Anti-trkb monoclonal antibodies and methods of use thereof

Abstract

Antibodies that specifically bind to TrkB and methods of use thereof are disclosed. Agonist antibodies having neuroprotective effects, as shown by their ability to enhance retinal ganglion cell survival in vitro, are disclosed, and these agonist antibodies can be used in the treatment of conditions such as eye diseases, including glaucoma. In addition, other neuronal diseases or conditions can also benefit from treatment with these agonist antibodies, including those characterized in part by neuronal damage. In certain embodiments, the present invention includes antibodies that bind to TrkB and mediate cell signaling. The antibodies of the present invention can be fully human, non-naturally occurring antibodies formulated with an excipient for injection.

Description

Invention Field

[0001] This invention relates to monoclonal antibodies against the antigen myosin receptor kinase B (TrkB). More specifically, this invention relates to compositions comprising anti-TrkB monoclonal antibodies and methods of using these antibodies. Background Technology

[0002] Tropomyosin receptor kinase B (TrkB) belongs to the family of single-transmembrane receptor tyrosine kinases, including TrkA and TrkC. These receptor kinases mediate the activity of neurotrophic proteins, which are essential for neuronal survival and development. Neurotrophic proteins 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). (Lo, KY et al., J. Biol. Chem., 280:41744-52 (2005)).

[0003] TrkB is a high-affinity receptor for BDNF (Minichiello, et al., Neuron 21:335-45 (1998)), but it is also known to bind NT4 / 5. BDNF binding to trkB leads to receptor dimerization, resulting in autophosphorylation of specific tyrosine residues on the receptor and activation of signaling pathways involving mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K), and phospholipase C-γ (PLC-γ). (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)). Upon binding to BDNF, TrkB mediates various functions of neurotrophic proteins, including neuronal differentiation and survival.

[0004] Since TrkB plays a major role in neuronal survival, differentiation, and function, TrkB agonists have the potential to treat many neurodegenerative and metabolic diseases.

[0005] Certain TrkB agonists have been described in US2010 / 0150914; US2003 / 0157099; US2010 / 0196390 and US2017 / 0157099. However, there is still a need to identify and develop additional TrkB agonists that, in addition to exhibiting neuronal survival and neuroprotective properties such as those described herein, provide improved specificity.

[0006] Invention Summary

[0007] This invention provides isolated monoclonal antibodies and their antigen-binding fragments that specifically bind to tropomyosin receptor kinase B (TrkB). The isolated antibodies and antigen-binding fragments of this invention can be used to treat diseases and conditions related to TrkB activity or expression.

[0008] In its broadest aspect, the present invention provides anti-TrkB agonist antibodies that activate TrkB and promote neuronal survival. These antibodies can be used to improve neurological function and treat any disease or condition characterized in part by cellular degeneration, including neuronal damage and / or chronic neurodegenerative diseases associated with nervous system injury.

[0009] In some implementations, anti-TrkB antibodies can be used to treat a variety of eye diseases or conditions and can be formulated for intraocular or intravitreal delivery to treat eye diseases such as, but not limited to, glaucoma.

[0010] The antibodies of the present invention may be full-length (e.g., IgG1 or IgG4 antibodies) or may contain only antigen-binding portions (e.g., Fab, F(ab')2 or scFv fragments), and may be modified to affect functionality, such as eliminating residual effector functions (Reddy et al., 2000, J. Immunol. 164: 1925-1933).

[0011] Exemplary anti-TrkB antibodies of the present invention are listed in Tables 1 and 2 herein. Table 1 lists 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 lists the nucleic acid sequence identifiers of the HCVR, LCVR, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the exemplary anti-TrkB antibody.

[0012] The present invention provides an antibody or antigen-binding fragment thereof that specifically binds to TrkB, comprising HCVR, wherein the HCVR comprises an amino acid sequence selected from any HCVR amino acid sequence listed in Table 1 or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with it.

[0013] The present invention provides an antibody or antigen-binding fragment thereof that specifically binds to TrkB, comprising an LCVR containing an amino acid sequence selected from any LCVR amino acid sequence listed in Table 1 or substantially similar to such an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0014] The present invention also provides antibodies or antigen-binding fragments thereof that specifically bind to TrkB, comprising HCVR and LCVR amino acid sequence pairs (HCVR / LCVR), wherein the HCVR / LCVR pair comprises any HCVR amino acid sequence listed in Table 1 and any LCVR amino acid sequence listed in Table 1 paired therewith. According to certain embodiments, the present invention provides antibodies or antigen-binding fragments thereof comprising the HCVR / LCVR amino acid sequence pairs contained in any exemplary anti-TrkB antibody listed in Table 1.

[0015] Therefore, in a first aspect, the present invention provides isolated antibodies or antigen-binding fragments thereof that specifically bind to tropomyosin receptor kinase B (TrkB), wherein the antibody or antigen-binding fragment comprises: three heavy chain complementarity-determining regions (CDRs) (HVR1, HCDR2, and HCDR3) contained in a heavy chain variable region (HCVR), the heavy chain variable region comprising the amino acid sequences shown in Table 1, or sequences substantially similar to them having at least 90% sequence identity; and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained in a light chain variable region (LCVR), the light chain variable region comprising the amino acid sequences shown in Table 1, or sequences substantially similar to them having at least 90% sequence identity.

[0016] In one embodiment, the anti-TrkB antibody or its antigen-binding fragment exhibits one or more properties selected from the following:

[0017] - It is an agonist antibody;

[0018] -Measured by surface plasmon resonance at 25°C or 37°C, with K values ​​less than approximately 200 nM. D Combined with human TrkB;

[0019] -Binding with human TrkB with a dissociation half-life (t1 / 2) greater than about 10 minutes, as measured by surface plasmon resonance at 25°C or 37°C;

[0020] - In cells engineered to express human TrkB, in the absence of brain-derived neurotrophic factor (BDNF), activation of human TrkB signaling, EC 50 It ranges from approximately 35 to 82 pM;

[0021] -When injected with human TrkB receptor homozygous (TrkB) hu / hu In the hippocampus of mice, TrkB phosphorylation is enhanced;

[0022] -When injected with human TrkB receptor homozygous (TrkB) hu / hu In mice, it promoted weight loss;

[0023] - Increased retinal ganglion cell (RGC) survival when evaluated in a humanized TrkB rat optic nerve transection model;

[0024] -Activate the MAPK / ERK and PI3K / Akt signaling pathways;

[0025] -Increases the survival of neurons in vitro; and

[0026] -Blocks the binding of TrkB to BDNF and / or NT-4, IC 50 Less than 5 nM.

[0027] In one embodiment, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to tropomyosin receptor kinase B (TrkB), wherein the antibody or antigen-binding fragment thereof comprises: (a) a complementarity-determining region (CDR) of a heavy chain variable region (HCVR) comprising the amino acid sequence shown in Table 1; and (b) a complementarity-determining region (CDR) of a light chain variable region (LCVR) comprising the amino acid sequence shown in Table 1.

[0028] In one embodiment, the antibody that specifically binds to TrkB or its antigen-binding fragment comprises: three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained in any HCVR sequence selected from SEQ ID NOs: 2, 18, 34, 49, 59, and 68 or a substantially similar sequence having at least 90% sequence identity with it; and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained in any LCVR sequence selected from SEQ ID NOs: 10, 26, 42, 53, 63, and 72 or a substantially similar sequence having at least 90% sequence identity with it.

[0029] In one embodiment, the isolated antibody or its antigen-binding fragment that specifically binds to TrkB comprises HCVR having an amino acid sequence selected from SEQ ID NO: 2, 18, 34, 49, 59 and 68.

[0030] 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 SEQ ID NO: 10, 26, 42, 53, 63 and 72.

[0031] In one embodiment, the isolated antibody or its antigen-binding fragment that specifically binds to TrkB comprises HCVR and LCVR, the HCVR having an amino acid sequence selected from SEQ ID NOs: 2, 18, 34, 49, 59 and 68, and the LCVR having an amino acid sequence selected from SEQ ID NOs: 10, 26, 42, 53, 63 and 72.

[0032] In one embodiment, the 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 SEQ ID NO: 2 / 10, 18 / 26, 34 / 42, 49 / 53, 59 / 63 and 68 / 72.

[0033] In one embodiment, the 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 SEQ ID NO: 2 / 10, 18 / 26, and 34 / 42.

[0034] In one embodiment, the isolated antibody or its antigen-binding fragment that specifically binds to TrkB comprises an HCVR / LCVR amino acid sequence pair selected from SEQ ID NO: 2 / 10, 18 / 26, 34 / 42, 49 / 53, 59 / 63 and 68 / 72.

[0035] In one embodiment, the isolated antibody or its antigen-binding fragment that specifically binds to TrkB comprises HCVR / LCVR amino acid sequence pairs selected from SEQ ID NO: 2 / 10, 18 / 26, and 34 / 42.

[0036] The present invention also provides an antibody or antigen-binding fragment thereof that specifically binds to TrkB, comprising a heavy chain CDR1 (HCDR1) comprising an amino acid sequence selected from any HCDR1 amino acid sequence listed in Table 1 or substantially similar to it having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0037] The present invention also provides an antibody or antigen-binding fragment thereof that specifically binds to TrkB, comprising a heavy chain CDR2 (HCDR2) comprising an amino acid sequence selected from any HCDR2 amino acid sequence listed in Table 1 or a sequence substantially similar to it having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0038] The present invention also provides an antibody or antigen-binding fragment thereof that specifically binds to TrkB, comprising a heavy chain CDR3 (HCDR3) comprising an amino acid sequence selected from any HCDR3 amino acid sequence listed in Table 1 or substantially similar to it having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0039] The present invention also provides an antibody or antigen-binding fragment thereof that specifically binds to TrkB, comprising a light chain CDR1 (LCDR1) comprising an amino acid sequence selected from any LCDR1 amino acid sequence listed in Table 1 or substantially similar to such an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0040] The present invention also provides an antibody or antigen-binding fragment thereof that specifically binds to TrkB, comprising a light chain CDR2 (LCDR2) comprising an amino acid sequence selected from any LCDR2 amino acid sequence listed in Table 1 or substantially similar to such an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0041] The present invention also provides an antibody or antigen-binding fragment thereof that specifically binds to TrkB, comprising a light chain CDR3 (LCDR3) comprising an amino acid sequence selected from any LCDR3 amino acid sequence listed in Table 1 or substantially similar to such an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0042] The present invention also provides antibodies or antigen-binding fragments thereof that specifically bind to TrkB, comprising an HCDR3 and LCDR3 amino acid sequence pair (HCDR3 / LCDR3), wherein the HCDR3 / LCDR3 pair comprises any HCDR3 amino acid sequence listed in Table 1 and any LCDR3 amino acid sequence listed in Table 1 paired therewith. According to certain embodiments, the present invention provides antibodies or antigen-binding fragments thereof comprising the HCDR3 / LCDR3 amino acid sequence pair contained in any exemplary anti-TrkB antibody listed in Table 1. In some embodiments, the HCDR3 / LCDR3 amino acid sequence pair is selected from: 8 / 16, 24 / 32, 40 / 48, 52 / 56, 62 / 66, and 71 / 75.

[0043] The present invention also provides antibodies that specifically bind to TrkB or antigen-binding fragments thereof, which comprise a set of six CDRs (i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3) contained in any of the exemplary anti-TrkB antibodies listed in Table 1. In some embodiments, the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are selected from: (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) SEQ ID NOs: 60, 61, 62, 64, 65, 66; and (f) SEQ ID NOs: 69, 70, 71, 73, 74, 75.

[0044] In one related embodiment, 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 antibody amino acid sequence pair as defined in Table 1. For example, the present invention includes an antibody that specifically binds to TrkB or an antigen-binding fragment thereof, comprising the amino acid sequence sets HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 contained in the following HCVR / LCVR amino acid sequence pairs selected from: 2 / 10, 18 / 26, 34 / 42, 49 / 53, 59 / 63, and 68 / 72. Methods and techniques for identifying CDRs in HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs in the specific HCVR and / or LCVR amino acid sequences disclosed herein. Exemplary conventional methods that can be used to identify CDR boundaries include, for example, the Kabat definition, the Chothia definition, and the AbM definition. Generally, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia methods. See, for example, 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 86:9268-9272 (1989). Public databases can also be used to identify CDR sequences within antibodies.

[0045] In one embodiment, the present invention provides an isolated antibody or antigen-binding fragment thereof that specifically binds to TrkB, comprising:

[0046] a) The HCDR1 domain has an amino acid sequence selected from SEQ ID NO: 4, 20, 36, 50, 60 and 69;

[0047] b) The HCDR2 domain has an amino acid sequence selected from SEQ ID NOs:6,22,38,51,61 and70;

[0048] c) The HCDR3 domain has an amino acid sequence selected from SEQ ID NOs:8,24,40,52,62 and71;

[0049] d) The LCDR1 domain has an amino acid sequence selected from SEQ ID NOs:12,28,44,54,64 and73;

[0050] e) The LCDR2 domain has an amino acid sequence selected from SEQ ID NOs:14,30,46,55,65 and74; and

[0051] f) The LCDR3 domain has an amino acid sequence selected from SEQ ID NOs:16,32,48,56,66 and 75.

[0052] In one embodiment, the present invention provides an isolated antibody or antigen-binding fragment thereof that specifically binds to TrkB, comprising:

[0053] a) The HCDR1 domain has an amino acid sequence selected from SEQ ID NO: 4, 20, and 36;

[0054] b) The HCDR2 domain has an amino acid sequence selected from SEQ ID NO: 6, 22 and 38;

[0055] c) The HCDR3 domain has an amino acid sequence selected from SEQ ID NO: 8, 24 and 40;

[0056] d) The LCDR1 domain has an amino acid sequence selected from SEQ ID NO: 12, 28 and 44;

[0057] e) The LCDR2 domain has an amino acid sequence selected from SEQ ID NO: 14, 30, and 46; and

[0058] f) The LCDR3 domain has an amino acid sequence selected from SEQ ID NO: 16, 32 and 48.

[0059] In one embodiment, the isolated antibody or its antigen-binding fragment comprises:

[0060] The following set of six CDRs (HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) are selected: (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) SEQ ID NOs: 60-61-62-64-65-66; and (f) SEQ ID NOs: 69-70-71-73-74-75.

[0061] In one embodiment, the isolated antibody or antigen-binding fragment thereof that binds to TrkB comprises an antibody or antigen-binding fragment thereof that competes with a reference antibody for binding to TrkB, wherein the reference antibody comprises an HCVR / LCVR amino acid sequence pair selected from the following: SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 49 / 53, 59 / 63 and 68 / 72.

[0062] In one embodiment, the isolated antibody or antigen-binding fragment thereof that binds to TrkB comprises an antibody or antigen-binding fragment thereof that binds to the same epitope as a reference antibody, wherein the reference antibody comprises an HCVR / LCVR amino acid sequence pair selected from the following: SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 49 / 53, 59 / 63 and 68 / 72.

[0063] In one embodiment, the isolated antibody or its antigen-binding fragment is measured at surface plasmon resonance at 25°C or 37°C with a Kc of less than about 300 nM. D Combined with human TrkB.

[0064] In one embodiment, the isolated antibody or its antigen-binding fragment is measured at surface plasmon resonance at 25°C or 37°C with a Kc of less than about 200 nM. D Combined with human TrkB.

[0065] In one embodiment, the isolated antibody or its antigen-binding fragment is measured at surface plasmon resonance at 25°C or 37°C with a Kc of less than about 150 nM. D Combined with human TrkB.

[0066] In one embodiment, the isolated antibody or its antigen-binding fragment is measured at surface plasmon resonance at 25°C or 37°C with a Kc of less than about 50 nM. D Combined with human TrkB.

[0067] In one embodiment, the isolated antibody or its antigen-binding fragment is measured at surface plasmon resonance at 25°C or 37°C with a Kc of less than about 100 pM. D Combined with human TrkB.

[0068] In one embodiment, the isolated antibody or its antigen-binding fragment binds to human TrkB with a dissociation half-life (t1 / 2) greater than about 10 minutes, measured by surface plasmon resonance at 25°C or 37°C.

[0069] In one embodiment, the isolated antibody or its antigen-binding fragment binds to human TrkB at a t1 / 2 of more than about 40 minutes, as measured by surface plasmon resonance at 25°C or 37°C.

[0070] In one embodiment, the isolated antibody or its antigen-binding fragment binds to human TrkB at a t1 / 2 of more than about 120 minutes, as measured by surface plasmon resonance at 25°C or 37°C.

[0071] In one embodiment, the isolated antibody or its antigen-binding fragment that binds to TrkB is in the absence of BDNF in engineered TrkB-expressing cells at EC50 concentrations of less than about 100 pM. 50 Activates human TrkB signal transduction.

[0072] In one embodiment, the isolated antibody or its antigen-binding fragment that binds to TrkB is in the absence of BDNF in engineered TrkB-expressing cells at approximately 35 pM to approximately 82 pM EC. 50 Activates human TrkB signal transduction.

[0073] In one implementation, in engineered cells expressing TrkB, in the presence of BDNF, isolated antibodies or antigen-binding fragments of TrkB that bind to TrkB enhance activation of human TrkB signaling, and their EC50... 50 Less than approximately 100 pM.

[0074] In one embodiment, a separate antibody or antigen-binding fragment thereof that binds to TrkB, when injected into the hippocampus of a humanized TrkB mouse, shows activation of TrkB, as evidenced by increased TrkB phosphorylation.

[0075] In one embodiment, isolated antibodies or antigen-binding fragments thereof that bind to TrkB show activation of the MAPK / ERK and PI3K / Akt signaling pathways, as demonstrated after incubation of agonist anti-TrkB antibodies with primary mouse cortical neurons.

[0076] In one implementation, the isolated antibody or its antigen-binding fragment that binds to TrkB enhances / increases the survival of retinal ganglion cells, as demonstrated in a TrkB-humanized rat optic nerve transection model.

[0077] In one embodiment, the isolated antibody or its antigen-binding fragment that binds to TrkB enhances / increases the in vitro survival of neuronal cells.

[0078] In one implementation, a segregated antibody or its antigen-binding fragment that binds to TrkB promotes weight loss in humanized TrkB mice.

[0079] In one implementation, a segregated antibody or its antigen-binding fragment that binds to TrkB promotes a reduction in fat mass in humanized TrkB mice.

[0080] In one implementation, the isolated antibody or its antigen-binding fragment that binds to TrkB promotes a reduction in food and water consumption in humanized TrkB mice.

[0081] In one implementation, a segregated antibody or its antigen-binding fragment that binds to TrkB promotes increased motility activity in humanized TrkB mice.

[0082] In one embodiment, the present invention provides an anti-TrkB antibody or its antigen-binding fragment, which specifically binds to TrkB and blocks the binding of TrkB to BDNF, IC 50 Less than approximately 5 nM.

[0083] In one embodiment, the present invention provides an anti-TrkB antibody or its antigen-binding fragment, which specifically binds to TrkB and blocks the binding of TrkB to BDNF, IC 50 Less than approximately 500 pM.

[0084] In one embodiment, the present invention provides an anti-TrkB antibody or its antigen-binding fragment that specifically binds to TrkB and blocks the binding of TrkB to BDNF, IC 50 Less than approximately 200 pM.

