonic stem cells and microinjecting them into blastocysts. The chimeric mice are then reared to produce homozygous offspring that express human antibodies. The genetically modified mice are immunized in a normal manner with all or part of a selected antigen, for example, the antigen (e.g., FAM19A1). Antigen-directed monoclonal antibodies can be obtained from genetically modified mice immunized using conventional hybridoma technology. The human immunoglobulin transgenes possessed by the genetically modified mice are rearranged during B cell differentiation, followed by class switching and somatic mutation. This allows for the production of therapeutically useful IgG, IgA, IgM, and IgE antibodies using such technology. For an overview of such technology for human antibody production, see Lonberg N & Huszar D (1995) Int Rev Immunol 13:65-93. For a detailed discussion of such techniques for producing human antibodies and human monoclonal antibodies, and protocols for producing such antibodies, see, for example, International Publication Nos. WO98 / 24893, WO96 / 34096 and WO96 / 33735; and U.S. Patent Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318 and 5,939,598.An example of a mouse capable of producing human antibodies is the XENOMOUSE. TM (Abgenix, Inc.; U.S. Patent Nos. 6,075,181 and 6,150,184), HUAB-MOUSE TM (Mederex, Inc. / Gen Pharm; U.S. Patent Nos. 5,545,806 and 5,569,825), TRANS CHROMO MOUSE TM (Kirin) and KM MOUSE TM See (Medarex / Kirin).

[0326] Human antibodies that specifically bind to FAM19A (e.g., human FAM19A1) can be produced using antibody libraries derived from human immunoglobulin sequences by a variety of methods known in the art, including the phage display method described above. See also U.S. Patent Nos. 4,444,887, 4,716,111 and 5,885,793; and International Publication Nos. WO98 / 46645, WO98 / 50433, WO98 / 24893, WO98 / 16654, WO96 / 34096, WO96 / 33735 and WO91 / 10741.

[0327] In some manner, 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. These mouse-human hybridomas can then be screened to determine whether they secrete human monoclonal antibodies that immunospecifically bind to a target antigen (e.g., human FAM19A1). These methods are publicly known and described in the art (see, e.g., Shinmoto H et al., (2004) Cytotechnology 46:19-23; Naganawa Y et al., (2005) Human Antibodies 14:27-31).

[0328] [VII. Methods for manipulating antibodies] As discussed above, the anti-FAM19A1 antibody or its antigen-binding moiety having the VH and VL sequences disclosed herein can be used to generate a new anti-FAM19A1 antibody or its antigen-binding moiety by modifying the VH and / or VL sequences or the constant region attached thereto. In other embodiments described herein, the structural features of the anti-FAM19A1 antibody described herein are used to generate a structurally related anti-FAM19A1 antibody that possesses at least one functional property of the antibody described herein, such as binding to human FAM19A1. For example, the starting material for the method described herein is the VH and / or VL sequences or one or more CDR regions thereof provided herein. It is not necessary to actually manufacture (i.e., express as a protein) an antibody having one or more of the VH and / or VL sequences or one or more CDR regions thereof provided herein in order to generate the modified antibody. Instead, the information contained in the aforementioned sequence is used as a starting material to generate a "second-generation" sequence derived from the original sequence, and then the "second-generation" sequence is manufactured and expressed as a protein.

[0329] Therefore, this specification is: (a)(i) Provide the CDR1, CDR2 and / or CDR3 sequences shown in Table 4 or the heavy chain variable region sequences including the heavy chain variable region CDR1, CDR2 and / or CDR3 shown in Table 6; and (ii) Provide the CDR1, CDR2 and / or CDR3 sequences shown in Table 5 or the light chain variable region sequences including the light chain variable region CDR1, CDR2 and / or CDR3 shown in Table 7; (b) Modify the sequence of the heavy chain variable region and / or the sequence of the light chain variable region to generate the sequence of at least one modified antibody or antigen-binding moiety; (c) The present invention provides a method for producing an anti-FAM19A1 antibody or its antigen-binding portion, which includes expressing the modified antibody or antigen-binding portion sequence as a protein.

[0330] Using standard molecular biology techniques, modified antibody or antigen-binding sequences can be manufactured and expressed.

[0331] In some manner, the antibody or antigen-binding moiety coded by the modified antibody or antigen-binding moiety sequence possesses one, some, or all of the functional properties of the anti-FAM19A1 antibody described herein. Non-limiting examples of such properties include: (1) For example, BIACORE TM Or when measured by ELISA, K D The property of binding to soluble human FAM19A1 with a concentration of 10 nM or less; (2) For example, BIACORE TM Or when measured by ELISA, K D The property of binding to membrane-bound human FAM19A1 with a concentration of 10 nM or less; (3) Properties that promote the differentiation of nerve cells; (4) The property of increasing neurite growth in differentiated nerve cells; (5) Properties that reduce, reverse, and / or prevent one or more symptoms associated with glaucoma; (6) Properties that improve retinal potential (e.g., demonstrated by increased rhythmic ripple); (7) Properties that reduce and / or restore the loss of retinal ganglion cells (e.g., observed in glaucoma patients); (8) Properties that reduce, reverse, and / or prevent one or more symptoms associated with neuropathic pain; (9) Characteristics that increase the delay time and / or threshold to external stimuli; and (10) Properties that increase and / or regulate sensory nerve conduction velocity; including

[0332] In certain manners of the methods for manipulating the antibodies described herein, mutations may be randomly or selectively introduced along all or part of the anti-FAM19A1 antibody coding sequence, and the resulting modified anti-FAM19A1 antibody can be screened for binding activity and / or other functional properties described herein. Mutation methods are described in the art. For example, Short's PCT Publication WO02 / 092780 describes a method for generating and screening antibody mutations using saturated mutagenesis, synthetic ligation assembly, or a combination thereof. In contrast, Lazar et al.'s PCT Publication WO03 / 074679 describes a method using computer screening methods to optimize the physicochemical properties of an antibody.

[0333] [VIII. Cells and Vectors] In certain embodiments, this specification provides cells (e.g., host cells) that express (e.g., by recombination) an antibody described herein that specifically binds to FAM19A1 (e.g., human FAM19A1), a related polynucleotide, and an expression vector. This specification provides vectors (e.g., expression vectors) that contain polynucleotides comprising a nucleotide sequence coding an anti-FAM19A1 antibody or fragment for recombinant expression in host cells, e.g., mammalian cells. This specification also provides host cells containing such vectors for recombinant expression of an anti-FAM19A1 antibody (e.g., human or humanized antibody). In certain embodiments, this specification provides a method for producing an antibody described herein, comprising expressing such an antibody from a host cell.

[0334] Recombinant expression of antibodies described herein (e.g., full-length antibodies, heavy chains and / or light chains or single-chain antibodies) that specifically bind to FAM19A1 (e.g., human FAM19A1) involves the preparation of an expression vector containing a polynucleotide coding the antibody. Once polynucleotides coding the antibody molecule, the heavy chain and / or light chain or fragment thereof (e.g., heavy chain and / or light chain variable domain) described herein are obtained, a vector for the production of the antibody molecule can be prepared by recombinant DNA technology using techniques well known in the art. Thus, a method for producing a protein by expressing a polynucleotide containing a nucleotide sequence coding an antibody or antibody fragment (e.g., light chain or heavy chain) is described herein. Expression vectors containing the antibody or antibody fragment (e.g., light chain or heavy chain) coding sequence and appropriate transcription and translation control signals can be prepared using methods well known to those skilled in the art. These methods include, for example, in vitro recombinant DNA technology, synthesis technology and in vivo genetic recombination. Furthermore, this specification provides a replicable vector comprising an antibody molecule, a heavy or light chain of an antibody, a variable domain or fragment thereof of a heavy or light chain of an antibody, or a nucleotide sequence coding a heavy or light chain CDR operably linked to a promoter. Such a vector may, for example, contain a nucleotide sequence coding the constant region of the antibody molecule (see, for example, International Publication Nos. WO86 / 05807 and WO89 / 01036, and U.S. Patent No. 5,122,464, whose full texts are included herein for reference), and the variable domain of the antibody may be cloned into such a vector for the expression of the entire heavy chain, the entire light chain, or all of the entire heavy and light chains.

