Polypeptide inhibitors and their uses

Abstract

The present invention provides a polypeptide capable of specifically binding to FAM19A5 protein, thereby inhibiting the interaction between a member of the LRRC4 protein family and the FAM19A5 protein. In some aspects, the polypeptide comprises a FAM19A5-binding domain of a member of the LRRC4 protein family. The present invention also provides a method for promoting neurite outgrowth by administering a polypeptide described herein.

Description

Detailed Description of the Invention

[0001] [Technical field] This PCT application claims priority to U.S. Provisional Application No. 63 / 219,670, filed July 8, 2021, the entire contents of which are incorporated herein by reference.

[0002] References to sequence listings submitted electronically using EFS-Web The entire contents of the sequences submitted electronically to this application as an .XML file (Name: 3763.018PC01_Seqlisting_ST26.xml, Size: 159,089 bytes; Creation Date: July 7, 2022) are incorporated by reference into this application.

[0003] The present invention provides polypeptides (eg, isolated polypeptides) that can specifically inhibit, reduce, and / or dissociate the interaction between a member of the LRRC4 protein family and a FAM19A5 protein.

[0004] [Background technology] Mammalian neurons continuously project neurites, including axons and dendrites, to form synapses with other neurons, muscles, and blood vessels. At the same time, neurons retract neurites to dismantle unnecessary synapses (e.g., those that have not been used for an extended period of time). The balance between synapse gain and loss is important for healthy central and peripheral nervous systems.

[0005] However, abnormal synapse loss can occur due to various factors (e.g., aging, cytotoxic microenvironment, acute damages, genetic mutations). Such increased synapse loss is associated with a variety of neurological disorders, including mental retardation, schizophrenia, autism spectrum disorder, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, Huntington's disease, prion diseases, neuropathic pain, spinal cord injury, and stroke (see, e.g., Hayashi-Takagi, Neurosci Res 114:3-8 (Jan. 2017); Wang et al, Prog Neuropsychopharmacol Biol Psychiatry 84 (Pt B):398-415 (Jun. 2018); Jha et al, J Alzheimer s Dis 57(4):1017-1039 (2017); Mitoma et al., Int J Mol Sci 21(14):4936 (Jul. 2020); and Brose et al, Biochem Soc Trans 38(2):443-4 (Apr. 2010).

[0006] The underlying causes of neurological disorders are not always fully understood, and therefore the majority of treatment options currently focus on treating symptoms associated with the disorder. And, even when available, treatments may have side effects and / or limited efficacy. Thus, there is a need for alternative, more effective treatment options for neurological disorders, such as those associated with abnormal loss of synapses.

[0007] Summary of the Invention [Means for solving the problems] The present invention provides an isolated polypeptide comprising, consisting of, or consisting essentially of a domain of a leucine-rich repeat containing 4 ("LRRC4") protein family member capable of binding to family with sequence similarity 19, member A5 ("FAM19A5"), wherein the polypeptide is shorter than a corresponding full-length LRRC4 protein family member (SEQ ID NO:4; SEQ ID NO:5; or SEQ ID NO:6).

[0008] In some aspects, the FAM19A5 binding domain is about 10 to 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, or about 23 amino acids in length. In some aspects, the FAM19A5 binding domain is about 10 amino acids in length.

[0009] In some aspects, the FAM19A5 binding domain can inhibit, reduce, and / or dissociate the interaction between a FAM19A5 protein and a LRRC4 protein family member.

[0010] In some aspects, the FAM19A5 binding domain comprises an amino acid sequence (N-terminus to C-terminus) having the following formula: A-(T / S)-B (Formula I): where (i) A comprises X1-(T / S)-(Y / F)-F-X5; 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; (ii) B comprises (V / I)-TV-(E / V); (V / I) is valine (V) or isoleucine (I); (E / V) is glutamic acid (E) or valine (V).

[0011] In some aspects, the FAM19A5 binding domain comprises an amino acid sequence (N-terminus to C-terminus) having the following formula: A-(T / S)-B (Formula I): where (i) A comprises (Y / W / M)-(T / Y)-(Y / W)-(F / Y / W)-(T / Y); (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); (ii) B includes X7-(T / S / Y)-X9-X10; 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); X10 is glutamic acid (E), aspartic acid (D), isoleucine (I), tyrosine (Y), phenylalanine (F), methionine (M), or tryptophan (W).

[0012] The present invention also provides an isolated polypeptide comprising an amino acid sequence (from N-terminus to C-terminus) having the following formula: X1-X2-X3-F-X5-T-X7-TV-X10 (Formula II): 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; 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.

[0013] The present invention further provides an isolated polypeptide comprising an amino acid sequence (N-terminus to C-terminus) of the following formula: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10 (Formula III): 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; 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.

[0014] 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.

[0015] In some aspects, the polypeptides described herein comprise the amino acid sequence presented as SEQ ID NO:29 (YTYFTTVTVE). In some aspects, the polypeptides consist of the amino acid sequence presented as SEQ ID NO:29 (YTYFTTVTVE). In some aspects, the polypeptides comprise the amino acid sequence presented as SEQ ID NO:20 (NYSFFTTVTVETTEISPEDTTRK). In some aspects, the polypeptides consist of the amino acid sequence presented as SEQ ID NO:20 (NYSFFTTVTVETTEISPEDTTRK). In some aspects, the polypeptides comprise the amino acid sequence presented as SEQ ID NO:30 (YSFFTTVTVE). In some aspects, the polypeptides consist of the amino acid sequence presented as SEQ ID NO:30 (YSFFTTVTVE). In some aspects, the polypeptides comprise the amino acid sequence presented as SEQ ID NO:21 (NFSYFSTVTVETMEPSQDERTTR). In some aspects, the polypeptides consist of the amino acid sequence presented as SEQ ID NO:21 (NFSYFSTVTVETMEPSQDERTTR). In some aspects, the polypeptide comprises the amino acid sequence listed as SEQ ID NO: 31 (FSYFSTVTVE). In some aspects, the polypeptide consists of the amino acid sequence listed as SEQ ID NO: 31 (FSYFSTVTVE).

[0016] In some aspects, the polypeptide comprises the amino acid sequence listed as SEQ ID NO: 18 (GYTYFTTVTVETLETQPGEE). In some aspects, the polypeptide consists of the amino acid sequence listed as SEQ ID NO: 18 (GYTYFTTVTVETLETQPGEE). In some aspects, amino acid residues T12 and L13 are modified (e.g., substituted) relative to the corresponding residues in SEQ ID NO: 18. In some aspects, the polypeptide comprises the amino acid sequence listed as any one of SEQ ID NOs: 123-142. In some aspects, the polypeptide consists of the amino acid sequence listed as any one of SEQ ID NOs: 123-142. In some aspects, one or more of the amino acid residues are of the D-amino acid type.

[0017] In some aspects, the polypeptide comprises the amino acid sequence listed as SEQ ID NO: 17 (GYTYFTTVTVETLETQ). In some aspects, the polypeptide consists of the amino acid sequence listed as SEQ ID NO: 17 (GYTYFTTVTVETLETQ). In some aspects, the polypeptide comprises the amino acid sequence listed as SEQ ID NO: 19 (GYTYFTTVTVETLETQPGEKEPPGPTTD). In some aspects, the polypeptide consists of the amino acid sequence listed as SEQ ID NO: 19 (GYTYFTTVTVETLETQPGEKEPPGPTTD).

[0018] In some aspects, the polypeptide comprises the amino acid sequence listed as SEQ ID NO: 143 (GYTYFTTVTVETLETQPGEEA). In some aspects, the polypeptide consists of the amino acid sequence listed as SEQ ID NO: 143 (GYTYFTTVTVETLETQPGEEA). In some aspects, amino acid residues T12 and L13 are modified (e.g., substituted) relative to the corresponding residues in SEQ ID NO: 143. In some aspects, the polypeptide comprises the amino acid sequence listed as any one of SEQ ID NOs: 123-149. In some aspects, the polypeptide consists of the amino acid sequence listed as any one of SEQ ID NOs: 123-149.

[0019] In some aspects, the amino acid at X2 is phosphorylated or O-glycosylated.

[0020] In some aspects, any of the polypeptides provided herein is linked to a moiety. In some aspects, the moiety can increase one or more of the following properties of the polypeptide: (1) binding affinity to FAM19A5 protein, (2) solubility, (3) resistance to degradation from proteases and / or peptidases, (4) suitability for in vivo administration, (5) ability to inhibit FAM19A5-LRRC4 protein family member interaction, or (6) any combination of (1)-(5). In some aspects, the moiety comprises a juxta-membrane sequence of an LRRC4 protein family member. In some aspects, the juxta-membrane comprises the sequence set forth in SEQ ID NO: 151 (LDEVMKTTK) or SEQ ID NO: 152 (IDEVMKTTK). In some aspects, the juxtamembrane region consists of the sequence provided in SEQ ID NO: 151 (LDEVMKTTK) or SEQ ID NO: 152 (IDEVMKTTK).

[0021] The present invention also provides an isolated polypeptide comprising an amino acid sequence having at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% sequence identity with the amino acid sequence set forth in SEQ ID NO:29, wherein the amino acid sequence is capable of binding to a FAM19A5 protein, thereby inhibiting, reducing, and / or dissociating the interaction between a FAM19A5 protein and a member of the LRRC4 protein family.

[0022] The present invention provides an isolated polypeptide comprising an amino acid sequence that is at least about 70%, at least about 75%, 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%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:4, or SEQ ID NO:6, respectively, and that contains at least one amino acid sequence modification relative to the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:4, or SEQ ID NO:6, respectively, wherein the polypeptide is capable of binding to a FAM19A5 protein, thereby inhibiting, reducing, and / or dissociating the interaction between a FAM19A5 protein and a member of the LRRC4 protein family.

[0023] In some aspects, the at least one amino acid modification increases binding of the polypeptide to FAM19A5 protein. In some aspects, the at least one amino acid modification increases stability of the polypeptide. In some aspects, the increase in binding and / or stability increases the ability of the polypeptide to inhibit, reduce, and / or dissociate an interaction between a FAM19A5 protein and a LRRC4 protein family member. In some aspects, the ability of the polypeptide to inhibit, reduce, and / or dissociate an interaction between a FAM19A5 protein and a LRRC4 protein family member 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 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, or at least about 50-fold compared to a corresponding polypeptide lacking the at least one amino acid modification.

[0024] In some aspects, the amino acid residue at position 453 of SEQ ID NO:5 (e.g., position 5 of SEQ ID NO:29) is T or modified with S or Y. In some aspects, the amino acid residue at position 454 of SEQ ID NO:5 (e.g., position 6 of SEQ ID NO:29) is T or modified with S or Y. In some aspects, the amino acid residue at position 449 of SEQ ID NO:5 (e.g., position 1 of SEQ ID NO:29) is Y or modified with F, V, L, I, W, or M. In some aspects, the amino acid residue at position 450 of SEQ ID NO:5 (e.g., position 2 of SEQ ID NO:29) is T or modified with S or Y. In some aspects, the amino acid residue at position 451 of SEQ ID NO:5 (e.g., position 3 of SEQ ID NO:29) is Y or modified with F or W. In some aspects, the amino acid residue at position 452 of SEQ ID NO:5 (e.g., position 4 of SEQ ID NO:29) is F or modified with Y or W. In some aspects, the amino acid residue at position 455 of SEQ ID NO:5 (e.g., position 7 of SEQ ID NO:29) is V or is modified with I, Y, F, L, W, or M. In some aspects, the amino acid residue at position 456 of SEQ ID NO:5 (e.g., position 8 of SEQ ID NO:29) is T or is modified with S or Y. In some aspects, the amino acid residue at position 457 of SEQ ID NO:5 (e.g., position 9 of SEQ ID NO:29) is V or is modified with I, Y, F, L, W, or M. In some aspects, the amino acid residue at position 458 of SEQ ID NO:5 (e.g., position 10 of SEQ ID NO:29) is E or is modified with D, V, I, Y, F, M, or W.

[0025] In some aspects, one or more amino acid residues of the polypeptide are in the D-form. In some aspects, the D-form is at either or both the N-terminus or C-terminus.

[0026] In some aspects, a polypeptide described herein (as provided above) is linked to a moiety. In some aspects, the moiety can increase one or more of the following properties of the polypeptide: (1) binding affinity to FAM19A5 protein, (2) solubility, (3) resistance to degradation from proteases and / or peptidases, (4) suitability for in vivo administration, (5) ability to inhibit FAM19A5-LRRC4 protein family member interaction, or (6) any combination of (1)-(5). In some aspects, the moiety comprises a juxtamembrane sequence of an LRRC4 protein family member. In some aspects, the juxtamembrane sequence comprises the sequence provided as SEQ ID NO: 151 (LDEVMKTTK) or SEQ ID NO: 152 (IDEVMKTTK). In some aspects, the juxtamembrane sequence consists of the sequence provided as SEQ ID NO: 151 (LDEVMKTTK) or SEQ ID NO: 152 (IDEVMKTTK).

[0027] In some aspects, the polypeptides described herein do not include the transmembrane and / or intracellular domains of a LRRC4 protein family member, hi some aspects, the polypeptides are capable of competing with a member of the LRRC4 protein family for binding to the FAM19A5 protein.

[0028] For any of the above-mentioned polypeptides, in some aspects the LRRC4 protein family member comprises a LRRC4 protein, a LRRC4B protein, a LRRC4C protein, or a combination thereof.

[0029] The invention also provides molecules comprising any of the polypeptides described herein. In some aspects, the molecules further comprise 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, the one or more additional amino acids are hydrophilic amino acids. In some aspects, the one or more additional amino acids are D-amino acids.

[0030] In some aspects, a molecule comprises any of the polypeptides described herein, wherein the N-terminus, C-terminus, or both the N-terminus and C-terminus of the polypeptide comprise a modification that increases the stability of the polypeptide, in some aspects, the modification comprises Fmoc, PEGylation, acetylation, methylation, cyclization, or a combination thereof.

[0031] In some aspects, the molecule comprising a polypeptide described herein is a fusion protein. In some aspects, the molecule further comprises a half-life extending moiety. In some aspects, the half-life extending moiety comprises Fc, albumin, albumin binding polypeptide, Pro / Ala / Ser (PAS), C-terminal peptide of the beta subunit of human chorionic gonadotropin (CTP), polyethylene glycol (PEG), long unstructured hydrophilic sequences of amino acids (XTEN), hydroxyethyl starch (HES), albumin binding small molecule, or a combination thereof.

[0032] The invention provides nucleic acids encoding any of the polypeptides or molecules of the invention. In some aspects, the nucleic acid is DNA or RNA. In some aspects, the nucleic acid is mRNA. In some aspects, the nucleic acid comprises a nucleic acid analog.

[0033] The invention provides a vector comprising any of the nucleic acids described herein.The invention provides a cell comprising any of the vectors described herein.The invention provides a protein conjugate comprising any of the polypeptides described herein linked to an agent.

[0034] The present invention provides a composition comprising any of the polypeptides, molecules, nucleic acids, vectors, cells or protein conjugates described herein. In some aspects, the composition further comprises a pharma- ceutically acceptable carrier.

[0035] The invention provides kits comprising any of the polypeptides, molecules, nucleic acids, vectors, cells or protein conjugates described herein and instructions for use.

[0036] The present invention also provides a method for producing a polypeptide capable of inhibiting, reducing and / or dissociating an interaction between a FAM19A5 protein and a member of the LRRC4 protein family, the method comprising culturing a cell described herein under suitable conditions such that the polypeptide is produced. In some aspects, the method further comprises isolating the produced polypeptide.

[0037] The present invention provides methods of increasing neurite outgrowth and / or synaptogenesis in a neuron comprising contacting the neuron with an ectodomain of a LRRC4 protein family member or a fragment thereof capable of binding to a FAM19A5 protein. In some aspects, the ectodomain comprises the amino acid sequence set forth in SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6. In some aspects, the fragment thereof comprises any of the polypeptides described herein.

[0038] The present invention provides a method of increasing neurite outgrowth and / or synaptogenesis in a neuron comprising contacting the neuron with any of the polypeptides, molecules, nucleic acids, vectors, cells, protein conjugates, or compositions described herein. In some aspects, the contacting step occurs in vivo in a subject in need thereof. In such aspects, the method can include administering the polypeptide to the subject prior to the contacting step. In some aspects, the contacting step occurs ex vivo.

[0039] In some aspects, the contacting increases neurite outgrowth in the neuron 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 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, or at least about 50-fold compared to neurite outgrowth in a corresponding neuron not contacted with any polypeptide, molecule, nucleic acid, vector, cell, protein conjugate, or composition described herein. In some aspects, the contacting increases synaptogenesis in the neuron 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 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, or at least about 50-fold compared to synaptogenesis in a corresponding neuron not contacted with any polypeptide, molecule, nucleic acid, vector, cell, protein conjugate, or composition described herein.

[0040] In some aspects, the increased neurite outgrowth and / or synaptogenesis reduces one or more symptoms associated with a disease or condition selected from amyotrophic lateral sclerosis (ALS), Alzheimer's disease, glaucoma, diabetic retinopathy, neuropathic pain, spinal cord injury, traumatic brain injury, stroke, Parkinson's disease, or a combination thereof.

[0041] The invention provides a method of inhibiting or reducing complex formation between a FAM19A5 protein and a LRRC4 protein family member in a subject in need thereof, comprising administering to the subject any of the polypeptides, molecules, nucleic acids, vectors, cells, protein conjugates, or compositions described herein.

[0042] In some aspects, complex formation between the FAM19A5 protein and the LRRC4 protein family member 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 at least about 100% after administration. In some aspects, the reduction in complex formation between the FAM19A5 protein and the LRRC4 protein family member increases the activity of the LRRC4 protein family member in the subject. In some aspects, the reduction in complex formation between the FAM19A5 protein and the LRRC4 protein family member reduces one or more symptoms associated with a disease or condition selected from amyotrophic lateral sclerosis (ALS), Alzheimer's disease, glaucoma, diabetic retinopathy, neuropathic pain, spinal cord injury, traumatic brain injury, stroke, Parkinson's disease, or a combination thereof.

[0043] The present invention also provides a method of treating a disease or condition in a subject in need thereof comprising the step of administering to the subject any one of the polypeptides, molecules, nucleic acids, vectors, cells, protein conjugates or compositions described herein, wherein the disease or condition is selected from amyotrophic lateral sclerosis (ALS), Alzheimer's disease, glaucoma, diabetic retinopathy, neuropathic pain, spinal cord injury, traumatic brain injury, stroke, Parkinson's disease, or a combination thereof.

[0044] BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A-1D show the activity of different members of the LRRC4 protein family (i.e., LRRC4C, LRRC4, and LRRC4B proteins) binding to FAM19A5 protein as measured using co-immunoprecipitation (Figures 1A and 1B) or immunofluorescence assay (Figures 1C and 1D).

[0045] In Figure 1A, cell lysates (from HEK293 cells expressing FLAG-tagged LRRC4C, LRRC4, or LRRC4B proteins and treated with recombinant FAM19A5 protein) were immunoprecipitated with anti-FLAG antibody, and the immunoprecipitated proteins were immunoblotted with anti-FLAG (top row) and anti-FAM19A5(3-2) (bottom row) antibodies. In Figure 1B, cell lysates (from HEK293 cells expressing FLAG-tagged LRRC4B proteins and treated with recombinant FAM19A5 protein) were immunoprecipitated with human IgG ("IgG") or anti-FAM19A5(1-65) antibodies ("1-65"). The immunoprecipitated proteins were immunoblotted with anti-FLAG (top row) and anti-FAM19A5(3-2) (bottom row) antibodies. In Fig. 1C, HEK293 cells expressing FLAG-tagged LRRC4B protein were treated with recombinant FAM19A5 protein and immunostained with anti-FLAG and anti-FAM19A5(3-2) antibodies. In Fig. 1D, primary cortical neurons were treated with recombinant FAM19A5 protein and immunostained with anti-FAM19A5(3-2) and anti-LRRC4B antibodies. In Fig. 1C and Fig. 1D, nuclei were stained with Hoechst33342. Furthermore, the images provided in the second (FAM19A5 protein staining alone), third (LRRC4B protein staining alone), and fourth (overlay of FAM19A5 and LRRC4B staining) columns of Fig. 1C and Fig. 1D are enlarged views of the boxed regions in the images provided in the first row. Colocalized signals are indicated by arrowheads. Scale bar = 30 μm.

[0046] Figures 2A-D show the binding of LRRC4B protein to isoform 1 and isoform 2 of FAM19A5 protein measured using immunofluorescence (Figures 2A and 2B) or co-immunoprecipitation assays (Figures 2C and 2D). Figure 2A provides immunofluorescence data showing the interaction between LRRC4B protein and FAM19A5 isoform 1. Figure 2B provides immunofluorescence data showing the interaction between LRRC4B protein and FAM19A5 isoform 2. In Figure 2C, cell lysates from co-transfected HEK293 cells were immunoprecipitated with anti-FLAG antibody and then immunoblotted with anti-FLAG (top row) and anti-FAM19A5(3-2) (bottom row) antibodies. In Figure 2D, cell lysates from co-transfected HEK293 cells were immunoprecipitated with the following antibodies: (i) human IgG antibody ("IgG"); (ii) anti-FAM19A5(1-65) antibody ("1-65"); or (iii) anti-FAM19A5(3-2) antibody ("3-2"). Immunoprecipitated proteins were immunoblotted with anti-FLAG (top row) and anti-FAM19A5(3-2) (bottom row) antibodies.

[0047] 3A and 3B show binding of different LRRC4B protein deletion constructs to FAM19A5 protein. FIG. 3A provides a schematic diagram of different domains of LRRC4B protein and shows the domains included in the different deletion constructs. The LRRC4B domains shown include: "SP" = signal peptide; "LRR" = leucine-rich repeat; "IG" = immunoglobulin-like C2 type; "Thr" = threonine-rich; "TM" = transmembrane; and "PB" = PSD95 binding. The column under "Binding" indicates whether a particular LRRC4B protein fragment bound to FAM19A5 protein: "O" = bound; "X" = not bound; "ND" = not confirmed. FIG. 3B shows binding of different LRRC4B protein deletion constructs to FAM19A5 protein as measured using a co-immunoprecipitation assay.

[0048] Figures 4A and 4B show the binding of FAM19A5 protein to the ectodomain of LRRC4 protein family members as measured using ELISA. Figure 4A provides data showing the binding of FAM19A5 protein to the full-length ectodomain of LRRC4 (amino acids 39-527 of SEQ ID NO:1; i.e., SEQ ID NO:4) ("1"), LRRC4B (amino acids 36-527 of SEQ ID NO:2; i.e., SEQ ID NO:5) ("2") and LRRC4C (amino acids 45-527 of SEQ ID NO:3; i.e., SEQ ID NO:6) ("3") proteins. Figure 4B provides the following data showing binding of FAM19A5 protein to different fragments of LRRC4B protein: (a) amino acids 453-576 of SEQ ID NO:2 (i.e., SEQ ID NO:7); (b) amino acids 484-576 of SEQ ID NO:2 (i.e., SEQ ID NO:8); (c) amino acids 482-576 of SEQ ID NO:2 (i.e., SEQ ID NO:9); (d) amino acids 482-497 of SEQ ID NO:2 (i.e., SEQ ID NO:10); and (e) amino acids 498-576 of SEQ ID NO:2 (i.e., SEQ ID NO:11).

[0049] 5A and 5B show binding of FAM19A5 protein to the following protein fragments of LRRC4 protein family members: (1) LRRC4 (amino acids 451-483 of SEQ ID NO:1) (i.e., SEQ ID NO:12); (2) LRRC4C (amino acids 451-484 of SEQ ID NO:3) (i.e., SEQ ID NO:13); and (3) LRRC4B (amino acids 484-522 of SEQ ID NO:2) (i.e., SEQ ID NO:14). FIG. 5A provides a schematic diagram of the distinct domains present within the LRRC4 protein family members, including the amino acid sequences of the protein fragments tested. The domains represented include: "SP" = signal peptide; "LRR" = leucine-rich repeats; "IG" = immunoglobulin-like C2 type; "Thr" = threonine-rich; "TM" = transmembrane; and "PB" = PSD95 binding. The column under "Binding" indicates whether a particular LRRC4B protein fragment bound to FAM19A5 protein: "O" = bound; "X" = not bound; "ND" = not confirmed. Figure 5B shows the interaction between FAM19A5 protein and different LRRC4 family protein fragments. Cell lysates were immunoprecipitated with anti-FLAG antibody, and the immunoprecipitated proteins were immunoblotted with anti-FLAG (upper gel) and anti-FAM19A5(3-2) (lower gel) antibodies.

[0050] FIG. 6 shows the activity of three different peptide fragments containing the YTYFTTVTVETLE (SEQ ID NO: 15) sequence of LRRC4B protein binding to FAM19A5 protein: (1) "FB-16" = 16 amino acids long (SEQ ID NO: 17); (2) "FB-20" = 20 amino acids long (SEQ ID NO: 18); and (3) "FB-28" = 28 amino acids long (SEQ ID NO: 19).

[0051] FIG. 7 shows the activity of the LRRC4B peptide fragment (amino acids 453-576 of SEQ ID NO:2) (i.e., SEQ ID NO:7) (bottom row) in inducing dissociation of the interaction between FAM19A5 (isoform 2) and full-length LRRC4B protein in HEK293 cells as measured using immunofluorescence microscopy. HEK293 cells treated with a mutant form of the LRRC4B peptide fragment (containing alanine substitutions at positions 488 and 489 of SEQ ID NO:2) (i.e., SEQ ID NO:16) ("MT") were used as a control. The boxed images (bottom row, see fourth box from the left) were enlarged as images stained with anti-hIgG alone (top row) and both anti-hIgG and anti-FLAG antibodies (bottom row). The filled arrowheads in the enlarged images indicate the FAM19A5 signal dissociated from LRRC4B. The unfilled arrowheads indicate LRRC4B(453-576)-hFc, where LRRC4B is present. Scale bar = 30 μm.

[0052] 8A and 8B provide competitive inhibition assay data comparing the ability of different LRRC4B peptide fragments to inhibit binding of FAM19A5 protein to the full-length ectodomain of LRRC4B protein (i.e., amino acids 36-576 of SEQ ID NO:2) (SEQ ID NO:5). Figure 8A provides data for the following LRRC4B peptide fragments: (1) LRRC4B (amino acids 453-576 of SEQ ID NO:2) (SEQ ID NO:7); (2) LRRC4B mutant (amino acids 453-576 of SEQ ID NO:2 with AA mutations at positions 488 and 489) (SEQ ID NO:16); (3) LRRC4B (amino acids 484-576 of SEQ ID NO:2) (SEQ ID NO:8); (4) LRRC4B (amino acids 482-576 of SEQ ID NO:2) (SEQ ID NO:9); (5) LRRC4B (amino acids 482-497 of SEQ ID NO:2) (SEQ ID NO:10); and (6) LRRC4B (amino acids 498-576 of SEQ ID NO:1) (SEQ ID NO:11). FIG. 8B provides competitive inhibition assay data showing the activity of (1) FB-28, (2) FB-20, and (3) FB-16 peptides (described in FIG. 6) in inhibiting the binding of FAM19A5 protein to the full-length ectodomain of LRRC4B protein.

[0053] Figures 9A-9C compare the activity of different LRRC4B peptide fragments in inhibiting the binding of FAM19A5 protein to the full-length ectodomain of different members of the LRRC4 protein family: LRRC4 (amino acid residues 39-572 of SEQ ID NO:1) (i.e., SEQ ID NO:4), LRRC4B (amino acid residues 36-576 of SEQ ID NO:2) (i.e., SEQ ID NO:5), and LRRC4C (amino acid residues 345-527 of SEQ ID NO:6). The distinct LRRC4B peptide fragments presented include: (1) LRRC4B (amino acids 453-576 of SEQ ID NO:2) (SEQ ID NO:7); (2) LRRC4B mutant (amino acids 453-576 of SEQ ID NO:2 with AA mutations at positions 488 and 489) (i.e., SEQ ID NO:16); and (3) FB-20 (i.e., a 20-amino acid long peptide fragment containing the YTYFTTVTVETLE sequence of the LRRC4B protein; GYTYFTTVTVETLETQPGEE; SEQ ID NO:18).

[0054] 10A and 10B compare the activity of the FBC4-23 and FBC4C-23 peptide fragments in inhibiting binding of FAM19A5 protein to the full-length ectodomain of LRRC4B protein (FIG. 10A) or to the threonine-rich domain of LRRC4B protein (i.e., amino acids 453-576 of SEQ ID NO:2; i.e., SEQ ID NO:7) (FIG. 10B). The FBC4C-23 peptide fragment contains the FAM19A5 binding domain of LRRC4C protein (bold and italics) and has the following sequence:

[0055] [ka]

[0056] The FBC4C-23 peptide fragment contains the FAM19A5 binding domain of the LRRC4C protein (bold and italicized) and has the following sequence:

[0057] [ka]

[0058] The FB-20 peptide (see FIG. 6) was also used for comparison purposes.

[0059] 11A and 11B show the activity of different FB-20 peptide fragment variants that inhibit binding of FAM19A5 protein to the full-length ectodomain of LRRC4B protein (FIG. 11A) or to an LRRC4B protein fragment containing the FAM19A5 binding domain (i.e., amino acids 453-576 of SEQ ID NO:2; SEQ ID NO:7) (FIG. 11B). The different FB-20 variants are: (1) FB-ml ldC, (2) FB-mlOdC, (3) FB-m9dC, (4) FB-m8dC, (5) FB-m7dC, (6) FB-m6dC, (7) FB-mlOdN, (8) FB-m9dN, (9) FB-m8dN, and (10) FB-m7dN. As described in Example 6, each FB-20 variant contained one or more amino acid deletions, YTYFTTVTVETLE (SEQ ID NO: 15), at the C-terminus or N-terminus of the LRRC4B protein domain capable of binding to the FAM19A5 protein. The specific amino acid sequences of the FB-20 variants are provided in Table 9.

