EA043771B1 - ANTIBODIES TO TRKB - Google Patents

Description

Field of technology to which the invention relates

The present invention generally relates to anti-TrkB agonistic antibodies for diagnostic and therapeutic use, and in particular to humanized anti-TrkB agonistic antibodies. More specifically, anti-TrkB agonistic antibodies and methods of use for the treatment of various diseases or disorders are described. Pharmaceutical compositions and kits containing an anti-TrkB agonist antibody are also described.

Prerequisites for creating the invention

Tropomyosin receptor kinase B (TrkB), also known as tyrosine receptor kinase B, or BDNF/NT-3 growth factor receptor or neurotrophic tyrosine kinase receptor type 2, is a protein that in humans is encoded by the NTRK2 gene (Genbank ID: 4915). TrkB is a receptor for brain-derived neurotrophic factor (BDNF).

Neurotrophic tyrosine kinase receptor B (TrkB; gene symbol: NTRK2) is expressed by retinal neurons and glial cells. In the normal retina, TrkB signaling counteracts cellular stress and promotes cell survival. In eye diseases such as diabetic retinopathy or geographic atrophy, there is loss and functional impairment of retinal neurons and glial cells, causing visual disturbances and loss of vision. Activation of TrkB signaling above basal levels (which is reduced in diabetic retinopathy) may counteract the loss and functional impairment of neurons and glial cells, thereby improving visual function. In addition, activation of TrkB has the potential to regenerate lost synaptic connections in the diseased eye, thereby promoting the restoration of visual function. Upon ligand binding, TrkB undergoes homodimerization followed by autophosphorylation. Depending on the phosphorylation sites (Y516, Y702, Y706, Y707, or Y817), different signaling pathways are activated, including the activity of PLCy1 or different subforms of AKT and ERK, which regulate various overlapping signaling cascades, including axonal/neurite growth, increased synaptic plasticity, or increased cell survival.

Agonistic antibodies to TrkB have been described in US 20100196390 and US 20100150914, and their use has also been proposed for the treatment of, for example, Charcot-Marie-Tooth disease or diabetes.

However, there remains a significant need for new effective agonistic antibodies to TrkB that can be used to activate the TrkB pathway and thus offer the possibility of their application for therapeutic interventions, for example, in neurodegenerative and psychiatric disorders.

The essence of the invention

The present invention provides monoclonal antibodies that specifically bind to human TrkB. In one aspect, the antibodies of the present invention have agonist activity and induce phosphorylation and/or activation of TrkB. In another aspect, the antibodies of the present invention are useful, for example, in the treatment of diseases of the eye and retina, such as geographic atrophy caused by age-related macular degeneration, diabetic retinopathy, glaucoma and/or diabetic macular edema.

In another aspect, the present invention provides an anti-TrkB antibody, particularly a humanized anti-TrkB antibody, having one or more of the properties described herein below.

In another aspect, the anti-TrkB antibody of the present invention binds with high affinity to human TrkB. In one embodiment related to this aspect, the anti-TrkB antibody of the present invention binds to human TrkB at a K _D <10 nM. In another embodiment, the anti-TrkB antibody of the present invention binds to human TrkB with a KD <5 nM.

In another aspect, the anti-TrkB antibody of the present invention activates TrkB with high efficiency. In one embodiment related to this aspect, the anti-TrkB antibody of the present invention activates human TrkB with an EC ₅₀ <100 pM. In a further embodiment, the anti-TrkB antibody of the present invention activates human TrkB with an EC ₅₀ <50 pM.

In another aspect, the anti-TrkB antibody of the present invention is more effective at inducing activation of downstream TrkB signaling pathways compared to the natural TrkB ligand, BDNF. In a further aspect, the anti-TrkB antibody of the present invention regulates gene expression through the TrkB-mediated signaling pathway in a comparable manner to BDNF.

In a further aspect, the anti-TrkB antibody of the present invention binds a novel epitope in the extracellular domain of human TrkB. In a further aspect, the anti-TrkB antibody of the present invention does not cross-react with TrkB from other species, and in particular does not cross-react with rodent TrkB.

In one aspect, the anti-TrkB antibody of the present invention can be formulated at high concentrations for intravitreal injection into the eye.

In a further aspect, the anti-TrkB antibody of the present invention has a low risk of immunogenicity as measured by the method described in Example 5.

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In another aspect, the CDR sequences of the anti-TrkB antibody of the present invention have low sequence affinities as determined by the method described in Example 4.

In yet another aspect, the anti-TrkB antibody of the present invention does not reduce BDNF-induced ERK phosphorylation.

In a further aspect, the anti-TrkB antibody of the present invention is specific for phosphorylation and/or activation of TrkB and does not non-specifically phosphorylate/activate TrkA or TrkC. In another aspect, the anti-TrkB antibody does not bind nonspecifically to human VEGF.

Further aspects include polynucleotide molecule(s) encoding antibodies of the present invention, expression vectors and viral vectors, as well as host cells containing such polynucleotide molecule(s), and methods for preparing antibodies of the present invention. The present invention further provides therapeutic applications of the antibodies of the present invention, particularly for retinal/eye diseases.

In one embodiment, the present invention provides an anti-TrkB antibody or antigen-binding fragment thereof comprising:

a light chain variable region containing the amino acid sequence of SEQ ID NO: 48 (L-CDR1); amino acid sequence SEQ ID NO: 49 (L-CDR2); and amino acid sequence SEQ ID NO: 50 (L-CDR3); and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 51 (H-CDR1); amino acid sequence SEQ ID NO: 52 (H-CDR2); and the amino acid sequence of SEQ ID NO: 53 (H-CDR3), or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 55 (H-CDR1); amino acid sequence SEQ ID NO: 56 (H-CDR2); and the amino acid sequence of SEQ ID NO: 57 (H-CDR3), or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 58 (H-CDR1); amino acid sequence SEQ ID NO: 59 (H-CDR2); and the amino acid sequence of SEQ ID NO: 60 (H-CDR3).

In one embodiment, the present invention provides an anti-TrkB antibody or antigen-binding fragment thereof comprising:

a variable light chain and a variable heavy chain comprising the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 12, respectively, a variable light chain and a variable heavy chain comprising the amino acid sequences of SEQ ID NO: 3 and SEQ ID NO: 13, respectively, a variable light chain and a variable heavy chain comprising the amino acid sequences of SEQ ID NO: 4 and SEQ ID NO: 14, respectively, a variable light chain and a variable heavy chain comprising the amino acid sequences of SEQ ID NO: 5 and SEQ ID NO: 15, respectively, a variable light chain and a variable heavy chain comprising the amino acid sequences of SEQ ID NO: 6 and SEQ ID NO: 16, respectively, a variable light chain and a variable heavy chain comprising the amino acid sequences of SEQ ID NO: 7 and SEQ ID NO: 17, respectively, a variable light chain and a variable heavy chain comprising the amino acid sequences of SEQ ID NO: 8 and SEQ ID NO: 18, respectively, a variable light chain and a variable heavy chain comprising the amino acid sequences of SEQ ID NO: 9 and SEQ ID NO: 19, respectively, or a variable light chain and a variable heavy chain comprising the amino acid sequences of SEQ ID NO: 10 and SEQ ID NO: 20, respectively.

In one embodiment, the present invention provides an anti-TrkB antibody or antigen-binding fragment thereof comprising:

a light chain containing the amino acid sequence of SEQ ID NO: 21, and a heavy chain containing the amino acid sequence of SEQ ID NO: 22 or 23, a light chain containing the amino acid sequence of SEQ ID NO: 24, and a heavy chain containing the amino acid sequence of SEQ ID NO: 24 25 or 26, a light chain containing the amino acid sequence of SEQ ID NO: 27, and a heavy chain containing the amino acid sequence of SEQ ID NO: 28 or 29, a light chain containing the amino acid sequence of SEQ ID NO: 30, and a heavy chain containing the amino acid sequence SEQ ID NO: 31 or 32, a light chain comprising the amino acid sequence of SEQ ID NO: 33, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 34 or 35, a light chain comprising the amino acid sequence of SEQ ID NO: 36, and a heavy chain containing the amino acid sequence of SEQ ID NO: 37 or 38, a light chain containing the amino acid sequence of SEQ ID NO: 39, and a heavy chain,

- 2 043771 containing the amino acid sequence SEQ ID NO: 40 or 41, a light chain containing the amino acid sequence SEQ ID NO: 42, and a heavy chain containing the amino acid sequence SEQ ID NO: 43 or 44, or a light chain containing the amino acid sequence SEQ ID NO: 45, and a heavy chain containing the amino acid sequence of SEQ ID NO: 46 or 47.

In a particularly preferred embodiment, the anti-TrkB antibody is a humanized anti-TrkB antibody.

In one embodiment, the present invention provides an anti-TrkB antibody or antigen-binding fragment thereof for medical use.

In one embodiment, the present invention provides an anti-TrkB antibody or antigen-binding fragment thereof for use in the treatment of retinal/ocular diseases.

In one embodiment, the present invention provides an anti-TrkB antibody or antigen-binding fragment thereof for use in the treatment of neuronal/neural eye or retinal diseases.

In one embodiment, the present invention provides an anti-TrkB antibody or antigen-binding fragment thereof for use in the treatment of geographic atrophy.

In one embodiment, the present invention provides an anti-TrkB antibody or antigen-binding fragment thereof for use in the treatment of age-related macular degeneration or diabetic retinopathy.

In one embodiment, the present invention provides a pharmaceutical composition comprising an anti-TrkB antibody or antigen-binding fragment thereof and a pharmaceutically acceptable carrier.

In one embodiment, the present invention provides an anti-TrkB antibody or antigen-binding fragment thereof, or a pharmaceutical composition comprising an anti-TrkB antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof is administered via the parenteral route, intravenous route, intravitreal route, or subcutaneous route. .

In one embodiment, the present invention provides isolated polynucleotide or polynucleotides comprising a sequence encoding a light chain or light chain variable region of an antibody or an antigen binding fragment thereof and a heavy chain or variable region of an antibody heavy chain or an antigen binding fragment thereof.

In one embodiment, the present invention provides an expression vector containing isolated polynucleotide or polynucleotides encoding a light chain or light chain variable region of an antibody or an antigen binding fragment thereof and a heavy chain or variable region of an antibody heavy chain or an antigen binding fragment thereof.

In one embodiment, the present invention provides a viral vector containing isolated polynucleotide or polynucleotides encoding a light chain or light chain variable region of an antibody or an antigen binding fragment thereof and a heavy chain or variable region of an antibody heavy chain or an antigen binding fragment thereof.

In one embodiment, the present invention provides a host cell containing an expression vector or isolated polynucleotide or polynucleotides encoding a light chain or light chain variable region of an antibody or an antigen binding fragment thereof and a heavy chain or heavy chain variable region of an antibody or its antigen-binding fragment.

In one embodiment, the present invention provides a method for producing an anti-TrkB antibody or antigen-binding fragment thereof, comprising:

obtaining a host cell containing an expression vector or isolated polynucleotide or polynucleotides encoding a light chain or light chain variable region of an antibody or an antigen binding fragment thereof and a heavy chain or variable region of an antibody heavy chain or an antigen binding fragment thereof; and culturing the host cell.

In one embodiment, a method of producing an anti-TrkB antibody or an antigen-binding fragment thereof further comprises reconstituting and purifying the anti-TrkB antibody or an antigen-binding fragment thereof.

In one embodiment, the present invention provides an anti-TrkB antibody or antigen-binding fragment thereof that binds to at least one amino acid residue within amino acid regions 92-112, 130-143 and/or 205-219 of the extracellular domain of human TrkB with sequence SEQ ID NO: 54.

In one embodiment, the present invention provides an anti-TrkB antibody or antigen-binding fragment thereof that binds to at least one amino acid residue within the amino acid regions 92-112 and 130-143; or 92-112 and 205-219; or 130-143 and 205-219; or 92-112 and 130-143 and 205-219 of the extracellular domain of human TrkB with the sequence SEQ ID NO: 54.

In one embodiment, the present invention provides an anti-TrkB antibody or

- 3 043771 antigen-binding fragment, which binds to at least one amino acid residue within any of the above combinations and additionally also to at least one amino acid residue within amino acid regions 313-330 and/or 348-367 of the TrkB extracellular domain human with the sequence SEQ ID NO: 54.

In one embodiment, the present invention provides an anti-TrkB antibody or antigen-binding fragment thereof that binds to at least one amino acid residue within the amino acid regions 92-112, 130-143, 205-219, 313-330, and 348- 367 extracellular domain of human TrkB with the sequence SEQ ID NO: 54.

Brief description of the figures

Fig. 1. Epitope mapping of BDNF, 277 antibody, C2 or C20 antibodies to the extracellular domain of human TrkB.

Fig. 2. qPCR expression data (Y-axis) for regulated genes (X-axis) in relation to synaptic plasticity on SH-SY5Y cells after treatment with BDNF, IgG1 control (anti-2,4,6-trinitrophenyl, anti-TNP) or D003 antibody.

Fig. 3. Graph of the concentration of 277-antibody in the eye compartments, as indicated, and plasma after intravenous injection of 1 mg of 277-antibody into the eyes of rabbits depending on time.

Fig. 4. Calculated ocular PK of IgG1 antibodies. The Y-axis shows the vitreal concentration and the X-axis shows the days after IVT application. The time after intravenous injection to achieve a vitreal concentration equivalent to EC ₅₀ is indicated by arrows.

Fig. 5. General design of the study, including groups of animals and time dynamics. At week 5, a baseline ERG (before IVT administration) was assessed to determine the extent of hyperglycemia-induced neural dysfunction in the retina. Group 1 and Group 2 received intravenous injections of an IgG1 control antibody to 2,4,6-trinitrophenyl (anti-TNP), which does not cause any effects on retinal function. Group 3 eyes were treated with C2 (an anti-TrkB agonist surrogate antibody). Retinal function was assessed again at week 7 (post-IRT).

Fig. 6. Changes in rod-mediated b-wave latency relative to the average latency of the control group, at baseline (before IVT) and 1 week after IVT administration (see Fig. 5 for the general study design). Nondiabetic control group 1 (black, n=23 eyes studied) and hyperglycemic group 2 (indicated by dots, n=21 eyes studied) received IVT injections using control IgG1. Group 3 received IVT treatment using C2 (n=24 eyes studied). Data are mean + ATP. nd, unreliable; *, p<0.05; **, p<0.01; ***, p<0.001; the differences before IVT between hyperglycemic groups 2 and 3 and control group 1 are significant (statistical data are not shown conventionally; one-way analysis of variance with Tukey’s multiple comparison test).

Fig. 7. Photosensitivity, S, mediated by rod b-waves, normalized to the average photosensitive sensitivity of the control group, S _control , at baseline (before IVT) or 1 week after IVT administration (see Fig. 5 regarding the general study design). Nondiabetic control group 1 (black, n=23 eyes studied) and hyperglycemic group 2 (red, n=20 eyes studied) received IVT injections using control IgG1. Group 3 received IVT treatment using C2 (n=24 eyes studied). Data are mean + ATP. nd, unreliable; *, p<0.05; **, p<0.01; ***, p<0.001; the differences before IVT between hyperglycemic groups 2 and 3 and control group 1 are significant (statistical data are not shown conventionally; one-way analysis of variance with Tukey’s multiple comparison test).

Fig. 8. Rod-mediated a-wave responses evoked by a light flash of 0.1 cds/m ² , R, normalized to the average response of controls, at baseline (before IVT) or 1 week after IVT administration (see Fig. 5 for overall research design). Nondiabetic control group 1 (black, n=23 eyes studied) and hyperglycemic group 2 (red, n=20 eyes studied) received IVT injections using control IgG1. Group 3 received IVT treatment using C2 (n=24 eyes studied). Data are mean + ATP. nd, unreliable; *, p<0.05; **, p<0.01; ***, p<0.001; the differences before IVT between hyperglycemic groups 2 and 3 and control group 1 are significant (statistical data are not shown conventionally; one-way analysis of variance with Tukey’s multiple comparison test).

Fig. 9. Changes in the latency of the UV cone-mediated b-wave relative to the average latency of the control group, at baseline (before IVT) and 1 week after IVT administration (see Fig. 5 for the general design of the study). Nondiabetic control group 1 (black, n=24 eyes studied) and hyperglycemic group 2 (red, n=22 eyes studied) received IVT injections using control IgG1. Group 3 received IVT treatment using C2 (n=24 eyes studied). Data are mean + ATP. nd, unreliable; *, p<0.05; **, p<0.01; ***, p<0.001; the differences before IVT between hyperglycemic groups 2 and 3 and control group 1 are significant (statistical data are not shown conventionally; one-way analysis of variance with Tukey’s multiple comparison test).

Fig. 10. Changes in the latency of the M-type cone-mediated b-wave relative to the average latency of the control group, at baseline (before IVT) and 1 week after IVT administration (see Fig. 5 for the general study design). Nondiabetic control group 1 (black, n=24 eyes studied) and hyperglycemic group 2 (red, n=22 eyes studied) received IVT injections using control IgG1. Group 3 received IVT treatment using C2 (n=24 eyes studied). Data are mean + ATP. nd, unreliable; *, p<0.05; **, p<0.01; ***, p<0.001; the differences before IVT between hyperglycemic groups 2 and 3 and control group 1 are significant (statistical data are not shown conventionally; one-way analysis of variance with Tukey’s multiple comparison test).

Fig. 11. Photosensitivities, S, UV of cone-mediated b-waves, normalized to the average photosensitive sensitivities of the control groups, S _control , at baseline (before IVT) or 1 week after IVT administration (see Fig. 5 for the general study design). Nondiabetic control group 1 (black, n=22 eyes studied) and hyperglycemic group 2 (red, n=22 eyes studied) received IVT injections using control IgG1. Group 3 received IVT treatment using C2 (n=21 eyes studied). Data are mean + ATP. nd, unreliable; *, p<0.05; **, p<0.01; ***, p<0.001; the differences before IVT between hyperglycemic groups 2 and 3 and control group 1 are significant (statistical data are not shown conventionally; one-way analysis of variance with Tukey’s multiple comparison test).

Fig. 12. Amplitudes of saturating oscillations, _Rmax , mediated by M-type cone b-waves, normalized to the average amplitude of saturating oscillations of controls, _Rmax , _control , at baseline (before IVT) or 1 week after IVT administration (see Fig. 5 regarding the general design of the study). Nondiabetic control group 1 (black, n=24 eyes studied) and hyperglycemic group 2 (red, n=22 eyes studied) received IVT injections using control IgG1. Group 3 received IVT treatment using C2 (n=24 eyes studied). Data are mean + ATP. nd, unreliable; *, p<0.05; **, p<0.01; ***, p<0.001; the differences before IVT between hyperglycemic groups 2 and 3 and control group 1 are significant (statistical data are not shown conventionally; one-way analysis of variance with Tukey’s multiple comparison test).

Fig. 13. ERK phosphorylation in CHO cells expressing the human TrkB receptor after stimulation with (A) BDNF, 277 antibody, or a combination of 1 nM BDNF with increasing concentrations of 277 antibody or (B) BDNF, C2 antibody, or a combination of 1 nM BDNF with increasing concentrations of C2 antibody. Data (symbols) are presented as means ± SES (for some points, the error bars are shorter than the height of the symbol); connecting lines represent nonlinear regression (log(agonist) vs. response (three parameters)). One representative example from a series of three experiments with independent cell batches is shown; n.d., non-significant difference between 1 nM BDNF alone and 1 nM BDNF with the indicated concentrations of 277 antibody. #p<0.001 1 nM BDNF separately rel. 1 nM BDNF with the indicated concentrations of C2 antibody; one-way analysis of variance with multiple comparison test.

Fig. 14. Binding of 277 antibody or C2 antibody to human or rat VEGF in an in vitro ELISA assay. Data (symbols) are presented as means ± SAR (note: error bars are shorter than symbol height); connecting lines represent nonlinear regression (log(agonist) vs. response (three parameters)). One representative example from a series of five independent experiments is shown. #p<0.01 hVEGF-C2 rel. hVEGF-IgG1 isotype at the indicated concentrations (one-sample t test).

Detailed Description of the Invention

Definitions

The general structure of antibodies or immunoglobulins is well known to those skilled in the art; these molecules are heterotetrameric glycoproteins, typically with a molecular weight of approximately 150,000 Daltons, consisting of two identical light (L) chains and two identical heavy (H) chains. Each light chain is covalently linked to a heavy chain by a single disulfide bond to form a heterodimer, and a heterotrimeric molecule is formed by a covalent disulfide bond between two identical heterodimer heavy chains. Although the light and heavy chains are linked by a single disulfide bond, the number of disulfide bonds between the two heavy chains varies depending on the immunoglobulin isotype. Each heavy and light chain also has evenly distributed intrachain disulfide bridges. Each heavy chain has an amino-terminal variable region ( _VH =variable heavy chain), followed by three or four constant domains ( _CH1 , _CH2 , _CH3 and _CH4 ), as well as a hinge region between _CH1 and _CH2 . Each light chain has two regions, an amino-terminal variable region (V _L =variable light chain) and a carboxy-terminal constant domain ( _CL ). The V _L region is non-covalently linked to the VH region, and the CL domain is usually covalently linked to the C _H1 domain through a disulfide bond. It is likely that certain amino acid residues form the interface between the variable regions of the light and heavy chains (Chothia et al., J. Mol. Biol. 186, 1985, pp. 651-663).

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Certain regions in the variable regions vary significantly among different antibodies, i.e. they are hypervariable. These hypervariable regions contain residues that are directly involved in binding and determine the specificity of each particular antibody with respect to its specific antigenic determinant. Hypervariability in the variable regions, both light chain and heavy chain, is concentrated in three segments, which are designated as complementarity determining regions (hypervariable regions) (CDR) or hypervariable loops (HVL). CDRs are determined by sequence comparison according to the method described by Kabat et al., in: Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md., 1991, a HVL is structurally determined based on the three-dimensional structure of the variable region according to the method described by Chothia and Lesk, J. Mol. Biol. 196, 1987, cc. 901-917. If the two methods result in some differences in identifying CDRs, then structure-based identification is preferred. According to Cabot nomenclature, CDR-L1 is located at approximately residues 24-34, CDR-L2 at approximately residues 50-56, and CDR-L3 at approximately residues 89-97 in the light chain variable region; CDR-H1 is located at approximately residues 31-35, CDR-H2 at approximately residues 50-65, and CDR-H3 at approximately residues 95-102 in the heavy chain variable region. Thus, CDR1, CDR2, CDR3 of the heavy and light chains determine unique and functional properties specific to a given antibody.

