Use of anti-FAM19A5 antagonists for the treatment of hearing impairment

Abstract

The present invention relates to the use of FAM19A5 antagonists (e.g., anti-FAM19A5 antibodies or LRRC4 family mimetic molecules) to treat a wide range of hearing disorders. The present invention also provides methods of using such antagonists to improve one or more properties of ribbon synapses.

Description

Detailed Description of the Invention

[0001] [Technical Field] CROSS-REFERENCE TO RELATED APPLICATIONS This PCT application claims priority to U.S. Provisional Application No. 63 / 486,187, filed February 21, 2023, and U.S. Provisional Application No. 63 / 595,942, filed November 3, 2023, each of which is incorporated herein by reference in its entirety.

[0002] Reference to an electronically submitted sequence listing The contents of the Sequence Listing have been submitted electronically with this application (Name: 3763_022PC02_Seqlisting_ST26.xml; Size: 340,155 bytes; Creation Date: Feb. 20, 2024), and are hereby incorporated by reference in their entirety.

[0003] Technical Field The present invention provides methods for treating hearing impairment in a subject (e.g., a human) using an antagonist (e.g., an antibody or an LRRC4 family mimetic molecule) that specifically targets family with sequence similarity 19, member A5 (FAM19A5), or a composition comprising the antagonist.

[0004] [Background technology] Hearing impairment is a major global health problem affecting more than 275 million people worldwide, with significant social and economic impacts. The incidence of hearing loss is rapidly increasing due to factors such as increased noise exposure and an aging population. Currently, there is a significant unmet medical need, as there are no approved pharmaceutical treatments. In particular, there is a need to provide effective methods for the prevention and subsequent treatment of hearing loss that allow for immediate and long-term maintenance of preventative and / or therapeutic benefits.

[0005] Summary of the Invention The present invention provides methods for treating and / or preventing hearing impairment in a subject in need thereof, comprising administering to the subject an antagonist (FAM19A5 antagonist) against a family with sequence similarity 19, member A5 (FAM19A5) protein. In some aspects, the hearing impairment comprises sensorineural hearing loss. In some aspects, the sensorineural hearing loss is associated with tinnitus. In some aspects, the sensorineural hearing loss is not associated with tinnitus. In some aspects, the sensorineural hearing loss comprises ototoxic drug-induced hearing loss, noise-induced hearing loss, presbycusis, sudden hearing loss, or a combination thereof.

[0006] The present invention also provides methods for reducing and / or preventing ribbon synapse damage in the inner ear of a subject in need thereof, comprising administering to the subject an antagonist (FAM19A5 antagonist) against the family with sequence similarity 19, member A5 (FAM19A5) protein. In some aspects, the ribbon synapse damage comprises a decrease in the number of ribbon synapses when compared to a matched subject without ribbon synapse damage in the inner ear (e.g., a matched subject with normal hearing). In some aspects, the ribbon synapse damage comprises abnormal ribbon synapse function.

[0007] The present invention provides a method for inducing ribbon synapse formation in the inner ear of a subject in need thereof, comprising administering to the subject an antagonist (FAM19A5 antagonist) against the family with sequence similarity 19, member A5 (FAM19A5) protein.

[0008] In some aspects, after the administration, the number of ribbon synapses is increased in the inner ear of the subject as compared to a reference subject (e.g., the subject before administration and / or a matched subject that has not received the administration). In some aspects, the number of ribbon synapses is increased by at least about 1-fold, at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, or at least about 50-fold as compared to the reference subject.

[0009] The present invention provides a method for improving ribbon synapse function in the inner ear of a subject in need thereof, comprising administering to the subject an antagonist (FAM19A5 antagonist) against the Family with Sequence Similarity 19, Member A5 (FAM19A5) protein.

[0010] In some aspects, the function includes the ability to release one or more neurotransmitters in response to signals from inner hair cells. In some aspects, after the administration, ribbon synapse function is improved by at least about 1-fold, at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, or at least about 50-fold.

[0011] The present invention also provides methods for reducing expression and / or activity of an antagonist to Family with sequence similarity 19, member A5 (FAM19A5) protein (FAM19A5 antagonist) in spiral ganglion neurons in a subject in need thereof, comprising administering to the subject an antagonist to FAM19A5 protein (FAM19A5 antagonist). In some aspects, following said administration, the expression and / or activity of FAM19A5 protein in spiral ganglion neurons of the subject is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%.

[0012] In connection with each of the provided methods, in some aspects, the subject has a hearing impairment. In some aspects, the hearing impairment comprises sensorineural hearing loss. In some aspects, the sensorineural hearing loss is associated with tinnitus. In some aspects, the sensorineural hearing loss is not associated with tinnitus. In some aspects, the sensorineural hearing loss comprises ototoxic drug-induced hearing loss, noise-induced hearing loss, presbycusis, sudden hearing loss, or a combination thereof.

[0013] In connection with any of the methods provided herein, in some aspects, the FAM19A5 antagonist comprises (i) an antisense oligonucleotide, siRNA, shRNA, miRNA, dsRNA, aptamer, PNA (peptide nucleic acid), or a vector comprising the same; (ii) an antibody or antigen-binding fragment thereof that specifically binds to FAM19A5 protein (an "anti-FAM19A5 antibody"); (iii) a polynucleotide encoding the anti-FAM19A5 antibody; (iv) a leucine-rich repeat-containing 4 (LRRC4) family mimetic molecule; (v) an LRRC4 family peptide; or (vi) any combination of (i) to (v).

[0014] In some aspects, the anti-FAM19A5 antibody exhibits a property selected from the following: (a) a K of 10 nM or less as measured by enzyme-linked immunosorbent assay (ELISA). D (b) binds to soluble human FAM19A5 with a K of 10 nM or less as measured by ELISA D (c) all of (a) and (b); and (c) all of (a) and (b).

[0015] In some aspects, the anti-FAM19A5 antibody cross-competes with a reference antibody for binding to a human FAM19A5 epitope, and the reference antibody is selected from the group consisting of each of the antibodies in Tables 2-5. In some aspects, the anti-FAM19A5 antibody comprises a heavy chain CDR1, CDR2, and CDR3, and a light chain CDR1, CDR2, and CDR3, wherein (i) the heavy chain CDR1 comprises a CDR1 selected from the group consisting of each CDR1 in Table 6; (ii) the heavy chain CDR2 comprises a CDR2 selected from the group consisting of each CDR2 in Table 6; (iii) the heavy chain CDR3 comprises a CDR3 selected from the group consisting of each CDR3 in Table 6; (iv) the light chain CDR1 comprises a CDR1 selected from the group consisting of each CDR1 in Table 7; (v) the light chain CDR2 comprises a CDR2 selected from the group consisting of each CDR2 in Table 7; and / or (vi) the light chain CDR3 comprises a CDR3 selected from the group consisting of each CDR3 in Table 7. In some aspects, (i) the heavy chain CDR1 comprises the sequence of SEQ ID NO: 220, (ii) the heavy chain CDR2 comprises the sequence of SEQ ID NO: 221, (iii) the heavy chain CDR3 comprises the sequence of SEQ ID NO: 16, (iv) the light chain CDR1 comprises the sequence of SEQ ID NO: 225, (v) the light chain CDR2 comprises the sequence of SEQ ID NO: 228, and (vi) the light chain CDR3 comprises the sequence of SEQ ID NO: 227.

[0016] In some aspects, the anti-FAM19A5 antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein (i) the VH comprises a VH selected from the group consisting of each VH in Table 8; and / or (ii) the VL comprises a VL selected from the group consisting of each VL in Table 9. In some aspects, the VH comprises the sequence of SEQ ID NO:235, and the VL comprises the sequence of SEQ ID NO:240.

[0017] In some aspects, the anti-FAM19A5 antibody comprises a Fab, a Fab', a F(ab')2, an Fv, or a single-chain Fv (scFv). In some aspects, the anti-FAM19A5 antibody is selected from the group consisting of IgG1, IgG2, IgG3, IgG4, and variants thereof. In some aspects, the anti-FAM19A5 antibody is an IgG2, an IgG4, or a combination thereof. In some aspects, the anti-FAM19A5 antibody comprises an IgG2 / IgG4 isotype antibody. In some aspects, the anti-FAM19A5 antibody is a chimeric antibody, a human antibody, or a humanized antibody.

[0018] In connection with any of the methods provided herein, in some aspects, the FAM19A5 antagonist is an LRRC4 family mimetic molecule, and the LRRC4 family mimetic molecule can inhibit, reduce, and / or dissociate the interaction between the FAM19A5 protein and a member of the LRRC4 protein family. In some aspects, the LRRC4 family mimetic molecule is not an antibody or an antigen-binding fragment thereof. In some aspects, the LRRC4 family mimetic molecule comprises a polypeptide. In some aspects, the LRRC4 family mimetic molecule comprises a small molecule.

[0019] In some aspects, the LRRC4 family mimetic molecule is a small molecule of Formula I, or a pharmaceutically acceptable salt thereof:

[0020] [ka]

[0021] where: (i) R1, R2 and R3 are independently hydrogen, fluoro, chloro, bromo, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl, isopentyl, fluoromethyl, difluoromethyl, trifluoromethyl, 2-fluoroethyl, 1-fluoroethyl, 2,2-difluoroethyl, 1,2-difluoroethyl, 1,1-difluoroethyl, 2,2,2-trifluoroethyl, methoxy, ethoxy, n-propyloxy, isopropyloxy, n-butoxy, trifluoromethoxy, difluoromethyl, ... selected from fluoromethoxy, fluoromethoxy, acetyl, propionyl, n-butanoyl, isobutanoyl, n-pentanoyl, nitro, amino, N-methylamino, N-ethylamino, N,N-propylamino, N,N-dimethylamino, N-acetylamino, N-propionylamino, N-(trifluoroacetyl)amino, formyl, hydroxy, methylthio, ethylthio, n-propylthio, methylsulfonyl, ethylsulfonyl, n-propylsulfonyl, phenyl, hydroxymethyl, 1-hydroxyethyl, and 2-hydroxyethyl; (ii)

[0022] [ka]

[0023] is a single or double bond; (iii) Z is a linear or branched (C1-C8) alkyl, a linear or branched (C2-C8) alkenyl, a linear or branched (C2-C8) alkynyl, a (C3-C8) cycloalkyl, a (C5-C8) cycloalkenyl, a (3- to 8-membered) heterocycloalkyl, a (C7-C 14 )bicycloalkyl, (C7-C 14 ) bicycloalkenyl, (7- to 14-membered) heterobicycloalkyl, (C6-C 10 )aryl, (5- to 10-membered)heteroaryl, and —CH—C(O)—CH═CH—Q (wherein Q is (C3-C8)cycloalkyl, (C5-C8)cycloalkenyl, (3- to 8-membered)heterocycloalkyl, (C6-C10 )aryl and (5- to 6-membered)heteroaryl; each cycloalkyl, cycloalkenyl, heterocyclylalkyl, aryl, and heteroaryl may be independently substituted with 1, 2, 3, 4, or 5 substituents selected from (C1-C6)alkoxy, (C1-C6)alkyl, halo, (C1-C6)haloalkoxy, nitro, amino, N-methylamino, N-ethylamino, N,N-propylamino, N,N-dimethylamino, formyl, and hydroxy; and (iv) L is a single, double or triple bond; The LRRC4 family mimetic molecule is not a small molecule selected from the following or a pharmaceutically acceptable salt thereof:

[0024] [ka]

[0025] In some aspects, the LRRC4 family mimetic molecule is a small molecule of Formula II, or a pharmaceutically acceptable salt thereof:

[0026] [ka]

[0027] where: (i) R1, R2 and R3 are hydrogen, fluoro, chloro, bromo, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl, isopentyl, fluoromethyl, difluoromethyl, trifluoromethyl, 2-fluoroethyl, 1-fluoroethyl, 2,2-difluoroethyl, 1,2-difluoroethyl, 1,1-difluoroethyl, 2,2,2-trifluoroethyl, methoxy, ethoxy, n-propyloxy, isopropyloxy, n-butoxy, trifluoromethoxy, difluoromethoxy independently selected from thio, fluoromethoxy, acetyl, propionyl, n-butanoyl, isobutanoyl, n-pentanoyl, nitro, amino, N-methylamino, N-ethylamino, N,N-propylamino, N,N-dimethylamino, N-acetylamino, N-propionylamino, N-(trifluoroacetyl)amino, formyl, hydroxy, methylthio, ethylthio, n-propylthio, methylsulfonyl, ethylsulfonyl, n-propylsulfonyl, phenyl, hydroxymethyl, 1-hydroxyethyl, and 2-hydroxyethyl; (ii) Z is a linear or branched (C1-C8) alkyl, a linear or branched (C2-C8) alkenyl, a linear or branched (C2-C8) alkynyl, a (C3-C8) cycloalkyl, a (C5-C8) cycloalkenyl, a (3- to 8-membered) heterocycloalkyl, a (C7-C 14 )bicycloalkyl, (C7-C 14 ) bicycloalkenyl, (7- to 14-membered) heterobicycloalkyl, (C6-C 10 )aryl, (5- to 10-membered)heteroaryl, and —CH═CH—Q (wherein Q is (C3-C8)cycloalkyl, (C5-C8)cycloalkenyl, (3))8-membered)heterocycloalkyl, (C6-C 10)aryl and (5- to 6-membered)heteroaryl; each cycloalkyl, cycloalkenyl, heterocyclylalkyl, aryl, and heteroaryl may be substituted by 1, 2, 3, 4, or 5 substituents selected from (C1-C6)alkoxy, (C1-C6)alkyl, halo, (C1-C6)haloalkoxy, nitro, amino, N-methylamino, N-ethylamino, N-N-propylamino, N,N-dimethylamino, formyl, and hydroxy; and (iii) L is a single, double or triple bond.

[0028] In some aspects, the LRRC4 family mimetic molecule is selected from the following structural formulas or a pharmaceutically acceptable salt thereof:

[0029] [ka]

[0030] In some aspects, the LRRC4 family mimetic molecule is a small molecule of Formula III, or a pharmaceutically acceptable salt thereof:

[0031] [ka]

[0032] where: (i) R1, R2 and R3 are hydrogen, fluoro, chloro, bromo, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl, isopentyl, fluoromethyl, difluoromethyl, trifluoromethyl, 2-fluoroethyl, 1-fluoroethyl, 2,2-difluoroethyl, 1,2-difluoroethyl, 1,1-difluoroethyl, 2,2,2-trifluoroethyl, methoxy, ethoxy, n-propyloxy, isopropyloxy, n-butoxy, trifluoromethoxy, difluoromethoxy independently selected from thio, fluoromethoxy, acetyl, propionyl, n-butanoyl, isobutanoyl, n-pentanoyl, nitro, amino, N-methylamino, N-ethylamino, N,N-propylamino, N,N-dimethylamino, N-acetylamino, N-propionylamino, N-(trifluoroacetyl)amino, formyl, hydroxy, methylthio, ethylthio, n-propylthio, methylsulfonyl, ethylsulfonyl, n-propylsulfonyl, phenyl, hydroxymethyl, 1-hydroxyethyl, and 2-hydroxyethyl; (ii) Z is a linear or branched (C1-C8) alkyl, a linear or branched (C2-C8) alkenyl, a linear or branched (C2-C8) alkynyl, -Y-(C3-C8) cycloalkyl, -Y-(C5-C8) cycloalkenyl, -Y-(3- to 8-membered) heterocycloalkyl, -Y-(C7-C 14 )bicycloalkyl, -Y-(C7-C 14 )bicycloalkenyl, -Y-(7- to 14-membered)heterobicycloalkyl, -Y-(C6-C 10 )aryl and -Y-(5 to 10 membered)heteroaryl, wherein Y is a bond or a C1-C3 linear or branched alkylene, and wherein said cycloalkyl, cycloalkenyl, heterocyclylalkyl, aryl, and heteroaryl may be substituted by 1, 2, 3, 4, or 5 substituents independently selected from C1-C6 alkoxy, C1-C6 alkyl, halo, C1-C6 haloalkoxy, nitro, amino, N-methylamino, N-ethylamino, N-N-propylamino, N,N-dimethylamino, formyl, and hydroxy; (iii) L is a single, double or triple bond, and (iv) n is 0 or 1.

[0033] In some aspects, the LRRC4 family mimetic molecule is selected from the following structural formulas or a pharmaceutically acceptable salt thereof:

[0034] [ka]

[0035] In some aspects, the LRRC4 family mimetic molecule is a polypeptide comprising, consisting of, or consisting essentially of a domain of an LRRC4 protein family member, wherein the domain is capable of binding to a FAM19A5 protein, and the polypeptide is shorter than the corresponding full-length LRRC4 protein family member (SEQ ID NO:6; SEQ ID NO:9; or SEQ ID NO:84).

[0036] In some aspects, the FAM19A5 binding domain is about 10 to about 23 amino acids in length.

[0037] In some aspects, the LRRC4 family mimetic molecule comprises an amino acid sequence having the following formula (from N-terminal to C-terminal): A-(T / S)-B (Formula IV), where: (i) A comprises X1-(T / S)-(Y / F)-F-X5; where: X1 is tyrosine (Y), phenylalanine (F), valine (V), leucine (L) or isoleucine (I); (T / S) is threonine (T) or serine (S); (Y / F) is tyrosine (Y) or phenylalanine (F); and X5 is any amino acid; (ii) B comprises (V / I)-TV-(E / V); where: (V / I) is valine (V) or isoleucine (I); and (E / V) is glutamic acid (E) or valine (V).

[0038] In some aspects, the LRRC4 family mimetic molecule comprises an amino acid sequence having the following formula (from N-terminal to C-terminal): A-(T / S)-B (Formula IV), where: (i) A comprises (Y / W / M)-(T / Y)-(Y / W)-(F / Y / W)-(T / Y); where: (Y / W / M) is tyrosine (Y), tryptophan (W) or methionine (M); (T / Y) is threonine (T) or tyrosine (Y); (Y / W) is tyrosine (Y) or tryptophan (W); and (F / Y / W) is phenylalanine (F), tyrosine (Y), or tryptophan (W); (ii) B comprises X7-(T / S / Y)-X9-X10; where: X7 is valine (V), tyrosine (Y), phenylalanine (F), leucine (L), tryptophan (W), or methionine (M); (T / S / Y) is threonine (T), serine (S), or tyrosine (Y); X9 is valine (V), isoleucine (I), tyrosine (Y), phenylalanine (F), leucine (L), tryptophan (W), or methionine (M); and X10 is glutamic acid (E), aspartic acid (D), isoleucine (I), tyrosine (Y), phenylalanine (F), methionine (M), or tryptophan (W).

[0039] In some aspects, the LRRC4 family mimetic molecule comprises an amino acid sequence having the following formula (from N-terminal to C-terminal): X1-X2-X3-F-X5-T-X7-TV-X10 (Formula V), where: X1 is Y, F, V, L or I; X2 is T or S; X3 is Y or F; X5 is any amino acid; X7 is V or I; and / or X10 is E or V.

[0040] In some aspects, the LRRC4 family mimetic molecule comprises an amino acid sequence having the following formula (from N-terminal to C-terminal): X1-X2-X3-X4-X5-X6-X7-X8-X9-X10 (Formula VI), where: X1 is Y, F, V, L, I, W or M; X2 is T, S or Y; X3 is Y, F or W; X4 is F, Y or W; X5 is any amino acid (e.g., T, S, or Y); X6 is T, S or Y; X7 is V, I, Y, F, L, W or M; X8 is T, S or Y; X9 is V, I, Y, F, L, W or M; and / or X10 is E, D, V, I, Y, F, M or W.

[0041] In some aspects, X1 is Y, F, V, L, or I. In some aspects, X2 is T or S. In some aspects, X3 is Y or F. In some aspects, X4 is F. In some aspects, X5 is T or S. In some aspects, X6 is T. In some aspects, X7 is V or I. In some aspects, X8 is T. In some aspects, X9 is V. In some aspects, X10 is E or V. In some aspects, the amino acid at X2 is phosphorylated or O-glycosylated.

[0042] In some aspects, the LRRC4 family mimetic molecule comprises, consists essentially of, or consists of the sequence of SEQ ID NO: 48 (GYTYFTTVTVETLETQ). In some aspects, the LRRC4 family mimetic molecule comprises, consists essentially of, or consists of the sequence of SEQ ID NO: 47 (GYTYFTTVTVETLETQPGEE). In some aspects, the LRRC4 family mimetic molecule comprises, consists essentially of, or consists of the sequence of SEQ ID NO: 49 (GYTYFTTVTVETLETQPGEKEPPGPTTD). In some aspects, the LRRC4 family mimetic molecule comprises, consists essentially of, or consists of the sequence of SEQ ID NO: 20 (NYSFFTTVTVETTEISPEDTTRK). In some aspects, the LRRC4 family mimetic molecule comprises, consists essentially of, or consists of the sequence of SEQ ID NO: 78 (NFSYFSTVTVETMEPSQDERTTR). In some aspects, the LRRC4 family mimetic molecule comprises, consists essentially of, or consists of the sequence of SEQ ID NO: 44 (YTYFTTVTVE). In some aspects, the LRRC4 family mimetic molecule comprises, consists essentially of, or consists of the sequence of SEQ ID NO: 45 (YSFFTTVTVE). In some aspects, the LRRC4 family mimetic molecule comprises, consists essentially of, or consists of the sequence of SEQ ID NO: 46 (FSYFSTVTVE). In some aspects, the LRRC4 family mimetic molecule comprises, consists essentially of, or consists of the amino acid sequence of any one of SEQ ID NOs: 44-80. In some aspects, one or more amino acid residues are in the D-amino acid form.

[0043] In connection with any of the methods provided herein, in some aspects, the FAM19A5 antagonist is administered to the subject via intracochlear injection, intravestibular injection, intravenous administration, or intratympanic administration. In some aspects, the subject is a human. In connection with any of the methods provided herein, in some aspects, the method further comprises administering an additional therapeutic agent to the subject.

[0044] Some aspects of the present invention relate to a means for treating hearing impairment in a subject in need thereof, comprising a pharmaceutical composition comprising an antagonist (FAM19A5 antagonist) against a family member A5 (FAM19A5) protein with sequence similarity 19 and a pharmaceutically acceptable carrier. In some aspects, the hearing impairment comprises sensorineural hearing loss. In some aspects, the sensorineural hearing loss is accompanied by or does not involve tinnitus. In some aspects, the sensorineural hearing loss comprises ototoxic drug-induced hearing loss, noise-induced hearing loss, presbycusis, sudden hearing loss, or a combination thereof. In some aspects, the FAM19A5 antagonist comprises (i) an antisense oligonucleotide, siRNA, shRNA, miRNA, dsRNA, aptamer, PNA (peptide nucleic acid), or a vector comprising the same; (ii) an antibody or antigen-binding fragment thereof that specifically binds to FAM19A5 protein (an "anti-FAM19A5 antibody"); (iii) a polynucleotide encoding the anti-FAM19A5 antibody; (iv) a leucine-rich repeat-containing 4 (LRRC4) family mimetic molecule; (v) an LRRC4 family peptide; or (vi) any combination of (i) to (v).

[0045] Some aspects of the present invention relate to methods for treating hearing impairment in a subject in need thereof, comprising administering to the subject a means for antagonizing a family with sequence similarity 19, member A5 (FAM19A5) protein and a pharmaceutically acceptable carrier. In some aspects, the hearing impairment comprises sensorineural hearing loss. In some aspects, the sensorineural hearing loss is accompanied by or without tinnitus. In some aspects, the sensorineural hearing loss comprises ototoxic drug-induced hearing loss, noise-induced hearing loss, presbycusis, sudden hearing loss, or a combination thereof. In some aspects, the means comprises an antagonist against the FAM19A5 protein. In some aspects, the FAM19A5 antagonist comprises (i) an antisense oligonucleotide, siRNA, shRNA, miRNA, dsRNA, aptamer, PNA (peptide nucleic acid), or a vector comprising the same; (ii) an antibody or antigen-binding fragment thereof that specifically binds to FAM19A5 protein (an "anti-FAM19A5 antibody"); (iii) a polynucleotide encoding the anti-FAM19A5 antibody; (iv) a leucine-rich repeat-containing 4 (LRRC4) family mimetic molecule; (v) an LRRC4 family peptide; or (vi) any combination of (i) to (v).

[0046] BRIEF DESCRIPTION OF THE DRAWINGS Figure 1: Schematic diagram of the cochlear sensory epithelium with inner and outer hair cells and their afferent innervation, as revealed by histology immunostained for nerve filament and synaptic ribbon proteins. Inner hair cells (IHCs) act as mechanoelectric transducers, stimulating the sensory fibers of the cochlear nerve by releasing neurotransmitters, while outer hair cells (OHCs) act as biological motors that amplify the movement of the sensory epithelium.

[0047] Figure 2 shows FAM19A5 gene expression in spiral ganglion neurons of embryonic day 15.5 (E15.5) mice measured by in situ hybridization (see bottom row, right image). For comparison, the expression of the following genes in the same spiral ganglion neurons is also shown: Atoh1 (hair cell marker; see top row, left image), Nef1 (spiral ganglion neuron marker; see top row, right image), LRRC4B (bottom row, left image), and LRRC4C (bottom row, middle image).

[0048] Figure 3 shows FAM19A5 gene expression in spiral ganglion neurons of 7-day-old (P7) mice (top and bottom rows, see right-most images) measured by in situ hybridization at 10x magnification (top row) and 20x magnification (bottom row). For comparison, expression of Nefl (spiral ganglion neuron markers, LRRC4B and LRRC4C genes) is also shown.

[0049] Figure 4 shows the expression of FAM19A5 and LRRC4B genes in spiral ganglion neurons of P7 mice (measured using in situ hybridization). Expression of each gene is displayed at 10x (upper box) and 20x magnification (lower box).

[0050] Figures 5a to 5c show the effects of FAM19A5 antagonist administration on hearing in mice. Auditory brainstem responses (ABRs) following administration of the FAM19A5 antagonist were measured to assess the effects on hearing. Figure 5a shows a schematic diagram of the experimental design. As shown, anti-FAM19A5 antibody (1-30) or a control human IgG antibody was intravenously administered to mice once a week (30 mg / kg per dose). ABRs (i.e., hearing tests) were measured before the initial administration of the antibody and at 1 day, 2 weeks, and 8 weeks after antibody administration. Figure 5b compares ABR thresholds in mice before the initial administration of anti-FAM19A5 antibody (box) or control IgG antibody (circle). Figure 5c compares ABR thresholds in mice 2 weeks after the initial administration of anti-FAM19A5 antibody (box) or control IgG antibody (circle). In all of Figures 5b and 5c, ABR thresholds were measured at frequencies ranging from 0 kHz to 64 kHz.

[0051] Figures 6a to 6f show the therapeutic effects of a FAM19A5 antagonist on auditory brainstem response (ABR) thresholds in a mouse model of noise-induced hearing loss. Figure 6a is a schematic diagram of the experimental design. As shown, anti-FAM19A5 antibody (1-30) or a control human IgG antibody was administered intravenously to mice once a week (30 mg / kg per dose) for a total of five doses. The first dose of antibody was administered one day before noise exposure. To induce noise-induced hearing loss, animals underwent a 30-minute 105 dB transient threshold shift (TTS). ABRs (i.e., hearing tests) were measured before and at various time points after the initial antibody administration. Figures 6b, 6c, 6d, and 6e compare ABR thresholds between mice administered control human IgG (circles) or anti-FAM19A5 antibody (squares) or before noise (triangles) at the following time points: (i) before TTS exposure, (ii) 1 day after TTS exposure, (iii) 2 weeks after TTS exposure, (iv) 4 weeks after TTS exposure, and (v) 8 weeks after TTS exposure. ABR thresholds were measured at frequencies ranging from 4 kHz to 64 kHz.

[0052] Figures 7a to 7d show the therapeutic effect of a FAM19A5 antagonist on distortion product otoacoustic emission (DPOAE) thresholds in a mouse model of noise-induced hearing loss. The overall experimental design was the same as that described in Figure 6a. DPOAE threshold data were measured (i) before TTS exposure (Figure 7a), (ii) 1 day after TTS exposure (Figure 7b), (iii) 2 weeks after TTS exposure (Figure 7c), and (iv) 4 weeks after TTS exposure (Figure 7d) after antibody administration. DPOAE thresholds were measured in the frequency range from approximately 4 kHz to approximately 32 kHz.

[0053] Figures 8a and 8b show the effect of a FAM19A5 antagonist on distortion product otoacoustic emissions (DPOAE) amplitude in a mouse model of noise-induced hearing loss. The overall experimental design was identical to that described in Figure 6a. DPOAE amplitude data were measured after antibody administration 2 weeks after TTS exposure. Figure 8a compares the mean DPOAE amplitude values ​​as a function of various noise levels for each of the following groups: (i) pre-noise (triangles), (ii) control antibody treatment (circles), and (iii) anti-FAM19A5 treatment (squares). Figure 8b shows the spectral level profiles for each group.

[0054] [Figure 9] This diagram illustrates the process by which stimuli generated in the cochlea are transmitted to the auditory nerve, and the signal is then transmitted through the brainstem to the auditory cortex. Before reaching the auditory cortex, the signal is transmitted through the auditory nerve (AN), cochlear nucleus (CN), superior olivary complex (SOC), lateral lemniscus (LL), and inferior colliculus (IC).

[0055] Figures 10a and 10b illustrate how the auditory brainstem response (ABR) can be used to measure sound wavelengths through signal amplitude. Figure 10a shows how sound wavelengths pass sequentially from the cochlea to the auditory cortex, flowing into the auditory nerve (AN), cochlear nucleus (CN), superior olivary complex (SOC), lateral lemniscus (LL), and inferior colliculus (IC). For the ABR results, the amplitude and latency of the first wavelength were measured for each of the AN, CN, SOC, LL, and IC. Figure 10b shows an exemplary wave I amplitude profile as a function of noise level (i.e., tone burst level).

[0056] Figures 11a and 11b show the effect of a FAM19A5 antagonist on ABR I / O function in a mouse model of noise-induced hearing loss. The overall experimental design is the same as that described in Figure 6a. Figure 11a shows wave I amplitude before noise (left graph) and 2 weeks after noise (right graph). Figure 11b shows wave I amplitude 4 weeks after noise. Figures 11a and 11b show the following animal groups: (i) control antibody treatment (circles) and (ii) anti-FAM19A5 antibody treatment (squares). For comparison, wave I amplitude at 2 and 4 weeks before noise exposure ("BN") are also shown.

[0057] Figures 12a and 12b show the therapeutic effects of anti-FAM19A5 antibody (1-30) and control human IgG antibody administration on immunohistochemistry (IHC) analysis in the mid-cochlear region 8 weeks after transient threshold shift (TTS) (i.e., noise-induced). Figure 12a shows that CtBP2, a marker of cochlear ribbon synapses, increased in IHC after administration of anti-FAM19A5 antibody (right panel) compared to the control hIgG antibody (left panel). Figure 12b shows that the number of ribbon bodies per cell increased in IHC (first panel) and OHC (second panel) after administration of anti-FAM19A5 antibody (second panel) compared to the hIgG (first panel) and pre-noise groups (last panel).

[0058] Figures 13a-e show the therapeutic effect of a FAM19A5 antagonist (administered 2 hours after noise induction) on auditory brainstem response (ABR) thresholds in a mouse model of noise-induced hearing loss. The overall design was similar to Figure 6a, except that the antibody was administered to animals 2 hours after noise induction. Figure 13a shows the results before noise exposure. Figure 13b shows the results 1 day after noise exposure. Figure 13c shows the results 2 weeks after noise exposure. Figure 13d shows the results 4 weeks after noise exposure. Figure 13e shows the results 8 weeks after noise exposure. ABR thresholds were measured at frequencies ranging from 4 kHz to 64 kHz.

[0059] Figures 14a and 14b show the effect of a FAM19A5 antagonist on ABR I / O function in a mouse model of noise-induced hearing loss. Figure 14a compares wave I amplitude (first graph) and wave IV amplitude (second graph) in animals treated with anti-FAM19A5 antibody (squares), the pre-noise group (triangles), or a control IgG antibody (circles) two weeks after noise exposure. Figure 14b compares wave I amplitude (first graph) and wave IV amplitude (second graph) in animals treated with anti-FAM19A5 antibody (squares), the pre-noise group (triangles), or a control IgG antibody (circles) four weeks after noise exposure. Tone bursts were measured from 0 dB to 90 dB.