[0060] 12A and 12B show the activity of different FB-20 peptide fragment variants with alanine (A) or asparagine (N) substitutions that inhibit FAM19A5 protein binding to the full-length ectodomain of LRRC4B protein (FIG. 12A) or to the LRRC4B protein fragment containing the FAM19A5 binding domain (i.e., amino acids 453-576 of SEQ ID NO:2; SEQ ID NO:7) (FIG. 12B). As described in Example 7, alanine or asparagine substitutions were independently introduced into the FB-20 peptide fragment at one of the amino acid residues in the LRRC4B protein domain capable of binding to FAM19A5 protein, i.e., YTYFTTVTVETLE (SEQ ID NO:15). The specific amino acid sequences of the FB-20 variants are provided in Table 10. For each FB-20 peptide variant in the figure (excluding FB-20[12-L] and FB-20[13-E]), the first bar is the alanine substitution and the second bar is the asparagine substitution. Only alanine substitutions are displayed in mutants FB-20[12-L] and FB-20[13-E].

[0061] Figures 13A and 13B show the transcription levels of FAM19A5 family members (Figure 13A) or LRRC4B and PTPRF genes (Figure 13B) in mouse hippocampal cultures. Primary hippocampal neurons from mouse brains were cultured in vitro for 15 days at postnatal day 1 as described in Example 8. Transcription levels of distinct genes were measured 1, 3, 7, 10 and 15 days after initial culture and quantified using RNA-seq analysis. In Figure 13A, for each day indicated, the first, second and third bars (from left to right) correspond to FAM19A1, FAM19A2 and FAM19A5, respectively. FAM19A3 and FAM19A4 transcripts were not detected. Data are mean ± SEM of triplicates.

[0062] 14A-14D show the activity of LRRC4B peptide fragments (amino acid residues 453-576 of SEQ ID NO:2; SEQ ID NO:7) in promoting neurite outgrowth of mouse primary cortical neurons in vitro at various concentrations (x-axis) (0.006-60 nM). Mouse primary cortical neurons (postnatal day 1) were treated with LRRC4B protein fragments 1 and 2 days after initial culture as described in Example 8, and the following were immunostained with beta-tubulin III antibody and quantified at day 3: (i) mean total neurite outgrowth (FIG. 14A), (ii) number of primary dendrites (FIG. 14B), (iii) number of branch points (FIG. 14C), and (iv) number of secondary neurites (FIG. 14D). Data are presented as mean±SEM. Statistical significance was assessed using one-way ANOVA with Bonferroni post-hoc test; a, P<0.01 vs. vehicle control group.

[0063] 15A-C show the efficacy of LRRC4B peptide fragment (amino acid residues 453-576 of SEQ ID NO:2; SEQ ID NO:7) on the expression of synaptophysin (SYP; presynaptic marker) and PSD95 (postsynaptic marker) in mouse hippocampal neurons. FIG. 15A and FIG. 15B show the total fluorescence intensity for SYN and PSD-95, respectively, in dendrites / neurites of hippocampal neurons with LRRC4B peptide fragment (6 or 60 nM) as measured using IMARIS software (IMARIS 9.0 Bitplane, Switzerland). FIG. 15C shows the number of colocalized voxels between SYP and PSD95 signals in dendrites / neurites of treated hippocampal neurons. In each of Figures 15A-C, vehicle ("Veh") and LRRC4B peptide fragment mutant (MT) (60 nM) (i.e., containing alanine substitutions at positions 488 and 489 of SEQ ID NO:2; SEQ ID NO:16) were used as controls. As described elsewhere herein, LRRC4B MT was unable to bind to FAM19A5 protein. Data are presented as mean ± SEM. The number of neurons used for quantifying the fluorescence intensity is indicated in brackets in the bar graphs. Statistical significance was assessed using one-way ANOVA with Bonferroni post-hoc test. a, P<0.05 vs Veh; b, P<0.05 vs LRRC4B MT (60 nM).

[0064] 16A-16C show the ability of the LRRC4B peptide fragment (amino acid residues 453-576 of SEQ ID NO:2; "WT") (i.e., SEQ ID NO:7) to promote synapse formation in the hippocampal CA1 of APP / PS1 mice. As also described in Example 8, APP / PS1 mice were treated with the LRRC4B peptide fragment (30 mg / kg; intravenous administration) for 4 consecutive weeks, and then synapse formation was assessed by fluorescence microscopy using antibodies against SYP and PSD95. Control animals were either untreated ("cont") or treated with a mutant LRRC4B peptide fragment (60 nM) (i.e., containing alanine substitutions at positions 488 and 489 of SEQ ID NO:2; SEQ ID NO:16). FIG. 16A provides representative fluorescence micrographs. FIG. 16B and FIG. 16C show SYP and PSD95 intensity, respectively.

[0065] Figures 17A-C show the activity of the LRRC4B peptide fragment (amino acid residues 453-576 of SEQ ID NO:2; "WT"; SEQ ID NO:7) in promoting synaptogenesis in the hippocampal CA3 of APP / PS1 mice. The animals were treated and analyzed as described in Figures 16A-C. Figure 17A provides representative fluorescent micrographs. Figures 17B and 17C show SYP and PSD95 intensity, respectively.

[0066] Figures 18A-E show neurite outgrowth in mouse primary cortical neurons treated in vitro with FB-16, FB-20, and FB-28 peptides (described in Figure 6). Primary cortical neurons were treated for 2 days, and neurite outgrowth was assessed on day 3 by immunostaining with anti-beta-tubulin III antibody. Figure 18A provides representative microscopy images from each treatment group. Figures 18B-E show (i) the average length of total neurite outgrowth, (ii) the number of primary dendrites, (iii) the number of branch points, and (iv) the number of secondary neurites, respectively. Data are presented as mean ± SEM. Statistical significance was assessed using one-way analysis of variance and Bonferroni post-hoc test; a, P<0.01 vs control group (CTRL).

[0067] Figures 19A-C show increased expression of synaptophysin (SYP; presynaptic marker) and PSD95 (postsynaptic marker) in mouse primary hippocampal neurons treated with FB-16, FB-20 and FB-28 peptides in vitro (described in Figure 6). Figures 19A and 19B show total fluorescence intensity for SYN and PSD-95, respectively, in dendrites / neurites of hippocampal neurons measured using IMARIS software (IMARIS 9.0 Bitplane, Switzerland). Figure 19C shows the number of colocalized voxels between SYP and PSD95 signals in dendrites / neurites of treated hippocampal neurons. Data are presented as mean ± SEM. The number of neurons used for quantification of fluorescence intensity is indicated in brackets of the bar graphs. Statistical significance was assessed using one-way analysis of variance and Bonferroni post-hoc test; *, P < 0.05 vs CTRL; **, P < 0.05 vs CTRL.

[0068] FIG. 20 provides sequences of domains of interest (i.e., capable of binding to FAM19A5 protein) in LRRC4 protein family members across different vertebrate species.

[0069] Figures 21A and 21B provide the effect of different amino acid modifications on the binding affinity of the LRRC4B fragment as assessed by in silico residue scanning of the FAM19A5-LRRC4 family complex using the Schrodinger platform. Figure 21A provides the predicted Gibbs free energy change upon alanine substitution at each amino acid residue of the FB-20 fragment (SEQ ID NO: 18). Figure 21B provides the predicted Gibbs free energy change for the top 20 FB-20 double mutants (containing amino acid substitutions at residues T12 and L13 of SEQ ID NO: 18) with improved affinity for the FAM19A5 protein. The sequences for the proposed FB-20 double mutants are provided in Example 9 (Table 12).

[0070] Figures 22A-C show the activity of different FB-21 peptide mutants binding to FAM19A5 protein. Figure 22A provides a comparison of the inhibitory effect of the following FB-21 peptide fragments on the interaction between hFc-fused hLRRC4B and rcFAM19A5, as confirmed by competitive inhibition assay: (1) wild-type FB-21 (SEQ ID NO: 143), (2) FB-21(P12Y13) (SEQ ID NO: 144), (3) FB-21(H12F13) (SEQ ID NO: 145), (4) FB-21(Q12R13) (SEQ ID NO: 146), (5) FB-21(W12Y13) (SEQ ID NO: 147), (6) FB-21(M12R13) (SEQ ID NO: 148), and (7) FB-21(I12F13) (SEQ ID NO: 149). FIG. 22B shows a comparison of the inhibitory effects of the following FB-21 peptide fragments on the interaction between HIS0TEV LRRC4B and rcFAM19A5 proteins, as determined by competitive inhibition assay: (1) FB-21 (wild-type) (SEQ ID NO: 143), (2) FB-21(W12Y13) (SEQ ID NO: 147), (3) FB-21(D12Y13) (SEQ ID NO: 131), (4) FB-21(F12F13) (SEQ ID NO: 132), (5) FB-21(H12Y13) (SEQ ID NO: 133), (6) FB-21(D12F13) (SEQ ID NO: 135), and (7) FB-21(D12I13) (SEQ ID NO: 136). FIG. 22C provides results for the following FB-21 peptide fragments containing D-amino acids at the amino and carboxyl termini and L-amino acids at all other residues: (1) d-type FB-21 ("dFB-21"), (2) a d-type FB-21 peptide with a juxtamembrane (JM) sequence ("dFB-JM-31"), (3) a d-type FB-21 peptide sequence with a BBB-penetrating sequence at each end ("dFB-BBB-39"), and (4) a d-type FB-21 mutant peptide with a DY alternation and an additional JM sequence ("dFB-DY-JM31").

[0071] Figure 22D provides the sequences of the different members of the LRRC4 family (i.e., LRRC4, LRRC4B and LRRC4C proteins). The following domains are boxed: (1) FAM19A5 binding domain ("FB"); (2) juxtamembrane domain ("JM"), and (3) transmembrane domain ("TM").

[0072] Figures 23A-D show the efficacy of different FB-21 peptide fragments described herein against amyloid beta-induced synapse loss in mouse primary neurons. Figure 23A provides representative images for PSD95 (top row), SYP (middle row), and merged (bottom row) of hippocampal neurons treated with FB-21, FB-13-JM, or FB-BBB-39 (all at 6.6 nM; see Figure 22C for a description of the different FB-21 peptide fragments tested). Nuclei of cells were stained with Hoechst (blue). Scale bar = 50 μm. Figure 23B provides a comparison of the number of colocalized voxels between SYP and PSD95 signal dendrites / neurites of hippocampal neurons treated with FB-21, FB-13-JM, or FB-BBB-39 (all at 6.6 nM). The number of colocalized voxels was calculated by IMARIS software (left panel, IMARIS 9.0 Bitplane, Switzerland). Figures 23C and 23D provide a comparison of the total fluorescence intensity for PSD95 and SYN, respectively, in dendrites / neurites of hippocampal neurons treated with FB-21, FB-13-JM or FB-BBB-39 (all 6.6 nM) as measured using IMARIS. Data are presented as mean ± SEM. Statistical significance was assessed using one-way ANOVA with Bonferroni post-hoc test; *, P<0.05, and *, P<0.01 vs NT.

[0073] FIG. 24A and FIG. 24B show the effect of exemplary FB-21 peptide fragments described herein (i.e., dFB-dWY-JM31 and dFB-DY-JM31) on promoting neurite outgrowth of primary mouse spinal motor neurons. FIG. 24A provides representative merged images of non-treated (NT) or FB-21 peptide fragment-treated spinal motor neurons immunostained with Tau-5 antibody. Neuronal cell bodies were stained and detected by Hoechst (blue). Scale bar=100 μm. FIG. 24B provides a quantitative comparison of the average total neurite length of primary spinal motor neurons from different treatment groups. Data are presented as mean±SEM. Statistical significance was assessed using one-way ANOVA with Bonferroni post-hoc test; *, P<0.01 vs NT.

[0074] Figures 25A and 25B show the efficacy of the FB-21 peptide variants described herein (dFB-dWY-JM31) against 6-OHDA-induced cell death in LUHMES cells. Figure 25A provides a quantitative comparison of luminescence expression after treatment with FB-21 peptide variants with or without 6-OHDA treatment. Figure 25B provides a quantitative comparison of luminescence expression after treatment with FB-21 peptide variants with 6-OHDA treatment. Data are presented as mean ± SEM. Statistical significance was assessed using one-way ANOVA with Bonferroni post-hoc test; *, P<0.01 vs NT.

[0075] Figures 26A and 26B show the efficacy of an exemplary FB-21 peptide variant (dFB-dDY-JM31) described herein in a chronic constriction injury (CCI) rat model. Figure 26A provides a comparison of paw withdrawal thresholds in response to mechanical alloynia at various time points after CCI induction in mice treated with vehicle control (circles) or FB-21 peptide variants (squares). Data are presented as mean ± SEM. Figure 26B provides a comparison of the overall area under the curve (AUC) for the data provided in Figure 26A. Statistical analysis for AUC was performed with a one-tailed unpaired t-test; *, p<0.05. FIG. 27 shows the efficacy of the FB-21 peptide variant described herein (dFB-dDY-JM31) on regulating retinal dysfunction and neural oscillations. Retinal conductance (electroretinogram, ERG) was recorded to measure the electrical signal emitted from the retina in response to a flash of light using a diabetic retinopathy mouse model (db / db). ERG amplitude of the b-wave measured between groups; heterogenous wild type (WT, db / +, black), DR control group (db / db, red) and dFB-dDY-JM31 treated DR (blue). Data are presented as mean ± SEM. Statistical analysis was performed by one-way ANOVA followed by Bonferroni multiple comparison test; ***, p<0.001, **, p<0.01.

[0076] Figures 28A and 28B show the efficacy of the FB-21 peptide variant (dFB-dWY-JM-31) described herein in a traumatic brain injury mouse model. Figure 28A provides representative Hoechst staining for each group. Figure 28B provides a quantitative comparison of lesion volumes based on the data provided in Figure 28A. Data are presented as mean ± SEM. Statistical analysis was performed with two-tailed unpaired t-test; ***, p<0.001.

[0077] [Mode for carrying out the invention] The present invention provides polypeptides (e.g., isolated polypeptides) capable of inhibiting, reducing and / or dissociating the binding between FAM19A5 protein and LRRC4 protein family members. Specifically, the present application is the first to demonstrate that FAM19A5 protein can bind to LRRC4 protein family members and inhibit the activity of LRRC4 protein family members. The polypeptides of the present invention comprise, consist of, or essentially consist of a domain of a LRRC4 protein family member (i.e., LRRC4, LRRC4B, or LRRC4C) capable of specifically binding to FAM19A5 protein. By inhibiting, reducing and / or dissociating the interaction between FAM19A5 and LRRC4 protein family members, the polypeptides of the present invention can restore the activity of endogenous LRRC4 protein family members. Additional aspects of the present invention are provided throughout the present application.

[0078] To facilitate understanding of the subject matter disclosed herein, a number of terms and phrases are defined. Additional definitions are provided throughout the detailed description.

[0079] I. Definition Throughout this specification, the term "a" or "an" entity means one or more of that entity. For example, "a polypeptide" is understood to refer to one or more polypeptides. Thus, the terms "a," "one or more," and "at least one" may be used interchangeably herein.

[0080] Also, "and / or" as used herein should be considered as a specific disclosure of each of the two specified features or components without the other. Thus, the term "and / or" as used herein in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A alone" and "B alone." Similarly, the term "and / or" as used in phrases such as "A, B and / or C" is intended to include 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 alone; B alone; and C alone.

[0081] Where an aspect is described herein with the term "comprising", it is understood that similar aspects described with the terms "consisting of" and / or "essentially consisting of" are also provided.

[0082] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art related to the present invention. For example, references such as The Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., Academic Press; and Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press provide those skilled in the art with a general dictionary of many of the terms used herein.

[0083] Units, prefixes, and symbols are expressed in SI (System International de Unites) accepted format. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, amino acid sequences are written from left to right in the amino to carboxy orientation. The headings provided herein are not intended to limit the various aspects of the invention, which may have been had by reference to the specification as a whole. Thus, the terms defined below are more fully defined by reference to the specification as a whole.

[0084] The term "about" is used herein to mean approximately, roughly, roughly, or in the region. 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 a numerical value above or below the set forth value, for example, by a variance of 10%, either up or down (which may be higher or lower).

[0085] The term "leucine-rich repeat containing 4 protein family" or "LRRC4 protein family (including derivatives)" refers to a family of proteins that have been described as master synaptic organizers, playing a role in various stages of neural circuit formation, including neuronal migration, neurite outgrowth, formation of synaptic contacts and functional assembly (see, e.g., Woo et al., Mol Cell Neurosci 42(1):1-10 (September 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 derivatives thereof). LRRC4 protein family members generally contain nine leucine-rich repeat (LRR) domains flanking the LRR N- and C-terminal ends (see FIG. 3A). Such LRR domains are known to interact with the fibronectin type III domain of presynaptic receptor protein tyrosine phosphatase (RPTP) proteins (e.g., Won et al., Mol Cells 2010;10:1111-1122, 2011). 41(7):622-630 (Jul. 2018). The LRR domain is followed by an immunoglobulin-like C2 type (IG) and a threonine (Thr)-rich domain, which together form the extracellular portion of the LRRC4 protein family members. Unlike the 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, the LRRC4 protein family members further contain a transmembrane (TM) domain and a postsynaptic density-binding (PB) domain at the C-terminus of the protein.

[0086] In humans, the gene encoding the LRRC4 protein is located on chromosome 7 (nucleotides 128,027,071-128,032,107 of GenBank Accession Number NC_000007.14; minus strand orientation). Synonyms for the LRRC4 protein are known, non-limiting examples 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 provided in Table 1 (below). The full length ectodomain of the LRRC4 protein corresponds to amino acid residues 39-527 of SEQ ID NO:1 (i.e., SEQ ID NO:4). Unless otherwise indicated, the term "LRRC4 protein (including synonyms thereof)" includes any variant or isoform of the LRRC4 protein that is naturally expressed by a cell.

[0087] [Table 1]

[0088] In humans, the gene encoding the LRRC4B protein is located on chromosome 19 (nucleotides 50,516,892-50,568,435 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 for the LRRC4B protein is 713 amino acids long and is provided in Table 2 (below). The full-length ectodomain of the LRRC4B protein corresponds to amino acid residues 36-576 of SEQ ID NO:2 (i.e., SEQ ID NO:5). Unless otherwise specified, the term "LRRC4B protein (including synonyms thereof)" includes any variant or isoform of the LRRC4B protein that is naturally expressed by a cell.

[0089] [Table 2]

[0090] In humans, the gene encoding the LRRC4C protein is located on chromosome 11 (nucleotides 40,107,066-41,460,419 of GenBank Accession Number NC_000011.10; minus strand orientation). Synonyms of the LRRC4C protein are known, and non-limiting examples include "NGL-1", "Netrin-G1 ligand" and "KIAA1580". The amino acid sequence for the LRRC4C protein is 640 amino acids in length and is provided in Table 3 (below). The full length ectodomain of the LRRC4C protein corresponds to amino acids 45-527 of SEQ ID NO:3 (i.e., SEQ ID NO:6). Unless otherwise indicated, the term "LRRC4C protein (including synonyms thereof)" includes any variant or isoform of the LRRC4C protein that is naturally expressed by a cell.

[0091] [Table 3]

[0092] As used herein, the term "FAM19A5 binding domain" refers to a segment / fragment of a LRRC4 protein family member capable of binding to the FAM19A5 protein. 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 expressed primarily in the brain and spinal cord. FAM19A5 is also known as "TAFA5" or "chemokine-like protein TAFA-5."

[0093] In humans, the gene encoding FAM19A5 is located on chromosome 22. There are multiple human FAM19A5 (UniProt:Q7Z5A7) isoforms that are said to be produced by alternative splicing. Isoform 1 (UniProt:Q7Z5A7-1) consists of 132 amino acids, isoform 2 (UniProt:Q7Z5A7-2) consists of 125 amino acids, and isoform 3 (UniProt:Q7Z5A7-3) consists of 53 amino acids. Human FAM19A5 protein is said to exist in membrane-bound and water-soluble (secreted) forms. Isoform 1 is considered to be a membrane protein with one transmembrane domain. Isoform 2, reported in the literature (Tang TY et al., Genomics 83(4):727-34(2004)) as a secreted protein (water-soluble), contains a signal peptide at amino acid positions 1-25. Isoform 1 is considered to be a membrane protein and is predicted based on EST data. Table 4 (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 an LRRC4 protein family member) can inhibit binding of FAM19A5 isoform 1, isoform 2, and / or isoform 3 to an LRRC4 protein family member.

[0094] [Table 4]

[0095] The term "endogenous" as 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 polypeptides of the present invention differ (structurally and / or functionally) from endogenous LRRC4 protein family members.

[0096] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., a polypeptide comprising a FAM19A5 binding domain) and its binding partner (e.g., a FAM19A5 protein). Unless otherwise specified, "binding affinity" as used herein refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair. The affinity of a molecule X (e.g., a polypeptide described herein that comprises a FAM19A5 binding domain of a LRRC4 protein family member) for its partner Y (e.g., FAM19A5) is generally measured by the dissociation constant (K D Affinity can be expressed as the equilibrium dissociation constant (K D ) and the equilibrium association constant (K A ), which may be measured and / or expressed in a number of ways known in the art, including but not limited to. D is k off / k on and expressed as molar concentration (M), whereas K A is k on / k off It is calculated as the quotient of k on means, for example, the binding rate constant of an antibody to an antigen, and k off means, for example, dissociation of an antibody against an antigen. on and k off may be measured by techniques known to those of skill in the art such as immunoassays (eg, enzyme-linked immunosorbent assay (ELISA)), BIACORE, or kinetic exclusion assay (KinExA).

[0097] As used herein, the terms "specifically bind," "specifically recognize," "specific binding," "selective binding," and "selectively bind" are similar terms and refer to the binding of a molecule (e.g., a polypeptide domain that contains FAM19A5 binding) to an antigen (e.g., a FAM19A5 protein), as would be understood by one of skill in the art. For example, a molecule that specifically binds to an antigen is generally capable of binding to other peptides or polypeptides with a lower affinity, as measured, for example, by immunoassays, BIACORE, KinExA3000 instruments (Sapidyne Instruments, Boise, ID), or other known assays. In some aspects, a molecule that specifically binds to an antigen exhibits a K A 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 The antigen binds to the antigen having the formula:

[0098] The term "antigen" as used herein means any natural or synthetic immunogenic substance, such as a protein, peptide or hapten. As is evident from the present invention, the antigen may be the FAM19A5 protein or a fragment thereof.

[0099] A polypeptide (e.g., as described herein) that "competes with another protein for binding to a target" refers to a polypeptide 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 proteins compete with each other for binding to a target, i.e., whether and to what extent a polypeptide described herein inhibits the binding of a naturally occurring member of the LRRC4 protein family to the FAM19A5 protein, may be determined using known competition experiments. In some aspects, a polypeptide described herein competes to inhibit the binding of a naturally occurring member of the LRRC4 protein family to the FAM19A5 protein 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%. Competitive assays may be performed as described herein or, for example, in the literature (Ed Harlow and David Lane, Cold Spring Harb Protoc; 2006; doi:10.1101 / pdb.prot4277 or Chapter 11 of "Using Antibodies" by Ed Harlow and David Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA).

[0100] Other competitive binding assays that can be used with the present invention include: solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see Stahli et al., Methods in Enzymology 9:242 (1983)); solid-phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614 (1986)); solid-phase direct label assay, solid-phase direct label sandwich assay (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid-phase direct label RIA using 1-125 label (see Morel et al., Mol. Immunol. 25(1):7 (1988)); solid-phase direct biotin-avidin EIA (see Cheung et al., J. Immunol. 25(1):7 (1988)); al., Virology 176:546 (1990)); and directly labeled RIA. (see Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)).

[0101] As used herein, the term "naturally occurring" or "naturally occurring" refers to the fact that an entity (e.g., a protein) can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that may be isolated from a natural source and has not been intentionally modified by man in a laboratory is naturally occurring. As further described elsewhere herein, the polypeptides useful in the present invention are not naturally occurring.

[0102] A "polypeptide" refers to a chain containing at least two contiguously linked amino acid residues, with no upper limit to the length of the chain. One or more amino acid residues of a protein may contain modifications such as, but not limited to, glycosylation, phosphorylation, or disulfide bond formation. A "protein" may include one or more polypeptides.

[0103] The term "nucleic acid" or "nucleic acid molecule" as used herein is intended to include DNA molecules and RNA molecules. A nucleic acid molecule may be single-stranded or double-stranded and may be cDNA.

[0104] The term "vector" as used herein is intended to mean a nucleic acid molecule capable of transporting additional nucleic acids to which it has been linked. 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, into which additional DNA segments can be ligated into the viral genome. Certain vectors (e.g., bacterial vectors having a bacterial origin of replication 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) may be integrated into the genome of the host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Certain vectors are also capable of directing the expression of genes to which they are operably 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 used herein, "plasmid" and "vector" may be used interchangeably as the plasmid is the most commonly used form of vector. However, other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions are also included.

[0105] The term "recombinant host cell" (or simply "host cell"), as used herein, is intended to refer to a cell that contains a nucleic acid that is not naturally present in the cell, and may be a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer to not only the particular subject cell, but also the progeny of such a cell. Because certain modifications may occur in successive generations due to mutation or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0106] As used herein, "administering" refers to the physical introduction of an agent (e.g., a polypeptide or molecule described herein) or a composition containing an agent to a subject using any of a variety of methods and delivery systems known to those of skill in the art. Non-limiting examples of administration routes available in the prior art include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal or other parenteral administration routes, such as injection or infusion. The term "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, intracheal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, as well as in vivo electroporation. Alternatively, the polypeptides or molecules described herein may be administered parenterally, such as via topical, epidermal or mucosal routes of administration, e.g., intranasally, orally, vaginally, rectally, sublingually or topically. Administration may also be, for example, once, multiple times and / or over one or more extended periods of time.

[0107] As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, etc.

[0108] As used herein, the term "neuron" includes electrically excitable cells that process and transmit information by electrical and chemical signals. Neurons are the primary components of the brain and spinal cord of the CNS and the ganglia of the peripheral nervous system (PNS) and can be connected to each other to form a neuronal network. A typical neuron is composed of a cell body (soma), dendrites, and an axon. The soma of a neuron contains the nucleus. The dendrites of a neuron are cell extensions with numerous branches from which most input to the neuron originates. Axons are finer cable-like processes that extend from the cell body and transmit neural signals away from the cell body and transmit certain types of information back to the cell body.

[0109] As used herein, the term "therapeutically effective amount" refers to an amount of a substance (e.g., a polypeptide or molecule described herein) that is effective, alone or in combination with other therapeutic agents, to "treat" a disease or disorder, which reduces or alleviates the risk, latency, likelihood or occurrence of a disease or disorder (e.g., a neurological disease described herein). A "therapeutically effective amount" includes an amount of a substance or therapeutic agent that provides some improvement or benefit to a subject having or at risk of having a disease or disorder (e.g., a neurological disease described herein). Thus, a "therapeutically effective amount" is an amount that reduces the risk, latency, likelihood or occurrence of a disease, provides a reduction or partial reduction of a disorder, reduces at least one indicator, or reduces at least one clinical symptom of a disease or disorder.

[0110] II. Polypeptides The present invention provides polypeptides (e.g., isolated polypeptides) capable of inhibiting, reducing and / or dissociating the interaction between FAM19A5 protein and LRRC4 protein family members. As demonstrated for the first time herein (see, e.g., Example 1), FAM19A5 protein exhibits high binding affinity to all members of the LRRC4 protein family. The present invention further demonstrates that a specific domain of an LRRC4 protein family member is primarily responsible for binding to FAM19A5 protein. Thus, in some aspects, the polypeptides described herein comprise, consist of, or consist essentially of a domain of an LRRC4 protein family member, said domain being capable of binding to FAM19A5 protein (also referred to as a "FAM19A5 binding domain"). In some aspects, the polypeptide comprises a FAM19A5 binding domain. In some aspects, the polypeptide consists of a FAM19A5 binding domain. In some aspects, the polypeptide consists essentially of a FAM19A5 binding domain.

[0111] As will be apparent from the present invention, the polypeptides described herein include one or more features that differ (structurally and / or functionally) from naturally occurring members of the LRRC4 protein family. For example, in some aspects, the polypeptides include 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, for example, increasing the stability and / or binding affinity of the polypeptide to the FAM19A5 protein. In some aspects, the polypeptides of the invention include a FAM19A5 binding domain, but lack one or more other domains of the LRRC4 protein family member. For example, in some aspects, the polypeptides described herein include a FAM19A5 binding domain, but do not include a transmembrane domain of the LRRC4 protein family member. In some aspects, the polypeptides include a FAM19A5 binding domain, but do not include an intracellular domain (e.g., a postsynaptic density binding (PB) domain) of the LRRC4 protein family member. In some aspects, the polypeptide comprises a FAM19A5 binding domain, but does not comprise all of the transmembrane and intracellular domains. Thus, in some aspects, the polypeptides described herein are shorter than naturally occurring LRRC4 protein family members. Also, when performing biological activities (e.g., during neural circuit formation), each member of the LRRC4 protein family (LRRC4, LRRC4B, and LRRC4C) interacts with a ligand (netrin-G2, receptor tyrosine phosphatase LAR, and netrin-G1) (see, e.g., Li et al., Mol Cancer 13:266 (Dec. 2014)). Because the polypeptides of the present invention do not comprise all of the domains of the LRRC4 protein family members, 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 LRRC4 protein family members.Instead, in some aspects, by inhibiting, reducing and / or dissociating the interaction between FAM19A5 and members of the LRRC4 protein family, the polypeptides of the invention may free endogenous LRRC4 family proteins to carry out their native biological functions.

