Anti-trkb monoclonal antibodies and methods of use

Abstract

Antibodies that specifically bind to TrkB and methods for using the same are described. Neuroprotective agonist antibodies are described, as indicated by their effect on enhancing the survival of retinal ganglion cells in vitro, and these agonist antibodies may be used to treat eye disorders, such as glaucoma. Additionally, other neuronal diseases or disorders, including those characterized by partial neuronal damage, benefit from treatment using these agonist antibodies. In certain embodiments, the present invention comprises antibodies that bind to TrkB and mediate cell signaling. The antibodies of the present invention may be whole human, non-natural antibodies formulated as injectable excipients.

Description

Technology Field

[0001]

[0001] The present invention relates to anti-tropomyosin-receptor-kinase B (TrkB) monoclonal antibodies. More specifically, the present invention relates to a composition comprising anti-TrkB monoclonal antibodies and a method of using these antibodies. Background Technology

[0002]

[0002] Tropomyosin receptor kinase B (TrkB) belongs to a family of single transmembrane receptor tyrosine kinases, including TrkA and TrkC. These receptor kinases mediate the activity of neurotrophins, which are required for neuronal survival and development. Neurotrophins include, but are not limited to, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 / 5 (NT-4 / 5). See the literature (Reference: Lo, KY et al., J. Biol. Chem., 280:41744-52 (2005)).

[0003]

[0003] TrkB is a high-affinity receptor for BDNF (see Minichiello, et al., Neuron 21:335-45 (1998)), but it is also known to bind to NT4 / 5. Binding of BDNF to trkB causes the receptor to dimerize, inducing autophosphorylation of specific tyrosine residues on the receptor and activation of signaling pathways including mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K), and phospholipase C-γ (PLC-γ) (References: Jing, et al. Neuron 9:1067-1079 (1992); Barbacid, J. Neurobiol. 25:1386-1403 (1994); Bothwell, Ann. Rev. Neurosci. 18:223 253 (1995); Segal and Greenberg, Ann. Rev. Neurosci. 19:463 489 (1996); Kaplan and Miller, Curr. Opinion Neurobiol. 10:381 391 (2000)). After binding to BDNF, TrkB mediates the multiple effects of neurotophorins, including neuronal differentiation and survival.

[0004]

[0004] Because TrkB plays a major role in neuronal survival, differentiation, and function, TrkB agonists may have therapeutic potential for treating a number of neurodegenerative and metabolic disorders.

[0005]

[0005] Certain TrkB agonists are described in the literature (references: US2010 / 0150914; US2003 / 0157099; US2010 / 0196390 and US2017 / 0157099). However, there is still a need for the identification and development of additional TrkB agonists that not only exhibit neuronal viability and neuroprotective properties similar to those described herein but also provide improved specificity.

[0006] A brief summary of the invention

[0007]

[0006] The present invention provides an isolated monoclonal antibody that specifically binds to tropomyosin-receptor-kinase B (TrkB) and an antigen-binding fragment thereof. The isolated antibody and antigen-binding fragment of the present invention are useful for treating diseases and disorders associated with TrkB activity or expression.

[0008]

[0007] In the broadest aspect thereof, the present invention provides anti-TrkB agonist antibodies that activate TrkB and promote neuronal survival. These antibodies can be used to improve neuronal function and treat any disease or disorder characterized by partial cellular degeneration, including neuronal damage associated with neurological damage and / or chronic neurodegenerative diseases.

[0009]

[0008] In certain embodiments, the anti-TrkB antibody may be useful for treating various eye diseases or disorders, but is not limited to, eye diseases such as glaucoma, and may be formulated for intraocular or intravitreal delivery.

[0010]

[0009] The antibody of the present invention may be the full length (e.g., IgG1 or IgG4 antibody) or may contain only the antigen-binding portion (e.g., Fab, F(ab')2 or scFv fragment), and may be modified to affect functionality, e.g., to remove residual effector function (see Reddy et al., 2000, J. Immunol. 164:1925-1933).

[0011]

[0010] Exemplary anti-TrkB antibodies of the present invention are listed in Tables 1 and 2 of this application. Table 1 presents the amino acid sequence identifiers of the heavy chain variable region (HCVR), light chain variable region (LCVR), heavy chain complementarity determining region (HCDR1, HCDR2, and HCDR3), and light chain complementarity determining region (LCDR1, LCDR2, and LCDR3) of the exemplary anti-TrkB antibody. Table 2 presents the nucleic acid sequence identifiers of the HCVR, LCVR, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the exemplary anti-TrkB antibody.

[0012]

[0011] The present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to TrkB comprising HCVR, comprising an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with respect thereto.

[0013]

[0012] In addition, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to TrkB, comprising an LCVR comprising an amino acid sequence selected from any of the LCVR amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with respect thereto.

[0014]

[0013] Additionally, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to TrkB, comprising an HCVR and LCVR amino acid sequence pair (HCVR / LCVR) comprising any HCVR amino acid sequence listed in Table 1 paired with any LCVR amino acid sequence listed in Table 1. According to a specific embodiment, the present invention provides an antibody or an antigen-binding fragment thereof comprising an HCVR / LCVR amino acid sequence pair contained within any exemplary anti-TrkB antibody listed in Table 1.

[0015]

[0014] Accordingly, in a first aspect, the present invention provides an isolated antibody or an antigen-binding fragment thereof that specifically binds to tropomyosin receptor kinase B (TrkB), wherein the antibody or the antigen-binding fragment thereof comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) comprising an amino acid sequence as presented in Table 1 or a substantially similar sequence having at least 90% sequence identity therewith; and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) comprising an amino acid sequence as presented in Table 1 or a substantially similar sequence having at least 90% sequence identity therewith.

[0016]

[0015] In one embodiment, the anti-TrkB antibody or its antigen-binding fragment exhibits one or more properties selected from the group consisting of:

[0017] (a) It is an agonist antibody;

[0018] (b) K less than approximately 200 nM when measured by surface plasmon resonance at 25°C or 37°C D Combine with person TrkB;

[0019] (c) Binds to human TrkB with a dissociation half-life (t½) of more than 10 minutes when measured by surface plasmon resonance at 25°C or 37°C;

[0020] (d) EC in the range of about 35 to 82 pM 50 Activating human TrkB signaling in the absence of brain-derived neurotrophic factor (BDNF) in cells processed to express human TrkB;

[0021] (e) Human TrkB receptor (TrkB hu / hu When injected into the hippocampus of mice homozygous for ), it promotes TrkB phosphorylation;

[0022] (f) Human TrkB receptor (TrkB hu / hu When injected into mice homozygous for ), it promotes body weight loss;

[0023] (g) Increased retinal ganglion cell (RGC) survival as evaluated in an optic nerve transection model in humanized TrkB rats;

[0024] (h) Activate the MAPK / ERK and PI3K / Akt signaling pathways;

[0025] (i) increase the survival of neuronal cells in vitro;

[0026] (j) ICs less than 5nM 50 It blocks the binding of TrkB to BDNF and / or NT-4.

[0027]

[0016] In one embodiment, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to tropomyosin-receptor-kinase B (TrkB), wherein the antibody or the antigen-binding fragment thereof comprises: (a) a complementation determining region (CDR) of a heavy chain variable region (HCVR) comprising an amino acid sequence as presented in Table 1; and (b) a CDR of a light chain variable region (LCVR) comprising an amino acid sequence as presented in Table 1.

[0028]

[0017] In one embodiment, an antibody that specifically binds to TrkB or an antigen-binding fragment thereof comprises three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained in any one of an HCVR sequence selected from the group consisting of SEQ ID NOs 2, 18, 34, 49, 59, and 68 or a substantially similar sequence having at least 90% sequence identity therewith; and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained in any one of an LCVR sequence selected from the group consisting of SEQ ID NOs 10, 26, 42, 53, 63, and 72 or a substantially similar sequence having at least 90% sequence identity therewith.

[0029]

[0018] In one embodiment, an isolated antibody or its antigen-binding fragment that specifically binds to TrkB comprises HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs 2, 18, 34, 49, 59 and 68.

[0030]

[0019] In one embodiment, the isolated antibody or its antigen-binding fragment that specifically binds to TrkB further comprises an LCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs 10, 26, 42, 53, 63 and 72.

[0031]

[0020] In one embodiment, the isolated antibody or its antigen-binding fragment that specifically binds to TrkB comprises an HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs 2, 18, 34, 49, 59 and 68; and an LCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs 10, 26, 42, 53, 63 and 72.

[0032]

[0021] In one embodiment, an isolated antibody or its antigen-binding fragment that specifically binds to TrkB comprises a CDR of an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs 2 / 10, 18 / 26, 34 / 42, 49 / 53, 59 / 63 and 68 / 72.

[0033]

[0022] In one embodiment, an isolated antibody or its antigen-binding fragment that specifically binds to TrkB comprises a CDR of an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs 2 / 10, 18 / 26, and 34 / 42.

[0034]

[0023] In one embodiment, an isolated antibody or its antigen-binding fragment that specifically binds to TrkB comprises an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs 2 / 10, 18 / 26, 34 / 42, 49 / 53, 59 / 63 and 68 / 72.

[0035]

[0024] In one embodiment, an isolated antibody or its antigen-binding fragment that specifically binds to TrkB comprises an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs 2 / 10, 18 / 26, and 34 / 42.

[0036]

[0025] In addition, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to TrkB, comprising a heavy chain CDR1 (HCDR1) comprising an amino acid sequence selected from any of the HCDR1 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with respect thereto.

[0037]

[0026] In addition, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to TrkB, comprising a heavy chain CDR2 (HCDR2) comprising an amino acid sequence selected from any of the HCDR2 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with respect thereto.

[0038]

[0027] In addition, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to TrkB, comprising a heavy chain CDR3 (HCDR3) comprising an amino acid sequence selected from any of the HCDR3 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with respect thereto.

[0039]

[0028] In addition, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to TrkB, comprising a light chain CDR1 (LCDR1) comprising an amino acid sequence selected from any of the LCDR1 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with respect thereto.

[0040]

[0029] In addition, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to TrkB, comprising a light chain CDR2 (LCDR2) comprising an amino acid sequence selected from any of the LCDR2 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with respect thereto.

[0041]

[0030] In addition, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to TrkB, comprising a light chain CDR3 (LCDR3) comprising an amino acid sequence selected from any of the LCDR3 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with respect thereto.

[0042]

[0031] Additionally, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to TrkB, comprising an HCDR3 and LCDR3 amino acid sequence pair (HCDR3 / LCDR3) comprising any of the HCDR3 amino acid sequences listed in Table 1 paired with any of the LCDR3 amino acid sequences listed in Table 1. According to a specific embodiment, the present invention provides an antibody or an antigen-binding fragment thereof comprising an HCDR3 / LCDR3 amino acid sequence pair contained within any exemplary anti-TrkB antibody listed in Table 1. In a specific embodiment, the HCDR3 / LCDR3 amino acid sequence pair is selected from the group consisting of 8 / 16, 24 / 32, 40 / 48, 52 / 56, 62 / 66, and 71 / 75.

[0043]

[0032] Additionally, the present invention provides an antibody that specifically binds to TrkB or an antigen-binding fragment thereof comprising a set of six CDRs (i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3) contained in any exemplary anti-TrkB antibodies listed in Table 1. In a specific embodiment, the set of amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 is selected from the group consisting of: (a) SEQ ID NOs 4, 6, 8, 12, 14, 16; (b) SEQ ID NOs 20, 22, 24, 28, 30, 32; (c) SEQ ID NOs 36, 38, 40, 44, 46, 48; (d) SEQ ID NOs 50, 51, 52, 54, 55, 56; (e) sequence numbers 60, 61, 62, 64, 65, 66; and (f) sequence numbers 69, 70, 71, 73, 74, 75.

[0044]

[0033] In a related embodiment, the present invention provides an antibody or its antigen-binding fragment that specifically binds to TrkB, comprising a set of six CDRs (i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3) contained within an HCVR / LCVR amino acid sequence pair as defined by any exemplary anti-TrkB antibody listed in Table 1. For example, the present invention comprises an antibody or its antigen-binding fragment that specifically binds to TrkB, comprising a set of amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 contained within an HCVR / LCVR amino acid sequence pair selected from the group consisting of 2 / 10, 18 / 26, 34 / 42, 49 / 53, 59 / 63, and 68 / 72. Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are widely known in the art and can be used to identify CDRs within specific HCVR and / or LCVR amino acid sequences disclosed herein. Exemplary methods that can be used to identify CDR boundaries include, for example, Kabat definitions, Chothia definitions, and AbM definitions. Generally, Kabat definitions are based on sequence variability, Chothia definitions are based on the location of structural loop regions, and AbM definitions are a compromise between the Kabat and Chothia approaches. For example, the literature (Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al. , J. Mol. Biol. 273 :927-948 (1997); and Martin et al. , Proc. Natl. Acad. Sci. USA 86 Refer to :9268-9272 (1989). Public databases can also be used to identify CDR sequences within antibodies.

[0045]

[0034] In one embodiment, the present invention provides an isolated antibody that specifically binds to TrkB or an antigen-binding fragment thereof comprising:

[0046] (a) an HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs 4, 20, 36, 50, 60 and 69;

[0047] (b) an HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs 6, 22, 38, 51, 61 and 70;

[0048] (c) HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs 8, 24, 40, 52, 62 and 71;

[0049] (d) an LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs 12, 28, 44, 54, 64 and 73;

[0050] (e) an LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs 14, 30, 46, 55, 65 and 74; and

[0051] (f) LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs 16, 32, 48, 56, 66 and 75.

[0052]

[0035] In one embodiment, the present invention provides an isolated antibody that specifically binds to TrkB or an antigen-binding fragment thereof comprising:

[0053] (a) an HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs 4, 20 and 36;

[0054] (b) an HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs 6, 22 and 38;

[0055] (c) an HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs 8, 24 and 40;

[0056] (d) an LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs 12, 28 and 44;

[0057] (e) an LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs 14, 30 and 46; and

[0058] (f) LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs 16, 32 and 48.

[0059]

[0036] In one embodiment, the isolated antibody or its antigen-binding fragment comprises a set of six CDRs (HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) selected from the group consisting of: (a) SEQ ID NO. 4-6-8-12-14-16; (b) SEQ ID NO. 20-22-24-28-30-32; (c) SEQ ID NO. 36-38-40-44-46-48; (d) SEQ ID NO. 50-51-52-54-55-56; (e) SEQ ID NO. 60-61-62-64-65-66; and (f) SEQ ID NO. 69-70-71-73-74-75.

[0060]

[0037] In one embodiment, the isolated antibody or its antigen-binding fragment that binds to TrkB comprises an antibody or its antigen-binding fragment that competes with the reference antibody for binding to TrkB, wherein the reference antibody comprises an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs 2 / 10, 18 / 26, 34 / 42, 49 / 53, 59 / 63 and 68 / 72.

[0061]

[0038] In one embodiment, the isolated antibody or its antigen-binding fragment that binds to TrkB comprises an antibody or its antigen-binding fragment that binds to the same epitope as that of a reference antibody, wherein the reference antibody comprises an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs 2 / 10, 18 / 26, 34 / 42, 49 / 53, 59 / 63 and 68 / 72.

[0062]

[0039] In one embodiment, the isolated antibody or its antigen-binding fragment has a K of less than about 300 nM when measured by surface plasmon resonance at 25°C or 37°C. D It binds to the human TrkB.

[0063]

[0040] In one embodiment, the isolated antibody or its antigen-binding fragment has a K of less than about 200 nM when measured by surface plasmon resonance at 25°C or 37°C. D It binds to the human TrkB.

[0064]

[0041] In one embodiment, the isolated antibody or its antigen-binding fragment has a K of less than about 150 nM when measured by surface plasmon resonance at 25°C or 37°C. D It binds to the human TrkB.

[0065]

[0042] In one embodiment, the isolated antibody or its antigen-binding fragment has a K of less than about 50 nM when measured by surface plasmon resonance at 25°C or 37°C. D It binds to the human TrkB.

[0066]

[0043] In one embodiment, the isolated antibody or its antigen-binding fragment has a K of less than about 100 pM when measured by surface plasmon resonance at 25°C or 37°C. D It binds to the human TrkB.

[0067]

[0044] In one embodiment, the isolated antibody or its antigen-binding fragment binds to human TrkB with a dissociation half-life (t½) of more than 10 minutes when measured by surface plasmon resonance at 25°C or 37°C.

[0068]

[0045] In one embodiment, the isolated antibody or its antigen-binding fragment binds to human TrkB for more than 40 minutes when measured by surface plasmon resonance at 25°C or 37°C.

[0069]

[0046] In one embodiment, the isolated antibody or its antigen-binding fragment binds to human TrkB for more than 120 minutes when measured by surface plasmon resonance at 25°C or 37°C.

[0070]

[0047] In one embodiment, an isolated antibody binding to TrkB or its antigen-binding fragment inhibits human TrkB signaling in cells engineered to express TrkB in the absence of BDNF at an EC of less than about 100 pM. 50 Activate it as.

[0071]

[0048] In one embodiment, an isolated antibody binding to TrkB or an antigen-binding fragment thereof inhibits human TrkB signaling in cells engineered to express TrkB in the absence of BDNF at an EC range of about 35 pM to about 82 pM. 50 Activate it as.

[0072]

[0049] In one embodiment, an isolated antibody binding to TrkB or its antigen-binding fragment activates human TrkB signaling in the presence of BDNF in cells engineered to express TrkB at an EC of less than about 100 pM 50 It promotes...

[0073]

[0050] In one embodiment, an isolated antibody binding to TrkB or its antigen-binding fragment demonstrates TrkB activation, as indicated by an increase in TrkB phosphorylation when injected into the hippocampus of a humanized TrkB mouse.

[0074]

[0051] In one embodiment, an isolated antibody binding to TrkB or its antigen-binding fragment demonstrates activation of the MAPK / ERK and PI3K / Akt signaling pathways, as demonstrated after incubating primary mouse cortical neurons with an agonist anti-TrkB antibody.

[0075]

[0052] In one embodiment, an isolated antibody binding to TrkB or its antigen-binding fragment promotes and / or increases the survival of retinal ganglion cells as shown in an optic nerve transection model in TrkB humanized rats.

[0076]

[0053] In one embodiment, an isolated antibody or its antigen-binding fragment that binds to TrkB promotes and / or increases the survival of neuronal cells in vitro.

[0077]

[0054] In one embodiment, an isolated antibody binding to TrkB or its antigen-binding fragment promotes body weight loss in humanized TrkB mice.

[0078]

[0055] In one embodiment, an isolated antibody binding to TrkB or its antigen-binding fragment promotes a loss of body fat mass in humanized TrkB mice.

[0079]

[0056] In one embodiment, an isolated antibody binding to TrkB or its antigen-binding fragment promotes a reduction in food and water consumption in humanized TrkB mice.

[0080]

[0057] In one embodiment, an isolated antibody binding to TrkB or its antigen-binding fragment promotes an increase in locomotor activity in humanized TrkB mice.

[0081]

[0058] In one embodiment, the present invention specifically binds to TrkB and has an IC of less than about 5 nM 50 Provides an anti-TrkB antibody or its antigen-binding fragment that blocks TrkB binding to BDNF.

[0082]

[0059] In one embodiment, the present invention specifically binds to TrkB and has an IC of less than about 500 pM. 50 Provides an anti-TrkB antibody or its antigen-binding fragment that blocks TrkB binding to BDNF.

[0083]

[0060] In one embodiment, the present invention specifically binds to TrkB and has an IC of less than about 200 pM. 50 Provides an anti-TrkB antibody or its antigen-binding fragment that blocks TrkB binding to BDNF.

[0084]

[0061] In a second aspect, the present invention provides a nucleic acid molecule encoding an anti-TrkB antibody or a portion thereof. For example, the present invention provides a nucleic acid molecule encoding any HCVR amino acid sequence listed in Table 1; in a specific embodiment, said nucleic acid molecule comprises a polynucleotide sequence selected from any HCVR nucleic acid sequence listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with respect thereto.

[0085]

[0062] Additionally, the present invention provides a nucleic acid molecule encoding any LCVR amino acid sequence listed in Table 1; in a specific embodiment, the nucleic acid molecule comprises a polynucleotide sequence selected from any LCVR nucleic acid sequence listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with respect thereto.

[0086]

[0063] Additionally, the present invention provides a nucleic acid molecule encoding any HCDR1 amino acid sequence listed in Table 1; in a specific embodiment, the nucleic acid molecule comprises a polynucleotide sequence selected from any HCDR1 nucleic acid sequence listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with respect thereto.

