EA044580B1 - MONOCLONAL ANTI-TRKB ANTIBODIES AND METHODS OF THEIR APPLICATION - Google Patents

Description

Field of technology to which the invention relates

The present invention relates to monoclonal antibodies against tropomyosin tyrosine kinase receptor B (TrkB). More specifically, the invention relates to compositions containing monoclonal anti-TrkB antibodies, and methods of using these antibodies.

State of the art

Tropomyosin tyrosine kinase receptor B (TrkB) belongs to a family of distinct transmembrane receptor tyrosine kinases that includes TrkA and TrkC. These receptor kinases mediate the activity of neurotrophins, which are essential for the survival and development of neurons. Neurotrophins include, but are not limited to, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4/5 (NT-4/5). (Lo, K Y et al., J. Biol. Chem., 280:41744-52(2005)).

TrkB is a high affinity receptor for BDNF (Minichiello, et al., Neuron 21: 335-45 (1998)), but is also known to bind NT4/5. Binding of BDNF to trkB causes dimerization of the receptor, which leads to 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 neurotrophin effects that include neuronal differentiation and survival.

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

Some TrkB agonists have been described in US 2010/0150914; US 2003/0157099; US 2010/0196390 and US 2017/0157099. However, there is still a need to identify and develop additional TrkB agonists that provide improved specificity in addition to exhibiting neuronal survival and neuroprotective properties, such as those described herein.

Summary of the invention

The present invention relates to isolated monoclonal antibodies and antigen binding fragments thereof that specifically bind to tropomyosin tyrosine kinase receptor B (TrkB). The isolated antibodies and antigen binding fragments of the invention are used to treat diseases and disorders associated with TrkB activity or expression.

Broadly, the invention relates to TrkB agonist antibodies that activate TrkB and promote neuronal survival. These antibodies can be used to improve nerve function and treat any disease or disorder characterized in part by cellular degeneration, including nerve cell damage associated with damage to the nervous system and/or chronic neurodegenerative diseases.

In some embodiments, anti-TrkB antibodies may be used to treat various ocular diseases or disorders and may be formulated for intraocular or intravitreal delivery to treat ocular diseases such as, among others, glaucoma.

Antibodies of the invention may be full length (eg an IgG1 or IgG4 antibody) or may contain only an antigen binding portion (eg a Fab fragment, F(ab') ₂ or scFv) and may be modified to affect functionality, e.g. to eliminate residual effector functions (Reddy et al., 2000, J. Immunol. 164:1925-1933).

Representative anti-TrkB antibodies of the present invention are listed in table. 1 and 2 in this document. In table 1 shows the amino acid sequence identifiers of the heavy chain variable regions (HCVR), light chain variable regions (LCVR), heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3), and light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) of typical anti-TrkB antibodies. In table Figure 2 shows the nucleic acid sequence identifiers HCVR, LCVR, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of typical anti-TrkB antibodies.

The present invention relates to antibodies or antigen-binding fragments thereof that specifically bind to TrkB, including HCVR containing an amino acid sequence selected from any of the HCVR amino acid sequences listed in table. 1, or a substantially similar sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to these sequences.

The present invention also relates to antibodies or antigen-binding fragments thereof that specifically bind to TrkB, including LCVR containing an amino acid sequence selected from any of the LCVR amino acid sequences listed in table. 1, or a substantially similar sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to these sequences.

The present invention also provides antibodies or antigen-binding fragments thereof that specifically bind to TrkB containing a pair of amino acid sequences HCVR and LCVR (HCVR/LCVR), containing any of the amino acid sequences of HCVR listed in table. 1, in combination with any of the LCVR amino acid sequences listed in table. 1. In accordance with some embodiments, the present invention provides antibodies or antigen binding fragments thereof comprising the HCVR/LCVR amino acid sequence pair contained in any of the exemplary anti-TrkB antibodies listed in Table. 1.

Accordingly, in a first aspect, the invention provides an isolated antibody or antigen binding fragment thereof that specifically binds to tropomyosin tyrosine kinase receptor B (TrkB), wherein the antibody or antigen binding fragment thereof comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2 and HCDR3 ), contained in the heavy chain variable region (HCVR), containing the amino acid sequence shown in table. 1, or a substantially similar sequence that is at least 90% identical to this sequence; and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained in a light chain variable region (LCVR) containing the amino acid sequence shown in table. 1, or a substantially similar sequence that is at least 90% identical to this sequence.

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

(a) is an agonistic antibody;

(b) binds to human TrkB with a K _D of less than about 200 nM, as determined by surface plasmon resonance at 25°C or at 37°C;

(c) binds to human TrkB with a dissociation half-life (tV ₂ ) greater than about 10 minutes, as determined by surface plasmon resonance at 25°C or at 37°C;

(d) activates human TrkB signaling in the absence of brain-derived neurotrophic factor (BDNF) in cells engineered to express human TrkB, with an EC ₅₀ ranging from 35 to 82 pM;

(e) enhances TrkB phosphorylation when injected into the hippocampus of mice homozygous for the human TrkB receptor (TrkB ^hu/hu );

(f) promotes weight loss when injected into mice homozygous for the human TrkB receptor (TrkB ^hu/hu );

(g) increases retinal ganglion cell (RGC) survival, as assessed in an optic nerve transection model in TrkB humanized rats;

(h) activates the MAPK/ERK and PI3K/Akt signaling pathways;

(i) increases the survival of neuronal cells in vitro; and (j) blocks TrkB binding to BDNF and/or NT-4 with an IC ₅₀ of less than 5 nM.

In one embodiment, the invention provides an antibody or antigen binding fragment thereof that specifically binds to tropomyosin tyrosine kinase receptor B (TrkB), wherein the antibody or antigen binding fragment thereof comprises: (a) heavy chain variable region (HCVR) complementarity determining regions (CDRs) ), containing the amino acid sequence indicated in table. 1; and (b) a light chain variable region (LCVR) CDR containing the amino acid sequence as shown in table. 1.

In one embodiment, an antibody or antigen-binding fragment thereof that specifically binds TrkB contains three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained in any of the HCVR sequences selected from the group consisting of SEQ ID NO: 2, 18, 34, 49, 59 and 68, or a substantially similar sequence that is at least 90% identical to this sequence; and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained in any of the LCVR sequences selected from the group consisting of SEQ ID NOs: 10, 26, 42, 53, 63 and 72, or a substantially similar sequence that, is at least 90% identical to this sequence.

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

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

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

In one embodiment, an isolated antibody or antigen binding fragment thereof that specifically binds TrkB comprises a CDR of the amino acid sequence pair HCVR/LCVR selected from the group consisting of SEQ ID NO: 2/10, 18/26, 34/42, 49/53, 59/63 and

68/72.

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

In one embodiment, an isolated antibody or antigen-binding fragment thereof that specifically binds TrkB comprises a pair of amino acid sequences HCVR/LCVR selected from the group consisting of SEQ ID NO: 2/10, 18/26, 34/42, 49 /53, 59/63 and 68/72.

In one embodiment, the isolated antibody or antigen-binding fragment thereof that specifically binds TrkB contains the amino acid sequence pair HCVR/LCVR selected from the group consisting of SEQ ID NOs: 2/10, 18/26 and 34/42.

The present invention also provides antibodies or antigen-binding fragments thereof that specifically bind TrkB, including a heavy chain CDR1 (HCDR1) containing an amino acid sequence selected from any of the HCDR1 amino acid sequences listed in table. 1, or a substantially similar sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to that sequence.

The present invention also provides antibodies or antigen-binding fragments thereof that specifically bind TrkB, including a heavy chain CDR2 (HCDR2) containing an amino acid sequence selected from any of the HCDR2 amino acid sequences listed in table. 1, or a substantially similar sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to that sequence.

The present invention also provides antibodies or antigen-binding fragments thereof that specifically bind TrkB, including a heavy chain CDR3 (HCDR3) containing an amino acid sequence selected from any of the HCDR3 amino acid sequences listed in table. 1, or a substantially similar sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to that sequence.

The present invention also provides antibodies or antigen-binding fragments thereof that specifically bind TrkB, including a light chain CDR1 (LCDR1) comprising an amino acid sequence selected from any of the LCDR1 amino acid sequences listed in table. 1, or a substantially similar sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to that sequence.

The present invention also provides antibodies or antigen-binding fragments thereof that specifically bind TrkB, including a light chain CDR2 (LCDR2) containing an amino acid sequence selected from any of the LCDR2 amino acid sequences listed in the table, or a substantially similar sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to this sequence.

The present invention also provides antibodies or antigen-binding fragments thereof that specifically bind TrkB, including a light chain CDR3 (LCDR3) containing an amino acid sequence selected from any of the LCDR3 amino acid sequences listed in table. 1, or a substantially similar sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to that sequence.

The present invention also provides antibodies or antigen-binding fragments thereof that specifically bind to TrkB, including a pair of amino acid sequences HCDR3 and LCDR3 (HCDR3/LCDR3) containing any of the amino acid sequences of HCDR3 listed in table. 1, in combination with any of the LCDR3 amino acid sequences listed in table. 1. In accordance with some embodiments, the present invention provides antibodies or antigen binding fragments thereof comprising the HCDR3/LCDR3 amino acid sequence pair contained in any of the exemplary anti-TrkB antibodies listed in Table. 1. In some embodiments, the HCDR3/LCDR3 amino acid sequence pair is selected from the group consisting of: 8/16, 24/32, 40/48, 52/56, 62/66, and 71/75.

The present invention also provides antibodies or antigen-binding fragments thereof that specifically bind TrkB, comprising a combination of six CDRs (i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3) contained in any of the typical anti-TrkB antibodies listed. 044580 listed in the table. 1. In some embodiments, a combination of amino acid sequences

HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 selected from the group consisting of: (a) SEQ ID NO: 4,

6, 8, 12, 14, 16; (b) SEQ ID NO: 20, 22, 24, 28, 30, 32; (c) SEQ ID NO: 36, 38, 40, 44, 46, 48; (d) SEQ ID NO:

50, 51, 52, 54, 55, 56; (e) SEQ ID NO: 60, 61, 62, 64, 65, 66; and (f) SEQ ID NO: 69, 70, 71, 73, 74, 75.

In a similar embodiment, the present invention provides antibodies or antigen-binding fragments thereof that specifically bind TrkB, comprising a combination of six CDRs (i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3) contained in the amino acid sequence pair HCVR/LCVR, in accordance with the definition of any of the typical anti-TrkB antibodies listed in table. 1. For example, the present invention includes antibodies or antigen-binding fragments thereof that specifically bind to TrkB, comprising a combination of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 amino acid sequences contained in the HCVR/LCVR amino acid sequence pair selected from the group consisting of : 2/10, 18/26, 34/42, 49/53, 59/63 and 68/72. Methods and techniques for identifying CDRs in HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs in said HCVR and/or LCVR amino acid sequences described herein. Typical conventions that can be used to identify CDR boundaries include, for example, the Kabat definition, the Chothia definition, and the antibody model definition. In general, 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 approaches. 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). Databases are also available to identify CDR sequences in antibodies.

In one embodiment, the invention provides an isolated antibody or antigen binding fragment thereof that specifically binds TrkB, comprising:

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

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

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

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

(e) an LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 30, 46, 55, 65 and 74; and (f) an LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 32, 48, 56, 66 and 75.

In one embodiment, the invention provides an isolated antibody or antigen binding fragment thereof that specifically binds TrkB, comprising:

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

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

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

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

(e) an LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 30 and 46; and (f) an LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 32 and 48.

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

In one embodiment, an isolated antibody or antigen-binding fragment thereof that binds to TrkB includes an antibody or antigen-binding fragment thereof that competes for binding to TrkB with a reference antibody, wherein the reference antibody comprises an HCVR/LCVR amino acid sequence pair selected from the group, consisting of SEQ ID NO: 2/10, 18/26, 34/42, 49/53, 59/63 and 68/72.

In one embodiment, the isolated antibody or antigen binding fragment thereof that binds TrkB includes an antibody or antigen binding fragment thereof that binds to the same epitope as the reference antibody, wherein the reference antibody comprises a pair of amino acid sequences HCVR/ LCVR selected from the group consisting of SEQ ID NO: 2/10,

18/26, 34/42, 49/53, 59/63 and 68/72.

In one embodiment, the isolated antibody or antigen binding fragment thereof binds to human TrkB with a K _D of less than about 300 nM, as determined by surface plasmon resonance at 25 or 37°C.

In one embodiment, the isolated antibody or antigen binding fragment thereof binds to human TrkB with a K _D of less than about 200 nM, as determined by surface plasmon resonance at 25 or 37°C.

In one embodiment, the isolated antibody or antigen binding fragment thereof binds to human TrkB with a K _D of less than about 150 nM, as determined by surface plasmon resonance at 25 or 37°C.

In one embodiment, the isolated antibody or antigen binding fragment thereof binds to human TrkB with a K _D of less than about 50 nM, as determined by surface plasmon resonance at 25 or 37°C.

In one embodiment, the isolated antibody or antigen binding fragment thereof binds to human TrkB with a K _D of less than about 100 pM, as determined by surface plasmon resonance at 25 or 37°C.

In one embodiment, the isolated antibody or antigen-binding fragment thereof binds to human TrkB with a dissociation half-life (tV ₂ ) greater than about 10 minutes, as determined by surface plasmon resonance at 25 or 37°C.

In one embodiment, the isolated antibody or antigen binding fragment thereof binds to human TrkB with tV ₂ greater than about 40 minutes, as determined by surface plasmon resonance at 25 or 37°C.

In one embodiment, the isolated antibody or antigen binding fragment thereof binds to human TrkB with a t ^1/2 greater than about 120 _minutes , as determined by surface plasmon resonance at 25 or 37°C.

In one embodiment, an isolated antibody or antigen binding fragment thereof that binds TrkB activates human TrkB signaling in the absence of BDNF in cells engineered to express TrkB at an EC ₅₀ of less than about 100 pM.

In one embodiment, an isolated antibody or antigen binding fragment thereof that binds TrkB activates human TrkB signaling in the absence of BDNF in cells engineered to express TrkB at an EC ₅₀ ranging from about 35 pM to about 82 pM.

In one embodiment, an isolated antibody or antigen binding fragment thereof that binds TrkB enhances activation of human TrkB signaling in the presence of BDNF in cells engineered to express TrkB with an EC ₅₀ of less than about 100 pM.

In one embodiment, an isolated antibody or antigen-binding fragment thereof that binds TrkB, which when injected into the hippocampus of humanized TrkB mice, exhibits TrkB activation as evidenced by increased TrkB phosphorylation.

In one embodiment, an isolated antibody or antigen-binding fragment thereof that binds TrkB exhibits activation of the MAPK/ERK and PI3K/Akt signaling pathways, as demonstrated after incubation of primary mouse cortical neurons with an agonistic anti-TrkB antibody.

In one embodiment, an isolated antibody or an antigen-binding fragment thereof that binds to TrkB enhances/increases the survival of retinal ganglion cells, as demonstrated in an optic nerve transection model in TrkB humanized rats.

In one embodiment, the isolated antibody or antigen-binding fragment thereof, which binds to TrkB, enhances the survival of neuronal cells in vitro.

In one embodiment, an isolated antibody or antigen-binding fragment thereof that binds to TrkB helps reduce the weight of humanized TrkB mice.

In one embodiment, an isolated antibody or antigen-binding fragment thereof that binds to TrkB promotes fat loss in humanized TrkB mice.

In one embodiment, an isolated antibody or antigen-binding fragment thereof that binds to TrkB helps reduce food and water intake in humanized TrkB mice.

In one embodiment, an isolated antibody or antigen-binding fragment thereof that binds to TrkB promotes increased locomotor activity in humanized TrkB mice.

In one embodiment, the invention provides an anti-TrkB antibody or antigen binding fragment thereof that specifically binds to TrkB and blocks the binding of TrkB to BDNF with an IC ₅₀ of less than about 5 nM.

- 5 044580

In one embodiment, the invention provides an anti-TrkB antibody or antigen-binding fragment thereof that specifically binds to TrkB and blocks TrkB from binding to

BDNF with an IC ₅₀ of less than about 500 pM.

In one embodiment, the invention provides an anti-TrkB antibody or antigen binding fragment thereof that specifically binds to TrkB and blocks the binding of TrkB to BDNF with an IC ₅₀ of less than about 200 pM.

In a second aspect, the present invention relates to nucleic acid molecules encoding anti-TrkB antibodies or portions thereof. For example, the present invention relates to nucleic acid molecules encoding any of the HCVR amino acid sequences listed in table. 1; in some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table. 2, or a substantially similar sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to that sequence.

The present invention also relates to nucleic acid molecules encoding any of the LCVR amino acid sequences listed in table. 1; in some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table. 2, or a substantially similar sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to that sequence.

The present invention also relates to nucleic acid molecules encoding any of the HCDR1 amino acid sequences listed in table. 1; in some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR1 nucleic acid sequences listed in Table. 2, or a substantially similar sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to that sequence.

The present invention also relates to nucleic acid molecules encoding any of the HCDR2 amino acid sequences listed in table. 1; in some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR2 nucleic acid sequences listed in Table. 2, or a substantially similar sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to that sequence.

The present invention also relates to nucleic acid molecules encoding any of the HCDR3 amino acid sequences listed in table. 1; in some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR3 nucleic acid sequences listed in Table. 2, or a substantially similar sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to that sequence.

The present invention also relates to nucleic acid molecules encoding any of the LCDR1 amino acid sequences listed in table. 1; in some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR1 nucleic acid sequences listed in Table. 2, or a substantially similar sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to that sequence.

The present invention also relates to nucleic acid molecules encoding any of the LCDR2 amino acid sequences listed in table. 1; in some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR2 nucleic acid sequences listed in Table. 2, or a substantially similar sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to that sequence.

The present invention also relates to nucleic acid molecules encoding any of the LCDR3 amino acid sequences listed in table. 1; in some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR3 nucleic acid sequences listed in Table. 2, or a substantially similar sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to that sequence.

The present invention also provides nucleic acid molecules encoding HCVR, wherein HCVR contains a combination of three CDRs (i.e., HCDR1, HCDR2, HCDR3), wherein the combination of amino acid sequences HCDR1, HCDR2, HCDR3 corresponds to the requirements of any of the typical anti-TRKB antibodies, listed in table. 1.

The present invention also provides nucleic acid molecules encoding LCVR, wherein LCVR contains a combination of three CDRs (i.e., LCDR1, LCDR2, LCDR3), wherein the combination of amino acid sequences LCDR1, LCDR2, LCDR3 is in accordance with the definition of any of the typical anti -TRKB antibodies listed in table. 1.

The present invention also relates to nucleic acid molecules encoding both HCVR and LCVR, where HCVR contains the amino acid sequence of any of the HCVR amino acid sequences listed in table. 1, and where LCVR includes the amino acid sequence of any of the LCVR amino acid sequences listed in table. 1. In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table. 2, or a substantially similar sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to this sequence, and a polynucleotide sequence, selected from any of the LCVR nucleic acid sequences listed in table. 2, or a substantially similar sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to that sequence. In some embodiments, in accordance with this aspect of the invention, the nucleic acid molecule encodes HCVR and LCVR, where both HCVR and LCVR are derived from the same anti-TrkB antibody specified in table. 1.

In a third aspect, the present invention provides recombinant expression vectors capable of expressing a polypeptide comprising an anti-TrkB antibody heavy or light chain variable region. For example, the present invention includes recombinant expression vectors containing any of the above nucleic acid molecules, that is, nucleic acid molecules encoding any of the HCVR, LCVR and/or CDR sequences as specified in table. 1. Also included within the scope of the present invention are the host cells into which such vectors have been introduced, as well as methods for producing antibodies or portions thereof by culturing the host cells under conditions allowing the production of antibodies or antibody fragments and recovering the antibodies and fragments thereof produced Thus.

The present invention includes anti-TrkB antibodies having a modified glycosylation profile. In some embodiments, a modification may be used to remove unwanted glycosylation sites, or an antibody lacking a fucose moiety present on the oligosaccharide chain, for example, to enhance antibody-dependent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC) . 277:26733). In other applications, modification of galactosylation can be carried out to modify complement dependent cytotoxicity (CDC).

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

In a related aspect, the invention provides a composition that 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 preferably combined with an anti-TrkB antibody. The second therapeutic agent may be used to alleviate at least one symptom of a neurodegenerative disease or disorder.

In a fifth aspect, the invention relates to a method of enhancing TrkB-mediated biological activity, comprising contacting TrkB with a biologically effective amount of an agonistic anti-TrkB antibody set forth in Table. 1, or contact of TrkB with a pharmaceutical composition containing a biologically effective amount of an agonistic anti-TrkB antibody presented in table. 1.

In some embodiments, the biological activity is neuronal protection or neuronal survival, and neuronal protection or neuronal survival is enhanced by contact of TrkB with an agonistic anti-TrkB antibody.

In some embodiments, the biological activity is neuroprotection and survival of retinal ganglion cells (RGC).

In a sixth aspect, the invention provides therapeutic methods for treating a disease or disorder associated with TrkB activity or expression, or at least one symptom associated with the disease or disorder, using an anti-TrkB antibody or antigen-binding portion of an antibody of the invention. Therapeutic methods in accordance with this aspect of the invention include administering a therapeutically effective amount of a pharmaceutical composition containing an antibody or antigen binding fragment of an antibody of the invention to a subject in need of such treatment. A treatable disorder is any disease or condition that is regressed, improved, inhibited or prevented by targeting TrkB and/or by activating TrkB-mediated cellular signaling.