[0335] An expression vector can be transmitted to a cell (e.g., a host cell) by conventional techniques, and the resulting cell can then be cultured by conventional techniques to produce an antibody described herein (e.g., an antibody comprising one or more of the VH and / or VL or VH and / or VL CDR of the anti-FAM19A1 antibody described herein) or a fragment thereof. Thus, this specification provides a host cell containing a polynucleotide, the polynucleotide coding an antibody described herein or a fragment thereof, its heavy chain or light chain or fragment thereof, or a single-chain antibody described herein, operably linked to a promoter for the expression of such a sequence in the host cell. In certain embodiments, a vector individually coding all of the heavy and light chains for the expression of a double-chain antibody can be co-expressed in a host cell for the expression of the entire immunoglobulin molecule, as described in detail later. In certain embodiments, a host cell contains a vector containing a polynucleotide coding all or a fragment thereof of the heavy and light chains of an antibody described herein. In certain embodiments, a host cell contains two distinct vectors: a first vector containing a polynucleotide coding for the heavy chain or heavy chain variable region or a fragment thereof of the antibody described herein, and a second vector containing a polynucleotide coding for the light chain or light chain variable region or a fragment thereof of the antibody described herein. In certain embodiments, the first host cell contains the first vector containing a polynucleotide coding for the heavy chain or heavy chain variable region or a fragment thereof of the antibody described herein, and the second host cell contains the second vector containing a polynucleotide coding for the light chain or light chain variable region of the antibody described herein. In certain embodiments, the heavy chain / heavy chain variable region expressed by the first cell associates with the light chain / heavy chain variable region of the second cell to form the anti-FAM19A1 antibody or its antigen-binding fragment described herein. In certain embodiments, this specification provides a population of host cells including such a first host cell and such a second host cell.

[0336] In certain aspects, this specification provides a group of vectors comprising a first vector containing a polynucleotide coding the light chain / light chain variable region of the anti-FAM19A1 antibody described herein, and a second vector containing a polynucleotide coding the heavy chain / heavy chain variable region of the anti-FAM19A1 antibody described herein.

[0337] A variety of host-expression vector systems can be used to express the antibody molecules described herein. Such host-expression systems correspond to a vehicle that generates and subsequently purifies the coding sequence of interest, or to cells that can express the antibody molecules described herein in situ upon transformation or translocation with a suitable nucleotide coding sequence. These include microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacon telophage DNA, plasmid DNA, or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors containing antibody coding sequences (e.g., baculovirus); 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 containing antibody coding sequences (e.g., Ti plasmid); or mammalian cell systems (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK) possessing recombinant expression structures containing promoters derived from mammalian cell genomes (e.g., metallothionein promoter) or promoters derived from mammalian viruses (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter). This includes, but is not limited to, cells (293, NSO, PER.C6, VERO, CRL7030, HsS78Bst, HeLa, NIH3T3, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSCl, BSC40, YB / 20, and BMT10). In a particular manner, cells for expressing the antibodies or antigen-binding fragments described herein include CHO cells, e.g., CHO GS SYSTEM TMThese are (Lonza) CHO cells. In some embodiments, the cells used to express the antibodies described herein are human cells, e.g., human cell lines. In some embodiments, the mammalian expression vector is POPTIVEC TM or pcDNA3.3. In some embodiments, bacterial cells such as Escherichia coli or eukaryotic cells (e.g., mammalian cells) are used for the expression of recombinant antibody molecules, particularly for the expression of the entire recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary (CHO) cells, along with vectors such as the major intermediate early gene promoter element of 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 some embodiments, the expression of the nucleotide sequence coding the antibodies described herein that immunospecifically bind to FAM19A1 (e.g., human FAM19A1) is regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

[0338] In bacterial systems, numerous expression vectors can be advantageously selected based on the intended use of the antibody molecule being expressed. For example, when attempting to mass-produce such antibodies to create pharmaceutical compositions of antibody molecules, vectors that direct the expression of a high level of readily purifiable fusion protein product are preferred. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruether U & Mueller-Hill B (1983) EMBO J 2:1791-1794); pIN vector (Inouye S & Inouye M (1985) Nuc Acids Res 13:3101-3109; Van Heeke G & Schuster SM (1989) J Biol Chem 24:5503-5509), in which the antibody coding sequence is individually ligated into the vector in a configuration having a lac Z coding region to produce a fusion protein. For example, a pGEX vector can be used to express an exogenous polypeptide as a fusion protein via glutathione 5-transferase (GST). Generally, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to matrix glutathione agarose beads, followed by elution in the presence of free glutathione. The pGEX vector is configured to include a thrombin or factor Xa protease cleavage site, and the cloned target gene product can be released from the GST moiety.

[0339] In insect systems, for example, AcNPV (Autographa californica nuclear polyhedron virus) can be used as a vector for expressing foreign genes. The virus grows in armyworm (Spodoptera frugiperda) cells. The antibody coding sequence can be individually cloned into a non-essential region of the virus (e.g., a polyhedrin gene) and may be under the control of the AcNPV promoter (e.g., a polyhedrin promoter).

[0340] In mammalian host cells, numerous viral expression systems can be used. When using adenovirus as an expression vector, the antibody coding sequence of interest can be ligated to the adenovirus transcription / translation regulatory complex, e.g., the late promoter and tripartite reader sequence. This chimeric gene can then be inserted into the adenovirus genome by recombination in vitro or in vivo. Insertion into a non-essential region of the viral genome (e.g., region E1 or E3) can generate a viable recombinant virus capable of expressing antibody molecules in an infected host (see, e.g., Logan J & Shenk T (1984) PNAS 81(12):3655-9). Specific start signals may also be required for efficient translation of the inserted antibody coding sequence. These signals include an ATG start codon and adjacent sequences. Furthermore, the start codon must align with the reading frame of the desired coding sequence to enable translation of the entire insertion. These exogenous translational control signals and start codons can have a wide variety of origins, both natural and synthetic. Expression efficiency can be increased by including appropriate transcriptional enhancers, transcription termination factors, etc. (see, for example, Bitter G et al., (1987) Methods Enzymol. 153:516-544).

[0341] Furthermore, host cell strains can be selected that regulate the expression of the inserted sequence or that deform and process the gene product in a desired, specific manner. Such deformation (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for protein function. Different host cells have characteristic yet specific mechanisms for post-translational processing and deformation of proteins and gene products. Appropriate cell lines or host systems can be selected to enable the correct deformation and processing of expressed foreign proteins. For this purpose, eukaryotic host cells possessing appropriate cellular mechanisms for processing primary transcripts and for glycosylation and phosphorylation of gene products can be used. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, Hela, MDCK, HEK 293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT20, T47D, NSO (mouse myeloma cell line that does not produce any immunoglobulin chains in the body), CRL7030, COS (e.g., COS 1 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 manner, the anti-FAM19A1 antibodies described herein are produced in mammalian cells such as CHO cells.

[0342] In some manner, the antibodies or their antigen-binding moieties described herein have reduced or no fucose content. Such antibodies can be produced using techniques known to those skilled in the art. For example, such antibodies can be expressed in cells with insufficient or absent fucosylation ability. In certain cases, cell lines in which both alleles of 1,6-fucosyltransferase are knocked out can be used to produce antibodies or their antigen-binding moieties with reduced fucose content. The POTELLIGENT® system (Lonza) is an example of such a system that can be used to produce antibodies or their antigen-binding moieties with reduced fucose content.

[0343] Stable expression cells can be created to produce recombinant proteins in high yield over long periods. For example, cell lines that stably express the anti-FAM19A1 antibody or its antigen-binding moiety as described herein can be manipulated. In a specific manner, the cells provided herein associate and stably express the light chain / light chain variable domain and heavy chain / heavy chain variable domain that form the antibody or its antigen-binding moiety as described herein.

[0344] In certain configurations, host cells can be transformed using DNA and selection markers controlled by appropriate expression regulatory elements (e.g., promoters, enhancers, sequences, transcription termination factors, polyadenylation sites, etc.) instead of using expression vectors containing viral replication origins. After introduction of exogenous DNA / polynucleotides, the engineered cells can be grown in concentrated medium for 1-2 days, and then switched to selective medium. The selection markers in recombinant plasmids confer resistance to selection, allowing cells to stably integrate and grow the plasmids into these chromosomes, forming foci that can be cloned and expanded into cell lines. This method is advantageously applicable to engineer cell lines expressing the anti-FAM19A1 antibody or its antibody-binding moiety as described herein. Such engineered cell lines may be particularly useful for screening and evaluating compositions that directly or indirectly interact with antibody molecules.