[0087]

[0064] Additionally, the present invention provides a nucleic acid molecule encoding any HCDR2 amino acid sequence listed in Table 1; in a specific embodiment, the nucleic acid molecule comprises a polynucleotide sequence selected from any HCDR2 nucleic acid sequence listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with respect thereto.

[0088]

[0065] Additionally, the present invention provides a nucleic acid molecule encoding any HCDR3 amino acid sequence listed in Table 1; in a specific embodiment, the nucleic acid molecule comprises a polynucleotide sequence selected from any HCDR3 nucleic acid sequence listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with respect thereto.

[0089]

[0066] Additionally, the present invention provides a nucleic acid molecule encoding any LCDR1 amino acid sequence listed in Table 1; in a specific embodiment, the nucleic acid molecule comprises a polynucleotide sequence selected from any LCDR1 nucleic acid sequence listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with respect thereto.

[0090]

[0067] Additionally, the present invention provides a nucleic acid molecule encoding any LCDR2 amino acid sequence listed in Table 1; in a specific embodiment, the nucleic acid molecule comprises a polynucleotide sequence selected from any LCDR2 nucleic acid sequence listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with respect thereto.

[0091]

[0068] Additionally, the present invention provides a nucleic acid molecule encoding any LCDR3 amino acid sequence listed in Table 1; in a specific embodiment, the nucleic acid molecule comprises a polynucleotide sequence selected from any LCDR3 nucleic acid sequence listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with respect thereto.

[0092]

[0069] In addition, the present invention provides a nucleic acid molecule encoding HCVR, wherein the HCVR comprises a set of three CDRs (i.e., HCDR1, HCDR2, HCDR3), and the set of amino acid sequences of HCDR1, HCDR2, HCDR3 is as defined by any exemplary anti-TRKB antibody listed in Table 1.

[0093]

[0070] In addition, the present invention provides a nucleic acid molecule encoding LCVR, wherein the LCVR comprises a set of three CDRs (i.e., LCDR1, LCDR2, LCDR3), and the set of amino acid sequences of LCDR1, LCDR2, LCDR3 is as defined by any exemplary anti-TRKB antibody listed in Table 1.

[0094]

[0071] Additionally, the present invention provides a nucleic acid molecule encoding both HCVR and LCVR, wherein the HCVR comprises an amino acid sequence among any HCVR amino acid sequence listed in Table 1, and the LCVR comprises an amino acid sequence among any LCVR amino acid sequence listed in Table 1. In a specific embodiment, the nucleic acid molecule comprises a polynucleotide sequence selected from any HCVR nucleic acid sequence listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity therewith, and a polynucleotide sequence selected from any LCVR nucleic acid sequence listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity therewith. In a specific embodiment according to the above aspect of the present invention, the nucleic acid molecule encodes HCVR and LCVR, wherein both HCVR and LCVR are derived from the same anti-TrkB antibody listed in Table 1.

[0095]

[0072] In a third aspect, the present invention provides a recombinant expression vector capable of expressing a polypeptide comprising a variable region of the heavy or light chain of an anti-TrkB antibody. For example, the present invention comprises a recombinant expression vector comprising any of the aforementioned nucleic acid molecules, namely, a nucleic acid molecule encoding any of the HCVR, LCVR, and / or CDR sequences as listed in Table 1. Additionally, within the scope of the present invention are methods for producing an antibody or a portion thereof by culturing the host cells into which such a vector has been introduced, as well as said host cells under conditions that enable the production of the antibody or antibody fragment, and by recovering the antibody and antibody fragment thus produced.

[0096]

[0073] The present invention comprises an anti-TrkB antibody having a modified glycosylation pattern. In some embodiments, modifications to remove undesirable glycosylation sites, or, for example, antibodies lacking fucose residues present on the oligosaccharide chain to increase antibody-dependent cytotoxicity (ADCC) function may be useful (cf. Shield et al. (2002) JBC 277:26733). In another application, modifications of galactosylation may be made to modify complement-dependent cytotoxicity (CDC).

[0097]

[0074] In a fourth aspect, the present invention provides a pharmaceutical composition comprising at least one antibody of the present invention or an antigen-binding fragment thereof that specifically binds to TrkB, and a pharmaceutically acceptable carrier.

[0098]

[0075] In a related aspect, the present invention features a composition which is a combination of an anti-TrkB antibody and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent advantageously combined with the anti-TrkB antibody. The second therapeutic agent may be useful for alleviating at least one symptom of a neurodegenerative disease or disorder.

[0099]

[0076] In a fifth aspect, the present invention provides a method for enhancing biological activity mediated by TrkB, the method comprising contacting TrkB with a biologically effective amount of an agonist anti-TrkB antibody of Table 1 or contacting TrkB with a biologically effective amount of an agonist anti-TrkB antibody of Table 1.

[0100]

[0077] In a specific embodiment, the biological activity is neuronal protection or neuronal survival, and neuronal protection and neuronal survival are enhanced upon contact between TrkB and the agonist anti-TrkB antibody.

[0101]

[0078] In a specific embodiment, the biological activity is neuroprotection and survival of retinal ganglion cells (RGCs).

[0102]

[0079] In a sixth aspect, the present invention provides a therapeutic method for treating a disease or disorder associated with TrkB activity or expression, or at least one symptom associated with said disease or disorder, by using the anti-TrkB antibody of the present invention or the antigen binding portion of the antibody. The therapeutic method according to the above aspect of the present invention comprises administering a therapeutically effective amount of a pharmaceutical composition comprising the antibody of the present invention or the antigen binding portion of the antibody to a subject in need thereof. The disorder to be treated is any disease or condition that is improved, alleviated, inhibited, or prevented by targeting TrkB and / or activating TrkB-mediated cell signaling.

[0103]

[0080] In one embodiment, the anti-TrkB antibody of the present invention may provide a method for preventing damage or death of retinal neurons. In one embodiment, the anti-TrkB antibody of the present invention may provide a method for treating pathological diseases in which retinal degeneration has occurred. In one embodiment, the anti-TrkB antibody of the present invention may provide a method for preventing degeneration of retinal cells by treating a surviving eye before or after exposure to ocular surgery, light, or other environmental trauma. In one embodiment, the anti-TrkB antibody of the present invention may provide a method for preventing photoreceptor damage and degeneration in a surviving eye. In one embodiment, the anti-TrkB antibody of the present invention may provide a method for protecting retinal neurons without inducing side effects due to cross-reactivity with other receptors such as p75 receptors. In one embodiment, the anti-TrkB antibody of the present invention may provide a method for allowing damaged photoreceptors to be recovered or regenerated.

[0104]

[0081] In a specific embodiment, the disease or disorder to be treated with the antibody of the present invention is a disease or disorder of the eye selected from the group consisting of glaucoma, diabetic retinopathy, age-related macular degeneration, ischemic optic neuropathy, optic neuritis, retinal ischemia, photoreceptor degeneration, retinitis pigmentosa, Leber congenital amaurosis, Leber hereditary optic neuropathy, Usher Syndrome, Stargardt disease, and retinal artery or vein occlusion.

[0105]

[0082] Other pathological conditions that can be treated with one or more anti-TrkB antibodies of the present invention include retinal detachment, photic retinopathies, surgery-induced retinopathy (mechanically or photo-induced), toxic retinopathy, retinopathy of prematurity, viral retinopathy such as AIDS-associated CMV or HIV retinopathy; uveitis; ischemic retinopathy due to venous or arterial occlusion or other vascular disorders, retinopathy due to trauma to the eye or penetrating lesions, peripheral vitreoretinopathy or inherited retinal degeneration.

[0106]

[0083] In one embodiment, the eye disease or disorder to be treated with the anti-TrkB antibody agonist of the present invention is glaucoma.

[0107]

[0084] A seventh aspect of the invention provides a method for reducing body weight in a subject, said method comprising administering to the subject a pharmaceutical composition comprising the TrkB agonist antibody of Table 1, or an antibody or an antigen-binding fragment thereof.

[0108]

[0085] In a related aspect, the present invention provides a method for reducing body fat mass in a subject, said method comprising administering to the subject a pharmaceutical composition comprising the TrkB agonist antibody of Table 1, or an antibody or an antigen-binding fragment thereof.

[0109]

[0086] The eighth aspect of the invention provides a method for promoting neuronal survival in a subject, said method comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of the TrkB agonist antibody of Table 1, or a therapeutically effective amount of the antibody or an antigen-binding fragment thereof.

[0110]

[0087] In one embodiment, the method described above can be achieved by administering an agonist anti-TrkB antibody or an antigen-binding fragment thereof to a subject requiring it, wherein the agonist anti-TrkB antibody comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) having an amino acid sequence as presented in Table 1 or a substantially similar sequence having at least 90% sequence identity therewith; and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) having an amino acid sequence as presented in Table 1 or a substantially similar sequence having at least 90% sequence identity therewith.

[0111]

[0088] In one embodiment, the method of the present invention may be achieved by administering the agonist TrkB antibody of the present invention, wherein the antibody or its antigen-binding fragment comprises three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained in any one of an HCVR sequence selected from the group consisting of SEQ ID NOs 2, 18, 34, 49, 59 and 68 or a substantially similar sequence having at least 90% sequence identity therewith; and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained in any one of an LCVR sequence selected from the group consisting of SEQ ID NOs 10, 26, 42, 53, 63 and 72 or a substantially similar sequence having at least 90% sequence identity therewith.

[0112]

[0089] In one embodiment, the antibody or its antigen-binding fragment comprises an HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs 2, 18, 34, 49, 59 and 68.

[0113]

[0090] In one embodiment, the antibody or its antigen-binding fragment comprises an LCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs 10, 26, 42, 53, 63 and 72.

[0114]

[0091] In one embodiment, the antibody or its antigen-binding fragment comprises an HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs 2, 18, 34, 49, 59 and 68; and an LCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs 10, 26, 42, 53, 63 and 72.

[0115]

[0092] In one embodiment, the antibody or its antigen-binding fragment comprises a CDR of an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs 2 / 10, 18 / 26, 34 / 42, 49 / 53, 59 / 63 and 68 / 72.

[0116]

[0093] In one embodiment, the antibody or its antigen-binding fragment comprises an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs 2 / 10, 18 / 26, 34 / 42, 49 / 53, 59 / 63 and 68 / 72.

[0117]

[0094] In one embodiment, the antibody or its antigen-binding fragment comprises the following:

[0118] (a) an HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs 4, 20, 36, 50, 60 and 69;

[0119] (b) an HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs 6, 22, 38, 51, 61 and 70;

[0120] (c) HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs 8, 24, 40, 52, 62 and 71;

[0121] (d) an LCDR1 domain having an amino acid sequence selected from the group consisting of sequence numbers 12, 28, 44, 54, 64 and 73;

[0122] (e) an LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs 14, 30, 46, 55, 65 and 74; and

[0123] (f) LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs 16, 32, 48, 56, 66 and 75.

[0124]

[0095] In one embodiment, the antibody or its antigen-binding fragment comprises a set of six CDRs (HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) selected from the group consisting of: (a) SEQ ID NO. 4-6-8-12-14-16; (b) SEQ ID NO. 20-22-24-28-30-32; (c) SEQ ID NO. 36-38-40-44-46-48; (d) SEQ ID NO. 50-51-52-54-55-56; (e) SEQ ID NO. 60-61-62-64-65-66; and (f) SEQ ID NO. 69-70-71-73-74-75.

[0125]

[0096] In one embodiment, the disease or disorder to be treated with an anti-TrkB antibody is obesity and any complication resulting from obesity.

[0126]

[0097] Any disease or disorder associated with TrkB activity or expression is considered to be compliant with treatment using the antibody of the present invention. These diseases may include any disease in which cell degradation is evident, for example, in a neurodegenerative state or after nerve damage.

[0127]

[0098] Other embodiments will become apparent from the following detailed description of the invention. Brief explanation of the drawing

[0128]

[0099] Fig. 1 This shows a Western blot evaluating total TRKB and phospho-TRKB levels in homozygous humanized TRKB mice at 1, 4, and 18 hours after direct hippocampal injection of the TRKB agonist antibody H4H9816P2 or an isotype control antibody.

[0100] Fig. 2 This shows that H4H9816P2, a TrkB agonist antibody, activates the downstream pathways of MAPK / ERK and PI3K / Akt. The figure above shows Western blots of phospho-TrkB, total TrkB, phospho-Akt, total AKT, phospho-ERK, and total ERK at 15 minutes and 2 hours after treatment of primary cortical neurons isolated from homozygous humanized TRKB mouse pups 1 day after birth using various TrkB agonist antibodies or BDNF.

[0101] Fig. 3 This shows that doses of three TrkB agonist antibodies increase the survival of SH-SY5Y cells in vitro in a dose-dependent manner. The isotype control antibody had no effect on cell survival.

[0102] Fig. 4 is TrkB hu / hu This shows the pharmacokinetic profile of the anti-TrkB agonist antibody H4H9816P2 in mice and wild-type mice. A single subcutaneous dose of 10 mg / kg was administered to mice on Day 0. Total serum concentrations of H4H9816P2 were measured using the Gyros immunoassay. Data points at 6 hours, and on Days 1, 2, 3, 6, 9, 16, 21, and 30 after administration indicate the mean antibody concentrations. The total antibody concentration of H4H9816P2 is TrkB hu / hu The mice are represented by black solid circles, and the wild-type mice by solid black squares. The data represent the mean. + Plot in SD.

[0103] Fig. 5It consists of Figures 5a and 5b, which demonstrate the ability of an anti-mouse TrkB monoclonal antibody to block the interaction between mouse or rat TrkB and its ligand BDNF (brain-derived neurotrophic factor). Using an ELISA-based method, the binding of mouse TrkB.hFc (Figure 5a with two graphs) and rat TrkB.mmh (Figure 5b with two graphs) to plate-coated BDNF was evaluated in the presence of concentration ranges of anti-mouse TrkB and isotype control mAbs. In Figure 5a (with two graphs), the inset is at an EC of 780 pM. 50 The dose-response curve of the binding of mouse TrkB.hFc (REGN2277) to BDNF is shown with values. In Fig. 5b (with two graphs), the inset is 2.2 nM EC 50 The values ​​show the dose-response curve of the binding of rat TrkB.mmh (REGN1808) to BDNF. Molar concentration (M) indicates the antibody concentration for the mAb. Error bars represent the standard deviation. Specific details for implementing the invention

[0129]

[0104] Before describing the present invention, it should be understood that the invention is not limited to the specific methods and experimental conditions described, as methods and conditions may vary. Furthermore, the terms used herein are intended only to describe specific embodiments and are not intended to be limiting, as the scope of the invention will be limited only by the appended claims.

[0130]

[0105] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. Where the term “about” as used herein is used to refer to a specific cited numerical value, it means that said value may vary up to 1% or less of the mentioned value. For example, as used herein, the expression “about 100” includes 99 and 101, and all values ​​in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0131]

[0106] Any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the invention, but preferred methods and materials will be described below. All patents, applications, and non-patent publications mentioned herein are incorporated herein by reference in their entirety.

[0132] definition

[0133]

[0107] Additionally, the expression "TrkB," known as “tropomyosin receptor kinase B,” etc., refers to a human receptor (unless otherwise indicated as another species) comprising an amino acid sequence as presented in amino acid residues 32 to 430 of Approval No. NP_001018074.1. Human TrkB containing a myc-myc-hexahistidine tag is represented as SEQ ID NO. 76 (having amino acid residues 1 to 399 which are human TrkB and amino acid residues 400 to 427 which are a myc-myc-hexahistidine tag). Other formats containing human TrkB proteins are described herein, which include SEQ ID NO. 77, human TrkB (residues 1 to 399) having a mouse Fc region (residues 400 to 632); It includes SEQ ID NO. 78, which is a human TrkB (residues 1 to 399) having a human Fc region (residues 400 to 626). Mouse TrkB comprises an amino acid sequence as presented in amino acid residues 32 to 429 of Approval No. NP_001020245. Mouse TrkB containing a myc-myc-hexahistidine tag is represented as SEQ ID NO. 79 (having amino acid residues 1 to 398 which are mouse TrkB and amino acid residues 399-426 which are a myc-myc-hexahistidine tag). Other formats containing mouse TrkB proteins are described herein, which include SEQ ID NO. 80, which is a mouse TrkB (residues 1 to 398) having a mouse Fc region (residues 399-631); It includes SEQ ID NO. 81, which is a mouse TrkB (residues 1 to 398) having a human Fc region (residues 399 to 625). Rabbit TrkB includes amino acid sequences as presented in amino acid residues 32 to 430 of approval number XP_002721319.1. Rabbit TrkB containing a myc-myc-hexahistidine tag is represented as SEQ ID NO. 82 (having amino acid residues 1 to 399 which are rabbit TrkB and amino acid residues 400-427 which are a myc-myc-hexahistidine tag).Other formats containing rabbit TrkB protein are described herein, including SEQ ID NO. 83, which is rabbit TrkB (residues 1 to 399) having a mouse Fc region (residues 400 to 632). Rat TrkB comprises an amino acid sequence as presented in Recognition No. NP_036863.1, with amino acid residues 32 to 429. Rat TrkB containing a myc-myc-hexahistidine tag is represented as SEQ ID NO. 84 (having amino acid residues 1 to 398 which are mouse TrkB and amino acid residues 399-426 which are a myc-myc-hexahistidine tag). Other formats containing rat TrkB protein are described herein, including SEQ ID NO. 85, which is rat TrkB (residues 1 to 398) having a mouse Fc region (residues 399 to 631). Rhesus macaque (Macaca mulata(. Macaca mulatta TrkB is represented as SEQ ID NO. 95 (amino acids 32 to 838 of approval number NP_001248226.1) and is a cynomolgus monkey (Macaca pasciculariis ( Macaca fascicularis TrkB is represented as sequence number 96 (amino acids 32 to 838 of approval number XP_005582102.1).

[0134]

[0108] The human "TrkA" protein is represented as SEQ ID NO. 86 having amino acids 1 to 375 which are TrkA (amino acids 34 to 414 of approval number NP_001012331.1 with V263L, C300S), amino acids 376 to 378 which are GPG linkers, and amino acids 379 to 605 which are human Fc.

[0135]

[0109] The human "TrkC" protein is represented by SEQ ID NO. 87 having amino acids 1 to 398 which are TrkC (amino acids 32 to 429 of approval number NP_001012338.1) and amino acids 399 to 426 which are myc-myc-his tags.

[0136]

[0110] The mouse "TrkC" protein is represented as SEQ ID NO. 88 having amino acids 1 to 398 which are TrkC (amino acids 32 to 429 of approval number NP_032772.3) and amino acids 399 to 426 which are myc-myc-his tags.

[0137]

[0111] The synomolgus monkey "TrkC" protein is represented as SEQ ID NO. 89 having amino acids 1 to 398 which are TrkC (amino acids 32 to 429 of approval number XP_015308837.1) and amino acids 399 to 426 which are myc-myc-his tags.

[0138]

[0112] In certain cases, cell lines expressing a TrkB protein comprising the transmembrane and cytoplasmic domains as well as the ecto domain of the TrkB protein were prepared. For example, SEQ ID NO. 91 is a human TrkB protein containing all three domains contained in amino acids 32 to 822 of Approval No. NP_001018074.1 or Uniprot Q16620-1, having a transmembrane / cytoplasmic domain defined by amino acids 1 to 398, which are the ecto domain, and amino acid residues 399 to 790. In one case, a TrkB cell line expressing mouse TrkB (the TrkB cell line of Approval No. NP_032771.1, amino acids 32 to 476; also refer to SEQ ID NO. 92) was prepared. In another case, cell lines expressing a chimeric TrkB protein were prepared having the ecto domain of mouse TrkB of origin of approval number NP_001020245.1 (also refer to SEQ ID NO. 93) or Uniprot number P15209-1 (amino acids 32 to 429) and the transmembrane and cytoplasmic domain of human TrkB (amino acids 431 to 822 of approval number NP_001018074.1 (also refer to SEQ ID NO. 91)). African green monkey (Chlorocebus sabaeus ( Chlorocebus sabaeus A cell line expressing TrkB (amino acids 32 to 822 of approval number XP_007967815.1 (see also SEQ ID number 94)) was also prepared.

[0139]

[0113] The terms “brain-derived neurotrophic factor” or “BDNF” refer to a ligand for TrkB, and the amino acid sequence of BDNF is represented by SEQ ID NO. 90 (isoform A1-120 having amino acids 129-247 of Approval No. NP_733928.1 with Met added to the N-terminus). In a specific embodiment described herein, the source of BDNF is from the manufacturer (R & D Systems, 248-BD / CF).