In one embodiment, the anti-TrkB antibodies of the invention may provide a method for preventing damage or death of retinal neurons. In one embodiment, the anti-TrkB antibodies of the invention may provide a method of treating pathological diseases,

- 7 044580 in which retinal degeneration occurs. In one embodiment, the anti-TrkB antibodies of the invention may provide a method of treating a living eye before or after ophthalmic surgery, exposure to light, or other exogenous trauma, thereby preventing degeneration of retinal cells. In one embodiment, the anti-TrkB antibodies of the invention may provide a method of preventing photoreceptor damage and degeneration in a living eye. In one embodiment, the anti-TrkB antibodies of the invention may provide a method of protecting retinal neurons without causing side effects, possibly due to cross-reactivity with other receptors, such as the p75 receptor. In one embodiment, the anti-TrkB antibodies of the invention may provide a method for allowing damaged photoreceptors to repair or regenerate.

In some embodiments, the disease or disorder to be treated by an antibody of the invention is an ocular disease or disorder selected from the group consisting of glaucoma, diabetic retinopathy, age-related macular degeneration, ischemic optic neuropathy, optic neuritis, retinal ischemia, photoreceptor degeneration, retinitis pigmentosa, Leber congenital amaurosis, Leber hereditary optic neuropathy, Usher syndrome, Stargard disease, and retinal artery or vein occlusion.

Other pathological conditions amenable to treatment by one or more anti-TrkB antibodies of the invention include retinal detachments, photic retinopathy caused by surgery (mechanical or light-induced), toxic retinopathy, retinopathy of prematurity, viral retinopathy such as cytomegalovirus or HIV-related retinopathy. with AIDS; uveitis; ischemic retinopathy caused by venous or arterial occlusion or other vascular disorder, retinopathy caused by trauma or penetrating ocular lesions, peripheral vitreoretinopathy or hereditary retinal degenerations.

In one embodiment, the ocular disease or disorder to be treated with an agonistic anti-TrkB antibody of the invention is glaucoma.

The seventh aspect of the invention involves providing a reduction in body weight in a subject, the method comprising administering to the subject a TrkB agonist antibody described in table. 1, or a pharmaceutical composition containing an antibody or an antigen-binding fragment thereof.

In a related aspect, the invention relates to a method of providing a reduction in adipose tissue mass in a subject, comprising administering a TrkB agonist antibody described in table. 1, or a pharmaceutical composition containing an antibody or an antigen-binding fragment thereof.

An eighth aspect of the invention relates to a method of promoting neuronal survival in a subject, comprising administering to the subject a therapeutically effective amount of a TrkB agonist antibody set forth in Table. 1, or a pharmaceutical composition containing a therapeutically effective amount of an antibody or an antigen-binding fragment thereof.

In one embodiment, the methods described above can be carried out by administering an agonistic anti-TrkB antibody or an antigen binding fragment thereof to a subject in need of such administration, wherein the agonistic anti-TrkB antibody comprises three heavy chain complementarity determining regions (CDRs) (HCDR1 , HCDR2 and HCDR3), contained in the heavy chain variable region (HCVR), containing the amino acid sequence presented in table. 1, or a substantially similar sequence that is at least 90% identical to this sequence; and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained in a light chain variable region (LCVR) containing the amino acid sequence shown in table. 1, or a substantially similar sequence that is at least 90% identical to this sequence.

In one embodiment, the methods of the invention can be carried out by administering a TrkB agonist antibody of the invention, wherein the antibody or antigen binding fragment thereof comprises three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained in any of the HCVR sequences selected from the group consisting of SEQ ID NOs: 2, 18, 34, 49, 59 and 68, or a substantially similar sequence that is at least 90% identical to this sequence; and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained in any of the LCVR sequences selected from the group consisting of SEQ ID NOs: 10, 26, 42, 53, 63 and 72, or a substantially similar sequence that, is at least 90% identical to this sequence.

In one embodiment, the antibody or antigen binding fragment thereof comprises HCVRs having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 49, 59 and 68.

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

In one embodiment, the antibody or antigen binding fragment thereof comprises an HCVR having an amino acid sequence selected from the group consisting of SEQ ID

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NO: 2, 18, 34, 49, 59 and 68; and LCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 53, 63 and 72.

In one embodiment, the antibody or antigen binding fragment thereof comprises

CDR of a pair of amino acid sequences HCVR/LCVR selected from the group consisting of

SEQ ID NO: 2/10, 18/26, 34/42, 49/53, 59/63 and 68/72.

In one embodiment, the antibody or antigen binding fragment thereof comprises a pair of amino acid sequences HCVR/LCVR selected from the group consisting of SEQ ID NO: 2/10, 18/26, 34/42, 49/53, 59/63 and 68 /72.

In one embodiment, the antibody or antigen binding fragment thereof comprises:

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

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

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

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

(e) an LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 30, 46, 55, 65 and 74; and (f) an LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 32, 48, 56, 66 and 75.

In one embodiment, the antibody or antigen binding fragment thereof comprises a combination of six CDRs (HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) selected from the group consisting of: (a) SEQ ID NO: 4-6-8- 12-14-16; (b) SEQ ID NO: 20-22-24-28-30-32; (c) SEQ ID NO: 36-38-4044-46-48; (d) SEQ ID NO: 50-51-52-54-55-56; (e) SEQ ID NO: 60-61-62-64-65-66; and (f) SEQ ID NO: 69-7071-73-74-75.

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

It is contemplated that any disease or disorder associated with TrkB activity or expression will be treatable with an antibody of the invention. These diseases can include any disease in which there is cell degradation, such as a neurodegenerative condition or after nerve damage.

Other embodiments will become apparent from a review of the following complete description.

Brief description of the figures

Fig. 1. Shown are Western blots assessing total TRKB levels and phospho-TRKB levels in homozygous humanized TRKB mice 1 h, 4 h, and 18 h after direct hippocampal injection of TRKB agonist antibody H4H9816P2 or isotype control antibody.

Fig. 2. The TrkB agonist antibody, H4H9816P2, has been shown to activate the downstream portions of the MAPK/ERK and PI3K/Akt signaling pathway. In fig. shows Western blots of phospho-TrkB, total TrkB, phosphoAkt, total AKT, phospho-ERK, and total ERK 15 min and 2 h after treatment of primary cortical neurons obtained on postnatal day 1 from homozygous humanized TRKB mice with various agonist antibodies TrkB or BDNF.

Fig. 3. It was shown that three TrkB agonist antibodies, depending on the dose, increased the survival of SH-SY5Y cells in vitro. Isotype control antibodies had no effect on cell survival.

Fig. 4. Pharmacokinetic profiles of the agonistic anti-TrkB antibody H4H9816P2 in TrkB and wild-type mice are shown. Mice were administered a single subcutaneous dose of 10 mg/kg on day 0. Serum concentrations of total H4H9816P2 were measured by the Gyros immunoassay. The dose data points at 6 hours, 1, 2, 3, 6, 9, 16, 21, and 30 days indicate the mean antibody concentration. Total concentrations of H4H9816P2 antibodies are represented by black solid circles for TrkBhu/hu mice and solid black squares for wild-type mice. Data are presented as mean + standard deviation.

Fig. 5. Includes FIG. 5A and FIG. 5B, which shows the ability of mouse monoclonal anti-TrkB antibodies to block the interaction of mouse or rat TrkB with its ligand BDNF (brain-derived neurotrophic factor). ELISA-based methods were used to assess the binding of (Figure 5A with two graphs) mouse TrkB.hFc and (Figure 5B with two graphs) rat TrkB.mmh to plate-coating BDNF at various concentrations of anti-mouse TrkB antibody and monoclonal antibodies (mAb) isotype control. In fig. 5A with two graphs shows a dose-response curve describing the binding of mouse TrkB.hFc (REGN2277) to BDNF with an EC5 value of _0.780 pM. In fig. 5B with two graphs shows a dose-response curve describing the binding of rat TrkB.mmh (REGN1808) to BDNF with an EC50 value _of 2.2 nM. Molarity (M) indicates the antibody concentration for the mAb. The whiskers correspond to the standard deviation.

Full description

Before considering the present invention, it should be understood that this invention is not limited to the specific methods and experimental conditions described, since such methods and conditions may vary. It should also be understood that the terminology used herein is intended to describe specific embodiments only and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention relates. The term about, as used herein, when used in relation to a specific numerical value given, means that the value may differ from the given value by no more than 1%. For example, as used herein, the expression about 100 includes 99 and 101 and all values in between (eg, 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and substances similar or equivalent to those described herein may be used in the practice or testing of the present invention, preferred methods and substances are described herein. All patents, applications and non-patent publications set forth in this specification are incorporated herein by reference in their entirety.

Definitions

The expression TrkB and the like, also known as tropomyosin tyrosine kinase receptor B, refers to a human receptor (unless designated as belonging to another species) containing the amino acid sequence represented at amino acid residues 32 to 430 with accession number NP_001018074.1. Human TrkB containing a myc-myc-hexahistidine tag is shown as SEQ ID NO: 76 (amino acid residues 1-399 correspond to human TrkB and amino acid residues 400-427 correspond to the myc-myc-hexahistidine tag). Other formats containing human TrkB proteins are described herein, including SEQ ID NO: 77, corresponding to human TrkB (residues 1-399) with a mouse Fc region (residues 400-632); and SEQ ID NO: 78, corresponding to human TrkB (residues 1-399) with the human Fc region (residues 400-626). The TrkB target contains an amino acid sequence represented in amino acid residues 32 to 429 with accession number NP_001020245. Mouse TrkB containing a myc-myc-hexahistidine tag is shown as SEQ ID NO: 79 (amino acid residues 1-398 correspond to mouse TrkB, and amino acid residues 399-426 correspond to the myc-myc-hexahistidine tag). Other formats containing mouse TrkB proteins are described herein, including SEQ ID NO: 80 corresponding to mouse TrkB (residues 1-398) with a mouse Fc region (residues 399-631); and SEQ ID NO: 81, corresponding to mouse TrkB (residues 1-398) with the human Fc region (residues 399-625). Rabbit TrkB contains an amino acid sequence represented in amino acid residues 32 to 430 with accession number XP_002721319.1. Rabbit TrkB containing the myc-myc-hexahistidine tag is shown as SEQ ID NO: 82 (amino acid residues 1-399 correspond to rabbit TrkB, and amino acid residues 400-427 correspond to the myc-myc-hexahistidine tag). Other formats containing rabbit TrkB proteins are described herein, including SEQ ID NO: 83, corresponding to rabbit TrkB (residues 1399) with a mouse Fc region (residues 400-632). Rat TrkB contains the amino acid sequence represented at amino acid residues 32 to 429 with accession number NP_036863.1. Rat TrkB containing the myc-myc-hexahistidine tag is shown as SEQ ID NO: 84 (with amino acid residues 1-398 corresponding to rat TrkB and amino acid residues 399-426 corresponding to the myc-myc-hexahistidine tag). Other formats containing rat TrkB proteins are described herein, including SEQ ID NO: 85 corresponding to rat TrkB (residues 1-398) with the mouse F region (residues 399-631). Rhesus monkey (Macaca mulatta) TrkB is shown as SEQ ID NO: 95 (amino acids 32 to 838 with accession number NP_001248226.1), and Cynomolgus monkey (Macaca flavicularis) TrkB is shown as SEQ ID NO: 96 (amino acids 32 to 838 ) with access number ХР_005582102.1).

The human TrkA protein is presented as SEQ ID NO: 86, with amino acids 1-375 corresponding to TrkA (amino acids 34-414 with accession number NP_001012331.1 with V263L, C300S), amino acids 376-378 corresponding to the GPG linker and amino acids 379-605 corresponding Human Fc.

The human TrkC protein is shown as SEQ ID NO: 87, with amino acids 1-398 corresponding to TrkC (amino acids 32-429 with accession number NP_001012338.1) and amino acids 399-426 corresponding to the myc-myc-histidine tag.

The mouse TrkC protein is shown as SEQ ID NO: 88, where amino acids 1-398 correspond to TrkC (amino acids 32-429 with accession number NP_032772.3) and amino acids 399-426 correspond to the myc-myc-histidine tag.

The cynomolgus TrkC protein is shown as SEQ ID NO: 89, where amino acids 1-398 correspond to TrkC (amino acids 32-429 with accession number XP_015308837.1) and amino acids 399-426 correspond to the myc-myc-histidine tag.

- 10 044580

In some cases, cell lines were obtained that expressed TrkB proteins, including the ectodomain, as well as the transmembrane and cytoplasmic domains of the TrkB protein. For example, SEQ ID NO: 91 is a human TrkB protein containing all three domains contained in amino acids 32-822 (accession number NP_001018074.1) or Uniprot Q16620-1, with amino acids 1398 being the ectodomain and the transmembrane/cytoplasmic region being defined , approximately, amino acid residues 399-790. In one case, a TrkB cell line was generated that expresses mouse TrkB (amino acids 32-476 with accession number NP_032771.1; see also SEQ ID NO: 92). In another case, a cell line was generated that expresses a chimeric TrkB protein with the mouse TrkB ectodomain of amino acids 32-429 with accession number NP_OO 1020245.1 (see also SEQ ID NO: 93) or Uniprot database number P15209-1, and transmembrane and human TrkB cytoplasmic domains (amino acids 431-822 with accession number NP_001018074.1 (see also SEQ ID NO: 91)). A cell line was also generated that expresses African green monkey (Chlorocebus sabaeus) TrkB (amino acids 32-822 with accession number XP_007967815.1 (see also SEQ ID NO: 94)).

The term brain-derived neurotrophic factor or BDNF refers to the ligand for TrkB, and the amino acid sequence of BDNF is shown in SEQ ID NO: 90 (isoform A 1-120, with amino acids 129-247 with accession number NP_733928.1 with the addition of Met at the N-terminus ). In some of the experiments described herein, the supplier of BDNF is R&D Systems, 248-BD/CF.

All references to proteins, polypeptides and protein fragments herein are intended to refer to the human variant of the corresponding protein, polypeptide or protein fragment unless specifically stated to be non-human. Thus, the expression TrkB means human TrkB unless specified as non-human species, eg monkey TrkB, mouse TrkB, rat TrkB, etc.

As used herein, the expression anti-TrkB antibody includes both monovalent antibodies with a single specificity and bispecific antibodies comprising a first arm that binds TrkB and a second arm that binds a second (target) antigen, wherein the anti-TrkB arm comprises any of HCVR/LCVR or CDR sequences, as indicated in table. 1 in this document. Anti-TrkB antibody expression also includes antibody-drug conjugates (ADCs) containing an anti-TrkB antibody or antigen-binding portion thereof conjugated to a drug or toxin (ie, a cytotoxic agent). Anti-TrkB antibody expression also includes antibody-radionuclide conjugates (ARCs) containing an anti-TrkB antibody or antigen-binding portion thereof conjugated to a radionuclide.

As used herein, the term anti-TrkB antibody means any antigen-binding molecule or molecular complex containing at least one complementarity determining region (CDR) that specifically binds or interacts with TrkB or a region of TrkB. The term antibody includes immunoglobulin molecules containing four polypeptide chains, two heavy (H) chains and two light (L) chains linked by disulfide bonds, as well as their multimers (for example, IgM). Each heavy chain contains a heavy chain variable region (abbreviated here as HCVR or VH) and a heavy chain constant region. The heavy chain constant region includes three domains, CH1, CH2 and CH3. Each light chain contains a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region contains one domain (CL1). The V _H and V _L regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), inserted into regions that are more conserved, called framework regions (FR). Each VH and VL consists of three CDRs and four FRs, located from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In various embodiments of the invention, the anti-TrkB antibody FR (or antigen binding region thereof) may be identical to human germline sequences or may be naturally or artificially modified. The amino acid consensus sequence can be determined based on parallel analysis of two or more CDRs.

As used herein, the term antibody also includes antigen-binding fragments of full-length antibody molecules. The terms antigen binding region of an antibody, antigen binding fragment of an antibody, and the like as used herein include any enzymatically produced, synthetic or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding antibody fragments can be prepared, for example, from whole antibody molecules using any suitable standard techniques, such as proteolytic digestion or recombinant genetic engineering techniques involving manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA is known and publicly available, for example from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated using chemical methods or using molecular biology techniques, for example, to arrange one or more variable and/or constant domains in a suitable configuration or to introduce codons, create cysteine residues, modifications, insertions or deletions of amino acids, etc.

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 molecules (scFv); (vi) dAb fragments; and (vii) minimal recognition components consisting of amino acid residues that mimic the hypervariable region of an antibody (eg, an isolated complementarity determining region (CDR), such as the CDR3 peptide), or the FR3-CDR3FR4 restricted peptide. Other engineered molecules such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, tri-bodies, tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies, divalent nanobodies, etc.) , small modular protein (SMIP) immunopharmaceuticals and shark IgNAR variable domains are also included in the expression antigen binding fragment used herein.

The antigen binding fragment of an antibody typically contains at least one variable domain. The variable domain can be of any size or amino acid composition and will typically contain at least one CDR adjacent or in frame to one or more framework sequences. In antigen binding fragments having a VH domain associated with a VL domain, the VH and VL domains may be arranged relative to each other in any suitable order. For example, the variable region may be dimeric and contain VH-VH, VH-VL or VL-VL dimers. Alternatively, the antigen binding fragment of the antibody may comprise a monomeric _VH or VL domain.

In some embodiments, an antigen binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found in an antigen binding fragment of an antibody of the present invention include: (i) VH-CH1; (ii) VH-CH2; (iii) VH-CH3; (iv) VH-CH1-CH2; (v) VH-CH1-CH2-CH3; (vi) V _H Ch2-Ch3; (vii) Vh-Cl; (viii) Vl-Ch1; (ix) Vl-Ch2; (x) Vl-Ch3; (xi) Vl-Ch1-Ch2; (xii) VL-Ch1-Ch2-Ch3; (xiii) V _l -C _h 2-C _h 3; and (xiv) V _L -C _L . In any configuration of variable and constant domains, including any of the typical configurations listed above, the variable and constant domains can either be directly linked to each other or can be linked through a full or partial hinge or linker region. The hinge region may consist of at least 2 (eg, 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 in a single polypeptide molecule. In addition, the antigen binding fragment of an antibody of the present invention may contain a homodimer or heterodimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with each other and/or with one or more monomeric V _H or V _L domains (eg via disulfide bond(s)).

For full antibody molecules, antigen binding fragments may be monospecific or multispecific (eg, bispecific). A polyspecific antigen-binding antibody fragment will typically contain at least two different variable domains, with each variable domain capable of specifically binding to a different antigen or to a different epitope on the same antigen. Any multispecific antibody format, including examples of bispecific antibody formats described herein, can be adapted for use in the context of an antigen binding fragment of an antibody of the present invention using routine techniques available in the art.

In some cases, it may be desirable to antagonize TrkB, for example, to inhibit the growth or proliferation of, for example, a neuronal tumor cell. However, the antibodies of the present invention act as agonistic antibodies that serve as neuronal survival enhancers and neuroprotectors. The antibodies of the present invention may function by enhancing the interaction between TrkB and its ligand, BDNF. Alternatively, the antibodies of the invention may mediate TrkB signaling through a mechanism that does not involve enhancing the interaction of TrkB with its ligand.

The term human antibody as used herein is intended to include non-naturally occurring human antibodies. The term includes antibodies that are recombinantly produced in a non-human mammal or in cells of a non-human mammal. The term is not intended to include antibodies isolated or generated in humans.

Antibodies of the invention, in some embodiments, may be recombinant and/or non-naturally occurring human antibodies. The term recombinant human antibody as used herein includes all human antibodies that are produced, expressed, created or isolated by recombinant means, for example, antibodies expressed using a recombinant expression vector introduced into a host cell (described further below ), antibodies derived from a combinatorial library of human recombinant antibodies (described further below), antibodies isolated from an animal (eg mouse) that is transgenic for human immunoglobulin genes (see, for example, Taylor et al. (1992) Nucl Acids Res. 20: 6287-6295), or antibodies produced, expressed, generated or isolated by any other means that involve splicing human immunoglobulin gene sequences into other DNA sequences. In some embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, if an animal transgenic for human Ig sequences is used, to somatic mutagenesis in vivo) and thus the amino acid sequences of the VH and _VL regions of the recombinant antibodies are sequences that, since they are isolated from and are close to human germline VH and _VL sequences, they cannot naturally exist in the germline human antibody repertoire in vivo.

Human antibodies can exist in two forms, which are associated with heterogeneity of the hinge region. In one form, the immunoglobulin molecule contains a stable four-chain construct of about 150-160 kDa in which the dimers are held together by heavy chain disulfide bond interactions. In the second form, the dimers are not linked by interchain disulfide bonds and a molecule of about 75-80 kDa is formed, composed of a covalently linked light and heavy chain (half-antibody). These forms are extremely difficult to separate, even after affinity purification.

The frequency of occurrence of the second form in different intact IgG isotypes is due to, but is not limited to, structural differences associated with the isotype of the hinge region of that antibody. Substitution of a single amino acid 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 using the human IgG1 hinge. The present invention covers antibodies having one or more mutations in the hinge region, the C _H 2 or C _H 3 region, which may be desirable, for example, in production, to improve the yield of the desired form of the antibody.