[0112] As described herein, the polypeptide of the present invention comprises at least the FAM19A5 binding domain of the LRRC4 protein family member. Unless otherwise specified, the total length of the FAM19A5 binding domain is not particularly limited, as long as the domain can bind 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 about 10 to 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, or about 23 amino acids in length. In some aspects, the FAM19A5 binding domain of the LRRC4 protein family member is about 10 amino acids in length.

[0113] In some aspects, the FAM19A5 binding domain of the polypeptides described herein comprises an amino acid sequence (N-terminus to C-terminus) having the following formula: A-(T / S)-B (Formula I) (SEQ ID NO:25): wherein (i) "A" includes X1-(T / S)-(Y / F)-F-X5, and (ii) "B" includes (V / I)-TV-(E / V); 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); (E / V) is glutamic acid (E) or valine (V).

[0114] In some aspects, the FAM19A5 binding domain of the polypeptides described herein comprises an amino acid sequence (N-terminus to C-terminus) having the following formula: A-(T / S)-B (Formula I) (SEQ ID NO:26): where (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; (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); X10 is glutamic acid (E), aspartic acid (D), isoleucine (I), tyrosine (Y), phenylalanine (F), methionine (M), or tryptophan (W).

[0115] In some aspects, a polypeptide described herein (e.g., comprising a FAM19A5 binding domain of a LRRC4 protein family member) comprises an amino acid sequence (N-terminus to C-terminus) having the following formula: X1-X2-X3-F-X5-T-X7-TV-X10 (Formula II) (SEQ ID NO: 27): 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; 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.

[0116] In some aspects, a polypeptide of the invention comprises an amino acid sequence (N-terminus to C-terminus) of the following formula: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10 (Formula III) (SEQ ID NO: 28): wherein XI 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 a FAM19A5 protein, thereby inhibiting, reducing and / or dissociating the interaction between the FAM19A5 protein and a member of the LRRC4 protein family.

[0117] For any of the above polypeptides, 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)-(x). 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.

[0118] In some aspects, the polypeptides described herein comprise a FAM19A5 binding domain of a LRRC4 protein family member, the FAM19A5 binding domain comprising the amino acid sequence set forth as SEQ ID NO:29 (YTYFTTVTVE) with 1, 2, 3, 4, 5, or 6 amino acids that differ (e.g., substituted) from each other in the amino acid sequence. In some aspects, the polypeptides described herein comprise a FAM19A5 binding domain of a LRRC4 protein family member, the FAM19A5 binding domain comprising the amino acid sequence set forth as SEQ ID NO:29 (YTYFTTVTVE) with 1, 2, 3, 4, 5, or 6 amino acids that differ (e.g., substituted) from each other in the amino acid sequence. In some aspects, the polypeptides described herein comprise a FAM19A5 binding domain of a LRRC4 protein family member, wherein the FAM19A5 binding domain essentially consists of the amino acid sequence set forth in SEQ ID NO:29 (YTYFTTVTVE), with 1, 2, 3, 4, 5, or 6 amino acids that differ (e.g., are substituted) from each other in the amino acid sequence.

[0119] In some aspects, a polypeptide of the invention comprises a FAM19A5 binding domain, which comprises the amino acid sequence set forth as SEQ ID NO:29 (YTYFTTVTVE). In some aspects, the FAM19A5 binding domain consists essentially of the amino acid sequence set forth as SEQ ID NO:29 (YTYFTTVTVE). As determined herein, the amino acid sequence set forth as SEQ ID NO:29 (YTYFTTVTVE) corresponds to the FAM19A5 binding domain of the LRRC4B protein.

[0120] In some aspects, the polypeptides described herein comprise a FAM19A5 binding domain, which comprises the amino acid sequence set forth as SEQ ID NO:30 (YSFFTTVTVE). In some aspects, the FAM19A5 binding domain consists essentially of the amino acid sequence set forth as SEQ ID NO:30 (YSFFTTVTVE). As determined herein, the amino acid sequence set forth as SEQ ID NO:30 (YSFFTTVTVE) corresponds to the FAM19A5 binding domain of the LRRC4 protein.

[0121] In some aspects, a polypeptide useful herein comprises a FAM19A5 binding domain, which comprises the amino acid sequence set forth as SEQ ID NO: 31 (FSYFSTVTVE). In some aspects, the FAM19A5 binding domain consists essentially of the amino acid sequence set forth as SEQ ID NO: 31 (FSYFSTVTVE). As determined herein, the amino acid sequence set forth as SEQ ID NO: 31 (FSYFSTVTVE) corresponds to the FAM19A5 binding domain of the LRRC4C protein.

[0122] As described herein, the FAM19A5 binding domain of LRRC4 protein family members is largely conserved among vertebrates (see, e.g., FIG. 20). Thus, without being bound by any one theory, one or more amino acid residues of the amino acid sequence set forth in any one of SEQ ID NOs: 29 (YTYFTTVTVE), 30 (YSFFTTVTVE) and 31 (FSYFSTVTVE) may be replaced with an amino acid that is present at that residue in another vertebrate. Examples of such replacements are provided elsewhere in the present invention (see, e.g., FIG. 20).

[0123] Additionally, in some aspects, one or more amino acid residues of the amino acid sequence set forth in any one of SEQ ID NOs: 29 (YTYFTTVTVE), 30 (YSFFTTVTVE), and 31 (FSYFSTVTVE) may be substituted with an amino acid that shares similar biochemical properties. For example, in the amino acid sequence set forth in SEQ ID NO: 29 (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 having a similar hydroxyl group (OH) in its side chain (e.g., S or Y). In some aspects, the Y at position 3 may be substituted with another amino acid having a common aromatic ring in its side chain that can participate in van der Waals interactions (e.g., F or W). 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 T at position 6 may be substituted with an amino acid such as S or Y. In some aspects, the V at position 7 may be substituted with another amino acid having a side chain with a large hydrophobic volume (e.g., I, Y, F, L, W, or M). In some aspects, the T at position 8 may be substituted with another amino acid such as S or Y. In some aspects, the V at position 9 may be substituted with I, Y, F, L, W, or M. In some aspects, the E at position 10 may be substituted with another amino acid having an acidic side chain (e.g., I, Y, F, M, or W).

[0124] In some aspects, a polypeptide of the invention comprises an amino acid sequence that is 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 set forth in SEQ ID NO:29 (YTYFTTVTVE), and the polypeptide is capable of binding to a FAM19A5 protein. In some aspects, the polypeptide is capable of inhibiting, reducing, and / or dissociating the interaction between a FAM19A5 protein and a member of the LRRC4 protein family.

[0125] In some aspects, a polypeptide of the present invention 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 set forth in SEQ ID NO:5, and the polypeptide is capable of binding to FAM19A5 protein, thereby inhibiting, reducing, and / or dissociating the interaction between FAM19A5 protein and the LRRC4 protein family. In some aspects, a polypeptide of the invention 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 set forth in SEQ ID NO:4, and the polypeptide is capable of binding to FAM19A5 protein, thereby inhibiting, reducing, and / or dissociating the interaction between FAM19A5 protein and the LRRC4 protein family. In some aspects, a polypeptide of the invention 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 set forth in SEQ ID NO:6, and the polypeptide is capable of binding to FAM19A5 protein, thereby inhibiting, reducing, and / or dissociating the interaction between FAM19A5 protein and the LRRC4 protein family.

[0126] As will be apparent from the present invention, in some aspects, the polypeptides described herein (e.g., comprising a FAM19A5 binding domain of a LRRC4 protein family member) comprise one or more amino acid modifications. In some aspects, the one or more amino acid modifications can increase the binding affinity of the polypeptide to the FAM19A5 protein. Thus, in some aspects, the binding affinity of the polypeptides described herein to the 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, relative to a control (e.g., corresponding to a naturally occurring LRRC4 protein family member or no amino acid modifications). In some aspects, the one or more amino acid modifications can increase the stability of the polypeptide. Thus, in some aspects, the stability of a polypeptide 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 control (e.g., a polypeptide having no amino acid modifications or corresponding to a naturally occurring member of the LRRC4 protein family).

[0127] In some aspects, one or more amino acid modifications can improve the activity of the polypeptides described herein that inhibit the interaction between the FAM19A5 protein and a member of the LRRC4 protein family (e.g., by increasing the degree of binding substitution and / or stability). Thus, in some aspects, the activity of the polypeptides described herein that 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 control (e.g., a naturally occurring member of the LRRC4 protein family that does not have an amino acid modification).

[0128] Non-limiting examples of amino acid modifications useful in the present invention are provided herein (see, e.g., Section III of the present invention). For example, in some aspects, the polypeptides described herein comprise one of the FAM19A5 binding domains of a member of the LRRC4 protein family, i.e., YTYFTTVTVE (SEQ ID NO:29), YSFFTTVTVE (SEQ ID NO:30), or FSYFSTVTVE (SEQ ID NO:31), 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, the polypeptides useful in the present 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. As identified herein (see, e.g., Example 9), in some aspects, one or more amino acids differ from the amino acid present at a particular residue in a naturally occurring LRRC4 protein family member.

[0129] For example, in some aspects, a polypeptide described herein comprises an amino acid sequence as presented as SEQ ID NO: 18 (GYTYFTTVTVETLETQPGEE) with one or more amino acid modifications (e.g., substitutions). In some aspects, a polypeptide described herein comprises an amino acid sequence as presented as SEQ ID NO: 18 (GYTYFTTVTVETLETQPGEE) with two amino acid modifications (e.g., substitutions). In some aspects, the amino acid modifications are at residues T12 and L13 of SEQ ID NO: 18. In some aspects, a polypeptide described herein comprises an amino acid sequence as presented as SEQ ID NO: 18 (GYTYFTTVTVETLETQPGEE) with one or more amino acid modifications (e.g., substitutions). In some aspects, a polypeptide described herein comprises an amino acid sequence as presented as SEQ ID NO: 18 (GYTYFTTVTVETLETQPGEE) with two amino acid modifications (e.g., substitutions). In some aspects, the amino acid modifications are at residues T12 and L13 of SEQ ID NO: 18. In some aspects, the polypeptides described herein consist essentially of the amino acid sequence set forth as SEQ ID NO: 18 (GYTYFTTVTVETLETQPGEE) with one or more amino acid modifications (e.g., substitutions). In some aspects, the polypeptides described herein consist essentially of the amino acid sequence set forth as SEQ ID NO: 18 (GYTYFTTVTVETLETQPGEE) with two amino acid modifications (e.g., substitutions). In some aspects, the amino acid modifications are at residues T12 and L13 of SEQ ID NO: 18.

[0130] In some aspects, the polypeptides described herein comprise the amino acid sequence presented as SEQ ID NO: 17 (GYTYFTTVTVETLETQ) with one or more amino acid modifications (e.g., substitutions). In some aspects, the polypeptides described herein comprise the amino acid sequence presented as SEQ ID NO: 17 (GYTYFTTVTVETLETQ) with two amino acid modifications (e.g., substitutions). In some aspects, the amino acid modifications are at residues T12 and L13 of SEQ ID NO: 17. In some aspects, the polypeptides described herein comprise the amino acid sequence presented as SEQ ID NO: 17 (GYTYFTTVTVETLETQ) with one or more amino acid modifications (e.g., substitutions). In some aspects, the polypeptides described herein comprise the amino acid sequence presented as SEQ ID NO: 17 (GYTYFTTVTVETLETQ) with two amino acid modifications (e.g., substitutions). In some aspects, the amino acid modifications are at residues T12 and L13 of SEQ ID NO: 17. In some aspects, the polypeptides described herein consist essentially of the amino acid sequence set forth as SEQ ID NO: 17 (GYTYFTTVTVETLETQ) with one or more amino acid modifications (e.g., substitutions). In some aspects, the polypeptides described herein consist essentially of the amino acid sequence set forth as SEQ ID NO: 17 (GYTYFTTVTVETLETQ) with two amino acid modifications (e.g., substitutions). In some aspects, the amino acid modifications are at residues T12 and L13 of SEQ ID NO: 17.

[0131] In some aspects, the polypeptides described herein comprise the amino acid sequence presented as SEQ ID NO: 19 (GYTYFTTVTVETLETQPGEKEPPGPTTD) with one or more amino acid modifications (e.g., substitutions). In some aspects, the polypeptides described herein comprise the amino acid sequence presented as SEQ ID NO: 19 (GYTYFTTVTVETLETQPGEKEPPGPTTD) with two amino acid modifications (e.g., substitutions). In some aspects, the amino acid modifications are at residues T12 and L13 of SEQ ID NO: 19. In some aspects, the polypeptides described herein comprise the amino acid sequence presented as SEQ ID NO: 19 (GYTYFTTVTVETLETQPGEKEPPGPTTD) with one or more amino acid modifications (e.g., substitutions). In some aspects, the polypeptides described herein comprise the amino acid sequence presented as SEQ ID NO: 19 (GYTYFTTVTVETLETQPGEKEPPGPTTD) with two amino acid modifications (e.g., substitutions). In some aspects, the amino acid modifications are at residues T12 and L13 of SEQ ID NO: 19. In some aspects, the polypeptides described herein consist essentially of the amino acid sequence set forth as SEQ ID NO: 19 (GYTYFTTVTVETLETQPGEKEPPGPTTD) with one or more amino acid modifications (e.g., substitutions). In some aspects, the polypeptides described herein consist essentially of the amino acid sequence set forth as SEQ ID NO: 19 (GYTYFTTVTVETLETQPGEKEPPGPTTD) with two amino acid modifications (e.g., substitutions). In some aspects, the amino acid modifications are at residues T12 and L13 of SEQ ID NO: 19.

[0132] In some aspects, the polypeptides described herein comprise the amino acid sequence presented as SEQ ID NO: 143 (GYTYFTTVTVETLETQPGEEA) with one or more amino acid modifications (e.g., substitutions). In some aspects, the polypeptides described herein comprise the amino acid sequence presented as SEQ ID NO: 143 (GYTYFTTVTVETLETQPGEEA) with two amino acid modifications (e.g., substitutions). In some aspects, the amino acid modifications are at residues T12 and L13 of SEQ ID NO: 143. In some aspects, the polypeptides described herein comprise the amino acid sequence presented as SEQ ID NO: 143 (GYTYFTTVTVETLETQPGEEA) with one or more amino acid modifications (e.g., substitutions). In some aspects, the polypeptides described herein comprise the amino acid sequence presented as SEQ ID NO: 143 (GYTYFTTVTVETLETQPGEEA) with two amino acid modifications (e.g., substitutions). In some aspects, the amino acid modifications are at residues T12 and L13 of SEQ ID NO: 143. In some aspects, the polypeptides described herein consist essentially of the amino acid sequence set forth as SEQ ID NO: 143 (GYTYFTTVTVETLETQPGEEA) with one or more amino acid modifications (e.g., substitutions). In some aspects, the polypeptides described herein consist essentially of the amino acid sequence set forth as SEQ ID NO: 143 (GYTYFTTVTVETLETQPGEEA) with two amino acid modifications (e.g., substitutions). In some aspects, the amino acid modifications are at residues T12 and L13 of SEQ ID NO: 143.

[0133] In some aspects, a polypeptide described herein (e.g., comprising a FAM19A5 binding domain of LRRC4B) comprises the amino acid sequence GYTYFTTVTVEPYETQPGEE (SEQ ID NO: 123). In some aspects, a polypeptide described herein (e.g., comprising a FAM19A5 binding domain of LRRC4B) consists of the amino acid sequence GYTYFTTVTVEPYETQPGEE (SEQ ID NO: 123). In some aspects, a polypeptide described herein (e.g., comprising a FAM19A5 binding domain of LRRC4B) consists essentially of the amino acid sequence GYTYFTTVTVEPYETQPGEE (SEQ ID NO: 123).

[0134] In some aspects, a polypeptide described herein (e.g., comprising a FAM19A5 binding domain of LRRC4B) comprises the amino acid sequence GYTYFTTVTVEMRETQPGEE (SEQ ID NO: 124). In some aspects, a polypeptide described herein (e.g., comprising a FAM19A5 binding domain of LRRC4B) consists of the amino acid sequence GYTYFTTVTVEMRETQPGEE (SEQ ID NO: 124). In some aspects, a polypeptide described herein (e.g., comprising a FAM19A5 binding domain of LRRC4B) consists essentially of the amino acid sequence GYTYFTTVTVEMRETQPGEE (SEQ ID NO: 124).

[0135] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) has the amino acid sequence

[0136] [ka]

[0137] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) comprises the amino acid sequence

[0138] [ka]

[0139] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) comprises the amino acid sequence

[0140] [ka]

[0141] It is mandatory to configure

[0142] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) has the amino acid sequence

[0143] [ka]

[0144] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) comprises the amino acid sequence

[0145] [ka]

[0146] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) comprises the amino acid sequence

[0147] [ka]

[0148] It is mandatory to configure

[0149] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) has the amino acid sequence

[0150] [ka]

[0151] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) comprises the amino acid sequence

[0152] [ka]

[0153] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) comprises the amino acid sequence

[0154] [ka]

[0155] It is mandatory to configure

[0156] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) has the amino acid sequence

[0157] [ka]

[0158] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) comprises the amino acid sequence

[0159] [ka]

[0160] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) comprises the amino acid sequence

[0161] [ka]

[0162] It is mandatory to configure

[0163] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) has the amino acid sequence

[0164] [ka]

[0165] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) comprises the amino acid sequence

[0166] [ka]

[0167] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) comprises the amino acid sequence

[0168] [ka]

[0169] It is mandatory to configure

[0170] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) has the amino acid sequence

[0171] [ka]

[0172] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) comprises the amino acid sequence

[0173] [ka]

[0174] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) comprises the amino acid sequence

[0175] [ka]

[0176] It is mandatory to configure

[0177] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) has the amino acid sequence

[0178] [ka]

[0179] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) comprises the amino acid sequence

[0180] [ka]

[0181] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) comprises the amino acid sequence

[0182] [ka]

[0183] It is mandatory to configure

[0184] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) has the amino acid sequence

[0185] [ka]

[0186] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) comprises the amino acid sequence

[0187] [ka]

[0188] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) comprises the amino acid sequence

[0189] [ka]

[0190] It is mandatory to configure

[0191] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) has the amino acid sequence

[0192] [ka]

[0193] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) comprises the amino acid sequence

[0194] [ka]

[0195] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) comprises the amino acid sequence

[0196] [ka]

[0197] It is mandatory to configure

[0198] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) has the amino acid sequence

[0199] [ka]

[0200] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) comprises the amino acid sequence

[0201] [ka]

[0202] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) comprises the amino acid sequence

[0203] [ka]

[0204] It is mandatory to configure

[0205] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) has the amino acid sequence

[0206] [ka]

[0207] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) comprises the amino acid sequence

[0208] [ka]

[0209] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) comprises the amino acid sequence

[0210] [ka]

[0211] It is mandatory to configure

[0212] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) has the amino acid sequence

[0213] [ka]

[0214] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) comprises the amino acid sequence

[0215] [ka]

[0216] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) comprises the amino acid sequence

[0217] [ka]

[0218] It is mandatory to configure

[0219] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) has the amino acid sequence

[0220] [ka]

[0221] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) comprises the amino acid sequence

[0222] [ka]

[0223] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) comprises the amino acid sequence

[0224] [ka]

[0225] It is mandatory to configure

[0226] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) has the amino acid sequence

[0227] [ka]

[0228] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) comprises the amino acid sequence

[0229] [ka]

[0230] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) comprises the amino acid sequence

[0231] [ka]

[0232] It is mandatory to configure

[0233] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) has the amino acid sequence

[0234] [ka]

[0235] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) comprises the amino acid sequence

[0236] [ka]

[0237] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) comprises the amino acid sequence

[0238] [ka]

[0239] It is mandatory to configure

[0240] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) has the amino acid sequence

[0241] [ka]

[0242] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) comprises the amino acid sequence

[0243] [ka]

[0244] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) comprises the amino acid sequence

[0245] [ka]

[0246] It is mandatory to configure

[0247] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) has the amino acid sequence

[0248] [ka]

[0249] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) comprises the amino acid sequence

[0250] [ka]

[0251] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) comprises the amino acid sequence

[0252] [ka]

[0253] It is mandatory to configure

[0254] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) has the amino acid sequence

[0255] [ka]

[0256] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) comprises the amino acid sequence

[0257] [ka]

[0258] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) comprises the amino acid sequence

[0259] [ka]

[0260] It is mandatory to configure

[0261] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) has the amino acid sequence

[0262] [ka]

[0263] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) comprises the amino acid sequence

[0264] [ka]

[0265] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) comprises the amino acid sequence

[0266] [ka]

[0267] It is mandatory to configure

[0268] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) has the amino acid sequence

[0269] [ka]

[0270] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) comprises the amino acid sequence

[0271] [ka]

[0272] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) comprises the amino acid sequence

[0273] [ka]

[0274] It is mandatory to configure

[0275] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) has the amino acid sequence

[0276] [ka]

[0277] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) comprises the amino acid sequence

[0278] [ka]

[0279] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) comprises the amino acid sequence

[0280] [ka]

[0281] It is mandatory to configure

[0282] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) has the amino acid sequence

[0283] [ka]

[0284] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) comprises the amino acid sequence

[0285] [ka]

[0286] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) comprises the amino acid sequence

[0287] [ka]

[0288] It is mandatory to configure

[0289] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) has the amino acid sequence

[0290] [ka]

[0291] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) comprises the amino acid sequence

[0292] [ka]

[0293] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) comprises the amino acid sequence

[0294] [ka]

[0295] It is mandatory to configure

[0296] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) has the amino acid sequence

[0297] [ka]

[0298] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) comprises the amino acid sequence

[0299] [ka]

[0300] In some aspects, a polypeptide described herein (e.g., comprising the FAM19A5 binding domain of LRRC4B) comprises the amino acid sequence

[0301] [ka]

[0302] It is mandatory to configure

[0303] Without being bound to any one theory, in some aspects, the polypeptides described herein may compete with naturally occurring members of the LRRC4 protein family. In some aspects, the polypeptides described herein exhibit one or more properties (e.g., increased binding affinity and / or stability) such that they may outcompete naturally occurring members of the LRRC4 protein family for binding to the FAM19A5 protein.

[0304] III. Molecules Also provided herein are molecules comprising any of the above described polypeptides (e.g., comprising a FAM19A5-binding domain of a LRRC4 protein family member). As used herein, the term "molecule" is not particularly limited, so long as the molecule maintains the activity of the polypeptide (e.g., inhibits, reduces and / or dissociates the interaction between the FAM19A5 protein and a member of the LRRC4 protein family). Non-limiting examples of molecules useful in the present invention include antibodies (or antigen-binding portions thereof), small molecules, peptides, proteins, or combinations thereof.

[0305] In some aspects, the molecules described herein comprise one or more moieties that may improve 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 molecules comprise (i) any of the polypeptides described herein, and (ii) one or more additional amino acids or compounds 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, the 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 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.

[0306] In some aspects, a molecule comprises (i) a polypeptide having an amino acid sequence listed as SEQ ID NO:29 (YTYFTTVTVE), and (ii) at least one additional amino acid at the N-terminus of the polypeptide. In some aspects, a molecule comprises (i) a polypeptide having an amino acid sequence listed as SEQ ID NO:29 (YTYFTTVTVE), and (ii) at least one additional amino acid at the C-terminus of the polypeptide. In some aspects, a molecule comprises (i) a polypeptide having an amino acid sequence listed as SEQ ID NO:29 (YTYFTTVTVE), and (ii) at least one additional amino acid at both the N-terminus and the C-terminus. In some aspects, a molecule useful in the invention comprises an amino acid sequence listed as SEQ ID NO:18 (GYTYFTTVTVETLETQPGEE). In some aspects, a molecule consists essentially of an amino acid sequence listed as SEQ ID NO:18 (GYTYFTTVTVETLETQPGEE). In some aspects, a molecule useful in the invention comprises the amino acid sequence presented as SEQ ID NO: 17 (GYTYFTTVTVETLETQ). In some aspects, a molecule consists of the amino acid sequence presented as SEQ ID NO: 17 (GYTYFTTVTVETLETQ). In some aspects, a molecule consists essentially of the amino acid sequence presented as SEQ ID NO: 17 (GYTYFTTVTVETLETQ). In some aspects, a molecule useful in the invention comprises the amino acid sequence presented as SEQ ID NO: 19 (GYTYFTTVTVETLETQPGEKEPPGPTTD). In some aspects, a molecule consists essentially of the amino acid sequence presented as SEQ ID NO: 19 (GYTYFTTVTVETLETQPGEKEPPGPTTD). In some aspects, a molecule consists essentially of the amino acid sequence presented as SEQ ID NO: 19 (GYTYFTTVTVETLETQPGEKEPPGPTTD).

[0307] In some aspects, a molecule useful in the invention comprises (i) a polypeptide having an amino acid sequence as provided in SEQ ID NO:30 (YSFFTTVTVE), and (ii) at least one additional amino acid at the N-terminus of the polypeptide. In some aspects, a molecule comprises (i) a polypeptide having an amino acid sequence as provided in SEQ ID NO:30 (YSFFTTVTVE), and (ii) at least one additional amino acid at the C-terminus of the polypeptide. In some aspects, a molecule comprises (i) a polypeptide having an amino acid sequence as provided in SEQ ID NO:30 (YSFFTTVTVE), and (ii) at least one additional amino acid at both the N-terminus and the C-terminus. In some aspects, a molecule useful in the invention comprises the amino acid sequence as provided in SEQ ID NO:20 (NYSFFTTVTVETTEISPEDTTRK). In some aspects, a molecule consists essentially of the amino acid sequence as provided in SEQ ID NO:20 (NYSFFTTVTVETTEISPEDTTRK).

[0308] In some aspects, a molecule useful in the invention comprises (i) a polypeptide having an amino acid sequence as provided in SEQ ID NO:31 (FSYFSTVTVE), and (ii) at least one additional amino acid at the N-terminus of the polypeptide. In some aspects, a molecule comprises (i) a polypeptide having an amino acid sequence as provided in SEQ ID NO:31 (FSYFSTVTVE), and (ii) at least one additional amino acid at the C-terminus of the polypeptide. In some aspects, a molecule comprises (i) a polypeptide having an amino acid sequence as provided in SEQ ID NO:31 (FSYFSTVTVE), and (ii) at least one additional amino acid at both the N-terminus and the C-terminus. In some aspects, a molecule useful in the invention comprises the amino acid sequence as provided in SEQ ID NO:21 (NFSYFSTVTVETMEPSQDERTTR). In some aspects, a molecule consists essentially of the amino acid sequence as provided in SEQ ID NO:21 (NFSYFSTVTVETMEPSQDERTTR).

[0309] In some aspects, a polypeptide of a molecule described herein comprises an amino acid sequence that is 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 set forth in SEQ ID NO:29 (YTYFTTVTVE), which is capable of binding to a FAM19A5 protein, and which 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.

[0310] In some aspects, a polypeptide of a molecule described herein comprises an amino acid sequence that is 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 set forth in SEQ ID NO:29 (YTYFTTVTVE), which is capable of binding to a FAM19A5 protein, and which 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 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 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 by a linker. In some aspects, the linker is a peptide linker.

[0311] In some aspects, the one or more additional amino acids added to the N-terminus and / or C-terminus may comprise 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 may comprise D-amino acids. Without being bound to any one theory, in some aspects, adding one or more D-amino acids to the N-terminus and / or C-terminus of a polypeptide can improve the durability of the 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 blood of a subject. Thus, as evidenced herein (see, e.g., Example 10), in some aspects, a polypeptide useful in the present invention may comprise both D-amino acids and L-amino acids. For example, in some aspects, a polypeptide described herein comprises a D-amino acid at the N-terminus and L-amino acids at all other amino acid residues. In some aspects, a polypeptide described herein comprises 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.

[0312] Further, as described herein, in some aspects, the above-mentioned molecules include polypeptides having the amino acid sequence set forth in any one of SEQ ID NOs: 29 (YTYFTTVTVE), 30 (YSFFTTVTVE), and 31 (FSYFSTVTVE), which have 1, 2, 3, 4, 5, or 6 amino acids that differ (e.g., are substituted) from said amino acid sequence.

[0313] In some aspects, the 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 combinations thereof. In some aspects, the polypeptide can be cyclized to increase stability. Such modifications can be made by any suitable method known in the art.

[0314] As described elsewhere herein, in some aspects, a molecule useful in the present invention comprises a FAM19A5 binding domain of a 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 to the FAM19A5 protein). As demonstrated herein (see, e.g., Example 10), Applicants have determined that the addition of a juxtra-membrane sequence of a LRRC4 protein family member can greatly improve the binding affinity of the molecule to the FAM19A5 protein. The juxtra-membrane sequence is highly conserved among LRRC4 family members and is provided as SEQ ID NO: 151 (LDEVMKTTK) (LRRC4 and LRRC4B) and SEQ ID NO: 152 (IDEVMKTTK) (LRRC4C) (see FIG. 22D).