[0140]

[0114] All references to proteins, polypeptides, and protein fragments in this document are intended to refer to the human versions of the respective proteins, polypeptides, or protein fragments, unless explicitly specified as originating from a non-human species. Accordingly, the expression "TrkB" refers to human TrkB unless otherwise specified as originating from a non-human species, e.g., "Monkey TrkB," "Mouse TrkB," "Rat TrkB," etc.

[0141]

[0115] The expression “anti-TrkB antibody” as used herein comprises both a monovalent antibody having single specificity and a bispecific antibody comprising a first arm binding to TrkB and a second arm binding to a second (target) antigen, wherein the anti-TrkB arm comprises any HCVR / LCVR or CDR sequence as presented in Table 1 of the present invention. The expression “anti-TrkB antibody” also comprises an anti-TrkB antibody conjugated to a drug or toxin (i.e., a cytotoxic agent) or an antibody-drug conjugate (ADC) comprising an antigen-binding portion thereof. The expression “anti-TrkB antibody” also comprises an anti-TrkB antibody conjugated to a radionuclide or an antibody-radionuclide conjugate (ARC) comprising an antigen-binding portion thereof.

[0142]

[0116] The term “anti-TrkB antibody” as used herein means any antigen-binding molecule or molecular complex comprising at least one complementarity determining region (CDR) that specifically binds to or interacts with TrkB or a portion of TrkB. The term “antibody” comprises an immunoglobulin molecule comprising four polypeptide chains and a multimer thereof (e.g., IgM) in which two heavy (H) chains and two light (L) chains are interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (HCVR or V in this application). HIt includes the heavy chain invariant region (abbreviated as) and the heavy chain invariant region. The heavy chain invariant region consists of three domains C H 1, C H 2 and C H 3. Each light chain includes a light chain variable region (LCVR or V in this application). L It includes a domain (abbreviated as) and a light chain invariant region. The light chain invariant region is a single domain (C L Includes 1). V H and V L The region can be further subdivided into a more conserved region referred to as the Framework Region (FR) and a hypervariable region referred to as the Complementarity Decision Region (CDR) that is scattered. Each V H and V L It consists of three CDRs and four FRs aligned from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the invention, the FRs of the anti-TrkB antibody (or its antigen-binding site) may be identical to human germline sequences or may be naturally or artificially modified. The amino acid consensus sequence may be defined based on the parallel analysis of two or more CDRs.

[0143]

[0117] The term “antibody” as used herein also includes antigen-binding fragments of full-length antibody molecules. Terms such as “antigen-binding portion” of an antibody, “antigen-binding fragment” of an antibody as used herein include polypeptides or glycoproteins that may be obtained by an enzyme that specifically binds to an antigen to form a complex, or that are synthetic or genetically processed. The antigen-binding fragment of an antibody may be derived from the whole antibody molecule using any suitable standard technique, such as proteolytic digestion or recombinant genetic processing techniques, which include, for example, the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA may be known and / or readily available from, for example, commercial sources, DNA libraries (including, for example, phage-antibody libraries), or synthesized. The DNA may be sequenced and manipulated by chemical or molecular biological techniques, for example, to align one or more variable and / or constant domains into a suitable configuration, to introduce codons, to generate cysteine ​​residues, to modify, add, or delete amino acids, etc.

[0144]

[0118] Non-limiting examples of antigen-binding fragments include the following: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-stranded Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues mimicking the hypervariable region of an antibody (e.g., a separated complementarity determining region (CDR), e.g., CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Other processed molecules, e.g., domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodidies, tetrabodies, minibodies, nanobodies (e.g. .Monovalent nanobodies, divalent nanobodies, etc.), small modular immunotherapies (SMIPs), and Sharq variable IgNAR domains are also included in the expression “antigen-binding fragment” as used herein.

[0145]

[0119] The antigen-binding fragment of the antibody typically comprises at least one variable domain. The variable domain may be a domain of any size or amino acid composition and will generally include at least one CDR adjacent to or forming a backbone with at least one backbone sequence. V L V combined with the domain H In the antigen-binding fragment having a domain, V H and V L Domains can be positioned in any suitable arrangement relative to one another. For example, the variable region can be a dimer and V H -V H , V H -V L or V L -V L It contains a dimer. Alternatively, the antigen-binding fragment of the antibody is a monomeric V H or V L It may contain a domain.

[0146]

[0120] In a specific embodiment, the antigen-binding fragment of the antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found within the antigen-binding fragment of the antibody of the present invention include: (i) V H -C H 1; (ii) V H -C H 2; (iii) V H -C H 3; (iv) V H -C H 1-C H 2; (v) V H -C H 1-C H2-C H 3; (vi) V H -C H 2-C H 3; (vii) V H -C L ; (viii) V L -C H 1; (ix) V L -C H 2; (x) V L -C H 3; (xi) V L -C H 1-C H 2; (xii) V L -C H 1-C H 2-C H 3; (xiii) V L -C H 2-C H 3; and (xiv) V L -C L In any configuration of variable and constant domains comprising any exemplary configurations listed above, the variable and constant domains may be directly connected to each other or connected by a complete or partial hinge or linker region. The hinge region may consist of at least two amino acids (e.g., 5, 10, 15, 20, 40, 60, or more), which induce flexible or semi-flexible binding between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, the antigen-binding fragments of the antibody of the present invention are each and / or one or more monomeric V H or V L It may include homodimers or heterodimers (or other polymers) of any variable and invariant domain configurations listed above, which are non-covalently bonded to the domain (e.g., by disulfide bond(s)).

[0147]

[0121] With respect to the entire antibody molecule, the antigen-binding fragment may be unispecific or multispecific (e.g., bispecific). A multispecific antigen-binding fragment of an antibody typically comprises at least two different variable domains, wherein each variable domain may specifically bind to a separate antigen or specifically bind to a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody format disclosed herein, may be adapted for use associated with the antigen-binding fragment of the antibody of the present invention using routine techniques available in the art.

[0148]

[0122] In certain cases, for example, it may be required to antagonize TrkB to inhibit the growth or proliferation of neuronal tumor cells. However, the antibody of the present invention acts as an agonist antibody that acts as a neuronal survival enhancer and a neuroprotective agent. The antibody of the present invention may function by enhancing the interaction between TrkB and the ligand BDNF. Alternatively, the antibody of the present invention may mediate TrkB signaling through a mechanism that does not involve enhancing the interaction between its ligand and TrkB.

[0149]

[0123] The term “human antibody” as used herein is intended to include human antibodies that do not exist naturally. The term includes antibodies produced by recombinant cells from non-human mammals or from non-human mammals. The term is not intended to include antibodies isolated from human subjects or produced from human subjects.

[0150]

[0124] The antibodies of the present invention may be recombinant and / or naturally occurring human antibodies in some embodiments. The term “recombinant human antibody” as used herein is intended to include any human antibody produced, expressed, generated, or isolated by recombinant means, e.g., an antibody expressed using a recombinant expression vector transfected into a host cell (further described below), an antibody isolated from a library of recombinant human antibodies (further described below), an antibody isolated from an animal (e.g., mouse) genetically transfected for a human immunoglobulin gene (see reference: e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or an antibody produced, expressed, generated, or isolated by any other means including splicing of a human immunoglobulin gene sequence into another DNA sequence. In a specific embodiment, the recombinant human antibody is applied for in vitro mutagenesis (or in vivo somatic mutagenesis where an animal genetically modified for the human Ig sequence is used) and thus, the V of the recombinant antibody H and V L The amino acid sequence of the region is human germline V H and V L It is a sequence that is related to the sequence but may not naturally exist within the in vivo human antibody germline repertoire.

[0151]

[0125] Human antibodies can exist in two forms associated with hinge heterogeneity. In one form, the immunoglobulin molecule comprises a stable four-chain composition of approximately 150 to 160 kDa, wherein the dimers are held together by interchain heavy chain disulfide bonds. In a second form, the dimers are not bound via interchain disulfide bonds, and a molecule of approximately 75-80 kDa is formed, consisting of covalently coupled light and heavy chains (half-antibody). These forms were extremely difficult to separate even after affinity purification.

[0152]

[0126] Among various intact IgG isotypes, the frequency of the appearance of the second form is due to, but is not limited to, structural differences associated with the hinge domain isotype of the antibody. A single amino acid substitution in the hinge domain of the human IgG4 hinge can significantly reduce the appearance of the second form (see: Angal et al. (1993) Molecular Immunology 30:105) to levels typically observed using the human IgG1 hinge. To improve the yield of the desired antibody form, the present invention [addresses], for example, the hinge, C that may be required in manufacturing H 2 or C H It includes antibodies having one or more mutations within 3 regions.

[0153]

[0127] Terms such as “specifically bind” or “specifically bind to” mean that an antibody or its antigen-binding fragment forms a complex with a relatively stable antigen under physiological conditions. Specific binding is at least about 1 x 10⁻⁶ -6 It can be characterized by an equilibrium dissociation constant less than or equal to M (e.g., a smaller K D(indicates a tighter bond). Methods for determining whether two molecules bind specifically are widely known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, etc. As described herein, antibodies that bind specifically to TrkB were identified by surface plasmon resonance, for example, BIACORE™. Furthermore, multispecific antibodies that bind to the TrkB protein and one or more additional antigens, or bispecific antibodies that bind to two different regions of TrkB, are considered to be "specifically binding" antibodies as used herein.

[0154]

[0128] The antibodies of the present invention may be isolated antibodies. As used herein, “isolated antibodies” means antibodies identified, isolated, and / or recovered from at least one component of its natural environment. For example, antibodies isolated or removed from at least one component of an organism, or from tissues or cells where antibodies are naturally present or naturally produced, are “isolated antibodies” for the purposes of the present invention. Isolated antibodies also include homologous antibodies within recombinant cells. Isolated antibodies are antibodies applied in at least one purification or isolation step. Depending on specific embodiments, isolated antibodies may be substantially free of other cellular materials and / or chemicals.

[0155]

[0129] The anti-TrkB antibodies disclosed herein may include one or more amino acid substitutions, insertions, and / or deletions within the backbone and / or CDR regions of the heavy and light chain variable domains. Such mutations can be easily identified by comparing the amino acid sequences disclosed herein with, for example, sequences available from public antibody sequence databases. Once obtained, antibodies and antigen-binding fragments containing one or more mutations can be easily tested for one or more desired properties, such as enhanced binding specificity, increased binding affinity, improved or enhanced antagonist or agonist biological properties (in some cases), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in the general manner described above are incorporated into the present invention.

[0156]

[0130] Additionally, the present invention comprises an anti-TrkB antibody comprising a variant of any HCVR, LCVR, and / or CDR amino acid sequence disclosed herein having one or more conservative substitutions. For example, the present invention comprises an anti-TrkB antibody having an HCVR, LCVR, and / or CDR amino acid sequence having conservative amino acid substitutions, for example, 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc., for any HCVR, LCVR, and / or CDR amino acid sequence presented in Table 1 of the present invention.

[0157]

[0131] The term “epitope” refers to an epitope that interacts with a specific antigen-binding site within a variable region of an antibody molecule known as a paratope. A single antigen may have more than one epitope. Therefore, different antibodies may bind to different regions on the antigen and have different biological effects. Epitopes can be morphological or linear. Morphological epitopes are generated by spatially adjacent amino acids of different segmental origins of a linear polypeptide chain. Linear epitopes are generated by adjacent amino acid residues in the polypeptide chain. In certain circumstances, epitopes may include a moiety of saccharide, phosphoryl, or sulfonyl groups on the antigen.

[0158]

[0132] When referring to a nucleic acid or a fragment thereof, the terms “substantial identity” or “substantially identical” indicate that when optimally aligned with another nucleic acid (or its complementary strand) by appropriate nucleotide insertion or deletion, nucleotide sequence identity exists in at least about 95% of the nucleotide bases, and more preferably at least about 96%, 97%, 98%, or 99%, when measured by any algorithm for widely known sequence identity such as FASTA, BLAST, or GAP as discussed below. A nucleic acid molecule having substantial identity with respect to a reference nucleic acid molecule may, in certain cases, encode a polypeptide having an amino acid sequence identical or substantially similar to that of the polypeptide encoded by the reference nucleic acid molecule.

[0159]

[0133] As applied to polypeptides, the terms "substantial similarity" or "substantially similar" mean that when two peptide sequences are optimally aligned, such as by the program GAP or BESTFIT using default gap weights, they share at least 95% sequence identity, and more preferably at least 98% or 99% sequence identity. Preferably, non-identical residue positions differ by conservative amino acid substitutions. "Conservative amino acid substitution" is the substitution of an amino acid residue by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions will not substantially alter the functional properties of the protein. If two or more amino acid sequences differ from each other due to conservative substitutions, the percentage sequence identity or degree of similarity may be upscaled to compensate for the conservative nature of the substitutions. Methods for performing such adjustments are widely known to those skilled in the art. Refer to the literature (Pearson (1994) Methods Mol. Biol. 24: 307-331, incorporated herein by reference). Examples of amino acid groups having side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartate and glutamate; and 7) sulfur-containing side chains: cysteine ​​and methionine. Desirable conservative amino acid substituents are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine.Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in the literature incorporated herein by reference [Gonnet et al. (1992) Science 256: 1443-1445]. A “moderately conservative” substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0160]

[0134] Sequence similarity for polypeptides, also referred to as sequence identity, is typically measured using sequencing software. Protein analysis software matches similar sequences using measurements of similarity assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software includes programs such as Gap and Bestfit, which can be used to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides of different organism species, or between a wild-type protein and its mutate, using default parameters. Literature ( for example Refer to GCG version 6.1. Polypeptide sequences may also be compared using FASTA with default or recommended parameters, which is a program within GCG version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides query search, alignment for the best overlap regions between search sequences, and % sequence identity (see Pearson (2000) above). Another preferred algorithm for comparing the sequences of the present invention with a database containing multiple sequences from different organisms is the computer program BLAST using default parameters, in particular BLASTP or TBLASTN. For example, the literature (Altschul et al.(1990) J. Mol. Biol. 215:403-410 and Altschul et al. See (1997) Nucleic Acids Res. 25:3389-402 (cited herein by reference).

[0161] Biological characteristics of antibodies

[0162]

[0135] The present invention relates to K of less than approximately 200 nM when measured by surface plasmon resonance at 25°C or 37°C. D The invention comprises an anti-TrkB antibody that binds to human TrkB. According to specific embodiments, the invention comprises K at a concentration of less than about 600 pM, less than about 300 pM, less than about 200 pM, less than about 150 pM, less than about 100 pM, less than about 80 pM, less than about 50 pM, less than about 40 pM, less than about 30 pM, less than about 20 pM, less than about 10 pM, less than about 5 pM, less than about 3 pM, or less than about 1 pM. D It includes an anti-TrkB antibody that binds to human TrkB.

[0163]

[0136] The present invention comprises an anti-TrkB antibody that binds to human TrkB with a dissociation half-life (t½) greater than about 10 minutes when measured by surface plasmon resonance at 25°C or 37°C. According to specific embodiments, the present invention comprises an anti-TrkB antibody that binds to human TrkB with a t½ greater than about 20 minutes, greater than about 50 minutes, greater than about 100 minutes, greater than about 120 minutes, greater than about 150 minutes, greater than about 300 minutes, greater than about 350 minutes, greater than about 400 minutes, greater than about 450 minutes, greater than about 500 minutes, greater than about 550 minutes, greater than about 600 minutes, greater than about 700 minutes, greater than about 800 minutes, greater than about 900 minutes, greater than about 1000 minutes, greater than about 1100 minutes, or greater than about 1200 minutes.

[0164]

[0137] The present invention comprises an anti-TrkB antibody that may or may not bind to monkey TrkB, or mouse or rat TrkB. When an antibody as used herein is tested in an antigen binding assay such as surface plasmon resonance, the antibody is greater than about 1000 nM K in said assay. D If it indicates or does not indicate any antigen binding, it “does not bind” to a specific antigen (e.g., monkey, mouse, or rat TrkB). Another screening format that can be used to determine whether an antibody binds to or does not bind to a specific antigen according to the above aspects of the present invention is ELISA.

[0165]

[0138] The present invention relates to an EC of less than about 100 pM. 50 It includes an anti-TrkB antibody that activates human TrkB signaling in cells processed to express a TrkB receptor. Using the assay format described in Example 5 or a substantially similar assay format, EC 50 The value can be calculated as the concentration of antibody required to activate TrkB-mediated signaling to the observed maximum half signal. Accordingly, depending on a specific embodiment, the present invention provides an EC of less than about 500 pM, less than about 400 pM, less than about 300 pM, less than about 200 pM, less than about 100 pM, less than about 90 pM, less than about 80 pM, less than about 70 pM, less than about 60 pM, less than about 50 pM, less than about 40 pM, less than about 30 pM, less than about 20 pM, less than about 10 pM, or less than about 5 pM when measured using the assay format or substantially similar assay described in Example 5 of this application. 50 It includes an anti-TrkB antibody that mediates human TrkB signaling in cells processed to express a TrkB receptor in the presence or absence of BDNF.

[0166]

[0139] The present invention includes an anti-TrkB antibody that activates the TrkB receptor, as indicated by TrkB phosphorylation after direct hippocampal injection in mice humanized to express the human TrkB receptor as shown in Example 6.

[0167]

[0140] The present invention comprises an anti-TrkB antibody that promotes weight loss in mice humanized to express a human TrkB receptor. The antibody of the present invention also acts to promote the loss of body fat mass and increase gait activity while reducing food and water intake (see Example 7).

[0168]

[0141] The antibody of the present invention promotes the survival of retinal ganglion cells (RGCs) in rats humanized to express the human TrkB receptor when tested in an optic nerve transection model. Refer to Example 8.

[0169]

[0142] The antibody of the present invention activates downstream pathways MAPK / EPK and PI3K / Akt as indicated by exposure of primary mouse cortical neurons obtained from humanized TrkB mice to the antibody of the present invention (see Example 9).

[0170]

[0143] The anti-TrkB antibody of the present invention also promotes the survival of SH-SY5Y cells in a dose-dependent manner, as shown in Example 10.

[0171]

[0144] The present invention relates to an IC of less than about 5 nM 50 It includes anti-TrkB antibodies that block TrkB binding to BDNF. For example, as shown in Example 12, all three antibodies tested blocked >50% of mouse or rat TrkB binding to BDNF. Using the assay format described in Example 12 or a substantially similar assay format, IC 50The value can be calculated as the concentration of antibody required to block TrkB binding to BDNF when compared to the maximum signal observed in the absence of the antibody. Accordingly, depending on a specific embodiment, the present invention, when measured using the assay format described herein in Example 12 or a substantially similar assay, has an IC of less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, less than about 900 pM, less than about 800 pM, less than about 700 pM, less than about 600 pM, about 500 pM, less than about 400 pM, less than about 300 pM, less than about 200 pM, less than about 100 pM, less than about 90 pM, less than about 80 pM, less than about 70 pM, less than about 60 pM, less than about 50 pM, less than about 40 pM, less than about 30 pM, or less than about 20 pM. 50 It comprises an anti-TrkB antibody that blocks TrkB binding to BDNF. In one embodiment, the TrkB antibody of the present invention has an IC50 range of about 180 pM to about 4 nM. 50 It blocks the binding of TrkB to BDNF.

[0172]

[0145] Where described in terms of being “measured by surface plasmon resonance,” the binding characteristics of the antibody of the present invention (e.g., any binding characteristics mentioned herein) mean that the relevant binding characteristics belonging to the interaction between the antibody and the antigen are measured using a surface plasmon resonance instrument (e.g., Biacore® instrument, GE Healthcare) using standard Biacore assay conditions or substantially similar assay formats as illustrated in Examples 3 and 4 herein. In certain embodiments, the binding parameters are measured at 25°C, and in other embodiments, the binding parameters are measured at 37°C.

[0173]

[0146] The present invention comprises an antibody that specifically binds to TrkB or an antigen-binding fragment thereof, comprising an HCVR and / or LCVR comprising an amino acid sequence selected from any HCVR and / or LCVR amino acid sequence listed in Table 1.

[0174]

[0147] The antibody of the present invention may have one or more of the biological characteristics mentioned above or any combination thereof. The above list of biological characteristics of the antibody of the present invention is not intended to be exhaustive. Other biological characteristics of the antibody of the present invention will become apparent to those skilled in the art from a review of this specification, including the examples herein.

[0175] Epitope Mapping and Related Techniques

[0176]

[0148] The epitope to which the antibody of the present invention binds may consist of a single continuous sequence of three or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) of the TrkB protein. Alternatively, the epitope may consist of a number of discontinuous amino acids (or amino acid sequences) of TrkB. In some embodiments, the epitope is located on or near the surface of TrkB, for example, in a domain that interacts with its ligand BDNF. In other embodiments, the epitope is located on or near the surface of TrkB that does not interact with the TrkB ligand, for example, on the surface of TrkB where the antibody, when bound to the epitope, does not interfere with the interaction between TrkB and its ligand.