[0085] In a second aspect, the present invention provides nucleic acid molecules encoding anti-TrkB antibodies or portions thereof. For example, the present invention provides nucleic acid molecules encoding any HCVR amino acid sequence listed in Table 1; in some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any HCVR nucleic acid sequence listed in Table 2, or a sequence substantially similar to it having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0086] The present invention also provides nucleic acid molecules encoding any LCVR amino acid sequence listed in Table 1; in some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any LCVR nucleic acid sequence listed in Table 2, or a sequence substantially similar to it having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0087] The present invention also provides nucleic acid molecules encoding any HCDR1 amino acid sequence listed in Table 1; in some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any HCDR1 nucleic acid sequence listed in Table 2, or a sequence substantially similar to it having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0088] The present invention also provides nucleic acid molecules encoding any HCDR2 amino acid sequence listed in Table 1; in some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any HCDR2 nucleic acid sequence listed in Table 2, or a sequence substantially similar to it having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0089] The present invention also provides nucleic acid molecules encoding any HCDR3 amino acid sequence listed in Table 1; in some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any HCDR3 nucleic acid sequence listed in Table 2, or a sequence substantially similar to it having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0090] The present invention also provides nucleic acid molecules encoding any LCDR1 amino acid sequence listed in Table 1; in some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any LCDR1 nucleic acid sequence listed in Table 2, or a sequence substantially similar to it having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0091] The present invention also provides nucleic acid molecules encoding any LCDR2 amino acid sequence listed in Table 1; in some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any LCDR2 nucleic acid sequence listed in Table 2, or a sequence substantially similar to it having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0092] The present invention also provides nucleic acid molecules encoding any LCDR3 amino acid sequence listed in Table 1; in some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any LCDR3 nucleic acid sequence listed in Table 2, or a sequence substantially similar to it having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0093] The present invention also provides a nucleic acid molecule encoding HCVR, wherein the HCVR comprises a set of three CDRs (i.e., HCDR1, HCDR2, HCDR3), wherein the amino acid sequence set of HCDR1, HCDR2, HCDR3 is defined by any of the exemplary anti-TRKB antibodies listed in Table 1.

[0094] The present invention also provides a nucleic acid molecule encoding an LCVR, wherein the LCVR comprises a set of three CDRs (i.e., LCDR1, LCDR2, LCDR3), wherein the amino acid sequence set of LCDR1, LCDR2, LCDR3 is defined by any of the exemplary anti-TRKB antibodies listed in Table 1.

[0095] The present invention also provides nucleic acid molecules encoding both HCVR and LCVR, wherein the HCVR comprises an amino acid sequence of any HCVR amino acid sequence listed in Table 1, and wherein the LCVR comprises an amino acid sequence of any LCVR amino acid sequence listed in Table 1. In some embodiments, 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 with it, 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 with it. In some embodiments of this aspect of the 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.

[0096] In a third aspect, the present invention provides a recombinant expression vector capable of expressing a polypeptide comprising a heavy or light chain variable region of an anti-TrkB antibody. For example, the present invention includes a recombinant expression vector comprising any of the aforementioned nucleic acid molecules, i.e., nucleic acid molecules encoding any HCVR, LCVR, and / or CDR sequences listed in Table 1. The present invention also includes a host cell incorporating the vector of the present invention, and a method for producing an antibody or a portion thereof by culturing the host cell under conditions allowing for the production of antibodies or antibody fragments and recovering the antibodies and antibody fragments.

[0097] This invention includes anti-TrkB antibodies with modified glycosylation patterns. In some embodiments, modifications to remove unwanted glycosylation sites may be useful, or antibodies lacking the fucose moiety present on the oligosaccharide chain may be useful, for example, to enhance antibody-dependent cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277:26733). In other applications, galactosylation may be modified to modify complement-dependent cytotoxicity (CDC).

[0098] In a fourth aspect, the present invention provides a pharmaceutical composition comprising at least one antibody of the present invention that specifically binds to TrkB or an antigen-binding fragment thereof and a pharmaceutically acceptable carrier.

[0099] In a related aspect, the present invention is characterized by 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 that is advantageously combined with the anti-TrkB antibody. The second therapeutic agent can be used to relieve at least one symptom of a neurodegenerative disease or condition.

[0100] In a fifth aspect, the present invention provides a method for enhancing TrkB-mediated biological activity, the method comprising contacting TrkB with a biologically effective amount of a Table 1 agonist anti-TrkB antibody, or contacting TrkB with a pharmaceutical composition containing a biologically effective amount of a Table 1 agonist anti-TrkB antibody.

[0101] In some implementations, the biological activity is neuroprotection or neuronal survival, and neuroprotection or neuronal survival is enhanced when TrkB is contacted with an agonist anti-TrkB antibody.

[0102] In some implementations, the biological activity is neuroprotection and survival of retinal ganglion cells (RGCs).

[0103] In a sixth aspect, the present invention provides a method of treatment using an anti-TrkB antibody or an antigen-binding portion of an antibody of the present invention for treating a disease or condition related to TrkB activity or expression, or at least one symptom related to such a disease or condition. The method of treatment according to this aspect of the invention comprises administering to a subject in need a therapeutically effective amount of a pharmaceutical composition comprising an antibody of the present invention or an antigen-binding fragment of an antibody. The disease treated can be any disease or condition that can be improved, alleviated, suppressed, or prevented by targeting TrkB and / or by activating TrkB-mediated cell signaling.

[0104] In one embodiment, the anti-TrkB antibody of the present invention can provide a method for preventing retinal neuronal damage or death. In one embodiment, the anti-TrkB antibody of the present invention can provide a method for treating a pathological disease in which retinal degeneration occurs. In one embodiment, the anti-TrkB antibody of the present invention can provide a method for treating a living eye before or after ophthalmic surgery, or before or after exposure to light or other environmental trauma, thereby preventing retinal cell degeneration. In one embodiment, the anti-TrkB antibody of the present invention can provide a method for preventing photoreceptor damage and degeneration in a living eye. In one embodiment, the anti-TrkB antibody of the present invention can provide a method for protecting retinal neurons without causing side effects that may be caused by cross-reactivity with other receptors such as the p75 receptor. In one embodiment, the anti-TrkB antibody of the present invention can provide a method for restoring or regenerating damaged photoreceptors.

[0105] In some embodiments, the disease or condition to be treated with the antibody of the present invention is selected from 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.

[0106] Other pathological conditions that can be treated with one or more anti-TrkB antibodies of the present invention include retinal detachment, photoretinopathy, surgically induced retinopathy (mechanical or photoinduced), toxic retinopathy, retinopathy of prematurity, viral retinopathy such as AIDS-related CMV or HIV retinopathy; uveitis; ischemic retinopathy caused by venous or arterial occlusion or other vascular diseases, retinopathy caused by ocular trauma or penetrating injury, peripheral vitreoretinopathy, or hereditary retinal degeneration.

[0107] In one embodiment, the eye disease or condition to be treated with the agonist anti-TrkB antibody of the present invention is glaucoma.

[0108] A seventh aspect of the invention provides a method for achieving weight loss in a subject, the method comprising administering to the subject a TrkB agonist antibody as listed in Table 1 or a pharmaceutical composition comprising the antibody or an antigen-binding fragment thereof.

[0109] In a related aspect, the present invention provides a method for achieving a reduction in fat mass in a subject, the method comprising administering to the subject a TrkB agonist antibody as listed in Table 1 or a pharmaceutical composition comprising the antibody or an antigen-binding fragment thereof.

[0110] An eighth aspect of the invention provides a method for promoting neuronal survival in a subject, the method comprising administering to the subject a therapeutically effective amount of a TrkB agonist antibody from Table 1, or a pharmaceutical composition comprising a therapeutically effective amount of the antibody or an antigen-binding fragment thereof.

[0111] In one embodiment, the above method can be achieved by administering an agonist anti-TrkB antibody or its antigen-binding fragment to a subject in need of it, wherein the agonist anti-TrkB antibody comprises: three heavy chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained in a heavy chain variable region (HCVR), the heavy chain variable region comprising the amino acid sequences shown in Table 1, or sequences substantially similar to them having at least 90% sequence identity; and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained in a light chain variable region (LCVR), the light chain variable region comprising the amino acid sequences shown in Table 1, or sequences substantially similar to them having at least 90% sequence identity.

[0112] In one embodiment, the method of the present invention can be implemented 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) included in the following HCVR sequences, wherein the HCVR sequence is selected from any one of SEQ ID NO: 2, 18, 34, 49, 59, and 68, or a substantially similar sequence having at least 90% sequence identity with it; and three light chain CDRs (LCDR1, LCDR2, and LCDR3) included in the following LCVR sequences, wherein the LCVR sequence is selected from any one of SEQ ID NO: 10, 26, 42, 53, 63, and 72, or a substantially similar sequence having at least 90% sequence identity with it.

[0113] In one embodiment, the antibody or its antigen-binding fragment comprises an HCVR having an amino acid sequence selected from SEQ ID NO: 2, 18, 34, 49, 59 and 68.

[0114] In one embodiment, the antibody or its antigen-binding fragment comprises an LCVR having an amino acid sequence selected from SEQ ID NO: 10, 26, 42, 53, 63 and 72.

[0115] In one embodiment, the antibody or its antigen-binding fragment comprises an HCVR having an amino acid sequence selected from SEQ ID NO: 2, 18, 34, 49, 59 and 68; and an LCVR having an amino acid sequence selected from SEQ ID NO: 10, 26, 42, 53, 63 and 72.

[0116] In one embodiment, the antibody or its antigen-binding fragment comprises a CDR from the HCVR / LCVR amino acid sequence pairs selected from SEQ ID NOs:2 / 10,18 / 26,34 / 42,49 / 53,59 / 63 and 68 / 72.

[0117] In one embodiment, the antibody or its antigen-binding fragment comprises an HCVR / LCVR amino acid sequence pair selected from SEQ ID NOs:2 / 10,18 / 26,34 / 42,49 / 53,59 / 63 and 68 / 72.

[0118] In one embodiment, the antibody or its antigen-binding fragment comprises:

[0119] a) Having an HCDR1 domain with an amino acid sequence selected from SEQ ID NO: 4, 20, 36, 50, 60 and 69;

[0120] b) Having an HCDR2 domain with an amino acid sequence selected from SEQ ID NO: 6, 22, 38, 51, 61 and 70;

[0121] c) Having an HCDR3 domain with an amino acid sequence selected from SEQ ID NO: 8, 24, 40, 52, 62 and 71;

[0122] d) Having an LCDR1 domain containing an amino acid sequence selected from SEQ ID NO: 12, 28, 44, 54, 64 and 73;

[0123] e) Having an LCDR2 domain containing an amino acid sequence selected from SEQ ID NO: 14, 30, 46, 55, 65 and 74; and

[0124] f) Having an LCDR3 domain having an amino acid sequence selected from SEQ ID NO: 16, 32, 48, 56, 66 and 75.

[0125] 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 following: (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) SEQ ID NOs: 60-61-62-64-65-66; and (f) SEQ ID NOs: 69-70-71-73-74-75.

[0126] In one embodiment, the disease or condition to be treated with the anti-TrkB antibody of the present invention is obesity, and any complications arising from obesity.

[0127] It is conceivable that any disease or condition associated with TrkB activity or expression is suitable for treatment with the antibodies of this invention. These diseases may include any disease with significant cellular degeneration, such as in neurodegenerative diseases or after nerve injury.

[0128] Other implementation methods will become apparent upon reading the following detailed description. Brief description of the attached diagram

[0130] Figure 1 Western blot analysis showed that, at 1 hour, 4 hours, and 18 hours after direct hippocampal injection of the TRKB agonist antibody H4H9816P2 or the isotype control antibody, total TRKB and phosphate-TRKB levels were assessed in homozygous humanized TRKB mice.

[0131] Figure 2 The figure shows that the TrkB agonist antibody H4H9816P2 activates downstream pathways of MAPK / ERK and PI3K / Akt. The figure also shows Western blots of phosphate-TrkB, total TrkB, phosphate-Akt, total AKT, phosphate-ERK, and total ERK at 15 minutes and 2 hours after treatment with various TrkB agonist antibodies or BDNF from homozygous humanized TRKB mouse pups on day 1 of life.

[0132] Figure 3 The results showed that the three TrkB agonist antibodies increased the in vitro survival rate of SH-SY5Y cells in a dose-dependent manner. The isotype control antibody had no effect on cell survival.

[0133] Figure 4 The results showed that the anti-TrkB agonist antibody H4H9816P2 was effective against TrkB. hu / hu Pharmacokinetic curves in mice and wild-type mice. Mice were administered a single subcutaneous dose of 10 mg / kg on day 0. Serum concentrations of total H4H9816P2 were measured using the Gyros immunoassay. Data points at 6 hours, 1, 2, 3, 6, 9, 16, 21, and 30 days post-administration indicate mean antibody concentrations. For TrkB hu / hu For mice, the total antibody concentration of H4H9816P2 is represented by solid black circles, while for wild-type mice it is represented by solid black squares. Data are plotted as mean ± SD.

[0134] Figure 5. By Figure 5A and Figure 5B composition, Figure 5A and Figure 5B This study demonstrated the ability of anti-mouse TrkB monoclonal antibodies to block the interaction between mouse or rat TrkB and its ligand BDNF (brain-derived neurotrophic factor). An ELISA-based method was used to assess mouse TrkB.hFc in the presence of anti-mouse TrkB and isotype control mAbs at specific concentration ranges. Figure 5A (Two images) and rat TrkB.mmh ( Figure 5B (Two images) combined with a flat plate covered by BDNF. Figure 5A The inset in the two figures shows the dose-response curve of BDNF binding in mouse TrkB.hFc (REGN2277), EC 50 The value is 780pM. Figure 5B The inset in the two figures shows the dose-response curve of rat TrkB.mmh (REGN1808) binding to BDNF, EC 50 The value is 2.2 nM. Molar concentration (M) represents the antibody concentration of the mAb. Error bars represent the standard deviation. Invention Details

[0136] Before describing the invention, it should be understood that the invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the invention, as the scope of the invention is limited only by the appended claims.

[0137] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. As used herein, the term “about”, when applied to a particular numerical value, means that the value may differ from the stated value by no more than 1%. For example, as used herein, the expression “about 100” includes 99 and 101, as well as all values ​​in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0138] While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of the invention, preferred methods and materials are described hereafter. All patents, applications, and non-patent publications referenced in this specification are incorporated herein by reference in their entirety.

[0139] definition

[0140] The designation "TrkB," also known as "tropomyosin receptor kinase B," refers to the human receptor (unless specified as being from another species) containing the amino acid sequence shown in amino acid residues 32 to 430 of accession number NP_001018074.1. Human TrkB containing the myc-myc-hexahistidine tag is shown as SEQ ID NO: 76 (where amino acid residues 1-399 are human TrkB and amino acid residues 400-427 are the myc-myc-hexahistidine tag). Other forms of the human TrkB protein are described herein, including SEQ ID NO: 77, which is human TrkB (residues 1-399) with the mouse Fc region (residues 400-632); and SEQ ID NO: 78, which is human TrkB (residues 1-399) with the human Fc region (residues 400-626). Mouse TrkB contains the amino acid sequence shown in amino acid residues 32 to 429 of accession number NP_001020245. Mouse TrkB containing the myc-myc-hexahistidine tag is shown as SEQ ID NO: 79 (where amino acid residues 1-398 are mouse TrkB, and amino acid residues 399-426 are the myc-myc-hexahistidine tag). Other forms containing mouse TrkB protein are described herein, including SEQ ID NO: 80, which is mouse TrkB (residues 1-398) with the mouse Fc region (residues 399-631); and SEQ ID NO: 81, which is mouse TrkB (residues 1-398) with the human Fc region (residues 399-625). Rabbit TrkB contains the amino acid sequence shown in accession number XP_002721319.1, which contains amino acid residues 32-430. Rabbit TrkB containing the myc-myc-hexahistidine tag is shown as SEQ ID NO: 82 (where amino acid residues 1-399 are rabbit TrkB, and amino acid residues 400-427 are the myc-myc-hexahistidine tag). This article describes other forms of the rabbit TrkB protein, including SEQ ID NO: 83, which is the rabbit TrkB (residues 1-399) with the mouse Fc region (residues 400-632). The rat TrkB contains the amino acid sequence shown in accession number NP_036863.1, which contains amino acid residues 32-429. The rat TrkB containing the myc-myc-hexahistidine tag is shown as SEQ ID NO: 84 (where amino acid residues 1-398 are rat TrkB and amino acid residues 399-426 are the myc-myc-hexahistidine tag). This article describes other forms of the rat TrkB protein, including SEQ ID NO: 85, which is the rat TrkB (residues 1-398) with the mouse Fc region (residues 399-631).The rhesus macaque (Macaca mulatta) TrkB is shown as SEQ ID NO: 95 (amino acids 32 to 838 of accession number NP_001248226.1), and the cynomolgus macaque (Macaca fascicularis) TrkB is shown as SEQ ID NO: 96 (amino acids 32 to 838 of accession number XP_005582102.1).

[0141] The human “TrkA” protein is shown in SEQ ID NO: 86, wherein amino acids 1-375 are TrkA (amino acids 34-414 of accession number NP_001012331.1, which have V263L and C300S), amino acids 376-378 are GPG linkers, and amino acids 379-605 are human Fc.

[0142] The human “TrkC” protein is shown in SEQ ID NO: 87, wherein amino acids 1-398 are TrkC (amino acids 32-429 of accession number NP_001012338.1), and amino acids 399-426 are myc-myc-his tags.

[0143] The mouse “TrkC” protein is shown in SEQ ID NO: 88, where amino acids 1-398 are TrkC (amino acids 32-429 of accession number NP_032772.3), and amino acids 399-426 are myc-myc-his tags.

[0144] The cynomolgus monkey “TrkC” protein is shown in SEQ ID NO: 89, wherein amino acids 1-398 are TrkC (amino acids 32-429 of accession number XP_015308837.1), and amino acids 399-426 are myc-myc-his tags.

[0145] In some cases, cell lines expressing the TrkB protein were prepared, the TrkB protein comprising the extracellular domain and transmembrane and cytoplasmic domains of the TrkB protein. For example, SEQ ID NO: 91 is the human TrkB protein, which contains all three domains contained in amino acids 32-822 of accession number NP_001018074.1 or Uniprot Q16620-1, wherein amino acids 1-398 are the extracellular domain and the transmembrane / cytoplasmic region is defined by approximately amino acid residues 399-790. In one case, a TrkB cell line expressing mouse TrkB (amino acids 32-476 of accession number NP_032771.1; see also SEQ ID NO: 92) was prepared. In another example, a cell line expressing a chimeric TrkB protein was prepared, the chimeric TrkB protein having the extracellular domain of mouse TrkB (amino acids 32-429 from accession number NP_001020245.1 (see also SEQ ID NO: 93) or Uniprot P15209-1) and the transmembrane and cytoplasmic domains of human TrkB (amino acids 431-822 from accession number NP_001018074.1 (see also SEQ ID NO: 91)). A cell line expressing African green monkey (Chlorocebus sabaeus) TrkB (amino acids 32-822 from accession number XP_007967815.1 (see also SEQ ID NO: 94)) was also prepared.

[0146] The term "brain-derived neurotrophic factor" or "BDNF" refers to the ligand of TrkB. The amino acid sequence of BDNF is shown in SEQ ID NO: 90 (isotype A1-120, amino acids 129-247 of accession number NP_733928.1, with an N-terminal addition of Met). In some of the experiments described herein, BDNF was derived from R&D Systems, 248-BD / CF.