The term specific binding or specifically binds to or the like means that the antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding may have an equilibrium dissociation constant of at least about 1 x 10'6 M or less (eg, a lower K _D value indicates stronger binding). Methods for determining the binding specificity of two molecules are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. As described herein, antibodies that specifically bind to TrkB have been identified by surface plasmon resonance, such as BIACORE™. In addition, multispecific antibodies that bind to the TrkB protein and one or more additional antigens, or bi-specific antibodies that bind to two different regions of TrkB, are nevertheless considered to be antibodies that specifically bind in the context of this document.

The antibodies of the invention may be isolated antibodies. As used herein, the term isolated antibody means an antibody that has been identified and separated and/or recovered from at least one component of its natural environment. For example, an antibody that has been separated or removed from at least one component of the body or from a tissue or cell in which the antibody exists or is naturally produced is an isolated antibody within the scope of the present invention. The isolated antibody also includes the antibody in situ in the recombinant cell. Isolated antibodies are antibodies that have been subjected to at least one purification or isolation step. In accordance with some embodiments, the isolated antibody may be substantially free of other cellular material and/or chemicals.

The anti-TrkB antibodies described herein may contain one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains. Such mutations can be readily identified by comparing the amino acid sequences described herein with sequences available, for example, from publicly available antibody sequence databases. Once produced, antibodies and antigen binding fragments that contain one or more mutations can be readily assayed for the presence of one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as appropriate). cases), reduced immunogenicity, etc. Antibodies and antigen binding fragments produced by this general method are included in the present invention.

The present invention also includes anti-TrkB antibodies containing variants of any of the HCVR, LCVR and/or CDR amino acid sequences described herein with one or more conservative substitutions. For example, the present invention includes anti- 13 044580

TrkB antibodies containing the amino acid sequences HCVR, LCVR and/or CDR, for example, with or less, 8 or less, 6 or less, 4 or less, etc. conservative amino acid substitutions relative to any of the amino acid sequences HCVR, LCVR and/or CDR listed in table. 1 in this document.

The term epitope refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, known as a paratope. A single antigen may have more than one epitope. Accordingly, different antibodies can bind to different sites on the antigen and can have different biological effects. Epitopes can be conformational or linear. A conformational epitope is produced by spatially adjacent amino acids from different segments of a linear polypeptide chain. A linear epitope is an epitope produced by adjacent amino acid residues in a polypeptide chain. Under certain circumstances, the epitope may include moieties of saccharides, phosphoryl groups, or sulfonyl groups of the antigen.

The term substantial identity or substantially identical, when referring to a nucleic acid or fragment thereof, indicates that, when optimally matched to suitable nucleotide insertions or deletions with another nucleic acid molecule (or its complementary strand), there is a nucleotide sequence identity of at least at least about 95%, or 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 . A nucleic acid molecule having substantial identity with a reference nucleic acid molecule may, in some cases, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

As applied to polypeptides, the term substantially identical or substantially identical means that two peptide sequences, when optimally aligned, for example by the GAP or BESTFIT programs using default deletion weights, have at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity. Preferably, positions of residues that are not identical are distinguished by conservative amino acid substitutions. A conservative amino acid substitution is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (eg, charge or hydrophobicity). In general, a conservative amino acid substitution does not significantly change the functional properties of the protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percentage sequence identity or degree of identity may be adjusted upward to correct for the conservative properties of the substitution. Means for making this adjustment are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24: 307-331, incorporated herein by reference. Examples of groups of amino acids that have side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine and tryptophan; (5) main side chains: lysine, arginine and histidine; (6) acid side chains: aspartate and glutamate, and (7) sulfur side chains: cysteine and methionine. Preferred groups of conservative amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate and asparagine-glutamine. Alternatively, a conservative replacement is any change that has a positive value in the PAM250 log-likelihood matrix described in Gonnet et al. (1992) Science 256: 1443-1445, incorporated herein by reference. A moderately conservative replacement is any change that has a non-negative value when estimated using the PAM250 log-likelihood function matrix.

Sequence similarity for polypeptides, also called sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using similarity measurements assigned to various substitutions, deletions and other modifications, including amino acid substitutions. For example, GCG software contains programs such as Gap and Bestfit that can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species or between a wild-type protein and its mutein. See, for example, GCG Version 6.1. Polypeptide sequences can also be compared by FASTA using the default or recommended parameters program in GCG Version 6.1. FASTA (eg FASTA2 and FASTA3) provides

- 14 044580 matches and percent sequence identity of the best match regions between the query sequences and the search sequences (Pearson (2000) supra). Another preferred algorithm when comparing a sequence of the invention to a database containing a large number of sequences from different organisms is the BLAST computer program, in particular 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. 25:3389402, each of which is incorporated herein by reference.

Biological characteristics of antibodies

The present invention includes anti-TrkB antibodies that bind to human TrkB with a KD of less than about 200 nM, as determined by surface plasmon resonance at 25°C or 37°C. In accordance with some embodiments, the invention includes anti-TrkB antibodies that bind to human TrkB with a K _D value of less than about 600 pM, less than about 300 pM, less than about 200 pM, less than about 150 pM, less than about 100 pM, less than about 80 pM, less than about 50 pM, less than about 40 pM, less than about 30 pM, less than about 20 pM, less than about 10 pM, less than about 5 pM, less than about 3 pM or less than about 1 pM.

The present invention includes anti-TrkB antibodies that bind to human TrkB with a dissociation half-life (t1/ ₂ ) greater than about 10 minutes, as determined by surface plasmon resonance at 25°C or 37°C. In accordance with some embodiments, the invention includes anti-TrkB antibodies that bind to human TrkB with a t ^1/2 greater than about ₂₀ minutes, greater than about 50 minutes, greater than about 100 minutes, greater than about 120 min, more than about 150 min, more than about 300 min, more than about 350 min, more than about 400 min, more than about 450 min, more than about 500 min, more than about 550 min , 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.

The present invention includes anti-TrkB antibodies that may or may not bind to monkey, mouse or rat TrkB. As used herein, an antibody does not bind to a particular antigen (e.g., monkey, mouse, or rat TrkB if the antibody, when tested in an antigen binding assay such as surface plasmon resonance, exhibits a K _D greater than about 1000 nM or does not exhibit any antigen binding in such an assay.Another assay format that can be used to determine whether an antibody binds or does not bind to a particular antigen, according to this aspect of the invention, is an ELISA.

The present invention includes anti-TrkB antibodies that activate human TrkB signaling in cells engineered to express a TrkB receptor with an EC ₅₀ of less than about 100 pM. Using the assay format described in Example 5, or a substantially similar assay format, the EC ₅₀ value can be calculated as the concentration of antibody required to activate TrkB-mediated signaling to the half-maximal signal observed. Thus, in accordance with some embodiments, the invention includes anti-TrkB antibodies that mediate human TrkB signaling in cells engineered to express the TrkB receptor in the presence or absence of BDNF with an EC ₅₀ of less than about 500 pM, less than about 400 pM , less than about 300 pM, less than about 200 pM, less than about 100 pM, less than about 90 pM, less than about 80 pM, less than about 70 pM, less than about 60 pM, less than about 50 pM, less 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 determined using the assay format described in Example 5 herein, or a substantially similar assay.

The present invention includes anti-TrkB antibodies that activate the TrkB receptor, as demonstrated by phosphorylation of TrkB following direct hippocampal injection into mice humanized to express the human TrkB receptor, as shown in Example 6.

The present invention includes anti-TrkB antibodies that promote weight loss in mice humanized to express the human TrkB receptor. The antibodies of the invention also serve to accelerate fat loss and increase locomotor activity in these mice while reducing food and water intake (see Example 7).

Antibodies of the invention promote the survival of retinal ganglion cells (RGCs) in rats humanized to express the human TrkB receptor in an optic nerve transection model. See example 8.

Antibodies of the invention activate downstream portions of the MAPK/ERK and PI3K/Akt signaling pathway, as demonstrated by exposure of primary murine cortical neurons derived from TrkB humanized mice to antibodies of the invention (see Example 9).

The agonistic anti-TrkB antibodies of the invention also promote the survival of SHSY5Y cells in a dose-dependent manner, as shown in Example 10.

The present invention includes anti-TrkB antibodies that block the binding of TrkB to

- 15 044580

BDNF has an IC ₅₀ of less than about 5 nM. For example, as shown in Example 12, all three antibodies tested blocked >50% of mouse or rat TrkB binding to BDNF. Using the assay format described in Example 12, or a substantially similar assay format, the IC ₅₀ value can be calculated as the concentration of antibody required to block TrkB binding to BDNF compared to the maximum signal observed in the absence of antibody. Thus, in accordance with some embodiments, the invention includes anti-TrkB antibodies that block the binding of TrkB to BDNF with an IC ₅₀ of less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, less than about 900 pM, less than about 800 pM, less than about 700 pM, less than about 600 pM, 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 determined using the assay format described in Example 12 herein, or a substantially similar assay. In one embodiment, the anti-TrkB antibodies of the invention block the binding of TrkB to BDNF with an IC ₅₀ ranging from about 180 pM to about 4 nM.

The binding characteristic of an antibody of the invention (e.g., any of the binding characteristics specified herein above), when described in terms of measurement by surface plasmon resonance, means that the corresponding binding characteristic relating to the interaction between the antibody and the antigen is determined using the instrument surface plasmon resonance (eg, Biacore® instrument, GE Healthcare), using standard Biacore assay conditions as shown in Examples 3 and 4, or a substantially similar assay format. In some embodiments, binding parameters are determined at 25°C, while in other embodiments, binding parameters are determined at 37°C.

The present invention includes antibodies or antigen-binding fragments thereof that specifically bind to TrkB, including HCVR and/or LCVR, comprising an amino acid sequence selected from any of the amino acid sequences of HCVR and/or LCVR listed in table. 1.

Antibodies of the present invention may have one or more of the above biological characteristics or any combination thereof. The above list of biological characteristics of antibodies according to the invention is not exhaustive. Other biological characteristics of the antibodies of the present invention will be apparent to one skilled in the art from a review of the present disclosure, including the worked examples provided herein.

Epitope mapping and related technologies

The epitope to which the antibodies of the present invention bind may consist of one contiguous sequence of 3 or more (for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids of the TrkB protein. Alternatively, the epitope may consist of multiple non-contiguous 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 a surface of TrkB that does not interact with a TrkB ligand, for example, at a location on the surface of TrkB at which an antibody, when bound to such an epitope, does not interfere with the interaction between TrkB and its ligand.

Various methods known to those skilled in the art can be used to determine whether an antibody reacts with one or more amino acids in a polypeptide or protein. Typical methods include, for example, routine epitope cross-competition assays such as those described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY), analysis of alanine scan mutants, peptide blotting (Reineke, 2004, Methods Mol Biol 248: 443-463) and peptide cleavage assay. In addition, techniques such as epitope removal, epitope extraction, and chemical modification of antigens can be used (Tomer, 2000, Protein Science 9: 487-496). Another method that can be used to identify amino acids in a polypeptide with which an antibody reacts is hydrogen-deuterium exchange, detected by mass spectrometry. In general terms, the hydrogen-deuterium exchange method involves deuterium labeling a protein of interest, followed by binding of an antibody to the deuterium-labeled protein. The protein/antibody complex is then transferred to water to exchange hydrogen with deuterium on all residues except the antibody-protected residues (which remain deuterium-labeled). After dissociation of the antibody, the target protein is subjected to protease digestion and mass spectrometric analysis, thereby identifying deuterium-labeled residues that correspond to the specific amino acids with which the antibody interacts. See, for example, Ehring (1999) Analytical Biochemistry 267(2):252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A.

The present invention includes anti-TrkB antibodies that bind to the same epitope as any of the specific exemplary antibodies described herein (e.g., antibodies containing any of the amino acid sequences set forth in Table 1 herein). ). Likewise, the present invention also includes anti-TrkB antibodies that compete for binding to TrkB with any of the exemplary specific antibodies described herein (eg, antibodies containing any of the amino acid sequences set forth in Table 1 herein).

Whether an antibody binds to the same epitope or competes for binding with a reference anti-TrkB antibody can be readily determined using routine methods known in the art and exemplified herein. For example, to determine whether a test antibody binds to the same epitope as a reference anti-TrkB antibody of the invention, the reference antibody is allowed to bind to the TrkB protein. The ability of the test antibody to bind to the TrkB molecule is then assessed. If the test antibody is able to bind to TrkB after binding saturation with the reference anti-TrkB antibody, it can be concluded that the test antibody, rather than the reference anti-TrkB antibody, binds to a different epitope. On the other hand, if the test antibody is unable to bind to the TrkB molecule after saturating binding to the reference anti-TrkB antibody, then the test antibody may bind to the same epitope as the epitope bound to the reference anti-TrkB antibody of the invention. Additional routine experiments (eg, peptide mutation and binding assays) can then be performed to confirm whether the observed lack of binding of the test antibody is actually due to binding to the same epitope as the reference antibody, or whether the lack of observed binding is due to steric blocking (or another phenomenon). Experiments of this nature can be performed using ELISA, RIA, Biacore, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art. In accordance with some embodiments of the present invention, two antibodies bind to the same (or overlapping) epitope if, for example, a 1-, 5-, 10-, 20-, or 100-fold excess of one antibody inhibits the binding of the other, according to at least 50%, but preferably 75%, 90% or even 99%, as determined in a competitive binding assay (see, for example, Junghans et al., Cancer Res. 1990:50:1495-1502). Alternatively, two antibodies are considered to bind to the same epitope if substantially all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other. Two antibodies are considered to have overlapping epitopes only if a subset of amino acid mutations that weaken or eliminate the binding of one antibody weaken or eliminate the binding of the other.

To determine whether an antibody competes for binding (or cross-competes for binding) with a reference anti-TrkB antibody, the binding method described above is carried out in two directions: in the first direction, the reference antibody is allowed to bind to the TrkB protein under saturating conditions, followed by an assessment of the binding of the test antibody with the TrkB molecule. In the second direction, the test antibody is allowed to bind to the TrkB molecule under saturation conditions, followed by assessment of the binding of the reference antibody to the TrkB molecule. If in both directions only the first (saturating) antibody is able to bind to the TrkB molecule, then it is concluded that the test antibody and the reference antibody are competing for binding to TrkB (see, for example, the assay format described in Example 4, in which the TrkB protein is captured on the sensor tips, and the TrkB-coated sensor tips are treated with the reference antibody [mAb-1] and the test anti-TrkB antibody [mAb-2] sequentially and in both binding orders). As one skilled in the art will appreciate, an antibody that competes for binding to a reference antibody may not necessarily bind to the same epitope as the reference antibody, but may sterically block binding of the reference antibody by binding to an overlapping or adjacent epitope.

Obtaining human antibodies

The anti-TrkB antibodies of the present invention may be fully human, but not naturally occurring antibodies. Methods for generating monoclonal antibodies, including fully human monoclonal antibodies, are known in the art. Any such known methods can be used in the context of the present invention to obtain human antibodies that specifically bind to human TrkB.

Using VELOCIMMUNE® technology (see, for example, US 6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®) or any other known method for producing monoclonal antibodies, chimeric antibodies with high affinity for the allergen, having a human variable region and a mouse constant region, are first isolated. VELOCIMMUNE® technology involves the creation of a transgenic mouse having a genome containing human heavy and light chain variable regions operably linked to endogenous mouse constant region loci such that the mouse produces an antibody containing the human variable region and the mouse constant region in response to antigenic stimulation . DNA encoding the heavy and light chain variable regions of this antibody is isolated and operably linked to DNA encoding the human heavy and light chain constant regions. This DNA is then expressed in a cell capable of fully expressing

- 17 044580 human antibody.

Typically, a VELOCIMMUNE® mouse is challenged with the antigen of interest and lymphatic cells (such as B cells) from the mice that express the antibodies are isolated. Lymphatic cells can be fused with a myeloma cell line to produce immortalized hybridoma cell lines, and such hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific for the antigen of interest. DNA encoding the heavy chain and light chain variable regions can be isolated and linked to the desired isotype heavy chain and light chain constant regions. Such an antibody protein may be produced in a cell, such as a CHO cell. Alternatively, DNA encoding antigen-specific chimeric antibodies or light and heavy chain variable domains can be isolated directly from antigen-specific lymphocytes.

As described in the experimental section below, high affinity chimeric antibodies that are isolated with a human variable region and a mouse constant region are characterized and selected to provide the desired characteristics including affinity, selectivity, epitope, etc. The mouse constant regions are then replaced with the desired human constant region to generate a fully human antibody of the invention, eg, wild type or modified IgG1 or IgG4. Although the selected constant region may vary depending on the particular application, the characteristics of antigen binding with high affinity and target specificity are located in the variable region.

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 can be used in circumstances where anti-TrkB antibodies can only bind to human TrkB but will not cross-react with mouse or rat TrkB. Some examples in the present invention were performed using mice and rats that have been genetically modified to express human trkB. Any method known to those skilled in the art can be used to produce such TrkB humanized mice and rats.

In general, the antibodies of the present invention have very high affinities, typically with a K _D of about 10 ^-12 to about 10 ^-9 M when measured by antigen binding as either solid-phase immobilized or solution-phase antibodies.

Bioequivalents

The anti-TrkB antibodies and antibody fragments of the present invention include proteins having amino acid sequences that differ from those of the described antibodies, but which retain the ability to bind human TrkB. Such variant antibodies and antibody fragments contain one or more additions, deletions or substitutions of amino acids compared to the original sequence, but exhibit biological activity that is substantially equivalent to the activity of the described antibodies. Likewise, DNA sequences encoding an anti-TrkB antibody of the present invention include sequences that include one or more additions, deletions or substitutions of nucleotides relative to the described sequence, but which encode an anti-TrkB antibody or antibody fragment that is substantially bioequivalent to an anti-TrkB antibody. -TrkB antibody or antibody fragment of the invention. Examples of such variants of amino acid and DNA sequences are discussed above.

Two antigen-binding proteins or antibodies are considered bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives whose rate and extent of absorption do not show significant differences when administered at the same molar dose under similar experimental conditions, either as a single dose or multiple doses . Some antibodies will be considered equivalents or pharmaceutical alternatives if they are equivalent in their extent of absorption, but not in their rate of absorption, and may still be considered bioequivalent because such differences in rate of absorption are intentional and reflected in the labeling and are not fundamentally significant to achieving effective concentrations of the drug in the body, for example, with long-term use, and are considered, from a medical point of view, to be insignificant for the particular drug being studied.

In one embodiment, two antigen binding proteins are bioequivalent if there are no clinically significant differences in their safety, purity and potency.

In one embodiment, two antigen-binding proteins are bioequivalent if a patient can be switched one or more times between the reference product and the biologic product without an expected increase in the risk of adverse effects, including a clinically significant change in immunogenicity or reduction in efficacy compared to continued therapy without such. translation.

In one embodiment, two antigen binding proteins are bioequivalent if they both act by a common mechanism or mechanisms of action for the condition or conditions of use, where such mechanisms are known.

Bioequivalence can be demonstrated by in vivo and in vitro methods. Bioec measurements

- 18 044580 vivalence include, for example, (a) an in vivo study in humans or other mammals in which the concentration of an antibody or its metabolites is measured in blood, plasma, serum or other biological fluid as a function of time; (b) an in vitro study that is consistent with and reasonably predictive of in vivo human bioavailability data; (c) an in vivo study in humans or other mammals in which the relevant acute pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) a well-controlled clinical trial that establishes the safety, efficacy or bioavailability or bioequivalence of the antibody.

Bioequivalent variants of the anti-TrkB antibodies of the invention can be constructed, for example, by making various substitutions of residues or sequences, or removing terminal or internal residues or sequences that do not affect biological activity. For example, cysteine residues not essential for biological activity can be removed or replaced with other amino acids to prevent the formation of unnecessary or inappropriate intramolecular disulfide bonds upon renaturation. In other contexts, bioequivalent antibodies may include variants of anti-TrkB antibodies containing amino acid substitutions that modify the glycosylation characteristics of the antibodies, such as mutations that eliminate or remove glycosylation.

Species selectivity and interspecific cross-reactivity

The present invention, in some embodiments, provides anti-TrkB antibodies that bind to human TrkB, but not to TrkB from other species. The present invention also includes anti-TrkB antibodies that bind to human TrkB and to TrkB of one or more non-human species. For example, anti-TrkB antibodies of the invention may bind to human TrkB and may or may not bind, as appropriate, to one or more TrkBs from mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat , sheep, cow, horse, camel, cynomolgus monkey, monkey, rhesus monkey or chimpanzee. In accordance with some exemplary embodiments of the present invention, anti-TrkB antibodies are provided that specifically bind to human TrkB but do not bind or weakly bind to mouse or rat TrkB.

Multispecific antibodies

Antibodies of the present invention may be monospecific or multispecific (eg, bispecific). Polyspecific antibodies may be specific for different epitopes of a single target polypeptide or may contain antigen binding domains specific for 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 antibodies of the present invention may be linked to or co-expressed with another functional molecule, such as another peptide or protein. For example, an antibody or fragment thereof may be operably linked (e.g., by chemical binding, genetic fusion, non-covalent association, or otherwise) 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.

The present invention includes bispecific antibodies in which one portion of the immunoglobulin binds human TrkB and the other portion of the immunoglobulin is specific for a second antigen. The TrkB binding arm may contain any of the amino acid sequences HCVR/LCVR or CDR, as indicated in table. 1 in this document.