[0060] Figures 15a-15e show the dose-dependent effects of FAM19A5 antagonist administration on ABR thresholds in a mouse model of noise-induced hearing loss. Figure 15a is a schematic diagram of the experimental design. As shown, one day after noise exposure, each animal was administered various concentrations of anti-FAM19A5 antibody (1-30) (0 mg / kg, 3 mg / kg, 7 mg / kg, 15 mg / kg, and 30 mg / kg). After noise exposure, control animals were administered human anti-IgG antibody (30 mg / kg). ABR thresholds were measured one day after noise exposure and two weeks after noise exposure. Figure 15b compares ABR thresholds across multiple treatment groups in the frequency range of 0 kHz to 64 kHz on day one after noise exposure. Figure 15c shows the same data as Figure 15b, but presented as a bar graph. Figure 15d compares ABR thresholds across multiple treatment groups in the frequency range of 0 kHz to 64 kHz on week two after noise exposure. Figure 15e shows the same data as Figure 15c in a bar graph. Figures 15b-15e also show ABR thresholds before noise exposure ("BN"). In Figures 15c and 15e, for each frequency group shown, the first bar (from the left) corresponds to before noise exposure ("BN"), the second bar corresponds to animals treated with a control anti-IgG antibody ("hIgG"). The third bar corresponds to animals treated with 0 mg / kg anti-FAM19A5 antibody, the fourth bar corresponds to animals treated with 3 mg / kg anti-FAM19A5 antibody, the fifth bar corresponds to animals treated with 7 mg / kg anti-FAM19A5 antibody, the sixth bar corresponds to animals treated with 15 mg / kg anti-FAM19A5 antibody, and the final bar corresponds to animals treated with 30 mg / kg anti-FAM19A5 antibody.

[0061] Figures 16a and 16b compare the therapeutic effects of FAM19A5 antagonist administration on mice of various ages (10 weeks, 6 months, and 12 months). In each figure, the effects of the FAM19A5 antagonist on hearing loss in normal and aged mice were compared through ABR threshold (left graph), wave I amplitude (middle graph), and DPOAE (right graph). Figure 16a compares 6-month-old mice with 10-week-old mice. Figure 16b compares 12-month-old mice with 10-week-old mice. ABR thresholds were measured at frequencies ranging from 2 kHz to 64 kHz. Tone bursts were measured at 0 dB to 90 dB. DPOAE amplitudes were measured at F2 frequencies ranging from 4 kHz to 32 kHz.

[0062] Figures 17a to 17d show the therapeutic effect of FAM19A5 antagonist (1-30) on auditory brainstem response (ABR) thresholds in an aging mouse model. Figure 17a is a schematic diagram of the experimental design. As shown, anti-FAM19A5 antibody (1-30) or a control human IgG antibody was administered intravenously to mice (1 year old) once a week (30 mg / kg per dose) for a total of eight doses. ABR (i.e., hearing test) was measured before the initial antibody administration and at 1 and 2 months after administration. Figure 17b shows the results before injection. Figure 17c shows the results after 1 month of administration. Figure 17d shows the results after 2 months of administration. ABR thresholds were measured at frequencies ranging from 4 kHz to 64 kHz.

[0063] Figures 18a to 18c show the therapeutic effect of FAM19A5 antagonist (1-30) through comparison of wave I amplitude profiles in an aging mouse model. Mice were treated as described in Figure 17a. Figure 18a shows the results before injection. Figure 18b shows the results one month after injection. Figure 18c shows the results two months after injection. Tone bursts were measured from 0 dB to 90 dB.

[0064] Figure 19 shows Western blot results for the binding of monoclonal antibody clone 1-65 to epitope fragments F1 to F6 (lanes 3 to 8, respectively). FAM19A5-Cκ (lane 1), PSA-Cκ (lane 2), peptide NDV-BSA (lane 9), and BSA (lane 10) were used as controls. The sizes of the various antigens used are indicated on the right side of the blot. The amount of antigen used per well was 300 ng. The primary antibody used for Western blot was 1-65-scFv-rabbit-Fc-SSS (2 μg / mL), and the secondary antibody used in the experiment was anti-rabbit IgG (Fc-specific)-HRP (1:4000).

[0065] Figures 20a and 20b show ELISA results for the binding of various anti-FAM19A5 antibodies to epitope fragments F1 to F6. Figure 20a shows the results for anti-FAM19A5 antibodies 1-65, 2-13, and 3-2. For the 3-2 antibody, two isotypes are displayed: human IgG1 ("h3-2") and mouse IgG1 ("m3-2"). Figure 20b shows the results for the P2-C12 antibody. For each antibody, the first, second, third, fourth, fifth, and sixth bars (starting from the left) show binding to epitope fragments F1, F2, F3, F4, F5, and F6, respectively. The rightmost bar (black) shows the positive control (His-tagged FAM19A5 protein). The exact OD values ​​are displayed above each bar.

[0066] Figures 21a and 21b show ELISA results related to the binding of anti-FAM19A5 antibodies 1-65 (Figure 21a) and P2-C12 (Figure 21b) to eight different FAM19A5 fragment 5 mutant peptides (F5-1 to F5-8). The exact OD values ​​are indicated above each bar.

[0067] Figures 22a-c show ELISA results related to the binding of various anti-FAM19A5 antibodies to FAM19A5 mutants M1-M8. Figure 22a shows results for anti-FAM19A5 antibodies 1-65, 1-28, 2-13, and 3-2. Figure 22b shows results for anti-FAM19A5 antibodies 13B4, 13F7, and 15A9. Figure 22c shows results for anti-FAM19A5 antibodies P1-A08, P1-F02, P1-G09, P2-A01, P2-A03, P2-C12, P2-F07, and P2-F11. In Figures 22a-c, the eight bars for each antibody correspond to mutants M1-M8 (moving from left to right).

[0068] Figures 23a and 23b: Figure 23a is a schematic diagram of a two-site sandwich ELISA assay used to evaluate cross-competition between different anti-FAM19A5 antibodies. Figure 23b shows the results of cross-competition assays for six different anti-FAM19A5 antibodies: 1-65, P2-A03, P2-F11, 13B4, 2-13, and 3-2. The term "S / N" refers to the signal-to-noise ratio, measured as follows: [OD at 10 ng / mL antigen] / [OD at 0 ng / mL antigen]. Gray boxes indicate cross-competition (S / N ratio less than 2).

[0069] [Figures 24a to 24c] ELISA results related to binding of anti-FAM19A5 antibodies 3-2 and 1-28 to 13 different FAM19A5 epitope F2 fragment mutant peptides: (i) F2-01-BSA (#1), (ii) F2-02-BSA (#2), (iii) F2-03-BSA (#3), (iv) F2-04-BSA (#4), (v) F2-05-BSA A (#5), (vi) F2-06-BSA (#6), (vii) F2-07-BSA (#7), (viii) F2-08-BSA (#8), (ix) F2-09-BSA (#9), (x) F2-10-BSA (#10), (xi) F2-11-BSA (#11), (xii) F2-12-BSA (#12), and (xiii) F2-13-BSA (#13). Figure 24a shows the results for the 3-2 antibody, which has a human IgG1 isotype. Figure 24b shows the results for the 3-2 antibody, which has a mouse IgG1 isotype. Figure 24c shows the results for the 1-28 antibody. The exact OD values ​​are indicated above each bar.

[0070] Figures 25a to 25j show the results of alanine scanning analysis, revealing specific amino acid residues within the epitope F2 fragment that are important for binding of various 3-2 antibody mutants to the FAM19A5 protein. Figures 25a, 25b, 25c, and 25d show the results for the following antibodies produced in HEK293F cells: (A) wild-type 3-2 antibody, (B) 1-30 antibody, (C) 1-32 antibody, and (D) 6-10 antibody. Figures 25e, 25f, 25g, 25h, 25i, and 25j show the results for the following antibodies produced in CHO cells: (E) 1-17 antibody, (F) 1-30 antibody, (G) 1-32 antibody, (H) 4-11 antibody, (I) 6-10 antibody, and (J) low-PI antibody. Mutant peptides were generated, each with a single amino acid residue in epitope fragment F2 substituted with alanine, as described in Example 7. Antibody binding to the various mutant peptides was measured using ELISA.

[0071] [Fig. 26] The three-dimensional structure of the FAM19A5 protein and the location of the main binding epitope for the 2-13 antibody.

[0072] Figures 27a and 27b show the ability of a FAM19A5 antagonist (e.g., an anti-FAM19A5 antibody) described herein to reverse the FAM19A5-induced inhibition of neurite outgrowth in mouse primary spiral ganglion neurons. Figure 27a shows representative confocal microscopy images of primary spiral ganglion neurons treated with one of the following: (1) control antibody (human IgG) alone, (2) 10 μM FAM19A5 protein alone, or (3) 10 μM FAM19A5 protein and 100 nM anti-FAM19A5 antibody (1-30). Scale bar = 200 μm. Figure 27b compares the average total length of neurites in primary spiral ganglion neurons quantified using Image J in Figure 27a. Number of spiral ganglion neurons per group: control = 18, FAM19A5 = 19, FAM19A5 + 1-30 = 19. Data are shown as mean ± SEM. Statistical significance was assessed using one-way ANOVA and Tukey's multiple comparison test. *, P<0.05; **, P<0.01 (vs. FAM19A5).

[0073] Figures 28a-h show the ability of FAM19A5 antagonists described herein (e.g., anti-FAM19A5 antibodies and FAM19A5-targeting peptides) to promote neurite outgrowth in primary mouse spiral ganglion neurons. Figures 28a, 28c, 28e, and 28g are representative confocal microscopy images of primary spiral ganglion neurons treated with a control (human IgG) antibody or various FAM19A5 antagonists described herein. The FAM19A5 antagonists are as follows: 1-30 antibody (Figure 28a), 2-13 antibody (Figure 28c), 1-65 SS01 antibody (Figure 28e), and dFB-DY-JM31 peptide (Figure 28f). In Figures 28a and 28g, the scale bars represent 100 μm. In Figures 28c and 28g, the scale bars represent 50 μm. Figures 28b, 28d, 28f, and 28h compare the average total neurite length of the first-order spiral ganglion neurons in Figures 28a, 28c, 28e, and 28g, respectively, quantified using Image J. The number of spiral ganglion neurons per group was: (1) control = 22, (2) 1-30 = 19; (2) control = 26, (3) 2-13 = 27; (3) control = 29, (4) 1-65 SS01 = 26; (4) control = 30, (5) dFB-DY-JM31 = 38. Data are presented as mean ± SEM. Statistical analysis was performed using an independent-samples t-test to assess significance. * indicates P<0.05, and ** indicates P<0.01 compared to the control group.

[0074] Figures 29a and 29b show the ability of the FAM19A5 antagonist described herein to reverse the FAM19A5-induced decrease in the number of CtBP2 puncta in inner hair cells of mouse cochlear explants. Figure 29a shows representative confocal microscopy images of CtBP2 in cochlear explants treated with one of the following: (1) control (human IgG) antibody alone, (2) 1 μM FAM19A5 protein alone, or (3) 1 μM FAM19A5 protein and anti-FAM19A5 antibody (1-30). Scale bar = 20 μm. Figure 29b compares the number of CtBP2 puncta in inner hair cells of the cochlear explants in Figure 29a, quantified using Image J. The number of inner hair cells calculated per group = 15. Data represent the mean ± SEM. Statistical significance was assessed using one-way ANOVA and Tukey's multiple comparison test. ***, P<0.001; ****, P<0.0001 (vs. FAM19A5).

[0075] Figures 30a and 30b show the ability of a FAM19A5 antagonist (e.g., an anti-FAM19A5 antibody) described herein to promote synaptogenesis in inner hair cells of mouse cochlear explants. Figure 30a shows representative stereomicroscope images showing the expression of ribbon synapse marker (CtBP2) in inner hair cells of mouse cochlear explants treated with control (human IgG) antibody or 1 μM anti-FAM19A5 antibody (1-30). Scale bar = 20 μm. Figure 30b shows a comparison of the number of CtBP2 puncta quantified using Image J in inner hair cells of the mouse cochlear explants in Figure 30a. The number of inner hair cells calculated per group = 15. Data represent the mean ± SEM. Statistical significance was assessed using an unpaired samples t-test. **, P < 0.01 (vs. control group).

[0076] [Mode for Carrying Out the Invention] Generally, the present invention relates to methods for treating hearing impairment. More specifically, the present invention provides a method for treating hearing impairment in a subject in need thereof, comprising administering to the subject an antagonist that specifically targets FAM19A5 (FAM19A5 antagonist). The present invention is the first to demonstrate that hearing can be improved by modulating FAM19A5 activity (e.g., in spiral ganglion neurons). Furthermore, as described later herein, the FAM19A5 antagonists provided by the present invention can be used to reduce / reduce ribbon synapse damage or induce ribbon synapse formation in the inner ear of a subject (e.g., suffering from hearing impairment). Additional aspects are provided throughout the present invention.

[0077] To facilitate understanding of the subject matter disclosed herein, a number of terms and phrases are defined. Additional definitions are presented throughout the detailed description.

[0078] I. Definition Throughout this specification, the term "a" or "an" means that there is one or more of the subject matter. For example, "a molecule" is understood to refer to one or more molecules. Thus, "a," "one or more," and "at least one" can be used interchangeably herein.

[0079] Also, in this specification, "and / or" should be considered to refer to each of the two features or components in question, either independently or together. Thus, in this specification, the term "and / or" used in a phrase such as "A and / or B" is intended to include "A and B," "A or B," "A" (single) and "B" (single). Similarly, the term "and / or" used in a phrase such as "A, B and / or C" is intended to include each of the following aspects: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (single); B (single); and C (single).

[0080] The term "about" is used herein to mean approximately, roughly, roughly, or in the range. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values ​​set forth. In general, the term "about" can modify the stated numerical value by a variance above and below (e.g., 10% more or less).

[0081] The term "hearing impairment" as used herein refers to any disease, disorder, or condition associated with hearing impairment. In some aspects, the hearing impairment is associated with partial hearing loss. For example, compared to a reference subject (e.g., a matched subject not suffering from hearing impairment), the hearing ability of a person suffering from a hearing impairment (e.g., as described herein) 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%, at least about 90%, or about 100%. In some aspects, the hearing impairment is associated with complete hearing loss. A subject's hearing ability can be assessed using any suitable method known in the art. Non-limiting examples of methods that can be used to assess whether a subject suffers from a hearing impairment include pure tone audiometry (PTA), extended tone audiometry (ETA), speech intelligibility scale (SDS), word recognition in noise (WIN), tinnitus functional index (TFI), and tinnitus intensity (TL). In some aspects, hearing impairments useful in the present invention include sensorineural hearing loss. As used herein, the term "sensorineural hearing loss" refers to hearing loss caused by impairment of the sound-sensing function of the cochlea or dysfunction of the auditory nerve or central nervous system, which transmits auditory stimuli to the brain. Additional non-limiting examples of hearing impairments relevant to the present invention are provided elsewhere herein.

[0082] As used herein, the term "tinnitus" refers to the perception of a hallucinated sound in the absence of external auditory stimuli. Two major types of tinnitus include (1) "objective tinnitus," which is caused by sounds originating elsewhere in the body (e.g., turbulence in the head or muscle contractions); and (2) "subjective tinnitus," which is the perception of meaningless sounds in the absence of any physical sound. Unlike subjective tinnitus, which is heard only by the person experiencing it, objective tinnitus occurs infrequently and can be heard by an observer. Tinnitus can manifest in a variety of forms, such as buzzing, ringing, or other various sounds. Tinnitus can occur in one or both ears, either persistently or sporadically. As will become apparent from the present invention, in some aspects, hearing disorders (e.g., sensorineural hearing loss) that can be treated with the present invention are associated with tinnitus. In some aspects, hearing disorders (e.g., sensorineural hearing loss) that can be treated with the present invention are not associated with tinnitus.

[0083] As used herein, the term "ribbon synapse" refers to a type of neural synapse characterized by the presence of an electronically integrated structure (i.e., a "synaptic ribbon") containing synaptic vesicles (containing various neurotransmitters) close to the active zone (i.e., the neurotransmitter release site). Ribbon synapses are characterized by tight vesicle-calcium channel junctions, which facilitate rapid neurotransmitter release and sustained signal transmission. Ribbon synapses respond to stepwise changes in membrane potential and undergo cycles of extracellular efflux and intracellular import. Unless otherwise specified, ribbon synapses as used herein refer to those that play a role related to hearing. Within the auditory system, ribbon synapses are generally located at the site of connection between inner hair cells (IHCs) and spiral ganglion neurons (Wei M., et al., Neural Plast 2020:8815990 (Nov. 2020)).

[0084] As used herein, the term "auditory system" refers to the sensory system for hearing in a subject (e.g., a human subject).

[0085] The term "inner hair cell" (IHC) refers to a type of "cochlear hair cell" (i.e., a sensory cell of the auditory system). At least in humans, cochlear hair cells consist of one row of inner hair cells and three rows of outer hair cells. The inner hair cells are the actual sensory receptors (transducing sound vibrations in the cochlear fluid into electrical signals), and 95% of the auditory nerve fibers going to the brain originate from this subpopulation.

[0086] The term "neuron" includes electrically excitable cells that process and transmit information through electrical and chemical signals. A typical neuron consists of a cell body (soma), dendrites, and axons (also referred to herein as "nerve fibers"). The soma (cell body) of a neuron contains the nucleus. The dendrites of a neuron are cell extensions with many branches where most of the input to the neuron originates. The axons are finer, cable-like extensions that extend from the soma and transmit nerve signals from the soma and specific types of information back to the soma.

[0087] As used herein, the term "nerve fiber" refers to a part of a neuron, i.e., an axon process, which carries action potentials from one end of the neuron to the other. In some aspects, nerve fibers include "cochlear nerve fibers." Cochlear nerve fibers originate from neurons in the spiral ganglion and project peripherally to cochlear hair cells and centrally to the cochlear nucleus in the brainstem. Cochlear nerve fibers mediate hearing.

[0088] As used herein, the term "spiral ganglion" refers to the sensory ganglion of the cochlear nerve. The cells of the spiral ganglion send fibers peripherally to the cochlear hair cells and centrally to the cochlear nucleus in the brainstem.

[0089] As used herein, the term "cochlea" refers to the spiral cavity of the inner ear that resembles the cochlea and contains nerve endings essential for hearing. The cochlea contains three fluid-filled chambers: the scala tympani, the scala vestibuli (containing perilymph), and the scala intermedius (containing endolymph). The scala tympani and scala vestibuli are adjacent to each other and meet at the edge of the cochlea, the foramen cochlearis. The stapes transmits vibrations to the oval window (oval window) on the outside of the cochlea, vibrating the perilymph in the scala vestibuli. The perilymph vibrates the endolymph in the scala intermedius, triggering the movement of hair bundles of acoustic sensory cells, which convert vibrations into electrical potentials. Hair cells are arranged in four rows within the organ of Corti along the entire length of the cochlear coil. Three rows are composed of outer hair cells (OHCs) and one row is composed of inner hair cells (IHCs). IHCs provide the primary neural output of the cochlea. On the other hand, outer hair cells receive neural input primarily from the brain, which influences their motility as part of the cochlear mechanical preamplifier.

[0090] The term "family with sequence similarity 19, member A5" or "FAM19A5" refers to a protein that belongs to the TAFA family of five highly homologous proteins (also known as the FAM19 family) and is predominantly expressed in the brain and spinal cord. FAM19A5 is also known as "TAFA5" or "chemokine-like protein TAFA-5."

[0091] In humans, the gene encoding FAM19A5 is located on chromosome 22. Multiple human FAM19A5 (UniProt:Q7Z5A7) isoforms exist, which are thought to be generated by alternative splicing: isoform 1 (UniProt:Q7Z5A7-1), consisting of 132 amino acids; isoform 2 (UniProt:Q7Z5A7-2), consisting of 125 amino acids; and isoform 3 (UniProt:Q7Z5A7-3), consisting of 53 amino acids. Human FAM19A5 protein is known to exist in membrane-bound and soluble (secreted) forms. Isoform 1 is considered a membrane protein with a single transmembrane domain. Isoform 2, reported as a secreted (soluble) protein in Tang TY et al., Genomics 83(4):727-34 (2004), contains a signal peptide at amino acid positions 1–25. Isoform 1 is considered a membrane protein and is predicted based on EST data. Table 1 (below) provides the amino acid sequences of the three known human FAM19A5 isoforms. Unless otherwise specified, the term "FAM19A5" includes any variant or isoform of the FAM19A5 protein that is naturally expressed by a cell. Thus, in some aspects, a polypeptide described herein (e.g., comprising a FAM19A5-binding domain of a member of the LRRC4 protein family) can inhibit binding of FAM19A5 isoform 1, isoform 2, and / or isoform 3 to a member of the LRRC4 protein family.

[0092] [Table 1]

[0093] The term "antagonist against FAM19A5" or "FAM19A5 antagonist" refers to any antagonist that suppresses the expression and / or activity of FAM19A5. Such antagonists may be peptides, nucleic acids, or chemical compounds. More specifically, the antagonist may be an antisense oligonucleotide, siRNA, shRNA, miRNA, dsRNA, aptamer, or PNA (peptide nucleic acid) targeting FAM19A5, or a vector containing the same. In some aspects, the antagonist may be an antibody that specifically binds to the FAM19A5 protein, or an antigen-binding portion thereof ("anti-FAM19A5 antibody"). As described later in this specification, in some aspects, FAM19A5 antagonists useful herein include a polynucleotide encoding an anti-FAM19A5 antibody.

[0094] As described later in this specification, in some aspects, FAM19A5 antagonists include mimetic molecules that can specifically target FAM19A5, thereby inhibiting, reducing, and / or dissociating the binding between FAM19A5 and LRRC4 protein family members (i.e., "LRRC4 family mimetic molecules"). A "mimetic molecule" refers to a molecule that is structurally and / or functionally similar to another molecule (a "reference molecule"). For example, in some aspects, a mimetic molecule can share a partial structure or sequence with a reference molecule, thereby allowing the mimetic molecule to exhibit one or more characteristics of the reference molecule. However, as will be apparent from the present invention, structural or sequence similarity is not always required. In some aspects, a mimetic molecule may be structurally different but behave in a manner similar to the reference molecule. As described herein, in some aspects, a mimetic molecule includes a small molecule. In some aspects, a mimetic molecule includes a peptide. In some aspects, a mimetic molecule is not an antibody or an antigen-binding portion thereof.

[0095] As is apparent from the present invention, the LRRC4 family mimetic molecules described herein share certain properties with members of the LRRC4 protein family but differ (structurally and / or functionally) from naturally occurring members of the LRRC4 protein family. For example, in some aspects, LRRC4 family mimetic molecules useful in the present invention include small molecules. As further described elsewhere herein, in some aspects, an LRRC4 family mimetic molecule comprises a polypeptide, the polypeptide comprising, consisting of, or consisting essentially of a domain of a member of the LRRC4 protein family, the domain capable of binding to FAM19A5 protein (also referred to herein as a "FAM19A5-binding domain"). In such aspects, the polypeptide can comprise one or more amino acid substitutions within the FAM19A5-binding domain. As described elsewhere herein, in some aspects, such amino acid substitutions can improve one or more properties of the polypeptide, e.g., increase the stability and / or binding affinity of the polypeptide for FAM19A5 protein. In some aspects, the polypeptide comprises a FAM19A5-binding domain but lacks one or more other domains of a member of the LRRC4 protein family. For example, in some aspects, a polypeptide comprises a FAM19A5-binding domain but does not comprise a transmembrane domain. In some aspects, a polypeptide comprises a FAM19A5-binding domain but does not comprise an intracellular domain (e.g., a postsynaptic substantia densa junction (PB) domain) of a member of the LRRC4 protein family. In some aspects, a polypeptide comprises a FAM19A5-binding domain but does not comprise both a transmembrane domain and an intracellular domain. Thus, in some aspects, the polypeptides described herein are shorter than naturally occurring members of the LRRC4 protein family.Furthermore, in carrying out biological activities (e.g., neural circuit formation), each member of the LRRC4 protein family (LRRC4, LRRC4B, and LRRC4C) interacts with their ligands (netrin-G2, receptor tyrosine phosphatase LAR, and netrin-G1, respectively). See, for example, Li et al., Mol Cancer 13:266 (Dec. 2014). Because the polypeptides of the present invention do not contain all domains of an LRRC4 protein family member, in some aspects, the polypeptides do not bind to LRRC4 protein family ligands but instead specifically target the FAM19A5 protein. Thus, in some aspects, the polypeptides described herein do not replace endogenous members of the LRRC4 protein family. Instead, in some aspects, by inhibiting, reducing, and / or dissociating the interaction between FAM19A5 and each member of the LRRC4 protein family, the polypeptides of the present invention free endogenous LRRC4 family proteins to carry out their natural biological activities.

[0096] Unless otherwise indicated, the term "FAM19A5 antagonist" includes both anti-FAM19A5 antibodies and LRRC4 family mimetic molecules.

[0097] The term "leucine-rich repeat-containing 4 protein family" or "LRRC4 protein family" (including derivatives thereof) refers to a family of proteins that are core synaptic regulators and have been described to play roles in various stages of neural circuit formation, including neuronal migration, neurite outgrowth, synaptic contact formation, and functional connectivity. See, e.g., Woo et al., Mol Cell Neurosci 42(1):1-10 (Sep. 2009). The LRRC4 protein family includes three members: (1) LRRC4, (2) LRRC4B, and (3) LRRC4C (collectively referred to herein as "LRRC4 protein family members" or "LRRC4 protein family members" (or their derivatives)). Members of the LRRC4 protein family generally contain nine leucine-rich repeat (LRR) domains flanking the N- and C-termini of the LRRs. These LRR domains are known to interact with the fibronectin type III domain of presynaptic receptor protein tyrosine phosphatase (RPTP) proteins. See, for example, Won et al., Mol Cells 41(7):622-630 (Jul. 2018). The LRR domain is linked to an immunoglobulin-like C2 (IG) and threonine (Thr)-rich domain, which together form the extracellular portion of LRRC4 protein family members. Unlike other members, the LRRC4B protein has an extra glycine (Gly)-rich domain between the IG and Thr-rich domains. In addition to the extracellular portion, LRRC4 protein family members further contain a transmembrane (TM) domain at the C-terminus of the protein and a postsynaptic basal junction (PB) domain.

[0098] In humans, the gene encoding the LRRC4 protein is located on chromosome 7 (nucleotides 128,027,071 to 128,032,107 of GenBank Accession Number NC_000007.14; minus-strand orientation). Synonyms for the LRRC4 protein include: "nasopharyngeal carcinoma-associated gene 14 protein," "brain tumor-associated protein BAG," "Netrin-G2 ligand," "NAG14," "NGL-2," and "BAG." The amino acid sequence of the LRRC4 protein is 653 amino acids in length and is listed in Table 2 (below). The full-length ectodomain of the LRRC4 protein corresponds to amino acid residues 39 to 527 of SEQ ID NO: 4 (i.e., SEQ ID NO: 6). Unless otherwise indicated, the term "LRRC4 protein" (including its synonyms) includes any variant or isoform of the LRRC4 protein that is naturally expressed by a cell.

[0099] [Table 2]

[0100] In humans, the gene encoding the LRRC4B protein is located on chromosome 19 (nucleotides 50,516,892 to 50,568,435 of GenBank Accession Number NC_000019.10; minus-strand orientation). Synonyms for the LRRC4B protein are known, and non-limiting examples include "Netrin-G3 ligand," "LRIG4," "NGL-3," "HSM," and "DKFZp761A179." The amino acid sequence of the LRRC4B protein is 713 amino acids in length and is listed in Table 3 (below). The full-length ectodomain of the LRRC4B protein corresponds to amino acid residues 36 to 576 of SEQ ID NO: 7 (SEQ ID NO: 9). Unless otherwise indicated, the term "LRRC4B protein" (including its synonyms) includes any variant or isoform of the LRRC4B protein naturally expressed by cells.

[0101] [Table 3]

[0102] In humans, the gene encoding the LRRC4C protein is located on chromosome 11 (nucleotides 40,107,066 to 41,460,419 of GenBank Accession Number NC_000011.10; minus-strand orientation). Synonyms for the LRRC4C protein are known, examples of which include "NGL-1," "Netrin-G1 ligand," and "KIAA1580." The amino acid sequence for the LRRC4C protein is 640 amino acids in length and is listed in Table 4 (below). The full-length ectodomain of the LRRC4C protein corresponds to amino acid residues 45 to 527 of SEQ ID NO: 82 (SEQ ID NO: 84). Unless otherwise indicated, the term "LRRC4C protein" (including its synonyms) includes any variant or isoform of the LRRC4C protein that is naturally expressed by a cell.

[0103] [Table 4] JPEG2026507379000013.jpg11169

[0104] As used herein, the term "FAM19A5 binding domain" refers to a segment / fragment of a member of the LRRC4 protein family that is capable of binding to the FAM19A5 protein.

[0105] The term "alkenyl," as used herein, refers to a group containing hydrogen and carbon and containing at least one carbon-carbon double bond.

[0106] The term "alkoxy," as used herein, refers to an alkyl group attached to the parent molecular moiety through an oxygen atom.

[0107] As used herein, the term "alkyl" refers to a group that contains hydrogen and carbon and does not contain any double bonds.

[0108] The term "alkynyl," as used herein, refers to a group containing hydrogen and carbon and containing at least one carbon-carbon triple bond.

[0109] The term "amino" as used herein refers to --NH.sub.2.

[0110] The term "bicycloalkenyl," as used herein, refers to a fused, spirocyclic, or bridged bicyclic hydrocarbon ring system containing at least one double bond.

[0111] The term "bicycloalkyl," as used herein, refers to a fused, spirocyclic, or bridged bicyclic cycloalkyl ring.

[0112] The term "cycloalkenyl," as used herein, refers to an unsaturated non-aromatic monocyclic hydrocarbon ring system containing zero heteroatoms. Representative examples of cycloalkenyl groups include, but are not limited to, cyclopentenyl, cyclohexenyl, cycloheptenyl, and cyclooctenyl.

[0113] The term "cycloalkyl," as used herein, refers to a saturated monocyclic hydrocarbon ring system having 0 heteroatoms. Representative examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0114] The term "formyl" as used herein refers to -CHO.

[0115] The terms "halo" and "halogen" as used herein refer to Cl, Br, I, or F.

[0116] The term "haloalkoxy," as used herein, refers to a haloalkyl group attached to the parent molecular moiety through an oxygen atom.

[0117] The term "haloalkyl," as used herein, refers to an alkyl group substituted with one, two, three, or four halogen atoms.

[0118] The term "heteroaryl," as used herein, refers to an aromatic ring containing one, two, or three heteroatoms independently selected from nitrogen, oxygen, and sulfur. Representative examples of heteroaryl groups include, but are not limited to, furyl, imidazolyl, pyrazolyl, pyridinyl, pyrrolyl, thiazolyl, and thienyl.

[0119] The term "heterobicycloalkyl," as used herein, refers to a non-aromatic bicyclic ring system containing one, two, three, or four heteroatoms independently selected from nitrogen, oxygen, and sulfur, and optionally containing one or more double bonds. The heterobicycloalkyl groups herein can be attached to the parent molecular moiety through any carbon or nitrogen atom within the group.

[0120] The term "heterocycloalkyl," as used herein, refers to a non-aromatic ring containing one, two, three, or four heteroatoms independently selected from nitrogen, oxygen, and sulfur, optionally containing one or more double bonds. Heterocycloalkyl groups herein can be attached to the parent molecular moiety through any carbon or nitrogen atom within the group. Representative examples of heterocycloalkyl groups include, but are not limited to, morpholinyl, piperazinyl, piperidinyl, tetrahydrofuranyl, tetrahydropyranyl, and thiomorpholinyl.

[0121] The terms "antibody" and "antibody" are used interchangeably herein as terms of the art and refer to a molecule having an antigen-binding site that specifically binds to an antigen. As used herein, these terms include whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") or single chain thereof. In some aspects, an "antibody" refers to a glycoprotein comprising at least two heavy (H) and light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. In some aspects, an "antibody" refers to a single-chain antibody comprising a single variable domain, e.g., a VHH domain. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. In certain naturally occurring antibodies, the heavy chain constant region is composed of three domains: CH1, CH2, and CH3. In certain naturally occurring antibodies, each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain, CL.

[0122] The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which may be separated by more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of antibodies can 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.

[0123] "Kabat numbering" and similar terms are art-recognized and refer to a system for numbering amino acid residues in the heavy and light chain variable regions of an antibody, or antigen-binding portions thereof. In some aspects, the CDRs of an antibody may be determined by the Kabat numbering system (see, e.g., Kabat EA & Wu TT (1971) Ann NY Acad Sci 190:382-391, and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system, the CDRs in an antibody heavy chain molecule are typically located at amino acid positions 31-35, which can optionally include one or two additional amino acids following 35 (designated 35A and 35B in the Kabat numbering system) (CDR1), amino acid positions 50-65 (CDR2), and amino acid positions 95-102 (CDR3). Using the Kabat numbering system, the CDRs in an antibody light chain molecule are typically located at amino acid positions 24-34 (CDR1), amino acid positions 50-56 (CDR2), and amino acid positions 89-97 (CDR3). In some aspects, the CDRs of the antibodies described herein were determined according to the Kabat numbering system.