[0147] Unless explicitly stated to be from a non-human species, all references to proteins, peptides, and protein fragments herein are intended to refer to the human form of the corresponding protein, peptide, or protein fragment. Therefore, the expression "TrkB" refers to human TrkB unless specifically stated to be from a non-human species, such as "monkey TrkB," "mouse TrkB," "rat TrkB," etc.

[0148] As used herein, the term "anti-TrkB antibody" includes monovalent antibodies with single specificity, as well as bispecific antibodies 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 shown in Table 1 herein. The term "anti-TrkB antibody" also includes antibody-drug conjugates (ADCs) comprising an anti-TrkB antibody or its antigen-binding moiety conjugated to a drug or toxin (i.e., a cytotoxic agent). The term "anti-TrkB antibody" also includes antibody-radionium conjugates (ARCs) comprising an anti-TrkB antibody or its antigen-binding moiety conjugated to a radionuclide.

[0149] As used herein, the term "anti-TrkB antibody" refers to any antigen-binding molecule or molecular complex containing at least one complementarity-determining region (CDR) that specifically binds to or interacts with TrkB or a portion of TrkB. The term "antibody" includes immunoglobulin molecules comprising four polypeptide chains (two heavy (H) chains and two light (L) chains) interconnected by disulfide bonds, and their polymers (e.g., IgM). Each heavy chain contains a heavy chain variable region (abbreviated herein as HCVR or V). H The heavy-chain constant region contains three structural domains, C H 1,C H 2 and C H 3. Each light chain contains a light chain variable region (abbreviated as LCVR or V in this document). L The light chain constant region contains a structural domain (C) and a light chain constant region. L 1). V H and V L The region can be further subdivided into hypervariable regions, called complementarity-determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the invention, the FRs of the anti-TrkB antibody (or its antigen-binding portion) may be identical to the human germline sequence, or may be natural or artificially modified. The amino acid common sequence can be defined based on a side-by-side analysis of two or more CDRs.

[0150] As used herein, the term "antibody" also includes the antigen-binding fragment of a full-length antibody molecule. As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, etc., include any enzymatically available, synthetic, or genetically engineered polypeptide or glycoprotein capable of specifically binding an antigen to form a complex. Antigen-binding fragments of antibodies can be derived from, for example, intact antibody molecules using any suitable standard technique, such as proteolytic digestion or recombinant genetic engineering, wherein said recombinant genetic engineering involves manipulating and expressing DNA encoding variable and optionally constant regions of the antibody. Such DNA is known and / or readily available from, for example, commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. DNA can be sequenced and manipulated by chemical methods or by using molecular biology techniques, for example, arranging one or more variable and / or constant domains into suitable conformations, or introducing codons, generating cysteine ​​residues, modifying, adding, or deleting amino acids, etc.

[0151] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues of a hypervariable region of a mimic antibody (e.g., a complementary determinant region (CDR) separated from a CDR3 peptide) or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single-domain antibodies, domain-deficient antibodies, chimeric antibodies, CDR-transplanted antibodies, diabodies, triabodies, tetrabodies, mini-antibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also included in the term "antigen-binding fragments" as used herein.

[0152] Antigen-binding fragments of antibodies typically contain at least one variable domain. Variable domains can have any size or amino acid composition and typically contain at least one CDR adjacent to or conforming to one or more frame sequences. In antigen-binding fragments having a VH domain that binds to a VL domain, the VH and VL domains can be positioned relative to each other in any suitable arrangement. For example, the variable region can be a dimer and contain VH-VH, VH-VL, or VL-VL dimers. Alternatively, the antigen-binding fragment of an antibody can contain a monomeric VH or VL domain.

[0153] In some embodiments, the antigen-binding fragment of the antibody may include at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of the variable and constant domains that may be present in 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 H 2-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 the variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be directly connected to each other or connected via all or part of a hinge or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids, resulting in a flexible or semi-flexible connection between adjacent variable and / or constant domains within a single polypeptide molecule. Furthermore, the antigen-binding fragment of the antibody of the present invention may comprise a homodimer or heterodimer (or other multimer) of any of the variable and constant domain configurations listed above, in a manner that is non-covalently bound to each other and / or to one or more monomeric VH or VL domains (e.g., via disulfide bonds).

[0154] Like complete antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically contain at least two distinct variable domains, each capable of specifically binding to a single antigen or a different epitope on the same antigen. Using conventional techniques available in the art, any multispecific antibody form, including the exemplary bispecific antibody forms disclosed herein, can be adapted to the antigen-binding fragments of the antibodies of this invention.

[0155] In some cases, it may be necessary to antagonize TrkB, for example, to inhibit the growth or proliferation of neuronal tumor cells. However, the antibodies of the present invention act as agonist antibodies, serving as enhancers of neuronal survival and neuroprotective agents. The antibodies of the present invention can function by enhancing the interaction between TrkB and its ligand BDNF. Alternatively, the antibodies of the present invention can mediate TrkB signaling through mechanisms that do not involve enhancing the interaction between TrkB and its ligand.

[0156] As used herein, the term "human antibody" is intended to include human antibodies that are not naturally occurring. The term includes antibodies recombinantly generated in the cells of non-human mammals. The term is not intended to include antibodies isolated or generated from human subjects.

[0157] In some embodiments, the antibodies of the present invention may be recombinant and / or non-naturally occurring human antibodies. As used herein, the term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, generated, or isolated by a recombinant manner, such as antibodies expressed using a recombinant expression vector transfected into host cells (described further below), antibodies isolated from a recombinant combined human antibody library (described further below), antibodies isolated from transgenic animals (e.g., mice) containing human immunoglobulin genes (see, for example, Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, generated, or isolated by any other method of splicing human immunoglobulin gene sequences to other DNA sequences. In some embodiments, such recombinant human antibodies are mutagenized in vitro (or, when using transgenic animals with human Ig sequences, in vivo somatic cell mutagenization), so that the amino acid sequences of the VH and VL regions of the recombinant antibody, although related to human germline VH and VL sequences, may not be naturally present in an in vivo human antibody germline library.

[0158] Human antibodies can exist in two forms associated with hinge heterogeneity. In one form, the immunoglobulin molecule comprises a stable four-chain construct of approximately 150-160 kDa, where dimers are linked together by interchain heavy-chain disulfide bonds. In the second form, the dimers are not linked by interchain disulfide bonds, forming a molecule of approximately 75-80 kDa composed of covalently coupled light and heavy chains (half-antibodies). These forms are difficult to separate even after affinity purification.

[0159] The frequency of the second form in various intact IgG isotypes is due to, but not limited to, structural differences associated with the hinge region isotype of the antibody. A single amino acid substitution in the hinge region of the human IgG4 hinge can significantly reduce the occurrence of the second form (Angal et al. (1993) Molecular Immunology 30:105) to levels typically observed with the human IgG1 hinge. This invention includes antibodies with one or more mutations in the hinge, CH2, or CH3 regions, which may be desirable, for example, in production, to improve the yield of the desired antibody form.

[0160] The terms "specific binding" or "specific binding" refer to the formation of a relatively stable complex between an antibody or its antigen-binding fragment and the antigen under physiological conditions. Specific binding can occur through at least approximately 1 x 10-1 -6 Characterized by M or a smaller equilibrium dissociation constant (e.g., a smaller K). D (Indicating a tighter binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, etc. As described herein, surface plasmon resonance, such as BIACORE, has been used... TM Antibodies that specifically bind to TrkB were identified. Furthermore, as used herein, multispecific antibodies that bind to the TrkB protein and one or more other antigens, or bispecific antibodies that bind to two different regions of TrkB, are still considered to be "specifically binding" antibodies.

[0161] The antibodies of this invention can be isolated antibodies. As used herein, "isolated antibody" means an antibody that has been identified and isolated and / or recovered from at least one component of its natural environment. For example, for the purposes of this invention, an antibody that has been isolated or removed from at least one component, tissue, or cell of an organism that naturally exists or naturally produces the antibody is an "isolated antibody." Isolated antibodies also include in situ antibodies within recombinant cells. Isolated antibodies are antibodies that have undergone at least one purification or isolation step. According to some embodiments, isolated antibodies may be substantially free of other cellular material and / or chemicals.

[0162] The anti-TrkB antibodies disclosed herein may contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the variable domains of the heavy and light chains. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein with sequences available from, for example, public antibody sequence databases. Once obtained, one or more desired properties of antibody-antigen binding fragments containing one or more mutations can be readily tested, such as increased binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as applicable), reduced immunogenicity, etc. Antibody-antigen binding fragments obtained in this general manner are included within the scope of this invention.

[0163] The present invention also includes anti-TrkB antibodies comprising a variant of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein having one or more conserved substitutions. For example, the present invention includes anti-TrkB antibodies having HCVR, LCVR, and / or CDR amino acid sequences, wherein the HCVR, LCVR, and / or CDR amino acid sequences have, for example, 10 or fewer, 8 or fewer, 6 or fewer, or 4 or fewer conserved amino acid substitutions relative to any HCVR, LCVR, and / or CDR amino acid sequences listed in Table 1 herein.

[0164] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site, known as a complementary site, within the variable region of an antibody molecule. A single antigen can have more than one epitope. Therefore, different antibodies can bind to different regions of the antigen and can have different biological effects. Epitopes can be conformational or linear. Conformational epitopes are generated from spatially adjacent amino acids of different segments of a linear polypeptide chain. Linear epitopes are generated from adjacent amino acid residues in a polypeptide chain. In some cases, an epitope may include portions of a sugar, phosphoryl, or sulfonyl group on the antigen.

[0165] The terms "substantially identical" or "substantially the same," when referring to a nucleic acid or fragment thereof, mean that when compared with another nucleic acid (or its complementary strand) with appropriate nucleotide insertions or deletions for optimal alignment, there is nucleotide sequence identity in at least about 95%, more preferably at least about 96%, 97%, 98%, or 99% of the nucleotide bases, as measured by any well-known sequence identity algorithm, such as FASTA, BLAST, or Gap, discussed below. In some cases, a nucleic acid molecule substantially identical to a reference nucleic acid molecule may encode a polypeptide with the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0166] When applied to peptides, the term "substantially similar" or "substantially similar" means that two peptide sequences share at least 95% sequence identity, and more preferably at least 98% or 99%, when optimally aligned, for example, using the GAP or BESTFIT procedure and default vacancy weights. Preferably, the dissimilar residue positions are distinguished by conserved amino acid substitutions. A "conserved amino acid substitution" is the substitution of one amino acid residue by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conserved amino acid substitutions do not substantially alter the functional properties of the protein. In cases where two or more amino acid sequences are dissimilar to each other by conserved substitutions, the percentage sequence identity or similarity can be adjusted upwards to correct for the conservatism of the substitution. The means of making such adjustments are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24:307-331, which is incorporated herein by reference. Examples of amino acid groups with side chains having similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic-hydroxy 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: aspartic acid and glutamic acid; and (7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conserved amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is any change with a positive value in the PAM250 log-likelihood matrix as disclosed in Gonnet et al. (1992) Science 256:1443-1445, which is incorporated herein by reference. A “moderately conservative” substitution is any change with a non-negative value in the PAM250 log-likelihood matrix.

[0167] Sequence analysis software is typically used to measure the sequence similarity, also known as sequence identity, of peptides. Protein analysis software uses similarity measures assigned to various substitutions, deletions, and other modifications, including conserved amino acid substitutions, to match similar sequences. For example, GCG software includes programs such as Gap and Bestfit, which can be used with default parameters to determine sequence homology or sequence identity between closely related peptides (e.g., homologous peptides from different species) or between wild-type proteins and their mutant counterparts. See, for example, GCG version 6.1. Peptide sequences can also be compared using FASTA with default or recommended parameters (programs in GCG version 6.1). FASTA (e.g., FASTA2 and FASTA3) provides alignment and percentage sequence identity of the best overlapping regions between the query and search sequences (Pearson (2000), ibid.). Another preferred algorithm when comparing the sequences of this invention with databases containing large numbers of sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, for example, Altschul et al. (1990) J.Mol.Biol. 215: 403-410 and Altschul et al. (1997) Nucleic Acids Res. 25: 3389-402, each of which is incorporated herein by reference.

[0168] Biological characteristics of antibodies

[0169] This invention includes an anti-TrkB antibody, which, through surface plasmon resonance measurements at 25°C or 37°C, achieves a Kc concentration of less than approximately 200 nM. D Combined with TrkB. According to certain embodiments, the present invention includes K with a value 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 Anti-TrkB antibodies that bind to human TrkB.

[0170] This invention includes anti-TrkB antibodies that bind to human TrkB, having a dissociation half-life (t1 / 2) greater than about 10 minutes, as measured by surface plasmon resonance at 25°C or 37°C. According to certain embodiments, the invention includes anti-TrkB antibodies that bind to human TrkB with a t1 / 2 of 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.

[0171] This invention includes anti-TrkB antibodies that may or may not bind to monkey TrkB, or mouse or rat TrkB. As used herein, the antibody "does not bind" to a specific antigen (e.g., monkey, mouse, or rat TrkB) if: when tested in an antigen binding assay such as surface plasmon resonance, the antibody exhibits a Kc greater than about 1000 nM. D Alternatively, it may not show any antigen binding in the assay. According to this aspect of the invention, another assay that can be used to determine whether an antibody binds to a specific antigen is ELISA.

[0172] This invention includes an anti-TrkB antibody that activates human TrkB signaling in cells engineered to express the TrkB receptor, EC. 50 Less than approximately 100 pM. EC can be measured using the measurement method described in Example 5 or a substantially similar method. 50 The value is calculated as the antibody concentration required to activate TrkB-mediated signaling to half of the observed maximum signal. Therefore, according to some embodiments, the present invention includes an anti-TrkB antibody that mediates TrkB signaling in cells engineered to express the TrkB receptor, with or without BDNF, and EC 50 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, as measured using the measurement method described in Example 5 of this document or a substantially similar measurement method.

[0173] The present invention includes an anti-TrkB antibody that activates the TrkB receptor, as shown by TrkB phosphorylation following direct hippocampal injection in humanized mice expressing the human TrkB receptor, as illustrated in Example 6.

[0174] This invention includes an anti-TrkB antibody that promotes weight loss in mice humanized to express the human TrkB receptor. The antibody of this invention can also be used to promote a reduction in fat mass and an increase in physical activity in these mice, while reducing food and water intake (see Example 7).

[0175] When tested in a optic nerve transection model, the antibody of the present invention promoted the survival of retinal ganglion cells (RGCs) in rats with humanized expression of the human TrkB receptor. See Example 8.

[0176] The antibodies of this invention activate the downstream pathways MAPK / ERK and PI3K / Akt, as demonstrated by exposing primary mouse cortical neurons obtained from humanized TrkB mice to the antibodies of this invention (see Example 9).

[0177] As shown in Example 10, the agonist anti-TrkB antibody of the present invention also promotes the survival of SH-SY5Y cells in a dose-dependent manner.

[0178] This invention includes an anti-TrkB antibody, which has an IC50 concentration of less than about 5 nM. 50 Blocking the binding of TrkB to BDNF. For example, as shown in Example 12, all three antibodies tested blocked >50% of the binding of TrkB to BDNF in mice or rats. Using the assay described in Example 12 or a substantially similar assay, IC50 can be... 50 The value is calculated as the antibody concentration required to block the binding of TrkB to BDNF, compared to the maximum signal observed in the absence of the antibody. Therefore, according to certain embodiments, the present invention includes an anti-TrkB antibody that blocks the binding of TrkB to BDNF, IC50. 50 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, 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, or less than about 20 pM, as measured using the assay form described in Example 12 of this document or a substantially similar assay. In one embodiment, the TrkB antibody of the present invention is expressed at an IC50 concentration of about 180 pM to about 4 nM. 50Block the binding of TrkB to BDNF.

[0179] When disclosed as "measured by surface plasmon resonance," the binding characteristics of the antibodies of the present invention (e.g., any binding characteristics mentioned above herein) refer to the relevant binding characteristics involving the interaction between the antibody and the antigen measured using a surface plasmon resonance instrument (e.g., The instrument (GE Healthcare) is used to measure the parameters using the standard Biacore assay conditions or substantially similar assay formats shown in Examples 3 and 4 of this document. In some embodiments, the parameters are measured at 25°C, while in other embodiments, the parameters are measured at 37°C.

[0180] The present invention includes an antibody that specifically binds to TrkB or an antigen-binding fragment thereof, which comprises HCVR and / or LCVR, wherein the HCVR and / or LCVR comprises an amino acid sequence selected from any HCVR and / or LCVR amino acid sequence listed in Table 1.

[0181] The antibodies of the present invention may have one or more of the above-described biological properties, or any combination thereof. The above list of biological properties of the antibodies of the present invention is not intended to be exhaustive. Other biological characteristics of the antibodies of the present invention will be apparent to those skilled in the art upon reading this disclosure, including the embodiments herein.

[0182] Epitope plotting and related techniques

[0183] The epitope for antibody binding in this invention can consist of a single continuous sequence of three or more amino acids (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 can consist of multiple non-continuous 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, at a location on the TrkB surface where binding to the epitope at that location does not interfere with the interaction between TrkB and its ligand.

[0184] Various techniques known to those skilled in the art can be used to determine whether an antibody interacts with "one or more amino acids" within a peptide or protein. Exemplary techniques include, for example, conventional cross-blocking assays, such as... AntibodiesThe assays described by Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY) include alanine scanning mutation analysis, peptide blotting (Reineke, 2004, Methods Mol Biol 248:443-463), and peptide cleavage analysis. Alternatively, methods such as epitope excision, epitope extraction, and antigen chemical modification can be used (Tomer, 2000, Protein Science 9:487-496). Another method for identifying amino acids within a peptide that interact with the antibody is the detection of hydrogen / deuterium exchange via mass spectrometry. Generally, the hydrogen / deuterium exchange method involves deuterating the target protein and then binding the antibody to the deuterated protein. Next, the protein / antibody complex is transferred to water to allow hydrogen-deuterium exchange to occur at all residues except those protected by the antibody (which remain deuterated). After antibody dissociation, the target protein is cleaved by protease and analyzed by mass spectrometry to reveal the deuterated residues corresponding to the specific amino acids that interact with the antibody. See, for example, Ehring (1999) Analytical Biochemistry 267(2):252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A.

[0185] This invention includes anti-TrkB antibodies that bind to the same epitope as any specific exemplary antibody described herein (e.g., an antibody comprising any amino acid sequence listed in Table 1 herein). Similarly, this invention also includes anti-TrkB antibodies that compete with any specific exemplary antibody described herein (e.g., an antibody comprising any amino acid sequence listed in Table 1 herein) for binding to TrkB.

[0186] By using conventional methods known in the art and illustrated herein, it can be readily determined whether an antibody binds to the same epitope as or competes with a reference anti-TrkB antibody. For example, to determine whether a test antibody binds to the same epitope as the reference anti-TrkB antibody of the present invention, the reference antibody is bound to the TrkB protein. Next, the ability of the test antibody to bind to the TrkB molecule is evaluated. If the test antibody binds to TrkB after saturation binding with the reference anti-TrkB antibody, it can be concluded that the test antibody binds to a different epitope than the reference anti-TrkB antibody. On the other hand, if the test antibody does not bind to the TrkB molecule after saturation binding with the reference anti-TrkB antibody, the test antibody may bind to the same epitope as the reference anti-TrkB antibody. Other conventional experiments (e.g., peptide mutation and binding assays) can then be performed to confirm whether the observed lack of test antibody binding is actually due to binding to the same epitope as the reference antibody, or whether steric hindrance (or other phenomena) is the cause of the unobserved binding. Such experiments can be performed using ELISA, RIA, Biacore, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art. According to certain embodiments of the invention, in a competitive binding assay, if, for example, one antibody inhibits the binding of another antibody by 1, 5, 10, 20, or 100 times excess, the binding of the other antibody by at least 50%, but preferably 75%, 90%, or even 99%, then the two antibodies bind to the same (or overlapping) epitopes (see, for example, Junghans et al., Cancer Res. 1990:50:1495-1502). Alternatively, if substantially all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other antibody, then the two antibodies are considered to bind to the same epitope. If only a subset of the amino acid mutations that reduce or eliminate the binding of one antibody reduces or eliminates the binding of the other antibody, then the two antibodies are considered to have “overlapping epitopes.”