The three CDRs in each of the heavy and light chains are separated by framework regions (FRs) that contain sequences that are less prone to variability. Starting from the amino terminus towards the carboxy terminus, the variable regions of the heavy and light chains of the FRs and CDRs are ordered as follows: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The largely β-sheet conformation of FR allows the CDRs on each strand to be in close proximity to each other, as well as to the CDRs from the other strand. The resulting conformation is inherent to the antigen-binding site (see Kabat et al., NIH Publ. No. 91-3242, vol. I, 1991, pp. 647-669), although it is not necessary that all CDR residues directly participate in antigen binding.

The FR residues and Ig constant domains are not directly involved in antigen binding, but are involved in antigen-associated and/or antigen-mediated effector function. Some FR residues are likely to have a profound effect on antigen binding in at least three ways: by noncovalently binding directly to the epitope, by interacting with one or more CDR residues, and by influencing the interface between the heavy and light chains. Constant domains are not directly involved in antigen binding, but mediate various Ig effector functions, such as antibody involvement in antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cellular phagocytosis (ADCP).

The light chains of vertebrate immunoglobulins are classified into one or two distinct classes, kappa (k) and lambda (λ), based on the amino acid sequence of the constant domain. By comparison, mammalian immunoglobulin heavy chains fall into one of five major classes based on the sequence of constant domains: IgA, IgD, IgE, IgG, and IgM. IgG and IgA are further divided into subclasses (isotypes), for example, IgG1, _IgG2 , _IgG3 , _IgG4 , IgA1 and _IgA2 , respectively. The heavy chain constant domains that correspond to different classes of immunoglobulins are designated α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of native immunoglobulin classes are well known.

As used herein, the terms antibody, anti-TrkB antibody, humanized anti-TrkB antibody, and humanized anti-TrkB antibody variant are used in their broadest sense and refer, in particular, to monoclonal antibodies (including full-length monoclonal antibodies), multispecific antibodies (e.g., bispecific antibodies), antibodies with minor modifications, such as N- or C-terminal truncations, and antibody fragments, such as variable regions and other regions of antibodies, that have the desired biological activity, such as the ability to bind to TrkB.

The term monoclonal antibody (mAb) refers to an antibody from a population of essentially homogeneous antibodies; those. Individual antibodies in a given population are identical except for naturally occurring mutations, which may be present in very small quantities. Monoclonal antibodies are highly specific; their target is an individual antigenic determinant, an epitope. Thus, the adjective monoclonal is a distinctive feature of a substantially homogeneous population of antibodies that are targeted by an identical epitope, and the concept is not limited to the requirement that the antibodies be produced by any particular method. It should be obvious that monoclonal antibodies can be obtained using any technique or methodology known in the art; including, for example, the hybridoma method (Kohler et al., Nature 256, 1975, p. 495), or recombinant DNA methods known in the art (see, for example, US 4816567), or methods for isolating monoclonal ones obtained using recombinant DNA antibodies using phage antibody libraries that are described in

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Clackson et al., Nature 352, 1991, pp. 624-628 and Marks et al., J. Mol. Biol. 222, 1991, pp. 581-597.

Chimeric antibodies consist of the heavy and light chain variable regions of antibodies from one species (eg, from a non-human mammal, such as a mouse) and the light and heavy chain constant regions of antibodies from another species (eg, human), and can be made by joining sequences DNA encoding antibody variable regions from a first species (eg, mouse), with DNA sequences of antibody constant regions from a second species (eg, human), and transforming the host with an expression vector containing the related sequences, allowing the host to produce the chimeric antibody. Alternatively, a chimeric antibody may also be an antibody in which one or more heavy and/or light chain regions or domains are identical to, homologous to, or a variant of the corresponding sequence in a monoclonal antibody from another immunoglobulin class or isotype or from a consensus or germline sequence . Chimeric antibodies can include fragments of these antibodies, provided that the antibody fragment has the desired biological activity of the parent antibody, such as the ability to bind to the same epitope (see, for example, US 4816567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81, 1984, pp. 6851-6855).

The terms antibody fragment, antigen-binding fragment, anti-TrkB antibody fragment, humanized anti-TrkB antibody fragment, and humanized anti-TrkB antibody variant fragment refer to the portion of a full-length anti-TrkB antibody that retains the variable region or its functional ability, e.g., binding specificity to a TrkB epitope. Examples of antibody fragments include (but are not limited to) Fab-, Fab'-, F(ab')2-, Fd-, Fv-, scFv- and scFv-Fc fragment, dimeric antibody (diabody), linear antibody , single chain antibody, minibody, dimeric antibody formed from antibody fragments, and multispecific antibodies formed from antibody fragments.

Full-length antibodies can be treated with enzymes such as papain or pepsin to produce the desired antibody fragments. Papain digestion is used to produce two identical antigen-binding fragments, called Fab fragments, each with one antigen-binding site, with the remainder designated the Fc fragment. The Fab fragment also contains a light chain constant domain and a heavy chain C _H1 domain. After treatment with pepsin, an F(ab') ₂ fragment is obtained, which carries two antigen-binding centers and still retains the ability to cross-link with antigen.

Fab' fragments differ from Fab fragments by the presence of additional residues, including one or more cysteine residues from the antibody hinge region at the C-terminus of the C _H1 domain. F(ab') ₂ antibody fragments are pairs of Fab' fragments linked via cysteine residues in the hinge region. Other chemical combinations of antibody fragments are known.

The Fv fragment contains a complete antigen recognition and antigen binding center, consisting of a dimer including the variable region of one heavy and one light chain in close non-covalent association. In this configuration, the three CDRs of each variable region interact with a specific antigen binding site on the surface of the VH-V _L dimer. In general, six CDRs determine the specificity of an antibody for antigen binding.

A single chain Fv or scFv antibody fragment is a single chain Fv variant containing the VH and VL regions of the antibody, the regions being present on the same polypeptide chain. Single-chain Fv has the ability to recognize and bind to antigen. The scFv polypeptide may optionally also contain a polypeptide linker located between the VH and VL regions to facilitate the formation of the three-dimensional structure required for antigen binding by the scFv (see, for example, Pluckthun, in: The Pharmacology of monoclonal Antibodies, vol. 113, edited by Rosenburg and Moore, Springer-Verlag, New York, 1991, pp. 269-315).

Other recognition capable antibody fragments include fragments that contain a pair of tandem Fd segments (V _H -C _H1 -V _H -C _H1 ) forming a pair of antigen binding sites. These linear antibodies can be bispecific or monospecific, as described, for example, in Zapata et al., Protein Eng. 8(10), 1995, cc. 1057-1062.

A humanized antibody or humanized antibody fragment is a specific type of chimeric antibody that includes a variant amino acid sequence of an immunoglobulin or fragment thereof that has the ability to bind to a predetermined antigen and that contains one or more FRs that have the amino acid sequence substantially corresponding to the sequence of a human immunoglobulin, and one or more CDRs that have(s) an amino acid sequence that substantially matches the amino acid sequence of a non-human immunoglobulin. Said non-human amino acid sequence, often referred to as an import sequence, is typically derived from the import region of an antibody, in particular the variable region. In general, a humanized antibody includes at least a CDR or HVL of a non-human antibody inserted between the FRs of a human heavy or light chain variable domain.

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The present invention describes specific humanized antibodies to TrkB that contain CDRs derived from the murine leader D003 inserted between the FR sequences of the human germline heavy and light chain variable regions. It will be apparent that certain residues of the murine FR can be imported to provide the function of humanized antibodies, and therefore certain residues of the human germline heavy and light chain variable region sequences are modified to be the same as in the corresponding murine sequence.

In one aspect, a humanized anti-TrkB antibody comprises substantially all of at least one and typically two variable regions (such as those included in Fab-, Fab'-, F(ab') _2- , Fabc- and Fv fragments) in which all or substantially all of the CDRs correspond to the CDRs of non-human immunoglobulins, and according to the present invention, the CDRs are murine D003 leader sequences, and the FRs are FRs that have a consensus or germline sequence of a human immunoglobulin. In another aspect of the invention, the humanized anti-TrkB antibody also includes at least a portion of the Fc region of an immunoglobulin, typically a human immunoglobulin. Typically, the antibody must contain both a light chain and at least a heavy chain variable region. The antibody may also optionally include one or more of the following regions: _CH1 , hinge region, _CH2 , _CH3 and/or _CH4 heavy chain.

The humanized anti-TrkB antibody may be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgG1, IgG2, _IgG3 , _IgG4 , IgA1 and IgA2. For example, the constant domain may be a complement-fixing constant domain, where it is desired that the humanized antibody exhibit cytotoxic activity and the isotype is typically IgG1. In cases where such cytotoxic activity is not desired, the constant domain may be another isotype, for example IgG2. An alternative humanized anti-TrkB antibody may contain sequences from more than one class or isotype of immunoglobulins, and the selection of specific constant domains to optimize the desired effector functions is within the skill of ordinary skill in the art. Specific embodiments of the present invention are antibodies that are IgG1 isotype antibodies, and more particularly IgG1 isotype antibodies in which effector functions are disabled.

The FR and CDR or HVL of a humanized anti-TrkB antibody do not need to exactly match the parent sequences. For example, one or more residues in an import CDR or HVL or in a consensus or germline FR sequence may be altered (eg, by mutagenesis) by substitution, insertion, or deletion such that the resulting amino acid residue is not identical to the original residue at the corresponding position of any parental sequence, but the antibody nevertheless retains function, such as the ability to bind TrkB. The change generally should not be extensive and should be a conservative change. Typically, at least 75% of the residues, more often at least 90% and most often more than 95% or more than 98% or more than 99%, of the humanized antibody must match the residues of the parent consensus sequence or germline FR sequence and import CDR sequences.

Immunoglobulin residues that affect the interface between the heavy and light chain variable regions (VL-VH interface) are residues that affect the proximity or orientation of the two chains relative to each other. Specific residues that may be involved in interchain interactions are VL residues 34, 36, 38, 44, 46, 87, 89, 91, 96, and 98 and VH residues 35, 37, 39, 45, 47, 91, 93. 95, 100 and 103 (according to the numbering system proposed by Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1987)). US 6,407,213 also discusses that residues such as _VL residues 43 and 85 and VH residues 43 and 60 may be involved in this interaction. Although these residues are specific to human IgG, they may be considered for other species. Important residues of the antibody that are expected to be involved in interchain interactions are selected for replacement in the consensus sequence.

The terms consensus sequence and consensus antibody refer to an amino acid sequence that contains the most frequently occurring amino acid residue at each position in all immunoglobulins of any class, isotype or subunit structure, for example, the variable region of a human immunoglobulin. The consensus sequence may be based on immunoglobulins of a specific species or multiple species. By consensus sequence, structure, or antibody is meant the human consensus sequence specified in particular embodiments of the invention and refers to the amino acid sequence that contains the most frequently occurring residues at each position in all human immunoglobulins of a particular class, isotype, or subunit structure. Thus the consensus sequence contains

- 8 043771 amino acid sequence that has at each position an amino acid that is present in one or more known immunoglobulins, but which may not be an exact copy of the complete amino acid sequence of any individual immunoglobulin. The consensus sequence variable region is not derived from any naturally occurring antibody or immunoglobulin (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md., 1991) and its variants. The heavy and light chain FR consensus sequences and variants thereof are valuable sequences for the production of humanized antibodies to TrkB (see, for example, US 6037454 and 6054297).

Human germline sequences are naturally present in the human antibody population. The combination of these germline genes creates antibody diversity. Antibody light chain germline sequences are derived from conserved human kappa or lambda germline v and j genes. Likewise, heavy chain sequences are derived from the germline v, d, and j genes (LeFranc M-P and LeFranc G, The Immunoglobulin Facts Book, Academic Press, 2001).

An isolated antibody is an antibody that has been identified and separated and/or purified from a component of its natural environment. Contaminants in the natural environment of an antibody are substances that may interfere with the diagnostic or therapeutic use of the antibody and may be enzymes, hormones, or other proteinaceous or nonproteinaceous solutes. In one aspect of the invention, the isolated antibody must be an antibody purified to a degree greater than 95% by weight.

An isolated antibody is an antibody in situ within the recombinant cells in which it is produced if no component of the antibody's natural environment is present. However, typically, the isolated antibody is obtained through at least one purification step during which the recombinant cellular material is removed.

The term antibody performance refers to factors/properties that describe an antibody's recognition of an antigen or the effectiveness of an antibody in vivo. Changes in the amino acid sequence of an antibody can affect properties of the antibody, such as folding, and can affect physical factors such as the initial rate of binding of the antibody to the antigen ( _ka ), the dissociation constant of the antibody from the antigen ( _kd ), the affinity constant of the antibody for the antigen (Kd), antibody conformation, protein stability and antibody half-life.

As used herein, identical or percent identical to two or more nucleotide or polypeptide sequences refers to two or more sequences or subsequences that are the same or have a certain percentage of the same nucleotide or amino acid residue when compared and aligned for maximum consistency. To determine percent identity, sequences are aligned for optimal comparison (eg, gaps can be introduced into the sequence of a first amino acid or nucleotide sequence for optimal comparative analysis of the primary structure with a second amino acid or nucleotide sequence). The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence contains the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions in the sequences (ie, % identity = number of identical positions/total number of positions (eg, overlapping positions) x 100). In some embodiments, the two sequences being compared are the same length after introducing, if necessary, gaps in the sequence (eg, excluding additional sequence extending beyond the sequences being compared). For example, when variable region sequences are compared, leader and/or constant domain sequences are not considered. When comparing the sequences of two sequences, the corresponding CDR refers to the CDR located at the same position in both sequences (eg, CDR-H1 of each sequence).

Determining the percent identity or percent similarity between two sequences can be done using a mathematical algorithm. A preferred example of a mathematical algorithm that can be used to compare two sequences is, but is not limited to, the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87, 1990, cc. 2264-2268, modified as described in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90, 1993, cc. 5873-5877. This algorithm is included in the NBLAST and XBLAST programs described in Altschul et al., J. Mol. Biol. 215, 1990, cc. 403-410. Nucleotide BLAST analysis can be performed using the NBLAST program, score = 100, word length = 12, to obtain nucleotide sequences homologous to the nucleic acid encoding the protein of interest.

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Protein BLAST analysis can be performed using the XBLAST program, score = 50, word length = 3, to obtain amino acid sequences homologous to the protein of interest. To perform comparative analyzes of the primary structure including gaps, the Gapped BLAST program described in Altschul et al., Nucleic Acids, 25, 1997, cc. 3389-3402. Alternatively, PSI-Blast can be used to perform an iterative search to determine distant relationships between molecules (Id.). When using the BLAST, Gapped BLAST, and PSI-Blast programs, you can use the default settings of the corresponding programs (for example, XBLAST and NBLAST). Another preferred example, but not limited to, of a mathematical algorithm that can be used for sequence comparison is the algorithm described in Myers and Miller, CABIOS, 1989. This algorithm is included in the ALIGN program (version 2.0), which is part of the software package for comparative analysis of the primary structure of GCG sequences. When ALIGN is used to compare amino acid sequences, the PAM120 weighted residue table, gap length penalty of 12, and gap penalty of 4 can be used. Additional algorithms for sequence analysis are known in the art and include ADVANCE and ADAM, described in Torellis and Robotti, Comput. . Appl. Biosci. 10, 1994, cc. 3-5; and FASTA, described in Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85, 1988, cc. 2444-2448. In the FASTA program, the ktup parameter denotes a control parameter that specifies the sensitivity and speed of the search. If ktup=2, then similar regions in the two sequences to be compared are identified by looking at pairs of aligned residues; if ktup=1, then the individual aligned amino acids are evaluated. Protein sequences can have a ktup value of 2 or 1, and DNA sequences can have values ranging from 1 to 6. Alternatively, comparative primary structure analysis of protein sequences can be performed using the CLUSTAL W algorithm described in HIiggins et al., Methods Enzymol. 266, 1996, cc. 383-402.

A nucleic acid sequence is operably linked if it is located in a functional relationship with another nucleic acid sequence. For example, a nucleic acid presequence or secretory leader sequence is operably linked to a nucleic acid encoding a polypeptide if it is expressed as a precursor protein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects transcription of the sequence; or the ribosome binding site is operably linked to the coding sequence if it is positioned in such a way as to facilitate translation. In general, operably linked means that the DNA sequences that are linked are contiguous and, in the case of a secretory leader sequence, contiguous and in reading frame. However, enhancers are not necessarily contiguous. Linking can be accomplished by ligation at suitable restriction sites. If such sites do not exist, then synthetic oligonucleotide adapters or linkers can be used.

As used herein, the expressions cell, cell line and cell culture are used interchangeably and all such designations include their progeny. Thus, transformants and transformed cells include the primary cells in question and the cultures derived from them, regardless of the number of transfers.

The term "mammal to be treated" refers to any animal that belongs to the class of mammals, including humans, domestic and farm animals, as well as zoo, sporting or pet animals such as dogs, horses, cats, cows, etc. .d. Preferably the mammal is a human.

A disorder, as used herein, is any condition that would benefit from treatment with the humanized anti-TrkB antibody described herein. It includes chronic and acute disorders or diseases, including those pathological conditions that predispose a mammal to the disorder. Non-limiting examples or disorders treated herein include ocular or retinal disorders.

As used herein, the term TrkB-related disorder or TrkB-related disease refers to a condition in which modification or activation of cells expressing TrkB is indicated. Disorders associated with TrkB include diseases and disorders such as age-related macular degeneration, geographic atrophy, diabetic retinopathy, diabetic macular edema, retinal pigmentary degeneration, hereditary retinal dystrophy, hereditary macular degeneration, myopic degeneration, retinal vein occlusions, retinal artery occlusions, endophthalmitis, uveitis, cystoid macular edema, choroidal neovascular membrane due to any retinal disease, optical neuropathies, glaucoma, retinal detachment, toxic retinopathy, radiation retinopathy, and traumatic retinopathy, as well as prodromal and mild to moderate Alzheimer's disease, delayed disease progression in patients with Alzheimer's disease, Huntington's disease, Parkinson's disease, major depressive disorder, schizophrenia, cognitive impairment associated with schizophrenia, prevention of first episode of psychosis in individuals with attenuated psychosis, prevention of recurrence in patients with schizophrenia, treatment-resistant depression, and metabolic diseases such as hyperphagia, obesity or metabolic syndrome.

The term intravitreal injection has a common meaning in the art and refers to the introduction of an antibody to TrkB or an antigen-binding fragment thereof into the vitreous body of a patient.

The term subcutaneous administration refers to the introduction of an anti-TrkB antibody or antigen-binding fragment thereof under the skin of a diseased animal or human, preferably into a pocket between the skin and underlying tissue, by a relatively slow, sustained injection from a drug-containing reservoir. Pinching or pulling the skin upward and away from the underlying tissue can create a pocket.

The term subcutaneous infusion refers to the introduction of a drug into the skin of a diseased animal or human, preferably into a pocket between the skin and the underlying tissue, by a relatively slow, sustained injection from a drug-containing reservoir over a period of time of, but not limited to, 30 minutes. or less or 90 minutes or less. Optionally, the infusion can be accomplished by subcutaneously implanting a drug delivery pump implanted under the skin of a diseased animal or human, wherein the pump delivers the drug in a predetermined amount over a predetermined period of time of 30 minutes, 90 minutes, or a period of time corresponding duration of the treatment regimen.

The term subcutaneous bolus refers to the administration of a drug under the skin of a diseased animal or human, wherein the duration of administration of the drug as a bolus is less than about 15 minutes; in another object of the invention, less than 5 minutes, and in another object of the invention, for less than 60 s. In a further aspect of the invention, administration is carried out into a pocket between the skin and the underlying tissue, where the pocket can be created by pinching or pulling the skin up and down from the underlying tissue.

The term therapeutically effective amount is used to refer to the amount of an anti-TrkB antibody or antigen-binding fragment thereof that reduces or alleviates one or more symptoms of the disorder being treated. The active substance is used in such a way that in the specified amount it has a beneficial effect on the patient. In one aspect, the therapeutically effective amount has a neuroprotective or neuroregenerative effect. In another aspect, a therapeutically effective amount refers to the target concentration in the patient's serum at which it appears effective, for example, in slowing the progression of a disease. Efficacy can be assessed in conventional ways depending on the condition being treated. For example, in eye/retinal diseases or disorders that are characterized by cells expressing TrkB, efficacy can be measured by determining the response rate, such as vision recovery, or by assessing the latency to disease progression.

The terms treatment and therapy and the like, as used herein, refer to therapeutic, as well as prophylactic or suppressive measures for a disease or disorder that result in any clinically important or beneficial effect, including (but not limited to) relief or weakening of one or more symptoms, regression, slowing or cessation of the development of a disease or disorder. Thus, for example, treatment includes administration of an anti-TrkB antibody or an antigen-binding fragment thereof before or after the onset of a symptom of a disease or disorder, resulting in the prevention or elimination of one or more symptoms of the disease or disorder. In another example, the concept refers to the administration of an anti-TrkB antibody or an antigen-binding fragment thereof after the clinical manifestation of a disease to combat symptoms of the disease. Moreover, as used herein, the term treatment or therapy refers to the administration of an anti-TrkB antibody or an antigen-binding fragment thereof after the onset and development of clinical symptoms, where the administration has an effect on clinical parameters of the disease or disorder, such as the degree of tissue damage or the number and distribution of metastases , regardless of whether treatment leads to relief of the disease or not. In addition, if the use of the compositions of the invention, either alone or in combination with another therapeutic agent, relieves or alleviates at least one of the symptoms of the disorder being treated compared with the specified symptom without the use of the composition containing an anti-TrkB antibody or an antigen-binding fragment thereof, the result should be considered as effective treatment of the required disorder, whether or not all symptoms of the disorder are relieved.

The term package insert refers to instructions typically included in commercial packaging of therapeutic products that contain information regarding indications, methods of use, administration, contraindications, and/or precautions for use.

- 11 043771 changes in these therapeutic products.