[0124] The terms "Kabat amino acid position numbering," "Kabat position," and grammatical variations thereof refer to the numbering system used for the heavy or light chain variable domain of each antibody as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using such a numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening or insertion of the FW or CDR of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and each inserted residue after heavy chain FW residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat). See Table 5.

[0125] [Table 5]

[0126] The Kabat numbering of residues can be determined for a given antibody by aligning the homologous regions of the antibody sequence with the "standard" Kabat-numbered sequence. Meanwhile, Chothia numbering indicates the location of structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). When numbering using the Kabat numbering system, the end of the Chothia CDR-H1 loop varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering system places insertions at H35A and H35B. If 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 regions represent a compromise between the Kabat CDRs and Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software.

[0127] IMGT (ImMunoGenetics) also provides a numbering system for immunoglobulin variable regions, including CDRs. See, for example, Lefranc, MP et al., Dev. Comp. Immunol. 27:55-77 (2003), incorporated herein by reference. The IMGT numbering system facilitates comparison of variable and CDR regions across all species based on the alignment of over 5,000 sequences, structural data, and characterization of hypervariable loops. According to the IMGT numbering system, VH-CDR1 is located at positions 26-35, VH-CDR2 is located at positions 51-57, VH-CDR3 is located at positions 93-102, VL-CDR1 is located at positions 27-32, VL-CDR2 is located at positions 50-52, and VL-CDR3 is located at positions 89-97.

[0128] An antibody 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., IgG1, IgG2, IgG3, and IgG4 in humans; IgG1, IgG2a, IgG2b, and IgG3 in mice) of immunoglobulin molecules. Immunoglobulins (e.g., IgG1) exist in many isotypes, which differ from each other by at most a few amino acids. The antibodies disclosed herein can be derived from any one of the commonly known isotypes, classes, subclasses, and isotypes. In some aspects, the antibodies described herein are of the IgG1, IgG2, IgG3, or IgG4 subclass, or any hybrid thereof. In some aspects, the antibodies belong to the human IgG1, IgG2, or IgG4 subclass.

[0129] "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; single-chain antibodies; camelized antibodies; affibodies; anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), and single domain antibodies (sdAbs), which include binding molecules comprised of a single monomeric variable antibody domain (e.g., a VH domain or a VL domain) that is fully capable of antigen binding. Harmen M M and Haard H J Appl Microbiol Biotechnol. 77(1):13-22 (2007)).

[0130] As used herein, 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 FAM19A5). Such "fragments" are, for example, between about 8 and about 1500 amino acids in length, suitably between about 8 and about 745 amino acids, suitably about 8 to about 300, e.g., about 8 to about 200 amino acids, or about 10 to about 50 or 100 amino acids in length. It has been found that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of individual binding fragments encompassed by the term antibody, for example, the "antigen-binding portion" of the anti-FAM19A5 antibody described herein, include: (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) an F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of one arm of an antibody, disulfide-linked to an Fv (sdFv); (v) a dAb fragment consisting of the VH domain (Ward et al., (1989) Nature 341:544-546); (vi) an isolated complementarity-determining region (CDR); or (vii) a combination of two or more isolated CDRs that can be selectively linked by a synthetic linker. Alternatively, the two domains of an Fv fragment, VL and VH, are encoded by separate genes but can be linked by a synthetic linker using recombinant methods to form a single protein chain. In this case, the VL and VH regions pair to form a monovalent molecule (also known as a single-chain Fv (scFv); see, e.g., Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as intact antibodies.Antigen-binding portions can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins.

[0131] 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 either the light or heavy chain, typically the amino-terminal 110 to 120 amino acids in a mature heavy chain and approximately 90 to 115 amino acids in a mature light chain, which vary extensively in sequence between antibodies and are used to determine the binding and specificity of a particular antibody to a particular antigen. Sequence variability is concentrated in regions known as complementarity-determining regions (CDRs), with the more conserved regions of a variable domain referred to as framework regions (FRs).

[0132] Without intending to be limited to a particular mechanism or theory, it is believed that the light and heavy chain CDRs are primarily responsible for antibody-antigen interaction and specificity. In some aspects, the variable regions are human variable regions. In some aspects, the variable regions comprise rodent or murine CDRs and human framework regions (FRs). In some aspects, the variable regions are primate (e.g., non-human primate) variable regions. In some aspects, the variable regions comprise rodent or murine CDRs and primate (e.g., non-human primate) framework regions (FRs).

[0133] As used herein, the term "heavy chain" (HC) as used in connection with antibodies can refer to any of the distinct types, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the constant domain, which give rise to the IgA, IgD, IgE, IgG, and IgM classes of antibodies, respectively, including the subclasses of IgG (e.g., IgG1, IgG2, IgG3, and IgG4).

[0134] As used herein, the term "light chain" (LC) in connection with an antibody can refer to any distinct type, e.g., kappa (κ) or lambda (λ), based on the amino acid sequence of the constant domain. Light chain amino acid sequences are well known in the art. In some aspects, the light chain is a human light chain.

[0135] The terms "VL" and "VL domain" are used interchangeably to refer to the light chain variable region of an antibody.

[0136] The terms "VH" and "VH domain" are used interchangeably to refer to the heavy chain variable region of an antibody.

[0137] As used herein, an "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 FAM19A5 is substantially free of antibodies that specifically bind to antigens other than FAM19A5). However, an isolated antibody that specifically binds to an epitope of FAM19A5 may cross-react with other FAM19A5 proteins of other species.

[0138] "Binding affinity" generally refers to the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, "binding affinity" herein refers to the intrinsic binding affinity that reflects a 1:1 interaction between each member of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for an entity Y is generally determined by the dissociation constant (K D The affinity can be expressed as the equilibrium dissociation constant (K D ) and the equilibrium binding constant (K A ) can be measured and / or expressed in a variety of ways, including but not limited to. D is k off / k on It is calculated from the ratio of K A is k on / k off It is calculated from the ratio of k onrepresents the binding rate constant of an antibody to an antigen, and k off indicates, for example, the dissociation of an antibody and an antigen. on and k off can be determined by techniques known to those skilled in the art, such as immunoassays (e.g., enzyme-linked immunosorbent assay, BIACORE®, or kinetic exclusion assay (KinExA)).

[0139] As used herein, the terms "specifically bind," "specifically recognize," "specific binding," "selective binding," and "selectively bind" are similar terms and refer to a molecule (e.g., a FAM19A5 antagonist described herein) that binds to an antigen (e.g., a FAM19A5 protein), and such binding is understood by one of skill in the art. For example, a molecule that specifically binds to an antigen can also bind to other peptides or polypeptides, but generally with lower affinity, as measured, for example, by immunoassay, BIACORE®, KinExA 3000 instrument (Sapidyne Instruments, Boise, ID), or other analytical methods known in the art. In some aspects, a molecule that specifically binds to an antigen has a K A K that is at least about 2 logs, at least about 2.5 logs, at least about 3 logs, or at least about 4 logs greater than A and binds to the antigen.

[0140] As used herein, the term "antigen" refers to any natural or synthetic immunogenic substance, such as a protein, peptide, or hapten. The antigen can be FAM19A5 or a fragment thereof.

[0141] The term "epitope," as used herein, is a term of art that refers to a localized region of an antigen to which an antibody can specifically bind. An epitope can be, for example, contiguous amino acids of a polypeptide (a linear or contiguous epitope), or an epitope can be assembled from, for example, two or more non-contiguous regions of a polypeptide or each polypeptide (a structural, non-linear, discontinuous, or non-contiguous epitope). Epitopes formed from contiguous amino acids generally survive exposure to denaturing solvents, while epitopes formed by tertiary folding generally are lost upon treatment with denaturing solvents. An epitope generally includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20 amino acids in a unique spatial conformation. Methods for determining which epitope a given antibody binds (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation analysis, in which the reactivity of a given antibody (e.g., an anti-FAM19A5 antibody) with overlapping or adjacent peptides (e.g., from FAM19A5) is tested. Methods for determining the spatial conformation of epitopes include techniques known in the art and described herein, such as X-ray crystallography, two-dimensional nuclear magnetic resonance, and HDX-MS (see, for example, the epitope mapping protocol in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996)).

[0142] In the context of two or more antibodies, the term "binds to the same epitope" means that the antibodies bind to the same segment of amino acid residues as determined by a given method. Techniques for determining whether each antibody binds to the "same epitope on FAM19A5" with the antibodies described herein include epitope mapping methods such as X-ray analysis of antigen:antibody complex crystals, which provide atomic resolution of the epitope, and hydrogen / deuterium exchange mass spectrometry (HDX-MS). Other methods monitor antibody binding 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 often considered indicative of epitope components. Computational combinatorial methods for epitope mapping can also be used. These methods rely on the ability of the antibody of interest to affinity isolate specific short peptides from a combinatorial phage display peptide library. Antibodies with identical VH and VL or identical CDR1, 2, and 3 sequences are predicted to bind to the same epitope.

[0143] Each antibody that "competes with another antibody for binding to a target" means each antibody that inhibits (partially or completely) the binding of the other antibody to a target. 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 a target, can be determined using known competition experiments. In some aspects, an antibody competes with the other antibody and inhibits its binding 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 the "blocking antibody" (i.e., the cold antibody that is first incubated with the target). Competition analysis can be performed, for example, by the method described in Chapter 11 "Using Antibodies" by Ed Harlow and David Lane, Cold Spring Harbor Protoc; 2006; doi:10.1101 / pdb.prot4277 or Ed Harlow and David Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA 1999. Competing antibodies may bind to the same epitope, overlapping epitopes, or adjacent epitopes (e.g., as evidenced by steric hindrance).

[0144] The term "endogenous" when used to describe a member of the LRRC4 protein family refers to an LRRC4 family protein that is naturally present in a subject. As described herein, the mimetic molecules of the present invention differ (structurally and / or functionally) from endogenous LRRC4 protein family members.

[0145] A molecule (e.g., an LRRC4 family mimetic molecule) that "competes with other proteins for binding to a target" refers to a molecule that inhibits (partially or completely) the binding of another protein (e.g., a naturally occurring member of the LRRC4 protein family) to a target. Whether two compounds compete with each other for binding to a target, i.e., whether and to what extent an LRRC4 family mimetic molecule described herein inhibits the binding of a naturally occurring member of the LRRC4 protein family to FAM19A5 protein, can be determined using known competition experiments. In some aspects, the LRRC4 family mimetic molecules described herein compete with each naturally occurring member of the LRRC4 protein family for binding to FAM19A5 protein and inhibit it by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%. Competition analyses can be performed by the methods described herein or, for example, by the methods described in Chapter 11 "Using Antibodies" in Ed Harlow and David Lane, Cold Spring Harbor Protoc; 2006; doi:10.1101 / pdb.prot4277, or Ed Harlow and David Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA 1999.

[0146] Other competitive binding assays that can be applied to the present invention include solid-phase direct or indirect radioimmunoassays (RIAs), solid-phase direct or indirect enzyme immunoassays (EIAs), and sandwich competitive assays (see Stahl et al., Methods in Enzymology 9:242 (1983)); solid-phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614 (1986)); solid-phase direct label assays, solid-phase direct label sandwich assays (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid-phase direct label RIA using 1-125 labels (see Morel et al., Mol. Immunol. 25(1):7 (1988)); and solid-phase direct biotin-avidin EIA (see Cheung et al., Virology 176:546 (1990)); directly labeled RIA (see Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)).

[0147] As used herein, the terms "naturally occurring" or "naturally occurring" mean that an object (e.g., a protein) can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including a virus) that can be isolated from a natural source and has not been intentionally modified by humans in a laboratory is naturally occurring. As further described elsewhere herein, the LRRC4 family mimetic molecules useful in the present invention are not naturally occurring.

[0148] "Polypeptide" refers to a chain comprising at least two consecutively linked amino acid residues, with no upper limit to the length of the chain. One or more amino acid residues within the protein may contain modifications such as, but not limited to, glycosylation, phosphorylation, or disulfide bond formation. A "protein" may include one or more polypeptides.

[0149] As used herein, the terms "nucleic acid" or "nucleic acid molecule" are intended to include DNA molecules and RNA molecules. A nucleic acid molecule may be single-stranded or double-stranded, and may be cDNA.

[0150] The term "vector," as used herein, refers to a nucleic acid molecule capable of transporting another nucleic acid linked thereto. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors (e.g., bacterial vectors derived from replicating bacteria and episomal mammalian vectors) are capable of autonomous replication in a host cell into which they are introduced. Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell when introduced into the host cell, and thereby be replicated along with the host genome. Certain vectors are also capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. As the plasmid is the most commonly used form of vector, "plasmid" and "vector" can be used interchangeably herein. However, other forms of expression vectors that serve equivalent functions, such as viral (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses) vectors, are also included.

[0151] As used herein, the term "recombinant host cell" (or simply "host cell") is intended to refer to a cell containing a nucleic acid that is not naturally occurring within the cell, such as a cell into which a recombinant expression vector has been introduced. Such terms should be understood to refer not only to the particular subject cell but also to the progeny of such a cell. Because certain variations can occur in successive generations due to mutation or environmental influences, such progeny may not be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0152] As used herein, "administration" refers to the physical introduction of a molecule (e.g., a FAM19A5 antagonist) or a composition containing the molecule into a subject using any of a variety of methods and delivery systems known to those of skill in the art. Non-limiting examples of available administration routes include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, or other parenteral routes, e.g., by injection or infusion. The phrase "parenteral administration," as used herein, generally refers to modes of administration other than enteral and topical administration by injection, and includes, but is not limited to, intravenous, intraperitoneal, intramuscular, intraarterial, intraspinal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, and in vivo electroporation. Alternatively, the molecules described herein (e.g., FAM19A5 antagonists) can be administered by routes other than parenteral, e.g., topical, epidermal, or mucosal routes of administration, such as intranasal, oral, vaginal, rectal, sublingual, or topical routes, and administration can be, for example, single, multiple, and / or over one or more extended periods of time.

[0153] As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, and worms.

[0154] As used herein, the terms "treat" and "treatment" refer to any type of intervention or process performed on a subject, or the administration of an active ingredient to a subject, for the purpose of reversing, alleviating, ameliorating, inhibiting, or slowing the progression, development, severity, or recurrence of symptoms, complications, conditions, or biochemical markers associated with a disease, or preventing the progression, development, severity, or recurrence of such symptoms, complications, conditions, or biochemical markers. Treatment can be performed on subjects with a disease or on subjects who do not have a disease (e.g., for prophylaxis).

[0155] II. Treatment method The present specification discloses a method for treating hearing impairment in a subject, comprising administering to a subject in need thereof an antagonist (e.g., an isolated monoclonal antibody or an antigen-binding fragment thereof) that specifically binds to FAM19A5 (also referred to herein as a "FAM19A5 antagonist"). The present specification also discloses a means for treating hearing impairment in a subject, comprising a pharmaceutical composition comprising an antagonist against the family with sequence similarity 19, member A5 (FAM19A5) protein (FAM19A5 antagonist) and a pharmaceutically acceptable carrier. The present invention also provides a method for treating hearing impairment in a subject in need thereof, comprising administering such a means to the subject to treat the hearing impairment. As described later in the specification, in some aspects, the FAM19A5 antagonist comprises an anti-FAM19A5 antibody described in Tables 6 to 9. In some aspects, the FAM19A5 antagonist comprises an LRRC4 family mimetic molecule described herein.

[0156] As described and demonstrated herein, a wide range of hearing disorders can be treated using the FAM19A5 antagonists described herein (e.g., anti-FAM19A5 antibodies, LRRC4 family mimetic molecules, or both). In some aspects, hearing disorders that can be treated with the present invention include sensorineural hearing loss. Unless otherwise indicated, any sensorineural hearing loss known in the art can be treated with the present invention. In some aspects, the sensorineural hearing loss is associated with tinnitus. In some aspects, the sensorineural hearing loss is not associated with tinnitus. Non-limiting examples of sensorineural hearing loss that can be treated with the present invention include ototoxic drug-induced hearing loss, noise-induced hearing loss, presbycusis, sudden hearing loss, hearing loss associated with Meniere's disease, autoimmune hearing loss, ischemic hearing loss, hearing loss associated with head injury, hereditary hearing loss, hearing loss associated with viral or bacterial infection, hearing loss due to damage or impairment of the organ of Corti, or a combination thereof. In some aspects, sensorineural hearing loss that can be treated includes ototoxic drug-induced hearing loss. In some aspects, sensorineural hearing loss that can be treated includes noise-induced hearing loss. In some aspects, noise-induced hearing loss can be caused by long-term exposure to noise, for example, loud music, heavy equipment or machinery, airplanes, bombing, or other people. In some aspects, sensorineural hearing loss that can be treated includes presbycusis. In some aspects, sensorineural hearing loss that can be treated includes sudden hearing loss. In some aspects, sensorineural hearing loss that can be treated includes hearing loss associated with disease (e.g., Meniere's disease). In some aspects, sensorineural hearing loss that can be treated includes autoimmune hearing loss. In some aspects, sensorineural hearing loss that can be treated includes ischemic hearing loss. In some aspects, sensorineural hearing loss that can be treated includes hearing loss associated with head injury. In some aspects, sensorineural hearing loss that can be treated includes hereditary hearing loss. In some aspects, sensorineural hearing loss that can be treated includes hearing loss associated with a viral or bacterial infection (e.g., labyrinthitis or meningitis). In some aspects, sensorineural hearing loss that may be treated includes hearing loss due to damage or impairment of the organ of Corti.

[0157] Optimal hearing depends on effective synaptic transmission at the ribbon synapses of auditory hair cells (Nouvian R., et al., J Membr Biol 209(2-3):153-65 (Feb.-Mar. 2006); and LoGiudice L. and Matthews, G., Neuroscientist 15(4):380-391 (Aug. 2009)). Furthermore, abnormal ribbon synaptic function is associated with many hearing disorders (Yuan X., et al., Drug Des Devel Ther 14:268502693 (Jul. 2020); and Moser T., et al., Otol Neurotol 34(6):995-1004 (Aug. 2013)). Without being limited to a particular theory, in some aspects, the FAM19A5 antagonists provided by the present invention (e.g., anti-FAM19A5 antibodies, LRRC4 family mimetic molecules, or both) can promote the reduction and / or improvement of ribbon synapse function. Thus, some aspects of the present invention relate to methods for reducing and / or preventing ribbon synapse damage in the inner ear of a subject in need thereof, comprising administering to the subject a FAM19A5 antagonist (e.g., anti-FAM19A5 antibodies, LRRC4 family mimetic molecules, or both). In some aspects, after administration of a FAM19A5 antagonist, ribbon synaptic damage 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%, at least about 90%, or about 100% compared to a reference subject (e.g., a subject before administration of the FAM19A5 antagonist and / or a matched subject not receiving the FAM19A5 antagonist).

[0158] Unless otherwise specified, "ribbon synapse damage" refers to abnormalities in ribbon synapses in a subject's inner ear. In some aspects, the damage is structural. For example, in some aspects, ribbon synapse damage includes a reduction in the number of ribbon synapses in the inner ear. As is apparent from the present invention, in some aspects, a reduction in the number of ribbon synapses in the inner ear is associated with hearing impairment. In some aspects, a subject experiences ribbon synapse damage, where the number of ribbon synapses in the inner ear 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%, at least about 90%, or about 100% compared to a reference subject (e.g., a matched subject not experiencing hearing impairment). Accordingly, some aspects of the present invention relate to methods of reducing and / or preventing ribbon synapse loss in the inner ear of a subject in need thereof, comprising administering to the subject a FAM19A5 antagonist described herein (e.g., an anti-FAM19A5 antibody, an LRRC4 family mimetic molecule, or both).

[0159] In some aspects, after administration of a FAM19A5 antagonist (e.g., an anti-FAM19A5 antibody, an LRRC4 family mimetic molecule, or both), the number of ribbon synapses in the subject's inner ear is increased relative to a reference subject (e.g., the subject before administration of the FAM19A5 antagonist and / or a matched subject who has not received the FAM19A5 antagonist). Accordingly, some aspects of the present specification relate to methods of increasing the number of ribbon synapses in a subject's inner ear (e.g., by inducing ribbon synapse formation), comprising administering to the subject a FAM19A5 antagonist (e.g., an anti-FAM19A5 antibody, an LRRC4 family mimetic molecule, or both) described herein. In some aspects, the number of ribbon synapses 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%, at least about 90%, or at least about 100% or more after administration compared to a reference subject. In some aspects, the number of ribbon synapses increases by at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, or at least about 8-fold after administration compared to a reference subject, resulting in an increase of at least about 9-fold, at least about 10-fold, at least about 12.5-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold after administration. Without being limited to a particular theory, in some aspects, an increase in the number of ribbon synapses in the inner ear is associated with improved hearing.

[0160] In some aspects, the ribbon synapse impairment is functional. For example, in some aspects, the ribbon synapse impairment includes reduced signal transmission following an auditory stimulus (e.g., sound vibration). In some aspects, the signal transmission is reduced by at least 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%, or about 100% compared to a reference subject (e.g., a matched subject not suffering from a hearing impairment). Thus, some aspects of the present invention relate to methods for improving one or more functions (e.g., signal transmission) of ribbon synapses in the inner ear of a subject, comprising administering to the subject a FAM19A5 antagonist provided by the present invention (e.g., an anti-FAM19A5 antibody, an LRRC4 family mimetic molecule, or both). In some aspects, after the administration, one or more functions of ribbon synapses are improved 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%, or at least about 100% or more compared to a reference subject (e.g., a subject before administration of a FAM19A5 antagonist and / or a corresponding subject not receiving the FAM19A5 antagonist). In some aspects, after said administration, the number of ribbon synapses is increased by at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 12.5-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold relative to a reference subject. Without being limited to a particular theory, in some aspects, improvement in the function of one or more ribbon synapses in a subject's inner ear is associated with improved hearing.

[0161] In some aspects, ribbon synapse damage is structural (e.g., a reduction in the number of ribbon synapses in the inner ear) and functional (e.g., a reduction in signal transmission following auditory stimulation). For example, in some aspects, after administering a FAM19A5 antagonist (e.g., an anti-FAM19A5 antibody, an LRRC4 family mimetic molecule, or both) to a subject in need thereof, both the number and one or more functions of ribbon synapses in the subject's inner ear are improved (e.g., increased or strengthened).

[0162] As described herein, in some aspects, hearing impairments that can be treated with the present invention are associated with increased FAM19A5 activity. For example, in some aspects, spiral ganglion neurons of subjects suffering from hearing impairment exhibit increased FAM19A5 expression. Unless otherwise specified, increased FAM19A5 expression can occur at the gene level (e.g., FAM19A5 mRNA) and / or protein level. In some aspects, FAM19A5 expression in spiral ganglion neurons of subjects suffering from hearing impairment 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%, at least about 90%, or at least about 100% or more compared to a reference subject (e.g., a matched subject not suffering from hearing impairment). In some aspects, the expression of FAM19A5 is increased by at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 12.5-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold relative to spiral ganglion neurons of a reference subject. As will be apparent to one of skill in the art, increased expression of FAM19A5 can be associated with FAM19A5 activity. Thus, in some aspects, FAM19A5 activity in spiral ganglion neurons of a subject suffering from a hearing impairment 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%, at least about 90%, or at least about 100% or more compared to a reference subject (e.g., a matched subject not suffering from a hearing impairment).In some aspects, FAM19A5 activity in spiral ganglion neurons of a subject suffering from a hearing impairment is increased by at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 12.5-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold compared to a reference subject.

[0163] Without being limited to a particular theory, in some aspects, administration of a FAM19A5 antagonist described herein (e.g., an anti-FAM19A5 antibody, an LRRC4 family mimetic molecule, or both) reduces the expression and / or activity of FAM19A5 in spiral ganglion neurons. As is evident from the present invention, in some aspects, reducing the expression and / or activity of FAM19A5 in spiral ganglion neurons can promote improved hearing. Thus, some aspects of the present invention relate to methods of reducing the expression and / or activity of FAM19A5 in spiral ganglion neurons in a subject in need thereof, comprising administering to the subject a FAM19A5 antagonist described herein (e.g., an anti-FAM19A5 antibody, an LRRC4 family mimetic molecule, or both). In some aspects, after administration of a FAM19A5 antagonist (e.g., an anti-FAM19A5 antibody, an LRRC4 family mimetic molecule, or both), the expression and / or activity of FAM19A5 in spiral ganglion neurons of the subject is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%.

[0164] As demonstrated herein, in some aspects, the FAM19A5 antagonists described herein (e.g., anti-FAM19A5 antibodies, LRRC4 family mimetic molecules, or both) can increase neurite outgrowth of neurons (e.g., spiral ganglion neurons). As used herein, the term "neurite outgrowth" refers to the process by which new processes (axons, dendrites, or both) are generated as neurons grow. Thus, some aspects of the present invention relate to methods for increasing neurite outgrowth of spiral ganglion neurons in a subject in need thereof, comprising administering to the subject a FAM19A5 antagonist described herein (e.g., anti-FAM19A5 antibodies, LRRC4 family mimetic molecules, or both). In some aspects, neurite outgrowth is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% after administration of the FAM19A5 antagonist. In some aspects, neurite outgrowth is increased by at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 12.5-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold after administration of the FAM19A5 antagonist. In some aspects, a FAM19A5 antagonist described herein (e.g., an anti-FAM19A5 antibody, an LRRC4 family mimetic molecule, or both) can reverse the inhibition of neurite outgrowth induced by FAM19A5.

[0165] In some aspects, the FAM19A5 antagonists described herein (e.g., anti-FAM19A5 antibodies, LRRC4 family mimetic molecules, or both) can promote synaptogenesis. As used herein, the term "synaptogenesis" refers to the process by which synapses are formed between individual neurons (e.g., individual spiral ganglion neurons). Thus, in some aspects, the present invention provides methods for increasing synaptogenesis in a subject in need thereof, comprising administering to the subject a FAM19A5 antagonist described herein (e.g., anti-FAM19A5 antibodies, LRRC4 family mimetic molecules, or both). In some aspects, after administration of the FAM19A5 antagonist, synaptogenesis is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%. In some aspects, administration of the FAM19A5 antagonist results in an increase in synaptogenesis of at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 12.5-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold. In some aspects, the FAM19A5 antagonists described herein can reverse the suppression of synaptogenesis induced by FAM19A5.

[0166] As described elsewhere herein, in any one of the methods provided herein, the FAM19A5 antagonist (e.g., an anti-FAM19A5 antibody, an LRRC4 family mimetic molecule, or both) can be administered to a subject using any suitable route of administration known in the art. In some aspects, the FAM19A5 antagonist (e.g., an anti-FAM19A5 antibody, an LRRC4 family mimetic molecule, or both) is administered to a subject via intracochlear injection, intravestibular infusion, intravenous administration, intratympanic administration, or a combination thereof. In some aspects, the FAM19A5 antagonist (e.g., an anti-FAM19A5 antibody, an LRRC4 family mimetic molecule, or both) is administered to a subject via intracochlear injection. In some aspects, the FAM19A5 antagonist (e.g., an anti-FAM19A5 antibody, an LRRC4 family mimetic molecule, or both) is administered to a subject via intravestibular injection. In some aspects, the FAM19A5 antagonist (e.g., an anti-FAM19A5 antibody, an LRRC4 family mimetic molecule, or both) is administered to a subject via intravenous administration. In some aspects, the FAM19A5 antagonist (e.g., an anti-FAM19A5 antibody, an LRRC4 family mimetic molecule, or both) is administered to a subject via intratympanic administration.

[0167] In some aspects, any one of the methods provided herein (e.g., provided above) can further include administering an additional therapeutic agent to the subject. For example, some aspects of the present invention include treating hearing impairment in a subject in need thereof, comprising administering to the subject a FAM19A5 antagonist (e.g., an anti-FAM19A5 antibody, an LRRC4 family mimetic molecule, or both) and an additional therapeutic agent. Non-limiting examples of such additional therapeutic agents are known in the art (e.g., standard of care, e.g., steroids for the treatment of sudden hearing loss). Dosages and administration of one or more additional therapeutic agents are known in the art, for example, as indicated in the product labeling of the respective drugs.

[0168] When a FAM19A5 antagonist (e.g., an anti-FAM19A5 antibody, an LRRC4 family mimetic molecule, or both) is administered to a subject together with an additional therapeutic agent, in some aspects, the FAM19A5 antagonist and the additional therapeutic agent can be administered to a subject simultaneously. For example, in some aspects, the FAM19A5 antagonist (e.g., an anti-FAM19A5 antibody, an LRRC4 family mimetic molecule, or both) and the additional therapeutic agent can be administered to a subject as a single composition. In some aspects, the FAM19A5 antagonist (e.g., an anti-FAM19A5 antibody, an LRRC4 family mimetic molecule, or both) and the additional therapeutic agent can be administered simultaneously but as separate compositions. In some aspects, the FAM19A5 antagonist (e.g., an anti-FAM19A5 antibody, an LRRC4 family mimetic molecule, or both) and the additional therapeutic agent can be administered to a subject sequentially. For example, in some aspects, the FAM19A5 antagonist (e.g., an anti-FAM19A5 antibody, an LRRC4 family mimetic molecule, or both) is administered to the subject prior to administration of the additional therapeutic agent. In some aspects, the FAM19A5 antagonist (e.g., an anti-FAM19A5 antibody, an LRRC4 family mimetic molecule, or both) is administered to the subject after administration of the additional therapeutic agent.

[0169] III. FAM19A5 antagonists One or more FAM19A5 antagonists can be used in the methods of the present invention. In some aspects, the FAM19A5 antagonist includes an antisense oligonucleotide, siRNA, shRNA, miRNA, dsRNA, aptamer, PNA (peptide nucleic acid) that specifically targets FAM19A5, or a vector containing the same. In some aspects, the FAM19A5 antagonist includes an anti-FAM19A5 antibody, a polynucleotide encoding an anti-FAM19A5 antibody, or a vector containing the polynucleotide. Thus, in some aspects, the FAM19A5 antagonist useful in the methods provided herein includes an antisense oligonucleotide that specifically targets FAM19A5. In some aspects, the FAM19A5 antagonist includes an siRNA that specifically targets FAM19A5. In some aspects, the FAM19A5 antagonist includes an shRNA that specifically targets FAM19A5. In some aspects, the FAM19A5 antagonist includes an miRNA that specifically targets FAM19A5. In some aspects, the FAM19A5 antagonist comprises a dsRNA that specifically targets FAM19A5. In some aspects, the FAM19A5 antagonist comprises an aptamer that specifically targets FAM19A5. In some aspects, the FAM19A5 antagonist comprises a PNA that specifically targets FAM19A5. In some aspects, the FAM19A5 antagonist comprises an anti-FAM19A5 antibody. In some aspects, the FAM19A5 antagonist comprises a polynucleotide encoding an anti-FAM19A5 antibody. In some aspects, the FAM19A5 antagonist useful in the present invention comprises an LRRC4 family mimetic molecule.

[0170] As is apparent from the present invention, FAM19A5 antagonists useful in the present invention can be characterized by specific structural and / or functional features. For example, in some aspects, the FAM19A5 antagonists described herein specifically bind to human FAM19A5, including soluble FAM19A5 and membrane-bound FAM19A5. In addition to specific binding to soluble and / or membrane-bound human FAM19A5, the FAM19A5 antagonists described herein also have (a) a K D(b) Can bind to soluble human FAM19A5 with a K of 10 nM or less? D can bind to membrane-bound human FAM19A5; both (a) and (b) can be achieved.

[0171] In some aspects, FAM19A5 antagonists suitable for the methods disclosed herein cross-compete for binding to human FAM19A5 with anti-FAM19A5 antibodies comprising the CDRs or variable regions disclosed herein. Non-limiting examples of such anti-FAM19A5 antibodies are provided elsewhere herein.

[0172] For example, in some aspects, the FAM19A5 antagonists described herein cross-compete for binding to human FAM19A5 with a reference antibody comprising the heavy and light chain CDRs of one or more of the following antibodies: 1-65, 3-2, 2-13, 1-28, P2-C12, 13B4, 13F7, 15A9, P1-A03, P1- A08, P1-F02, P2-A01, P2-A03, P2-F07, P2-F11, SS01-13, SS01-13-s5, S5-2.GKNG, 1-7A-IT, L ow-PI, 1-30, 1-17, 1-32, 4-11, 6-10, 2-13D, 2-13D-37, 2-13D-37-1.5W-41, 2-13D-37-3W-16. In some aspects, FAM19A5 antagonists useful in the present invention cross-compete for binding to human FAM19A5 with a reference antibody, the reference antibody comprising one or more heavy and light chain variable regions of each of the following antibodies: 1-65, 3-2, 2-13, 1-28, P2-C12, 13B4, 13F7, 15A9, P1-A03, P1-A08, P 1-F02, P2-A01, P2-A03, P2-F07, P2-F11, SS01-13, SS01-13-s5, S5-2.GKNG, 1-7A-IT, Low- PI, 1-30, 1-17, 1-32, 4-11, 6-10, 2-13D, 2-13D-37, 2-13D-37-1.5W-41, 2-13D-37-3W-16.