[0177]

[0149] It is possible to determine whether an antibody interacts with "one or more amino acids" within a polypeptide or protein using various techniques known to those skilled in the art. Exemplary techniques are, for example, referenced in the literature (see: AntibodiesThis includes conventional cross-blocking assays as described in Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY), alanine scanning mutation analysis, peptide blot analysis (Reference: Reineke, 2004, Methods Mol Biol 248:443-463), and peptide cleavage analysis. Additionally, methods such as epitope excision, epitope extraction, and chemical modification of the antigen may also be used (Reference: Tomer (2000) Prot. Sci. 9: 487-496). Another method that can be used to identify amino acids within a polypeptide with which an antibody interacts is hydrogen / deuterium exchange, which is detected by mass spectrometry. Generally, the hydrogen / deuterium exchange method comprises the step of deuterium-labeling the protein of interest and then binding an antibody to the deuterium-labeled protein. Subsequently, the protein / antibody complex is added to water to allow hydrogen-deuterium exchange to occur in all residues except those protected by the antibody (remaining in a deuterium-labeled state). After the dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectroscopic analysis to identify the deuterium-labeled residues corresponding to the specific amino acids with which the antibody interacts. For example, the literature (Ehring (1999) Analytical Biochemistry 267(2):252-259; Engen and Smith (2001) Anal. Chem. 73 Refer to :256A-265A).

[0178]

[0150] The present invention comprises an anti-TrkB antibody that binds to the same epitope as any specific exemplary antibody described herein (e.g., an antibody comprising any amino acid sequence as presented in Table 1 of the present invention). Additionally, the present invention comprises an anti-TrkB antibody that competes to bind to TrkB with any specific exemplary antibody described herein (e.g., an antibody comprising any amino acid sequence as presented in Table 1 of the present invention).

[0179]

[0151] A person skilled in the art can easily determine whether an antibody binds to or competes for binding to the same epitope as the reference anti-TrkB antibody by using ordinary methods known in the art and exemplified herein. For example, to determine whether a test antibody binds to the same epitope as the standard anti-TrkB antibody of the present invention, the reference antibody is made to bind to the TrkB protein. Next, the ability of the test antibody to bind to the TrkB molecule is evaluated. If the test antibody can bind to TrkB after saturating binding with the standard anti-TrkB antibody, it can be concluded that the test antibody binds to an epitope different from that of the standard anti-TrkB antibody. On the other hand, if the test antibody cannot bind to the TrkB molecule after saturating binding with the reference anti-TrkB antibody, the test antibody may bind to the same epitope as that bound by the reference anti-TrkB antibody of the present invention. Subsequently, additional general experiments (e.g., peptide mutation and binding assays) can be performed to determine whether the lack of binding observed in the test antibody is actually due to binding to the same epitope as the standard antibody, or whether stereochemical blockade (or another phenomenon) is the cause of the observed lack of binding. These types of experiments may be performed using ELISA, RIA, Viacor, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the industry. According to a specific embodiment of the present invention, two antibodies bind to the same (or overlapping) epitope, for example, in a 1-, 5-, 10-, 20-, or 100-fold excess of one antibody, when measured in a competitive binding assay, inhibits the binding of the other by at least 50% but preferably 75%, 90%, or even 99% (see reference: e.g., Junghans et al., Cancer Res. 1990:50:1495-1502).Alternatively, two antibodies are considered to bind to the same epitope if all amino acid mutations within the antigen that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other antibody. Two antibodies are considered to have a “redundant epitope” if only a subset of amino acid mutations that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other.

[0180]

[0152] To determine whether the antibody competes for binding with the reference anti-TrkB antibody (or cross-competes for binding), the binding method described above is performed in two directions: in the first direction, the reference antibody is bound to the TrkB protein under saturation conditions, and then the binding of the test antibody to the TrkB molecule is evaluated. In the second direction, the reference antibody is bound to the TrkB molecule under saturation conditions, and then the binding of the reference antibody to the TrkB molecule is evaluated. In both directions, if only the first (saturated) antibody is able to bind to the TrkB molecule, it is concluded that the test antibody and the reference antibody compete for binding to TrkB (see reference: e.g., the assay format described in Example 4 of the present invention, wherein the TrkB protein is captured onto a sensor tip and the TrkB-coated sensor tip is treated with the reference antibody [mAb-1] and subsequently with the test anti-TrkB antibody [mAb-2] in both binding order). As those skilled in the art would know, antibodies competing for binding to a reference antibody may not necessarily bind to the same epitope as the standard antibody, but may bind to overlapping or adjacent epitopes and structurally block the binding of the reference antibody.

[0181] Production of human antibodies

[0182]

[0153] The anti-TrkB antibody of the present invention may be a completely human antibody, but may also be an antibody that does not exist naturally. Methods for producing monoclonal antibodies, including a completely human monoclonal antibody, are known in the art. Any of these known methods may be used to produce a human antibody that specifically binds to human TrkB in connection with the content of the present invention.

[0183]

[0154] Using VELOCIMMUNE® technology [e.g., see US Patent No. 6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®] or any other known method for generating monoclonal antibodies, a high-affinity chimeric antibody to an allergen having a human variable region and a mouse constant region is initially isolated. The VELOCIMMUNE® technique involves generating a genetically modified mouse having a genome containing human heavy and light chain variable regions operably linked to an endogenous mouse constant region locus so that the mouse produces an antibody containing the human variable region and the mouse constant region in response to stimulation by the antigen. The DNA encoding the variable regions of the heavy and light chains of the antibody is isolated and operably linked to the DNA encoding the human heavy and light constant regions. Next, said DNA is expressed in cells capable of expressing a complete human antibody.

[0184]

[0155] Generally, VELOCIMMUNE® mice are immunized with the antigen of interest, and lymphoid cells (e.g., B cells) are recovered from the antibody-expressing mice. These lymphoid cells can be fused with a myeloma cell line to produce an immortal hybridoma cell line, and these hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific to the antigen of interest. DNA encoding variable regions of the heavy and light chains can be isolated and ligated to desired homomorphic regions of the heavy and light chains. These antibody proteins can be produced in cells such as CHO cells. Alternatively, antigen-specific chimeric antibodies or DNA encoding variable domains of the light and heavy chains can be isolated directly from antigen-specific lymphocytes.

[0185]

[0156] As described in the experimental section below, isolated high-affinity chimeric antibodies having a human variable region and a mouse constant region were characterized and selected for desirable features including affinity, selectivity, epitopes, etc. Then, the mouse constant regions were replaced with the desired human constant region to prepare the complete human antibody of the present invention, e.g., wild-type or modified IgG1 or IgG4. Although the selected constant region may vary depending on the specific application, high-affinity antigen binding and target-specificity features remain in the variable region.

[0186]

[0157] In certain embodiments, it may be desirable to test an anti-human TrkB antibody in mice or rats processed to express a human TrkB receptor. These mice or rats may be advantageous in situations where the anti-TrkB antibody can bind only to human TrkB but does not cross-react with mouse or rat TrkB. In the present invention, certain embodiments were performed using mice and rats genetically modified to express human TrkB. Any method known to those skilled in the art may be used to produce the TrkB humanized mice and rats.

[0187]

[0158] Generally, the antibody of the present invention has a very high affinity, typically about 10 when measured by binding to an antigen immobilized in a solid or solution phase. -12 to about 10 -9 M's K D has

[0188] raw deer

[0189]

[0159] The anti-TrkB antibodies and antibody fragments of the present invention comprise proteins having amino acid sequences that differ from those of the antibodies described above but possess the ability to bind to human TrkB. These variant antibodies and antibody fragments include one or more additions, deletions, or substitutions of amino acids compared to the parent sequence, but exhibit biological activity essentially equivalent to that of the antibodies described herein. Likewise, the anti-TrkB antibody-coding DNA sequence comprises a sequence encoding an anti-TrkB antibody or antibody fragment that includes one or more additions, deletions, or substitutions of nucleotides compared to the disclosed sequence, but is essentially bioequivalent to the anti-TrkB antibody or antibody fragment of the present invention. Examples of the variant amino acids and DNA sequences are discussed above.

[0190]

[0160] Two antigen-binding proteins or antibodies are considered bioequivalent if, for example, when administered at the same molar dose, whether as a single dose or multiple doses, under similar experimental conditions, they do not show a significant difference in absorption rate and degree of absorption. Some antibodies will be considered equivalent or pharmaceutical substitutes if they are equivalent in degree of absorption but not in absorption rate, and may nevertheless be considered bioequivalent because this difference in absorption rate is intentional, reflected in labeling, is not essential for achieving an effective in vivo drug concentration, for example, during chronic use, and is considered medically insignificant for the specific drug product being studied.

[0191]

[0161] In one embodiment, the two antigen-binding proteins are bioequivalent when there is no clinically significant difference in their safety, purity, and efficacy.

[0192]

[0162] In one embodiment, the two antigen-binding proteins are bioequivalent if no clinically significant change in immunogenicity or increased risk of adverse effects, including reduced efficacy, is expected compared to a continuous treatment without such switching, even if the patient may switch between the reference product and the biological product one or more times.

[0193]

[0163] In one embodiment, two antigen-binding proteins are bioequivalent if they both act to the extent that such mechanisms are known by common mechanisms of action for the conditions of use or conditions.

[0194]

[0164] Bioequivalence can be demonstrated by in vivo and in vitro methods. Measurements of bioequivalence include, for example, (a) in vivo tests in humans or other mammals that measure the concentration of an antibody or its metabolite as a function of time in blood, plasma, serum, or other biological body fluids; (b) in vitro tests that correlate with and reasonably predict human bioavailability data; (c) in vivo tests in humans or other mammals that measure the appropriate short-term pharmacological effect of the antibody (or its target) as a function of time; and (d) well-controlled clinical tests that establish the safety, efficacy, or bioavailability or bioequivalence of the antibody.

[0195]

[0165] Bioequivalent variants of the anti-TrkB antibody of the present invention may be constructed, for example, by performing various substitutions of residues or sequences, or by deleting terminal or internal residues or sequences that are not required for biological activity. For example, cysteine ​​residues that are not essential for biological activity may be deleted or replaced with other amino acids to prevent the formation of unnecessary or incorrect intramolecular disulfide bridges upon regeneration. In other cases, bioequivalent antibodies may include anti-TrkB antibody variants comprising amino acid changes that modify the glycosylation properties of the corresponding antibody, for example, mutations that eliminate or remove glycosylation.

[0196] Species selectivity and species cross-reaction

[0197]

[0166] According to specific embodiments, the present invention provides an anti-TrkB antibody that binds to human TrkB but does not bind to TrkB of other species origin. The present invention also comprises an anti-TrkB antibody that binds to human TrkB and TrkB of one or more non-human species origins. For example, the anti-TrkB antibody of the present invention may bind to human TrkB and, in some cases, may or may not bind to mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cattle, horse, camel, cynomologus, marmoset, rhesus, or chimpanzee TrkB. According to specific exemplary embodiments of the present invention, an anti-TrkB antibody that specifically binds to human TrkB but does not bind to or only weakly binds to mouse or rat TrkB is provided.

[0198] Multispecific antibodies

[0199]

[0167] The antibodies of the present invention may be monospecific or multispecific (e.g., bispecific). Multispecific antibodies may be specific to different epitopes of a single target polypeptide or may comprise antigen-binding domains specific to more than one target polypeptide. For example, see the literature (Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al See ., 2004, Trends Biotechnol. 22:238-244). The anti-TrkB antibody of the present invention may be linked to another functional molecule, e.g., another peptide or protein, or may be co-expressed with it. For example, an antibody or a fragment thereof may be functionally linked (e.g. by chemical coupling, genetic fusion, non-covalent association, or others) to one or more other molecular entities, such as another antibody or antibody fragment, to produce a bispecific or multispecific antibody having a second binding specificity.

[0200]

[0168] The present invention comprises a two-specific antibody, wherein one arm of the immunoglobulin binds to human TrkB and the other arm of the immunoglobulin is specific to a second antigen. The TrkB-binding arm may comprise any HCVR / LCVR or CDR amino acid sequence as presented in Table 1 of the present invention.

[0201]

[0169] An exemplary bispecific antibody format that can be used in connection with the present invention is a first immunoglobulin (Ig) C H 3rd domain and 2nd Ig C H It involves the use of 3 domains, wherein the first and second Ig C H The three domains differ from each other by at least one amino acid, and at least one amino acid difference reduces the binding of the bispecific antibody to protein A compared to a bispecific antibody without amino acid differences. In one embodiment, the first Ig C H The 3rd domain binds to protein A and the 2nd Ig C H Domain 3 contains mutations that reduce or abolish Protein A binding, such as the H95R variant (by IMGT exon numbering; H435R according to EU numbering). 2 C H 3 may additionally include a Y96F variant (by IMGT; according to the EU, Y436F). 2 C HAdditional variations that may be found within 3 include the following: for IgG1 antibodies, D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT; according to the EU, D356E, L358M, N384S, K392N, V397M, and V422I); for IgG2 antibodies, N44S, K52N, and V82I (by IMGT; according to the EU, N384S, K392N, and V422I); and for IgG4 antibodies, Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (by IMGT; according to the EU, Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I). Changes in the above-mentioned specific antibody format are considered to be within the scope of the present invention.

[0202]

[0170] Other exemplary bispecific formats that may be used in connection with the present invention include, without limitation, scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, Quadroma, knob-into-hole, common light chains (e.g., common light chains having knob-into-holes), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody, IgG1 / IgG2, dual-functional Fab (DAF)-IgG, and Mab 2This includes bispecific formats (for details on these formats, refer, for example, to the literature [Klein et al. 2012, mAbs 4:6, 1-11] and the references cited therein). Bispecific antibodies can also be constructed using peptide / nucleic acid configurations, for example, here, a site-specific antibody-oligonucleotide conjugate is generated using non-natural amino acids with orthogonal chemical reactivity, which then self-assembles into a multimeric complex having a limited composition, binding valence, and geometry. (References, e.g., Kazane et al ., J. Am. Chem. Soc . [ Epub : Dec. 4, 2012 ).

[0203] Therapeutic formulations and administration

[0204]

[0171] The present invention provides a pharmaceutical composition comprising the anti-TrkB antibody of the present invention or an antigen-binding fragment thereof. The pharmaceutical composition of the present invention is formulated with suitable carriers, excipients, and other agents that provide improved transport, delivery, tolerance, etc. A number of suitable formulations may be found in formulations known to all pharmaceutical chemistry: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid-containing vesicles (e.g., LIPOFECTIN™, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion carbowax (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. Also refer to the literature (Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-311).

[0205]

[0172] The dose of the antibody administered to a patient may vary depending on the patient's age and size, target disease, pathological condition, route of administration, etc. The preferred dose is typically calculated based on body weight or body surface area. In adult patients, it may be advantageous to administer the antibody of the present invention intravenously as a single dose of about 0.01 to about 20 mg / kg body weight normally, more preferably about 0.02 to about 7, about 0.03 to about 5, or about 0.05 to about 3 mg / kg body weight. Depending on the severity of the disease, the frequency and duration of treatment may be adjusted. The effective dose and schedule for administering the anti-TrkB antibody may be determined empirically; for example, the patient's course may be monitored by periodic evaluation and the dose adjusted accordingly. Furthermore, interspecies scaling of the dose may be performed using methods widely known in the art (e.g., Mordenti et al. , 1991, Pharmaceut. Res. 8 :1351).

[0206]

[0173] Various delivery systems are known and the pharmaceutical composition of the present invention may be administered using such systems, for example, by liposomal encapsulation, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, or by receptor-mediated endocytosis (see reference: e.g., Wu et al., 1987, J. Biol. Chem. 262: 4429-4432). Methods of administration include, but are not limited to, intravitreal, intraocular, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition may be administered by any convenient route, for example, by infusion or bolus injection, by absorption through the epithelial or mucocutaneous walls (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered in combination with other biological active agents. Administration may be systemic or local.

[0207]

[0174] The pharmaceutical composition of the present invention may be delivered subcutaneously or intravenously using standard needles and syringes. Additionally, regarding subcutaneous delivery, a pen delivery device is applied to deliver the pharmaceutical composition of the present invention. Such a pen delivery device may be reusable or disposable. Reusable pen delivery devices generally use a replaceable cartridge containing the pharmaceutical composition. Once all the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily disposed of and replaced with a new cartridge containing the pharmaceutical composition. After doing so, the pen delivery device can be reused. Disposable pen delivery devices do not have replaceable cartridges. Rather, disposable pen delivery devices are pre-filled with the pharmaceutical composition maintained in a reservoir within the device. Once the pharmaceutical composition in the reservoir is emptied, the entire device is discarded.

[0208]

[0175] Various reusable pens and automatic injection delivery devices are used for the subcutaneous delivery of the pharmaceutical composition of the present invention. To give just a few examples, AUTOPEN™ [manufactured by Owen Mumford, Inc., Woodstock, UK], DISETRONIC™ pen [manufactured by Disetronic Medical Systems, Bergdorf, Switzerland], HUMALOG MIX 75 / 25™ pen, HUMALOG™ pen, HUMALIN 70 / 30™ pen [manufactured by Eli Lilly and Co., Indianapolis, Indiana, USA], NOVOPEN™ I, II, and III [manufactured by Novo Nordisk, Copenhagen, Denmark], NOVOPEN JUNIOR™ (manufactured by Novo Nordisk, Copenhagen, Denmark), BD™ pen [manufactured by Becton Dickinson, Franklin Lake, New Jersey, USA], OPTIPEN™, Includes, but is not limited to, OPTIPEN PRO™, OPTIPEN STARLET™ and OPTICLIK™ [manufactured by Sanofi-Aventis, Frankfurt, Germany]. Examples of disposable pen delivery devices applied for the subcutaneous delivery of the pharmaceutical composition of the present invention include, to name only, SOLOSTAR™ pen (Sanofi-Aventis), FLEXPEN™ (Novo Nordisk) and KWIKPEN™ (Eli Lilly), SURECLICK™ auto-injector [manufactured by Amgen, Thousand Oaks, California, USA], PENLET™ [manufactured by Haselmeier, Stuttgart, Germany], EPIPEN [manufactured by Dey, LP] and HUMIRA™ pen [manufactured by Abbott Labs, Abbott Park, Illinois, USA], but are not limited thereto.

[0209]

[0176] In certain situations, the pharmaceutical composition may be delivered to a controlled-release system. In one embodiment, a pump may be used (see reference: Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, a polymeric material may be used; see reference (Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida). In another embodiment, the controlled-release system may be located adjacent to the target of the composition and thus requires only a fraction of the systemic dose (see reference, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled-release systems are discussed in a review by reference (Langer, 1990, Science 249:1527-1533).

[0210]

[0177] Injectable preparations may include administration forms for intravenous, intravitreal, intraocular, subcutaneous, intradermal, and intramuscular injection, drip infusion, etc. These injectable preparations may be manufactured by methods known to the public. For example, injectable preparations for example,The above-described antibody or its salt can be prepared by dissolving, suspending, or emulsifying it in a sterile aqueous or oil medium commonly used for injection. Examples of aqueous media for injection include physiological saline, isotonic solutions containing glucose, and other adjuvants, which may be used in combination with suitable solubilizers, such as alcohol (e.g., ethanol), polyalcohol (e.g., propylene glycol, polyethylene glycol), nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As oil media, examples include sesame oil, soybean oil, etc., which may be used in combination with solubilizers such as benzyl benzoate, benzyl alcohol, etc. Thus, the prepared injection solution is preferably filled into a suitable ampoule.

[0211]

[0178] Advantageously, the above-described pharmaceutical composition for oral or parenteral use is prepared in a unit dose formulation adapted to the dose of the active ingredient. Such unit dose formulations include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the antibody contained therein is generally about 5 to about 500 mg per unit dose formulation; particularly in the injectable form, it is preferable that the antibody be contained in about 5 to about 100 mg, and in other formulations in about 10 to about 250 mg.

[0212] Therapeutic Uses of Antibodies

[0213]

[0179] The present invention comprises administering a therapeutic composition comprising an anti-TrkB antibody (e.g., an anti-TrkB antibody comprising any HCVR / LCVR or CDR sequence as presented in Table 1 of the present invention) to a subject in need thereof. The therapeutic composition may comprise any one or more anti-TrkB antibodies or antigen-binding fragments thereof described herein, and a pharmaceutically acceptable carrier or diluent.

[0214]

[0180] The antibody of the present invention is particularly useful for the treatment, prevention, and / or improvement of any disease or disorder associated with or mediated by TrkB expression or activity. Neurological function can be improved using the TrkB agonist antibody of the present invention, and any disease or pathological condition characterized by partial cell degradation, in particular, neuronal damage or neuronal degeneration, e.g., acute neurological injury or chronic neurodegenerative disease, can be treated or prevented using the same.