Antibodies

The antibodies described and disclosed herein are anti-TrkB antibodies, particularly humanized anti-TrkB antibodies, as well as compositions and articles containing anti-TrkB antibodies according to the present invention. Antigen-binding antibody fragments to TrkB have also been described. Antibodies to TrkB and antigen-binding fragments thereof can be used to treat various diseases or disorders that are characterized by reduced activity of the TrkB pathway. The anti-TrkB antibody and an antigen-binding fragment thereof each include at least a component that specifically recognizes a TrkB epitope.

For initial characterization, the mouse anti-TrkB leader D003 was selected based on its much better antibody performance. A library of variants was created by placing the mouse CDR leader sequence into the FR of the human heavy and light chain consensus variable domains, and by constructing the FR with various changes.

This resulted in various humanized heavy and light chain variable sequences as shown below:

VL Sequences.

D003 VL (mouse leader), variable light chain, SEQ ID NO: 1

DIVMSOSPSSLAVSVGEKVTMSCKSSOSLLYSSNOKNYLAWYOQKPGOSPKLLI

YWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCOQYYSYPYTFGGGT

KLEIK

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277-gr_VL, (humanized) variable light chain, SEQ ID NO: 2 DIVMTOSPDSLAVSLGERATINCKSSOSLLYSSNOKNYLAWYOQKPGOPPKLLI YWASTRESGVPDRFSGSGSGTDFTLTISSLOAEDVAVYYCOQYYSYPYTFGOGT KLEIK

277-33_VL: (humanized) variable light chain, SEQ ID NO: 3 DIVMTOSPDSLAVSLGERATISCKSSOSLLYSSNOKNYLAWYOQKPGOPPKLLIY WASTRESGVPDRFSGSGSGTDFTLTISSLOAEDVAVYYCOQYYSYPYTFGGGTK LEIK

277-35 VL: (humanized) variable light chain, SEQ ID NO: 4 DIVMTOSPDSLAVSLGERATISCKSSOSLLYSSNOKNYLAWYOQKPGOPPKLLIY WASTRESGVPDRFSGSSGTDFTLTISSLOAEDVAVYYCOQYYSYPYTFGGGTK LEIK

277-42_VL, (humanized) variable light chain, SEQ ID NO: 5 DIVMTOSPDSLAVSLGERATINCKSSOSLLYSSNOKNYLAWYOQKPGOPPKLLI YWASTRESGVPDRFSGSGSGTDFTLTISSLOAEDVAVYYCOQYYSYPYTFGGGT KLEIK

277-44 VL, (humanized) variable light chain, SEQ ID NO: 6 DIVMTOSPDSLAVSLGERATINCKSSOSLLYSSNOKNYLAWYOQKPGOSPKLLI YWASTRESGVPDRFSGSGSGTDFTLTISSLOAEDVAVYYCQQYYSYPYTFGOGT KLEIK

277-48 VL, (humanized) variable light chain, SEQ ID NO: 7 DIVMTOSPDSLAVSLGERATINCKSSOSLLYSSNOKNYLAWYOQKPGOPPKLLI YWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPYTFGOGT KLEIK

277-51_VL, (humanized) variable light chain, SEQ ID NO: 8 DIVMTOSPDSLAVSLGERATISCKSSOSLLYSSNOKNYLAWYOQKPGOPPKLLIY WASTRESGVPDRFSGSGSGTDFTLTISSLOAEDVAVYYCOQYYSYPYTFGGGTK LEIK

- 13 043771

277-64 VL, (humanized) variable light chain, SEQ ID NO: 9 DIVMTOSPDSLAVSLGERATISCKSSOSLLYSSNQKNYLAWYOOKPGOPPKLLIY WASTRESGVPDRFSGSGSGTDFTLTISSLOAEDVAVYYCOQYYSYPYTFGOGTK LEIK

277-67_VL, (humanized) variable light chain, SEQ ID NO: 10 DIVMTOSPDSLAVSLGERATINCKSSOSLLYSSNQKNYLAWYOOKPGOSPKLLI YWASTRESGVPDRFSGSGSGTDFTLTISSLOAEDVAVYYCOQYYSYPYTFGOGT KLEIK

VH SEQUENCES

D003 VH, (mouse leader) variable heavy chain, SEQ ID NO: AND OVOLOQSGAELAKPGASVKMSCKASGYTFTGYWMHWVKORPGOGLEWIGYI NPSTDYTEYNOKFKDKATLTADK S S S T A YMQL SSLTSEDSAVYYCARSRTGNY WGQGTTLTVSS

277-gr_VH, (humanized) variable heavy chain, SEQ ID NO: 12 OVOLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVROAPGOGLEWMGYI NPSTDYTEYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSRTGNY WGQGTLVTVSS

277-33 VH, (humanized) variable heavy chain, SEQ ID NO: 13 OVOLVOSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQRPGOGLEWIGYIN PSTDYTEYNQKFKDRVTLTRDTS T S T V YMEL S SLTSEDTAVYYCARSRTGNYW GQGTTVTVSS

277-35 VH, (humanized) variable heavy chain, SEQ ID NO: 14 OVOLVOSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRORPGOGLEWIGYIN PSTDYTEYNQKFKDRVTLTRDTS T S TV YMEL S SLRSEDTAVYYCARSRTGNYW GQGTTVTVSS

277-42 VH, (humanized) variable heavy chain, SEQ ID NO: 15 OVOLVOSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQRPGOGLEWIGYIN PSTDYTEYNQKFKDRVTLTRDTS T S TV YMEL S SLRSEDTAVYYCARSRTGNYW GQGTTVTVSS

- 14 043771

277-44_VH, (humanized) variable heavy chain, SEQ ID NO: 16 OVOLVOSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGOGLEWIGYIN PSTDYTEYNQKFKDRVTMTRDT S T S T V YMEL S SLT SEDTAVYYCARSRTGNYW GQGTTVTVSS

277-48_VH, (humanized) variable heavy chain, SEQ ID NO: 17 OVOLVOSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQRPGOGLEWIGYIN PSTDYTEYNQKFKDRVTMTRDTS T S TV YMEL S SLRSEDTAVYYCARSRTGNYW GQGTTVTVSS

277-51_VH, (humanized) variable heavy chain, SEQ ID NO: 18 OVOLVOSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQRPGOGLEWIGYIN PSTDYTEYNQKFKDRATLTRDTS T S TV YMEL S SLRSEDTAVYYCARSRTGNYW GQGTTVTVSS

277-64_VH, (humanized) variable heavy chain, SEQ ID NO: 19

OVOLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVROAPGOGLEWIGYIN PSTDYTEYNQKFKDRVTMTRDTS T S TV YMEL S SLRSEDTAVYYCARSRTGNYW GQGTTVTVSS

277-67_VH, (humanized) variable heavy chain, SEQ ID NO: 20 OVOLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVROAPGOGLEWIGYIN PSTDYTEYNQKFKDRVTMTRDTS T S TV YMEL S SLRSEDTAVYYCARSRTGNYW GQGTTVTVSS

The underlined sequences correspond to the CDR regions of the light and heavy chain variable regions.

The humanized anti-TrkB antibodies of the present invention are antibodies that have light and heavy chain sequences as shown in the table below. IgG1-KO mutants were created by introducing two mutations into the Fc region, Leu232Ala and Leu233Ala, to reduce effector function.

Table 1

Antibody Subsequence SEQ ID NO: 277-gr (Light chain, IgGl) DIVMTQSPDS LAVS L GE RATINCKSSQSLLYSSNQK NYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSG SGTDFTLTISSLQAEDVAVYYCQQYYSYPYTFGQGT KLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LS STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN 21

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RGEC 277-gr (Heavy chain, IgGl) QVQLVQSGAEVKKРGASVKVSСKASGYTFTGYWMHW VRQAPGQGLE WMGYINPSTDYTEYNQKFKDRVTMTR DTSTSTVYMELSSLRSEDTAVYYCARSRTGNYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSL GTQTYICNVNHKPSNTKVDKRVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPG 22 277-gr (Heavy chain, IgGl-KO) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHW VRQAPGQGLE WMGYINPSTDYTEYNQKFKDRVTMTR DTSTSTVYMELSSLRSEDTAVYYCARSRTGNYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLG TQTYICNVNHKPSNTKVDKRVEPK SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPG 23 277-33 (Light chain, IgGl) DIVM T Q S P D S LAVS L GE RATIS CKSSQSLLYSSNQK NYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSG SGTDFTLTISSLQAEDVAVYYCQQYYSYPYTFGGGT KLEIKRTVAAPSVEIFPPSDEQLKSGTASVVCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC 24 277-33 (Heavy chain, IgGl) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHW VRQRPGQGLEWIGYINPSTDYTEYNQKFKDRVTLT R DTSTSTVYMELSSLTSEDTAVYYCARSRTGNYWGQG TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGT QTYICNVNHKPSNTKVDKRVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPG 25 277-33 (Heavy chain, IgGl-KO) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHW VRQRPGQGLEWIGYINPSTDYTEYNQKFKDRVTLT R DTSTSTVYMELSSLTSEDTAVYYCARSRTGNYWGQG 26

- 16 043771

ТTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVV SVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPG 277-35 (Light chain, IgGl) DIVMTQSPDS LAVS L GE RATIS CKSSQSLLYSSNQK NYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSG SGTDFTLTISSLQAEDVAVYYCQQYYSYPYTFGGGT KLEIKRTVAAPSVEIFPPSDEQLKSGTASVVCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC 27 277-35 (Heavy chain, IgGl) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHW VRQRPGQGLEWIG YINPSTDYTEYNQKFKDRVT L T R DTSTSTVYMELSSLRSEDTAVYYCARSRTGNYWGQG TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLG TQTYICNVNHKPSNTKVDKRVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPG 28 277-35 (Heavy chain, IgGl-KO) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHW VRQRPGQGLEWIG YINPSTDYTEYNQKFKDRVT L T R DTSTSTVYMELSSLRSEDTAVYYCARSRTGNYWGQG TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLG TQTYICNVNHKPSNTKVDKRVEPK SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPG 29 277-42 (Light chain, IgGl) DIVMTQSPDSLAVSLGERATINCKSSQSLLYSSNQK NYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSG SGTDFTLTISSLQAEDVAVYYCQQYYSYPYTFGGGT KLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFN RGEC thirty

- 17 043771

277-42 (Heavy chain, IgGl) QVQLVQSGAEVKKРGASVKVSСKASGYTFTGYWMHW VRQRPGQGLEWIGYINPSTDYTEYNQKFKDRVTLT R DTSTSTVYMELSSLRSEDTAVYYCARSRTGNYWGQG TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSL GTQTYICNVNHKPSNTKVDKRVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPG 31 277-42 (Heavy chain, IgGl-KO) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHW VRQRPGQGLEWIGYINPSTDYTEYNQKFKDRVTLT R DTSTSTVYMELSSLRSEDTAVYYCARSRTGNYWGQG TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGT QTYICNVNHKPSNTKVDKRVEPK SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPG 32 277-44 (Light chain, IgGl) DIVMTQSPDS LAVS L GE RATINCKSSQSLLYSSNQK NYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSG SGTDFTLTISSLQAEDVAVYYCQQYYSYPYTFGQGT KLEIKRTVAAPSVEIFPPSDEQLKSGTASVVCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC 33 277-44 (Heavy chain, IgGl) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHW VRQAPGQGLEWIGYINPSTDYTEYNQKFKDRVTMTR DTSTSTVYMELSSLTSEDTAVYYCARSRTGNYWGQG TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGT QTYICNVNHKPSNTKVDKRVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPG 34 277-44 (Heavy chain, IgGl-KO) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHW VRQAPGQGLEWIGYINPSTDYTEYNQKFKDRVTMTR DTSTSTVYMELSSLTSEDTAVYYCARSRTGNYWGQG TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV 35

- 18 043771

ALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPG 277-48 (Light chain, IgGl) DIVMTQSPDSLAVSLGERATINC KSSQSLLYSSNQK NYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSG SGTDFTLTISSLQAEDVAVYYCQQYYSYPYTFGQGT KLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC 36 277-48 (Heavy chain, IgGl) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHW VRQRPGQGLEWIGYINPSTDYTEYNQKFKDRVTMTR DTSTSTVYMELSSLRSEDTAVYYCARSRTGNYWGQG TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGT QTYICNVNHKPSNTKVDKRVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPG 37 277-48 (Heavy chain, IgGl-KO) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHW VRQRPGQGLEWIGYINPSTRUTEYNQKFKDRVTMTR DTSTSTVYMELSSLRSEDTAVYYCARSRTGNYWGQG TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGT QTYICNVNHKPSNTKVDKRVEPK SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPG 38 277-51 (Light chain, IgGl) DIVM T Q S P D S LAVS L GE RATIS CKSSQSLLYSSNQK NYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSG SGTDFTLTISSLQAEDVAVYYCQQYYSYPYTFGGGT KLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY S LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC 39 277-51 (Heavy QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHW 40

- 19 043771

chain, IgGl) VRQRPGQGLEWIGYINPSTDYTEYNQKFKDRATL T R DTSTSTVYMELSSLRSEDTAVYYCARSRTGNYWGQG TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHN HYTQKSLSLSPG 277-51 (Heavy chain, IgGl-KO) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHW VRQRPGQGLEWIGYINPSTDYTEYNQKFKDRATL T R DTSTSTVYMELSSLRSEDTAVYYCARSRTGNYWGQG TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLG TQTYICNVNHKPSNTKVDKRVEPK SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPG 41 277-64 (Light chain, IgGl) DIVM T Q S P D S LAVS L GE RATIS CKSSQSLLYSSNQK NYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSG SGTDFTLTISSLQAEDVAVYYCQQYYSYPYTFGQGT KLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC 42 277-64 (Heavy chain, IgGl) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHW VRQAPGQGLEWIGYINPSTDYTEYNQKFKDRVTMTR DTSTSTVYMELSSLRSEDTAVYYCARSRTGNYWGQG TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGT QTYICNVNHKPSNTKVDKRVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPG 43 277-64 (Heavy chain, IgGl-KO) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHW VRQAPGQGLEWIGYINPSTDYTEYNQKFKDRVTMTR DTSTSTVYMELSSLRSEDTAVYYCARSRTGNYWGQG TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV 44

- 20 043771

ALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPG 277-67 (Light chain, IgGl) DIVM T Q S P D S LAVS L GE RATINСKSSQSLLYSSNQK NYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSG SGTDFTLTISSLQAEDVAVYYCQQYYSYPYTFGQGT KLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSK DSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC 45 277-67 (Heavy chain, IgGl) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHW VRQAPGQGLEWIGYINPSTDYTEYNQKFKDRVTMTR DTSTSTVYMELSSLRSEDTAVYYCARSRTGNYWGQG TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGT QTYICNVNHKPSNTKVDKRVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPG 46 277-67 (Heavy chain, IgGl-KO) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHW VRQAPGQGLEWIGYINPSTDYTEYNQKFKDRVTMTR DTSTSTVYMELSSLRSEDTAVYYCARSRTGNYWGQG TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGT QTYICNVNHKPSNTKVDKRVEPK SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPG 47 277 L-CDR1 KSSQSLLYSSNQKNYLA 48 277 L-CDR2 WASTRES 49 277 L-CDR3 QQYYSYPYT 50 277 H-CDR1 (CCG) GYTFTGYWMH 51 277 H-CDR2 (CCG) YINPSTDYTEYNQKFKD 52 277 H-CDR3 (CCG) SRTGNY 53 277 H-CDR1 (Cabot) GYWMH 55 277 H-CDR2 (Cabot) YINPSTDYTEYNQKFKD 56 277 H-CDR3 (Cabot) SRTGNY 57 277 H-CDR1 (Chotia) GYTFTGY 58 277 H-CDR2 (Chotia) NPSTDY 59 277 H-CDR3 (Chotia) SRTGNY 60

The CDRs presented above, as assigned by the Computing Group (CCG), are underlined (Almagro et al., Proteins 2011; 79:3050-3066 and Maier et al., Proteins 2014; 82:1599-1610). Sequences defined according to Cabot's nomenclature are in bold and those defined according to the system

- 21 043771 Chotia numbering, in italics.

In one aspect, the anti-TrkB antibody of the present invention binds to human TrkB at a K _D <10 nM. In a further aspect, the anti-TrkB antibody of the present invention binds to human TrkB at a K _D <5 nM. In a still further aspect, the anti-TrkB antibody of the present invention binds to human TrkB at a K _D of approximately 1 nM. The binding affinities of antibodies to TrkB can be determined according to the method described in Example 6.

In another aspect, the anti-TrkB antibody of the present invention induces phosphorylation and/or activation of TrkB with high efficiency. In one aspect, the anti-TrkB antibody of the present invention phosphorylates human TrkB with an EC _{50 of} <100 pM. In a further aspect, the anti-TrkB antibody of the present invention phosphorylates human TrkB with an EC _{50 of} <50 pM. In a further aspect, the anti-TrkB antibody of the present invention phosphorylates human TrkB with an EC ₅₀ <40 pM. In a further aspect, the anti-TrkB antibody of the present invention phosphorylates human TrkB with an EC ₅₀ <30 pM. In a further aspect, the anti-TrkB antibody of the present invention phosphorylates human TrkB with an EC ₅₀ of approximately 20 pM.

In another aspect, the anti-TrkB antibody of the present invention is more effective at inducing activation of downstream TrkB signaling pathways compared to the native TrkB ligand BDNF. The effectiveness of anti-TrkB antibodies can be determined in differentiated SH-SY5Y cells according to the method described in Example 8.

In a further aspect, the anti-TrkB antibody of the present invention induces gene expression comparable to the natural TrkB ligand BDNF. Stimulation with agonistic antibodies to TrkB increases the expression of ARC, VGF, EGR1 mRNA, which are markers for increased synaptic plasticity and therefore indicators that antibodies to TrkB act like a suitable BDNF ligand to modulate neuronal function, i.e., increase neuronal survival and /or synaptic plasticity. Gene expression patterns can be determined according to the method described in Example 10.

In a still further aspect, the anti-TrkB antibody of the present invention protects neurons, glial cells and/or neurovascular unit in the retina of patients, for example, with age-related macular degeneration, geographic atrophy or diabetic retinopathy by stimulating TrkB-dependent survival signaling pathways and thereby providing thus, a neuroprotective effect.

In another aspect, the anti-TrkB antibody of the present invention regenerates axons/dendrites and/or synapses in the retina after the onset of disease, such as age-related macular degeneration, geographic atrophy, or diabetic retinopathy, thereby providing neuroregeneration.

In a further aspect, the anti-TrkB antibody of the present invention has a low risk of immunogenicity and low sequence affinity relative to its CDR sequences. Immunogenicity and heterogeneity are two important aspects of a therapeutic antibody. Measurements of such aspects can be accomplished using methods as described in Examples 4 and 5. Antibodies of the present invention have been carefully designed to have minimal or no immunogenicity and/or heterogeneity.

In yet another aspect, the anti-TrkB antibody of the present invention does not reduce BDNF-induced ERK phosphorylation. To this end, ERK phosphorylation can be measured, for example, in CHO cells expressing human TrkB, as described in Example 13. This may indicate that ERK phosphorylation induced by endogenously expressed BDNF is not reduced by administration of the anti-TrkB antibody of the invention. This may also mean that the anti-TrkB antibody of the invention does not reduce ERK phosphorylation when administered before, simultaneously or subsequently after exogenously administered BDNF. In some cases, anti-TrkB antibody does not compete with or reduce BDNF-induced ERK phosphorylation compared to BDNF induction alone.

In a further aspect, the anti-TrkB antibody of the present invention is specific for phosphorylation and/or activation of TrkB and does not non-specifically phosphorylate and/or activate TrkA or TrkC.

In another aspect, the anti-TrkB antibody does not bind nonspecifically to human VEGF and/or rat VEGF. Binding nonspecifically in the present context means that the anti-TrkB antibody of the invention does not bind to human VEGF as measured, for example, by ELISA as described in Example 14. In one embodiment, the absence of nonspecific binding of the anti-TrkB antibody can be determined by measuring statistically significant difference (eg, one-sample t test, p<0.05) in binding to human VEGF between the anti-TrkB antibody and the appropriate IgG isotype control antibody. In particular, the lack of nonspecific binding of the anti-TrkB antibody of the invention can be measured by determining any differences in binding to human VEGF between the anti-TrkB antibody and an IgG1 isotype control antibody. The anti-TrkB antibody of the invention does not differ significantly in terms of lack of binding

- 22 043771 with human VEGF compared to a similar concentration of IgGl isotype control antibody up to a concentration of approximately 0.3 nM, 0.4 nM, 0.5 nM, 0.6 nM, 0.7 nM, 0.8 nM, 0 ,9 nM, 1 nM, 5 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 110 nM, 120 nM, 130 nM, 140 nM, 150 nM, 160 nM, 170 nM, 180 nM, 190 nM or 200 nM. In an alternative embodiment, the absence of non-specific binding of the anti-TrkB antibody of the invention can be measured by determining the IC ₅₀ value of binding of the anti-TrkB antibody to any one of the human or rat VEGFs. In particular, the anti-TrkB antibody of the invention will exhibit a human or rat VEGF binding _IC50 value greater than about 0.9 μM, preferably greater than 1 μM, greater than 2 μM, greater than 3 μM, greater than 5 μM, greater than 10 μM, greater than 20 μM , above 30 µM, above 40 µM, above 50 µM, above 100 µM, above 150 µM, above 200 µM, above 250 µM, above 300 µM, above 350 µM, above 400 µM, above 450 µM, above 500 µM, above 550 µM, above 600 µM, above 650 µM, above 700 µM, above 750 µM, above 800 µM, above 850 µM, or above 900 µM.

Humanization and amino acid sequence variants

Further variants of anti-TrkB antibodies and antibody fragments can be designed based on the set of CDRs identified in the mouse leader D003. In these variant amino acid sequences of anti-TrkB antibodies and antibody fragments, the CDRs are understood to remain unchanged, but the surrounding regions, such as the FR regions, can be engineered. Anti-TrkB antibody amino acid sequence variants can be generated by introducing appropriate nucleotide changes into the anti-TrkB antibody DNA or by peptide synthesis. These variants include, for example, deletions and/or insertions and/or substitutions of residues in the amino acid sequences of the anti-TrkB antibodies exemplified herein. Any combination of deletions, insertions and substitutions is used to obtain the final construct, provided that the final construct has the required characteristics. Amino acid changes may also alter post-translational processing of the humanized anti-TrkB antibody or variant thereof, such as changing the number or position of glycosylation sites.