[0173] In some aspects, a FAM19A5 antagonist useful in the present invention cross-competes for binding to human FAM19A5 with a reference antibody, the reference antibody comprising a heavy chain variable region (VH) CDR1, CDR2, and CDR3, and a light chain variable region (VL) CDR1, CDR2, and CDR3, wherein (i) the VH CDR1 of the reference antibody comprises any one of the amino acid sequences of SEQ ID NOs: 11, 14, 17, 20, 89, 95, 101, 107, 113, 119, 125, 131, 137, 143, 149, 218, 220, 222, and 22; (ii) the VH CDR1 of the reference antibody comprises any one of the amino acid sequences of SEQ ID NOs: 11, 14, 17, 20, 89, 95, 101, 107, 113, 119, 125, 131, 137, 143, 149, 218, 220, 222, and 22; (iii) the VH CDR3 of the reference antibody comprises any one of the amino acid sequences of SEQ ID NOs: 13, 16, 19, 22, 91, 97, 103, 109, 115, 121, 127, 133, 139, 145, 151, 246, and 247; and (iv) the VL CDR3 of the reference antibody comprises any one of the amino acid sequences of SEQ ID NOs: 13, 16, 19, 22, 91, 97, 103, 109, 115, 121, 127, 133, 139, 145, 151, 246, and 247. (v) the VL CDR1 of the reference antibody comprises any one of the amino acid sequences of SEQ ID NOs: 23, 26, 29, 32, 92, 98, 104, 110, 116, 122, 128, 134, 140, 146, 152, 208, 225, and 248; (v) the VL CDR2 of the reference antibody comprises any one of the amino acid sequences of SEQ ID NOs: 24, 27, 30, 33, 93, 99, 105, 111, 117, 123, 129, 135, 141, 147, 153, 209, 213, 224, 226, 228, 229, 230, and 232; and / or (vi) the VL CDR2 of the reference antibody. CDR3 comprises any one of the amino acid sequences of SEQ ID NOs: 25, 28, 31, 34, 94, 100, 106, 112, 118, 124, 130, 136, 142, 148, 154, 227, 231 and 233.

[0174] In some aspects, the FAM19A5 antagonists described herein bind to the same FAM19A5 epitope as a reference antibody. In some aspects, the FAM19A5 antagonist and the reference antibody both bind to one or more of the FAM19A5 epitopes described herein (e.g., comprising the amino acid sequence of any one of SEQ ID NOS: 89-94). In some aspects, the FAM19A5 antagonists described herein bind to the same FAM19A5 epitope as a reference antibody, and the reference antibody comprises one or more heavy and light chain CDRs of each of the following antibodies: 1-65, 3-2, 2-13, 1-28, P2-C12, 13B4, 13F7, 15A9, P1-A03, P1-A08, and P1-C12. -F02, P2-A01, P2-A03, P2-F07, P2-F11, SS01-13, SS01-13-s5, S5-2.GKNG, 1-7A-IT, Low- PI, 1-30, 1-17, 1-32, 4-11, 6-10, 2-13D, 2-13D-37, 2-13D-37-1.5W-41, 2-13D-37-3W-16. In some aspects, the FAM19A5 antagonist binds to the same FAM19A5 epitope as a reference antibody, wherein the reference antibody comprises the heavy and light chain variable regions of one or more of the following antibodies: 1-65, 3-2, 2-13, 1-28, P2-C12, 13B4, 13F7, 15A9, P1-A03, P1-A08, P1-F02. , P2-A01, P2-A03, P2-F07, P2-F11, SS01-13, SS01-13-s5, S5-2.GKNG, 1-7A-IT, Low-PI , 1-30, 1-17, 1-32, 4-11, 6-10, 2-13D, 2-13D-37, 2-13D-37-1.5W-41, 2-13D-37-3W-16.

[0175] In some aspects, the FAM19A5 antagonists described herein bind to the same FAM19A5 epitope as a reference antibody, wherein the reference antibody comprises a heavy chain variable region (VH) CDR1, CDR2, and CDR3, and a light chain variable region (VL) CDR1, CDR2, and CDR3, wherein (i) the VH CDR1 of the reference antibody comprises any one of the amino acid sequences of SEQ ID NOs: 11, 14, 17, 20, 89, 95, 101, 107, 113, 119, 125, 131, 137, 143, 149, 218, 220, 222, and 223; and (ii) the VH CDR1 of the reference antibody comprises any one of the amino acid sequences of SEQ ID NOs: 11, 14, 17, 20, 89, 95, 101, 107, 113, 119, 125, 131, 137, 143, 149, 218, 220, 222, and 223. (iii) the VH CDR3 of the reference antibody comprises any one of the amino acid sequences of SEQ ID NOs: 13, 16, 19, 22, 91, 97, 103, 109, 115, 121, 127, 133, 139, 145, 151, 246, and 247; and (iv) the VL CDR3 of the reference antibody comprises any one of the amino acid sequences of SEQ ID NOs: 13, 16, 19, 22, 91, 97, 103, 109, 115, 121, 127, 133, 139, 145, 151, 246, and 247. (v) the VL CDR1 of the reference antibody comprises any one of the amino acid sequences of SEQ ID NOs: 23, 26, 29, 32, 92, 98, 104, 110, 116, 122, 128, 134, 140, 146, 152, 208, 225, and 248; (v) the VL CDR2 of the reference antibody comprises any one of the amino acid sequences of SEQ ID NOs: 24, 27, 30, 33, 93, 99, 105, 111, 117, 123, 129, 135, 141, 147, 153, 209, 213, 224, 226, 228, 229, 230, and 232; and / or (vi) the VL CDR2 of the reference antibody. CDR3 comprises any one of the amino acid sequences of SEQ ID NOs: 25, 28, 31, 34, 94, 100, 106, 112, 118, 124, 130, 136, 142, 148, 154, 227, 231 and 233.

[0176] In some aspects, FAM19A5 antagonists provided herein bind to FAM19A5 (e.g., human FAM19A5) with 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or even greater affinity than other proteins in the FAM19A5 series, as measured, for example, by immunoassay (e.g., ELISA), surface plasmon resonance, or dynamic exclusion assay. In some aspects, FAM19A5 antagonists provided herein bind to FAM19A5 (e.g., human FAM19A5) without cross-reactivity with other proteins in the FAM19A5 series, as measured, for example, by immunoassay.

[0177] III.A. Exemplary Anti-FAM19A5 Antibodies In some aspects, FAM19A5 antagonists that can be used in the methods provided herein include antibodies. In some aspects, specific antibodies that can be used in the methods disclosed herein include 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 the variable region or CDR sequences. The amino acid sequences for the VH and VL CDRs of various exemplary anti-FAM19A5 antibodies are provided in Tables 6 and 7, respectively. The CDRs for the following antibodies were identified using the Kabat numbering system (see above): 1-65, 3-2, 2-13, 1-28, P2-C12, 13B4, 13F7, 15A9, P1-A03, P1-A08, P1-F02, P2-A01, P2-A03, P2-F07, P2-F11, SS01-13, SS01-13-s5, and S5-2.GKNG. The CDRs for the following antibodies were identified using the IMGT numbering system (see above): 1-7A-IT, Low-PI, 1-30, 1-17, 1-32, 4-11, 6-10, 2-13D, 2-13D-37, 2-13D-37-1.5W-41, and 2-13D-37-3W-16. The VH and VL amino acid sequences of different anti-FAM19A5 antibodies of the present invention are presented in Tables 8 and 9, respectively.

[0178] [Table 6] JPEG2026507379000016.jpg255164JPEG2026507379000017.jpg54169

[0179] [Table 7] JPEG2026507379000019.jpg255164JPEG2026507379000020.jpg16169

[0180] [Table 8] JPEG2026507379000022.jpg255164JPEG2026507379000023.jpg255164JPEG2026507379000024.jpg35169

[0181] [Table 9] JPEG2026507379000026.jpg255164JPEG2026507379000027.jpg190169

[0182] Thus, in some aspects, anti-FAM19A5 antibodies useful in the present invention comprise heavy and light chain variable regions, wherein the heavy chain variable region comprises the amino acid sequence of any one of SEQ ID NOs: 35-38, 155-165, 211, 214, 234-237, and 249-251 (see Table 8). In some aspects, anti-FAM19A5 antibodies useful in the present invention comprise the CDRs of the heavy chain variable region selected from the group consisting of SEQ ID NOs: 35-38, 155-165, 211, 214, 234-237, and 249-251 (see Table 8).

[0183] In some aspects, anti-FAM19A5 antibodies useful in the present invention comprise heavy and light chain variable regions, and the light chain variable region comprises the amino acid sequence of any one of SEQ ID NOs: 39-42, 166-176, 212, 215, and 238-245 (see Table 9). In some aspects, anti-FAM19A5 antibodies useful in the present invention comprise the CDRs of the light chain variable region selected from the group consisting of SEQ ID NOs: 39-42, 166-176, 212, 215, and 238-245 (see Table 9).

[0184] In some aspects, anti-FAM19A5 antibodies that can be used with the methods provided herein comprise a heavy chain variable region CDR selected from the group consisting of SEQ ID NOs: 35-38, 155-165, 211, 214, 234-237, and 249-251, and a light chain variable region CDR selected from the group consisting of SEQ ID NOs: 39-42, 166-176, 212, 215, and 238-245.

[0185] In some aspects, anti-FAM19A5 antibodies that can be used with the methods provided herein comprise a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises amino acids that are 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 of SEQ ID NOs: 35-38, 155-165, 211, 214, 234-237, or 249-251 (see Table 8). In some aspects, an anti-FAM19A5 antibody comprises a heavy chain variable region and a light chain variable region, wherein the light chain variable region comprises an amino acid sequence 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 of SEQ ID NOs: 39-42, 166-176, 212, 215, or 238-245 (see Table 9). In some aspects, an anti-FAM19A5 antibody useful in the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence 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 of SEQ ID NO: 35-38, 155-165, 211, 214, 234-237, or 249-251, and the light chain variable region comprises an amino acid sequence 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 of SEQ ID NO: 39-42, 166-176, 212, 215, or 238-245.

[0186] In some aspects, the anti-FAM19A5 antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL): (1) VH and VL comprise the sequences of SEQ ID NOs: 35 and 39, respectively; (2) VH and VL comprise the sequences of SEQ ID NOs: 36 and 40, respectively; (3) VH and VL comprise the sequences of SEQ ID NOs: 37 and 41, respectively; (4) VH and VL comprise the sequences of SEQ ID NOs: 38 and 42, respectively; (5) VH and VL comprise the sequences of SEQ ID NOs: 155 and 166, respectively; (6) VH and VL comprise the sequences of SEQ ID NOs: 156 and 167, respectively; (7) VH and VL comprise the sequences of SEQ ID NOs: 157 and 168, respectively; (8) VH and VL comprise the sequences of SEQ ID NOs: 158 and 169, respectively; (9) VH and VL comprise the sequences of SEQ ID NOs: 159 and 170, respectively; (10) VH and VL comprise the sequences of SEQ ID NOs: 160 and 171, respectively; (11) VH and VL comprise the sequences of SEQ ID NOs: 161 and 172, respectively; (12) VH and VL comprise the sequences of SEQ ID NOs: 162 and 173, respectively; (13) VH and VL comprise the sequences of SEQ ID NOs: 163 and 174, respectively; (14) VH and VL comprise the sequences of SEQ ID NOs: 164 and 175, respectively; (15) VH and VL comprise the sequences of SEQ ID NOs: 165 and 176, respectively; (16) VH and VL comprise the sequences of SEQ ID NOs: 211 and 212, respectively; (17) VH and VL comprise the sequences of SEQ ID NOs: 214 and 215, respectively; (18) VH and VL comprise the sequences of SEQ ID NOs: 249 and 215, respectively; (19) VH and VL comprise the sequences of SEQ ID NOs: 234 and 238, respectively; (20) VH and VL comprise the sequences of SEQ ID NOs: 235 and 239, respectively; (21) VH and VL comprise the sequences of SEQ ID NOs: 235 and 240, respectively; (22) VH and VL comprise the sequences of SEQ ID NOs: 235 and 241, respectively; (23) VH and VL comprise the sequences of SEQ ID NOs: 235 and 242, respectively; (24) VH and VL comprise the sequences of SEQ ID NOs: 235 and 243, respectively; (25) VH and VL comprise the sequences of SEQ ID NOs: 235 and 244, respectively; (26) VH and VL comprise the sequences of SEQ ID NOs: 236 and 245, respectively; (27) VH and VL comprise the sequences of SEQ ID NOs: 250 and 245, respectively; (28) VH and VL comprise the sequences of SEQ ID NOs: 237 and 245, respectively; or (29) VH and VL comprise the sequences of SEQ ID NOs: 251 and 245, respectively.

[0187] In some aspects, an anti-FAM19A5 antibody comprises a VH and a VL, wherein the VH comprises the amino acid sequence of SEQ ID NO: 35 and the VL comprises the amino acid sequence of SEQ ID NO: 39. In some aspects, an anti-FAM19A5 antibody comprises a VH and a VL, wherein the VH comprises the amino acid sequence of SEQ ID NO: 36 and the VL comprises the amino acid sequence of SEQ ID NO: 40. In some aspects, an anti-FAM19A5 antibody comprises a VH and a VL, wherein the VH comprises the amino acid sequence of SEQ ID NO: 37 and the VL comprises the amino acid sequence of SEQ ID NO: 41. In some aspects, an anti-FAM19A5 antibody comprises a VH and a VL, wherein the VH comprises the amino acid sequence of SEQ ID NO: 38 and the VL comprises the amino acid sequence of SEQ ID NO: 42. In some aspects, an anti-FAM19A5 antibody comprises a VH and a VL, wherein the VH comprises the amino acid sequence of SEQ ID NO: 155 and the VL comprises the amino acid sequence of SEQ ID NO: 166. In some aspects, an anti-FAM19A5 antibody comprises a VH and a VL, wherein the VH comprises the amino acid sequence of SEQ ID NO: 156 and the VL comprises the amino acid sequence of SEQ ID NO: 167. In some aspects, an anti-FAM19A5 antibody comprises a VH and a VL, wherein the VH comprises the amino acid sequence of SEQ ID NO: 157 and the VL comprises the amino acid sequence of SEQ ID NO: 168. In some aspects, an anti-FAM19A5 antibody comprises a VH and a VL, wherein the VH comprises the amino acid sequence of SEQ ID NO: 158 and the VL comprises the amino acid sequence of SEQ ID NO: 169. In some aspects, an anti-FAM19A5 antibody comprises a VH and a VL, wherein the VH comprises the amino acid sequence of SEQ ID NO: 159 and the VL comprises the amino acid sequence of SEQ ID NO: 170. In some aspects, an anti-FAM19A5 antibody comprises a VH and a VL, wherein the VH comprises the amino acid sequence of SEQ ID NO: 160 and the VL comprises the amino acid sequence of SEQ ID NO: 171. In some aspects, an anti-FAM19A5 antibody comprises a VH and a VL, wherein the VH comprises the amino acid sequence of SEQ ID NO: 161 and the VL comprises the amino acid sequence of SEQ ID NO: 172. In some aspects, an anti-FAM19A5 antibody comprises a VH and a VL, wherein the VH comprises the amino acid sequence of SEQ ID NO: 162 and the VL comprises the amino acid sequence of SEQ ID NO: 173. In some aspects, an anti-FAM19A5 antibody comprises a VH and a VL, wherein the VH comprises the amino acid sequence of SEQ ID NO: 163 and the VL comprises the amino acid sequence of SEQ ID NO: 174.In some aspects, an anti-FAM19A5 antibody comprises a VH and a VL, wherein the VH comprises the amino acid sequence of SEQ ID NO: 164 and the VL comprises the amino acid sequence of SEQ ID NO: 175. In some aspects, an anti-FAM19A5 antibody comprises a VH and a VL, wherein the VH comprises the amino acid sequence of SEQ ID NO: 165 and the VL comprises the amino acid sequence of SEQ ID NO: 176. In some aspects, an anti-FAM19A5 antibody comprises a VH and a VL, wherein the VH comprises the amino acid sequence of SEQ ID NO: 211 and the VL comprises the amino acid sequence of SEQ ID NO: 212. In some aspects, an anti-FAM19A5 antibody comprises a VH and a VL, wherein the VH comprises the amino acid sequence of SEQ ID NO: 214 and the VL comprises the amino acid sequence of SEQ ID NO: 215. In some aspects, an anti-FAM19A5 antibody comprises a VH and a VL, wherein the VH comprises the amino acid sequence of SEQ ID NO: 249 and the VL comprises the amino acid sequence of SEQ ID NO: 215. In some aspects, an anti-FAM19A5 antibody comprises a VH and a VL, wherein the VH comprises the amino acid sequence of SEQ ID NO: 234 and the VL comprises the amino acid sequence of SEQ ID NO: 238. In some aspects, an anti-FAM19A5 antibody comprises a VH and a VL, wherein the VH comprises the amino acid sequence of SEQ ID NO: 235 and the VL comprises the amino acid sequence of SEQ ID NO: 239. In some aspects, an anti-FAM19A5 antibody comprises a VH and a VL, wherein the VH comprises the amino acid sequence of SEQ ID NO: 235 and the VL comprises the amino acid sequence of SEQ ID NO: 240. In some aspects, an anti-FAM19A5 antibody comprises a VH and a VL, wherein the VH comprises the amino acid sequence of SEQ ID NO: 235 and the VL comprises the amino acid sequence of SEQ ID NO: 241. In some aspects, an anti-FAM19A5 antibody comprises a VH and a VL, wherein the VH comprises the amino acid sequence of SEQ ID NO: 235 and the VL comprises the amino acid sequence of SEQ ID NO: 242. In some aspects, an anti-FAM19A5 antibody comprises a VH and a VL, wherein the VH comprises the amino acid sequence of SEQ ID NO: 235 and the VL comprises the amino acid sequence of SEQ ID NO: 243. In some aspects, an anti-FAM19A5 antibody comprises a VH and a VL, wherein the VH comprises the amino acid sequence of SEQ ID NO: 235 and the VL comprises the amino acid sequence of SEQ ID NO: 244. In some aspects, an anti-FAM19A5 antibody comprises a VH and a VL, wherein the VH comprises the amino acid sequence of SEQ ID NO: 236 and the VL comprises the amino acid sequence of SEQ ID NO: 245.In some aspects, an anti-FAM19A5 antibody comprises a VH and a VL, wherein the VH comprises the amino acid sequence of SEQ ID NO: 250 and the VL comprises the amino acid sequence of SEQ ID NO: 245. In some aspects, an anti-FAM19A5 antibody comprises a VH and a VL, wherein the VH comprises the amino acid sequence of SEQ ID NO: 237 and the VL comprises the amino acid sequence of SEQ ID NO: 245. In some aspects, an anti-FAM19A5 antibody comprises a VH and a VL, wherein the VH comprises the amino acid sequence of SEQ ID NO: 251 and the VL comprises the amino acid sequence of SEQ ID NO: 245.

[0188] In some aspects, the anti-FAM19A5 antibody comprises: (1) 2-13 heavy chain CDR1, CDR2, and CDR3, or a combination thereof, and / or 2-13 light chain CDR1, CDR2, and CDR3, or any combination thereof; (2) 3-2 heavy chain CDR1, CDR2, and CDR3, or a combination thereof, and / or 3-2 light chain CDR1, CDR2, and CDR3, or any combination thereof; (3) 1-65 heavy chain CDR1, CDR2, and CDR3, or a combination thereof, and / or 1-65 light chain CDR1, CDR2, and CDR3, or any combination thereof; (4) heavy chain CDR1, CDR2 and CDR3, or a combination thereof, of 1-28, and / or light chain CDR1, CDR2 and CDR3, or any combination thereof; (5) heavy chain CDR1, CDR2 and CDR3, or a combination thereof, of P2-C12, and / or light chain CDR1, CDR2 and CDR3, or any combination thereof; (6) heavy chain CDR1, CDR2 and CDR3 of 13B4, or a combination thereof. (7) heavy chain CDR1, CDR2 and CDR3 of 13F7, or a combination thereof, and / or light chain CDR1, CDR2 and CDR3 of 13F7, or any combination thereof; (8) heavy chain CDR1, CDR2 and CDR3 of 15A9, or a combination thereof, and / or light chain CDR1, CDR2 and CDR3 of 15A9, or any combination thereof; (9) heavy chain CDR1, CDR2 and CDR3 of P1-A03 (10) heavy chain CDR1, CDR2 and CDR3 of P1-A08, or a combination thereof, and / or light chain CDR1, CDR2 and CDR3 of P1-A08, or any combination thereof; (11) heavy chain CDR1, CDR2 and CDR3 of P1-F02, or a combination thereof, and / or light chain CDR1, CDR2 and CDR3 of P1-F02, or any combination thereof;(12) heavy chain CDR1, CDR2 and CDR3 of P2-A01, or a combination thereof, and / or light chain CDR1, CDR2 and CDR3 of P2-A01, or any combination thereof; (13) heavy chain CDR1, CDR2 and CDR3 of P2-A03, or a combination thereof, and / or light chain CDR1, CDR2 and CDR3 of P2-A03, or any combination thereof; (14) heavy chain CDR1, CDR2 and CDR3 of P2-F07, or a combination thereof, and / or light chain CDR1, CDR2 and CDR3 of P2-F07 (15) heavy chain CDR1, CDR2 and CDR3 of P2-F11, or a combination thereof, and / or light chain CDR1, CDR2 and CDR3 of F2-F11, or any combination thereof; (16) heavy chain CDR1, CDR2 and CDR3 of SS01-13, or a combination thereof, and / or light chain CDR1, CDR2 and CDR3 of SS01-13, or any combination thereof; (17) heavy chain CDR1, CDR2 and CDR3 of SS01-13-s5, or a combination thereof, and (18) heavy chain CDR1, CDR2 and CDR3 of S5-2.GKNG, or a combination thereof, and / or light chain CDR1, CDR2 and CDR3 of S5-2.GKNG, or any combination thereof; (19) heavy chain CDR1, CDR2 and CDR3 of 1-7A-IT, or a combination thereof, and / or light chain CDR1, CDR2 and CDR3 of 1-7A-IT, or any combination thereof; (20) Low-PI (21) heavy chain CDR1, CDR2 and CDR3, or a combination thereof, and / or low-PI light chain CDR1, CDR2 and CDR3, or any combination thereof; (22) heavy chain CDR1, CDR2 and CDR3, or a combination thereof, and / or light chain CDR1, CDR2 and CDR3, or any combination thereof, from 1 to 30; (23) heavy chain CDR1, CDR2 and CDR3, or a combination thereof, from 1 to 17, and / or light chain CDR1, CDR2 and CDR3, or any combination thereof;(23) 1-32 heavy chain CDR1, CDR2 and CDR3, or a combination thereof, and / or 1-32 light chain CDR1, CDR2 and CDR3, or any combination thereof; (24) 4-11 heavy chain CDR1, CDR2 and CDR3, or a combination thereof, and / or 4-11 light chain CDR1, CDR2 and CDR3, or any combination thereof; (25) 6-10 heavy chain CDR1, CDR2 and CDR3, or a combination thereof, and / or 6-10 light chain CDR1, CDR2 and CDR3, or any combination thereof; (26) 2-13D heavy chain CDR1, CDR2 and CDR3, or a combination thereof, and / or 2-13D light chain CDR1, CDR2 and CDR3, or (27) the heavy chain CDR1, CDR2, and CDR3 of 2-13D-37, or a combination thereof, and / or the light chain CDR1, CDR2, and CDR3 of 2-13D-37, or any combination thereof; (28) the heavy chain CDR1, CDR2, and CDR3 of 2-13D-37-1.5W-41, or a combination thereof, and / or the light chain CDR1, CDR2, and CDR3 of 2-13D-37-1.5W-41, or any combination thereof; or (29) the heavy chain CDR1, CDR2, and CDR3 of 2-13D-37-3W-16, or a combination thereof, and / or the light chain CDR1, CDR2, and CDR3 of 2-13D-37-3W-16, or any combination thereof. The amino acid sequences of VH CDR1, CDR2 and CDR3 for the various anti-FAM19A5 antibodies disclosed in the present invention are presented in Table 6. The amino acid sequences of VL CDR1, CDR2 and CDR3 for the various anti-FAM19A5 antibodies disclosed in the present invention are presented in Table 7.

[0189] In some aspects, anti-FAM19A5 antibodies useful in the present invention comprise (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 11, (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 12, (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 13, (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 23, (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 24, and (f) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 25. In some aspects, anti-FAM19A5 antibodies useful in the present invention comprise (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 14, (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 15, (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 16, (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 26, (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and (f) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 28. In some aspects, anti-FAM19A5 antibodies useful in the present invention comprise (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 17, (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 18, (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 19, (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 29, (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and (f) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 31. In some aspects, anti-FAM19A5 antibodies useful in the present invention comprise (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 20, (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 21, (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 22, (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 32, (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 33, and (f) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 34.In some aspects, anti-FAM19A5 antibodies useful in the present invention comprise (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 89, (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 90, (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 91, (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 92, (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 93, and (f) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 94. In some aspects, anti-FAM19A5 antibodies useful in the present invention comprise (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 95, (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 96, (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 97, (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 98, (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 99, and (f) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 100. In some aspects, an anti-FAM19A5 antibody useful in the present invention comprises (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 101, (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 102, (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 103, (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 104, (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 105, and (f) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 106. In some aspects, an anti-FAM19A5 antibody useful in the present invention comprises (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 107, (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 108, (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 109, (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 110, (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 111, and (f) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 112.In some aspects, an anti-FAM19A5 antibody useful in the present invention comprises (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 113, (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 114, (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 115, (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 116, (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 117, and (f) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 118. In some aspects, an anti-FAM19A5 antibody useful in the present invention comprises (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 119, (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 120, (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 121, (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 122, (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 123, and (f) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 124. In some aspects, an anti-FAM19A5 antibody useful in the present invention comprises (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 125, (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 126, (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 127, (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 128, (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 129, and (f) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 130. In some aspects, an anti-FAM19A5 antibody useful in the present invention comprises (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 131, (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 132, (c) a VH CDR comprising the amino acid sequence of SEQ ID NO: 133, (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 134, (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 135, and (f) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 136.In some aspects, an anti-FAM19A5 antibody useful in the present invention comprises (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 137, (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 138, (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 139, (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 140, (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 141, and (f) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 142. In some aspects, an anti-FAM19A5 antibody useful in the present invention comprises (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 143, (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 144, (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 145, (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 146, (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 147, and (f) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 148. In some aspects, an anti-FAM19A5 antibody useful in the present invention comprises (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 149, (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 150, (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 151, (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 152, (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 153, and (f) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 154. In some aspects, an anti-FAM19A5 antibody useful in the present invention comprises (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 17, (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 18, (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 19, (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 208, (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 209, and (f) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 31.In some aspects, an anti-FAM19A5 antibody useful in the present invention comprises (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 17, (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 252, (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 19, (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 208, (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 213, and (f) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 31. In some aspects, an anti-FAM19A5 antibody useful in the present invention comprises (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 17, (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 253, (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 19, (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 208, (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 213, and (f) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 31. In some aspects, an anti-FAM19A5 antibody useful in the present invention comprises (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 218, (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 219, (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 16, (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 26, (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 224, and (f) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 28. In some aspects, an anti-FAM19A5 antibody useful in the present invention comprises (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 220, (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 221, (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 16, (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 225, (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 226, and (f) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 227.In some aspects, an anti-FAM19A5 antibody useful in the present invention comprises (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 220, (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 221, (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 16, (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 225, (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 228, and (f) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 227. In some aspects, an anti-FAM19A5 antibody useful in the present invention comprises (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 220, (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 221, (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 16, (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 26, (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 229, and (f) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 227. In some aspects, an anti-FAM19A5 antibody useful in the present invention comprises (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 220, (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 221, (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 16, (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 26, (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 228, and (f) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 227. In some aspects, an anti-FAM19A5 antibody useful in the present invention comprises (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 220, (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 221, (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 16, (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 26, (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 230, and (f) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 231.In some aspects, an anti-FAM19A5 antibody useful in the present invention comprises (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 220, (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 221, (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 16, (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 26, (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 232, and (f) SEQ ID NO: 233. In some aspects, an anti-FAM19A5 antibody useful in the invention comprises (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 222, (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 12, (c) a VH CDR comprising the amino acid sequence of SEQ ID NO: 13, (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 248, (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 24, and (f) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 25. In some aspects, an anti-FAM19A5 antibody useful in the present invention comprises (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 223, (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 12, (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 13, (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 248, (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 24, and (f) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 25. In some aspects, an anti-FAM19A5 antibody useful in the present invention comprises (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 223, (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 12, (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 246, (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 248, (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 24, and (f) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 25. In some aspects, an anti-FAM19A5 antibody useful in the present invention comprises (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 223, (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 12, (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 247, (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 248, (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 24, and (f) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 25.

[0190] Non-limiting examples of anti-FAM19A5 antibodies that can be used in the present invention are provided in US20220372122A1, US20200299373A1, US20210054062A1, and US20220144932A1, each of which is incorporated by reference in its entirety herein.

[0191] In some aspects, anti-FAM19A5 antibodies useful in the invention bind to at least one epitope of mature human FAM19A5 (also referred to herein as a "FAM19A5 epitope"), e.g., as determined by antibody binding to a fragment of human FAM19A5. In some aspects, a FAM19A5 epitope useful in the invention comprises an amino acid sequence at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98%, at least about 99%, or about 100% identical in sequence to SEQ ID NO: 178, 179, 180, 181, 182, or 183, or a fragment thereof.

[0192] Thus, in some aspects, anti-FAM19A5 antibodies useful in the invention bind to a FAM19A5 epitope comprising an amino acid sequence at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical in sequence to SEQ ID NO: 178 or a fragment thereof. In some aspects, the FAM19A5 epitope comprises SEQ ID NO: 178 or a fragment thereof. In some aspects, the FAM19A5 epitope consists of SEQ ID NO: 178 or a fragment thereof. In some aspects, anti-FAM19A5 antibodies bind to a FAM19A5 epitope comprising an amino acid sequence at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical in sequence to SEQ ID NO: 179 or a fragment thereof. In some aspects, the FAM19A5 epitope comprises the sequence of SEQ ID NO: 179 or a fragment thereof. In some aspects, the FAM19A5 epitope consists of the sequence of SEQ ID NO: 179 or a fragment thereof. In some aspects, an anti-FAM19A5 antibody binds to a FAM19A5 epitope comprising an amino acid sequence at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical in sequence to the sequence of SEQ ID NO: 180 or a fragment thereof. In some aspects, the FAM19A5 epitope comprises the sequence of SEQ ID NO: 180 or a fragment thereof. In some aspects, the FAM19A5 epitope consists of the sequence of SEQ ID NO: 180 or a fragment thereof. In some aspects, the anti-FAM19A5 antibody binds to a FAM19A5 epitope comprising an amino acid sequence at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical in sequence to SEQ ID NO: 181 or a fragment thereof. In some aspects, the FAM19A5 epitope comprises the sequence of SEQ ID NO: 181 or a fragment thereof. In some aspects, the FAM19A5 epitope consists of the sequence of SEQ ID NO: 181 or a fragment thereof.In some aspects, an anti-FAM19A5 antibody binds to a FAM19A5 epitope comprising an amino acid sequence at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical in sequence to SEQ ID NO: 182 or a fragment thereof. In some aspects, the FAM19A5 epitope comprises the sequence of SEQ ID NO: 182 or a fragment thereof. In some aspects, the FAM19A5 epitope consists of the sequence of SEQ ID NO: 182 or a fragment thereof. In some aspects, an anti-FAM19A5 antibody binds to a FAM19A5 epitope comprising an amino acid sequence at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical in sequence to SEQ ID NO: 183 or a fragment thereof. In some aspects, the FAM19A5 epitope comprises the sequence of SEQ ID NO: 183 or a fragment thereof. In some aspects, the FAM19A5 epitope consists of the sequence of SEQ ID NO: 183 or a fragment thereof.

