Use of anti-fam19a1 antagonists for treating central nervous system diseases

Abstract

The present disclosure relates to a method of treating a disease or disorder associated with a central nervous system (CNS) dysfunction. Also provided is a method of diagnosing and / or identifying a subject having a CNS dysfunction. Also provided are FAM19A1 antagonists useful with the present disclosure.

Description

[0001] Cross-references to related applications

[0002] This PCT application claims priority to U.S. Provisional Application No. 62 / 984,166, filed March 2, 2020, the entire contents of which are incorporated herein by reference.

[0003] Refer to the electronic submission sequence list

[0004] The entire contents of the sequence list submitted electronically in the ASCII text file (name: 3763.017PC01_SeqListing_ST25.txt; size: 32,234 bytes; creation date: March 1, 2021) submitted with this application are incorporated herein by reference.

[0005] Government Support Statement

[0006] This work (Grant No. C0558337) was supported by the Industry-Academia-Research Collaborative Research and Development Project funded by the Korea Ministry of SMEs and Startups in 2017. Technical Field

[0007] This disclosure provides: antagonists (e.g., antibodies) that specifically bind to sequence-similar family 19A1 members (FAM19A1), compositions containing such antagonists, and methods of using such antagonists to prevent and / or treat central nervous system diseases and abnormalities. Background Technology

[0008] Diseases and disorders of the central nervous system (CNS) include a heterogeneous group of diseases, often with unknown etiologies and pathogenesis. Currently, there are no cures for some CNS diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and traumatic brain injury (TBI), neuropathic pain, and glaucoma. In fact, in most cases, existing treatments for CNS diseases offer relatively small symptom relief benefits and are essentially palliative. Furthermore, with global population growth and increased life expectancy, the number of people suffering from CNS diseases and disorders is expected to increase further. See Feigin, VL et al., in Lancet Neurology (Lancet Neurol 16(11): 877-897(2017)). Therefore, there remains a need for more effective treatments for CNS-related diseases and disorders. Summary of the Invention

[0009] This article provides an antagonist that specifically binds to a sequence-similar family 19 A1 member ("FAM19A1 antagonist") for therapeutic purposes. In some aspects, the FAM19A1 antagonist is capable of treating diseases or disorders in subjects of need.

[0010] In some aspects, the disease or disorder includes central nervous system (CNS) related diseases or disorders. In some aspects, the CNS related diseases or disorders are associated with abnormal neural circuits. In some aspects, the CNS related diseases or disorders include: mood disorder, psychiatric disorder, or both. In some respects, the aforementioned CNS-related diseases or disorders include: anxiety, depression, post-traumatic stress disorder (PTSD), bipolar disorder, attention deficit / hyperactivity disorder (ADHD), autism, schizophrenia, neuropathic pain, glaucoma, addiction, arachnoid cyst, catalepsy, encephalitis, epilepsy / seizures, locked-in syndrome, meningitis, migraine, multiple sclerosis, myelopathy, Alzheimer's disease, Huntington's disease, and Parkinson's disease. Amyotrophic lateral sclerosis (ALS), Batten disease, Tourette's syndrome, traumatic brain injury, cerebrospinal damage, stroke, tremors (essential or Parkinsonian), dystonia, intellectual disability, brain tumor, or a combination thereof.

[0011] In some respects, the CNS-related disorders or disturbances are: anxiety disorder, depression, PTSD, or a combination thereof. In some respects, the FAM19A1 antagonist can improve one or more symptoms associated with anxiety disorder and / or depression (e.g., improve the subject's locomotion activity and / or improve the subject's responsiveness to external stress).

[0012] In some respects, glaucoma is a CNS-related disease or disorder that can be treated with this disclosure. In some respects, the FAM19A1 antagonist can reduce, alleviate, or inhibit inflammation associated with glaucoma. In some respects, the FAM19A1 antagonist can improve retinal potentials in the retina. In some aspects, the glaucoma is selected from the group consisting of: open-angle glaucoma, angle-closure glaucoma, normal-tension glaucoma (NTG), congenital glaucoma, secondary glaucoma, pigmentary glaucoma, pseudoexfoliative glaucoma, traumatic glaucoma, neovascular glaucoma, irido corneal endothelial syndrome, uveitic glaucoma, and combinations thereof. In some respects, the glaucoma is associated with: optic nerve injury in the subject, loss of retinal ganglion cells (RGCs), elevated intraocular pressure (IOP), impaired blood-retina barrier, and / or increased microglia activity in the retina and / or optic nerve. In some respects, the glaucoma is caused by mechanical damage to the optic nerve head and / or increased inflammation in the retina and / or optic nerve.

[0013] In some aspects, the FAM19A1 antagonist can delay the onset of retinal nerve cell degeneration in a subject. In some aspects, the FAM19A1 antagonist can reduce the loss of retinal ganglion cells in the retina of a subject and / or restore the number of retinal ganglion cells. In some aspects, the loss of retinal ganglion cells 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 reference values ​​(e.g., corresponding values ​​in subjects who have not received the FAM19A1 antagonist or corresponding values ​​in subjects before administration of the FAM19A1 antagonist). In some aspects, the number of retinal ganglion cells recovers 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 reference value (e.g., the corresponding value in a subject not receiving the FAM19A1 antagonist or the corresponding value in a subject before administration of the FAM19A1 antagonist). In some aspects, the FAM19A1 antagonist is able to protect the neural connections of the inner plexus layer of the subject's retina.

[0014] In some respects, the CNS-related diseases or disorders that can be treated with the FAM19A1 antagonists disclosed in this article are neuropathic pain.

[0015] In some aspects, the FAM19A1 antagonist can increase the threshold or latency of a subject in need of an external stimulus. In some aspects, the external stimulus is a mechanical stimulus. In some aspects, the external stimulus is a thermal stimulus. In some aspects, the threshold or latency to the external stimulus is 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 a reference value (e.g., the corresponding value in a subject who has not received the FAM19A1 antagonist or the corresponding value in a subject before the administration of the FAM19A1 antagonist).

[0016] In some aspects, the FAM19A1 antagonist is capable of increasing or modulating sensory nerve conduction velocity in a subject with a need. In some aspects, the sensory nerve conduction velocity is 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 a reference value (e.g., the corresponding value in a subject who has not received the FAM19A1 antagonist or the corresponding value in a subject before administration of the FAM19A1 antagonist).

[0017] In some aspects, the neuropathic pain is central or peripheral neuropathic pain. In some aspects, the neuropathic pain is associated with: physical injury, infection, diabetes, cancer treatment, alcoholism, amputation, weakness of muscles in the back, legs, hips, or face, trigeminal neuralgia, multiple sclerosis, shingles, spinal surgery, or any combination thereof. In some aspects, the neuropathic pain includes: carpal tunnel syndrome, central pain syndrome, degenerative disc disease, diabetic neuropathy, phantom limb pain, postherpetic neuralgia (shingles), pudendal neuralgia, sciatica, low back pain, trigeminal neuralgia, or any combination thereof. In some respects, the neuropathic pain is caused by nerve compression. In some respects, the diabetic neuropathy is diabetic peripheral neuropathy. In some respects, the neuropathic pain is sciatica.

[0018] In some respects, the FAM19A1 antagonist can modulate or improve central nervous system function in subjects with a need. In some respects, the central nervous system function includes: limbic system-related functions, olfactory system-related functions, sensory system-related functions, visual system-related functions, or combinations thereof.

[0019] In some aspects, the FAM19A1 antagonist can reduce the expression levels of FAM19A1 mRNA and / or FAM19A1 protein in brain regions. In some aspects, the brain regions include: the cerebral cortex, hippocampus, hypothalamus, midbrain, prefrontal cortex, amygdala (e.g., the lateral amygdaloid nucleus and the basomedial amygdaloid nucleus), piriform cortex, anterior olfactory nucleus, lateral entorhinal cortex, habenula, or combinations thereof.

[0020] In some aspects, the FAM19A1 antagonist can reduce the expression levels of FAM19A1 mRNA and / or FAM19A1 protein in the retinal region. In some aspects, the retinal region includes a ganglion cell layer (GCL) or an inner plexiform layer (INL).

[0021] In some respects, the FAM19A1 antagonist can reduce the expression levels of FAM19A1 mRNA and / or FAM19A1 protein in the spinal cord region. In some respects, the spinal cord region includes the dorsal horn.

[0022] In some respects, the expression level of the FAM19A1 protein and / or the expression level of 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 reference values ​​(e.g., the corresponding values ​​in subjects who have not received the FAM19A1 antagonist or in subjects before administration of the FAM19A1 antagonist).

[0023] In some respects, the FAM19A1 antagonist can regulate, induce, or increase the differentiation of neural stem cells in subjects in need.

[0024] In some aspects, the FAM19A1 antagonist can increase neurite outgrowth in differentiated neural stem cells compared to a reference value (e.g., the corresponding value in a subject who has not received the FAM19A1 antagonist or the corresponding value in a subject before administration of the FAM19A1 antagonist). In some aspects, the neurite outgrowth is 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 reference value.

[0025] This application also discloses a method for diagnosing central nervous system (CNS) dysfunction in a subject requiring diagnosis, the method comprising: contacting a sample of the subject with a FAM19A1 antagonist and measuring the FAM19A1 protein level or FAM19A1 mRNA level in the sample. This application further provides a method for identifying a subject with central nervous system (CNS) dysfunction, the method comprising: contacting a sample of the subject with a FAM19A1 antagonist and measuring the FAM19A1 protein level or FAM19A1 mRNA level in the sample.

[0026] In some respects, the contact and the measurement are performed in vitro.

[0027] In some aspects, the CNS functions include: limbic system-related functions, olfactory system-related functions, sensory system-related functions, visual system-related functions, or combinations thereof. In some aspects, abnormalities in the CNS functions are related to abnormal neural circuits.

[0028] In some respects, abnormalities in central nervous system function are associated with CNS-related diseases or disorders. In some respects, these CNS-related diseases or disorders include: mood disorders, mental disorders, or both. In some respects, these CNS-related diseases or disorders include: anxiety disorders, depression, post-traumatic stress disorder (PTSD), bipolar disorder, attention deficit / hyperactivity disorder (ADHD), autism, schizophrenia, neuropathic pain, glaucoma, addiction, arachnoid cysts, hypnosis, encephalitis, epilepsy / seizures, locked-in syndrome, meningitis, migraine, multiple sclerosis, myelopathy, Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Barten's disease, tic disorders, traumatic brain injury, spinal cord injury, stroke, tremor (primary or Parkinson's disease), dystonia, intellectual disability, brain tumors, or combinations thereof. In some respects, the CNS-related disorder or disorder is anxiety, depression, PTSD, or a combination thereof. In other respects, the CNS-related disorder or disorder is glaucoma, neuropathic pain, or both.

[0029] In some aspects, the aforementioned abnormality in central nervous system function is associated with an increase in the FAM19A1 mRNA level and / or the FAM19A1 protein level in the sample compared to a reference value (e.g., the corresponding value in a sample from a subject without central nervous system dysfunction (e.g., a healthy subject)). In some aspects, the increase in the FAM19A1 protein level and / or the FAM19A1 mRNA level compared to the reference value is 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.

[0030] In some aspects, the aforementioned abnormality in central nervous system function is associated with a decrease in the FAM19A1 mRNA level and / or the FAM19A1 protein level in the sample compared to a reference value (e.g., the corresponding value in a sample from a subject without central nervous system dysfunction (e.g., a healthy subject)). In some aspects, the FAM19A1 protein level and / or the FAM19A1 mRNA level decreases 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 reference value.

[0031] In some aspects, the FAM19A1 protein level is measured by: immunohistochemistry, Western ink dot assay, radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), radioimmunodiffusion, immunoprecipitation assay, Euler's immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, FACS, protein microarray, or a combination thereof. In some aspects, the FAM19A1 mRNA level is measured by: reverse transcription polymerase chain reaction (RT-PCR), real-time polymerase chain reaction, Northern ink dot assay, or a combination thereof.

[0032] In some respects, the sample includes: tissue, cells, blood, serum, plasma, saliva, urine, cerebrospinal fluid (CSF), or a combination thereof.

[0033] In some aspects, the diagnostic or identification methods disclosed herein further include administering a FAM19A1 antagonist to the subject if the FAM19A1 protein level and / or the FAM19A1 mRNA level increases compared to the reference value. In some aspects, the diagnostic or identification methods disclosed herein further include administering an agonist targeting FAM19A1 (“FAM19A1 agonist”) if the FAM19A1 protein level and / or the FAM19A1 mRNA level decreases compared to the reference value.

[0034] In some aspects, the FAM19A1 agonist is the FAM19A1 protein. In some aspects, the FAM19A1 antagonist is: an antisense oligonucleotide, siRNA, shRNA, miRNA, dsRNA, aptamer, PNA, or a vector comprising the thereof that specifically targets FAM19A1. In some aspects, the FAM19A1 antagonist is: an anti-FAM19A1 antibody, a polynucleotide encoding the anti-FAM19A1 antibody, a vector containing the polynucleotide, a cell containing the polynucleotide, or any combination thereof. In some aspects, the FAM19A1 antagonist is an anti-FAM19A1 antibody.

[0035] In some respects, the subjects were male.

[0036] This article provides an anti-FAM19A1 antibody or its antigen-binding fragment (“anti-FAM19A1 antibody”) that exhibits properties selected from the following: (a) having a K+ concentration of 10 nM or less as measured by ELISA. D(b) binds to soluble human FAM19A1; and (c) contains K, as measured by ELISA, 10 nM or less. D The membrane-bound human FAM19A1 binds; or (c), (a) and (b) both.

[0037] In some respects, the anti-FAM19A1 antibody cross-competes with reference antibodies comprising heavy chain CDR1, CDR2, and CDR3, as well as light chain CDR1, CDR2, and CDR3, to bind to human FAM19A1 epitopes.

[0038] (i) wherein the heavy chain CDR1 comprises the amino acid sequence shown in SEQ ID NO: 10, the heavy chain CDR2 comprises the amino acid sequence shown in SEQ ID NO: 11, the heavy chain CDR3 comprises the amino acid sequence shown in SEQ ID NO: 12, the light chain CDR1 comprises the amino acid sequence shown in SEQ ID NO: 13, the light chain CDR2 comprises the amino acid sequence shown in SEQ ID NO: 14, and the light chain CDR3 comprises the amino acid sequence shown in SEQ ID NO: 15;

[0039] (ii) wherein the heavy chain CDR1 comprises the amino acid sequence shown in SEQ ID NO: 4, the heavy chain CDR2 comprises the amino acid sequence shown in SEQ ID NO: 5, the heavy chain CDR3 comprises the amino acid sequence shown in SEQ ID NO: 6, the light chain CDR1 comprises the amino acid sequence shown in SEQ ID NO: 7, the light chain CDR2 comprises the amino acid sequence shown in SEQ ID NO: 8, and the light chain CDR3 comprises the amino acid sequence shown in SEQ ID NO: 9;

[0040] (iii) wherein the heavy chain CDR1 comprises the amino acid sequence shown in SEQ ID NO: 16, the heavy chain CDR2 comprises the amino acid sequence shown in SEQ ID NO: 17, the heavy chain CDR3 comprises the amino acid sequence shown in SEQ ID NO: 18, the light chain CDR1 comprises the amino acid sequence shown in SEQ ID NO: 19, the light chain CDR2 comprises the amino acid sequence shown in SEQ ID NO: 20, and the light chain CDR3 comprises the amino acid sequence shown in SEQ ID NO: 21; or

[0041] (iv) wherein the heavy chain CDR1 comprises the amino acid sequence shown in SEQ ID NO: 22, the heavy chain CDR2 comprises the amino acid sequence shown in SEQ ID NO: 23, the heavy chain CDR3 comprises the amino acid sequence shown in SEQ ID NO: 24, the light chain CDR1 comprises the amino acid sequence shown in SEQ ID NO: 25, the light chain CDR2 comprises the amino acid sequence shown in SEQ ID NO: 26, and the light chain CDR3 comprises the amino acid sequence shown in SEQ ID NO: 27.

[0042] In some respects, the anti-FAM19A1 antibody binds to the same FAM19A1 epitope as a reference antibody comprising heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3.

[0043] (i) wherein the heavy chain CDR1 comprises the amino acid sequence shown in SEQ ID NO: 10, the heavy chain CDR2 comprises the amino acid sequence shown in SEQ ID NO: 11, the heavy chain CDR3 comprises the amino acid sequence shown in SEQ ID NO: 12, the light chain CDR1 comprises the amino acid sequence shown in SEQ ID NO: 13, the light chain CDR2 comprises the amino acid sequence shown in SEQ ID NO: 14, and the light chain CDR3 comprises the amino acid sequence shown in SEQ ID NO: 15;

[0044] (ii) wherein the heavy chain CDR1 comprises the amino acid sequence shown in SEQ ID NO: 4, the heavy chain CDR2 comprises the amino acid sequence shown in SEQ ID NO: 5, the heavy chain CDR3 comprises the amino acid sequence shown in SEQ ID NO: 6, the light chain CDR1 comprises the amino acid sequence shown in SEQ ID NO: 7, the light chain CDR2 comprises the amino acid sequence shown in SEQ ID NO: 8, and the light chain CDR3 comprises the amino acid sequence shown in SEQ ID NO: 9;

[0045] (iii) wherein the heavy chain CDR1 comprises the amino acid sequence shown in SEQ ID NO: 16, the heavy chain CDR2 comprises the amino acid sequence shown in SEQ ID NO: 17, the heavy chain CDR3 comprises the amino acid sequence shown in SEQ ID NO: 18, the light chain CDR1 comprises the amino acid sequence shown in SEQ ID NO: 19, the light chain CDR2 comprises the amino acid sequence shown in SEQ ID NO: 20, and the light chain CDR3 comprises the amino acid sequence shown in SEQ ID NO: 21; or

[0046] (iv) wherein the heavy chain CDR1 comprises the amino acid sequence shown in SEQ ID NO: 22, the heavy chain CDR2 comprises the amino acid sequence shown in SEQ ID NO: 23, the heavy chain CDR3 comprises the amino acid sequence shown in SEQ ID NO: 24, the light chain CDR1 comprises the amino acid sequence shown in SEQ ID NO: 25, the light chain CDR2 comprises the amino acid sequence shown in SEQ ID NO: 26, and the light chain CDR3 comprises the amino acid sequence shown in SEQ ID NO: 27.

[0047] In some respects, the anti-FAM19A1 antibody binds to at least one epitope selected from the group consisting of D112, M117, A119, T120, N122, and combinations thereof.

[0048] In some aspects, the anti-FAM19A1 antibody comprises heavy chains CDR1, CDR2, and CDR3, and light chains CDR1, CDR2, and CDR3, wherein the heavy chain CDR3 comprises the amino acid sequence shown in SEQ ID NO: 12, 6, 18, or 24. In some aspects, the heavy chain CDR1 comprises the amino acid sequence shown in SEQ ID NO: 10, 4, 16, or 22. In further aspects, the heavy chain CDR2 comprises the amino acid sequence shown in SEQ ID NO: 11, 5, 17, or 23. In some aspects, the light chain CDR1 comprises the amino acid sequence shown in SEQ ID NO: 13, 7, 19, or 25. In some aspects, the light chain CDR2 comprises the amino acid sequence shown in SEQ ID NO: 14, 8, 20, or 26. In some aspects, the light chain CDR3 comprises the amino acid sequence shown in SEQ ID NO: 15, 9, 21, or 27.

[0049] In some aspects, the anti-FAM19A1 antibody comprises heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3, wherein

[0050] (i) The heavy chains CDR1, CDR2 and CDR3 each comprise the amino acid sequences shown in SEQ ID NOs: 10-12, and the light chains CDR1, CDR2 and CDR3 each comprise the amino acid sequences shown in SEQ ID NOs: 13-15;

[0051] (ii) The heavy chains CDR1, CDR2 and CDR3 each comprise the amino acid sequences shown in SEQ ID NOs: 4-6, and the light chains CDR1, CDR2 and CDR3 each comprise the amino acid sequences shown in SEQ ID NOs: 7-9;

[0052] (iii) The heavy chains CDR1, CDR2, and CDR3 each comprise the amino acid sequences shown in SEQ ID NOs: 16-18, and the light chains CDR1, CDR2, and CDR3 each comprise the amino acid sequences shown in SEQ ID NOs: 19-21; or

[0053] (iv) The heavy chains CDR1, CDR2 and CDR3 each comprise the amino acid sequences shown in SEQ ID NOs: 22-24, and the light chains CDR1, CDR2 and CDR3 each comprise the amino acid sequences shown in SEQ ID NOs: 25-27.

[0054] In some aspects, the anti-FAM19A1 antibody comprises: a heavy chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 30, 28, 32 or 34; and / or a light chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 31, 29, 33 or 35.

[0055] In some aspects, the anti-FAM19A1 antibody comprises: a heavy chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 30; and a light chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 31. In some aspects, the anti-FAM19A1 antibody comprises: a heavy chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 28; and a light chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 29. In some aspects, the anti-FAM19A1 antibody comprises: a heavy chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 32; and a light chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 33. In some aspects, the anti-FAM19A1 antibody comprises: a heavy chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 34; and a light chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 35.

[0056] In some aspects, 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 listed in SEQ ID NO: 30, 28, 32, or 34; and / or wherein the VL comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence listed in SEQ ID NO: 31, 29, 33, or 35.

[0057] In some aspects, the anti-FAM19A1 antibody is a chimeric antibody, a human antibody, or a humanized antibody. In some aspects, the anti-FAM19A1 antibody includes: Fab, Fab', F(ab')2, Fv, or single-chain Fv (scFv). In some aspects, the anti-FAM19A1 antibody is selected from the group consisting of: IgG1, IgG2, IgG3, IgG4, variants thereof, and any combination thereof. In some aspects, the anti-FAM19A1 antibody is an IgG1 antibody. In some aspects, the anti-FAM19A1 antibody includes: a constant region without Fc function.

[0058] In some aspects, the anti-FAM19A1 antibody is linked to a pharmaceutical agent to form an immunoconjugate. In other aspects, the anti-FAM19A1 antibody is formulated with a pharmaceutically acceptable carrier.

[0059] In some respects, the FAM19A1 antagonist useful for the methods disclosed herein is the anti-FAM19A1 antibody provided herein.

[0060] In some respects, the FAM19A1 antagonist is administered intravenously, orally, parenterally, intrathecally, intraventricularly, pulmonaryly, intramuscularly, subcutaneously, intravitreally, or intraventricularly. In some respects, the subject is a human.

[0061] This document also provides a nucleic acid comprising a nucleotide sequence encoding the anti-FAM19A1 antibody of this disclosure. This disclosure also provides a vector comprising the nucleic acid disclosed herein and one or more promoters operatively linked to said nucleic acid. This disclosure further provides a cell comprising the nucleic acid or vector described herein. This document discloses a composition comprising: the anti-FAM19A1 antibody of this disclosure, and a vector. This disclosure provides a kit comprising: the anti-FAM19A1 antibody of this disclosure, and instructions for use.

[0062] This article provides a method for producing an anti-FAM19A1 antibody, the method comprising: culturing the cells disclosed herein under appropriate conditions, and isolating the anti-FAM19A1 antibody. Attached Figure Description

[0063] Figure 1 ELISA results of binding to positive polyscFv-phage antibody pools in each round of bio-panning as described in Example 2 are shown. The shown positive polyscFv-phage antibody pools include those from: (i) round 1 bio-panning (“1'Fc”), (ii) round 2 bio-panning (“2'mFc”), and (iii) round 3 bio-panning (“3'Fc”). M13 phage #38 and the entire library were used as controls. For each of the shown scFv-phage antibody libraries, binding to FAM19A1-Fc, FAM19A1-mFc, and non-FAM19A1 protein (ITGA6-Fc) is shown from left to right.

[0064] Figure 2 The results of ELISA showing the binding of a single monomeric scFv-phage clone isolated from the third round of biopanning to the FAM19A1 protein are shown. The clones shown include (from left to right): 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 of the antibody clones, the left bar represents binding to FAM19A1-Fc. For each of the antibody clones, the bar on the right represents the binding to the negative control (non-FAM19A1-Fc).

[0065] Figure 3BstNI fingerprinting analysis of different monomeric scFv-phage clones isolated from the third round of biopanning is shown. The clones shown 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 specifically bind to FAM19A1 (i.e., do not bind to control proteins other than FAM19A1) with high affinity: 1A11, 1C1, 2G7, and 3A8. Antibody clones 2C9, 5A3, and 2E1 do not specifically bind to FAM19A1, are not monophages, or bind to FAM19A1 with low affinity, respectively.

[0066] Figure 4 ELISA results for binding to both FAM19A1 and non-FAM19A1 proteins are shown for different monomeric scFv-phage clones. For each clone, 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. These three FAM19A1 proteins differ only in the tag used to detect binding.

[0067] Figures 5a, 5b, 5c, and 5d illustrate the different characteristics of the anti-FAM19A1IgG1 antibody produced as described in Example 3. Figure 5A A schematic diagram of the construction of the expression vector for producing the anti-FAM19A1 IgG1 antibody is provided. Figure 5B The purity and migration rate of the antibody, as confirmed by SDS-PAGE analysis, are provided. Figure 5C It provides productivity data. Figure 5D An analysis of the antibody's binding to the FAM19A1 protein, measured by ELISA, is provided. The table below the figure provides the Kd values. Figure 5B , Figure 5C and Figure 5D The anti-FAM19A1 IgG1 antibody shown includes clones 1A11, 1C1, 2G7, and 3A8.

[0068] Figure 6 ELISA results of different anti-FAM19A1 antibody clones binding to FAM19A1 mutants M1-M7 are shown. Wild-type FAM19A1 and PBS were used as controls. 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).

[0069] Figure 7 The expression of FAM19A1 mRNA in different mouse tissues is shown. The tissues shown include those from different brain regions (i.e., the cerebral cortex, cerebellum, midbrain, spinal cord, hippocampus, olfactory bulb, hypothalamus, and pituitary gland) and peripheral tissues (i.e., the heart, liver, spleen, stomach, small intestine, testes, kidneys, and lungs). The brain regions are shown within the boxes.

[0070] Figure 8A , Figure 8B , Figure 8C , Figure 8D , Figure 8E , Figure 8F , Figure 8G and Figure 8H The expression of FAM19A1 in FAM19A1 LacZ knock-in (KI) mice is shown in the example. Figure 8A A schematic diagram of the FAM19A1 LacZ KI mouse gene construct is provided. The LacZ gene sequence is inserted after the start codon of exon 2 of the FAM19A1 gene. This gene construct is expressed using the native FAM19A1 promoter. Due to the poly-A tail following the LacZ sequence, the resulting product is β-galactosidase without any part of the FAM19A1 protein. Therefore, homozygous FAM19A1 LacZ KI mice are considered to be 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'-glycosylphosphotransferase gene; pA, poly-A tail. Figure 8BGenomic DNA PCR results comparing FAM19A1 (243 bp) and β-galactosidase (343 bp) in wild-type, FAM19A1 LacZ KI(+ / -), and FAM19A1 LacZ KI(- / -) animals are provided. Figure 8C RT-PCR results comparing FAM19A1 expression in the cortex (CTX) and hippocampus (HIP) of different animals are provided. Figure 8D A comparison of the expression of endogenous FAM19A1 protein in the cortex (CTX) and hippocampus (HIP) of different animals using FAM19A1-specific antibodies is provided. Exposure times during development with ECL solution are given: 30 minutes for FAM19A1 and 1 minute for β-actin. Figure 8E (cortex) and Figure 8F (Haima) provided support for Figure 8D Quantitative analysis of the results shown. Figure 8G The expression of FAM19A1 mRNA and protein in various brain regions of wild-type animals is shown. Exposure times during development with ECL solution are shown: 30 minutes for FAM19A1 and 1 minute for β-actin. Regions shown include: (i) cortex (CTX), (ii) hippocampus (HIP), (iii) olfactory bulb (OB), (iv) cerebellum (CB), (v) thalamus + hypothalamus (TH+HYP), (vi) midbrain (MB), and (vii) pons (PO). Figure 8H Provided for Figure 8G Quantitative analysis of the results shown. Figure 8E , Figure 8F and Figure 8H In this context, "au" refers to any unit. Data are expressed as mean ± standard error (SEM) of that mean. One-way ANOVA and Bonferroni post-hoc tests showed that, compared to WT, **p < 0.01 and ***p < 0.001.

[0071] Figure 9A , Figure 9B and Figure 9C The whole brain X-gal staining of FAM19A1 LacZ KI(- / -) mice at different developmental stages is shown. Figure 9A A comparison of β-galactosidase expression in WT and FAM19A1 LacZ KI mice at day 12.5 of embryonic development (E12.5) is provided. Figure 9B The expression of β-galactosidase in FAM19A1 LacZ KI mice at embryonic days 14.5, 16.5, and 18.5 is shown. Figure 9CThe expression of β-galactosidase in FAM19A1 LacZ KI mice at 0.5, 2.5, 7.5, 14.5, and 56.6 days postnatal is shown. Figure 9A , Figure 9B and Figure 9C In this context, the scale is 2 millimeters.

[0072] Figure 10A and Figure 10B X-gal staining of the brains of FAM19A1 LacZ knock-in (KI) mice during the embryonic and postnatal periods was provided. Figure 10A The detection of X-gal signal in coronal brain slices is shown at day 14.5 (E14.5) and day 18.5 (E18.5) of embryonic development. Figure 10BThe detection of X-gal signals in different regions is shown at 0.5 days (P0.5), 7.5 days (P7.5), and 14.5 days (P14.5) after birth. ACo, anterior cortical amygdaloid nucleus; Amy, amygdala; AO, anterior olfactory nucleus; Au, auditory cortex; BMA, basomedial amygdaloid nucleus, 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, optic nerve layer of the superior colliculus. (layer); PF, pontine flexure; PMCo, posteromedial cortical amygdaloid nucleus; Pn, pontine nuclei; PrL, prelimbic cortex; RMC, red nucleus, magnocellular part; S, somatosensory cortex; V, visual cortex.

[0073] Figure 11A , Figure 11B and Figure 11C The expression patterns of FAM19A1 in the brains of different adult mice are shown. Figure 11A In the study, X-gal deposits (red) were detected in a subset of cortical L2-3 CUX1-positive neurons (green) from FAM19A1 LacZ KI mice. Figure 11BIn the study, β-galactosidase (green) was found in the L5 CTIP2-positive neuronal (magenta) subset of the cortex of FAM19A1 LacZ KI mice. Arrows indicate cortical marker cells expressing X-gal or β-galactosidase. Figure 11CThis image shows X-gal staining of coronal brain sections from adult mice, as determined by immunohistochemistry. Different panels represent different brain regions in the respective animals. AO, anterior olfactory nucleus; Apir, amygdala piriform transition area; BLA, basomedial amygdaloid nucleus; BLP, basolateral amygdaloid nucleus, posterior part; CEn, entorhinal cortex; CPf, piriform cortex; D3V, dorsal 3rd ventricle; FrA, frontal association cortex; L2-3, cortical layers 2-3; L5, cortical layer 5; CA1, 2, and 3, regions of CA1, CA2, and CA3 in the hippocampus; LaDL, lateral amygdala. LHb, lateral habenular nucleus; LO, lateral orbital cortex; LS, lateral septal nucleus; LV, lateral ventricle; MGN, medial geniculate nucleus; MO, medial orbital cortex; Op, superior colliculus optic nerve layer; PMCo, posteromedial cortical amygdaloid nucleus; PrL, prelimbic cortex; Py, pyramidal cell layer of hippocampus; RG, retrosplenial granular cortex; VO, ventral orbital cortex.