An exemplary bispecific antibody format that may be used in the context of the present invention includes the use of a first immunoglobulin (Ig) C _H 3 domain and a second Ig C _H 3 domain, wherein the first and second Ig 3 C _H 3 domains are different from each other by at least , one amino acid, and wherein the at least one amino acid difference impairs binding of the bispecific antibody to Protein A compared to a bispecific antibody that lacks the amino acid difference. In one embodiment, the first C _H 3 Ig domain binds to protein A, and the second C _H 3 Ig domain contains a mutation that weakens or eliminates protein A binding, such as the H95R modification (according to IMGT exon numbering; H435R in according to EU numbering). The second CH3 may further contain the modification Y96F (according to IMGT; Y436F according to EU). Additional modifications that may be found in the second CH3 include: D16E, L18M, N44S, K52N, V57M and V82I (according to IMGT; D356E, L358M, N384S, K392N, V397M and V422I according to EU) in the case of IgG1 antibodies ; N44S, K52N and V82I (IMGT; N384S, K392N and V422I from EU) for IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q and V82I (according to IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q and V422I according to EU) for IgG4 antibodies. The bispecific antibody format options described above are included within the scope of the present invention.

Other exemplary bispecific formats that may be used in the context of the present invention include, but are not limited to, for example, bispecific formats based on

- 19 044580 diantibody-based or scFv-based, IgG-scFv fusions, double variable domain (DVD)-Ig, quadra, peak-to-valve, common light chain (e.g. common light chain with peak-to-valley, etc. ), CrossMab, CrossFab, (SEED)-body, leucine zipper, Duobody, IgG1/IgG2, Dual-acting Fab (DAF)-IgG, and Mab bispecific formats (see, e.g., Klein et al., 2012, mAbs 4:6 , 1-11 and the links provided therein for an overview of the above formats). Bispecific antibodies can also be constructed using the conjugation of peptides and nucleic acids, for example, when non-naturally occurring amino acids with orthogonal chemical reactivity are used to produce site-specific antibody-nucleotide conjugates, which then self-assemble into multimeric complexes with a defined composition, valency and geometric shape. (See, for example, Kazane et al., J. Am. Chem. Soc. [Epub: Dec. 4, 2012]).

Therapeutic composition and administration

The invention relates to pharmaceutical compositions containing anti-TrkB antibodies or antigen-binding fragments thereof according to the present invention. The pharmaceutical compositions of the invention are formulated with suitable carriers, excipients and other agents that provide improved transport, delivery, tolerability and the like. Many suitable formulations can be found in a reference book known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These compositions include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid-containing (cationic or anionic) vesicles (eg, LIPOFECTIN™, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorbent pastes, emulsions oil-in-water and water-in-oil types, carbowax emulsions (polyethylene glycols 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) J Pharm Sci Technol 52:238–311.

The dose of antibody administered to a patient may vary depending on the age and size of the patient, the target disease, conditions, route of administration, and the like. The preferred dose is usually determined based on body weight or body surface area. For an adult patient, intravenous administration of an antibody of the present invention may be effective, typically in a single dose of from about 0.01 to about 20 mg/kg body weight, more preferably from about 0.02 to about 7, from about 0.03 to about 5 or from about 0.05 to about 3 mg/kg body weight. Depending on the severity of the condition, the frequency and duration of treatment may be adjusted. Effective doses and schedules of administration of anti-TrkB antibodies can be determined experimentally; for example, the patient's disease course can be monitored through periodic assessment and the dose adjusted accordingly. In addition, interspecies dose adjustment can be accomplished by methods well known in the art (eg, Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

Various delivery systems are well known that can be used to administer the pharmaceutical composition of the invention, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see, for example, Wu et al., 1987, J Biol Chem 262: 4429-4432). Routes of administration include, but are not limited to, intravitreal, intraocular, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucosal linings (for example, oral mucosa, rectal and intestinal mucosa, etc.) and can be administered together with other biologically active agents. Administration may be systemic or local.

The pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously using a standard needle and syringe. Moreover, with respect to subcutaneous delivery, a pen-type delivery device is easily used in delivering the pharmaceutical composition of the present invention. This pen-type delivery device can be reusable or disposable. A reusable pen-type delivery device typically uses a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition contained within the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen-type delivery device can then be reused. The disposable pen-type delivery device does not have a replaceable cartridge. Preferably, the disposable pen-type delivery device comes pre-filled with a pharmaceutical composition held in a reservoir within the device. Once the pharmaceutical composition is depleted in the reservoir, the entire device is disposed of.

Various reusable delivery devices such as pen and auto-injector find use in the subcutaneous delivery of the pharmaceutical composition of the present invention. Examples

- 20 044580 includes, but is not limited to, AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25™ pen, syringe -HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™ and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany), to name a few. Examples of a disposable pen-type delivery device having use in the subcutaneous delivery of a pharmaceutical composition of the present invention include, but are not limited to, the SOLOSTAR™ pen (sanofi-aventis), FLEXPEN™ (Novo Nordisk) and KWIKPEN™ (Eli Lilly), SURECLICKTM auto-injector (Amgen, Thousand Oaks, CA), PENLETTM (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, L.P.), and HUMIRATM pen (Abbott Labs, Abbott Park IL), to name a few.

In certain situations, the pharmaceutical composition may be delivered in a controlled release system. In one embodiment, a pump can be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14: 201). In another embodiment, polymeric substances may 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 in close proximity to the target of the composition, and therefore only a portion of the systemic dose will be required (see, for example, Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are reviewed in Langer, 1990, Science 249: 1527-1533.

Injectable preparations may include dosage forms for intravenous, intravitreal, intraocular, subcutaneous, intradermal and intramuscular injections, drip infusions, etc. These injectable preparations can be prepared by generally known methods. For example, injectable preparations can be prepared, for example, by dissolving, suspending or emulsifying the antibody or a salt thereof described above in a sterile aqueous or oily medium commonly used for injection. As the aqueous injection medium, there are, for example, physiological saline solution, isotonic solution containing glucose and other auxiliary agents, etc., which can be used in combination with a suitable solubilizing agent such as alcohol (eg ethanol), polyalcohol ( e.g. propylene glycol, polyethylene glycol), nonionic surfactant [e.g. polysorbate 80, HCO-50 (adduct of hydrogenated castor oil and polyoxyethylene (50 mol))], etc. As the oil medium, for example, sesame oil, soybean oil, etc. are used, which can be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus obtained is preferably filled into a suitable ampoule.

Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared in dosage forms at a unit dose corresponding to the dosage of the active ingredients. Such unit dose dosage forms include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the above antibody contained is typically from about 5 to about 500 mg per unit dose dosage form; particularly in injection form, it is preferred that said antibody be contained in an amount of from about 5 to about 100 mg and from about 10 to about 250 mg for other dosage forms.

Therapeutic uses of antibodies

The present invention provides methods comprising administering to a subject in need of such administration a therapeutic composition comprising an anti-TrkB antibody (e.g., an anti-TrkB antibody containing any of the HCVR/LCVR or CDR sequences as set forth in Table 1 herein ). The therapeutic composition may contain any one or more anti-TrkB antibodies or antigen-binding fragments thereof described herein and a pharmaceutically acceptable carrier or diluent.

The antibodies of the invention are used, among other things, for the treatment, prevention and/or amelioration of any disease or disorder associated with or mediated by the expression or activity of TrkB. The TrkB agonist antibodies of the invention may be used to improve nervous system function and may be used to treat or prevent any disease or condition that is characterized in part by cell degradation, in particular nerve cell damage or nerve cell degeneration, such as acute nervous system injury or chronic neurodegenerative disease. disease.

The present invention provides methods for treating or preventing a disease or visual impairment by administering to a patient in need of such treatment an anti-TrkB antibody or an antigen binding fragment thereof, as described elsewhere herein.

In one embodiment, the anti-TrkB antibodies of the invention may provide a method for preventing damage or death of retinal neurons. In one embodiment,

- 21 044580 anti-TrkB antibodies of the invention may provide a method of treating pathological diseases in which retinal degeneration occurs. In one embodiment, the anti-TrkB antibodies of the invention may provide a method of treating the eye before or after eye surgery, light exposure, or other environmental trauma, thereby preventing degeneration of retinal cells. In one embodiment, the anti-TrkB antibodies of the invention may provide a method for preventing photoreceptor damage and degeneration in the eye. In one embodiment, the anti-TrkB antibodies of the invention may provide a method of protecting retinal neurons without causing side effects. In one embodiment, the anti-TrkB antibodies of the invention may provide a method for allowing damaged photoreceptors to repair or regenerate.

In some embodiments, eye diseases that can be treated using one or more anti-TrkB antibodies of the invention may be selected from the group consisting of glaucoma, diabetic retinopathy, age-related macular degeneration or other maculopathies, ischemic optic neuropathy, optic neuritis nerve, retinal ischemia, photoreceptor degeneration, retinal pigmentary dystrophy, Leber congenital amaurosis, Leber hereditary optic neuropathy, Usher syndrome, Stargard disease and retinal artery or vein occlusion.

Other pathological conditions amenable to treatment by one or more anti-TrkB antibodies of the invention include retinal detachments, photic retinopathy, surgically induced retinopathy (mechanical or light-induced), toxic retinopathy, retinopathy of prematurity, viral retinopathy such as cytomegalovirus or HIV AIDS-related retinopathy; uveitis; ischemic retinopathy caused by venous or arterial occlusion or other vascular disorder, retinopathy caused by trauma or penetrating ocular lesions, peripheral vitreoretinopathy or hereditary retinal degenerations.

In one embodiment, the anti-TrkB antibodies of the invention may be formulated for intraocular or intravitreal delivery.

The present invention also relates to methods of treating other diseases or disorders of the central or peripheral nervous system, such as stroke or traumatic brain injury. In addition, since the antibodies of the present invention promote the survival of neurons and act as neuroprotectors, any one or more of these agonist antibodies may be effective in treating a patient suffering from a disease or disorder of the nervous system, where the survival of neurons is of paramount importance in cure or recovery cellular damage caused by damage to the nervous system or caused by a disease that has a serious effect on the nervous system, including neurodegenerative diseases.

In the context of the treatment methods described herein, an anti-TrkB antibody may be administered as monotherapy (ie, as a single therapeutic agent) or in combination with one or more additional therapeutic agents.

Combination therapy and compositions

The present invention includes compositions and therapeutic compositions comprising any of the anti-TrkB antibodies described herein in combination with one or more additional therapeutically active components, and methods of treatment comprising administering such combinations to subjects in need of such treatment.

The anti-TrkB antibodies of the present invention may be combined and/or administered in combination with one or more additional therapeutically active components selected from the group consisting of: a drug that helps lower intraocular pressure (IOP-lowering drug), a neurotrophin, and a factor antagonist vascular endothelial growth hormone (VEGF), such as a VEGF decoy such as aflibercept (EYLEA®). Other drugs that can be combined with the TrkB antibodies of the invention include, but are not limited to, a prostaglandin analogue (eg, ZIOPTAN™, XALATAN®), a beta blocker (eg, TIMOPTIC XE®, ISTALOL®, BETOPTIC®S); alpha-2 adrenergic agonist (eg, apraclonidine), carbonic anhydrase inhibitors (eg, TRUSOPT®, AZOPT®), cholinergic agent (eg, ISOPTO® CARPINE, PILOPINE HS® gel), or combination therapy (beta blocker plus carbonic anhydrase inhibitor, e.g. COMBIGAN™, COSOPT®).

Anti-TrkB antibodies of the invention can also be administered and/or combined in combination with antivirals, antibiotics, analgesics, antioxidants, COX inhibitors and/or NSAIDs. Anti-TrkB antibodies may also be used in combination with other therapies, including stem cell therapy, glaucoma filtration surgery, laser surgery, or gene therapy.

Additional therapeutically active component(s), such as any of the above agents or derivatives thereof, may be administered immediately before, simultaneously with, or shortly after administration of the anti-TrkB antibody of the present invention; (for the purposes of the present invention such

- 22 044580 administration regimens are considered to be the administration of an anti-TrkB antibody in combination with an additional therapeutically active component). The present invention includes pharmaceutical compositions in which the anti-TrkB antibody of the present invention is combined with one or more additional therapeutically active component(s) as described elsewhere herein.

Administration modes

In accordance with some embodiments of the present invention, multiple doses of an anti-TrkB antibody (or a pharmaceutical composition comprising a combination of an anti-TrkB antibody and any of the additional therapeutically active agents specified herein) can be administered to a subject over a period of time. Methods according to this aspect of the invention involve sequentially administering to a subject multiple doses of an anti-TrkB antibody of the invention. As used herein, sequential administration means that each dose of anti-TrkB antibody is administered to a subject at different times, eg on different days separated by a predetermined interval (eg, hours, days, weeks or months). The present invention provides methods that include sequentially administering to a patient one initial dose of an anti-TrkB antibody, then one or more secondary doses of an anti-TrkB antibody, and then, optionally, one or more tertiary doses of an anti-TrkB antibody.

The terms initial dose, secondary doses and tertiary doses refer to the time sequence of administration of the anti-TrkB antibody of the invention. Thus, the starting dose is the dose that is administered at the beginning of the treatment regimen (also called the base dose); secondary doses are doses that are administered after the initial dose; and tertiary doses are doses that are administered after secondary doses. The initial, secondary and tertiary doses may contain the same amount of anti-TrkB antibody, but may generally differ from each other in terms of frequency of administration. However, in some embodiments, the amount of anti-TrkB antibody contained in the initial, secondary and/or tertiary doses varies (eg, is adjusted depending on the situation) during the course of treatment. In some embodiments, two or more (eg, 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen as loading doses followed by subsequent doses that are administered at a less frequent schedule (eg, maintenance doses).

In some exemplary embodiments of the present invention, each secondary and/or tertiary dose is administered from 1 to 26 (eg, 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 after the immediately preceding dose. The phrase immediately preceding dose as used herein means, in a multiple dosing sequence, the dose of anti-TrkB antibody that is administered to a patient before administration of the immediately next dose in the sequence, with no intervening doses.

Methods according to this aspect of the invention may include administering to the patient any number of secondary and/or tertiary doses of anti-TrkB antibody. For example, in certain embodiments, only one secondary dose is administered to the patient. In other embodiments, the patient is administered two or more (eg, 2, 3, 4, 5, 6, 7, 8 or more) secondary doses. Likewise, in certain embodiments, only one tertiary dose is administered to the patient. In other embodiments, the patient is administered two or more (eg, 2, 3, 4, 5, 6, 7, 8 or more) tertiary doses. The administration schedule may be continued indefinitely throughout the life of a particular subject or as long as such treatment is therapeutically necessary or beneficial.

In embodiments involving multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1-2 weeks or 1-2 months after the immediately preceding dose. Likewise, in embodiments involving multiple tertiary doses, each tertiary dose can be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2-12 weeks after the immediately preceding dose. In some embodiments of the invention, the frequency with which secondary and/or tertiary doses are administered to a patient may vary over the course of treatment. The frequency of administration may also be adjusted by the physician during the course of treatment depending on the needs of the individual patient after clinical examination.

The present invention includes dosing regimens in which 2 to 6 loading doses are administered to a patient at a first frequency (eg, once a week, once every two weeks, once every three weeks, once a month, once every two months, and etc.) followed by administration of two or more maintenance doses to the patient at a lower frequency. For example, according to this aspect of the invention, if loading doses are administered at a frequency of once per month, then maintenance doses may be administered to the patient once every six weeks, once every two months, once every three months, or the like.

- 23 044580

Diagnostic applications of antibodies

Anti-TrkB antibodies 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 fragment thereof can be used to diagnose a condition or disease characterized by aberrant expression (eg, overexpression, underexpression, lack of expression, etc.) of TrkB. Typical diagnostic assays for TrkB may include, for example, contacting a sample obtained from a patient with an anti-TrkB antibody of the invention, wherein the anti-TrkB antibody is labeled with a detectable label or reporter molecule. Alternatively, an unlabeled anti-TrkB antibody may be used for diagnostic purposes in combination with a secondary antibody that is itself detectably labeled. The detectable label or reporter molecule may be a radioisotope such as H, C, P, S or I; a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate or rhodamine; or an enzyme such as alkaline phosphatase, beta-galactosidase, horseradish peroxidase or luciferase. Specific typical 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).

Samples that can be used in TrkB diagnostic assays in accordance with the present invention include any tissue or fluid sample obtained from a patient that contains detectable amounts of TrkB protein or fragments thereof under normal or pathological conditions. Typically, TrkB levels in a particular sample obtained from a healthy patient (eg, a patient not suffering from a disease or condition associated with abnormal TrkB levels or activity) will be measured to initially establish a baseline or standard TrkB level. This baseline TrkB level can then be compared to TrkB levels measured in samples obtained from individuals suspected of having a TrkB-related disease or condition.

Examples

The following examples are provided for the purpose of providing those skilled in the art with complete disclosure and description of how to make and use the methods and compositions of the invention, and are not intended to limit the scope of what the inventors believe to be their invention. Efforts have been made to ensure accuracy in terms of parameters used (eg quantities, temperature, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise noted, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, room temperature is about 25° C., and pressure is at or near atmospheric pressure.

Example 1. Production of human antibodies to TrkB.

Human antibodies to TrkB were generated in a mouse containing DNA encoding the variable regions of the heavy and kappa light chains of human immunoglobulin light chain. In one embodiment, human antibodies are generated in VELOCIMMUNE® mice. In one embodiment, Veloclmmune® (VI) mice were immunized with human TrkB(ecto)mFc (SEQ ID NO: 77). In one embodiment, Veloclmmune® (VI) mice were immunized with mouse TrkB(ecto)mFc (SEQ ID NO: 80). The antibody response was monitored by a TrkB-specific immunoassay. For example, sera were analyzed for titers of specific antibodies to purified full-length TrkB. Antibody-producing clones were isolated using B-cell sorting technology (BST) and hybridoma methods. For example, when the desired immune response was achieved, splenocytes were collected and fused with mouse myeloma cells, maintaining their viability and forming a hybridoma cell line. Hybridoma cell lines were screened and selected to identify cell lines that produce TrkB-specific antibodies. Specific anti-mouse TrkB antibodies were prepared in this manner and designated M2aM14173N, M2aM14178N, and M2aM14179N.

An-TrkB antibodies were also isolated directly from antigen-positive mouse B cells without fusion with myeloma cells, as described in US Pat. No. 7,582,298, which is incorporated herein by reference in its entirety. Using this method, several fully human anti-TrkB antibodies (ie, antibodies containing human variable domains and human constant domains) have been produced; Typical antibodies produced in this manner were designated H4H9780P, H4H9814P, and H4H9816P2.

The biological properties of the exemplary antibodies produced in accordance with the methods of this example are described in detail in the examples below.

Example 2. Amino acid sequences and nucleotide sequences of the variable region of the heavy and light chains.

In table 1a shows the amino acid sequence identifiers of the heavy and light chain variable regions and the CDRs of selected anti-TrkB antibodies according to the invention. In table 1b shows the amino acid sequence identifiers for the full-length heavy and light chains of selected anti-TrkB antibodies according to the invention. The corresponding nucleic acid sequence identifiers for selected anti-TrkB antibodies according to the invention are shown in table. 2.

Table 1a

Amino acid sequence identifiers

Designated not antibodies NS VR NS DR1 HCD R2 HCD R3 LCV R LCD R1 LCD R2 LCD R3 mlg G2a Light chain constant region N4N9780R 2 4 6 8 10 12 14 16 N4N9814R 18 20 22 24 26 28 thirty 32 N4N9816R2 34 36 38 40 42 44 46 48 М2аМ1417 3N 49 50 51 52 53 54 55 56 57 58 М2аМ1417 59 60 61 62 63 64 65 66 57 67 8N М2аМ1417 9N 68 69 70 71 72 73 74 75 57 58

Table 1b

Antibody designation Full size heavy chain Full Length Light Chain N4N9780R 99 100 N4N9814R 101 102 N4N9816R2 103 104

table 2

Nucleic acid sequence identifiers

Antibody designation VH HCDR 1 HCDR 2 HCDR 3 VK LCDR1 LCDR2 LCDR 3 N4N9780R 1 3 5 7 9 eleven 13 15 N4N9814R 17 19 21 23 25 27 29 31 N4N9816R2 33 35 37 39 41 43 45 47

Antibodies are generally designated herein according to the following nomenclature: an Fc prefix (eg, H4H, H2M, etc.) followed by a numerical identifier (eg, 9780, 9816, etc., as shown in Table 1 or 2), followed by the suffix P, P2, or N. The H4H prefix when designating antibodies indicates a specific isotype of the Fc region of the antibody. Thus, in accordance with this nomenclature, the antibody may be referred to herein, for example, as H4H9780P, which indicates the Fc region of human IgG4, and M2aM14179N, for example, indicates the Fc region of mouse IgG2a. Variable regions are fully human if they are designated by the first H in the antibody designation. The prefix M denotes the mouse variable region. As one skilled in the art will appreciate, an antibody having a particular Fc isotype can be converted to an antibody with a different Fc isotype (e.g., a mouse IgG1 Fc antibody can be converted to a human IgG4 antibody, etc.), but in any case, variable domains (including CDRs), which are designated by the numeric identifiers shown in table. 1 or 2 will remain the same and the antigen binding properties are expected to be identical or substantially the same regardless of the nature of the Fc domain.

Example 3 Binding kinetics of anti-TrkB monoclonal antibodies binding to various TrkB reagents measured using the Biacore system at 25°C and 37°C.