[0193] In some aspects, the anti-FAM19A5 antibody binds to at least one FAM19A5 epitope identified as EP6, EP7, and / or EP8, where EP6 comprises, consists essentially of, or consists of the amino acids KTKQWCDML (SEQ ID NO: 189), EP7 comprises, consists essentially of, or consists of the amino acids GCDLLINR (SEQ ID NO: 190), and EP8 comprises, consists essentially of, or consists of the amino acids TCTQPGGR (SEQ ID NO: 191). In some aspects, the anti-FAM19A5 antibody binds only to EP6, EP7, and / or EP8. In some aspects, the anti-FAM19A5 antibody additionally binds to an additional FAM19A5 epitope selected from the group consisting of SEQ ID NO: 184 (also referred to herein as "EP1"), SEQ ID NO: 185 (also referred to herein as "EP2"), SEQ ID NO: 186 (also referred to herein as "EP3"), SEQ ID NO: 187 (also referred to herein as "EP4"), SEQ ID NO: 188 (also referred to herein as "EP5"), and any combination thereof. In some aspects, the anti-FAM19A5 antibody, or antigen-binding portion thereof, binds to at least one FAM19A5 epitope identified as EP2, EP4, and / or EP8, wherein EP2 comprises, consists essentially of, or consists of the amino acids DSSQP (SEQ ID NO: 185), EP4 comprises, consists essentially of, or consists of the amino acids ARCACRK (SEQ ID NO: 187), and EP8 comprises, consists essentially of, or consists of the amino acids TCTQPGGR (SEQ ID NO: 191). In some aspects, the at least one epitope has an amino acid sequence that is at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to EP2, EP4, or EP8. In some aspects, an anti-FAM19A5 antibody, or antigen-binding portion thereof, binds only to EP2. In some aspects, an anti-FAM19A5 antibody, or antigen-binding portion thereof, binds to EP4 and EP8.In some aspects, the anti-FAM19A5 antibody or antigen-binding portion thereof binds to one or more FAM19A5 epitopes selected from the group consisting of SEQ ID NO: 184 (i.e., EP1), SEQ ID NO: 186 (i.e., EP3), SEQ ID NO: 188 (i.e., EP5), SEQ ID NO: 189 (i.e., EP6), and SEQ ID NO: 190 (i.e., EP7), and any combination thereof.

[0194] In some aspects, the anti-FAM19A5 antibody binds to at least one epitope having the amino acid sequence of QLAAGTCEIVTLDR (amino acid residues 32-45 of SEQ ID NO:178 or SEQ ID NO:2), or a fragment located within said at least one epitope (e.g., a fragment located within the amino acid sequence of SEQ ID NO:178, e.g., an epitope having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 amino acids of SEQ ID NO:178). In some aspects, the anti-FAM19A5 antibody binds to at least one epitope having the amino acid sequence of CDMLPCLEGEGCDLLINRSG (amino acid residues 90-109 of SEQ ID NO:182 or SEQ ID NO:2), or a fragment located within said at least one epitope (e.g., a fragment located within the amino acid sequence of SEQ ID NO:182, e.g., an epitope having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids of SEQ ID NO:182). In some aspects, the anti-FAM19A5 antibody binds to at least one epitope having the amino acid sequence of NRSGWTCTQPGGRIKTTTVS (amino acid residues 106-125 of SEQ ID NO:183 or SEQ ID NO:2), or a fragment located within said at least one epitope (e.g., a fragment located within the amino acid sequence of SEQ ID NO:183, e.g., an epitope having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids of SEQ ID NO:183).

[0195] In some aspects, the anti-FAM19A5 antibody binds to at least one epitope having the amino acid sequence of TLDRDSSQPRRTIARQTARC (amino acid residues 42-61 of SEQ ID NO:179 or SEQ ID NO:2), or a fragment located within said at least one epitope (e.g., a fragment located within the amino acid sequence of SEQ ID NO:179, e.g., an epitope having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids of SEQ ID NO:179). In some aspects, anti-FAM19A5 antibodies useful in the present invention bind to one or more amino acids corresponding to amino acid residues 44 to 52 of SEQ ID NO:2 (i.e., DRDSSQPRR; SEQ ID NO:192), e.g., amino acid residues 45, 46, 50, 51, and 52 (i.e., RD---PRR), amino acid residues 45, 50, 51, and 52 (i.e., R----PRR), or amino acid residues 43, 50, and 51 (i.e., R----PR). In some aspects, anti-FAM19A5 antibodies bind to one or more amino acids corresponding to amino acid residues 46 to 51 of SEQ ID NO:2 (i.e., DSSQPR; SEQ ID NO:196), e.g., amino acid residues 46, 50, and 52 (i.e., D---PR), or amino acid residues 46, 47, 48, and 50 (i.e., DSS-P).

[0196] In some aspects, an anti-FAM19A5 antibody binds to at least one epitope having the amino acid sequence of TARCACRKGQIAGTTRAPA (amino acid residues 58-77 of SEQ ID NO:180 or SEQ ID NO:2), or a fragment located within said at least one epitope (e.g., a fragment located within the amino acid sequence of SEQ ID NO:180, e.g., an epitope having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids of SEQ ID NO:180). In some aspects, an anti-FAM19A5 antibody binds to one or more amino acids corresponding to amino acid residues 63-75 of SEQ ID NO:2 (i.e., CRKGQIAGTTRAR; SEQ ID NO:199).

[0197] In some aspects, an anti-FAM19A5 antibody binds to at least one epitope having the amino acid sequence ARPACVDARIIKTKQWCDML (amino acid residues 74-93 of SEQ ID NO: 181 or SEQ ID NO: 2), or a fragment located within said at least one epitope (e.g., a fragment located within the amino acid sequence of SEQ ID NO: 181, e.g., an epitope having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids of SEQ ID NO: 181). In some aspects, an anti-FAM19A5 antibody that can be used in the present invention binds to one or more amino acids corresponding to amino acid residues 76-89 of SEQ ID NO: 2 (i.e., PACVDARIIKTKQW; SEQ ID NO: 200). In some aspects, the one or more additional FAM19A5 epitopes are chosen from QLAAGTCEIVTLDR (SEQ ID NO: 178, epitope F1), TLDRDSSQPRRTIARQTARC (SEQ ID NO: 179, epitope F2), TARCACRKGQIAGTTRARPA (SEQ ID NO: 180, epitope F3), ARPACVDARIIKTKQWCDML (SEQ ID NO: 181, epitope F4), CDMLPCLEGEGCDLLINRSG (SEQ ID NO: 182, epitope F5), NRSGWTCTQPGGRIKTTTVS (SEQ ID NO: 183, epitope F6), or a fragment located within the amino acid sequence of SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, or SEQ ID NO: 183, or any combination thereof.

[0198] Fragments located within the amino acid sequence of SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, or SEQ ID NO: 183 include fragments having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids of any one of SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, or SEQ ID NO: 183. In some aspects, the anti-FAM19A5 antibodies, or antigen-binding portions thereof, of the invention bind to any one of the one or more additional epitopes in their native form (i.e., undenatured). In some aspects, the anti-FAM19A5 antibody, or antigen-binding portion thereof, binds to both glycosylated and non-glycosylated versions of the one or more additional FAM19A5 epitopes.

[0199] In some aspects, anti-FAM19A5 antibodies useful in the present invention can bind to multiple FAM19A5 epitopes (e.g., those described herein). In some aspects, anti-FAM19A5 antibodies bind to at least two different FAM19A5 epitopes described herein (e.g., SEQ ID NOs: 178-183 or fragments thereof). In some aspects, anti-FAM19A5 antibodies bind to at least three different FAM19A5 epitopes described herein. In some aspects, anti-FAM19A5 antibodies bind to at least four different FAM19A5 epitopes described herein. In some aspects, anti-FAM19A5 antibodies bind to at least five different FAM19A5 epitopes described herein.

[0200] In some aspects, the anti-FAM19A5 antibodies of the invention bind to SEQ ID NO: 179 in its native form (i.e., undenatured) or a fragment thereof. In some aspects, the anti-FAM19A5 antibodies, or antigen-binding portions thereof, bind to both glycosylated and non-glycosylated human FAM19A5.

[0201] III.B Exemplary LRRC4 Family Mimetic Molecules As described later herein, in some aspects, FAM19A5 antagonists useful in the present invention include LRRC4 family mimetic molecules.

[0202] When the LRRC4 family mimetic molecule comprises a small molecule compound, in some aspects the mimetic molecule has the following structural formula or a pharmaceutically acceptable salt thereof:

[0203] [ka]

[0204] where: (i) R1, R2 and R3 are independently hydrogen, fluoro, chloro, bromo, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl, isopentyl, fluoromethyl, difluoromethyl, trifluoromethyl, 2-fluoroethyl, 1-fluoroethyl, 2,2-difluoroethyl, 1,2-difluoroethyl, 1,1-difluoroethyl, 2,2,2-trifluoroethyl, methoxy, ethoxy, n-propyloxy, isopropyloxy, n-butoxy, trifluoromethoxy, difluoromethyl, ... selected from fluoromethoxy, fluoromethoxy, acetyl, propionyl, n-butanoyl, isobutanoyl, n-pentanoyl, nitro, amino, N-methylamino, N-ethylamino, N,N-propylamino, N,N-dimethylamino, N-acetylamino, N-propionylamino, N-(trifluoroacetyl)amino, formyl, hydroxy, methylthio, ethylthio, n-propylthio, methylsulfonyl, ethylsulfonyl, n-propylsulfonyl, phenyl, hydroxymethyl, 1-hydroxyethyl, and 2-hydroxyethyl; (ii)

[0205] [ka]

[0206] is a single or double bond; (iii) Z is a linear or branched (C1-C8) alkyl, a linear or branched (C2-C8) alkenyl, a linear or branched (C2-C8) alkynyl, a (C3-C8) cycloalkyl, a (C5-C8) cycloalkenyl, a (3- to 8-membered) heterocycloalkyl, a (C7-C 14 )bicycloalkyl, (C7-C 14 ) bicycloalkenyl, (7- to 14-membered) heterobicycloalkyl, (C6-C 10 )aryl, (5- to 10-membered)heteroaryl, and —CH—C(O)—CH═CH—Q (wherein Q is (C3-C8)cycloalkyl, (C5-C8)cycloalkenyl, (3- to 8-membered)heterocycloalkyl, (C6-C 10 )aryl and (5- to 6-membered)heteroaryl; each cycloalkyl, cycloalkenyl, heterocyclylalkyl, aryl, and heteroaryl may be independently substituted with 1, 2, 3, 4, or 5 substituents selected from (C1-C6)alkoxy, (C1-C6)alkyl, halo, (C1-C6)haloalkoxy, nitro, amino, N-methylamino, N-ethylamino, N,N-propylamino, N,N-dimethylamino, formyl, and hydroxy; and (iv) L is a single, double or triple bond.

[0207] In some aspects, R1, R2, and R3 are selected from hydrogen, hydroxy, methoxy, ethoxy, n-propyloxy, isopropyloxy, n-butoxy, trifluoromethoxy, difluoromethoxy, and fluoromethoxy. In some aspects, R1, R2, and R3 are selected from hydrogen, hydroxy, methoxy, ethoxy, n-propyloxy, isopropyloxy, and n-butoxy. In some aspects, R1, R2, and R3 are selected from hydrogen, hydroxy, and methoxy. In some aspects, R1 and R2 are selected from hydroxy and methoxy, and R3 is hydrogen.

[0208] In some aspects, Z is selected from linear or branched (C1-C8) alkyl, linear or branched (C2-C8) alkenyl, linear or branched (C2-C8) alkynyl, and -CH-C(O)-CH=CH-Q (where Q is (C6-C 10 )aryl and (5- to 6-membered)heteroaryl; wherein the aryl and heteroaryl may be independently substituted with one, two, or three substituents independently selected from (C1-C6)alkoxy, (C1-C6)alkyl, halo, (C1-C6)haloalkoxy, and hydroxy. In some aspects, Z is selected from linear or branched (C1-C8)alkyl, linear or branched (C2-C8)alkenyl, and -CH-C(O)-CH=CH-Q (where Q may be substituted with one, two, or three substituents independently selected from (C1-C6)alkoxy, (C1-C6)alkyl, halo, (C1-C6)haloalkoxy, and hydroxyl). 10 ) aryl).

[0209] In some aspects, the LRRC4 family mimetic molecule is a small molecule of Formula I: (i) R1, R2 and R3 are selected from hydrogen, hydroxy, methoxy, ethoxy, n-propyloxy, iso-propyloxy, n-butoxy, trifluoromethoxy, difluoromethoxy and fluoromethoxy; (ii)

[0210] [ka]

[0211] is a single or double bond; (iii) Z is a linear or branched (C1-C8) alkyl, a linear or branched (C2-C8) alkenyl, a linear or branched (C2-C8) alkynyl, and -CH-C(O)-CH=CH-Q (wherein Q is (C6-C 10) aryl and (5- to 6-membered) heteroaryl; wherein said aryl and heteroaryl may be substituted with 1, 2, or 3 substituents independently selected from (C1-C6) alkoxy, (C1-C6) alkyl, halo, (C1-C6) haloalkoxy, and hydroxy; and (iv) L is a double or triple bond. In some aspects, the LRRC4 family mimetic molecule is a small molecule of Formula I: (i) R1, R2 and R3 are selected from hydrogen, hydroxy, methoxy, ethoxy, n-propyloxy, iso-propyloxy and n-butoxy; (ii)

[0212] [ka]

[0213] is a single or double bond; (iii) Z is a linear or branched (C1-C8) alkyl, a linear or branched (C2-C8) alkenyl, and -CH-C(O)-CH=CH-Q (wherein Q can be substituted with one, two, or three substituents independently selected from (C1-C6) alkoxy, (C1-C6) alkyl, halo, (C1-C6) haloalkoxy, and hydroxy (C6-C 10 ) aryl); and (iv) L is a double or triple bond.

[0214] In some aspects, the LRRC4 family mimetic molecule is selected from the following structural formulas or a pharmaceutically acceptable salt thereof:

[0215] [ka]

[0216] In some aspects, the LRRC4 family mimetic molecule has the following structural formula or a pharmaceutically acceptable salt thereof:

[0217] [ka]

[0218] In some aspects, the LRRC4 family mimetic molecule has the following structural formula or a pharmaceutically acceptable salt thereof:

[0219] [ka]

[0220] In some aspects, the LRRC4 family mimetic molecule has the following structural formula or a pharmaceutically acceptable salt thereof:

[0221] [ka]

[0222] In some aspects, the LRRC4 family mimetic molecule has the following structural formula or a pharmaceutically acceptable salt thereof:

[0223] [ka]

[0224] In some aspects, the LRRC4 family mimetic molecule has the following structural formula or a pharmaceutically acceptable salt thereof:

[0225] [ka]

[0226] where: (i) R1, R2 and R3 are independently hydrogen, fluoro, chloro, bromo, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl, isopentyl, fluoromethyl, difluoromethyl, trifluoromethyl, 2-fluoroethyl, 1-fluoroethyl, 2,2-difluoroethyl, 1,2-difluoroethyl, 1,1-difluoroethyl, 2,2,2-trifluoroethyl, methoxy, ethoxy, n-propyloxy, isopropyloxy, n-butoxy, trifluoromethoxy, difluoromethyl, ... selected from fluoromethoxy, fluoromethoxy, acetyl, propionyl, n-butanoyl, isobutanoyl, n-pentanoyl, nitro, amino, N-methylamino, N-ethylamino, N,N-propylamino, N,N-dimethylamino, N-acetylamino, N-propionylamino, N-(trifluoroacetyl)amino, formyl, hydroxy, methylthio, ethylthio, n-propylthio, methylsulfonyl, ethylsulfonyl, n-propylsulfonyl, phenyl, hydroxymethyl, 1-hydroxyethyl, and 2-hydroxyethyl; (ii)

[0227] [ka]

[0228] is a single or double bond; (iii) Z is a linear or branched (C1-C8) alkyl, a linear or branched (C2-C8) alkenyl, a linear or branched (C2-C8) alkynyl, a (C3-C8) cycloalkyl, a (C5-C8) cycloalkenyl, a (3- to 8-membered) heterocycloalkyl, a (C7-C 14 )bicycloalkyl, (C7-C 14 ) bicycloalkenyl, (7- to 14-membered) heterobicycloalkyl, (C6-C 10 )aryl, (5- to 10-membered)heteroaryl, and —CH—C(O)—CH═CH—Q (wherein Q is (C3-C8)cycloalkyl, (C5-C8)cycloalkenyl, (3- to 8-membered)heterocycloalkyl, (C6-C 10)aryl and (5- to 6-membered)heteroaryl; each cycloalkyl, cycloalkenyl, heterocyclylalkyl, aryl, and heteroaryl may be independently substituted by 1, 2, 3, 4, or 5 substituents selected from (C1-C6)alkoxy, (C1-C6)alkyl, halo, (C1-C6)haloalkoxy, nitro, amino, N-methylamino, N-ethylamino, N,N-propylamino, N,N-dimethylamino, formyl, and hydroxy; (iv) L is a single, double or triple bond, and The LRRC4 family mimetic molecule is not selected from the following structural formulas or pharmaceutically acceptable salts thereof:

[0229] [ka]

[0230] In some aspects, the LRRC4 family mimetic molecule has the following structural formula or a pharmaceutically acceptable salt thereof:

[0231] [ka]

[0232] where: (i) R1, R2 and R3 are hydrogen, fluoro, chloro, bromo, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl, isopentyl, fluoromethyl, difluoromethyl, trifluoromethyl, 2-fluoroethyl, 1-fluoroethyl, 2,2-difluoroethyl, 1,2-difluoroethyl, 1,1-difluoroethyl, 2,2,2-trifluoroethyl, methoxy, ethoxy, n-propyloxy, isopropyloxy, n-butoxy, trifluoromethoxy, difluoromethoxy independently selected from thio, fluoromethoxy, acetyl, propionyl, n-butanoyl, isobutanoyl, n-pentanoyl, nitro, amino, N-methylamino, N-ethylamino, N,N-propylamino, N,N-dimethylamino, N-acetylamino, N-propionylamino, N-(trifluoroacetyl)amino, formyl, hydroxy, methylthio, ethylthio, n-propylthio, methylsulfonyl, ethylsulfonyl, n-propylsulfonyl, phenyl, hydroxymethyl, 1-hydroxyethyl, and 2-hydroxyethyl; (ii) Z is a linear or branched (C1-C8) alkyl, a linear or branched (C2-C8) alkenyl, a linear or branched (C2-C8) alkynyl, a (C3-C8) cycloalkyl, a (C5-C8) cycloalkenyl, a (3- to 8-membered) heterocycloalkyl, a (C7-C 14 )bicycloalkyl, (C7-C 14 ) bicycloalkenyl, (7- to 14-membered) heterobicycloalkyl, (C6-C 10 )aryl, (5- to 10-membered)heteroaryl, and —CH═CH—Q (wherein Q is (C3-C8)cycloalkyl, (C5-C8)cycloalkenyl, (3- to 8-membered)heterocycloalkyl, (C6-C 10)aryl and (5- to 6-membered)heteroaryl; each cycloalkyl, cycloalkenyl, heterocyclylalkyl, aryl, and heteroaryl may be substituted by 1, 2, 3, 4, or 5 substituents selected from (C1-C6)alkoxy, (C1-C6)alkyl, halo, (C1-C6)haloalkoxy, nitro, amino, N-methylamino, N-ethylamino, N-N-propylamino, N,N-dimethylamino, formyl, and hydroxy; and (iii) L is a single, double or triple bond.

[0233] In some aspects, R1, R2, and R3 are selected from hydrogen, fluoro, chloro, bromo, hydroxy, methoxy, ethoxy, n-propyloxy, isopropyloxy, n-butoxy, trifluoromethoxy, difluoromethoxy, fluoromethoxy, amino, N-methylamino, N-ethylamino, N,N-propylamino, and N,N-dimethylamino. In some aspects, R1, R2, and R3 are selected from hydrogen, fluoro, chloro, bromo, hydroxy, methoxy, ethoxy, n-propyloxy, amino, N-methylamino, N-ethylamino, N,N-propylamino, and N,N-dimethylamino. In some aspects, R1, R2, and R3 are selected from hydrogen, fluoro, hydroxy, methoxy, and N,N-dimethylamino. In some aspects, R1 and R2 are selected from fluoro, hydroxy, methoxy, and N,N-dimethylamino, and R3 is hydrogen.

[0234] In some aspects, Z is selected from linear or branched (C1-C8) alkyl, linear or branched (C2-C8) alkenyl, linear or branched (C2-C8) alkynyl, and -CH=CH-Q (where Q is (C6-C 10)aryl and (5- to 6-membered)heteroaryl; wherein the aryl and heteroaryl may be substituted with one, two, or three substituents independently selected from (C1-C6)alkoxy, (C1-C6)alkyl, halo, (C1-C6)haloalkoxy, and hydroxy. In some aspects, Z is selected from linear or branched (C1-C8)alkyl, linear or branched (C2-C8)alkenyl, and -CH=CH-Q (where Q may be substituted with one, two, or three substituents independently selected from (C1-C6)alkoxy, (C1-C6)alkyl, halo, (C1-C6)haloalkoxy, and hydroxyl). 10 ) aryl).

[0235] In some aspects, the LRRC4 family mimetic molecule is a small molecule of Formula II: (i) R1, R2, and R3 are selected from hydrogen, fluoro, chloro, bromo, hydroxy, methoxy, ethoxy, n-propyloxy, isopropyloxy, n-butoxy, trifluoromethoxy, difluoromethoxy, fluoromethoxy, amino, N-methylamino, N-ethylamino, N-N-propylamino, and N,N-dimethylamino. In some aspects, R1, R2, and R3 are selected from hydrogen, fluoro, chloro, bromo, hydroxy, methoxy, ethoxy, n-propyloxy, amino, N-methylamino, N-ethylamino, N-N-propylamino, and N,N-dimethylamino; (ii) Z is a linear or branched (C1-C8) alkyl, a linear or branched (C2-C8) alkenyl, a linear or branched (C2-C8) alkynyl, and -CH=CH-Q (wherein Q is (C6-C 10 ) aryl and (5- to 6-membered) heteroaryl; wherein said aryl and heteroaryl may be substituted with 1, 2, or 3 substituents independently selected from (C1-C6) alkoxy, (C1-C6) alkyl, halo, (C1-C6) haloalkoxy, and hydroxy; and (iii) L is a double bond.

[0236] In some aspects, the LRRC4 family mimetic molecule is a small molecule of Formula II: (i) R1, R2 and R3 are selected from hydrogen, fluoro, chloro, bromo, hydroxy, methoxy, ethoxy, n-propyloxy, amino, N-methylamino, N-ethylamino, N,N-propylamino and N,N-dimethylamino; (ii) Z is a straight-chain or branched (C1-C8) alkyl, a straight-chain or branched (C2-C8) alkenyl, and -CH=CH-Q (wherein Q can be substituted with one, two, or three substituents independently selected from (C1-C6) alkoxy, (C1-C6) alkyl, halo, (C1-C6) haloalkoxy, and hydroxy (C6-C 10 ) aryl); and (iii) L is a double bond.

[0237] In some aspects, the LRRC4 family molecule is selected from the following structural formulas or a pharmaceutically acceptable salt thereof:

[0238] [ka]

[0239] In some aspects, the LRRC4 family mimetic molecule has the following structural formula or a pharmaceutically acceptable salt thereof:

[0240] [ka]

[0241] In some aspects, the LRRC4 family mimetic molecule has the following structural formula or a pharmaceutically acceptable salt thereof:

[0242] [ka]

[0243] In some aspects, the LRRC4 family mimetic molecule has the following structural formula or a pharmaceutically acceptable salt thereof:

[0244] [ka]

[0245] In some aspects, the LRRC4 family mimetic molecule has the following structural formula or a pharmaceutically acceptable salt thereof:

[0246] [ka]

[0247] In some aspects, the LRRC4 family mimetic molecule has the following structural formula or a pharmaceutically acceptable salt thereof:

[0248] [ka]

[0249] In some aspects, the LRRC4 family mimetic molecule has the following structural formula or a pharmaceutically acceptable salt thereof:

[0250] [ka]

[0251] In some aspects, the LRRC4 family mimetic molecule has the following structural formula or a pharmaceutically acceptable salt thereof:

[0252] [ka]

[0253] where: (i) R1, R2 and R3 are hydrogen, fluoro, chloro, bromo, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl, isopentyl, fluoromethyl, difluoromethyl, trifluoromethyl, 2-fluoroethyl, 1-fluoroethyl, 2,2-difluoroethyl, 1,2-difluoroethyl, 1,1-difluoroethyl, 2,2,2-trifluoroethyl, methoxy, ethoxy, n-propyloxy, isopropyloxy, n-butoxy, trifluoromethoxy, difluoromethoxy independently selected from thio, fluoromethoxy, acetyl, propionyl, n-butanoyl, isobutanoyl, n-pentanoyl, nitro, amino, N-methylamino, N-ethylamino, N,N-propylamino, N,N-dimethylamino, N-acetylamino, N-propionylamino, N-(trifluoroacetyl)amino, formyl, hydroxy, methylthio, ethylthio, n-propylthio, methylsulfonyl, ethylsulfonyl, n-propylsulfonyl, phenyl, hydroxymethyl, 1-hydroxyethyl, and 2-hydroxyethyl; (ii) Z is a linear or branched (C1-C8) alkyl, a linear or branched (C2-C8) alkenyl, a linear or branched (C2-C8) alkynyl, -Y-(C3-C8) cycloalkyl, -Y-(C5-C8) cycloalkenyl, -Y-(3- to 8-membered) heterocycloalkyl, -Y-(C7-C 14 )bicycloalkyl, -Y-(C7-C 14 )bicycloalkenyl, -Y-(7- to 14-membered)heterobicycloalkyl, -Y-(C6-C 10 )aryl and -Y-(5 to 10 membered)heteroaryl, wherein Y is a bond or a C1-C3 linear or branched alkylene, and wherein said cycloalkyl, cycloalkenyl, heterocyclylalkyl, aryl, and heteroaryl may be substituted by 1, 2, 3, 4, or 5 substituents independently selected from C1-C6 alkoxy, C1-C6 alkyl, halo, C1-C6 haloalkoxy, nitro, amino, N-methylamino, N-ethylamino, N-N-propylamino, N,N-dimethylamino, formyl, and hydroxy; (iii) L is a single, double or triple bond, and (iv) n is 0 or 1.

[0254] In some aspects, R1, R2, and R3 are selected from hydrogen, hydroxy, methoxy, ethoxy, n-propyloxy, isopropyloxy, n-butoxy, trifluoromethoxy, difluoromethoxy, and fluoromethoxy. In some aspects, R1, R2, and R3 are selected from hydrogen, hydroxy, methoxy, ethoxy, n-propyloxy, isopropyloxy, and n-butoxy. In some aspects, R1, R2, and R3 are selected from hydrogen, hydroxy, and methoxy. In some aspects, R1 and R2 are selected from hydroxy and methoxy, and R3 is hydrogen.

[0255] In some aspects, Z is selected from the group consisting of -Y-(C3-C8)cycloalkyl, -Y-(C5-C8)cycloalkenyl, -Y-(3- to 8-membered)heterocycloalkyl, -Y-(C7-C 14 )bicycloalkyl, -Y-(C7-C 14 )bicycloalkenyl, -Y-(7- to 14-membered)heterobicycloalkyl, -Y-(C6-C 10 )aryl, and -Y-(5 to 10 membered)heteroaryl, where Y is a bond or C1-C3 linear or branched alkylene, and the cycloalkyl, cycloalkenyl, heterocyclylalkyl, aryl, and heteroaryl may be independently substituted with 1, 2, 3, 4, or 5 substituents selected from C1-C6 alkoxy, C1-C6 alkyl, halo, C1-C6 haloalkoxy, nitro, amino, N-methylamino, N-ethylamino, N-N-propylamino, N,N-dimethylamino, formyl, and hydroxy. In some aspects, Z is selected from -Y-(C6-C 10 )aryl and -Y-(5 to 10 membered)heteroaryl, where Y is a bond or a C1-C3 linear or branched alkylene, and the aryl and heteroaryl can be substituted with 1, 2, or 3 substituents independently selected from C1-C6 alkoxy and halo.

[0256] In some aspects, the LRRC4 family mimetic molecule is a small molecule of Formula III: (i) R1, R2 and R3 are selected from hydrogen, hydroxy, methoxy, ethoxy, n-propyloxy, iso-propyloxy, n-butoxy, trifluoromethoxy, difluoromethoxy and fluoromethoxy; (ii) Z is -Y-(C3-C8)cycloalkyl, -Y-(C5-C8)cycloalkenyl, -Y-(3- to 8-membered)heterocycloalkyl, -Y-(C7-C 14 )bicycloalkyl, -Y-(C7-C 14 )bicycloalkenyl, -Y-(7- to 14-membered)heterobicycloalkyl, -Y-(C6-C 10 )aryl, and -Y-(5 to 10 membered)heteroaryl, wherein Y is a bond or C1-C3 linear or branched alkylene, and the cycloalkyl, cycloalkenyl, heterocyclylalkyl, aryl, and heteroaryl may be independently substituted with 1, 2, 3, 4, or 5 substituents selected from C1-C6 alkoxy, C1-C6 alkyl, halo, C1-C6 haloalkoxy, nitro, amino, N-methylamino, N-ethylamino, N-N-propylamino, N,N-dimethylamino, formyl, and hydroxy; and (iii) L is a triple bond.

[0257] In some aspects, the LRRC4 family mimetic molecule is a small molecule of Formula III: (i) R1, R2 and R3 are selected from hydrogen, hydroxy, methoxy, ethoxy, n-propyloxy, iso-propyloxy and n-butoxy; (ii) Z is -Y-(C6-C 10 ) aryl and -Y-(5- to 10-membered)heteroaryl, where Y is a bond or a C1-C3 linear or branched alkylene, and the aryl and heteroaryl may be substituted with 1, 2, or 3 substituents independently selected from C1-C6 alkoxy and halo; and (iii) L is a triple bond.

[0258] In some aspects, the LRRC4 family mimetic molecule is selected from the following structural formulas or a pharmaceutically acceptable salt thereof:

[0259] [ka]

[0260] In some aspects, the LRRC4 family mimetic molecule has the following structural formula or a pharmaceutically acceptable salt thereof:

[0261] [ka]

[0262] In some aspects, the LRRC4 family mimetic molecule has the following structural formula or a pharmaceutically acceptable salt thereof:

[0263] [ka]

[0264] In some aspects, the LRRC4 family mimetic molecule has the following structural formula or a pharmaceutically acceptable salt thereof:

[0265] [ka]

[0266] As described herein, when an LRRC4 family mimetic molecule useful in the present invention comprises a polypeptide, in some aspects, the polypeptide comprises at least a FAM19A5-binding domain of an LRRC4 protein family member. Unless otherwise specified, the overall length of the FAM19A5-binding domain is not particularly limited, as long as the domain is capable of binding to the FAM19A5 protein. In some aspects, the FAM19A5 binding domain is at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, or at least about 30 amino acids in length. In some aspects, the FAM19A5 binding domain is between about 10 and about 23 amino acids in length. In some aspects, the FAM19A5 binding domain is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 22, or about 23 amino acids in length. In some aspects, the FAM19A5 binding domain of an LRRC4 family mimetic molecule is about 10 amino acids in length.

[0267] In some aspects, the polypeptide of the LRRC4 family mimetic molecule comprises an amino acid sequence having the following formula (from N-terminal to C-terminal): A-(T / S)-B (Formula IV) (SEQ ID NO: 317), wherein: (i) "A" includes X1-(T / S)-(Y / F)-F-X5, and (ii) "B" includes (V / I)-TV-(E / V), where: X1 is tyrosine (Y), phenylalanine (F), valine (V), leucine (L) or isoleucine (I); (T / S) is threonine (T) or serine (S); (Y / F) is tyrosine (Y) or phenylalanine (F); X5 is any amino acid; (V / I) is valine (V) or isoleucine (I); and (E / V) is glutamic acid (E) or valine (V).

[0268] In some aspects, the polypeptides of the LRRC4 family mimetic molecules described herein comprise an amino acid sequence having the following formula (from N-terminal to C-terminal): A-(T / S)-B (Formula IV) (SEQ ID NO: 318), wherein: (i) "A" includes (Y / W / M)-(T / Y)-(Y / W)-(F / Y / W)-(T / Y), and (ii) "B" includes X7-(T / S / Y)-X9-X10, where: (Y / W / M) is tyrosine (Y), tryptophan (W) or methionine (M); (T / Y) is threonine (T) or tyrosine (Y); (Y / W) is tyrosine (Y) or tryptophan (W); (F / Y / W) is phenylalanine (F), tyrosine (Y), or tryptophan (W); X7 is valine (V), tyrosine (Y), phenylalanine (F), leucine (L), tryptophan (W), or methionine (M); (T / S / Y) is threonine (T), serine (S), or tyrosine (Y); X9 is valine (V), isoleucine (I), tyrosine (Y), phenylalanine (F), leucine (L), tryptophan (W), or methionine (M); and X10 is glutamic acid (E), aspartic acid (D), isoleucine (I), tyrosine (Y), phenylalanine (F), methionine (M), or tryptophan (W).