In some embodiments, the present invention includes anti-TrkB antibodies or antibody fragments thereof having a variable light chain and a variable heavy chain, wherein the amino acid sequence of the variable light chain and the amino acid sequence of the variable heavy chain are at least 80%, at least 90% the same %, at least 95%, at least 98%, or at least 99% identical to the amino acid sequences presented in SEQ ID NO: 2 and 12, or 3 and 13, or 4 and 14, or 5 and 15, or 6 and 16, or 7 and 17, or 8 and 18, or 9 and 19, or 10 and 20, respectively.

In some embodiments, the present invention includes anti-TrkB antibodies or antibody fragments thereof having a light chain and a heavy chain, wherein the amino acid sequence of the light chain and the amino acid sequence of the heavy chain are at least 80%, at least 90%, at least at least 95%, at least 98%, or at least 99% identical to the amino acid sequences presented in SEQ ID NO: 21 and 22 or 23; 24 and 25 or 26; 27 and 28 or 29; 30 and 31 or 32; 33 and 34 or 35; 36 and 37 or 38; 39 and 40 or 41; 42 and 43 or 44; 45 and 46 or 47 respectively.

In a further embodiment, the present invention includes anti-TrkB antibodies that compete for binding to TrkB with any of the following antibodies.

An antibody having a light chain containing the amino acid sequence of SEQ ID NO: 21 and a heavy chain containing the amino acid sequence of SEQ ID NO: 22 or 23.

An antibody having a light chain containing the amino acid sequence of SEQ ID NO: 24 and a heavy chain containing the amino acid sequence of SEQ ID NO: 25 or 26.

An antibody having a light chain containing the amino acid sequence of SEQ ID NO: 27 and a heavy chain containing the amino acid sequence of SEQ ID NO: 28 or 29.

An antibody having a light chain containing the amino acid sequence of SEQ ID NO: 30 and a heavy chain containing the amino acid sequence of SEQ ID NO: 31 or 32.

An antibody having a light chain containing the amino acid sequence of SEQ ID NO: 33 and a heavy chain containing the amino acid sequence of SEQ ID NO: 34 or 35.

An antibody having a light chain containing the amino acid sequence of SEQ ID NO: 36 and a heavy chain containing the amino acid sequence of SEQ ID NO: 37 or 38.

An antibody having a light chain containing the amino acid sequence of SEQ ID NO: 39 and a heavy chain containing the amino acid sequence of SEQ ID NO: 40 or 41.

An antibody having a light chain containing the amino acid sequence of SEQ ID NO: 42 and a heavy chain containing the amino acid sequence of SEQ ID NO: 43 or 44.

An antibody having a light chain containing the amino acid sequence of SEQ ID NO: 45 and a heavy chain containing the amino acid sequence of SEQ ID NO: 46 or 47.

Another type of producing an amino acid variant of an antibody is to change the original glycosylation pattern of the antibody. The term change in the context of this application means the deletion of one or more carbohydrate moieties present in the antibody and/or the addition of one or more

- 23 043771 several glycosylation sites not previously present in the antibody.

In some aspects, the present invention includes nucleic acid molecules that encode amino acid sequence variants of the anti-TrkB antibodies described herein. Nucleic acid molecules encoding variant amino acid sequences of the anti-TrkB antibody are prepared using various methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or production by oligonucleotide mutagenesis (or site-directed mutagenesis), PCR mutagenesis, and cassette mutagenesis of a previously obtained variant or non-variant. variant version of the anti-TrkB antibody.

In certain embodiments, the anti-TrkB antibody is an antibody fragment. There are techniques that have been developed to obtain antibody fragments. Fragments can be obtained by proteolytic cleavage of intact antibodies (see, for example, Morimoto et al., Journal of Biochemical and Biophysical Methods 24, 1992, pp. 107-117 and Brennan et al., Science 229, 1985, p. 81). Alternatively, fragments can be produced directly in recombinant host cells. For example, Fab'-SH fragments can be isolated directly from E. coli and chemically cross-linked to produce F(ab') ₂ fragments (see, for example, Carter et al., Bio/Technology 10, 1992, cc. 163167) . According to another approach, F(ab') ₂ fragments can be isolated directly from a recombinant host cell culture. Other techniques for preparing antibody fragments will be apparent to one skilled in the art.

Antibodies to TrkB and their antigen-binding fragments may include modifications.

In certain embodiments, it may be desirable to use an anti-TrkB antibody fragment rather than an intact antibody. It may be desirable to modify an antibody fragment to increase its serum half-life. This can be accomplished, for example, by incorporating a salvage receptor binding epitope into an antibody fragment. In one method, a suitable region of the antibody fragment can be changed (eg, mutated), or the epitope can be incorporated into a peptide tag, which is then fused to the antibody fragment at either end or in the middle, for example, by DNA synthesis or peptide synthesis. See, for example, WO 96/32478.

In other embodiments, the present invention includes covalent modifications of anti-TrkB antibodies. Covalent modifications include modification of cysteinyl residues, histidyl residues, lysinyl and amino-terminal residues, arginyl residues, tyrosyl residues, carboxyl side groups (aspartyl or glutamyl), glutaminyl and asparaginyl residues, or seryl or threonyl residues. Another type of covalent modification involves chemical or enzymatic cross-linking of glycosides to the antibody. These modifications can be accomplished by chemical synthesis or by enzymatic or chemical cleavage of the antibody, if possible. To introduce other types of covalent modifications into the antibody molecule, interaction of labeled amino acid residues of the antibody with an organic derivatizing agent that can react with selected side chains or amino-terminal or carboxy-terminal residues can be used.

Removal of any carbohydrate moieties present on the antibody can be accomplished chemically or enzymatically. Chemical deglycosylation is described in Hakimuddin et al., Arch. Biochem. Biophys. 259, 1987, p. 52 and in Edge et al., Anal. Biochem., 118, 1981, p. 131. Enzymatic cleavage of carbohydrate moieties on antibodies can be accomplished using endo- and exoglycosidases according to the method described in Thotakura et al., Meth. Enzymol., 138, 1987, p. 350.

Another type of suitable covalent modification involves coupling the antibody to one of a variety of non-protein polymers, such as, for example, polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, using the methods described in one or more of US 4,640,835, US 4,496,689, US 4,301,144, US 4,670,417. US 4791192 and US 4179337.

Epitope binding

The antibodies of the invention specifically bind to native or recombinant human TrkB. Antibodies of the present invention recognize a specific epitope of the TrkB antigen and a TrkB epitope. In particular, the antibodies of the invention bind to an epitope in the extracellular domain of human TrkB with the sequence SEQ ID NO: 54.

The extracellular domain of human TrkB essentially includes the following sequence (SEQ ID NO. 54):

- 24 043771

CPTSCKCSASRIWCSDPSPGIVAFPRLEPNSVDPENITEIFIANQKRLEIINEDDVE AYVGLRNLTIVDSGLKFVAHKAFLKNSNLQHINFTRNKLTSLSRKHFRHLDLSE LILVGNPFTCSCDIMWIKTLQEAKSSPDTQDLYCLNESSKNIPLANLQIPNCGLPS ANLAAPNLTVEEGKSITLSCSVAGDPVPNMYWDVGNLVSKHMNETSH TQGSLR ITNISSDDSGKQISCVAENLVGEDQDSVNLTVHFAPTITFLESPTSDHHWCIFTV KGNPKPALQWFYNGAILNESKYICTKIHVTNHTEYHGCLQLDNPTHMNNGDYT LIAKNEYGKDEKQISAHFMGWPGIDDGANPNYPDVIYEDYGTAANDIGDTTNRS NEIPSTDVTDKTGREH

As used herein, the terms TrkB antigen epitope and TrkB epitope refer to a molecule (eg, peptide) or fragment of a molecule capable of binding to an anti-TrkB antibody or antigen-binding fragment thereof. These terms further include, for example, a TrkB antigenic determinant that is recognized by any of the antibodies or antibody fragments of the present invention that has a combination of light and heavy chain CDRs selected from the light chain CDRs set forth in SEQ ID NOs 48-50 and the heavy chain CDRs. chains presented in SEQ ID NO: 51-53.

TrkB antigen epitopes may be included in proteins, protein fragments, peptides or the like. Epitopes are most often proteins, short oligopeptides, oligopeptide mimics (ie, organic compounds that mimic the antibody-binding properties of the TrkB antigen), or combinations thereof.

Antibodies or antibody fragments of the present invention have been found to bind to unique epitopes in the extracellular domain of human TrkB. Preferably, the anti-TrkB antibody or antigen-binding fragment thereof binds to at least one amino acid residue within amino acid regions 92-112, 130-143 and/or 205-219 of the human TrkB extracellular domain of SEQ ID NO: 54.

In one embodiment, the present invention provides an anti-TrkB antibody or antigen-binding fragment thereof that binds to at least one amino acid residue within the amino acid regions 92-112 and 130-143; or 92-112 and 205-219; or 130-143 and 205-219; or 92-112 and 130-143 and 205-219 of the extracellular domain of human TrkB with the sequence SEQ ID NO: 54.

In one embodiment, the present invention provides an anti-TrkB antibody or antigen-binding fragment thereof that binds to at least one amino acid residue within any of the above combinations of amino acid regions and additionally also to at least one amino acid residue within the amino acid regions 313-330 and/or 348-367 of the extracellular domain of human TrkB with the sequence SEQ ID NO: 54.

In one embodiment, the present invention provides an anti-TrkB antibody or antigen-binding fragment thereof that binds to at least one amino acid residue within the amino acid regions 92-112, 130-143, 205-219, 313-330, and 348- 367 extracellular domain of human TrkB with the sequence SEQ ID NO: 54.

Thus, in the context of epitope binding, the expression binds within the amino acid regions X-Y... means that the anti-TrkB antibody or antigen binding fragment thereof binds to at least one amino acid residue within the amino acid region specified in the sequence.

If, for example, an anti-TrkB antibody or an antigen-binding fragment thereof binds to at least one amino acid residue within the amino acid regions 92-112 or 130-143, then this means that the anti-TrkB antibody or an antigen-binding fragment thereof binds to at least one amino acid residue residue either within amino acid regions 92-112 or 130-143.

In a further example, if the anti-TrkB antibody or antigen-binding fragment thereof binds to at least one amino acid residue within the amino acid regions 92-112 and 130143, then the anti-TrkB antibody or antigen-binding fragment thereof binds to at least one amino acid residue within amino acid region 92-112 and also binds to at least one amino acid residue within amino acid region 130143 SEQ ID NO: 54.

In another aspect, the anti-TrkB antibody or antigen-binding fragment thereof binds to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% of the amino acid residues within the amino acid regions 92-112, 130-143 and/or 205-219 of the extracellular domain of human TrkB with the sequence SEQ ID NO: 54.

In one embodiment, the present invention provides an anti-TrkB antibody or antigen-binding fragment thereof that binds to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85 %, 90%, 95%, or 100% of amino acid residues within amino acid regions 92-112 and 130-143; or 92-112 and 205-219; or 130-143 and 205-219; or 92-112 and 130-143 and 205-219

- 25 043771 extracellular domain of human TrkB with the sequence SEQ ID NO: 54.

In one embodiment, the present invention provides an anti-TrkB antibody or antigen-binding fragment thereof that binds to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85 %, 90%, 95%, or 100% of amino acid residues within any of the above combinations of amino acid regions and additionally also with at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80 %, 85%, 90%, 95%, or 100% of the amino acid residues within amino acid regions 313-330 and/or 348-367 of the human TrkB extracellular domain of SEQ ID NO: 54.

In one embodiment, the present invention provides an anti-TrkB antibody or antigen-binding fragment thereof that binds to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85 %, 90%, 95%, or 100% of the amino acid residues within the amino acid regions 92-112, 130-143, 205-219, 313-330, and 348-367 of the human TrkB extracellular domain of SEQ ID NO: 54.

If, for example, an anti-TrkB antibody or antigen-binding fragment thereof binds to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% of amino acid residues within amino acid regions 92-112 or 130-143, this means that the anti-TrkB antibody or antigen binding fragment thereof binds to at least 10%, 20%, 30%, 40%, 50%, 60% , 70%, 80%, 85%, 90%, 95%, or 100% of amino acid residues within the amino acid region 92-112 or binds to at least 10%, 20%, 30%, 40%, 50%, 60 %, 70%, 80%, 85%, 90%, 95%, or 100% of amino acid residues within the amino acid region 130-143.

In a further example, if the anti-TrkB antibody or antigen-binding fragment thereof binds to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% of the amino acid residues within the amino acid regions 92-112 and 130-143, this means that the anti-TrkB antibody or antigen binding fragment thereof binds to at least 10%, 20%, 30%, 40%, 50%, 60 %, 70%, 80%, 85%, 90%, 95%, or 100% of amino acid residues within amino acid region 92112 and also binds to at least 10%, 20%, 30%, 40%, 50%, 60 %, 70%, 80%, 85%, 90%, 95%, or 100% of amino acid residues within the amino acid region 130-143.

Without wishing to be bound by theory, it is believed that by binding to novel TrkB epitopes, the antibody exhibits its far superior properties in activating TrkB.

Polynucleotides, vectors, host cells and recombinant methods

Other embodiments include isolated polynucleotides, which include an anti-TrkB antibody coding sequence, vectors and host cells containing the polynucleotides, and recombinant techniques for producing the antibody. The isolated polynucleotides may encode any desired form of anti-TrkB antibody, including, for example, full-length monoclonal antibodies, Fab, Fab', F(ab') ₂ , and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from fragments antibodies.

The polynucleotide(s) that contain a sequence encoding an anti-TrkB antibody, or a fragment or chain thereof, may be coupled to one or more regulatory or control sequences, as known in the art, and can be contained in suitable expression vectors or host cells, as known in the art. Each of the polynucleotide molecules encoding the heavy or light chain variable domains can be independently paired with a polynucleotide sequence encoding a constant domain, such as a human constant domain, enabling the production of intact antibodies. Alternatively, the polynucleotides, or portions thereof, may be conjugated together to provide a template for the production of a single chain antibody.

For recombinant production, a polynucleotide encoding an antibody is inserted into a replicable vector for cloning (DNA amplification) or expression. Various suitable vectors for expression of recombinant antibodies are available. Vector components typically include, but are not limited to, one or more of the following components: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

Antibodies to TrkB can also be prepared as fusion polypeptides in which the antibody is fused to a heterologous polypeptide, such as a signal sequence or other polypeptide having a specific cleavage site at the amino terminus of the mature protein or polypeptide. The heterologous signal sequence is typically selected from a sequence that is recognized and processed (ie, cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize or process the anti-TrkB antibody signal sequence, the signal sequence can be replaced with a prokaryotic signal sequence. The signal sequence may be, for example, alkaline phosphatase, penicillinase, lipoprotein, thermostable enterotoxin II leader sequences, and the like. For secretion by yeast, the native signal sequence may be replaced, for example, by a leader sequence derived from yeast alpha factor invertase (including

- 26 043771 leader sequences of the a-factors of Saccharomyces and Kluyveromyces), acid phosphatase, glucoamylase of C. albicans or the signal sequence described in WO 90/13646. In mammalian cells, mammalian signal sequences as well as viral secretory leader sequences, for example the herpes simplex gD signal sequence, can be used. The DNA for such a precursor region is ligated in frame to DNA encoding a humanized anti-TrkB antibody.

Expression and cloning vectors contain a nucleotide sequence that enables the vector to replicate in one or more selected host cells. In general, in cloning vectors, this sequence is the sequence that enables the vector to replicate independently of host chromosomal DNA, and includes origins of replication or autonomously replicating sequences. Such sequences are well known for various bacteria, yeasts and viruses. The origin of replication from plasmid pBR322 is suitable for most gram-negative bacteria, the origin of replication is 2-υ. Plasmids are suitable for yeast, and various viral origins of replication (SV40, polyoma, adenovirus, VSV, and BPV) are suitable as cloning vectors in mammalian cells. In general, the origin of replication component is not necessary for mammalian expression vectors (the SV40 origin of replication can typically be used only because it contains an early promoter).

Expression and cloning vectors may contain a gene that encodes a selectable marker to facilitate identification of expression. Typical selectable marker genes encode proteins that confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate or tetracycline, or alternatively, complement axotrophic deficiencies, or in other alternatives, supplement specific nutrients that are not present in complex culture media, for example, the gene encoding the D-alanine racemate for Bacilli.

One example of a selection scheme uses a drug to stop the growth of a host cell. Those cells that are successfully transformed with the heterologous gene produce the drug resistance protein and therefore survive the selection scheme. Examples of such dominant selection are the use of the drugs neomycin, mycophenolic acid and hygromycin. Common selectable markers for mammalian cells are those that provide the ability to identify cells competent to accept a nucleic acid encoding a humanized anti-TrkB antibody, such as DHFR (dihydrofolate reductase), thymidine kinase, metallothionein-I and -II (such as the primate metallothionein genes), adenosine deaminase, ornithine decarboxylase, and others. Cells transformed by the DHFR-selectable gene are first identified by culturing all transformants in a culture medium that contains methotrexate (Mtx), a competitive antagonist of DHFR. A suitable host cell used, if wild-type DHFR is used, is a Chinese hamster ovary (CHO) cell line deficient in DHFR activity (eg, DG44).

Alternatively, host cells (preferably wild-type hosts that contain endogenous DHFR) transformed or cotransformed with DNA sequences encoding an anti-TrkB antibody, wild-type DHFR protein, and another selectable marker such as aminoglycoside 3'-phosphotransferase (APH) can be selected by growing the cells in a medium containing a selectable agent for the selectable marker, such as an aminoglycoside antibiotic, eg kanamycin, neomycin, or G418. see, for example, US patent 4,965,199.

When recombinant production is carried out in a yeast cell as a host cell, the TRP1 gene present in the yeast plasmid YRp7 (Stinchcomb et al., 1979, Nature 282: 39) can be used as a selectable marker. The TRP1 gene provides a selection marker for a mutant yeast strain that lacks the ability to grow on tryptophan, such as ATCC No. 44076 or PEP4-1 (Jones, 1977, Genetics 85:12). Subsequently, the presence of the trp1 lesion in the host yeast cell genome provides an efficient environment for detecting transformation by growth in the absence of tryptophan. Likewise, Leu2p-deficient yeast strains such as ATCC 20,622 and 38,626 are complemented with known plasmids carrying the LEU2 gene.

Additionally, vectors derived from the 1.6 μm circular plasmid pKD1 can be used to transform Kluyveromyces yeast. Alternatively, an expression system for the industrial production of recombinant bovine chymosin for K. lactis has been described (Van den Berg, 1990, Bio/Technology 8:135). Stable multicopy expression vectors for the secretion of mature recombinant human serum albumin using commercial Kluyveromyces strains have also been described (Fleer et al., 1991, Bio/Technology 9:968-975).

Expression and cloning vectors typically contain a promoter that is recognized by the host and is operably linked to a nucleic acid molecule encoding an anti-TrkB antibody or polypeptide chain thereof. Promoters suitable for use with prokaryotic hosts include the phoA promoter, the β-lactamase and lactose promoter systems, the alkaline phosphatase, the tryptophan (trp) promoter system, and hybrid promoters such as the tac promoter.

Other known bacterial promoters are also suitable. Promoters for use in bacterial systems will also contain a Shine-Dalgamo sequence.

S.D.), functionally linked to DNA encoding a humanized antibody to TrkB.

Many eukaryotic promoter sequences are known. In fact, all eukaryotic genes have an AT-rich region located approximately 25-30 bp upstream of the transcription start site. Another sequence, 70-80 bases upstream from the start of transcription of many genes, is the CNCAAT region, where N can be any nucleotide. At the 3' end of most eukaryotic genes there is an AATAAAA sequence, which can be a signal for the addition of a poly-A tail to the 3' end of the coding sequence. All of these sequences are suitably inserted into expression vectors.

Examples of suitable promoter sequences for use with yeast hosts include promoters for 3-phosphoglycerate kinase or other hydrolytic enzymes such as enolase, glyceraldehyde 3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.

Inducible promoters have the added benefit of transcription under the control of growth conditions. These include the yeast promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, derivative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for the utilization of maltose and galactose. Suitable vectors and promoters for use for expression in yeast are further described in EP 73,657. Yeast enhancers are also advantageously used with yeast promoters.

Transcription of anti-TrkB antibody from vectors in mammalian host cells is controlled, for example, by promoters derived from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus , retrovirus, hepatitis B virus and simian virus 40 (SV40), from heterologous mammalian promoters, for example, actin promoter or immunoglobulin promoter, or from heat shock promoters, provided that such promoters are compatible with host cell systems.

The SV40 early and late promoters are readily obtained as an SV40 restriction fragment, which also contains the SV40 origin of replication. The immediate early promoter of human cytomegalovirus is readily obtained as a HindIII E restriction fragment. A system for expressing DNA in mammalian hosts using bovine papillomavirus as a vector is described in US Pat. No. 4,419,446. A modification of this system is described in US Patent No. 4,601,978. See also Reyes et al., 1982, Nature 297:598-601, which describes the expression of human p-interferon cDNA in mouse cells under the control of the thymidine kinase promoter from herpes simplex virus. Alternatively, the Rous sarcoma virus long terminal repeat can be used as a promoter.

Another useful element that can be used in a recombinant expression vector is an enhancer sequence, which is used to enhance transcription of DNA encoding an anti-TrkB antibody by higher eukaryotes. Currently, many enhancer sequences are known from mammalian genes (eg, globin, elastase, albumin, α-fetoprotein, and insulin). However, typically an enhancer from a eukaryotic cell virus is used. Examples include the SV40 late origin enhancer (100-270), cytomegalovirus early promoter enhancer, late polyoma enhancer, and adenoviral enhancers. Also see Yaniv, 1982, Nature 297:17-18 for a description of enhancer elements for activation of eukaryotic promoters. The enhancer can be spliced into the vector at a position 5' or 3' to the anti-TrkB antibody coding sequence, but is preferably located at a site 5' from the promoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insects, plants, animals, humans or nucleated cells from other multicellular organisms) may also contain sequences necessary for transcription termination and for mRNA stabilization. Such sequences are publicly available from the 5' and sometimes 3' untranslated regions of eukaryotic or viral DNA or to DNA. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the anti-TrkB antibody. One useful transcription termination component is the bovine growth hormone polyadenylation site. See WO94/11026 and the expression vector described therein. In some embodiments, anti-TrkB antibodies can be expressed using the CHEF system. (See, for example, US Pat. No. 5,888,809; the contents of which are incorporated herein by reference.)