Equilibrium dissociation constants (KD values) for TrkB binding to purified monoclos

- 25 044580 nal anti-TrkB antibodies were determined using a real-time surface plasmon resonance biosensor using a Biacore 4000 instrument. All binding studies were carried out in 10 mM Hepes pH 7.4, 150 mM NaCl, 3 mM EDTA and 0 .05% v/v Surfactant Tween-20 (working buffer HBS-ET) at 25°C and 37°C. The Biacore sensor surface was first derivatized by amine coupling with the F(ab') ₂ fragment of a goat anti-human Fcy-specific polyclonal antibody (Jackson ImmunoResearch Laboratories, #109-006-098) or a rabbit polyclonal anti-mouse Fc antibody (GE Healthcare #BR-1008 -38) to capture monoclonal anti-TrkB antibodies. Binding studies were performed with the following TrkB reagents; human TrkB extracellular domain expressed by a C-terminal myc-myc-hexahistidine tag (hTRKB.mmH; SEQ ID NO: 76; accession number NP_001018074.1), mouse TrkB extracellular domain expressed by a C-terminal myc-myc-hexahistidine tag (mTRKB.mmH ; SEQ ID NO: 79; accession number NP_001020245), rat TrkB extracellular domain expressed by a C-terminal myc-mychexahistidine tag (rTRKB.mmH; SEQ ID NO: 84; accession number NP_036863.1), and human TrkB extracellular domain expressed C-terminal Fc-tagged mouse IgG2a (hTrkB-mFc; SEQ ID NO: 77; accession number NP_001018074.1). Various concentrations of TrkB reagents were first prepared in HBS-ET working buffer (100 nM - 1.23 nM; 3-fold serial dilution) and injected onto the surface of human Fc-captured monoclonal anti-TrkB antibody for 4 min at speed flow rate of 30 μl/min, while the dissociation of the TrkB reagent bound to the monoclonal antibody was monitored for 10 min in FfflS-ET running buffer. Kinetic association (k _a ) and dissociation (k d ) rate constants were determined by fitting real-time binding sensorgrams to a mass transfer-limited 1:1 binding model using Scrubber 2.0c curve processing software. Equilibrium dissociation binding constants (K _D ) and dissociation half-lives (t1/ ₂ ) were calculated from the kinetic rate constants as follows:

kd 1п(2)

K _D (M) = ^ka and 1½ (min) = 60 * fed

The kinetic parameters of binding of hTrkB.mmH, mTrkB.mmH, rTrkB.mmH or hTrkB-mFc with various monoclonal anti-TrkB antibodies according to the invention at 25°C and 37°C are shown in table. 3-10.

Summary of results:

At 25°C, monoclonal anti-TrkB antibodies bound to hTrkB.mmH with K _D values ranging from 545 to 41.3 nM, as shown in Table. 3. At 37°C, monoclonal antibodies bound to hTrkB.mmH with K _D values ranging from 2.28 to 135 nM, as shown in table. 4.

At 25°C, monoclonal anti-TrkB antibodies bound to hTrkB-mFc with K _D values ranging from 31.1 μM to 4.48 nM, as shown in Table. 5. At 37°C, monoclonal anti-TrkB antibodies bound to hTrkB-mFc with K _D values ranging from 73.3 pm to 3.46 nM, as shown in table. 6.

At 25°C, the monoclonal anti-TrkB reference antibody, designated herein as H1M8037C (see US2010/0196390, antibody designated C2; see also the amino acid sequences SEQ ID NO: 97 and 98 for the reference heavy and light chain, respectively ), bound to mTrkB.mmH with a K _D value of 40.8 nM, as shown in Table. 7. Anti-TrkB antibodies according to the invention did not bind to mTrkB.mmH at 25°C, as shown in table. 7. At 37°C, the reference monoclonal anti-TrkB antibody bound to mTrkB.mmH with a K _D value of 94.1 nM, as shown in Table. 8. Anti-TrkB antibodies according to the invention did not bind to mTrkB.mmH at 37°C, as shown in table. 8.

At 25°C, the reference monoclonal anti-TrkB antibody bound to rTrkB.mmH with a K _D value of 31.8 nM, as shown in Table. 9. Anti-TrkB antibodies according to the invention did not bind to rTrkB.mmH at 25°C, as shown in table. 9. At 37°C, the reference monoclonal anti-TrkB antibody bound to rTrkB.mmH with a K _D value of 87.5 nM, as shown in Table. 10. Anti-TrkB antibodies of the invention did not bind to rTrkB.mmH at 37°C, as shown in table. 10.

- 26 044580

Table 3

Kinetic parameters of binding of hTrkB.mmH to monoclonal anti-TrkB antibodies at 25°C.

Captured analyte Quantity of analyte captured (RU) 100 nM hTrkB.mm N binding (RU) k _a (l/Ms) kd(l/s) _KD (M) bDmin) N4N9780R 116±2.8 20 4.00E+04 8.67E-04 2D7E-08 13 N4N9814R 93 ±2.1 63 E26E+06 6.86E-04 5D5E-10 17 N4N9816R 2 138 ± 1.5 thirty 6D4E+04 8.45E-04 E38E-08 14 N1M8037 C* reference antibody 757 23 4.65E+04 E92E-03 4DZE-08 6

*indicates that the mAb was captured using a surface immobilized with anti-mFc antibodies

Table 4

Kinetic parameters of hTrkB.mmH binding to monoclonal anti-TrkB antibodies at 37°C.

Captured Quantities YuONM k _a (l/Ms) ka(l/s) _KD (M) b/Dmin) th analyte O captured analyte (RU) bTgkV. mmH communication (RU) N4N9780R 64 ± 1.9 14 5.71E+04 3.64E-03 6.38E-08 3.2 N4N9814R 52 ± 1.2 32 2.05E+06 4.68E-03 2.28E-09 2.5 N4N9816R2 80 ± 2.3 12 5.28E+04 3.74E-03 7.08E-08 building Н1М8037С* antibody comparisons 815.11 28 6DZE+04 8.31E-03 E35E-07 1.4

*indicates that the mAb was captured using a surface immobilized with anti-mFc antibodies

- 27 044580

Table 5

Kinetic parameters of hTrkB-mFc binding to monoclonal anti-TrkB antibodies at 25°C.

Captured analyte Amount of other analyte captured (RU) 100nM hTrkBmFc connection (RU) k _a (l/Ms) kd(l/s) _KD (M) G.(min) N4N9780R 105 ± 1.6 14 3.74E+04 1.68E-04 4.48E-09 69 N4N9814R 83 ± 1.3 69 1.91E+06 5.95E-05 3.11E-11 194 N4N9816R2 131 ± 1 25 6.52E+04 7.98E-05 1.22E-09 145 Н1М8037С* antibody comparisons 753 41 8.80E+04 9.55E-05 1.09E-09 121

*indicates that the mAb was captured using a surface immobilized with anti-mFc antibodies

Table 6

Kinetic parameters of hTrkB.mFc binding to monoclonal anti-TrkB antibodies at 37°C.

Captured analyte Quantity of analyte captured (RU) 100nM hTrkB-mFc coupling (RU) k _a (1/ Ms) kd(l/s) _KD (M) G.(min) N4N9780R 56 ± 1 14 6.65E +04 2.22E- 04 3.33E-09 52 N4N9814R 46 ± 1 42 2.83E +06 2.08E- 04 7,ЗЗЭ-11 56 N4N9816R2 71 ± 1.4 14 6.95E +04 2.40E- 04 3.46E-09 48 Н1М8037С* antibody comparisons 814.46 58 2.35E +05 2.92E- 04 1.24E-09 40

*indicates that the mAb was captured using a surface immobilized with anti-mFc antibodies

- 28 044580

Table 7

Kinetic parameters of mTrkB.mmH binding to monoclonal anti-TrkB antibodies at 25°C.

Captured analyte Quantity of analyte captured (RU) 100nM shTgkV. mm II connection (RU) k _a (1/ Ms) kd(l/s) _KD (M) bDmin) N4N9780R 111 ±0.7 -1 N.B. N.B. N.B. N.B. N4N9814R 88 ± 0.7 -2 N.B. N.B. N.B. N.B. N4N9816R2 135 ±0.6 0 N.B. N.B. N.B. N.B. Н1М8037С* antibody comparisons 755 26 4D2E +04 l,68E-03 4.08E- 08 7

*indicates that the mAb was captured using a surface immobilized with anti-mFc antibodies

Table 8

Kinetic parameters of mTrkB.mmH binding to monoclonal anti-TrkB antibodies at 37°C.

Captured analyte Amount of analyte captured (RU) 100nM shTgkWtsh II connection (RU) k _a (l/Ms) kd(l/s) _KD (M) bDmin) H4H9780P 60 ± 0.7 1 N.B. N.B. N.B. N.B. H4H9814P 49 ± 0.6 0 N.B. N.B. N.B. N.B. H4H9816P2 75 ± 0.9 0 N.B. N.B. N.B. N.B. H1M8037C* antibody comparisons 815.95 31 7D0E+04 6.96E-03 9.41E- 08 1.7

*indicates that the mAb was captured using a surface immobilized with anti-mFc antibodies

- 29 044580

Table 9

Kinetic parameters of binding of rTrkB.mmH to monoclonal anti-TrkB antibodies at 25°C.

Captured analyte Quantity of analyte captured (RU) YuONM rTrkB.mmH communication (RU) ka(l/Ms) ka(l/s) _KD (M) b/ ₂ (min) N4N9780R 108 ±0.7 -1 N.B. N.B. N.B. N.B. N4N9814R 86 ± 0.4 -1 N.B. N.B. N.B. N.B. N4N9816R2 134 ±0.3 0 N.B. N.B. N.B. N.B. Н1М8037С* antibody comparisons 756 26 5D6E+04 l,60E-03 ZDOE- 08 7

*indicates that the mAb was captured using a surface immobilized with anti-mFc antibodies

Table 10

Kinetic parameters of binding of rTrkB.mmH to monoclonal anti-TrkB antibodies at 37°C.

Captured analyte Quantity of analyte captured (RU) YuONM rTrkB.mm N connection (RU) ka(l/Ms) kd(l/s) _KD (M) b/ ₂ (min) H4H9780P 59 ±0.2 0 N.B. N.B. N.B. N.B. H4H9814P 48 ± 0.5 -1 N.B. N.B. N.B. N.B. H4H9816P2 74 ± 0.6 0 N.B. N.B. N.B. N.B. H1M8037C* antibody comparisons 815.45 32 7.93E+04 6.94E-03 8.75E- 08 1.7

*indicates that the mAb was captured using a surface immobilized with anti-mFc antibodies

Example 4: Binding kinetics of surrogate monoclonal anti-TrkB antibodies to various TrkB reagents measured at 25°C using the Biacore system.

Equilibrium dissociation constants ( _KD values) for TrkB binding to purified monoclonal anti-TrkB antibodies were determined using a real-time surface plasmon resonance biosensor using a Biacore T200 instrument. All binding studies were performed in 10 mM Hepes at pH 7.4, 150 mM NaCl, 3 mM EDTA and 0.05% v/v. Surfactant Tween-20 (working buffer HBS-ET) at 25°C. The Biacore sensor surface was first derivatized by amine coupling with a rabbit polyclonal anti-mouse Fc antibody (GE Healthcare #BR-1008-38) to capture monoclonal anti-TrkB antibodies. Binding studies were performed with the following TrkB reagents; extracellular domain of human TrkB expressed by a Terminal myc-myc-hexahistidine tag (hTrkB.mmH; SEQ ID NO: 76; NP_001018074.1), extracellular domain of mouse TrkB expressed by a C-terminal myc-myc-hexahistidine tag (mTRKB.mmH; SEQ ID NO: 79; NP_001020245) and the extracellular domain of rat TrkB expressed by a C-terminal myc-myc-hexahistidine tag (rTRKB.mmH; SEQ ID NO: 84; XP_002721319.1). Various concentrations of TrkB reagents were first prepared in HBS-ET working buffer (90 nM 3.33 nM; 3-fold serial dilution) and injected onto the surface of the Fc-captured anti-TrkB monoclonal antibody target for 4 min at a flow rate of 50 μl /min, while the dissociation of the TrkB reagent bound to the monoclonal antibody was monitored for 10 min in HBS-ET running buffer. Kinetic association ( _ka ) and dissociation (kd) rate constants were determined by fitting real-time binding sensorgrams to a mass transfer-limited 1:1 binding model using Scrubber 2.0 s curve processing software. Equilibrium dissociation binding constants ( _KD ) and dissociation half-lives ( ^t1 / ₂ ) were calculated from the kinetic rate constants as follows:

kd 1п(2)

Kd (M) = k ^a ₉ and 1½ (min) = 60 * fed

The kinetic parameters of binding of hTrkB.mmH, mTrkB.mmH or rTrkB.mmH with various monoclonal anti-TrkB antibodies according to the invention at 25°C are shown in table. 11-13.

Results:

At 25°C, the surrogate monoclonal anti-TrkB antibodies of the invention do not show binding to hTrkB.mmH, as shown in table. eleven.

At 25°C, the surrogate monoclonal anti-TrkB antibodies of the invention bound to mTrkB.mmH with K _D values ranging from 2.39 nM to 32.4 nM, as shown in table. 12.

At 25°C, the surrogate monoclonal anti-TrkB antibodies of the invention bound to rTrkB.mmH with K _D values ranging from 2.56 nM to 26.9 nM, as shown in table. 13.

Table 11

Kinetic parameters of binding of hTrkB.mmH to monoclonal anti-TrkB antibodies at 25°C.

Captured analyte Quantity of analyte captured (RU) 90nM hTrkB.mm II connection (RU) ka(l/Ms) kd(l/s) _KD (M) ti/ ₂ (MH H) M2aM14173N 230 ±0.8 0 N.B. N.B. N.B. N.B. M2aM14178N 125 ±0.1 -1 N.B. N.B. N.B. N.B. M2aM14179N 396 ±2.1 -2 N.B. N.B. N.B. N.B.

Table 12

Kinetic parameters of mTrkB.mmH binding to monoclonal anti-TrkB antibodies at 25°C.

Captured analyte Quantity of analyte captured (RU) 90nM mTrkB.m mH connection (RU) k _a (l/Ms) kd(l/s) _KD (M) Mmin) M2aM14173N 236 ± 1.1 33 7.41E+04 6.30E-04 8.49E- 09 18 M2aM14178N 126 ±0.3 12 L03E+05 2.47E-04 2.39E- 09 47 M2aM14179N 416 ±4.3 32 1D5E+05 3.72E-03 3.24E- 08 3.1

- 31 044580

Table 13

Kinetic parameters of binding of rTrkB.mmH to monoclonal ________________anti-TrkB antibodies at 25°C_______________

Captured analyte Quantity of analyte captured (RU) 90nM rTrkB.mm H connection (RU) ka(l/Ms) ka(l/s) _KD (M) B/ ₂ (min) М2аМ14173 N М2аМ14178 233 ± 0.7 33 7.90E+04 6.32E-04 8.00E-09 18 N 125 ±0.3 eleven 9.00E+04 2.30E-04 2.56E-09 50 М2аМ14179 N 404 ± 2.5 33 1.48E+05 3.98E-03 2.69E-08 2.9

Example 5 Bioassay with HEK293/SRE-luc/hTrkB and HEK293/SRE-luc/mTrkB(Ecto)-hTrkB (TM-Cyto) cells.

A bioassay was developed to detect TrkB activation using a luciferase reporter gene under the control of a serum response element (SRE) and a ligand, brain-derived neurotrophic factor (BDNF, R&D Systems). HEK293 cell lines were generated that stably express a luciferase reporter (SRE response element luciferase, SRE luciferase, SA Bioscience, #CLS-010L) with the extracellular domain of either human TrkB (hTrkB, amino acids 32-429 NP_001018074.1 or database no. Uniprot Q16620-1), or mouse TrkB fused to the transmembrane and cytoplasmic domains of human TrkB (mTrkB, amino acids 32-429 NP_001020245.1 or Uniprot database number P15209-1, fused to hTrkB, amino acids 431-822). Stable cell lines HEK293/SRE-Luc/hTrkB and HEK293/SRELuc/mTrkB were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, nonessential amino acids, penicillin/streptomycin/glutamine, 1 μg/ml puromycin and 500 μg /ml G418.

To perform the bioassay, cells were seeded into 96-well lytic plates at a concentration of 20,000 cells/well in Opti-MEM™ medium supplemented with 0.1% FBS, penicillin/streptomycin and L-glutamine, and then incubated at 37°C in 5% CO2 overnight. The next morning, human brain-derived neurotrophic factor (BDNF) or antibodies were serially diluted from 100 nM to 0.002 nM (plus a sample containing only buffer without ligand) and added to the cells to determine activation of the TrkB signaling pathway. Serially diluted antibodies were also probed in the presence of 100 pM BDNF (R&D Systems, 248-BD/CF). The cells were then incubated for 5.5 h at 37°C in the presence of 5% CO2. Luciferase activity was measured after addition of OneGlo reagent (Promega) using a Victor X instrument (Perkin Elmer). The results were analyzed using non-linear regression (four-parameter logistic) using Prism 5 software (GraphPad) to obtain EC ₅₀ and IC ₅₀ values. Maximal antibody activation was determined using the following equation:

(Maximum luminescence signal intensity (RLU) achieved by the antibody) - (Intensity Max.

luminescence signal (RLU) achieved without BDNF) activation in = * 100 (Maximum luminescence signal intensity (RLU) achieved by BDNF) - (luminescence signal intensity (RLU) achieved without BDNF)

Summary of results and conclusions:

As shown in table. 14, three anti-TrkB antibodies according to the invention H4H9816P2, H4H9814P, H4H9780P showed activation of the human TrkB signaling pathway in HEK293/SRE-luc/hTrkB cells without BDNF with an EC ₅₀ value of 35-82 pM with a maximum activation in the range of 88-92%. The three antibodies of the invention were also tested in the presence of 100 pM BDNF. While 100 pM BDNF showed 56% activation in the presence of an irrelevant control mAb, control mAb2, the antibodies of the invention showed greater activation with an EC ₅₀ value of 45-76 pM with maximum activation in the range of 77-80%. Three anti-TrkB antibodies of the present invention showed no activation

- 32 044580 mouse TrkB signaling in HEK293/SRE-luc/mTrkB cells in the absence of BDNF or in the presence of 100 pM BDNF. The control mAb1, anti-TrkB reference antibody H1M8037C, showed activation of human TrkB signaling with EC ₅₀ 76 pM with a maximum activation of 78% and mouse TrkB with EC ₅₀ 43 pM with maximum activation 85% without BDNF. In the presence of 100 pM BDNF, control mAb 1 activated human TrkB signaling with an _{EC50 of} 110 pM with a maximum activation of 79% and mouse TrkB with an EC50 of 42 pM with a maximum activation of 69%, which is greater than the activation of control mAb 2 with 100 pM BDNF. Control mAb 2, an irrelevant human IgG4 antibody, showed no activation in the absence or presence of 100 pM BDNF.

As shown in table. 15, three anti-TrkB antibodies of the invention, M2aM14173N, M2aM14178N, M2aM14179N, showed activation of the mouse TrkB signaling pathway in HEK293/SRE-luc/mTrkB cells in the absence of BDNF with EC ₅₀ from 34 to 190 pM with maximum activation ranging from 76 to 190 pM 94%. The three antibodies of the invention were also tested in the presence of 100 pM BDNF. While 100 pM BDNF showed 60% activation in the presence of an irrelevant isotype control mAb, control mAb4, the antibodies of the invention showed greater activation with an EC50 of 17-100 pM with maximum activation in the range of 67-75%. The three anti-TrkB antibodies of the invention did not show activation of the human TrkB signaling pathway in HEK293/SRE-luc/hTrkB cells in the absence of BDNF or in the presence of 100 pM BDNF. Control mAb 1 showed activation of the human TrkB signaling pathway with EC ₅₀ 57 pM with maximum activation of 80% and mouse TrkB with EC ₅₀ 43 pM with maximum activation 85% without BDNF. In the presence of 100 μM BDNF, control mAb 1 activated human TrkB EC ₅₀ 110 pM with a maximum activation of 79% and mouse TrkB EC ₅₀ 42 pM with maximum activation 69%, which is greater than the activation of control mAb 4 with 100 pM BDNF . Control mAb 3 and control mAb 4, irrelevant mouse IgG2a isotype control antibodies, did not show any activation in the absence or presence of 100 pM BDNF.