[0187] To determine whether an antibody competes for binding (or cross-competitively binds) with a reference anti-TrkB antibody, the binding method described above is performed in two directions: In the first direction, the reference antibody is allowed to bind 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 test antibody is allowed to bind to the TrkB molecule under saturation conditions, and then the binding of the reference antibody to the TrkB molecule is evaluated. If only the first (saturated) antibody can bind to the TrkB molecule in both directions, it can be concluded that the test antibody and the reference antibody compete for binding to TrkB (e.g., see the assay format described in Example 4, where the TrkB protein is captured on a sensor tip, and the TrkB-coated sensor tip is treated sequentially with the reference antibody [mAb-1] and the test anti-TrkB antibody [mAb-2] in two binding sequences). As will be understood by those skilled in the art, the antibody competing for binding to the reference antibody may not necessarily bind to the same epitope as the reference antibody, but may spatially block the binding of the reference antibody by binding to overlapping or adjacent epitopes.

[0188] Preparation of human antibodies

[0189] The anti-TrkB antibody of the present invention may be a fully human but not naturally occurring antibody. Methods for producing monoclonal antibodies, including fully human monoclonal antibodies, are known in the art. Any such known method may be used in the present invention to prepare human antibodies that specifically bind to human TrkB.

[0190] use Technology (see, for example, US 6,596,541, Regeneron Pharmaceuticals, (or any other known method for producing monoclonal antibodies) can first isolate a high-affinity chimeric antibody against the allergen, which has a human variable region and a mouse constant region. The technology involves the generation of transgenic mice with a genome containing human heavy and light chain variable regions operatively linked to endogenous mouse constant region loci, thereby enabling the mice to produce antibodies containing both human variable and mouse constant regions in response to antigen stimulation. DNA encoding the antibody heavy and light chain variable regions is isolated and efficiently linked to DNA encoding the human heavy and light chain constant regions. The DNA is then expressed in cells capable of expressing fully human antibodies.

[0191] generally, Mice are challenged with a target antigen, and lymphocytes (e.g., B cells) are recovered from mice expressing antibodies. Lymphocytes can be fused with myeloma cell lines to prepare immortalized hybridoma cell lines, and these hybridoma cell lines can be screened and selected to identify hybridoma cell lines that produce antibodies specific to the target antigen. DNA encoding the variable regions of the heavy and light chains can be isolated and ligated to the desired isotype constant regions of the heavy and light chains. Such antibody proteins can be produced in cells such as CHO cells. Alternatively, DNA encoding antigen-specific chimeric antibodies or variable domains of the light and heavy chains can be directly isolated from antigen-specific lymphocytes.

[0192] As described in the experimental section below, the isolated high-affinity chimeric antibodies containing human variable regions and mouse constant regions are characterized and selected for desired features, including affinity, selectivity, epitopes, etc. The mouse constant regions are then replaced with the desired human constant regions to generate the fully human antibodies of this invention, such as wild-type or modified IgG1 or IgG4. While the selected constant regions can vary depending on the specific application, high-affinity antigen binding and target-specific features are present in the variable regions.

[0193] In some embodiments, it may be desirable to test anti-human TrkB antibodies in mice or rats that have been engineered to express the human TrkB receptor. These mice or rats may be advantageous where the anti-TrkB antibody may bind only to human TrkB without cross-reacting with mouse or rat TrkB. Some embodiments of the invention have been carried out using genetically modified mice and rats to express human trkB. Such humanized TrkB mice and rats can be generated using any method known to those skilled in the art.

[0194] Typically, when measured by binding to antigens immobilized on a solid phase or in a solution phase, the antibodies of the present invention exhibit very high affinity, typically around 10. -12 To about 10 -9 M of K D .

[0195] bioequivalent

[0196] The anti-TrkB antibodies and antibody fragments of the present invention cover proteins having an amino acid sequence that differs from the amino acid sequence of the described antibody but retains the ability to bind human TrkB. When compared to the parental sequence, such variant antibodies and antibody fragments contain the addition, deletion, or substitution of one or more amino acids but exhibit substantially equivalent biological activity to the described antibody. Similarly, the DNA sequences encoding anti-TrkB antibodies of the present invention cover sequences that contain the addition, deletion, or substitution of one or more nucleotides compared to the disclosed sequences but encode anti-TrkB antibodies or antibody fragments substantially bioequivalent to the anti-TrkB antibodies or antibody fragments of the present invention. Examples of such variant amino acid and DNA sequences have been discussed above.

[0197] Two antigen-binding proteins or antibodies are considered bioequivalent if, for example, they are drug equivalents or drug substitutes, and when administered at the same molar dose (single or multiple doses) under similar experimental conditions, there is no significant difference in their absorption rate and extent. If some antibodies are equivalent in extent of absorption but not in rate of absorption, these antibodies can be considered equivalents or drug substitutes and can still be considered bioequivalent because such differences in absorption rate can be intentional and reflected on the label, are not necessary for achieving effective bodily drug concentrations (e.g., with long-term use), and can be considered medically secondary for the specific drug product under investigation.

[0198] In one implementation, two antigen-binding proteins are bioequivalent if they do not differ clinically in terms of safety, purity, and potency.

[0199] In one implementation, the two antigen-binding proteins are bioequivalent if a patient can switch between the reference product and the biological product once or multiple times without an expected increase in the risk of adverse reactions, including clinically significant changes in immunogenicity or reduced effectiveness, compared to continuous treatment without such switching.

[0200] In one implementation, if two antigen-binding proteins act on (one or more) conditions of use through one or more co-action mechanisms, they are bioequivalent to the extent that the mechanisms are known.

[0201] Bioequivalence can be demonstrated through in vivo and in vitro methods. Bioequivalence measurements include, for example, (a) in vivo studies in humans or other mammals, wherein the concentration of the antibody or its metabolites in blood, plasma, serum, or other biological fluids is measured over time; (b) in vitro studies that have been correlated with in vivo bioavailability data and can be used to reasonably predict such in vivo bioavailability data; (c) in vivo studies in humans or other mammals, wherein appropriate acute pharmacological effects of the antibody (or its target) are measured over time; and (d) determinations in clinical trials that establish good controls over the safety, efficacy, or bioavailability or bioequivalence of the antibody.

[0202] Bioequivalent variants of the anti-TrkB antibody of the present invention can be constructed, for example, by various substitutions of residues or sequences, or by deletion of terminal or internal residues or sequences not required for biological activity. For example, cysteine ​​residues not essential for biological activity can be deleted or substituted with other amino acids to prevent the formation of unnecessary or incorrect intramolecular disulfide bonds during renaturation. In other cases, the bioequivalent antibody may comprise an anti-TrkB antibody variant comprising amino acid changes that modify the glycosylation characteristics of the antibody, such as mutations that eliminate or remove glycosylation.

[0203] Species selectivity and species cross-reactivity

[0204] According to certain embodiments, the present invention provides anti-TrkB antibodies that bind to human TrkB but not to TrkB of other species. The present invention also includes anti-TrkB antibodies that bind to human TrkB and one or more TrkBs of non-human species. For example, the anti-TrkB antibodies of the present invention may bind to human TrkB and, depending on the circumstances, may bind to or not bind to one or more of the following TrkBs: mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomolgus monkey, marmoset, macaque, or chimpanzee TrkB. According to certain exemplary embodiments of the present invention, anti-TrkB antibodies are provided that specifically bind to human TrkB but do not bind to mouse or rat TrkB or only weakly bind to them.

[0205] Multispecific antibodies

[0206] The antibodies of the present invention can be monospecific or multispecific (e.g., bispecific). Multispecific antibodies can be specific to different epitopes of a target polypeptide, or can contain antigen-binding domains specific to more than one target polypeptide. See, for example, Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238-244. The anti-TrkB antibody of the present invention can be linked to or co-expressed with another functional molecule, such as another peptide or protein. For example, the antibody or a fragment thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent binding, or other means) to one or more other molecular entities, such as another antibody or antibody fragment, to produce a bispecific or multispecific antibody with a second binding specificity.

[0207] This invention includes a bispecific antibody, wherein one arm of an immunoglobulin binds to human TrkB, while the other arm of the immunoglobulin is specific for a second antigen. The TrkB binding arm may contain any HCVR / LCVR or CDR amino acid sequence as shown in Table 1 herein.

[0208] An exemplary form of bispecific antibody that can be used in this invention involves the use of a first immunoglobulin (Ig) C H 3 structural domains and second Ig C H 3 structural domains, where the first and second IgC H The three domains differ from each other by at least one amino acid, wherein this at least one amino acid difference reduces the binding of the bispecific antibody to protein A compared to a bispecific antibody lacking this amino acid difference. In one embodiment, the first IgC H 3-domain binding protein A, second IgC H Domain 3 contains mutations that reduce or eliminate protein A binding, such as the H95R modification (according to IMGT exon numbering; according to EUR numbering, H435R). Second C H 3 may further include Y96F modifications (according to IMGT; according to EU, Y436F). In the second C HOther modifications that may exist in 3 include: for IgG1 antibodies, D16E, L18M, N44S, K52N, V57M, and V82I (according to IMGT; according to EU, D356E, L358M, N384S, K392N, V397M, and V422I); for IgG2 antibodies, N44S, K52N, and V82I (IMGT; according to EU, N384S, K392N, and V422I); for IgG4 antibodies, Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (according to IMGT; according to EU, Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I). Variations of the above-described bispecific antibody forms are considered within the scope of this invention.

[0209] Other exemplary bispecific forms that may be used in this invention include, but are not limited to, scFv-based or biantibody-based bispecific forms, IgG-scFv fusions, dual variable domain (DVD)-Ig, Quadroma, knots-into-holes, common light chains (e.g., common light chains with knots-into-holes, etc.), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody, IgG1 / IgG2, dual-action Fab (DAF)-IgG and Mab 2 Bispecific forms (for a review of the above forms, see, for example, Klein et al. 2012, mAbs 4:6, 1-11 and the references cited therein). Bispecific antibodies can also be constructed using peptide / nucleic acid conjugations, for example, in which non-natural amino acids with orthogonal chemical reactivity are used to generate site-specific antibody-oligonucleotide conjugations, which then self-assemble into multimeric complexes with defined composition, titer, and geometry. (See, for example, Kazane et al., J.Am.Chem.Soc. [Epub: Dec.4, 2012]).

[0210] Therapeutic preparations and administration

[0211] This invention provides pharmaceutical compositions comprising the anti-TrkB antibody of the present invention or its antigen-binding fragment. The pharmaceutical compositions of the present invention are formulated with suitable carriers, excipients, and other agents that provide improved transfer, delivery, tolerability, etc. Many suitable formulations can be found in all pharmaceutical chemists' known formulations: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, and lipid-containing (cationic or anionic) vesicles (e.g., LIPOFECTIN). TM 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. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) JPharm Sci Technol 52:238-311.

[0212] The antibody dosage administered to a patient can vary depending on the patient's age and size, the target disease, condition, route of administration, etc. Preferred dosages are typically calculated based on body weight or body surface area. In adult patients, it is advantageous to administer the antibody of the invention intravenously in a single dose of about 0.01 to about 20 mg / kg body weight, 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. The frequency and duration of treatment can be adjusted according to the severity of the condition. Effective doses and schedules for administering anti-TrkB antibodies can be determined empirically; for example, patient progression can be monitored through regular assessments, and the dose adjusted accordingly. Furthermore, interspecies dose scaling can be performed using methods well known in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

[0213] Various delivery systems are known and can be used to administer the pharmaceutical compositions of the present invention, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, and receptor-mediated endocytosis (see, for example, Wu et al. 1987, J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intravitreal, intraocular, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions can be administered via any convenient route, such as by infusion or high-dose injection, absorption through epithelial or mucosal skin linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered co-administered with other bioactive agents. Administration can be systemic or local.

[0214] The pharmaceutical compositions of the present invention can be delivered subcutaneously or intravenously using standard needles and syringes. Alternatively, regarding subcutaneous delivery, pen delivery devices can be readily used to deliver the pharmaceutical compositions of the present invention. Such pen delivery devices can be reusable or disposable. Reusable pen delivery devices typically utilize 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 discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In disposable pen delivery devices, there is no replaceable cartridge. Instead, the disposable pen delivery device is pre-filled with the pharmaceutical composition, which is contained in a reservoir within the device. Once the pharmaceutical composition in the reservoir is emptied, the entire device is discarded.

[0215] Many reusable pens and auto-injector delivery devices can be used for subcutaneous delivery of the pharmaceutical compositions of the present invention. Examples include, but are not limited to, AUTOPEN. TM (Owen Mumford,Inc.,Woodstock,UK),DISETRONIC TM Pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25 TM Pen, HUMALOG TM Pen, HUMALIN 70 / 30 TM Pen(Eli Lilly and Co.,Indianapolis,IN),NOVOPEN TM I,II and III(Novo Nordisk,Copenhagen,Denmark),NOVOPEN JUNIOR TM (Novo Nordisk,Copenhagen,Denmark),BD TMPen(Becton Dickinson,Franklin Lakes,NJ),OPTIPEN TM OPTIPEN PRO TM OPTIPEN STARLET TM , and OPTICLIK TM (sanofi-aventis, Frankfurt, Germany), etc. Examples of disposable pen delivery devices that can be used for subcutaneous delivery of the pharmaceutical compositions of the present invention include, but are not limited to: SOLOSTAR. TM Pen (sanofi-aventis), FLEXPEN TM (Novo Nordisk), and KWIKPEN TM (Eli Lilly), SURECLICK TM Automatic injectors (Amgen, Thousand Oaks, CA), PENLET TM (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA TM Pens (Abbott Labs, Abbott Park IL), etc.

[0216] In some cases, the drug composition can be delivered using a controlled-release system. In one embodiment, a pump can be used (see Langer, ibid.; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, a polymeric material can be used; see, Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, the controlled-release system can be placed near the target of the composition, thereby requiring only a portion of the systemic dose (see, for example, Goodson, 1984, Medical Applications of Controlled Release, ibid., vol. 2, pp. 115-138). Other controlled-release systems are discussed in the review in Langer, 1990, Science 249:1527-1533.

[0217] Injectable formulations may include dosage forms for intravenous, intravitreal, intraocular, subcutaneous, intradermal, and intramuscular injection, infusion, etc. These injectable formulations can be prepared by known methods. For example, they can be prepared by dissolving, suspending, or emulsifying the aforementioned antibodies or their salts in a sterile aqueous or oily medium conventionally used for injection. Aqueous media for injection include, for example, physiological saline, isotonic solutions containing glucose and other adjuvants, which can be used in combination with suitable solubilizers, such as alcohols (e.g., ethanol), polyols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants [e.g., polysorbate 80, HCO-50 (a polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]. Oily media can be used, for example, sesame oil, soybean oil, etc., which can be used in combination with solubilizers such as benzyl benzoate, benzyl alcohol, etc. The resulting injections are preferably packaged in suitable ampoules.

[0218] Advantageously, the above-described pharmaceutical compositions for oral or parenteral use are formulated into unit-dose dosage forms suitable for delivering a dose of the active ingredient. Such unit-dose dosage forms include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the antibody contained is typically about 5 to about 500 mg per unit-dose dosage form; particularly in the form of injection, the antibody content is preferably about 5 to about 100 mg, and about 10 to about 250 mg for other dosage forms.

[0219] Therapeutic applications of antibodies

[0220] This invention includes a method comprising administering to a subject in need a therapeutic composition comprising an anti-TrkB antibody (e.g., an anti-TrkB antibody comprising any HCVR / LCVR or CDR sequence as listed in Table 1 herein). The therapeutic composition may comprise any one or more anti-TrkB antibodies disclosed herein or antigen-binding fragments thereof, and a pharmaceutically acceptable carrier or diluent.

[0221] The antibodies of the present invention are particularly useful for the treatment, prevention, and / or improvement of any disease or condition related to or mediated by TrkB expression or activity. The TrkB agonist antibodies of the present invention can be used to improve neurological function and can be used to treat or prevent any disease or condition partially characterized by cellular degeneration, particularly diseases or conditions characterized by nerve cell damage or degeneration, such as acute nervous system injury or chronic neurodegenerative diseases.

[0222] The present invention includes methods for treating or preventing eye diseases or conditions, wherein an anti-TrkB antibody or an antigen-binding fragment thereof, as disclosed elsewhere herein, is administered to a patient in need of such treatment.

[0223] In one embodiment, the anti-TrkB antibody of the present invention can provide a method for preventing damage or death of retinal neurons. In one embodiment, the anti-TrkB antibody of the present invention can provide a method for treating a pathological disease in which retinal degeneration occurs. In one embodiment, the anti-TrkB antibody of the present invention can provide a method for treating the eye before or after ophthalmic surgery, exposure to light, or other environmental trauma to prevent retinal cell degeneration. In one embodiment, the anti-TrkB antibody of the present invention can provide a method for preventing damage and degeneration of photoreceptors in the eye. In one embodiment, the anti-TrkB antibody of the present invention can provide a method for protecting retinal neurons without inducing side effects. In one embodiment, the anti-TrkB antibody of the present invention can provide a method for restoring or regenerating damaged photoreceptors.

[0224] In some embodiments, the eye diseases that can be treated by using one or more of the anti-TrkB antibodies of the present invention may be selected from: glaucoma, diabetic retinopathy, age-related macular degeneration or other 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.

[0225] Other pathological conditions that can be treated with one or more anti-TrkB antibodies of the present invention include retinal detachment, photoretinopathy, surgically induced retinopathy (mechanical or photoinduced), toxic retinopathy, retinopathy of prematurity, viral retinopathy such as CMV or HIV retinopathy associated with AIDS; uveitis; ischemic retinopathy caused by venous or arterial occlusion or other vascular diseases, retinopathy caused by ocular trauma or penetrating injury, peripheral vitreoretinopathy, or hereditary retinal degeneration.

[0226] In one embodiment, the anti-TrkB antibody of the present invention can be formulated for intraocular or intravitreal delivery.

[0227] The present invention also provides methods for treating other central or peripheral nervous system diseases or conditions, such as stroke or traumatic brain injury. Furthermore, since the antibodies of the present invention promote neuronal survival and act as neuroprotective agents, any one or more of these agonist antibodies may prove beneficial in treating patients with nervous system diseases or conditions in which neuronal survival is crucial for the recovery or repair of cellular damage caused by or resulting from nervous system impairment, and wherein the diseases are those that have a significant impact on the nervous system (including neurodegenerative diseases).

[0228] In the treatment methods described herein, anti-TrkB antibodies may be administered as a monotherapy (i.e., as the sole treatment agent) or in combination with one or more other treatment agents.