[0315] Thus, in some aspects, the molecules described herein comprise the FAM19A5 binding domain of the LRRC4 protein (i.e., YSFFTTVTVE; SEQ ID NO:30) and the juxtamembrane sequence designated as SEQ ID NO:151 (LDEVMKTTK). In some aspects, the molecules described herein comprise the FAM19A5 binding domain of the LRRC4 protein (i.e., YSFFTTVTVE; SEQ ID NO:30) and the juxtamembrane sequence designated as SEQ ID NO:152 (IDEVMKTTK). In some aspects, the molecules described herein comprise the FAM19A5 binding domain of the LRRC4B protein (i.e., YTYFTTVTVE; SEQ ID NO:29) and the juxtamembrane sequence designated as SEQ ID NO:151 (LDEVMKTTK). In some aspects, the molecules described herein comprise the FAM19A5 binding domain of the LRRC4 protein (i.e., YTYFTTVTVE; SEQ ID NO:29) and the juxtamembrane sequence designated as SEQ ID NO:152 (IDEVMKTTK). In some aspects, the molecules described herein comprise the FAM19A5 binding domain of the LRRC4B protein (i.e., FSYFSTVTVE; SEQ ID NO:31) and the juxtamembrane sequence designated SEQ ID NO:151 (LDEVMKTTK). In some aspects, the molecules described herein comprise the FAM19A5 binding domain of the LRRC4 protein (i.e., FSYFSTVTVE; SEQ ID NO:31) and the juxtamembrane sequence designated SEQ ID NO:152 (IDEVMKTTK). In some aspects, the juxtamembrane is added to the C-terminus of the molecule.

[0316] As will be apparent from the present invention, any of the modifications described herein (e.g., amino acid substitutions, addition of juxtamembrane sequences, D-amino acids) to improve one or more properties of a molecule may be used in combination. For example, in some aspects, a molecule (e.g., a polypeptide) useful in the present invention comprises: (i) an amino acid sequence as set forth in SEQ ID NO: 18 (GYTYFTTVTVETLETQPGEE) with amino acid modifications at residues T12 and L13; and (ii) a juxtamembrane sequence at the C-terminus of the molecule (e.g., SEQ ID NO: 151 or SEQ ID NO: 152). In some aspects, a molecule (e.g., a polypeptide) useful in the present invention comprises: (i) an amino acid sequence as set forth in SEQ ID NO: 18 (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 juxtamembrane sequence at the C-terminus of the molecule (e.g., SEQ ID NO: 151 or SEQ ID NO: 152). In some aspects, a molecule (e.g., a polypeptide) useful in the invention comprises: (i) the amino acid sequence set forth in SEQ ID NO: 17 (GYTYFTTVTVETLETQ) with amino acid modifications at residues T12 and L13; and (ii) a juxtamembrane sequence at the C-terminus of the molecule (e.g., SEQ ID NO: 151 or SEQ ID NO: 152). In some aspects, a molecule (e.g., a polypeptide) useful in the invention comprises: (i) the amino acid sequence set forth in SEQ ID NO: 17 (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 juxtamembrane sequence at the C-terminus of the molecule (e.g., SEQ ID NO: 151 or SEQ ID NO: 152). In some aspects, a molecule (e.g., a polypeptide) useful in the invention comprises: (i) the amino acid sequence set forth in SEQ ID NO: 19 (GYTYFTTVTVETLETQPGEKEPPGPTTD) with amino acid modifications at residues T12 and L13; and (ii) a juxtamembrane sequence at the C-terminus of the molecule (e.g., SEQ ID NO: 151 or SEQ ID NO: 152).In some aspects, a molecule (e.g., a polypeptide) useful in the invention comprises: (i) the amino acid sequence provided as SEQ ID NO: 19 (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 juxtamembrane sequence at the C-terminus of the molecule (e.g., SEQ ID NO: 151 or SEQ ID NO: 152). In some aspects, a molecule (e.g., a polypeptide) useful in the invention comprises: (i) the amino acid sequence provided as SEQ ID NO: 143 (GYTYFTTVTVETLETQPGEEA) with amino acid modifications at residues T12 and L13; and (ii) a juxtamembrane sequence at the C-terminus of the molecule (e.g., SEQ ID NO: 151 or SEQ ID NO: 152). In some aspects, a molecule (e.g., a polypeptide) useful in the invention comprises: (i) the amino acid sequence set forth in SEQ ID NO: 143 (GYTYFTTVTVETLETQPGEEA) with amino acid modifications at residues T12 and L13; (ii) D-amino acids at the N-terminus and / or C-terminus; and (iii) a juxtamembrane sequence at the C-terminus of the molecule (e.g., SEQ ID NO: 151 or SEQ ID NO: 152).

[0317] In some aspects, the molecules described herein (e.g., including polypeptides comprising a FAM19A5 binding domain of a member of the LRRC4 family of proteins) may include one or more additional peptides that allow them to be specifically targeted to different tissues, for example, when administered to a subject. For example, in some aspects, the molecules described herein include a peptide that allows the molecule to penetrate across the blood-brain barrier (also referred to herein as the "BBB shuttle"). Examples of such BBB shuttles are known in the art. Non-limiting examples are provided in Table 5 (below) (see, e.g., Oller-Salvia et al., Chem Soc Rev 45:4690 (2016)).

[0318] [Table 5]

[0319] The nomenclature for cyclic peptides (&) is aligned with the three-letter amino acid code described in the literature (Spengler et al., J Pept Res 65:550-555 (2005)); [Dap] stands for diaminopropionic acid.

[0320] In some aspects, molecules useful in the present invention include fusion proteins. For example, in some aspects, the molecules described herein may include (i) any polypeptide of the present invention, and (ii) a half-life extending moiety. Any suitable half-life extending moiety known in the art can be used to generate the fusion proteins of the present invention. Non-limiting examples of such half-life extending moieties include: Fc, albumin, albumin binding polypeptide, Pro / Ala / Ser (PAS), C-terminal peptide of the beta subunit of human chorionic gonadotropin (CTP), polyethylene glycol (PEG), long unstructured hydrophilic sequences of amino acids (XTEN), hydroxyethyl starch (HES), albumin binding small molecules, or combinations thereof.

[0321] In some aspects, the molecules described herein (e.g., including any one of the polypeptides described herein that can inhibit, reduce, and / or dissociate the interaction of a FAM19A5 protein with a LRRC4 protein family member) include protein-drug conjugates. For example, in some aspects, the polypeptides may be conjugated to a therapeutic agent, such as a therapeutic agent useful for treating a disease or disorder.

[0322] The protein-drug conjugates described herein may be prepared by methods known in the art. In some aspects, the conjugation methods produce substantially (or nearly) non-immunogenic linkages, such as peptide- (i.e., amide-), sulfide-, (sterically hindered), disulfide-, hydrazone-, and ether linkages. These linkages are largely non-immunogenic and exhibit substantial stability in serum (see, e.g., Senter, PD, Curr. Opin. Chem. Biol. 13 (2009) 235-244; WO2009 / 059278; WO95 / 17886, the entire contents of which are incorporated herein by reference).

[0323] Depending on the biochemical nature of the moiety and the polypeptide, different conjugation strategies may 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 modifying naturally occurring amino acids with amino acids that have reactivity orthogonal to the reactivity of other functional groups present. For example, specific cysteines within rare sequence contexts may be enzymatically converted from aldehydes (see, e.g., Frese, MA, and Dierks, T., ChemBioChem. 10 (2009) 425-427). It is also possible to obtain specific amino acid modifications by using specific enzymatic reactivities of natural amino acids and specific enzymes in a given sequence (see, for example, Taki, M. et al., Prot. Eng. Des. Sel. 17 (2004) 119-126; Gautier, A. et al., Chem. Biol. 15 (2008) 128-136; and Protease-catalyzed formation of CN bond is used by Bordusa, F., Highlights in Bioorganic Chemistry (2004) 389-403).

[0324] Site-specific reactions and covalent couplings may also be achieved by selective reaction of the terminal amino acid with an appropriate modification reagent. The reactivity of N-terminal cysteines with benzonitrile may be used to achieve site-specific covalent bonds (see, e.g., Ren, H. et al., Angew. Chem. Int. Ed. Engl. 48 (2009) 9658-9662). Native chemical ligation may also rely on a C-terminal cysteine ​​residue (see, e.g., Taylor, E. Vogel; Imperiali, B, Nucleic Acids and Molecular Biology (2009), 22 (Protein Engineering), 65-96).

[0325] The moiety may also be a synthetic peptide or peptidomimetic. In this case, the polypeptide may be chemically synthesized and amino acids with orthogonal chemical reactivity may be integrated during such synthesis (see, for example, de Graaf, AJ et al., Bioconjug. Chem. 20 (2009) 1281-1295). To obtain a single labeled polypeptide, the conjugate with 1:1 stoichiometry may be separated from other conjugation by-products by chromatography. This procedure may be facilitated by dye-labeled binding pair members and charged linkers. By using such kind of labeled and highly negatively charged binding pair members, single binding polypeptides are easily separated from unlabeled polypeptides and polypeptides with one or more linkers, since the charge and molecular weight differences can be used for separation. Fluorescent dyes may be useful for purifying the conjugate from unbound components, such as labeled monovalent binding agents.

[0326] IV. Pharmaceutical Compositions The invention also provides compositions comprising a polypeptide described herein (e.g., comprising a FAM19A5-binding domain of an LRRC4 protein family member) (or a molecule, nucleic acid, vector, cell, protein conjugate described herein) having a predetermined 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 non-toxic to recipients at the volumes and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as 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. These include proteins such as albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN, PLURONICS, or polyethylene glycol (PEG).

[0327] In some aspects, pharmaceutical compositions useful in the present invention include any of the polypeptides, molecules, nucleic acids, vectors, cells, protein conjugates, and optionally one or more additional prophylactic or therapeutic agents described herein in a pharma- ceutically acceptable carrier. In some aspects, pharmaceutical compositions include any of the polypeptides, molecules, nucleic acids, vectors, cells, protein conjugates, and optionally one or more additional prophylactic or therapeutic agents described herein in a pharma- ceutically acceptable carrier. In some aspects, the polypeptides, molecules, nucleic acids, vectors, cells, protein conjugates described herein are the only active ingredients contained in the pharmaceutical composition. The pharmaceutical compositions described herein may be useful for inhibiting, reducing, and / or dissociating the interaction between FAM19A5 protein and LRRC4 protein family members. As described elsewhere herein, inhibiting, reducing, and / or dissociating the interaction between FAM19A5 protein and LRRC4 protein family members can improve neural circuit formation (e.g., by promoting neurite outgrowth and synapse formation).

[0328] Pharmaceutically acceptable carriers used in parenteral formulations include aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents, and other pharma- ceutically acceptable substances. Examples of aqueous vehicles include sodium chloride injection, infusion injection, isotonic dextrose injection, sterile water injection, dextrose and lactate infusion injection. Nonaqueous parenteral vehicles include vegetable fixed oils, cottonseed oil, corn oil, sesame oil, and peanut oil. Antimicrobial agents in bacteriostatic or fungistatic concentrations may be added to parenteral formulations packaged in multi-dose containers including phenol or cresol, mercurial, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride and benzethonium chloride. Isotonicity agents include sodium chloride and dextrose. Buffers include phosphates and citrates. Antioxidants include sodium bisulfate. Topical anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcellulose, hydroxypropyl methylcellulose and polyvinylpyrrolidone. Emulsifying agents include polysorbate 80 (TWEEN 80). Metal ion 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.

[0329] The pharmaceutical composition may be formulated for any route of administration to a subject. Specific examples of routes of administration include intranasal, oral, parenterally, intrathecally, intracerebroventricularly, pulmonary, subcutaneously, or intraventricularly. Parenteral administration, characterized by subcutaneous, intramuscular, or intravenous injection, is also contemplated herein. Injectables may be prepared in conventional forms, such as liquid solutions or suspensions, solid forms suitable for dissolving or suspending in liquid prior to injection, or emulsions. Injectables, solutions, and emulsions also contain one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol, or ethanol. In addition, if desired, the pharmaceutical compositions to be administered may also contain minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, and other formulating agents, such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, and cyclodextrins.

[0330] Formulations for parenteral administration of the polypeptides described herein include sterile solutions for injection, sterile dry soluble products such as lyophilized powders, sterile dry suspensions ready to be combined with a solvent immediately prior to use, including subcutaneous tablets, sterile suspensions for injection, sterile dry insoluble products ready to be combined with a vehicle immediately prior to use, and sterile emulsions. The solutions may be aqueous or non-aqueous.

[0331] For intravenous administration, 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.

[0332] Topical mixtures containing the polypeptides described herein are prepared as described for local and systemic administration. The resulting mixtures may be solutions, suspensions, emulsions, etc., and may be formulated as creams, gels, ointments, emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes, foams, aerosols, irrigations, sprays, suppositories, bandages, skin patches, or other dosage forms suitable for topical administration.

[0333] Pharmaceutical compositions (including, for example, any of the polypeptides, molecules, nucleic acids, vectors, cells, or protein conjugates described herein) may be formulated as aerosols for local application, such as inhalation (see, for example, U.S. Patent Nos. 4,044,126, 4,414,209, and 4,364,923). Such formulations for administration to the respiratory tract may be in the form of an aerosol or solution for a nebulizer, or in the form of a fine powder for inhalation, alone or in combination with an inert carrier such as lactose. In this case, the particles of the formulation may have diameters of less than about 50 microns, for example, less than about 10 microns, in some aspects.

[0334] Pharmaceutical compositions (e.g., comprising any of the polypeptides, molecules, nucleic acids, vectors, cells or protein conjugates described herein) may be formulated in the form of gels, creams and lotions for topical or local application, e.g., topical application to the skin and mucous membranes, for application to the eye or for intrastriatal or intraspinal application. Topical administration is contemplated for transdermal delivery and / or administration to the eye or mucous membranes or inhalation therapy. Nasal solutions of the antibodies may also be administered, alone or together with other pharmaceutically acceptable excipients.

[0335] Transdermal patches, including iontophoretic and electrophoretic devices, are well known to those of skill in the art and may be used to administer any of the polypeptides, molecules, nucleic acids, vectors, cells or protein conjugates described herein. For example, such patches are disclosed in U.S. Patent Nos. 6,267,983, 6,261,595, 6,256,533, 6,167,301, 6,024,975, 6,010715, 5,985,317, 5,983,134, 5,948,433, and 5,860,957.

[0336] In some aspects, the pharmaceutical compositions described herein are lyophilized powders that can be reconstituted for administration as solutions, emulsions, and other mixtures. They may also be reconstituted and formulated as solids or gels. Lyophilized powders are prepared by dissolving any of the polypeptides, molecules, nucleic acids, vectors, cells, or protein conjugates described herein or pharma- ceutically acceptable derivatives thereof in a suitable solvent. In some aspects, the lyophilized powders are sterile. The solvent can contain excipients that improve stability or other pharmacological components of the powder or reconstituted solution made from the powder. Excipients that can be used include, but are not limited to, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable formulations. The solvent can also contain a buffer such as citrate, sodium or potassium phosphate, or other buffers known to those skilled in the art. In some aspects, the buffer is at approximately neutral pH. Subsequent sterile filtration of the solution following lyophilization under standard conditions known to those skilled in the art provides the desired formulation. In some aspects, the resulting solution may be sorted into vials for lyophilization. Each vial may contain a single dose or multiple doses of a compound (e.g., any of a polypeptide, molecule, nucleic acid, vector, cell, or protein conjugate). The lyophilized powder may be stored under appropriate conditions, such as at about 4° C. to room temperature.

[0337] This lyophilized powder is reconstituted with water for injection to provide a parenteral dosage form. For reconstitution, the lyophilized powder is added to sterile water or other suitable carrier. The exact amount will vary depending on the compound selected. Such amounts may be empirically determined.

[0338] In some aspects, pharmaceutical compositions comprising any of the polypeptides, molecules, nucleic acids, vectors, cells, or protein conjugates described herein may also be formulated to target specific tissues, receptors, or other regions of the body of a subject (see, e.g., U.S. Pat. Nos. 6,316,652, 6,274,552, 6,271,359, 6,253,872, 6,1 (See Nos. 3,985, 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).

[0339] Compositions to be used for in vivo administration can be sterilized, in some aspects, this can be accomplished, for example, by filtration through sterile filtration membranes.

[0340] V. 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 the polypeptides or molecules described herein, e.g., fusion proteins. The nucleic acids may be present in whole cells, cell lysates, 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 include modified nucleotide analogs. The nucleic acids are "isolated" or "substantially pure" nucleic acids when purified from other cellular components or other contaminants, e.g., other cellular nucleic acids (e.g., other chromosomal DNA, chromosomal DNA linked to naturally isolated DNA) or proteins, by standard techniques including alkaline / SDS treatment, CsCl banding, column chromatography, restriction enzymes, agarose gel electrophoresis, and others well known in the art (see F. Ausubel, et al., ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York). 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 may be obtained using standard molecular biology techniques known in the art.

[0341] In some aspects, the invention provides vectors comprising an isolated nucleic acid molecule encoding a polypeptide or molecule described herein, e.g., a fusion protein. Suitable vectors of the invention include, but are not limited to, expression vectors, viral vectors, and plasmid vectors. In some aspects, the vector is a viral vector.

[0342] As used herein, the term "expression vector" refers to any nucleic acid structure that contains the necessary elements for the transcription and translation of an inserted coding sequence, or, in the case of RNA viral vectors, the necessary elements for replication and translation, when introduced into an appropriate host cell. Expression vectors may include plasmids, phagemids, viruses, and derivatives thereof.

[0343] As used herein, "viral vector" includes, but is not limited to, nucleic acid sequences from the following viruses: retroviruses, e.g., Moloney murine leukemia virus, Harvey murine sarcoma virus, murine mammary tumor virus, and Rous sarcoma virus; lentiviruses; adenoviruses; adeno-associated viruses; SV40 viruses; polyomaviruses; Epstein-Barr viruses; papilloma viruses; herpes viruses; vaccinia viruses; polio viruses; and RNA viruses such as retroviruses. Certain viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced with the gene of interest. Non-cytopathic viruses include retroviruses, whose life cycle involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cell DNA.

[0344] In some aspects, the vector is derived from an adeno-associated virus. In some aspects, the vector is derived from a lentivirus. Examples of lentivirus vectors are disclosed in WO9931251, WO9712622, WO9817815, WO9817816 and WO9818934, the entire contents of which are incorporated herein by reference.

[0345] Other vectors include plasmid vectors (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989). In recent years, plasmid vectors have proven to be particularly advantageous for transferring genes to cells in vivo, since they cannot replicate and integrate within the host genome. However, those plasmids that have promoters compatible with the host cell are capable of expressing peptides from genes operably encoded within the plasmid. Some commonly used plasmids that are commercially available include pBR322, pUC18, pUC19, the various pcDNA plasmids, pRC / CMV, the various pCMV plasmids, pSV40, and pBlueScript. Additional examples of specific plasmids are pcDNA3.1, catalog number V79020; pcDNA3.1 / hygro, catalog number V87020; pcDNA4 / myc-His, catalog number V86320; and pBudCE4.1, catalog number V53220, all from Invitrogen, Carlsbad, Calif. Plasmids may also be custom designed using standard molecular biology techniques to remove and / or add specific segments of DNA.

[0346] Further included in the present invention are methods of producing the polypeptides or molecules described herein, such as fusion proteins. In some aspects, such methods may include expressing the polypeptides or molecules, such as fusion proteins, in cells that contain a nucleic acid molecule encoding the polypeptides or molecules described herein. Host cells that contain these nucleotide sequences are included herein. Non-limiting examples of host cells that can be used include immortal hybridoma cells, NS / 0 myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, human amniotic fluid-derived cells (CapT cells), COS cells, or combinations thereof.

[0347] VI. Kit The invention also provides kits comprising one or more of the polypeptides, molecules, nucleic acids, vectors, cells, protein conjugates, or compositions described herein. In some aspects, provided herein are pharmaceutical packs or kits comprising one or more of the components of the pharmaceutical compositions described herein, e.g., one or more polypeptides provided herein, and one or more containers filled with optional instructions for use. In some aspects, the kits contain a pharmaceutical composition described herein and any prophylactic or therapeutic agents as described herein.

[0348] VII. Method of the Invention As demonstrated herein, the polypeptides, molecules, nucleic acids, vectors, cells, protein conjugates or compositions of the invention are useful for inhibiting, reducing and / or dissociating the interaction between FAM19A5 protein and a member of the LRRC4 protein family. Thus, in some aspects, a method for inhibiting, reducing and / or dissociating the complex formation between FAM19A5 protein and a member of the LRRC4 protein family in a subject in need thereof comprises administering to the subject an effective amount of any of the polypeptides, molecules, nucleic acids, vectors, cells, protein conjugates or compositions described herein. In some aspects, after administration, formation of the complex 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 at least about 100% compared to a control group (e.g., the corresponding value in the subject before administration or the value in the non-administered subject of interest).

[0349] As described elsewhere in the present invention, the binding of FAM19A5 protein to LRRC4 protein family members can suppress the activity of LRRC4 protein family members. For example, in some aspects, the formation of FAM19A5-LRRC4 family protein complexes can lead to impaired neural circuit formation, resulting in an imbalance in the dynamic gain and loss of synapses, which is essential for the healthy function of neurons in the central and peripheral nervous systems.

[0350] Thus, in some aspects, a decrease in the formation of a complex between the FAM19A5 protein and a LRRC4 protein family member can increase the activity of the LRRC4 protein family member. In some aspects, after administration, the activity of the LRRC4 protein family member 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 control group (e.g., a corresponding value in a pre-administration subject or a corresponding value in a non-administered subject). Non-limiting examples of such activities can include neurite outgrowth, neuronal migration, and the formation and functional assembly of synaptic contacts.

[0351] As will be apparent from the present invention, in some aspects, the present invention relates to a method of increasing neurite outgrowth and / or synaptogenesis in a neuron, comprising contacting the neuron with any of the polypeptides, molecules, nucleic acids, vectors, cells, protein conjugates, or compositions described herein. In some aspects, the contacting occurs in vivo (e.g., in a subject in need thereof). In such aspects, the method may further comprise administering any of the polypeptides, molecules, nucleic acids, vectors, cells, protein conjugates, or compositions to the subject prior to the contacting. In some aspects, the contacting occurs ex vivo. In some aspects, the contacting increases neurite outgrowth in the neuron 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 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, or at least about 50-fold compared to a control (e.g., neurite outgrowth in a corresponding neuron not contacted with any of the polypeptides, molecules, nucleic acids, vectors, cells, protein conjugates, or compositions described herein). In some aspects, the contacting increases synaptogenesis in the neurons 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 10-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 a control group (e.g., synaptogenesis in a corresponding neuron not contacted with any of the polypeptides, molecules, nucleic acids, vectors, cells, protein conjugates, or compositions described herein).

[0352] In some aspects, any therapeutic effect of a polypeptide, molecule, nucleic acid, vector, cell, protein conjugate or composition described herein (e.g., a decrease in complex formation between FAM19A5 protein and the LRRC4 protein family, an increase in neurite outgrowth and / or synaptogenesis) can reduce one or more symptoms of a disease or condition, such as those associated with impaired neural circuit formation.

[0353] Thus, in some aspects, the present invention relates to a method of treating a disease or condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any of the polypeptides, molecules, nucleic acids, vectors, cells, proteins, conjugates, or compositions described herein, wherein the disease or condition is selected from amyotrophic lateral sclerosis (ALS), Alzheimer's disease, glaucoma, diabetic retinopathy, neuropathic pain, spinal cord injury, traumatic brain injury, stroke, Parkinson's disease, or a combination thereof. As further described below, in some aspects, provided herein is a method of treating amyotrophic lateral sclerosis (ALS), comprising administering to a subject any of the polypeptides, molecules, nucleic acids, vectors, cells, protein conjugates, or compositions described herein. In some aspects, the present invention provides a method of treating Alzheimer's disease, comprising administering to a subject any of the polypeptides, molecules, nucleic acids, vectors, cells, protein conjugates, or compositions described herein. In some aspects, the invention provides a method of treating glaucoma comprising administering to a subject any of the polypeptides, molecules, nucleic acids, vectors, cells, protein conjugates, or compositions described herein. In some aspects, the invention provides a method of treating diabetic retinopathy comprising administering to a subject any of the polypeptides, molecules, nucleic acids, vectors, cells, protein conjugates, or compositions described herein. In some aspects, the invention provides a method of treating neuropathic pain comprising administering to a subject any of the polypeptides, molecules, nucleic acids, vectors, cells, protein conjugates, or compositions described herein. In some aspects, the invention provides a method of treating spinal cord injury comprising administering to a subject any of the polypeptides, molecules, nucleic acids, vectors, cells, protein conjugates, or compositions described herein. In some aspects, the invention provides a method of treating traumatic brain injury comprising administering to a subject any of the polypeptides, molecules, nucleic acids, vectors, cells, protein conjugates, or compositions described herein.In some aspects, the invention provides a method of treating stroke comprising administering to a subject any of the polypeptides, molecules, nucleic acids, vectors, cells, protein conjugates or compositions described herein.In some aspects, the invention provides a method of treating Parkinson's disease comprising administering to a subject any of the polypeptides, molecules, nucleic acids, vectors, cells, protein conjugates or compositions described herein.

[0354] Accordingly, some aspects of the present invention relate to a method of treating ALS in a subject in need thereof, comprising administering to the subject any of the polypeptides, nucleic acids, vectors, cells, protein conjugates, or compositions described herein. In some aspects, ALS that may be treated by the present invention includes sporadic ALS, familial ALS, or both. As used herein, the term "sporadic" ALS refers to ALS where the occurrence of ALS is not associated with any family dynamics. Approximately 90% or more of ALS diagnoses involve sporadic ALS. As used herein, the term "familial" ALS refers to ALS that occurs more than once in a family, implying a genetic component to the disease. In some aspects, ALS that may be treated by the present invention includes primary lateral sclerosis (PLS). PLS can affect the upper motor neurons of the arms and legs. However, lower motor neuron symptoms develop within four years of onset in more than 75% of individuals with overt PLS, until which time PLS cannot be definitively diagnosed. PLS has a better prognosis than classical ALS because it progresses more slowly, causes less functional loss, does not affect breathing ability, and does not cause severe weight loss. In some aspects, ALS involves progressive muscular atrophy (PMA). PMA can affect the lower motor neurons of the arms and legs. Although PMA is associated with a longer survival on average compared to classical ALS, it still progresses to other spinal cord regions over time, eventually leading to respiratory failure and death. Upper motor neuron signs can occur later in the PMA process, in which case the diagnosis can be changed to classical ALS.

[0355] In some aspects, administration of any of the therapeutic agents described herein (e.g., a polypeptide, molecule, nucleic acid, vector, cell, protein conjugate, or composition described herein) can ameliorate one or more symptoms associated with ALS. Non-limiting examples of such symptoms include: difficulty walking or performing normal daily activities; stumbling; weakness of limbs; slurred speech; difficulty swallowing; muscle pain and spasms; inappropriate crying, laughing, or yawning; dementia; cognitive and behavioral changes; and combinations thereof.

[0356] As demonstrated by the present invention (see, e.g., Example 11), in some aspects, the present invention provides a method of treating Alzheimer's disease in a subject in need thereof, comprising administering any of the polypeptides, molecules, nucleic acids, vectors, cells, protein conjugates, or compositions. Without being bound to any one theory, in some aspects, treating Alzheimer's disease comprises reducing amyloid beta (Aβ) plaque load in a subject (e.g., suffering from Alzheimer's disease). As described herein, "amyloid beta plaque" refers to all forms of abnormal deposition of amyloid beta, including large aggregates and small clusters of small amyloid beta peptides, and may include any modification of amyloid beta peptides. Amyloid beta (Aβ) plaques are known to cause neuronal changes, such as, for example, synaptic organization, synaptic morphology, synaptic density, synaptic conductance loss, dendritic diameter changes, dendritic length changes, spine density changes, spine area changes, spine length changes, or spine head diameter changes. In some aspects, increased Aβ plaque load can cause synapse loss in neurons. Thus, in some aspects, the present invention provides a method of reducing synapse loss in neurons, comprising contacting the neurons with any of the polypeptides, molecules, nucleic acids, vectors, cells, protein conjugates, or compositions described herein. In some aspects, the contacting can occur in vivo. In some aspects, the contacting can occur ex vivo.

[0357] Also, as demonstrated by the present invention (see, e.g., Example 13), in some aspects, the present invention provides a method of treating Parkinson's disease in a subject in need thereof, comprising administering any of the polypeptides, molecules, nucleic acids, vectors, cells, protein conjugates, or compositions described herein. The term "Parkinson's disease" (PD) as used herein refers to a neurodegenerative disorder characterized by widespread degeneration of dopaminergic neurons in the nigrostriatal system, eliciting motor and non-motor signs (i.e., symptoms). Non-limiting examples of motor and non-motor signs of PD are provided elsewhere herein. Proteinopathy (α-synuclein abnormal aggregation) is a hallmark of PD. Other exemplary hallmarks of PD include dopaminergic neuron damage, mitochondrial dysfunction, neuroinflammation, proteostasis (e.g., autophagic removal of damaged proteins and organelle glial dysfunction), and combinations thereof.

[0358] As demonstrated herein (see, e.g., Example 14), in some aspects, the therapeutic agents provided herein (e.g., any polypeptide, molecule, nucleic acid, vector, cell, protein conjugate or composition described herein) are useful for increasing the threshold or latency to an external stimulus (e.g., mechanical and / or thermal stimulus) in a subject in need thereof. Thus, in some aspects, after administration, the subject has a higher threshold to an external stimulus compared to a control group (e.g., a corresponding subject that has not received a polypeptide described herein). As used herein, the term "threshold to an external stimulus" refers to the amount of pressure (from an external stimulus) before the subject reacts to the stimulus (e.g., by moving away).