[0345] Numerous selection systems are available, but are not limited to, those containing the herpes simplex virus thymidine kinase (Wigler M et al., (1977) Cell 11(1):223-32), hypoxanthine guanine phosphoribosyltransferase (Szybalska EH & Szybalski W (1962) PNAS 48(12):2026-2034), and adenine phosphoribosyltransferase (Lowy I et al., (1980) Cell 22(3):817-23) genes, which can be used in tk-, hgprt-, or aprt- cells, respectively. Furthermore, anti-metabolite resistance can be used as a basis for selection for genes such as: dhfr conferring methotrexate resistance (Wigler M et al., (1980) PNAS 77(6):3567-70; O'Hare K et al., (1981) PNAS 78:1527-31); gpt conferring mycophenolate resistance (Mulligan RC & Berg P (1981) PNAS 78(4):2072-6); neo conferring aminoglycoside G-418 resistance (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 (Santerre RF et al., (1984) Gene 30(1-3):147-56), which confers hygromycin resistance.Methods commonly known in the field of recombinant DNA technology are typically applicable 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 Chapters 12 and 13, Dracopoli NC et al., (eds.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); and Colbere-Garapin F et al., (1981) J Mol Biol 150:1-14, the full text of which is included herein by reference.

[0346] The expression level of antibody molecules can be increased by vector amplification (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) for further consideration). When a marker in an antibody-expressing vector system is amplified, increasing the level of the inhibitor present in the host cell culture can increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, antibody production can also increase (Crouse GF et al., (1983) Mol Cell Biol 3:257-66).

[0347] The host cell may be simultaneously transfused by two or more expression vectors described herein, namely a first vector coding for a heavy chain polypeptide and a second vector coding for a light chain polypeptide. The two vectors may contain the same selection marker that enables the same expression of the heavy chain and light chain polypeptides. The host cell may be simultaneously transfused by different amounts of the two or more expression vectors. For example, the host cell may be transfused by any one of the following ratios of the first expression vector and the second expression vector: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:12, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50.

[0348] In contrast, a single vector capable of coding and expressing all heavy and light chain polypeptides can be used. In such a situation, the light chain must be located ahead of the heavy chain to prevent excessive release of toxic heavy chain (Proudfoot NJ (1986) Nature 322:562-565; and Kohler G (1980) PNAS 77:2197-2199). The sequences coding the heavy and light chains may include cDNA or genomic DNA. The expression vector may be monocistronic or multicistronic. A multicistronic nucleic acid structure can code 2, 3, 4, 5, 6, 7, 8, 9, 10 or more genes / nucleotide sequences, or in the range of 2-5, 5-10, or 10-20. For example, a bisistronic nucleic acid structure may include, in order, a promoter, a first gene (e.g., the heavy chain of the antibody described herein), and a second gene (e.g., the light chain of the antibody described herein). In such a vector, the transcription of the two genes may be driven by the promoter, while the translation of mRNA from the first gene may be driven by a cap-dependent scanning mechanism, and the translation of mRNA from the second gene may be driven by a cap-independent mechanism, such as IRES.

[0349] Once the antibody molecules described herein are produced by recombinant expression, they can be purified by any method known in the art for the purification of immunoglobulin molecules, such as chromatography (e.g., ion exchange, affinity, particularly protein A and later, affinity for specific antigens, and sizing column chromatography), centrifugation, differential isosolubility, or other standard techniques for protein purification. Furthermore, the purification of the antibodies described herein can be facilitated by fusing them to heterologous polypeptide sequences described herein or otherwise known in the art.

[0350] In certain manner, the antibodies or their antigen-binding moieties described herein are isolated or purified. Generally, the isolated antibodies are substantially free of other antibodies whose antigen specificity differs from that of the isolated antibody. For example, in certain manner, the antibody formulations described herein are substantially free of cellular material and / or chemical precursors. The term “substantially free of cellular material” includes antibody formulations that are isolated from cells or produced by recombination and isolated from the cellular components of cells. Thus, antibodies substantially free of cellular material include antibody formulations that contain less than about 30%, 20%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (on a dry weight basis) of heterologous proteins (also referred herein as “contaminating proteins”) and / or variants of the antibody, such as different post-translational modified forms of the antibody or other different modified forms of the antibody (or antibody-binding moiety). When the antibody is produced by recombinant DNA, the antibody generally contains substantially no culture medium, i.e., the culture medium accounts for less than about 20%, 10%, 2%, 1%, 0.5%, or 0.1% of the volume of the protein preparation. When the antibody is produced by chemical synthesis, the antibody generally contains substantially no chemical precursors or other chemical substances, i.e., it is separated from chemical precursors or other chemical substances involved in protein synthesis. Thus, such antibody preparations contain less than about 30%, 20%, 10%, or 5% (on a dry weight basis) of chemical precursors or compounds other than the antibody of interest. In some embodiments, the antibodies described herein are separated or purified.

[0351] [IX. Diagnosis] As described above, the FAM19A1 antagonists described herein (e.g., anti-FAM19A1 antibodies) can be used for diagnostic purposes, including sample testing and in vivo imaging, for which an immunoconjugate can be formed by conjugating the antibody (or its binding portion) to a suitable detectable formulation. For diagnostic purposes, a suitable formulation is a detectable label containing a radioisotope for whole-body imaging and a radioisotope, enzyme, fluorescent label, and other suitable antibody tag for sample testing.

[0352] The detectable labels include particulate labels containing metal sols such as colloidal gold, for example, peptide chelating agents of type N2S2, N3S, or N4. 125 or Tc 99 This includes not only chromophores containing isotopes, fluorescent markers, luminescent markers, phosphorescent markers, etc., but also enzymatic labels that convert a given substrate into a detectable marker, and polynucleotide tags that can be identified after amplification by, for example, a polymerization enzyme chain reaction, and can be any of the various types currently used in the field of in vitro diagnostics. Suitable enzymatic labels include horseradish peroxidase and alkaline phosphatase. For example, the label may be not only adamantylmethoxyphosphoryloxyphenyldioxetane (AMPPD) or disodium 3-(4-(methoxyspiro1,2-dioxetane-3,2'-(5'-chloro)tricyclo3.3.1.1 3,7-decane-4-yl)phenylphosphate (CSPD), but also an alkaline phosphatase enzyme detected by measuring the presence or formation of chemiluminescence after chelation of a 1,2-dioxetane substrate such as CDP and CDP-STAR®, or other luminescent substrates well known to those skilled in the art, such as terbium(III) and europium(III). The detection means is determined by the selected label. The appearance of the label or its reaction product can be obtained by standard procedures using the naked eye or by instruments such as a spectrophotometer, illuminometer, or fluorometer, provided that the label is a fine particle but accumulates at an appropriate level.

[0353] Furthermore, FAM19A1 antagonists described herein (e.g., anti-FAM19A1 antibodies) may be conjugated to therapeutic agents to form immunoconjugates such as antibody-drug conjugates (ADCs). Appropriate therapeutic agents include formulations capable of treating CNS dysfunction or diseases and disorders associated with such dysfunction (e.g., glaucoma or neuropathic pain). Non-limiting examples of such therapeutic agents are provided throughout this disclosure.

[0354] Immunoconjugates can be prepared by methods known to the art. In some forms, the conjugation method results in substantially (or almost) non-immunogenic bindings, such as peptide-(i.e., amide-), sulfide-, (sterically hindered), disulfide-, hydrazon-, and ether bindings. These bindings are mostly non-immunogenic and exhibit considerable stability in serum (see, e.g., Senter, PD, Curr. Opin. Chem. Biol. 13(2009) 235-244; WO2009 / 059278; WO95 / 17886).

[0355] Depending on the biochemical attributes of the moiety and antibody, different conjugation strategies can be used. If the moiety is naturally occurring or a recombinant of 50 to 500 amino acids, standard procedures describing chemical methods for synthesizing the protein conjugate are available in textbooks and can be easily followed by those skilled in the art (e.g., Hackenberger, CPR, and Schwarzer, D., An gew. Chem. Int. Ed. Engl. 47 (2008) 10030-10074). In some embodiments, a reaction is used between a cysteine ​​residue in the antibody or moiety and the maleinimid moiety. This is a particularly suitable coupling chemical method when, for example, the Fab or Fab'-fragment of the antibody is used. In contrast, in some embodiments, coupling is performed at the C-terminus of the antibody or moiety. Protein modifications, such as C-terminal deformation of Fab fragments, can be carried out as described, for example, in Sunbul, M. and Yin, J., Org. Biomol. Chem. 7 (2009) 3361-3371.