Summary table of data:

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Table 14

Activation of HEK293/SRE-Luc/hTrkB and HEK293/SRE-Luc/mTrkB cells by anti-TrkB antibodies

Cells BDNF HEK293/SRE-luc/hTrkB 1.0E-10 HEK293/SRE-luc/mTrkB 7.4E-11 Ligand Absence BDNF lOOnMBDNF Absence BDNF lOOnMBDNF Antibody EU50 (M) Akti ration (%) EU50 (M) Akti ration (%) EU50 (M) Activation (%) EU50 (M) Activation (%) N4N9780R 8.2E- eleven 88 7.6E- eleven 77 No activation 0 No AI activation 55 N4N9814R 3.5E- eleven 92 4.9E- eleven 80 No activation 0 No AI activation 54 N4N9816R2 6,ZE- eleven 90 4,SE- II 78 No activation 0 No AI activation 58 Control mAb 1 (H1M8037C reference antibody) 7.6E- eleven 78 1DE- 10 79 4,ZE- eleven 85 4.2E-11 69 Negative isotype control mAb 2 No activation 3 No activation 56 No activation 0 No AI activation 54

Table 15

Activation of HEK293/SRE-Luc/hTrkB and HEK293/SRE-Luc/mTrkB cells by anti-TrkB antibodies (mock)

Cells HEK293/SRE-luc/hTrkB HEK293/SRE-luc/mTrkB BDNF 1,ZE-10 1.0E-10 7DE-11 1DE-10 Ligand Absence BDNF YuOPM BDNF Absence BDNF lOOnMBDNF Antibody EU50 (M) Akti vation EU50 (M) Assets ation EU50 (M) Assets ation EU50 (M) Aktiva tion

- 34 044580

(%) (%) (%) (%) Μ2αΜ14173Ν No activation 2 No AI activation 59 3.6E- eleven 94 1.7E-P 74 Μ2αΜ14178Ν No activation 0 No AI activation 52 1.9E- 10 76 1.0E-10 67 Μ2αΜ14179Ν No activation 4 No AI activation 52 3.4E- eleven 87 2DE-11 75 Control mAb 1 (H1M8037C reference antibody) 5.7E- eleven 80 1DE-10 79 4,ZE- eleven 85 4.2E-11 69 Negative isotypically th control wаb 3 No activation 0 Not researched Vans Not researched Not researched Not researched Not researched Vans Not researched Negative isotype control wаb 4 Not researched Not researched No AI activation 61 No activation 0 No AI activation 60

Example 6. In vivo comparison of the effects of TrkB agonist antibodies H4H9816P2 and IgG4 isotopic control REGN1945 on TrkB phosphorylation in the brain after stereotactic injection of TrkB ^hu/hu mice.

To determine the effect of the TrkB agonist antibody of the invention, H4H9816P2, on the kinetics of TrkB activation, a TrkB phosphorylation kinetic assay was performed following direct injection into the hippocampus in mice homozygous for the human TrkB receptor rather than the mouse TrkB receptor (referred to as TrkB ^hu/hu mice). . TrkB ^hu/hu mice (N=48) received bilateral stereotactic injections of either 2 μl of vehicle (PBS) or REGN1945, referred to herein as isotype control IgG4 antibodies (final concentration 27.5 mg/ml), or agonist antibodies TrkB H4H9816P2 (final concentration 27.5 mg/ml)) to the hippocampus -2 mm posterior and +1.5 mm lateral to bregma. To minimize tissue damage, injections and needle removal were performed gradually at 5-minute intervals. TrkB ^hu/hu mice were then killed by CO ₂ euthanasia at approximately 30 min, 1 hr, 4 hr, or 18 hr post-injection. Terminal hemorrhage was performed by cardiac puncture to collect blood, and the mice were then transcardially perfused with cold heparinized saline. The brain was carefully removed from the skull, and a 2-mm portion of the tissue surrounding the injection site was excised, collected in an Eppendorf tube, and stored on ice. Next, part of the brain was lysed in 300 μl of RIPA lysis buffer (ThermoFisher Scientific, Cat # 89901) containing 2x protease and phosphatase inhibitors (ThermoFisher Scientific, Cat #

- 35 044580

78444) and stored on ice. The lysed tissue was then homogenized for further processing, aliquoted and stored at -80°C.

To assess TrkB phosphorylation in brain tissue, immunoprecipitation and Western blotting were performed. Anti-human TrkB antibody H4H10108N, which does not compete for binding with H4H9816P2, was coupled to NHS-activated Sepharose beads (prepared according to the manufacturer's protocol; GE Healthcare, Cat # 17-0906) and washed three times with Dulbecco's phosphate-buffered saline (DPBS). ) to remove remaining preservative solution. Homogenized brain lysates were thawed on ice and diluted to a concentration of 1 mg/ml (brain weight to buffer volume) in a buffer consisting of 1% NP-40, 0.1% Tween-20, protease and phosphatase inhibitors in TBST. Protein concentration in homogenized brain lysate was quantified by standard protein content assay (PCA) according to the manufacturer's instructions (Thermo Scientific Pierce, Cat #23225). For every 100 μg of protein, 15 μl of NHS-Sepharose-activated beads coupled with anti-human TrkB antibody (H4H10108N) was added to the brain lysate solution and the mixture was incubated overnight at 4°C with gentle shaking at 20 rpm (rotator Thermo). The next day, samples were centrifuged at 1000xg for one minute and then the supernatant was carefully removed. The beads were then washed twice with 400 μl of TRIS-buffered saline (Bio-Rad, Cat # 1706435) with 1% Tween-20 (Sigma Aldrich, Cat # P9416) (TBST). After careful aspiration of the wash buffer, 60 μl of 0.1% trifluoroacetic acid (TFA; Sigma-Aldrich, T62200) in water at pH 3.0 was added to each sample. The solution was stirred and left for 2 minutes, then collected and transferred to a separate tube. This process was repeated with another 0.1% TFA volume of 60 μl at pH 3.0. Next, the two 0.1% TFA solutions for each sample were combined and 2 μl of 1 M Tris-HCl (ThermoFisher Scientific, Cat # 15567-027) was added at pH 8.5.

The solution was dried using a flash vacuum and then resuspended and reconstituted with a mixture of 20 μl of 1x Laemmli buffer (Bio-Rad, Cat # 1610737) and 355 nM 2-mercaptoethanol (BME; Gibco, Cat # 21985-023). Samples were boiled at 95°C for 10 min and loaded into an electrophoresis chamber with a 10-well module, 4-15% Tris-glycine Mini-Protean gel (Bio-Rad, Cat # 4561086). After electrophoresis, protein samples were transferred from the Tris-glycine gel to a PVDF membrane (Bio-Rad, Cat # 170-4156) using the Trans-Blot Turbo Transfer System (Bio-Rad, Cat # 1704156) for 30 min at a constant speed of 1. 3 A and 25 V. After transfer, the membrane was blocked with 2.5% milk (Bio-Rad, Cat # 170-6406) in TBST for one hour at room temperature and then probed overnight with either anti-phospho- TrkB antibody (Novus, Cat #NB100-92656) diluted 1:1000 in 2.5% BSA, or primary anti-TrkB antibody (Cell Signaling, Cat #4603) diluted 1:1000 in 2.5% milk in TBST buffer at 4°C on a shaker at 30 rpm. The next day, blots were washed with TBST and incubated with horseradish peroxidase-conjugated rabbit IgG antibody (Jackson, Cat #111-035-144) at 1:1000 in 1% milk in TBST buffer for 1 hour at room temperature. The blots were then washed again, developed in ECL solution (PerkinElmer, Inc. Cat # RPN2106), and subsequent images taken every 30 s.

Summary of results and conclusions:

Immunoprecipitation and subsequent Western blotting of protein obtained from TrkB ^hu/hu mouse brain lysates demonstrated that hippocampal phosphorylation of TrkB was detected in mice injected with the TrkB agonist antibody, H4H9816P2, but not in mice treated with vehicle or isotype control antibodies. , as shown in Fig. 1. Among the multiple time points calculated, TrkB phosphorylation peaked 4 h after stereotactic injection in H4H9816P2-treated mice. Phosphorylation of TrkB was also detected by Western blotting at 18 h postdose in some but not all mice. Conversely, injection of vehicle and isotype control IgG4 antibodies did not induce TrkB phosphorylation at any time point. Western blotting also showed that total TrkB receptor levels were reduced in some, but not all TrkB ^hu/hu mice treated with H4H9816P2 compared with vehicle and isotype control mice. Total TrkB levels appear to be slightly reduced in H4H9816P2-treated subjects at 18 hours post-dose. Thus, these results indicate that direct injection of the TrkB agonist antibody, H4H9816P2, induces phosphorylation of hippocampal TrkB receptors in TrkB ^hu/hu mice.

Example 7. In vivo comparison of the effect of the H4H9816 antibody and the REGN1945 isotype control on body weight and metabolism in TrkB ^hu/hu mice.

To determine the effect of the TrkB agonist antibody of the invention, H4H9816P2, on the weight and composition of body tissues, a metabolic study of the expression of the human TrkB receptor instead of the mouse TrkB receptor (TrkB ^hu/hu mice) was carried out in homozygous mice after a single subcutaneous injection of antibodies. TrkB ^hu/hu mice (20-week-old males) were first moved from a group cage to individual cages for 2 weeks of acclimation. After this period, mice were moved into metabolic cages (CLAMS, Columbus Instruments) to assess changes in intake.

- 36 044580 information about food and water, physical activity, calorie consumption and respiration after the administration of antibodies. Regular powdered feed was stored in a floor chamber on a spring scale (Mettler Toledo, PL602E) to measure feed intake by changing total feed mass. Water was available through the top valve in the cage, and water consumption was measured by monitoring volume changes in the pressure line (Oxymax®/CLAMS Liquid Unit). The CLAMS cell's metabolic machinery measured each of these parameters at continuous 16-18 minute intervals throughout the study. Metabolic data were analyzed in individual measurements and summarized into 24-hour intervals comprising one complete dark-light cycle using OXYMAX®/CLAMS software (Columbus instruments, v5.35). After habituation to the cages for two weeks, TrkB ^hu/hu mice received a single subcutaneous dose of 50 mg/kg of either the TrkB agonist antibody, H4H9816P2, or an isotype control IgG4 antibody in PBS at pH 7.2. A group of untreated control TrkB ^hu/hu mice did not receive the injection. Mice were weighed immediately before dosing and 24, 48, 72, 96, and 120 h after dosing. To determine body tissue composition, nuclear magnetic resonance relaxometry, also called quantitative magnetic resonance, was performed on each mouse using an EchoMRITM-500 analyzer (EchoMRI LLC). Before dosing, mice were placed in a clear plastic holder and inserted into an NMR-MRI device to determine each subject's lean tissue mass, adipose tissue mass, and hydration status. Measurements were taken for each mouse for 0.5-3.2 minutes, and repeated approximately 120 hours after dosing.

Summary of results and conclusions:

To determine whether a single subcutaneous injection of H4H9816P2 causes weight loss in TrkB ^hu/hu mice, body weight was monitored daily. Pre-dose, there were no significant differences in the mean body weight of the three treatment groups, with each group having a mean pre-dose body weight of 28.39-29.85 g (Table 16). However, at 48 hours post-dose, H4H9816P2-treated TrkB ^hu/hu mice had lost an average of 1.70 g, or 5.96% of pre-dose body weight. At the same time, in TrkB ^hu/hu mice that did not receive treatment and in TrkB ^hu/hu u mice treated with isotype control antibodies, their body weights increased by 1.79-2.37% of pre-dose weight. TrkB ^hu/hu mice treated with H4H9816P2 continued to lose weight throughout the study, and at 72 and 96 hours post-dose, these mice had lost an average of 8.42% and 11.80% of their pre-dose body weight, respectively. At 120 hours post-dose, TrkB ^hu/hu mice treated with H4H9816P2 lost an average of 12.67% of their pre-dose body weight. However, untreated and isotype control TrkB ^hu/hu mice did not experience predose weight loss throughout the study. Because body weight in TrkB ^hu/hu mice treated with H4H9816P2 was significantly reduced compared with untreated and isotype control at 48, 72, 96, and 120 h post-dose, the TrkB agonist antibody H4H9816P2 was found to cause significant loss body weight in TrkB ^hu/hu mice.

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Table 16

Body weight of TrkB ^hu/hu mice after administration of a dose of the TrkB agonist antibody H4H9816P2

Experimental Average Average Average Average Average Average tall weight weight weight weight weight weight group body organism organism organism organism body before a (d) through a (d) through a (d) through a (d) through (d) through introduction 24 hours 48 h 72 h 96 h 120 hours after doses (g) after after after after introduction (±standard introduction introduction introduction introduction doses new doses doses doses doses (±standard deviation (±standard (±standard (±standard (±standard new ) precise precise precise precise deviation rejected rejected rejected rejected ) e) e) e) e) Percentage Percentage Percentage Percentage Percentage Percentage e oh oh oh oh e change changes changes changes changes change masses e masses e masses e masses e masses masses body organism organism organism organism body before and before and before and before and before before introduction introduction introduction introduction introduction introduction doses (+/- doses (+/- doses (+/- doses (+/- doses (+/- doses (+/- SD) SD) SD) SD) SD) SD) Not 28.85 29.69 29.36 29.32 29.29 28.88 receiving (+/- 0.81) (+/- 0.97) (+/- 1,Yu) (+/- 1.29) (+/- 1,Yu) (+/- 1.04) I'm treating N/A +2.91% +1.79%(+/ +1.65%(+/ +1.54%(+/ +0.10%(+/- (n=3) (+/- 0.62) - 1.62) - 2.24) - 1.22) 1.05) Isotypic 29.21(+/- 30.27(+/- 29.90(+/- 30.08(+/- 29.87(+/- 29.69(+/- cue 2.68) 2.51) 2.63) 2.69) 2.52) 2.68) control N/A +3.61%(+/ +2.37%(+/ +2.98%(+/ +2.25%(+/ +1.65%(+/- (n=4) - 1.68) - 1.50) - 1.09) - 1.56) 0.81) N4N9816R2 28.39(+/- 27.87(+/- 26.69(+/- 26.00*(+/- 25.04**(+ 24.79**(+/- (n=4) 1.35) 1.29) 0.87) 0.98) /- 1.03) 1.36) N/A - - - - -12.67%(+/- 1.83%(+/- 5.96%(+/- 8.42%(+/- 11.80%(+/ 1.66) 0.56) 1.88) 1.85) - 1.52)

Note: statistical significance is indicated, determined by two-way ANOVA with Tukey's post hoc multiple comparisons method (*=p<0.05, **=p<0.01, ***=p<0.001, ** **=p<0.0001, compared with the isotype control group: TrkB ^hu/hu mice treated with 50 mg/kg isotype control antibody.

The effect of injection of the TrkB agonist antibody H4H9816P2 on body tissue composition was also measured by pre- and post-dose NMR-MRI for each subject. Before dosing, the three TrkB ^hu/hu treatment groups did not show any significant differences in fat mass.

-38 044580 tissue or lean tissue mass, since each group had an average of 4.19-4.75 g of fat tissue mass and 21.32-21.70 g of lean tissue mass (Table 17). However, after administration of antibodies, TrkB ^hu/hu mice treated with H4H9816P2 lost an average of 48.90% of their total body fat mass over the course of the study (Table 17). Untreated and TrkB ^hu/hu isotype control antibody-treated mice lost an average of 8.49% and 9.48% of their pre-dose adipose tissue mass, respectively, which was significantly less than that of H4H9816P2-treated subjects (Table 17). Additionally, TrkB ^hu/hu mice treated with H4H9816P2 lost an average of 7.84% of their lean tissue mass throughout the study, which was significantly greater than the 2.41% and 1.75% average lean tissue mass before dosing. , lost in the groups that did not receive treatment and were treated with isotype control antibodies, respectively (Table 17). Thus, the described loss of body weight can be explained by the significant loss of adipose tissue mass and moderate loss of lean tissue mass after injection of the TrkB agonist antibody H4H9816P2 TrkB ^hu/hu mice.

Table 17

Composition of body tissues in TrkB ^hu/hu mice after administration of a dose of the TrkB agonist antibody H4H9816P2

Experiment mental group Mean pre-dose adipose tissue mass (%) (±standard deviation) Average adipose tissue mass (%) 120 hours after dosing (±standard deviation) Change in mean fat mass (%) 120 hours post-dose (±standard deviation) Average lean tissue mass BEFORE dose administration (%) (±standard deviation) Mean lean tissue mass (%) at 120 hours post-dose (±standard deviation) Change in mean lean tissue mass (%) at 120 hours post-dose (±standard deviation) Not getting it treatment (n=3) 4.65(+/- 0.32) 4.27(+/- 0.55) -8.49(+/- 7.18) 21.45 (+/- 0.79) 20.94(+/- 0.98) -2.41(+/- 1.81) Isotype control (n=4) 4.75(+/- 2.98) 4.40(+/- 2.98) -9.48(+/- 6.00) 21.70(+/- 0.50) 21.32(+/- 0.35) -1.75(+/- 0.98) N4N9816 P2 (n=4) 4.19(+/- 1.15) 2.14(+/0.64) 48 90**** (+/- 5.06) 21.32 (+/- 1.87) 19.64 (+/- 1.69) -7.84***(+/0.94)

Note: statistical significance is indicated as determined by one-way Kruskal-Wallis analysis of variance with Tukey's post hoc multiple comparisons method (*=p<0.05, **=p<0.01, ***=p<0.001, ****= p<0.0001, compared with the isotype control group: TrkB ^hu/hu mice treated with 50 mg/kg isotype control antibody.

In addition to assessing the effects of injection of the TrkB agonist antibody H4H9816P2 on body mass and tissue composition in TrkB ^hu/hu mice, the cellular metabolic machinery was continuously measured for food intake, fluid intake, and locomotor activity. Before dosing, TrkB ^hu/hu mice consumed an average of 3.49 to 3.73 g of food per day. However, within 24 hours after dosing, TrkB ^hu/hu mice treated with H4H9816P2 had a significant reduction in food intake, to 2.20 g per day. The average food intake of TrkB ^hu/hu mice treated with H4H9816P2 did not exceed 2.49 g per day during the remainder of the study, while untreated and isotype control antibody-treated TrkB ^hu/hu mice consistently consumed an average of 3 .62-4.07 g of feed per day (Table 18).

Likewise, there were no significant differences in daily water intake between treatment groups before dosing. TrkB ^hu/hu mice consumed an average of 4.67-5.55 ml of water per day in each treatment group (Table 19). Following dosing, TrkB ^hu/hu mice treated with H4H9816P2 decreased their water intake to 2.05-3.24 ml of water per day. This was significantly lower than that observed in TrkB ^hu/hu mice, which consistently consumed 4.50-5.77 ml of water per day throughout the study (Table 19). Thus, injection of the TrkB agonist antibody, H4H9816P2, appeared to provide a significant reduction in food and water intake in TrkB ^hu/hu mice compared with untreated and isotype control mice.

Table 18

Consumption of TrkB ^hu/hu food in mice after dosing with the TrkB agonist antibody H4H9816P2

Experimental group Average total feed intake (g) during the period 0-24 hours before dosing (±standard deviation) Mean total feed intake (g) 0-24 hours post-dose (±SD) Average total feed intake (g) in the period 24-48 hours after dose administration (±standard deviation) Average total feed intake (g) during the period 48-72 hours after dose administration (±standard deviation) Average total feed intake (g) during the period 72-96 hours after dose administration (±standard deviation) Not receiving treatment (n=3) 3.51(+/- 0.53) 3.98(+/- 0.08) 3.76(+/- 0.19) 3.62(+/- 0.35) 3.91(+/- 0.18) Isotype control (n=4) 3.73(+/- 0.48) 4.07(+/- 0.23) 3.99(+/- 0.17) 3.89(+/- 0.22) 3.80(+/- 0.22) N4N981 6Р2 (n=4) 3.49(+/- 1.07) 2.20* ***(+/0.16) 2.08* ***(+/- 0.36) 2.18****(+/- 0.37) 2.49***(+/- 0.47)

Note: statistical significance is indicated as determined by one-way Kruskal-Wallis analysis of variance with Tukey's post hoc multiple comparisons method (*=p<0.05, **=p<0.01, ***=p<0.001, ****= p<0.0001, compared with the isotype control group: TrkB ^hu/hu mice treated with 50 mg/kg isotype control antibody.

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Table 19

Water consumption of TrkB ^hu/hu mice after dosing with the TrkB agonist antibody H4H9816P2

Average Average Average Average Average general general general general general consumption consumption consumption consumption consumption water (ml) in water (ml) in water (ml) in water (ml) in water (ml) in Experiment period 0-24 period 0-24 period 24-48 period 48-72 period 72-96 ntal h to h after h after h after h after group introduction introduction introduction introduction introduction doses doses doses doses doses (±standard (±standard (±standard (±standard (±standard e e e e e deviation) deviation) deviation) deviation) deviation) Not receiving 4.79 (+/- 5.42 (+/- 4.96 (+/- 4.57 (+/- 4.88 (+/- I'm treating 0.21) 0.94) 0.91) 0.56) 0.32) (n=3) Isotypical skiy 5.55 (+/- 4.50 (+/- 5.08 (+/- 5.09 (+/- 5.77 (+/- control 1.23) 1.08) 1.39) 1.10) 1.62) (n=4) N4N9816R 4.67 (+/- 2.25** (+/- 3.24* (+/- 2.05*** (+/- 2.25**** (+/- 2(n=4) 1.13) 0.55) 1.10) 0.29) 0.24)

Note: statistical significance is indicated as determined by one-way Kruskal-Wallis analysis of variance with Tukey's post hoc multiple comparisons method (*=p<0.05, **=p<0.01, ***=p<0.001, ****= p<0.0001, compared with the isotype control group: TrkB ^hu/hu mice treated with 50 mg/kg isotype control antibody.