[0359] As will be apparent to one of skill in the art, such a therapeutic effect may be useful in treating one or more symptoms associated with neuropathic pain. Thus, in some aspects, the present invention provides a method of treating, preventing, or ameliorating neuropathic pain in a subject in need thereof, comprising administering to the subject any one of the polypeptides, molecules, nucleic acids, vectors, cells, protein conjugates, or compositions of the present invention.

[0360] In some aspects, neuropathic pain is central neuropathic pain, i.e., pain due to injury or damage affecting any level of the CNS, including the central somatosensory nervous system (e.g., brain injury and spinal cord injury), or associated with or resulting from a disease or disorder such as stroke, multiple sclerosis, or lateral medullary infarction. In some aspects, neuropathic pain is peripheral neuropathic pain, pain due to injury or damage affecting any level of the peripheral nervous system (e.g., damage to motor nerves, sensory nerves, autonomic nerves, or a combination thereof), or associated with or resulting from a disease or disorder.

[0361] In some aspects, the therapeutic agents provided herein (e.g., any polypeptide, molecule, nucleic acid, vector, cell, protein conjugate, or composition described herein) may be useful for treating retinopathies. In some aspects, retinopathies that may be treated by the present invention include diabetic retinopathy. "Diabetic retinopathy" includes all types of diabetic retinopathy, including, but not limited to, non-proliferative diabetic retinopathy (NPDR), proliferative diabetic retinopathy (PDR), diabetic maculopathy, and diabetic macular edema. Without being bound to any one theory, in some aspects, treating a retinopathies (e.g., diabetic retinopathy) includes improving the electroretinogram in a subject in need thereof. In some aspects, improved retinal potential comprises increased values ​​for A-wave, B-wave and / or oscillatory potential as compared to a control group (e.g., a matched subject not treated with a polypeptide described herein). It will be apparent to one of skill in the art that treating retinal pathology may be useful in treating other types of eye disorders, including, but not limited to, treating macular degeneration and glaucoma.

[0362] In some aspects, the methods described herein (e.g., increasing neurite outgrowth) may include administering to the subject an additional therapeutic agent. The additional therapeutic agent may include any known agent for treating and / or alleviating one or more symptoms associated with any of the above-mentioned indications. In some aspects, the additional therapeutic agent includes an acetylcholinesterase inhibitor. In some aspects, the additional therapeutic agent includes a dopamine agonist. In some aspects, the additional therapeutic agent includes a dopamine receptor antagonist. In some aspects, the additional therapeutic agent includes an antipsychotic. In some aspects, the additional therapeutic agent includes a monoamine oxidase (MAO) inhibitor. In some aspects, the additional therapeutic agent includes a catechol O-methyltransferase (COMT) inhibitor. In some aspects, the additional therapeutic agent includes an N-methyl-D-aspartate (NMDA) receptor antagonist. In some aspects, the additional therapeutic agent comprises an immunomodulatory agent. In some aspects, the additional therapeutic agent comprises an immunosuppressant.

[0363] Non-limiting examples of such formulations include: tetrabenazine (XENAZINE), antipsychotic drugs such as haloperidol (HALODOL), chlorpromazine, risperidone (rRIDPERDAL), quetiapine (SEROQUEL), levodopa (with or without carbidopa) (LODOSYN), dopamine agonists such as pramipexole (MIRAPEX), ropinirole (REQUIP), and rotigotine (NEUPRO), and apromorphine (APMO). rphine (Apokyn), selegiline (ELDEPRYL, ZELAPAR), rasagiline (AZILECT), entacapone (COMTAN), benztropine (COGENTIN), trihexyphenidyl, amantadine, donepezil (ARICEPT), galantamine (RAZADYNE), rivastigmine (EXELON), glatiramer acetate acetate (COPAXONE), dimethyl fumarate (TECFIDERA), fingolimod (GILENYA), teriflunomide (AUBAGIO), natalizumab (TYSABRI), alemtuzumab (LEMTRADA), mitoxantrone (NOVANTRONE), riluzole (RILUTEK), physostigmine salicylate (ANTILIRIUM), physostigmine sulfatesulfate (ESERINE), metrifonate, neostigmine, ganstigmine, pyridostigmine (MESTINON), ambenonium (MYTELASE), demarcarium, Debio 9902 (ZT-1); Debiopharmladostigil, NP-0361, tacrine (COGNEX), tolserine, velnacrine maleate, memoquin, huperzine A (HUP-A); NeuroHitech, phenserine, edrophonium (ENLON, TENSILON), INM-176, apomorphine (APOKYN), bromocriptine (PARLODEL), cabergoline (DOSTINEX), dihydrexidine, dihydroergocryptine, fenoldopam (CORLOPAM), lisuride (DOPERGIN), terguride spergolide (PERMAX), piribedil (TRIVASTAL, TRASTAL), quinpirole, SKF-82958 (GlaxoSmithKline), cariprazine, pardoprunox, sarizotan, chlorpromazine, fluphenazineloxzpine, resperidone, thioridazine, thiothixene, trifluoperazine, 7-hydroxyamoxapine, droperidol (INAPSINE, DRIDOL, DROPLETAN), domperidone (MOTILIUM), L-741742, L-745870, raclopride, SB-277011A, SCH-23390, ecopipam, SKF-83566, metoclopramide (REGLAN), lurasidone (also known as LATUDA, SM-13496; Dainippon Sumitomo, aripiprazole (ABILIFY), chlorpromazine (THORAZINE), iloperidone (FANAPTA), flupentixol decanoate (DEPIXOL, FLUANXOL), reserpine (SERPLAN), pimozide (ORAP), fluphenazine decanoate, fluphenazine hydrochloridehydrochloride), prochlorperazine (COMPRO), asenapine (SAPHRIS), loxapine (LOXITANE), molindone (MOBAN), perphenazine, thioridazine, thiothixine, trifluoperazine (STELAZINE), ramelteon, Clozapine (CLOZARIL), norclozapine (ACP-104), paliperidone (INVEGA), melperone, olanzapine (ZYPREXA), talnetant, amisulpride, ziprasidone (GEODON), blonanserin (LONASEN), ACP-103 (Acadia Pharmaceuticals, selegiline hydrochloride (I-deprenyl, ELDEPRYL, ZELAPAR), dimethylselegilene, brofaromine, phenelzine (NARDIL), tranylcypromine (PARNATE), moclobemide (AURORIX, MANERIX), befloxatone, safinamide, isocarboxazid (MARPLAN), nialamide (NIAMID), iproniazide (MARSILID, IPROZID, IPRONID), CHF-3381 (ChiesiFarmaceutici, iproclozide, toloxatone (HUMORYL, PERENUM), bifemelane, desoxypeganine, harmine (telepathine, or banasterine), harmaline, linezolid (ZYVOX, ZYVOXID), pargyline (EUDATIN, SUPIRDYL), nitecapone, tolcapone (TASMAR), tropolone, memantine (NAMENDA, AXURA, EBIXA), amantadine (SYMMET REL), acamprosate (CAMPRAL), besonprodil, ketamine (KETALAR), delucemine, dexanabinol, dexefaroxan, dextromethorphan, dextrorphan, traxoprodil, CP-283097, himantane, idantadol, ipenoxazone, L-701252 (Merck), lancicemine, levorphanol (DROMORAN), LY-233536 and LY-235959 (bothLilly, methadone (DOLOPHINE), neramexane, perzinfotel, phencyclidine, tianeptine (STABLON), dizocilpine (MK-801), EAB-318 (Wyeth), ibogaine, voacangine, tiletamine, aptiganel (CERESOTAT), gavestenel, remacimide ), MBP-8298 (synthetic myelin basic protein peptide), roquinimex (LINOMIDE), laquinimod (ABR-215062 and SAIK-MS), ABT-874 (human anti-IL-12 antibody; Abbott), rituximab (RITUXAN), leflunomide, ciclesonide, daclizumab (ZENAPAX), methotrexate (TREXALL, RHEUMATREX), suplatast tosilate tosilate, mycophenolate mofetil (CELLCEPT), mycophenolate sodium (MYFORTIC), azathioprine (AZASAN, IMURAN), mercaptopurine (PURI-NETHOL), cyclophosphamide (NEOSAR, CYTOXAN7), voclosporin, PUR-118, AMG 357, AMG 811, BCT197, chlorambucil (LEUKERAN), cladribine (LEUSTATIN, MYLINAX), alpha-fetoprotein, etanercept (ENBREL), leflunomide, Ciclesonide chloroquine, hydroxychloroquine, d-penicillamine, auranofin, sulfasalazine, sodium gold thiomalate aurothiomalate, cyclosporine, cromolyn, infliximab, adalimumab, certolizumab, pegol, golimumab, rituximab, ocrelizumab, ofatumumab, 4-benzyloxy-5-((5-undecyl-2H-pyrrol-2-ylidene)methyl)-2,2'-bi-1H-pyrrole (PNU-156804), and combinations thereof.

[0364] In some aspects, any polypeptide, molecule, nucleic acid, vector, cell, protein conjugate, or composition described herein is administered intravenously, orally, parenterally, transthecally, intrathecally, intra-cerebroventricularly, pulmonarily, subcutaneously, intradermally, intramuscularly, or intraventricularly.

[0365] The following examples are offered by way of illustration and not by way of limitation.

[0366] Working Example Example 1: Interaction analysis between FAM19A5 and LRRC4 protein family members To better understand the interaction between FAM19A5 and LRRC4 protein family members, HEK293 cells were modified to express FLAG-tagged members of the LRRC4 protein family, i.e., LRRC4C protein, LRRC4 protein, or LRRC4B protein. HEK293 cells or primary cortical neurons were then treated with recombinant FAM19A5 protein (1 μM) for 30 min, and binding between FAM19A5 protein and other members of the LRRC4 protein family was assessed using both co-immunoprecipitation and immunofluorescence analysis.

[0367] For co-immunoprecipitation assay, cell lysates from different FAM19A5-treated HEK293 cells were collected and immunoprecipitated with anti-FLAG antibody, anti-FAM19A5(1-65) antibody, or human IgG antibody (control group). The immunoprecipitated proteins were immunoblotted with anti-FLAG and anti-FAM19A5(3-2) antibodies. For immunofluorescence assay, HEK293 cells treated with recombinant FAM19A5 protein were immunostained with anti-FAM19A5(3-2) (to detect FAM19A5 protein expression) and anti-FLAG antibody (to detect LRRC4 protein family members). Primary cortical neurons treated with recombinant FAM19A5 protein were immunostained with anti-FAM19A5(3-2) and anti-LRRC4B antibodies. Nuclei were stained with Hoechst33342 (blue).

[0368] As shown in Figure 1A, anti-FLAG antibody could co-immunoprecipitate FAM19A5 protein. Similarly, as shown in Figure 1B, anti-FAM19A5(1-65) antibody could specifically co-immunoprecipitate LRRC4B protein. Similar results were observed using immunofluorescence analysis. In both LRRC4B-expressing HEK293 cells and primary cortical neurons, FAM19A5 protein was mostly associated with dendrite-like processes or neurites where LRRC4B protein was highly expressed (see Figure 1C and Figure 1D), implying an interaction between LRRC4 protein family (e.g., LRRC4B) and FAM19A5 protein members.

[0369] Next, to assess whether the above results were specific to a particular isoform of the FAM19A5 protein, HEK293 cells were co-transfected with cDNA encoding FLAG-tagged LRRC4B protein and cDNA encoding FAM19A5 protein isoform 1 or isoform 2. Binding was then assessed using both immunofluorescence and co-immunoprecipitation assays.

[0370] For immunofluorescence assay, co-transfected HEK293 cells were immunostained with anti-FAM19A5(1-65) and anti-FLAG antibodies to confirm the subcellular localization of FAM19A5 (isoform 1 or 2) and LRRC4B proteins, respectively. Nuclei were stained with Hoechst33342 (blue). For co-immunoprecipitation analysis, cell lysates from co-transfected HEK293 cells were immunoprecipitated with anti-FLAG, anti-FAM19A5(1-65), anti-FAM19A5(3-2) or human IgG antibodies (control group). Immunoprecipitated proteins were immunoblotted with anti-FLAG and anti-FAM19A5(3-2) antibodies.

[0371] Similar to previous results, in co-transfected HEK293 cells, the two isoforms of FAM19A5 protein were found to be highly co-localized with LRRC4B protein, especially in vesicular-type spots near the plasma membrane and in dendrite-like processes (see Figures 2A and 2B). Similarly, immunoprecipitation with anti-FLAG and anti-FAM19A5(1-65) antibodies confirmed the interaction between LRRC4B protein and both isoforms of FAM19A5 protein. For example, anti-FLAG antibody was able to co-immunoprecipitate both isoforms 1 and 2 of FAM19A5 protein (see Figure 2C). Similarly, anti-FAM19A5(1-65) antibody was able to co-immunoprecipitate LRRC4B protein (see Figure 2D). Similarly, anti-FAM19A5(3-2) antibody was unable to co-immunoprecipitate LRRC4B protein. Without being bound to any one theory, the differences observed from the 1-65 and 3-2 anti-FAM19A5 antibodies may be due to their binding epitopes, as these antibodies are known to bind to other epitopes within the FAM19A5 protein (see US Publ. 2020 / 0299373, the entire contents of which are incorporated herein by reference).

[0372] In summary, the above results confirm an interaction between the FAM19A5 protein and other members of the LRRC4 protein family (e.g., LRRC4B). As described herein, the polypeptides of the invention may be useful in some aspects for modulating biological activities associated with such interactions by inhibiting, reducing and / or dissociating such interactions.

[0373] Example 2: Identification of the FAM19A5 protein binding domain of the LRRC4B protein To identify the specific motif or domain of LRRC4B protein responsible for binding to FAM19A5 protein, various FLAG-tagged LRRC4B deletion constructs were generated (see Table 6 below). HEK293 cells were transfected with different deletion constructs and then treated with recombinant FAM19A5 protein as described in Example 1. Then, cell lysates from different HEK293 cells were immunoprecipitated with anti-FLAG antibody. The immunoprecipitated proteins were immunoblotted with anti-FLAG and anti-FAM19A5(3-2) antibodies.

[0374] [Table 6] JPEG2024528351000082.jpg240169JPEG2024528351000083.jpg14169

[0375] As shown in Figures 3A and 3B, all deletion constructs containing the threonine-rich domain ("Thr" in Figure 3A) of LRRC4B protein were able to bind to FAM19A5 protein at various levels. These structures include: LRRC4B(36-713) (i.e., structure #1); LRRC4B(157-713) (i.e., structure #2); LRRC4B(230-713) (i.e., structure #3); LRRC4B(364-713) (i.e., structure #4); LRRC4B(453-713) (i.e., structure #5); LRRC4B(36-576) (i.e., structure #7); LRRC4B(364-576) (i.e., structure #10); LRRC4B(453-576) (i.e., structure #11); and LRRC4B(484-576) (i.e., structure #12). In particular, the amino acid sequence at positions 484-497 of the LRRC4B protein was shown to bind to the FAM19A5 protein and play an important role in the binding, as a deletion construct containing amino acids 484-576 (i.e., construct #12) but not a deletion construct containing amino acids 498-576 (i.e., construct #13) did not (see Figures 3A and 3B).

[0376] To confirm the co-immunoprecipitation results, an ELISA assay was then used to measure the binding of FAM19A5 protein to the full-length ectodomain of LRRC4 protein family members or various LRRC4B ectodomain protein fragments. Specifically, ELISA plates were coated with one of the following LRRC4B ectodomain proteins bound to human Fc (100 nM / well): (1) the full-length ectodomain of LRRC4 protein (amino acid residues 39-527 of SEQ ID NO:1) (SEQ ID NO:4); (2) the full-length ectodomain of LRRC4B protein (amino acids 36-576 of SEQ ID NO:2; i.e., construct #7 in Table 6) (SEQ ID NO:5); (3) the full-length ectodomain of LRRC4C protein (amino acids 45-527 of SEQ ID NO:3) (SEQ ID NO:6); (4) the LRRC4B ectodomain fragment (sequence #1, #2, #3, #4, #5, #6, #7, #8, #9, #10, #11, #12, #13, #14, #15, #16, #17, #18, #19, #20, #21, #22, #23, #24, #25, #26, #27, #28, #29, #30, #31, #32, #33, #34, #35, #36, #37, #38, #39, #40, #41, #42, #43, #44, #45, #46, #50, #51, #52, #63, #64, #75, #86, #97, #108, #119, #129, #130, #14, #15, #16, #17, #18, #21 (5) LRRC4B ectodomain fragment (amino acids 484-576 of SEQ ID NO:2; i.e., construct #12 of Table 6) (SEQ ID NO:8); (6) LRRC4B ectodomain fragment (amino acids 482-576 of SEQ ID NO:2) (SEQ ID NO:9); (7) LRRC4B ectodomain fragment (amino acids 482-497 of SEQ ID NO:2) (SEQ ID NO:10); and (8) LRRC4B ectodomain fragment (amino acids 498-576 of SEQ ID NO:2; i.e., construct #13 of Table 6) (SEQ ID NO:11). Recombinant FAM19A5 protein (0.005, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5 and 10 nM) was then added to the relevant wells and the plates were incubated at 37° C. for 1 hour. The amount of FAM19A5 protein bound to LRRC4B was then detected using HRP-conjugated anti-FAM19A5(1-65) antibody.

[0377] As shown in Figure 4A. The full-length ectodomains of all members of the LRRC4 protein family (i.e., LRRC4, LRRC4B and LRRC4C proteins) could seek out FAM19A5 protein at various levels. The full-length ectodomains of LRRC4 and LRRC4B proteins bound to FAM19A5 protein with EC50 of 0.48 nM and 0.64 nM, respectively. No binding saturation was observed for full-length LRRC4C protein at 10 nM concentration of FAM19A5. And consistent with the co-immunoprecipitation analysis, the LRRC4B ectodomain protein fragment containing the sequence at positions 484-497 of SEQ ID NO:2 had a significant affinity for FAM19A5, whereas the ectodomain protein fragment lacking this sequence (i.e., LRRC4B(498-576)) failed to bind to FAM19A5 (see Figure 4B).

[0378] As additional confirmation, three synthetic polypeptides were constructed that contained amino acids 484-497 of SEQ ID NO:2 (i.e., YTYFTTVTVETLET; SEQ ID NO:65): (1) FB-16 (GYTYFTTVTVETLETQ; SEQ ID NO:17), (2) FB-20 (GYTYFTTVTVETLETQPGEE; SEQ ID NO:18), and (3) FB-28 (GYTYFTTVTVETLETQPGEKEPPGPTTD; SEQ ID NO:19). The peptides differ in overall length. The ability of these polypeptides to bind to recombinant FAM19A5 protein was assessed using an ELISA assay as described above. As shown in FIG. 4B, each of FB-16, FB-20, and FB-28 was able to bind to recombinant FAM19A5 protein with high affinity, similar to the LRRC4B ectodomain protein fragment that contained amino acids 484-497 of SEQ ID NO:2 (i.e., SEQ ID NO:65).

[0379] In summary, the above results suggest the importance of an amino acid sequence within positions 484-497, specifically positions 484-493 (i.e., YTYFTTVTVE; SEQ ID NO:29) of the LRRC4B protein (i.e., SEQ ID NO:65) in binding to the FAM19A5 protein (also referred to as the "FAM19A5 binding domain"). As shown in Figure 20, this sequence is generally evolutionarily conserved in the LRRC4 protein family among different vertebrates.

[0380] Example 3: Identification of FAM19A5 protein binding domains in different members of the LRRC4 protein family As described in Example 2, all members of the LRRC4 protein family could bind to FAM19A5 protein at various levels. Therefore, the amino acid sequences of LRRC4B, LRRC4 and LRRC4C proteins were aligned to compare the binding domains. As shown in FIG. 5A, the amino acid sequence of 484-522 position of LRRC4B was compared with the amino acid sequence of the corresponding position of LRRC4 and LRRC4C proteins, and showed many similarities. Therefore, to evaluate whether the corresponding positions of LRRC4 and LRRC4C are important in the binding of these proteins to FAM19A5 protein, cDNA encoding (i) LRRC4C protein fragment (amino acids 354-527 of SEQ ID NO:3; SEQ ID NO:66) or (ii) LRRC4 protein fragment (amino acids 353-527 of SEQ ID NO:1; SEQ ID NO:67) was engineered (see Table 7). HEK293 cells were transfected to express one of the protein fragments and then treated with recombinant FAM19A5 protein as described in Example 1. Next, cell lysates from different HEK293 cells were immunoprecipitated with anti-FLAG antibody, and the immunoprecipitated proteins were immunoblotted with anti-FLAG and anti-FAM19A5(3-2) antibodies.

[0381] [Table 7]

[0382] As shown in Figure 5B, both LRRC4C and the LRRC4 peptide fragment were able to bind to the FAM19A5 protein, highlighting the similarities in the binding domains of different members of the LRRC4 family.

[0383] Example 4: Analysis of the role of the FAM19A5 binding domain in suppressing the interaction of FAM19A5 and LRRC4B proteins Since the LRRC4B protein fragment containing the binding domain described in Example 2 (e.g., LRRC4B(453-576); i.e., construct #11 in Table 6) could bind to FAM19A5 protein with high affinity, such protein fragment could compete with naturally occurring LRRC4B protein for binding to FAM19A5 protein, and thus dissociate the FAM19A5-LRRC4 protein family complex. Briefly, HEK293 cells expressing both FAM19A5 isoform 2 and LRRC4B protein were treated (i.e., cultured in vitro) with LRRC4B(453-576)-hFc or mutant LRRC4B(453-576)-hFc (containing alanine substitutions at positions 488 and 489 of SEQ ID NO:2; SEQ ID NO:16) protein fragment for 30 minutes. Cells were then immunostained with anti-FAM19A5(1-65) and anti-LRRC4B antibodies to confirm the expression of FAM19A5 and full-length LRRC4B proteins, respectively. Anti-hIgG antibody was used to confirm the expression of hFc-fused LRRC4B protein fragments. Nuclei were stained with Hoechst33342.

[0384] As previously observed (see Figures 2A and 2B), when FAM19A5 protein bound to full-length LRRC4B protein, the complex was highly co-localized, especially at the plasma membrane and dendrite-like processes. In cells treated with LRRC4B(453-576)-hFc, FAM19A5 was almost separated from full-length LRRC4B protein (see Figure 7, bottom row). In contrast, in HEK293 cells treated with mutant LRRC4B(453-576)-hFc, FAM19A5 protein remained almost bound to full-length LRRC4B protein, indicating the importance of the FAM19A5 family binding domain identified in Example 2.

[0385] Next, to further evaluate the role that the binding domain of the LRRC4B protein has on the interaction between the FAM19A5 protein and LRRC4 protein family members, a competitive inhibition assay was used to determine whether the different LRRC4B deletion constructs from Example 2 could inhibit FAM19A5 binding to the full-length ectodomain of the LRRC4B protein (i.e., amino acids 36-576 of SEQ ID NO:2; SEQ ID NO:5). Briefly, 100 nM of the full-length ectodomain of LRRC4B protein was used to coat plates, and then recombinant FAM19A5 protein (5 ng / mL) was added to the plates in combination with the following LRRC4B deletion constructs (increasing concentrations): (1) LRRC4B(453-576) (i.e., construct #11 in Table 6); (2) LRRC4B(453-576) mutant (containing alanine substitutions at positions 488 and 489 of SEQ ID NO:2; SEQ ID NO:16); (3) LRRC4B(484-576) (i.e., construct #12 in Table 6); (4) LRRC4B(482-576); (5) LRRC4B(482-497); and (6) LRRC4B(498-576). After incubating the plates at 37° C., the amount of FAM19A5 bound to the coated LRRC4B ectodomain protein was measured using HRP-conjugated anti-FAM19A5(1-65) antibody.

[0386] As shown in Figure 8A, LRRC4B(453-576) could inhibit FAM19A5 protein from binding to the coated full-length LRRC4B ectodomain protein. Other LRRC4B protein fragments including amino acid residues 484-497 of SEQ ID NO:2 (i.e., the binding domain of LRRC4B protein; SEQ ID NO:65) could also inhibit the interaction between FAM19A5 and the full-length LRRC4B ectodomain protein (see LRRC4B(484-576), LRRC4B(482-576) and LRRC4B(482-497)). Similar results were observed from synthetic peptides FB-16, FB-20 and FB-28 (see Figure 8B). In contrast, an LRRC4B protein fragment lacking amino acid residues 484-497 of SEQ ID NO:2 was much less able to inhibit the interaction (see LRRC4B mutant and LRRC4B(498-576)) (see FIG. 8A).

[0387] In summary, the above results demonstrate that peptide fragments (eg, synthetic) containing the binding domain of the LRRC4B protein may be useful in inhibiting the formation of the FAM19A5-LRRC4B protein complex.

[0388] Example 5: Analysis of the role of the binding domain of the LRRC4 protein family in suppressing FAM19A5-LRRC4 protein family complex formation As described in Example 3, it has been observed that there are many similarities in the binding domains of different members of the LRRC4 family. Therefore, to assess whether a polypeptide containing the LRRC4B binding domain can inhibit other members of the LRRC4 protein family from binding to the FAM19A5 protein, ELISA plates were coated with one of the following proteins (100 nM / well): (1) the ectodomain of the LRRC4 protein (amino acid residues 39-527 of SEQ ID NO:1; SEQ ID NO:4); (2) the full-length ectodomain of the LRRC4B protein (amino acids 36-576 of SEQ ID NO:2; i.e., construct #7 of Table 6; SEQ ID NO:5); and (3) the full-length ectodomain of the LRRC4C protein (amino acids 45-527 of SEQ ID NO:3; SEQ ID NO:6). Recombinant FAM19A5 protein (5 ng / mL) was then added together with one of the following: (i) LRRC4B(484-576) protein fragment, (ii) LRRC4B(453-576,AA) protein fragment, and (iii) synthetic FB-20 peptide. After incubating the plates at 37°C, the amount of FAM19A5 protein bound to the coated LRRC4, LRRC4B, or LRRC4C protein was measured using anti-FAM19A5(1-65) antibody.

[0389] As shown in Figures 9A-C, both the LRRC4B (484-576) protein fragment and the synthetic FB-20 peptide were able to inhibit FAM19A5 protein binding to coated LRRC4 and LRRC4C proteins, and consistent with previous data, the LRRC4B fragment with alanine substitutions at positions 488 and 489 of SEQ ID NO:2, i.e., the LRRC4B mutant (SEQ ID NO:16), had minimal effect.

[0390] Next, to evaluate whether polypeptides containing the binding domain of LRRC4 or LRRC4C protein can have a similar inhibitory effect on LRRC4B protein binding, the following synthetic peptides were constructed: (1) FBC4-23 (containing the binding domain of LRRC4 protein, i.e., YSFFTTVTVETTE); and (2) FBC4C-23 (containing the binding domain of LRRC4C protein, i.e., FSYFSTVTVETME). Next, the ability of the peptides to inhibit the binding of LRRC4 protein family members to FAM19A5 protein was evaluated using a competitive inhibition assay. Briefly, plates were coated with 100 nM of LRRC4B protein fragment #1 (amino acids 36-576 of SEQ ID NO:2; SEQ ID NO:5) or LRRC4B protein fragment #2 (amino acids 453-576 of SEQ ID NO:2; SEQ ID NO:7). Next, recombinant FAM19A5 protein (5 ng / mL for plates coated with LRRC4B fragment #1; 1 ng / mL for plates coated with LRRC4B fragment #2) was added to the plates together with 20 nM of FBC4-23, FBC4C-23 and FB-20 peptides. After incubation of the plates at 37° C., the amount of FAM19A5 protein bound to the coated LRRC4B protein fragments was evaluated using HRP-conjugated anti-FAM19A5(1-65) antibody.

[0391] As shown in Figures 10A and 10B; and Table 8 (bottom), all three peptides (i.e., FB-20, FBC4-23, and FBC4C-23) greatly reduced the interaction of FAM19A5 protein with the coated LRRC4B protein fragment.

[0392] [Table 8]

[0393] In summary, the above results demonstrate that peptides containing the FAM19A5-binding domain of any member of the LRRC4 protein family can inhibit the interaction between FAM19A5 and LRRC4B proteins, thus demonstrating the importance of the conserved properties of the FAM19A5-binding domain in the LRRC4 protein family.

[0394] Example 6: Identification of the minimal FAM19A5 binding domain sequence required to inhibit the interaction between FAM19A5 protein and LRRC4 protein family members Next, ten FB-20 peptide variants were constructed by sequentially deleting one or more amino acids at the N-terminus or C-terminus to identify the minimal sequence required to inhibit the interaction between FAM19A5 and LRRC4 protein family members (see Table 9 for FB-20 peptides). Next, a competitive inhibition assay was used to evaluate the ability of the different FB-20 peptide variants to inhibit the interaction between FAM19A5 and LRRC4B protein. Again, plates were coated with 100 nM of LRRC4B protein fragment #1 (amino acids 36-576 of SEQ ID NO:2; SEQ ID NO:5) or LRRC4B protein fragment #2 (amino acids 453-576 of SEQ ID NO:2; SEQ ID NO:7). Recombinant FAM19A5 protein (5 ng / mL for plates coated with LRRC4B fragment #1; 1 ng / mL for plates coated with LRRC4B fragment #2) was then added to the plates along with 20 nM of the different peptides described above.