[0356] Generally, site-directed reactions and covalent bonding are based on converting native amino acids into amino acids with reactivity orthogonal to the reactivity of other functional groups present. For example, certain cysteines in rare sequence contexts can be enzymatically converted to aldehydes (see Frese, MA and Dierks, T., ChemBioChem. 10 (2009) 425-427). Desired amino acid deformations can also be obtained using the specific enzymatic reactivity of native amino acids and specific enzymes in a given sequence context (e.g., Taki, M. et al., Prot.Eng.Des.Sel. 17 (2004) 119-126; Gautier, A. et al., Chem.Biol. 15 (2008) 128-136; and Protease-catalyzed formation of CN bonds is used by Bordusa, F., Highlights in Bioorganic Chemistry (2004) 389-403).

[0357] Furthermore, site-directed reactions and covalent bonding can be achieved by appropriate deformatives and selective reactions of terminal amino acids. Site-directed covalent bonding can be obtained using the reactivity of benzonitrile with N-terminal cysteine ​​(see Ren, H. et al., Angew. Chem. Int. Ed. Engl. 48 (2009) 9658-9662). In addition, natural chemical ligation may depend on the C-terminal cysteine ​​residue (Taylor, E. Vogel; Imperiali, B, Nucleic Acids and Molecular Biology (2009), 22 (Protein Engineering), 65-96).

[0358] EP 1 074 563 describes a conjugation method based on a faster reaction between cysteine ​​located within a series of positively charged amino acids and cysteine ​​located within a series of negatively charged amino acids.

[0359] The aforementioned moiety may be a synthetic peptide or a peptide mimetic. When polypeptides are chemically synthesized, orthogonal, chemically reactive amino acids can be introduced during such synthesis (see, for example, de Graaf, AJet al., Bioconjug. Chem. 20(2009) 1281-1295). A wide variety of orthogonal functional groups can be introduced into synthetic peptides, albeit in an unstable manner, so linking such peptides to a linker is a standard chemical method.

[0360] To obtain a single-labeled polypeptide, conjugates having a 1:1 stoichiometric ratio can be separated from other conjugation byproducts by chromatography. This process can be facilitated by using dye-labeled binding pair members and charged linkers. By using such types of labeling and strongly negatively charged binding pair members, the difference in charge and molecular weight can be utilized for separation, so that the single-conjugated polypeptide can be easily separated from unlabeled polypeptides and polypeptides having more than one linker. The fluorescent dye may be useful for purifying the complex from unbound components such as labeled monovalent binders.

[0361] [X. Pharmaceutical Compositions] This specification provides compositions comprising a FAM19A1 antagonist described herein (e.g., anti-FAM19A1 antibody) having a desired degree of purity in a physiologically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences (1990), Mack Publishing Co., Easton, PA). Acceptable carriers, excipients, or stabilizers are nontoxic to the recipient at the dose and concentration used and include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, phenol, butyl, or benzyl alcohol, alkylparabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); low molecular weight (less than about 10 residues) polypeptides; serum albumin, gelatin It includes proteins such as nucleotides or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0362] In some embodiments, the pharmaceutical composition comprises, within a pharmaceutically acceptable carrier, an antibody or its antigen-binding fragment, a bispecific molecule or immune complex, and optionally one or more additional prophylactic or therapeutic agents. In certain embodiments, the pharmaceutical composition comprises, within a pharmaceutically acceptable carrier, an effective amount of the antibody or its antigen-binding fragment, and optionally one or more additional prophylactic or therapeutic agents. In some embodiments, the antibody is the sole active ingredient in the pharmaceutical composition. The pharmaceutical compositions described herein may be useful, for example, in the treatment of diseases or disorders associated with CNS dysfunction by reducing FAM19A1 activity.

[0363] Pharmacokinetically acceptable carriers used in parenteral formulations include aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspensions and dispersants, emulsifiers, metal ion sequestering agents, chelating agents, and other pharmacokinetically acceptable substances. Examples of aqueous vehicles include sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, dextrose, and Ringer's lactate injection. Non-aqueous parenteral vehicles include plant-derived fixative oils, cottonseed oil, corn oil, sesame oil, and peanut oil. Antimicrobial agents at bacteriostatic or fungiostatic concentrations may be added to parenteral formulations packaged in multi-dose containers, including phenol or cresol, mercury-containing substances, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoates, thimerosal, benzalkonium chloride, and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. The buffering agent contains phosphate and citrate. The antioxidant contains sodium bisulfate. The local anesthetic contains procaine hydrochloride. The suspension and dispersant contains sodium carboxymethylcellulose, hydroxypropyl methylcellulose, and polyvinylpyrrolidone. The emulsifier contains Polysorbate 80 (TWEEN® 80). The metal ion sequestering or chelating agent contains EDTA. The pharmaceutical carrier also contains ethyl alcohol, polyethylene glycol, and propylene glycol for water-miscible vehicles; and sodium hydroxide, hydrochloric acid, citric acid, or lactic acid for pH adjustment.

[0364] Pharmaceutical compositions can be formulated for any route of administration. Specific examples include intranasal, oral, parenteral, intrathecal, intraventricular, pulmonary, subcutaneous, or intraventricular administration. Parenteral administration characterized by subcutaneous, intramuscular, or intravenous injection is also considered herein. Injectable preparations may be prepared in conventional forms as liquid solutions or suspensions, as solid forms suitable for solutions or suspensions from liquids before injection, or as emulsions. Injectable preparations, solutions, and emulsions may also contain one or more excipients. Suitable excipients include, for example, water, saline, dextrose, glycerol, or ethanol. If necessary, the pharmaceutical composition to be administered may also contain small amounts of non-toxic adjuncts such as wetting or emulsifying agents, pH buffers, stabilizers, solubility enhancers, and formulations such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, and cyclodextrin.

[0365] Antibody parenteral administration formulations include immediate sterile solutions for injection, tablets for subcutaneous injection, sterile dried soluble products such as lyophilized powders that can be immediately combined with a solvent immediately before use, immediate sterile suspensions for injection, sterile dried insoluble products that can be immediately combined with a vehicle immediately before use, and sterile emulsions. The solutions may be aqueous or non-aqueous.

[0366] Suitable carriers for intravenous administration include physiological saline or phosphate-buffered saline (PBS), and solutions containing thickeners and solubilizers such as glucose, polyethylene glycol, polypropylene glycol, and mixtures thereof.

[0367] Topical mixtures containing antibodies are prepared as described for topical and systemic administration. The resulting mixture may be a solution, suspension, or emulsion, and may be formulated as a cream, gel, ointment, emulsion, solution, elixir, lotion, suspension, tincture, paste, foam, aerosol, irrigation, spray, suppository, bandage, skin patch, or any other dosage form suitable for topical administration.

[0368] The anti-FAM19A1 antibodies described herein can be formulated, for example, as topical aerosols for inhalation (see, for example, U.S. Patent Nos. 4,044,126, 4,414,209 and 4,364,923 describing steroid delivery aerosols useful for the treatment of inflammatory diseases, particularly asthma). These airway administration formulations may be in the form of aerosols or solutions for nebulizers, or as fine powders for inhalation, either alone or in combination with an inactive carrier such as lactose. In this case, the particles of the formulations have a diameter of less than 50 microns in some embodiments and less than 10 microns in certain embodiments.

[0369] The antibodies or antigen-binding fragments described herein may be formulated for topical or local application to the eyes, such as topical application to the skin and mucous membranes, in the form of gels, creams, and lotions, or for intratracisternal or intraspinal application. Topical administration may also be considered for transdermal delivery, and for ocular or mucosal administration or inhalation therapy. Nasal solutions of the antibodies may be administered alone or in combination with other pharmaceutically acceptable excipients.

[0370] Transdermal patches containing ionography 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. Patent Nos. 6,267,983, 6,261,595, 6,256,533, 6,167,301, 6,024,975, 6,010,715, 5,985,317, 5,983,134, 5,948,433 and 5,860,957.