To determine the effects of antibody treatment on activity, locomotor activity was analyzed using OXYMAX®/CLAMS software (Columbus instruments, v5.35), which continuously measured the total number of x-plane movements for each mouse. One mouse exhibited predose hyperactivity and was excluded from postdose statistical analysis. While untreated subjects treated with isotype control antibodies consistently recorded an average of 11,000-15,000 ambulations per day throughout the study, TrkB ^hu/hu mice treated with H4H9816P2 recorded 28,260 ambulations between 24-48 hours post-administration dose and recorded 21,193 and 27,028 movements in subjects from 48-72 and 72-96 hours after dosing, respectively (Table 20). TrkB ^hu/hu mice treated with H4H9816P2 exhibited a greater total number of ambulations at each time point after antibody administration, suggesting that hyperactivity is an additional effect of H4H9816P2 injection. Taken together, these effects suggest that a single subcutaneous injection of the TrkB agonist antibody, H4H9816P2, caused significant changes in body weight, body tissue composition, metabolism, and locomotor activity in TrkB ^hu/hu mice.

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Table 20

Locomotor activity of TrkB ^hu/hu mice after administration of a dose of the TrkB agonist antibody H4H9816P2

Experimental Average Average Average Average Average tall quantity quantity quantity quantity quantity group total total total total total mobile mobile movement mobile movement th (signals) th (signals) th (signals) in th th (signals) in in the period 0- in the period 0- period 24-48 (signals) in period 72-96 24 hours before 24 hours after h after period 48- h after introduction introduction introduction 72 hours after introduction doses doses doses introduction doses (±standard (±standard (±standard doses (±standard oh oh e (±standard e deviation) deviation) deviation) oh deviation) deviation) Not 16562(+/- 14692(+/- 14387(+/- 13279(+/- 12525(+/- receiving treatment (n=3) 3380) 2792) 6126) 3607) 4121) Isotypic 18105(+/- 13380(+/- 13049(+/- 11371(+/- 11468(+/- control (REGN1945) (n=4) 4085) 2730) 3376) 2552) 2088) N4N9816R2 13292(+/- 16575(+/- 28260(+/- 21193(+/- 27028*(+/- (n=4) 5294) 6836) 19874) 6668) 10969)

Note: statistical significance is indicated as determined by one-way Kruskal-Wallis analysis of variance with Tukey's post hoc multiple comparisons method (*=p<0.05, **=p<0.01, ***=p<0.001, ****= p<0.0001, compared with the isotype control group: TrkBhu/hu mice treated with 50 mg/kg isotype control antibody.

Example 8: Optic Nerve Transection Model to Determine the Effect of Anti-TrkB Antibodies on Retinal Ganglion Cell (RGC) Survival.

All procedures were performed in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Use of Animals in Ophthalmic and Vision Research and Regeneron Pharmaceutical Inc. guidelines. IACUC. Adult female TrkB humanized rats (Velocigene, Regeneron Pharmaceutical Inc.) aged 8-10 weeks, each weighing 200-250 g, were used. All surgical procedures on rats were performed under general anesthesia using an intraperitoneal injection of ketamine (63 mg/kg) and xylazine (6.0 mg/kg). An ophthalmic ointment containing erythromycin (0.5%, Bausch & Lomb) was used to protect the cornea.

Intraorbital optic nerve axotomy and intravitreal injection

The left optic nerve (ON) was treated intraorbitally with its meninges exposed. The optic nerve was cut approximately 1.5 mm behind the eyeball. Care was taken to ensure that the blood supply to the retina was not compromised. Intravitreal injections were performed directly behind the pars plana of the ciliary body using an elongated glass pipette connected to a 50-μl Hamilton syringe. Measures were taken to prevent injury to the lens. Rats with any significant postoperative complications (eg, retinal ischemia, cataract) were excluded from further study. Animals were distributed into different experimental groups. One control group received intravitreal injections of 3 μl

- 42 044580 isotype control REGN1945 (46.6 μg/μl); the other group received an injection of 3 μl of the human anti-TrkB antibody H4H9816P2 (45.7 μg/μl) 3 and 10 days after optic nerve axotomy.

In another experiment, the dose-dependent effect of the anti-human TrkB antibody H4H9816P2 was studied.

Homozygous TrkB humanized rats, 1–9 months of age, received an intravitreal injection of 3 μl anti-human TrkB antibody

H4H9816P2 (0.01, 0.1, 1 or 10 μg/μl) or isotype control REGN1945 (10 μg/μl) 3 and 10 days after optic nerve axotomy.

Immunohistochemical staining and counting of viable retinal ganglion cells (RGCs)

Brn3a (brain-specific homeobox/PM domain protein 3A) was used as a marker of retinal ganglion cell (RGC) survival because it has been shown to be an effective and reliable method for selectively labeling viable RGCs in whole-mount retinas after optic nerve injury (Nadal-Nicolas FM , Jimenez-Lopez M, Sobrado-Calvo P, Nieto-Lopez L, Canovas-Martinez I, Salinas-Navarro M, Vidal-Sanz M, Agudo M., Invest Ophthalmol Vis Sci. 2009 Aug;50(8):3860- 8). For Brn3a immunostaining, retinas were blocked in 10% normal donkey serum and 0.5% Triton X-100 for 1 h, then incubated in the same medium with anti-Brn3a antibody (1:400; Cat #: sc-31984, Santa Cruz) 2 hours at room temperature. After additional washes, retinas were incubated with Alexa594-conjugated donkey anti-goat secondary antibody (1:400; Cat #: A-11058, Invitrogen) overnight at 4°C.

Summary of results and conclusions:

To evaluate the effect of TrkB agonist antibody on RGC survival in vivo, we used a complete optic nerve transection model. A TrkB agonist antibody (H4H9816P2) or an isotype control antibody (negative control) was administered 3 and 10 days after surgery. Animals were sacrificed 14 days after axotomy. RGC density in the uninjured contralateral eye was similar to the three TrkB genotypes, averaging about 1600 per ^mm2 , as shown in Table 1. 21 The density of surviving RGCs was assessed in whole‐mount retinas by Brn3a staining. It was noted that in homozygous TrkB humanized rats, the TrkB agonist antibody (H4H9816P2) significantly (p < 0.01, Mann-Whitney test) increased RGC survival compared to controls (685 ± 106 versus 255 ± 66 RGCs per mm ² ). In heterozygous TrkB humanized rats, there was also a significant (p < 0.05, Mann-Whitney test) survival effect caused by the TrkB agonist antibody (444 ± 90 versus 208 ± 50 RGCs per mm ² ). In TrkB wild-type rats, a small but non-significant increase in RGC number was observed with the TrkB agonist antibody compared to the isotype control (Table 22). In a dose-response study, RGC density was quantified in whole-mount retinas using Brn3a staining 14 days after axotomy. There was a clear dose-dependent effect of the TrkB agonist antibody. Compared with the control antibody group (168 ± 43 RGCs per ^mm2 ), the TrkB agonist antibody (H4H9816P2) significantly (p < 0.01, one-way ANOVA with Tukey's post hoc test) increased RGC survival in the 3 μg injected group ( 564±124 RGCs per ^mm2 ) or 30 μg (543±242 RGCs per ^mm2 ). There was no difference between the 3 mcg and 30 mcg groups. In the groups injected with 0.03 μg (202 ± 96 RGCs per mm ² ) or 0.3 μg (337 ± 210 RGCs per mm ² ), there was only a trend, but not a significant increase in RGC survival (Table 23). In conclusion, the TrkB agonist antibody H4H9816P2 showed significantly increased RGC survival in TrkB ^hu/hu and TrkB ^hu/hu rats.

Conclusion:

TrkB agonist antibody (H4H9816P2) dose-dependently significantly increased RGC survival in the TrkB humanized rat.

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Table 21

Data on the number of RGCs (RGC/mm ² ) in the uninjured control eye

TRKB genotypes

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

Table 22

Data on the number of RGCs (RGC/mm ² ) after damage to the optic nerve

H4H9816P2 Isotype control antibody A:Y1 A:Y2 A:Y3 A:Y4 A:Y5 B:Y1 B:Y2 B:Y3 B:Y4 B:Y5 Hu/H u 790.1 737.1 756.3 587.8 555.7 322.8 295.0 286.9 171.3 197.9 Hu/+ 530.4 457.5 522.9 390.6 319.2 231.0 184.6 265.1 151.3 +/+ 320.9 355.5 256.9 342.7 112.3

Table 23

RGC count data (RGC/mm ² ) in a dose-response study

Control antibody N4N9816R2 30 mcg/intravit 0.03 mcg/intravitrea 0.3 mcg/intravitre 3 mcg/intravitre 30 mcg/intravitreal really flax ally ally really 222.3 162.7 341.7 637.9 493.0 205.9 190.2 269.1 686.4 613.5 131.7 127.7 292.2 533.2 557.6 136.3 174.0 252.8 574.7 227.1 144.3 163.8 128.3 334.4 954.3 392.6 740.9 620.6 411.3

Example 9. Effect of anti-TrkB antibodies on activation of Akt and Erk signaling pathways.

All procedures were performed in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Use of Animals in Ophthalmic and Vision Research and Regeneron Pharmaceutical Inc. guidelines. 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 effect of TrkB agonist antibody on Akt and Erk downstream signaling pathways (p-Akt, p-Erk1/2). Primary cortical neurons from humanized TrkB mice

- 44 044580 on postnatal day 1 (P1) were cultured for 4 days (DIV-4) in NeuralQ Basal Medium (Global Stem, cat. # GSM-9420) supplemented with GS21 Neural Supplement (Global Stem, cat. # GSM -3100), Glutamax (Invitrogen, cat. # 35050-061) and a mixture of penicillin and streptomycin. Cells were treated with TrkB agonist antibodies: H4H9816P-L1 (10 μg/ml), H4H9780P-L1 (10 μg/ml), H4H9814P-L1 (10 μg/ml), isotype control IgG4 REGN1945 (10 μg/ml), control antibody H1M8037C-L1 (10 μg/ml), BDNF (1 μg/ml), for 15 min or 2 h. Western blotting was performed to determine whether there was a difference between agonists in the maintenance and intensity of downstream signaling. Treated cells were washed and cleared in cold PBS containing 1% protease and phosphatase inhibitors (Sigma). Protein concentration was determined by the Bradford method (Pierce). Samples (50 μg) were separated by SDS-PAGE on 3-8% Tris-acetate reducing gels (Novex) and transferred to a nitrocellulose membrane (Bio-Rad).

The membrane was incubated for 1 hour in a blocking solution containing 5% milk and 0.1% Tween 20, pH 7.6. Then incubated overnight at 4°C in blocking buffer containing 5% BSA, 0.1% Tween-20 and rabbit anti-phospho-Trk (Cell Signaling, cat # 9141, 1:500), rabbit anti-phospho-Akt ( Cell Signaling, cat #9271, 1:1000) or rabbit anti-phospho-ERK1/2t antibodies (Sigma, cat #E7028, 1:5000). Labeled proteins were then visualized by incubation with horseradish peroxidase (HRP)-conjugated goat, mouse, or rabbit IgG antibodies, followed by visualization with chemiluminescent substrate for HRP (Pierce). To determine the amounts of total TrkB, MAPK, or Akt present in each track, nitrocellulose membranes were stripped of antibodies in stripping buffer (Pierce) for 20 min and incubated with rabbit anti-TrkB (Cell Signaling, cat # 4603, 1:1000). , rabbit anti-Erk1/2 (Cell Signaling, cat #06-182, 1:1000) or rabbit anti-Akt antibody (Cell Signaling, cat #9272, 1:1000) and then visualized in the manner described above. Beta actin (Sigma, cat #A5316, 1:20000 and GAPDH (Sigma, cat #G9295) were used as loading controls.

Summary of results and conclusions:

As shown in Figure 2, 15 min after incubation, while all TrkB agonist antibodies showed activation of the MAPK/ERK and PI3K/Akt pathway, only BDNF and H4H9814P showed phosphorylation of TrkB. At 2 h postincubation, all TrkB agonist antibodies showed TrkB activation.

Example 10. Effect of agonistic anti-TrkB antibodies on the survival of SH-SY5Y cells.

Cultivation of human neuroblastoma SH-SY5Y cells in vitro:

Cells of the neuroblastoma line SH-SY5Y (Sigma ATCC # 94030304, cat # 11C016) were seeded in a nutrient medium containing a mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F12 substrate (Invitrogen cat # 11330), a mixture of penicillin and streptomycin (Invitrogen cat. # 15140), PBS 10% (Invitrogen cat # 10082-147) at 37°C in 5% CO ₂ . At passage, 23-27 cells were seeded in 96-well plates in differentiation medium containing 10 µM all-trans retinoic acid (Alfa Aesar cat. #44540), DMEM:F12 mixture (Invitrogen cat #11330), penicillin-streptomycin mixture (Invitrogen cat #15140), PBS 10% (Invitrogen cat #10082-147). Cells (30 K/well) were differentiated for 4 days. Antibodies were screened in a survival bioassay in which the culture was replaced with serum-free differentiation medium (100 μl/well) containing varying doses of antibodies (100-0.01 μg/ml). After 2 days, CCK8 reagent (Dojindo, cat #CK04) (10 µl/well) was added, plates were incubated for 3-4 hours, optical density (OD) was measured at 450 nm (Victor or FlexStation III) to determine the percentage surviving cells. Data were normalized to serum-free medium without treatment. Serum-free treatment without antibodies corresponds to 100% survival.

Summary of results and conclusions:

As shown in FIG. 3 and in table. 23, all TrkB agonist antibodies of the invention showed a significant dose-dependent increase in SH-SY5Y cell survival compared to the negative isotype control antibody (p < 0.0001 by two-way ANOVA).

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Table 24

Average % survival Dose mcg/ml H4H9816P-L1 H4H9780P-L1 H4H9814P-L1 REGN1945-L14 (negative isotype control) 100 116.7 119.7 114.5 97.1 10 122.3 125.3 116.6 97.7 1 120.2 121.7 116.0 92.5 0.1 113.9 116.4 116.3 92.9 0.01 98.0 103.0 97.0 93.6

Example 11. Evaluation of the pharmacokinetics of anti-TrkB antibodies in humanized TrkB mice and WT mice.

The pharmacokinetics of anti-TrkB antibodies H4H9816P2 were assessed in humanized TrkB mice (mice homozygous for human TrkB expression, TrkB ^hu/hu ) and wild-type (WT) mice. Groups contained 5 mice per mouse strain. All mice received a single subcutaneous (SC) dose of 10 mg/kg. Blood samples were collected 6 hours 1, 2, 3, 6, 9, 16, 21 and 30 days after dosing. Serum was obtained from the blood and frozen at -80°C until the study.

Circulating antibody concentrations were determined by anti-human total IgG4/hIgG1 antibody assay using GyroLab xPlore™ (Gyros, Uppsala, Sweden). Briefly, biotinylated mouse anti-human IgG4/IgG1-specific monoclonal antibody (REGN2567), diluted to 100 μg/ml in antibody dilution buffer (0.05% Tween-20 + PBS), was captured on a Gyrolab Bioaffy 200 CD, which contained columns for affinity chromatography preloaded with streptavidin-coated particles (Dynospheres™). 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 REGN2567 antibody-coated CD affinity chromatography columns, run at room temperature, was detected by adding 0.5 μg/ml Alexa-647-conjugated mouse monoclonal anti-human kappa chain antibody (REGN654) diluted in detection (Rexxip F buffer); and the resulting fluorescent signal was recorded in response units (RU) using the GyroLab xPlore instrument. Sample concentrations were determined by interpolation from a standard curve, which was fitted using a 5-parameter logistic fit using Gyrolab Evaluator software. Average concentrations from two replicate experiments were used for subsequent pharmacokinetic (PK) analysis.

Pharmacokinetic parameters were determined by noncompartmental analysis (NCA) using Phoenix® WinNonlin® Version 6.3 software (Certara, LP, Princeton, NJ) and an extravascular dosing model. Using the corresponding mean concentration values for each antibody, all pharmacokinetic parameters, including the observed maximum serum concentration (Cmax), the estimated observed half-life (t1/ ₂ ) and the area under the concentration versus time to last measured concentration curve (AUC _last ) were determined using the linear trapezoidal rule with linear interpolation and uniform weighting.

Summary of results and conclusions:

Following subcutaneous administration of 10 mg/kg anti-TrkB antibody, H4H9816P2, similar maximum antibody concentrations (C _max ) were observed on days 1 or 2 in both TrkB ^hu/hu and WT mice (135 and 131 μg/ml, respectively, see table 26). At day 9, H4H9816P2 showed greater clearance in TrkB ^hu/hu mice than in WT mice, indicating a target-mediated effect. At day 30, antibody concentrations were approximately 35-fold lower in TrkB ^hu/hu mice. Antibody exposure (AUClast) for H4H9816P2 in WT mice was ~1.7 times higher than in TrkB ^hu/hu mice (1730 and 1020 d*μg/ml, respectively). WT mice also showed an approximately 3-fold increase in half-life (T1/2) compared to TrkBhu/hu mice (8.4 and 2.9 days, respectively).

A summary of data on total anti-TrkB antibody concentrations is presented in Table. 25, the average pharmacokinetic parameters are presented in table. 26, and the average total antibody concentrations as a function of time are shown in FIG. 4.

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Table 25

Mean (+SD) serum total IgG concentrations after a single subcutaneous injection of 10 mg/kg H4H9816P2 TrkB ^hu/hu mice and wild-type mice as a function of time

Antibody Time (d) Total mAb concentration in mouse serum 10 mg/kg Average value (µg/ml) +/-SD TrkB ^hu/hu mice 0.25 72.42 4.06 1 132.0 18.0 2 124.9 15.9 3 113.4 11.8 6 78.72 9.98 9 37.74 14.0 16 5,592 4.97 21 2,060 2.11 thirty 0.447 0.506 Wild type mice 0.25 56.73 14.5 1 120.8 6.26 2 131.2 7.54 3 125.7 7.46 6 101.9 11.4 9 75.94 7.06 16 42.61 16.1 21 27.75 16.9 thirty 15.52 13.0

Abbreviations: time=time in days after a single dose; d=day of study; 8П=standard deviation.

Table 26

Summary of Pharmacokinetic Parameters

Parameter Units N4N9816R2 TrkB ^hu/hu mouse Wild type mouse Sorry µg/ml 135 ± 15 131 ±7.5 Tschakh day 1.4 ±0.56 2.0 ±0 T1/2 day 2.94 ± 1.1 8.36 ±3.9 AUCl _ast dmkg/ml 1020±150 1730±310

Pharmacokinetic parameters were obtained from mean concentration-time profiles. Tch and AUCi _as t are based on concentrations up to day 30.

Abbreviations: _Cmax = peak concentration; AiC=area under the concentration-time curve; AUCi _as t=AUC, calculated from time zero to the time of the last sample collection at which the concentration is above the lower limit of quantitation; T^final elimination half-life; T _max = time after antibody administration when maximum serum concentration is reached

Example 12. The ability of monoclonal antibodies against mouse TrkB to block the interaction between mouse or rat TrkB and its ligand BDNF (brain tissue-derived neurotrophic factor).

Anti-mouse TrkB monoclonal antibodies (mAbs) were generated by immunizing TrkB humanized mice with mouse TrkB protein. The three main mAbs identified in this immunization were: M2aM14173N, M2aM14178N and M2aM14179N. The primary mAbs of the invention were characterized by their ability to block the interaction of mouse or rat TrkB with plate-bound BDNF in a blocking ELISA.

The experiments were carried out using the following procedure. Human BDNF was applied at a concentration of 0.5 μg/ml (to block mouse TrkB.hFc interaction) or 0.3 μg/ml (to block rat TrkB.mmh interaction) in PBS on a 96-well microtiter plate and incubated overnight at 4°C. Nonspecific binding sites were then blocked using a 5% (w/v) solution of BSA in PBS (assay buffer). In a 96-well dilution plate, 850 pM mouse TrkB.hFc or rat TrkB.mmh was mixed with threefold serially diluted anti-mouse TrkB and control antibodies. The final antibody concentrations ranged from 1.69 pM to 100 nM. The protein-antibody mixture was incubated at room temperature 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 original study data. ELISA plates were incubated at room temperature for 1 hour and then washed with plate washing solution. Mouse plate-bound TrkB.hFc was detected using an HRP-conjugated goat anti-human Fcy fragment recognition antibody (Jackson Immunoresearch), and rat TrkB.mmh was detected using an HRP-conjugated anti-histidine antibody (Qiagen). The plates were incubated with detection antibody for 1 h at room temperature and then washed with plate wash solution. Analytical plates were developed with TMB colorimetric substrates according to the procedure recommended by the manufacturer.

The absorbance at 450 nm for each well was recorded and plotted as a function of antibody concentration. Data were analyzed by GraphPad Prism software using a four-parameter logistic equation with an 11-point dose-response curve, and IC ₅₀ values were calculated. The calculated IC ₅₀ value, defined as the concentration of antibody required to reduce TrkB binding to BDNF by 50%, was used as an indicator of blocking efficiency. The percentage of blocking, at the maximum concentration of the test antibody, was calculated as an indicator of the ability of the antibodies to block the binding of TrkB to BDNF on the plate relative to the original study data. The binding signal of 850 pM mouse or rat TrkB in the absence of antibody was defined as 100% binding or 0% blocking. The initial signal of the assay buffer alone was defined as 0% binding or 100% blocking.

Summary of results and conclusions:

The ability of anti-mouse TrkB antibodies to block the binding of mouse or rat TrkB to BDNF was assessed using blocking enzyme-linked immunosorbent assays.