- 28 043771

Suitable host cells for cloning or expressing DNA in the vectors of the present invention are prokaryotic, yeast or higher eukaryotic cells as described above. Suitable prokaryotes for this purpose include eubacteria, such as gram-negative or gram-positive organisms, e.g. Enterobacteriaceae, such as Escherichia, e.g. E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g. Salmonella typhimurium, Serratia, for example, Serratia marcescans, and Shigella, as well as Bacilli, such as B. subtilis and B. licheniformis (for example, B. licheniformis 41 P, disclosed in DD 266,710, published April 12, 1989), Pseudomonas, such as P. aeruginosa , and Streptomyces. One of the preferred hosts for cloning E. coli is E. coli 294 (ATCC 31,446), although other strains such as E. coli B, E. coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are also suitable. . These examples are illustrative and not restrictive.

In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable hosts for cloning or expressing vectors encoding an anti-TrkB antibody. Saccharomyces cerevisiae, or the common baker's yeast, is the most commonly used lower eukaryotic microorganism host. However, various other genera, species and strains are publicly available and useful herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts, such as, for example, K. lactis, K. fragilis (ATCC 12.424), K. bulgaricus (ATCC 16.045), K. wickeramii (ATCC 24.178), K. waltii (ATCC 56.500), K. drosophilarum (ATCC 36.906 ), K. thermotolerans, and K. marxianus; yarrowia (EP 402.226); Pichia pastors (EP 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi such as, for example, Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.

Suitable host cells for expressing glycosylated anti-TrkB antibodies are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells, including, for example, various baculovirus strains and variants and corresponding insect-permitted host cells from organisms such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori (silkworm). Various viral strains are publicly available for transfection, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses can be used particularly for transfection of Spodoptera frugiperda cells.

Plant cell cultures of cotton, corn, potatoes, soybeans, petunia, tomato and tobacco can also be used as hosts.

Anti-TrkB antibodies or antigen-binding fragments thereof according to the invention can also be incorporated into viral vectors, that is, a polynucleotide encoding an anti-TrkB antibody or antigen-binding fragment thereof is introduced into a viral vector and then expressed in the patient after infection with the virus.

In another aspect, the expression of antibodies to TrkB is carried out in vertebrate cells. Propagating vertebrate cells in culture (tissue culture) has become a common procedure and techniques are widely available. Examples of suitable mammalian host cell lines include the monkey kidney line CV1 transformed with SV40 (COS-7, ATCC CRL 1651), human embryonic kidney lines (293 or 293 cells subcloned for growth in suspension culture, (Graham et al., 1977, J. Gen Virol. 36: 59), newborn hamster kidney cells (BHK, ATCC CCL 10), Chinese hamster ovary cells/-DHFR1 (CHO, Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77: 4216; e.g. DG44), mouse Sertoli cells (TM4, Mather, 1980, Biol. Reprod. 23:243-251), monkey kidney cells (CV1 ATCC CCL 70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical carcinoma cells (HELA, ATCC CCL 2), dog kidney cells (MDCK, ATCC CCL 34), Buffalo rat kidney cells (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human liver cells (Hep G2, HB 8065), mouse mammary tumor (MMT 060562, ATCC CCL51), TR1 cells (Mather et al., 1982, Annals N.Y. Acad. Sci. 383: 44-68) , MRC 5 cells, FS4 cells, and a human hepatoma line (Hep G2).

Host cells are transformed with the expression or cloning vectors described above to produce anti-TrkB antibodies and cultured in a suitable growth medium modified, if appropriate, to induce promoters, select transformants, or amplify genes encoding the desired sequences.

The host cells used to produce the anti-TrkB antibodies described herein can be cultured in a variety of culture media. Commercially available media such as Ham F10 (Sigma-Aldrich Co., St. Louis, Mo.), Minimal Essential Medium (MEM), (Sigma-Aldrich Co.), RPMI-1640 (Sigma-Aldrich Co.), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma-Aldrich Co.) are suitable for culturing host cells. Additionally, any of the media described in one or more of the following: Ham et al., 1979, Meth. Enz. 58: 44, Barnes et al., 1980, Anal Biochem 102: 255, US Patent No. 4,767,704, US Patent No. 4,657,866, US Patent No. 4,927,762, US Patent No. 4,560,655, US Patent No. 5,122,469, WO 90/103430, and WO 87/00195, can be used in

- 29 043771 as a culture medium for host cells. Any of these media may be supplemented, if necessary, with hormones and/or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES ), nucleotides (such as adenosine and thymidine), antibiotics (such as gentamicin), trace minerals (defined as inorganic compounds typically present in final concentrations in the micromolar range), and glucose or an equivalent energy source. Other additives may also be included in suitable concentrations as will be apparent to one skilled in the art. Culture conditions, such as temperature, pH, and others, are conditions that have previously been used for the host cell selected for expression and will be understood by one skilled in the art.

Using recombinant techniques, the antibody can be produced intracellularly, into the periplasmic space, or directly secreted into the culture medium. If the antibody is produced intracellularly, the cells can be disrupted to release the protein as a first step. Particulate waste, either host cells or lysed fragments, can be removed, for example, by centrifugation or ultrafiltration. Carter et al., 1992, Bio/Technology 10:163-167 describes a procedure for isolating antibodies that are secreted into the periplasmic space of E. coli. Briefly, the cell mass is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for approximately 30 minutes. Cellular debris can be removed by centrifugation. If the antibody is secreted into the culture medium, the supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the above steps to inhibit proteolysis and antibiotics may be included to prevent the growth of incidental contaminants. Various methods can be used to isolate an antibody from a host cell.

The antibody composition prepared from cells can be purified, for example, by hydroxyapatite chromatography, gel electrophoresis, dialysis and affinity chromatography, where a typical purification technique is affinity chromatography. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies that are based on human gamma1, gamma2, or gamma4 heavy chains (see, eg, Lindmark et al., 1983 J. Immunol. Meth. 62:1-13). Protein G is recommended for all murine isotypes and for human gamma3 (see, eg, Guss et al., 1986 EMBO J. 5:1567-1575). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are also available. Mechanically stable matrices such as controlled pore glass or poly(styrene divinyl)benzene offer the possibility of faster flow rates and shorter processing times, which can be achieved with agarose. If the antibody includes a C _H3 domain, then Bakerbond ABX™ resin (J. T. Baker, Phillipsburg, NJ) is suitable for purification. Other technologies for protein purification are also available, such as ion exchange column fractionation, ethanol precipitation, reverse phase HPLC, silica chromatography, heparin chromatography SEPHAROSE™ chromatography on anion or cation exchange resin (such as a polyaspartic acid), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation, depending on the antibody being reduced.

After any preparatory purification step(s), the mixture containing the antibody of interest and contaminants can be subjected to hydrophobic interaction chromatography at low pH using an elution buffer at a pH in the range of 2.5-4.5, typically carried out at low salt concentrations (eg, from about 0-0.25 M salt).

The invention also relates to nucleic acids that hybridize under relaxed, mild or stringent conditions, as defined herein, with all or part (for example, a part that encodes a variable region) of a nucleotide sequence that represents the isolated polynucleotide(s). ) sequence(s) encoding an anti-TrkB antibody or antibody fragment. The hybridizing region of the hybridizing nucleic acid typically consists of at least 15 (eg, 20, 25, 30, or 50) nucleotides. The hybridizing region of the hybridizing nucleic acid is at least 80% identical, such as at least 90%, at least 95%, or at least 98% identical to the sequence of the region or all of the nucleic acid encoding an anti-TrkB polypeptide (e.g. , heavy chain or light chain variable region), or its complement. Hybridizing nucleic acids of the type described herein can be used, for example, as a cloning probe, a primer, for example a PCR primer, or a diagnostic probe.

In some embodiments, the present invention includes isolated polynucleotide(s) comprising sequences that encode an antibody or antibody fragment having/having a variable light chain and a variable heavy chain, wherein the amino acid sequence of the variable light chain chains and amino acid sequence of the variable heavy chain are at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequences set forth in SEQ ID NO: 2 and 12, or 3 and 13, or 4 and 14, or 5 and 15, or 6 and 16, or 7 and 17, or 8 and

18, or 9 and 19, or 10 and 20, respectively.

In some embodiments, the present invention includes isolated polynucleotide(s), including sequences that encode an antibody or antibody fragment having/having a light chain and a heavy chain, wherein the light chain amino acid sequence and the heavy chain amino acid sequence are at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequences presented in SEQ ID NO: 21 and 22 or 23; 24 and 25 or 26; 27 and 28 or 29; 30 and 31 or 32; 33 and 34 or 35; 36 and 37 or 38; 39 and 40 or 41; 42 and 43 or 44; 45 and 46 or 47, respectively.

In these anti-TrkB antibodies and antibody fragments, it is intended that the nucleic acid sequence encoding the CDRs remain unchanged (unchanged with respect to the amino acid they encode, possible DNA sequence equivalents due to the degeneracy of the genetic code), but the surrounding regions, e.g., FR regions, can be designed.

Non-therapeutic uses

The antibodies presented herein can be used as affinity-based purification agents. In this process, antibodies are immobilized on a solid phase, such as a Protein A resin, using methods well known in the art. The immobilized antibody is contacted with a sample containing the TrkB protein (or fragment thereof) to be purified, and the support is then washed with a suitable solvent which should remove substantially all of the material in the sample except the TrkB protein bound to the immobilized antibody. Finally, the support is washed with another suitable solvent, which should separate the TrkB protein from the antibody.

Antibodies to TrkB can also be used in diagnostic assays to detect and/or quantify TrkB protein, for example, by determining the expression of TrkB in specific cells, tissues or serum.

In some embodiments, it is advantageous, for example for diagnostic purposes, to label an antibody with a detectable moiety. Numerous detectable tags are known and available, such as radioisotopes, fluorescent tags, enzyme substrate tags, and the like. The label can be indirectly conjugated to the antibody using various known techniques. For example, an antibody can be conjugated to biotin, and any of the above three broad categories of labels can be conjugated to avidin, or vice versa. Biotin selectively binds to avidin and therefore the label can be conjugated to the antibody indirectly. Alternatively, to achieve label-antibody indirect conjugation, the antibody can be conjugated to a small hapten (such as digoxin) and one of the various types of labels above conjugated to an anti-hapten antibody (for example, an anti-digoxin antibody). In this way, indirect conjugation of the label with the antibody can be achieved.

Examples of radioisotope labels are ³⁵ S, ¹⁴ C, ¹²⁵ I, ³ H and ¹³¹ I. An antibody can be labeled with a radioisotope using techniques described, for example, in Current Protocols in Immunology, vols. 1 and 2, 1991, ed. Coligen et al., Wiley-Interscience, New York, NY, 1991. Radioactivity can be measured, for example, using a scintillation counter.

Examples of fluorescent tags are known tags based on rare earth metal chelates (europium chelates), or tags based on fluorescein and its derivatives, rhodamine and its derivatives, dansyl, lissamine, phycoerythrin and Texas red. Fluorescent tags can be conjugated to the antibody using known techniques, for example, those described in Current Protocols in Immunology, supra. Fluorescence can be quantified using a fluorimeter.

Various well-characterized enzyme-substrate tags are known in the art (see, for example, the review given in US 4,275,149). The enzyme typically catalyzes a chemical change in a chromogenic substrate, which can be assessed using a variety of techniques. For example, the change may be a change in the color of the substrate, which can be assessed spectrophotometrically. Alternatively, the enzyme can change the fluorescence or chemiluminescence of the substrate. Fluorescence quantification techniques are described above. As a result of a chemical reaction, an electronically excited state arises in the chemiluminescent substrate, as a result of which it can emit light, which can be assessed, for example, using a chemiluminometer, or it can act as an energy donor for a fluorescent acceptor.

Examples of enzymatic tags are luciferases such as firefly luciferase and bacterial luciferase (US 4737456), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (such as glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, etc. Methods for conjugating enzymes with antibodies are described, for example, in O'Sullivan et al., Methods for the Preparation of Enzyme-Antibody Conjugates for use in Enzyme

Immunoassay, in: Methods in Enzym., ed. J. Langone and N. Van Vunakis, Academic press, N.Y., 73,

1981, cc. 147-166.

Examples of enzyme-substrate combinations are, for example: horseradish peroxidase (HRPO) and hydrogen peroxidase as substrate, wherein the hydrogen peroxidase oxidizes a dye precursor such as orthophenylenediamine (OPD) or 3,3',5,5'-tetramethylbenzidine hydrochloride (TMB); alkaline phosphatase (AP) and para-nitrophenylphosphonate as a chromogenic substrate; and βD-galactosidase (β-D-Gal) and a chromogenic substrate, such as para-nitrophenyl-β-D-galactosidase, or a fluorogenic substrate, such as 4-methylumbelliferyl-β-D-galactosidase.

Numerous other enzyme-substrate combinations are known to those skilled in the art. A general overview is presented, for example, in US 4275149 and US 4318980.

In other embodiments, the anti-TrkB antibody is used unlabeled and detected using a labeled antibody coupled to the anti-TrkB antibody.

The antibodies provided herein can be used in any known assay, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays (see, for example, Zola, Monoclonal Antibodies: A Manual of Techniques, ed. in CRC Press, Inc., 1987, pp. 147-158).

Diagnostic kits

An anti-TrkB antibody may be used as part of a diagnostic kit, which is a package that contains a combination of reagents in predetermined quantities, in combination with instructions for performing the diagnostic assay. If the antibody is labeled with an enzyme, the kit may include substrates and cofactors required by the enzyme, such as a precursor substrate that forms a detectable chromophore or fluorophore. In addition, other additives such as stabilizers and buffers (eg, blocking buffer or lysis buffer) and the like may be included. The relative amounts of the various reagents can vary widely, providing concentrations of reagents in solution that greatly optimize the sensitivity of the assay. The reagents may be dry powders, typically lyophilized, including excipients which, when dissolved, should form a reagent solution having the appropriate concentration.

Therapeutic applications and TrkB-related disorders

In another embodiment, an anti-TrkB antibody (or a functional fragment thereof) described herein is useful for treating various disorders associated with TrkB expression as described herein.

The anti-TrkB antibody or antigen-binding fragment thereof is administered by any suitable route, including intravitreal, oral, parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Additionally, the anti-TrkB antibody is suitably administered by pulsatile infusion, primarily with decreasing doses of the antibody. In one aspect, dosing is carried out by injection, most preferably intravenous or subcutaneous injection, including whether the treatment is short-term or long-term. Preferably, the anti-TrkB antibody is administered by intravitreal injection into the eye.

To prevent or treat a disease, the appropriate dose of antibody will depend on a number of factors, such as the type of disease being treated, the severity and progression of the disease, whether the antibody is being administered for prophylactic or therapeutic purposes, prior therapy, the patient's medical history and response to the antibody, and from the order of the attending physician. The antibody can be administered to the patient once or through a series of treatments.

As used herein, the term suppression has the same meaning as the terms alleviation and relief, and refers to the attenuation or reduction of one or more characteristics of a disease.

The antibody composition can be formulated, dosed and administered in accordance with good clinical practice. Important factors in this regard include the specific disorder being treated, the clinical condition of the individual patient, the cause of the disorder, the site of administration of the agent, the method of administration, the schedule of administration, and other factors known to medical practitioners. In this regard, the therapeutically effective amount of antibody to be administered is adjusted to be the minimum amount necessary to prevent, alleviate, or treat a disorder associated with TrkB expression.

The antibody is optionally, but in certain cases, formulated in combination with one or more agents currently used to prevent or treat the disorder in question. The effective amount of these other agents depends on the amount of anti-TrkB antibody present in the formulation, the type of disorder or treatment, and other factors noted above. They are typically administered at the same dosages and routes of administration in which they were previously administered, or at a dose that is from about 1% to about 99% of their intended dosage.

The TrkB agonistic antibodies of the present invention stimulate TrkB signaling. Without wishing to be bound by theory, it is believed that TrkB signaling is beneficial in protecting photoreceptors, other retinal neurons, including ganglion cells, and retinal pigment epithelial cells from cell death in the retina. Therefore, agonistic antibodies to TrkB may protect retinal neurons such as photoreceptors from degeneration, that is, prevent neurodegeneration, and also enhance neuronal survival and/or plasticity, ultimately counteracting vision loss associated with either reduced TrkB activity and/or or increased cellular stress or apoptotic activity. Thus, the antibodies are useful for treating eye diseases or retinal diseases, especially neurodegenerative retinal diseases or eye diseases. Such diseases include, but are not limited to, age-related macular degeneration, geographic atrophy, diabetic retinopathy, diabetic macular edema, retinal pigmentary degeneration, hereditary retinal dystrophy, hereditary macular degeneration, myopic degeneration, retinal vein occlusions, retinal artery occlusions, endophthalmitis, uveitis, cystoid macular edema, choroidal neovascular membrane due to any of the retinal diseases, optic neuropathies, glaucoma, retinal detachment, toxic retinopathy, radiation retinopathy and traumatic retinopathy.

The antibodies are also useful for the treatment of diseases of the central nervous system, such as prodromal and mild to moderate Alzheimer's disease, delayed disease progression in patients with Alzheimer's disease, Huntington's disease, Parkinson's disease, major depressive disorder, schizophrenia, cognitive impairment associated with schizophrenia, prevention first episode of psychosis in individuals with attenuated psychosis, preventing recurrence in patients with schizophrenia or treatment-resistant depression.

In another embodiment, the antibodies can be used to treat hearing loss, particularly cisplatin-induced hearing loss, as well as noise-induced hearing loss and age-related hearing loss.

In particular, the anti-TrkB agonistic antibodies of the present invention are indicated for the treatment of non-proliferative and/or proliferative diabetic retinopathy and/or diabetic macular edema in addition to standard treatment: Retinal neuronal dysfunction and neurodegeneration are one of the major pathological events in diabetic retinopathy and diabetic macular edema spots (at all stages of the disease) that ultimately cause vision loss and visual dysfunction. Activation of TrkB would counteract the loss and functional impairment of retinal neurons and thus help maintain normal vision, reduce loss of visual function in disease, and potentially help restore visual function.

In addition, there is a potential application of TrkB activation as an adjunct to anti-VEGF treatment, which would significantly enhance the therapeutic benefits of the latter, since anti-VEGF only targets vascular dysfunction in the eye and not the neuronal/glial cell system; additionally, long-term anti-VEGF treatment may cause neurodegenerative side effects. The approach of activating TrkB as a complement to anti-VEGF would reduce such neurodegenerative side effects.

Antibodies to TrkB or antigen binding fragments thereof are particularly useful for the treatment or prevention of retinal disorders such as diabetic retinopathy, diabetic macular edema, geographic atrophy and glaucoma.

In a further aspect, antibodies to TrkB or antigen binding fragments thereof are also useful for the treatment of metabolic diseases such as hyperphagia, obesity and metabolic syndrome.

In another aspect, the anti-TrkB antibodies of the present invention may be used in a method of treating and/or preventing a TrkB-associated disorder, particularly geographic atrophy, which includes administering a therapeutically effective amount of an anti-TrkB antibody of the invention to an individual suffering from geographic atrophy, thereby reducing one or more symptoms of geographic atrophy.

In a still further aspect, anti-TrkB agonistic antibodies may be used in a method of treating and/or preventing geographic atrophy, which includes administering a therapeutically effective amount of an anti-TrkB agonist antibody to an individual suffering from geographic atrophy, thereby reducing one or more symptoms of geographic atrophy.

Pharmaceutical compositions and their administration

A composition containing an anti-TrkB antibody or an antigen-binding fragment thereof can be administered to a subject who suffers from, or is at risk of developing, an eye or retinal disease. The invention further provides the use of an anti-TrkB antibody or an antigen-binding fragment thereof for

- 33 043771 preparation of a medicinal product for the prevention or treatment of TrkB disease. The term subject, as used herein, refers to any mammalian patient to which an anti-TrkB antibody or antigen-binding fragment thereof can be administered, including, for example, humans and non-human mammals such as primates, rodents and dogs. Subjects, including humans in particular, can be treated using the methods presented herein. The anti-TrkB antibody or antigen-binding fragment thereof can be administered either alone or in combination with other compositions.

Preferred antibodies for use in such pharmaceutical compositions are antibodies that are a humanized antibody or antibody fragments having a variable light chain and a variable heavy chain with the amino acid sequence set forth in SEQ ID NOs: 2 and 12, or 3 and 13, or 4 and 14, or 5 and 15, or 6 and 16, or 7 and 17, or 8 and 18, or 9 and 19, or 10 and 20, respectively.

Also covered are antibodies for use in such pharmaceutical compositions that are a humanized antibody or antibody fragments having a variable light chain and a variable heavy chain with an amino acid sequence of at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 2 and 12, or 3 and 13, or 4 and 14, or 5 and 15, or 6 and 16, or 7 and 17, or 8 and 18, or 9 and 19, or 10 and 20, respectively.

Further preferred antibodies for use in such pharmaceutical compositions are those comprising a humanized antibody or antibody fragments having the light chain and heavy chain amino acid sequence of SEQ ID NO: 21 and 22 or 23; 24 and 25 or 26; 27 and 28 or 29; 30 and 31 or 32; 33 and 34 or 35; 36 and 37 or 38; 39 and 40 or 41; 42 and 43 or 44; 45 and 46 or 47, respectively.

Further preferred antibodies for use in such pharmaceutical compositions are those that are a humanized antibody or antibody fragments having at least 80%, at least 90%, or at least 95% amino acid sequence of a light chain and a heavy chain. , at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 21 and 22 or 23; 24 and 25 or 26; 27 and 28 or 29; 30 and 31 or 32; 33 and 34 or 35; 36 and 37 or 38; 39 and 40 or 41; 42 and 43 or 44; 45 and 46 or 47 respectively.