[0229] Combination therapies and formulations

[0230] The present invention includes compositions and therapeutic formulations comprising any of the anti-TrkB antibodies described herein combined with one or more other therapeutically active ingredients, and treatment methods comprising administering such combinations to subjects in need of them.

[0231] The anti-TrkB antibody of the present invention can be formulated and / or administered in combination with one or more other therapeutically active ingredients selected from: drugs that help lower intraocular pressure (IOP-lowering drugs), neurotrophic proteins, and antagonists of vascular endothelial growth factor (VEGF), such as VEGF traps, such as aflibercept. Other drugs that can be combined with the TrkB antibody of the present invention include, but are not limited to, prostaglandin analogs (e.g., zioptanol). TM , ), β-receptor blockers (e.g. ); α-2 adrenergic agonists (e.g., apraclonidine), carbonic anhydrase inhibitors (e.g. ), cholinergic drugs (e.g.) (gel) or combination therapy (β-blocker plus carbonic anhydrase inhibitor, such as COMBIGAN) TM , ).

[0232] The anti-TrkB antibody of the present invention can also be administered in combination with and / or co-formulated with antiviral agents, antibiotics, analgesics, antioxidants, COX inhibitors, and / or NSAIDs. The anti-TrkB antibody can also be used in combination with other types of therapies, including stem cell therapy, glaucoma filtration surgery, laser surgery, or gene therapy.

[0233] Other therapeutically active ingredients (one or more), such as any of the agents listed above or their derivatives, may be administered before, simultaneously with, or shortly after the administration of the anti-TrkB antibody of the present invention; (for the purposes of this disclosure, such administration is considered to be a “combined” administration of the anti-TrkB antibody and other therapeutically active ingredients). The present invention includes pharmaceutical compositions wherein the anti-TrkB antibody of the present invention is formulated with one or more other therapeutically active ingredients as described elsewhere herein.

[0234] Application plan

[0235] According to certain embodiments of the invention, multiple doses of anti-TrkB antibody (or a pharmaceutical composition comprising an anti-TrkB antibody and any other therapeutically active agent mentioned herein) may be administered to a subject over a predetermined time period. A method according to this aspect of the invention comprises sequentially administering multiple doses of the anti-TrkB antibody of the invention to a subject. As used herein, “sequentially administering” means that each dose of the anti-TrkB antibody is administered to the subject at different time points, for example, on different days separated by predetermined intervals (e.g., hours, days, weeks, or months). The invention includes a method comprising sequentially administering to a patient a single initial dose of anti-TrkB antibody, followed by one or more second doses of anti-TrkB antibody, and optionally followed by one or more third doses of anti-TrkB antibody.

[0236] The terms "initial dose," "second dose," and "third dose" refer to the order in which the anti-TrkB antibody of the present invention is administered. Thus, the "initial dose" is the dose given at the start of the treatment regimen (also known as the "baseline dose"); the "second dose" is the dose given after the initial dose; and the "third dose" is the dose given after the second dose. The initial, second, and third doses may all contain the same amount of anti-TrkB antibody, but they may typically differ from each other in terms of dosing frequency. However, in some embodiments, the amount of anti-TrkB antibody contained in the initial, second, and / or third doses differs from each other during treatment (e.g., adjusted upwards or downwards as appropriate). In some embodiments, two or more doses (e.g., 2, 3, 4, or 5) are administered at the start of the treatment regimen as a "loading dose," followed by subsequent doses (e.g., "maintenance doses") at a lower frequency.

[0237] In certain exemplary embodiments of the invention, each second and / or third dose is administered 1-26 weeks (e.g., 1, 11 / 2, 2, 21 / 2, 3, 31 / 2, 4, 41 / 2, 5, 51 / 2, 6, 61 / 2, 7, 71 / 2, 8, 81 / 2, 9, 91 / 2, 10, 101 / 2, 11, 111 / 2, 12, 121 / 2, 13, 131 / 2, 14, 141 / 2, 15, 151 / 2, 16, 161 / 2, 17, 171 / 2, 18, 181 / 2, 19, 191 / 2, 20, 201 / 2, 21, 211 / 2, 22, 221 / 2, 23, 231 / 2, 24, 241 / 2, 25, 251 / 2, 26, 261 / 2, or more weeks) immediately following the previous dose. As used herein, the phrase “immediately preceding dose” refers to the dose of anti-TrkB antibody administered to a patient in a sequence of multiple dosings, wherein the dose is administered to the patient in the sequence prior to the next dose, with no intermediate doses administered in between.

[0238] Methods according to this aspect of the invention may include administering any number of second and / or third doses of anti-TrkB antibody to a patient. For example, in some embodiments, only a single second dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8 or more) second doses are administered to the patient. Similarly, in some embodiments, only one third dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8 or more) third doses are administered to the patient. The administration regimen may be performed indefinitely for the lifetime of a particular subject, or until the treatment is no longer therapeutically necessary or beneficial.

[0239] In embodiments involving multiple second doses, each second dose may be administered at the same frequency as the other second doses. For example, each second dose may be administered to the patient 1-2 weeks or 1-2 months after the immediate preceding dose. Similarly, in embodiments involving multiple third doses, each third dose may be administered at the same frequency as the other third doses. For example, each third dose may be administered to the patient 2 to 12 weeks after the immediate preceding dose. In some embodiments of the invention, the frequency of administration of the second and / or third doses to the patient may vary throughout the treatment regimen. During treatment, the administration frequency may also be adjusted by the physician based on the individual patient's needs following a clinical examination.

[0240] The present invention includes an administration regimen in which two to six loading doses are administered to a patient at a first frequency (e.g., once a week, once every two weeks, once every three weeks, once a month, once every two months, etc.), followed by two or more maintenance doses administered to the patient at a lower frequency. For example, according to this aspect of the invention, if the loading dose is administered once a month, the maintenance dose can be administered to the patient once every six weeks, once every two months, once every three months, etc.

[0241] Diagnostic applications of antibodies

[0242] The anti-TrkB antibody of the present invention can also be used to detect and / or measure TrkB or TrkB-expressing cells in a sample, for example, for diagnostic purposes. For example, an anti-TrkB antibody or a fragment thereof can be used to diagnose a condition or disease characterized by abnormal expression of TrkB (e.g., overexpression, underexpression, lack of expression, etc.). An exemplary diagnostic assay for TrkB may include, for example, 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 marker or reporter molecule. Alternatively, an unlabeled anti-TrkB antibody may be combined with a second antibody that is itself detectably labeled for diagnostic applications. The detectable marker or reporter molecule may be a radioactive isotope, for example… 3 H, 14C, 32 P, 35 S, or 125 I; a fluorescent or chemiluminescent component, such as fluorescein isothiocyanate or rhodamine; or an enzyme, such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, or luciferase. Specific exemplary assays that can be used to detect or measure TrkB in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

[0243] Samples that can be used for the TrkB diagnostic assay according to the present invention include any tissue or body fluid sample available from a patient under normal or pathological conditions, containing a detectable amount of TrkB protein or fragments thereof. Typically, TrkB levels are measured in specific samples obtained from healthy patients (e.g., patients without a disease or condition associated with abnormal TrkB levels or activity) to initially establish a baseline or standard TrkB level. This baseline TrkB level can then be compared with TrkB levels measured in samples obtained from individuals suspected of having a TrkB-related disease or condition. Example

[0244] The following examples are provided to provide those skilled in the art with a complete disclosure and description of how to implement and use the methods and compositions of the present invention, and these examples are not intended to limit the scope of the invention. Efforts have been made to ensure the accuracy of the figures used (e.g., quantities, temperatures, etc.), but certain experimental errors and deviations should be taken into account. Unless otherwise stated, parts are parts by weight, molecular weight is the average molecular weight, temperature is degrees Celsius, room temperature is approximately 25°C, and pressure is atmospheric pressure or close to atmospheric pressure.

[0245] Example 1: Human antibodies producing TrkB

[0246] Human antibodies against TrkB were generated in mice containing DNA encoding the variable regions of the human immunoglobulin heavy and κ light chains. In one embodiment, the human antibody is... Produced in mice. In one implementation, (VI) Mice were immunized with human TrkB(ecto)mFc (SEQ ID NO:77). In one implementation, (VI) Mice were immunized with mouse TrkB(ecto)mFc (SEQ ID NO:80). Antibody immune responses were monitored using TrkB-specific immunoassays. For example, the titer of specific antibodies against purified full-length TrkB in serum was measured. Antibody-producing clones were isolated using B-cell sorting (BST) and hybridoma methods. For example, after obtaining the desired immune response, spleen cells were harvested and fused with mouse myeloma cells to maintain their viability and form hybridoma cell lines. Hybridoma cell lines were screened and selected to identify cell lines producing TrkB-specific antibodies. Certain anti-mouse TrkB antibodies were generated in this manner and named M2aM14173N, M2aM14178N, and M2aM14179N.

[0247] As described in U.S. Patent 7,582,298, anti-TrkB antibodies were also isolated directly from antigen-positive mouse B cells without fusing with myeloma cells, the entire contents of which are incorporated herein by reference. Using this method, several fully human anti-TrkB antibodies (i.e., antibodies having human variable and human constant domains) were obtained; exemplary antibodies produced in this manner were named H4H9780P, H4H9814P, and H4H9816P2.

[0248] The biological characteristics of the exemplary antibodies generated according to the method of this embodiment are described in detail in the embodiments set forth below.

[0249] Example 2: Amino acid and nucleotide sequences of the variable regions of the heavy and light chains

[0250] Table 1a lists the amino acid sequence identifiers of the variable regions and CDRs of the heavy and light chains of the anti-TrkB antibodies selected in this invention. Table 1b lists the amino acid sequence identifiers of the full-length heavy and light chains of the anti-TrkB antibodies selected in this invention. Table 2 lists the corresponding nucleic acid sequence identifiers of the anti-TrkB antibodies selected in this invention.

[0251] Table 1a: Amino acid sequence identifiers

[0252]

[0253] Table 1b

[0254] Ab name Full-length heavy chain Full-length light chain H4H9780P 99 100 H4H9814P 101 102 H4H9816P2 103 104

[0255] Table 2: Nucleic Acid Sequence Identifiers

[0256]

[0257] Antibodies are typically referred to in this document using the following nomenclature: an Fc prefix (e.g., "H4H", "H2M", etc.), followed by a numerical identifier (e.g., "9780", "9816", etc., as shown in Tables 1 or 2), and then a suffix "P", "P2", or "N". The H4H prefix in the antibody name indicates a specific Fc region isotype of the antibody. Therefore, according to this nomenclature, antibodies may be referred to in this document as, for example, "H4H9780P" (indicating the human IgG4 Fc region) and, for example, M2aM14179N (indicating the mouse IgG2a Fc region). Variable regions are fully human and are indicated by the first "H" in the antibody name. The "M" prefix indicates the mouse variable region. As will be understood by those skilled in the art, antibodies having a specific Fc isotype can be converted into antibodies having a different Fc isotype (e.g., antibodies having mouse IgG1 Fc can be converted into antibodies having human IgG4, etc.), but in any case, the variable domains (including CDRs) (indicated by the numerical identifiers shown in Table 1 or 2) will remain unchanged, and the binding properties with the antigen are expected to be the same or substantially similar, regardless of the nature of the Fc domains.

[0258] Example 3. Biacore binding kinetics of anti-TrkB monoclonal antibody with different TrkB reagents were measured at 25°C and 37°C.

[0259] The equilibrium dissociation constant (K) of TrkB binding to purified anti-TrkB monoclonal antibody. DThe values ​​were determined using a Biacore 4000 instrument with a real-time surface plasmon resonance biosensor. 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 running buffer). The Biacore sensor surface was first derivatized to capture anti-TrkB monoclonal antibodies by amine-conjugated goat anti-human Fcγ specific polyclonal antibody (Jackson ImmunoResearch Laboratories, #109-006-098) or rabbit anti-mouse Fc polyclonal antibody (GE Healthcare #BR-1008-38). The following TrkB reagents were used in binding studies: human TrkB extracellular domain expressed with a C-terminal myc-myc-hexahistidine tag (hTRKB.mmH; SEQ ID NO: 76; accession number NP_001018074.1), mouse TrkB extracellular domain expressed with a C-terminal myc-myc-hexahistidine tag (mTRKB.mmH; SEQ ID NO: 79; accession number NP_001020245), rat TrkB extracellular domain expressed with a C-terminal myc-myc-hexahistidine tag (rTRKB.mmH; SEQ ID NO: 84; accession number NP_036863.1), and human TrkB extracellular domain expressed with a C-terminal mouse IgG2aFc tag (hTrkB-mFc; SEQ ID NO: 77; accession number NP_001018074.1). First, different concentrations of TrkB reagent (100 nM–1.23 nM; 3-fold serial dilutions) were prepared in HBS-ET running buffer. Then, the reagent was injected at a flow rate of 30 μL / min onto the surface of anti-human Fc-captured anti-TrkB monoclonal antibody for 4 minutes. The dissociation of the TrkB-bound monoclonal antibody was monitored in HBS-ET running buffer for 10 minutes. Using Scrubber 2.0c curve fitting software, the real-time binding sensor map was fitted to a 1:1 binding model with mass transport limitation to determine the kinetic binding (kk). a ) and dissociation (k d Rate constant. Combined with the dissociation equilibrium constant (K) D The dissociation half-life (t1 / 2) is calculated from the kinetic rate constant as follows:

[0260] and

[0261] Tables 3 to 10 show the binding kinetic parameters of hTrkB.mmH, mTrkB.mmH, rTrkB.mmH, or hTrkB-mFc to different anti-TrkB monoclonal antibodies of the present invention at 25°C and 37°C.

[0262] Summary of results:

[0263] As shown in Table 3, at 25°C, the KD values ​​of anti-TrkB monoclonal antibody binding to hTrkB.mmH ranged from 545 pM to 41.3 nM. As shown in Table 4, at 37°C, the KD values ​​of anti-TrkB monoclonal antibody binding to hTrkB.mmH ranged from 2.28 nM to 135 nM.

[0264] As shown in Table 5, at 25°C, the KD values ​​of anti-TrkB monoclonal antibody binding to hTrkB-mFc ranged from 31.1 pM to 4.48 nM. As shown in Table 6, at 37°C, the KD values ​​of anti-TrkB monoclonal antibody binding to hTrkB-mFc ranged from 73.3 pM to 3.46 nM.

[0265] As shown in Table 7, at 25°C, the comparative anti-TrkB monoclonal antibody (referred to herein as H1M8037C) (see US2010 / 0196390, antibody named C2; for the amino acid sequences of the heavy and light chains of the comparative antibody, see SEQ ID NO: 97 and 98, respectively) binds to mTrkB.mmH with a KD value of 40.8 nM. As shown in Table 7, the anti-TrkB antibody of the present invention does not bind to mTrkB.mmH at 25°C. As shown in Table 8, at 37°C, the comparative anti-TrkB monoclonal antibody binds to mTrkB.mmH with a KD value of 94.1 nM. As shown in Table 8, at 37°C, the anti-TrkB antibody of the present invention does not bind to mTrkB.mmH.

[0266] As shown in Table 9, at 25°C, the comparative anti-TrkB monoclonal antibody bound to rTrkB.mmH had a KD value of 31.8 nM. As shown in Table 9, at 25°C, the anti-TrkB antibody of the present invention did not bind to rTrkB.mmH. As shown in Table 10, at 37°C, the comparative anti-TrkB monoclonal antibody bound to rTrkB.mmH had a KD value of 87.5 nM. As shown in Table 10, at 37°C, the anti-TrkB antibody of the present invention did not bind to rTrkB.mmH.

[0267] Table 3: Binding kinetic parameters of hTrkB.mmH and TrkB monoclonal antibody at 25℃.

[0268]

[0269] *Indicates that mAb uses an anti-mFc immobilized surface for capture.

[0270] Table 4: Binding kinetic parameters of hTrkB.mmH and TrkB monoclonal antibody at 37℃.

[0271]

[0272] *Indicates that mAb uses an anti-mFc immobilized surface for capture.

[0273] Table 5: Binding kinetic parameters of hTrkB-mFc and TrkB monoclonal antibody at 25℃

[0274]

[0275] *Indicates that mAb uses an anti-mFc immobilized surface for capture.

[0276] Table 6: Binding kinetic parameters of hTrkB.mFc to TrkB monoclonal antibody at 37℃.

[0277]

[0278] *Indicates that mAb uses an anti-mFc immobilized surface for capture.

[0279] Table 7: Binding kinetic parameters of mTrkB.mmH binding to TrkB monoclonal antibody at 25℃.

[0280]

[0281] *Indicates that mAb uses an anti-mFc immobilized surface for capture.

[0282] Table 8: Binding kinetic parameters of mTrkB.mmH binding to TrkB monoclonal antibody at 37℃.

[0283]

[0284] *Indicates that mAb uses an anti-mFc immobilized surface for capture.

[0285] Table 9: Binding kinetic parameters of rTrkB.mmH to TrkB monoclonal antibody at 25℃.

[0286]

[0287] *Indicates that mAb uses an anti-mFc immobilized surface for capture.

[0288] Table 10: Binding kinetic parameters of rTrkB.mmH to TrkB monoclonal antibody at 37℃.

[0289]

[0290] *Indicates that mAb uses an anti-mFc immobilized surface for capture.

[0291] Example 4. Biacore binding kinetics of the substitute anti-TrkB monoclonal antibody with different TrkB reagents were measured at 25°C.

[0292] The equilibrium dissociation constant (K) of TrkB binding to purified anti-TrkB monoclonal antibody. D The values ​​were determined using a Biacore T200 instrument with a real-time surface plasmon resonance biosensor. 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 running buffer). The Biacore sensor surface was first derivatized with rabbit anti-mouse Fc polyclonal antibody (GE Healthcare #BR-1008-38) via amine conjugation to capture anti-TrkB monoclonal antibody. The following TrkB reagents were used in binding studies: human TrkB extracellular domain with C-terminal myc-myc-hexahistidine tag expression (hTrkB.mmH; SEQ ID NO:76; NP_001018074.1), mouse TrkB extracellular domain with C-terminal myc-myc-hexahistidine tag expression (mTRKB.mmH; SEQ ID NO:79; NP_001020245), and rat TrkB extracellular domain with C-terminal myc-myc-hexahistidine tag expression (rTRKB.mmH; SEQ ID NO:84; XP_002721319.1). First, different concentrations of TrkB reagent (90 nM–3.33 nM; 3-fold serial dilutions) were prepared in HBS-ET running buffer. Then, the reagent was injected at a flow rate of 50 μL / min onto the surface of anti-TrkB monoclonal antibody captured by mouse Fc for 4 minutes. The dissociation of the TrkB-bound monoclonal antibody was monitored in HBS-ET running buffer for 10 minutes. Using Scrubber 2.0c curve fitting software, the real-time binding sensor map was fitted to a 1:1 binding model with mass transport limitation to determine the kinetic binding (kk). a ) and dissociation (k d Rate constant. Combined with the dissociation equilibrium constant (K) D The dissociation half-life (t1 / 2) is calculated from the kinetic rate constant as follows:

[0293] and

[0294] Tables 11 to 13 show the binding kinetic parameters of hTrkB.mmH, mTrkB.mmH, or rTrkB.mmH to different anti-TrkB monoclonal antibodies of the present invention at 25°C.

[0295] result:

[0296] As shown in Table 11, at 25°C, the anti-TrkB monoclonal antibody, an alternative of the present invention, did not show binding to hTrkB.mmH.