[0371] In certain embodiments, the pharmaceutical compositions comprising the anti-FAM19A1 antibody described herein are solutions, emulsions, and other mixtures, and are lyophilized powders that can be reconstituted for administration. The lyophilized powder may also be reconstituted and formulated as a solid or gel. The lyophilized powder is prepared by dissolving the antibody (e.g., anti-FAM19A1 antibody) or a pharmaceutically acceptable derivative thereof in a suitable solvent. In certain embodiments, the lyophilized powder is sterile. The solvent may contain excipients that improve the stability or other pharmacological components of the powder or the reconstituted solution prepared from the powder. Available excipients include, but are not limited to, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable formulations. The solvent may also contain buffers such as citrate, sodium phosphate, or potassium phosphate, or other such buffers known to those skilled in the art, in certain embodiments, with approximately neutral pH. Next, the solution is sterilized and filtered, and then freeze-dried under standard conditions known to those skilled in the art to provide the desired dosage form. In some forms, the obtained solution can be distributed into freeze-drying vials. Each vial may contain a single dose or multiple doses of the compound. The freeze-dried powder can be stored under suitable conditions such as about 4°C to room temperature.

[0372] This invention provides a dosage form for parenteral administration by reconstituting such lyophilized powder with sterile water for injection. For reconstitution, the lyophilized powder is added to sterile water or another suitable carrier. The exact amount varies depending on the selected compound. Such an amount can be determined empirically.

[0373] Furthermore, the antibodies or their antigen-binding fragments, bispecific molecules, immune complexes described herein, and other compositions provided herein may be formulated to target specific tissues, receptors, or other body regions to be treated. Such targeting methods are well known to many people skilled in the art. In this specification, any such targeting method is considered for use in the present compositions. For non-limiting examples of targeting methods, see, for example, U.S. Patent Nos. 6,316,652, 6,274,552, 6,271,359, 6,253,872, 6,139,865, 6,131,570, 6,120,751, 6,071,495, 6,060,082, 6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, 5,900,252, 5,840,674, 5,759,542 and 5,709,874, whose full texts are included herein by reference.

[0374] Compositions used for intra vivo administration may be sterile. This can be easily achieved, for example, by filtration through a sterile filtration membrane.

[0375] [XI. Kit] This specification provides diagnostic or therapeutic kits comprising one or more antibodies or antigen-binding fragments thereof as described herein. In certain embodiments, this specification provides a pack or kit comprising one or more containers filled with one or more components of the pharmaceutical compositions described herein, such as one or more antibodies or antigen-binding fragments thereof, and optionally an instruction manual. In some embodiments, the kit contains the compositions described herein and any prophylactic or therapeutic agents described herein.

[0376] [Examples] (Example 1: Anti-FAM19A1 antibody screening) Y-Biologics (Daejeon, South Korea) has a phage-scFv antibody library containing 1-3 × 10⁻¹⁶ antibodies. 10 It consists of 10 distinct sets of libraries with diversity, totaling 1 × 10 11Diversity is formed. Biopanning was performed to screen for related antibodies. Briefly, immunoadsorption tubes were coated with FAM19A1-Fc and FAM19A1-mFc proteins (Y-Biologics, Daejeon, South Korea) and then blocked. After phage infection, human scFv library cells (10) were incubated at 30°C for 16 hours. 10 Library phages were prepared by culturing (variety), concentrating with PEG, and then suspending in PBS buffer. Next, the library phages were added to immunoadsorption tubes and cultured at room temperature for 2 hours. After culturing, the tubes were washed with 1×PBS / T and 1×PBS to elute only scFv phages bound to the antigen (FAM19A1 protein). Using the pool of positive phages, E. coli was infected for additional amplification and biopanning. The above process was repeated, and biopanning was performed up to a total of three times. For each amplification, phages were screened and selected for their high affinity for the FAM19A1 protein.

[0377] (Example 2: Selection of anti-FAM19A1 antibody clone) Polyphage ELISA was performed to investigate the specificity of the positive poly-scFv-phage antibody pool from each round of biopanning. ELISA immunoplates were coated with ITGA6-Fc protein or FAM19A1-4-Fc protein. The phage antibody pool from Example 1 was then added to the plates, and ELISA was performed directly. M13 phage #38 (unlabeled antibody-oriented) was used as the negative control group.

[0378] As shown in Figure 1, the scFv-phage antibody pool in round 3 of biopanning was successfully enriched with anti-FAM19A1 phage antibody.

[0379] Next, based on the polyphage ELISA results, approximately 1000 single clones were selected from tertiary biopanning that showed high binding affinity. These single clones were cultured in 96-well plates and infected with helper phages. Then, mono-scFv phages were transferred to immunosuppressor plates coated with FAM19A1-Fc protein, and ELISA was performed directly. To clarify that binding was specific to FAM19A1, immunosuppressor plates coated with ITGA6-Fc protein (non-specific antigen control group) were also used.

[0380] As shown in Figure 2, it was revealed that the mono-scFv-phage clone binds only to FAM19A1-Fc, confirming the specificity of the scFv-phage clone.

[0381] Next, to group the selected positive scFv-phage clones, colony PCR was performed using a primer set capable of amplifying scFv. After processing the amplified PCR samples with BstNI, the samples were developed (run) on an 8% DNA polyacrylamide gel, and antibody diversity was assessed.

[0382] As shown in Figure 3, based on the PCR fragmentation results, the positive scFv-phage clones could be divided into seven groups. All of these were previously shown to bind strongly to FAM19A1-Fc but not to ITGA6-Fc.

[0383] Next, to confirm that the sc-Fv phage in each of the seven groups was not bound to other antigens, additional ELISA binding analysis (described above) was performed using additional antigens (C-Fc, hRAGE-Fc, CD58-Fc, ITGA6-Fc, and AIRTR).

[0384] As shown in Figure 4, of the seven clones tested, clones 1A11, 1C1, 2G7, and 3A8 showed the least binding to the non-FAM19A1 antigen. Sequence analysis revealed that all four of these clones possessed unique amino acid sequences.

[0385] (Example 3: Production of anti-FAM19A1 IgG1 antibody) To convert the four selected monoclonal phage antibodies from scFv to human IgG, the variable regions of the heavy and light chains of each phage antibody were subcloned into an expression vector containing the constant regions of the heavy and light chains. See Figure 5A. Next, the plasmids containing the heavy and light chains were co-transferred into HEK 293F cells for 6 days. Then, the antibodies produced for 6 days were purified using protein A affinity chromatography. After purification, the antibodies were separated through glycine buffer, and the final resuspension buffer was changed to PBS. The purified antibodies were quantified by BCA and nanodrop. The purity and mobility of the purified proteins were confirmed by SDS-PAGE analysis after loading 5 μg of each of the four antibodies under reducing and non-reducing conditions. As shown in Figure 5B, the four anti-FAM19A1 antibody clones had a size of approximately 150 kDa or larger under non-reducing conditions. Antibody production yields ranged from approximately 11 mg / L (2G7 clone) to 90.5 mg / L (1C1 clone) (Figure 5C).

[0386] The affinity of the four anti-FAM19A1 antibody clones was also evaluated using ELISA. As shown in Figure 5D, clone 1C1 showed the highest affinity for FAM19A1 at all concentrations tested.

[0387] (Example 4: Epitope mapping analysis) To further characterize the anti-FAM19A1 antibody clones, epitope mapping analysis was performed. Briefly, the amino acid sequences of different FAM19 family members (i.e., FAM19A1-5) were aligned, and seven regions where the FAM19A1 protein's amino acid sequence differed most significantly from other members of the FAM19A family (i.e., FAM19A2-5) were identified. The amino acid sequences of these regions were replaced with the consensus sequences of the corresponding regions for the FAM19A2-5 proteins to produce M1-M7 mutations. See Table 10.

[0388] [Table 10] To evaluate binding, ELISA plates were coated with 500 ng of mutant M1-M7 or wild-type FAM19A1 protein overnight at 4°C, followed by two washes with 1×PBS. The plates were then blocked with blocking buffer (100 μL / well) at room temperature for 1 hour. Next, different anti-FAM19A1 antibody clones (1A11, 1C1, D6, E1, and F41H5; 1 μg) were added to the appropriate wells of the ELISA plate, and the plates were incubated at room temperature for 1 hour. After washing the plates, anti-hKappa-HRP antibody (1:2000) was added to the wells, and the plates were incubated at room temperature for 30 minutes. A color change reaction was induced by the addition of TMB substrate. This reaction was terminated using 50 μL of sulfuric acid (2N H2SO4), and the degree of color change was detected by absorption at 450 nm with a reference wavelength of 620 nm using a 96-well microplate reader (Molecular Device).