[0074] Figure 12FAM19A1 expression in the adult mouse brain, spinal cord, and dorsal root ganglia (as shown by X-gal staining) is presented. Plate AG shows X-gal stained coronal sections of different brain regions from FAM19A1 LacZ knock-in (KI) heterozygous mice. Plates H and I show X-gal stained coronal sections of different brain regions from homozygous FAM19A1 LacZ KI mice. Plate JM shows X-gal stained coronal sections of the spinal cord from heterozygous FAM19A1 LacZ KI mice. Plate N shows X-gal stained dorsal root ganglia from heterozygous FAM19A1 LacZ KI mice.3V, third ventricle; 7N, facial nucleus; cp, cerebral peduncle; DC, dorsal cochlear nucleus; Ecu, external cuneate nucleus; ic, internal capsule; IPDL, interpeduncular nucleus, dorsolateral subnucleus; lfp, longitudinal fasciculus of the pons; LPO, lateral preoptic area; LRt, lateral reticular nucleus; ml, medial lemniscus; MPOM, medial preoptic nucleus, medial part; MMeMC, medial vestibular nucleus, magnocellular part. (part); MMePC, medial vestibular nucleus, parvicellular part; Pn, pontine nuclei; Po, posterior thalamic nuclear group; Pr, prepositus nucleus; py, pyramidal tract; RtTg, reticuloreticular nucleus of the pons; Sp5I, spinal trigeminal nucleus, interpolar part; Sp5O, spinal trigeminal nucleus, oral part; SpVe, spinal vestibular nucleus; SuVe, superior vestibular nucleus; VMH, ventromedial hypothalamic nucleus nucleus); X, X nucleus (nucleus X).

[0075] Figure 13 The expression of FAM19A1 mRNA in the brains of developing and mature wild-type (WT) rats is shown using in situ hybridization with a FAM19A1 mRNA probe. The rat age for each brain slice shown is provided in the lower right corner of each panel: (i) day 14.5 of embryonic development (E14.5), (ii) day 16.5 of embryonic development (E16.5), (iii) day 18.5 of embryonic development (E18.5), (iv) day 0.5 postnatal (P0.5), (v) day 7.5 postnatal (P7.5), (vi) day 14.5 postnatal (P14.5), (vii) day 21.5 postnatal (P21.5), and (viii) different views of the adult brain (sagittal, horizontal, and coronal planes). Amy, amygdala; AO, anterior olfactory nucleus; Cer, cerebellum; CTX, cerebral cortex; Hb, pineal ligament; Hip, hippocampus; Mes, midbrain; SC, spinal cord; Tel, telencephalon; Th, thalamus.

[0076] Figure 14 A table is provided showing the number and percentage of offspring produced from heterozygous FAM19A1 LacZ KI parents.

[0077] Figure 15A , Figure 15B , Figure 15C , Figure 15D , Figure 15E , Figure 15F , Figure 15G , Figure 15H , Figure 15I and Figure 15J A comparison of morphological differences between wild-type and FAM19A1 LacZ knock-in (KI) mice is provided. Figure 15A and Figure 15B The changes in body weight of male and female mice with age are shown separately. Figure 15C Whole-mount views of the brains of WT and FAM19A1(- / -) adult mice are provided. Figure 15D The motor cortex is shown in Nissl-stained brain tissue from WT, heterozygous FAM19A1 LacZ KI (FAM19A1+ / -), and homozygous FAM19A1 LacZ KI (FAM19A1- / -) mice. Figure 15E , Figure 15F and Figure 15G The total brain length, cerebral cortex length, and brain width of adult mice with WT (n=9), FAM19A1+ / - (n=8), and FAM19A1- / - (n=8) were provided respectively. Figure 15H , Figure 15I and Figure 15J Cortical thicknesses of the motor, somatosensory, and visual cortices in adult mice of WT (n=5), FAM19A1+ / - (n=5), and FAM19A1- / - (n=4) groups were provided. Data are expressed as mean ± standard error of the mean (SEM). One-way or two-way ANOVA and Bonferroni post-hoc tests were performed. Compared with WT or FAM19A1+ / -, *p<0.05, **p<0.01, ***p<0.001.

[0078] Figure 16A and Figure 16B Estimated cortical volumes in the cerebral cortex of wild-type (WT), FAM19A1+ / -, and FAM19A1- / - adult mice are provided. Figure 16A ) and estimate the total number of nerve cells ( Figure 16B The data are compared as mean ± standard error of mean (SEM).

[0079] Figure 17A , Figure 17B , Figure 17C , Figure 17D , Figure 17E and Figure 17F Cortical thickness comparisons were provided for wild-type, FAM19A1+ / -, and FAM19A1- / - adult mice. Figure 17A , Figure 17B and Figure 17C The cortical thicknesses of the motor, somatosensory, and visual cortices are shown separately. Figure 17D , Figure 17E and Figure 17F The thickness ratios of each cortical layer in the motor, somatosensory, and visual cortices to the total cortical thickness are shown separately. Different cortical layers are plotted on the X-axis. For each cortical layer mentioned on the X-axis, the bar charts (from left to right) represent wild-type (WT), FAM19A1+ / -, and FAM19A1- / - adult mice, respectively. Data are expressed as mean ± standard error of the mean (SEM). One-way ANOVA was performed, and compared with WT mice, *p<0.05, **p<0.01, ***p<0.001.

[0080] Figure 18A , Figure 18B , Figure 18C and Figure 18D A comparison of neuronal cell density in the motor cortex of wild-type, FAM19A1+ / -, and FAM19A1- / - adult mice is provided. Figure 18A In this study, NeuN was used as a marker for neuronal cells. Different cortical layers were identified as L1, L2-3, L4, L5, and L6. Figure 18B , Figure 18C and Figure 18D They provided Figure 18A The quantitative comparison of neuronal cell density, volume, and total number of NeuN-positive cells in each cortical layer is shown. Figure 18B , Figure 18C and Figure 18D In the graph, for each cortical layer described on the (X-axis), the bar charts (from left to right) represent wild-type (WT), FAM19A1+ / -, and FAM19A1- / - adult mice, respectively. Data are expressed as mean ± standard error of mean (SEM).

[0081] Figure 19A , Figure 19B , Figure 19C , Figure 19D and Figure 19E A comparison of the number of glial cells in the motor cortex of wild-type, FAM19A1+ / -, and FAM19A1- / - adult mice is provided. Figure 19A In this study, GFAP (green) positive astrocytes and Iba1 (red) positive microglia were detected in the motor cortex. Figure 19B Oligodendrocytes (exemplary positive cells indicated by white arrows) were found in the motor cortex. Figure 19C , Figure 19D and Figure 19E The numbers of GFAP-positive cells, Iba1-positive cells, and Olig2-positive cells in the motor cortex are shown respectively. Figure 19C , Figure 19D and Figure 19E In the graph, for each cortical layer described on the (X-axis), the bar charts (from left to right) represent wild-type (WT), FAM19A1+ / -, and FAM19A1- / - adult mice, respectively. Data are expressed as mean ± standard error of mean (SEM).

[0082] Figure 20A , Figure 20B , Figure 20C , Figure 20D , Figure 20E , Figure 20F , Figure 20G and Figure 20H A comparison of hyperactivity was provided for wild-type, FAM19A1+ / -, and FAM19A1- / - adult mice. Figure 20A and Figure 20B The total time and total walking distance in the open arm measured in the elevated maze (EPM) test are shown separately. Figure 20C and Figure 20DThe total time and total walking distance at the center are shown separately in the Open Ground Test (OFT). Figure 20E It provides simple line tracing of animal movement within the OFT testing arena. Figure 20F Shows the percentage of immobility time measured in the tail suspension test (TST). Figure 20G and Figure 20H Spontaneous changes and total walking distances measured in the Y-maze test are shown separately. Data are expressed as mean ± standard error of mean (SEM). *p < 0.05, **p < 0.01, ***p < 0.001 compared to WT or FAM19A1+ / -, by one-way ANOVA and Bonferroni post-hoc test.

[0083] Figure 21A , Figure 21B , Figure 21C , Figure 21D , Figure 21E and Figure 21F A comparison of short-term and long-term memory formation in wild-type, FAM19A1+ / -, and FAM19A1- / - adult mice is provided. Figure 21A , Figure 21B and Figure 21C The total exploration time, object preference, and discrimination index are shown separately in the short-term memory novelty recognition (NOR) test. Figure 21D , Figure 21E and Figure 21F The total exploration time, object preference, and discrimination index, as measured in the long-term memory NOR test, are shown separately. Figure 21A , Figure 21B , Figure 21D and Figure 21E In the diagram, for each animal group, the left bar chart represents the results for familiar objects, and the right bar chart represents the results for novel objects. Data are expressed as mean ± standard error of the mean (SEM). One-way ANOVA and Bonferroni post-hoc tests showed *p < 0.05, **p < 0.01, and ***p < 0.001 compared to WT or FAM19A1+ / -.

[0084] Figure 22A , Figure 22B , Figure 22C and Figure 22D A comparison of fear responses in wild-type, FAM19A1+ / -, and FAM19A1- / - adult mice is provided. Figure 22A This shows the fear conditions during the fear learning phase of Pavlov's fear conditioning test. Arrows indicate the relevant groups. Figure 22B and Figure 22C The results of situational and auditory memory tests are shown separately, which were conducted 24 hours after the fear acquisition phase. Figure 22D Results of the innate fear test using synthetic fox feces odor and 2,5-dihydro-2,4,5-trimethylthiazoline (TMT) are shown. Arrows indicate relevant groups. Data are presented as mean ± standard error of mean (SEM). *p<0.05, **p<0.01, ***p<0.001 compared to WT were determined by two-way ANOVA with Bonferroni post-hoc test or Student's t-test.

[0085] Figure 23A and Figure 23B A comparison of FAM19A1 and FAM19A5 expression in the brain of FAM19A1 LacZ KI and FAM19A5 LacZ KI mice is provided (as evidenced by β-galactosidase expression). Figure 23A This is a schematic diagram of the FAM19A5 LacZKI mouse gene construction. The LacZ gene sequence was inserted via homologous recombination. The resulting product is a fusion of FAM19A5 and β-galactosidase. Figure 23B The expression of FAM19A1 (left) and FAM19A5 (right) in the brain is shown. The regions shown include: L2-3 (cortical layers 2 and 3); L5b (cortical layer 5b); CA1 (hippocampal CA1 area); CA2 (hippocampal CA2 area); CA3 (hippocampal CA3 area); DG (dentate gyrus); CC (corpus callosum); CTX (cortex); TH (thalamus); fi (fimbria of the hippocampus). Scale bar = 500 μm.

[0086] Figure 24 A comparison of neurite outgrowth in differentiated neurons treated with the following methods in adult mouse neural stem cells is provided: (i) control IgG antibody (left panel); (ii) anti-FAM19A1 antibody (middle panel); or (iii) FAM19A1 protein (right panel).

[0087] Figure 25The image shows a comparison of intraocular pressure in glaucoma-induced animals treated with human IgG1 (“hIgG”; hollow squares) or anti-FAM19A1 antibody (“FAM19A1 Ab”; solid squares). Normal healthy animals (i.e., those without glaucoma induction) are also shown. The control group was used as a reference. Intraocular pressure was measured on days 0, 14, and 28 after glaucoma induction. Data are expressed as mean ± SD. "***" indicates a statistically significant difference compared to the control group without glaucoma (P < 0.001).

[0088] Figure 26 This paper compares the oscillatory potential grades in glaucoma-induced animals treated with human IgG1 (“hIgG”) or anti-FAM19A1 antibody (“FAM19A1Ab”). Normal healthy animals (i.e., without glaucoma induction) (“no glaucoma”) were used as controls. Data are expressed as mean ± SD. “***” indicates a statistically significant difference compared to the no glaucoma control group (P<0.001). “###” indicates a statistically significant difference compared to the hIgG group (P<0.001).

[0089] Figure 27A and Figure 27B This diagram shows a comparison of the number of retinal ganglion cells (“RGCs”) in glaucoma-induced animals treated with human IgG1 (“hIgG”) or anti-FAM19A1 antibody (“FAM19A1 Ab”). Normal healthy animals (i.e., those without glaucoma induction) (“unaffected”) were used as controls. Figure 27A The absolute number of RGC cells is shown. Data are expressed as mean ± SD. "***" indicates a statistically significant difference compared to the control group (P < 0.001). "##" indicates a statistically significant difference compared to the hIgG group (P < 0.01). Figure 27B Fluorescence images of the retinal ganglion cell layer from representative animals in each group are shown (magnified 100x).

[0090] Figure 28 This study compares the paw retraction thresholds in rats treated with saline (hollow squares) or anti-FAM19A1 antibody (solid squares) induced by chronic contractile injury (CCI). Normal healthy animals (i.e., without CCI induction) (“uninfected”) served as controls. Paw retraction thresholds were measured on days 7, 14, and 21 following CCI induction. Data are presented as mean ± SD. “#” indicates a statistically significant difference compared to the saline group (P < 0.05).

[0091] Figure 29This paper compares the rotarod latency (the time required for an animal to fall off a rotarod-treadmill, as described in the example) in rats treated with saline (hollow squares) or anti-FAM19A1 antibody (solid squares) induced by chronic contractile injury (CCI). Normal healthy animals (i.e., without CCI induction) (“unaffected”) were used as controls. Latency was measured at days 7, 14, and 21 following CCI induction. Data are expressed as mean ± SD. “#” indicates a statistically significant difference compared to the saline group (P < 0.05).

[0092] Figure 30A , Figure 30B , Figure 30C , Figure 30D , Figure 30E and Figure 30F This study demonstrates the effects of anti-FAM19A1 treatment on neurite outgrowth and dendritic branching in primary mouse hippocampal neurons. Figure 30A and Figure 30B This illustrates the outward growth of neurites via immunohistochemistry in mouse hippocampal neurons treated with either a vehicle-control or anti-FAM19A1 antibody. Figure 30C The average total length of the neural neurite is shown (μm). Figure 30D This shows the number of primary neurites. Figure 30E Shows the number of branch points. Figure 30F It provides the number of secondary neurites. In Figures 30C to 30F In this study, data are expressed as mean ± SD. "*" indicates a statistically significant difference (P < 0.05). Figure 30A and Figure 30B In the diagram, the scale is 20 μm.

[0093] Figure 31This study illustrates the analgesic effect of the anti-FAM19A1 monoclonal antibody (A1-1C1) in CCI-induced mechanical allodynia. The analgesic effect is shown as the number of paw withdrawal responses (“withdrawal response frequency percentage”) after 10 applications of filaments to each hind paw at 10-second intervals. Animals treated with the human IgG control antibody (solid circles) and normal healthy animals (i.e., without CCI induction) (hollow circles) served as controls. Paw withdrawal responses were measured on days 6, 10, 13, 17, and 20 post-CCI induction. Data are expressed as mean ± SD. “*” indicates a statistically significant difference (P < 0.01). Arrows indicate when the anti-FAM19A1 antibody was administered (i.e., days 7 and 14 post-CCI induction).

[0094] Figure 32 This study illustrates the analgesic effect of anti-FAM19A1 monoclonal antibody (A1-1C1) in CCI-induced thermal hyperalgesia. The analgesic effect is shown as withdrawal latency (how long it takes for animals to withdraw their paws in response to a thermal stimulus). Animals treated with human IgG control antibody in response to CCI (solid circles) and normal healthy animals (i.e., without CCI induction) (hollow circles) served as controls. Paw withdrawal responses were measured at days 6, 10, 13, 17, and 20 post-CCI induction. Data are expressed as mean ± SD. Arrows indicate when the anti-FAM19A1 antibody was administered (i.e., days 7 and 14 post-CCI induction). Detailed Implementation

[0095] What is disclosed herein is an antagonist (e.g., a monoclonal antibody) that specifically binds to a member of the human sequence similarity family 19 A1 (FAM19A1) and exhibits one or more of the properties disclosed herein.

[0096] To facilitate understanding of the content disclosed herein, some terms and phrases have been defined. Further definitions are provided throughout the detailed description.

[0097] I. Definition

[0098] In this disclosure, the term "a" or "an" refers to one or more of the entities; for example, "an antibody" is understood to represent one or more antibodies. Therefore, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.

[0099] Furthermore, the term “and / or” as used herein should be considered as specifically disclosing each of two particular features or components, regardless of the presence of the other. Therefore, the term “and / or” as used herein 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 the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0100] It should be understood that when the term "comprising" is used to describe an aspect wherever it is used in this document, other similar aspects described as "consisting of" and / or "consisting essentially of" are also provided.

[0101] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art in connection with this disclosure. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd edition, 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, revised edition, 2000, Oxford University Press provide general dictionaries for those skilled in the art of the use of many of the terms used in this disclosure.

[0102] Units, prefixes, and symbols are all represented in their International System of Units (SI) accepted form. Numerical ranges include the numbers defining the ranges. Unless otherwise stated, amino acid sequences are written from left to right with the orientation from amino to carboxyl. The headings provided herein are not intended to limit any aspect of this disclosure, which can be obtained by referring to the full text of this specification. Therefore, the terms defined immediately thereafter are defined more completely by referring to this specification in its entirety.

[0103] The term “about” in this text means approximately, roughly, around, or within its range. When the term “about” is used in conjunction with a numerical range, it modifies the range by extending the boundaries above and below the stated numerical value. Generally, the term “about” can modify a numerical value to be higher or lower than the stated value by a variation of, for example, 10% upward or downward (higher or lower).

[0104] 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), a group of five highly homologous proteins. These proteins contain conserved cysteine ​​residues at fixed positions and are distantly related to MIP-1alpha, a member of the CC-chemokine family. FAM19A1 is primarily expressed in the central nervous system (brain and spinal cord). See example. FAM19A1 is also known as TAFA1 or chemokine-like protein TAFA-1.

[0105] In humans, the gene encoding FAM19A1 is located on chromosome 3. Human FAM19A1 (UniProt: Q7Z5A9) has two potential isoforms: isoform 1 (UniProt: Q7Z5A9-1), consisting of 133 amino acids; and isoform 2 (UniProt: A0A087X2J7), consisting of 52 amino acids and predicted based on EST data. The amino acid sequences of these two known human FAM19A1 isoforms are provided in Table 1 below.

[0106] Table 1. Amino acid sequences of FAM19A1 isomers

[0107]

[0108] The term “FAM19A1” includes any variant or isoform of FAM19A1 naturally expressed by cells. Therefore, the antagonists (e.g., antibodies) described herein may cross-react with different isoforms within the same species (e.g., different isoforms of human FAM19A1) or with FAM19A1 from species other than humans (e.g., mouse FAM19A1). Alternatively, the antibody may be specific to human FAM19A1 and not exhibit any cross-reactivity with other species. FAM19A1 or any variants and isoforms thereof may be isolated from cells or tissues that naturally express them or generated through recombinant synthesis. The GenBank accession number for the polynucleotide encoding human FAM19A1 is NM_213609.3, and its sequence is as follows:

[0109] Table 2. Nucleotide sequence of FAM19A1 (isoform 1)

[0110]

[0111] The term "antagonist against a FAM19A1 protein" or "FAM19A1 antagonist" refers to all antagonists that inhibit the expression of the FAM19A1 protein. Such antagonists can be peptides, nucleic acids, or compounds. In some aspects, FAM19A1 antagonists include: antisense oligonucleotides, siRNA, shRNA, miRNA, dsRNA, aptamers, PNAs (peptide nucleic acids), or vectors including them that target FAM19A1. In other aspects, FAM19A1 antagonists include: antibodies that specifically bind to the FAM19A1 protein, or antigen-binding fragments thereof.

[0112] The term "agonist against a FAM19A1 protein" or "FAM19A1 agonist" refers to all agonists that promote the expression of the FAM19A1 protein and / or have the same biological function as the FAM19A1 protein, thereby increasing the activity of FAM19A1. In some respects, a FAM19A1 agonist is a FAM19A1 protein.

[0113] The terms “antibody” and “antibodies” are terms used in this art and are used interchangeably herein, referring to a molecule having an antigen-binding site that can specifically bind to an antigen. As used herein, the term includes the entire antibody and any antigen-binding fragment (i.e., an “antigen-binding fragment”) or a single chain thereof. In some aspects, “antibody” refers to a glycoprotein comprising—interconnected by disulfide bonds—at least two heavy (H) chains and two light (L) chains, or an antigen-binding fragment thereof. 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 some naturally occurring antibodies, the heavy chain constant region consists of three domains: CH1, CH2, and CH3. In some naturally occurring antibodies, each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of a structural domain CL.

[0114] The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which contain more conserved regions called frame regions (FRs). Each VH and VL 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 the antigen. The constant regions of the antibody mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0115] The term "Kabat number" and similar terms are generally accepted in the art to refer to a system for numbering amino acid residues in the variable regions of the heavy and light chains of an antibody or its antigen-binding fragment. In some respects, the CDR of an antibody can be determined according to the Kabat numbering system (see, for example, Kabat EA & Wu TT (1971), Ann NY Acad Sci 190:382-391 and Kabat EA et al. (1991), Sequences of Proteins of Immunological Interest, 5th Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system, CDRs within antibody heavy chain molecules are typically located at amino acid positions 31 to 35 (optionally including one or two additional amino acids), after 35 (referred to as 35A and 35B in the Kabat numbering scheme) (CDR1), at amino acid positions 50 to 65 (CDR2), and at amino acid positions 95 to 102 (CDR3). Using the Kabat numbering system, CDRs within antibody light chain molecules are typically located at amino acid positions 24 to 34 (CDR1), at amino acid positions 50 to 56 (CDR2), and at amino acid positions 89 to 97 (CDR3). In some respects, the CDRs of the antibodies described herein have been determined according to the Kabat numbering scheme.

[0116] The phrases “amino acid position numbering as in Kabat,” “Kabat position,” and their grammatical variations refer to the numbering system for heavy chain or light chain variable domains used in antibody compilation, as described in Kabat et al., *Sequences of Proteins of Immunological Interest*, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering system, the actual linear amino acid sequence can contain fewer or additional amino acids, corresponding to shortening or insertion of the FW or CDR of the variable domain. For example, a heavy chain variable domain could include a single amino acid insertion following residue 52 of H2 (according to residue 52a in Kabat) and inserted residues following residue 82 of the heavy chain FW (e.g., residues 82a, 82b, and 82c, etc., according to Kabat). See Table 3.

[0117] Table 3

[0118]

[0119]

[0120] For a given antibody, the Kabat number of residues can be determined by comparing the homologous region of the antibody sequence with the “standard” Kabat numbering sequence. Chothia, on the other hand, refers to the location of the structural loop (Chothia and Lesk, *Journal of Molecular Biology* 196: 901-917 (1987)). When numbering using the Kabat numbering convention, the end of the Chothia CDR-H1 loop varies between H32 and H34, depending on the loop length (this is because the Kabat numbering scheme places the insertion sites at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable region represents a compromise between Kabat CDRs and the Chothia structural loop and is used by the Oxford Molecular AbM antibody modeling software.

[0121] IMGT (ImMunoGeneTics) also provides a numbering system for the variable regions (including CDRs) of immunoglobulins. See, for example, Lefranc, MP, et al., *Dev. Comp. Immunol.* 27:55-77 (2003), which is incorporated herein by reference. The IMGT numbering system is based on alignments, structural data, and characteristics of hypervariable loops from over 5,000 sequences and allows for easy comparison of variable regions and CDRs across all species. According to the IMGT numbering scheme, VH-CDR1 is 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.

[0122] For all heavy chain constant region amino acid positions discussed in this disclosure, the numbering is based on the EU index first described by Edelman et al. in Proc. Natl. Acad. Sci. USA 63(1): 78-85 in 1969, which describes the amino acid sequence of the myeloma protein EU, the first human lgG1 to be sequenced. This EU index by Edelman et al. is also listed in Sequences of Proteins of Immunological Interest, 5th Edition, United States Public Health Service, National Institutes of Health, Bethesda, Kabat et al. (1991). Therefore, the phrases “EU index listed in Kabat” or “EU index of Kabat” and “according to the EU index listed in Kabat, position…” and their grammatical variations refer to: residue numbering based on the human lgG1 EU antibody of Edelman et al. (as listed in Kabat 1991).

[0123] The numbering system for the amino acid sequences of the variable domains (both heavy and light chains) and the light chain constant region is listed in Kabat 1991.

[0124] Antibodies can be any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgD, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., human IgG1, IgG2, IgG3, and IgG4; and mouse IgG1, IgG2a, IgG2b, and IgG3). Immunoglobulins, such as IgG1, exist in several allotypes, which differ from each other by at most a few amino acids. The antibodies disclosed herein can be derived from any commonly known isotype, class, subclass, or heterotype. In some respects, the antibodies described herein belong to the IgG1, IgG2, IgG3, or IgG4 subclass or any hybrid thereof. In some respects, these antibodies belong to the human IgG1 subclass or the human IgG2 or human IgG4 subclass.

[0125] "Antibody" includes, for example, naturally occurring and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human and non-human antibodies; fully synthetic antibodies; single-chain antibodies; monospecific antibodies; multispecific antibodies (including bispecific antibodies); tetrameric antibodies comprising two heavy chain and two light chain molecules; antibody light chain monomers; antibody heavy chain monomers; antibody light chain dimers, antibody heavy chain dimers; antibody light chain-antibody heavy chain pairs; intrabodies; heteroconjugate antibodies; monovalent antibodies; camelized antibodies; affybodies; anti-idiotype (anti-Id) antibodies (including, for example, anti-anti-Id antibodies); and single-domain antibodies (sdAbs) comprising a binding molecule consisting of a single monomeric variable antibody domain (e.g., a VH domain or a VL domain) fully capable of antigen binding. Harmen MM and Hard HJ, Appl Microbiol Biotechnol. 77(1): 13-22 (2007).

[0126] The terms "antigen-binding portion" and "antigen-binding fragment" of an antibody are used interchangeably and refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., human FAM19A1). Such "fragments" are, for example, between about 8 and about 1500 amino acids in length, appropriately between about 8 and about 745 amino acids in length, appropriately between about 8 and about 300 amino acids in length, for example between about 8 and about 200 amino acids, or about 10 to about 50 or 100 amino acids in length. It has been shown that the antigen-binding function of an antibody can be accomplished by fragments of a full-length antibody. Examples of binding fragments included in the “antigen-binding portion” of an antibody include, for example, (i) a Fab fragment, a monovalent fragment consisting of VL, VH, CL, and CH1 domains; (ii) an F(ab')2 fragment, a bivalent fragment consisting of two Fab fragments connected by a disulfide bridge in the hinge region; (iii) an Fd fragment consisting of VH and CH1 domains; (iv) an Fv fragment consisting of VL and VH domains of a single arm of the antibody, and a disulfide-linked Fvs (sdFv); (v) a dAb fragment (Ward et al., Nature 341:544-546 (1989)) consisting of a VH domain; and (vi) separate complementarity-determining regions (CDRs) or (vii) combinations of two or more separate CDRs, which may optionally be linked by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by different genes, they can be linked together using a recombinant approach via synthetic linkers that allow them to be prepared into single protein chains, where the VL and VH regions pair to form monovalent molecules (called single-chain Fv (scFv)); see, for example, Bird et al., Science 242:423-426 (1988); and Huston et al., Proceedings of the National Academy of Sciences of the United States of America (Proc. Natl. Acad. Sci. USA) 85:5879-5883 (1988). Such single-chain antibodies are also included in the term "antigen-binding moiety" of antibodies. These antibody fragments are obtained using conventional techniques known to those skilled in the art and are screened for utility in the same manner as intact antibodies. The antigen-binding moiety can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

[0127] As used herein, the terms “variable region” and “variable domain” are used interchangeably and are common in the art. A variable region typically refers to a portion of an antibody, generally a portion of the light or heavy chain, typically about 110 to 120 amino acids at the amino terminus in the mature heavy chain and about 90 to 115 amino acids in the mature light chain. These regions exhibit significant sequence differences between antibodies and are responsible for the binding and specificity of a particular antibody to its specific antigen. Sequence variability is concentrated in regions called complementarity-determining regions (CDRs), while more highly conserved regions within a variable domain are called framework regions (FRs).

[0128] Without being bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for antibody-antigen interactions and specificity. In some respects, the variable region is the human variable region. In some respects, the variable region includes rodent or mouse CDRs and human frame regions (FRs). In some respects, the variable region is the primate (e.g., non-human primate) variable region. In some respects, the variable region includes rodent or mouse CDRs and primate (e.g., non-human primate) frame regions (FRs).

[0129] As used herein, the term “heavy chain” (HC) when referring to antibodies can refer to any different type of amino acid sequence based on a constant structural domain, such as alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), which produce IgA, IgD, IgE, IgG, and IgM antibodies, including subclasses of IgG such as IgG1, IgG2, IgG3, and IgG4.

[0130] As used herein, the term "light chain" (LC), when used to refer to an antibody, can refer to any different type of amino acid sequence based on a constant domain, such as kappa (κ) or lambda (λ). Light chain amino acid sequences are well known in the art. In some specific respects, the light chain is the human light chain.

[0131] The terms “VL” and “VL domain” are used interchangeably and refer to the variable region of the light chain of an antibody.

[0132] The terms “VH” and “VH domain” are used interchangeably and refer to the variable region of the heavy chain of an antibody.

[0133] As used herein, the terms “constant region” and “constant domain” are interchangeable and have their usual meaning in the art. A constant region is an antibody portion, such as the carboxyl-terminal portion of a light chain and / or heavy chain, which does not directly participate in antibody-antigen binding but can exhibit various effector functions, such as interaction with Fc receptors. The constant regions of immunoglobulin molecules typically have a more conserved amino acid sequence than the variable domains of immunoglobulins.

[0134] The “Fc region” (crystallizable fragment region), “Fc domain”, or simply “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. Therefore, the Fc region includes the constant region of the antibody, excluding the first constant region immunoglobulin domains (e.g., CH1 or CL). In IgG, IgA, and IgD antibody isotypes, the Fc region comprises two identical protein fragments derived from the second (CH2) and third (CH3) constant domains of the two antibody heavy chains; the IgM and IgE Fc regions comprise three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. For IgG, the Fc region includes immunoglobulin domains Cγ2 and Cγ3, as well as the hinge between Cγ1 and Cγ2. Although the boundaries of the Fc region of the immunoglobulin heavy chain may vary, the human IgG heavy chain Fc region is generally defined as extending from an amino acid residue at position C226 or P230 (or an amino acid between these two) to the carboxyl terminus of the heavy chain, where the numbering is based on the EU index as in Kabat. The CH2 domain of the human IgG Fc region extends from about amino acid 231 to about amino acid 340, while the CH3 domain is located on the C-terminal side of the Cm domain of the Fc region, extending from about amino acid 341 to about amino acid 447 of IgG. As used herein, the Fc region can be the native sequence Fc, including any allotype variant, or variant Fc (e.g., a non-naturally occurring Fc). Fc can also refer to the isolated region or the region in the context of a protein polypeptide containing the Fc, such as "Fc-containing binding protein," also known as "Fc fusion protein" (e.g., antibody or immunoadhesin).