[0297] As shown in Table 12, at 25°C, the KD values ​​of the anti-TrkB monoclonal antibody, an alternative of the present invention, bound to mTrkB.mmH ranged from 2.39 nM to 32.4 nM.

[0298] As shown in Table 13, at 25°C, the KD values ​​of the anti-TrkB monoclonal antibody, an alternative of the present invention, bound to rTrkB.mmH ranged from 2.56 nM to 26.9 nM.

[0299] Table 11: Binding kinetic parameters of hTrkB.mmH and TrkB monoclonal antibody at 25℃.

[0300]

[0301] Table 12: Binding kinetic parameters of mTrkB.mmH binding to TrkB monoclonal antibody at 25℃.

[0302]

[0303] Table 13: Binding kinetic parameters of rTrkB.mmH to TrkB monoclonal antibody at 25℃.

[0304]

[0305] Example 5. Bioassays using HEK293 / SRE-luc / hTrkB and HEK293 / SRE-luc / mTrkB(Ecto)-hTrkB(TM-Cyto) cells.

[0306] A bioassay was developed to detect TrkB activation using a luciferase reporter gene and its ligand brain-derived neurotrophic factor (BDNF, R&D Systems) under the control of a serum response element (SRE). The HEK293 cell line was generated, stably expressing the luciferase reporter gene (SRE-luciferase, SRE-luc, SA Bioscience, #CLS-010L) and human TrkB (hTrkB, amino acids 32-429 of NP_001018074.1 or Uniprot Q16620-1) or the extracellular domain of mouse TrkB fused with the transmembrane and cytoplasmic domains of human TrkB (mTrkB, amino acids 32-429 of NP_001020245.1 or Uniprot P15209-1, fused with hTrkB, amino acids 431-822). 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 μg / mL puromycin, and 500 μg / mL G418.

[0307] For bioassays, cells were seeded at 20,000 cells / well into 96-well assay plates supplemented with Opti-MEM containing 0.1% FBS, penicillin / streptomycin, and L-glutamine. TM Cells were incubated overnight at 37°C in 5% CO2. The next morning, human BDNF or antibody was serially diluted from 100 nM to 0.002 nM (plus a sample containing only buffer but no ligand) and added to cells to determine TrkB signaling activation. The serially diluted antibody was also tested with 100 pM BDNF (R&D Systems, 248-BD / CF). Cells were then incubated at 37°C for 5.5 h in the presence of 5% CO2. Luciferase activity was measured after the addition of OneGlo reagent (Promega) using a Victor X instrument (Perkin Elmer). Results were analyzed using nonlinear regression (4-parameter logistics) with Prism 5 software (GraphPad) to obtain EC5 values. 50 and IC 50 Value. The maximum activation of the antibody is calculated as follows:

[0308]

[0309] Results summary and conclusions:

[0310] As shown in Table 14, the three anti-TrkB antibodies of this invention, H4H9816P2, H4H9814P, and H4H9780P, demonstrated activation of human TrkB signaling in HEK293 / SRE-luc / hTrkB cells in the absence of BDNF, EC 50 The concentration was 35–82 pM, with a maximum activation range of 88–92%. The three antibodies of the present invention were also tested in the presence of 100 pM BDNF. Although the antibodies of the present invention showed 56% activation in the presence of an irrelevant control mAb (control mAb2), they showed further activation, EC50%. 50 The concentration was 45-76 pM, with a maximum activation range of 77-80%. In the absence of BDNF or in the presence of 100 pM BDNF, none of the three anti-TrkB antibodies of this invention showed activation of mouse TrkB signaling in HEK293 / SRE-luc / mTrkB cells. In the absence of BDNF, control mAb 1 (anti-TrkB comparative antibody H1M8037C) showed activation of human TrkB signaling, with 76 pM EC50. 50 78% maximum activation, and showed activation of mouse TrkB with 43 pM EC. 50 85% maximum activation. In the presence of 100 pM BDNF, compared with mAb 1 activating human TrkB signaling, EC 50 The value was 110 pM, with a maximum activation of 79%, while for mouse TrkB, EC... 50 The concentration was 42 pM, with a maximum activation of 69%, which was greater than the activation induced by control mAb 2 in the presence of 100 pM BDNF. In the absence or presence of 100 pM BDNF, control mAb 2 (an unrelated human IgG4 antibody) showed no activation.

[0311] As shown in Table 15, the three anti-TrkB antibodies of this invention, M2aM14173N, M2aM14178N, and M2aM14179N, activated mouse TrkB signaling in HEK293 / SRE-luc / mTrkB cells in the absence of BDNF, and EC 50 The concentrations were 34–190 pM, with maximum activation ranging from 76–94%. The three antibodies of this invention were also tested in the presence of 100 pM BDNF. In the presence of an irrelevant isotype control mAb (control mAb4), 100 pM BDNF showed 60% activation, while the antibodies of this invention showed further activation, EC50%. 50The concentration was 17–100 pM, with a maximum activation of 67–75%. In the absence of BDNF or in the presence of 100 pM BDNF, none of the three anti-TrkB antibodies of this invention showed activation of human TrkB signaling in HEK293 / SRE-luc / hTrkB cells. In the absence of BDNF, control mAb 1 showed activation of human TrkB signaling, EC1... 50 The value was 57 pM, with a maximum activation of 80%; while for mouse TrkB, EC... 50 The concentration was 43 pM, with a maximum activation of 85%. In the presence of 100 pM BDNF, compared to mAb 1 activating human TrkB signaling, EC... 50 The value was 110 pM, with a maximum activation of 79%, while for mouse TrkB, EC... 50 The concentration was 42 pM, with a maximum activation of 69%, which was greater than the activation of control mAb 4 in the presence of 100 pM BDNF. Neither control mAb 3 nor control mAb 4 (an unrelated mouse IgG2a isotype control antibody) showed any activation in the absence or presence of 100 pM BDNF.

[0312] Summary of table data:

[0313] Table 14: Activation of HEK293 / SRE-Luc / hTrkB and HEK293 / SRE-Luc / mTrkB cells induced by anti-TrkB antibody

[0314]

[0315] Table 15: Activation of HEK293 / SRE-Luc / hTrkB and HEK293 / SRE-Luc / mTrkB cells induced by anti-TrkB antibody (alternative).

[0316]

[0317] Example 6. In TrkB hu / hu Following stereotactic injection into mice, the effects of the TrkB agonist antibody H4H9816P2 and the IgG4 isotype control REGN1945 on TrkB phosphorylation in the brain were compared in vivo.

[0318] To determine the effect of the TrkB agonist antibody H4H9816P2 of the present invention on TrkB activation kinetics, homozygous mice with human TrkB receptor replaced by mouse TrkB receptor (referred to as TrkB mice) were used. hu / hu In mice, the time-course study of TrkB phosphorylation following direct hippocampal injection was investigated. hu / huMice (N=48) received bilateral stereotactic injections into the hippocampus of either 2 μL of solvent (PBS), REGN1945 (used here as an IgG4 isotype control antibody) (final concentration 27.5 mg / mL), or the TrkB agonist antibody H4H9816P2 (final concentration 27.5 mg / mL), at -2 mm posterior and +1.5 mm lateral to the anterior fontanelle. To minimize tissue damage, the injection and needle withdrawal were performed gradually over 5 minutes. TrkB agonists were then euthanized by CO2 at approximately 30 minutes, 1 hour, 4 hours, or 18 hours post-injection. hu / hu Mice. Blood was collected via cardiac puncture during the final blood draw, followed by transcardiac perfusion of mice with cold heparinized saline. The brain was carefully removed from the skull, and the area around the injection site was dissected within 2 mm. 3 Tissue sections were collected in Eppendorf tubes and stored on ice. Brain sections were then 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 homogenate was then used for further processing, aliquoted, and stored at -80°C.

[0319] Immunoprecipitation and Western blotting were performed to assess TrkB phosphorylation in brain tissue. Anti-human TrkB antibody H4H10108N, which does not compete with H4H9816P2 for binding, was conjugated to NHS-activated agarose 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 homogenized brain lysate was thawed on ice and diluted to a concentration of 1 mg / mL (brain weight: buffer volume) in a buffer consisting of 1% NP-40, 0.1% Tween-20, protease, and phosphatase inhibitors from TBST. Protein concentrations in the homogenized brain lysate were quantified using a standard BCA assay according to the manufacturer's instructions (Thermo Scientific Pierce, Cat#23225). For every 100 μg of protein, 15 μL of NHS-activated agarose beads containing anti-human TrkB antibody (H4H10108N) was added to the brain lysis buffer, and the mixture was incubated overnight at 4 °C with gentle shaking at 20 rpm (Thermo rotator). The next day, the sample was centrifuged at 1000 x g for 1 minute, and the supernatant was carefully removed. The beads were then washed twice with 400 μL of Tris-buffered saline (Bio-Rad, Cat#1706435) (TBST) containing 1% Tween-20 (Sigma Aldrich, Cat#P9416). After carefully aspirating the wash buffer, 60 μL of 0.1% trifluoroacetic acid (TFA; Sigma-Aldrich, T62200) at pH 3.0 in water was added to each sample. The solutions were mixed and allowed to stand for two minutes, then collected and transferred to separate test tubes. This procedure was repeated with another 60 μL of 0.1% TFA at pH 3.0. The two 0.1% TFA solutions for each sample were then combined and 2 μL of 1M Tris-HCl at pH 8.5 (ThermoFisher Scientific, Cat#15567-027) was added.

[0320] The solution was dried using high-speed vacuum drying, then resuspended and reduced with a mixture of 20 μL 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 min and then loaded onto a 10-well Mini-Protean 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) using a Trans-Blot Turbo transfer system (Bio-Rad, Cat#1704156) for 30 min at a constant rate of 1.3 A and 25 V. After transfer, the membrane was blocked at room temperature with 2.5% milk in TBST (Bio-Rad, Cat#170-6406), and then probed overnight at 4°C on a shaker at 30 rpm with anti-TrkB phosphate antibody diluted 1:1000 in 2.5% BSA solution (Novus, Cat#NB100-92656) or anti-TrkB primary antibody diluted 1:1000 in 2.5% milk TBST (Cell Signaling, Cat#4603). The next day, the blot was washed with TBST and incubated at room temperature for 1 hour with anti-rabbit IgG antibody conjugated with horseradish peroxidase (Jackson, Cat#111-035-144) (1:1000 in 1% milk in TBST). The blot was then washed again, developed with ECL solution (PerkinElmer, Inc., Cat#RPN2106), and then imaged every 30 seconds.

[0321] Results summary and conclusions:

[0322] Originating from TrkB hu / hu Immunoprecipitation and subsequent Western blotting of proteins from mouse brain lysates revealed hippocampal TrkB phosphorylation in mice injected with the TrkB agonist antibody H4H9816P2, but not in mice treated with solvent or isotype control antibodies. Figure 1As shown in the figure. At the time points evaluated, TrkB phosphorylation peaked at 4 hours post-stereotactic injection in mice injected with H4H9816P2. In some, but not all, mice also showed TrkB phosphorylation by Western blotting at 18 hours post-administration. Conversely, at any time point, injection of the solvent and the IgG4 isotype control antibody did not induce TrkB phosphorylation. Western blotting also indicated that, relative to the solvent and isotype control treatments, TrkB phosphorylation was significantly higher in some, but not all, mice administered H4H9816P2. hu / hu In mice, total TrkB receptor levels were downregulated. At 18 hours post-drug administration, total TrkB levels appeared to be slightly downregulated in individuals treated with H4H9816P2. Therefore, these results suggest that TrkB receptor levels are downregulated. hu / hu Direct injection of the TrkB agonist antibody H4H9816P2 into mice can induce phosphorylation of the hippocampal TrkB receptor.

[0323] Example 7. In vivo comparison of H4H9816 and isotype control REGN1945 antibodies against TrkB hu / hu Effects on mouse body weight and metabolism.

[0324] To determine the effects of the TrkB agonist antibody H4H9816P2 of the present invention on body weight and composition, homozygous mice expressing the human TrkB receptor instead of the mouse TrkB receptor (TrkB) were subjected to a single subcutaneous antibody injection. hu / hu Metabolic studies in mice. First, TrkB... hu / hu Male mice (20 weeks old) were transferred from group housing to individual housing for two weeks to acclimatize. Afterward, the mice were transferred to metabolic cages (CLAMS, Columbus Instruments) to assess changes in food and water consumption, movement, energy expenditure, and respiration following antibody administration. Plain powdered food was stored on a floor-mounted spring scale (MettlerToledo, PL602E) to measure food consumption by changes in total food weight. Water was obtained through a cage-top nozzle, and changes in pump line volume were tracked. Intake was measured using a Liquid Unit. Throughout the study, the CLAMS metabolic cages measured each of these parameters at continuous 16-18 minute intervals. CLAMS software (Columbus Instruments, v5.35) was used to analyze metabolic data in each individual measurement and summarize them in 24-hour intervals containing a complete light-dark cycle. After two weeks of acclimatization in cages, TrkB... hu / huMice received a single 50 mg / kg subcutaneous dose of either the TrkB agonist antibody H4H9816P2 or the IgG4 isotype control antibody in PBS at pH 7.2. A group of naive control TrkB antibodies were also administered. hu / hu Mice were not injected. Mice were weighed before administration and at 24, 48, 72, 96, and 120 hours after administration. Body composition of each mouse was measured using EchoMRI. TM The -500 analyzer (EchoMRI LLC) performed nuclear magnetic resonance relaxation (NMR), also known as quantitative magnetic resonance. Before administration, mice were placed in transparent plastic containers and inserted into the NMR-MRI device to measure each subject's lean mass, fat mass, and hydration status. Measurements were taken for each mouse over a period of 0.5–3.2 minutes, and again approximately 120 hours after administration.

[0325] Results summary and conclusions:

[0326] Perform daily weight monitoring to determine if a single subcutaneous injection of H4H9816P2 is effective in TrkB. hu / hu In mice, it induced weight loss. Before administration, there was no significant difference in mean body weight among the three treatment groups, with mean pre-administration weights of 28.39–29.85 g in each group (Table 16). However, 48 hours after administration, TrkB mice treated with H4H9816P2 showed significant weight loss. hu / hu Mice lost an average of 1.70 grams, or 5.96% of their pre-drug body weight. At the same time point, naive and isotype control antibody-treated TrkB mice showed... hu / hu Mice gained 1.79-2.37% of their pre-drug body weight. TrkB mice treated with H4H9816P2... hu / hu Mice continued to lose weight throughout the study period, reaching an average loss of 8.42% and 11.80% of their pre-drug body weight at 72 and 96 hours post-drug administration, respectively. At 120 hours post-drug administration, TrkB mice treated with H4H9816P2 showed... hu / hu Mice lost an average of 12.67% of their pre-drug body weight. In contrast, throughout the study, naive and isotype control TrkB mice showed a significantly lower weight loss. hu / hu No mice showed any decrease in body weight prior to administration. At 48, 72, 96, and 120 hours post-administration, TrkB mice treated with H4H9816P2 showed... hu / hu The mice showed a significant decrease in body weight compared to naive and isotype-controlled mice, thus confirming that the TrkB agonist antibody H4H9816P2 induces TrkB... hu / hu The mice experienced a significant reduction in body weight.

[0327] Table 16: TrkB agonist antibody H4H9816P2 after administration of TrkB hu / hu Mouse weight

[0328]

[0329] Note: Statistical significance is shown by two-way ANOVA and Tukey's multiple comparison post-hoc test (* = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001, compared with the isotype control group: TrkB administered with 50 mg / kg isotype control antibody). hu / hu Mice.

[0330] The effects of TrkB agonist antibody H4H9816P2 injection on body composition were also measured by performing NMR-MRI on each subject before and after administration. Before administration, TrkB... hu / hu No significant differences were observed in fat mass or lean body mass among the three treatment groups of mice, with mean fat mass of 4.19–4.75 g and lean body mass of 21.32–21.70 g per group (Table 17). However, after antibody administration, mice were given TrkB of H4H9816P2 during the study. hu / hu Mice lost an average of 48.90% of their total body fat (Table 17). TrkB mice treated with naive and isotype control antibodies... hu / hu Mice lost an average of 8.49% and 9.48% of their pre-drug fat mass, respectively, which was significantly less than those treated with H4H9816P2 (Table 17). Furthermore, throughout the study, TrkB mice treated with H4H9816P2 showed significantly lower fat content. hu / hu Mice lost an average of 7.84% of their lean body mass, which was significantly greater than the average pre-drug lean body mass losses of 2.41% and 1.75% in the naive and isotype control antibody treatment groups, respectively (Table 17). Therefore, in TrkB hu / hu The weight loss reported in mice after injection of the TrkB agonist antibody H4H9816P2 can be explained by a significant reduction in fat mass and a moderate reduction in lean body mass.

[0331] Table 17: TrkB agonist antibody H4H9816P2 after administration of TrkB hu / hu Mouse body composition

[0332]

[0333] Note: Statistical significance is shown by post-hoc tests of Kruskal-Wallis one-way ANOVA and Tukey multiple comparisons (* = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001, compared with the isotype control group: TrkB administered with 50 mg / kg of isotype control antibody). hu / hu Mice.

[0334] In addition to evaluating the effect of TrkB agonist antibody H4H9816P2 injection on TrkB hu / hu In addition to the effects on mouse body weight and composition, food intake, water consumption, and locomotor activity were continuously measured using metabolic cages. Before administration, TrkB... hu / hu Mice consumed an average of 3.49–3.73 g of food per day. However, within 24 hours of administration, TrkB mice treated with H4H9816P2 showed… hu / hu The mice's food intake was significantly reduced to 2.20 grams per day. Throughout the remainder of the study, TrkB mice treated with H4H9816P2... hu / hu The average food intake of mice did not exceed 2.49 grams per day, while that of naive and isotype antibody-treated TrkB mice was significantly higher. hu / hu Mice consistently consumed an average of 3.62–4.07 g of food per day (Table 18).

[0335] Similarly, there were no significant differences in daily water intake among the treatment groups before drug administration. Within each treatment group, TrkB... hu / hu Mice consumed an average of 4.67–5.55 mL of water per day (Table 19). Following drug administration, TrkB mice treated with H4H9816P2... hu / hu The mice's water intake decreased to 2.05–3.24 mL per day. This was significantly lower than that of TrkB mice treated with nausea and isotype control antibodies. hu / hu Mice consumed 4.50–5.77 mL of water daily throughout the study (Table 19). Therefore, injection of the TrkB agonist antibody H4H9816P2 appears to induce TrkB... hu / hu The mice's food and water intake was significantly reduced compared to naive and isotype controls.

[0336] Table 18: TrkB agonist antibody H4H9816P2 after administration of TrkB agonist antibody H4H9816P2 hu / hu Food consumption of mice

[0337]

[0338] Note: Statistical significance is shown by post-hoc tests of Kruskal-Wallis one-way ANOVA and Tukey multiple comparisons (* = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001, compared with the isotype control group: TrkB administered with 50 mg / kg of isotype control antibody). hu / hu Mice.

[0339] Table 19: TrkB agonist antibody H4H9816P2 after administration of TrkB agonist antibody H4H9816P2 hu / hu Water consumption in mice

[0340]

[0341]

[0342] Note: Statistical significance is shown by post-hoc tests of Kruskal-Wallis one-way ANOVA and Tukey multiple comparisons (* = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001, compared with the isotype control group: TrkB administered with 50 mg / kg of isotype control antibody). hu / hu Mice.