[0215]

[0181] The present invention includes a method for treating or preventing diseases or disorders of the eye by administering an anti-TrkB antibody or an antigen-binding fragment thereof, as described elsewhere in this invention, to a patient in need thereof.

[0216]

[0182] In one embodiment, the anti-TrkB antibody of the present invention may provide a method for preventing damage or death of retinal neurons. In one embodiment, the anti-TrkB antibody of the present invention may provide a method for treating pathological diseases in which retinal degeneration has occurred. In one embodiment, the anti-TrkB antibody of the present invention may provide a method for preventing degeneration of retinal cells by treating the eye before or after exposure to ocular surgery, light, or other environmental trauma. In one embodiment, the anti-TrkB antibody of the present invention may provide a method for preventing damage and degeneration of photoreceptors in the eye. In one embodiment, the anti-TrkB antibody of the present invention may provide a method for preventing retinal neurons without inducing side effects. In one embodiment, the anti-TrkB antibody of the present invention may provide a method for recovering or regenerating damaged photoreceptors.

[0217]

[0183] In a specific embodiment, the eye disease that can be treated with the anti-TrkB antibody of the present invention may be selected from the group consisting of glaucoma, diabetic retinopathy, age-related macular degeneration or other macular diseases, ischemic optic neuropathy, optic neuritis, retinal ischemia, photoreceptor degeneration, retinitis pigmentosa, Leber congenital amaurosis, Leber hereditary optic neuropathy, Usher Syndrome, Stargardt disease, and retinal artery or vein occlusion.

[0218]

[0184] Other pathological conditions that can be treated with one or more anti-TrkB antibodies of the present invention include retinal detachment, photic retinopathies, surgery-induced retinopathy (mechanically or photo-induced), toxic retinopathy, retinopathy of prematurity, viral retinopathy such as AIDS-associated CMV or HIV retinopathy; uveitis; ischemic retinopathy due to venous or arterial occlusion or other vascular disorders, retinopathy due to trauma to the eye or penetrating lesions, peripheral vitreoretinopathy or inherited retinal degeneration.

[0219]

[0185] In one embodiment, the anti-TrkB antibody of the present invention may be formulated for delivery into the eye or vitreous humor.

[0220]

[0186] The present invention also provides a method for treating other central or peripheral nervous system diseases or disorders, such as stroke or traumatic brain injury. Additionally, since the antibodies of the present invention act to promote neuronal survival and function as neuroprotective agents, any one or more of these agonist antibodies can demonstrate benefit in treating patients suffering from nervous system diseases or disorders, where neuronal survival is most critical in the recovery or repair of cell damage caused by diseases having a major effect on the nervous system, including neurodegenerative diseases.

[0221]

[0187] In connection with the treatment methods described herein, the anti-TrkB antibody may be administered as monotherapy (i.e., as a therapeutic agent only) or in combination with one or more additional therapeutic agents.

[0222] Combination therapy and formulations

[0223]

[0188] The present invention comprises a composition and a therapeutic agent comprising any anti-TrkB antibody described herein combined with one or more additional therapeutically active ingredients, and a therapeutic method comprising administering said composition to a subject in need thereof.

[0224]

[0189] The anti-TrkB antibody of the present invention may be co-formulated with and / or administered in combination with one or more additional therapeutically active ingredient(s) selected from the group consisting of: drugs that help lower intraocular pressure (IOP-lowering drugs), neurotrophins, and antagonists of vascular endothelial growth factor (VEGF), e.g., VEGF traps, e.g., aflibercept (EYLEA®). Other drugs that may be combined with the TrkB antibody of the present invention are prostaglandin analogs (e.g., ZIOPTAN™, XALATAN®), beta-blockers (e.g., TIMOPTIC XE®, ISTALOL®, BETOPTIC®S); alpha-2 adrenergic agonists (e.g., apraclonidine), carbonic anhydrase inhibitors (e.g. , TRUSOPT®, AZOPT®), cholinergic agents (e.g., ISOPTO®CARPINE, PILOPINE HS® gel), or combined therapeutic agents (beta-blockers + carbonic anhydrase inhibitors, e.g., COMBIGAN™, COSOPT®), but not limited thereto.

[0225]

[0190] The anti-TrkB antibody of the present invention may also be administered in combination with antiviral agents, antibiotics, analgesics, antioxidants, COX inhibitors, and / or NSAIDs, or co-formulated with them. The anti-TrkB antibody may also be used in conjunction with other types of therapeutic regimens, including stem cell therapy, glaucoma filtration surgery, laser surgery, or gene therapy.

[0226]

[0191] Additional therapeutic active ingredient(s) may be administered immediately, simultaneously with, or immediately after the administration of the anti-TrkB antibody of the present invention (for the purposes of the present disclosure, the administration regimen considers the administration of the anti-TrkB antibody “combined” with the additional therapeutic active ingredient). The present invention comprises a pharmaceutical composition in which the anti-TrkB antibody of the present invention is co-formulated with one or more of the additional therapeutic active ingredient(s) as described in other parts of the present invention.

[0227] Administration

[0228]

[0192] According to a specific embodiment of the present invention, multiple doses of an anti-TrkB antibody (or a pharmaceutical composition comprising a combination of the anti-TrkB antibody and any additional therapeutic activator mentioned herein) may be administered to a subject requiring them for a limited period. A method according to the above aspect of the present invention comprises the step of administering multiple doses of the anti-TrkB antibody of the present invention to a subject in succession. As used herein, the term “in succession” means administering each dose of the anti-TrkB antibody to the subject at different times, for example, on different dates with a predetermined interval (e.g., hours, days, weeks, or months). The present invention comprises a method comprising administering to a subject sequentially one or more secondary doses of the anti-TrkB antibody and optionally one or more tertiary doses of the anti-TrkB antibody following a single initial dose of the anti-TrkB antibody.

[0229]

[0193] The terms “initial dose,” “secondary dose,” and “tertiary dose” refer to the chronological order of administration of the anti-TrkB antibody of the present invention. Accordingly, the “initial dose” is the dose administered at the initiation of the therapeutic regimen (also referred to as the “baseline dose”); the “secondary dose” is the dose administered after the initial dose; and the “tertiary dose” is the dose administered after the second dose. The initial, secondary, and tertiary doses may all contain the same amount of anti-TrkB antibody, but may generally differ from one another in terms of the frequency of administration. However, in certain embodiments, the amount of anti-TrkB antibody contained in the initial, secondary, and / or tertiary doses differs from one another during the course of treatment (e.g., appropriately up- or down-adjusted). In a specific embodiment, two or more doses (e.g., two, three, four, or five) are administered as a “loading dose” at the start of the treatment regimen, and then subsequent doses are administered at a lower frequency (e.g., “maintenance dose”).

[0230]

[0194] In a specific exemplary embodiment of the present invention, each second and / or third dose is given for 1 hour to 26 weeks after the immediately preceding dose (e.g., 1, 1½, 2, 2½, 3, 3½, 4, 4½, 5, 5½, 6, 6½, 7, 7½, 8, 8½, 9, 9½, 10, 10½, 11, 11½, 12, 12½, 13, 13½, 14, 14½, 15, 15½, 16, 16½, 17, 17½, 18, 18½, 19, 19½, 20, 20½, 21, 21½, 22, 22½, 23, 23½, 24, 24½, 25, 25½, Administer after 26, 26½ hours or more. As used herein, the term “immediate preceding dose” means a dose of anti-TrkB antibody administered to the patient immediately before the next dose in sequence without an intervening dose in a sequence of multiple doses.

[0231]

[0195] A method according to the above aspect of the present invention may include the step of administering any number of secondary and / or tertiary doses of an anti-TrkB antibody to a patient. For example, in a specific embodiment, only a single secondary dose is administered to the patient. In another embodiment, two or more secondary doses (e.g., 2, 3, 4, 5, 6, 7, 8 or more) are administered to the patient. Additionally, in a specific embodiment, only a single tertiary dose is administered to the patient. In another embodiment, two or more tertiary doses (e.g., 2, 3, 4, 5, 6, 7, 8 or more) are administered to the patient. The administration regimen may be performed indefinitely over the lifetime of a specific subject or until the treatment is no longer therapeutically required or beneficial.

[0232]

[0196] In an embodiment comprising multiple secondary doses, each secondary dose may be administered at the same frequency as other secondary doses. For example, each secondary dose may be administered to the patient 1 to 2 weeks or 1 to 2 months after the previous preceding dose. Similarly, in an embodiment comprising multiple tertiary doses, each tertiary dose may be administered at the same frequency as other tertiary doses. For example, each tertiary dose may be administered to the patient 2 to 12 weeks after the previous preceding dose. In specific embodiments of the present invention, the frequency at which secondary and / or tertiary doses are administered to the patient may vary during the course of the treatment regimen. Additionally, the frequency of administration may be adjusted during the course of treatment by the attending physician according to the individual patient's needs after the clinical trial.

[0233]

[0197] The present invention allows for 2 to 6 loading doses to be administered to a patient at a first frequency (e.g., once a week, once every 2 weeks, once every 3 weeks, once every 1 month, once every 2 months, etc.), followed by 2 or more maintenance doses to be administered to the patient at a lower frequency. For example, according to the above aspect of the invention, when the loading dose is administered at a frequency of once a month, the maintenance dose may be administered to the patient at a frequency of once every 6 weeks, once every 2 months, once every 3 months, etc.

[0234] Diagnostic uses of antibodies

[0235]

[0198] The anti-TrkB antibody of the present invention may be used, for example, to detect and / or measure TrkB or TrkB-expressing cells in a sample for diagnostic purposes. For example, an anti-TrkB antibody or a fragment thereof may be used to diagnose a pathological condition or disease characterized by abnormal expression of TrkB (e.g., overexpression, low expression, absence of expression, etc.). An exemplary diagnostic test for TrkB may include, for example, the step of contacting a sample obtained from a patient with the anti-TrkB antibody of the present invention, wherein the anti-TrkB antibody is labeled with a detectable label or reporter molecule. Alternatively, an unlabeled anti-TrkB antibody may be used for diagnostic purposes in combination with a secondary antibody that is itself detectably labeled. The detectable label or reporter molecule is, for example, 3 H, 14 C, 32 P, 35 S or 125It may be a radioactive isotope such as I; a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate or rhodamine; or an enzyme such as alkaline phosphatase, β-galactosidase, horseradish peroxidase or luciferase. Specific exemplary assays that may be used to detect or measure TrkB in samples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

[0236]

[0199] Samples that can be used for the TrkB diagnostic test according to the present invention include any tissue or body fluid sample obtainable from a patient containing an amount of TrkB protein or a fragment thereof detectable under normal or pathological conditions. Generally, a baseline or standard level of TrkB will first be established by measuring the TrkB level in a specific sample obtained from a healthy patient (a patient not suffering from a disease or pathological condition associated with abnormal TrkB). The said baseline level of TrkB can be compared with the level of TrkB measured in a sample obtained from an individual suspected of having a TrkB-related disease or pathological condition.

[0237] Examples

[0238]

[0200] The following examples are presented to provide those skilled in the art with a complete description and technique of the scope of manufacturing and using the methods and compositions of the present invention, and are not intended to limit the scope of what the inventors consider to be their invention. Although efforts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, temperatures, etc.), some experimental error and deviation must be taken into account. Unless otherwise indicated, parts are by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, room temperature is about 25°C, and pressure is atmospheric pressure or near atmospheric pressure.

[0239] Example 1: Production of human antibodies against TrkB

[0240]

[0201] Human antibodies against the TrkB protein were generated in mice containing DNA encoding the human immunoglobulin heavy chain and kappa light chain variable regions. In one embodiment, the human antibody was VELOCIMMUNE ® It was generated in mice. In one embodiment, VelocImmune® (VI) mice were immunized with human TrkB(ecto)mFc (SEQ No. 77). In one embodiment, VelocImmune® (VI) mice were immunized with mouse TrkB(ecto)mFc (SEQ No. 80). The antibody immune response was monitored by TrkB-specific immunoassay. For example, serum was tested for specific antibody titers against purified full-length TrkB. Antibody-producing clones were isolated using both B-cell sorting technology (BST) and the hybridoma method. For example, when the desired immune response was achieved, splenocytes were harvested and fused with mouse myeloma cells to preserve their viability and form a hybridoma cell line. The hybridoma cell line was screened and selected to identify cell lines that produce TrkB-specific antibodies. Specific anti-mouse TrkB antibodies were generated in the manner described above and designated as M2aM14173N, M2aM14178N, and M2aM14179N.

[0241]

[0202] Additionally, anti-TrkB antibodies were isolated directly from antigen-positive mouse B cells without fusing them to myeloma cells, as described in U.S. Patent No. 7,582,298 (the full text of which is incorporated herein by reference). Using the above method, several complete human anti-TrkB antibodies (i.e., antibodies having a human variable domain and a human constant domain) were obtained; exemplary antibodies produced in this manner were designated as H4H9780P, H4H9814P, and H4H19816P2.

[0242]

[0203] The biological characteristics of the exemplary antibodies produced according to the method of the present embodiment are described in detail in the examples presented below.

[0243] Example 2: Amino acid and nucleotide sequences in the heavy and light chain variable regions

[0244]

[0204] Table 1a presents amino acid sequence identifiers for the heavy and light chain variable regions and CDRs of the selected anti-TrkB antibody of the present invention. Table 1b presents amino acid sequence identifiers for the full-length heavy and light chains of the selected anti-TrkB antibody of the present invention. Corresponding nucleic acid sequence identifiers for the selected anti-TrkB antibody of the present invention are presented in Table 2.

[0245] [Table 1a]

[0246]

[0247] [Table 1b]

[0248]

[0249] [Table 2]

[0250]

[0251]

[0205] Antibodies are typically referred to herein according to the following nomenclature: an Fc prefix (e.g., "H4H," "H2M," etc.), a numerical identifier (e.g., "9780," "9816," etc. as shown in Table 1 or 2), and a suffix "P," "P2," or "N." In the designation of an antibody, the H4H prefix indicates the isotype of a specific Fc region for said antibody. Thus, according to this nomenclature, an antibody may be referred to herein, for example, as "H4H9780P," which indicates the human IgG4 Fc region and M2aM14179N, and, for example, the mouse IgG2a Fc region. The variable region is fully human, as indicated by the first "H" in the antibody designation. The 'M' prefix indicates the mouse variable region. As anyone skilled in the art would know, an antibody having a specific Fc isotype can be converted into an antibody having a different Fc isotype (e.g., an antibody having mouse IgG1 Fc can be converted into an antibody having human IgG4, etc.), but in any case, the variable domain (including the CDR)—indicated by the numerical identifier shown in Table 1 or 2—will remain the same, and the binding properties to the antigen are expected to be the same or substantially similar regardless of the properties of the Fc domain.

[0252] Example 3. Biacore binding kinetics for anti-TrkB monoclonal antibodies binding to different TrkB reagents measured at 25°C and 37°C

[0253]

[0206] Equilibrium dissociation constant (K) for TrkB binding to purified anti-TrkB monoclonal antibody DThe values ​​were determined using a real-time surface plasmon resonance biosensor with a BiaCore 4000 instrument. All binding studies were performed at 25°C and 37°C in 10 mM Hepes pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.05% v / v surfactant Tween-20 (HBS-ET development buffer). The BiaCore sensor surface was first derivatized by amine coupling with an F(ab')2 fragment goat anti-human Fcγ specific polyclonal antibody (Jackson ImmunoResearch Laboratories, #109-006-098) or a rabbit anti-mouse Fc polyclonal antibody (GE Healthcare #BR-1008-38) to capture the anti-TrkB monoclonal antibody. Binding studies were performed on the following TrkB reagents: a human TrkB extracellular domain expressed with a C-terminal myc-myc-hexahistidine tag (hTRKB.mmH; SEQ NO. 76; Approval No. NP_001018074.1), a mouse TrkB extracellular domain expressed with a C-terminal myc-myc-hexahistidine tag (mTRKB.mmH; SEQ NO. 79; Approval No. NP_001020245), a rat TrkB extracellular domain expressed with a C-terminal myc-myc-hexahistidine tag (rTRKB.mmH; SEQ NO. 84; Approval No. NP_036863.1), and a human TrkB extracellular domain expressed with a C-terminal mouse IgG2a Fc tag (hTrkB-mFc; SEQ NO. 77; Approval No. NP_001018074.1). TrkB reagents of different concentrations were first prepared in HBS-ET development buffer (100 nM–1.23 nM; 3-fold serial dilution) and injected onto the surface of anti-human Fc-captured anti-TrkB monoclonal antibodies at a flow rate of 30 μL / min for 4 minutes, and the dissociation of the monoclonal antibody-bound TrkB reagent was monitored in HBS-ET development buffer for 10 minutes. Kinetic binding ( Ka ) and Harry (K dThe rate constants were determined by fitting real-time coupled sensorgrams to a mass transport-restricted 1:1 coupled model using Scrubber 2.0c curve fitting software. The coupled dissociation equilibrium constant (KD) and dissociation half-life (t½) were calculated from the kinetic rate constants as follows:

[0254]

[0255]

[0207] Binding kinetic parameters for the binding of hTrkB.mmH, mTrkB.mmH, rTrkB.mmH, or hTrkB-mFc to different anti-TrkB monoclonal antibodies of the present invention at 25℃ and 37℃ are shown in Tables 3 to 10.

[0256] Results Summary:

[0257]

[0208] At 25°C, anti-TrkB monoclonal antibodies bound to hTrkB.mmH with KD values ​​ranging from 545 pM to 41.3 nM as shown in Table 3. At 37°C, monoclonal antibodies bound to hTrkB.mmH with KD values ​​ranging from 2.28 nM to 135 nM as shown in Table 4.

[0258]

[0209] At 25°C, the anti-TrkB monoclonal antibodies bound to hTrkB-mFc with KD values ​​ranging from 31.1 pM to 4.48 nM, as shown in Table 5. At 37°C, the anti-TrkB monoclonal antibodies bound to hTrkB-mFc with KD values ​​ranging from 73.3 pM to 3.46 nM, as shown in Table 6.

[0259]

[0210] At 25°C, the comparator anti-TrkB monoclonal antibody referred herein as H1M8037C (reference: US2010 / 0196390, antibody designated as C2; also refer to SEQ ID NOs 97 and 98 for the respective comparator heavy chain and light chain, amino acid sequences) bound to mTrkB.mmH with a KD value of 40.8 nM as shown in Table 7. The anti-TrkB antibody of the present invention did not bind to mTrkB.mmH at 25°C as shown in Table 7. At 37°C, the comparator anti-TrkB monoclonal antibody bound to mTrkB.mmH with a KD value of 94.1 nM as shown in Table 8. The anti-TrkB antibody of the present invention did not bind to mTrkB.mmH at 37°C as shown in Table 8.

[0260]

[0211] At 25°C, the comparator anti-TrkB monoclonal antibodies bound to rTrkB.mmH with a KD value of 31.8 nM, as shown in Table 9. The anti-TrkB antibody of the present invention did not bind to rTrkB.mmH at 25°C, as shown in Table 9. At 37°C, the comparator anti-TrkB monoclonal antibody bound to rTrkB.mmH with a KD value of 87.5 nM, as shown in Table 10. The anti-TrkB antibody of the present invention did not bind to rTrkB.mmH at 37°C, as shown in Table 10.