[0135] The “native sequence Fc region” or “native sequence Fc” includes an amino acid sequence identical to that of an Fc region found in nature. Native sequence human Fc regions include the native sequence human IgG1 Fc region; the native sequence human IgG2 Fc region; the native sequence human IgG3 Fc region; and the native sequence human IgG4 Fc region, as well as their naturally occurring variants. Native sequence Fc includes various allotypes of Fes (see, for example, Jefferis et al., Monoclonal Antibodies (mAbs) 1:1 (2009); Vidarsson G. et al., Frontiers in Immunology 5:520 (2014).

[0136] An Fc receptor, or FcR, is a receptor that binds to the Fc region of an immunoglobulin. FcRs that bind to IgG antibodies include receptors of the FcγR family, including allelic variants and alternatively spliced ​​forms. The FcγR family includes 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. In terms of the type of activating Fc receptor it binds to, it is considered comparable to mouse IgG2a. Conversely, human IgG4 elicits the smallest Fc effector function. (Vidarsson G. et al., Frontiers in Immunology 5:520, published online October 20, 2014).

[0137] Constant regions can be manipulated, for example, through recombinant techniques, to eliminate one or more effector functions. An "effector function" refers to the interaction between the antibody Fc region and an Fc receptor or ligand, or the resulting biochemical event. Exemplary "effector functions" include C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, FcγR-mediated effector functions such as ADCC and antibody-dependent cell-mediated phagocytosis (ADCP), and downregulation of cell surface receptors (e.g., B cell receptor; BCR). Such effector functions typically 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" encompasses constant regions mediated by the Fc region that have reduced or no one or more effector functions.

[0138] The effector function of an antibody can be reduced or avoided through various methods. Sub-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 composed of monomeric VH or VL domains). Alternatively, so-called aglycosylated antibodies can be generated by removing sugars linked to specific residues in the Fc region to reduce the antibody's effector function while preserving other valuable properties of the Fc region (e.g., prolonged half-life and heterodimerization). Aglycosylated antibodies can be generated, for example, by deleting or altering the residues linked to the sugar, enzymatically removing the sugar, generating the antibody in cells cultured with glycosylation inhibitors, or by expressing the antibody in cells that cannot glycosylate proteins (e.g., bacterial host cells). See, for example, U.S. Publication No. 20120100140. Another approach is to utilize Fc regions from IgG subclasses with reduced effector function; for example, IgG2 and IgG4 antibodies are characterized by lower Fc effector function levels than IgG1 and IgG3. The residues closest to the hinge region in the CH2 domain of the Fc portion are responsible for the antibody's effector function because it contains largely overlapping binding sites for C1q (complement) and the IgG-Fc receptor (FcγR) on effector cells of the innate immune system. (Vidarsson G. et al., Frontiers in Immunology 5:520 (2014)). Therefore, antibodies with reduced Fc effector function or without Fc effector function can be prepared by generating, for example, a chimeric Fc region comprising, the CH2 domain of an IgG antibody from the IgG4 isotype and the CH3 domain of an IgG antibody from the IgG1 isotype, or a chimeric Fc region comprising a hinge region from IgG2 and a CH2 region from IgG4 (see, for example, Lau C et al., *Journal of Immunology* 191:4769-4777 (2013)), or an Fc region having mutations that alter Fc effector function (e.g., reduced or absent Fc function). Such mutated Fc regions are known in the art. See, for example, U.S. Publication No. 20120100140 and the U.S. and PCT applications cited therein, and An et al., *Monoclonal Antibodies (mAbs)* 1:6,572-579 (2009).

[0139] The term "hinge," "hinge domain," "hinge region," or "antibody hinge region" refers to a domain within the heavy chain constant region that connects the CH1 and CH2 domains, including the upper, middle, and lower parts of the hinge (Roux et al., *Journal of Immunology* 161:4083 (1998)). The hinge provides varying degrees of flexibility between the antibody's binding and effector regions and also provides sites for intermolecular disulfide bonds between the two heavy chain constant regions. As used herein, for all IgG isotypes, the hinge begins at Glu216 and terminates at Gly237 (Roux et al., *Journal of Immunology* 161:4083 (1988)). The sequences of the hinges for wild-type IgG1, IgG2, IgG3, and IgG4 are known in the art. See, for example, Kabat EA et al., Sequences of Proteins of Immunological Interest, 5th Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; Vidarsson G. et al., Front Immunol. 5:520 (published online on October 20, 2014).

[0140] The term "CH1 domain" refers to a heavy chain constant region that connects a variable domain to a hinge within the heavy chain constant domain. As used herein, the CH1 domain begins at A118 and terminates at V215. The term "CH1 domain" includes the wild-type CH1 domain, as well as its naturally occurring variants (e.g., allotypes). The CH1 domain sequences of IgG1, IgG2, IgG3, and IgG4 (including wild-type and allotypes) are known in the art. See, for example, Kabat EA et al., (1991) ibid. and Vidarsson G. et al., Frontiers in Immunol. 5:520 (published online October 20, 2014). Example CH1 domains include CH1 domains with mutations that alter antibody biological activity (e.g., half-life), as described, for example, in U.S. Patent Publication No. 20120100140 and the U.S. Patents and Publications cited therein, as well as PCT publications.

[0141] The term "CH2 domain" refers to the heavy chain constant region, which connects the hinge to the CH3 domain within the heavy chain constant region. As used herein, the CH2 domain begins at P238 and ends at K340. The term "CH2 domain" includes the wild-type CH2 domain, as well as its naturally occurring variants (e.g., allotypes). The CH2 domain sequences of IgG1, IgG2, IgG3, and IgG4 (including wild-type and allotypes) are known in the art. See, for example, Kabat EA et al., (1991) ibid. and Vidarsson G. et al., Frontiers in Immunol. 5:520 (published online October 20, 2014). Example CH2 domains include CH2 domains with mutations that alter antibody biological activity (e.g., half-life and / or reduced Fc effector function), as described, for example, in U.S. Patent Publication No. 20120100140 and the U.S. Patents and Publications cited therein, as well as PCT publications.

[0142] The term "CH3 domain" refers to the heavy chain constant region, which is the C-terminus of the CH2 domain within the heavy chain constant region. As used herein, the CH3 domain begins at G341 and ends at K447. The term "CH3 domain" includes the wild-type CH3 domain, as well as its naturally occurring variants (e.g., allotypes). The CH3 domain sequences of IgG1, IgG2, IgG3, and IgG4 (including wild-type and allotypes) are known in the art. See, for example, Kabat EA et al., (1991) ibid. and Vidarsson G. et al., Frontiers in Immunol. 5:520 (published online October 20, 2014). Example CH3 domains include CH3 domains with mutations that alter antibody biological activity (e.g., half-life), as described, for example, in U.S. Patent Publication No. 20120100140 and the U.S. Patents and Publications cited therein, as well as PCT publications.

[0143] As used in this article, “isotype” refers to the class of antibodies encoded by the heavy chain constant region gene (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibodies).

[0144] “Allotype” refers to a naturally occurring variant within a specific allotype group that differs in a few amino acids (see, for example, Jefferis et al. (2009), Monoclonal Antibodies (mAbs) 1:1). The antibodies described herein can be of any allotype. Allotypes of IgG1, IgG2, IgG3, and IgG4 are known in the art. See, for example, Kabat EA et al. (1991), ibid.; Vidarsson G. et al., Front Immunol. 5:520 (published online October 20, 2014); and Lefranc MP, Monoclonal Antibodies (mAbs) 1:4, 1-7 (2009).

[0145] The phrases “an antibody recognizing an antigen” and “an antibody specific for an antigen” are used interchangeably with the term “antibody that binds specifically to an antigen” in this document.

[0146] As used herein, "isolated antibody" refers to an antibody that is substantially free of other antibodies with 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 the FAM19A1 epitope may be cross-reactive with other FAM19A1 proteins from different species.

[0147] "Binding affinity" generally refers to the strength of the sum of interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise stated, as used herein, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y can generally be expressed by the dissociation constant (K0). D Affinity can be expressed in various ways known in the art, including but not limited to the equilibrium dissociation constant (K). D ) and equilibrium association constant (K A K D It is k off / k on The quotient is calculated and expressed as molar concentration (M), while K A It is k on / k off It is calculated from the quotient. K onThis refers to, for example, the rate constant of antibody-antigen binding, while K... off This refers to, for example, the dissociation of antibodies and antigens. on and k off It can be determined using techniques known to those skilled in the art, such as immunoassays (e.g., enzyme-linked immunosorbent assay (ELISA)), BIAcore, etc. TM Or kinetic exclusion measurement

[0148] As used herein, the terms “specifically binds,” “specifically recognizes,” “specific binding,” “selective binding,” and “selectively binds” are similar terms in the context of antibodies and refer to molecules (e.g., antibodies) that bind to antigens (e.g., epitopes or immune complexes) in a manner understood by those skilled in the art. For example, molecules that specifically bind to antigens may bind to other peptides or polypeptides, generally with lower affinity, for example, by immunoassays, BIAcore, etc. TM , The assay is determined by a 3000 instrument (Sapidyne Instruments, Boise, ID) or other assays known in the art. In some respects, molecules that specifically bind to the antigen bind to the antigen, while their K... A The value is at least 2 logs, 2.5 logs, 3 logs, 4 logs, or greater than the K value when the molecule binds to another antigen. A .

[0149] Antibodies typically bind specifically to their homologous antigens with high affinity, which is achieved through 10... -5 Up to 10 -11 M or a lower dissociation constant (K) D ) reflects. Anything greater than approximately 10 -4 M of K D Generally, this is considered to indicate non-specific binding. As used herein, an antibody that "specifically binds" to an antigen refers to an antibody that binds with high affinity to the antigen and substantially the same antigen, meaning that when measured by, for example, immunoassays (e.g., ELISA) or surface plasmon resonance (SPR) techniques on a BIACORE surface... TM When performing immunoassays using a predetermined antigen in the 2000 instrument, it possesses K D 10 -7 M or lower, preferably 10 -8 M or lower, or even better, 10-9 M or lower, with the best option at 10 -8 M and 10 -10 M or lower, but will not bind to irrelevant antigens with high affinity.

[0150] As used herein, the term "antigen" refers to any natural or synthetic immunogenic substance, such as a protein, peptide, or hapten. An antigen may be FAM19A1 or a fragment thereof.

[0151] "Epitope" is a term in the art and refers to a localized region of an antigen to which an antibody can specifically bind. An epitope can be, for example, a series of amino acids of a polypeptide (linear or continuous epitope), or it can be, for example, a combination of two or more discontinuous regions from one or more polypeptides (conformational, nonlinear, discontinuous, or non-continuous epitope). Epitopes formed from continuous amino acids are typically, but not always, preserved upon exposure to denaturing solvents, while epitopes formed from ternary folding are typically lost upon treatment with denaturing solvents. Epitopes typically comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20 amino acids in a unique spatial conformation. Several methods for determining which epitopes are bound by a given antibody (i.e., epitope mapping) are known in the art and include, for example, immunoblotting and immunoprecipitation assays, in which overlapping or sequential peptides from (e.g., from FMAM19A5) are tested for reactivity with a given antibody (e.g., anti-FAM19A1 antibody). Some methods for determining the spatial conformation of epitopes include those techniques in the art and those described herein, such as X-ray crystallography, two-dimensional nuclear magnetic resonance, and HDX-MS (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GEMorris, Ed. (1996)).

[0152] In some respects, antibody-bound epitopes can be determined by, for example, NMR spectroscopy, X-ray diffraction crystallography, ELISA assays, hydrogen / deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography-electrospray mass spectrometry), array-based oligopeptide scanning assays, and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). For X-ray crystallography, crystallization can be accomplished using any method known in the art (e.g., Giege R et al. Acta Crystallographica D: Biol Crystallography 50(Pt 4): 339-350 (1994); McPherson A. Eur J Biochem 189: 1-23 (1990); Chayen NE. Structure 5: 1269-1274 (1997); McPherson A. J Biol Chem 251: 6300-6303 (1976)). Antibody: Antigen crystals can be studied using well-known X-ray diffraction techniques and computer software such as X-PLOR (Yale University, 1992, published by Molecular Simulations, Inc.; see, for example, *Meth Enzymol* (1985), Vol. 114 and 115, edited by Wyckoff HW et al.; 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*). The crystallization was refined using Crystallogr (Pt10): 1316-1323 (2000). Mutation mapping studies can be performed using any method known to those skilled in the art.See, for example, descriptions of mutagenesis techniques, including alanine scanning mutagenesis, in the Journal of Biochemistry, 270 (1995): 1388-1394, by Cunningham BC & Wells JA, Science, 244: 1081-1085 (1989).

[0153] The term "epitope mapping" refers to the process of identifying molecular determinants used for antibody-antigen recognition.

[0154] The term "binds to the same epitope" when referring to two or more antibodies means that the antibodies bind to the same amino acid residue segment, as determined by a given method. Techniques for determining whether an antibody binds to the "same epitope on FAM19A1" as described herein include, for example, epitope mapping methods, such as X-ray analysis of crystals of antigen:antibody complexes, which provides atomic resolution and hydrogen / deuterium exchange mass spectrometry (HDX-MS) of the epitope. Other methods monitor the binding of antibodies to antigen fragments or mutated variants of the antigen, where loss of binding due to modification of amino acid residues within the antigen sequence is generally considered an indicator of epitope components. Additionally, computational combinatorial methods for epitope mapping can also be used. These methods rely on the ability of the relevant antibody to affinity isolate specific short peptides from combinatorial phage display peptide libraries. Antibodies with the same VH and VL or the same CDR1, 2 and 3 sequences are expected to bind to the same epitopes.

[0155] An antibody that “compete with another antibody for binding to a target” refers to an antibody that (partially or completely) inhibits the binding of another antibody to a target. Known competition assays can be used to determine whether two antibodies compete with each other for binding to a target, i.e., whether and to what extent one antibody inhibits the binding of the other antibody to the target. In some respects, one antibody competes with another for binding to a target and inhibits the binding of that other antibody to the target by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. The level of inhibition or competition can vary depending on which antibody is a “blocking antibody” (i.e., a cold antibody that is first incubated with the target). Competitive assays can be performed as described, for example, in Ed Harlow and David Lane's Cold Spring Harb Protocol, 2006; doi: 10.1101 / pdb.prot4277 or in Chapter 11 of Ed Harlow and David Lane's Using Antibodies (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA 1999). Competitive antibodies bind to the same epitope, overlapping epitopes, or adjacent epitopes (e.g., as demonstrated by steric hindrance).

[0156] Other competitive binding assays include: solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competitive assay (see Stahli et al., Methods in Enzymology 9:242 (1983)); solid-phase direct biotin-avidin EIA (see Kirkland et al., Journal of Immunology 137:3614 (1986)); solid-phase direct labeling assay, solid-phase direct labeling sandwich assay (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor). Press (1988)); solid-phase direct-labeled RIA using 1-125 label (see Morel et al., Molecular Immunology 25(1):7 (1988)); solid-phase direct biotin-avidin EIA (Cheung et al., Virology 176:546 (1990)); and directly labeled RIA (Moldenhauer et al., Scandinavian Journal of Immunology 32:77 (1990)).

[0157] A "bispecific" or "bifunctional antibody" is an artificial hybrid antibody that has two distinct heavy / light chain pairs and two distinct binding sites. Bispecific antibodies can be produced by a variety of methods, including hybridoma fusion or Fab fragment linkage. See, for example, Songsivilai & Lachmann, *Clin. Exp. Immunol.* 79:315-321 (1990); Kostelny et al., *Journal of Immunology* 148, 1547-1553 (1992).

[0158] The term "monoclonal antibody," as used herein, refers to: an antibody, or an antibody composition, that exhibits single binding specificity and affinity for a specific epitope—where all antibodies exhibit single binding specificity and affinity for the specific epitope. Therefore, the term "human monoclonal antibody" refers to an antibody or antibody composition that exhibits single binding specificity and has variable and optional constant regions derived from human germline immunoglobulin sequences. In some aspects, human monoclonal antibodies are produced by hybridomas containing B cells derived from transgenic nonhuman animals (e.g., transgenic mice) whose genomes include human heavy chain and light chain transgenes fused with immortalized cells.

[0159] The term "recombinant human antibody," as used herein, includes all human antibodies prepared, expressed, created, or isolated by recombinant means, such as: (a) antibodies isolated from transgenic or transchromosomally transgenic animals (e.g., mice) or hybridomas prepared therefrom; (b) antibodies isolated from host cells transformed to express antibodies (e.g., from transfectomas); (c) antibodies isolated from recombinant, combined libraries of human antibodies; and (d) antibodies prepared, expressed, created, or isolated by any other method involving the splicing of human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies include variable and constant regions utilizing specific human germline immunoglobulin sequences encoded by germline genes, but include subsequent rearrangements and mutations, for example, that occur during antibody maturation. As is known in the art (see, for example, Lonberg, Nature Biotech. 23(9): 1117-1125 (2005)), the variable region contains an antigen-binding domain—encoded by various genes rearranged to form antibodies specific to foreign antigens. In addition to rearrangements, the variable region can be further modified by multiple single-amino acid alterations (called somatic mutations or hypermutations) to increase the antibody's affinity for foreign antigens. The constant region will be altered in further responses to the antigen (i.e., isotype conversion). Therefore, rearranged and somatically mutated nucleic acid molecules encoding light and heavy chain immunoglobulin polypeptides in response to antigens cannot have sequence identity with the original nucleic acid molecule, but will be substantially identical or similar (i.e., have at least 80% identity).

[0160] A "human" antibody (HuMAb) is an antibody with a variable region, wherein both the framework region and the CDR region are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, that constant region is also derived from a human germline immunoglobulin sequence. Antibodies described herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, the term "human antibody," as used herein, is not intended to include antibodies in which a CDR sequence derived from another mammalian species (e.g., mouse) has been grafted onto human framework sequences. The terms "human" antibody and "fully human" antibody are used synonymously.

[0161] A "humanized" antibody is an antibody in which some, most, or all of the amino acids outside the CDR domain of a non-human antibody are replaced by corresponding amino acids derived from human immunoglobulins. In some respects, some, most, or all of the amino acids outside the CDR domain are replaced by amino acids derived from human immunoglobulins, while some, most, or all of the amino acids within one or more CDR regions remain unchanged. Minor additions, deletions, insertions, substitutions, or modifications of amino acids are permitted as long as they do not abrogate the antibody's ability to bind to a specific antigen. Humanized antibodies retain antigen specificity similar to that of the original antibody.

[0162] A "chimeric antibody" is an antibody in which the variable region is derived from one species and the constant region is derived from another species. For example, the variable region is derived from a mouse antibody and the constant region is derived from a human antibody.

[0163] The term "cross-reacts," as used herein, refers to the ability of the antibodies described herein to bind to FAM19A1 from different species. For example, an antibody described herein that binds to human FAM19A15 may also bind to FAM19A1 from another species (e.g., mouse FAM19A1). As used herein, cross-reactivity can be measured by detecting specific reactivity with purified antigen in a binding assay (e.g., SPR, ELISA) or in binding to or otherwise functionally interacting with cells that physiologically express FAM19A1. Methods for determining cross-reactivity include: standard binding assays as described herein (e.g., using Biacore). TM 2000SPR instrument (Biacore AB, Uppsala, Sweden) for Biacore TM Surface plasmon resonance (SPR) analysis, or flow cytometric technique.

[0164] The term "naturally-occurring," when applied to the subject matter here, refers to the fact that the subject matter can be found in nature. For example, a polypeptide or polynucleotide sequence present in an organism (including viruses) that has been isolated from a natural source and has not been intentionally modified by humans in a laboratory is naturally-occurring.

[0165] A "polypeptide" is a chain consisting of at least two consecutively linked amino acid residues, with no upper limit on chain length. One or more amino acid residues in a protein may contain modifications, such as, but not limited to, glycosylation, phosphorylation, or disulfide bond formation. A "protein" may include one or more polypeptides.

[0166] The term "nucleic acid molecule," as used herein, is intended to include both DNA and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded and can be cDNA.

[0167] The term "vector," as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is the "plasmid," which refers to a circular double-stranded DNA loop in which an additional DNA segment can be linked. Another type of vector is a viral vector, in which an additional DNA segment can be linked to a viral genome. Some vectors are capable of autonomous replication in the host cells to which they are introduced (e.g., bacterial vectors with bacterial origins of replication and attachable mammalian vectors). Other vectors (e.g., non-attached mammalian vectors) can integrate into the host cell's genome after introduction into the host cell, thereby replicating along with the host genome. Furthermore, some vectors are capable of directing the expression of genes operatively linked to them. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Typically, expression vectors used for recombinant DNA technologies are in the form of plasmids. In this specification, "plasmid" and "vector" are used interchangeably because plasmids are the most commonly used form of vector. However, other forms of expression vectors are also included, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which provide equivalent functionality.

[0168] The term "recombinant host cell" (or simply "host cell"), as used herein, is intended to refer to cells that include nucleic acids not naturally present in cells, and may be cells into which recombinant expression vectors have been introduced. It should be understood that this term refers not only to the specific test cell but also to the progeny of such cells. Because certain modifications may occur in subsequent generations due to mutations or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0169] The term "linked," as used herein, refers to the association of two or more molecules. This link can be covalent or non-covalent. Linkage can also be genetic (i.e., recombinant fusion). Such links can be achieved using a variety of techniques recognized in the art, such as chemical conjugation and recombinant protein production.

[0170] The term "therapeutically effective amount," as used herein, refers to an amount of medicine, alone or in combination with another therapeutic agent, that is effective in "treating" a subject's CNS-related disease or disorder, or in reducing the risk, potential, likelihood, or occurrence of a CNS-related disease or disorder. "Therapeutically effective amount" includes the amount of medicine or therapeutic agent that provides some improvement or benefit to a subject who has a CNS-related disease or disorder or is at risk of having a CNS-related disease or disorder. Therefore, a "therapeutically effective" amount means an amount that reduces the risk, potential, likelihood, or occurrence of a CNS-related disease or disorder, or provides relief, mitigation, and / or reduction of at least one indicator of a CNS-related disease or disorder and / or reduction of at least one clinical symptom. Non-limiting examples of CNS-related diseases or disorders are provided elsewhere in this disclosure.

[0171] The terms “treat,” “treating,” and “treatment,” as used herein, refer to any type of intervention or procedure performed on a subject, or the administration of an active agent to them, with the aim of reversing, reducing, alleviating, inhibiting, slowing, or preventing the progression, development, severity, or recurrence of disease-related symptoms, complications, conditions, or biochemical indicators. The subject of treatment may be a person with the disease or a person without the disease (e.g., for prevention).

[0172] The term “central nervous system” (CNS), as used herein, refers to the portion of the nervous system that includes the bran and spinal cord. The CNS may additionally include the retina and optic nerves (cranial nerve II), and the olfactory nerves (cranial nerve I) and olfactory epithelium. The brain is the main control module of the CNS and is broadly divided into four lobes: (1) the temporal lobe (important for processing sensory input and giving it emotional meaning; building long-term memory and some language perception); (2) the occipital lobe (the brain’s visual processing area, containing the visual cortex); (3) the parietal lobe (integrating sensory information, including touch, spatial awareness and navigation; language perception); and (4) the frontal lobe (containing most dopamine-sensitive neurons, thus involved in attention, reward, short-term memory, motivation, and planning).

[0173] The brain can be further divided into different regions: (1) basal ganglia (involved in controlling voluntary movement, procedural learning, and deciding which motor activities to perform; diseases affecting this region include Parkinson's disease and Huntington's disease); (2) cerebellum (involved in precise motor control, language, and attention; damage to this region can lead to motor control disorders, i.e., ataxia); (3) Broca's area (involved in language processing; damage to this area can lead to language disorders); (4) corpus callosum (a wide band of nerve fibers connecting the left and right hemispheres; it is known that children with dyslexia have a smaller corpus callosum); (5) medulla oblongata. (6) The hypothalamus (which secretes various neurohormones and affects body temperature control, thirst, and hunger); (7) The thalamus (which receives sensory and motor inputs and transmits information to other parts of the cerebral cortex); and (8) The amygdala (located in the temporal lobe and involved in decision-making, memory, and emotional responses).

[0174] The term “spinal cord,” as used herein, refers to a long, tubular structure of neural tissue that extends from the medulla oblongata in the brainstem to the lumbar region. Non-limiting examples of spinal cord function include: (1) connecting most of the peripheral nervous system to the brain and being responsible for transmitting signals between the brain and surrounding tissues; and (2) acting as a small coordination center responsible for some simple reflexes, such as the withdrawal reflex.

[0175] The term "neuron," as used herein, includes a neuron and one or more of its parts (e.g., the neuronal cell body, axon, or dendrite). The term "neuron" refers to a nerve cell that includes a central cell body or neuronal soma and two types of extensions or processes: dendrites, through which most neuronal signals are transmitted to the cell body, and axons, through which most neuronal signals are transmitted from the cell body to effector cells (such as target neurons or muscles).

[0176] The term "neurite" refers to any protrusion (e.g., axon or dendrite) that originates from the cell body of a neuron.

[0177] "Administering," as used herein, means the physical introduction of a therapeutic agent or a composition containing a therapeutic agent into a subject using various methods and delivery systems known to those skilled in the art. Different routes of administration for the antibodies described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, or other parenteral administration routes, such as by injection or infusion. The phrase "parenteral administration," as used herein, refers to a mode of administration other than enteral and local administration, typically by injection, including but not limited to intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intracardiac, intradermal, tracheal, intratracheal, pulmonary, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraventricular, intravitreal, epidural, and intrasternal injections and infusions, as well as in vivo electroporation. Alternatively, the antibodies described herein may be administered via non-enteric routes, such as local, epidermal, or mucosal administration routes, such as intranasal, oral, vaginal, rectal, sublingual, or local administration. Application may be performed, for example, once, multiple times, and / or over one or more extended time periods.

[0178] The term "diagnosis," as used herein, refers to a method for identifying a subject suitable for treatment by determining or predicting whether a patient has a particular disease or condition. A person skilled in the art can make a diagnosis based on one or more diagnostic markers (e.g., FAM19A1), wherein the presence, absence, quantity, or change in quantity of the diagnostic marker indicates the presence, severity, or absence of a condition. In some aspects, increased expression of FAM19A5 in a biological sample from a subject indicates a CNS-related disease or disorder. The term "diagnosis" does not mean the ability to determine the presence or absence of a particular disease with 100% accuracy, or even the ability to make a given process or outcome more likely than not occurring. Rather, a person skilled in the art will understand that the term "diagnosis" means an increased likelihood of the presence of a certain disease or disorder in a subject. In some aspects, the term "diagnosis" includes one or more diagnostic methods for identifying a subject with a CNS-related disease or disorder. Non-limiting examples of CNS-related diseases or disorders are provided elsewhere in this disclosure.

[0179] Compositions for diagnosing CNS dysfunction include: a pharmaceutical agent for measuring the protein level of FAM19A1 or the level of nucleic acid (e.g., mRNA) encoding FAM19A1 in a sample of a subject in need (e.g., suspected of having CNS dysfunction). Such pharmaceutical agents include: oligonucleotides having a sequence complementary to FAM19A1 mRNA, or nucleic acid probes, primers, and antibodies, or antigen-binding fragments thereof, that specifically bind to FAM19A1 protein.

[0180] As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, including non-human primates, sheep, dogs, cattle, chickens, amphibians, reptiles, etc. As described herein (e.g., examples), the beneficial effects of the FAM19A1 antagonists of this disclosure are not sex-dependent. Therefore, in some aspects, subjects who may benefit from the FAM19A1 antagonists of this disclosure (e.g., improvement in CNS function or treatment of CNS-related diseases or disorders) are male subjects. In some aspects, the term "male" refers to an individual possessing both X and Y chromosomes. In some aspects, subjects who may benefit from the FAM19A1 antagonists of this disclosure (e.g., improvement in CNS function or treatment of CNS-related diseases or disorders) are female subjects. In some aspects, the term "female" refers to an individual possessing both X chromosomes. In some respects, subjects who may benefit from the FAM19A1 antagonists disclosed herein (e.g., improvement in CNS function or treatment of CNS-related diseases or disorders) include both male and female subjects.

[0181] 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 ganglia of the brain and spinal cord (CNS) and the peripheral nervous system (PNS), and they interconnect to form neural networks. A typical neuron consists of a cell body (soma), dendrites, and an axon. The cell body (cell body) of a neuron contains the nucleus. The dendrites of a neuron are branched extensions of the cell, where most of the neuron's input occurs. The axon is a finer, cable-like projection extending from the cell body, carrying neural signals out of the cell body and returning certain types of information to the cell body. The term "promoting neuronal regeneration" includes stimulating, promoting, increasing, or activating the growth of neurons, preferably after injury or damage.

[0182] The term “glaucoma” refers to a group of eye-related diseases and / or conditions characterized by the progressive loss of retinal ganglion cells and optic nerve atrophy. Glaucoma may be associated with: optic nerve damage, loss of retinal ganglion cells (“RGCs”), high intraocular pressure (“IOP”), impaired blood-retinal barrier, and / or increased microglial cell activity in the retina and / or optic nerve of the subject. Glaucoma may be asymptomatic or present with the following eye-related symptoms: burning or stinging sensation, tearing, dryness, fatigue, blurred / dimmed vision, tunneling vision, difficulty seeing in the daytime, difficulty seeing in the dark, halos around lights, and / or blindness. (Lee et al., *Arch Ophthalmol* 116:861-866 (1998)). The term “glaucoma” includes any and all types of glaucoma (regardless of cause) and any and all symptoms of glaucoma.

[0183] The term "glaucoma" includes, but is not limited to, the following types of glaucoma: open-angle glaucoma, angle-closure glaucoma, normal-tension glaucoma ("NTG"), congenital glaucoma, secondary glaucoma, pigmentary glaucoma, pseudoexfoliative glaucoma, traumatic glaucoma, neovascular glaucoma, iridocorneal endothelial syndrome, and / or uveitis-related glaucoma. Some risk factors include: elevated intraocular pressure, genetic susceptibility (e.g., family history of glaucoma, certain ethnicities), age, diabetes, hypertension, and / or physical injury to the eye.

[0184] The term "inflammation" refers to the complex response of the innate immune system in vascularized tissue, involving the accumulation and activation of immune cells (such as microglia) and plasma proteins at sites of injury and / or damage. In healthy individuals, the blood-retinal barrier provides a tight barrier preventing substances from flowing freely from the blood to the retina, and vice versa. However, in patients with glaucoma, this barrier is impaired. This vascular dysregulation restricts blood flow to the optic disc and allows the production of various inflammatory mediators (e.g., TNF-α, IL-6, IL-9, IL-10, and nitric oxide) that can freely migrate to the optic disc.

[0185] As used in this article, 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 at the seventh week of development and is composed of retinal ganglion cell axons and glial cells. The optic nerve extends from the optic disc to the thalamus and continues as the optic tract to the lateral geniculate nucleus, the forebrain nucleus, and the superior colliculus. (Selhorst, JB et al., Semin Neurol 29(1):29-35 (2009)).