[0343] To determine the effect of antibody treatment on activity, by Motion was analyzed using software (Columbus Instruments, v5.35), in which the total number of x-plane movements for each mouse was continuously measured. One mouse exhibited hyperactivity prior to administration and was removed from the post-administration statistical analysis. Throughout the study, subjects treated with naive and allotype antibodies consistently recorded an average of 11,000–15,000 movements per day, while TrkB treated with H4H9816P2 recorded significantly more. hu / hu 28,260 migrations were recorded between 24 and 48 hours post-administration, and 21,193 and 27,028 migrations were recorded between 48 and 72 hours post-administration, respectively (Table 20). TrkB treated with H4H9816P2 hu / hu Mice showed increased total movement counts at each time point following antibody administration, indicating that hyperactivity was another effect of H4H9816P2 injection. Taken together, these effects suggest that a single subcutaneous injection of the TrkB agonist antibody H4H9816P2 significantly reduces TrkB activity. hu / hu It caused significant changes in body weight, body composition, metabolism, and movement in mice.

[0344] Table 20: TrkB agonist antibody H4H9816P2 after administration of TrkB agonist antibody H4H9816P2 hu / hu mouse movement

[0345]

[0346] Note: Statistical significance is shown by post-hoc tests of Kruskal-Wallis one-way ANOVA and Tukey multiple comparisons (* = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001, compared with the isotype control group: TrkB administered with 50 mg / kg of isotype control antibody). hu / hu Mice.

[0347] Example 8. Determining the effect of anti-TrkB antibody on retinal ganglion cell (RGC) survival using an optic nerve transection model.

[0348] All procedures were performed in accordance with ARVO's statement on animal use in ophthalmology and vision studies and Regeneron Pharmaceutical Inc. IACUC. Adult female TrkB humanized rats (Velocigene, Regeneron Pharmaceutical Inc.), aged 8–10 weeks and weighing 200–250 g each, were used. All surgical procedures on the rats were performed under general anesthesia via intraperitoneal injection of ketamine (63 mg / kg) and toluidine (6.0 mg / kg). An eye ointment containing erythromycin (0.5%, Bausch & Lomb) was used to protect the cornea.

[0349] Intraorbital optic nerve axon transection and intravitreal injection

[0350] The left optic nerve (ON) was exposed intraorbitally, and its dura mater was opened. The ON was transected approximately 1.5 mm posterior to the eyeball. Care was taken to avoid damaging the blood supply to the retina. Intravitreal injections were performed immediately posterior to the pars plana of the ciliary body using a stretch glass pipette attached to a 50 μl Hamilton syringe. Care was taken not to damage the lens. Rats with any major postoperative complications (e.g., retinal ischemia, cataracts) were excluded from further analysis. Animals were assigned to different experimental groups. On days 3 and 10 post-ON axon transection, 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 anti-human TrkB antibody H4H9816P2 (45.7 μg / μl).

[0351] In another experiment, the dose-response of anti-human TrkB antibody H4H9816P2 was tested. Homozygous TrkB humanized rats aged 1–9 months received intravitreal injections of 3 μL of 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 axonal transection.

[0352] Immunohistochemical staining and counting of live RGCs

[0353] Brn3a (brain-specific homeobox / POU domain protein 3A) has been used as a marker of surviving retinal ganglion cells (RGCs) because it has been shown to be an effective and reliable method for selectively labeling live RGCs in the retinal whole mounts after ON injury (Nadal-Nicolás FM, Jiménez-López M, Sobrado-Calvo P, Nieto-López L, Cánovas-Martínez I, Salinas-Navarro M, Vidal-Sanz M, Agudo M., Invest Ophthalmol Vis Sci. 2009 Aug; 50(8):3860-8). To perform immunostaining for Brn3a, the retina was blocked for 1 hour in 10% normal donkey serum and 0.5% Triton X-100, and then incubated at room temperature in the same medium for 2 hours with Brn3a antibody (1:400; Cat#:sc-31984, Santa Cruz). After further washing, the retina was incubated overnight at 4°C with Alexa594-conjugated donkey anti-goat secondary antibody (1:400; Cat#:A-11058, Invitrogen).

[0354] Results summary and conclusions:

[0355] To assess the effect of TrkB agonist antibodies on in vivo RGC survival, we 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 14 days after axonal transection. As shown in Table 21, RGC density was similar in the undamaged contralateral eye across the three TrkB genotypes, averaging approximately 1600 / mm². 2The density of surviving red globulins (RGCs) was assessed in the whole retina using Brn3a staining. In homozygous TrkB humanized rats, the TrkB agonist antibody (H4H9816P2) was observed to significantly (p < 0.01, Mann-Whitney test) increase RGC survival (685 ± 106 vs. 255 ± 66 RGCs / mm²) compared to control. 2 In heterozygous TrkB humanized rats, the TrkB agonist antibody also exhibited a significant (p < 0.05, Mann-Whitney test) survival effect (444 × 90 vs. 208 × 50 RGC / mm). 2 In wild-type TrkB rats, the TrkB agonist antibody showed a slight but insignificant increase in the number of retinoid cells (RGCs) compared to the isotype control (Table 22). In dose-response experiments, the RGC density in the entire retina was quantified using Brn3a staining 14 days after axonal transection. The TrkB agonist antibody showed a clear dose-response. Compared to the antibody control group (168 ± 43 RGCs / mm²), the overall retinal RGC density was significantly higher. 2 The TrkB agonist antibody (H4H9816P2) showed a significant (p < 0.01, one-way ANOVA and Tukey post-hoc test) effect at 3 μg / injection (564 + / - 124 RGC / mm). 2 ) or 30ug / injection (543+ / -242RGC / mm 2 The 0.03ug group showed improved RGC survival. There was no difference between the 3 and 30ug groups. Each injection administered 0.03ug (202+ / -96 RGCs / mm²). 2 ) or 0.3ug (337+ / -210RGCs / mm 2 In the group with TrkB agonist antibody H4H9816P2, RGC survival showed an increasing trend but was not significant (Table 23). In summary, the TrkB agonist antibody H4H9816P2 has an increasing effect on TrkB... hu / hu and TrkB hu / + A significant increase in RGC survival was observed in rats.

[0356] in conclusion:

[0357] The TrkB agonist Ab (H4H9816P2) significantly increased RGC survival in humanized TrkB rats in a dose-dependent manner.

[0358] Table 21. RGC quantification in undamaged control eyes (RGCs / mm) 2 TRKB genotype

[0359] hu / hu hu / + + / + 1637.3 1720.4 1636.3 1551.5 2064.6 1670.2 1651.4 1738.8 1873.4 1628.2 2029.8 1725.4 1804.7 1929.6 1973.4 1741.3 1645.9 1739.7 1761.5 1698.8 1787.5 1862.5 1914.0 1779.4

[0360] Table 22 Quantitative analysis of RGCs after optic nerve injury (RGCs / mm) 2 )

[0361]

[0362] Table 23 RGC quantification in dose-response studies (RGCs / mm) 2 )

[0363]

[0364] Example 9. Effects of anti-TrkB antibody on Akt and Erk signaling pathways

[0365] All procedures were performed in accordance with ARVO's statement on animal use in ophthalmology and vision studies and Regeneron Pharmaceutical Inc. IACUC. Primary mouse cortical neurons were isolated and cultured from humanized TrkB mice (MAID 7139) (Nat Protoc. 2012 Sep; 7(9):1741-54. doi:10.1038 / nprot.2012.099). Western blotting (WB) was performed to determine the effects of the TrkB agonist Ab on downstream pathways of Akt and Erk (p-Akt, p-Erk1 / 2). Primary cortical neurons from humanized TrkB mouse pups at day 1 (P1) were cultured for 4 days (DIV-4) in NeuralQ basal medium (Global Stem, cat.#GSM-9420) supplemented with GS21 neuron supplement (Global Stem, cat.#GSM-3100), Glutamax (Invitrogen, cat.#35050-061), and penicillin / streptomycin. Cells were treated with the following substances: TrkB agonist Abs: H4H9816P-L1 (10ug / ml), H4H9780P-L1 (10ug / ml), H4H9814P-L1 (10ug / ml), IgG4 isotype control REGN1945 (10ug / ml), control antibody H1M8037C-L1 (10ug / ml), and BDNF (1ug / ml) for 15 minutes or 2 hours. Western blotting was performed to determine whether agonists differentiated in the maintenance and intensity of downstream signal transduction. Treated cells were washed and scraped into cold PBS containing 1% protease and phosphatase inhibitors (Sigma). Protein concentrations were determined using the Bradford protein assay (Pierce). Samples (50 μg) were separated by SDS-PAGE on 3–8% Tris-acetic acid reducing gels (Novex) and transferred to nitrocellulose membranes (Bio-Rad).

[0366] The membrane was incubated for 1 hour in a blocking solution containing 5% milk and 0.1% Tween-20 (pH 7.6). Then, it was incubated overnight at 4°C in a blocking buffer containing 5% BSA, 0.1% Tween-20, and rabbit antiphosphokinase Trk (Cell Signaling, cat. #9141, 1:500), rabbit antiphosphokinase Akt (Cell Signaling, cat. #9271, 1:1000), or rabbit antiphosphokinase ERK1 / 2 antibody (Sigma, cat. #E7028, 1:5000). Subsequently, the labeled protein was revealed by incubation with anti-goat, mouse, or rabbit IgG conjugated with horseradish peroxidase (HRP) followed by development with a chemiluminescent substrate of HRP (Pierce). To determine the total amount of TrkB, MAPK, or Akt present in each lane, antibodies on the nitrocellulose membrane were stripped in peeling buffer (Pierce) for 20 minutes, then 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), followed by visualization as described above. β-actin (Sigma, cat. #A5316, 1:20000) and GAPDH (Sigma, cat. #G9295) were used as sample loading controls.

[0367] Results summary and discussion:

[0368] like Figure 2 As shown, at 15 minutes after incubation, although all TrkB agonist Abs showed activation of the MAPK / ERK and PI3K / Akt pathways, only BDNF and H4H9814P showed TrkB phosphorylation. After 2 hours of incubation, all TrkB agonist Abs showed TrkB activation.

[0369] Example 10. Effect of agonist anti-TrkB antibody on SH-SY5Y cell survival

[0370] In vitro culture of the human neuroblastoma SH-SY5Y cell line:

[0371] Neuroblastoma cell line SH-SY5Y (Sigma ATCC#94030304, cat.#11C016) was seeded in a growth medium containing DMEM:F12 (Invitrogen cat.#11330), Pen / Strep (Invitrogen cat.#15140), and 10% FBS (Invitrogen cat.#10082-147) and incubated at 37°C in 5% CO2. At passages 23-27, cells were seeded into differentiation medium in 96-well plates 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 differentiated (30 kJ / well) for 4 days. Antibodies were screened using a survival bioassay in which the culture was changed to serum-free differentiation medium (100 μL / well) containing different doses of antibody (100–0.01 μg / ml). After 2 days, CCK8 (Dojindo, cat.#CK04) reagent (10 μL / well) was added, and the plates were incubated for 3–4 hours. OD (Victor or FlexStation III) was measured at 450 nm to determine the percentage of surviving cells. The data were normalized relative to untreated serum-free medium. Antibody-free serum-free treatment = 100% survival.

[0372] Results summary and conclusions:

[0373] like Figure 3 As shown in Table 24, all the TrkB agonist antibodies of the present invention showed a significant dose-dependent increase in the survival of SH-SY5Y cells compared with the negative isotype control antibody (by two-dimensional ANOVA, p < 0.0001).

[0374] Table 24

[0375]

[0376] Example 11. Pharmacokinetic evaluation of anti-TrkB antibody in humanized TrkB and WT mice

[0377] In humanized TrkB mice (human TrkB expression homozygous mice, TrkB... hu / huThe pharmacokinetics of the anti-TrkB antibody H4H9816P2 were evaluated in both 4-type and wild-type (WT) mice. Each mouse strain contained 5 mice. All mice received a single subcutaneous (SC) dose of 10 mg / kg. Blood samples were collected at 6 hours and on days 1, 2, 3, 6, 9, 16, 21, and 30 post-administration. Blood was processed into serum and frozen at -80°C until analysis.

[0378] Using GyroLab xPlore TM (Gyros, Uppsala, Sweden) Circulating antibody concentrations were determined by total human IgG4 / hIgG1 antibody analysis. In summary, 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 captured on a Gyrolab Bioaffy 200 CD containing affinity columns (Dynospheres) pre-loaded with streptavidin-coated beads. TM The standard used for calibration in this assay was H4H9816P at concentrations ranging from 0.488 to 2000 ng / mL in dilution buffer (0.5% BSA + PBS) containing 0.1% normal mouse serum (NMS). Serum samples were diluted 1:100 in antibody dilution buffer. Human IgG captured on an affinity column coated with anti-REGN2567 on CD at room temperature was detected by adding 0.5 μg / mL of mouse anti-human kappa monoclonal antibody (REGN654) conjugated with Alexa-647, diluted in detection buffer (Rexxip F buffer); and the resulting fluorescence signal was recorded in response units (RU) using a GyroLab xPlore instrument. Sample concentrations were determined by extrapolation from a standard curve fitted using a 5-parameter logic curve fitting method using Gyrolab Evaluator software. The average concentration from two replicate experiments was used for subsequent pharmacokinetic analysis.

[0379] use Software version 6.3 (Certara, LP, Princeton, NJ) and an extravascular administration model were used to determine PK parameters via non-compartmental analysis (NCA). For each antibody, the corresponding average concentration value was used for all PK parameters, including the observed maximum serum concentration (C). max ), Observed estimated half-life (t 1 / 2 ), and the area under the curve (AUC) of concentration vs. time to reach the final measurable concentration. last The linear trapezoidal rule, linear inference, and uniform weighting are used to determine the value.

[0380] Results summary and conclusions:

[0381] Following administration of 10 mg / kg subcutaneous anti-TrkB antibody H4H9816P2, on day 1 or 2, TrkB... hu / hu Similar peak antibody concentrations (C0) were observed in WT mice (135 μg / mL and 131 μg / mL, respectively, see Table 26). max By day 9, compared to WT mice, H4H9816P2 showed better performance in TrkB mice. hu / hu Steeper drug clearance was observed in mice, indicating a target-mediated effect. In TrkB hu / hu In mice, antibody concentrations decreased by approximately 35-fold on day 30. Antibody exposure (AUC) to H4H9816P2 in WT mice. last ) compared to TrkB hu / hu The levels observed in mice were approximately 1.7 times higher (1730 and 1020 d*μg / mL, respectively). WT mice also showed a longer half-life (T0). 1 / 2 ) compared to TrkB hu / hu The survival time in mice increased approximately threefold (8.4 days and 2.9 days, respectively).

[0382] Table 25 summarizes the data on total anti-TrkB antibody concentration, and Table 26 describes the average pK parameters. Figure 4 The relationship between average total antibody concentration and time is shown.

[0383] Table 25: Over time, in TrkB hu / hu Mean serum total IgG concentration (±SD) after a single subcutaneous injection of 10 mg / kg H4H9816P2 in wild-type mice and other mice.

[0384]

[0385] Abbreviations: Time = number of days after a single dose; d = study day; SD = standard deviation.

[0386] Table 26: Summary of Pharmacokinetic Parameters

[0387]

[0388] The PK parameter is derived from the average concentration versus time curve. T 1 / 2 and AUC last Based on the concentration up to day 30.

[0389] Abbreviation: C max = Peak concentration; AUC = Area under the concentration-time curve; AUC last = AUC calculated from time zero to the final positive concentration; T1 / 2 =Terminal elimination half-life; T max =Time after antibody administration to reach maximum serum concentration

[0390] Example 12: The ability of anti-mouse TrkB monoclonal antibody to block the interaction between mouse or rat TrkB and its ligand BDNF (brain-derived neurotrophic factor).

[0391] Anti-mouse TrkB monoclonal antibodies (mAbs) were generated by immunizing TrkB-humanized mice with mouse TrkB protein. Three lead mAbs were identified from this immunization: M2aM14173N, M2aM14178N, and M2aM14179N. The ability of these lead mAbs to block the interaction between mouse or rat TrkB and plate-bound BDNF was characterized in a blocking ELISA.

[0392] The following procedure was used for experiments. Human BDNF was coated onto 96-well microtiter plates in PBS at a concentration of 0.5 μg / mL (for blocking mouse TrkB.hFc interaction) or 0.3 μg / mL (for blocking rat TrkB.mmh interaction) and incubated overnight at 4°C. Non-specific binding sites were then blocked with a 5% (w / v) solution of BSA in PBS (assay buffer). In 96-well dilution plates, 850 pM of mouse TrkB.hFc or rat TrkB.mmh was mixed with three-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. The pre-bound mixture was then transferred in duplicate to BDNF-coated microtiter plates. A control containing only assay buffer was included to calculate the baseline for the assay. The ELISA plates were incubated at RT for 1 hour and then washed with plate washing buffer. Mouse TrkB.hFc conjugated with HRP was detected using a goat anti-human Fcγ fragment-specific antibody (Jackson Immunoresearch), and rat TrkB.mmh was detected using an HRP-conjugated antihistamine (Qiagen). The plates were incubated with the detection antibodies at RT for 1 hour, followed by washing with plate washing buffer. Color development of the detection plates was performed using TMB colorimetric substrate according to the manufacturer's recommended procedure.

[0393] The absorbance at 450 nm for each well was recorded and plotted as a function of antibody concentration. The data were analyzed using a four-parameter logistic equation on the 11-point dose-response curves using GraphPadPrism software, and the IC50 was calculated. 50 Value. Calculated IC 50The value (defined as the antibody concentration required to reduce TrkB binding to BDNF by 50%) is used as an indicator of blocking efficacy. The percentage of block at the maximum concentration of the test antibody, relative to the assay baseline, is calculated as an indicator of the antibody's ability to block TrkB binding to BDNF on the plate. In the absence of antibody, the binding signal of 850 pM mouse or rat TrkB is defined as 100% binding or 0% blocking. The baseline signal of the assay buffer alone is defined as 0% binding or 100% blocking.

[0394] Results Summary and Conclusions

[0395] The ability of anti-mouse TrkB antibodies to block the binding of mouse or rat TrkB to BDNF was evaluated using a blocking ELISA.

[0396] The results of the blocking are summarized in Table 27 and Figure 5A In B, the percentage of blockade for all antibodies is reported, calculated at the highest antibody concentration tested (100 nM). Antibodies showing IC50 only against mice or rats with >50% blockade of TrkB binding to BDNF were reported. 50 Value. Among the three antibodies of this invention, the anti-mouse TrkB mAb, M2aM14178N, blocked >50% of the binding of mouse and rat TrkB protein to BDNF. M2aM14178N blocked the binding of 850pM mouse TrkB.hFc, IC50 value. 50 The concentration was 426 pM, with a % blockade rate of 84.4%. M2aM14178N blocked the binding of 850 pM rat TrkB.mmh to BDNF, with an IC50 concentration of 426 pM and an IC50 percentage point (IC50). 50 The value was 184 pM, with a % blockade of 89.5%. M2aM14173N showed a 29.7% blockade on the binding of BDNF to TrkB.hFc in mice at 850 pM. M2aM14173N showed an 80.7% blockade on the binding of BDNF to TrkB.mmh in rats at 850 pM, IC50 value 89.5%. 50 The value was 3.81 nM. M2aM14179N blocked the binding of BDNF to 11.6% of mouse TrkB.hFc. M2aM14179N showed increased binding to BDNF in rat TrkB.mmh at concentrations greater than 1 nM.