[0395] After the plates were incubated at 37° C., the amount of FAM19A5 protein bound to the coated LRRC4B protein fragments was assessed using HRP-conjugated anti-FAM19A5 (1-65) antibody.

[0396] As shown in Figures 11A and 11B; and Table 9, a peptide fragment containing the first 10 amino acids of the LRRC4B protein binding domain (i.e., YTYFTTVTVE; SEQ ID NO:29) could significantly inhibit the interaction between FAM19A5 and the coated LRRC4B protein fragment (see "FB-m11dC" and "FB-m10dC"). In contrast, peptide fragments lacking one or more amino acids at positions 1-10 of the LRRC4B protein binding domain could not significantly inhibit the FAM19A5-LRRC4B protein interaction (see "FB-m10dC", "FB-m9dC", "FB-m8dC", "FB-m7dC", "FB-m6dC", "FB-m10dN", "FB-m9dN", "FB-m8dN" and "FB-m7dN").

[0397] [Table 9]

[0398] The above results indicate the importance of at least the first 10 amino acid residues of the FAM19A5-binding domain of LRRC4 protein family members in inhibiting, reducing and / or dissociating the interaction between LRRC4 protein family members and FAM19A5 proteins.

[0399] Example 7: Identification of important FAM19A5 binding domain residues for inhibiting the interaction between LRRC4 protein family members and FAM19A5 protein To identify critical amino acid residues, multiple FB-20 peptide mutants were constructed by substituting individual residues in the core binding domain (i.e., YTYFTTVTVETLE; SEQ ID NO: 15) with alanine (A) or asparagine (N) (see Table 10). The ability of these FB-20 peptide mutants to inhibit the interaction between LRRC4B and FAM19A5 proteins was then assessed using a competitive inhibition assay as described in Examples 3 and 4.

[0400] As shown in Figures 12A and 12B; and Table 10, FB-20 peptide mutants with alanine or asparagine substitutions at positions 5, 11, 12, and 13 of the core binding domain were still able to significantly inhibit the interaction between FAM19A5 and LRRC4B proteins. In contrast, alanine or asparagine substitutions at positions 1, 2, 3, 4, 6, 7, 8, 9, and 10 significantly reduced the ability of the peptide to inhibit LRRC4B protein from binding to FAM19A5. This indicates the importance of such amino acid positions within the core binding domain in inhibiting, reducing, and / or dissociating the interaction between FAM19A5 and LRRC4 protein family members.

[0401] [Table 10]

[0402] Example 8: Analysis of the therapeutic efficacy of polypeptides containing the binding domain of a member of the LRRC4 protein family To evaluate the therapeutic potential of the polypeptides described herein, the transcript levels of FAM19A5, LRRC4B, and PTPRF (the postsynaptic partner of LRRC4B) were assessed in primary hippocampal neurons (derived from mouse brains at postnatal day 1) at various time points after culture using RNA sequencing. As shown in FIG. 13A, FAM19A5 transcript levels were significantly higher than other members of the FAM19 family even at day 1 after culture and remained high until day 15 after culture. Similarly, as shown in FIG. 13B, primary hippocampal neurons also showed high transcript levels of LRRC4B and PTPRF, which were also maintained until at least day 15 after culture. The high expression levels of these genes in primary neurons imply that they have important roles in various aspects of neurogenesis.

[0403] Next, primary cortical neurons (postnatal day 1) were cultured in vitro with various concentrations (0.006-60 nM) of LRRC4B(453-576) protein fragment (i.e., amino acid residues 453-576 of SEQ ID NO:2; SEQ ID NO:7), and the effect on neurite outgrowth was evaluated by immunostaining the cells with anti-beta-tubulin III antibody 3 days after initial culture. Cells cultured with DMSO ("Veh") were used as the control group.

[0404] As shown in Figures 14A-14D, primary cortical neurons treated with the LRRC4B (453-576) protein fragment were observed to have increased neurite outgrowth in a dose-dependent manner. For example, compared to the control group, neurons treated with the LRRC4B protein fragment showed increased neurite length (Figure 14A), increased numbers of primary and secondary neurites (Figures 14B and 14D, respectively), and increased numbers of branching points (Figure 14C). Increased neurite outgrowth was also observed when the FB-16, FB-20, and FB-28 peptides were used instead of the LRRC4B (453-576) protein fragment (see Figures 18A-18E). It is believed that all of the FB-16, FB-20, and FB-28 peptides have similar positive effects on neurite outgrowth.

[0405] Secondly, it is well known that neurites growing from other neurites can differentiate into axons to form presynapses. Other neurites remain as small neurites and differentiate into dendrites to form postsynapses. Therefore, we also evaluated whether the LRRC4B(453-576) protein fragment could affect presynapse and postsynapse formation. Briefly, mouse primary hippocampal neurons were cultured in vitro with the LRRC4B(453-576) protein fragment (6 or 60 nM). Control cells were treated with DMSO ("Veh") or a mutant LRRC4B(453-576) protein fragment (containing alanine substitutions at positions 488 and 489) that cannot bind to FAM19A5 protein. Then, the expression level of synaptophysin (SYP; presynaptic marker) was evaluated 3 and 6 days after the initial culture. Seven days after initial culture, the expression level of postsynaptic density 95 (PSD95; a postsynaptic marker) was assessed.

[0406] As shown in Figures 15A and 15B, LRRC4B(453-576) protein fragment (at both concentrations) increased both SYP and PSD95 expression in neurons, indicating that the observed increase in neurite outgrowth may increase synaptogenesis. As shown in Figure 15C, the number of puncta co-labeled with SYP and PSD95 increased in peptide-treated mouse primary hippocampal neurons, indicating presynaptic and postsynaptic fusion. Similar results were observed with FB-16, FB-20 and FB-28 peptides (60 nM for each peptide) in Figures 19A-C.

[0407] To further confirm the effect on neurite outgrowth in vivo, APP / PS1 mice (Alzheimer's mouse model) were used. APP / PS1 mice showed synapse loss in the CA1 of the hippocampus at 4 months after birth, with a 50% reduction in puncta co-labeled for presynaptic and postsynaptic markers such as SYP and PSD95 (see Hong et al., Science 352(6286):712-716 (May 2016)). Such synapse and neuronal loss is likely associated with impaired spatial learning and memory abilities (see Yoshiyama et al., Neuron 53:337-351 (2007)). The CA1 of the hippocampus is the main destination for inputs going from the EC to the hippocampus. Information from the EC reaches the CA1 through two major pathways. Among them, one pathway is the direct perforation pathway from EC to CA1, and the other pathway is the indirect pathway using a triple-connection circuit from EC to gyrus (first order synapse), CA3 (second order synapse), and CA1 (third order synapse). Therefore, we investigated how administration of LRRC4B (453-576) protein fragment (containing the FAM19A5-binding domain of LRRC4B protein) affects synaptic connectivity in the hippocampus, especially in the CA1 and CA3 regions. Briefly, APP / PS1 mice were treated with (i) wild-type LRRC4B (amino acid residues 453-576 of SEQ ID NO:2) protein fragment (SEQ ID NO:7) or (ii) mutant LRRC4B protein fragment (i.e., containing alanine substitutions at positions 488 and 489 of SEQ ID NO:2) (SEQ ID NO:16).

[0408] As shown in Figures 16A-C and 17A-C, APP / PS1 mice treated with wild-type LRRC4B protein fragments showed increased SYP and PSD95 immunoreactivity in CA1 and CA3 of mice compared to mutant LRRC4B protein fragments, with levels similar to those observed from untreated normal animals ("Cont").

[0409] In summary, the above results indicate that any peptide containing the core binding domain of a LRRC4 protein family member can act as a decoy receptor for FAM19A5, thus interfering with the inhibitory effect of FAM19A5 protein on the activity (e.g., neuritogenesis and synaptogenesis) of LRRC4 protein family members.

[0410] Example 9: In silico residue scanning of FAM19A5-LRRC4 family complexes using the Schrödinger platform To further characterize the residues that play a role in the interaction between FAM19A5 protein and LRRC4 family members, in silico alanine scanning was performed at every single non-alanine residue in the FAM19A5-LRRC4 family member complex using SCHRODINGER BIOLUMINATE, and the change in Gibbs free energy was identified to indicate the binding affinity for each amino acid residue. Specifically, all non-alanine residues in FB-20 (i.e., GYTYFTTVTVETLETQPGEE; SEQ ID NO: 18), a fragment of the LRRC4B protein that contains the FAM19A5 binding domain, were mutated to alanine. The sequences for the different FB-20 peptide variants are provided in Table 11 (below).

[0411] [Table 11]

[0412] As shown in FIG. 21A (and consistent with previous data - see, e.g., Example 7), certain residues of the FB-20 peptide fragment (e.g., residues Y2 to E11) were considered to be important in the interaction between the FAM19A5 protein and LRRC4B, since the free energy change increased significantly when alanine mutations were introduced into these residues. Similarly, certain residues (e.g., residues T12 and L13) were considered to play a minimal role, since alanine substitution of such residues did not significantly alter the protein-peptide binding affinity.

[0413] Next, to evaluate whether the binding affinity of the FB-20 peptide fragments could be improved, the T12 and L13 residues (thought to play a minimal role in the interaction between FAM19A5 protein and LRRC4B) were replaced with all other possible amino acids, and then the binding affinity was confirmed using Schrodinger Bioluminescence. Since histidine can have three different molecular structures in the protonation state (abbreviated as HIP, HID, and HIE; HIP: +1 charge, δ- and ε-nitrogen protonated; HID: neutral, δ-nitrogen protonated, HIE: neutral, ε-nitrogen protonated), each residue may be replaced with 21 other amino acids. Thus, double mutations on both T12 and L13 generated 441 mutations. The sequences for the top 20 FB-20 peptide double mutants (T12 and L13) predicted to improve the binding affinity between FAM19A5 and LRRC4B are provided in Table 12 (below).

[0414] [Table 12]

[0415] As shown in FIG. 21B, LRRC4B peptide fragments containing specific T12 / L13 double mutants (eg, T12P-L13Y and T12I-L13F) exhibited increased binding affinity to FAM19A5 protein.

[0416] The above results further confirmed that specific amino acid residues (e.g., Y2-E11) of the LRRC4B peptide fragment are important for binding to the FAM19A5 protein. The above results further demonstrated that the binding affinity of the LRRC4B peptide fragment can be improved, for example, by mutating amino acid residues that do not naturally play an important role in binding, thereby helping to stabilize the interaction between the polypeptides described herein (including the FAM19A5-binding domain of the LRRC4 protein family member) and the FAM19A5 protein.

[0417] Example 10: Binding affinity analysis of various FB-21 peptide mutants The in silico analysis provided in Example 9 highlighted that certain T12 and L13 double mutants may be important in improving the binding affinity of the polypeptides of the present invention to the FAM19A5 protein. Therefore, the ability of wild-type FB-21 peptide (which is identical to the FB-20 peptide described herein, except that the FB-21 peptide contains an additional alanine at the C-terminus) and various FB-21 mutants to have an inhibitory effect on hFc-fused hLRRC4B and FAM19A5 complex formation was tested. Specifically, the sequences for the different FB-21 peptide fragments tested are provided in Table 13 (below). Briefly, plates were coated with 100 nM LRRC4B(453-576,TT / TT)-hFc and then incubated with 1 ng / mL rFAM19A5 and increasing concentrations of various FB-21 peptide fragments (0.03, 0.1, 0.3, 1, 3, 10, 30, 100, 300, and 1000 nM) at 37° C. LRRC4B-bound FAM19A5 concentrations were measured using HRP-conjugated 1-65 antibody.

[0418] [Table 13]

[0419] As shown in Figure 22A (and consistent with the data provided in Example 9), several of the FB-21 peptide mutants tested were able to inhibit the interaction between hFc-fused hLRRC4B and FAM19A5 proteins (see Table 14 for IC50 of inhibition). For example, the ability of the FB-21 (W12Y13) mutant to disrupt the LRRC4B-FAM19A5 complex was increased by 2.9-fold compared to wild-type FB-21.

[0420] [Table 14]

[0421] To further evaluate the inhibitory effect of FB-21 mutants, the ability of additional FB-21 mutants described in Example 9 was tested for their ability to inhibit the interaction between FAM19A5 and LRRC4B proteins. Briefly, plates were coated with 100 nM His-TEV LRRC4B, and then 1 ng / mL rFAM19A5 was incubated at 37° C. in the presence of increasing concentrations (0.3, 1, 3, 10, 30, 100, 300, 1000, 3000, and 10000 nM) of FB-21 peptide fragments. LRRC4B-bound FAM19A5 concentrations were measured using HRP-conjugated 1-65 SS01 antibody. As shown in FIG. 22B (see Table 15 for IC50), most (but not all) of the FB-21 mutations improved their ability to inhibit the interaction between LRRC4B and recombinant FAM19A5 protein. For example, the FB-21(D12Y13) mutation was 2.4- and 7-fold more effective in dissociating LRRC4B-FAM19A5 complex formation compared to FB-21 and FB-21(W12Y13), respectively.

[0422] [Table 15]

[0423] Next, to assess whether other properties of the polypeptides described herein (e.g., solubility, protection from proteases and peptidases, and in vivo administration) could be improved, the following additional FB-21 peptide mutants were constructed and tested for their ability to inhibit the interaction between FAM19A5 protein and LRRC4B: (1) a d-type FB-21 peptide ("dFB-21"), (2) a d-type FB-21 peptide with a juxtamembrane (JM) sequence ("dFB-JM-31"), (3) a d-type FB-21 peptide with a BBB-penetrating sequence at each end of the sequence ("dFB-BBB-39"), and (4) a d-type FB-21 mutant peptide with a DY alternation and an additional JM sequence ("dFB-DY-JM31"). The sequence for the d-form FB-21 peptide is provided as SEQ ID NO: 153 (nYTYFTTVTVETLETQPGEEa; lower case amino acids represent the D-form of the amino acid and upper case amino acids represent the L-form of the amino acid). The sequence for dFB-BBB-39 is provided as SEQ ID NO: 154 (nYTYFTTVTVETLETQPGEEALRKLRKRLLLRKLRKRLl; lower case amino acids represent the D-form of the amino acid and upper case amino acids represent the L-form of the amino acid). The sequence for dFB-JM-31 is provided as SEQ ID NO: 155 (nYTYFTTVTVETLETQPGEEALDEVMKTTKa; lower case amino acids represent the D-form of the amino acid and upper case amino acids represent the L-form of the amino acid). The sequence for dFB-DY-JM31 is presented as SEQ ID NO: 156 (nYTYFTTVTVEDYETQPGEEALDEVMKTTKa; lowercase amino acids represent D-forms of amino acids, uppercase amino acids represent L-forms of amino acids). The sequence for dFB-DY-JM31 is presented as SEQ ID NO: 156 (nYTYFTTVTVEDYETQPGEEALDEVMKTTKa; lowercase amino acids represent D-forms of amino acids, uppercase amino acids represent L-forms of amino acids). The overall experimental method was the same as described above. As shown in FIG. 22D, the JM sequence is a conserved motif in the juxtamembrane region of LRRC4 family genes.As shown in FIG. 22C, FB-21 containing the JM-motif containing mutant peptide with T12L13 to D12Y13 substitution was 4-fold more effective at inhibiting complex formation than wild-type FB-21.

[0424] As can be seen from the above results, the specific modifications described herein (e.g., amino acid substitutions at residues T12 and L13 of the FAM19A5-binding domain of LRRC4 family members; and the addition of a juxtamembrane motif in LRRC4 family members) improve the activity of the polypeptides of the invention to inhibit the interaction between the FAM19A5 protein and a LRRC4 protein family member.

[0425] Example 11: Effect of polypeptides containing the FAM19A5-binding domain of LRRC4 protein family members on amyloid beta-induced synapse loss Alzheimer's disease (AD) is closely related to amyloid-β (Aβ) metabolic disorder. To evaluate whether the polypeptide provided herein (i.e., comprising the FAM19A5 binding domain of LRRC4 protein family member) can have any therapeutic effect on AD, the effect of various FB-21 peptide fragments described herein on synaptic deformation induced by toxic Aβ oligomers and subsequent structural recovery was evaluated. Specifically, the following FB-21 peptide fragments were tested: FB-21, FB-JM-31 and FB-BBB-39 (see Example 10).

[0426] As shown in Figure 23B, it can be seen that the colocalized voxels for PSD95 and synaptophysin are significantly increased upon treatment with the only FB peptide with a JM sequence when co-treated with toxic Aβ oligomers. However, no significant changes were observed in PSD95 and synaptophysin intensity (see Figures 23C and 23D). These results highlight the structural conservation and neuroprotective properties of the polypeptides provided herein (i.e., containing the FAM19A5 binding domain of the LRRC4 protein family member) against toxic Aβ oligomers.

[0427] Example 12: Effect of a polypeptide containing the FAM19A5-binding domain of a LRRC4 protein family member on neurite outgrowth Spinal cord injury (SCI) is a spinal cord injury below the level of injury that induces temporary or permanent changes in motor and / or sensory and / or autonomic functions in the body parts served by the spinal cord. In most cases, the injury occurs due to physical trauma such as falls, car accidents, or sports injuries, but it can also occur due to non-traumatic causes such as infections and tumors. To evaluate the regenerative capacity of motor neurons after injuries such as SCI, the therapeutic effect of the polypeptides described herein on spinal motor neurons was evaluated. Briefly, mouse spinal motor neurons sampled on postnatal day 1 were treated with 10 nM of FB-21 peptide fragments (dFB-dWY-JM31 and dFB-DY-JM31) at 1 and 2 DIV and immunostained with Tau-5 antibody at 3 DIV. Non-treated cells ("NT") were used as the control group.

[0428] As shown in Figures 24A and 24B, an increase in the overall neurite length of spinal motor neurons was observed in SCI-induced mice treated with the LRRC4B peptide fragments described herein. Such results further highlight the therapeutic potential of the polypeptides described herein (i.e., comprising the FAM19A5-binding domain of the LRRC4 protein family member) for the treatment of SCI.

[0429] Example 13: Effect of polypeptides containing the FAM19A5-binding domain of LRRC4 protein family members on 6-OHDA-induced cell death Parkinson's disease (PD) is a long-term degenerative disorder of the central nervous system that primarily affects the motor system through the degeneration of dopaminergic neurons. To evaluate possible neuroprotective potential against PD, the effects of the polypeptides described herein on neurodegeneration and cell death of dopaminergic neurons (often seen in PD) were evaluated. Briefly, Lund human mesencephalic (LUHMES) cells were differentiated into dopaminergic neurons and treated with 6-OHDA (a known neurotoxin that induces PD-like degeneration of dopaminergic neurons) alone or in combination with various doses (10, 30 and 100 nM) of FB-21 peptide fragment (dFB-dWY-JM31) for 12 hours. Some LUHMES cells were treated with FB-21 peptide fragment alone (i.e., without 6-OHDA treatment). Luminescence expression was then measured using a CellTiter-Glo assay.

[0430] As shown in FIG. 25, treatment with FB-21 peptide (dFB-dWY-JM31) and 6-OHDA showed a dose-dependent reversal of LUHMES cell viability, highlighting the potential use of the polypeptides described herein (i.e., containing the FAM19A5-binding domain of the LRRC4 protein family member) as novel therapeutic agents for the treatment of PD.

[0431] Example 14: Effects of polypeptides containing the FAM19A5-binding domain of LRRC4 protein family members on neuropathic pain Neuropathic pain is an intractable disease induced by nerve damage or injury in the peripheral and central nervous systems. The pain is generally described as a burning sensation, and the affected area is often sensitive to touch. To evaluate the analgesic effect of the polypeptides described herein under neuropathic conditions, a chronic constriction injury (CCI) animal model was used. Briefly, after CCI induction, animals were intrathecally injected with vehicle or 50 μg of the FB-21 peptide variant (dFB-dDY-JM31) twice weekly for five times. Mechanical allodymia was then measured with the Von Frey test at 8, 11, 15, and 18 days after CCI induction.

[0432] As shown in Figure 26A, in CCI-induced rats treated with FB-21 peptide fragment (dFB-dDY-JM31), there was an increased paw withdrawal threshold (PWT) at all time points evaluated compared to the vehicle group. The significant difference between treated and control animals was much more clear when the overall results were converted to area under the curve (AUC) (see Figure 26B). These results demonstrate that administration of the polypeptides provided herein (e.g., dFB-dDY-JM31) can be useful in reversing mechanical allodynia induced by CCI, highlighting their potential use as an analgesic agent.

[0433] Example 15: Effects of polypeptides containing the FAM19A5-binding domain of LRRC4 protein family members on retinal dysfunction and regulation of neural oscillations To evaluate the therapeutic effect of the polypeptides provided herein (i.e., comprising the FAM19A5-binding domain of the LRRC4 protein family member) on retinal dysfunction (e.g., induced by diabetic retinopathy (DR)), the amplitudes of bipolar cell and Muller cell-associated b-waves were measured from transgenic diabetic model mice (db / db) by electroretinogram (ERG) examination. Vehicle or 10 μg of dFB-dDY-JM31 was administered weekly by intravitreal (ivt) injection from 12 to 18 weeks.

[0434] As shown in Figure 27, db / db control animals showed significantly decreased b-wave amplitude compared to wild-type litters (db / +). However, when db / db control animals were treated with the FB-21 peptide fragment (dFB-dWY-JM31), the b-wave amplitude was significantly increased. Such results highlight the therapeutic potential of the polypeptide of the present invention (i.e., containing the FAM19A5 binding domain of the LRRC4 protein family member) against retinal dysfunction, including that induced by DR.

[0435] Example 16: Effect of a polypeptide containing the FAM19A5-binding domain of a LRRC4 protein family member on brain injury due to traumatic brain injury To further evaluate the therapeutic effects of the polypeptides described herein, a mouse model of traumatic brain injury (i.e., cold-induced TBI) was used. Approximately 24 hours after TBI induction, the animals were treated (by intranasal administration) with vehicle control or dFB-dWY-JM31 peptide (100 μg). Brain tissue was then harvested and stained with Hoechst 24 hours after treatment administration.

[0436] As shown in Figure 28, compared to TBI-control animals (i.e., treated with vehicle control after TBI induction), TBI animals treated with FB-21 peptide fragments showed a significant reduction in lesion volume. These results demonstrate that the polypeptide of the present invention (e.g., dFB-dWY-JM31) can reduce brain lesion size induced by TBI, indicating its potential as a therapeutic agent for TBI.

[0437] Example 17: Methods and Materials The examples provided herein (see above) use one or more of the following methods: Aβ42 production Aβ42 (#20276) peptide was purchased from AnaSpec (Fremont, USA). A lyophilized aliquot (1 mg) of Aβ42 peptide was dissolved in 80 μl of 1% NH 4 After dissolving in OH, it was dissolved in 920 μl of sterile phosphate-buffered saline (PBS) to obtain a stock solution with a concentration of 1 mg / ml (stored at -20°C in 100 μl aliquots). Working Aβ solutions were prepared 1 day before treatment by diluting the stock concentration to 100 nM final Aβ peptide concentration in Neurobasal medium (Gibco, Life Technologies, USA). The working solutions were incubated at 4°C for 24 hours to obtain the oligomeric state as described in Zheng et al., Amyloid 20(1):13-20 (2013), the entire contents of which are incorporated herein by reference. On the day of use, the working solutions were centrifuged at 14000 g for 10 min at 4°C to purify the oligomeric Aβ fraction from the fibrils.

[0438] Primary hippocampal neuronal culture Primary hippocampal neurons were prepared from postnatal (day 1) C57BL / 6 (Nara Biotech, Seoul, Korea) pups as described previously (Beaudoin et al., Nature protocols 7(9):1741-1754 (2012), the entire contents of which are incorporated herein by reference). Briefly, cortices were dissected in Hank's buffered salt solution (HBSS) (Invitrogen, Carlsbad, CA, USA) and digested with 2.5% trypsin for 15 min at 37°C. The supernatant was removed and the tissue was washed with HBSS. The tissue was gently triturated and dissociated cells were plated on poly-D-lysine-coated glass coverslips in 60 mm culture dishes at 8 × 10 per dish in minimum Eagle's medium (MEM) supplemented with 0.5% glucose, 1 mM fibric acid, 1.2 mM L-glutamine, and 12% fetal bovine serum. 5 Six hours after plating, the medium was replaced with Neurobasal medium (Invitrogen, Carlsbad, CA, USA) supplemented with 2% B-27 and 0.5 mM L-glutamine. Cells were incubated at 4 °C for 2 h in a 5% CO 2 The plates were maintained in a -humified incubator at 37°C. Neurobasal medium was replaced by half every 3 or 4 days.

[0439] Primary spinal motoneuron culture Primary spinal motor neurons were prepared from postnatal (day 1) C57BL / 6 (Nara Biotech, Seoul, Korea) pups as previously described by Eldeiry et al., JoVE (Journal of Visualized Expeirments) 125:255856 (2017), the entire contents of which are incorporated herein by reference. Briefly, spinal cords were dissected in Dulbecco's phosphate-buffered saline (DPBS) (Gibco, Life Technologies, USA) and digested with papain (2.5 mg / ml) at 30°C for 30 min. The supernatant was removed by centrifugation, and the tissue was washed with Hibernate A (Gibco, Life Technologies, USA) supplemented with 2% B-27 and 0.5 mM L-glutamine. The tissue was gently triturated and the dissociated cells were plated in Neurobasal medium (Invitrogen, Carlsbad, CA, USA) supplemented with 2% B-27 and 0.5 mM L-glutamine onto poly-D-lysine and laminin (Thermofisher Scientific, USA)-coated glass coverslips in 12-well plates at 3 × 10 cells per well. 5 The cells were seeded with 100x100 cells / well. The cells were incubated in 5% CO 2 -humified incubator maintained at 37 °C.

[0440] immunostaining Primary neurons were fixed with 4% paraformaldehyde (PFA) at the appropriate DIV. The cells were blocked with 3% bovine serum albumin (BSA) and 0.1% Triton X-100 in phosphate-buffered saline (PBS) for 1 h at room temperature. Then, the cells were treated with primary antibodies overnight at 4°C. The primary antibodies used in this study were mouse anti-Tau5 (Invitrogen, California, United States), rabbit anti-PSD95 (Invitrogen), and mouse anti-synaptophysin (Sigma). After multiple washes with PBS, the appropriate fluorescent-conjugated secondary antibodies were treated with Hoechst33342 (Invitrogen) for 30 min at room temperature. Then, cell images were obtained using a confocal microscope (Leica, Wetzlar, Germany).

[0441] Quantitative analysis of synaptogenesis Hippocampal neurons were treated with 6.6 nM FB-21, 6.6 nM FB-13-JM, and 6.6 nM FB-13-BBBX2 at 14, 17, and 20 DIV and immunostained for SYP, a presynaptic marker protein, and PSD95, a postsynaptic marker protein, to confirm the level of synaptogenesis at 21 DIV. To quantify the fluorescence intensity of SYN and PSD95 and the number of colocalized voxels between SYN and PSD95 signals, z-stack confocal images at a depth of 3 μm were converted into 3D images using IMARIS software (IMARIS9.0, Bitplane AG, Zurich, Switzerland). The "Surface tool" of the IMARIS software was used to exclude all signals detected from neuronal cell bodies, and the number of colocalized voxels between SYN and PSD95 signals in neurites was calculated using the "Coloc tool." The total fluorescence intensity for SYN and PSD95 in the neurites was then obtained.

[0442] Quantitative analysis of neurite outgrowth Mouse hippocampal neurons were treated with LRRC4B peptides at 1 and 2 DIV to measure neurite outgrowth. Three different parameters were measured: total neurite length, number of primary and secondary neurites. Neurons were stained with beta-tubulin III at 3 DIV and then neurite length and branching points were measured using Fiji (Image J, NIH, Bethesda). Individual neurons were manually selected and such parameters were calculated using the Simple neurite tracer plugin. Mouse spinal motor neurons sampled at postnatal day 1 were treated with 10 nM NS101 and LRRC4B-peptides (dFB-dWY-JM31 and dFB-DY-JM31) at 1 and 2 DIV and immunostained with Tau-5 antibody at 3 DIV. Total neurite length and number of imaged somas were measured with the Neurology Image J plugin.

[0443] LUHMES cell culture and differentiation LUHMES human neuronal precursor cells were obtained from ATCC (CRL2927) as previously described (Harischandra et al., Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease 1866(4):165533 (2020), the entire contents of which are incorporated herein by reference). Briefly, plastic culture plates were pre-coated overnight with 50 μg / mL poly-l-ornithine (Sigma Aldrich) and cultured overnight with 1 μg / mL fibronectin (Sigma) after washing twice with cell culture grade water (Invitrogen) at the end of the culture. Cells were cultured in a 24-well plate maintained at 37°C and CO2-free with growth medium composed of Advanced DMEM (Dulbecco's modified Eagle's medium) / F12, N-2 supplement (1x), 2 mM L-glutamine and 40 ng / mL recombinant basic fibroblast growth factor (bFGF) (Sigma). 2The cells were grown in an incubator. During growth, half of the medium was replaced every other day, and the cells were enzymatically dissociated with 0.025% trypsin and subcultured when the cultures reached 80% confluency. Briefly, 3.5 × 10 6 Cells were seeded onto T75 flasks pre-coated with growth medium and cultured for 24 h. The next day, the medium was replaced with freshly prepared differentiation medium and cultured for 48 h to induce differentiation. Differentiation medium consisted of Advanced DMEM / F12, N-2 supplement (1x), 2 mM L-glutamine, 1 mM dibutyryl cAMP, 1 μg / mL tetracycline, and 2 ng / mL recombinant human GDNF (R&D Systems). At the end of the 48-h culture, cells were detached with 0.025% trypsin / EDTA and cultured at 1.5 × 10 cells / ml in differentiation medium. 5 individual cells / cm 2 The cells were replated onto pre-coated plates at a cell density of 100 x 100. Differentiation medium was replaced every other day after cells were replated, and all experiments were performed on day 5 of differentiation unless otherwise stated.