It is understood that in these anti-TrkB antibodies and antibody fragments, the amino acid sequence of the CDR remains unchanged, but the surrounding regions, eg, FR regions, can be engineered.

Various delivery systems are known and can be used to administer an anti-TrkB antibody or an antigen-binding fragment thereof. Routes of administration include, but are not limited to, intravitreal, eye drops, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The anti-TrkB antibody or antigen-binding fragment thereof may be administered, for example, by infusion, bolus, or injection, and may be administered in conjunction with other biologically active agents. Administration may be systemic or local. In preferred embodiments, administration is by intravitreal injection. Preparations for such injections can be prepared, for example, in pre-filled syringes.

The anti-TrkB antibody or antigen-binding fragment thereof can be administered in the form of pharmaceutical compositions containing a therapeutically effective amount of the anti-TrkB antibody or antigen-binding fragment thereof and one or more pharmaceutically compatible ingredients.

According to exemplary embodiments of the invention, the pharmaceutical composition is prepared according to conventional procedures to provide a pharmaceutical composition suitable for intravenous or subcutaneous administration to humans. Typically, compositions for injection are solutions in sterile isotonic aqueous buffer. If necessary, the pharmaceutical composition may also contain a solubilizing agent and a local anesthetic, such as lidocaine, to relieve pain at the injection site. Typically, the ingredients are supplied either separately or mixed to form a unit dose of the drug, for example, as a dry lyophilized powder or a water-free concentrate in a sealed container such as an ampoule or sachet, indicating the amount of active ingredient. In cases where the pharmaceutical agent is intended for administration by infusion, it can be filled into infusion bottles containing sterile water or pharmaceutical grade saline solution. In cases where the pharmaceutical agent is administered by injection, an ampoule of sterile water for injection or saline may be used so that the ingredients can be mixed before administration.

In addition, the pharmaceutical composition can be included in a pharmaceutical kit that contains (a) a container containing an anti-TrkB antibody or antigen binding fragment thereof in lyophilized form and (b) a second container containing a pharmaceutically acceptable diluent (eg, sterile water) for injection. A pharmaceutically acceptable diluent can be used to reconstitute or dilute the lyophilized anti-TrkB antibody or antigen binding fragment thereof. Optionally, the specified container(s) may be accompanied by a notice in a form approved by an official agency authorizing the manufacture, use, or sale of pharmaceuticals or biological products, where the notice reflects the agency's approval of the manufacture, use, or sale for the purpose of administration to humans.

The amount of anti-TrkB antibody or antigen-binding fragment thereof that is effective in treating or preventing a TrkB-related disorder can be determined by conventional clinical research methods. In addition, in vitro assays may not necessarily be used, the results of which may help determine optimal dosage limits. The exact dose to be used in the formulation should also depend on the route of administration and the stage of the disorder, and should be based on the recommendations of the treating physician and the circumstances specific to each patient. Effective doses can be determined by extrapolation from dose response curves obtained from in vitro experiments or animal model test systems.

For example, the toxicity and therapeutic efficacy of an anti-TrkB antibody or antigen-binding fragment thereof can be determined in cell cultures or experimental animals by standard pharmaceutical methods used to determine the ED ₅₀ (therapeutically effective dose for 50% of a population). Preferred is an anti-TrkB antibody or an antigen-binding fragment thereof that exhibits a high therapeutic index.

Data obtained from cell culture assays and animal studies can be used to determine dose limits for human use. Typically, the dose of anti-TrkB antibody or antigen-binding fragment thereof is within the range of circulating concentrations that include the ED50 while exhibiting little or no toxicity. The dose can be varied within these limits depending on the dosage form used and the route of administration used. For any anti-TrkB antibody or antigen-binding fragment thereof used in the method, a therapeutically effective dose may initially be determined based on the results of cell culture assays. The dose can be determined based on data obtained in animal models, reaching plasma concentration limits that include the IC ₅₀ (i.e., the concentration of the test compound at which half the maximum inhibition of symptoms is achieved) based on data obtained in culture cells. Such information can be used to more accurately determine suitable doses for humans. Plasma concentrations can be determined, for example, by high performance liquid chromatography, ELISA, etc.

For intravitreal injection of anti-TrkB antibody, longer treatment intervals are generally preferred. Due to their improved efficacy, the anti-TrkB antibodies of the present invention can be administered at longer intervals.

In one embodiment, the anti-TrkB antibody is administered every 6 weeks, preferably every 7 weeks, also preferably every 8 weeks, further preferably every 9 weeks, more preferably every 10 weeks, further preferably every 11 weeks, and more preferably every 12 weeks . In a further preferred embodiment, the anti-TrkB antibody is administered once every 3 months.

In one embodiment, the anti-TrkB antibody is administered to a subject in need thereof at an initial dose of 1-2000 mg. In another embodiment, optionally one or more subsequent doses are administered, each containing 1-2000 mg of anti-TrkB antibody.

Due to the fact that the volume that can be injected into the eye is significantly limited, it is extremely important that the anti-TrkB antibody can be formulated in high concentrations. In addition, the effectiveness of the anti-TrkB antibody is extremely important since an effective antibody can exert its effect even at very low doses and therefore prolongs activity as well as treatment intervals.

The antibodies of the present invention can be formulated at extremely high doses, which include, but are not limited to, 20 mg/ml, 30 mg/ml, 40 mg/ml, 50 mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml or 100 mg/ml.

The typical dosage that may be administered to a patient is approximately 3 mg/eye. Typical buffer components that can be used for such a formulation include, for example, sodium acetate, PS20 and trehalose dihydrate.

In one embodiment, the anti-TrkB antibody is formulated with 10 mM histidine buffer, 240 mM sucrose, 0.02% w/v. polysorbate 20 at pH 5.5 with a final protein concentration of 60 mg/ml.

In some embodiments, pharmaceutical compositions comprising an anti-TrkB antibody or an antigen-binding fragment thereof may further include a therapeutic agent, conjugated or unconjugated to a binding agent. An anti-TrkB antibody or an antigen-binding fragment thereof can be co-administered in combination with one or more therapeutic agents to treat or prevent a TrkB-associated disease. For example, combination therapy may include anti-VEGF, anti-PDGF or anti-ANG2.

Such combination therapy may have additive or synergistic effects on disease features (eg, symptom severity, number of symptoms, or relapse rates).

When using combination administration treatment regimens, in a particular embodiment of the invention, the anti-TrkB antibody or antigen-binding fragment thereof is administered simultaneously with the therapeutic agent. In another specific embodiment of the invention, the therapeutic agent is administered before or after administration of the anti-TrkB antibody or antigen-binding fragment thereof, for from 1 hour to several months, for example, for at least 1 hour, 5 hours, 12 hours, days, weeks, a month or three months before or after administration of an anti-TrkB antibody or antigen-binding fragment thereof.

Products

Another object of the invention is an article containing substances suitable for the treatment of the disorders described above. The product includes a container and a label. Acceptable containers include, for example, bottles, vials, syringes and laboratory tubes. Containers can be made from various materials such as glass or plastic. The container contains a composition that is effective for treating a particular condition and may have a sterile access opening. For example, the container may be a bag or vial for an intravenous solution having a closure device permeable to a hypodermic injection needle. The active substance in the composition is an antibody to TrkB or its antigen-binding fragment. The label on or associated with the container states that the composition is used to treat the selected condition. The article may further include a second container containing a pharmaceutically acceptable buffer, such as phosphate buffered saline, Ringer's solution, and dextrose solution. It may additionally include other substances whose presence is desirable from a commercial and consumer point of view, including other buffers, diluents, filters, needles, syringes and package inserts with instructions for use.

The invention is described below using the examples provided, which are not intended to limit the scope of the invention.

Examples

1. Creation of an antibody (immunization).

B cells from mice during the immunization complex were screened using a proprietary single B cell platform technology. Anti-TrkB binders were minimized after testing to determine activity, potency, and efficiency in an in-vitro TrkB phosphorylation assay.

2. Obtaining humanized antibodies.

The mouse leader D003 was selected for further optimization. The mouse leader component had a variable light chain corresponding to SEQ ID NO: 1 and a variable heavy chain corresponding to SEQ ID NO: 11. To optimize the TrkB sequence of the mouse leader D003, the most similar human germline antibody sequences were selected, IGKV41*01, KJ2 for mild chains and IGHV1-46*03, HJ6 for heavy chain. We created a library of options. This resulted in the humanized light chain variable sequences shown in SEQ ID NOs: 2-10 and the heavy chain variable sequences shown in SEQ ID NOs: 12-20.

table 2

277-gr VL, (humanized) variable light chain SEQ ID NO 2 277-33 VL: (humanized) variable light chain SEQ ID NO 3 277-35 VL: (humanized) variable light chain SEQ ID NO 4 277-42 VL, (humanized) variable light chain SEQ ID NO 5 277-44 VL, (humanized) variable light chain SEQ ID NO 6 277-48 VL, (humanized) variable light chain SEQ ID NO 7 277-51 VL, (humanized) variable light chain SEQ ID NO 8 277-64 VL, (humanized) variable light chain SEQ ID NO 9 277-67 VL, (humanized) variable light chain SEQ ID NO 10

- 36 043771

277-gr_VH, (humanized) variable heavy chain SEQ ID NO: 12 277-33_VH: (humanized) variable heavy chain SEQ ID NO: 13 277-3 5_VH: (humanized) variable heavy chain SEQ ID NO: 14 277-42_VH, (humanized) variable heavy chain SEQ ID NO: 15 277-44_VH, (humanized) variable heavy chain SEQ ID NO: 16 277-48_VH, (humanized) variable heavy chain SEQ ID NO: 17 277-51 VH, (humanized) variable heavy chain SEQ ID NO: 18 277-64_VH, (humanized) variable heavy chain SEQ ID NO: 19 277-67_VH, (humanized) variable heavy chain SEQ ID NO: 20

Exemplary humanized antibodies of the present invention are antibodies that have light and heavy chain sequences as indicated in the table below. IgG1KO has two mutations in the Fc region, Leu234Ala and Leu235Ala, to reduce effector function.

Table 3

277-gr (Light chain, IgGl) 21 277-gr (Heavy chain, IgGl) 22 277-gr (Heavy chain, IgGl-KO) 23 277-33 (Light chain, IgGl) 24 277-33 (Heavy chain, IgGl) 25 277-33 (Heavy chain, IgGl-KO) 26 277-35 (Light chain, IgGl) 27 277-35 (Heavy chain, IgGl) 28 277-35 (Heavy chain, IgGl-KO) 29 277-42 (Light chain, IgGl) thirty 277-42 (Heavy chain, IgGl) 31 277-42 (Heavy chain, IgGl-KO) 32 277-44 (Light chain, IgGl) 33 277-44 (Heavy chain, IgGl) 34 277-44 (Heavy chain, IgGl-KO) 35 277-48 (Light chain, IgGl) 36 277-48 (Heavy chain, IgGl) 37 277-48 (Heavy chain, IgGl-KO) 38 277-51 (Light chain, IgGl) 39 277-51 (Heavy chain, IgGl) 40 277-51 (Heavy chain, IgGl-KO) 41 277-64 (Light chain, IgGl) 42 277-64 (Heavy chain, IgGl) 43 277-64 (Heavy chain, IgGl-KO) 44 277-67 (Light chain, IgGl) 45 277-67 (Heavy chain, IgGl) 46 277-67 (Heavy chain, IgGl-KO) 47 277 L-CDR1 48 277 L-CDR2 49 277 L-CDR3 50 277 H-CDR1 51 277 H-CDR2 52 277 H-CDR3 53

3. Information about the epitope.

Materials.

Water (Sigma Aldrich, part number 37877-4L).

Acetonitrile (Sigma Aldrich, part number 34998-4L).

Formic acid (Fluka, catalog number 94318).

Urea (Sigma Aldrich, part number 51456-500G).

TCEP-HCl - 10 g (Thermo Scientific - Pierce, catalog number 20491).

Dibasic sodium phosphate (Sigma Aldrich, part number S7907-100G).

Monobasic sodium phosphate (Sigma Aldrich, part number S8282-500G).

Pre-column ACQUITY UPLC VEN C18 VanGuard, 130A, 1.7 microns, 2.1 mm X 5 mm (Waters Technolo

- 37 043771 gies Corp, 186003975).

Poroszyme® I immobilized pepsin cartridge, 2.1 mm x 30 mm (Life Technologies Corp, 2313100).

Acquity UPLC column VEN C18 1.7 µm, 1 mm X 50 mm (Waters, 186002344).

Solvent A: 0.1% Formic acid/99% water/1% acetonitrile.

Solvent B: 0.1% Formic acid / 5% water / 95% acetonitrile.

Aqueous buffer: H ₂ O 10 mM sodium phosphate pH 7.4.

Deuterium buffer: D2O 10 mM sodium phosphate pH 7.4.

Quenching buffer: water, 8M urea, 0.4M TCEP-HCl.

In control samples for epitope mapping, antigen was run with and without antibody. To determine the list of antigenic peptides, a first run was first performed in this puncture using aqueous buffer instead of deuterium buffer. 4 μl of sample was mixed with 40 μl of deuterium buffer. This mixture was incubated at 20°C for several time points (1, 2 and 4 min). After this, 40 μl of the mixture was transferred to 40 μl of 4°C quenching buffer (4 M Urea, 0.4 M Tser-HCl) and mixed. 60 μl of quenching protein was injected, where it was digested on a pepsin column for 2 minutes by washing with 200 μl/ml solvent A: 0.1% Formic acid/99% water/1% acetonitrile. Subsequent peptides were desalted on a Vanguard precolumn for 3 minutes. Pepsin-digested peptides were sent to a reverse-phase VEN C18 column inside a temperature-controlled compartment column/valve. A gradient solvent system was used consisting of: solvent A: 0.1% Formic acid/99% water/1% acetonitrile and solvent B: 0.1% Formic acid/5% water/95% acetonitrile. The percentage of solvent B was increased from 10% to 15% after 5.1 minutes, to 50% after 11 minutes, to 90% after 11.5 minutes, holding until 12.5 minutes, to 0% B after 13 minutes, holding until 14 minutes. Chromatographic separation occurred at 4°C with a flow rate of 180 μL/min. After chromatographic separation, the sample was fed to a Thermo Scientific Orbitrap Fusion mass spectrometer operating in positive electrospray ionization mode. The method used included CID and ETD activation types in identifying reference peptides using a separation of 120k, a minimum signal of 10k, a separation width of 1.0, and a normalized collision energy of 35.0V. The RF level of the S-lenses was set to 60%. For control peptides, the data acquisition type was profiles for full MS scans and centroid for CID MS/mS data. For deuterated samples, no MS/MS was collected. Data were collected over the mass range of 280–1800 Da. For analysis of raw LC-MS/ms fragmentation data, control samples (with CID and ETD MS/ms) were analyzed using Proteome Discover 1.4 (Thermo Scientific) and PMi Byonic (Protein Metrics) against a given sequence to generate a peptide list and time holding. The raw data files were pre-processed and converted to ASCII format using proprietary SHARC software. The identified peptides were then noted and the data summarized using our own developed SHAFT software. Epitopes were determined based on differences in the average mass shift induced by binding after deuterium labeling. At the peptide level, protection greater than 0.4 Da was considered significant.

Epitope mapping results are shown for BDNF, 277 antibodies, (C2) (US20100196390), and (C20) (US20100150914) in FIG. 1. Specific binding sites for each human TrkB extracellular domain molecule with SEQ ID NO: 54 are highlighted in dark gray. Compared to the natural BDNF ligand, 277 antibodies bind to other novel epitopes.

4. Susceptibility sequences in CDRs.

CDR sequences were checked for the presence of any potential exposures such as N-glycosylation sites, strong deamidation motifs (NG, NS, NH, NA, ND, NT, NN), aspartate isomerization motifs (DG), fragmentation motifs (DG, DS), cysteine. These amino acids or motifs may undergo a chemical reaction and impart undesirable heterogeneity to the product, also with the potential to negatively affect target binding and function. For these reasons, it is preferable to remove such amino acids or motifs (if present) from the CDR.

The CDRs of the mouse leader and humanized sequences did not contain such potential susceptibility motifs.

Table 4

Antibody D003/277 C2 S20 29D7 or TAM-163 Exposures 0 2 3 6

5. Immunogenicity.

Sequence immunogenicity was assessed in silico using an algorithm licensed from Epivax. The EpiMatrix Treg-adj score takes into account T cell epitopes and Treg epitopes. The lower the immunogenicity score, the less likely the sequence is to be immunogenic. In general, a negative score is considered to indicate a low risk of immunogenicity, while a positive score is considered to indicate potential immunogenicity. ______________________________________________________________ Table 5

VL Epivax evaluation VH Epivax evaluation 277-gr VL -24 277-gr VH -46 277-33 VL -31 277-33 VH -32 277-35 VL -31 277-35 VH -26 277-42 VL -27 277-42 VH -26 277-44 VL -14 277-44 VH -49 277-48 VL -24 277-48 VH -29 277-51 VL -31 277-51 VH -22 277-64 VL -28 277-64 VH -44 277-67 VL -14 277-67 VH -44 C2 VL -2 C2 VH -21 C20 VL +9 C20 VH +20 TAM163VL -51 TAM163VH -19

6. Affinity.

Surface plasmon resonance (SPR) technology was used to determine the binding affinity of 277 antibodies, C2 (US 20100196390), and C20 (US 20100150914) and TAM-163 (WO 2015173756) to Hu-TrkB-His. The experiment was carried out on a ProteOn XPR36 device.

Method: The running buffer for this experiment and all dilutions (except where specifically noted) were done in PBS-T-EDTA with 0.01% Tween20 [100 µL 100% Tween20 was added to 2 L PBS-T-EDTA to obtain final concentration of Tween 20 0.01%]. The GLM sensor chip was normalized and preconditioned according to the manufacturer's instructions. The sensor chip was activated using an equilibrated EDC/s-NHS mixture in a horizontal direction for 300 s at a flow rate of 30 μL/min and immobilized with Recombinant Protein A/G (60 μg/ml in 10 mM acetate pH 4.5) in a horizontal direction. direction for 300 s at a flow rate of 30 μl/min, which resulted in ~7100-7130 EO Protein A/G on the surface. The sensor chip was deactivated using 1 M ethanolamine HCl in a horizontal direction for 300 s at a flow rate of 30 μL/min. The sensor chip was stabilized for 18 s with 0.85% phosphoric acid at a flow rate of 100 μL/min 3 times horizontally and 3 times vertically. 277-antibody (0.6 μg/ml), C2 (2.1 μg/ml), C20 (1.2 μg/ml), and TAM-163 (5 μg/ml) were captured vertically onto the surface of Protein A/G for 70 s at a flow rate of 30 μL/min, resulting in capture levels of ∼615, 1390, 780, and 3380 EO, respectively. The baseline was stabilized by injecting PBS-T-EDTA for 60 s at a flow rate of 100 μL/min horizontally and then a second injection of PBS-T-EDTA for 60 s at a flow rate of 100 μL/min and dissociating for 120 s horizontally. The analyte (Hu-TrkB-His) was injected horizontally over the captured antibody for 600 s at a flow rate of 30 μL/min and dissociated for 1800 s. The analyte concentrations were 0 nM, 1.23 nM, 3.7 nM, 11.11 nM, 33.33 nM, and 100 nM. The surface was regenerated by injecting 0.85% phosphoric acid for 18 s at a flow rate of 100 μL/min once horizontally and once vertically. PBS-TEDTA was injected for 60 s at a flow rate of 100 μL/min once vertically. Crosstalk (interactions with the sensor surface) and blank data (PBS-T-EDTA with 0.01% Tween20 or 0 nM analyte) were subtracted from the raw data. The sensorgrams were then fitted globally to 1:1 Langmuir binding, yielding association (ka), dissociation (kd) and affinity ( _KD ) values.

277 antibodies bind to Hu-TrkB-His with a ka of 1.93 X 10 ⁵ 1/Ms, KD of 3.51 X 10 ^-4 1/s, and KD of 1.2 nM. C2 binds to Hu-TrkB-His with a ka of 2.32 X 10 ⁴ 1/Ms, KD of 3.39 X 10 ^-4 1/s, and KD of 10.5 nM. C20 binds to HuTrkB-His with a KA of 1.29 X 10 ⁵ 1/Ms, a KD of 6.13 X 10 ^-3 1/s, and a KD _of 47.7 nM. TAM-163 does not bind to 100 nM HuTrkB-His.

Table 6

Human TrkB

mAb k _a (l/ms) k _d (1/s) K _D (nM) TAM-163 anti-TrkB does not bind at 100 nM C20_chimeric anti-TrkB huIgGlKO EX00077781 1.29E+05 6DZE-OZ 47.7 anti-TrkB C2_HuIgGlko (EX00077780) 2.32E+04 3.39E-04 10.5 Anti-TRKB 277 antibodies 1.93E+05 3.51E-04 1.19

7. Nonspecific binding.

A nonspecific binding assay was developed using biosensor technology to determine whether novel biological entity (NBE) candidates would have significant binding to unrelated charged proteins. In this assay, antibodies passed over two SPR surfaces, one coated with an unrelated negatively charged protein and the other coated with

- 39 043771 unrelated positively charged protein. When an NBE candidate exhibits significant nonspecific binding to these surfaces, it is likely that the cause of binding is the presence of positively or negatively charged surface sites on the candidate. Non-specific binding of NBE candidates may result in poor pharmacokinetics (PK) and biodistribution and may also negatively impact subsequent manufacturability. The results of this analysis were used in the context of the risk assessment for this project. The purpose of this study was to reduce the risk of poor PK, lack of distribution in the target tissue and difficulty in subsequent manufacturing processability.

The objective was to determine whether 277 antibodies would exhibit nonspecific binding or not exhibit nonspecific binding by collecting 277 antibody binding sensorgrams and comparing them with those of controls to categorize nonspecific binding as favorable (green), acceptable (yellow), or unfavorable (red). ).

Method.

The experiment was carried out on a Biacore T200. Dilution, surface preparation, and binding experiments were performed at 25°C in 1X HBS-EP buffer prepared from 10X HBS-EP. The flow rate for both the immobilization protocol and the binding experiment was 5 μL/min.