[0261] [Table 3]

[0262]

[0263] [Table 4]

[0264]

[0265] [Table 5]

[0266]

[0267] [Table 6]

[0268]

[0269] [Table 7]

[0270]

[0271] [Table 8]

[0272]

[0273] [Table 9]

[0274]

[0275] [Table 10]

[0276]

[0277] Example 4. Biacore binding kinetics for surrogate anti-TrkB monoclonal antibodies binding to different TrkB reagents measured at 25°C

[0278]

[0212] Equilibrium dissociation constant (K) for TrkB binding to purified anti-TrkB monoclonal antibody DThe value was determined using a real-time surface plasmon resonance biosensor equipped with a BiaCore T200 instrument. All binding studies were performed at 25°C in 10 mM Hepes pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.05% v / v surfactant Tween-20 (HBS-ET development buffer). The surface of the BiaCore sensor was first derivatized by amine coupling with a rabbit anti-mouse Fc polyclonal antibody [GE, Healthcare # BR-1008-38] to capture the anti-TrkB monoclonal antibody. Binding studies were performed on the following TrkB reagents: a human TrkB extracellular domain expressed with a C-terminal myc-myc-hexahistidine tag (hTRKB.mmH; SEQ NO. 76; NP_001018074.1), a mouse TrkB extracellular domain expressed with a C-terminal myc-myc-hexahistidine tag (mTRKB.mmH; SEQ NO. 79; NP_001020245), and a rat TrkB extracellular domain expressed with a C-terminal myc-myc-hexahistidine tag (rTRKB.mmH; SEQ NO. 84; XP_002721319.1). TrkB reagents of different concentrations were first prepared in HBS-ET development buffer (90 nM–3.33 nM; 3-fold serial dilution) and injected onto the surface of anti-mouse Fc-captured anti-TrkB monoclonal antibodies at a flow rate of 50 μL / min for 4 minutes, and the dissociation of the monoclonal antibody-bound TrkB reagent was monitored in HBS-ET development buffer for 10 minutes. Kinetic binding ( Ka ) and Harry (K d The rate constant was determined by fitting a real-time coupled sensorgram to a mass transport-restricted 1:1 coupled model using Scrubber 2.0c curve fitting software. The coupled dissociation equilibrium constant (K D ) and the dissociation half-life (t½) were calculated from the kinetic rate constants as follows:

[0279]

[0280]

[0213] Binding kinetic parameters for the binding of hTrkB.mmH, mTrkB.mmH, or rTrkB.mmH to different anti-TrkB monoclonal antibodies of the present invention at 25°C are shown in Tables 11 to 13.

[0281] result:

[0282]

[0214] At 25°C, the surrogate anti-TrkB monoclonal antibody of the present invention did not bind to hTrkB.mmH as shown in Table 11.

[0283]

[0215] At 25°C, the surrogate anti-TrkB monoclonal antibodies of the present invention bound to mTrkB.mmH with KD values ​​ranging from 2.39 nM to 32.4 nM as shown in Table 12.

[0284]

[0216] At 25°C, the surrogate anti-TrkB monoclonal antibodies of the present invention bound to rTrkB.mmH with KD values ​​ranging from 2.56 nM to 26.9 nM as shown in Table 13.

[0285] [Table 11]

[0286]

[0287] [Table 12]

[0288]

[0289] [Table 13]

[0290]

[0291] Example 5. Bioassay using HEK293 / SRE-luc / hTrkB and HEK293 / SRE-luc / mTrkB(Ecto)-hTrkB (TM-Cyto) cells.

[0292]

[0217] A bioassay was developed to detect the activation of TrkB using a luciferase reporter gene under the control of serum response factor (SRE) and ligand, brain-derived neurotrophic factor (BDNF, R & D Systems). HEK293 cell lines were prepared to stably express the luciferase reporter (SRE response factor-luciferase, SRE-luc, SA Bioscience, #CLS-010L) together with human TrkB (hTrkB, amino acids 32-429 of NP_001018074.1 or Uniprot number Q16620-1) or the mouse TrkB extracellular domain fused to the transmembrane and cytoplasmic domains of human TrkB (hTrkB, amino acids 431-822 fused to NP_001020245.1 or Uniprot number P15209-1 mTrkB, amino acids 32-429). Stable cell lines, HEK293 / SRE-Luc / hTrkB and HEK293 / SRE-Luc / mTrkB, were maintained in DMEM supplemented with 10% FBS, non-essential amino acids, penicillin / streptomycin / glutamine, 1 □g / mL puromycin, and 500 □g / mL G418.

[0293]

[0218] For biopsy, cells were placed in Opti-MEM supplemented with 0.1% FBS, penicillin / streptomycin, and L-glutamine. TM 20,000 cells / well were seeded into 96-well assay plates and subsequently incubated overnight at 37°C in 5% CO2. The following morning, human BDNF or antibodies were serially diluted from 100 nM to 0.002 nM (+ buffer containing only the sample without ligand) and added to the cells to determine the activation of TrkB signaling. The serially diluted antibodies were also tested using 100 pM BDNF (R & D Systems, 248-BD / CF). The cells were then incubated at 37°C for 5.5 hours in the presence of 5% CO2. Luciferase activity was measured using a Victor X instrument (Perkin Elmer) after the addition of OneGlo reagent (Promega). The results were analyzed using non-linear regression (4-parameter logistic regression) with Prism 5 software (GraphPad) to determine EC 50 and IC 50 The value was obtained. The maximum activation of the antibody was calculated using the following:

[0294]

[0295] Results Overview and Conclusion:

[0296]

[0219] As shown in Table 14, the three anti-TrkB antibodies of the present invention, H4H9816P2, H4H9814P, and H4H9780P, demonstrated activation of human TrkB signaling in HEK293 / SRE-luc / hTrkB cells in the absence of BDNF, and EC 50s The concentration is 35–82 pM, and the maximum activity ranges from 88 to 92%. The three antibodies of the present invention were also tested in the presence of 100 pM BDNF. In contrast, 100 pM BDNF showed 56% activation in the presence of unrelated control mAb and control mAb2, while the antibodies of the present invention showed additional activation and EC 50 The concentration was 45–76 pM, and the maximum activation was 77–80%. The three anti-TrkB antibodies of the present invention did not demonstrate activation of mouse TrkB signaling in HEK293 / SRE-luc / mTrkB cells in the absence of BDNF or in the presence of 100 pM BDNF. The control mAb 1, anti-TrkB comparator antibody H1M8037C, demonstrated activation of human TrkB signaling in the absence of BDNF, and EC 50 It was 76 pM and the maximum activation was 78%, showing the activation of mouse TrkB and EC 50It was 43 pM and the maximum activation was 85%. In the presence of 100 pM BDNF, control mAb 1 activated human TrkB signaling and EC 50 It is 110 pM and maximum activation is 79%, mouse TrkB was activated and EC 50 The concentration was 42 pM and the maximum activation was 69%, exceeding the activation by control mAb 2 in the presence of 100 pM BDNF. Control mAb 2 and unrelated human IgG4 antibodies showed no activation in the absence or presence of 100 pM BDNF.

[0297]

[0220] As shown in Table 15, the three anti-TrkB antibodies of the present invention, M2aM14173N, M2aM14178N, and M2aM14179N, demonstrated activation of mouse TrkB signaling in HEK293 / SRE-luc / mTrkB cells in the absence of BDNF, and EC 50s The concentration is 34–190 pM, and the maximum activity ranges from 76 to 94%. The three antibodies of the present invention were also tested in the presence of 100 pM BDNF. While 100 pM BDNF showed 60% activation in the presence of unrelated isotype control mAb and control mAb4, the antibodies of the present invention showed additional activation and EC 50 The concentration was 17–100 pM, and the maximum activation ranged from 67 to 75%. The three anti-TrkB antibodies of the present invention did not demonstrate activation of human TrkB signaling in HEK293 / SRE-luc / hTrkB cells in the absence of BDNF or in the presence of 100 pM BDNF. The control mAb 1 demonstrated activation of human TrkB signaling in the absence of BDNF, and EC 50 It was 57 pM and the maximum activation was 80%, showing the activation of the mouse TrkB and EC 50 It was 43 pM and the maximum activation was 85%. In the presence of 100 pM BDNF, control mAb 1 activated human TrkB signaling and EC50 It is 110 pM and maximum activation is 79%, mouse TrkB was activated and EC 50 The concentration was 42 pM and the maximum activation was 69%, exceeding the activation by control mAb 4 in the presence of 100 pM BDNF. Control mAb 3 and control mAb 4, and unrelated human IgG2a isotype control antibodies showed no activation in the absence or presence of 100 pM BDNF.

[0298] Table-based data summary:

[0299] [Table 14]

[0300]

[0301] [Table 15]

[0302]

[0303] Example 6. TrkB hu / hu In vivo comparison of the effects of the TrkB agonist antibody H4H9816P2 and the IgG4 isotype control REGN1945 on TrkB phosphorylation in the brain after stereotactic injection in mice

[0304]

[0221] To determine the effect of H4H9816P2, the TrkB agonist antibody of the present invention, on TrkB activation kinetics, a time-course study of TrkB phosphorylation after direct hippocampal injection was conducted on homozygous mice (TrkB) for the human TrkB receptor instead of the mouse TrkB receptor. hu / hu Performed in (referred to as mouse). TrkB hu / hu Mice (N=48) were administered 2 μL of vehicle (PBS), REGN1945 (final concentration of 27.5 mg / mL) as an IgG4 isotype control antibody (final concentration), or TrkB agonist antibody H4H9816P2 (final concentration of 27.5 mg / mL) by bilateral stereotactic injection into the hippocampus at -2 mm anterior and +1.5 mm posterior to the parietal region. To minimize tissue damage, both injection and needle removal were performed gradually at 5-minute intervals. TrkB hu / huMice were subsequently sacrificed by CO2 euthanasia at approximately 30 minutes, 1 hour, 4 hours, or 18 hours after injection. Final blood collection was performed via cardiac puncture to collect blood, followed by perfusion of the mice with chilled heparinized saline via transcardiac perfusion. The brain was carefully removed from the skull, and 2 mm around the injection site 3 The sections were dissected, collected in Eppendorf tubes, and stored on ice. Subsequently, the brain sections were lysed in 300 µL of RIPA lysis buffer (ThermoFisher Scientific, Cat#89901) containing 2x protease and phosphatase inhibitors (ThermoFisher Scientific, Cat#78444) and stored on ice. The lysed tissue was then lysed and titrated for further processing and stored at -80°C.

[0305]

[0222] To evaluate TrkB phosphorylation in brain tissue, immunoprecipitation and Western blot were performed. The anti-human TrkB antibody H4H10108N, which does not compete for binding with H4H9816P2, was coupled to NHS-activated Cephalos beads (prepared using the manufacturer's protocol; GE Healthcare, Cat# 17-0906) and washed three times with DPBS to remove any residual preservation solution. The lysed brain lysates were thawed on ice and diluted to a concentration of 1 mg / mL (brain weight relative to buffer volume) in a buffer composed of 1% NP-40, 0.1% Tween-20, protease, and phosphatase inhibitors in TBST. The protein concentration of the lysed brain lysates was quantified by performing a standard BCA assay (Thermo Scientific Pierce, Cat# 23225) according to the manufacturer's instructions. For every 100 µg of protein, 15 µL of anti-human TrkB antibody (H4H10108N) NHS-activated Sepharose beads were added to the brain lysate solution, and the mixture was incubated overnight at 4°C with gentle shaking at 20 rpm (thermo rotator). The next day, the samples were centrifuged at 1000 xg for 1 minute, and the supernatant was then carefully removed. The beads were then washed twice with 400 µL of Tris-buffered saline (Bio-Rad, Cat#1706435) containing 1% Tween-20 (Sigma Aldrich, Cat#P9416) (TBST). After carefully aspirating the wash buffer, 60 µL of 0.1% trifluoroacetic acid (TFA; Sigma-Aldrich, T62200) in water at pH 3.0 was added to each sample. The solution was mixed and left to stand for 2 minutes before being collected and transferred to a separate tube. The above process was repeated with another 60 µL of 0.1% TFA at pH 3.0. Subsequently, the two 0.1% TFA solutions for each sample were combined and the pH was adjusted to 8.2 uL of 1M Tris-HCl (ThermoFisher Scientific, Cat#15567-027) was added at 5.

[0306]

[0223] The solution was dried using a speed vacuum, subsequently resuspended, and reduced with a mixture of 20 µL of 1x Laemmli buffer (Bio-Rad, Cat#1610737) and 355 nM 2-mercaptoethanol (BME; Gibco, Cat#21985-023). The sample was boiled at 95°C for 10 minutes and loaded onto a 10-well, small-protein 4-15% Tris-glycine gel (Bio-Rad, Cat#4561086). After electrophoresis, the protein sample was transferred from the Tris-glycine gel to a PVDF membrane (Bio-Rad, Cat#170-4156) via a Trans-Blot Turbo Transfer System (Bio-Rad, Cat#1704156) at a constant rate of 1.3 A and 25 V for a 30-minute process. After delivery, the membrane was blocked with 2.5% milk (Bio-Rad, Cat#170-6406) in TBST for 1 hour at room temperature, and then detected overnight on a shaker at 30 rpm at 4°C with an anti-phospho-TrkB antibody (Novus, Cat#NB100-92656) diluted 1:1000 in a solution of 2.5% BSA or an anti-TrkB primary antibody (Cell Signaling, Cat#4603) diluted 1:1000 in 2.5% milk TBST. The next day, the blot was washed with TBST and incubated with an anti-rabbit IgG antibody conjugated to horseradish peroxidase (Jackson, Cat#111-035-144) in 1% milk in TBST for 1 hour at room temperature. Next, the blot was washed again and developed with an ECL solution (PerkinElmer, Inc. Cat# RPN2106), and subsequently, image exposures were acquired every 30 seconds.

[0307] Results Overview and Conclusion:

[0308]

[0224] TrkB hu / hu Immunoprecipitation and subsequent Western blotting of proteins derived from mouse brain lysates, as shown in Figure 1, were detectable in mice injected with the TrkB agonist antibody H4H9816P2, but not in mice treated with vehicle or isotype control antibodies. Among the time points evaluated, TrkB phosphorylation peaked at 4 hours after stereotactic injection in mice administered H4H9816P2. TrkB phosphorylation was detected by Western blotting at 18 hours after administration in some mice, but not all. Conversely, injection of the vehicle and IgG4 isotype control antibodies did not induce TrkB phosphorylation at any time point. Western blotting also revealed that total TrkB receptor levels were higher in all TrkB-administered mice compared to those treated with vehicle and isotype controls. hu / hu It was pointed out that downregulation occurred in some mice, although not in mice. Total TrkB levels were found to be weakly downregulated in H4H9816P2-treated subjects at 18 hours after administration. Therefore, these results suggest that the direct injection of the TrkB agonist antibody H4H9816P2 [causes] TrkB hu / hu It indicates that it induces phosphorylation of the hippocampal TrkB receptor in mice.

[0309] Example 7. I TrkB hu / hu In vivo comparison of the effects of H4H9816 and isotype control REGN1945 antibodies on body weight and metabolism in mice.

[0310]

[0225] To determine the effect of H4H9816P2, the TrkB agonist antibody of the present invention, on body weight and composition, homozygous mice (TrkB) with respect to the expression of human TrkB receptors instead of mouse TrkB receptors. hu / hu Metabolic studies of mice) were performed after injection of a single subcutaneous antibody. TrkB hu / huMice (male, 20 weeks of age) were first transferred from group cages to single-cage housings for a 2-week acclimatization period. After this period, the mice were transferred to metabolic cages (CLAMS, Columbus Instruments) to evaluate changes in food and water consumption, gait, energy expenditure, and respiration following antibody administration. Regular powdered food was stored in a bottom chamber on a spring-loaded scale (Mettler Toledo, PL602E), and food consumption was measured by changes in total food weight. Water was accessible through the top snout of the cage, and intake was measured by tracking changes in pump-line volume (Oxymax ® / CLAMS Liquid Unit). The CLAMS metabolic cage measured each of these parameters at continuous intervals of 16 to 18 minutes throughout the study period. Metabolic data were analyzed as a single measurement, and OXYMAX ® / CLAMS software (Columbus instruments, v5.35) was used to summarize at 24-hour intervals including one complete dark and light cycle. After acclimatizing to the cage for 2 weeks, TrkB hu / hu Mice were administered a single subcutaneous dose of 50 mg / kg of either the TrkB agonist antibody H4H9816P2 or an IgG4 isotype control antibody in PBS at pH 7.2. Pure control TrkB hu / hu The mouse group was not injected. Mice were weighed immediately before administration and at 24, 48, 72, 96, and 120 hours after administration. To measure the body composition of each mouse, nuclear magnetic resonance relaxation measurements, also referred to as quantitative magnetic resonance, were performed using EchoMRI. TMThe procedure was performed using a -500 analyzer (EchoMRI LLC). Before administration, mice were placed in clear plastic holders and inserted into an NMR-MRI device to measure lean body mass, body fat, and hydration status of each subject. Measurements were performed over a period of 0.5–3.2 minutes per mouse, and measurements were taken again approximately 120 hours after administration.

[0311] Results Overview and Conclusion:

[0312]

[0226] By performing daily weight monitoring, a single subcutaneous injection of H4H9816P2 TrkB hu / hu It was determined whether it induces body weight loss in mice. There was no significant difference in mean body weight among the three treatment groups prior to administration, and each had an mean pre-administration body weight of 28.39 to 29.85 g (Table 16). At 48 hours after administration, however, H4H9816P2-treated TrkB hu / hu Mice lost an average of 1.70 g or 5.96% of their pre-administration body weight. At the same time point, pure and isotype control antibody-treated TrkB hu / hu Mice showed an increase of 1.79–2.37% of their pre-administration body weight. H4H9816P2-treated TrkB hu / hu Mice continued to lose body weight throughout the entire study period, and at 72 and 96 hours after administration, these mice lost an average of 8.42% and 11.80% of their pre-administration body weight, respectively. At 120 hours after administration, H4H9816P2-treated TrkB hu / hu Mice lost an average of 12.67% of their pre-administration body weight. Conversely, pure and isotype control-treated TrkB hu / hu Mice did not show any weight loss from pre-administration body weight throughout the study. H4H9816P2-treated TrkB hu / huAs body weight in mice was significantly reduced at 48, 72, 96, and 120 hours after administration compared to both pure and isotype controls, the TrkB agonist antibody H4H9816P2 TrkB hu / hu It was determined that it induced significant weight loss in mice.

[0313] [Table 16]

[0314]

[0315]

[0316]

[0227] The effect of TrkB agonist antibody H4H9816P2 injection on body composition was also measured by performing NMR-MRI on each subject before and after administration. Before administration, TrkB hu / hu The three treatment groups of mice showed no significant differences in body fat or lean body mass, and each group had an average body fat of 4.19–4.75 g and a lean body mass of 21.32–21.70 g (Table 17). However, after antibody administration, TrkB administered with H4H9816P2 hu / hu Mice lost an average of 48.90% of their total body fat over the study period (Table 17). Pure and isotype control antibody-treated TrkB hu / hu The mice lost an average of 8.49% and 9.48% of their pre-administration body fat, respectively, which was significantly less than that of the H4H9816P2-treated subjects (Table 17). Additionally, the H4H9816P2-treated TrkB hu / hu Mice lost an average of 7.84% of their lean body mass throughout the study, which was significantly higher than the average pre-administration lean body mass loss of 2.41% and 1.75% lost by the pure and isotype control antibody-treated groups, respectively (Table 17). As such, the described body weight loss is TrkB hu / huThis can be explained by the significant loss of body fat and slight loss of lean body mass after injection of the TrkB agonist antibody H4H9816P2 in mice.

[0317] [Table 17]

[0318]

[0319]

[0228] TrkB hu / hu In addition to evaluating the effects of TrkB agonist antibody H4H9816P2 injection on mouse body weight and composition, dietary intake, drinking, and gait activity were measured sequentially using a metabolic cage. Before administration, TrkB hu / hu Mice consumed an average of 3.49–3.73 g of food per day. Within 24 hours of administration, however, H4H9816P2-treated TrkB hu / hu Mice significantly reduced their food intake to 2.20 g of food per day. H4H9816P2-treated TrkB hu / hu The average level of food intake in mice did not exceed 2.49 kg of food per day throughout the remainder of the study, and pure and isotype antibody-treated TrkB hu / hu Mice consistently consumed an average of 3.62 to 4.07 g of food per day (Table 18).

[0320]

[0229] Similarly, there was no significant difference in daily water consumption between the pre-treatment groups. TrkB hu / hu Mice in each treatment group consumed an average of 4.67–5.55 mL of water per day (Table 19). After administration, H4H9816P2-treated TrkB hu / hu Mice had their water intake reduced to 2.05–3.24 mL per day. This corresponds to pure and isotype control antibody-treated TrkB, which consistently consumed 4.50–5.77 mL per day throughout the study. hu / huIt was significantly lower than in mice (Table 19). Therefore, injection of the TrkB agonist antibody H4H9816P2 compared TrkB with both pure and isotype controls. hu / hu It was found to significantly reduce the intake of both food and water in mice.

[0321] [Table 18]

[0322]

[0323] [Table 19]

[0324]

[0325]

[0230] To determine the effect of antibody treatment on activity, walking exercise was OXYMAX ® Analysis was performed using CLAMS software (Columbus Instruments, v5.35), which sequentially measured the total number of x-plane moves for each mouse. One mouse exhibited hyperactivity prior to administration and was excluded from the statistical analysis after administration. Pure and isotype antibody-treated subjects registered an average of 11,000–15,000 moves per day throughout the study, whereas H4H9816P2-treated TrkB hu / hu H4H9816P2-treated TrkB registered 28,260 moves at 24 to 48 hours after administration, and 21,193 and 27,028 moves at 48 to 72 hours and 72 to 96 hours after administration, respectively (Table 20). hu / hu Mice registered a higher total number of moves at each time point after antibody administration, suggesting that hyperactivity is an additional effect of the H4H9816P2 injection. In combination, these effects suggest that a single subcutaneous injection of the TrkB agonist antibody H4H9816P2 is an additional effect of TrkB hu / hu This suggests that significant changes in body weight, body composition, metabolism, and gait were induced in mice.