[0269] In some aspects, LRRC4 family mimetic molecules useful in the present invention include polypeptides comprising an amino acid sequence having the following formula (N-terminal to C-terminal): X1-X2-X3-F-X5-T-X7-T V-X10 (Formula V) (SEQ ID NO: 319), wherein: X1 is Y, F, V, L or I; X2 is T or S; X3 is Y or F; X5 is any amino acid; X7 is V or I; and / or X10 is E or V, The polypeptide is capable of binding to the FAM19A5 protein, thereby inhibiting, reducing and / or dissociating the interaction between the FAM19A5 protein and a member of the LRRC4 protein family.

[0270] In some aspects, the LRRC4 family mimetic molecules described herein include polypeptides comprising an amino acid sequence having the following formula (from N-terminal to C-terminal): X1-X2-X3-X4-X5-X6-X7-X8-X9-X10 (Formula VI) (SEQ ID NO: 320), wherein: X1 is Y, F, V, L, I, W or M; X2 is T, S or Y; X3 is Y, F or W; X4 is F, Y or W; X5 is any amino acid (e.g., T, S, or Y); X6 is T, S or Y; X7 is V, I, Y, F, L, W or M; X8 is T, S or Y; X9 is V, I, Y, F, L, W or M; and / or X10 is E, D, V, I, Y, F, M or W, The polypeptide is capable of binding to the FAM19A5 protein, thereby inhibiting, reducing and / or dissociating the interaction between the FAM19A5 protein and a member of the LRRC4 protein family.

[0271] For any of the above LRRC4 family mimetic molecules, in some aspects, (i) X1 is Y, F, V, L, or I; (ii) X2 is T or S; (iii) X3 is Y or F; (iv) X4 is F; (v) X5 is T or S; (vi) X6 is T; (vii) X7 is V or I; (viii) X8 is T; (ix) X9 is V; (x) X10 is E or V; or (xi) any combination of (i) through (x) is also possible. In some aspects, X1 is Y, F, V, L, or I. In some aspects, X2 is T or S. In some aspects, X3 is Y or F. In some aspects, X4 is F. In some aspects, X5 is T or S. In some aspects, X6 is T. In some aspects, X7 is V or I. In some aspects, X8 is T. In some aspects, X9 is V. In some aspects, X10 is E or V. In some aspects, the amino acid at position X2 is phosphorylated. In some aspects, the amino acid at position X2 is O-glycosylated.

[0272] In some aspects, the polypeptides of the LRRC4 family mimetic molecules described herein comprise an amino acid sequence that differs (e.g., by substitution) by one, two, three, four, five, or six amino acids from the amino acid sequence of SEQ ID NO: 44 (YTYFTTVTVE). In some aspects, the polypeptides of the LRRC4 family mimetic molecules disclosed herein consist of an amino acid sequence that differs (e.g., by substitution) by one, two, three, four, five, or six amino acids from the amino acid sequence of SEQ ID NO: 44 (YTYFTTVTVE). In some aspects, the polypeptides of the LRRC4 family mimetic molecules disclosed herein consist essentially of an amino acid sequence that differs (e.g., by substitution) by one, two, three, four, five, or six amino acids from the amino acid sequence of SEQ ID NO: 44 (YTYFTTVTVE).

[0273] In some aspects, the polypeptide of the LRRC4 family mimetic molecule comprises the amino acid sequence of SEQ ID NO: 44 (YTYFTTVTVE). In some aspects, the polypeptide of the LRRC4 family mimetic molecule consists of the amino acid sequence of SEQ ID NO: 44 (YTYFTTVTVE). In some aspects, the polypeptide of the LRRC4 family mimetic molecule consists essentially of the amino acid sequence of SEQ ID NO: 44 (YTYFTTVTVE). As demonstrated herein, the amino acid sequence of SEQ ID NO: 44 (YTYFTTVTVE) corresponds to the FAM19A5-binding domain of the LRRC4B protein.

[0274] In some aspects, the polypeptide of the LRRC4 family mimetic molecule comprises the amino acid sequence of SEQ ID NO: 45 (YSFFTTVTVE). In some aspects, the polypeptide of the LRRC4 family mimetic molecule consists of the amino acid sequence of SEQ ID NO: 45 (YSFFTTVTVE). In some aspects, the polypeptide of the LRRC4 family mimetic molecule consists essentially of the amino acid sequence of SEQ ID NO: 45 (YSFFTTVTVE). As demonstrated herein, the amino acid sequence of SEQ ID NO: 45 (YSFFTTVTVE) corresponds to the FAM19A5-binding domain of the LRRC4 protein.

[0275] In some aspects, the polypeptide of the LRRC4 family mimetic molecule comprises the amino acid sequence of SEQ ID NO: 46 (FSYFSTVTVE). In some aspects, the polypeptide of the LRRC4 family mimetic molecule consists of the amino acid sequence of SEQ ID NO: 46 (FSYFSTVTVE). In some aspects, the polypeptide of the LRRC4 family mimetic molecule consists essentially of the amino acid sequence of SEQ ID NO: 46 (FSYFSTVTVE). As demonstrated herein, the amino acid sequence of SEQ ID NO: 46 (FSYFSTVTVE) corresponds to the FAM19A5-binding domain of the LRRC4C protein.

[0276] As described herein, the FAM19A5-binding domain of LRRC4 protein family members is generally conserved among vertebrates. Thus, without being limited to a particular theory, one or more amino acid residues in any one of the amino acid sequences of SEQ ID NOs: 44 (YTYFTTVTVE), 45 (YSFFTTVTVE), and 46 (FSYFSTVTVE) can be substituted with an amino acid present at that residue in another vertebrate. Examples of such substitutions are provided herein.

[0277] In some aspects, one or more amino acid residues in any one of the amino acid sequences of SEQ ID NO: 44 (YTYFTTVTVE), 45 (YSFTTVTVE), and 46 (FSYFSTVTVE) may be substituted with an amino acid that shares similar biochemical properties. For example, in some aspects, in the amino acid sequence of SEQ ID NO: 44 (YTYFTTVTVE), the Y at position 1 may be substituted with another hydrophobic amino acid (e.g., F, V, L, I, W, or M). In some aspects, the T at position 2 may be substituted with another amino acid (e.g., S or Y) that has a similar hydroxyl (OH) group in its side chain. In some aspects, the Y at position 3 may be substituted with another amino acid (e.g., F or W) that has a common aromatic ring in its side chain that can participate in van der Waals interactions. In some aspects, the F at position 4 may be substituted with an amino acid such as Y or W. In some aspects, the T at position 5 may be substituted with an amino acid such as S or Y. In some aspects, the V at position 6 may be substituted with an amino acid such as S or Y. In some aspects, the V at position 7 can be substituted with another amino acid having a bulky hydrophobic side chain (e.g., I, Y, F, L, W, or M). In some aspects, the T at position 8 can be substituted with another amino acid such as S or Y. In some aspects, the V at position 9 can be substituted with another amino acid such as I, Y, F, L, W, or M. In some aspects, the E at position 10 can be substituted with another amino acid having an acidic side chain (e.g., I, Y, F, M, or W).

[0278] In some aspects, the LRRC4 family mimetic molecule polypeptides described herein comprise an amino acid sequence 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% identical to the amino acid sequence of SEQ ID NO:44 (YTYFTTVTVE), wherein the polypeptide is capable of binding to a FAM19A5 protein and inhibiting, reducing, and / or dissociating the interaction between the FAM19A5 protein and a member of the LRRC4 protein family. In some aspects, the LRRC4 family mimetic molecule polypeptide comprises an amino acid sequence at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO:9. In this case, the polypeptide can bind to the FAM19A5 protein and inhibit, reduce, and / or dissociate the interaction between the FAM19A5 protein and a member of the LRRC4 protein family. In some aspects, the polypeptide of the LRRC4 family mimetic molecule comprises an amino acid sequence that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 6. In this case, the polypeptide can bind to the FAM19A5 protein, thereby inhibiting, reducing, and / or dissociating the interaction between the FAM19A5 protein and a member of the LRRC4 protein family.In some aspects, a polypeptide of the LRRC4 family mimetic molecule comprises an amino acid sequence at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 84, wherein the polypeptide is capable of binding to a FAM19A5 protein, thereby inhibiting, reducing, and / or dissociating the interaction between the FAM19A5 protein and a member of the LRRC4 protein family.

[0279] As apparent from the present invention, in some aspects, the polypeptides of the LRRC4 family mimetic molecules described herein (e.g., comprising the FAM19A5-binding domain of an LRRC4 protein family member) contain one or more amino acid modifications. In some aspects, the one or more amino acid modifications can increase the binding affinity of the LRRC4 family mimetic molecule for FAM19A5 protein. Thus, in some aspects, the binding affinity of the LRRC4 family mimetic molecules described herein for FAM19A5 protein is increased by at least about 0.5-fold, at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold compared to a reference molecule (e.g., a corresponding LRRC4 family mimetic molecule without the amino acid modifications, or a naturally occurring member of the LRRC4 protein family). In some aspects, the one or more amino acid variations can improve the stability of the LRRC4 family mimetic molecule. Thus, in some aspects, the stability of the LRRC4 family mimetic molecules described herein is increased by at least about 0.5-fold, at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold relative to a reference (e.g., a corresponding LRRC4 family mimetic molecule without the amino acid variation, or a naturally occurring member of the LRRC4 protein family).

[0280] In some aspects, the one or more amino acid modifications can improve the ability of the LRRC4 family mimetic molecules described herein to inhibit the interaction between the FAM19A5 protein and a member of the LRRC4 protein family (e.g., by increasing binding affinity and / or stability). Thus, in some aspects, the ability of the LRRC4 family mimetic molecule to inhibit the interaction between the FAM19A5 protein and a member of the LRRC4 protein family is increased by at least about 0.5-fold, at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold compared to a reference (e.g., a corresponding LRRC4 family mimetic molecule without the amino acid modification(s), or a naturally occurring member of the LRRC4 protein family).

[0281] Non-limiting examples of amino acid variations useful in the present invention are provided throughout this specification. For example, in some aspects, the LRRC4 family mimetic molecules described herein comprise one of the FAM19A5-binding domains of the LRRC4 protein family members, i.e., YTYFTTVTVE (SEQ ID NO: 44), YSFFTTVTVE (SEQ ID NO: 45), or FSYFSTVTVE (SEQ ID NO: 46), and one or more amino acids at the N-terminus, C-terminus, or both the N-terminus and C-terminus of the polypeptide. In some aspects, polypeptides useful in the invention comprise at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, or at least about 20 additional amino acids at the N-terminus of the polypeptide. In some aspects, the polypeptide comprises at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, or at least about 20 additional amino acids at the C-terminus of the polypeptide.In some aspects, the polypeptide comprises: (i) at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, or at least about 20 additional amino acids at the N-terminus of the polypeptide; and (ii) at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, or at least about 20 additional amino acids at the C-terminus of the polypeptide. In some aspects, the one or more amino acids differ from the amino acid present at a particular residue in a naturally occurring LRRC4 protein family member.

[0282] For example, in some aspects, the polypeptides described herein comprise an amino acid sequence having one or more amino acid variations (e.g., substitutions) in the amino acid sequence of SEQ ID NO:47 (GYTYFTTVTVETLETQPGEE). In some aspects, the polypeptides described herein comprise an amino acid sequence having two amino acid variations (e.g., substitutions) in the amino acid sequence of SEQ ID NO:47 (GYTYFTTVTVETLETQPGEE). In some aspects, the amino acid variations are at residues T12 and L13 of SEQ ID NO:47. In some aspects, the polypeptides described herein consist of an amino acid sequence having one or more amino acid variations (e.g., substitutions) in the amino acid sequence of SEQ ID NO:47 (GYTYFTTVTVETLETQPGEE). In some aspects, the polypeptides described herein consist of an amino acid sequence having two amino acid variations (e.g., substitutions) in the amino acid sequence of SEQ ID NO:47 (GYTYFTTVTVETLETQPGEE). In some aspects, the amino acid variations are at residues T12 and L13 of SEQ ID NO:47. In some aspects, the polypeptides described herein consist essentially of the amino acid sequence of SEQ ID NO: 47 (GYTYFTTVTVETLETQPGEE) with one or more amino acid variations (e.g., substitutions). In some aspects, the polypeptides described herein consist essentially of the amino acid sequence of SEQ ID NO: 47 (GYTYFTTVTVETLETQPGEE) with two amino acid variations (e.g., substitutions). In some aspects, the amino acid variations are at residues T12 and L13 of SEQ ID NO: 47.

[0283] In some aspects, the polypeptides described herein comprise an amino acid sequence having one or more amino acid variations (e.g., substitutions) in the amino acid sequence of SEQ ID NO:48 (GYTYFTTVTVETLETQ). In some aspects, the polypeptides described herein comprise an amino acid sequence having two amino acid variations (e.g., substitutions) in the amino acid sequence of SEQ ID NO:48 (GYTYFTTVTVETLETQ). In some aspects, the amino acid variations are at residues T12 and L13 of SEQ ID NO:48. In some aspects, the polypeptides described herein consist of an amino acid sequence having one or more amino acid variations (e.g., substitutions) in the amino acid sequence of SEQ ID NO:48 (GYTYFTTVTVETLETQ). In some aspects, the polypeptides described herein consist of an amino acid sequence having two amino acid variations (e.g., substitutions) in the amino acid sequence of SEQ ID NO:48 (GYTYFTTVTVETLETQ). In some aspects, the amino acid variations are at residues T12 and L13 of SEQ ID NO:48. In some aspects, the polypeptides described herein consist essentially of the amino acid sequence of SEQ ID NO: 48 (GYTYFTTVTVETLETQ) with one or more amino acid variations (e.g., substitutions). In some aspects, the polypeptides described herein consist essentially of the amino acid sequence of SEQ ID NO: 48 (GYTYFTTVTVETLETQ) with two amino acid variations (e.g., substitutions). In some aspects, the amino acid variations are at residues T12 and L13 of SEQ ID NO: 48.

[0284] In some aspects, the polypeptides described herein comprise an amino acid sequence having one or more amino acid variations (e.g., substitutions) in the amino acid sequence of SEQ ID NO:49 (GYTYFTTVTVETLETQPGEKEPPGPTTD). In some aspects, the polypeptides described herein comprise an amino acid sequence having two amino acid variations (e.g., substitutions) in the amino acid sequence of SEQ ID NO:49 (GYTYFTTVTVETLETQPGEKEPPGPTTD). In some aspects, the amino acid variations are at residues T12 and L13 of SEQ ID NO:49. In some aspects, the polypeptides described herein consist of an amino acid sequence having one or more amino acid variations (e.g., substitutions) in the amino acid sequence of SEQ ID NO:49 (GYTYFTTVTVETLETQPGEKEPPGPTTD). In some aspects, the polypeptides described herein consist of an amino acid sequence having two amino acid variations (e.g., substitutions) in the amino acid sequence of SEQ ID NO:49 (GYTYFTTVTVETLETQPGEKEPPGPTTD). In some aspects, the amino acid variations are present at residues T12 and L13 of SEQ ID NO: 49. In some aspects, the polypeptides described herein consist essentially of the amino acid sequence of SEQ ID NO: 49 (GYTYFTTVTVETLETQPGEKEPPGPTTD) with one or more amino acid variations (e.g., substitutions). In some aspects, the polypeptides described herein consist essentially of the amino acid sequence of SEQ ID NO: 49 (GYTYFTTVTVETLETQPGEKEPPGPTTD) with two amino acid variations (e.g., substitutions). In some aspects, the amino acid variations are present at residues T12 and L13 of SEQ ID NO: 49.

[0285] In some aspects, the polypeptides described herein comprise an amino acid sequence having one or more amino acid variations (e.g., substitutions) in the amino acid sequence of SEQ ID NO:50 (GYTYFTTVTVETLETQPGEEA). In some aspects, the polypeptides described herein comprise an amino acid sequence having two amino acid variations (e.g., substitutions) in the amino acid sequence of SEQ ID NO:50 (GYTYFTTVTVETLETQPGEEA). In some aspects, the amino acid variations are at residues T12 and L13 of SEQ ID NO:50. In some aspects, the polypeptides described herein consist of an amino acid sequence having one or more amino acid variations (e.g., substitutions) in the amino acid sequence of SEQ ID NO:50 (GYTYFTTVTVETLETQPGEEA). In some aspects, the polypeptides described herein consist of an amino acid sequence having two amino acid variations (e.g., substitutions) in the amino acid sequence of SEQ ID NO:50 (GYTYFTTVTVETLETQPGEEA). In some aspects, the amino acid variations are at residues T12 and L13 of SEQ ID NO:50. In some aspects, the polypeptides described herein consist essentially of the amino acid sequence of SEQ ID NO: 50 (GYTYFTTVTVETLETQPGEEA) with one or more amino acid variations (e.g., substitutions). In some aspects, the polypeptides described herein consist essentially of the amino acid sequence of SEQ ID NO: 50 (GYTYFTTVTVETLETQPGEEA) with two amino acid variations (e.g., substitutions). In some aspects, the amino acid variations are at residues T12 and L13 of SEQ ID NO: 50.

[0286] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) comprises the amino acid sequence of GYTYFTTVTVEPYETQPGEE (SEQ ID NO: 51). In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) consists of the amino acid sequence of GYTYFTTVTVEPYETQPGEE (SEQ ID NO: 51). In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) consists essentially of the amino acid sequence of GYTYFTTVTVEPYETQPGEE (SEQ ID NO: 51).

[0287] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) comprises the amino acid sequence of GYTYFTTVTVEMRETQPGEE (SEQ ID NO: 52). In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) consists of the amino acid sequence of GYTYFTTVTVEMRETQPGEE (SEQ ID NO: 52). In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) consists essentially of the amino acid sequence of GYTYFTTVTVEMRETQPGEE (SEQ ID NO: 52).

[0288] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) comprises the amino acid sequence of GYTYFTTVTVEIFETQPGEE (SEQ ID NO: 53). In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) consists of the amino acid sequence of GYTYFTTVTVEIFETQPGEE (SEQ ID NO: 53). In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) consists essentially of the amino acid sequence of GYTYFTTVTVEIFETQPGEE (SEQ ID NO: 53).

[0289] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) comprises the amino acid sequence of GYTYFTTVTVEHFETQPGEE (SEQ ID NO: 54). In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) consists of the amino acid sequence of GYTYFTTVTVEHFETQPGEE (SEQ ID NO: 54). In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) consists essentially of the amino acid sequence of GYTYFTTVTVEHFETQPGEE (SEQ ID NO: 54).

[0290] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) comprises the amino acid sequence of GYTYFTTVTVEWYETQPGEE (SEQ ID NO: 55). In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) consists of the amino acid sequence of GYTYFTTVTVEWYETQPGEE (SEQ ID NO: 55). In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) consists essentially of the amino acid sequence of GYTYFTTVTVEWYETQPGEE (SEQ ID NO: 55).

[0291] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) comprises the amino acid sequence of GYTYFTTVTVEQRETQPGEE (SEQ ID NO: 56). In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) consists of the amino acid sequence of GYTYFTTVTVEQRETQPGEE (SEQ ID NO: 56). In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) consists essentially of the amino acid sequence of GYTYFTTVTVEQRETQPGEE (SEQ ID NO: 56).

[0292] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) comprises the amino acid sequence of GYTYFTTVTVEWFETQPGEE (SEQ ID NO: 57). In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) consists of the amino acid sequence of GYTYFTTVTVEWFETQPGEE (SEQ ID NO: 57). In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) consists essentially of the amino acid sequence of GYTYFTTVTVEWFETQPGEE (SEQ ID NO: 57).

[0293] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) comprises the amino acid sequence of GYTYFTTVTVEERETQPGEE (SEQ ID NO: 58). In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) consists of the amino acid sequence of GYTYFTTVTVEERETQPGEE (SEQ ID NO: 58). In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) consists essentially of the amino acid sequence of GYTYFTTVTVEERETQPGEE (SEQ ID NO: 58).

[0294] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) comprises the amino acid sequence of GYTYFTTVTVEDYETQPGEE (SEQ ID NO: 59). In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) consists of the amino acid sequence of GYTYFTTVTVEDYETQPGEE (SEQ ID NO: 59). In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) consists essentially of the amino acid sequence of GYTYFTTVTVEDYETQPGEE (SEQ ID NO: 59).

[0295] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) comprises the amino acid sequence of GYTYFTTVTVEFFETQPGEE (SEQ ID NO: 60). In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) consists of the amino acid sequence of GYTYFTTVTVEFFETQPGEE (SEQ ID NO: 60). In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) consists essentially of the amino acid sequence of GYTYFTTVTVEFFETQPGEE (SEQ ID NO: 60).

[0296] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) comprises the amino acid sequence of GYTYFTTVTVEHYETQPGEE (SEQ ID NO: 61). In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) consists of the amino acid sequence of GYTYFTTVTVEHYETQPGEE (SEQ ID NO: 61). In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) consists essentially of the amino acid sequence of GYTYFTTVTVEHYETQPGEE (SEQ ID NO: 61).

[0297] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) comprises the amino acid sequence of GYTYFTTVTVEMMETQPGEE (SEQ ID NO: 62). In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) consists of the amino acid sequence of GYTYFTTVTVEMMETQPGEE (SEQ ID NO: 62). In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) consists essentially of the amino acid sequence of GYTYFTTVTVEMMETQPGEE (SEQ ID NO: 62).

[0298] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) comprises the amino acid sequence of GYTYFTTVTVEDFETQPGEE (SEQ ID NO: 63). In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) consists of the amino acid sequence of GYTYFTTVTVEDFETQPGEE (SEQ ID NO: 63). In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) consists essentially of the amino acid sequence of GYTYFTTVTVEDFETQPGEE (SEQ ID NO: 63).

[0299] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) comprises the amino acid sequence of GYTYFTTVTVEDIETQPGEE (SEQ ID NO: 64). In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) consists of the amino acid sequence of GYTYFTTVTVEDIETQPGEE (SEQ ID NO: 64). In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) consists essentially of the amino acid sequence of GYTYFTTVTVEDIETQPGEE (SEQ ID NO: 64).

[0300] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) comprises the amino acid sequence of GYTYFTTVTVELIETQPGEE (SEQ ID NO: 65). In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) consists of the amino acid sequence of GYTYFTTVTVELIETQPGEE (SEQ ID NO: 65). In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) consists essentially of the amino acid sequence of GYTYFTTVTVELIETQPGEE (SEQ ID NO: 65).

[0301] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) comprises the amino acid sequence of GYTYFTTVTVEEIETQPGEE (SEQ ID NO: 66). In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) consists of the amino acid sequence of GYTYFTTVTVEEIETQPGEE (SEQ ID NO: 66). In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) consists essentially of the amino acid sequence of GYTYFTTVTVEEIETQPGEE (SEQ ID NO: 66).

[0302] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) comprises the amino acid sequence of GYTYFTTVTVEAFETQPGEE (SEQ ID NO: 67). In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) consists of the amino acid sequence of GYTYFTTVTVEAFETQPGEE (SEQ ID NO: 67). In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) consists essentially of the amino acid sequence of GYTYFTTVTVEAFETQPGEE (SEQ ID NO: 67).

[0303] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) comprises the amino acid sequence of GYTYFTTVTVEHHETQPGEE (SEQ ID NO: 68). In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) consists of the amino acid sequence of GYTYFTTVTVEHHETQPGEE (SEQ ID NO: 68). In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) consists essentially of the amino acid sequence of GYTYFTTVTVEHHETQPGEE (SEQ ID NO: 68).

[0304] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) comprises the amino acid sequence of GYTYFTTVTVEPFETQPGEE (SEQ ID NO: 69). In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) consists of the amino acid sequence of GYTYFTTVTVEPFETQPGEE (SEQ ID NO: 69). In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) consists essentially of the amino acid sequence of GYTYFTTVTVEPFETQPGEE (SEQ ID NO: 69).

[0305] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) comprises the amino acid sequence of GYTYFTTVTVEDWETQPGEE (SEQ ID NO: 70). In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) consists of the amino acid sequence of GYTYFTTVTVEDWETQPGEE (SEQ ID NO: 70). In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) consists essentially of the amino acid sequence of GYTYFTTVTVEDWETQPGEE (SEQ ID NO: 70).

[0306] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) comprises the amino acid sequence of GYTYFTTVTVEPYETQPGEEA (SEQ ID NO: 71). In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) consists of the amino acid sequence of GYTYFTTVTVEPYETQPGEEA (SEQ ID NO: 71). In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) consists essentially of the amino acid sequence of GYTYFTTVTVEPYETQPGEEA (SEQ ID NO: 71).

[0307] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) comprises the amino acid sequence of GYTYFTTVTVEHFETQPGEEA (SEQ ID NO: 72). In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) consists of the amino acid sequence of GYTYFTTVTVEHFETQPGEEA (SEQ ID NO: 72). In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) consists essentially of the amino acid sequence of GYTYFTTVTVEHFETQPGEEA (SEQ ID NO: 72).

[0308] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) comprises the amino acid sequence of GYTYFTTVTVEQRETQPGEEA (SEQ ID NO: 73). In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) consists of the amino acid sequence of GYTYFTTVTVEQRETQPGEEA (SEQ ID NO: 73). In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) consists essentially of the amino acid sequence of GYTYFTTVTVEQRETQPGEEA (SEQ ID NO: 73).

[0309] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) comprises the amino acid sequence of GYTYFTTVTVEWYETQPGEEA (SEQ ID NO: 74). In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) consists of the amino acid sequence of GYTYFTTVTVEWYETQPGEEA (SEQ ID NO: 74). In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) consists essentially of the amino acid sequence of GYTYFTTVTVEWYETQPGEEA (SEQ ID NO: 74).

[0310] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) comprises the amino acid sequence of GYTYFTTVTVEMRETQPGEEA (SEQ ID NO: 75). In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) consists of the amino acid sequence of GYTYFTTVTVEMRETQPGEEA (SEQ ID NO: 75). In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) consists essentially of the amino acid sequence of GYTYFTTVTVEMRETQPGEEA (SEQ ID NO: 75).

[0311] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) comprises the amino acid sequence of GYTYFTTVTVEIFETQPGEEA (SEQ ID NO: 76). In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) consists of the amino acid sequence of GYTYFTTVTVEIFETQPGEEA (SEQ ID NO: 76). In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5-binding domain of LRRC4B) consists essentially of the amino acid sequence of GYTYFTTVTVEIFETQPGEEA (SEQ ID NO: 76).

[0312] In some aspects, the LRRC4 family mimetic molecules described herein comprise one or more components capable of improving the ability of the polypeptide to inhibit the interaction between the FAM19A5 protein and a member of the LRRC4 protein family. For example, in some aspects, the molecule comprises (i) any polypeptide described herein and (ii) one or more additional amino acids at the N-terminus of the polypeptide, the C-terminus of the polypeptide, or both the N-terminus and C-terminus of the polypeptide. In some aspects, molecules useful in the invention comprise at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, or at least about 20 additional amino acids at the N-terminus of the polypeptide. In some aspects, the molecule comprises at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, or at least about 20 additional amino acids at the C-terminus of the polypeptide.In some aspects, the molecule comprises (i) at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least 19, or at least about 20 additional amino acids at the N-terminus of the polypeptide. (ii) comprises at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least 19, or at least about 20 additional amino acids at the C-terminus of the polypeptide.

[0313] In some aspects, an LRRC4 family mimetic molecule comprises (i) a polypeptide having the amino acid sequence of SEQ ID NO: 44 (YTYFTTVTVE); and (ii) at least one additional amino acid at the N-terminus of the polypeptide. In some aspects, an LRRC4 family mimetic molecule comprises (i) a polypeptide having the amino acid sequence of SEQ ID NO: 44 (YTYFTTVTVE); and (ii) at least one additional amino acid at the C-terminus of the polypeptide. In some aspects, an LRRC4 family mimetic molecule comprises (i) a polypeptide having the amino acid sequence of SEQ ID NO: 44 (YTYFTTVTVE); and (ii) at least one additional amino acid at both the N-terminus and the C-terminus. In some aspects, an LRRC4 family mimetic molecule useful in the present invention comprises the amino acid sequence of SEQ ID NO: 47 (GYTYFTTVTVETLETQPGEE). In some aspects, an LRRC4 family mimetic molecule consists of the amino acid sequence of SEQ ID NO: 47 (GYTYFTTVTVETLETQPGEE). In some aspects, the LRRC4 family mimetic molecule consists essentially of the amino acid sequence of SEQ ID NO: 47 (GYTYFTTVTVETLETQPGEE). In some aspects, the LRRC4 family mimetic molecule useful in the present invention comprises the amino acid sequence of SEQ ID NO: 48 (GYTYFTTVTVETLETQ). In some aspects, the LRRC4 family mimetic molecule consists essentially of the amino acid sequence of SEQ ID NO: 48 (GYTYFTTVTVETLETQ). In some aspects, the LRRC4 family mimetic molecule useful in the present invention comprises the amino acid sequence of SEQ ID NO: 49 (GYTYFTTVTVETLETQPGEKEPPGPTTD). In some aspects, the LRRC4 family mimetic molecule consists essentially of the amino acid sequence of SEQ ID NO: 49 (GYTYFTTVTVETLETQPGEKEPPGPTTD). In some aspects, the LRRC4 family mimetic molecule consists essentially of the amino acid sequence of SEQ ID NO: 49 (GYTYFTTVTVETLETQPGEKEPPGPTTD).

[0314] In some aspects, an LRRC4 family mimetic molecule useful in the present invention comprises (i) a polypeptide having the amino acid sequence of SEQ ID NO: 45 (YSFFTVTVE); and (ii) at least one additional amino acid at the N-terminus of the polypeptide. In some aspects, an LRRC4 family mimetic molecule comprises (i) a polypeptide having the amino acid sequence of SEQ ID NO: 45 (YSFFTVTVE); and (ii) at least one additional amino acid at the C-terminus of the polypeptide. In some aspects, an LRRC4 family mimetic molecule comprises (i) a polypeptide having the amino acid sequence of SEQ ID NO: 45 (YSFFTTVTVE); and (ii) at least one additional amino acid at both the N-terminus and the C-terminus. In some aspects, an LRRC4 family mimetic molecule useful in the present invention comprises the amino acid sequence of SEQ ID NO: 77 (NYSFFTTVTVETTEISPEDTTRK). In some aspects, an LRRC4 family mimetic molecule consists of the amino acid sequence of SEQ ID NO: 77 (NYSFFTTVTVETTEISPEDTTRK). In some aspects, the LRRC4 family mimetic molecule consists essentially of the amino acid sequence of SEQ ID NO: 77 (NYSFFTTVTVETTEISPEDTTRK).

[0315] In some aspects, an LRRC4 family mimetic molecule useful in the present invention comprises (i) a polypeptide having the amino acid sequence of SEQ ID NO: 46 (FSYFSTVTVE); and (ii) at least one additional amino acid at the N-terminus of the polypeptide. In some aspects, an LRRC4 family mimetic molecule comprises (i) a polypeptide having the amino acid sequence of SEQ ID NO: 46 (FSYFSTVTVE); and (ii) at least one additional amino acid at the C-terminus of the polypeptide. In some aspects, an LRRC4 family mimetic molecule comprises (i) a polypeptide having the amino acid sequence of SEQ ID NO: 46 (FSYFSTVTVE); and (ii) at least one additional amino acid at both the N-terminus and the C-terminus. In some aspects, an LRRC4 family mimetic molecule useful in the present invention comprises the amino acid sequence of SEQ ID NO: 78 (NFSYFSTVTVETMEPSQDERTTR). In some aspects, an LRRC4 family mimetic molecule consists of the amino acid sequence of SEQ ID NO: 78 (NFSYFSTVTVETMEPSQDERTTR). In some aspects, the LRRC4 family mimetic molecule consists essentially of the amino acid sequence of SEQ ID NO: 78 (NFSYFSTVTVETMEPSQDERTTR).

[0316] In some aspects, the LRRC4 family mimetic molecule polypeptide comprises an amino acid sequence 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% identical to the amino acid sequence of SEQ ID NO:44 (YTYFTTVTVE), wherein the polypeptide is capable of binding to a FAM19A5 protein, and the amino acid sequence further comprises one or more hydrophobic amino acids at the N-terminus. In some aspects, the hydrophobic amino acids comprise at least 2 amino acids, at least 3 amino acids, at least 4 amino acids, at least 5 amino acids, at least 6 amino acids, at least 7 amino acids, at least 8 amino acids, at least 9 amino acids, at least 10 amino acids, at least 15 amino acids, at least 20 amino acids, at least 25 amino acids, at least 30 amino acids, at least 35 amino acids, at least 40 amino acids, at least 45 amino acids, or at least 50 amino acids at the N-terminus.