[0186] The term "optic nerve damage" refers to an alteration in the normal structure or function of the optic nerve. This alteration can result from any disease, disorder, or injury, including glaucoma. Alterations in normal optic nerve function include any change in the optic nerve's ability to function properly, such as transmitting visual information from the retina to the brain. Functional changes can manifest themselves as, for example, visual field loss, impaired central visual acuity, abnormal color vision, and so on. Examples of structural changes include loss of nerve fibers in the retina, abnormal cupping of the optic nerve, and / or loss of cells in the retinal ganglion cell layer. As used herein, "optic nerve damage" may include optic nerve injury to one or both optic nerves of a subject.

[0187] Retinal ganglion cells (RGCs) are a specific type of neuron located near the innermost layer of the retina (the ganglion cell layer). These cells play a crucial role in transmitting visual information gathered from photoreceptors to the brain. (Sanes et al., *Annu RevNeurosci* 38:221-46 (2015)). RGCs can vary greatly in size, connectivity, and response to visual stimuli, but they all share a defining characteristic: long axons extending into the brain.

[0188] The term "intraocular pressure" (IOP) refers to the pressure maintained within the eye. The anterior chamber of the eye is surrounded by the cornea, iris, pupil, and lens. It is filled with aqueous humor, a watery fluid responsible for supplying oxygen and nutrients to the cornea and lens. The aqueous humor provides the necessary pressure to help maintain the shape of the eye. When the normal secretion of aqueous humor is interrupted, intraocular pressure is affected.

[0189] The term “microglia or microglial cells” refers to a class of glial cells found within the retina. These cells behave similarly to macrophages and constantly investigate foreign antigens and / or damage in the surrounding microenvironment. Once this is recognized, microglia are activated and respond rapidly to foreign antigens and / or damage by engulfing any potentially harmful debris to limit the damage, secreting inflammatory mediators, and interacting with other immune cells to generate an effective immune response. (Kettenmann et al., Physiol Rev. 91(2): 461-553 (2011)). Activated microglia differ from those in a resting state in that they exhibit 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 cell death. (Ashwell et al., Visual Neuroscience 2(5): 437-448 (1989)).

[0190] The term "neuropathic pain" refers to pain caused by any level of damage, impairment, and / or dysfunction affecting the central nervous system (CNS) and / or peripheral nervous system. The term "neuropathic pain" includes any and all types of neuropathic pain (regardless of their cause), and any and all symptoms of neuropathic pain.

[0191] Neuropathic pain includes central neuropathic pain and peripheral neuropathic pain. As used herein, the term "central neuropathic pain" refers to pain caused by 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 caused by damage or infection of peripheral sensory nerves.

[0192] Symptoms of neuropathic pain can include persistent / chronic pain, spontaneous pain, and allodynia (e.g., a painful response to stimuli that are normally painless), hyperalgesia (e.g., an aggravated response to painful stimuli that normally cause only mild discomfort, such as pricking), hyperesthesia (e.g., excessive physical sensitivity to stimuli, particularly those on the skin), or hyperpathia (e.g., a transient discomfort turning into prolonged, severe pain). In some respects, symptoms can be persistent and persist even after the underlying cause (if any) has been resolved. Merck Manual, Neuropathic Pain, available at merckmanuals.com / professional / neurologic-disorders / pain / neuropathic-pain; Campbell JN and Meyer RA, Neuron 52(1):77-92 (2006).

[0193] As used in this article, "mononeuropathy" is a peripheral neuropathy involving loss of motor or sensory sensation in a specific area due to damage or destruction of a single peripheral nerve or group of nerves. Mononeuropathy is most commonly caused by injury or trauma to a localized area, such as prolonged pressure / compression on a single nerve. However, certain systemic diseases, such as polyneuropathy, can also cause mononeuropathy. In some cases, localized injury or trauma leads to the destruction of the myelin sheath (covering) of the nerve or parts of the nerve cells (axons), which can slow or block the conduction of impulses through the nerve. In other cases, mononeuropathy can affect any part of the body. Examples of mononeuropathy include, but are not limited to, sciatic nerve dysfunction, common peroneal nerve dysfunction, radial nerve dysfunction, ulnar nerve dysfunction, intracranial mononeuropathy VI, intracranial mononeuropathy VII, intracranial mononeuropathy III (compression type), cranial mononeuropathy III (diabetic type), axillary nerve dysfunction, carpal tunnel syndrome, femoral nerve dysfunction, tibial nerve dysfunction, Bell's palsy, thoracic outlet syndrome, carpal tunnel syndrome, and sixth (abducens) nerve palsy. Finnerup NB et al., Pain 157(8): 1599-1606 (2016); National Institute of Neurological Disorders and Stroke, Peripheral Neuropathy Fact Sheet, available at ninds.nih.gov / disorders / peripheralneuropathy / detail_peripheralneuropathy.htm.

[0194] As used in this article, "polyneuropathy" is a peripheral neuropathy involving loss of motor or sensory function in a specific area due to damage or destruction of multiple peripheral nerves. Polyneuropathy includes, but is not limited to, post-polio syndrome, post-mastectomy syndrome, diabetic neuropathy, alcoholic neuropathy, amyloidosis, toxins, HIV / AIDS, hypothyroidism, uremia, vitamin deficiencies, chemotherapy-induced pain, 2',3'-didexoycytidine (ddC) treatment, Guillain-Barré syndrome, or Fabry's disease. Finnerup NB et al., Pain 157(8): 1599-1606 (2016); National Institute of Neurological Disorders and Stroke, Peripheral Neuropathy Fact Sheet, available at ninds.nih.gov / isorders / peripheralneuropathy / detail_peripheralneuropathy.htm.

[0195] The term “neuropathic pain associated with disease or disorder” refers to neuropathic pain that is associated with, caused by or resulting from a disease or disorder (e.g., those disclosed herein).

[0196] II. The method disclosed herein

[0197] Methods of treating diseases or disorders

[0198] This document discloses FAM19A1 antagonists (e.g., antibodies) that can be used for treatment (e.g., for treating diseases or disorders). As described herein, FAM19A1 is expressed in many regions of the CNS (e.g., neural circuits), and it is believed that abnormal FAM19A1 expression may lead to impairment of normal CNS function. As shown in 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 further found that the beneficial effects of the disclosed FAM19A1 antagonists are independent of the sex of the subject. Therefore, in some aspects, the disclosed FAM19A1 antagonists can be used to treat male subjects (e.g., those with impaired CNS function). In some aspects, the subject is not a female subject. In some aspects, the disclosed FAM19A1 antagonists can be used to treat female subjects (e.g., those with impaired CNS function). In some aspects, the disclosed FAM19A1 antagonists can be used to treat both male and female subjects (e.g., those with impaired CNS function).

[0199] In some respects, the FAM19A1 antagonists disclosed herein can be used to treat CNS-related diseases or disorders. In some respects, the CNS-related diseases or disorders treatable with this disclosure are related to abnormal neural circuits. As used herein, the term "neural circuit" refers to a group of neurons interconnected by synapses that perform a specific function upon activation. Neural circuits can interconnect to form large-scale brain networks. Neural circuits are components of the brain's ability to correctly transmit information to neurons. Moreover, different neural circuits are generally associated with different functions. Abnormalities in one or more neural circuits can lead to impaired CNS function, as observed in various types of CNS-related diseases or disorders.

[0200] In some respects, CNS-related disorders or disturbances that can be treated with the FAM19A1 antagonists disclosed herein include: mood disorders, mental disorders, or both. In some respects, CNS-related disorders or disturbances that can be treated with this disclosure include: anxiety disorders, depression, post-traumatic stress disorder (PTSD), bipolar disorder, attention deficit / hyperactivity disorder (ADHD), autism, schizophrenia, neuropathic pain, glaucoma, addiction, arachnoid cysts, hypnosis, encephalitis, epilepsy / seizures, locked-in syndrome, meningitis, migraine, multiple sclerosis, myelopathy, Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Barten's disease, tic disorders, traumatic brain injury, spinal cord injury, stroke, tremor (primary or Parkinson's disease), dystonia, intellectual disability, brain tumors, or combinations thereof.

[0201] In some respects, CNS-related disorders or disturbances treatable with the FAM19A1 antagonist disclosed herein are mood disorders. As used herein, the term “mood disorder” refers to any type of mental illness affecting an individual’s emotional state. As used herein, the term “mood” refers to an individual’s internal emotional state. In some respects, mood disorders treatable with this disclosure may be characterized by a pervasive, prolonged, and ineffective exaggeration of emotions and feelings associated with behavioral, physiological, cognitive, neurochemical, and psychomotor dysfunctions. Mood disorders may be associated with persistent elevated mood (mania), persistent depressive mood, or mood that cycles between mania and depression. Mood disorders may be inherently hereditary and / or induced by secondary factors (e.g., illness, medication, drug treatment). Examples of mood disorders include, but are not limited to, major depressive disorder (MDD), bipolar disorder (BD), mild depression, persistent depression (dysthymia), seasonal affective disorder (SAD), psychotic depression, postpartum depression, transient recurrent depression, premenstrual anxiety disorder (PMDD), situational depression, atypical depression, anxiety disorder, and cyclic affective disorder.

[0202] The term "major depressive disorder" or "MDD" as used in this article refers to a mood disorder characterized by two or more major depressive episodes. Symptoms of MDD may include fatigue, feelings of worthlessness or guilt, difficulty concentrating or indecisiveness, insomnia or hypersomnia, markedly diminished interest or pleasure in almost all activities, restlessness, recurrent thoughts of death or suicide, and significant weight loss or gain (5% change in body weight). The diagnostic criteria for MDD are the same as for other mood disorders and can be found in, for example, in the Diagnostic and Statistical Manual of Mental Disorders (4th edition). It was found in the Diagnostic and Statistical Manual of Mental Disorders (DSM IV) and is very helpful in assessing subjects.

[0203] The term "bipolar disorder" (i.e., manic-depressive disorder) refers to a mood disorder characterized by alternating periods of extreme mood. People with bipolar disorder experience cycles of mood, typically ranging from excessive excitement or agitation (mania) to sadness and despair (depression), and then back to normal, with periods of normal mood in between. The diagnosis of bipolar disorder is described, for example, in DSM-IV. Categories of bipolar disorder include, but are not limited to, bipolar I (bipolar disorder with or without major depression) and bipolar II (bipolar disorder with major depression). As used herein, the term "mania" or "manic" refers to a disordered mental state of extreme excitement. The term "hypomania" refers to a less extreme manic episode of lower severity.

[0204] As can be seen from this disclosure, the FAM19A1 antagonists disclosed herein can be used to treat all types of mood disorders.

[0205] In some respects, the damage that can be treated with the FAM19A1 antagonists disclosed herein is related to the visual system. Therefore, in some respects, this document provides a method for treating glaucoma in a subject in need, comprising: administering a FAM19A1 antagonist to said subject. In some respects, the FAM19A1 antagonist useful to this disclosure is: an antisense oligonucleotide, siRNA, shRNA, miRNA, dsRNA, aptamer, PNA, or a carrier comprising thereof that specifically targets FAM19A1. In some respects, the FAM19A1 antagonist is: an anti-FAM19A1 antibody, a polynucleotide encoding said anti-FAM19A1 antibody, or a carrier comprising said polynucleotide. In some respects, the FAM19A1 antagonist binds to the FAM19A1 protein and reduces FAM19A1 activity. In further respects, the reduced FAM19A1 activity decreases, alleviates, or inhibits inflammation associated with glaucoma.

[0206] In some respects, FAM19A1 antagonists (e.g., anti-FAM19A1 antibodies) reduce the loss of retinal ganglion cells (RGCs) and / or restore the number of retinal ganglion cells in subjects (e.g., glaucoma patients). In some respects, the loss of retinal ganglion cells 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 reference values ​​(e.g., corresponding values ​​in subjects not receiving FAM19A1 antagonists or corresponding values ​​in subjects before administration of FAM19A1 antagonists). In some respects, the number of retinal ganglion cells recovered 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 reference values ​​(e.g., the corresponding values ​​in subjects who did not receive the FAM19A1 antagonist or in subjects before the administration of the FAM19A1 antagonist).

[0207] In some respects, 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 respects, the FAM19A1 antagonists protect the neural connections of the inner plexiform layer of the retina in the subjects (e.g., glaucoma patients). In some respects, a FAM19A1 antagonist inhibits inflammation around the optic disc by modulating the activation of microglia. In some respects, the FAM19A1 antagonists disclosed herein (e.g., anti-FAM19A1 antibodies) are now effective in reducing elevated intraocular pressure observed, for example, in glaucoma subjects. However, in some respects, FAM19A1 antagonists do increase and / or improve retinal potentials in subjects (e.g., glaucoma patients).

[0208] In some respects, glaucoma includes: open-angle glaucoma, angle-closure glaucoma, normal-tension glaucoma (“NTG”), congenital glaucoma, secondary glaucoma, pigmentary glaucoma, pseudoexfoliative glaucoma, traumatic glaucoma, neovascular glaucoma, iridocorneal endothelial syndrome, uveitis-related glaucoma, or a combination thereof. In some respects, glaucoma is associated with: optic nerve damage, loss of retinal ganglion cells (“RGC”), high intraocular pressure (“IOP”), impaired blood-retinal barrier, and / or increased microglial cell activity in the retina and / or optic nerve of the subject.

[0209] In some aspects, methods of treating glaucoma further include: administering one or more adjunctive agents for treating glaucoma. In some aspects, said adjunctive agent is a prostaglandin analog, such as... In some respects, the additional agent is an alpha agonist, such as P and In some further aspects, the additional agent is a carbonic anhydrase inhibitor, such as... and This adjunctive agent can be administered before, simultaneously with, or after the administration of the FAM19A1 antagonist.

[0210] In some respects, impairment of CNS function is related to the sensory system, particularly touch. Therefore, in some respects, this disclosure provides a method for treating, preventing, or alleviating neuropathic pain in a subject in need, comprising administering a FAM19A1 antagonist to the subject.

[0211] In some respects, neuropathic pain is central neuropathic pain, which is pain caused by or impaired by any level of damage or impairment affecting the CNS (e.g., brain injury and spinal cord injury) (including the central somatosensory nervous system), or pain caused by or associated with a disease or disorder (such as stroke, multiple sclerosis, or lateral myelopathy). In some respects, central neuropathic pain can be spontaneous or irritation-induced. In some respects, central neuropathic pain can involve dynamic mechanical allodynia and cold allodynia. Symptoms of central neuropathic pain include: sensations such as burning, tingling, shooting, squeezing, cold, numbness, and sensory disturbances are common (e.g., tingling, needle-like, cold, and pressure sensations). The distribution of central neuropathic pain ranges from small to large areas, such as in the periorbital region, or covering half of the body in the case of a stroke, or covering the lower half of the body in the case of a spinal cord injury, or involving one side of the face and the opposite side of the body or limbs. Central nervous system pain caused by spinal cord injury includes "at-level" pain and "below-level" pain. The former is pain felt in a segmental pattern at the level of injury, while the latter is pain felt below the level of injury. In some respects, the methods described can reduce, reverse, alleviate, relieve, inhibit, or slow down or prevent central nervous system pathological pain, pain-related symptoms, the underlying cause of pain, or a combination thereof.

[0212] In some respects, neuropathic pain is peripheral neuropathic pain, meaning pain caused by damage or impairment of the peripheral nervous system at any level (e.g., damage to motor nerves, sensory nerves, autonomic nerves, or a combination thereof), or pain caused by or associated with disease or disorder. Damage or impairment of motor nerves is associated with a range of symptoms such as muscle weakness (e.g., weakness of muscles in the back, legs, hips, or face), painful spasms and fasciitis (uncontrolled muscle twitching visible under the skin), muscle atrophy (severe atrophy of muscle size), and decreased reflexes. Sensory nerve damage leads to a variety of symptoms, including pain and hypersensitivity of pain receptors in the skin, resulting in abnormal pain sensations (e.g., severe pain caused by normally painless stimuli).

[0213] In some aspects, the methods of treating one or more types of neuropathic pain include administering a FAM19A1 antagonist (e.g., an anti-FAM19A1 antibody) to a subject in need. In some aspects, neuropathic pain treatable by the methods disclosed herein is neuralgia, including but not limited to trigeminal neuralgia (TN) (e.g., pain in the trigeminal nerve region of the face or mouth), atypical trigeminal neuralgia (ATN), occipital neuralgia, postherpetic neuralgia (e.g., pain unilaterally distributed in one or more spinal dermatomes or trigeminal ophthalmic divisions), peripheral nerve injury pain (e.g., pain in the area innervated by the affected nerve, typically distal to trauma, surgery, or compression), glossopharyngeal neuralgia (e.g., irritation of the ninth cranial nerve causing extreme pain in the back of the throat, tongue, and ear), sciatica, lower back pain, and atypical facial pain. In some aspects, the neuralgia is caused by or related to chemical irritation, inflammation, trauma (including surgery), compression of the nerve by nearby structures (e.g., tumors), or infection. In some aspects, the neuropathic pain is a deafferentation pain syndrome, including but not limited to brain or spinal cord injury, post-stroke pain, phantom pain, paraplegia, brachial plexus avulsion injuries, and lumbar radiculopathies. In some aspects, neuropathic pain is a complex regional pain syndrome (CRPS), including CRPS1 and CRPS2. In some aspects, symptoms associated with CRPS may include severe pain, changes in the nails, bones, and skin; and increased sensitivity to touch in the affected limb. In some aspects, neuropathic pain is a neuropathy (e.g., central or peripheral). Non-limiting examples of neuropathic pain include, for example, mononeuropathy and polyneuropathy.

[0214] In some respects, neuropathic pain is caused by or related to bodily injury, which includes, for example, (1) traumatic injury or damage, including nerve compression (e.g., nerve squeezing, nerve stretching, nerve entrapment, or incomplete nerve transsection); (2) spinal cord injury (e.g., spinal cord hemisection); (3) injury or damage to peripheral nerves (e.g., motor nerves, sensory nerves, or autonomic nerves, or combinations thereof); (4) limb amputation; contusion; inflammation (e.g., myelitis); or surgery; and (5) repetitive stress, including, for example, repetitive, clumsy, and / or forceful activities requiring prolonged movement of any group of joints (e.g., ulnar neuropathy and carpal tunnel syndrome). In some respects, the method treats neuropathic pain caused by or related to exposure to toxic agents.

[0215] In some respects, neuropathic pain is caused by or associated with one or more diseases or disorders, including, for example, (1) ischemic events (e.g., stroke or heart attack), (2) multiple sclerosis, (3) metabolic and / or endocrine diseases or disorders (e.g., diabetes, metabolic diseases, and acromegaly, a disease caused by excessive secretion of growth hormone and characterized by abnormal enlargement of some skeletal parts, including joints, leading to nerve entrapment and pain), (4) small vessel diseases that cause reduced oxygen supply to peripheral nerves, resulting in damage to nerve tissue (e.g., vasculitis, i.e., vascular inflammation), (5) autoimmune diseases (e.g., Sjogren's syndrome, lupus, rheumatoid arthritis, and acute inflammatory demyelinating neuropathy, also known as Guillain-Barré syndrome), (6) kidney disease, and (7) cancer or tumors (e.g., neoplastic tumors). (8) Infections (e.g., viral infections such as herpes zoster, Epstein-Barr virus, West Nile virus, cytomegalovirus, and herpes simplex virus, HIV, or bacterial infections such as Lyme disease, diphtheria, and leprosy), (9) inflammatory diseases, (10) peripheral nerve diseases (e.g., neuroma), (11) genetic diseases, whether hereditary or aplastic (e.g., Charcot-Marie-Tooth disorders), (12) mononeuropathy, (13) polyneuropathy, or combinations thereof. In some respects, the neuropathic pain is caused by or associated with diabetes (type I or type II). In some respects, the neuropathic pain described is diabetic peripheral neuropathy.

[0216] In some respects, neuropathic pain is caused by or associated with exposure to infectious agents, including, for example, tick-borne infections, varicella-zoster virus, Epstein-Barr virus, West Nile virus, cytomegalovirus, herpes simplex virus, HIV, or toxic agents (e.g., drugs, alcohol, heavy metals (e.g., lead, arsenic, mercury)), or industrial agents (e.g., solvents, glue fumes) and nitrous oxide).

[0217] In some respects, neuropathic pain is caused by or related to: physical injury, infection, diabetes, cancer treatment, alcoholism, amputation, multiple sclerosis, shingles, spinal surgery, sciatica (pain along the sciatic nerve), lower back pain, neuralgia such as trigeminal neuralgia (e.g., pain in the trigeminal nerve region of the face or mouth), neuropathic pain such as painful polyneuropathy (e.g., foot pain that can extend to involve the calf, thigh, and hand), or a combination thereof. In some respects, neuropathic pain is trigeminal neuralgia. In some respects, neuropathic pain is associated with weakness in the muscles of the back, legs, hips, or face. In some respects, neuropathic pain is caused by compression of nerves (e.g., nerves in the legs, feet, hips, or facial muscles). In some respects, neuropathic pain includes sciatic nerve injury. In some respects, the aforementioned neuropathic pain is sciatica.

[0218] In some aspects, the methods of this disclosure can reverse, reduce, alleviate, inhibit, slow down, or prevent one or more symptoms associated with neuropathic pain. Therefore, in one aspect, this disclosure provides a method for improving hyperalgesia in a subject of need, comprising administering to the subject an antagonist against FAM19A1. As used herein, the term “hyperalgesia” refers to an increased or aggravated response to painful stimuli (e.g., a needle prick or a hot plate). In some aspects, the hyperalgesia is in response to mechanical stimuli, such as a needle prick (mechanical hyperalgesia). In other aspects, the hyperalgesia is in response to thermal stimuli, such as a hot plate (thermal hyperalgesia). In some aspects, the subject of need has chronic contractile injury (e.g., sciatica). In some aspects, the subject of need has diabetic peripheral neuropathy.

[0219] In some respects, administering a FAM19A1 antagonist to a subject in need enables the subject to have a higher threshold to mechanical stimulation compared to a reference control group (e.g., a neuropathic pain subject who has not received a FAM19A1 antagonist). As used herein, the term "threshold to mechanical stimulation" refers to the amount of pressure (from the mechanical stimulation) before the subject responds to the stimulus (e.g., withdraws). Thus, a subject with a higher threshold can tolerate or resist more mechanical stimulation compared to a subject with a lower threshold. In some respects, the methods disclosed herein can increase a subject's threshold to mechanical stimulation 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 the FAM19A1 antagonist).

[0220] In some aspects, administration of a FAM19A1 antagonist to a subject in need increases the subject's latency to a thermal stimulus (e.g., a hot plate) compared to a reference control group (e.g., a neuropathic pain subject who has not received a FAM19A1 antagonist). In some aspects, the methods disclosed herein can increase the subject's latency to a thermal stimulus 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 the FAM19A15 antagonist).

[0221] In another aspect, this disclosure provides a method for improving sensory nerve conduction velocity in subjects with need. The term “sensory nerve conduction velocity” (SNCV) refers to the speed at which an electrical signal travels through a peripheral nerve. Healthy nerves transmit electrical signals faster and more forcefully than damaged nerves. See Chouhan S., J Clin Diagn Res 10(1): CC01-3 (2016). Therefore, tests that assist in measuring SNCV (e.g., sensory nerve conduction velocity tests) can be used to identify potential nerve damage and / or dysfunction in subjects. In some aspects, the methods disclosed herein can increase the SNCV of subjects with neuropathic pain 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 the FAM19A1 antagonist).

[0222] In some respects, methods of treating neuropathic pain may further include: administering additional medications for treating neuropathic pain. Non-limiting example medications for treating neuropathic pain include venlafaxine. Antiepileptic drugs such as carbamazepine Gabapentin (FDA approved for relieving trigeminal neuralgia pain) Approved for the treatment of postherpetic neuralgia (PHN), pain lasting one to three months after shingles recovery, and pregabalin. Approved for the treatment of PHN, diabetic neuropathic pain, and fibromyalgia, sodium channel blocking agents such as lidocaine, adrenergic drugs such as clonidine, phentolamine, phenoxybenzamine, reserveine, dexmedetomidine, opioids such as morphine, and antidepressants such as amitriptyline, imipramine, and duloxetine.

[0223] The dosage and administration of the one or more additional therapeutic agents are known in the art, for example, as indicated on the product label of each agent.

[0224] In some respects, the subjects being treated are non-human animals, such as rats or mice. In other respects, the subjects being treated are humans.

[0225] Methods to regulate or improve the function of the central nervous system (CNS)

[0226] Without being bound by any theory, in some aspects, the FAM19A1 antagonists disclosed herein can treat diseases or disorders by reducing and / or inhibiting FAM19A1 activity. In some aspects, the reduced and / or inhibited FAM19A1 activity can improve one or more functions of the central nervous system. Therefore, in some aspects, what is disclosed herein is a method for modulating or improving one or more functions of the central nervous system of a subject in need, the method comprising administering a FAM19A1 antagonist to the subject. In some aspects, the FAM19A1 antagonists useful to this disclosure are: antisense oligonucleotides, siRNAs, shRNAs, miRNAs, dsRNAs, aptamers, PNAs, or vectors comprising the like that specifically target FAM19A1. In some aspects, the FAM19A1 antagonists comprise: anti-FAM19A1 antibodies, polynucleotides encoding said anti-FAM19A15 antibodies, or vectors comprising said polynucleotides.

[0227] As mentioned above, most tissues within the CNS are involved in specific functions (although there is some overlap), and these functions can be categorized into different groups. Therefore, in some respects, central nervous system functions include: limbic system-related functions, olfactory system-related functions, sensory system-related functions, visual system-related functions, or combinations thereof.

[0228] As used herein, the term "limbic system related function" refers to activities associated with the limbic system. The term "limbic system" refers to the part of the brain that processes three key functions: emotion, memory, and arousal (or stimulation). In some respects, the limbic system includes the following brain regions: olfactory bulb, hippocampus, hypothalamus, amygdala, anterior thalamic nucleus, fornix, fornix column, mammillary body, septum pellucidum, hippocampal process, cingulate gyrus, parahippocampal gyrus, entorhinal cortex, pineal habenula, and limbic mesoencephal regions.

[0229] As used in this article, the term “olfactory system-related functions” refers to activities related to the olfactory system, which is the part of the sensory system used for smelling (olfaction).

[0230] As used herein, the term "sensory system-related function" refers to activities associated with the sensory system. The sensory system includes sensory neurons (including sensory receptor cells), neural pathways, and parts of the brain involved in sensory perception. In some respects, sensory system-related functions include hearing, touch, taste, balance, or combinations thereof.

[0231] As used herein, the term "visual system-related functions" refers to activities related to vision. The term "visual system" refers to a portion of the central nervous system involved in processing visual details (e.g., by detecting and interpreting information from visible light), and the formation of several non-image-related light response functions (e.g., pupillary light reflex (PLR) and circadian photoentrainment). The visual system performs complex tasks including: receiving light and forming monocular representations; establishing binocular perception from a pair of two-dimensional projections; identifying and classifying visual objects; assessing distances to and between objects; and guiding body movements associated with seen objects.

[0232] In conjunction with this disclosure, it has been shown that FAM19A1 is expressed in certain brain regions related to CNS function as disclosed herein. See, for example, Example 8. Without wishing to be bound by any particular mechanism or theory, aberrant expression of FAM19A1 may be a cause of CNS dysfunction.

[0233] In some respects, the 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 the CNS functions disclosed herein). In some respects, 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, pineal habenula, or combinations thereof.

[0234] In some respects, FAM19A1 antagonists (e.g., anti-FAM19A1 antibodies) reduce FAM19A1 protein expression and / or FAM19A1 mRNA expression in the retinal region. In some respects, the retinal region includes the ganglion cell layer (GCL) or the internal plexiform layer (INL).

[0235] In some respects, FAM19A1 antagonists (e.g., anti-FAM19A1 antibodies) reduce FAM19A1 protein expression and / or FAM19A1 mRNA expression in spinal cord regions. In some respects, these spinal regions include the dorsal horn.

[0236] In some aspects, following administration of a FAM19A1 antagonist (e.g., an anti-FAM19A1 antibody) disclosed herein, 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 reference values ​​(e.g., corresponding values ​​in subjects who have not received the FAM19A1 antagonist or corresponding values ​​in subjects before administration of the FAM19A1 antagonist). In some aspects, the reduced FAM19A1 protein expression and / or FAM19A1 mRNA expression is associated with improved CNS function.

[0237] Methods to regulate, induce, or increase neuronal differentiation

[0238] Neural stem cells (NSCs) possess the ability to continuously divide (i.e., self-renew) and differentiate into neurons, astrocytes, and oligodendrocytes in the central nervous system. The differentiation into neurons primarily occurs during the embryonic period, but the 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).

[0239] A balance in the number of neurons and glial cells (such as astrocytes) is crucial for maintaining normal CNS function. While astrocytes do have certain beneficial functions (e.g., providing structural support for neurons, secreting nerve growth factors, and helping to maintain the blood-brain barrier), excessive astrocyte formation can hinder neuronal regeneration and lead to inflammation-mediated damage to CNS tissues. See Myer et al., Brain 129:2761-2772 (2006); Chen and Swanson, JCereb Blood Flow Metab 23:137-149 (2003); Cunningham et al., Brain 128:1931-1942 (2005); and Faden, Curr Opin Neurol 15:707-712 (2002); see also USPub. No. 2015 / 0118230, the entire contents of which are incorporated herein by reference.

[0240] Gliosis is a phenomenon commonly occurring in various pathological processes of the central nervous system, caused by the excessive proliferation and activation of astrocytes due to neuronal damage. When the central nervous system is damaged, normal astrocytes become hypertrophic reactive astrocytes—which increase the production of an intermediate filament protein called glial fibrillary acidic protein (GFAP). Various glial cells, including reactive astrocytes, proliferate excessively after injury, forming a solid layer of cells called glialscar, a product of the healing process. This gliosis can be observed in degenerative brain diseases (including Huntington's disease, Parkinson's disease, and Alzheimer's disease), spinal cord injuries, and various pathological phenomena of the central nervous system (such as 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).

[0241] Therefore, without being bound by any particular mechanism or theory, CNS function can be improved by promoting and / or regulating the differentiation of neurons (e.g., from neural stem cells). Thus, the present invention provides a method for regulating, inducing, or increasing the differentiation and / or maturation of neurons in a desired subject, comprising administering a FAM19A1 antagonist (e.g., an anti-FAM19A1 antibody) to the subject. In some aspects, the FAM19A1 antagonist increases the outward growth of neurites of differentiated neural stem cells (i.e., neurons) compared to a reference value (e.g., a corresponding value in a subject who has not received the FAM19A1 antagonist or a corresponding value in a subject prior to the administration of the FAM19A1 antagonist). In some aspects, the outward growth of neurites 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 reference value.

[0242] Methods for diagnosing central nervous system (CNS) dysfunction

[0243] This article discloses a method for diagnosing CNS dysfunction in subjects in need, comprising 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. This article also discloses a method for identifying subjects with central nervous system dysfunction, comprising contacting a sample of the subject with a FAM19A1 antagonist and measuring the FAM19A1 protein level or FAM19A1 mRNA level in the sample.

[0244] The term "abnormality of central nervous system function" refers to the inability (or decline) to perform one or more functions related to the CNS. In some respects, central nervous system function includes: limbic system-related functions, olfactory system-related functions, sensory system-related functions, visual system-related functions, or combinations thereof.