[0389] As shown in Figure 6, the anti-FAM19A1 antibody clone 1C1, previously identified as having the highest FAM19A1 binding affinity, was unable to bind to the FAM19A1 mutant M6. As shown in Table 8 above, the M6 ​​mutant has substitutions at amino acid residues D112N, M117S, A119S, T120S, and N122H. This result suggests that these residues are important binding epitopes for the anti-FAM19A1 antibody clone 1C1.

[0390] (Example 5: FAM19A1 expression analysis) To further characterize the expression pattern of FAM19A1, FAM19A1 mRNA levels were measured in other mouse tissues using RT-PCR. Briefly, all RNAs were isolated from different brain regions (i.e., cerebral cortex, cerebellum, midbrain, spinal cord, hippocampus, olfactory bulb, hypothalamus, and pituitary gland) and peripheral tissues (i.e., heart, liver, spleen, stomach, small intestine, testes, kidneys, and lungs). RNA was isolated using the single-step acidic guanidinium thiocyanate-phenol-chloroform method, as previously described. See Chomczynski, P., et al., Anal Biochem 162(1):156-9 (1987). Next, 1 μm of each RNA sample was reverse transcribed with Maloney Murine Leukemia Virus (M-MLV) Reverse Transcriptase (Promega, Madison, WI). Next, the cDNA fractions were amplified using the following primers: (i) mFAM19A1_F:5'-ATG GCA ATG GTC TCT GCA-3'; and (ii) mFAM19A1_R:5'-TTA GGT TCT TGG GTG AAT-3'.

[0391] As shown in Figure 7, FAM19A1 mRNA was observed in all brain regions tested. However, in peripheral tissues, expression was either completely absent or relatively low compared to brain regions. This result suggests that FAM19A1 is primarily expressed in the central nervous system.

[0392] (Example 6: Development of FAM19A1 LacZ Knock-In (KI) mouse) To further analyze FAM19A1 expression and functional characteristics, we established transgenic mice in which a lacZ reporter was inserted into the FAM19A1 gene. Briefly, a LacZ sequence containing a targeted vector for FAM19A1 was constructed (Figure 8A), and this was transmitted to embryonic stem (ES) cells by electroporation. Genotyping analysis of the transgenic ES cells and chromosomal coefficients verified the incorporation of the target vector. The confirmed ES cells were injected into blastocysts and transferred to the uterus of female-receptor mice. Gonadal transmission tests were performed to ensure stable gonadal expression during chimera generation. The generated FAM19A1 LacZ KI chimeric mice were reverse-crossed onto a C57BL / 6J genetic background. The two strains were maintained by mating heterozygous male mice with wild-type C57BL / 6J female mice. To obtain homozygous FAM19A1 LacZ KI mice, heterozygous male mice were mated with heterozygous female mice.

[0393] According to this animal model, insertion of the lacZ gene, which is presumed to contain FAM19A1 (inserted directly after the start codon in exon 2 of the FAM19A1 gene, see Figure 8A), was expected to manifest as β-galactosidase expression instead of FAM19A1. Therefore, insertion of the target vector into both alleles of the FAM19A1 gene was predicted to manifest as complete removal of FAM19A1 expression. Mice in which the lacZ gene was inserted into both alleles were denoted as isozygous FAM19A1 LacZ Knock-In ("FAM19A1 LacZ KI(- / -)").

[0394] To confirm the deletion of the FAM19A1 gene at the genomic level, DNA PCR was performed using primers that specifically target the inserted LacZ gene sequence. Western blotting ("WB") and immunohistochemistry ("IHC") were performed using polyclonal anti-FAM19A1 and / or anti-β-galactosidase antibodies to confirm the complete deletion of the FAM19A1 gene at the protein level in these mice.

[0395] For WB analysis, adult mouse cortical and hippocampal regions were isolated and lysed in a buffer containing 50 mM Tris-HCl (pH 7.5), 0.1% sodium dodecyl sulfate (SDS), and a protein inhibitor cocktail tablet (Roche Applied Science). The protein content in the lysates was quantified using BioRad Bradford Protein Analysis Reagent (BioRad), and the proteins were separated from the SDS polyacrylamide gel. The separated proteins were transferred to a nitrocellulose blotting membrane using a Bio-Rad Trans-Blot electrophoresis apparatus (Richmond, CA), and the blot was blocked at RT for 30 minutes with Tris buffered saline containing 0.3% Tween 20 and 5% skim milk. The blot was cultured with the primary antibody for 3 hours, and then cultured with horseradish peroxidase-conjugated secondary antibody at room temperature for 1 hour. After applying GE Healthcare ECL reagent, the samples were exposed to X-ray film, and the immunoreaction bands were visualized. The antibodies and their dilution factors were as follows: rabbit polyclonal anti-FAM19A1 (laboratory-generated) at 1:500, β-actin (ab8227, Abcam) at 1:2000, and HRP-conjugated anti-rabbit (Jackson ImmunoResearch Laboratories, West Grove, PA) at 1:5000.

[0396] For IHC analysis, animals were permeated with 4% paraformaldehyde in phosphate-buffered saline (PBS). After separating the brain, it was post-fixed overnight in the same fixative. The brain was then cryoprotected with 30% sucrose in PBS and serially sectioned at 40 μm using Cryostat (Leica). The sections were blocked with 3% BSA and 0.1% Triton X-100 in PBS at room temperature (RT) for 30 minutes. After application of the primary antibody overnight at 4°C, a suitable fluorescently conjugated secondary antibody was applied with Hoechst 33342 (Invitrogen) at RT for 30 minutes. The antibodies and their dilutions were as follows: rabbit polyclonal anti-FAM19A1 (laboratory-generated) at 1:500, β-galactosidase (ab9391, Abcam) at 1:500, and fluorescently conjugated anti-rabbit and anti-chicken (Life Technologies) at 1:500. Images were obtained using a confocal microscope (TCS SP8, Leica).

[0397] As shown in Figure 8B, successful insertion of the LacZ gene was confirmed in FAM19A1 LacZ KI(- / -) animals via genomic DNA PCR. Considering that the inserted LacZ gene sequence contains not only a self-termination codon but also a poly-A tail, the final product of this gene configuration is intact β-galactosidase, which contains no part of FAM19A1. In FAM19A1 LacZ KI mice, disruption of the FAM19A1 gene was confirmed by RT-PCR (Figure 8C). Furthermore, as shown in Figures 8D to 8F, FAM19A1 protein expression was substantially reduced in heterozygous mice (FAM19A1 LacZ KI(+ / -)) compared to wild-type animals in all areas of the cortex (CTX) and hippocampus (HIP). FAM19A1 protein was not detected in homozygous mice (FAM19A1 LacZ KI(- / -)). Figures 8G and 8H confirm that disruption of FAM19A1 protein expression is directly associated with mRNA levels. These results confirm the complete deletion of the FAM19A1 gene in FAM19A1 LacZ KI(- / -) mice.

[0398] (Example 7: FAM19A1 expression in the brains of embryonic and postnatal mice) To further understand the function of FAM19A1, we evaluated the pattern and timing of FAM19A1 expression using X-gal staining, an enzymatic analysis method based on β-galactosidase activity. Since complete knockout of the FAM19A1 gene, which is initiated during development, induces deformation of brain structure and can alter the outcome, we used FAM19A1 LacZ KI(+ / -) (heterozygous) animals.

[0399] X-gal staining of embryos, postnatal brains, and adult eyes: For embryonic X-gal staining, pregnant mice were sacrificed by cervical luxation, and embryos were isolated. Whole embryos E12.5 were fixed at 4°C in 4% paraformaldehyde and 0.2% glutaraldehyde in PBS for 15 minutes. Embryos older than E14.5 were decapitated and the skin removed. The embryo heads were fixed at 4°C in the same fixative for 1-2 hours. For postnatal brains and adult eyes, the brains and eyes were separated from the skull and fixed at 4°C in the same fixative for 1-2 hours. Next, the fixed tissue was washed twice with PBS for 5 minutes each, incubated overnight, and then stained in the dark with X-gal stain in 0.1 M phosphate buffer at pH 7.3 for 24–48 hours at 37°C with 1 mg / ml X-Gal, 2 mM MgCl2, 5 mM EGTA, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 0.01% sodium deoxycholate, and 0.02% Nonidet-P40. The stained tissue was fixed overnight at 4°C with 4% paraformaldehyde in PBS, and whole brain images were obtained after washing.