[0326] [Table 20]

[0327]

[0328] Example 8. Optic nerve transection model to determine the effect of anti-TrkB on retinal ganglion cell (RGC) survival

[0329]

[0231] All procedures were performed at the Ophthalmic and Vision Research and the Regeneron Pharmaceutical Inc. IACUC in accordance with the ARVO statement regarding the use of animals. Mature female TrkB humanized rats (Velocigene, Regeneron Pharmaceutical Inc.) aged 8 to 10 weeks with a body weight of 200–250 g were used. All surgical procedures on the rats were performed under general analgesics using intraperitoneal injections of ketamine (63 mg / kg) and xylarzine (6.0 mg / kg). An eye ointment containing erythromycin (0.5%, Bausch & Lomb) was applied to protect the cornea.

[0330] Intraorbital optic nerve axonectomy and intravitreal injection

[0331]

[0232] The left optic nerve (ON) was exposed within the orbit, and its dura mater was opened. The ON was transected approximately 1.5 mm behind the globe. Care was taken to avoid damage to the blood supply to the retina. Intravitreal injection was performed just posterior to the pars plana using a pulled glass pipette connected to a 50 μl Hamilton syringe. Care was taken to avoid damaging the lens. Rats with any significant postoperative complications (e.g., retinal ischemia, cataracts) were excluded from further analysis. Animals were assigned to different test groups. One control group received an intravitreal injection of 3 μl of the isotype control REGN1945 (46.6 μg / μl); the other group received an injection of 3 μl of the anti-human TrkB antibody H4H9816P2 (45.7 μg / μl) on days 3 and 10 after ON axonectomy.

[0332]

[0233] In another experiment, the dose-response of the anti-human TrkB antibody H4H9816P2 was tested. Homozygous TrkB humanized rats aged 1 to 9 months were injected intravitreally with 3 μl of the anti-human TrkB antibody H4H9816P2 (0.01, 0.1, 1, or 10 μg / μl) or the isotype control REGN1945 (10 μg / μl) on days 3 and 10 after ON axonectomy.

[0333] Immunohistochemical staining and counting of viable RGCs

[0334]

[0234] Brn3a (brain-specific homeobox / POU domain protein 3A) was used as a marker for viable retinal ganglion cells (RGCs) because it has been shown to be an efficient and reliable method for the selective labeling of viable RGCs in whole-retinal loading after ON injury (Reference: Nadal--Nicolas FM, Jimenez-Lopez L, Canovas-Martinez I. Salinas-Navrro M, Agudo M., Invest Ophthalmol Vis Sci. 2009 Aug;50(8):3860-8). For immunostaining for Brn3a, the retina was blocked in 10% normal donkey serum and 0.5% Triton X-100 for 1 hour, followed by incubation in the same medium with Brn3a antibody (1:400; Cat#: sc-31984, Santa Cruz) at room temperature for 2 hours. After additional washing, the retina was incubated overnight at 4°C with Alexa594-conjugated donkey anti-goat secondary antibody (1:400; Cat#: A-11058, Invitrogen).

[0335] Results Overview and Conclusion:

[0336]

[0235] To evaluate TrkB agonist antibodies on in vivo RGC survival, the inventors used a complete optic nerve transection model. TrkB agonist antibodies (H4H9816P2) or isotype (negative control) antibodies were administered on days 3 and 10 post-surgery. Animals were euthanized on day 14 post-axonectomy. RGC density in the intact contralateral eye was mm² as shown in Table 21. 2 The average is approximately 1600 per retina, similar across the three TrkB genotypes. The density of viable RGCs was evaluated on whole-retinal loading using Brn3a staining. In homozygous TrkB humanized rats, the TrkB agonist antibody (H4H9816P2) was compared to the control (mm² 2 Compared to 685±106 RGCs versus 255±66 RGCs, RGC survival was significantly ( p <0.01, was observed to increase the Mann-Whitney test). In heterozygous TrkB humanized rats, also TrkB agonist antibodies (mm 2 Significant difference between 444±90 and 208±50 RGCs ( p There is a survival effect <0.05 (Mann-Whitney test). In wild-type TrkB rats, the number of RGCs with TrkB agonist antibodies increases slightly but not significantly compared to isotype controls (Table 22). In dose-response experiments, RGC density was quantified on whole-retinal loading using Brn3a staining 14 days after axonectomy. There is a clear dose-response with TrkB agonist antibodies. Compared to the antibody control group (mm 2 (168 ± 43 RGCs), TrkB agonist antibody (H4H9816P2) was significantly ( p <0.01, One-way ANOVA using post-Turk test) 3 µg (mm) per injection group 2 564 + / - 124 RGC) or 30 ug (mm 2It increased RGC survival in 543 + / - 242 RGCs per group. There was no difference between the 3 and 30 µg groups. 0.03 µg (mm²) per injection group. 2 202+ / - 96 RGC) or 0.3 ug (mm) per 2 In the group administered 337 + / - 210 RGCs, there was only a trend in RGC survival, but no significant increase (Table 23). In conclusion, the TrkB agonist antibody H4H9816P2 is TrkB hu / hu and TrkB hu / + It showed significantly increased RGC survival in rats.

[0337] conclusion:

[0338]

[0236] The dose of the TrkB agonist Ab (H4H9816P2) significantly increased RGC survival in humanized TrkB rats.

[0339] [Table 21]

[0340]

[0341] [Table 22]

[0342]

[0343] [Table 23]

[0344]

[0345] Example 9. Effects of anti-TrkB antibody on Akt and Erk signaling pathways

[0346]

[0237] All procedures were performed at the laboratory (Ophthalmic and Vision Research and the Regeneron Pharmaceutical Inc. IACUC) in accordance with the ARVO statement regarding the use of animals. Primary mouse cortical neurons were isolated and cultured from humanized TrkB mice (MAID 7139) (Reference: Nat Protoc. 2012 Sep;7(9):1741-54. doi: 10.1038 / nprot.2012.099). Western blot (WB) was performed to determine the effects of TrkB agonist antibodies on the downstream pathways of Akt and Erk (p-Akt, p-Erk1 / 2). Primary cortical neurons from humanized TrkB mouse pups 1 day after birth (P1) were cultured for 4 days (DIV-4) in NeuralQ basal medium (Global Stem, cat. # GSM-9420) supplemented with GS21 neuronal supplement (Global Stem, cat. # GSM-3100), Glutamax (Invitrogen, cat. # 35050-061), and penicillin / streptomycin. Cells were treated for 15 minutes or 2 hours with TrkB agonist antibodies: H4H9816P-L1 (10 µg / ml), H4H9780P-L1 (10 µg / ml), H4H9814P-L1 (10 µg / ml), IgG4 isotype control REGN1945 (10 µg / ml), control antibody H1M8037C-L1 (10 µg / ml), and BDNF (1 µg / ml). Western blots were performed to determine whether the agonists differed in downstream signaling maintenance and intensity. Treated cells were washed and exfoliated in cold PBS containing 1% protease and phosphatase inhibitor (Sigma). Protein concentrations were determined by the Bradford protein assay (Pierce). The sample (50 μg) was separated by SDS-PAGE in a 3-8% Tris-acetate reduced gel (Novex) and transferred to a nitrocellulose membrane (Bio-Rad).

[0347]

[0238] The membrane was incubated for 1 hour in a blocking solution containing 5% milk and 0.1% Tween-20 at pH 7.6. Subsequently, it was incubated overnight at 4°C in a blocking solution containing 5% BSA, 0.1% Tween-20, and rabbit anti-phospho-Trk (cell signaling, cat. # 9141, 1:500), rabbit anti-phospho-Akt (cell signaling, cat. # 9271, 1:1000), or rabbit anti-phospho-ERK1 / 2 antibody (Sigma, cat. # E7028, 1:5000). Subsequently, the labeled protein was incubated with horseradish peroxidase (HRP) conjugated anti-goat, mouse, or rabbit IgG, followed by visualization by developing with a chemiluminescent substrate for HRP (Pierce). To determine the total amount of TrkB, MAPK, or Akt present in each lane, nitrocellulose membranes were stripped of antibodies in stripping buffer (Pierce) for 20 minutes, incubated with rabbit anti-TrkB (cell signaling, cat. # 4603, 1:1000), rabbit anti-Erk1 / 2 (cell signaling, cat. # 06-182, 1:1000), or rabbit anti-Akt antibody (cell signaling, cat. # 9272, 1:1000), and then visualized as described above. Beta-actin (Sigma, cat. # A5316, 1:20000) and GAPDH (Sigma, cat. # G9295) were detected as sample loading controls.

[0348] Results Overview and Conclusion:

[0349]

[0239] As shown in Figure 2, at 15 minutes after incubation, all TrkB agonist antibodies induced activation of the MAPK / ERK and PI3K / Akt pathways, while only BDNF and H4H9814P showed TrkB phosphorylation. After 2 hours of incubation, all TrkB agonist antibodies showed TrkB activation.

[0350] Example 10. Effect of the agonist anti-TrkB antibody on the survival of SH-SY5Y cells

[0351] In vitro culture of human neuroblastoma SH-SY5Y cell line:

[0352]

[0240] Neuroblastoma cell line SH-SY5Y (Sigma ATCC # 94030304, cat. # 11C016) cells were seeded into growth medium containing DMEM:F12 (Invitrogen cat#11330), Pen / Strep (Invitrogen cat.# 15140), and 10% FBS (Invitrogen cat# 10082-147) at 37°C in 5% CO2. At passages 23–27, cells were seeded into 96-well plates in differentiation medium containing 10 µM all-trans retinoic acid (Alfa Aesar cat. #44540), DMEM:F12 (Invitrogen cat#11330), Pen / Strep (Invitrogen cat#15140), and 10% FBS (Invitrogen cat#10082-147). Cells (30K / well) were differentiated for 4 days. Antibodies were screened for viability biopsy, where the cultures were replaced with serum-free differentiation medium (100 µl / well) containing varying doses of antibodies (100–0.01 µg / ml). After 2 days, CCK8 (Dojindo, cat. #CK04) reagent was added (10 µl / well), and the plates were incubated for 3 to 4 hours. OD was measured at 450 nm (Victor or FlexStation III) to determine the percentage of viable cells. Data were standardized to serum-free medium without treatment. Serum-free treatment without antibodies = 100% viability.

[0353] Results Overview and Conclusion:

[0354]

[0241] As shown in Figure 3 and Table 23, all agonist TrkB antibodies of the present invention showed a significant dose-dependent increase in the survival of SH-SY5Y cells compared to negative isotype control antibodies (p<0.0001 by two-way ANOVA).

[0355] [Table 24]

[0356]

[0357] Example 11. Evaluation of pharmacokinetics of anti-TrkB antibody in humanized TrkB and WT mice

[0358]

[0242] Evaluation of the pharmacokinetics of the anti-TrkB antibody, H4H9816P2, was performed in humanized TrkB mice (homozygous mice for human TrkB expression, TrkB hu / hu The study was performed on ) and wild-type (WT) mice. The cohort contained 5 mice per mouse species. A single subcutaneous (SC) dose of 10 mg / kg was administered to all mice. Blood samples were collected 6 hours after administration and on days 1, 2, 3, 6, 9, 16, 21, and 30. The blood was processed into serum and frozen at -80°C until analysis.

[0359]

[0243] Circulating antibody concentrations were determined by total human IgG4 / hIgG1 antibody assay using GyroLab xPlore™ (Gyros, Uppsala, Sweden). Briefly, biotinylated mouse anti-human IgG4 / IgG1-specific monoclonal antibody (REGN2567) diluted to 100 μg / mL in antibody dilution buffer (0.05% Tween-20 + PBS) was applied to streptavidin-coated beads (Dynospheres ™ It was captured on a Gyrolab Bioaffy 200 CD containing an affinity column pre-loaded with ). The standard used for correction in the above assay was 0.488 to 2000 in dilution buffer (0.5% BSA+PBS) containing 0.1% normal mouse serum (NMS). H4H9816P at concentrations in the ng / mL range. Serum samples were diluted 1:100 in antibody dilution buffer. Human IgG captured on an anti-REGN2567-coated affinity column on a CD developed at room temperature was detected by adding 0.5 μg / mL Alexa-647-conjugated mouse anti-human kappa monoclonal antibody (REGN654) diluted in detection buffer (Rexxip F buffer); the obtained fluorescence signal was detected by GyroLab xPlore It was recorded as a reaction unit (RU) by the instrument. Sample concentrations were determined by interpolation from a standard curve fitted using a 5-parameter logistic curve fit with Gyrolab Evaluator software. The average concentration from two replicate experiments was used for subsequent PK analysis.

[0360]

[0244] The PK parameter is Phoenix ® WinNonlin ® It was determined by non-compartmental analysis (NCA) using software version 6.3 (Certara, LP, Princeton, NJ) and an extravascular administration model. Using the respective average concentration values ​​for each antibody, the maximum concentration observed in serum (C max ), observed and evaluated half-life (t 1 / 2 ), and area under the concentration curve (AUC) for the time to the last measurable concentration last ) was determined using the linear trapezoidal rule with linear interpolation and uniform weighing.

[0361] Results Overview and Conclusion:

[0362]

[0245] After administration of 10 mg / kg sc of the anti-TrkB antibody, H4H9816P2, a similar maximum concentration of the antibody (C max ) is TrkB hu / huH4H9816P2 was observed in both WT mice (135 and 131 mg / mL, respectively; see Table 26) by day 1 or 2. By day 9, H4H9816P2 was observed more than TrkB in WT mice. hu / hu Mice showed more rapid drug clearance, which indicates a target-mediated effect. The 30-day antibody concentration was TrkB hu / hu It was approximately 35 times lower in mice. Antibody exposure to H4H9816P2 in WT mice (AUC 마지막 ) is TrkB hu / hu It was ~1.7-fold higher than that observed in mice (1730 and 1020 d*µg / mL, respectively). WT mice also TrkB hu / hu Half-life (T) compared to mice (8.4 and 2.9 days, respectively) 1 / 2 It showed an increase of about three times in ).

[0363]

[0246] A summary of the data for total anti-TrkB antibody concentration is presented in Table 25. The average PK parameters are listed in Table 26, and the average total antibody concentration over time is shown in Figure 4.

[0364] [Table 25]

[0365]

[0366] Abbreviations: Time = Time in days after single-dose injection; d = Study days; SD = Standard deviation

[0367] [Table 26]

[0368]

[0369] Example 12: Ability of an anti-mouse TrkB monoclonal antibody to block the interaction between mouse or rat TrkB and its ligand BDNF (brain-derived neurotrophic factor).

[0370]

[0247] Anti-mouse TrkB monoclonal antibodies (mAbs) were produced by immunizing TrkB humanized mice with mouse TrkB protein. Three major mAbs identified from said immunization are M2aM14173N, M2aM14178N, and M2aM14179N. The major mAbs of the present invention are characterized by their ability to block the interaction between plate-bound BDNF and mouse or rat TrkB in a blocking ELISA.

[0371]

[0248] The experiment was performed using the following procedure. Human BDNF was coated onto 96-well microtiter plates at a concentration of 0.5 μg / mL in PBS (to block mouse TrkB.hFc interactions) or 0.3 μg / mL (to block rat TrkB.mmh interactions) and incubated overnight at 4°C. Non-specific binding sites were subsequently blocked using a 5% (w / v) solution of BSA in PBS (test buffer). In 96-well dilution plates, 850 pM mouse TrkB.hFc or rat TrkB.mmh was mixed with 3-fold serially diluted anti-mouse TrkB antibody and control antibody. The final antibody concentration ranged from 1.69 pM to 100 nM. The protein-antibody mixture was incubated at room temperature (RT) for 1 hour. Subsequently, the pre-conjugated mixture was delivered twice to BDNF-coated microtiter plates. A control group containing only the assay buffer was included to calculate the baseline for the assay. ELISA plates were incubated at RT for 1 hour and then washed with plate washing solution. Plate-bound mouse TrkB.hFc was detected with HRP-conjugated goat anti-human Fcγ fragment-specific antibody (Jackson Immunoresearch), and rat TrkB.mmh was detected with HRP-conjugated anti-histamine antibody (Qiagen). Plates were incubated with the detection antibodies at RT for 1 hour and then washed with plate washing solution. The assay plates were developed with TMB colorimetric substrate according to the manufacturer's recommended procedure.

[0372]

[0249] The absorbance at 450 nm for each well was recorded and plotted as a function of antibody concentration. Data were analyzed in GraphPad Prism software using a 4-parameter logistic regression for 11-point dose-response curves and IC 50The value was calculated. The calculated IC50, defined as the antibody concentration required to reduce TrkB binding to BDNF by 50%. 50 The value was used as an indicator of blocking efficacy. Percent blockade at the maximum concentration of the tested antibody was calculated as an indicator of the antibody's ability to block the binding of TrkB to BDNF on the plate relative to the baseline of the assay. In the absence of the antibody, the binding signal of 850 pM mouse and rat TrkB was defined as 100% binding or 0% blockade. The baseline signal of the assay buffer alone was defined as 0% binding or 100% blockade.

[0373] Summary of Results and Conclusion

[0374]

[0250] The ability of anti-mouse TrkB antibodies to block the binding of mouse or rat TrkB to BDNF was evaluated using a blocking ELISA.

[0375]

[0251] Blocking results are summarized in Table 27 and Figures 5a and b. % blocking was reported for all antibodies and calculated at the highest antibody concentration tested (100 nM). IC 50 Values ​​are presented only for antibodies that block >50% of mouse or rat TrkB binding to BDNF. Among the three antibodies of the present invention, the anti-mouse TrkB mAb, M2aM14178N, blocked >50% of both mouse and rat TrkB binding to BDNF. M2aM14178N had an IC50 of 426 pM. 50 and blocked the binding of 850 pM mouse TrkB.hFc with an 84.4% % block. M2aM14178N has an IC of 184 pM. 50 and blocked the binding of 850 pM rat TrkB.mmh to BDNF with an 89.5% block. M2aM14173N demonstrated a 29.7% block of 850 pM mouse TrkB.hFc binding to BDNF. M2aM14173N had an IC of 3.81 nM. 50At a value of 850 pM, it showed an 80.7% blockade of rat TrkB.mmh binding to BDNF. M2aM14179N blocked 11.6% of mouse TrkB.hFc binding to BDNF. M2aM14179N showed increased rat TrkB.mmh binding to BDNF at concentrations above 1 nM.

[0376]

[0252] The comparator anti-mouse TrkB mAb, H1M8037C, is an IC of 180 pM 50 850 pM mouse TrkB.hFc blocked the binding of TrkB.hFc to BDNF with a value and 91.5% blocking. H1M8037C had an IC of 1.42 nM. 50 850 pM rat TrkB.mmh blocked binding with a value and 83.3% % block. The mIgG2a isotype control mAb, REGN1097, did not show any blockade of mouse or rat TrkB under the same testing conditions. At concentrations above 10 nM, REGN1027 showed an increase in rat TrkB binding.

[0377] [Table 27]

[0378]

[0379] Example 13. Ability of an anti-human TrkB monoclonal antibody to block the interaction between human TrkB and its natural ligands human BDNF and NT4.

[0380]

[0253] The ability of anti-human TrkB antibodies designated as H4H9814P, H4H9816P2, and H4H9780P to block the binding of TrkB protein to plate-captured BDNF or NT-4 was measured using two competitive sandwich ELISAs. In the assay, various concentrations of anti-TrkB antibodies were pre-mixed with a certain amount of dimeric TrkB protein, and the reduction in the binding of TrkB to plate-immobilized BDNF or NT-4 due to the presence of the antibodies was calculated.

[0381]

[0254] The recombinant dimeric TrkB protein used in the experiment consisted of a human TrkB extracellular domain (aa Cys32-His430) portion (hTrkB-hFc; Accession # NP_006171.2, molecular weight 69,700 Daltons) expressed together with the Fc portion of human IgG1 at the c-terminus. The BDNF and NT-4 proteins consisted of the extracellular domains of human BDNF (aa His129-Arg247, Accession # P23560, R&D Systems) or NT-4 (aa Gly81-Ala210, Accession # P34130, R&D Systems), respectively. Two isotype antibody controls, an anti-Fel d 1 human IgG4 antibody, and an antibody specific to Fel d 1 antibody along with mouse IgG1 were included as controls for IgG background detection.