[0245] As described in other parts of this disclosure, many diseases or disorders affecting the CNS are associated with some degree of CNS dysfunction (e.g., a primary symptom associated with glaucoma is visual impairment). Therefore, in some respects, the methods disclosed herein can also be used to diagnose and / or identify subjects with CNS-related diseases or disorders. Non-limiting examples of such diseases or disorders include: glaucoma, neuropathic pain, addiction, arachnoid cysts, attention deficit hyperactivity disorder (ADHD), autism, bipolar disorder, hypnosis, depression, encephalitis, epilepsy / seizures, locked-in syndrome, meningitis, migraine, multiple sclerosis, myelopathy, Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Barten's disease, tic disorders, traumatic brain injury, post-traumatic stress disorder (PTSD), spinal cord injury, stroke, tremor (primary or Parkinson's disease), dystonia, schizophrenia, intellectual disability, and brain tumors. In some respects, abnormalities in the function of the central nervous system are associated with glaucoma or neuropathic pain.

[0246] In some aspects, methods for diagnosing and / or identifying subjects with central nervous system dysfunction include administering a FAM19A1 antagonist to the subject prior to the measurement, such that contact between the FAM19A1 antagonist and FAM19A1 occurs in vivo. In some aspects, both the contact and the measurement are performed in vitro.

[0247] In some aspects, the abnormality of central nervous system function is associated with an increase in the FAM19A1 protein level and / or the FAM19A1 mRNA level in the sample compared to the reference value (e.g., the corresponding value in a sample from a subject without central nervous system dysfunction (e.g., a healthy subject)). In some aspects, the increase in the FAM19A1 protein level and / or the FAM19A1 mRNA level compared to the reference value is 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.

[0248] In some respects, samples from subjects with central nervous system dysfunction showed increases in FAM19A1 protein levels and / or FAM19A1 mRNA levels of at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 15-fold, at least 20-fold, at least 25-fold, or at least 30-fold compared to reference values ​​(e.g., the corresponding values ​​in samples from subjects without central nervous system dysfunction, such as healthy subjects).

[0249] In some aspects, the abnormality of central nervous system function is associated with a decrease in the FAM19A1 protein level and / or the FAM19A1 mRNA level in the sample compared to a reference value (e.g., the corresponding value in a sample from a subject without central nervous system dysfunction (e.g., a healthy subject)). In some aspects, the FAM19A1 protein level and / or the FAM19A1 mRNA level is 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 reference value.

[0250] In some respects, samples from subjects with central nervous system dysfunction showed a decrease in FAM19A1 protein levels and / or FAM19A1 mRNA levels of at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 15-fold, at least 20-fold, at least 25-fold, or at least 30-fold compared to reference values ​​(e.g., the corresponding values ​​in samples from subjects without central nervous system dysfunction, such as healthy subjects).

[0251] In some respects, FAM19A1 protein levels are measured using the following methods: immunohistochemistry, Western blot, radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), radioimmunodiffusion, immunoprecipitation assay, Ouchterlony immunodiffusion method, rocket immunoelectrophoresis, tissue immunostaining method, complement fixation assay, FACS, protein chip, or combinations thereof. In some respects, FAM19A1 mRNA levels are measured using the following methods: reverse transcription polymerase chain reaction (RT-PCR), real-time polymerase chain reaction (RT-PCR), Northern blot, or combinations thereof. In some respects, FAM19A1 protein levels are measured using the assay of FAM19A1 antagonists (e.g., anti-FAM19A1 antibodies) disclosed herein.

[0252] In some respects, samples (e.g., in which FAM19A1 protein levels and / or FAM19A1 mRNA levels are measured) include: tissues, cells, blood, serum, plasma, saliva, urine, cerebrospinal fluid (CSF), or combinations thereof.

[0253] In some respects, methods for diagnosing and / or identifying subjects with central nervous system dysfunction further include administering a FAM19A1 antagonist to the subject if the FAM19A1 protein level and / or FAM19A1 mRNA level are increased compared to reference values ​​(e.g., corresponding values ​​in samples from subjects without central nervous system dysfunction (e.g., healthy subjects)).

[0254] In some respects, methods for diagnosing and / or identifying subjects with central nervous system dysfunction further include administering a FAM19A1 agonist to the subject if the FAM19A1 protein level and / or FAM19A1 mRNA level decreases compared to a reference value (e.g., the corresponding value in a sample of a subject without central nervous system dysfunction (e.g., a healthy subject)).

[0255] In some aspects, FAM19A1 agonists comprise the FAM19A1 protein. In some aspects, FAM19A1 antagonists comprise: an anti-FAM19A1 antibody, a polynucleotide encoding said anti-FAM19A1 antibody, a vector containing said polynucleotide, a cell containing said polynucleotide, or any combination thereof. In some aspects, FAM19A1 antagonists comprise: an antisense oligonucleotide, siRNA, shRNA, miRNA, dsRNA, aptamer, PNA, or a vector comprising the thereof that specifically targets FAM19A1. In some aspects, FAM19A1 antagonists are anti-FAM19A1 antibodies.

[0256] III. FAM19A1 antagonists

[0257] One or more FAM19A1 antagonists may be used in conjunction with this method. In some aspects, the FAM19A1 antagonist is: an antisense oligonucleotide, siRNA, shRNA, miRNA, dsRNA, aptamer, PNA (peptide nucleic acid), or a vector comprising thereof that specifically targets FAM19A1. In some aspects, the FAM19A1 antagonist is: an anti-FAM19A1 antibody, a polynucleotide encoding said anti-FAM19A1 antibody, or a vector comprising said polynucleotide.

[0258] 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 specifically binding to soluble and / or membrane-bound human FAM19A1, the antibodies described herein also: (a) at 10 nM or less K D (b) binds to soluble human FAM19A1; and (c) in 10 nM or less K D The membrane-bound human FAM19A1 is bound; or both (a) and (b).

[0259] In some respects, the anti-FAM19A1 antibodies disclosed herein bind specifically to soluble human FAM19A1 or membrane-bound human FAM19A1 with high affinity, said high affinity being, for example, K... D 10 -7 M or smaller, 10 -8M (10nM) or smaller, 10 - 9 M (1nM) or smaller, 10 -10 M (0.1nM) or smaller, 10 -11 M or smaller, or 10 -12 M or smaller, such as 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, 5X 10 -12 M, 10 -11 M, 5X 10 -11 M, 10 - 10 M, 5X10 -10 M, 10 -9 M, 5X 10 -9 M, 10 -8 M, 5X 10 -8 M, 10 -7 M, or 5 x 10 -7 M. Standard assays for assessing the binding ability of the antibody to human FAM19A1 of various species are known in the art, including, for example, ELISAs, Western ink dot assays, and RIAs. Suitable assays are described in detail in the examples. The binding kinetics of the antibody (e.g., binding affinity) can also be determined by standard assays known in the art (e.g., by ELISA, Biacore™ assay, or...). To evaluate.

[0260] In some respects, the anti-FAM19A1 antibody disclosed in this article uses K D This K binds to soluble human FAM19A1. D (For example, as measured by ELISA) is 10 -7 M or smaller, 10 -8 M (10nM) or smaller, 10 -9 M (1nM) or smaller, 10 -10 M or smaller, 10 -12 M to 10 -7 M, 10 -11 M to 10 -7 M, 10 -10 M to 10 -7 M, 10 -9 M to 10 -7 M, or 10 -8M to 10 -7 M. In some respects, anti-FAM19A1 antibodies at 10 nM or less K D When combined with soluble FAM19A1, this 10 nM or smaller K D For example, concentrations between 0.1 and 10 nM, between 0.1 and 5 nM, between 0.1 and 1 nM, between 0.5 and 10 nM, between 0.5 and 5 nM, between 0.5 and 1 nM, between 1 and 10 nM, between 1 and 5 nM, or between 5 and 10 nM. In some respects, anti-FAM19A1 antibodies are K D This K specifically binds to soluble human FAM19A1. D Approximately 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 approximately 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, or 9 nM, or approximately 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, or 90 nM, as determined by ELISA.

[0261] In some respects, anti-FAM19A1 antibodies use K D The K-cell receptor binds to human FAM19A1 bound to the membrane. D (For example, as determined by ELISA) is 10 -7 M or smaller, 10 -8 M (10nM) or smaller, 10 -9 M (1nM) or smaller, 10 -10 M or smaller, 10 -12 M to 10 -7 M, 10 -11 M to 10 -7 M, 10 -10 M to 10 -7 M, 10 -9 M to 10 -7 M, or 10 -8 M to 10 -7 M. In some respects, anti-FAM19A1 antibodies at (as determined by ELISA) 10 nM or less K D Human FAM19A1 specifically binds to membrane-bound K+, which is 10 nM or smaller. DFor example, between 0.1 and 10 nM, between 0.1 and 5 nM, between 0.1 and 1 nM, between 0.5 and 10 nM, between 0.5 and 5 nM, between 0.5 and 1 nM, between 1 and 10 nM, between 1 and 5 nM, or between 5 and 10 nM. In some respects, the anti-FAM19A1 antibody or its antigen-binding portion uses K... D The K-cell receptor binds to human FAM19A1 bound to the membrane. D Approximately 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 approximately 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, or 9 nM, or approximately 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, or 90 nM, as determined by ELISA.

[0262] In addition to the above, FAM19A1 antagonists (e.g., anti-FAM19A1 antibodies) exhibit one or more of the following functional properties:

[0263] (1) Promotes neuronal differentiation;

[0264] (2) Increase the outward growth of neurites in differentiated neurons;

[0265] (3) Reduce, reverse and / or prevent one or more symptoms associated with glaucoma;

[0266] (4) Improves retinal potentials (e.g., as demonstrated by an increase in oscillatory potentials);

[0267] (5) Reduce and / or restore the loss of retinal ganglion cells (e.g., as observed in glaucoma subjects);

[0268] (6) Reduce, reverse and / or prevent one or more symptoms associated with neuropathic pain;

[0269] (7) Increase the latency and / or threshold to external stimuli; and

[0270] (8) Increase and / or regulate the conduction velocity of sensory nerves.

[0271] Other functional properties of the anti-FAM19A1 antibody disclosed herein are provided throughout this application.

[0272] In some respects, the anti-FAM19A1 antibodies disclosed herein cross-competitive with anti-FAM19A1 antibodies containing the variable regions (e.g., 1C1, 1A11, 2G7, and 3A8) and CDRs disclosed herein to bind (or inhibit binding) human FAM19A1 epitopes.

[0273] In some aspects, the anti-FAM19A1 antibody of this disclosure inhibits the binding of a reference antibody comprising heavy chains CDR1, CDR2, and CDR3 and light chains CDR1, CDR2, and CDR3, (i) wherein the heavy chains CDR1, CDR2, and CDR3 of the reference antibody each comprise SEQ ID NOs: 10-12, and the light chains CDR1, CDR2, and CDR3 of the reference antibody each comprise the amino acid sequences listed in SEQ ID NOs: 13-15; (ii) wherein the heavy chains CDR1, CDR2, and CDR3 of the reference antibody each comprise the amino acid sequences shown in SEQ ID NOs: 4-6, and the light chains CDR1, CDR2, and CDR3 of the reference antibody each comprise the amino acid sequences shown in SEQ ID NOs: 7-9; (iii) wherein the heavy chains CDR1, CDR2, and CDR3 of the reference antibody each comprise the amino acid sequences shown in SEQ ID NOs: 10-12. (iv) wherein the amino acid sequences shown in NOs: 16-18, and the light chains CDR1, CDR2 and CDR3 of the reference antibody comprise the amino acid sequences shown in SEQ ID NO: 19-21; or (iv) wherein the heavy chains CDR1, CDR2 and CDR3 of the reference antibody each comprise the amino acid sequences shown in SEQ ID NO: 22-24, and the light chains CDR1, CDR2 and CDR3 of the reference antibody each comprise the amino acid sequences shown in SEQ ID NO: 25-27.

[0274] In some aspects, the reference antibody comprises: (a) a heavy and light chain variable region comprising the amino acid sequences shown in SEQ ID NOs: 30 and 31, respectively; (b) a heavy and light chain variable region comprising the amino acid sequences shown in SEQ ID NOs: 28 and 29, respectively; (c) a heavy and light chain variable region comprising the amino acid sequences shown in SEQ ID NOs: 32 and 33, respectively; or (d) a heavy and light chain variable region comprising the amino acid sequences shown in SEQ ID NOs: 34 and 35, respectively.

[0275] In some respects, the anti-FAM19A1 antibodies disclosed herein exhibit inhibition rates of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% against the binding of this reference antibody to human FAM19A1. Competitive antibodies bind to the same epitope, overlapping epitopes, or adjacent epitopes (e.g., as demonstrated by steric hindrance). Whether two antibodies compete with each other to bind to the target can be determined using competition experiments known in the art (such as RIA and EIA).

[0276] In some respects, the anti-FAM19A1 antibody binds to the same FAM19A1 epitope as the reference antibody disclosed herein, the reference antibody comprising heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3, (i) wherein the heavy chain CDR1 comprises the amino acid sequence shown in SEQ ID NO: 10, the heavy chain CDR2 comprises the amino acid sequence shown in SEQ ID NO: 11, the heavy chain CDR3 comprises the amino acid sequence shown in SEQ ID NO: 12, the light chain CDR1 comprises the amino acid sequence shown in SEQ ID NO: 13, the light chain CDR2 comprises the amino acid sequence shown in SEQ ID NO: 14, and the light chain CDR3 comprises the amino acid sequence shown in SEQ ID NO: 15; (ii) wherein the heavy chain CDR1 comprises the amino acid sequence shown in SEQ ID NO: 4, the heavy chain CDR2 comprises the amino acid sequence shown in SEQ ID NO: 5, the heavy chain CDR3 comprises the amino acid sequence shown in SEQ ID NO: 6, the light chain CDR1 comprises the amino acid sequence shown in SEQ ID NO: 7, and the light chain CDR2 comprises the amino acid sequence shown in SEQ ID NO: 6. (iii) wherein the heavy chain CDR1 comprises the amino acid sequence shown in SEQ ID NO: 8, the heavy chain CDR2 comprises the amino acid sequence shown in SEQ ID NO: 17, the heavy chain CDR3 comprises the amino acid sequence shown in SEQ ID NO: 18, the light chain CDR1 comprises the amino acid sequence shown in SEQ ID NO: 19, the light chain CDR2 comprises the amino acid sequence shown in SEQ ID NO: 20, and the light chain CDR3 comprises the amino acid sequence shown in SEQ ID NO: 21; or (iv) wherein the heavy chain CDR1 comprises the amino acid sequence shown in SEQ ID NO: 22, the heavy chain CDR2 comprises the amino acid sequence shown in SEQ ID NO: 23, the heavy chain CDR3 comprises the amino acid sequence shown in SEQ ID NO: 24, the light chain CDR1 comprises the amino acid sequence shown in SEQ ID NO: 25, the light chain CDR2 comprises the amino acid sequence shown in SEQ ID NO: 26, and the light chain CDR3 comprises the amino acid sequence shown in SEQ ID NO: 27. In some aspects, the reference antibody comprises: (i) a heavy chain variable domain comprising the amino acid sequence shown in SEQ ID NOs: 30, 28, 32 or 34, and (ii) a light chain variable domain comprising the amino acid sequence shown in SEQ ID NOs: 31, 29, 33 or 35.

[0277] In some aspects, the reference antibody comprises: (a) a heavy and light chain variable region comprising the amino acid sequences shown in SEQ ID NOs: 30 and 31, respectively; (b) a heavy and light chain variable region comprising the amino acid sequences shown in SEQ ID NOs: 28 and 29, respectively; (c) a heavy and light chain variable region comprising the amino acid sequences shown in SEQ ID NOs: 32 and 33, respectively; or (d) a heavy and light chain variable region comprising the amino acid sequences listed in SEQ ID NOs: 34 and 35, respectively.

[0278] 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, which provides atomic resolution and hydrogen / deuterium exchange mass spectrometry (HDX-MS) of the epitope; methods for monitoring the binding of antibodies to antigen fragments or antigen mutants, wherein binding loss due to modification of amino acid residues in the antigen sequence is generally considered an indicator of epitope components; and computational combinatorial methods for epitope mapping.

[0279] The anti-FAM19A1 antibody disclosed herein can bind to at least one epitope of mature human FAM19A1, as determined, for example, by binding of the antibody to a fragment of human FAM19A1. In some aspects, the anti-FAM19A1 antibody binds to at least one epitope selected from the group consisting of: D112, M117, A119, T120, N122, and combinations thereof.

[0280] In some respects, this document provides an anti-FAM19A1 antibody with an affinity for FAM19A1 that is 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or higher than its affinity for another protein in the FAM19A family, as measured by, for example, immunoassays (e.g., ELISA), surface plasmon resonance, or kinetic exclusion assays. In some respects, the anti-FAM19A1 antibody disclosed herein does not cross-react with another protein in the FAM19A family, as measured by, for example, immunoassays (e.g., ELISA), surface plasmon resonance, or kinetic exclusion assays.

[0281] In some respects, the anti-FAM19A1 antibody is not a native antibody or is not a naturally occurring antibody. For example, in some respects, the anti-FAM19A1 antibody disclosed herein has post-translational modifications that differ from those of naturally occurring antibodies, such as having more, fewer, or different types of post-translational modifications.

[0282] IV. Example of an anti-FAM19A1 antibody

[0283] Specific antibodies that can be used in the methods disclosed herein are: antibodies having the CDR and / or variable region sequences disclosed herein (e.g., monoclonal antibodies), and antibodies having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or at least 99% identity) with respect to their variable region or CDR sequence. Tables 6 and 7 provide the VH and VL amino acid sequences of different anti-FAM19A1 antibodies of this disclosure, respectively.

[0284] Table 4. Variable heavy chain CDR amino acid sequences (based on the IMGT system)

[0285]

[0286] Table 5. Variable light chain CDR amino acid sequences (based on the IMGT system)

[0287]

[0288] Table 6. Variable Heavy Chain Amino Acid Sequences

[0289]

[0290] Table 7. Variable light chain amino acid sequences

[0291]

[0292] In some aspects, the anti-FAM19A1 antibody of this disclosure includes heavy and light chain variable regions, wherein the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 30, 28, 32 or 34. In some aspects, the anti-FAM19A1 antibody of this disclosure includes CDRs of the heavy chain variable region selected from the group consisting of SEQ ID NOs: 30, 28, 32 and 34.

[0293] In some aspects, the anti-FAM19A1 antibodies disclosed herein include heavy and light chain variable regions, wherein the light chain variable regions include the amino acid sequences shown in SEQ ID NO: 31, 29, 33, or 35. In some aspects, the anti-FAM19A1 antibodies disclosed herein include CDRs of the light chain variable regions selected from the group consisting of SEQ ID NOs: 31, 29, 33, and 35.

[0294] In some respects, the anti-FAM19A1 antibody includes: CDRs of the heavy chain variable region selected from the group consisting of SEQ ID NOs: 30, 28, 32 and 34, and CDRs of the light chain variable region selected from the group consisting of SEQ ID NOs: 31, 29, 33 and 35.

[0295] In some aspects, an anti-FAM19A1 antibody includes heavy and light chain variable regions, (i) wherein the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 30, and wherein the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 31; (ii) wherein the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 28, and wherein the light chain variable region includes the amino acid sequence listed in SEQ ID NO: 29; (iii) wherein the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 32, and wherein the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 33; and (iv) wherein the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 34, and wherein the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 35.

[0296] In some aspects, the anti-FAM19A1 antibody includes heavy and light chain variable regions, wherein the heavy chain variable region includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence listed in SEQ ID NO: 30, 28, 32, or 34.

[0297] In some aspects, the anti-FAM19A1 antibody includes heavy and light chain variable regions, wherein the light chain variable regions comprise 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 listed in SEQ ID NO: 31, 29, 33, or 35.

[0298] In some aspects, the anti-FAM19A1 antibody includes heavy and light chain variable regions, wherein the heavy chain variable region comprises at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% of the amino acid sequence listed in SEQ ID NO: 30, 28, 32, or 34, and wherein the light chain variable region comprises at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% of the amino acid sequence listed in SEQ ID NO: 31, 29, 33, or 35.

[0299] In some respects, an anti-FAM19A1 antibody includes:

[0300] (a) The heavy and light chain variable regions, which respectively include the amino acid sequences shown in SEQ ID NOs: 30 and 31;

[0301] (b) Heavy and light chain variable regions, which respectively include the amino acid sequences shown in SEQ ID NOs: 28 and 29;

[0302] (c) Heavy and light chain variable regions, comprising the amino acid sequences shown in SEQ ID NOs: 32 and 33, respectively; or

[0303] (d) The heavy and light chain variable regions, which include the amino acid sequences shown in SEQ ID NOs: 34 and 35, respectively.

[0304] In some aspects, the anti-FAM19A1 antibody disclosed herein comprises: (i) the heavy chains CDR1, CDR2 and CDR3 of 1C1, or combinations thereof, and / or the light chains CDR1, CDR2 and CDR3 of 1C1, or any combination thereof; (ii) the heavy chains CDR1, CDR2 and CDR3 of 1A11, or combinations thereof, and / or the light chains CDR1, CDR2 and CDR3 of 1A11, or any combination thereof; (iii) the heavy chains CDR1, CDR2 and CDR3 of 2G7, or combinations thereof, and / or the light chains CDR1, CDR2 and CDR3 of 2G7, or any combination thereof; or (iv) the heavy chains CDR1, CDR2 and CDR3 of 3A8, or combinations thereof, and / or the light chains CDR1, CDR2 and CDR3 of 3A8, or any combination thereof. Table 4 provides the amino acid sequences of VH CDR1, CDR2, and CDR3 of the different anti-FAM19A1 antibodies disclosed herein. Table 5 provides the amino acid sequences of VL CDR1, CDR2, and CDR3 of the different anti-FAM19A1 antibodies disclosed herein.

[0305] In some respects, an antibody against FAM19A1 that specifically binds to human FAM19A1 includes:

[0306] (a) VH CDR1 containing the amino acid sequence listed in SEQ ID NO: 10;

[0307] (b) VH CDR2 containing the amino acid sequence listed in SEQ ID NO: 11; and / or

[0308] (c) VH CDR3 containing the amino acid sequence listed in SEQ ID NO: 12.

[0309] In some respects, an antibody may include one, two, or all three of the aforementioned VH CDRs.

[0310] In some respects, an anti-FAM19A1 antibody that specifically binds to human FAM19A1 includes:

[0311] (a) VL CDR1 containing the amino acid sequence listed in SEQ ID NO: 13;

[0312] (b) VL CDR2 containing the amino acid sequence listed in SEQ ID NO: 14; and / or

[0313] (c) VL CDR3 containing the amino acid sequence described in SEQ ID NO: 15.

[0314] In some respects, an antibody may include one, two, or all three of the aforementioned VL CDRs.

[0315] In some respects, an antibody against FAM19A1 that specifically binds to human FAM19A1 includes:

[0316] (a) VH CDR1 containing the amino acid sequence listed in SEQ ID NO: 10;

[0317] (b) VH CDR2 containing the amino acid sequence listed in SEQ ID NO: 11;

[0318] (c) VH CDR3 containing the amino acid sequence listed in SEQ ID NO: 12;

[0319] (d) VL CDR1 containing the amino acid sequence described in SEQ ID NO: 13;

[0320] (e) VL CDR2 containing the amino acid sequence listed in SEQ ID NO: 14; and / or

[0321] (f) VL CDR3 containing the amino acid sequence listed in SEQ ID NO: 15.

[0322] In some respects, an antibody against FAM19A1 that specifically binds to human FAM19A1 includes:

[0323] (a) VH CDR1 containing the amino acid sequence listed in SEQ ID NO: 4;

[0324] (b) VH CDR2 containing the amino acid sequence listed in SEQ ID NO: 5; and / or

[0325] (c) VH CDR3 containing the amino acid sequence listed in SEQ ID NO: 6.

[0326] In some respects, an antibody may include one, two, or all three of the aforementioned VH CDRs.

[0327] In some respects, an antibody against FAM19A1 that specifically binds to human FAM19A1 includes:

[0328] (a) VL CDR1 containing the amino acid sequence listed in SEQ ID NO: 7;

[0329] (b) VL CDR2 containing the amino acid sequence listed in SEQ ID NO: 8; and / or

[0330] (c) VL CDR3 containing the amino acid sequence listed in SEQ ID NO: 9.

[0331] In some respects, an antibody may include one, two, or all three of the aforementioned VL CDRs.

[0332] In some respects, an antibody against FAM19A1 that specifically binds to human FAM19A1 includes:

[0333] (a) VH CDR1 containing the amino acid sequence listed in SEQ ID NO: 4;

[0334] (b) VH CDR2 containing the amino acid sequence listed in SEQ ID NO: 5;

[0335] (c) VH CDR3 containing the amino acid sequence listed in SEQ ID NO: 6;

[0336] (d) VL CDR1 containing the amino acid sequence listed in SEQ ID NO: 7;

[0337] (e) VL CDR2 containing the amino acid sequence listed in SEQ ID NO: 8; and / or

[0338] (f) VL CDR3 containing the amino acid sequence described in SEQ ID NO: 9.

[0339] In some respects, an antibody against FAM19A1 that specifically binds to human FAM19A1 includes:

[0340] (a) VH CDR1 containing the amino acid sequence listed in SEQ ID NO: 16;

[0341] (b) VH CDR2 containing the amino acid sequence listed in SEQ ID NO: 17; and / or

[0342] (c) VH CDR3 containing the amino acid sequence listed in SEQ ID NO: 18.

[0343] In some respects, an antibody may include one, two, or all three of the aforementioned VH CDRs.

[0344] In some respects, an antibody against FAM19A1 that specifically binds to human FAM19A1 includes:

[0345] (a) VL CDR1 containing the amino acid sequence listed in SEQ ID NO: 19;

[0346] (b) VL CDR2 containing the amino acid sequence listed in SEQ ID NO: 20; and / or

[0347] (c) VL CDR3 containing the amino acid sequence described in SEQ ID NO: 21.

[0348] In some respects, an antibody may include one, two, or all three of the aforementioned VL CDRs.

[0349] In some respects, an anti-FAM19A1 antibody that specifically binds to human FAM19A1 includes:

[0350] (a) VH CDR1 containing the amino acid sequence listed in SEQ ID NO: 16;

[0351] (b) VH CDR2 containing the amino acid sequence listed in SEQ ID NO: 17;

[0352] (c) VH CDR3 containing the amino acid sequence listed in SEQ ID NO: 18;

[0353] (d) VL CDR1 containing the amino acid sequence described in SEQ ID NO: 19;

[0354] (e) VL CDR2 containing the amino acid sequence listed in SEQ ID NO: 20; and / or

[0355] (f) VL CDR3 containing the amino acid sequence listed in SEQ ID NO: 21.

[0356] In some respects, an anti-FAM19A1 antibody that specifically binds to human FAM19A1 includes:

[0357] (a) VH CDR1 containing the amino acid sequence listed in SEQ ID NO: 22;

[0358] (b) VH CDR2 containing the amino acid sequence listed in SEQ ID NO: 23; and / or

[0359] (c) VH CDR3 containing the amino acid sequence listed in SEQ ID NO: 24.

[0360] In some respects, an antibody may include one, two, or all three of the aforementioned VH CDRs.

[0361] In some respects, an antibody against FAM19A1 that specifically binds to human FAM19A1 includes:

[0362] (a) VL CDR1 containing the amino acid sequence listed in SEQ ID NO: 25;

[0363] (b) VL CDR2 containing the amino acid sequence listed in SEQ ID NO: 26; and / or

[0364] (c) VL CDR3 containing the amino acid sequence listed in SEQ ID NO: 27.

[0365] In some respects, an antibody may include one, two, or all three of the aforementioned VL CDRs.

[0366] In some respects, an antibody against FAM19A1 that specifically binds to human FAM19A1 includes:

[0367] (a) VH CDR1 containing the amino acid sequence listed in SEQ ID NO: 22;

[0368] (b) VH CDR2 containing the amino acid sequence listed in SEQ ID NO: 23;

[0369] (c) VH CDR3 containing the amino acid sequence listed in SEQ ID NO: 24;

[0370] (d) VL CDR1 containing the amino acid sequence described in SEQ ID NO: 25;

[0371] (e) VL CDR2 containing the amino acid sequence listed in SEQ ID NO: 26; and / or

[0372] (f) VL CDR3 containing the amino acid sequence listed in SEQ ID NO: 27.

[0373] The VH domain or one or more of its CDRs described herein can be linked to a constant domain to form a heavy chain, such as a full-length heavy chain. Similarly, the VL domain or one or more of its CDRs described herein can be linked to a constant domain to form a light chain, such as a full-length light chain. Full-length heavy chains and full-length light chains combine to form full-length antibodies.

[0374] Accordingly, in some aspects, the anti-FAM19A1 antibody comprises an antibody light chain and a heavy chain, for example, separate light and heavy chains. Regarding the light chain, in some aspects, the light chain of the antibody described herein is a kappa light chain. In some aspects, the light chain of the antibody described herein is a lambda light chain. In some aspects, the light chain of the antibody described herein is a human kappa light chain or a human lambda light chain. In some aspects, the antibody described herein that specifically binds to a FAM19A1 polypeptide (e.g., human FAM19A1) comprises a light chain that comprises any VL or VL CDR amino acid sequence described herein, and wherein the constant region of said light chain comprises the amino acid sequence of the constant region of a human kappa light chain. In some aspects, the antibody described herein that specifically binds to a FAM19A1 polypeptide (e.g., human FAM19A1) comprises a light chain that comprises the VL or VL CDR amino acid sequence described herein, and wherein the constant region of said light chain comprises the amino acid sequence of the constant region of a human lambda light chain. Non-limiting examples of human constant region sequences have been described in the art, for example, see U.S. Patent No. 5,693,780 and Kabat EA et al., (1991), ibid.

[0375] Regarding the heavy chain, in some aspects, 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 aspects, the heavy chain of the antibody may include a human alpha (α), delta (δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In some aspects, the antibody described herein that specifically binds to FAM19A1 (e.g., human FAM19A1) comprises a heavy chain that includes the VH or VH CDR amino acid sequence described herein, and wherein the constant region of the heavy chain includes the amino acid sequence of the constant region of a human gamma (γ) heavy chain. In some aspects, the antibody described herein that specifically binds to FAM19A1 (e.g., human FAM19A1) comprises a heavy chain that includes the VH or VH CDR amino acid sequence disclosed herein, and wherein the constant region of the heavy chain includes amino acids of human heavy chains described herein or known in the art. Non-limiting examples of human constant region sequences have been described in the art, for example, see U.S. Patent No. 5,693,780 and Kabat EA et al., (1991), ibid.

[0376] In some respects, the antibodies described herein that specifically bind to FAM19A1 (e.g., human FAM19A1) include a VL domain and a VH domain, the VL domain and VH domain including the VH or VH CDR and VL and VL CDR described herein, and 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 some aspects, the antibodies described herein that specifically bind to FAM19A1 (e.g., human FAM19A1) include a VL domain and a VH domain, which comprise any of the amino acid sequences described herein, and wherein the constant region comprises the amino acid sequence of the constant region of any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) immunoglobulin molecule, IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule. In some aspects, the constant region comprises the amino acid sequence of the constant region of human IgG, which is naturally occurring and includes subclasses (e.g., IgG1, IgG2, IgG3, or IgG4) and allotypes (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 aspects, the constant region comprises the amino acid sequence of the constant region of human IgG1, IgG2, IgG3, or IgG4 or variants thereof.