[0317] In some aspects, the LRRC4 family mimetic molecule polypeptide comprises an amino acid sequence 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% identical to the amino acid sequence of SEQ ID NO:44 (YTYFTTVTVE), wherein the polypeptide is capable of binding to a FAM19A5 protein, and the amino acid sequence further comprises one or more amino acids at the N-terminus and / or C-terminus. In some aspects, the one or more amino acids linked to the N-terminus and / or C-terminus comprise one or more amino acid sequences derived from a LRRC4B protein. In some aspects, the one or more amino acids linked to the N-terminus include at least 2 amino acids, at least 3 amino acids, at least 4 amino acids, at least 5 amino acids, at least 6 amino acids, at least 7 amino acids, at least 8 amino acids, at least 10 amino acids, at least 15 amino acids, at least 20 amino acids, at least 25 amino acids, at least 30 amino acids, at least 35 amino acids, at least 40 amino acids, at least 45 amino acids, or at least 50 amino acids at the N-terminus. In some aspects, the one or more amino acids linked to the C-terminus include at least 2 amino acids, at least 3 amino acids, at least 4 amino acids, at least 5 amino acids, at least 6 amino acids, at least 7 amino acids, at least 8 amino acids, at least 10 amino acids, at least 15 amino acids, at least 20 amino acids, at least 25 amino acids, at least 30 amino acids, at least 35 amino acids, at least 40 amino acids, at least 45 amino acids, or at least 50 amino acids at the C-terminus. In some aspects, the one or more amino acids linked to the N-terminus and / or C-terminus are linked via a linker, hi some aspects, the linker is a peptide linker.

[0318] In some aspects, the one or more additional amino acids added to the N-terminus and / or C-terminus can include any suitable amino acid known in the art. In some aspects, the one or more additional amino acids are hydrophilic amino acids. In some aspects, the one or more additional amino acids can include D-amino acids. Without being limited to a particular theory, in some aspects, adding one or more D-amino acids to the N-terminus and / or C-terminus of the polypeptide can improve the durability of the LRRC4 family mimetic molecule, for example, when administered to a subject. For example, the inclusion of D-amino acids can protect the polypeptide from protease and peptidase degradation in the subject's blood. Thus, in some aspects, polypeptides useful in the present invention can include both D-amino acids and L-amino acids. For example, in some aspects, the polypeptides described herein include a D-amino acid at the N-terminus and L-amino acids at all other amino acid residues. In some aspects, the polypeptides described herein include a D-amino acid at the C-terminus and L-amino acids at all other amino acid residues. In some aspects, the polypeptides described herein contain D-amino acids at both the N-terminus and C-terminus, and L-amino acids at all other amino acid residues.

[0319] As described herein, in some aspects, the LRRC4 family mimetic molecule comprises a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 44 (YTYFTTVTVE), 45 (YSFFTTVTVE), and 46 (FSYFSTVTVE), and having 1, 2, 3, 4, 5, or 6 amino acids that differ (e.g., by substitution) from the amino acid sequence.

[0320] In some aspects, LRRC4 family mimetic molecules useful in the present invention include additional modifications at the N-terminus, C-terminus, or both the N-terminus and C-terminus of the polypeptide, which can increase the stability of the polypeptide. For example, in some aspects, the N-terminus of the polypeptide is methylated. Non-limiting examples of additional modifications that can be made at the N-terminus and / or C-terminus include Fmoc, PEGylation, acetylation, or a combination thereof. In some aspects, the polypeptide can be cyclized to increase stability. Such modifications can be made using any suitable method known in the art.

[0321] As further described elsewhere herein, in some aspects, molecules useful in the present invention comprise a FAM19A5-binding domain of an LRRC4 family protein member and an additional moiety that can improve one or more properties of the molecule (e.g., the binding affinity of the molecule for the FAM19A5 protein). As demonstrated herein (see, e.g., Example 10), Applicants have determined that the addition of a membrane-proximal sequence to a member of the LRRC4 protein family can significantly improve the binding affinity of the molecule for the FAM19A5 protein. The membrane-proximal sequence is highly conserved among members of the LRRC4 family and is represented by SEQ ID NO: 79 (LDEVMKTTK) (LRRC4 and LRRC4B) and SEQ ID NO: 80 (IDEVMKTTK) (LRRC4C).

[0322] Thus, in some aspects, the molecules described herein comprise the FAM19A5 binding domain of the LRRC4 protein (i.e., YSFFTTVTVE; SEQ ID NO:45) and the membrane juxtamembrane sequence of SEQ ID NO:79 (LDEVMKTTK). In some aspects, the molecules described herein comprise the FAM19A5 binding domain of the LRRC4 protein (i.e., YSFFTTVTVE; SEQ ID NO:45) and the membrane juxtamembrane sequence of SEQ ID NO:80 (IDEVMKTTK). In some aspects, the molecules described herein comprise the FAM19A5 binding domain of the LRRC4B protein (i.e., YTYFTTVTVE; SEQ ID NO:44) and the membrane juxtamembrane sequence of SEQ ID NO:79 (LDEVMKTTK). In some aspects, the molecules described herein comprise the FAM19A5 binding domain of the LRRC4 protein (i.e., YTYFTTVTVE; SEQ ID NO:44) and the membrane juxtamembrane sequence of SEQ ID NO:80 (IDEVMKTTK). In some aspects, the molecules described herein comprise the FAM19A5 binding domain of the LRRC4B protein (i.e., FSYFSTVTVE; SEQ ID NO:46) and the membrane juxtamembrane sequence of SEQ ID NO:79 (LDEVMKTTK). In some aspects, the molecules described herein comprise the FAM19A5 binding domain of the LRRC4 protein (i.e., FSYFSTVTVE; SEQ ID NO:46) and the membrane juxtamembrane sequence of SEQ ID NO:80 (IDEVMKTTK). In some aspects, the membrane juxtamembrane sequence is added to the C-terminus of the molecule.

[0323] As will become apparent from the present invention, any of the modifications described herein (e.g., amino acid substitutions, addition of membrane-proximal sequences, D-amino acids) can be used in combination to improve one or more properties of a molecule. For example, in some aspects, a molecule (e.g., a polypeptide) useful in the present invention comprises (i) the amino acid sequence of SEQ ID NO:47 (GYTYFTTVTVETLETQPGEE) with amino acid modifications at residues T12 and L13; and (ii) a membrane-proximal sequence at the C-terminus of the molecule (e.g., SEQ ID NO:79 or SEQ ID NO:80). In some aspects, a molecule (e.g., a polypeptide) useful in the present invention comprises (i) the amino acid sequence of SEQ ID NO:47 (GYTYFTTVTVETLETQPGEE) with amino acid modifications at residues T12 and L13; (ii) D-amino acids at the N-terminus and / or C-terminus; and (iii) a membrane-proximal sequence at the C-terminus of the molecule (e.g., SEQ ID NO:79 or SEQ ID NO:80). In some aspects, molecules (e.g., polypeptides) useful in the invention comprise (i) the amino acid sequence of SEQ ID NO:48 (GYTYFTTVTVETLETQ) with amino acid modifications at residues T12 and L13; and (ii) a membrane-proximal sequence at the C-terminus of the molecule (e.g., SEQ ID NO:79 or SEQ ID NO:80). In some aspects, molecules (e.g., polypeptides) useful in the invention comprise (i) the amino acid sequence of SEQ ID NO:48 (GYTYFTTVTVETLETQ) with amino acid modifications at residues T12 and L13; (ii) D-amino acids at the N-terminus and / or C-terminus; and (iii) a membrane-proximal sequence at the C-terminus of the molecule (e.g., SEQ ID NO:79 or SEQ ID NO:80). In some aspects, a molecule (e.g., a polypeptide) useful in the invention comprises (i) the amino acid sequence of SEQ ID NO:49 (GYTYFTTVTVETLETQPGEKEPPGPTTD) with amino acid variations at residues T12 and L13; and (ii) a membrane-proximal sequence at the C-terminus of the molecule (e.g., SEQ ID NO:79 or SEQ ID NO:80).In some aspects, molecules (e.g., polypeptides) useful in the invention comprise (i) the amino acid sequence of SEQ ID NO:49 (GYTYFTTVTVETLETQPGEKEPPGPTTD) with amino acid modifications at residues T12 and L13; (ii) D-amino acids at the N-terminus and / or C-terminus; and (iii) a membrane-proximal sequence at the C-terminus of the molecule (e.g., SEQ ID NO:79 or SEQ ID NO:80). In some aspects, molecules (e.g., polypeptides) useful in the invention comprise (i) the amino acid sequence of SEQ ID NO:50 (GYTYFTTVTVETLETQPGEEA) with amino acid modifications at residues T12 and L13; and (ii) a membrane-proximal sequence at the C-terminus of the molecule (e.g., SEQ ID NO:79 or SEQ ID NO:80). In some aspects, a molecule (e.g., a polypeptide) useful in the present invention comprises (i) the amino acid sequence of SEQ ID NO: 50 (GYTYFTTVTVETLETQPGEEA) with amino acid variations at residues T12 and L13; (ii) D-amino acids at the N-terminus and / or C-terminus; and (iii) a membrane-proximal sequence (e.g., SEQ ID NO: 79 or SEQ ID NO: 80) at the C-terminus of the molecule.

[0324] In some aspects, the LRRC4 family mimetic molecules described herein can include one or more additional peptides, e.g., that allow the molecules to be specifically targeted to other tissues when administered to a subject. For example, in some aspects, the LRRC4 family mimetic molecules include a peptide that allows the molecules to cross the blood-brain barrier (also referred to herein as a "BBB shuttle"). Examples of such BBB shuttles are known in the art. Non-limiting examples are provided in Table 10 (below). See, e.g., Oller-Salvia et al., Chem Soc Rev 45:4690 (2016).

[0325] [Table 10]

[0326] The nomenclature of each cyclic peptide (&) follows the amino acid code of Spengler et al., J Pept Res 65:550-555 (2005); [Dap] means diaminopropionic acid.

[0327] In some aspects, LRRC4 family mimetic molecules useful in the present invention comprise fusion proteins. For example, in some aspects, the LRRC4 family mimetic molecules described herein can comprise (i) any polypeptide of the present invention and (ii) a half-life extending moiety. Fusion proteins of the present invention can be generated using any suitable half-life extending moiety known in the art. Non-limiting examples of such half-life extending moieties include Fc, albumin, albumin-binding polypeptide, Pro / Ala / Ser (PAS), C-terminal peptide of the β subunit of human chorionic gonadotropin (CTP), polyethylene glycol (PEG), long unstructured hydrophilic amino acid sequences (XTEN), hydroxyethyl starch (HES), albumin-binding small molecules, or combinations thereof.

[0328] In some aspects, the LRRC4 family mimetic molecule comprises a protein-drug conjugate. For example, in some aspects, the polypeptide of the LRRC4 family mimetic molecule can be conjugated to a therapeutic agent, such as a therapeutic agent useful for treating a disease or disorder.

[0329] The protein-drug conjugates described herein can be prepared by methods known in the art. In some aspects, substantially (or mostly) non-immunogenic linkages are obtained through conjugation, such as peptide (i.e., amide), sulfide, (sterically hindered), disulfide, hydrazone, and ether linkages. Such linkages are largely non-immunogenic and exhibit suitable stability in serum (see, e.g., Senter, PD, Curr. Opin. Chem. Biol. 13 (2009) 235-244; WO 2009 / 059278; WO 95 / 17886, each of which is incorporated herein by reference in its entirety).

[0330] Depending on the moiety and biochemical properties of each polypeptide, a variety of conjugation strategies can be used (see, e.g., Hackenberger, CPR, and Schwarzer, D., Angew. Chem. Int. Ed. Engl. 47 (2008) 10030-10074). In some aspects, site-specific reactions and covalent couplings are based on converting natural amino acids into amino acids with orthogonal reactivity relative to the reactivity of other functional groups present. For example, certain cysteines in rare sequence contexts can be enzymatically converted into aldehydes (see, Frese, MA, and Dierks, T., ChemBioChem. 10 (2009) 425-427). It is also possible to obtain desired amino acid modifications by utilizing the specific enzymatic reactivity of certain enzymes against natural amino acids in a given sequence context (see, for example, Taki, M. et al., Prot. Eng. Des. Sel. 17 (2004) 119-126; Gautier, A. et al., Chem. Biol. 15 (2008) 128-136; and Protease-catalyzed formation of C—N bonds is used by Bordusa, F., Highlights in Bioorganic Chemistry (2004) 389-403).

[0331] Site-specific reactions and covalent coupling can also be achieved through selective reaction of terminal amino acids with appropriate modification reagents. The reactivity of N-terminal cysteines with benzonitrile can be used to achieve site-specific covalent coupling (Ren, H. et al., Angew. Chem. Int. Ed. Engl. 48 (2009) 9658-9662). 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).

[0332] The moiety can also be a synthetic peptide or peptidomimetic. In this case, the polypeptide can be chemically synthesized, and amino acids with orthogonal chemical reactivity can be introduced during the synthesis process (see, for example, de Graaf, AJ et al., Bioconjug. Chem. 20 (2009) 1281-1295). To obtain a single-labeled polypeptide, a conjugate with a 1:1 stoichiometry can be separated from other conjugation by-products by chromatography. This process can be facilitated by using dye-labeled binding pair members and charged linkers. The use of such labeled, highly negatively charged binding pair members allows for separation based on charge and molecular weight differences, so that single-conjugated polypeptides can be easily separated from unlabeled polypeptides and polypeptides bearing one or more linkers. Fluorescent dyes, like labeled monovalent binding agents, can be useful for purifying conjugates from unbound components.

[0333] IV. Nucleic Acids, Vectors, and Host Cells Additional aspects described herein relate to one or more nucleic acid molecules (also referred to herein as "nucleic acids" or derivatives thereof) encoding a therapeutic agent (e.g., an LRRC4 family mimetic molecule and / or a FAM19A5 antagonist described herein). The nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. In some aspects, the nucleic acids are DNA and / or RNA sequences (e.g., mRNA). In some aspects, the nucleic acids comprise modified nucleotide analogs. A nucleic acid is "isolated" or "substantially pure" when it is purified from other cellular components or other contaminants, such as other nucleic acids (e.g., other chromosomal DNA, e.g., chromosomal DNA linked to naturally isolated DNA) or proteins of the cell using standard techniques, including alkali / SDS treatment, CsCl banding, column chromatography, restriction enzymes, agarose gel electrophoresis, and other techniques well known in the art. See F. Ausubel, et al., ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. In some aspects, the nucleic acid molecule may or may not contain intron sequences. In some aspects, the nucleic acid is a cDNA molecule. The nucleic acids described herein can be obtained using standard molecular biology techniques known in the art.

[0334] In some aspects, the present invention provides vectors comprising an isolated nucleic acid molecule encoding a therapeutic agent disclosed herein (e.g., an LRRC4 family mimetic molecule and / or a FAM19A5 antagonist described herein). Vectors suitable for the present invention include, but are not limited to, expression vectors, viral vectors, and plasmid vectors. In some aspects, the vector is a viral vector.

[0335] As used herein, "expression vector" means any nucleic acid construct that, when introduced into an appropriate host cell, contains the necessary elements for the transcription and translation of an inserted coding sequence, or, in the case of RNA viral vectors, the elements necessary for replication and translation. Expression vectors can include plasmids, phagemids, viruses, and their derivatives.

[0336] As used herein, the term "viral vector" includes, but is not limited to, nucleic acid sequences from the following viruses: retroviruses, such as Moloney murine leukemia virus, Harvey murine sarcoma virus, mouse mammary tumor virus, and Rous sarcoma virus; lentiviruses; adenoviruses; adeno-associated viruses; SV40 viruses; polyomaviruses; Epstein-Barr virus; papillomaviruses; herpesviruses; vaccinia viruses; polioviruses; and RNA viruses such as retroviruses. Certain viral vectors are based on non-cytopathic eukaryotic viruses in which nonessential genes have been replaced with a gene of interest. Non-cytopathic viruses include retroviruses, whose life cycle involves reverse transcription of genomic viral RNA into DNA, followed by integration of the provirus into host cell DNA.

[0337] In some aspects, the vector is derived from an adeno-associated virus. In some aspects, the vector is derived from a lentivirus. Examples of lentiviral vectors are disclosed in WO9931251, WO9712622, WO9817815, WO9817816, and WO9818934, each of which is incorporated herein by reference in its entirety.

[0338] Other vectors include plasmid vectors. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. Over the past few years, plasmid vectors have proven particularly advantageous for transferring genes to cells in vivo because they cannot replicate and integrate within the host genome. However, these plasmids, which have a promoter compatible with the host cell, are capable of expressing peptides from genes operably encoded within the plasmid. Plasmids commonly used by commercial suppliers include pBR322, pUC18, pUC19, various pcDNA plasmids, pRC / CMV, various pCMV plasmids, pSV40, and pBlueScript. Additional examples of specific plasmids include pcDNA3.1, catalog number V79020; pcDNA3.1 / hygro, catalog number V87020; pcDNA4 / myc-His, catalog number V86320; and pBudCE4.1, catalog number V53220, all of which are products of Invitrogen (Carlsbad, Calif.). Plasmids can also be custom designed to remove and / or add specific DNA fragments using standard molecular biology techniques.

[0339] V. Pharmaceutical Compositions The present invention provides compositions comprising a therapeutic agent (e.g., an LRRC4 family mimetic molecule, a FAM19A5 antagonist, or both, including nucleic acids, vectors, cells, or protein conjugates encoding and / or containing an LRRC4 family mimetic molecule and / or a FAM19A5 antagonist) having a desired degree of purity in a physiologically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol, and m-cresol); low molecular weight (less than about 10 residues) polypeptides; serum albumin; proteins such as guanylate, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0340] In some aspects, pharmaceutical compositions useful in the present invention comprise any of the therapeutic agents described herein (e.g., nucleic acids, vectors, cells, or protein conjugates encoding and / or comprising an LRRC4 family mimetic molecule and / or a FAM19A5 antagonist, including an LRRC4 family mimetic molecule, a FAM19A5 antagonist, or both) and, optionally, one or more additional prophylactic or therapeutic agents in a pharmaceutically acceptable carrier. In some aspects, pharmaceutical compositions comprise any of the therapeutic agents described herein (e.g., nucleic acids, vectors, cells, or protein conjugates encoding and / or comprising an LRRC4 family mimetic molecule and / or a FAM19A5 antagonist, including an LRRC4 family mimetic molecule, a FAM19A5 antagonist, or both) and, optionally, one or more additional prophylactic or therapeutic agents in a pharmaceutically acceptable carrier. In some aspects, a therapeutic agent described herein is the only active ingredient contained in the pharmaceutical composition.

[0341] Pharmaceutically acceptable carriers used in parenteral formulations include aqueous vehicles, non-aqueous vehicles, antibacterial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents, and other pharmaceutically acceptable substances. Examples of aqueous vehicles include sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, dextrose and lactated Ringer's injection. Non-aqueous parenteral vehicles include fixed vegetable oil, cottonseed oil, corn oil, sesame oil, and peanut oil. Antibacterial agents in bacteriostatic or fungistatic concentrations can be added to parenteral formulations packaged in multi-dose containers, including phenol or cresol, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride, and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. Buffers include phosphates and citrates. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcellulose, hydroxypropyl methylcellulose, and polyvinylpyrrolidone. Emulsifying agents include polysorbate 80 (TWEEN® 80). Sequestering or chelating agents include EDTA. Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol, and propylene glycol for water-miscible vehicles, and sodium hydroxide, hydrochloric acid, citric acid, or lactic acid for pH adjustment.

[0342] Pharmaceutical compositions can be formulated for any route of administration to a subject. Specific examples of administration routes include intranasal, oral, parenteral, intrathecal, intraventricular, pulmonary, subcutaneous, or intraventricular routes. Parenteral administration, characterized by subcutaneous, intramuscular, or intravenous injection, is also contemplated by the present invention. Injectables can be prepared in conventional forms, such as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or emulsions. In addition, such injectables, solutions, and emulsions contain one or more excipients. Suitable excipients are, for example, water, saline, glucose, glycerol, or ethanol. If necessary, the administered pharmaceutical composition can also contain minor amounts of nontoxic auxiliary substances, such as wetting or emulsifying agents, pH buffers, stabilizers, solubility enhancers, and other formulating agents, such as, for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, and cyclodextrins.

[0343] Formulations for parenteral administration of the therapeutic agents described herein (e.g., nucleic acids, vectors, cells, or protein conjugates encoding and / or containing LRRC4 family mimetic molecules and / or FAM19A5 antagonists, including LRRC4 family mimetic molecules, FAM19A5 antagonists, or both) include sterile solutions ready for injection, sterile and dried soluble products (including subcutaneous tablets) such as lyophilized powders ready for mixing with a solvent immediately before use, sterile suspensions ready for injection, sterile dry insoluble products ready for mixing with a vehicle immediately before use, and sterile emulsions. The solutions may be aqueous or non-aqueous.

[0344] If administered intravenously, suitable carriers include physiological saline or phosphate buffered saline (PBS) and solutions containing thickening and solubilizing agents such as glucose, polyethylene glycol, polypropylene glycol, and mixtures thereof.

[0345] Topical mixtures containing a therapeutic agent are prepared as described for local and systemic administration. The mixtures prepared may be solutions, suspensions, emulsions, etc., and may be formulated into creams, gels, ointments, emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes, foams, aerosols, washes, sprays, suppositories, bandages, skin patches, or other formulations suitable for topical administration.

[0346] Pharmaceutical compositions can be formulated as aerosols for topical application, such as inhalation (see, e.g., U.S. Patent Nos. 4,044,126, 4,414,209, and 4,364,923). These formulations for administration to the respiratory tract can be in the form of aerosols or solutions for nebulization, or in the form of fine powders for inhalation, and can be used alone or in combination with an inert carrier, such as lactose. In this case, the particles of the formulation can, in some aspects, have diameters of less than about 50 microns, e.g., less than about 10 microns.

[0347] Pharmaceutical compositions can be formulated for topical or local application, e.g., topical application to the skin and mucous membranes, including the eyes, in the form of gels, creams, and lotions, for application to the eye, or for intracisternal or intraspinal application. Topical administration is contemplated for transdermal delivery, administration to the eye or mucous membranes, or inhalation therapy. Nasal solutions of the antibody can also be administered alone or in combination with other pharmaceutically acceptable excipients.

[0348] Transdermal patches, including iontophoretic and electrophoretic devices, are well known to those of skill in the art and can be used to administer any of the therapeutic agents described herein. 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, 6,010,715, 5,985,317, 5,983,134, 5,948,433, and 5,860,957.

[0349] In some aspects, the pharmaceutical compositions described herein are lyophilized powders that can be reconstituted with solutions, emulsions, and other mixtures for administration. The pharmaceutical compositions may also be reconstituted and formulated in solid or gel form. The lyophilized powders are prepared by dissolving any of the therapeutic agents described herein or a pharmaceutically acceptable derivative thereof in a suitable solvent. In some aspects, the lyophilized powders are sterilized. The solvent may contain excipients that improve the stability or other pharmacological components of the powder or a reconstituted solution prepared from the powder. Excipients that may be used include, but are not limited to, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable formulations. The solvent may also contain a buffer such as citric acid, sodium, or potassium phosphate, or other buffers known to those of skill in the art. In some aspects, the pH of the buffer is approximately neutral. The solution is then sterile filtered and lyophilized under standard conditions known to those of skill in the art to provide the desired dosage form. In some aspects, the prepared solution can be apportioned into multiple lyophilization vials, each containing a single dose or multiple doses of any of the therapeutic agents described herein (e.g., LRRC4 family mimetic molecules, FAM19A5 antagonists, or both, including nucleic acids, vectors, cells, or protein conjugates encoding and / or containing LRRC4 family mimetic molecules and / or FAM19A5 antagonists). The lyophilized powder can be stored under appropriate conditions, such as at about 4°C to room temperature.

[0350] Reconstitution of this lyophilized powder with water for injection provides a formulation for use in parenteral administration. For reconstitution, the lyophilized powder is added to sterile water or other suitable carrier. Exact amounts will vary with the compound selected; such amounts can be determined empirically.

[0351] Additionally, in some aspects, pharmaceutical compositions containing any of the therapeutic agents described herein can be formulated to be targeted to a particular tissue, receptor, or other region of the body to be treated. Non-limiting examples of targeting methods can be found in, e.g., 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.

[0352] Compositions to be used for in vivo administration can be sterilized, hi some aspects, such sterilization can be accomplished, for example, by filtration through sterile filtration membranes.

[0353] VI. Kit The present invention provides kits comprising one or more FAM19A5 antagonists for treating hearing disorders (e.g., as described herein). In some aspects, the present invention provides packs or kits comprising one or more containers filled with one or more of the components of the compositions described herein, such as one or more FAM19A5 antagonists, and optionally instructions for use.

[0354] [Example] Example 1: Expression analysis of FAM19A5 and LRRC4 family members in the inner ear To begin evaluating the therapeutic efficacy of the FAM19A5 antagonists provided by the present invention for hearing loss, we assessed the expression of FAM19A5 and LRRC4 family members in the mouse auditory system, particularly in spiral ganglion neurons, which are known to interact with inner hair cells at synaptic ribbons in the cochlea (see Figure 1).

[0355] As shown in Figure 2, clear expression of FAM19A5, LRRC4B, and LRRC4C was observed in cochlear tissue from embryonic day 15.5 mice. Overlapping expression with Nefl (a marker for spiral ganglion neurons) and Atoh1 (a marker for inner hair cells) was observed, suggesting that FAM19A5, LRRC4B, and LRRC4C were expressed within synaptic ribbons, particularly in spiral ganglion neurons. Similar results were observed in 7-day-old mice (see Figures 3 and 4).

[0356] These results indicate a potential role for FAM19A5 and LRRC4 family members in synaptic activity (eg, formation, loss, and / or signal transmission) within the auditory system.

[0357] Example 2: Effect of FAM19A5 antagonist administration on hearing ability To assess whether FAM19A5 antagonist administration itself affects hearing, we intravenously administered anti-FAM19A5 antibody (1-30 antibody) or a control human IgG antibody to C57BL / 6 mice (8 weeks old). Both the anti-FAM19A5 antibody and the control antibody were administered at a dose of 30 mg / kg per unit dose once weekly for a total of 8 weeks (see Figure 5a). To assess hearing, auditory brainstem response (ABR) thresholds were measured before initial antibody administration and at 1 day, 2 weeks, and 8 weeks after antibody administration. Higher ABR thresholds at a specific frequency were expected to indicate impaired hearing at that frequency, while lower ABR thresholds at a specific frequency were expected to indicate normal hearing at that frequency.

[0358] As shown in Figure 5b, before antibody administration, animals in one of the treatment groups (i.e., anti-FAM19A5 antibody or control IgG antibody) showed similar ABR thresholds at all frequencies, and as shown in Figure 5c, ABR thresholds remained at similar levels two weeks after antibody administration.

[0359] Such results demonstrate that the FAM19A5 antagonists described herein themselves do not have any negative effects on hearing.

[0360] Example 3: Analysis of the therapeutic effect of FAM19A5 antagonists on hearing loss To evaluate whether the FAM19A5 antagonists described herein can treat hearing loss, we used a noise-induced hearing loss mouse model. Briefly, hearing loss was induced by exposing animals to a transient threshold shift (TTS) of 105 dB for 30 minutes. Anti-FAM19A5 antibody (1-30 antibody) or a control human IgG antibody was administered intravenously to C57BL / 6 mice (8 weeks old) once weekly for a total of five doses (30 mg / kg per dose). As shown in Figure 6a, the first dose of antibody was administered to mice one day before noise exposure. To assess the effect on hearing loss, ABR thresholds, DPOAE thresholds, and / or DPOAE amplitudes were measured at various time points (see "Hearing Test" in Figure 6a).

[0361] As expected, before noise exposure, animals in each treatment group did not show significant differences in the various hearing-related parameters evaluated (see Figures 6b and 7a). One day after TTS exposure, a clear increase in ABR thresholds was observed in all animals (see, e.g., Figure 6c). This confirmed that TTS exposure caused hearing impairment. However, the increase in ABR thresholds was much more dramatic in animals treated with control antibodies than in animals treated with anti-FAM19A5 antibodies. In general, at all time points evaluated, ABR thresholds in animals treated with anti-FAM19A5 antibodies were similar to the corresponding values ​​before TTS exposure (see, e.g., Figures 6c to 6e). For animals in the control group, the maximum increase in ABR thresholds was observed immediately after TTS exposure (e.g., day 1, see Figure 6c), eventually beginning to approach baseline levels (i.e., pre-TTS values) approximately two weeks after TTS exposure.

[0362] Furthermore, as shown in Figures 7a-7d, the anti-FAM19A5 antibody-treated group and the control IgG antibody-treated group did not show significant differences in DPOAE threshold. However, significant differences in DPOAE amplitude were observed between the anti-FAM19A5 antibody-treated group and the control IgG antibody-treated group at least 2 weeks after TTS exposure (see Figures 8a and 8b). More specifically, animals treated with anti-FAM19A5 antibody exhibited higher DPOAE amplitude at a frequency of approximately 18 kHz compared to animals treated with control antibody. Furthermore, as shown in Figure 12a, after administration of anti-FAM19A5 antibody, the expression of CtBP2, a marker of cochlear ribbon synapses, in IHCs was significantly increased compared to the control antibody. Figure 12b also shows that after administration of anti-FAM19A5 antibody, the number of ribbon bodies per cell increased in IHCs (first graph) and OHCs (second graph) compared to the hIgG and pre-noise groups. An increase in the number of cochlear ribbon synapses is associated with hearing function.

[0363] Next, we used the noise-induced hearing loss mouse model described above to evaluate whether the timing of anti-FAM19A5 antibody administration had an effect. Again, to induce hearing loss, each animal was exposed to a 105 dB transient threshold shift (TTS) for 30 minutes. Anti-FAM19A5 antibody (1-30 antibody) or a control human IgG antibody was administered intravenously once weekly for a total of five doses (30 mg / kg per dose). In this experiment, the first dose of antibody was administered to the animals two hours after noise exposure (as opposed to the previous experiment, where the antibody was administered one day before noise exposure). Each animal's hearing loss was assessed (e.g., by measuring ABR threshold, DPOAE threshold, and / or DPOAE amplitude) before noise exposure and at 1 day, 2 weeks, and 4 weeks after noise exposure.

[0364] As shown in Figures 13a-e, no significant differences were observed between animals treated with anti-FAM19A5 antibody and animals treated with control antibody with respect to ABR thresholds. However, as further described in Example 4, significant differences were observed between treatment groups at higher tone burst levels, particularly with respect to the amplitude of Wave I and Wave IV (see Figures 14a-b).

[0365] Example 4: Analysis of ABR waveforms after administration of a FAM19A5 antagonist To further understand the therapeutic effects of the FAM19A5 antagonists described herein on hearing loss, ABR waveform analysis was performed. As shown in Figure 9, sound enters the cochlea and is transmitted to the auditory nerve (AN), then through the brainstem to the auditory cortex via the cochlear nucleus (CN), superior olivary complex (SOC), lateral lemniscus (LL), and inferior colliculus (IC). ABR waveform analysis can be performed by first measuring the amplitude of the signal from the auditory nerve each time a wavelength passes through the AN, CN, SOC, LL, or IC, and then measuring the initial amplitude of the first wavelength and subsequent wavelengths through the latency period (see Figure 10a). An exemplary click wave I amplitude profile obtained in this manner is shown in Figure 10b.

[0366] As shown in Figure 11a, no significant differences in wave I amplitude were observed before TTS exposure (left graph). However, significant differences were observed at both 2 weeks (Figure 11a, right graph) and 4 weeks (Figure 11b) after noise exposure. Wave I is an example of signal summation from synapses. Wave I amplitude correlates with signal strength, and latency correlates with conductance.

[0367] The above results collectively demonstrate that the FAM19A5 antagonists described herein are useful for treating and / or preventing hearing loss. Example 5: Effect of FAM19A5 antagonist dose on therapeutic efficacy for hearing loss To evaluate whether the therapeutic effect of the FAM19A5 antagonist on hearing loss was dose-dependent, we used the noise-induced hearing loss mouse model described in Example 3. Briefly, mice were exposed to noise, and then anti-FAM19A5 antibody (1-30 antibody) or a control human IgG antibody was administered intravenously three times: (i) 1 day before noise exposure, (ii) 1 week after noise exposure, and (iii) 2 weeks after noise exposure. The anti-FAM19A5 antibody was administered at one of the following doses: 1 mg / kg, 3 mg / kg, 7 mg / kg, 15 mg / kg, and 30 mg / kg. ABR thresholds were measured before noise exposure, 1 day after noise exposure, and 2 weeks after noise exposure.