[0397] Comparative studies showed that the anti-tumor drug TrkB mAb H1M8037C blocked the binding of 850 pM TrkB.hFc to BDNF in mice, with an IC50 concentration of 100 mg / kg. 50 The value was 180 pM, and the % blockade was 91.5%. H1M8037C blocked the binding of 850 pM rat TrkB.mmh, IC50 value. 50The value was 1.42 nM, with a % blockade of 83.3%. Under the same assay conditions, the mIgG2a isotype control mAb REGN1097 showed no blocking effect on mouse or rat TrkB. At concentrations greater than 10 nM, REGN1027 showed increased rat TrkB binding.

[0398] Table 27: IC50 of anti-mouse TrkB blocking the binding of TrkB to BDNF in mice or rats 50 Summary of (M) values

[0399]

[0400] 100% blocking = OD450 nm value of wells containing HRP-conjugated secondary antibody in assay buffer (without mouse or rat TrkB protein).

[0401] 0% blocking = OD450 nm value of wells with HRP-conjugated secondary antibody in assay buffer in the presence of mouse or rat TrkB protein (without TrkB antibody).

[0402] The negative maximum blocking percentage indicates an increase in TrkB binding detected in the presence of the antibody.

[0403] Not calculated = IC50 value not quantified for antibodies that block <50% at the highest tested concentration.

[0404] Example 13. The ability of anti-human TrkB monoclonal antibody to block the interaction between human TrkB and its natural ligands human BDNF and NT4.

[0405] Two competitive sandwich ELISAs were used to measure the ability of anti-human TrkB antibodies, named H4H9814P, H4H9816P2, and H4H9780P, to block the binding of TrkB protein to plate-captured BDNF or NT-4. In the assays, various concentrations of anti-TrkB antibodies were premixed with constant amounts of dimeric TrkB protein, and the reduction in TrkB binding to BDNF or NT-4 immobilized on the plate due to the presence of the antibodies was calculated.

[0406] The recombinant TrkB protein used in the experiment consisted of the extracellular domain of human TrkB expressed at the C-terminus with human IgG1 in the Fc region (aa Cys32-His430) (hTrkB-hFc; accession number NP_006171.2, molecular weight 69,700 Daltons). BDNF and NT-4 proteins consisted of the extracellular domains of human BDNF (aa His129-Arg247, accession number #P23560, R&D Systems) or NT-4 (aa Gly81-Ala210, accession number #P34130, R&D Systems), respectively. As controls for IgG background detection, two isotype antibody controls were included: anti-Feld 1 human IgG4 antibody and an antibody specific to α-Feld 1 antibody containing mouse IgG1.

[0407] The following procedure was used for experiments. Human BDNF or NT-4 was coated overnight at 4°C in PBS at concentrations of 0.5 μg / mL or 2 μg / mL on 96-well microtiter plates. Non-specific binding sites were then blocked using BSA solution in PBS. Blocking solutions and dilution buffers containing 5% (w / v) BSA in PBS were used for BDNF coating assays, or 0.5% (w / v) BSA in PBS were used for NT-4 coating assays. On separate microtiter plates, a constant amount of 500 pM hTrkB-hFc protein was added to serially diluted antibody solutions (final concentrations ranging from 1.7 pM to 100 nM) and to solutions without antibody. (A constant concentration of hTrkB-hFc for antibody inhibition assays was selected from the near-midpoint of the linear portion of the corresponding binding curves of hTrkB-hFc to hBDNF or hNT-4 on the coated plates). After incubation at room temperature for one hour, an antibody-protein complex of hTrkB-hFc protein at a constant concentration of 500 pM was transferred to a microtiter plate coated with hBDNF or hNT-4. After incubation at room temperature for one hour, the wells were washed, and the binding of hTrkB-hFc to the plate was detected using a horseradish peroxidase-conjugated anti-human Fcγ fragment-specific goat polyclonal antibody (Jackson ImmunoResearch). The plate was then developed using TMB substrate solution (BD Biosciences) according to the manufacturer's recommendations, and the plate was read on a Victor plate reader (PerkinElmer). TM The absorbance at 450 nm was measured.

[0408] Prism TMData analysis was performed using an S-shaped dose-response model in the 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. The percentage of blocking at the maximum concentration of the antibody tested, relative to the assay baseline, was calculated as an indicator of the antibody's ability to block the binding of 500 pM hTrkB-hFc to hBDNF or hNT-4 on the plate. In the calculations, the binding signal of the 500 pM hTrkB-hFc sample in the absence of antibody was referred to as 100% binding or 0% blocking; the baseline signal of the buffer sample containing neither hTrkB-hFc nor antibody was referred to as 0% binding or 100% blocking.

[0409] Results summary and conclusions:

[0410] The ability of anti-TrkB antibodies to block the binding of TrkB to BDNF or NT-4 was evaluated using two competitive sandwich ELISAs. The binding of human TrkB-hFc to hBDNF or hNT-4 coated on 96-well microtiter plates was detected using an HRP-conjugated goat polyclonal antibody specific to the human Fcγ fragment, in the presence of serially diluted antibodies or without antibody controls. The IC50 value was calculated and used as a measure of the antibody's efficacy in blocking the binding of hTrkB-hFc to hBDNF or hNT-4. Furthermore, the maximum blocking effect of each antibody against 500 pM hTrkB-hFc at the highest tested concentration was calculated and compared.

[0411] The blocking results are summarized in Table 28. The percentage of blockade for all antibodies is reported, calculated at the highest tested antibody concentration of 100 nM. Negative percentage blockade indicates increased TrkB binding detected in the antibody's presence. IC50 values ​​are shown for antibodies blocking >50% of TrkB binding to BDNF or NT-4. For antibodies blocking <50%, the IC50 value is not quantitative and is reported as (-).

[0412] At the highest antibody concentration tested, one of the three anti-TrkB antibodies (H4H9780P) blocked >50% of the binding of hTrkB to BDNF or NT-4 ligands, with IC50 values ​​of 150 pM and 180 pM, respectively. At 100 nM, the blocking percentage was 93% for BDNF and 80% for NT-4. At 3.7 nM, the antibody blocked 99% of the binding of 500 pM hTrkB-hFc to NT-4. The decrease in the blocking percentage at the highest tested concentration may be attributed to the non-specific binding of H4H9780P to the microtiter plate and the fact that this binding was detected using an HRP-conjugated anti-human Fcγ fragment-specific polyclonal antibody.

[0413] Two of the three anti-TrkB antibodies (H4H9814P and H4H9816P2) and the irrelevant blocking control antibody blocked <50% of the binding of hTrkB to BDNF or NT-4. The comparative antibody blocked >50% of the binding of 500 pM hTrkB-hFc to both BDNF and NT-4.

[0414] Table 28

[0415]

[0416] (*) 99% blockade at a 3.7 nM H4H9780P antibody concentration.

[0417] Example 14. Octet cross-competition among different anti-hTrkB monoclonal antibodies

[0418] To assess whether the two antibodies competitively bind to their epitopes on hTrkB-mmh, binding competition between anti-hTrkB monoclonal antibodies was determined using real-time label-free biolayer interferometry on an Octet RED384 biosensor (Pall ForteBio Corp.). Cross-competition assays were performed at 25°C with 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), with plate shaking at 1000 rpm. All anti-hTrkB antibody and hTrkB-mmh solutions were prepared in Octet HBS-EP buffer. To assess whether the two antibodies could competitively bind to their epitopes on hTrkB-mmh, approximately [amount missing] μg / mL hTrkB-mmh was first captured from wells containing 50 μg / mL hTrkB-mmh on an anti-His-coated Octet biosensor probe for 5 minutes. hTrkB-mmh was captured at a wavelength of 0.14–0.24 nm. The Octet biosensor probe containing captured hTrkB-mmh was immersed in wells containing 50 μg / ml of primary anti-hTrkB monoclonal antibody (hereinafter referred to as mAb-1) for 5 minutes to saturate the probe, and then immersed in wells containing secondary anti-hTrkB monoclonal antibody (hereinafter referred to as mAb-2) for another 5 minutes. Between steps, the Octet biosensor probe was washed in HBS-EP buffer for 30 seconds. Real-time binding reactions were monitored during the experiment, and the binding reaction at the end of each step was recorded. The binding reactions of mAb-2 with hTrkB.mmh pre-conjugated with mAb-1 were compared, and the competitive / non-competitive behavior of different anti-hTrkB monoclonal antibodies was determined using a 60% inhibition threshold.

[0419] Table 29 clearly defines the relationships between antibodies that compete in both directions regardless of the binding sequence.

[0420] result:

[0421] Table 29: Cross-competition between anti-hTrkB antibody and human hTrkB.mmh binding.

[0422]

[0423] Example 15. Biacore binding kinetics of the substitute anti-mouse TrkB monoclonal antibody with different TrkB reagents were measured at 25°C.

[0424] To assess whether the two antibodies competitively bind to their respective epitopes on mTrkB-mmh, binding competition between anti-mTrkB monoclonal antibodies was determined using real-time, label-free biolayer interferometry on the Octet HTX biosensor (Pall ForteBio Corp.). Cross-competition assays were performed at 25°C with a plate 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 antibody and mTrkB-mmh solutions were prepared in Octet HBS-EP buffer. To assess whether the two antibodies could competitively bind to their respective epitopes on mTrkB-mmh, approximately [amount missing] μg / mL mTrkB-mmh was first captured from wells containing 20 μg / mL mTrkB-mmh for 5 minutes on an anti-His-coated Octet biosensor probe. mTrkB-mmh was captured at 0.20–0.27 nm. The Octet biosensor probe, containing captured mTrkB-mmh, was saturated by immersing it in wells containing 50 μg / ml of primary anti-mTrkB monoclonal antibody (hereinafter referred to as mAb-1) for 5 minutes, followed by immersion in wells containing secondary anti-mTrkB monoclonal antibody (hereinafter referred to as mAb-2) for another 3 minutes. Between steps, the Octet biosensor probe was washed in HBS-EP buffer for 30 seconds.

[0425] The binding reaction was monitored in real time during the experiment, and the binding reaction at the end of each step was recorded. The binding reaction of mAb-2 with mTrkB.mmh pre-complexed with mAb-1 was compared, and the competitive / non-competitive behavior of different anti-mTrkB monoclonal antibodies was determined using a 50% inhibition threshold.

[0426] Table 30 clearly defines the relationships between antibodies that compete in both directions regardless of the binding sequence.

[0427] result:

[0428] Table 30: Cross-competition between anti-mTrkB antibody and mouse hTrkB.mmh binding.

[0429]

[0430] Example 16: Octet Blocking: Blocking the binding of BDNF or NT-4 to TrkB with anti-human TrkB or anti-mouse TrkB antibodies.

[0431] Experiment 1.

[0432] The blocking effect of BDNF or NT-4 on the binding of anti-human TrkB or anti-mouse TrkB antibodies to TrkB was evaluated using the Octet HTX instrument based on real-time biolayer interferometry (BLI). The entire study was conducted 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 running buffer). All samples were dispensed into 384-well tilted plates, which were placed on an orbital vibrator at 1000 rpm. hTrkB.mFc was captured on the anti-mFc (AMC) Octet sensor, while hTrkB.hFc or mTrkB.hFc was captured on the anti-hFc (AHC) Octet sensor by immersing it in a well containing 10 μg / mL TrkB reagent for 2 minutes. The Octet biosensor, containing either hTrkB.mFc or hTrkB.hFc, was saturated by immersing it in wells containing 20 nM BDNF, hNT-4, or mNT-4 for 2 minutes. The Octet biosensor was then immersed in wells containing 300 nM of different TrkB mAbs for 4 minutes. The binding of TrkB mAbs to complexes of TrkB with BDNF, hNT-4, or mNT-4 was determined using Scrubber 2.0c analysis software.

[0433] As shown in Tables 31 and 32, the binding of any anti-human TrkB or anti-mouse TrkB antibody of the present invention was not blocked by BDNF and NT-4.

[0434] Table 31: Binding of anti-human TrkB monoclonal antibody to complexes of hTrkB-mFc and BDNF or human NT-4.

[0435]

[0436] Table 32: Binding of anti-mouse TrkB monoclonal antibody to complexes of mTrkB-hFc and BDNF or mouse NT-4.

[0437]

[0438] Experiment 2.

[0439] The blocking effect of anti-human TrkB or anti-mouse TrkB antibodies on the binding of BDNF or NT-4 to TrkB was evaluated using the Octet HTX instrument based on real-time biolayer interferometry (BLI). The entire study was conducted 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 running buffer). All samples were dispensed into 384-well tilted plates, which were placed on an orbital vibrator at 1000 rpm. hTrkB.mFc was captured on the anti-mFc (AMC) Octet sensor, while hTrkB.hFc or mTrkB.hFc was captured on the anti-hFc (AHC) Octet sensor by immersing it in a well containing 10 μg / mL TrkB reagent for 2 minutes. Octet biosensors containing captured hTrkB.mFc or hTrkB.hFc were saturated by immersing them in wells containing 300 nM of different TrkB mAbs for 4 minutes. The Octet biosensors were then immersed in wells containing 20 nM BDNF, hNT-4, or mNT-4 for 2 minutes. The binding of BDNF, hNT-4, or mNT-4 to TrkB and the complexes of different TrkB mAbs was determined using Scrubber 2.0c analysis software.

[0440] As shown in Table 33, one of the three anti-human TrkB antibodies of the present invention blocked the binding of BDNF and hNT-4. As shown in Table 34, one of the three anti-mouse TrkB antibodies of the present invention partially blocked the binding of BDNF and mNT-4.

[0441] Table 33: Binding of BDNF or human NT-4 with complexes of hTrkB-mFc and anti-human TrkB monoclonal antibody.

[0442]

[0443] Table 34: Binding of BDNF or mouse NT-4 to complexes of mTrkB-hFc and anti-mouse TrkB monoclonal antibody.

[0444]

[0445] This invention is not limited to the specific embodiments described herein. In fact, various modifications to the invention will be apparent to those skilled in the art, in addition to those described herein, based on the foregoing description and drawings. Such modifications are intended to fall within the scope of the appended claims.

Claims

1. An isolated antibody or antigen-binding fragment thereof that specifically binds to tropomyosin receptor kinase B (TrkB), wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region (HCVR) includes: (a) The heavy chain complementarity-determining region 1 of the amino acid sequence shown in SEQ ID NO:4, namely HCDR1. (b) The heavy chain complementarity-determining region 2, i.e., HCDR2, of the amino acid sequence shown in SEQ ID NO:6, and (c) The heavy chain complementarity-determining region 3 of the amino acid sequence shown in SEQ ID NO:8, namely HCDR3. The light chain variable region (LCVR) includes: (d) The light chain complementarity-determining region 1, i.e., LCDR1, of the amino acid sequence shown in SEQ ID NO:

12. (e) The light chain complementarity-determining region 2, namely LCDR2, of the amino acid sequence shown in SEQ ID NO:14, and (f) The light chain complementarity-determining region 3, namely LCDR3, of the amino acid sequence shown in SEQ ID NO:

16.

2. The isolated antibody or its antigen-binding fragment according to claim 1, wherein, The antibody or its antigen-binding fragment comprises the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 2 and the light chain variable region of the amino acid sequence shown in SEQ ID NO:

10.

3. The isolated antibody or its antigen-binding fragment according to claim 1 or 2, wherein, The antibody or its antigen-binding fragment was measured at a Kc of less than 300 nM by surface plasmon resonance at 25 °C or 37 °C. D Combined with human TrkB.

4. The isolated antibody or its antigen-binding fragment according to claim 3, wherein, The antibody or its antigen-binding fragment was measured at a Kc of less than 150 nM by surface plasmon resonance at 25 °C or 37 °C. D Combined with human TrkB.

5. The isolated antibody or its antigen-binding fragment according to claim 1 or 2, wherein, By measuring surface plasmon resonance at 25°C or 37°C, the antibody or its antigen-binding fragment binds to human TrkB with a dissociation half-life (t1 / 2) greater than 10 minutes.

6. The isolated antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof activates human TrkB signaling in engineered TrkB-expressing cells in the absence of BDNF, EC 50 Less than 100pM.

7. The isolated antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof enhances activation of human TrkB signaling in engineered TrkB-expressing cells in the presence of BDNF, EC 50 Less than 100pM.

8. The isolated antibody or its antigen-binding fragment according to claim 1 or 2, wherein, When injected into the hippocampus of humanized TrkB mice, the antibody or its antigen-binding fragment demonstrates TrkB activation, as indicated by increased TrkB phosphorylation.

9. The isolated antibody or its antigen-binding fragment according to claim 1 or 2, wherein, Incubation of primary mouse cortical neurons with an agonist anti-TrkB antibody demonstrated that the antibody or its antigen-binding fragments activated the MAPK / ERK and PI3K / Akt signaling pathways.

10. The isolated antibody or its antigen-binding fragment according to claim 1 or 2, wherein, As demonstrated in the TrkB humanized rat optic nerve transection model, the antibody or its antigen-binding fragment enhances the survival of retinal ganglion cells.

11. The isolated antibody or its antigen-binding fragment according to claim 1 or 2, wherein, The antibody or its antigen-binding fragment promoted weight loss in humanized TrkB mice.

12. The isolated antibody or its antigen-binding fragment according to claim 1 or 2, wherein, The antibody or its antigen-binding fragment promotes a reduction in fat mass in humanized TrkB mice.

13. The isolated antibody or its antigen-binding fragment according to claim 1 or 2, wherein, The antibody or its antigen-binding fragment promoted a reduction in food and water consumption in humanized TrkB mice.

14. The isolated antibody or its antigen-binding fragment according to claim 1 or 2, wherein, The antibody or its antigen-binding fragment promotes increased motility activity in humanized TrkB mice.

15. The isolated antibody or its antigen-binding fragment according to claim 1 or 2, wherein, The antibody or its antigen-binding fragment exhibits at least two of the following properties: a) is an agonist antibody; b) Measurements were taken at 25°C or 37°C using surface plasmon resonance, with K values ​​less than 200 nM. D Combined with human TrkB; c) The dissociation half-life (t1 / 2) of binding with human TrkB is greater than 10 minutes, as measured by surface plasmon resonance at 25°C or 37°C. d) In engineered cells expressing human TrkB, in the absence of brain-derived neurotrophic factor (BDNF), activation of human TrkB signaling, EC 50 Less than 100pM; e) When injected into the hippocampus of human TrkB receptor homozygous mice, TrkB phosphorylation is enhanced; f) When injected into human TrkB receptor homozygous mice, it promotes weight loss; g) Increased retinal ganglion cell (RGC) survival when evaluated in a humanized TrkB rat optic nerve transection model; h) Activate the MAPK / ERK and PI3K / Akt signaling pathways; i) Increase / enhance the in vitro survival of neurons; and j) Block the binding of TrkB to BDNF and / or NT4, IC 50 Less than 5 nM.

16. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1-15 and a pharmaceutically acceptable carrier.

17. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1-15 and a pharmaceutically acceptable diluent.

18. An isolated nucleic acid molecule comprising a polynucleotide sequence encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1-15.

19. A vector comprising the polynucleotide sequence of claim 18.

20. Cells expressing the vector of claim 19.

21. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-15 in the preparation of a medicament for treating glaucoma.

22. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-15 in the preparation of a medicament for achieving weight loss in a subject.

23. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-15 in the preparation of a medicament for achieving a reduction in body fat in a subject.

24. Use according to any one of claims 21-23, wherein the medicament is an injectable formulation.

Images