[0400] For x-gal stained sections, the entire stained brain or eye was freeze-protected in 30% sucrose in PBS and sectioned to 40 μm using Cryostat (Leica). Nuclear Fast Red (H-3403, VECTOR) was used as a counter-stain in some cases. Section images were acquired using a slide scanner (Axioscan Z1, Zeiss).

[0401] X-gal staining of adult brains: Animals were infused with 4% paraformaldehyde and 0.2% glutaraldehyde in phosphate buffer (PB). The brains were isolated and post-fixed in 0.2% glutaraldehyde in PB at 4°C for 24 hours. The brains were then cryoprotected with 30% sucrose in PBS and serially sectioned into 40 μm sections using Cryostat (Leica). The sectioned tissues were cultured in the dark at 37°C for 24–48 hours in an X-gal staining solution in 0.1 M phosphate buffer at pH 7.3, containing 1 mg / ml X-Gal, 2 mM MgCl2, 5 mM EGTA, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 0.01% sodium deoxycholate, and 0.02% Nonidet-P40. Images of the tissue sections were captured using a slide scanner (Axio scan Z1, Zeiss).

[0402] As shown in Figures 9A and 9B, FAM19A1 was first expressed in restricted cortical regions during early embryonic development (verified by positive β-galactosidase staining). There was no sign of FAM19A1 expression until embryonic day 12.5 (E12.5). However, from embryonic day 14.5 (E14.5), β-galactosidase activity (i.e., FAM19A1 expression) was observed in the ventral cortical region, with particularly strong expression in the rostral portion. These stained regions were thought to be the early piriform cortex (Cpf) and entorhinal cortex (Cen). See Figure 10A.

[0403] Postnatally, neocortical expression of FAM19A1 became more apparent. See Figure 9C. In the early postnatal period, FAM19A1 was first observed in the somatosensory, visual, and auditory cortical regions, and continued to be expressed in the piriform and entorhinal cortex. Over time, FAM19A1 expression expanded to other neocortical regions. Until postnatal day 14.5 (P14.5), neocortical β-galactosidase (i.e., FAM19A1) expression was specifically detected in the cortical layers. See Figure 10B. In addition, X-gal staining signals were detected in the limbic region, including the posteromedial cortical amygdala (PMCo), hippocampus, and amygdala. See Figure 10B. These results, given that the FAM19A1 expression pattern was specifically limited to the cortical layers and limbic regions, suggest that FAM19A1 may be expressed in neurons that differentiate during neurodevelopment. Furthermore, FAM19A1 has not been detected in stem cell-rich regions such as the ventricular zone and subventricular zone, suggesting that FAM19A1 may not be involved in NSC proliferation.

[0404] (Example 8: FAM19A1 expression in the brain of adult mice) To evaluate whether FAM19A1 plays a role in neuronal activity, we mapped the expression pattern of FAM19A1 in adult FAM19A1 LacZ KI heterozygous mice.

[0405] In the brains of adult mice, X-gal staining revealed that FAM19A1 was expressed in all cortical regions (Figure 9C). Immunohistochemistry using X-gal staining showed that X-gal precipitate and β-galactosidase were co-localized with CUX1, a pyramidal neuron marker for cortical layers 2-3 (L2-3), and CTIP2, a pyramidal neuron marker for cortical layer 5b (L5b), respectively. This indicates that FAM19A1 is expressed in a layer-specific manner, mainly in pyramidal neurons (Figures 11A, 11B, and 11C (Panel iv)). Furthermore, X-gal showed signaling in the corticospinal tract, including the internal capsule (ic), cerebral peduncle (cp), and pyramidal tract (py), suggesting the presence of FAM19A1 in pyramidal neurons of the primary motor cortex L5b (Figure 12, Panels G and I).

[0406] Furthermore, the presence of FAM19A1 in specific sensory circuits was investigated. In the olfactory neural circuit, β-galactosidase and FAM19A1 mRNA expression were hardly observed in the olfactory bulb (OB) (Figure 8F), but FAM19A1 protein was detected by Western blotting (Figures 8F and 8G). The detected FAM19A1 protein can be released from neurons in other olfactory-related brain regions, including the anterior olfactory nucleus (AO), CPf, and cortical amygdala, which show positive X-gal signals (Figures 8D, 8E, and 8H). In the visual neural circuit, β-galactosidase expression was not observed in the optic chiasm or lateral geniculate nucleus (LGN) of the visual neural circuit, but β-galactosidase expression was observed in both the optic layer of the superior colliculus (Op) and the visual cortex (Figure 11C, panel vii; Figure 9C), which suggests that FAM19A1 may be involved in superior colliculus-dependent visual information processing and eye movement control. Furthermore, β-galactosidase expression was observed in certain regions associated with auditory neural circuits, including the medial geniculate nucleus (MGN), the dorsal cochlear nucleus (DC), and the auditory cortex (Figure 11C, panel ix; Figure 12, panel E).

[0407] Clear expression of FAM19A1 was observed in the peripheral regions, including the hippocampus and amygdala. In the hippocampus, β-galactosidase was expressed in the CA region but not in the dentate gyrus (DG) (Figure 11C, panel iv). In CEn, β-galactosidase expression suggested that hippocampal FAM19A1 expression played a role in the hippocampal trisynaptic circuit (Figure 11C, panels iv and viii). β-galactosidase was expressed only in the basolateral nucleus, including the lateral amygdala (LaDL) and basomedial amygdala (BLA) (Figure 11C, panel v). Furthermore, FAM19A1 expression was detected in PMCo and the amygdala-piriform transition area (Apir), which are thought to be directly linked to BLA, Cen, and CPf (Figure 11C, panel vi).

[0408] β-galactosidase was also expressed in some hypothalamic nuclei, including the medial preoptic nucleus (MPOM), lateral preoptic area (LPO), and ventromedial hypothalamic nucleus (VMH) (Figure 12, panels B and C). As part of the limbic system, the hypothalamus is known to act as a mediator between the CNS and the endocrine system. Therefore, the data provided here suggest that FAM19A1 may contribute to endocrine homeostasis. The lateral septal nucleus (LS), another brain region extensively connected to the limbic region, also showed β-galactosidase expression (Figure 11C, panel iii).

[0409] In-situ hybridization using adult wild-type rat brains showed that FAM19A1 mRNA was detected in the upper and lower cortical layers, the CA region of the hippocampus, and the basolateral nucleus of the amygdala (Figure 13). This FAM19A1 mRNA expression pattern matched the β-galactosidase expression pattern in the brains of FAM19A1 LacZ KI mice, thereby confirming the FAM19A1 expression mapping observed using FAM19A1 LacZ KI mice (Figure 11C, panels ii, iv, and v). Furthermore, the observed FAM19A1 expression pattern was consistent with an open-source single-cell basis RNA-sequence analysis database for the brains of wild-type mice. In summary, these data suggest that FAM19A1 is primarily expressed in neurons, particularly pyramidal neurons, and may be involved in motor behavior, sensory information processing, and / or limbic system-related brain functions.

[0410] (Example 9: Comparison of morphological differences between wild-type and FAM19A1- / - animals) Since early deficiency of FAM19A1 can induce brain developmental abnormalities, we investigated the morphological differences between isozygous FAM19A1 LacZ KI (FAM19A1- / -), heterozygous FAM19A1 LacZ KI (FAM19A1+ / -), and wild-type mice.

[0411] For typical characteristics, FAM19A1- / - mice were born from heterozygous parents at approximately 24-25% Mendelian frequency and similar sex ratios (Figure 14). Immediately after birth, there were no clear differences in gross appearance between neonatal genotypes; however, FAM19A1- / - mice (all males and females) were significantly heavier than wild-type control animals (Figures 15A and 15B).

[0412] The total length and width of the adult brain were similar between wild-type (WT) and FAM19A1- / - mice (Figures 15C, 15E, and 15G), but the length of the cerebral cortex was even longer in FAM19A1- / - mice compared to WT mice (Figure 15F). Removal of genes specifically expressed in the cortical layer may lead to improper cortical layer assembly. However, in the FAM19A1- / - mice disclosed herein, the volume of the cerebral cortex was not affected (Figures 16A and 16B). Furthermore, no significant structural abnormalities were detected in the brain structure by overall observation of X-gal stained brain sections of FAM19A1- / - mice (data not shown). The thickness of all neocortical regions was not significantly reduced in FAM19A1- / - mice (Figures 15H, 15I, and 15J). However, in terms of the ratio of cortical layers, L4 in the visual cortex and L6 in the motor cortex were reduced in FAM19A1- / - compared to WT mice (Figures 17A-17F).