To prepare the surface for the nonspecific binding experiment, white lysozyme from chicken eggs and trypsin inhibitor type 1-S from soybean were manually bound to the S Series CM5 chip with a surface density of 3000-5000 EO using an amino immobilization kit according to the manufacturer's instructions. FC1 and FC2 were activated by injecting a 1:1 mixture of EDC and NHS for 7 minutes. The surface of chicken egg white lysozyme was prepared on FC2 by injecting 150 μg/ml chicken egg white lysozyme in 10 mM NaOAc, pH 5.5 for 5 minutes. FC1 and FC2 were deactivated by injection of 1 M ethanolamine-HCl for 7 minutes. FC1 was used as a reference surface. FC3 was activated by injecting a 1:1 mixture of EDC and HNS for 7 minutes. Soybean type 1-S trypsin inhibitor surface was prepared on FC3 by injecting 300 μg/ml soybean type 1-S trypsin inhibitor in 10 mM NaOAc, pH 4.0 buffer for 20 min followed by 7 min. injection of 1 M ethanolamine-HCl to deactivate the flow cell. Polyclonal anti-lysozyme antibody, anti-trypsin inhibitor antibody, and BI negative control were used as controls in the assay. Controls and antibodies were prepared at 1 μM in 1X HBS-EP buffer. Samples were injected over FC1, FC2 and FC3 surfaces for 10 min. association and 15 min. dissociation. The surfaces were regenerated between each binding cycle by injection for 1 min. 0.85% phosphoric acid and 30 s of 50 mM NaOH at a flow rate of 50 μl/min, followed by a stabilization period of 2 min. with 1X HBS-EP buffer flowing through all surfaces. Polyclonal anti-lysozyme antibody and anti-trypsin inhibitor antibody were injected over FC1, FC2 and FC3 at the beginning and end of the experiment. Data were collected using Biacore T200 Control Software version 2.0.1 and analyzed using Biacore T200 Evaluation Software version 3.0.

Antibody binding sensorgrams were compared with negative control BI sensorgrams to determine the level of nonspecific binding to the surfaces of chicken egg white lysozyme and soybean trypsin inhibitor type 1-S.

The data obtained showed that the 277 antibody does not bind significantly to unrelated charged proteins; specifically white lysozyme from chicken eggs and trypsin inhibitor type 1-S from soybeans. The 277 antibody is classified as favorable (green) compared to the binding response and shape of the curves compared to controls. Therefore, it can be assumed that the 277 antibody will have negligible risks regarding pharmacokinetics, biodistribution and formulation based on off-target charged species.

Table 7

Sample Surface -4400 EO Lysozyme Surface -2300 EO Trypsin inhibitor Anti-TRKB 277 antibodies No binding No binding

8. Activity (SY5Y) and efficiency.

Differentiation of SH-SY5Y cells to a neuronal phenotype.

SH-SY5Y cells (ATCC® CRL-2266™) were cultured in DMEM:F12 (Lonza #BE12-719F) with 15% fetal bovine serum in a 175 cm2 tissue culture flask (Corning #353112). For cell seeding, the flask was washed once with D-PBS (Lonza No. 17-512Q) and cells were detached using 37°C prewarmed Accutase solution (A6964 SIGMA). After incubation for 3 minutes at room temperature, the cells are attached using a 10 ml pipette and transferred to a 15 ml Falcon tube. Cells were counted and 6000 cells per 100 µl

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DMEM:F12 with 15% FBS per well was seeded into collagen-I-coated 96-well plates (BD Biosciences no. 354407). Cells were maintained in a humidified incubator at 37°C, 5% CO ₂ . After 8 hours, 100 μl of 6 μM retinoic acid solution (Sigma no. R2625) was added to each well. The final concentration of retinoic acid was 3 μM. After 48 hours and 96 hours, stimulation with additional retinoic acid is necessary. To do this, 100 μl of medium per well was removed and 100 μl of fresh medium with 6 μM retinoic acid was added. After differentiation for 7 days, SHSY5Y cells had a dopaminergic-like phenotype and were ready for use.

Stimulation and lysis of differentiated SH-SY5Y cells.

Well media was replaced with 80 μl of prewarmed DMEM-F12 with 0.2% BSA. The plate was incubated at room temperature for 15 minutes. Peptides and antibodies were diluted in DMEM-F12 with 0.2% BSA. All dilutions were prepared with low binding tips, tubes and plates. Cells were stimulated by adding 20 μl of the appropriate peptide/antibody for 45 min at room temperature. During incubation, a buffer for cell lysis was prepared in a 15 ml Falcon tube on ice: 1 ml 10x Triton X-100 lysis buffer 8.7 ml _H2O 1 full mini tablet, protease inhibitor 100 µl cocktail of phosphatase inhibitors 2 100 µl cocktail phosphatase inhibitors 3 100 µl 1 mM PMSF. After cell stimulation, the medium was removed and 30 μl of ice-cold lysis buffer was added to each well. The plate was covered with a top seal and incubated on ice for 20 min. After this, the contents of the tablet were mixed on an Eppendorf plate shaker at 500 rpm for 5 minutes.

For this step, all materials and solutions were kept on ice. A 96-well plate with lysed cells was centrifuged at 235 g for 10 s and stored on ice. AlphaLISA immunoassay buffer (PerkinElmer #AL000C) was prepared by diluting 260 μl of a 10x stock solution in 2340 μl H ₂ O. pNTRK2 acceptor beads (PerkinElmer #CUSM81822; beads coated with Perkin Elmer antibody from CellSignaling CST # 4621 clone C50F3) were diluted in buffer for AlphaLISA immunoassay to a final concentration of 10 μg/ml. NTRK2 biotin antibody (PerkinElmer #CUSM81820; tagged with PerkinElmer with CellSignaling antibody CST=#4609 clone C17F1) was diluted in AlphaLISA immunoassay buffer to a final concentration of 1 nM and streptavidin donor beads (PerkinElmer #6760002S) were diluted in AlphaLISA immunoassay buffer to a final concentration of 20 μg/ml. 5 μl of pNTRK2 acceptor beads and 2.5 μl of cell lysate per well were transferred to a white flat-bottomed 384-well small volume plate (Greiner #784075) and centrifuged at 235 g for 10 s and stored for 45 min at room temperature in darkness. After incubation, 2.5 μl of NTRK2 biotin antibody per well was added and the plate was centrifuged at 235 g for 10 s and stored for 45 minutes at room temperature in the dark. Finally, 2.5 μl of streptavidin donor beads per well was added and the plate was centrifuged at 235 g for 10 s and stored for 30 min at room temperature in the dark. Immediately thereafter, the plate was analyzed with an EnVision plate reader using the AlphaScreen protocol (570 nM filter #244 and mirror #444).

Determination of ERK1/2 phosphorylation.

Measurement of ERK 1/2 (Thr202/Tyr204) phosphorylation was carried out using 8 cells of cell lysate according to the manufacturer's instructions (AlphaScreen SureFire p-ERK 1/2 (Thr202/Tyr204) kit (PerkinElmer #TGRES10k))

Determination of AKT 1/2/3 phosphorylation.

Measurement of Akt 1/2/3 (Thr308) phosphorylation was carried out using 8 cells of cell lysate according to the manufacturer's instructions ((AlphaScreen SureFire p-Akt 1/2/3 (Thr308) kit (PerkinElmer no. TGRA3S10K)).

Several anti-TrkB antibodies of the invention were assayed to determine their potential activity to activate TrkB as well as trigger downstream signals. The study also included the natural ligand BDNF and antibodies C2 and C20. The anti-TrkB antibodies of the present invention are on average ten times more potent in activating TrkB compared to the natural ligand BDNF. When compared with antibodies C2 and C20, the anti-TrkB antibodies of the invention are on average three times to fifteen times more effective. The downstream signaling of pERK and AKT1/2/3 is entirely consistent with these measurements regarding TrkB activation and exhibits similar improved activity of the anti-TrkB antibodies of the invention.

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Table 8

Compound EC50 pM [mean ± CO1 TrkB-P BDNF 430 [±254] 277-gr (IgGl) 39 [±18] 277-gr (IgGl-KO) 46 [±27] 277-64 (IgGl) 53 [±41] 277-64 (IgGl-KO) 44 [±23] C2 134 [±72] C20 654 [±180] p-ERK 1/2 (Thr202/Tyr204) BDNF 301 [±279] 277-gr (IgGl) 30 [±12] 277-gr (IgGl-KO) 47 [±42] 277-64 (IgGl) 38 [±11] 277-64 (IgGl-KO) 56 [±72] C2 103 [±56] C20 238 [±36] ACT 1/2/3-P (T308) BDNF 255 [±179] 277-gr (IgGl) 23 [±0] 277-gr (IgGl-KO) 39 [±22] 277-64 (IgGl) 25 [±3] 277-64 (IgGl-KO) 22 [±1] C2 132 [±76] C20 430 [±111]

9. Gene expression.

Next generation sequencing analysis (data not shown) was performed on differentiated SH-SY5Y cells after stimulation with several TrkB agonists. To confirm the results, most of the regulated genes in relation to synaptic plasticity were selected and then the changes were analyzed by qPCR. ARC, VGF, EGR1 are known markers for synaptic plasticity. Synaptic plasticity is the process by which specific patterns of synaptic activity lead to changes in synaptic strength. In fig. 2 stimulation of differentiated SH-SY5Y cells with BDNF or agonistic antibodies increases the amount of ARC, VGF, EGR1 mRNA expression, which may be a marker for increased synaptic plasticity. NTRK2 mRNA expression was not affected by stimulation with BDNF or agonistic antibodies. The changes in expression patterns induced by BDNF and D003 are generally very similar. These results confirm those obtained from next generation sequencing analysis.

Specifically, SH-SY5Y cells were differentiated for 7 days in a 6-well culture plate (SIGMA no. Corning CLS3516). After differentiation, cells were stimulated in serum-free DMEM:F12 with 0.2% BSA with control IgG [10 nM], BDNF [10 nM] and D003 [10 nM] for 6 hours in a humidified incubator at 37°C, 5% CO2 . Cells were then washed once with prewarmed lx D-PBS and mRNA extraction was performed according to the manufacturer's instructions (Qiagen #74104 RNeasy Mini Kit). Transcription of mRNA to cDNA was performed according to the manufacturer's instructions (Qiagen no. 205310 QuantiTect Rev. Transcription Kit). The cDNA was used for TaqMan real-time PCR assays based on 5' nuclease probes (ThermoFischer Scientific) on a 7900HT Fast real-time PCR system (Applied Biosystems). Delta CT values were calculated based on the CT value for ASTV (CT = 18.18).

TaqMan Gene Expression Assays from ThermoFischer Scientific:

ACTB-actin beta: Hs01060665_g1; Cat. no. 4331182,

NTRK2-neurotrophic receptor tyrosine kinase 2: Hs0017881 1_ml; Cat. no. 4331182,

ARC-associated cytoskeleton activity-regulated protein: Hs01045540_g1; Cat. No. 4331182,

VGF-inducible nerve growth factor: Hs00705044_s1, Cat. No. 4331182 EGR1 -early growth response 1: Hs00152928_m1; Cat. no. 4331182.

10. Pharmacokinetic study of intravitreal administration (IV) in rabbits.

Female New Zealand White rabbits (1.7–2 kg body weight) were acclimatized for >15 days prior to study entry. Antibody 277 was prepared sterile at a concentration of 20 mg/ml and 1 mg (in 50 μl of buffer: 60 mM sodium acetate, 150 mM NaCl, pH 5.0) was injected bilaterally into the vitreous body of anesthetized animals. At different time points, 2 animals were euthanized and ocular tissues consisting of aqueous humor, vitreous, and retina were collected and frozen until analysis. Blood samples were collected at various time points (Fig. 3), centrifuged, and plasma frozen until analysis.

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Antibody quantification was performed using ELISA. For each tissue type, an antibody calibration curve was prepared (in triplicate ranging from 0.5-100 ng/mL) in identical sample buffer and included in each plate. Absorbance was measured at 405 nm using a SpectraMax 340PC-384 photometer and analysis of the implementation data using SoftMaxPro6.5.

The concentration of 277 antibody is shown in FIG. 3, showing the concentration in various compartments of the eye, as indicated, and plasma after intravenous injection of 1 mg of 277-antibody into the eyes of rabbits as a function of time. Estimated ocular and plasma half-lives are presented in the table below.

Table 9

ti/2 (DAYS) Vitreous body Intraocular fluid Retina Plasma 4.9 4.8 5.2 7.0

The estimated half-lives were 4.9, 4.8, 5.2 and 7.0 days in the vitreous, aqueous humor, retina and plasma, respectively. These half-lives are similar to those described in the literature for the clinically used recombinant humanized monoclonal IgG1 antibody Avastin (anti-VEGF, bevacizumab, Bakri et al., Opthalmology, 2007), which have also been validated experimentally in-house. These results were consistent with those expected, since intravitreal clearance of full-length IgG is mainly dependent on their molecular size, which is similar for our antibody 277 and Avastin. Thus, the human PK, including ocular half-life, of antibody 277 and Avastin is believed to be similar. The reported half-life in the human eye for Avastin is 9.73 ± 1.48 days (Hutton-Smith, 2016).

11. Frequency of intravenous injection of IgG1 antibodies.

Based on in vivo data obtained in the pharmacokinetic study (Example 10), as well as activity data obtained in SH-SY5Y cells, human ocular PK (vitreal concentration over time) of IgG1 antibodies was studied and calculated based on well-established single-compartment kinetics models relative to ocular clearance of biomolecules. For the calculations, the following parameters were used: half-life in the human eye t _1/2 =10 days, as described for Avastin, and also confirmed by the given in vivo data in example 10; molecular weight of 149 kDa as for full-length IgG1 antibodies, and the amount of i.t. injected antibody is 1 mg (see Fig. 4). Since ocular PK is primarily dependent on molecular weight, an identical ocular PK plot was obtained for all five antibodies.

To assess the frequency of iVT injection, it should be taken into account that in vivo efficacy can be maintained at vitreal concentrations > cellular EC ₅₀ as determined by TrkB phosphorylation assays in SH-SY5Y cells (Example 8). The time after IVT injection to achieve a vitreal concentration equivalent to EC ₅₀ is indicated by arrows in Fig. 4. Based on this analysis, the time to achieve this vitreal concentration after injection of 1 mg of antibody is summarized in the following table for each antibody.

Table 10

Compound Time AFTER IVT BEFORE REACHING _C Vitreous. = EU ₅₀ [days] 277-gr (IgGl) 154 277-gr (IgGl-KO) 151 277-64 (IgGl) 150 277-64 (IgGl-KO) 152 C2 136

For example, after injection of 1 mg C2 into the human eye, the vitreal concentration corresponding to the EC ₅₀ for C2 (Table 10) is achieved after 136 days (i.e., after 136 days, the next IVT injection is required). In comparison, in the case of 277-64 (IgG1), this time will be extended to 150 days after the injection. In this case, the next IVT injection will be needed 14 days later compared to C2. Taking into account that the minimum effective dose values 10 times higher than the EC ₅₀ will reduce the time between injections, however, the temporal difference in injection frequency between C2 and the other antibodies listed in Table. 10 above will remain identical.

The injectable volume that can be injected into the eye is highly limited. At the same time, repeated injections into the eyes are necessary to treat eye conditions. Improving the half-life of an antibody is one way to increase the time between injection intervals, but is stated to be primarily dependent on the molecular weight of the molecule. As demonstrated in vivo in Example 10 and in SH-SY5Y cells in Example 8, by providing an improved and more effective antibody, injection intervals can be lengthened, allowing patients to receive ocular injections less frequently.

12. Neuroprotective and neuroregenerative effects of TrkB agonist antibody in STZ-induced diabetic rats.

None of the 277 antibodies are cross-reactive in rodents, therefore, proof of concept and in vivo efficacy studies were performed using the TrkB activating antibody C2 as a surrogate antibody to test the hypothesis that TrkB activating approach I ) provides a neuroprotective effect ('neuroprotective approach'), and ii) provides reactivation of dysfunctional neurons/assemblies ('regenerative approach'). Both approaches are considered in several studies. As an animal disease model, a rat model of streptozotocin (STZ)-induced diabetes was used, which is characterized by loss of pancreatic beta cells, low insulin levels, stable hyperglycemia and subsequent neuronal dysfunction in the retina.

Animal research.

Male gray rats (BN rats) were obtained from Charles River (Germany). Animals were housed in groups of 2 in an IVC under temperature and humidity controlled conditions on a 12-hour day/night cycle (lights off from 6:00 pm to 6:00 am). They were fed ad libitum with pelleted diet no. 3438 from Provimi Kliba (Switzerland) and had free access to drinking water. Animals were acclimatized to their environment for one week before the start of the study. The rats were 6-8 weeks old at the beginning of the intravital phase of the study. Hyperglycemia was induced by IP injection of STZ (65 mg/kg body weight; Sigma no. S0130-1G). Animals that do not respond, or respond poorly, were not included in the study, that is, animals with blood glucose concentrations <20 mM on day 5 after STZ administration. Body weight and blood glucose levels were monitored regularly. Animals were dosed by a single i.v. injection of C2 (50 μg in 4.6 μl) or control anti-2,4,6-trinitrophenyl antibody (anti-TNP) (50 μg in 4.5 μl) on day 36 after STZ administration. Retinal function was assessed using electroretinography (ERG) recordings, and animals were sacrificed after the last recording using a lethal dose of intravenous phenobarbital.

Dosing and intravitreal injections.

The general scheme of the study is presented in Fig. 5. For IVT injection, rats were anesthetized using 2.5-3% isoflurane (Forene; Abbvie). A drop of 4 mg/mL oxybuprocaine hydrochloride (Novesine; Omnivision) was administered as a topical local anesthetic. 4.6 μl of C2 stock solution (10.9 mg/ml; buffer: 20 mM sodium citrate, 115 mM NaCl, pH 6.0) was injected i.t. into each eye of hyperglycemic rats (group 3, n=24 eyes, Fig. 5 ), resulting in an injected dose of ~50 μg/eye. As a control, 4.5 μl of anti-anti-2,4,6-trinitrophenyl stock solution (11.2 mg/ml; buffer 20 mM sodium citrate, 115 mM NaCl, pH 6.0) was injected i.t. into the eyes of nondiabetic and diabetic control rats (group 1, n=24 eyes, and group 2, n=22 eyes, Fig. 5), resulting in an injected dose of ~50 μg/eye. IVT injections were carried out under a dissecting loupe. The Antibody solution was delivered using a 34-gauge needle (attached to a 10-μL glass Hamilton syringe) into the vitreous just posterior to the limbus, and the overall quality of the injection was monitored by fundoscopy (using a Micron IV Retinal Imaging Microscope, Phoenix Research Labs).

Electroretinography.

Electroretinography (ERG) is a non-invasive electrophysiological technique for assessing light-induced electrical activity of various retinal neurons, and provides the ability to quantify various aspects of retinal function, such as low-light vision or color vision. ERG was measured as the change in potential between the corneal and reference electrodes using an Espion E3 ERG recording system (Diagnosys LLC). Before ERG recordings, animals were dark-adapted for at least 2 hours and anesthetized by intravenous injection of ketamine (Ketanest, ca. 100 mg/kg) and xylazine (Rompun, ca. 5 mg/kg). Animals were placed on a heated table to maintain a constant body temperature at 37°C. The pupils were dilated with 1% cyclopentolate-HCl and 2.5% phenylephrine. A drop of Methocel 2% solution (OmniVision) was placed on the cornea to prevent drying of the eye and the development of cataracts during recordings. Recordings were made simultaneously for both eyes using gold loop electrodes. The reference electrode was a toothless alligator clip, moistened with Methocel, and attached to the animal's cheek. For electrical grounding, a clamp was attached to the animal's tail. ERG signals were sampled at 1 kHz and recorded with cutoff modes of 0.15 Hz low frequency and 500 Hz high frequency. Light stimuli consisted of short, full-field flashes delivered by an array of light-emitting diodes (duration, <4 ms). All flashes were produced using a Ganzfeld stimulator (ColorDome; Diagnosys) either in the dark or against a steady green or red background light.

ERG protocols.

ERG responses were first recorded from dark-adapted animals (to isolate rod-mediated responses), then recorded from animals adapted to red background light (50 cd/ ^m2 , to isolate UV-cone-mediated ERG responses) and then adapted to green background light. (25.5 cd/ ^m2 , to highlight M-cone-mediated responses). In the case of dark-adapted ERGs, responses were evoked by series of flashes ranging from 1-10'5 to 0.1 cd-s/m ² . For flashes with luminescence of 1-10'5 and 3-10'5 cd-s/m ² , the responses of 20 experiments were averaged.

- 44 043771

For flashes ranging from 1 -10 ^-4 down to 0.05 cd-s/m ² , the responses of 10 trials were averaged, and for a final flash of 0.1 cd-s/m ² , 8 trials were averaged. The intervals between individual flashes were chosen to ensure complete retinal recovery from each flash (no evidence of flash-induced reduction in response amplitudes or reduction in latency). Based on this criterion, the inter-flash intervals were 2 s for 1 -10 ^-5 and 3-10 ^-5 cd-s/m ² flashes, 5 s for flashes in the range from 1 -10 ^-4 down to 0.05 cd- s/m ² , and 10 s for a flash of 0.1 cd-s/m ² .

For recordings of UV cone-mediated responses, animals were light-adapted to red background light for 2 min. Light responses were induced using UV flashes of 0.02, 0.04, 0.08, 0.17, 0.35, 0.83, 1.66, 2.90, and 4.15 μW/m ² . For recordings of M-cone-mediated responses, animals were light-adapted to a green background light for 2 min. Light responses were evoked using green flashes of 0.25, 0.1, 1.0, 5.0, 50, and 150 cd-s/ ^m2 . For UV and green flashes, responses from 10 trials were averaged with inter-flash intervals of 3 s.

Data analysis.