[0377] In some respects, the anti-FAM19A1 antibodies disclosed herein do not possess Fc effector functions, such as complement-dependent cytotoxicity (CDC) and / or antibody-dependent cellular phagocytosis (ADCP). Effector functions are mediated by the Fc region, with the residues closest to the hinge region in the CH2 domain of the Fc region responsible for antibody effector functions because it contains a large overlap of binding sites against C1q (complement) and IgG-Fc receptors (FcγR) on effector cells of the innate immune system. Furthermore, the Fc effector function levels of IgG2 and IgG4 antibodies are lower than those of IgG1 and IgG3 antibodies. The effector function of an antibody can be reduced or avoided by various methods known in the art, including: (1) using antibody fragments lacking an Fc region (e.g., Fab, F(ab')2, single-chain Fv (scFv), or sdAb composed of monomeric VH or VL domains); (2) generating non-glycosylated antibodies, which can be generated by: for example, deleting or altering sugar-linked residues, removing sugar by enzymatic methods, generating antibodies in cells in the presence of glycosylation inhibitors, or expressing antibodies in cells that cannot glycosylate proteins (e.g., bacterial host cells, see, for example, USPub. No. 20120100140); (3) employing an Fc region from an IgG subclass whose effector function has been reduced (e.g., the Fc region of an IgG2 or IgG4 antibody or a chimeric Fc region containing a CH2 domain of an IgG2 or IgG4 antibody, see, for example, USPub. No. 20120100140 and Lau). C. et al., J. Immunol. 191: 4769-4777 (2013); and (4) generating Fc regions with mutations that result in reduced or absent Fc function. See, for example, US Pub. No. 20120100140 and the US and PCT applications cited therein, and An et al., mAbs 1: 6,572-579 (2009).

[0378] Therefore, in some respects, the antigen-binding fragments disclosed herein are Fab, Fab', F(ab')2, Fv, single-chain Fv (scFv), or sdAb composed of monomeric VH or VL domains. Such antibody fragments are well known in the art and have been described above.

[0379] In some aspects, the anti-FAM19A1 antibodies disclosed herein include: an Fc region with reduced or absent Fc effector function. In some aspects, the constant region includes the amino acid sequence of the Fc region of human IgG2 or IgG4, and in some aspects, the anti-FAM19A1 antibody is an IgG2 / IgG4 isotype. In some aspects, the anti-FAM19A1 antibody includes: a chimeric Fc region comprising a CH2 domain from an IgG4 isotype IgG antibody and a CH3 domain from an IgG1 isotype IgG antibody; or a chimeric Fc region comprising a hinge region from IgG2 and a CH2 region from IgG4; or a mutated Fc region resulting in reduced or absent Fc function. Fc regions with reduced or absent Fc effector function include those known in the art. See, for example, Lau C. et al., J. Immunol. 191: 4769-4777 (2013); An et al., mAbs1: 6,572-579 (2009); and US Pub. No. 20120100140 and the U.S. patents and publications cited therein, as well as PCT publications. Those skilled in the art can readily fabricate Fc regions with reduced or no Fc effector functionality.

[0380] The anti-FAM19A1 antibody described herein can be used for diagnostic purposes, including sample detection and in vivo imaging. For this purpose, the antibody can be conjugated with a suitable detectable agent to form an immunoconjugate. Suitable formulations for diagnostic purposes include: detectable tags comprising radioisotopes for whole-body imaging, and radioisotopes, enzymes, fluorescent tags, and other suitable antibody tags for sample detection.

[0381] Detectable tags can be any of the various types used in the in vitro diagnostics field, including: microparticle tags containing metal sols such as colloidal gold; isotopes such as I125 or Tc99, presented for example with peptide chelators of the N2S2, N3S, or N4 type; chromophores including fluorescent, luminescent, and phosphorescent labels; enzyme tags that convert a given substrate into a detectable tag; and polynucleotide tags displayed after amplification (e.g., via polymerase chain reaction). Suitable enzyme tags include horseradish peroxidase, alkaline phosphatase, etc. For example, the label could be: alkaline phosphatase, which measures 1,2-dioxetane substrates such as adamantyl methoxy phosphoryloxy phenyl dioxetane (AMPPD), 3-(4-(methoxyspiro{l,2-dioxetane-3,2'-(5'-chloro)tricyclo{3.3.1.1 3,7}decan}-4-yl)phenyl phosphate (CSPD), and CDP and The presence or formation of chemiluminescence following conversion of other luminescent substrates well-known in the art (e.g., suitable lanthanide chelates such as terbium(III) and europium(III)) can be detected. The detection method is determined by the label chosen. When the label is granular and accumulates at an appropriate level, or when instruments such as spectrophotometers, luminometers, and fluorometers are used, the appearance of the label or its reaction products can be observed with the naked eye, all of which conform to standard practice.

[0382] Immunoconjugates can be prepared by methods known in the art. Preferably, the linkages produced by the coupling method are substantially (or nearly) non-immunogenic, for example, peptide- (i.e., amide-), sulfide-, (sterically hindered), disulfide-, hydrazone-, and ether linkages. These linkages are nearly non-immunogenic and exhibit reasonable stability in serum (see, for example, Senter, PD, Curr. Opin. Chem. 13 (2009) 235-244; WO2009 / 059278; WO95 / 17886).

[0383] Depending on the biochemical properties of the molecule and antibody, different coupling strategies can be employed. If the molecule is naturally occurring or recombinant, consisting of 50–500 amino acids, textbooks contain standard procedures for the synthesis of protein conjugates that can be readily followed by skilled technicians (e.g., see Hackenberger, CPR, and Schwarzer, D., Angew. Chem. Int. Ed. 47 (2008) 10030–10074). In some respects, maleimide groups are used in the reaction with antibody or cysteine ​​residues within the molecule. This is a particularly suitable coupling chemical reaction, for example, when using the Fab or Fab' fragment of the antibody. Additionally, in some respects, coupling can be performed with the C-terminus of the antibody or a moiety. C-terminal modifications of proteins, such as C-terminal modifications of Fab fragments, can be performed, for example, as described (Sunbul, M. and Yin, J., Org. Biomol. Biomol. Chem. 7 (2009) 3361–3371).

[0384] Generally, site-specific reactions and covalent coupling are based on converting native amino acids into reactive amino acids, the reactivity of which is orthogonal to the reactivity of other functional groups present. For example, within a rare sequence range, a specific cysteine ​​can be converted into an aldehyde by an enzyme (see Frese, MA, and Dierks, T., ChemBioChem. 10 (2009) 425-427). It is also possible to obtain the desired amino acid modification by utilizing the specific enzymatic reactivity of certain enzymes with native amino acids in a specific sequence background (see, for example, Taki, M. et al., Prot. Eng. Des. Sel. 17 (2004) 119-126; Gautier, A. et al., Chem. 15 (2008) 128-136; and Protease-catalyzed formation of C-N bonds is used by Bordusa, F., Highlights in Bioorganic Chemistry (2004) 389-403).

[0385] Site-specific reactions and covalent coupling can also be achieved through the selective reaction of terminal amino acids with appropriate modifying agents. The reactivity of N-terminal cysteine ​​with benzonitrile (see Ren, H. et al., Angew. Chem. Int. Ed. Engl. 48 (2009) 9658-9662) can be used to achieve site-specific covalent coupling. Native chemical ligation can also rely on C-terminal cysteine ​​residues (Taylor, E. Vogel; Imperiali, B., Nucleic Acids and Molecular Biology (2009), 22 (Protein Engineering), 65-96).

[0386] EP 1 074 563 describes a coupling method based on the rapid reaction of a cysteine ​​in a negatively charged amino acid segment with a cysteine ​​in a positively charged amino acid segment.

[0387] This molecular moiety can also be a synthetic peptide or a peptide mimic. If the peptide is chemically synthesized, amino acids with orthogonal chemical reactivity can be added during such synthesis (see, for example, de Graaf, AJ et al., Bioconjug. Chem. 20 (2009) 1281-1295). Since a large number of orthogonal functional groups are at stake and can be introduced into the synthetic peptide, the coupling of this peptide with the linker is a standard chemical reaction.

[0388] To obtain single-labeled peptides, conjugates with a 1:1 stoichiometry can be separated from other conjugation byproducts using chromatography. This process can be facilitated by using a dye-labeled binding pair member and a charged linker. By using this labeled, highly negatively charged binding pair member, monomeric conjugate peptides can be easily separated from unlabeled peptides and peptides carrying more than one linker, due to the difference in charge and molecular weight that can be used for separation. Fluorescent dyes can be used to purify complexes from unbound components, such as labeled monovalent binders.

[0389] V. Nucleic acid molecules

[0390] Another aspect described herein relates to one or more nucleic acid molecules that encode any of the antibodies or antigen-binding fragments described herein. The nucleic acid may be present in whole cells, cell lysates, or in partially or substantially purified forms. When the nucleic acid is isolated from other cellular components or other contaminants, such as other cellular nucleic acids (e.g., other chromosomal DNA, such as chromosomal DNA linked to isolated DNA in nature) or proteins, by standard techniques, including base / SDS treatment, CsCl banding, column chromatography, restriction enzymes, agarose gel electrophoresis, and other techniques well-known in the art, the nucleic acid is “isolated” or “rendered substantially pure.” See F. Ausubel et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987). The nucleic acid described herein may be, for example, DNA or RNA, and may or may not contain intron sequences. In some aspects, the nucleic acid is a cDNA molecule.

[0391] The nucleic acids described herein can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes, as described below), the light and heavy chains of cDNAs encoding the antibodies produced by the hybridomas can be obtained using standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display technology), the nucleic acids encoding the antibodies can be recovered from the library.

[0392] Certain nucleic acid molecules described herein are nucleic acid molecules encoding the VH and VL sequences of various anti-FAM19A1 antibodies disclosed herein. Example DNA sequences encoding the VH sequence of such antibodies are listed in SEQ ID NOs: 38, 36, 40, and 42. Table 8. Example DNA sequences encoding the VL sequence of such antibodies are listed in SEQ ID NOs: 39, 37, 41, and 43. Table 9.

[0393] Table 8. Variable Heavy Chain Polynucleotide Sequences

[0394]

[0395]

[0396] Table 9. Variable Light Chain Polynucleotide Sequences

[0397]

[0398]

[0399] Methods for manufacturing anti-FAM19A1 antibodies (e.g., those disclosed herein) may include expressing the relevant heavy and light chains of the antibody in a cell line containing nucleotide sequences encoding heavy and light chains and a signal peptide. Host cells containing these nucleotide sequences are included herein.

[0400] Once the DNA fragments encoding VH and VL segments are obtained, these fragments can be further manipulated using standard recombinant DNA techniques, such as converting variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these operations, one DNA fragment encoding VL or VH is operatively linked to another DNA fragment encoding a different protein, such as an antibody constant region or a flexible linker. The term "operative linking," as used herein, is intended to refer to the joining of two DNA fragments such that the amino acid sequences encoded by the two fragments remain in-frame.

[0401] Isolated DNA encoding the VH region can be converted into a full-length heavy chain gene by operably ligating the VH-encoding DNA to another DNA molecule encoding a heavy chain constant region (hinge, CH1, CH2, and / or CH3). Sequences of human heavy chain constant region genes are known in the art (see, for example, Kabat, E.A., et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. Heavy chain constant regions can be IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant regions, such as IgG2 and / or IgG4 constant regions. For Fab fragment heavy chain genes, the DNA encoding VH can be operably ligated to another DNA molecule encoding only the heavy chain CH1 constant region.

[0402] Isolated DNA encoding the VL region can be converted into a full-length light chain gene (and Fab light chain gene) by operatively linking the VL-encoded DNA to another DNA molecule encoding a light chain constant region (CL). Sequences of human light chain constant region genes are known in the art (see, for example, Kabat, E.A., et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region.

[0403] To generate the scFv gene, a DNA fragment encoding VH and VL is operatively linked to another fragment encoding a flexible linker, for example, encoding the amino acid sequence (Gly4-Ser)3, so that the VH and VL sequences can be expressed as a continuous single-stranded protein, with the VL and VH regions connected 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)).

[0404] In some respects, the vectors disclosed herein comprise an isolated nucleic acid molecule, said nucleic acid molecule comprising a nucleotide sequence encoding an antibody or an antigen-binding fragment thereof.

[0405] Vectors applicable to this disclosure include expression vectors, viral vectors, and plasmid vectors. In some aspects, the vectors are viral vectors.

[0406] As used herein, "expression vector" refers to any nucleic acid construct containing the necessary elements for transcription and translation of the inserted coding sequence, or, in the case of an RNA viral vector, the necessary elements for replication and translation when introduced into a suitable host cell. Expression vectors can include plasmids, bacteriophages, viruses, and their derivatives.

[0407] VI. Antibody Production

[0408] The anti-FAM19A1 antibody disclosed herein can be produced by any method of synthetic antibody production known in the art, such as chemical synthesis or recombinant expression techniques. Unless otherwise stated, the methods described herein employ conventional techniques from molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields within the art. These techniques are described, for example, in the references cited herein and are fully explained therein. See, for example, Maniatis T et al., (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Sambrook J et al., (1989), Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press; Sambrook J et al., (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel FM et al., Current Protocols in Molecular Biology, John Wiley&Sons (1987and annual updates); Approach, IRL Press; Birren B et al., (eds.) (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press.

[0409] In some respects, the antibodies described herein are antibodies prepared, expressed, created, or isolated by any means involving creation, such as synthesis, genetic engineering of DNA sequences (e.g., recombinant antibodies). In some respects, such antibodies include sequences (such as DNA sequences or amino acid sequences) that are not naturally present in an antibody population within an animal or mammal (such as a human).

[0410] In one aspect, this document provides a method for manufacturing an antibody or an antigen-binding fragment thereof that immune-specifically binds to FAM19A1 (e.g., human FAM19A1), the method comprising: culturing the cells or host cells described herein. In another aspect, this document provides a method for manufacturing an antibody or an antigen-binding fragment thereof that immune-specifically binds to FAM19A1 (e.g., human FAM19A1), the method comprising: expressing (e.g., recombinantly expressing) the antibody or antigen-binding fragment thereof using the cells or host cells described herein (e.g., cells or host cells containing a polynucleotide encoding the antibody described herein). In some aspects, the cells are isolated cells. In some aspects, exogenous polynucleotides have been introduced into the cells. In some aspects, the method further comprises the step of: purifying the antibody or antigen-binding fragment thereof obtained from the cells or host cells.

[0411] Methods for producing polyclonal antibodies are known in the art (see, for example, Chapter 11, Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel FM et al., eds., John Wiley and Sons, New York).

[0412] Monoclonal antibodies can be prepared using a variety of techniques known in the art, including hybridoma, recombinant, and phage display techniques, or combinations thereof. For example, monoclonal antibodies can be produced using hybridoma techniques, including those known in the art, see, for example, Harlow E & Lane D, Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory Press, 2nd ed. 1988); Hammerling GJ et al., Monoclonal Antibodies and T-Cell Hybridomas, 563 681 (Elsevier, NY, 1981). The term "monoclonal antibody" is not limited to antibodies produced by hybridoma techniques. For example, monoclonal antibodies can be recombinantly generated from host cells expressing exogenous antibodies or fragments thereof (e.g., the light and / or heavy chains of such antibodies).

[0413] In some aspects, a “monoclonal antibody,” as used herein, is an antibody produced by a single cell (e.g., a hybridoma or a host cell that produces a recombinant antibody), wherein the antibody binds immunospecifically to FAM19A1 (e.g., human FAM19A1), as determined, for example, by ELISA or other antigen-binding or competitive binding assays known in the art or the examples provided herein. In some aspects, a monoclonal antibody may be a chimeric antibody or a humanized antibody. In some aspects, a monoclonal antibody is a monovalent or multivalent (e.g., bivalent) antibody. In further aspects, a monoclonal antibody is a monospecific or multispecific antibody (e.g., a bispecific antibody). The monoclonal antibodies described herein may, for example, be prepared by the hybridoma method described in Kohler G & Milstein C (1975) Nature 256:495, or may, for example, be isolated from a phage library using the techniques described herein. Other methods for preparing clonal cell lines and monoclonal antibodies expressed therefrom are well known in the art (see, for example, Chapter 11, Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel FM et al., ibid.).

[0414] The methods for producing and screening specific antibodies using hybridoma technology are routine and well-known in the art. For example, in the hybridoma approach, mice or other suitable host animals, such as sheep, goats, rabbits, rats, hamsters, or macaques, are immunized to stimulate lymphocytes that produce or are capable of producing antibodies that will specifically bind to proteins used for immunization (e.g., human FAM19A1). Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusion agent, such as polyethylene glycol, to form 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 at Multiple Sites) technology (Kilpatrick KE et al., (1997) Hybridoma 16:381-9, incorporated herein by reference in its entirety).

[0415] In some respects, 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, by detecting antigen-specific antibodies in mouse serum, the mouse spleen is harvested and spleen cells are isolated. The spleen cells are then fused with any suitable myeloma cells, such as cells from the American Type Culture Collection (ATCC) (Manassas, VA) cell line SP20, using well-known techniques to form hybridomas. Hybridomas are selected and cloned through limited dilution. In other respects, lymph nodes from immunized mice are harvested and fused with NSO myeloma cells.

[0416] Hybridoma cells prepared in this way are seeded and grown in a suitable culture medium, preferably containing one or more substances that inhibit the growth or survival of unfused parental myeloma cells. For example, if the parental myeloma cells lack hypoxanthine-guanine nucleotide transferase (HGPRT or HPRT), the hybridoma culture medium will typically include hypoxanthine, aminopterin, and thymidine (HAT medium), which can prevent the growth of HGPRT-deficient cells.

[0417] Specifically, myeloma cells are used that can fuse efficiently, support stable high-level antibody production by selected antibody-producing cells, and are sensitive to media such as HAT. Among these myeloma cell lines are mouse myeloma lines, such as the NSO cell line or cell lines derived from MOPC-21 and MPC-11 mouse tumors, available from the Salk Institute Cell Distribution Center, San Diego, CA, USA, and SP-2 or X63-Ag8.653 cells available from the American Type Culture Collection, Rockville, MD, USA. Human myeloma and mouse-human atypical myeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor D (1984) J Immunol 133:3001-5; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

[0418] The culture medium for hybridoma cell growth is analyzed to produce monoclonal antibodies 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 by in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0419] Once it has been determined that hybridoma cells will produce antibodies with the desired specificity, affinity, and / or activity, the clones can be subcloned using a limiting dilution procedure and cultured using standard methods (Goding JW (Ed), Monoclonal Antibodies: Principles and Practices, ibid.). Suitable media for this purpose include, for example, D-MEM or RPMI 1640. Furthermore, hybridoma cells can be grown in animals as ascites tumors.

[0420] Subclonal secreted monoclonal antibodies can be appropriately separated from culture medium, ascites, or serum using conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0421] The antibodies described herein comprise antibody fragments that recognize a specific FAM19A1 (e.g., human FAM19A1) and can be generated by any technique known to those skilled in the art. For example, the Fab and F(ab')2 fragments described herein can be generated by protein cleavage of an immunoglobulin molecule using enzymes such as papain (which generates the Fab fragment) or pepsin (which generates the F(ab')2 fragment). The Fab fragment corresponds to one of the two identical arms of the antibody molecule and contains a complete light chain paired with the VH and CH1 domains of the heavy chain. The F(ab')2 fragment contains two antigen-binding arms of the antibody molecule linked by disulfide bonds in the hinge region.

[0422] Furthermore, the antibodies or antigen-binding fragments described herein can also be generated using various phage display methods known in the art. In phage display methods, a functional antibody structure is displayed on the surface of a phage particle carrying a polynucleotide sequence encoding the antibody. In particular, DNA sequences encoding the VH and VL domains are amplified from an animal cDNA library (e.g., a cDNA library of affected tissues from humans or non-humans, such as mice or chickens). The DNA encoding the VH and VL domains is recombined with an scFv linker by PCR and cloned into a phage vector. This vector is electroporated in *E. coli*, which is then infected with helper phages. The phages used in these methods are typically filamentous phages (including fd and M13), and the VH and VL domains are typically recombined and fused to gene III or gene VIII of the phage. Phages expressing antigen-binding domains that bind to specific antigens can be selected or recognized using antigens, for example, using labeled antigens or antigens bound to or captured by solid surfaces or beads. Examples of phage display methods that can be used to manufacture the antibodies described herein include those disclosed in the following literature: 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 WO 90 / 02809, WO 91 / 10737, WO92 / 01047, WO 92 / 18619, WO 93 / 1 1236, WO95 / 15982, WO 95 / 20401, and WO 97 / 13844; and U.S. Patent Nos. 5,698,426,5,223,409,5,403,484,5,580,717,5,427,908,5,750,753,5,821,047,5,571,698,5,427,908,5,516,637,5,780,225,5,658,727,5,733,743 and 5,969,108.

[0423] As described in the aforementioned references, after phage selection, antibody coding regions from phages can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen-binding fragments, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, for example, as described below. Techniques for recombinantly producing antibody fragments such as Fab, Fab', and F(ab')2 fragments can also be employed using methods known in the art, as disclosed in the following publications: PCT Publication No. WO 92 / 22324; Mullinax RL et al., (1992) BioTechniques 12(6): 864-9; Sawai H et al., (1995) Am J Reprod Immunol 34: 26-34; and Better M et al., (1988) Science 240: 1041-1043.

[0424] In one aspect, to generate the full antibody, the VH or VL sequence can be amplified from a template (e.g., an scFv clone) using PCR primers comprising the VH or VL nucleotide sequence, a restriction site, and flanking sequences protecting the restriction site. Using cloning techniques known to those skilled in the art, the PCR-amplified VH domain can be cloned into a vector expressing the VH constant region, and the PCR-amplified VL domain can be cloned into a vector expressing the VL constant region, such as the human kappa or lambda constant region. The VH and VL domains can also be cloned into a vector expressing the necessary constant region. The heavy chain conversion vector and the light chain conversion vector are then co-transfected into cell lines using techniques known to those skilled in the art to generate stable or transient cell lines expressing full-length antibodies (e.g., IgG).

[0425] A chimeric antibody is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules. For example, a chimeric antibody may comprise a variable region of a non-human animal (e.g., mouse, rat, or chicken) monoclonal antibody fused with a constant region of a human antibody. Methods for producing chimeric antibodies are known in the art. See, for example, Morrison SL (1985) Science 229:1202-7; Oi VT & Morrison SL (1986) BioTechniques 4:214-221; Gilles 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.

[0426] Humanized antibodies are capable of binding to a predetermined antigen and comprise: a framework region having an amino acid sequence substantially that of a human immunoglobulin, and CDRs having an amino acid sequence substantially that of a non-human immunoglobulin (e.g., mouse or chicken immunoglobulin). In some specific aspects, humanized antibodies also include at least a portion of the immunoglobulin constant region (Fc) (typically the constant region of a human immunoglobulin). The antibody may also include CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. Humanized antibodies can be selected from: any class 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 in the art, including but not limited to CDR grafting (European Patent No.: EP 239400; International Publication No.: WO 91 / 09967; and US Patent Nos.: 5,225,539, 5,530,101, and 5,585,089), bonding or repositioning (European Patent Nos. EP 592106 and EP 519596; Padlan EA (1991) Mol Immunol 28 (4 / 5): 489-498; Studnicka GM et al., (1994) Prot Engineering 7 (6): 805-814; and Roguska MA et al., (1994) PNAS 91: 969-973), chain tampering (chain shuffling (US Patent No. 5,565,332), and techniques disclosed in, for example, the following documents: US Patent No. 6,407,213, US Patent No. 5,766,886, International Publication No. WO 93 / 17105; Tan P et al., (2002) J Immunol 169: 1119-25; Caldas C et al., (2000) Protein Eng. 13(5): 353-60; Morea V et al., (2000) Methods 20(3): 267-79; Baca M et al., (1997) J Biol Chem 272(16): 10678-84; Roguska MA et al., (1996) Protein Eng 9(10). 895 904; Couto JR et al., (1995) Cancer Res. 55 (23 Supp): 5973s-5977s; Couto JR et al., (1995) Cancer Res 55 (8): 1717-22; Sandhu JS (1994) Gene 150 (2): 409-10 and Pedersen JT et al., (1994) J Mol Biol 235 (3): 959-73.See also U.S. Patent Application Publication No. US2005 / 0042664 A1 (February 24, 2005), the entire contents of which are incorporated herein by reference.

[0427] Methods for manufacturing multispecific (e.g., bispecific antibodies) have been 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.

[0428] Single-domain antibodies (e.g., 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. WO 94 / 04678, WO 94 / 25591 and WO 01 / 44301.

[0429] Furthermore, by using techniques well known to those skilled in the art, antibodies that bind specifically to the FAM19A1 antigen can be conversely utilized to generate anti-heteroantibodies against the “mimic” antigen. (See, for example, Greenspan NS & Bona CA (1989) FASEB J 7(5): 437-444; and Nissinoff A (1991) J Immunol 147(8): 2429-2438).

[0430] In certain aspects, the antibodies described herein that bind to the same epitopes of FAM19A1 (e.g., human FAM19A1) as the anti-FAM19A1 antibodies described herein are human antibodies or antigen-binding fragments thereof. In certain aspects, the antibodies described herein that competitively block (e.g., in a dose-dependent manner) the binding of the antibodies described herein (e.g., 1C1, 1A11, 2G7, and 3A8) to FAM19A1 (e.g., human FAM19A1) are human antibodies or antigen-binding fragments thereof.

[0431] Human antibodies can be produced using any method known in the art. For example, transgenic mice that cannot express functional endogenous immunoglobulins but can express human immunoglobulin genes can be used. In particular, human heavy chain and light chain immunoglobulin gene complexes can be introduced into mouse embryonic stem cells randomly or via homologous recombination. Alternatively, in addition to human heavy chain and light chain genes, human variable, constant, and diversity regions can be introduced into mouse embryonic stem cells. Mouse heavy chain and light chain immunoglobulin genes can be introduced into human immunoglobulin loci, either alone or simultaneously, via homologous recombination, rendering them nonfunctional. In particular, the loss of homology in the JH region can prevent the production of endogenous antibodies. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce allogeneic offspring expressing human antibodies. The transgenic mice are immunized in a normal manner with selected antigens, such as all or part of an antigen (e.g., FAM19A1). Monoclonal antibodies against this antigen can be obtained from the immunized transgenic mice using conventional hybridoma techniques. The human immunoglobulin transgenes carried by transgenic mice rearrange during B cell differentiation, followed by class switching and somatic mutations. Therefore, this technology can be used to produce therapeutic IgG, IgA, IgM, and IgE antibodies. For an overview of this technique for producing human antibodies, see Lonberg N & Huszar D (1995) Int RevImmunol 13:65-93. For a detailed discussion of this technique for producing human antibodies and human monoclonal antibodies, and the protocols for producing such antibodies, see, for example, WO 98 / 24893, WO 96 / 34096, and WO96 / 33735; and U.S. Patent Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, and 5,939,598. Examples of mice that can produce human antibodies include Xenomouse. TM (Abgenix, Inc.; U.S. Patent Nos. 6,075,181 and 6,150,184), HuaAb-Mouse TM (Mederex, Inc. / Gen Pharm; U.S. Patent Nos. 5,545,806 and 5,569,825), Trans Chromo Mouse TM (Qilin) ​​and KM Mouse TM (Medarex / Kirin).

[0432] Human antibodies that specifically bind to FAM19A1 (e.g., human FAM19A1) can be prepared by various methods known in the art, including the phage display method described above using an antibody library derived from human immunoglobulin sequences. See also U.S. Patent Nos. 4,444,887, 4,716,111, and 5,885,793; and International Publication Nos. WO 98 / 46645, WO 98 / 50433, WO 98 / 24893, WO 98 / 16654, WO 96 / 34096, WO 96 / 33735, and WO91 / 10741.

[0433] In some respects, mouse-human hybridomas can be used to produce human antibodies. 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 hybridomas can be screened to identify those that secrete human monoclonal antibodies that specifically bind to a target antigen (e.g., FAM19A1, such as human FAM19A1). Such methods are known and described in the art; see, for example, Shinmoto H et al., (2004) Cytechnology 46: 19-23; Naganawa Y et al., (2005) Human Antibodies 14: 27-31.

[0434] VII. Methods of Antibody Engineering

[0435] As described above, anti-FAM19A1 antibodies or their antigen-binding regions having the VH and VL sequences disclosed herein can be used to create new anti-FAM19A1 antibodies or their antigen-binding regions by modifying the VH and / or VL sequences, or by attaching constant regions thereto. Therefore, in another aspect described herein, the structural features of the anti-FAM19A1 antibodies described herein (e.g., 1C1, 1A11, 2G7, and 3A8) are used to create structurally related anti-FAM19A1 antibodies—which retain at least one functional property of the antibodies described herein (e.g., binding to human FAM19A1). For example, the starting material for this engineering method is the VH and / or VL sequences, or one or more CDR regions thereof, provided herein. To create engineered antibodies, it is not necessary to actually prepare (i.e., express as a protein) an antibody having one or more VH and / or VL sequences, or one or more CDR regions thereof, provided herein. Instead, the information contained in the sequence is used as starting material to create a “second-generation” sequence derived from the original sequence, which is then prepared and expressed as a protein.

[0436] Therefore, this article provides a method for preparing anti-FAM19A1 antibodies or their antigen-binding moiety, including:

[0437] (a) Provide: (i) a heavy chain variable region sequence, including the CDR1, CDR2 and / or CDR3 sequences listed in Table 4 or the CDR1, CDR2 and / or CDR3 sequences of the heavy chain variable regions listed in Table 6; and (ii) a light chain variable region sequence, including the CDR1, CDR2 and / or CDR3 sequences listed in Table 5 or the CDR1, CDR2 and / or CDR3 sequences of the light chain variable regions listed in Table 7.

[0438] (b) altering at least one amino acid residue in the heavy chain variable region sequence and / or the light chain variable region sequence to produce at least one altered antibody or antigen-binding moiety sequence; and

[0439] (c) Express the altered antibody or antigen-binding sequence as a protein.

[0440] Standard molecular biology techniques can be used to prepare and express altered antibody or antigen-binding motif sequences.

[0441] In some respects, the antibody encoded by the altered antibody or antigen-binding moiety sequence, or the antigen-binding moiety thereof, is an antibody that retains one, some, or all of the functional properties of the anti-FAM19A1 antibody described herein. Non-limiting examples of such properties include:

[0442] (1) With K of 10 nM or less D Binds to soluble human FAM19A1, for example, via BIACORE TM Or measured by ELISA;

[0443] (2) With K of 10 nM or less D Human FAM19A1 binds to the membrane, for example, as via BIACORE. TM Or measured by ELISA;

[0444] (3) Promotes neuronal differentiation;

[0445] (4) Increase the outward growth of neurites in differentiated neurons;

[0446] (5) Reduce, reverse and / or prevent one or more symptoms associated with glaucoma;

[0447] (6) Improves retinal potentials (e.g., as demonstrated by an increase in oscillatory potentials);

[0448] (7) Reduce and / or restore the loss of retinal ganglion cells (e.g., as observed in glaucoma subjects);

[0449] (8) Reduce, reverse and / or prevent one or more symptoms associated with neuropathic pain;

[0450] (9) Increase the latency and / or threshold to external stimuli; and

[0451] (10) Increase and / or regulate the conduction velocity of sensory nerves.