[0382]

[0255] The experiment was performed using the following procedure. Human BDNF or NT-4 was coated on 96-well microtiter plates at concentrations of 0.5 μg / mL or 2 μg / mL, respectively, in PBS overnight at 4°C. Non-specific binding sites were subsequently blocked using a BSA solution in PBS. The blocking solution and dilution buffer contained a 5% (w / v) solution of BSA in PBS for assays using the BDNF coat, or a 0.5% (w / v) solution of BSA in PBS for assays using the NT-4 coat. On separate microtiter plates, a fixed amount of 500 pM hTrkB-hFc protein was added to the serial dilutions of the antibody, with final concentrations ranging from 1.7 pM to 100 nM, and the solution was antibody-free. (The constant concentration of hTrkB-hFc for the antibody inhibition assay was selected from the approximate midpoint within the linear portion of the individual binding curves of hTrkB-hFc to plate-coated hBDNF or hNT-4). After incubation at room temperature for 1 hour, a constant concentration of 500 pM hTrkB-hFc protein and antibody-protein complex were delivered to microtiter plates coated with hBDNF or hNT-4. After incubation at room temperature for 1 hour, the wells were washed, and the plate-bound hTrkB-hFc was detected using an anti-human Fcγ fragment-specific goat polyclonal antibody conjugated with horseradish peroxidase (Jackson Immuno Research). The plates were then developed using TMB substrate solution (BD Biosciences) according to the manufacturer's recommendation, and the absorbance at 450 nm was measured using a Victor X4 plate reader (PerkinElmer TM It was measured on the )

[0383]

[0256] Data analysis was performed using an S-shaped dose-response model within Prism™ software (GraphPad). The calculated IC50 value, defined as the antibody concentration required to reduce the binding of hTrkB-hFc to hBDNF or hNT-4 by 50%, was used as an indicator of blocking efficacy. Percent block at the maximum concentration of the tested antibody was calculated as an indicator of the antibody's ability to block the binding of 500 pM hTrkB-hFc to hBDNF or hNT-4 relative to the baseline of the assay. In the above calculations, the binding signal of the sample of 500 pM hTrkB-hFc without the presence of the antibody was referred to as 100% binding or 0% block; the baseline signal of the sample in buffer of hTrkB-hFc or antibody absence was referred to as 0% binding or 100% block.

[0384] Results Overview and Conclusion:

[0385]

[0257] The ability of anti-TrkB antibodies to block the binding of TrkB to BDNF or NT-4 was evaluated using two competitive sandwich ELISAs. Binding of human TrkB-hFc to hBDNF or hNT-4 coated on 96-well microtiter plates in the presence of serially diluted antibodies or in the absence of antibody controls was detected using HRP-conjugated anti-human Fcγ fragment-specific goat polyclonal antibodies. IC50 values ​​were calculated and used as indicators of the efficacy of antibodies blocking the binding of hTrkB-hFc to hBDNF or hNT-4. Additionally, the maximum blockade of 500 pM hTrkB-hFc using each antibody at the highest tested concentration was calculated and compared.

[0386]

[0258] The blocking results are summarized in Table 28. % blocking was reported for all antibodies and calculated at the highest tested antibody concentration of 100 nM. Negative % blocking indicates an increase in TrkB binding detected in the presence of the antibody. IC50 values ​​are shown for antibodies that block >50% of TrkB binding to BDNF or NT-4. IC50 values ​​for antibodies that block <50% were reported as non-quantitative (-).

[0387]

[0259] At the highest concentration of the antibodies tested, one of the three anti-TrkB antibodies (H4H9780P) blocked the binding of hTrkB to BDNF or NT-4 ligands by >50%, with IC50 values ​​of 150 pM and 180 pM, respectively, and at 100 nM antibody, the percentage block was 93% for BDNF and 80% for NT-4. At 3.7 nM, the antibody blocked the binding of 500 pM hTrkB-hFc to NT-4 by 99%. The decrease in % block at the highest tested concentration may be attributed to the non-specific binding of H4H9780P to microtiter plates and the detection of such binding with HRP-conjugated anti-human Fcγ fragment-specific polyclonal antibodies.

[0388]

[0260] Three anti-TrkB antibodies (H4H9814P and H4H9816P2) and two of the unrelated blocking control antibodies blocked <50% of hTrkB binding to BDNF or NT-4. The comparator blocked >50% of 500 pM hTrkB-hFc binding to both BDNF and NT-4.

[0389] [Table 28]

[0390]

[0391] Example 14. Octet cross-competition between different anti-hTrkB monoclonal antibodies

[0392]

[0261] To evaluate whether the two antibodies compete with each other for binding to their epitopes on hTrkB-mmh, binding competition between the anti-hTrkB monoclonal antibodies was determined using real-time label-free bio-layer interferometry on an Octet RED384 biosensor (Pall ForteBio Corp.). Cross-competition experiments were performed at 25°C with plates shaken at 1000 rpm in 0.01 M HEPES pH 7.4, 0.15 M NaCl, 3.4 mM EDTA, 0.05% v / v surfactant Tween-20, and 0.1 mg / mL BSA (HBS-EP buffer). All tested anti-hTrkB antibodies and hTrkB-mmh solutions were prepared in Octet HBS-EP buffer. To evaluate whether two antibodies could compete with each other to bind to their respective epitopes on hTrkB-mmh, approximately ~0.14–0.24 nm of hTrkB-mmh was first captured on an anti-His coated octet biosensor tip for 5 minutes from a well containing 50 μg / mL of hTrkB-mmh. The hTrkB-mmh-captured octet biosensor tip was saturated by immersing it in a well containing 50 μg / mL of the first anti-hTrkB monoclonal antibody (referred to as mAb-1) for 5 minutes, followed by immersion in a well containing the second anti-hTrkB monoclonal antibody (referred to as mAb-2) for an additional 5 minutes. Between steps, the octet biosensor tip was washed in HBS-EP buffer for 30 seconds.

[0393]

[0262] Real-time binding reactions were monitored throughout the experiment, and binding reactions were recorded at the end of each step. The binding reactions of mAb-2 to mAb-1 and pre-conjugated hTrkB.mmh were compared, and the competitive / non-competitive behavior of different anti-hTrkB monoclonal antibodies was determined using a 60% inhibition threshold.

[0394]

[0263] Table 29 clearly defines the relationship between antibodies competing in both directions, independent of the order of binding.

[0395] result:

[0396] [Table 29]

[0397]

[0398] Example 15. Biacore binding kinetics of surrogate anti-mouse TrkB monoclonal antibodies binding to different TrkB reagents measured at 25°C

[0399]

[0264] To evaluate whether the two antibodies compete with each other for binding to their epitopes on mTrkB-mmh, binding competition between the anti-hTrkB monoclonal antibodies was determined using a real-time label-free bio-layer interferometer assay on an Octet HTX biosensor (Pall ForteBio Corp.). Cross-competition experiments were performed at 25°C with plates shaken at 1000 rpm in 0.01 M HEPES pH 7.4, 0.15 M NaCl, 3.4 mM EDTA, 0.05% v / v surfactant Tween-20, and 0.1 mg / mL BSA (HBS-EP buffer). All tested anti-mTrkB antibodies and mTrkB-mmh solutions were prepared in Octet HBS-EP buffer. To evaluate whether two antibodies could compete with each other to bind to their respective epitopes on mTrkB-mmh, approximately ~0.20–0.27 nm of mTrkB-mmh was first captured on an anti-His coated octet biosensor tip for 5 minutes from a well containing 20 μg / mL of mTrkB-mmh. The mTrkB-mmh-captured octet biosensor tip was saturated by immersing it in a well containing 50 μg / mL of the first anti-hTrkB monoclonal antibody (referred to as mAb-1) for 5 minutes, followed by immersion in a well containing the second anti-mTrkB monoclonal antibody (referred to as mAb-2) for an additional 3 minutes. Between steps, the octet biosensor tip was washed in HBS-EP buffer for 30 seconds.

[0400]

[0265] Real-time binding reactions were monitored throughout the experimental process, and binding reactions were recorded at the end of each step. The binding reactions of mAb-2 to mAb-1 and pre-conjugated mTrkB.mmh were compared, and the competitive / non-competitive behavior of different anti-mTrkB monoclonal antibodies was determined using a 50% inhibition threshold.

[0401]

[0266] Table 30 clearly defines the relationship between antibodies competing in both directions, independent of the order of binding.

[0402] result:

[0403] [Table 30]

[0404]

[0405] Example 16: Octet Blockade: Blockade of anti-human TrkB or anti-mouse TrkB antibodies from binding to TrkB by BDNF or NT-4

[0406] Experiment 1.

[0407]

[0267] The blockade of anti-human TrkB or anti-mouse TrkB antibodies against binding to TrkB by BDNF or NT-4 was evaluated using real-time bio-layer interferometry (BLI) based on the Octet HTX instrument. The overall study was performed at 25°C in 10 mM HEPES pH 7.4, 300 mM NaCl, 3 mM EDTA, 1 mg / mL BSA, 0.02% NaN3, and 0.05% v / v surfactant Tween-20 (HBS-EBT development buffer). All samples were dispensed into 384 tilted-well plates, and the plates were placed on a rotary shaker at a shaking speed of 1000 rpm. hTrkB.mFc was captured on an anti-mFc (AMC) octet sensor by immersing hTrkB.hFc or mTrkB.hFc in a well containing 10 μg / mL of TrkB reagent for 2 minutes. The octet biosensors captured with hTrkB.mFc or hTrkB.hFc were saturated by immersing them in a well containing 20 nM of BDNF, hNT-4, or mNT-4 for 2 minutes, followed by immersion in a well containing 300 nM of a different TrkB mAb for 4 minutes. The binding of TrkB mAb to the complex of TrkB and BDNF, hNT-4, or mNT-4 was determined using Scrubber 2.0c analysis software.

[0408]

[0268] The binding of any anti-human TrkB or anti-mouse TrkB antibody of the present invention was not blocked by both BDNF and NT-4, as reported in Tables 31 and 32.

[0409] [Table 31]

[0410]

[0411] [Table 32]

[0412]

[0413] Experiment 2.

[0414]

[0269] Blocking of BDNF or NT-4 from binding to TrkB by anti-human TrkB or anti-mouse TrkB antibodies was evaluated using a real-time bio-layer interferometer (BLI)-based Octet HTX instrument. The overall study was performed at 25°C in 10 mM HEPES pH 7.4, 300 mM NaCl, 3 mM EDTA, 1 mg / mL BSA, 0.02% NaN3, and 0.05% v / v surfactant Tween-20 (HBS-EBT development buffer). All samples were dispensed into 384 tilted-well plates, and the plates were placed on a rotary shaker at a shaking speed of 1000 rpm. hTrkB.mFc was captured on an anti-mFc (AMC) octet sensor by immersing hTrkB.hFc or mTrkB.hFc in a well containing 10 μg / mL of TrkB reagent for 2 minutes. The octet biosensor containing hTrkB.mFc or hTrkB.hFc was saturated by immersing it in a well containing 300 nM of a different TrkB mAb for 4 minutes, followed by immersing the octet biosensor in a well containing 20 nM of BDNF, hNT-4, or mNT-4 for 2 minutes. The binding of BDNF, hNT-4, or mNT-4 to the complex of TrkB and the different TrkB mAb was determined using Scrubber 2.0c analysis software.

[0415]

[0270] The binding of one of the three anti-human TrkB antibodies of the present invention blocked the binding of BDNF and hNT-4 as reported in Table 33. The binding of one of the three anti-mouse TrkB antibodies of the present invention partially blocked the binding of BDNF and mNT-4 as reported in Table 34.

[0416] [Table 33]

[0417]

[0418] [Table 34]

[0419]

[0420]

[0271] The present invention should not be limited to the scope of the specific embodiments described herein. More precisely, various modifications thereof other than those described herein will be apparent to those skilled in the art from the above detailed description and the accompanying drawings. Such modifications are intended to be within the scope of the appended claims.

Claims

Claim 1 An isolated antibody or its antigen-binding fragment that specifically binds to tropomyosin receptor kinase B (TrkB), wherein the antibody or its antigen-binding fragment comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 2, 18, 34, 49, 59, and 68; and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 10, 26, 42, 53, 63, and 72. Claim 2 The isolated antibody or its antigen-binding fragment according to claim 1, wherein the antibody or its antigen-binding fragment comprises a CDR of an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs 2 / 10, 18 / 26, 34 / 42, 49 / 53, 59 / 63 and 68 / 72. Claim 3 An isolated antibody or its antigen-binding fragment, comprising an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs 2 / 10, 18 / 26, 34 / 42, 49 / 53, 59 / 63 and 68 / 72, in accordance with claim 1 or 2. Claim 4 An isolated antibody or its antigen-binding fragment according to any one of claims 1 to 3, comprising: (a) an HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs 4, 20, 36, 50, 60 and 69; (b) an HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs 6, 22, 38, 51, 61, and 70; (c) an HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs 8, 24, 40, 52, 62, and 71; (d) an LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs 12, 28, 44, 54, 64, and 73; (e) an LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs 14, 30, 46, 55, 65, and 74; and (f) an LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs 16, 32, 48, 56, 66, and 75. Claim 5 An isolated antibody or its antigen-binding fragment according to any one of claims 1 to 4, wherein the antibody or its antigen-binding fragment comprises a set of six CDRs (HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) selected from the group consisting of (a) SEQ ID NO. 4-6-8-12-14-16; (b) SEQ ID NO. 20-22-24-28-30-32; (c) SEQ ID NO. 36-38-40-44-46-48; (d) SEQ ID NO. 50-51-52-54-55-56; (e) SEQ ID NO. 60-61-62-64-65-66; and (f) SEQ ID NO. 69-70-71-73-74-75. Claim 6 In any one of claims 1 to 5, the antibody or its antigen-binding fragment has a K of less than about 300 nM when measured by surface plasmon resonance at 25°C or 37°C. D An isolated antibody or its antigen-binding fragment that binds to human TrkB. Claim 7 In claim 6, K is selected from the group consisting of less than about 150 nM, less than about 50 pM, and less than about 100 pM when measured by surface plasmon resonance at 25°C or 37°C. D An isolated antibody or its antigen-binding fragment that binds to human TrkB. Claim 8 An isolated antibody or its antigen-binding fragment according to any one of claims 1 to 5, wherein the antibody or its antigen-binding fragment binds to human TrkB with a dissociation half-life (t½) selected from the group consisting of greater than about 10 minutes, greater than about 40 minutes, and greater than about 120 minutes when measured by surface plasmon resonance at 25°C or 37°C. Claim 9 In any one of claims 1 to 5, human TrkB signaling in cells processed to express TrkB with the antibody or its antigen-binding fragment at an EC of less than about 100 pM in the absence of BDNF. 50 An isolated antibody or its antigen-binding fragment that activates. Claim 10 In any one of claims 1 to 5, the activation of human TrkB signaling in the presence of BDNF in cells processed to express TrkB by the antibody or its antigen-binding fragment is less than about 100 pM EC 50 An isolated antibody or its antigen-binding fragment that enhances Claim 11 An isolated antibody or its antigen-binding fragment that demonstrates TrkB activation, as indicated by an increase in TrkB phosphorylation, when injected into the hippocampus of a humanized TrkB mouse, in any one of claims 1 to 5. Claim 12 An isolated antibody or its antigen-binding fragment according to any one of claims 1 to 5, wherein the antibody or its antigen-binding fragment demonstrates activation of the MAPK / ERK and PI3K / Akt signaling pathways, as demonstrated after incubating primary mouse cortical neurons with the agonist anti-TrkB antibody. Claim 13 An isolated antibody or its antigen-binding fragment according to any one of claims 1 to 12, wherein the antibody or its antigen-binding fragment promotes the survival of retinal ganglion cells as shown in an optic nerve transection model in TrkB humanized rats. Claim 14 An isolated antibody or its antigen-binding fragment according to any one of claims 1 to 12, wherein the antibody or its antigen-binding fragment promotes weight loss in humanized TrkB mice. Claim 15 An isolated antibody or its antigen-binding fragment according to any one of claims 1 to 12, wherein the antibody or its antigen-binding fragment promotes body fat loss in humanized TrkB mice. Claim 16 An isolated antibody or its antigen-binding fragment according to any one of claims 1 to 12, wherein the antibody or its antigen-binding fragment promotes a reduction in food and water consumption in humanized TrkB mice. Claim 17 An isolated antibody or its antigen-binding fragment according to any one of claims 1 to 12, wherein the antibody or its antigen-binding fragment promotes an increase in locomotor activity in humanized TrkB mice. Claim 18 In any one of claims 1 to 12, the isolated antibody or its antigen-binding fragment, wherein the antibody or its antigen-binding fragment exhibits one or more properties selected from the group consisting of: (a) an agonist antibody; and (b) K less than about 200 nM when measured by surface plasmon resonance at 25°C or 37°C. D (c) binds to human TrkB with a dissociation half-life (t½) greater than about 10 minutes when measured by surface plasmon resonance at 25°C or 37°C; and (d) human TrkB signaling at an EC of less than about 100 pM in the absence of brain-derived neurotrophic factor (BDNF) in cells processed to express human TrkB. 50 Activated by; (e) human TrkB receptor (TrkB hu / hu When injected into the hippocampus of mice homozygous for ) it enhances TrkB phosphorylation; (f) human TrkB receptor (TrkB hu / hu (g) promotes body weight loss when injected into mice homozygous for ) and increases retinal ganglion cell (RGC) survival as evaluated in an optic nerve transection model in humanized TrkB rats; (h) activates MAPK / ERK and PI3K / Akt signaling pathways; (i) promotes and / or increases neuronal cell survival in vitro; and (j) has an IC less than 5 nM 50 It blocks the binding of TrkB to BDNF and / or NT-4. Claim 19 A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 12, and a pharmaceutically acceptable carrier or diluent. Claim 20 An isolated nucleic acid molecule comprising a polynucleotide sequence encoding an anti-TrkB antibody or a fragment thereof according to any one of claims 1 to 12. Claim 21 A vector comprising the polynucleotide sequence of claim 20. Claim 22 Cells expressing the vector of claim 21. Claim 23 A use of a composition for enhancing biological activity mediated by TrkB, wherein the use comprises contacting TrkB with a biologically effective amount of an agonist anti-TrkB antibody according to any one of claims 1 to 12, or contacting TrkB with a pharmaceutical composition comprising a biologically effective amount of an agonist anti-TrkB antibody according to any one of claims 1 to 5. Claim 24 In paragraph 23, the above biological activity is neuronal protection or neuronal survival, and neuronal protection and neuronal survival are enhanced upon contact with TrkB and an agonist anti-TrkB antibody. Claim 25 In paragraph 23, the use of contact between TrkB and the agonist anti-TrkB antibody inducing neuroprotection and survival of retinal ganglion cells (RGCs). Claim 26 A use of a composition for treating or preventing a disease or disorder associated with TrkB activity or expression, or for improving at least one symptom associated with a disease or disorder associated with TrkB activity or expression, wherein the use comprises administering to a subject requiring treatment a pharmaceutical composition comprising a therapeutically effective amount of an agonist anti-TrkB antibody according to any one of claims 1 to 12 or a therapeutically effective amount of an agonist anti-TrkB antibody according to any one of claims 1 to 12. Claim 27 In paragraph 26, the above disease or disorder of the eye selected from the group consisting of glaucoma, diabetic retinopathy, age-related macular degeneration, ischemic optic neuropathy, optic neuritis, retinal ischemia, photoreceptor degeneration, retinitis pigmentosa, and retinal artery or vein occlusion, use. Claim 28 In Clause 27, the use where the above disease or disorder is glaucoma. Claim 29 A use of a composition for achieving weight loss in a subject, wherein the use comprises administering to the subject a TrkB agonist that is an antibody specific to TrkB or an antigen-binding fragment thereof according to any one of claims 1 to 5, or a pharmaceutical composition comprising said antibody or an antigen-binding fragment thereof. Claim 30 A use of a composition for achieving a reduction in body fat in a subject, wherein the use comprises administering to the subject a TrkB agonist that is an antibody specific to TrkB or an antigen-binding fragment thereof according to any one of claims 1 to 5, or a pharmaceutical composition comprising said antibody or an antigen-binding fragment thereof. Claim 31 A use of a composition for promoting neuronal survival in a subject, wherein the method comprises administering to the subject a TrkB agonist which is an antibody specific to TrkB or an antigen-binding fragment thereof according to any one of claims 1 to 5, or a pharmaceutical composition comprising said antibody or antigen-binding fragment. Claim 32 A use in which contact with TrkB is performed by injecting an injectable formulation comprising an antibody or an antigen-binding fragment thereof as claimed in any one of claims 1 to 12 into a patient, in any one of claims 23 to 31.

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