To determine ERG b-wave amplitudes (a positive depolarization response originating primarily from bipolar cells), ERG data were recorded and analyzed using MATLAB software (version R2014a; MathWorks). Oscillatory potentials (small high-frequency waves superimposed on the b-wave) were removed from the signals by 55 Hz fast Fourier transform low-pass filtering because oscillatory potentials can influence the amplitude and position of the b-wave peak, particularly under light-adapted conditions. The b-wave amplitude was calculated from the trough of the a-wave response (the negative deflection resulting primarily from photoreceptor activity) to the peak of the b-wave peak. b-wave latency was measured as the time after flash stimulation required to reach the peak of the b-wave.

b-wave amplitudes as a function of stimulus intensity were fitted using the following equation using a least-squares fitting procedure (GraphPad Prism, version 6.01): р тП ^R =7^ _} (Equation!) where R is the response amplitude, Rmax is amplitude of saturating oscillations, I flare intensity, I _h half-saturating flare intensity and n Hill coefficient.

Photosensitivity, S, was defined as the ratio between the amplitude of the saturating oscillations, _Rmax , and the half-saturating flash intensity, Ih.

S and Rma _x values were normalized to the mean values of non-diabetic controls obtained for week 5, before IVT, and for week 7, after IVT treatment, respectively.

To analyze the latency of rod-mediated b-waves, the average latency of non-diabetic control animals for b-waves evoked by bursts of 1-10 ^-5 , 3-10 ^-5 , 1-10 ^-4 and 2-10 ^-4 cd-s/m was determined. ² (the corresponding latency is very similar). For each study group, the relative change for the mean of the controls was calculated for the flash intensities indicated above and averaged to obtain the mean relative change in b-wave latency.

To analyze UV cone-mediated b-wave latency, relative latency changes to the control mean for b-wave responses evoked by flash intensities of 0.042, 0.083, 0.166, and 0.348 μW/cm ² were calculated and averaged.

To analyze the latency of the M-type cone-mediated b-wave, relative changes in latency to the control mean were calculated for b-wave responses evoked by flash intensities of 1, 5, 50, and 150 cd-s/m ² and averaged.

The rod-mediated a-wave response was determined at the peak of the a-wave (negative signal deflection immediately after the light stimulus) evoked by a 0.1 cd-s/m ² flash.

Results.

At week 5 after diabetes induction, baseline ERG recordings were performed to quantify the degree of neural dysfunction in the retina before IVT treatment (see also FIG. 5). Based on these ERG results, it was determined that the two diabetic (hyperglycemic) groups exhibited a similar distribution of retinal dysfunction (which received subsequent IVT treatment with either control IgG1 to 2,4,6-trinitrophenyl - group 2 - or TrkB agonist IgG1 C2 - group 3). In general, all IVT treatments were well tolerated and no side effects were observed. Blood glucose was stable at ~5 mM in nondiabetic control rats (group 1, n=12) during the study period. In STZ-induced diabetic animals (groups 2 and 3, each n=12), blood glucose concentrations reached levels of >20 mM ∼4 days after STZ treatment, and did not decrease below 20 mM during the study period. IVT treatment with C2 or control IgG1 to 2,4,6-trinitrophenyl did not have any effect on blood glucose levels.

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Rod-mediated b-wave latency.

b-wave latency is a measure of the rate of light-induced electrical response in the retina (mainly at the level of bipolar cells and photoreceptor-to-bipolar cell synaptic transmission). Latency is defined as the time required to reach response amplitude after stimulation with a flash of light. The effects of hyperglycemia and IVT treatment are summarized in FIG. 6. Changes in latency relative to the mean latency measured from control rats were calculated for data obtained before IVT treatment (ERG baseline assessment) and after IVT treatment. There is a strong increase in latency of ~14 ms in the two hyperglycemic groups before IVT treatment, indicating a hyperglycemia-induced slowing of b-wave response kinetics, that is, a reduced rate of rod-mediated processing in the retina. IVT injection of control IgG1 to 2,4,6-trinitrophenyl did not have any significant effects on latency in the nondiabetic control group and the hyperglycemic group (group 1, black bars, and group 2, dotted bars in Fig. 6, after IVT, respectively). However, iT treatment with the TrkB activator C2 apparently resulted in reduced latency (group 3, gray bar in Fig. 6, after iT). On average, IVT treatment with C2 causes a significant acceleration of b-wave responses by ~9 ms (ie, latency is reduced from 14 ms down to 5 ms).

Rod-mediated b-wave photosensitivity.

ERG responses of rod-mediated b-wave light sensitivity are an indicator of the number of neurons participating in rod-mediated signaling pathways in the retina and their sensitivity to light stimuli to produce depolarizing light responses. Light sensitivity was normalized to the average light sensitivity of control rats (group 1, before and after IHT treatment) and graphically presented in Fig. 7. Normalized photosensitivity decreased by ~40-50% in hyperglycemic animals (before IHT treatment, groups 2 and 3). IVT injection of control IgG1 did not have any significant effects on the photosensitivity of control rats (group 1) or hyperglycemic rats (group 2). It is extremely important that IVT treatment with the TrkB activator C2 significantly increased photosensitivity by ~30% (group 3, gray, after IVT compared with before IVT).

Rod-mediated a-wave responses.

The rod-mediated a-wave response is an indicator for the size of the rod photoreceptor light responses evoked by a flash of light. Rod-mediated a-wave responses were normalized to the mean values obtained from control rats (group 1, before and after IVT treatment) and are graphically presented in Fig. 8. Normalized amplitudes of saturating oscillations decreased by ~17% in hyperglycemic animals (groups 2 and 3, before IVT treatment). IVT injection of control IgG1 did not have any significant effects on normalized a-wave responses in control rats (group 1, black) or hyperglycemic rats (group 2, indicated by dots), where the latter group showed a further decrease in a-wave response during conducting research. However, iT treatment with the TrkB activator C2 (group 3, gray) can completely prevent the further decrease in a-wave responses observed for hyperglycemic group 2.

UV and M-cone-mediated b-wave latency.

Changes in latency relative to the mean latency measured for control rats were calculated for data obtained before IVT treatment (ERG baseline assessment) and after IVT treatment. This analysis was performed for UV cone b-wave latency (FIG. 9) and M-cone b-wave latency (FIG. 10). In the case of UV cone-mediated responses, there is a strong increase in latency of ~8 ms in the two hyperglycemic groups before IVT treatment, while the increase in M-cone-mediated latency is ~5 ms for the hyperglycemic rats. These statistically significant effects indicate a hyperglycemia-induced slowing of the kinetics of b-wave responses and a decrease in the rate of cone-mediated processing in the retina. IVT injection of control IgG1 did not have any significant effects on latency in the nondiabetic control group (group 1, black) or hyperglycemic group (group 2, dotted), for both UV and M-cone b-wave mediated latency. IVT treatment with C2 (group 3, gray) reduced latency by ~5 ms in the case of UV cones (corresponding to ~50% normalization of the kinetics of b-wave responses), and almost complete restoration of the kinetics of normal b-wave responses in the case of M-type cone-mediated responses b-waves

Cone-mediated b-wave UV photosensitivity.

Cone-mediated b-wave UV photosensitivity ERG responses were assessed as described for rod-mediated ERG. Light sensitivity was normalized to the average light sensitivity of control rats (group 1, before and after IHT treatment) and graphically presented in Fig. 11. Normalized photosensitivity decreased by ~50% in hyperglycemic animals at baseline (before IVT). IVT injection of control IgG1 did not have any significant effects on photosensitivity, while IVT treatment with C2 significantly increased light sensitivity.

- 46 043771 sensitivity to the level observed in healthy control rats (group 3, gray).

M-cone-mediated saturating b-wave response amplitude.

The effect of hyperglycemia on M-cone-mediated b-wave photosensitivity is less pronounced than that on UV-cone-mediated b-wave (at the time points studied). However, a significant deterioration in the amplitude of saturating oscillations of M-cone b-waves can be observed at week 5 after the onset of hyperglycemia (~15% reduction compared to non-diabetic control rats, Fig. 12). IVT treatment with C2 could restore the amplitude of saturating oscillations to the level of controls (group 3, gray), while treatment with control IgG1 did not have any significant effects (group 2, dots).

The main research findings are summarized below.

Activation of TrkB causes neuroprotection and preserves retinal function. Neuroprotective effects were demonstrated in a rat model of STZ-induced diabetes, displaying hyperglycemia-induced neural dysfunction. Activation of TrkB has been shown to prevent photoreceptor dysfunction.

Activation of TrkB induces restoration/improvement of neuronal function in the retina in a rat model of STZ-induced diabetes after prolonged hyperglycemia, such as improved light response latency (i.e., reduction in ERG b-wave latency), or improved rod- and cone-mediated light sensitivity.

In summary, pharmacological data provide support for the therapeutic concept that TrkB activation can improve/restore the function of various types of neuronal cells in the eye in various disease states.

Activation of TrkB also protects photoreceptors and the outer retina, as shown by a-wave ERG analysis, which is an indicator of photoreceptor light responses. Similar to the results obtained for the α-wave ERG, contrast sensitivity of the eyes of diabetic rats decreases during ongoing hyperglycemia, whereas iT treatment with the TrkB activator C2 completely preserves contrast sensitivity. In addition, the improvement in ERG light sensitivity to rod and cone b-wave mediated light responses as described above (reflecting light sensitivity of photoreceptor synaptic transmission onto bipolar cells) further strengthens the concept that TrkB activation may improve/protect outer retinal function, including photoreceptors.

13. BDNF function in combination treatments.

Cultivation of CHO cells expressing the human TrkB receptor.

CHO cells expressing the human TrkB receptor (hTrkB) (ThermoFisher Scientific, no. K1491) were cultured in DMEM (Lonza, no. BE12-604F) supplemented with 10% fetal bovine serum, glutamax, nonessential amino acids, 20 mM HEPES, 5 μg/ml blasticidin and 200 μg/ml zeocin. Twenty-four hours before stimulation, 5000 cells (30 μl) were seeded into each cavity of a clear 384-well tissue culture plate (BD Falcon, #353963) and incubated in a humidified incubator at 37°C and 5% CO ₂ .

Analysis of ERK phosphorylation in CHO cells by the hTrkB receptor.

Twenty-four hours after plating, the CHO/hTrkB cell supernatant was replaced with 40 μl of room temperature DMEM with 0.1% BSA but no other additional components. After 15 min, 10 μl of DMEM/0.1% BSA was added with increasing concentrations of BDNF (1E-13 to 3E-8 mol/L), 277-antibody (1E-13 to 2.025E-8 mol/L), or C2 -antibodies (1E-13 to 2.025E-8 mol/l) alone or combination with 1 nM BDNF with increasing concentrations of 277- or C2-antibodies (both 1E-13 to 2.025E-8 mol/l) in triplicate for stimulation ERK phosphorylation. After incubation for 45 min at room temperature, supernatants were removed and cells were lysed for 20 min on liquid ice in 20 μl of lysis buffer per well (1x Tryton lysis buffer (CellSignaling no. 9803-S) supplemented with complete mini protease inhibitor tablets (Roche No. 04693124001) and cocktails of phosphatase inhibitors 2 (Sigma No. P5726) and 3 (Sigma No. P0044), and 1 mM PMSF (Sigma No. 93482)). Five microliters of the resulting lysate was used to quantify ERK1/2 phosphorylation at T202/Y204 using a commercially available assay (Perkin Elmer #TGRES500), according to the manufacturer's instructions. Acceptor bead light emission representing ERK phosphorylation was recorded at 570 nm on a Perking Elmer EnVision microplate reader and prepared for presentation using GraphPad Prism (version 7), including raw data (mean ± SPE) and nonlinear regression (log(agonist)rel response (three parameters)).

As shown in FIG. 13,277 antibody does not reduce BDNF-induced ERK phosphorylation. Increasing concentrations of C2 antibody result in a decrease in BDNF-induced ERK phosphorylation down to the level of C2-induced ERK phosphorylation. This may have a significant impact on dosing of anti-TrkB antibody to the eye, where the antibody is administered at high saturating concentrations to prolong the time required before the next administration.

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Claims (23)

The 277 antibody can be administered at high doses without interfering with existing BDNF-induced ERK phosphorylation, while the C2 antibody reduces existing BDNF-induced ERK phosphorylation down to antibody levels. 14. Binding to human VEGF in vitro ELISA assay. For in vitro binding assays, MaxiSorp 96-well plates (Nunc #437111) were coated overnight at 4°C with 500 ng/ml human VEGF (R&D Systems #293-VE-050) or rat VEGF (R&D Systems #564 -RV-050) in carbonate/bicarbonate buffer (3.03 g/L Na ₂ CO ₃ and 6.00 g/L NaHCO ₃ , pH 9.6). Plates were washed three times with PBS-T (phosphate buffered saline (Invitrogen #70011-036) with 0.05% Tween20 (Sigma #P7949)) and then incubated with 1% BSA (Sigma #A3059) in PBS-T for three hours at room temperature to block nonspecific binding. Plates were washed three times with PBS-T and then incubated with a 1:4 serial dilution of 1 μM Avastin (Roche clinic package), C2 antibody, 277 antibody, or human IgG1 isotype control antibody in PBS-T overnight at 4°C WITH. The plates were washed once with PBS-T and then incubated with a 1:2000 dilution of Alexa fluor 647 goat anti-human IgG (Invitrogen #A21445) for two hours at room temperature. After three additional wash cycles with PBS-T, light emission at 647 nm (representing antibody binding to VEGF) was recorded on a Perking Elmer EnVision microplate reader. Data were prepared for presentation using GraphPad Prism (version 7), including raw data (mean ± SPE) and nonlinear regression (log(agonist) vs. response (three parameters)). As shown in FIG. 14 there is no statistically significant difference for human VEGF binding between the 277 anti-TrkB antibody and the IgG1 isotype control antibody at antibody concentrations as low as 0.25 μM (one sample t test, p>0.05). In contrast, a statistically significant difference for human VEGF binding between C2 and isotype control antibody was already observed at an antibody concentration of 0.24 nM (one sample t test, **p < 0.01). Such nonspecific binding to VEGF may cause, for example, undesirable side effects, make it difficult to control dosage for TrkB activation, or increase the amount of antibody required to achieve the desired effect. CLAIM 1. An antibody that specifically binds to tyrosine kinase receptor B (TrkB), or an antigen-binding fragment thereof, containing (containing): a) a light chain variable region containing the amino acid sequence of SEQ ID NO: 48 (L-CDR1); amino acid sequence SEQ ID NO: 49 (L-CDR2); and amino acid sequence SEQ ID NO: 50 (L-CDR3); And b) a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 51 (H-CDR1); amino acid sequence SEQ ID NO: 52 (H-CDR2); and the amino acid sequence of SEQ ID NO: 53 (H-CDR3), or c) a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 55 (H-CDR1); amino acid sequence SEQ ID NO: 56 (H-CDR2); and the amino acid sequence of SEQ ID NO: 57 (H-CDR3), or d) a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 58 (H-CDR1); amino acid sequence SEQ ID NO: 59 (H-CDR2); and the amino acid sequence of SEQ ID NO: 60 (H-CDR3). 2. An antibody to TrkB or an antigen-binding fragment thereof according to claim 1, where the antibody or an antigen-binding fragment thereof contains: a) a variable light chain containing an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO : 2, and a variable heavy chain containing an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 12, b) a variable light chain containing an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO : 3, and a variable heavy chain comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 13, c) a variable light chain containing an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO : 4, and variable severe - 48 043771 chain containing an amino acid sequence of at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 14, d) a variable light chain containing an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO : 5, and a variable heavy chain containing an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 15, e) a variable light chain containing an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO : 6, and a variable heavy chain containing an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 16, f) a variable light chain containing an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO : 7, and a variable heavy chain comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 17, g) a variable light chain containing an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO : 8, and a variable heavy chain comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 18, h) a variable light chain containing an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO : 9, and a variable heavy chain comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 19, or i) a variable light chain containing an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO : 10, and a variable heavy chain containing an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 20. 3. Antibody to TrkB according to claim 1, where the antibody contains: a) a light chain containing an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 21, and a heavy chain containing an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO : 22 or 23, b) a light chain containing an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 24, and a heavy chain containing an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO : 25 or 26, c) a light chain containing an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 27, and a heavy chain containing an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO : 28 or 29, d) a light chain containing an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 30, and a heavy chain containing an amino acid sequence of at least 80%, at least 90%, at least - 49 043771 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 31 or 32, e) a light chain containing an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 33, and a heavy chain containing an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 34 or 35, e) a light chain containing an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 36 , and a heavy chain containing an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 37 or 38, g) a light chain containing an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 39, and a heavy chain containing an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO : 40 or 41, h) a light chain containing an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 42, and a heavy chain containing an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO : 43 or 44, i) a light chain containing an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 45, and a heavy chain containing an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO : 46 or 47. 4. An antibody to TrkB or an antigen-binding fragment thereof according to claim 1, where the antibody or an antigen-binding fragment thereof contains: a) a variable light chain and a variable heavy chain containing the amino acid sequences SEQ ID NO: 2 and SEQ ID NO: 12, respectively, b) a variable light chain and a variable heavy chain containing the amino acid sequences SEQ ID NO: 3 and SEQ ID NO: 13, respectively, c) a variable light chain and a variable heavy chain containing the amino acid sequences SEQ ID NO: 4 and SEQ ID NO: 14, respectively, d) a variable light chain and a variable heavy chain containing the amino acid sequences SEQ ID NO: 5 and SEQ ID NO: 15, respectively, e) a variable light chain and a variable heavy chain containing the amino acid sequences of SEQ ID NO: 6 and SEQ ID NO: 16, respectively, f) a variable light chain and a variable heavy chain containing the amino acid sequences of SEQ ID NO: 7 and SEQ ID NO: 17, respectively, g) a variable light chain and a variable heavy chain containing the amino acid sequences SEQ ID NO: 8 and SEQ ID NO: 18, respectively, h) a variable light chain and a variable heavy chain containing the amino acid sequences of SEQ ID NO: 9 and SEQ ID NO: 19, respectively, or i) a variable light chain and a variable heavy chain containing the amino acid sequences of SEQ ID NO: 10 and SEQ ID NO: 20, respectively. 5. Antibody to TrkB according to claim 1, where the antibody contains: a) a light chain containing the amino acid sequence SEQ ID NO: 21, and a heavy chain containing the amino acid sequence SEQ ID NO: 22 or 23, b) a light chain containing the amino acid sequence SEQ ID NO: 24, and a heavy chain containing the amino acid sequence SEQ ID NO: 25 or 26, c) a light chain containing the amino acid sequence SEQ ID NO: 27, and a heavy chain containing the amino acid sequence SEQ ID NO: 28 or 29, d) a light chain containing the amino acid sequence SEQ ID NO: 30, and a heavy chain containing the amino acid sequence SEQ ID NO: 31 or 32, e) a light chain containing the amino acid sequence of SEQ ID NO: 33, and a heavy chain, - 50 043771 containing the amino acid sequence SEQ ID NO: 34 or 35, e) a light chain containing the amino acid sequence of SEQ ID NO: 36, and a heavy chain containing the amino acid sequence of SEQ ID NO: 37 or 38, g) a light chain containing the amino acid sequence SEQ ID NO: 39, and a heavy chain containing the amino acid sequence SEQ ID NO: 40 or 41, h) a light chain containing the amino acid sequence of SEQ ID NO: 42, and a heavy chain containing the amino acid sequence of SEQ ID NO: 43 or 44, or i) a light chain containing the amino acid sequence of SEQ ID NO: 45, and a heavy chain containing the amino acid sequence of SEQ ID NO: 46 or 47, respectively. 6. The anti-TrkB antibody of claim 1, wherein the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 21 and a heavy chain containing the amino acid sequence of SEQ ID NO: 22 or 23. 7. The anti-TrkB antibody of claim 1, wherein the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 24 and a heavy chain containing the amino acid sequence of SEQ ID NO: 25 or 26. 8. The anti-TrkB antibody of claim 1, wherein the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 27 and a heavy chain containing the amino acid sequence of SEQ ID NO: 28 or 29. 9. The anti-TrkB antibody of claim 1, wherein the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 30 and a heavy chain containing the amino acid sequence of SEQ ID NO: 31 or 32. 10. The anti-TrkB antibody of claim 1, wherein the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 33 and a heavy chain containing the amino acid sequence of SEQ ID NO: 34 or 35. 11. The anti-TrkB antibody of claim 1, wherein the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 36 and a heavy chain containing the amino acid sequence of SEQ ID NO: 37 or 38. 12. The anti-TrkB antibody of claim 1, wherein the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 39 and a heavy chain containing the amino acid sequence of SEQ ID NO: 40 or 41. 13. The anti-TrkB antibody of claim 1, wherein the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 42 and a heavy chain containing the amino acid sequence of SEQ ID NO: 43 or 44. 14. The anti-TrkB antibody of claim 1, wherein the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 45 and a heavy chain containing the amino acid sequence of SEQ ID NO: 46 or 47. 15. The use of an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 14 in medicine for the treatment of diseases that are characterized by reduced activity of the TrkB pathway. 16. Use of an antibody or an antigen-binding fragment thereof according to claim 15 for the treatment of eye or retinal diseases. 17. Use of an antibody or an antigen-binding fragment thereof according to claim 16 for the treatment of neuronal/neural diseases of the eye or retina. 18. Use of an antibody or an antigen-binding fragment thereof according to claim 16 for the treatment of geographic atrophy. 19. Use of an antibody or an antigen-binding fragment thereof according to claim 16 for the treatment of age-related macular degeneration, diabetic retinopathy, diabetic macular edema, retinal pigmentary degeneration, hereditary retinal dystrophy, hereditary macular degeneration, myopic degeneration, retinal vein occlusions, retinal artery occlusions, endophthalmitis , uveitis, cystoid macular edema, choroidal neovascular membrane due to any of the retinal diseases, optic neuropathies, glaucoma, retinal detachment, toxic retinopathy, radiation retinopathy and traumatic retinopathy. 20. Use of an antibody or an antigen-binding fragment thereof according to claim 15 for the treatment of neurodegenerative diseases. 21. Use according to claim 20 for the treatment of prodromal and mild to moderate Alzheimer's disease, delay of disease progression in patients with Alzheimer's disease, Huntington's disease, Parkinson's disease, major depressive disorder, schizophrenia, cognitive impairment associated with schizophrenia, prevention of first episode of psychosis in individuals with a weakened psychotic syndrome, preventing recurrence in patients with schizophrenia, treatment-resistant depression. 22. A pharmaceutical composition containing an antibody or an antigen-binding fragment according to any one of claims 1 to 14 and a pharmaceutically acceptable carrier. 23. A method for treating diseases that are characterized by reduced activity of the TrkB pathway, -