[0452] In certain aspects of the engineered antibody methods described herein, mutations can be randomly or selectively introduced along all or part of the coding sequence of the anti-FAM19A1 antibody, and the resulting modified anti-FAM19A1 antibodies can be screened for binding activity and / or other functional properties as described herein. Mutation methods have been described in the art. For example, Short's PCT publication WO02 / 092780 describes methods for creating and screening antibody mutations using saturated mutagenesis, synthetic linker assembly, or combinations thereof. Additionally, Lazar et al.'s PCT publication WO 03 / 074679 describes methods for optimizing the physiochemical properties of antibodies using computational screening methods.

[0453] VIII. Cells and Vectors

[0454] In some aspects, this document provides cells (e.g., host cells) that express (e.g., recombinantly) the antibodies described herein (or their antigen-binding fragments), which specifically bind to FAM19A1 (e.g., human FAM19A1) and associated polynucleotides and expression vectors. This document provides vectors (e.g., expression vectors) containing polynucleotides, which include nucleotide sequences encoding anti-FAM19A1 antibodies or fragments, for recombinant expression in host cells, such as mammalian cells. This document also provides host cells containing such vectors for recombinant expression of the anti-FAM19A1 antibodies described herein (e.g., human antibodies or humanized antibodies). In one particular aspect, this document provides methods for producing the antibodies described herein, including expressing such antibodies from host cells.

[0455] The recombinant expression of antibodies (e.g., full-length antibodies, heavy and / or light chains of antibodies, or single-chain antibodies as described herein) that specifically bind to FAM19A1 (e.g., human FAM19A1) involves constructing expression vectors containing polynucleotides encoding the antibody. Once a polynucleotide encoding an antibody molecule, the heavy and / or light chain of the antibody, or fragments thereof (such as variable domains of the heavy and / or light chains) is obtained, vectors for producing antibody molecules can be produced using techniques well-known in the art via recombinant DNA technology. Therefore, this document describes a method for preparing proteins by expressing polynucleotides containing nucleotide sequences encoding antibodies or antibody fragments (e.g., light or heavy chains). Methods well-known to those skilled in the art can be used to construct expression vectors containing sequences encoding antibodies or antibody fragments (e.g., light or heavy chains) and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA technology, synthetic techniques, and in vivo gene recombination. Also provided are reproducible vectors comprising nucleotide sequences encoding the antibody molecule described herein, the heavy or light chain of the antibody, a variable domain of the heavy or light chain of the antibody or a fragment thereof, or a CDR of the heavy or light chain, operably linked to a promoter. For example, such vectors may comprise nucleotide sequences encoding constant regions of the antibody molecule (see, for example, International Publications WO 86 / 05807 and WO 89 / 01036; and U.S. Patent No. 5,122,464, each of which is incorporated herein by reference), and variable domains of the antibody may be cloned into such vectors to express the entire heavy chain, the entire light chain, or the entire heavy and light chains.

[0456] Expression vectors can be transferred into cells (e.g., host cells) using conventional techniques, and the resulting cells can then be cultured using conventional techniques to produce the antibodies described herein (e.g., VH and / or VL of the anti-FAM19A1 antibody disclosed herein, or antibodies with one or more VH and / or VL CDRs) or fragments thereof. Therefore, what is provided herein is a host cell containing a polynucleotide encoding the antibody described herein or a fragment thereof, or a heavy or light chain thereof, or a fragment thereof, or a single-chain antibody described herein, operably linked to a promoter for expression of such a sequence in the host cell. In some aspects, for the expression of double-chain antibodies, vectors encoding the heavy and light chains can be co-expressed in the host cell to express the whole immunoglobulin molecule, as detailed below. In some aspects, the host cell contains a vector comprising polynucleotides encoding the heavy and light chains of the antibody described herein, or fragments thereof. In a specific aspect, a host cell contains two distinct vectors, the first vector comprising a polynucleotide encoding the heavy chain or a variable region of the heavy chain of the antibody described herein, or a fragment thereof, and the second vector comprising a polynucleotide encoding the light chain or a variable region of the light chain of the antibody described herein, or a fragment thereof. In some aspects, the first host cell includes a first vector comprising a polynucleotide or fragment thereof encoding a heavy chain or a heavy chain variable region of the antibody described herein, and the second host cell includes a second vector comprising a polynucleotide encoding a light chain or a light chain variable region of the antibody described herein. In specific aspects, the heavy chain / heavy chain variable region expressed by the first cell is associated with the light chain / light chain variable region of the second cell to form the anti-FAM19A1 antibody or its antigen-binding fragment described herein. In some aspects, this document provides a host cell population comprising such a first host cell and such a second host cell.

[0457] In some respects, this document provides a group of vectors comprising: a first vector containing a polynucleotide encoding a light chain / light chain variable region of the anti-FAM19A1 antibody described herein; and a second vector containing a polynucleotide encoding a heavy chain / heavy chain variable region of the anti-FAM19A1 antibody described herein.

[0458] Various host expression vector systems can be used to express the antibody molecules described herein. These host expression systems represent mediators that can produce and subsequently purify coding sequences of interest, but also represent cells that can express the antibody molecules described herein in situ when transformed or transfected with appropriate nucleotide coding sequences. These cells include, but are not limited to: microorganisms, such as bacteria transformed with recombinant phage DNA, plasmid DNA, or Cosmid DNA expression vectors containing antibody-encoding sequences (e.g., *Escherichia coli* and *Bacillus subtilis*); yeast transformed with recombinant yeast expression vectors containing antibody-encoding sequences (e.g., *Pichia* yeast); insect cell systems infected with recombinant viral expression vectors containing antibody-encoding sequences (e.g., baculovirus); plant cell systems (e.g., green algae, such as *Chlamydomonas reinhardtii*), which are infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors containing antibody-encoding sequences (e.g., Ti plasmid); or mammalian cell systems (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK 293, NSO, PER.C6, VERO, CRL7030, HsS78Bst, HeLa, and NIH). Cells including 3T3, HEK-293T, HepG2, SP210, Rl.l, BW, LM, BSC1, BSC40, YB / 20, and BMT10 carry recombinant expression constructs containing promoters from mammalian cell genomes (e.g., metallothionein promoters) or from mammalian viruses (e.g., adenovirus late promoters; vaccine virus 7.5K promoters). In one specific aspect, the cells used to express the antibodies described herein or their antigen-binding fragments are CHO cells, such as those from the CHO GS system. TM (Lonza) CHO cells. In some respects, the cells used to express the antibodies described herein are human cells, for example, human cell lines. In some respects, the mammalian expression vector is pOptiVEC. TMOr pcDNA3.3. In some respects, bacterial cells such as *Escherichia coli*, or eukaryotic cells (e.g., mammalian cells), particularly for expressing whole recombinant antibody molecules, are used for expressing recombinant antibody molecules. For example, mammalian cells such as Chinese hamster ovary (CHO) cells, combined with major intermediate early gene promoter elements of vectors such as human cytomegalovirus, are an effective antibody expression system (Foecking MK & Hofstetter H (1986) Gene 45:101-5; and Cockett MI et al., (1990) Biotechnology 8(7):662-7). In some respects, the antibodies described herein are produced by CHO cells or NSO cells. In some respects, the expression of the nucleotide sequence encoding the antibody described herein is regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter, which allows the antibody to bind immune-specifically to FAM19A1 (e.g., human FAM19A1).

[0459] In bacterial systems, expression vectors can be advantageously selected based on the intended use of the expressed antibody molecule. For example, when large-scale production of such antibodies is required, a vector pointing to the expression of easily purified, high-level fusion protein products may be ideal for generating pharmaceutical compositions of antibody molecules. Such vectors include, but are not limited to, the *E. coli* expression vector pUR278. *E. coli* expression vector pUR278 (Ruether U & Mueller-Hill B (1983) EMBO J 2: 1791-1794), in which the antibody coding sequence can be individually linked to the lacZ coding region to produce a fusion protein; 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); and analogues. For example, the pGEX vector can also be used to express exogenous peptides as fusion proteins with glutathione 5-transferase (GST). Generally, this fusion protein is soluble and can be adsorbed and bound to matrix glutathione agarose beads, then eluted in the presence of free glutathione, and is readily purified from lysed cells. The pGEX vector is designed to include cleavage sites for thrombin or factor Xa protease so that the cloned target gene product can be released from the GST molecule.

[0460] In insect systems, for example, *Autographa californica* nuclear polyhedrosis virus (AcNPV) can be used as a vector to express foreign genes. This virus is grown in *Spodoptera frugiperda* cells. Antibody-coding sequences can be cloned separately into non-essential regions of the virus (e.g., polyhedral genes) and placed under the control of AcNPV promoters (e.g., polyhedral promoters).

[0461] In mammalian host cells, several virus-based expression systems can be utilized. When using adenovirus as the expression vector, the antibody-coding sequence of interest can be linked to the adenovirus's transcription / translation control complex, such as the late promoter and the triplet leader sequence. This chimeric gene is then inserted into the adenovirus genome via in vitro or in vivo recombination. Insertion into non-essential regions of the viral genome (such as the E1 or E3 regions) will result in the survival of the recombinant virus and its ability to express antibody molecules in the infected host (see, for example, Logan J & Shenk T (1984) PNAS 81(12): 3655-9). Effective translation of the inserted antibody-coding sequence also requires specific initiation signals. These signals include the ATG start codon and adjacent sequences. Furthermore, the start codon must coincide with the reading frame of the desired coding sequence to ensure the translation of the entire insert. These exogenous translation control signals and start codons can be of various sources, including natural and synthetic. The efficiency of expression can be improved by adding appropriate transcriptional enhancing elements, transcription terminators, etc. (see, for example, Bitter G et al., (1987) Methods Enzymol. 153: 516-544).

[0462] Furthermore, a host cell strain can be selected that can regulate the expression of the inserted sequence or modify and process the gene product in a specific way as desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products are important for protein function. Different host cells have their own characteristics and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be selected to ensure the proper modification and processing of expressed foreign proteins. For this purpose, eukaryotic host cells with the correct cellular mechanisms for handling primary transcripts, glycosylation, and phosphorylation of gene products can be used. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, HeLa, MDCK, HEK 293, NIH3T3, W138, BT483, Hs578T, HTB2, BT20 and T47D, NSO (a mouse myeloma cell line that does not produce any immunoglobulin chains), CRL7030, COS (e.g., COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC 1, BSC40, YB / 20, BMT10 and HsS78Bst cells. In some respects, the anti-FAM19A1 antibody described herein is produced in mammalian cells such as CHO cells.

[0463] In some respects, 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, these antibodies can be expressed in cells lacking fucose capacity. In a specific example, an antibody or its antigen-binding moieties with reduced fucose content can be produced using a cell line with two alleles knocked out of 1,6-fucosyltransferase. The Lonza system is an example of such a system, which can be used to produce antibodies or their antigen-binding moieties with reduced fucose content.

[0464] To achieve long-term, high-yield production of recombinant proteins, stable expression cells can be generated. For example, cell lines that stably express the anti-FAM19A1 antibody and its antigen-binding moiety described herein can be designed. Specifically, the cells provided herein stably express light chain / light chain variable domains and heavy chain / heavy chain variable domains, which together form the antibody or its antigen-binding moiety described herein.

[0465] In some respects, instead of using expression vectors containing viral replication origins, it is preferable to transform host cells with DNA controlled by appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and selectable markers. After introducing foreign DNA / polynucleotides, engineered cells can be grown in enriched media for 1–2 days, followed by a change to a selective medium. The selectable markers in the recombinant plasmid confer resistance to selection and allow cells to stably integrate the plasmid into their chromosomes and grow to form foci, which can then be cloned and expanded into cell lines. This method can be advantageously used for engineered cell lines expressing the anti-FAM19A1 antibody described herein or its antibody-binding moiety. Such engineered cell lines are particularly useful in screening and evaluating compositions that interact directly or indirectly with antibody molecules.

[0466] Several selection systems can be used, including but not limited to: herpes simplex virus thymidine kinase (Wigler M et al., (1977) Cell 11(1): 223-32), hypoxanthine nucleotide transferase (Szybalska EH & Szybalski W (1962) PNAS 48(12): 2026-2034) and adenine phosphoribosyl transferase (Lowy I et al., (1980) Cell 22(3): 817-23) genes can be used in tk-, hgprt- or aplt- cells, respectively. In addition, resistance to antimetabolites can serve as a basis for selecting the following genes: dhfr, which confers resistance to methotrexate (Wigler M et al., (1980) PNAS 77(6): 3567-70; O'Hare K et al., (1981) PNAS 78: 1527-31); gpt, which confers resistance to mycophenolic acid (Mulligan RC & Berg P (1981) PNAS 78(4): 2072-6); neo, which confers resistance to aminoglycoside G-418 (Wu GY & Wu CH (1991) Biotherapy 3: 87-95; Tolstoshev P (1993) Ann Rev Pharmacol Toxicol 32: 573-596; Mulligan RC (1993) Science 260: 926-932; and Morgan RA & Anderson). WF (1993) AnnRev Biochem 62: 191-217; Nabel GJ & Feigner PL (1993) Trends Biotechnol 11(5): 211-5); and hygro, which confers resistance to hygromycin (Santerre RF et al., (1984) Gene 30(1-3): 147-56).Known methods in the field of recombinant DNA technology can be routinely used to select desired recombinant clones, as described in the following literature: 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 Dracopoli NC et al. (eds.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colbe-Garapin F et al. (1981) J Mol Biol 150: 1-14, the full text of which are included in this paper by reference.

[0467] The expression level of antibody molecules can be increased by vector amplification (see Bebbington CR & Henschel CCG, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, New York, 1987)). When a marker expressing an antibody in a vector system can be amplified, the increased level of inhibitors present in the host cell culture will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, antibody production will also increase (Crouse GF et al., (1983) Mol Cell Biol 3: 257-66).

[0468] Host cells can be co-transfected with two or more expression vectors described herein, the first vector encoding a heavy-chain-derived polypeptide and the second vector encoding a light-chain-derived polypeptide. Both vectors can contain the same selectable markers, enabling equal expression of the heavy-chain and light-chain polypeptides. Host cells can be co-transfected with varying numbers of the two or more expression vectors. For example, host cells can be transfected with the first and second expression vectors in any of the following ratios: 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.

[0469] Alternatively, a single vector can be used that encodes and expresses both heavy and light chain polypeptides. In this case, the light chain should be placed before the heavy chain to avoid an overdose of toxic free heavy chain (Proudfoot NJ (1986) Nature 322: 562-565; and Kohler G (1980) PNAS 77: 2197-2199). The coding sequences for the heavy and light chains can consist of cDNA or genomic DNA. The expression vector can be monocistronic or multicistronic. Multicistronic nucleic acid constructs can encode 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, or within the range of 2-5, 5-10, or 10-20 gene / nucleotide sequences. For example, a bicistronic nucleic acid construct can include, in the following order: promoter, first gene (e.g., the heavy chain of the antibody described herein), and second gene (e.g., the light chain of the antibody described herein). In such a vector, transcription of two genes can be driven by a promoter, while translation of the mRNA of the first gene can be achieved through a cap-dependent scanning mechanism, and translation of the mRNA of the second gene can be achieved through a cap-independent mechanism (e.g., via IRES).

[0470] Once the antibody molecules described herein are generated through recombinant expression, they can be purified using any method known in the art for purifying immunoglobulin molecules, such as chromatography (e.g., ion exchange, affinity, particularly by affinity for specific antigens following protein A, and gel column chromatography), centrifugation, differential solubility, or any other standard technique for purifying proteins. Furthermore, the antibodies described herein can be fused with heterologous polypeptide sequences described herein or known in the art to facilitate purification.

[0471] In some specific aspects, the antibodies or their antigen-binding portions described herein are isolated or purified. Generally, the isolated antibodies are substantially free of other antibodies that have a different antigen specificity than the isolated antibody. For example, in some aspects, the preparation of the antibodies described herein is substantially free of cellular material and / or chemical precursors. The language “substantially free of cellular material” includes the preparation of antibodies in which the antibody is separated from the cellular components of the cells in which it is isolated or recombined. Thus, antibodies substantially free of cellular material include antibody preparations having less than 30%, 20%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (by dry weight) of heterologous proteins (also referred to herein as “contaminating proteins”) and / or antibody variants, such as different post-translational modified forms of the antibody or other different versions of the antibody (or antibody-binding portions). When the antibody is recombined, it is also generally substantially free of culture medium, i.e., the culture medium constitutes less than 20%, 10%, 2%, 1%, 0.5%, or 0.1% of the volume of the protein preparation. When antibodies are produced through chemical synthesis, they are typically substantially free of chemical precursors or other chemicals; that is, they are isolated from the chemical precursors or other chemicals involved in protein synthesis. Therefore, in such antibody formulations, the content of chemical precursors or compounds, other than the antibody of interest, is less than about 30%, 20%, 10%, or 5% (on a dry weight basis). In some respects, the antibodies described herein are isolated or purified.

[0472] IX. Diagnosis

[0473] As described above, the FAM19A1 antagonists (e.g., anti-FAM19A1 antibodies) described herein can be used for diagnostic purposes (including sample testing and in vivo imaging). For this purpose, the antibody (or its binding portion) can be conjugated with a suitable detectable agent to form an immunoconjugate. For diagnostic purposes, suitable formulations include: a detectable tag comprising a radioisotope for whole-body imaging, and a radioisotope, enzyme, fluorescent tag, and other suitable antibody tag for sample testing.

[0474] The detectable tag can be any of the various types currently used in the in vitro diagnostics field, including: microparticle tags, such as metal sols like colloidal gold; isotopes like I... 125 or Tc 99Examples of suitable enzyme tags include those presented using peptide chelators of the N2S2, N3S, or N4 type; chromophores including fluorescent, luminescent, and phosphorescent labels; enzyme tags that convert a given substrate into a detectable label; and polynucleotide tags displayed after amplification (e.g., via polymerase chain reaction). Suitable enzyme tags include horseradish peroxidase and alkaline phosphatase. For example, a tag could be an alkaline phosphatase that measures 1,2-dioxolane substrates such as adamantane methoxyphosphoryloxyphenyl dioxolane (AMPPD), 3-(4-(methoxyspiro{1,2-dioxolane-3,2'-(5'-chloro)tricyclo{3.3.1.1 3,7}decane}-4-yl)phenyl phosphate (CSPD), and CDP and... The presence or formation of chemiluminescence following conversion of other luminescent substrates well known to those skilled in the art (e.g., chelates of suitable lanthanides, such as terbium(III) and europium(III)) is detected. The detection method is determined by the label chosen. When the label is granular and accumulates at an appropriate level, or when instruments such as spectrophotometers, luminometers, and fluorometers are used, the appearance of the label or its reaction products can be observed with the naked eye, all of which conform to standard practice.

[0475] The FAM19A1 antagonists described herein (e.g., anti-FAM19A1 antibodies) can also be conjugated with therapeutic agents to form immunoconjugates, such as antibody-drug conjugates (ADCs). Suitable therapeutic agents include those that can treat 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 in this report.

[0476] Immunoconjugates can be prepared by methods known in the art. In some respects, the linkages produced by the conjugation methods are substantially (or nearly) non-immunogenic, for example, peptide- (i.e., amide-), sulfide-, (sterically hindered), disulfide-, hydrazone-, and ether linkages. These linkages are nearly non-immunogenic and exhibit reasonable stability in serum (see, for example, Senter, PD, Curr. Opin. Chem. 13 (2009) 235-244; WO 2009 / 059278; WO 95 / 17886).

[0477] Depending on the biochemical properties of the molecule and antibody, different coupling strategies can be employed. If the molecule is naturally occurring or recombinant, consisting of 50 to 500 amino acids, textbooks contain standard procedures for the chemistry of synthesizing protein conjugates, which skilled technicians can easily follow (see, for example, Hackenberger, CPR, and Schwarzer, D., Angew. Chem. Int. Ed. Engl. 47 (2008) 10030-10074). In some respects, maleimide groups are used in the reaction with cysteine ​​residues within the antibody or molecule. This is a particularly suitable coupling chemistry in cases, for example, where the Fab or Fab' fragment of the antibody is used. Additionally, in some respects, coupling can be performed with the C-terminus of the antibody or molecule. C-terminal modifications of the protein, such as C-terminal modifications of the Fab fragment, can be performed, for example, as described in the following literature (Sunbul, M. and Yin, J., Org. Biomol. Biomol. Chem. 7 (2009) 3361-3371).

[0478] Generally, site-specific reactions and covalent coupling are based on converting native amino acids into reactive amino acids, which are orthogonal to the reactivity of other functional groups present. For example, within a rare sequence range, a specific cysteine ​​can be converted into an aldehyde by enzymatic action (see Frese, MA, and Dierks, T., ChemBioChem. 10 (2009) 425-427). Desired amino acid modifications can also be obtained by utilizing the specific enzymatic reactivity of certain enzymes with native amino acids in a specific sequence background (see, for example, Taki, M. et al., Prot. Eng. Des. Sel. 17 (2004) 119-126; Gautier, A. et al., Chem. 15 (2008) 128-136; and Protease-catalyzed formation of C—N bonds is used by Bordusa, F., Highlights in Bioorganic Chemistry (2004) 389-403).

[0479] Site-specific reactions and covalent coupling can also be achieved through the selective reaction of terminal amino acids with appropriate modifying agents. The reactivity of N-terminal cysteine ​​with benzonitrile (see Ren, H. et al., Angew. Chem. Int. Ed. Engl. 48 (2009) 9658-9662) can be used to achieve site-specific covalent coupling. Natural chemical linkages can also rely on C-terminal cysteine ​​residues (Taylor, E. Vogel; Imperiali, B., Nucleic Acids and Molecular Biology (2009), 22 (Protein Engineering), 65-96).

[0480] EP 1 074 563 describes a coupling method based on the rapid reaction of cysteine ​​in a negatively charged amino acid segment with cysteine ​​in a positively charged amino acid segment.

[0481] This molecular moiety can also be a synthetic peptide or a peptide mimic. If the peptide is chemically synthesized, amino acids with orthogonal chemical reactivity can be added during such synthesis (see, for example, de Graaf, AJ et al., Bioconjug. Chem. 20 (2009) 1281-1295). Since a large number of orthogonal functional groups are at risk and can be introduced into the synthetic peptide, the coupling of this peptide with the linker is a standard chemical reaction.

[0482] To obtain single-labeled peptides, conjugates with a 1:1 stoichiometry can be separated from other conjugation byproducts using chromatography. This process can be facilitated by using a dye-labeled binding pair member and a charged linker. Monomeric conjugate peptides can be readily separated from unlabeled peptides and peptides carrying more than one linker by using such labeled, highly negatively charged binding pair members, due to the difference in charge and molecular weight that can be used for separation. Fluorescent dyes can be used to purify complexes from unbound components, such as labeled monovalent binders.

[0483] X. Pharmaceutical Compositions

[0484] This document provides a composition comprising a disclosed FAM19A1 antagonist (e.g., an anti-FAM19A1 antibody) of desired purity in a physiologically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA). The acceptable carrier, excipient, or stabilizer is non-toxic to the recipient at the dose and concentration used and comprises: buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyl dimethyl benzyl ammonium chloride; hexamethyl ammonium chloride; benzalkonium chloride; phenol, butyl or benzyl alcohol; alkyl benzoates such as methyl or propyl benzoates; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); and low molecular weight (less than about 10). (Residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as... Or polyethylene glycol (PEG).

[0485] In some aspects, the pharmaceutical composition comprises: an antibody or antigen-binding fragment thereof described herein, a bispecific molecule or immunoconjugate, and optionally one or more additional prophylactic or therapeutic agents, in a pharmaceutically acceptable carrier. In some aspects, the pharmaceutical composition comprises: an effective amount of an antibody or antigen-binding fragment thereof described herein, and optionally one or more additional prophylactic or therapeutic agents, in a pharmaceutically acceptable carrier. In some aspects, the antibody is the only active ingredient included in the pharmaceutical composition. The pharmaceutical compositions described herein can be used to reduce the activity of FAM19A1, thereby treating diseases or disorders, such as those related to CNS dysfunction (e.g., glaucoma and neuropathic pain).

[0486] Pharmaceutically acceptable carriers for parenteral preparations include aqueous carriers, non-aqueous carriers, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifiers, sealing or chelating agents, and other pharmaceutically acceptable substances. Examples of aqueous carriers include sodium chloride injection, Ringer's solution, isotonic glucose injection, sterile water injection, glucose and milk Ringer's solution. Non-aqueous parenteral carriers include plant-derived fixed oils, cottonseed oil, corn oil, sesame oil, and peanut oil. Antimicrobial agents at bacteriostatic or bactericidal concentrations may be added to parenteral preparations packaged in multi-dose containers, including phenol or cresol, mercury, benzyl alcohol, chlorobutanol, methylparaben and propylparaben, thimerosal, benzalkonium chloride, and phenethylamine. Isotonic agents include sodium chloride and glucose. Buffers include phosphates and citrates. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspension and dispersant agents include sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose, and polyvinylpyrrolidone. Emulsifiers include polysorbate 80 (…). 80). Metal ion encapsulation or chelating agents include EDTA. Pharmaceutical carriers also include ethanol, polyethylene glycol, and propylene glycol for water-mixable carriers; and sodium hydroxide, hydrochloric acid, citric acid, or lactic acid for pH adjustment.

[0487] Pharmaceutical compositions can be formulated for any route of administration. Specific examples include intranasal, oral, parenteral, intrahepatic, intracerebral, intrapulmonary, subcutaneous, or intravenous administration. Parenteral administration, characterized by subcutaneous, intramuscular, or intravenous injection, is also considered herein. Injectable formulations can be prepared in conventional forms, such as liquid solutions or suspensions, solid forms suitable for dissolving or suspending in a liquid prior to injection, or emulsions. Injectable formulations, solutions, and emulsions also contain one or more excipients. Suitable excipients are, for example, water, saline, glucose, glycerol, or ethanol. In addition, if desired, the pharmaceutical composition to be administered may also contain small amounts of non-toxic excipients, such as wetting agents or emulsifiers, pH buffers, stabilizers, solubility enhancers, etc., such as sodium acetate, sorbitol monolaurate, triethanolamine oleate, and cyclodextrin.

[0488] Parenteral formulations include: sterile solutions available for injection, sterile dry solubles (e.g., lyophilized powders (including subcutaneous tablets) that can be bound to a solvent before use), sterile suspensions available for injection, sterile dry insolubles that can be bound to a medium before use, and sterile emulsions. Solutions may be aqueous or non-aqueous solutions.

[0489] If administered intravenously, suitable carriers include physiological saline or phosphate-buffered saline (PBS), as well as solutions containing thickeners and solubilizers (such as glucose, polyethylene glycol, and polypropylene glycol, and mixtures thereof).

[0490] The antibody-containing topical mixture is prepared as described for topical and systemic application. The resulting mixture may be a solution, suspension, emulsion, or the like, and may be formulated into creams, gels, ointments, lotions, solutions, elixirs, lotions, suspensions, tinctures, pastes, foams, aerosols, rinses, sprays, suppositories, bandages, skin patches, or any other formulation suitable for topical application.

[0491] The anti-FAM19A1 antibody described herein can be formulated as an aerosol for topical application, such as by inhalation (see, for example, U.S. Patent Nos. 4,044,126, 4,414,209, and 4,364,923, which describe aerosols for delivering steroids for the treatment of inflammatory diseases, particularly asthma). These formulations for inhalation can be in the form of an aerosol or solution for nebulizers, or as a fine powder for insufflation, used alone or in combination with an inert carrier such as lactose. In this case, the particle diameter of the formulation will be less than 50 micrometers in some respects and less than 10 micrometers in others.

[0492] The antibodies or antigen-binding fragments described herein can be formulated for topical or external use, such as for skin and mucous membranes, like the eyes, in the form of gels, creams, and lotions, and for ocular or intranasal or spinal applications. Topical application may be considered for transdermal administration, ocular or mucous membrane administration, or for inhalation therapy. Nasal solutions of the antibody can also be used alone or in combination with other pharmaceutically acceptable excipients.

[0493] Transdermal patches, including iontophoresis and electrophoresis devices, are well known to those skilled in the art and can be used to administer antibodies. Such patches are disclosed, for example, in U.S. Patent Nos. 6,267,983, 6,261,595, 6,256,533, 6,167,301, 6,024,975, 6,010715, 5,985,317, 5,983,134, 5,948,433, and 5,860,957, the full text of each of which is incorporated herein by reference.

[0494] In some respects, pharmaceutical compositions comprising the anti-FAM19A1 antibody described herein are lyophilized powders, which can be reconstituted into solutions, emulsions, and other mixtures for administration. They can also be reconstituted and formulated into solids or gels. The lyophilized powder is prepared by dissolving an antibody (such as the anti-FAM19A1 antibody) or a pharmaceutically acceptable derivative thereof in a suitable solvent. In some respects, the lyophilized powder is sterile. The solvent may contain an excipient that can improve the stability of the powder or reconstituted solution, or other pharmacological components prepared from the powder. Excipients that may be used include, but are not limited to, glucose, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose, or other suitable formulations. The solvent may contain a buffer, such as citrate, sodium phosphate, or potassium phosphate, or other such buffers known to those skilled in the art, and in some respects, approximately a neutral pH. The solution is then sterilely filtered and then lyophilized under standard conditions known to those skilled in the art to provide the desired formulation. In some respects, the resulting solution will be dispensed into vials for lyophilization. Each vial will contain a single or multiple doses of the compound. The freeze-dried powder can be stored under appropriate conditions, for example, at approximately 4°C to room temperature.

[0495] Recombining this lyophilized powder with water for injection provides a formulation for parenteral administration. For recombination, the lyophilized powder is added to sterile water or another suitable carrier. The precise amount depends on the compound chosen. This amount can be determined empirically.

[0496] The antibodies or antigen-binding fragments thereof, bispecific molecules or immune conjugates described herein, as well as other compositions provided herein, can also be formulated to target specific tissues, receptors, or other areas of the body of a subject to be treated. Many such targeting methods are known. All such targeting methods are contemplated herein for use in the compositions of the invention. 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, the full text of each of which is incorporated herein by reference.

[0497] Compositions intended for in vivo administration can be sterile. This can be easily achieved through filtration, for example, using a sterile filter membrane.

[0498] XI. Reagent Kit

[0499] This document provides a kit comprising one or more antibodies or antigen-binding fragments thereof described herein, wherein the kit is for diagnostic or therapeutic purposes. In some aspects, this document provides a reagent kit or reagent package comprising one or more containers filled with one or more components of the compositions described herein, such as one or more antibodies or antigen-binding fragments thereof provided herein, optionally, and instructions for use. In some aspects, the kit comprises the compositions described herein and any diagnostic, prophylactic, or therapeutic agent, such as those described herein.