The blocking results are shown in table. 27 and in fig. 5A and B. Percentage blocking is presented for all antibodies and calculated at the highest antibody concentration assayed (100 nM). IC ₅₀ values are shown only for antibodies that block >50% of mouse or rat TrkB binding to BDNF. Of the three antibodies of the present invention, the anti-mouse TrkB mAb, M2aM14178N, blocked >50% of mouse and rat TrkB protein binding to BDNF. M2aM14178N blocked the binding of 850 pM mouse TrkB.hFc with an IC value of _50,426 pM and a percentage blocking of 84.4%. M2aM14178N blocked the binding of 850 pM rat TrkB.mmh to BDNF with an IC ₅₀ value of 1 84 pM and a percentage blocking of 89.5%. M2aM14173N showed 29.7% blocking of 850 pM mouse TrkB.hFc binding to BDNF. M2aM14173N showed 80.7% blocking of 850 pM rat TrkB.mmh binding to BDNF with an IC ₅₀ value of 3.81 nM. M2aM14179N blocked 11.6% of mouse TrkB.hFc binding to BDNF. M2aM14179N showed an increase in rat TrkB.mmh binding to BDNF at concentrations that were greater than 1 nM.

A comparison mouse monoclonal antibody to TrkB, H1M8037C, blocked the binding of 850 pM mouse TrkB.hFc to BDNF with an IC ₅₀ value of 1 80 pM and a blocking percentage of 91.5%. H1M8037C blocked the binding of 850 pM rat TrkB.mmh with an IC ₅₀ value of 1.42 nM and a blocking percentage of 83.3%. The mIgG2a isotype control mAb, REGN1097, did not show any blocking of mouse or rat TrkB under similar study conditions. At concentrations above 10 nM, REGN1027 showed an increase in rat TrkB binding.

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Table 27

Final IC ₅₀ (M) values for blocking mouse or rat TrkB binding to BDNF anti-mouse TrkB antibodies

Antibody blocking binding 780 nM TrkB.hFc mice with BDNF coating plate Antibody blocking binding of 780 pM rat TrkB.hFc to BDNF plate coating AbPID 1C ₅₀ [M] % blocking with 100 nM antibody 1C ₅₀ [M] % blocking with 100 nM antibody Μ2αΜ14173Ν undefined 29.7 3.81E-09 80.7 Μ2αΜ14178Ν 4.26E-10 84.4 1.84E-10 89.5 Μ2αΜ14179Ν undefined 11.6 Not defined -38.1 H1M8037C (comparison antibody) 1.80E-10 91.5 1D2E-09 83.3 Negative isotype control (REGN1097) undefined No blocking Not defined -9.33

100% blocking = OD450 nm value in wells with HRP-conjugated secondary antibody in assay buffer only (without mouse or rat TrkB protein).

0% blocking = absorbance value at 450 nm in wells containing HRP-conjugated secondary antibody in assay buffer in the presence of mouse or rat TrkB protein (without TrkB antibody).

The percentage negative maximum flare indicates the increase in TrkB binding detected in the presence of the antibody.

not determined=IC ₅₀ values not quantified for antibody blocking <50% at the highest concentration tested.

Example 13. The ability of monoclonal antibodies against human TrkB to block the interaction between human TrkB and its natural ligands BDNF and human NT4.

The ability of anti-human TrkB antibodies, designated H4H9814P, H4H9816P2, and H4H9780P, to block the binding of TrkB protein to plate-captured BDNF or NT-4 was measured using two competitive sandwich ELISAs. In the studies, various concentrations of anti-TrkB antibody were premixed with a constant amount of TrkB dimeric protein, and the decrease in TrkB binding to plate-immobilized BDNF or NT-4 due to the presence of the antibody was calculated.

The recombinant dimeric TrkB protein used in the experiments consisted of a region of the human TrkB extracellular domain (aa Cys32-His430) expressed by the Fc region of human IgG1 at Cconc (hTrkB-hFc; accession number NP_006171.2, molecular weight 69700 daltons). The BDNF and NT-4 proteins consist of the extracellular domain of BDNF (aa His129-Arg247, Accession #P23560, R&D Systems) or human NT-4 (aa Gly81-Ala210, Accession #P34130, R&D Systems), respectively. Two isotype control antibodies, anti-Fel d 1 human IgG4 antibody and a-Fel d 1 mouse IgG1-specific antibody, were included as controls for detection of IgG background.

The experiments were carried out using the following procedure. Overnight at 4°C in PBS, a 96-well microtiter plate was coated individually with human BDNF or NT-4 at a concentration of 0.5 μg/ml or 2 μg/ml, respectively. Sites of nonspecific binding were subsequently blocked with a solution of BSA in PBS. The blocking solution and dilution buffer contained 5% w/v. BSA solution in PBS for analysis coated with BDNF or 0.5% w/v. BSA solution in PBS for analysis with NT-4 coating. On separate microtiter plates to serial ones

- 49,044,580 deniers of antibodies were added to a fixed amount, 500 pM, of hTrkB-hFc protein with final concentrations ranging from 1.7 pM to 100 nM and solutions without antibodies. (The fixed concentration of hTrkB-hFc for antibody inhibition assays was selected from the approximated midpoint on the linear portion of the individual hTrkB-hFc binding curves to hBDNF or hNT-4 plate coating). After one hour of incubation at room temperature, antibody-protein complexes with 500 pM fixed concentration of hTrkB-hFc protein were transferred to microtiter plates coated with hBDNF or hNT-4. After one hour of incubation at room temperature, wells were washed and plate-bound hTrkB-hFc was detected using anti-human Fcy fragment-specific goat polyclonal antibodies conjugated to horseradish peroxidase (Jackson ImmunoResearch). The plate was then developed using TMB substrate solution (BD Biosciences) according to the manufacturer's recommendations, and absorbance was measured at 450 nm on a Victor plate reader (PerkinElmer™).

Data analysis was performed using a sigmoidal dose-response model in Prism™ software (GraphPad). The calculated IC ₅₀ value, defined as the concentration of antibody required to reduce the binding of hTrkB-hFc to hBDNF or hNT-4 by 50%, was used as an indicator of blocking efficiency. The percentage of blocking, at the maximum concentration of the test antibody, was calculated as an indicator of the ability of the antibodies to block the binding of 500 pM hTrkB-hFc to hBDNF or hNT-4 on the plate relative to the original study data. When calculated, the binding signal of a 500 pM hTrkB-hFc sample without antibody corresponds to 100% binding or 0% blocking; and the initial sample buffer signal without hTrkB-hFc or antibody corresponds to 0% binding or 100% blocking.

Summary of results and conclusions:

The ability of anti-TrkB antibodies to block the binding of TrkB to BDNF or NT-4 was assessed using two competitive sandwich ELISAs. Binding of human TrkB-hFc to hBDNF or hNT-4 coated 96-well microtiter plates in the presence of serially diluted antibodies or without antibody controls was detected with HRP-conjugated anti-human Fcy fragment-specific goat polyclonal antibodies. IC ₅₀ values were calculated and used as an indicator of the activity of the antibody blocking the binding of hTrkB-hFc to hBDNF or hNT-4. In addition, the maximum blocking of 500 μM hTrkBhFc for each antibody at the highest concentration tested was calculated and compared.

The blocking results are presented in table. 28. Percentage blocking is presented for all antibodies and is calculated at the highest concentration of the test antibody, 100 nM. The percentage of negative blocking indicates the increase in TrkB binding detected in the presence of the antibody. IC ₅₀ values are shown for antibodies blocking >50% of TrkB binding to BDNF or NT-4. Non-quantifiable IC ₅₀ values for antibodies blocking <50% are indicated as (-).

At the highest concentration of the antibody tested, one (H4H9780P) of three anti-TrkB antibodies blocked >50% of hTrkB binding to ligands BDNF or NT-4 with IC ₅₀ values of 1 50 pM and 180 pM, respectively, with the percentage blocking at antibody concentration 100 nM, being 93% for BDNF and 80% for NT-4. At 3.7 nM, this antibody blocked the binding of 500 pM hTrkB-hFc to NT-4 by 99%. The decrease in blocking percentage at the highest concentrations tested may be due to nonspecific binding of H4H9780P to the microtiter plate and detection of this binding to HRP-conjugated anti-human Fcy fragment-specific polyclonal antibodies.

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

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Table 28

(*) 99% blocking at a concentration of antibody H4H9780P 3.7 nM.

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

To assess whether two antibodies compete with each other for binding to their epitopes on hTrkB-mmh, binding competition between anti-hTrkB monoclonal antibodies was determined using a real-time marker-free biolayer interferometry assay on an Octet RED384 biosensor (Pall ForteBio Corp. ). Cross-competition experiments were performed at 25°C in 0.01 M HEPES, pH 7.4, 0.15 M NaCl, 3.4 mM EDTA, 0.05% v/v. Surfactant Tween-20, 0.1 mg/ml BSA (HBS-EP buffer) with shaking the plate at 1000 rpm. All solutions of the studied anti-hTrkB antibodies and hTrkB-mmh were prepared in Octet HBS-EP buffer. To assess the ability of two antibodies to compete with each other for binding to their respective epitopes on hTrkBmmh, first approximately ∼0.14–0.24 nM hTrkB-mmh was captured onto anti-His antibody-coated ends of the Octet biosensor from wells containing 50 μg/ ml hTrkB-mmh for 5 min. Octet biosensor ends with captured hTrkB-mmh were saturated by immersing for 5 min in wells containing 50 μg/ml of the first monoclonal anti-hTrkB antibody (hereafter referred to as mAb-1), followed by immersion in wells containing the second monoclonal anti-HTrkB antibody (referred to as mAb-2) for an additional 5 min. Between steps, the ends of the Octet biosensor were washed in HBS-EP buffer for 30 s.

During the experiment, the binding response was monitored in real time, and the binding response was recorded at the end of each stage. The binding response of mAb-2 to hTrkB.mmh precomplexed with mAb-1 was compared and the competitive/non-competitive behavior of different anti-hTrkB monoclonal antibodies was determined using a 60% inhibition threshold.

In table 29 explicitly defines the relationships between antibodies competing in both directions, regardless of the order of binding.

Results:

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Table 29

Cross-competition of anti-hTrkB antibodies for binding to human hTrkB.mmh

mAb-1 mAb-2 N4N9814R N4N9816R2 N4N9814R N4N9816R2 N4N9814R N4N9816R2 N4N9780R N4N9780R N1M8037S N1M8037S (comparison antibody) (comparison antibody)

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

To assess whether two antibodies compete with each other for binding to their epitopes on mTrkB-mmh, competition for binding between monoclonal anti-mTrkB antibodies was determined using real-time label-free biolayer interferometry analysis on an Octet HTX biosensor (Pall ForteBio Corp.). Cross-competition experiments were performed at 25°C in 0.01 M HEPES, pH 7.4, 0.15 M NaCl, 3.4 mM EDTA, 0.05% v/v. Surfactant Tween-20, 0.1 mg/ml BSA (HBS-EP buffer) with shaking the plate at 1000 rpm. All solutions of the studied anti-mTrkB antibodies and mTrkB-mmh were prepared in Octet HBS-EP buffer. To assess the ability of two antibodies to compete with each other for binding to their respective epitopes on mTrkBmmh, first approximately -0.20-0.27 nm mTrkB-mmh was captured on anti-His antibody-coated ends of the Octet biosensor from wells containing 20 μg/ ml mTrkB-mmh for 5 min. The ends of the Octet biosensor with captured mTrkB-mmh were saturated by immersion for 5 min in wells containing 50 μg/ml of the first monoclonal anti-mTrkB antibody (hereinafter referred to as mTrkB-l), followed by immersion in wells containing the second monoclonal anti-mTrkB antibody (designated mAb-2) for an additional 3 min. Between steps, the ends of the Octet biosensor were washed in HBS-EP buffer for 30 s.

During the experiment, the binding response was monitored in real time, and the binding response was recorded at the end of each stage. The binding response of mAb-2 to mTrkB.mmh, which is precomplexed with mAb-l, was compared and the competitive/non-competitive behavior of various anti-mTrkB monoclonal antibodies was determined using a 50% inhibition threshold.

In table 30 explicitly defines the relationships between antibodies competing in both directions, regardless of the order of binding.

Results:

Table 30

Cross-competition of anti-mTrkB antibodies for binding to mouse hTrkB.mmh

mAb-1 mAb-2 M2aM14173N M2aM14178N M2aM14173N M2aM14178N M2aM14173N M2aM14178N M2aM14179N M2aM14179N H1M8037C (comparison antibody) H1M8037C (comparison antibody)

Example 16 Blocking Evaluation Using the Octet System: Blocking Anti-Human TrkB or Anti-Mouse TrkB Antibodies from Binding to TrkB by BDNF or NT-4.

Experiment 1.

Blocking anti-human TrkB antibodies or anti-mouse TrkB antibodies from binding to TrkB

-52044580 by BDNF or NT-4 was assessed using an Octet HTX real-time biolayer interferometry (BLI) assay. The entire study was performed 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 (mobile buffer HBS-EBT) at 25°C. All samples were dispensed into a 384-well tilt plate and placed on an orbital shaker with a shaking speed of 1000 rpm. hTrkB.mFc was captured on Octet sensors with anti-mFc antibody (AMC), while hTrkB.hFc or mTrkB.hFc was captured on Octet sensors with anti-hFc antibody (AHC) by immersion in wells containing 10 μg/ml TrkB reagents for 2 min. Octet biosensors with captured hTrkB.mFc or hTrkB.hFc were saturated by immersion in wells containing 20 nM BDNF, hNT-4, or mNT-4 for 2 min, followed by immersion of Octet biosensors in wells containing 300 nM of various TrkB mAbs for 4 min. The binding of TrbB mAb to the TrkB complex and BDNF, hNT-4, or mNT-4 was determined using Scrubber 2.0c analysis software.

The binding of any anti-human TrkB antibodies or anti-mouse TrkB antibodies of the present invention was not blocked by either BDNF or NT-4, as shown in table. 31 and table. 32.

Table 31

Binding of anti-human TrkB monoclonal antibodies to the hTrkB-mFc complex and human BDNF or NT-4

Human anti-TrkB antibody binding (nm) Grip surface hTrkB.mFc Capture level (nm) 0.29 ±0.01 mAb REGN1945 (negative isotype control) Lack of ligand 0.01 ±0.01 BDNF saturation (0.05 ± 0.007) nm 0.00±0.00 hNT-4- saturation (0.05 ± 0.006) nm 0.00±0.00 N4N9780R 0.18 ±0.01 0.20 ±0.014 0.2 ± 0.007 N4N9814R 0.18 ±0.01 0.22 ± 0.006 0.23 ± 0.00 N4N9816R2 0.19 ±0.00 0.26 ± 0.007 0.24 ± 0.007 hTrkB.hFc 0.47 ±0.01 Н1М8037С (comparison antibody) 0.38 ±0.01 0.07 ± 0.00 0.10 ±0.006

Binding values of anti-human TrkB antibodies to the complex of hTrkB.mFc or hTrkB.hFc with BDNF or hNT-4 are the average of the binding response measured using three independent Octet biosensors with standard deviation. Binding of BDNF or hNT-4 to hGFRa3.mFc does not inhibit the binding of any anti-human TrkB antibodies.

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Table 32

Binding of monoclonal antibodies against mouse TrkB to the mTrkB-hFc complex and mouse BDNF or NT-4.

Anti-mouse TrkB binding (nm) Surface capture Capture level (nm) mAb Lack of ligand BDNF Saturation (0.07 ± 0.006) nm mNT-4 Saturation (0.07 ± 0.005) nm mTrkB.hFc 0.47 ± 0.01 REGN1318 (negative isotype control) 0.01 ± 0.00 0.00±0.00 0.00 ± 0.006 M2aM14173N 0.42 ± 0.006 0.38 ±0.006 0.4 ± 0.00 M2aM14178N 0.47 ± 0.00 0.35 ± 0.007 0.41 ± 0.006 M2aM14179N 0.42 ± 0.006 0.44 ± 0.006 0.43 ± 0.006 Н1М8037С (comparison antibody) 0.42 ± 0.006 0.06 ± 0.006 0.11 ± 0.00

Binding values of anti-mouse TrkB antibodies to the complex of mTrkB.mFc with BDNF or mNT-4 are the average of the binding response measured using three independent Octet biosensors with standard deviation. Binding of BDNF or mNT-4 to mGFRa3.hFc did not inhibit the binding of any anti-mouse TrkB antibodies.

Experiment 2.

Blocking of BDNF or NT-4 from binding to TrkB by anti-human TrkB antibodies or anti-mouse TrkB antibodies was assessed using an Octet HTX real-time biolayer interferometry assay. The entire study was performed 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 (working buffer HBS-EBT) at 25°C. All samples were dispensed into a 384-well tilt plate and placed on an orbital shaker with a shaking speed of 1000 rpm. hTrkB.mFc was captured on Octet sensors with anti-mFc antibody (AMC), while hTrkB.hFc or mTrkB.hFc was captured on Octet sensors with anti-hFc antibody (AHC) by immersion in wells containing 10 μg/ml TrkB reagents for 2 min. Octet biosensors with captured hTrkB.mFc or hTrkB.hFc were saturated by immersion in wells containing 300 nM of various TrkB mAbs for 4 min, followed by immersion of Octet biosensors in wells containing 20 nM BDNF, hNT-4, or mNT-4 for 2 min. The binding of BDNF, hNT-4, or mNT-4 to the TrkB complex and various TrkB mAbs was determined using Scrubber 2.0c analysis software.

Binding of one of the three anti-human TrkB antibodies of the present invention blocked the binding of BDNF and hNT-4, as shown in table. 33. Binding of one of the three anti-mouse TrkB antibodies of the present invention partially blocked the binding of BDNF and mNT-4, as shown in table. 34.

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Table 33

Binding of human BDNF or NT-4 to the hTrkB-mFc complex and anti-human TrkB monoclonal antibodies.

BDNF binding hNT-4 binding Grip surface Capture level (nm) mAb Tie up not mAb (nm) BDNF Tie up ie mAb (nm) hNT-4 hTrkB.mFc 0.28 ± 0.01 absence of mAb N/A 0.05 ± 0.007 N/A 0.04 ± 0.005 REGN1945 (negative isotype 0.01 ± 0.01 0.04 ± 0.01 0.01 ± 0.01 0.033 ± 0.006 II control) N4N9780R 0.18 ±0.01 0.01 ± 0.00 0.18 ±0.01 0.007 ± 0.006 N4N9814R 0.18 ±0.01 0.05 ± 0.00 0.18 ±0.01 0.04 ± 0.00 N4N9816R2 0.19 ± 0.00 0.057 ± 0.006 0.18 ±0.01 0.043 ± 0.006 hTrkB.hFc 0.48 ± 0.01 Н1М8037С (comparison antibody) 0.38 ± 0.01 -0.017 ±0.006 0.39 ± 0.01 -0.013 ± 0.006

Binding values for BDNF or hNT-4 to the complex of hTrkB.mFc or hTrkB.hFc to anti-human TrkB antibodies are the average of the binding response measured using three independent Octet biosensors with standard deviation. Binding of H4H9780P to hGFRa3.mFc blocked the binding of BDNF and hNT-4.

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Claims (3)

Table 34 Binding of mouse BDNF or NT-4 to the mTrkB-hFc complex and anti-mouse TrkB monoclonal antibodies. BDNF binding hNT-4 binding Grip surface Capture level (nm) mAb mAb binding (nm) BDNF Tie up ie mAb (nm) hNT-4 mTrkB.hFc 0.48 ± 0.01 Lack of mAb Ν/Α 0.07 ± 0.01 Ν/Α 0.07 ± 0.005 REGN1318 (negative isotype control) 0.01 ± 0.00 0.06 ± 0.00 0.01 ±0.00 0.06 ± 0.00 M2aM14173N 0.42 ± 0.006 0.07 ± 0.00 0.413 ± 0.006 0.06 ± 0.00 M2aM14178N 0.47 ± 0.00 0.05 ± 0.00 0.47 ±0.01 0.033 ± 0.006 M2aM14179N 0.42 ± 0.006 0.07 ± 0.00 0.41 ± 0.00 0.06 ± 0.00 Н1М8037С (comparison antibody) 0.42 ± 0.006 -0.017 ±0.006 0.40 ± 0.00 -0.02 ± 0.00 The binding values of BDNF or mNT-4 to the mTrkB.hFc complex and anti-mouse TrkB antibodies represent the average of the binding response measured using three independent Octet biosensors with standard deviation. Binding of M2aM14178N to mTrkB.hFc partially blocked the binding of BDNF and mNT-4. The scope of the present invention should not be limited to the specific embodiments described herein. In fact, various modifications of the invention other than those presented herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are included within the scope of the appended claims. CLAIM 1. An isolated antibody or antigen-binding fragment thereof that specifically binds to tropomyosin tyrosine kinase receptor B (TrkB), wherein the antibody or antigen-binding fragment thereof contains a combination of six complementarity determining regions (HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) containing HCDR1 containing the amino acid sequence SEQ ID NO: 4; HCDR2 containing the amino acid sequence SEQ ID NO: 6; HCDR3 containing the amino acid sequence SEQ ID NO: 8; LCDR1 containing the amino acid sequence SEQ ID NO: 12; LCDR2 containing the amino acid sequence SEQ ID NO: 14; And LCDR3 containing the amino acid sequence SEQ ID NO: 16. 2. An isolated antibody or antigen-binding fragment thereof according to claim 1, containing a heavy chain variable region (HCVR) containing the amino acid sequence SEQ ID NO: 2; and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 10. 3. An isolated antibody or antigen-binding fragment according to claim 1 or claim 2, where the antibody or its -