[0368] As shown in Figures 15b and 15c, mice treated with the control antibody showed an increase in ABR thresholds at all frequencies on day 1 after noise exposure. Animals treated with the anti-FAM19A5 antibody showed a dose-dependent decrease in ABR thresholds across various frequencies. The greatest effect was observed in animals treated with 15 mg / kg and 30 mg / kg of the anti-FAM19A5 antibody. Two weeks after noise exposure, ABR thresholds in animals treated with the control antibody were slightly reduced compared to the levels observed on day 1 after noise exposure. However, ABR threshold levels were still higher than those measured before noise exposure. Significant decreases in ABR thresholds were still observed at many frequencies in animals treated with the anti-FAM19A5 antibody (e.g., at doses of 7 mg / kg, 15 mg / kg, or 30 mg / kg) (see Figures 15d and 15e).

[0369] These results indicate that the neural connections between hair cells and spiral ganglion neurons can be damaged by noise exposure, and further demonstrate that the FAM19A5 antagonists (e.g., anti-FAM19A5 antibodies) provided by the present invention can reverse such damage.

[0370] Example 6: Effect of administration of FAM19A5 antagonist on the treatment of presbycusis To begin evaluating the potential therapeutic effects of FAM19A5 antagonists on presbycusis, we compared various hearing-related parameters (e.g., ABR thresholds, DPOAE amplitudes, and ABR waveform analysis) in aged mice (6 and 12 months old) and normal-aged mice (10 weeks old).

[0371] As shown in Figure 16a, 6-month-old mice showed increased ABR thresholds at even higher frequencies (e.g., above 32 kHz) compared with normal aging mice. These differences were even more pronounced when normal aging mice were compared with 1-year-old mice (see Figure 16b). Similar results were observed when comparing wave I amplitude with DPOAE amplitude.

[0372] Next, to evaluate whether the FAM19A5 antagonists provided by the present invention can also treat presbycusis, aged mice (e.g., approximately 6 months to 1 year old) were used. As shown in Figure 17a, anti-FAM19A5 antibody (1-30 antibody) or a control human IgG antibody was administered intravenously to each animal every week for 8 weeks (30 mg / kg per dose). Various hearing-related parameters (e.g., ABR threshold, DPOAE amplitude, and ABR waveform analysis) were then evaluated 1 and 2 months after the initial administration.

[0373] As shown in Figure 17b, before anti-FAM19A5 antibody administration, no significant differences in ABR thresholds were observed among the animals. However, at 1 and 2 months after administration, ABR thresholds were significantly reduced in animals treated with anti-FAM19A5 antibody compared with control animals (see Figures 17c and 17d, respectively). Although the results regarding wave I amplitude were not statistically significant, the general trend was similar to that observed for ABR thresholds (i.e., wave I amplitude was reduced in animals treated with anti-FAM19A5 antibody) (see Figures 18a to 18c).

[0374] Such results demonstrate that the FAM19A5 antagonists (eg, anti-FAM19A5 antibodies) described herein may have a therapeutic effect on presbycusis.

[0375] Example 7: Epitope mapping analysis using FAM19A5 epitope fragments F1 to F6 Overlapping peptide fragments of human FAM19A5 protein (F1 to F6 epitope fragments; SEQ ID NOs: 178 to 183) were synthesized and then conjugated to BSA (SEQ ID NOs: 322 to 327). The binding of various anti-FAM19A5 antibodies to the BSA-conjugated peptide fragments F1 to F6 was determined by Western blot or ELISA analysis. For Western blot analysis, the BSA-conjugated FAM19A5 fragments F1 to F6 were separated by SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane using standard procedures. The membrane was incubated with an anti-FAM19A5 antibody (e.g., 1-65, 2 μg / mL, 1-65-scFv-rabbitFc-SSS), and the antigen-antibody complex was detected with an appropriate secondary antibody conjugated to horseradish peroxidase (anti-rabbit IgG (Fc-specific)-HRP, 1:4000 dilution). The following protocol was used for ELISA analysis: FAM19A5 fragments F1 to F6 (diluted at 1 μg / mL in 50 mM carbonate buffer (Biosesang) or at 20 μg / mL for high-concentration assays) were used to coat wells of a 96-well immunoplate (Thermo Scientific) overnight at 4°C (100 μL / well) and then washed twice with 1× PBS. The plate was then blocked with blocking buffer (100 μL / well) for 1 hour at room temperature. The relevant anti-FAM19A5 antibody was diluted in dilution buffer to 1 μg / mL (or 20 μg / mL for high-concentration assays) during a 1-hour incubation. After washing the plate (twice with 1× PBS), the diluted anti-FAM19A5 antibody was added to the appropriate wells, and the plate was incubated at room temperature for 1 hour. The plate was then washed a total of five times with wash buffer. Next, ODP substrate (prepared by dissolving one ODP tablet (O-phenylenediamine dihydrochloride, Thermo) in 9 mL of sterile deionized water and 1 mL of 10X peroxide-stable buffer (Theromo)) was added to each well, and the color change reaction was allowed to proceed for 10 minutes. The reaction was terminated by adding 100 μL of 2N H2SO4 (Daejung) to the well.The absorbance value of each well was detected at 492 nm using a 96-well microplate reader (Molecular Device).

[0376] As shown in Figures 19 and 20a, Western blot and ELISA analyses confirmed that anti-FAM19A5 antibody 1-65 strongly bound to fragment F5. Anti-FAM19A5 antibody P2-C12 also strongly bound to epitope fragment F5, but not significantly more so than the other fragments (F1 to F4 and F6) (see Figure 20b). On the other hand, anti-FAM19A5 antibody 3-2 did not bind to fragment F5. Instead, the 3-2 antibody strongly bound to epitope fragment F2 while minimizing binding to other fragments (see Figure 20a). This was also the case with anti-FAM19A5 1-28 antibody (data not shown). However, in contrast to the other antibodies, anti-FAM19A5 antibody 2-13 did not bind to any of the epitope fragments (see Figure 20a).

[0377] Next, to identify the specific amino acid residues in the epitope fragment F5 to which the 1-65 and P2-C12 antibodies bind, different amino acid residues in the F5 fragment were replaced with alanine as shown in Table 11 below. The mutated residues are underlined and in bold. The binding affinity of each anti-FAM19A5 antibody was measured using ELISA analysis as described above.

[0378] [Table 11]

[0379] Double underline: Cysteine ​​is reactive during peptide synthesis and was replaced with serine to reduce reactivity. Serine was chosen because it is structurally closest to cysteine. Such replacements are shown with double underlines.

[0380] As shown in Figure 21a, the 1-65 antibody could bind to mutant peptides #1, 5, 6, and 7 with similar affinity. However, when amino acid residues D13, L14, I16, and R18 of fragment F5 were mutated to alanine (numbering based on SEQ ID NO: 275 in Table 11), the 1-65 antibody could no longer bind to the peptide fragments. This suggests that these amino acid residues are important binding sites for the 1-65 antibody. On the other hand, the P2-C12 antibody showed high binding to mutant peptides #2, 3, 4, 5, and 7, but not to mutant peptides #1, 6, and 8. See Figure 21b. As shown in Table 11 above, mutant peptides #1, 6, and 8 contain alanine substitutions at amino acid residues G11, E10, and R18 (numbering based on SEQ ID NO: 275), suggesting that these amino acid residues are important binding sites for the P2-C12 antibody.

[0381] Next, to identify the specific amino acid residues in the epitope fragment F2 to which the 3-2 and 1-28 antibodies bind, different amino acid residues in the F2 fragment were replaced with alanine or valine, as shown in Table 12 below. The mutated residues are underlined and in bold. The binding affinity of the anti-FAM19A5 antibodies was measured using ELISA analysis as described above.

[0382] [Table 12]

[0383] As shown in Figure 25a, when amino acid residues R4, D5, P9, R10, or R11 were mutated to alanine, the binding ability of the 3-2 antibody to the epitope fragment F2 was significantly reduced. See Figures 24a and 24b. Similar analyses were performed on several deimmunized variants of the 3-2 antibody (see Example 8 for details on the deimmunization process). As shown in Figures 25b, 25c, 25d, 25f, 25g, 25i, and 25j, amino acid residues R4, P9, R10, and R11 were important for the binding of antibodies 1-30, 1-32, and 6-10 to FAM19A5. In the case of antibodies 1-17 and 4-11, amino acid residues R4, P9, and R10 were important (see Figures 25e and 25h).

[0384] In the case of the 1-28 antibody, strong binding was observed to the mutant F2 peptide fragments #1, 2, 6, and 8-13, while the binding strength of the 1-28 antibody to peptide fragments #3, 4, 5, and 7 was significantly reduced (see Figure 24c). These data suggest that the anti-FAM19A5 antibody 1-28 binds to FAM19A5 mainly at amino acid residues D5, S6, S7, and P9 (numbering is based on SEQ ID NO: 179 in Table 12) within the epitope F2 fragment. Example 8: Epitope mapping analysis using FAM19A5 mutants M1 to M8 To further characterize the binding epitope of the anti-FAM19A5 antibody disclosed herein, the amino acid sequences of different FAM19 family members (i.e., FAM19A1-5) were aligned. Based on this alignment, eight regions (M1 to M8) were identified in which the amino acid sequence of the FAM19A5 protein most significantly differs from other members of the FAM19A family (i.e., FAM19A1-4). The amino acid sequences of these regions were replaced with the consensus sequences of the corresponding regions for the FAM19A1-4 proteins. See Table 13 (mutated amino acid residues are shown in bold and underlined).

[0385] Phage expressing mutant FAM19A5 was prepared as follows. To prepare phage culture medium, 55.6 mL of 2 M glucose (D-(+)-glucose, Sigma), 5 mL of 1 M MgCl2 (magnesium chloride, Junsei), and 1 mL of 34 mg / mL chloramphenicol (Sigma) were added to 2xYT medium. Colonies obtained through monophage ELISA were selected and placed in 5 mL of prepared medium (2xYT-GMC) and cultured at 37°C for 16 hours in a shaking incubator (VS-8480, Vision). 100 μL of each culture was transferred individually to 10 mL of 2xYT-GMC and cultured at 37°C in an incubator until the OD600nm detection value reached 0.5. Once the OD600nm detection value reached 0.5, 5 mL of each culture was used as a sample for infection. After sample preparation, 50 μL of M1 helper phage was added to each sample and cultured at 37°C for 30 minutes without shaking, followed by an additional 30 minutes with shaking. Each culture was centrifuged at 3,850 rpm for 15 minutes using a microcentrifuge (Micro12, Hanil). The supernatant from the centrifuged culture was removed and stored separately and replaced with 5 mL of 2xYT medium containing 1 mL of 1 M IPTG (AG Scientific), 5 mL of 1 M MgCl2, 1 mL of 70 μg / μL kanamycin (Biopure), and 1 mL of chloramphenicol. The resulting pellet was completely dispersed in the newly added medium and then incubated at 30°C for 16 hours with shaking.

[0386] [Table 13]

[0387] As shown in Figure 22a, and consistent with previous data, anti-FAM19A5 antibody 1-65 failed to bind to FAM19A5 mutants M6, M7, and M8. The M6 ​​and M7 mutants contain amino acid mutations at sites corresponding to regions within the epitope fragment F5 of FAM19A5. See Table 13 (above).

[0388] The epitope analysis results for additional anti-FAM19A5 antibodies are presented in Figures 22b and 22c. The anti-FAM19A5 antibodies 13B4, 13F7, and 15A9 were unable to bind to the FAM19A5 mutant M8 and showed reduced binding to the mutant M7. The 13B4 antibody was also unable to bind to the FAM19A5 mutant M6 (see Figure 22b). Similarly, the antibodies P1-A08, P1-F02, P2-A01, P2-A03, P2-F07, and P2-F11 were unable to bind to the FAM19A5 mutant M8, and many of these antibodies also showed reduced binding to the mutant M7. The P2-C12 antibody differed from these antibodies in that it strongly bound to the FAM19A5 mutant M8. Instead, the P2-C12 antibody was unable to bind to the FAM19A5 mutant M6. This result is consistent with previous data demonstrating that epitope fragment F5 contains the critical FAM19A5 binding site for the P2-C12 antibody.

[0389] As shown in Figure 22a, the anti-FAM19A5 antibodies 1-28 and 3-2 failed to bind to the FAM19A5 mutant M2. As shown in Table 13 above, the M2 mutant has a substitution at amino acid residues 21 to 25 (i.e., epitope EP2), which corresponds to a region within the epitope fragment F2 of FAM19A5. This is consistent with previous data and confirms the importance of epitope fragment F2 for the binding of the 1-28 and 3-2 antibodies to FAM19A5.

[0390] The 2-13 antibody was confirmed to bind to FAM19A5 via an epitope completely different from that of the 1-28, 3-2, and 1-65 antibodies. As shown in Figure 22a, the anti-FAM19A5 antibody 2-13 exhibited intermediate and high binding to FAM19A5 mutants M1-M3 and M5-M7, but failed to bind to mutants M4 and M8. This suggests that epitopes EP4 and EP8 are important for the binding of the 2-13 antibody to FAM19A5. As previously discussed, given that the 2-13 antibody does not appear to bind to any of the FAM19A5 epitope fragments (see Figure 20a), these data suggest that the 2-13 antibody has a conformational epitope rather than a linear epitope.

[0391] Example 9: Cross-competition assay Next, to assess whether different anti-FAM19A5 antibodies with similar binding epitopes cross-compete with each other, a two-site sandwich ELISA was performed as described below (see Figure 23a).

[0392] First, the indicated anti-FAM19A5 antibody was diluted to a concentration of 10 μg / mL in 1× PBS. The diluted anti-FAM19A5 antibody (10 μg / mL in 1× PBS) ("capture antibody") was used to coat a 96-well plate (100 μL / well) for approximately 1 hour at 37° C. After incubation, the plate was washed a total of five times with wash buffer (0.01% Tween-20 / PBS; also known as 0.01% PBST), blocked with blocking solution (5% BSA / PBS; also known as 5% PBSA) (250 μL / well), and then left at 37° C. for 1 hour before being washed again. Next, 100 μg / mL of FAM19A5 antigen (diluted in PBS containing 5% BSA, 0.01% Tween-20; also referred to as 5% PBSAT; "dilution buffer") was added to each well, and the 96-well plate was incubated at 37° C. for 2 hours. After incubation, the plate was washed a total of five times with 0.01% PBST. After the final wash, the indicated biotinylated anti-FAM19A5 antibody ("detection antibody") (diluted at 1 μg / mL in 5% PBSAT) was added to the wells (100 μL volume), and the plate was further incubated at 37° C. for 1 hour. The plate was then washed again with 0.01% PBST (a total of five washes). Next, 100 μL of diluted (1 / 2000 in 5% PBSAT) streptavidin-HRP (1 mg / mL, Sigma, USA) was added to the wells, and the plate was incubated at room temperature for 30 minutes. The plate was then washed and treated with 100 μL of TMB substrate (followed by washing the plate and treating it with 100 μL of Lution (Thermo Fisher Scientific)). After further incubation at room temperature for 30 minutes, TMB substrate was added to induce a color change reaction. The reaction was terminated with 50 μL of sulfuric acid (2N H2SO4), and the degree of color change was detected by absorbance at 450 nm against a reference wavelength of 620 nm using a 96-well microplate reader (Molecular Devices).

[0393] As shown in Figure 23b, the anti-FAM19A5 antibodies 1-65, P2-A03, P2-F11, and 13B4 all cross-competed with each other. Similar to the 1-65 antibody, the P2-A03, P2-F11, and 13B4 antibodies all bind to FAM19A5 at epitopes M6, M7, and / or M8 (i.e., within the F5 and / or F6 epitope fragments) (data not shown). In contrast, antibodies 2-13 and 3-2 did not cross-compete with other anti-FAM19A5 antibodies, confirming previous epitope mapping analysis results showing that these antibodies primarily bind to the F2 epitope fragment.

[0394] Example 10: HDX-MX epitope mapping of antibody 2-13 To determine the binding epitope of human FAM19A5 for the clone 2-13 anti-FAM19A5 antibody, hydrogen / deuterium exchange mass spectrometry (HDX-MS) was performed as follows.

[0395] 1. Optimizing conditions to maximize FAM19A5 application range Before conducting epitope mapping experiments, we performed experiments without deuteration to generate a general peptide list for the FAM19A5 protein. We digested the FAM19A5 protein using offline and online pepsin digestion (Houde, D. et al., Methods Mol Biol 988:269-89 (2013)). For online digestion, we used a pepsin-immobilized column (~20°C) and adjusted the flow rate to control the reaction efficiency. For offline digestion, we manually digested the FAM19A5 protein with pepsin at 4°C for 5 minutes, then loaded the mixture onto a liquid chromatography column for analysis. To maximize protein coverage, we adjusted one or more of the following parameters: potassium phosphate, sodium phosphate, quenching time, reducing agent (TCEP), urea concentration, and pepsin concentration. The resulting raw mass spectrometry (MS) data were analyzed using Waters PLGS software to confirm maximum protein coverage. To confirm experimental reproducibility, each experimental condition was repeated at least twice to identify the peptides generated. Maximum coverage was observed using the following conditions: 1 M TCEP, 2 M urea (pH 2.66) and pepsin at a 1:2 concentration.

[0396] 2.FAM19A5 epitope mapping Maximum (100%) binding (K D To obtain a densitometric titer (i.e., 1 nM), the antigen-antibody complex samples were incubated for a minimum of 3 hours before hydrogen / deuterium labeling. After labeling, the volumes of each sample were made equal with equilibration buffer.

[0397] To initiate the labeling reaction, 2.5 μL of each prepared sample (antigen alone, antibody alone, or antigen-antibody complex) diluted 1:15 in labeling buffer was mixed with DO labeling solution (1.8 μM). The reaction was performed for various times: 0 min (no deuteration), 20 s, 10 min, 60 min, and 240 min. For the deuterium reaction, the prepared sample was mixed with equilibration buffer. At the end of the labeling reaction, the reaction was stopped using quenching buffer. The samples were then vortexed, immediately frozen in liquid nitrogen, and stored at -80°C until analysis.

[0398] Before analysis by mass spectrometry, stored frozen samples were thawed and then digested with pepsin on ice for approximately 5 minutes (i.e., the offline method described above) using the software program DynamX 3.0. TM (Waters) and the resulting relative deuterium levels were plotted against exchange time.

[0399] For each identified peptide, deuterium absorption data (single antigen / antibody v. antigen-antibody complex) were compared. Significant deuterium absorption was observed for each of the following peptide sequences: (i) ACRKGQIAGTTRARPAC (residues 37-53 of SEQ ID NO:304; i.e., SEQ ID NO:306), (ii) ACRKGQIAGTTRARPACVD (residues 37-55 of SEQ ID NO:304; i.e., SEQ ID NO:307), (iii) ACRKGQIAGTTRARPACVDA (residues 37-56 of SEQ ID NO:304; i.e., SEQ ID NO:308), (iv) ARIIKTKQWC (residues 56-65 of SEQ ID NO:304; i.e., SEQ ID NO:309), (v) ARII KTKQWCDM (residues 56 to 67 of SEQ ID NO:304; i.e., SEQ ID NO:310), (vi) ARIIKTKQWCDML (residues 56 to 68 of SEQ ID NO:304; i.e., SEQ ID NO:311), (vii) ARIIKTKQWCDMLPCL (residues 56 to 71 of SEQ ID NO:304; i.e., SEQ ID NO:312), (viii) RIIKTKQWCDM (residues 57 to 67 of SEQ ID NO:304; i.e., SEQ ID NO:313), and (ix) RIIKTKQWCDML (residues 57 to 68 of SEQ ID NO:304; i.e., SEQ ID NO:314).

[0400] Using the results above, a heat map was constructed to identify regions within the FAM19A5 protein with the greatest difference in deuterium absorption between the single antigen / antibody sample and the antigen-antibody complex. Amino acid residues 38-50 (CRKGQIAGTTRAR; i.e., SEQ ID NO: 199) and 51-64 (PACVDARIIKTKQW; i.e., SEQ ID NO: 200) of SEQ ID NO: 304 were identified as critical binding residues for the 2-13 antibody. Figure 26 shows the locations of these residues within the three-dimensional structure of the FAM19A5 protein.

[0401] Example 11: Additional Exemplary Materials and Methods To evaluate the therapeutic effects of the FAM19A5 antagonists on hearing loss described in Examples 12 and 13, the following materials and methods were used.

[0402] Spiral ganglion neuron culture Spiral ganglion neurons were cultured in a culture medium consisting of high-glucose (4.5 mg / mL) DMEM, 0.1 mg / mL penicillin, 0.1 mg / mL streptomycin, N2 supplement, and 10 μg / mL insulin. + ] = 5.4 mM, so this was expressed as "5K". + A depolarizing medium labeled "30K" was also used in which Na was replaced with an equimolar amount of K to increase the [Na saturation] to 30 mM.

[0403] Dissociated spiral ganglion cell cultures were prepared from postnatal day 4-5 (P4-5) cochleae and maintained using a procedure modified from that described in Bok et al., J Neurosci 23(3):777-87 (2003), which is incorporated herein by reference in its entirety. Cochleae were aseptically dissected from the temporal bone and immersed in ice-cold HBSS (Gibco). The bony cochlear capsule and spiral ligament were then removed. The connective tissue and organ of Corti surrounding the bony modiolus were removed, preserving the spiral ganglion neurons within the modiolus. Ca-free PBS containing 0.1% trypsin and 0.1% collagenase was used. 2+ -Mg 2+ After collecting ganglia in HBSS, enzymatic dissociation was performed for 20 minutes in a 37°C CO2 incubator. The cells were washed three times with 5K medium and gently triturated approximately 15 times with a pipette in 30K medium. The cells were then seeded onto 4-well glass slides coated with poly-D-lysine and laminin (Thermo Fisher Scientific) and maintained at 37°C in a humidified 5% CO2 incubator.

[0404] Cochlear explant culture Cochlear explants were cultured in a plating medium consisting of Neurobasal medium (Invitrogen), 1% N2 supplement, and 10 μg / mL ampicillin, plus 1% FBS.

[0405] Postnatal day 4-5 (P4-5) C57BL / 6 mice were sacrificed for cochlear dissection. The mouse head was bisected and brain tissue removed. The cochlea was carefully separated from the exoskeleton in ice-cold HBSS (5 mM HEPES / 1.3 mM CaCl2, 0.9 mM MgCl2, pH 7.4) or external solution (1.2 mM CaCl2, 1 mM MgCl2, and 5 mM HEPES in HBSS). The stria vascularis and spiral lamina were then removed, and the cochlea was cut into two to three sections. Reissner's membrane and tectorial membrane were then removed from the dissected tissue. The tissue was then transferred to a four-well glass slide coated with poly-D-lysine and laminin (Thermo Fisher Scientific). The cochlear explant tissue was positioned so that the hair cell region, from which the tectorial membrane had been removed, was facing upward. Explants were allowed to stabilize overnight in plating medium and maintained at 37°C in a humidified 5% CO2 incubator after a complete change of culture medium.

[0406] immunostaining Primary spiral ganglion neurons and cochlear explants were fixed with 4% paraformaldehyde (PFA) for 20 minutes at the appropriate days in vitro (DIV). Cells / tissues were blocked with 3% bovine serum albumin (BSA) and 0.1% Triton X-100 in phosphate-buffered saline (PBS) for 1 hour at room temperature. Primary antibodies were then applied to the cells / tissues overnight at 4°C. The primary antibodies used in this study were mouse anti-NF200 (Sigma) and mouse anti-CtBP2 (BD Bioscience). After multiple washes with PBS, appropriate fluorescently labeled secondary antibodies were applied for 1 hour at room temperature. Cells / tissues were then imaged using a confocal microscope (Leica).

[0407] Quantitative analysis of neurite outgrowth Neurite outgrowth was quantified by measuring the total length of neurites per neuron. At 2 or 3 days after initiation of transfer, neurons were stained with NF200 and neurite length was measured using Fiji (Image J). Individual neurons were manually selected, and neurite length was determined using the Simple Neurite Tracer plugin.

[0408] Quantitative analysis of synaptogenesis Mouse cochlear explant cultures were treated with FAM19A5, FAM19A5 and a FAM19A5 antagonist (e.g., the 1-30 antibody described herein), or a FAM19A5 antagonist (e.g., the 1-30 antibody described herein) alone at 1 DIV. To determine the level of synaptogenesis, the cells were immunostained for the ribbon synapse marker CtBP2 at 2 DIV. To quantify the number of CtBP2 puncta in individual inner hair cells, z-stack confocal images were exported to "multipage" mode, and the number of CtBP2 puncta in each individual inner hair cell was counted using the ROI manager and multipoint tools in Fiji (Image J). The number of CtBP2 puncta emanating from five individual inner hair cells was calculated per image, with three z-stack images measured for each treatment group.

[0409] statistical analysis All statistical analyses were performed using GraphPad Prism 8 (GraphPad Software Inc.), and data were presented as mean ± standard error of the mean (SEM). Statistical significance was assessed using Tukey's multiple comparison test and Student's t-test and / or one-way analysis of variance (ANOVA). A p value of less than 0.05 was considered statistically significant.

[0410] Example 12: Effect on neurite outgrowth inhibition induced by FAM19A5 Spiral ganglion neurons (SGNs) are core components of the auditory system. These specialized neurons serve as the primary conduits for transmitting electrical impulses generated by mechanosensory hair cells in the cochlea to the central auditory pathway in the brain. These functions include encoding and transmitting important auditory parameters, including sound frequency, intensity, and temporal characteristics. However, these neurons are vulnerable to damage in cases of sensorineural hearing loss, the most frequent form of hearing damage. To assess the regenerative potential of SGNs under hearing impairment, we evaluated the ability of the FAM19A5 antagonist described herein to reverse the FAM19A5-induced inhibition of neurite outgrowth in mouse primary spiral ganglion neurons.

[0411] Briefly, mouse primary spiral ganglion neurons (cultured and treated as described in Example 11) were treated with FAM19A5 protein (10 μM) alone or with anti-FAM19A5 antibody (1-30) (100 nM) at 0 days in vitro (DIV). Untreated cells (i.e., not treated with FAM19A5 protein or anti-FAM19A5 antibody) served as a control. Then, at 3 days in vitro, neurons were stained for the expression of neurofilament protein (NF200), and the average length of neurites was quantified.

[0412] As shown in Figures 27a and 27b, neurons treated with FAM19A5 protein showed a significant decrease in total neurite length compared to untreated cells (i.e., the "control" and "FAM19A5" groups). However, neurons treated with both anti-FAM19A5 antibody and FAM19A5 showed less reduction in neurite outgrowth and were similar to control neurons. These results suggest that co-treatment with a FAM19A5 antagonist described herein (e.g., an anti-FAM19A5 antibody, e.g., 1-30 antibody) effectively counteracted the inhibitory effect of FAM19A5 protein on neurite outgrowth in spiral ganglion neurons.

[0413] Example 13: Effect on neurite outgrowth promotion To further evaluate the effects on neurite outgrowth, primary mouse spiral ganglion neurons (isolated on postnatal day 4) were treated with one of the follo...

Claims

1. A pharmaceutical composition for treating or preventing hearing impairment, comprising (a) an antagonist to a family, member A5 (FAM19A5) protein having sequence similarity 19 (FAM19A5 antagonist), and (b) a pharmaceutically acceptable carrier, The FAM19A5 antagonist comprises (i) an antibody or antigen-binding fragment thereof that specifically binds to the FAM19A5 protein ("anti-FAM19A5 antibody"); (ii) a polynucleotide that encodes the anti-FAM19A5 antibody; or (iii) a vector containing the polynucleotide. The anti-FAM19A5 antibody comprises heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3. (1) The heavy chain CDR1 consists of the amino acid sequence of SEQ ID NO: 14, the heavy chain CDR2 consists of the amino acid sequence of SEQ ID NO: 15, the heavy chain CDR3 consists of the amino acid sequence of SEQ ID NO: 16, the light chain CDR1 consists of the amino acid sequence of SEQ ID NO: 26, the light chain CDR2 consists of the amino acid sequence of SEQ ID NO: 27, and the light chain CDR3 consists of the amino acid sequence of SEQ ID NO: 28, (2) The heavy chain CDR1 consists of the amino acid sequence of SEQ ID NO: 220, the heavy chain CDR2 consists of the amino acid sequence of SEQ ID NO: 221, the heavy chain CDR3 consists of the amino acid sequence of SEQ ID NO: 16, the light chain CDR1 consists of the amino acid sequence of SEQ ID NO: 225, the light chain CDR2 consists of the amino acid sequence of SEQ ID NO: 226, and the light chain CDR3 consists of the amino acid sequence of SEQ ID NO: 227, (3) The heavy chain CDR1 consists of the amino acid sequence of SEQ ID NO: 220, the heavy chain CDR2 consists of the amino acid sequence of SEQ ID NO: 221, the heavy chain CDR3 consists of the amino acid sequence of SEQ ID NO: 16, the light chain CDR1 consists of the amino acid sequence of SEQ ID NO: 225, the light chain CDR2 consists of the amino acid sequence of SEQ ID NO: 228, and the light chain CDR3 consists of the amino acid sequence of SEQ ID NO: 227, (4) The heavy chain CDR1 consists of the amino acid sequence of SEQ ID NO: 220, the heavy chain CDR2 consists of the amino acid sequence of SEQ ID NO: 221, the heavy chain CDR3 consists of the amino acid sequence of SEQ ID NO: 16, the light chain CDR1 consists of the amino acid sequence of SEQ ID NO: 26, the light chain CDR2 consists of the amino acid sequence of SEQ ID NO: 229, and the light chain CDR3 consists of the amino acid sequence of SEQ ID NO: 227, (5) The heavy chain CDR1 consists of the amino acid sequence of SEQ ID NO: 220, the heavy chain CDR2 consists of the amino acid sequence of SEQ ID NO: 221, the heavy chain CDR3 consists of the amino acid sequence of SEQ ID NO: 16, the light chain CDR1 consists of the amino acid sequence of SEQ ID NO: 26, the light chain CDR2 consists of the amino acid sequence of SEQ ID NO: 228, and the light chain CDR3 consists of the amino acid sequence of SEQ ID NO: 227, (6) The heavy chain CDR1 consists of the amino acid sequence of SEQ ID NO: 220, the heavy chain CDR2 consists of the amino acid sequence of SEQ ID NO: 221, the heavy chain CDR3 consists of the amino acid sequence of SEQ ID NO: 16, the light chain CDR1 consists of the amino acid sequence of SEQ ID NO: 26, the light chain CDR2 consists of the amino acid sequence of SEQ ID NO: 230, the light chain CDR3 consists of the amino acid sequence of SEQ ID NO: 231, or (7) A pharmaceutical composition in which the heavy chain CDR1 consists of the amino acid sequence of SEQ ID NO: 220, the heavy chain CDR2 consists of the amino acid sequence of SEQ ID NO: 221, the heavy chain CDR3 consists of the amino acid sequence of SEQ ID NO: 16, the light chain CDR1 consists of the amino acid sequence of SEQ ID NO: 26, the light chain CDR2 consists of the amino acid sequence of SEQ ID NO: 232, and the light chain CDR3 consists of the amino acid sequence of SEQ ID NO:

233.

2. The pharmaceutical composition according to claim 1, wherein the hearing impairment includes sensory nerve hearing loss.

3. (a) The sensory nerve hearing loss is accompanied by tinnitus; (b) The sensory nerve hearing loss is not accompanied by tinnitus; or, (c) The pharmaceutical composition according to claim 2, wherein the sensory nerve hearing loss includes ototoxic drug-induced hearing loss, noise-induced hearing loss, age-related hearing loss, sudden hearing loss, or a combination thereof.

4. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition (a) reduces or prevents damage to ribbon synapses in the inner ear of a subject requiring it; (b) induces the formation of ribbon synapses in the inner ear of a subject requiring it; or (c) improves the function of ribbon synapses in the inner ear of a subject requiring it.

5. The pharmaceutical composition according to claim 4, wherein the damage to the ribbon synapses includes (a) a reduction in the number of ribbon synapses compared to a corresponding subject without damage to the ribbon synapses in the inner ear; (b) abnormal function of the ribbon synapses; or (c) both of (a) and (b).

6. The pharmaceutical composition according to claim 4, wherein the function includes the ability to release one or more neurotransmitters in response to signals from inner hair cells.

7. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition reduces the expression or activity of the FAM19A5 protein in spiral ganglion neurons of a target requiring the same.

8. The pharmaceutical composition according to claim 1, wherein the anti-FAM19A5 antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL).

9. The pharmaceutical composition according to claim 8, wherein VH comprises the sequence of sequence number 235, and VL comprises the sequence of sequence number 240.

10. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is administered to a subject by intracochlear injection, intravestibular injection, intravenous administration, or intratympanic administration.

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