[0444] Quantitative analysis of cell viability Differentiated LUHMES cells were treated with 10, 30, and 100 nM dFB-dWY-JM31 and cell viability levels were measured against 5 μM 6-hydroxydopamine hydrobromide (Tocris) using the CellTiter-Glo Luminescent Cell Viability Assay (Promega). To measure cell viability levels, CellTiter-Glo reagent was added to the cell culture medium present in each well at a 1:1 ratio and the contents were mixed on an orbital shaker to induce cell lysis. The plates were incubated at room temperature for 10 min to stabilize the luminescence signal, and the luminescence signal was read on a microplate reader (Synergy H1, Biotek). Each experiment was performed in triplicate.

[0445] Chronic Constriction Injury (CCI) CCI was performed on normal healthy subjects during the habituation period. The first surgery day was set as Day 0. SD mice were removed from the anesthesia room and immobilized. After disinfecting the surgical site with povidone (betadine) and 70% alcohol, the skin of the left lower leg was incised (0.5 cm deep in the skin, approximately 3-4 cm parallel to the thigh). A small hole was made with tweezers, and a curved needle was inserted into the hole to separate the sciatic nerve and dullness. While observing under a microscope, the membranes (fascia) on both sides of the sciatic nerve were taken with tweezers and incised with fine scissors. The nerve was tied three times at 1 mm intervals with 4-0 sutures.

[0446] Paw Withdrawal Threshold (PWT) Measurement Each mouse was allowed to acclimate to the test environment for at least 30 minutes. To measure the 50% paw withdrawal threshold (PWT), von Frey filaments of 0.4, 0.6, 1.0, 2.0, 4.0, 6.0, 8.0, and 15.0 g were used. A 2.0 g von Frey filament was applied to the hindlimb of CCI mice for 4-5 seconds. If the mouse showed symptoms (raising the paw or twitching), a scaled-down 2.0 g was applied to the mouse. If the mouse did not show any symptoms, a scaled-up 2.0 g was applied to the mouse. In this manner, von Frey filaments of 0.4 g to 15.0 g were applied. At the time when an altered response occurred (the time when the mouse started to respond to the 2.0 g von Frey filament or did not start to respond to the 2.0 g von Frey filament), a 5-fold increase in stimulation was applied to obtain the PWT.

[0447] Electroretinogram (ERG) measurements (db / db) in a mouse model of diabetic retinopathy For the evaluation of diabetic retinal neurodegeneration, db / db and db / + mice were used (see, e.g., Bogdanov et al., PLoS One 9(5):e97302 (2014), the entire contents of which are incorporated herein by reference). ERG was recorded to measure the electrical signal emitted from the retina in response to a light flash. Each mouse underwent an ophthalmologic examination to test ERG according to International Society for Clinical Electrophysiology of Vision standards, and each mouse was acclimated in a dark room for 12 hours. Dark-adapted ERG was performed after injection (6 and 10 weeks). ERG amplitude of the b-wave was measured and was within -0.9 log cd sm -2 The b-waves induced by light intensities of 100-200 nm were compared between groups. ERG analysis was performed using the LabScribeERG (iWorx DataAcquisition Software) program.

[0448] Cold-Induced Traumatic Brain Injury (TBI) After anesthetizing the mice with isoflurane inhalation exposure, each mouse was stably positioned in a stereotaxic device. A 3.0 mm incision was made in the midline scalp. Cold-induced TBI was performed by applying the tip (2.5 mm) of a liquid nitrogen-cooled (-80°C) copper cylinder rod to the right frontal skull for 45 seconds to generate a cryogenic lesion (see, for example, Keskin et al., Neural regeneration research 12(5):761-764(2017), the entire contents of which are incorporated herein by reference). All animals were sacrificed by cardiac perfusion 48 hours after trauma and 24 hours after intranasal injection of vehicle or dFB-dWY-JM31 peptide.

[0449] Quantitative analysis of TBI brain lesions The brains of traumatized mice were removed, and brain sections were taken as a total of 8–9 consecutive coronal sections (20 μm thick) throughout the brain and stained with Hoechst (ThermoFisher, Waltham, MA, USA). The borders of the injured and non-injured regions were demarcated with the Image J software program (NIH, Bethesda, MD, USA). The injury area was assessed by subtracting the area of ​​the non-lesioned ipsilateral hemisphere from the area of ​​the contralateral side. The volume of injury was calculated by integrating these lesion areas. All 8–9 sections were measured individually, and the corresponding volumes were calculated.

[0450] statistical analysis All statistical analyses were performed using GraphPad Prism 5 (GraphPad Software Inc., California, United States) and data are presented as mean ± standard error of the mean (SEM). Statistical significance was assessed using Student's t-test and / or one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. p-values ​​less than 0.05 were considered statistically significant.

[0451] It should be understood that the Detailed Description section, and not the Summary and Abstract sections, are intended to be used when analyzing the claims. The Summary and Abstract sections may describe one or more but not all exemplary aspects of the invention contemplated by the inventor(s) and thus are not intended to limit the scope of the invention and the appended claims in any respect.

[0452] The present invention has been described in detail with the aid of functional building blocks illustrating the implementation of certain functions and relationships thereof. The boundaries of such functional building blocks have been arbitrarily defined herein for the convenience of description. Alternative boundaries may be defined so long as the specified functions and relationships thereof are appropriately performed.

[0453] The foregoing description of specific aspects is intended to fully express the general characteristics of the inventive subject matter so that others may easily modify and / or adapt it to various adaptations of such specific aspects without undue experimentation, applying knowledge of the art. Thus, without departing from the general concept of the invention, such adaptations and modifications are intended to be within the meaning and range of equivalents of the presented aspects based on the description and guidance presented herein. The phraseology or terminology of this specification should be understood as being illustrative and not limiting, and therefore the phraseology or terminology of this specification should be interpreted by skilled artisans in light of the description and guidance.

[0454] The breadth and scope of the present invention should not be limited by any of the above exemplary aspects, but should be defined only in accordance with the following claims and their equivalents.

[0455] All publications, patents, patent applications, internet sites and accession numbers / database sequences (including both polynucleotide and polypeptide sequences) cited herein are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, internet site or accession number / database sequence was specifically and individually indicated to be incorporated by reference. [Brief description of the drawings]

[0456] [Figure 1A]Figures 1A-1D show the activity of different members of the LRRC4 protein family (i.e., LRRC4C, LRRC4, and LRRC4B proteins) to bind to FAM19A5 protein, as measured by co-immunoprecipitation (Figures 1A and 1B) or immunofluorescence assay (Figures 1C and 1D). In Figure 1A, cell lysates (from HEK293 cells expressing FLAG-tagged LRRC4C, LRRC4, or LRRC4B proteins and treated with recombinant FAM19A5 protein) were immunoprecipitated with anti-FLAG antibody, and the immunoprecipitated proteins were immunoblotted with anti-FLAG (top row) and anti-FAM19A5(3-2) (bottom row) antibodies. [Figure 1B] Figures 1A-1D show the activity of different members of the LRRC4 protein family (i.e., LRRC4C, LRRC4, and LRRC4B proteins) binding to FAM19A5 protein measured using co-immunoprecipitation (Figures 1A and 1B) or immunofluorescence assay (Figures 1C and 1D). In Figure 1B, cell lysates (from HEK293 cells expressing FLAG-tagged LRRC4B protein and treated with recombinant FAM19A5 protein) were immunoprecipitated with human IgG ("IgG") or anti-FAM19A5(1-65) antibody ("1-65"). Immunoprecipitated proteins were immunoblotted with anti-FLAG (top row) and anti-FAM19A5(3-2) (bottom row) antibodies. [Figure 1C]Figures 1A-1D show the activity of different members of the LRRC4 protein family (i.e., LRRC4C, LRRC4, and LRRC4B proteins) binding to FAM19A5 protein, measured using co-immunoprecipitation (Figures 1A and 1B) or immunofluorescence assay (Figures 1C and 1D). In Figure 1C, HEK293 cells expressing FLAG-tagged LRRC4B protein were treated with recombinant FAM19A5 protein and immunostained with anti-FLAG and anti-FAM19A5 (3-2) antibodies. Furthermore, the images provided in the second (FAM19A5 protein staining alone), third (LRRC4B protein staining alone), and fourth (overlay of FAM19A5 and LRRC4B staining) columns of Figures 1C and 1D are enlargements of the boxed regions in the images provided in the first row. Colocalized signals are indicated by arrowheads. Scale bar = 30 μm. [Figure 1D] Figures 1A-1D show the activity of different members of the LRRC4 protein family (i.e., LRRC4C, LRRC4, and LRRC4B proteins) binding to FAM19A5 protein, measured using co-immunoprecipitation (Figures 1A and 1B) or immunofluorescence assay (Figures 1C and 1D). In Figure 1D, primary cortical neurons were treated with recombinant FAM19A5 protein and immunostained with anti-FAM19A5(3-2) and anti-LRRC4B antibodies. In Figures 1C and 1D, nuclei were stained with Hoechst33342. Furthermore, the images provided in the second (FAM19A5 protein staining alone), third (LRRC4B protein staining alone), and fourth (overlay of FAM19A5 and LRRC4B staining) columns of Figures 1C and 1D are enlargements of the boxed areas in the images provided in the first row. Colocalized signals are indicated by arrowheads. Scale bar = 30 μm. [Figure 2A]Figures 2A-2D show the binding of LRRC4B protein to FAM19A5 protein isoforms 1 and 2 as measured using immunofluorescence (Figures 2A and 2B) or co-immunoprecipitation assays (Figures 2C and 2D). Figure 2A provides immunofluorescence data showing the interaction between LRRC4B protein and FAM19A5 isoform 1. [Figure 2B] Figures 2A-2D show the binding of LRRC4B protein to FAM19A5 protein isoforms 1 and 2 as measured using immunofluorescence (Figures 2A and 2B) or co-immunoprecipitation assays (Figures 2C and 2D). Figure 2B provides immunofluorescence data showing the interaction between LRRC4B protein and FAM19A5 isoform 2. [Figure 2C] Figures 2A-D show the binding of LRRC4B protein to isoform 1 and isoform 2 of FAM19A5 protein measured using immunofluorescence (Figures 2A and 2B) or co-immunoprecipitation assays (Figures 2C and 2D). In Figure 2C, cell lysates from co-transfected HEK293 cells were immunoprecipitated with anti-FLAG antibody and then immunoblotted with anti-FLAG (top row) and anti-FAM19A5(3-2) (bottom row) antibodies. [Figure 2D] Figures 2A-D show the binding of LRRC4B protein to isoform 1 and isoform 2 of FAM19A5 protein measured using immunofluorescence (Figures 2A and 2B) or co-immunoprecipitation assays (Figures 2C and 2D). In Figure 2D, cell lysates from co-transfected HEK293 cells were immunoprecipitated with the following antibodies: (i) human IgG antibody ("IgG"); (ii) anti-FAM19A5(1-65) antibody ("1-65"); or (iii) anti-FAM19A5(3-2) antibody ("3-2"). Immunoprecipitated proteins were immunoblotted with anti-FLAG (top row) and anti-FAM19A5(3-2) (bottom row) antibodies. [Figure 3A]Figures 3A and 3B show binding of different LRRC4B protein deletion constructs to FAM19A5 protein. Figure 3A provides a schematic diagram of different domains of the LRRC4B protein, showing the domains included in the different deletion constructs. The LRRC4B domains shown include: "SP" = signal peptide; "LRR" = leucine-rich repeat; "IG" = immunoglobulin-like C2 type; "Thr" = threonine-rich; "TM" = transmembrane; and "PB" = PSD95 binding. The column under "Binding" indicates whether a particular LRRC4B protein fragment bound to FAM19A5 protein: "O" = bound; "X" = not bound; "ND" = not confirmed. [Figure 3B] Figures 3A and 3B show the binding of different LRRC4B protein deletion constructs to FAM19A5 protein, as measured using a co-immunoprecipitation assay. [Figure 4A] Figures 4A and 4B show the binding of FAM19A5 protein to the ectodomain of LRRC4 protein family members as measured using ELISA. Figure 4A provides data showing the binding of FAM19A5 protein to the full-length ectodomain of LRRC4 (amino acids 39-527 of SEQ ID NO:1; i.e., SEQ ID NO:4) ("1"), LRRC4B (amino acids 36-527 of SEQ ID NO:2; i.e., SEQ ID NO:5) ("2") and LRRC4C (amino acids 45-527 of SEQ ID NO:3; i.e., SEQ ID NO:6) ("3") proteins. [Figure 4B]Figures 4A and 4B show the binding of FAM19A5 protein to the ectodomain of LRRC4 protein family members measured using ELISA. Figure 4B provides data showing the binding of FAM19A5 protein to different fragments of LRRC4B protein: (a) amino acids 453-576 of SEQ ID NO:2 (i.e., SEQ ID NO:7); (b) amino acids 484-576 of SEQ ID NO:2 (i.e., SEQ ID NO:8); (c) amino acids 482-576 of SEQ ID NO:2 (i.e., SEQ ID NO:9); (d) amino acids 482-497 of SEQ ID NO:2 (i.e., SEQ ID NO:10); and (e) amino acids 498-576 of SEQ ID NO:2 (i.e., SEQ ID NO:11). [Figure 5A] 5A and 5B show binding of FAM19A5 protein to the following protein fragments of LRRC4 protein family members: (1) LRRC4 (amino acids 451-483 of SEQ ID NO:1) (i.e., SEQ ID NO:12); (2) LRRC4C (amino acids 451-484 of SEQ ID NO:3) (i.e., SEQ ID NO:13); and (3) LRRC4B (amino acids 484-522 of SEQ ID NO:2) (i.e., SEQ ID NO:14). FIG. 5A provides a schematic diagram of the distinct domains present within the LRRC4 protein family members, including the amino acid sequences of the protein fragments tested. The domains represented include: "SP" = signal peptide; "LRR" = leucine-rich repeats; "IG" = immunoglobulin-like C2 type; "Thr" = threonine-rich; "TM" = transmembrane; and "PB" = PSD95 binding. The column under "Binding" indicates whether a particular LRRC4B protein fragment bound to the FAM19A5 protein: "O" = bound; "X" = not bound; "ND" = not confirmed. [Figure 5B]5A and 5B show binding of FAM19A5 protein to the following protein fragments of LRRC4 protein family members: (1) LRRC4 (amino acids 451-483 of SEQ ID NO:1) (i.e., SEQ ID NO:12); (2) LRRC4C (amino acids 451-484 of SEQ ID NO:3) (i.e., SEQ ID NO:13); and (3) LRRC4B (amino acids 484-522 of SEQ ID NO:2) (i.e., SEQ ID NO:14). FIG. 5A provides a schematic diagram of the distinct domains present within the LRRC4 protein family members, including the amino acid sequences of the protein fragments tested. The domains represented include: "SP" = signal peptide; "LRR" = leucine-rich repeats; "IG" = immunoglobulin-like C2 type; "Thr" = threonine-rich; "TM" = transmembrane; and "PB" = PSD95 binding. The column under "Binding" indicates whether a particular LRRC4B protein fragment bound to the FAM19A5 protein: "O" = bound; "X" = not bound; "ND" = not confirmed. [Figure 6] FIG. 6 shows the activity of three different peptide fragments containing the YTYFTTVTVETLE (SEQ ID NO: 15) sequence of LRRC4B protein binding to FAM19A5 protein: (1) "FB-16" = 16 amino acids long (SEQ ID NO: 17); (2) "FB-20" = 20 amino acids long (SEQ ID NO: 18); and (3) "FB-28" = 28 amino acids long (SEQ ID NO: 19). [Figure 7]FIG. 7 shows the activity of the LRRC4B peptide fragment (amino acids 453-576 of SEQ ID NO:2) (i.e., SEQ ID NO:7) (bottom row) in inducing dissociation of the interaction between FAM19A5 (isoform 2) and full-length LRRC4B protein in HEK293 cells as measured using immunofluorescence microscopy. HEK293 cells treated with a mutant form of the LRRC4B peptide fragment (containing alanine substitutions at positions 488 and 489 of SEQ ID NO:2) (i.e., SEQ ID NO:16) ("MT") were used as a control. The boxed images (bottom row, see fourth box from the left) were enlarged as images stained with anti-hIgG alone (top row) and both anti-hIgG and anti-FLAG antibodies (bottom row). The filled arrowheads in the enlarged images indicate the FAM19A5 signal dissociated from LRRC4B. The unfilled arrowheads indicate LRRC4B(453-576)-hFc, where LRRC4B is present. Scale bar = 30 μm. [Figure 8A] 8A and 8B provide competitive inhibition assay data comparing the ability of different LRRC4B peptide fragments to inhibit binding of FAM19A5 protein to the full-length ectodomain of LRRC4B protein (i.e., amino acids 36-576 of SEQ ID NO:2) (SEQ ID NO:5). Figure 8A provides data for the following LRRC4B peptide fragments: (1) LRRC4B (amino acids 453-576 of SEQ ID NO:2) (SEQ ID NO:7); (2) LRRC4B mutant (amino acids 453-576 of SEQ ID NO:2 with AA mutations at positions 488 and 489) (SEQ ID NO:16); (3) LRRC4B (amino acids 484-576 of SEQ ID NO:2) (SEQ ID NO:8); (4) LRRC4B (amino acids 482-576 of SEQ ID NO:2) (SEQ ID NO:9); (5) LRRC4B (amino acids 482-497 of SEQ ID NO:2) (SEQ ID NO:10); and (6) LRRC4B (amino acids 498-576 of SEQ ID NO:1) (SEQ ID NO:11). [Figure 8B]Figures 8A and 8B provide competitive inhibition assay data comparing the activity of different LRRC4B peptide fragments to inhibit binding of FAM19A5 protein to the full-length ectodomain of LRRC4B protein (i.e., amino acids 36-576 of SEQ ID NO:2) (SEQ ID NO:5). Figure 8B provides competitive inhibition assay data showing the activity of (1) FB-28, (2) FB-20, and (3) FB-16 peptides (described in Figure 6) to inhibit FAM19A5 protein binding to the full-length ectodomain of LRRC4B protein. [Figure 9A] Figures 9A-9C compare the activity of different LRRC4B peptide fragments in inhibiting the binding of FAM19A5 protein to the full-length ectodomain of different members of the LRRC4 protein family: LRRC4 (amino acid residues 39-572 of SEQ ID NO:1) (i.e., SEQ ID NO:4), LRRC4B (amino acid residues 36-576 of SEQ ID NO:2) (i.e., SEQ ID NO:5), and LRRC4C (amino acid residues 345-527 of SEQ ID NO:6). The distinct LRRC4B peptide fragments presented include: (1) LRRC4B (amino acids 453-576 of SEQ ID NO:2) (SEQ ID NO:7); (2) LRRC4B mutant (amino acids 453-576 of SEQ ID NO:2 with AA mutations at positions 488 and 489) (i.e., SEQ ID NO:16); and (3) FB-20 (i.e., a 20-amino acid long peptide fragment containing the YTYFTTVTVETLE sequence of the LRRC4B protein; GYTYFTTVTVETLETQPGEE; SEQ ID NO:18). [Figure 9B]Figures 9A-9C compare the activity of different LRRC4B peptide fragments in inhibiting the binding of FAM19A5 protein to the full-length ectodomain of different members of the LRRC4 protein family: LRRC4 (amino acid residues 39-572 of SEQ ID NO:1) (i.e., SEQ ID NO:4), LRRC4B (amino acid residues 36-576 of SEQ ID NO:2) (i.e., SEQ ID NO:5), and LRRC4C (amino acid residues 345-527 of SEQ ID NO:6). The distinct LRRC4B peptide fragments presented include: (1) LRRC4B (amino acids 453-576 of SEQ ID NO:2) (SEQ ID NO:7); (2) LRRC4B mutant (amino acids 453-576 of SEQ ID NO:2 with AA mutations at positions 488 and 489) (i.e., SEQ ID NO:16); and (3) FB-20 (i.e., a 20-amino acid long peptide fragment containing the YTYFTTVTVETLE sequence of the LRRC4B protein; GYTYFTTVTVETLETQPGEE; SEQ ID NO:18). [Figure 9C] Figures 9A-9C compare the activity of different LRRC4B peptide fragments in inhibiting the binding of FAM19A5 protein to the full-length ectodomain of different members of the LRRC4 protein family: LRRC4 (amino acid residues 39-572 of SEQ ID NO:1) (i.e., SEQ ID NO:4), LRRC4B (amino acid residues 36-576 of SEQ ID NO:2) (i.e., SEQ ID NO:5), and LRRC4C (amino acid residues 345-527 of SEQ ID NO:6). The distinct LRRC4B peptide fragments presented include: (1) LRRC4B (amino acids 453-576 of SEQ ID NO:2) (SEQ ID NO:7); (2) LRRC4B mutant (amino acids 453-576 of SEQ ID NO:2 with AA mutations at positions 488 and 489) (i.e., SEQ ID NO:16); and (3) FB-20 (i.e., a 20-amino acid long peptide fragment containing the YTYFTTVTVETLE sequence of the LRRC4B protein; GYTYFTTVTVETLETQPGEE; SEQ ID NO:18). [Figure 10A]10A and 10B compare the activity of the FBC4-23 and FBC4C-23 peptide fragments in inhibiting binding of FAM19A5 protein to the full-length ectodomain of LRRC4B protein (FIG. 10A) or to the threonine-rich domain of LRRC4B protein (i.e., amino acids 453-576 of SEQ ID NO:2; i.e., SEQ ID NO:7) (FIG. 10B). The FBC4C-23 peptide fragment contains the FAM19A5 binding domain of LRRC4C protein (bold and italics) and has the following sequence:

[0457] [ka]

[0458] The FBC4C-23 peptide fragment contains the FAM19A5 binding domain of the LRRC4C protein (bold and italicized) and has the following sequence:

[0459] [ka]

[0460] The FB-20 peptide (see FIG. 6) was also used for comparison purposes. [Figure 10B] 10A and 10B compare the activity of the FBC4-23 and FBC4C-23 peptide fragments in inhibiting binding of FAM19A5 protein to the full-length ectodomain of LRRC4B protein (FIG. 10A) or to the threonine-rich domain of LRRC4B protein (i.e., amino acids 453-576 of SEQ ID NO:2; i.e., SEQ ID NO:7) (FIG. 10B). The FBC4C-23 peptide fragment contains the FAM19A5 binding domain of LRRC4C protein (bold and italics) and has the following sequence:

[0461] [ka]

[0462] The FBC4C-23 peptide fragment contains the FAM19A5 binding domain of the LRRC4C protein (bold and italicized) and has the following sequence:

[0463] [ka]