[0413] While these changes in cortical thickness may result from abnormal cellular structure, no significant differences were observed in the neuronal and glial cell populations of the cortical layer between FAM19A1- / - and WT mice (Figures 18A-18D and 19A-19E). Furthermore, there were no abnormalities in neuronal and glial cell morphology significant enough to warrant attention (data not shown). In summary, these findings suggest that overall FAM19A1 ablation reduced weight gain and slightly altered neocortical structure, but did not have a significant impact on the composition of cortical cell types.

[0414] (Example 10: Analysis of the role of FAM19A1 in excessive behavior) As previously described, FAM19A1 was expressed in many areas of the limbic system, including the anterior limbic cortex and amygdala (Figure 11C, panels i and v), which are known to be involved in emotion processing. Therefore, to evaluate the effects of FAM19A1 depletion on anxiety and depression, elevated cusp maze (EPM), open field (OFT), and tail suspension (TST) tests were performed using FAM19A1- / - mice, as detailed in previous examples. Male mice were used in particular.

[0415] The EPM (Epoch-Proof Maze) experiment was conducted as follows: The elevated cross maze (EPM) had four vertical arms and two open (5 × 30 cm) and two closed (5 × 30 cm) walls, each 20 cm high. The maze was 50 cm above the ground. Test animals were individually positioned in the center of the maze, facing one of the open arms, and allowed to explore freely for 15 minutes. Recorded video was analyzed using the ANY-Maze Video Tracking program (Stoelting, Illinois, USA). The number of times the animals entered an open arm, the time spent in an open arm, the number of times they crossed the center, and the total distance traveled were recorded. An entry was defined as when all four paws were positioned within the arm.

[0416] OFT was conducted as follows: An OFT apparatus measuring 40 cm wide (w) × 40 cm high (h) × 40 cm long (d) was constructed from opaque plastic. The test arena was defined as 30% of the central area and the surrounding boundary area. Experimental animals were individually placed in the center of the arena and their behavior was recorded for 10 minutes. The ratio of time taken and entry into the central area were scored, and the total distance traveled was determined using the ANY-Maze Video Tracking program (Stoelting).

[0417] The TST (Therapeutic Strategy) was performed as follows: Each mouse tail was individually suspended in a box (36.5 × 30.5 × 30.5 cm) for 6 minutes. The recorded video was analyzed using the ANY-Maze Video Tracking program (Stoelting). Immobility was defined as the mouse's termination of anxiety and escape attempts.

[0418] During the EPM test, FAM19A1- / - mice showed increased time spent on the open arm (Figure 20A) and increased total movement distance (Figure 20B) compared to WT mice. During OFT, the time spent in the center of the OFT arener was similar between FAM19A1- / - mice and WT mice, but the total movement distance was even higher in FAM19A1- / - mice (Figures 20C, 20D, and 20E). During TST, FAM19A1- / - mice showed lower immobility than WT mice (Figure 20F).

[0419] The results suggest that inhibiting FAM19A1 activity may increase its activity, which could be helpful in treating anxiety or depression-related disorders.

[0420] (Example 11: Analysis of the role of FAM19A1 in memory) Short-term memory (STM), particularly spatial operational memory, is known to involve interactions between CA1, CA3, and CEn in the hippocampus. As shown in Figure 11C (panels iv and viiii), FAM19A1 is highly expressed in these regions, suggesting a possible role for FAM19A1 in memory formation. To evaluate the potential role FAM19A1 may have in memory (both short-term and long-term), a Y-maze test was performed as follows: The Y-maze arena had three identical arms, each 30 cm long, 5 cm wide, and 20 cm high. Test animals were individually positioned in the center, and the arm entry sequence and total distance traveled were recorded over 5 minutes and analyzed using the ANY-maze video tracking program (Stoelting). The spontaneous change rate was calculated by dividing the number of attempts, including the number of times all three arms were entered (ABC, ACB, BAC, BCA, CAB, CBA), by the maximum possible change (corresponding to the total number of arms entered minus 2), and then multiplying by 100.

[0421] As shown in Figure 20G, no significant difference in spontaneous changes was observed between FAM19A1- / - and WT mice. However, total migration distance was significantly increased in FAM19A1- / - mice compared to the WT control group (Figure 20H). This result confirms the findings from the EPM and OFT tests (see Example 10), which suggests that inhibition of FAM19A1 may lead to increased activity.

[0422] Furthermore, a new object recognition (NOR) test was conducted to investigate any potential defects in object recognition memory. Briefly, the test arena measured 40 cm wide (w) × 40 cm high (h) × 40 cm long (d). A T-75 flask filled with sand and stacked plastic tiles (7 cm w × 13 cm h × 15 cm h) was used as the object. Mice were individually adapted to the object-free test arena for 10 minutes. The following day, two identical objects were placed in the arena during the acquisition phase, allowing each mouse 10 minutes to freely explore. In the acquisition phase, the minimum exploration time criterion for the two identical objects was 20 seconds. The testing phase was planned to take place 6 hours (for short-term memory tests) or 24 hours (for long-term memory tests) after acquisition. During the testing phase, all previously introduced and new objects were placed in the arena. Next, the mice were allowed 10 minutes to freely explore the arena. The acquisition and testing phases were recorded for analysis. The time required to explore each object was measured. Exploratory behavior was defined as showing interest in an object while sniffing it. ...

Claims

1. A monoclonal antibody or its antigen-binding fragment ("anti-FAM19A1 antibody") that specifically binds to a family, member A1 (FAM19A1) having sequence similarity 19, The aforementioned anti-FAM19A1 antibody is (a) When measured by ELISA, K Ｄ The property of binding to soluble human FAM19A1 with a concentration of 10 nM or less. (b) When measured by ELISA, K Ｄ The property of binding to membrane-bound human FAM19A1 with a concentration of 10 nM or less, or (c)(a) and (b) The characteristics selected from these are shown. The aforementioned anti-FAM19A1 antibody is (i) comprising a heavy chain variable domain containing the amino acid sequence shown in SEQ ID NO: 30, and a light chain variable domain containing the amino acid sequence shown in SEQ ID NO: 31; (ii) A heavy chain variable domain containing the amino acid sequence shown in SEQ ID NO: 28, and a light chain variable domain containing the amino acid sequence shown in SEQ ID NO: 29; (iii) comprising a heavy chain variable domain containing the amino acid sequence shown in SEQ ID NO: 32, and a light chain variable domain containing the amino acid sequence shown in SEQ ID NO: 33; or (iv) comprising a heavy chain variable domain containing the amino acid sequence shown in SEQ ID NO: 34, and a light chain variable domain containing the amino acid sequence shown in SEQ ID NO: 35; Anti-FAM19A1 antibody.

2. The anti-FAM19A1 antibody according to claim 1, wherein the anti-FAM19A1 antibody is a chimeric antibody or a human antibody.

3. The anti-FAM19A1 antibody according to claim 1, wherein the anti-FAM19A1 antibody comprises Fab, Fab', F(ab')2, Fv, or single-chain Fv(scFv).

4. The anti-FAM19A1 antibody according to claim 1, wherein the anti-FAM19A1 antibody is selected from the group consisting of IgG1, IgG2, IgG3, IgG4, and any combination thereof.

5. The anti-FAM19A1 antibody according to claim 1, further comprising a constant region lacking Fc function.

6. The anti-FAM19A1 antibody according to claim 1, wherein the anti-FAM19A1 antibody is linked to a formulation to form an immunoconjugate.

7. The anti-FAM19A1 antibody according to claim 1, wherein the anti-FAM19A1 antibody is formulated with a pharmaceutically acceptable carrier.

8. A nucleic acid comprising a nucleic acid sequence encoding the anti-FAM19A1 antibody according to claim 1.

9. A vector comprising the nucleic acid described in claim 8.

10. A cell comprising the nucleic acid described in claim 8 or the vector described in claim 9.

11. A composition comprising the anti-FAM19A1 antibody and carrier described in claim 1.

12. A method for producing an anti-FAM19A1 antibody, comprising the steps of culturing the cells described in claim 10 under appropriate conditions and separating the anti-FAM19A1 antibody.

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