[0500] Example

[0501] Example 1: Screening of anti-FAM19A1 antibodies

[0502] The phage-scFv antibody library from Y-Biologics in Daejon, Korea, consists of 10 different library sets, each with dimensions of 1-3 x 10⁻⁶. 10 The diversity forms a total of 1x10 11 The diversity of antibodies was assessed. To screen for relevant antibodies, a biopane was performed. In short, FAM19A1-Fc and FAM19A1-mFc proteins (Y-Biologics, Daejon, Korea) were used to coat immunoadsorption tubes, followed by blocking. After phage infection, human scFv library cells (containing 10...) were... 10 (To enhance diversity) phages were cultured at 30°C for 16 hours, concentrated with PEG, and then suspended in PBS buffer to prepare a phage library. The phages from the library were then added to immunotubes and incubated at room temperature for 2 hours. After incubation, the tubes were washed with 1X PBS / T and 1X PBS, removing only those scFv phages that bound the antigen (FAM19A1 protein). The positive phage pool was used to infect *E. coli* for additional amplification and biopanning. Three rounds of biopanning were performed by repeating the above process. Each amplification screened for phages with high affinity for the FAM19A1 protein.

[0503] Example 2: Selection of anti-FAM19A1 antibody clones

[0504] To investigate the specificity of the positive poly-scFv-phage antibody pools from each round of biopanning, a polyphage ELISA was performed. ELISA plates were coated with either ITGA6-Fc or FAM19A1-4-Fc protein. The phage antibody pools from Example 1 were then added to the plates, and ELISA was performed directly. M13 phage #38 (directed to an undisplayed antibody) was used as a negative control.

[0505] like Figure 1As shown, anti-FAM19A1 phage antibodies were successfully enriched in the scFv-phage antibody pool during the third round of bio-particle screening.

[0506] Next, based on the polyphage ELISA results, approximately 1000 monoclonal antibodies exhibiting high binding capacity were selected from the third round of biological panning. These monoclonal antibodies were cultured in 96-well plates and infected with helper phages. The monomeric scFv-phages were then transferred to immunoassay plates coated with FAM19A1-Fc protein for direct ELISA. To demonstrate the specificity of binding to FAM19A1, immunoassay plates coated with ITGA6-Fc protein (a non-specific antigen control) were also used.

[0507] like Figure 2 As shown, it was found that the monomeric scFv-phage clone only binds to FAM19A1-Fc, thus confirming the specificity of the scFv-phage clone.

[0508] Next, to group the selected positive scFv-phage clones, colony PCR was performed using a set of primers capable of amplifying scFv. The amplified PCR samples were treated with BstNI, and then antibody diversity was assessed by running these samples on an 8% DNA polyacrylamide gel.

[0509] like Figure 3 As shown, based on the PCR fragment analysis results, positive scFv-phage clones can be divided into 7 groups, all of which have previously been shown to bind strongly to FAM19A1-Fc but not to ITGA6-Fc.

[0510] Next, to confirm that the sc-Fv phage in each of the seven groups did not bind to other antigens, additional ELISA binding assays were performed using other antigens (C-Fc, hRAGE-Fc, CD58-Fc, ITGA6-Fc, and AIRTR) (as described above).

[0511] like Figure 4 As shown, among the seven clones tested, clones 1A11, 1C1, 2G7, and 3A8 showed the least binding to non-FAM19A1 antigens. Sequence analysis revealed that these four clones all had unique amino acid sequences.

[0512] Example 3: Production of anti-FAM19A1 IgG1 antibody

[0513] To convert the four selected monoclonal phage antibodies from scFv into 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 5AThe plasmids containing both heavy and light chains were then co-transfected into HEK 293F cells for 6 days. The antibodies produced during these 6 days were then purified using protein A affinity chromatography. After purification, the antibodies were separated using glycine buffer, and the resuspending buffer was replaced with PBS. The purified antibodies were quantified using BCA and nanotiter methods. Under both reducing and non-reducing conditions, 5 μg of each of the four antibodies were analyzed by SDS-PAGE to confirm the purity and migration rate of the purified proteins. Figure 5B As shown, the four anti-FAM19A1 antibody clones are approximately 150 kDa or larger under non-reducing conditions. Antibody yields ranged from approximately 11 mg / L (2G7 clone) to 90.5 mg / L (1C1 clone). Figure 5C ).

[0514] The affinity of the four anti-FAM19A1 antibody clones was also assessed using ELISA. Figure 5D As shown, at all tested concentrations, the 1C1 clone exhibited the greatest affinity for FAM19A1.

[0515] Example 4 Epitope Plotting Analysis

[0516] To further characterize the anti-FAM19A1 antibody clones, epitope mapping analysis was performed. In short, the amino acid sequences of different FAM19 family members (i.e., FAM19A1-5) were compared, and seven regions in the FAM19A1 protein with the greatest differences from other members of the FAM19A family (i.e., FAM19A2-5) were identified. The amino acid sequences in these regions were replaced with the consensus sequences of the corresponding regions in the FAM19A2-5 protein to generate the M1-M7 mutants. See Table 10.

[0517] Table 10. Amino acid sequences of wild-type FAM19A1 and M1-M7 mutants

[0518]

[0519] To assess binding, ELISA plates were plated with 500 ng of mutant M1-M7 or wild-type FAM19A1 protein at 4°C overnight, followed by two washes with 1X PBS. The plates were then blocked for 1 hour at room temperature with blocking buffer (100 μL / well). Different anti-FAM19A1 antibody clones (1A11, 1C1, D6, E1, and F41H5; 1 μg) were then added to the appropriate wells of the ELISA plates and incubated for 1 hour at room temperature. After washing, anti-hKappa-HRP antibody (1:2000) was added to the wells and incubated for 30 minutes at room temperature. A color change reaction was induced by adding TMB substrate. The reaction was stopped with 50 μL of sulfuric acid (2N H2SO4), and the degree of color change was detected using a 96-well microplate reader (Molecular Device) at 450 nm with a reference wavelength of 620 nm.

[0520] like Figure 6 As shown, the anti-FAM19A1 antibody clone 1C1, which previously exhibited the highest FAM19A1 binding affinity, failed to bind to the FAM19A1 mutant M6. As shown in Table 8 (above), the M6 ​​mutant contains substitutions at amino acid residues D112N, M117S, A119S, T120S, and N122H. This result indicates that these residues are important binding epitopes for the anti-FAM19A1 antibody clone 1C1.

[0521] Example 5: Expression analysis of FAM19A1

[0522] To better characterize the expression pattern of FAM19A1, RT-PCR was used to measure FAM19A1 mRNA levels in different mouse tissues. Briefly, total RNA was 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 a single-step guanidine thiocyanate-phenol-chloroform method, as previously described. See Chomczynski, P., et al., Anal Biochem 162(1):156-9 (1987). Then, 1 μm of each RNA sample was reverse transcribed using Maloney Murine Leukemia Virus (M-MLV) Reverse Transcriptase (Promega, Madison, WI). Next, the cDNA amplification 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'.

[0523] like Figure 7 As shown, FAM19A1 mRNA was observed in all tested brain regions. However, in peripheral tissues, expression was not observed or was relatively low compared to brain regions. This result indicates that FAM19A1 is primarily expressed in the central nervous system.

[0524] Example 6: Development of FAM19A1 LacZ gene knock-in (KI) mice

[0525] To further characterize the expression and function of FAM19A1, transgenic mice with a lacZ reporter gene inserted into the FAM19A1 gene were constructed. In short, a targeting vector containing the LacZ sequence of FAM19A1 was constructed. Figure 8A The target vector was transferred to embryonic stem (ES) cells via electroporation. Integration of the target vector was verified by genotyping and chromosome counting of the transgenic ES cells. Confirmed ES cells were injected into blastocysts and transferred to the uterus of female recipient mice. Germplasm transfer experiments were conducted in the first generation of chimeric mice to obtain stable germline expression. The resulting FAM19A1 LacZ KI chimeric mice were backcrossed into a C57BL / 6J genetic background. Both strains were maintained by mating heterozygous male mice with broad-type C57BL / 6J female mice. To obtain homozygous FAM19A1 LacZ KI mice, heterozygous male mice were mated with heterozygous female mice.

[0526] In current animal models, it is expected that the insertion will occur in the lacZ gene (after the start codon of exon 2 of the FAM19A1 gene, see below). Figure 8A This will result in the expression of beta-galactosidase, instead of FAM19A1, where FAM19A1 should be present. Therefore, it is expected that inserting the target vector into both alleles of the FAM19A1 gene will lead to the complete elimination of FAM19A1. Mice with the lacZ gene inserted into both alleles were designated as homozygous FAM19A1 LacZ knock-in (FAM19A1 LacZ Knock-In, i.e., "FAM19A1 LacZ KI(- / -)").

[0527] To confirm the deletion of the FAM19A1 gene at the genomic level, DNA PCR was performed using primers specifically targeting the inserted LacZ gene sequence. To confirm the complete deletion of the FAM19A1 gene at the protein level in these mice, Western ink dot assay (“WB”) and immunohistochemistry (“IHC”) were performed using polyclonal anti-FAM19A1 and / or anti-beta-galactosidase antibodies.

[0528] For Western blotting (WB) analysis, cortical and hippocampal regions of adult mice were isolated and lysed using a buffer containing 50 mM Tris-HCl (pH 7.5), 0.1% sodium dodecyl sulfate (SDS), and a protein inhibitor cocktail (Roche Applied Sciences). Protein content in the lysate was quantified using BioRad Bradford Protein Assay Reagent (BioRad) and resolved on an SDS-polyacrylamide gel. The lysed proteins were transferred to a nitrocellulose blot membrane in a Bio-Rad Trans-Blot electrophoresis system (Richmond, CA), and the blot was blocked at RT for 30 min in a triple-buffered saline solution containing 0.3% Tween 20 and 5% skim milk. The blot was incubated with primary antibodies for 3 h, followed by incubation at RT for 1 h with secondary antibodies bound to horseradish peroxidase. Immunoreaction bands were visualized by exposure to X-ray film after using GE Healthcare ECL reagents. The antibodies and their dilution factors are as follows: 1:500 rabbit polyclonal anti-FAM19A1 (laboratory-produced), 1:2000 β-actin (ab8227, Abcam), and 1:5000 HRP-conjugated anti-rabbit (Jackson ImmunoReserch Laboratories, WestGrove, PA).

[0529] For IHC analysis, animals were perfused with 4% paraformaldehyde in phosphate-buffered saline (PBS). Brains were isolated and post-fixed overnight in the same fixative. The brains were then cryoprotected with 30% sucrose and PBS, and serial transverse sections were prepared at 40 μm using Cryostat (Leica). Sections were blocked in PBS with 3% BSA and 0.1% Triton X-100 at room temperature (RT) for 30 minutes. Primary antibodies were applied overnight at 4°C, followed by application of appropriate fluorescently conjugated secondary antibodies at RT for 30 minutes using Hoechst 33342 (Invitrogen). Antibodies and their dilutions were as follows: 1:500 rabbit polyclonal anti-FAM19A1 (laboratory-produced), 1:500 β-galactosidase (ab9391, Abcam), and 1:500 fluorescently conjugated anti-rabbit and anti-chicken (Life Technologies). Images were obtained using a confocal microscope (TCS SP8, Leica).

[0530] like Figure 8BAs shown, genomic DNA PCR confirmed the successful insertion of the LacZ gene in FAM19A1 LacZ KI(- / -) animals. Given that the inserted LacZ gene sequence has its own stop codon and a poly-A tail, the final product of this gene construction is a complete β-galactosidase, without any part of FAM19A1. The interruption of the FAM19A1 gene in FAM19A1 LacZ KI mice was confirmed by RT-PCR. Figure 8C Moreover, such as Figures 8D to 8F As shown, compared with wild-type animals, FAM19A1 protein expression was significantly reduced in heterozygous mice (FAM19A1 LacZ KI(+ / -)) in the cerebral cortex (CTX) and hippocampus (HIP). FAM19A1 protein was not detected in homozygous mice (FAM19A1 LacZ KI(- / -)). Figure 8G and Figure 8H This study confirmed that the disruption of FAM19A1 protein expression is directly related to mRNA levels. These results confirm the complete deletion of the FAM19A1 gene in FAM19A1 LacZ KI(- / -) mice.

[0531] Example 7: Expression of FAM19A1 in the brains of embryonic and postnatal mice

[0532] To better understand the function of FAM19A1, X-gal staining (an enzymatic method based on beta-galactosidase activity) was used to assess the expression patterns and timing of FAM19A1. Because complete knockout of the FAM19A1 gene from the developmental stage may lead to distorted brain structures and thus alter the outcome, animals with FAM19A1 LacZ KI(+ / -) (heterozygous) were used.

[0533] X-gal staining of embryos, postnatal brains, and adult eyes: For embryonic X-gal staining, pregnant mice were killed by cervical dislocation, and the embryos were isolated. Whole embryos of E12.5 were fixed in PBS at 4°C for 15 minutes with 4% paraformaldehyde and 0.2% glutaraldehyde. For embryos of E14.5 and above, the head was cut open and the epidermis removed. The embryonic head was fixed in the same fixative at 4°C for 1–2 hours. For postnatal brains and adult eyes, the brain and eyes were separated from the skull and fixed in the same fixative at 4°C for 1–2 hours. The fixed tissue was washed twice with PBS for 5 minutes each time, and then stained with X-gal staining solution (1 mg / ml X-gal, 2 mM MgCl2, 5 mM EGTA, 5 mM potassium ferricyanide, 5 mM potassium ferricyanide, 0.01% sodium deoxycholate, 0.02% Nonidet-P40, 0.1 M phosphate buffer, pH 7.3) for 24-48 hours in the dark at 37°C. The stained tissue was then fixed overnight in PBS with 4% paraformaldehyde at 4°C, washed, and then whole-brain images were obtained.

[0534] For X-gal stained sections, the stained whole brain or eyes were cryoprotected with 30% sucrose in PBS and sectioned at 40 μm using Cryostat (Leica). Nuclear Fast Red (H-3403, VECTOR) was used as reverse staining in appropriate locations. Images of the sections were captured using a sliding scanner (Axio Scan Z1, Zeiss).

[0535] X-gal staining of adult brains: Animals were perfused with 4% paraformaldehyde and 0.2% glutaraldehyde in phosphate-buffered saline (PB). Brains were isolated and post-fixed in 0.2% glutaraldehyde in PB at 4°C for 24 hours. They were then cryoprotected in PBS with 30% sucrose and serially sectioned at 40 μm using Cryostat (Leica). The tissue sections were then incubated in X-gal staining solution (1 mg / ml, 2 mM MgCl2, 5 mM EGTA, 5 mM potassium ferricyanide, 0.01% sodium deoxycholate, 0.02% Nonidet-P40, pH 7.3, 0.1 M phosphate-buffered saline) in the dark at 37°C for 24–48 hours. Images of the sections were captured using a sliding scanner (Axio Scan Z1, Zeiss).

[0536] like Figure 9A and Figure 9BAs shown, FAM19A1 is initially expressed in limited cortical regions during early embryonic development (as evidenced by beta-galactosidase-positive staining). No signs of FAM19A1 expression are observed by day 12.5 (E12.5). However, from day 14.5 (E14.5), β-galactosidase activity (i.e., FAM19A1 expression) is observed in lateral cortical regions, with particularly strong expression in the rostral region. These stained areas are believed to be the premature piriform cortex (Cpf) and entorhinal cortex (Cen). Figure 10A .

[0537] After birth, neocortical expression of FAM19A1 becomes more pronounced. Figure 9C In the early postnatal period, FAM19A1 is initially observed in the somatosensory, visual, and auditory cortices, and is persistently expressed in the piriform cortex and entorhinal cortex. Over time, FAM19A1 expression expands to other neocortical areas. By day 14.5 postnatal (P14.5), neocortical β-galactosidase (i.e., FAM19A1) expression is detected in a cortex-specific manner. Figure 10B In addition, X-gal staining signals were found in the marginal regions, including the posteromedial cortical amygdala (PMCo), hippocampus, and amygdala. Figure 10B These results suggest that FAM19A1 is likely expressed in differentiated neurons during neural development, as its expression pattern is particularly restricted to the cortex and limbic system regions. Furthermore, FAM19A1 was not detected in stem cell-rich areas, such as the ventricular or subventricular regions, indicating that it may not be involved in NSC proliferation.

[0538] Example 8: Expression of FAM19A1 in the brain of adult mice

[0539] To assess whether FAM19A1 plays a role in neural activity, the expression pattern of FAM19A1 was mapped in adult FAM19A1 LacZ KI heterozygous mice.

[0540] In the adult mouse brain, X-gal staining showed that FAM19A1 was expressed in all cortical regions. Figure 9C Immunohistochemistry and X-gal staining showed that X-gal precipitates and β-galactosidase co-localized with CUX1, a pyramidal neuron marker in cortical layer 2-3 (L2-3), and CTIP2, a pyramidal neuron marker in cortical layer 5b (L5b), respectively. This indicates that FAM19A1 is primarily expressed in pyramidal neurons in a layer-specific manner. Figure 11A , Figure 11B and Figure 11C(Figure iv)). Furthermore, X-gal signals are emitted in the corticospinal tract, including the internal capsule (ic), cerebral infarction (cp), and pyramidal tract (py), further indicating the presence of FAM19A1 in pyramidal neurons of L5b in the primary motor cortex. Figure 12 (Figures G and I).

[0541] The presence of FAM19A1 in specific sensory circuits was also investigated. In the olfactory neural circuit, the expression of β-galactosidase and FAM19A1 mRNA was barely observed in the olfactory bulb (OB). Figure 8F However, the FAM19A1 protein was detected using the Western ink dot method. Figure 8F and Figure 8G The detected FAM19A1 protein may have been released from neurons in other olfactory-related brain regions, including the anterior olfactory nucleus (AO), CPf, and cortical amygdala, which exhibit X-gal signaling positivity. Figure 8D , Figure 8E and Figure 8H For the visual neural circuit, the thalamus and lateral geniculate nucleus (LGN) of the visual neural circuit do not express β-galactosidase, but the visual lamina and visual cortex of the superior colliculus (Op) do express β-galactosidase. Figure 11C , Drawing board vii; Figure 9C This suggests that FAM19A1 may be involved in superior colliculus-dependent visual information processing and eye movement control. β-galactosidase expression was also observed in several regions associated with auditory neural circuits, including the medial geniculate nucleus (MGN), dorsal cochlear nucleus (DC), and auditory cortex. Figure 11C , drawing board ix; Figure 12 (Figure E)

[0542] FAM19A1 is prominently expressed in marginal regions, including the hippocampus and amygdala. In the hippocampus, β-galactosidase is expressed in the CA region but not in the dentate gyrus (DG). Figure 11C (Figure iv). With the expression of β-galactosidase at CEn, the expression of hippocampal FAM19A1 may indicate the role of FAM19A1 in the hippocampal trisynaptic circuit. Figure 11C (Figures iv and viii). Between the amygdala nuclei, β-galactosidase is expressed only in the basolateral nuclei, including the lateral amygdala nucleus (LaDL) and the basolateral amygdala nucleus (BLA). Figure 11C (Figure v). Furthermore, FAM19A1 expression was detected in the PMCo and amygdala transition region (Apir). Figure 11C (Figure vi), these areas are considered to be directly related to BLA, Cen and CPf.

[0543] β-galactosidase is also expressed in some hypothalamic nuclei, including the medial preoptic nucleus (MPOM), lateral preoptic area (LPO), and lateral hypothalamic nucleus (VMH). Figure 12 (Figures B and C). As part of the limbic system, the hypothalamus is considered an intermediary between the central nervous system and the endocrine system. Therefore, the data presented here suggest that FAM19A1 may contribute to endocrine homeostasis. The lateral septal nucleus (LS), another brain region extensively connected to the limbic system, also shows expression of β-galactosidase (…). Figure 11C (Figure iii)

[0544] In situ hybridization of 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 basal layer of the amygdala. Figure 13 This FAM19A1 mRNA expression pattern is consistent with the expression pattern of β-galactosidase in the brains of FAM19A1 LacZKI mice, confirming the FAM19A1 expression profile observed in FAM19A1 LacZKI mice. Figure 11C (See Figures ii, iv, and v). Furthermore, the observed FAM19A1 expression patterns were consistent with open-source single-cell RNA sequencing databases of wild-type mouse brains. 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.

[0545] Example 9: Comparison of morphological differences between wild-type and FAM19A1- / - animals

[0546] Since early deficiency of FAM19A1 may lead to abnormal brain development, morphological differences were investigated in homozygous FAM19A1LacZKI (FAM19A1- / -), allosynthetic FAM19A1LacZKI (FAM19A1+ / -), and WT mice.

[0547] In general, at birth, FAM19A1- / - mice show a Mendelian frequency of approximately 24-25% from heterozygous parents, with similar sex ratios. Figure 14 No significant differences in appearance were observed between neonatal genotypes immediately after birth; however, FAM19A1- / - mice (both males and females) showed significantly lower body weight compared to wild-type controls. Figure 15A and Figure 15B ).

[0548] The total length and width of the adult brains of WT and FAM19A1- / - mice are similar. Figure 15C , Figure 15E and Figure 15G The cerebral cortex of FAM19A1- / - mice was longer than that of WT mice. Figure 15F The reduction of genes specifically expressed in the cortex can lead to abnormal cortical assemblies. However, in the FAM19A1- / - mice disclosed in this paper, the volume of the cerebral cortex remained unaffected. Figure 16A and Figure 16B Furthermore, a cursory examination of X-gal stained brain sections from FAM19A1- / - mice revealed no significant structural abnormalities (data not shown). The thickness of all neocortical regions in FAM19A1- / - mice was not significantly reduced. Figure 15H , Figure 15I and Figure 15J However, in terms of cortical proportions, compared to WT mice, FAM19A1- / - mice showed a decrease in both the L4 layer of the visual cortex and the L6 layer of the motor cortex. Figures 17A to 17F ).

[0549] Although these changes in cortical thickness may be a result of abnormal cellular structure, no significant differences in the neuronal and glial cell populations of the cortex were observed between FAM19A1- / - and WT mice. Figures 18A-18D (and 19A-19E). Furthermore, no significant abnormalities were observed in the morphology of neurons and glial cells (data not shown). In summary, these findings suggest that global FAM19A1 ablation reduces weight gain, mildly alters the structure of the neocortex, but does not significantly affect the cell type composition of the cortex.

[0550] Example 10: Analysis of the effect of FAM19A1 on ADHD

[0551] As previously mentioned, FAM19A1 is expressed in several regions of the limbic system, including the forelimb cortex and amygdala. Figure 11C The FAM19A1 mice (Figures i and v) are known to be involved in emotion processing. Therefore, to assess the effects of FAM19A1 depletion on anxiety and depression, the elevated maze (EPM), open field test (OFT), and tail suspension test (TST) were performed using FAM19A1- / - mice as described in the previous examples. In particular, male mice were used.

[0552] The EPM test was conducted as follows. The elevated maze (EPM) had four vertical arms, two open (5x30 cm) and two closed (5x30 cm) with walls 20 cm high. The maze was 50 cm above the ground. The test animals were placed individually 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, United States). The number of entries into the open arms, the time spent in the open arms, the number of times the center was crossed, and the total distance traveled were recorded. One entry was defined as having all four paws inside the arm.

[0553] OFT was conducted as follows. The OFT apparatus, measuring 40 x 40 x 40 cm, was made of opaque plastic. The test area was defined as 30% of the central area and surrounding boundary area. Test animals were placed individually in the center of the test area, and their behavior was recorded for 10 minutes. The time spent and the percentage of time spent in the central area were scored, and the total distance traveled was determined using an ANY-maze video tracking program (Stoelting).

[0554] TST was performed as follows: Mice were individually suspended by their tails in a box (36.5 x 30.5 x 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 ceasing agitation and attempts to escape.

[0555] In the EPM test, compared with WT mice, FAM19A1- / - mice spent less time in the open arm ( Figure 20A ) and the increase in total distance traveled ( Figure 20B In the OFT test, FAM19A1- / - mice and WT mice spent similar amounts of time in the center of the OFT test area, but FAM19A1- / - mice had a higher total distance traveled. Figure 20C , Figure 20D and Figure 20E On TST, FAM19A1- / - mice showed lower immobility than WT mice. Figure 20F ).

[0556] The above results indicate that inhibiting the activity of FAM19A1 can lead to increased activity, which in turn can help treat anxiety or depression-related disorders.

[0557] Example 11: Analysis of the effect of FAM19A1 on memory

[0558] It is known that short-term memory (STM), particularly spatial working memory, involves the interaction between CA1 and CA3 in the hippocampus and CEn. For example... Figure 11C As shown in Figures iv and viii, FAM19A1 is highly expressed in these regions, suggesting that FAM19A1 may play a role in memory formation. To assess the potential role of FAM19A1 in memory (short-term and long-term), a Y-maze test was conducted as follows. The Y-maze test area had three identical arms, 30 cm long, 5 cm wide, and 20 cm high walls. The test animals were placed individually in the center, and the order of entry into the arms and the total walking distance were recorded and analyzed over 5 minutes using the ANY-maze video tracking program (Stoelting). The percentage of spontaneous change was calculated by dividing the number of tests including entry into all three arms (ABC, ACB, BAC, BCA, CAB, CBA) by the maximum possible change (equivalent to the total number of arms entered minus 2) and multiplying by 100.

[0559] like Figure 20G As shown, no significant differences in spontaneous changes were observed between FAM19A1- / - and WT mice. However, compared with WT control animals, FAM19A1- / - mice showed a significant increase in total walking distance. Figure 20H This result confirms the findings of the EPM and OFT experiments (see Example 10), demonstrating that inhibition of FAM19A1 can lead to increased activity.

[0560] In addition, a novel object recognition (NOR) test was conducted to examine potential deficiencies in object recognition memory. In short, the test area was 40 cm wide × 40 cm high × 40 cm long. T-75 flasks filled with sand and stacked plastic bricks (7 cm wide x 13 cm h...

Claims

1. An anti-FAM19A1 antibody or its antigen-binding fragment, It exhibits characteristics selected from the following: (a) with K of 10 nM or less D It binds to soluble human FAM19A1 and is measured by ELISA; (b) with K of 10 nM or less D Binding to human FAM19A1 bound to the membrane, as measured by ELISA; or (c) Both (a) and (b) The anti-FAM19A1 antibody comprises heavy chains CDR1, CDR2, and CDR3, and light chains CDR1, CDR2, and CDR3, wherein... (i) The heavy chain CDR1 is composed of the amino acid sequence shown in SEQ ID NO: 10, the heavy chain CDR2 is composed of the amino acid sequence shown in SEQ ID NO: 11, the heavy chain CDR3 is composed of the amino acid sequence shown in SEQ ID NO: 12, the light chain CDR1 is composed of the amino acid sequence shown in SEQ ID NO: 13, the light chain CDR2 is composed of the amino acid sequence shown in SEQ ID NO: 14, and the light chain CDR3 is composed of the amino acid sequence shown in SEQ ID NO: 15; (ii) The heavy chain CDR1 is composed of the amino acid sequence shown in SEQ ID NO: 4, the heavy chain CDR2 is composed of the amino acid sequence shown in SEQ ID NO: 5, the heavy chain CDR3 is composed of the amino acid sequence shown in SEQ ID NO: 6, the light chain CDR1 is composed of the amino acid sequence shown in SEQ ID NO: 7, the light chain CDR2 is composed of the amino acid sequence shown in SEQ ID NO: 8, and the light chain CDR3 is composed of the amino acid sequence shown in SEQ ID NO: 9; (iii) The heavy chain CDR1 is composed of the amino acid sequence shown in SEQ ID NO: 16, the heavy chain CDR2 is composed of the amino acid sequence shown in SEQ ID NO: 17, the heavy chain CDR3 is composed of the amino acid sequence shown in SEQ ID NO: 18, the light chain CDR1 is composed of the amino acid sequence shown in SEQ ID NO: 19, the light chain CDR2 is composed of the amino acid sequence shown in SEQ ID NO: 20, and the light chain CDR3 is composed of the amino acid sequence shown in SEQ ID NO: 21; or (iv) The heavy chain CDR1 is composed of the amino acid sequence shown in SEQ ID NO: 22, the heavy chain CDR2 is composed of the amino acid sequence shown in SEQ ID NO: 23, the heavy chain CDR3 is composed of the amino acid sequence shown in SEQ ID NO: 24, the light chain CDR1 is composed of the amino acid sequence shown in SEQ ID NO: 25, the light chain CDR2 is composed of the amino acid sequence shown in SEQ ID NO: 26, and the light chain CDR3 is composed of the amino acid sequence shown in SEQ ID NO:

27.

2. The anti-FAM19A1 antibody according to claim 1, wherein, The anti-FAM19A1 antibody comprises: a heavy chain variable domain consisting of the amino acid sequence shown in SEQ ID NO: 30; and a light chain variable domain consisting of the amino acid sequence shown in SEQ ID NO:

31.

3. The anti-FAM19A1 antibody according to claim 1, wherein, The anti-FAM19A1 antibody comprises: a heavy chain variable domain consisting of the amino acid sequence shown in SEQ ID NO: 28; and a light chain variable domain consisting of the amino acid sequence shown in SEQ ID NO:

29.

4. The anti-FAM19A1 antibody according to claim 1, wherein, The anti-FAM19A1 antibody comprises: a heavy chain variable domain consisting of the amino acid sequence shown in SEQ ID NO: 32; and a light chain variable domain consisting of the amino acid sequence shown in SEQ ID NO:

33.

5. The anti-FAM19A1 antibody according to claim 1, wherein, The anti-FAM19A1 antibody comprises: a heavy chain variable domain consisting of the amino acid sequence shown in SEQ ID NO: 34; and a light chain variable domain consisting of the amino acid sequence shown in SEQ ID NO:

35.

6. The anti-FAM19A1 antibody according to claim 1, wherein it is a chimeric antibody, a human antibody, or a humanized antibody.

7. The anti-FAM19A1 antibody according to claim 1, comprising: Fab, Fab', F(ab')2, Fv, or single-chain Fv (scFv).

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

9. The anti-FAM19A1 antibody according to claim 8 is an IgG1 antibody.

10. The anti-FAM19A1 antibody according to claim 1, wherein, The anti-FAM19A1 antibody further includes a constant region that does not have Fc function.

11. A pharmaceutical composition comprising the anti-FAM19A1 antibody according to claim 1 and a pharmaceutically acceptable carrier.

12. A nucleic acid, comprising: The nucleotide sequence encoding the anti-FAM19A1 antibody of claim 1.

13. A carrier, comprising: The nucleic acid according to claim 12, and one or more promoters operatively linked to said nucleic acid.

14. A cell comprising: The nucleic acid according to claim 12 or the vector according to claim 13.

15. A pharmaceutical composition for treating central nervous system (CNS) related diseases or disorders, comprising the anti-FAM19A1 antibody of claim 1, the nucleic acid of claim 12, the vector of claim 13, the cell of claim 14, or any combination thereof.

16. The pharmaceutical composition according to claim 15, wherein, The CNS-related diseases or disorders are selected from anxiety disorders, depression, post-traumatic stress disorder (PTSD), bipolar disorder, attention deficit / hyperactivity disorder (ADHD), autism, schizophrenia, neuropathic pain, glaucoma, addiction, arachnoid cysts, hypnotics, encephalitis, epilepsy, locked-in syndrome, meningitis, migraine, multiple sclerosis, myelopathy, Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Barten's disease, tic disorders, traumatic brain injury, spinal cord injury, stroke, essential tremor, Parkinson's tremor, dystonia, intellectual disability, brain tumors, or combinations thereof.

17. A reagent kit comprising: The anti-FAM19A1 antibody according to claim 1, and instructions for use.

18. A method for producing an anti-FAM19A1 antibody, comprising: The cells according to claim 14 are cultured under appropriate conditions, and the anti-FAM19A1 antibody is isolated.

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