[0464] The FB-20 peptide (see FIG. 6) was also used for comparison purposes. [Figure 11A] 11A and 11B show the activity of different FB-20 peptide fragment variants that inhibit binding of FAM19A5 protein to the full-length ectodomain of LRRC4B protein (FIG. 11A) or to an LRRC4B protein fragment containing the FAM19A5 binding domain (i.e., amino acids 453-576 of SEQ ID NO:2; SEQ ID NO:7) (FIG. 11B). The different FB-20 variants are: (1) FB-ml ldC, (2) FB-mlOdC, (3) FB-m9dC, (4) FB-m8dC, (5) FB-m7dC, (6) FB-m6dC, (7) FB-mlOdN, (8) FB-m9dN, (9) FB-m8dN, and (10) FB-m7dN. As described in Example 6, each FB-20 variant contained one or more amino acid deletions, YTYFTTVTVETLE (SEQ ID NO: 15), at the C-terminus or N-terminus of the LRRC4B protein domain capable of binding to the FAM19A5 protein. The specific amino acid sequences of the FB-20 variants are provided in Table 9. [Figure 11B]11A and 11B show the activity of different FB-20 peptide fragment variants that inhibit binding of FAM19A5 protein to the full-length ectodomain of LRRC4B protein (FIG. 11A) or to an LRRC4B protein fragment containing the FAM19A5 binding domain (i.e., amino acids 453-576 of SEQ ID NO:2; SEQ ID NO:7) (FIG. 11B). The different FB-20 variants are: (1) FB-ml ldC, (2) FB-mlOdC, (3) FB-m9dC, (4) FB-m8dC, (5) FB-m7dC, (6) FB-m6dC, (7) FB-mlOdN, (8) FB-m9dN, (9) FB-m8dN, and (10) FB-m7dN. As described in Example 6, each FB-20 variant contained one or more amino acid deletions, YTYFTTVTVETLE (SEQ ID NO: 15), at the C-terminus or N-terminus of the LRRC4B protein domain capable of binding to the FAM19A5 protein. The specific amino acid sequences of the FB-20 variants are provided in Table 9. [Figure 12A] 12A and 12B show the activity of different FB-20 peptide fragment variants with alanine (A) or asparagine (N) substitutions that inhibit FAM19A5 protein binding to the full-length ectodomain of LRRC4B protein (FIG. 12A) or to the LRRC4B protein fragment containing the FAM19A5 binding domain (i.e., amino acids 453-576 of SEQ ID NO:2; SEQ ID NO:7) (FIG. 12B). As described in Example 7, alanine or asparagine substitutions were independently introduced into the FB-20 peptide fragment at one of the amino acid residues in the LRRC4B protein domain capable of binding to FAM19A5 protein, i.e., YTYFTTVTVETLE (SEQ ID NO:15). The specific amino acid sequences of the FB-20 variants are provided in Table 10. For each FB-20 peptide variant in the figure (excluding FB-20[12-L] and FB-20[13-E]), the first bar is the alanine substitution and the second bar is the asparagine substitution. Only alanine substitutions are displayed in mutants FB-20[12-L] and FB-20[13-E]. [Figure 12B]12A and 12B show the activity of different FB-20 peptide fragment variants with alanine (A) or asparagine (N) substitutions that inhibit FAM19A5 protein binding to the full-length ectodomain of LRRC4B protein (FIG. 12A) or to the LRRC4B protein fragment containing the FAM19A5 binding domain (i.e., amino acids 453-576 of SEQ ID NO:2; SEQ ID NO:7) (FIG. 12B). As described in Example 7, alanine or asparagine substitutions were independently introduced into the FB-20 peptide fragment at one of the amino acid residues in the LRRC4B protein domain capable of binding to FAM19A5 protein, i.e., YTYFTTVTVETLE (SEQ ID NO:15). The specific amino acid sequences of the FB-20 variants are provided in Table 10. For each FB-20 peptide variant in the figure (excluding FB-20[12-L] and FB-20[13-E]), the first bar is the alanine substitution and the second bar is the asparagine substitution. Only alanine substitutions are displayed in mutants FB-20[12-L] and FB-20[13-E]. [Figure 13A] Figures 13A and 13B show the transcription levels of FAM19A5 family members (Figure 13A) or LRRC4B and PTPRF genes (Figure 13B) in mouse hippocampal cultures. Primary hippocampal neurons from mouse brains were cultured in vitro for 15 days at postnatal day 1 as described in Example 8. Transcription levels of distinct genes were measured 1, 3, 7, 10 and 15 days after initial culture and quantified using RNA-seq analysis. In Figure 13A, for each day indicated, the first, second and third bars (from left to right) correspond to FAM19A1, FAM19A2 and FAM19A5, respectively. FAM19A3 and FAM19A4 transcripts were not detected. Data are mean ± SEM of triplicates. [Figure 13B]Figures 13A and 13B show the transcription levels of FAM19A5 family members (Figure 13A) or LRRC4B and PTPRF genes (Figure 13B) in mouse hippocampal cultures. Primary hippocampal neurons from mouse brains were cultured in vitro for 15 days at postnatal day 1 as described in Example 8. Transcription levels of distinct genes were measured 1, 3, 7, 10 and 15 days after initial culture and quantified using RNA-seq analysis. In Figure 13A, for each day indicated, the first, second and third bars (from left to right) correspond to FAM19A1, FAM19A2 and FAM19A5, respectively. FAM19A3 and FAM19A4 transcripts were not detected. Data are mean ± SEM of triplicates. [Figure 14A] 14A-14D show the activity of LRRC4B peptide fragments (amino acid residues 453-576 of SEQ ID NO:2; SEQ ID NO:7) in promoting neurite outgrowth of mouse primary cortical neurons in vitro at various concentrations (x-axis) (0.006-60 nM). Mouse primary cortical neurons (postnatal day 1) were treated with LRRC4B protein fragments 1 and 2 days after initial culture as described in Example 8, and the following were immunostained with beta-tubulin III antibody and quantified at day 3: (i) mean total neurite outgrowth (FIG. 14A), (ii) number of primary dendrites (FIG. 14B), (iii) number of branch points (FIG. 14C), and (iv) number of secondary neurites (FIG. 14D). Data are presented as mean±SEM. Statistical significance was assessed using one-way ANOVA with Bonferroni post-hoc test; a, P<0.01 vs. vehicle control group. [Figure 14B]14A-14D show the activity of LRRC4B peptide fragments (amino acid residues 453-576 of SEQ ID NO:2; SEQ ID NO:7) in promoting neurite outgrowth of mouse primary cortical neurons in vitro at various concentrations (x-axis) (0.006-60 nM). Mouse primary cortical neurons (postnatal day 1) were treated with LRRC4B protein fragments 1 and 2 days after initial culture as described in Example 8, and the following were immunostained with beta-tubulin III antibody and quantified at day 3: (i) mean total neurite outgrowth (FIG. 14A), (ii) number of primary dendrites (FIG. 14B), (iii) number of branch points (FIG. 14C), and (iv) number of secondary neurites (FIG. 14D). Data are presented as mean±SEM. Statistical significance was assessed using one-way ANOVA with Bonferroni post-hoc test; a, P<0.01 vs. vehicle control group. [Figure 14C] 14A-14D show the activity of LRRC4B peptide fragments (amino acid residues 453-576 of SEQ ID NO:2; SEQ ID NO:7) in promoting neurite outgrowth of mouse primary cortical neurons in vitro at various concentrations (x-axis) (0.006-60 nM). Mouse primary cortical neurons (postnatal day 1) were treated with LRRC4B protein fragments 1 and 2 days after initial culture as described in Example 8, and the following were immunostained with beta-tubulin III antibody and quantified at day 3: (i) mean total neurite outgrowth (FIG. 14A), (ii) number of primary dendrites (FIG. 14B), (iii) number of branch points (FIG. 14C), and (iv) number of secondary neurites (FIG. 14D). Data are presented as mean±SEM. Statistical significance was assessed using one-way ANOVA with Bonferroni post-hoc test; a, P<0.01 vs. vehicle control group. [Figure 14D]14A-14D show the activity of LRRC4B peptide fragments (amino acid residues 453-576 of SEQ ID NO:2; SEQ ID NO:7) in promoting neurite outgrowth of mouse primary cortical neurons in vitro at various concentrations (x-axis) (0.006-60 nM). Mouse primary cortical neurons (postnatal day 1) were treated with LRRC4B protein fragments 1 and 2 days after initial culture as described in Example 8, and the following were immunostained with beta-tubulin III antibody and quantified at day 3: (i) mean total neurite outgrowth (FIG. 14A), (ii) number of primary dendrites (FIG. 14B), (iii) number of branch points (FIG. 14C), and (iv) number of secondary neurites (FIG. 14D). Data are presented as mean±SEM. Statistical significance was assessed using one-way ANOVA with Bonferroni post-hoc test; a, P<0.01 vs. vehicle control group. [Figure 15A] 15A-15C show the efficacy of LRRC4B peptide fragment (amino acid residues 453-576 of SEQ ID NO:2; SEQ ID NO:7) on the expression of synaptophysin (SYP; presynaptic marker) and PSD95 (postsynaptic marker) in mouse hippocampal neurons. FIG. 15A and FIG. 15B show the total fluorescence intensity for SYN and PSD-95, respectively, in dendrites / neurites of hippocampal neurons with LRRC4B peptide fragment (6 or 60 nM) as measured using IMARIS software (IMARIS 9.0 Bitplane, Switzerland). In each of FIG. 15A-15C, vehicle ("Veh") and LRRC4B peptide fragment mutant (MT) (60 nM) (i.e., containing alanine substitutions at positions 488 and 489 of SEQ ID NO:2; SEQ ID NO:16) were used as controls. As described elsewhere herein, LRRC4B MT was not able to bind to FAM19A5 protein. Data are presented as mean ± SEM. The number of neurons used for quantifying the fluorescence intensity is indicated in brackets in the bar graphs. Statistical significance was assessed using one-way ANOVA with Bonferroni post-hoc test. a, P < 0.05 vs Veh; b, P < 0.05 vs LRRC4B MT (60 nM). [Figure 15B]15A-15C show the efficacy of LRRC4B peptide fragment (amino acid residues 453-576 of SEQ ID NO:2; SEQ ID NO:7) on the expression of synaptophysin (SYP; presynaptic marker) and PSD95 (postsynaptic marker) in mouse hippocampal neurons. FIG. 15A and FIG. 15B show the total fluorescence intensity for SYN and PSD-95, respectively, in dendrites / neurites of hippocampal neurons with LRRC4B peptide fragment (6 or 60 nM) as measured using IMARIS software (IMARIS 9.0 Bitplane, Switzerland). In each of FIG. 15A-15C, vehicle ("Veh") and LRRC4B peptide fragment mutant (MT) (60 nM) (i.e., containing alanine substitutions at positions 488 and 489 of SEQ ID NO:2; SEQ ID NO:16) were used as controls. As described elsewhere herein, LRRC4B MT was not able to bind to FAM19A5 protein. Data are presented as mean ± SEM. The number of neurons used for quantifying the fluorescence intensity is indicated in brackets in the bar graphs. Statistical significance was assessed using one-way ANOVA with Bonferroni post-hoc test. a, P < 0.05 vs Veh; b, P < 0.05 vs LRRC4B MT (60 nM). [Figure 15C]15A-15C show the efficacy of LRRC4B peptide fragment (amino acid residues 453-576 of SEQ ID NO:2; SEQ ID NO:7) on the expression of synaptophysin (SYP; presynaptic marker) and PSD95 (postsynaptic marker) in mouse hippocampal neurons. FIG. 15C shows the number of colocalized voxels between SYP and PSD95 signals in dendrites / neurites of treated hippocampal neurons. In each of FIGS. 15A-15C, vehicle ("Veh") and LRRC4B peptide fragment mutant (MT) (60 nM) (i.e., containing alanine substitutions at positions 488 and 489 of SEQ ID NO:2; SEQ ID NO:16) were used as controls. As described elsewhere herein, LRRC4B MT was unable to bind to FAM19A5 protein. Data are shown as mean±SEM. The number of neurons used for quantifying the fluorescence intensity is indicated in brackets of the bar graph. Statistical significance was evaluated using one-way ANOVA with Bonferroni post-hoc test. a, P < 0.05 vs Veh; b, P < 0.05 vs LRRC4B MT (60 nM). [Figure 16A] 16A-C show the ability of the LRRC4B peptide fragment (amino acid residues 453-576 of SEQ ID NO:2; "WT") (i.e., SEQ ID NO:7) to promote synapse formation in the hippocampal CA1 of APP / PS1 mice. As also described in Example 8, APP / PS1 mice were treated with the LRRC4B peptide fragment (30 mg / kg; intravenous administration) for 4 consecutive weeks, and then synapse formation was assessed by fluorescence microscopy using antibodies against SYP and PSD95. Control animals were either untreated ("cont") or treated with the mutant LRRC4B peptide fragment (60 nM) (i.e., containing alanine substitutions at positions 488 and 489 of SEQ ID NO:2; SEQ ID NO:16). FIG. 16A provides representative fluorescence micrographs. [Figure 16B]16A-16C show the ability of the LRRC4B peptide fragment (amino acid residues 453-576 of SEQ ID NO:2; "WT") (i.e., SEQ ID NO:7) to promote synapse formation in the hippocampal CA1 of APP / PS1 mice. As described in Example 8, APP / PS1 mice were treated with the LRRC4B peptide fragment (30 mg / kg; intravenous administration) for 4 consecutive weeks, and then synapse formation was evaluated by fluorescence microscopy using antibodies against SYP and PSD95. Control animals were either untreated ("cont") or treated with a mutant LRRC4B peptide fragment (60 nM) (i.e., containing alanine substitutions at positions 488 and 489 of SEQ ID NO:2; SEQ ID NO:16). FIG. 16B and FIG. 16C show SYP and PSD95 intensity, respectively. [Figure 16C] 16A-16C show the ability of the LRRC4B peptide fragment (amino acid residues 453-576 of SEQ ID NO:2; "WT") (i.e., SEQ ID NO:7) to promote synapse formation in the hippocampal CA1 of APP / PS1 mice. As described in Example 8, APP / PS1 mice were treated with the LRRC4B peptide fragment (30 mg / kg; intravenous administration) for 4 consecutive weeks, and then synapse formation was evaluated by fluorescence microscopy using antibodies against SYP and PSD95. Control animals were either untreated ("cont") or treated with a mutant LRRC4B peptide fragment (60 nM) (i.e., containing alanine substitutions at positions 488 and 489 of SEQ ID NO:2; SEQ ID NO:16). FIG. 16B and FIG. 16C show SYP and PSD95 intensity, respectively. [Figure 17A] Figures 17A-C show the activity of the LRRC4B peptide fragment (amino acid residues 453-576 of SEQ ID NO:2; "WT"; SEQ ID NO:7) in promoting synaptogenesis in the hippocampal CA3 of APP / PS1 mice. The animals were treated and analyzed as described in Figures 16A-C. Figure 17A provides representative fluorescent micrographs. [Figure 17B]Figures 17A-C show the activity of the LRRC4B peptide fragment (amino acid residues 453-576 of SEQ ID NO:2; "WT"; SEQ ID NO:7) in promoting synaptogenesis in the hippocampal CA3 of APP / PS1 mice. The animals were treated and analyzed as described in Figures 16A-C. Figures 17B and 17C show SYP and PSD95 intensity, respectively. [Figure 17C] Figures 17A-C show the activity of the LRRC4B peptide fragment (amino acid residues 453-576 of SEQ ID NO:2; "WT"; SEQ ID NO:7) in promoting synaptogenesis in the hippocampal CA3 of APP / PS1 mice. The animals were treated and analyzed as described in Figures 16A-C. Figures 17B and 17C show SYP and PSD95 intensity, respectively. [Figure 18A] Figures 18A-E show neurite outgrowth in mouse primary cortical neurons treated in vitro with FB-16, FB-20 and FB-28 peptides (described in Figure 6). Primary cortical neurons were treated for 2 days, and neurite outgrowth was assessed on day 3 by immunostaining with anti-beta tubulin III antibody. Figure 18A provides representative microscopy images from each treatment group. [Figure 18B] Figures 18A-E show neurite outgrowth in mouse primary cortical neurons treated in vitro with FB-16, FB-20, and FB-28 peptides (described in Figure 6). Primary cortical neurons were treated for 2 days, and neurite outgrowth was assessed on the third day by immunostaining with anti-beta-tubulin III antibody. Figures 18B-E show (i) the average length of total neurite outgrowth, (ii) the number of primary dendrites, (iii) the number of branch points, and (iv) the number of secondary neurites, respectively. Data are presented as mean ± SEM. Statistical significance was assessed using one-way ANOVA with Bonferroni post-hoc test; a, P<0.01 vs control group (CTRL). [Figure 18C]Figures 18A-E show neurite outgrowth in mouse primary cortical neurons treated in vitro with FB-16, FB-20, and FB-28 peptides (described in Figure 6). Primary cortical neurons were treated for 2 days, and neurite outgrowth was assessed on the third day by immunostaining with anti-beta-tubulin III antibody. Figures 18B-E show (i) the average length of total neurite outgrowth, (ii) the number of primary dendrites, (iii) the number of branch points, and (iv) the number of secondary neurites, respectively. Data are presented as mean ± SEM. Statistical significance was assessed using one-way ANOVA with Bonferroni post-hoc test; a, P<0.01 vs control group (CTRL). [Figure 18D] Figures 18A-E show neurite outgrowth in mouse primary cortical neurons treated in vitro with FB-16, FB-20, and FB-28 peptides (described in Figure 6). Primary cortical neurons were treated for 2 days, and neurite outgrowth was assessed on the third day by immunostaining with anti-beta-tubulin III antibody. Figures 18B-E show (i) the average length of total neurite outgrowth, (ii) the number of primary dendrites, (iii) the number of branch points, and (iv) the number of secondary neurites, respectively. Data are presented as mean ± SEM. Statistical significance was assessed using one-way ANOVA with Bonferroni post-hoc test; a, P<0.01 vs control group (CTRL). [Figure 18E] Figures 18A-E show neurite outgrowth in mouse primary cortical neurons treated in vitro with FB-16, FB-20, and FB-28 peptides (described in Figure 6). Primary cortical neurons were treated for 2 days, and neurite outgrowth was assessed on the third day by immunostaining with anti-beta-tubulin III antibody. Figures 18B-E show (i) the average length of total neurite outgrowth, (ii) the number of primary dendrites, (iii) the number of branch points, and (iv) the number of secondary neurites, respectively. Data are presented as mean ± SEM. Statistical significance was assessed using one-way ANOVA with Bonferroni post-hoc test; a, P<0.01 vs control group (CTRL). [Figure 19A]Figures 19A-C show increased expression of synaptophysin (SYP; presynaptic marker) and PSD95 (postsynaptic marker) in mouse primary hippocampal neurons treated with FB-16, FB-20 and FB-28 peptides in vitro (described in Figure 6). Figures 19A and 19B show total fluorescence intensity for SYN and PSD-95, respectively, in dendrites / neurites of hippocampal neurons measured using IMARIS software (IMARIS 9.0 Bitplane, Switzerland). Statistical significance was assessed using one-way ANOVA and Bonferroni post-hoc test; *, P<0.05 vs CTRL; **, P<0.05 vs CTRL. [Figure 19B] Figures 19A-C show increased expression of synaptophysin (SYP; presynaptic marker) and PSD95 (postsynaptic marker) in mouse primary hippocampal neurons treated with FB-16, FB-20 and FB-28 peptides in vitro (described in Figure 6). Figures 19A and 19B show total fluorescence intensity for SYN and PSD-95, respectively, in dendrites / neurites of hippocampal neurons measured using IMARIS software (IMARIS 9.0 Bitplane, Switzerland). Data are presented as mean ± SEM. The number of neurons used for quantifying the fluorescence intensity is indicated in brackets on the bar graphs. Statistical significance was assessed using one-way ANOVA with Bonferroni post-hoc test; *, P<0.05 vs CTRL; **, P<0.05 vs CTRL. [Figure 19C]Figures 19A-C show increased expression of synaptophysin (SYP; presynaptic marker) and PSD95 (postsynaptic marker) in mouse primary hippocampal neurons treated with FB-16, FB-20 and FB-28 peptides in vitro (described in Figure 6). Figure 19C shows the number of colocalized voxels between SYP and PSD95 signals in dendrites / neurites of treated hippocampal neurons. Data are presented as mean ± SEM. The number of neurons used for quantifying the fluorescence intensity is indicated in brackets on the bar graphs. Statistical significance was assessed using one-way ANOVA with Bonferroni post-hoc test; *, P<0.05 vs CTRL; **, P<0.05 vs CTRL. [Figure 20] FIG. 20 provides sequences of domains of interest (i.e., capable of binding to FAM19A5 protein) in LRRC4 protein family members across different vertebrate species. [Figure 21A] Figures 21A and 21B provide the effect of different amino acid modifications on the binding affinity of the LRRC4B fragment as assessed by in silico residue scanning of the FAM19A5-LRRC4 family complex using the Schrodinger platform. Figure 21A provides the predicted Gibbs free energy change upon alanine substitution at each amino acid residue of the FB-20 fragment (SEQ ID NO: 18). [Figure 21B] Figures 21A and 21B provide the effect of different amino acid modifications on the binding affinity of the LRRC4B fragment as assessed by in silico residue scanning of the FAM19A5-LRRC4 family complex using the Schrodinger platform. Figure 21B provides the predicted Gibbs free energy change for the top 20 FB-20 double mutants (containing amino acid substitutions at residues T12 and L13 of SEQ ID NO: 18) with improved affinity for the FAM19A5 protein. The sequences for the proposed FB-20 double mutants are provided in Example 9 (Table 12). [Figure 22A]Figures 22A-C show the activity of different FB-21 peptide mutants binding to FAM19A5 protein. Figure 22A provides a comparison of the inhibitory effect of the following FB-21 peptide fragments on the interaction between hFc-fused hLRRC4B and rcFAM19A5, as confirmed by competitive inhibition assay: (1) wild-type FB-21 (SEQ ID NO: 143), (2) FB-21(P12Y13) (SEQ ID NO: 144), (3) FB-21(H12F13) (SEQ ID NO: 145), (4) FB-21(Q12R13) (SEQ ID NO: 146), (5) FB-21(W12Y13) (SEQ ID NO: 147), (6) FB-21(M12R13) (SEQ ID NO: 148), and (7) FB-21(I12F13) (SEQ ID NO: 149). [Figure 22B] Figures 22A-C show the activity of different FB-21 peptide mutants binding to FAM19A5 protein. Figure 22B shows a comparison of the inhibitory effect of the following FB-21 peptide fragments on the interaction between HIS0TEV LRRC4B and rcFAM19A5 protein, as confirmed by competitive inhibition assay: (1) FB-21 (wild type) (SEQ ID NO: 143), (2) FB-21 (W12Y13) (SEQ ID NO: 147), (3) FB-21 (D12Y13) (SEQ ID NO: 131), (4) FB-21 (F12F13) (SEQ ID NO: 132), (5) FB-21 (H12Y13) (SEQ ID NO: 133), (6) FB-21 (D12F13) (SEQ ID NO: 135), and (7) FB-21 (D12I13) (SEQ ID NO: 136). [Figure 22C] Figures 22A-C show the activity of different FB-21 peptide mutants binding to FAM19A5 protein. Figure 22C provides results for the following FB-21 peptide fragments that contain D-amino acids at the amino and carboxyl termini and L-amino acids at all other residues: (1) d-FB-21 ("dFB-21"), (2) d-FB-21 peptide with a juxtamembrane (JM) sequence ("dFB-JM-31"), (3) d-FB-21 peptide sequence with a BBB-penetrating sequence at each end ("dFB-BBB-39"), and (4) d-FB-21 mutant peptide with a DY alternation and an additional JM sequence ("dFB-DY-JM31"). [Figure 22D] Figure 22D provides the sequences of the different members of the LRRC4 family (i.e., LRRC4, LRRC4B and LRRC4C proteins). The following domains are boxed: (1) FAM19A5 binding domain ("FB"); (2) juxtamembrane domain ("JM"), and (3) transmembrane domain ("TM"). [Figure 23A] Figures 23A-D show the efficacy of different FB-21 peptide fragments described herein against amyloid beta-induced synapse loss in mouse primary neurons. Figure 23A provides representative images for PSD95 (top row), SYP (middle row), and merged (bottom row) of hippocampal neurons treated with FB-21, FB-13-JM, or FB-BBB-39 (all at 6.6 nM; see Figure 22C for a description of the different FB-21 peptide fragments tested). Nuclei of cells were stained with Hoechst (blue). Scale bar = 50 μm. [Figure 23B] Figures 23A-D show the efficacy of different FB-21 peptide fragments described herein against amyloid beta-induced synapse loss in mouse primary neurons. Figure 23B provides a comparison of the number of colocalized voxels between SYP and PSD95 signal dendrites / neurites in hippocampal neurons treated with FB-21, FB-13-JM, or FB-BBB-39 (all at 6.6 nM). The number of colocalized voxels was calculated by IMARIS software (left panel, IMARIS 9.0 Bitplane, Switzerland). Data are presented as mean ± SEM. Statistical significance was assessed using one-way ANOVA with Bonferroni post-hoc test; *, P<0.05, and *, P<0.01 vs NT. [Figure 23C]Figures 23A-D show the efficacy of different FB-21 peptide fragments described herein against amyloid beta-induced synapse loss in mouse primary neurons. Figures 23C and 23D provide a comparison of total fluorescence intensity for PSD95 and SYN, respectively, in dendrites / neurites of hippocampal neurons treated with FB-21, FB-13-JM, or FB-BBB-39 (all at 6.6 nM) as measured using IMARIS. Data are presented as mean ± SEM. Statistical significance was assessed using one-way ANOVA with Bonferroni post-hoc test; *, P<0.05, and *, P<0.01 vs NT. [Figure 23D] Figures 23A-D show the efficacy of different FB-21 peptide fragments described herein against amyloid beta-induced synapse loss in mouse primary neurons. Figures 23C and 23D provide a comparison of total fluorescence intensity for PSD95 and SYN, respectively, in dendrites / neurites of hippocampal neurons treated with FB-21, FB-13-JM, or FB-BBB-39 (all at 6.6 nM) as measured using IMARIS. Data are presented as mean ± SEM. Statistical significance was assessed using one-way ANOVA with Bonferroni post-hoc test; *, P<0.05, and *, P<0.01 vs NT. [Figure 24A] Figures 24A and 24B show the effect of exemplary FB-21 peptide fragments described herein (i.e., dFB-dWY-JM31 and dFB-DY-JM31) on promoting neurite outgrowth of primary mouse spinal motor neurons. Figure 24A provides representative merged images of non-treated (NT) or FB-21 peptide fragment-treated spinal motor neurons immunostained with Tau-5 antibody. Neuronal cell bodies were stained and detected by Hoechst (blue). Scale bar = 100 μm. [Figure 24B]Figures 24A and 24B show the effect of exemplary FB-21 peptide fragments described herein (i.e., dFB-dWY-JM31 and dFB-DY-JM31) on promoting neurite outgrowth in primary mouse spinal motoneurons. Figure 24B provides a quantitative comparison of the mean total neurite length of primary spinal motoneurons from different treatment groups. Data are presented as mean ± SEM. Statistical significance was assessed using one-way ANOVA with Bonferroni post-hoc test; *, P<0.01 vs NT. [Figure 25A] Figures 25A and 25B show the efficacy of the FB-21 peptide variants described herein (dFB-dWY-JM31) against 6-OHDA-induced cell death in LUHMES cells. Figure 25A provides a quantitative comparison of luminescence expression after treatment with the FB-21 peptide variants with or without 6-OHDA treatment. Data are presented as mean ± SEM. Statistical significance was assessed using one-way ANOVA with Bonferroni post-hoc test; *, P<0.01 vs NT. [Figure 25B] Figures 25A and 25B show the efficacy of the FB-21 peptide variants described herein (dFB-dWY-JM31) against 6-OHDA-induced cell death in LUHMES cells. Figure 25B provides a quantitative comparison of luminescence expression after FB-21 peptide variants with 6-OHDA treatment. Data are presented as mean ± SEM. Statistical significance was assessed using one-way ANOVA with Bonferroni post-hoc test; *, P<0.01 vs NT. [Figure 26A] Figures 26A and 26B show the efficacy of an exemplary FB-21 peptide variant (dFB-dDY-JM31) described herein in a chronic constriction injury (CCI) rat model. Figure 26A provides a comparison of paw withdrawal thresholds in response to mechanical alloynia at various time points after CCI induction in mice treated with vehicle control (circles) or FB-21 peptide variants (squares). Data are presented as mean ± SEM. [Figure 26B]Figures 26A and 26B show the efficacy of an exemplary FB-21 peptide variant (dFB-dDY-JM31) described herein in a chronic constriction injury (CCI) rat model. Figure 26B provides a comparison of the area under the overall curve (AUC) for the data provided in Figure 26A. Statistical analysis for AUC was performed with a single-tailed unpaired t-test; *, p<0.05. [Figure 27] FIG. 27 shows the efficacy of the FB-21 peptide variant described herein (dFB-dDY-JM31) on regulating retinal dysfunction and neural oscillations. Retinal conductance (electroretinogram, ERG) was recorded to measure the electrical signal emitted from the retina in response to a flash of light using a diabetic retinopathy mouse model (db / db). ERG amplitude of the b-wave measured between groups; heterogenous wild type (WT, db / +, black), DR control group (db / db, red) and dFB-dDY-JM31 treated DR (blue). Data are presented as mean ± SEM. Statistical analysis was performed by one-way ANOVA followed by Bonferroni multiple comparison test; ***, p<0.001, **, p<0.01. [Figure 28A] Figures 28A and 28B show the efficacy of the FB-21 peptide variant (dFB-dWY-JM-31) described herein in a mouse model of traumatic brain injury. Figure 28A provides representative Hoechst staining of each group. [Figure 28B] Figures 28A and 28B show the efficacy of the FB-21 peptide variant (dFB-dWY-JM-31) described herein in a mouse model of traumatic brain injury. Figure 28B provides a quantitative comparison of lesion volumes based on the data provided in Figure 28A. Data are presented as mean ± SEM. Statistical analysis was performed with two-tailed unpaired t-test; ***, p<0.001.

Claims

**Claim 1** An isolated polypeptide comprising a domain of a leucine-rich repeat-containing 4 ("LRRC4") protein family member capable of binding to a family, member A5 ("FAM19A5") protein having an array similarity of 19, wherein the length of the polypeptide is shorter than that of the corresponding full-length LRRC4 protein family member. **Claim 2** The polypeptide according to claim 1, wherein the FAM19A5 binding domain comprises an amino acid sequence (from the N-terminus to the C-terminus) having the following chemical formula: A-(T / S)-B (Chemical formula I): wherein (a)(i) A comprises X1-(T / S)-(Y / F)-F-X5; 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; (ii) B comprises (V / I)-T-V-(E / V); (V / I) is valine (V) or isoleucine (I); (E / V) is glutamic acid (E) or valine (V); or (b)(i) A comprises (Y / W / M)-(T / Y)-(Y / W)-(F / Y / W)-(T / Y); (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); (ii) B comprises X7-(T / S / Y)-X9-X10; 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); X10 is glutamic acid (E), aspartic acid (D), isoleucine (I), tyrosine (Y), phenylalanine (F), methionine (M), or tryptophan (W).

3. An isolated polypeptide comprising the amino acid sequence (from N-terminus to C-terminus) of the following chemical formula, X1-X2-X3-X4-X5-X6-X7-X8-X9-X10 (Chemical Formula III): 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; 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, wherein the polypeptide is capable of binding to the FAM19A5 protein, whereby the interaction between the FAM19A5 protein and a member of the LRRRC4 protein family can be suppressed, reduced, and / or dissociated. An isolated polypeptide characterized by this.

4. (a) X1 is Y, F, V, L or I; (b) X2 is T or S; (c) X3 is Y or F; (d) X4 is F; (e) X5 is T or S; (f) X6 is T; (g) X7 is V or I; (h) X8 is T; (i) X9 is V; (j) X10 is E or V; or, (k) The polypeptide according to claim 3, characterized in that it is any combination of (a) to (j).

5. The polypeptide according to claim 1, characterized in that the FAM19A5 binding domain comprises the amino acid sequence presented as SEQ ID NO: 29, SEQ ID NO: 30 or SEQ ID NO:

31.

6. The polypeptide according to claim 5, characterized in that it comprises the amino acid sequence presented as SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, or SEQ ID NO:

149. **Claim 7**: The polypeptide according to claim 6, wherein one or more amino acid residues in the amino acid sequence of the FAM19A5 binding domain are (a) of the D - amino acid type, (b) phosphorylated, (c) O - glycosylated, or (d) any combination of (a) to (c). **Claim 8** An isolated polypeptide comprising an amino acid sequence having at least about 60% sequence identity with the amino acid sequence set forth in SEQ ID NO: 29, wherein the polypeptide is capable of binding to the FAM19A5 protein, whereby the interaction between the FAM19A5 protein and an LRRC4 protein family member can be inhibited, reduced and / or dissociated. **Claim 9** An isolated polypeptide comprising an amino acid sequence having at least about 70% sequence identity with the amino acid sequence set forth in SEQ ID NO: 5, SEQ ID NO: 4, or SEQ ID NO: 6, and comprising at least one amino acid modification with respect to each of the amino acid sequences set forth in SEQ ID NO: 5, SEQ ID NO: 4, or SEQ ID NO: 6, wherein the polypeptide is capable of binding to the FAM19A5 protein, whereby the interaction between the FAM19A5 protein and an LRRC4 protein family member can be inhibited, reduced and / or dissociated. **Claim 10** The polypeptide according to claim 9, wherein the at least one amino acid modification is (a) increasing the binding of the polypeptide to the FAM19A5 protein, (b) increasing the stability of the polypeptide, or (c) both (a) and (c). **Claim 11** A nucleic acid encoding the polypeptide according to any one of claims 1 to 10. **Claim 12** A vector comprising the nucleic acid according to claim 11. **Claim 13** A cell comprising the vector according to claim 12. **Claim 14** A protein conjugate comprising the polypeptide according to any one of claims 1 to 10 bound to a formulation. **Claim 15** A composition comprising the polypeptide according to any one of claims 1 to 10, the nucleic acid encoding the polypeptide, the vector comprising the nucleic acid, the cell comprising the vector, or the protein conjugate comprising the polypeptide bound to a formulation. **Claim 16** A kit comprising a polypeptide according to any one of claims 1 to 10, a nucleic acid encoding the polypeptide, a vector containing the nucleic acid, a cell containing the vector, or a protein conjugate containing the polypeptide and bound to a formulation, and an instruction manual.

17. A method for producing a polypeptide capable of suppressing, reducing, and / or dissociating the interaction between FAM19A5 protein and a member of the LRRC4 protein family, comprising culturing the cell of claim 13 under suitable conditions for producing the polypeptide.

18. A pharmaceutical composition for increasing neurite outgrowth or synapse formation in neurons, comprising contacting the neurons with a polypeptide according to any one of claims 1 to 10, a nucleic acid encoding the polypeptide, a vector containing the nucleic acid, a cell containing the vector, or a protein conjugate containing the polypeptide and bound to a formulation.

19. The increase in neurite outgrowth or synapse formation reduces one or more symptoms associated with a disease or condition selected from amyotrophic lateral sclerosis (ALS), Alzheimer's disease, glaucoma, diabetic retinopathy, neuropathic pain, spinal cord injury, traumatic brain injury, stroke, Parkinson's disease, or a combination thereof. The pharmaceutical composition according to claim 18.

20. A pharmaceutical composition for treating neurological diseases in a subject in need thereof, comprising administering to the subject a polypeptide according to any one of claims 1 to 10, a nucleic acid encoding the polypeptide, a vector containing the nucleic acid, a cell containing the vector, or a protein conjugate containing the polypeptide and bound to a formulation.

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