WO2023125485A1

WO2023125485A1 - TrkB ANTIBODY AND APPLICATION THEREOF

Info

Publication number: WO2023125485A1
Application number: PCT/CN2022/142194
Authority: WO
Inventors: Xiaoming Guan; Chen LV; Bai Lu; Xiaoyu Zhang
Original assignee: 4B Technologies (Beijing) Co., Limited
Priority date: 2021-12-28
Filing date: 2022-12-27
Publication date: 2023-07-06
Also published as: WO2023125485A9

Abstract

Provided are TrkB antibodies, compositions comprising such antibodies, and methods of using such antibodies for neuroprotection, enhancing cell survival, enhancing neural injury repairing, protecting neural cells from apoptosis and/or necroptosis in neural cells expressing TrkB or promoting sensorimotor function in a TrkB associated condition in a subject in need thereof.

Description

TrkB ANTIBODY AND APPLICATION THEREOF

BACKGROUND OF THE INVENTION

Neurodegenerative diseases are the most well-known neurological diseases yet with little advances in its medical intervention. As a devastating neurodegenerative disease, Alzheimer’s disease (AD) is a huge burden in a growing number of patients and families while the corresponding drug discovery is facing great challenges.

Among the Trk receptors, the role of TrkB has been well characterized in the central nervous system (CNS) . TrkB is widely distributed in the brain, including in the neocortex, hippocampus, striatum, olfactory formation and brainstem. Using brain-derived neurotrophic factor (BDNF) as a cognate ligand, the indispensable roles of TrkB in neuronal survival, differentiation and neuro regeneration have been shown in a number of neurodegenerative models, including stroke, spinal cord injury, axotomy and ALS. Brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5) are preferred ligands for TrkB.

To date, there exists no successful examples of therapeutics that act as agonists of TrkB. There is therefore a need to develop effective TrkB agonists.

SUMMARY OF THE INVENTION

The present disclosure provides TrkB agonist antibodies and use thereof. The TrkB antibodies of the present disclosure or the antigen-binding fragments thereof are useful for preventing and/or treating diseases and disorders associated with inappropriate expression or function of TrkB, the TrkB/BDNF signaling pathway, and/or the TrkB/NT4-5 signaling pathway.

In one aspect, the present application provides an antibody or an antigen binding fragment thereof, which specifically binds to TrkB and exhibits at least one of the following properties: 1) is capable of binding to TrkB with a K _D of less than 2.7E-06 M, as measured by Octet; 2) does not bind and/or does not activate human TrkA; 3) does not bind and/or does not activate human TrkC; 4) does not bind or and/or does not activate human p75; 5) is capable of activating TrkB; 5) is capable of activating ERK signaling pathway; 6) does not induce the reduction of body weight; 7) is capable of reducing the epileptiform discharge events and epileptiform discharge duration; and 8) has the ability of neuroprotection.

In the present application, the antibody or the antigen binding fragment thereof does not substantially compete with BDNF for binding to TrkB protein and is capable of selectively activating downstream signaling pathways. In the present application, the antibody or the antigen binding fragment thereof exhibits diminished effects on body weight loss and epileptogenic activities while maintaining neuroprotective function. In the present application, the unique functional properties (such as reduced effect on body weight and epileptogenesis) of the TrkB antibodies of the present disclosure are likely to result from their unique interactions with TrkB (e.g. lack of competition with BDNF binding) and subsequent impact on downstream signaling pathways.

In the present application, the TrkB antibody of the present disclosure is still capable of maintaining neuroprotective effect at high concentration.

In some embodiments, the antibody is selected from the group consisting of: a monoclonal antibody, a chimeric antibody, a humanized antibody, a fully human antibody and a bispecific antibody.

In some embodiments, the antigen binding fragment is selected from the group consisting of: a Fab fragment, a Fab’ fragment, a F (ab) 2 fragment, a Fv fragment, a ScFv and a VHH.

In some embodiments, the TrkB is human TrkB.

In some embodiments, the antibody or the antigen binding fragment comprises at least one Complementarity Determining Region (CDR) of a heavy chain variable region (VH) , the VH comprises the amino acid sequence as set for in SEQ ID NO: 100 or SEQ ID NO: 102.

In some embodiments, the antibody or the antigen binding fragment comprises at least one Complementarity Determining Region (CDR) of a heavy chain variable region (VH) , the VH comprises the amino acid sequence as set forth in any one of SEQ ID NOs: 15-22, 28-30, and 77-82.

In some embodiments, the antibody or the antigen binding fragment comprises at least one Complementarity Determining Region (CDR) of a light chain variable region (VL) , the VL comprises the amino acid sequence as set forth in SEQ ID NO: 101 or SEQ ID NO: 103.

In some embodiments, the antibody or the antigen binding fragment comprises at least one Complementarity Determining Region (CDR) of a light chain variable region (VL) , the VL comprises the amino acid sequence as set forth in any one of SEQ ID NOs: 23-27, and 31-33.

In some embodiments, the antibody comprises a heavy chain or a fragment thereof.

In some embodiments, the heavy chain or a fragment thereof comprises a heavy chain CDR1, and the heavy chain CDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 91 (X ₁YX ₂MH, wherein X ₁ is G, S, or T, X ₂ is T or W) .

In some embodiments, the heavy chain or a fragment thereof comprises a heavy chain CDR1, and the heavy chain CDR1 comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 3, 9, and 67.

In some embodiments, the heavy chain or a fragment thereof comprises a heavy chain CDR2, and the heavy chain CDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 92.

In some embodiments, the heavy chain or a fragment thereof comprises a heavy chain CDR2, and the heavy chain CDR2 comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 93-94.

In some embodiments, the heavy chain or a fragment thereof comprises a heavy chain CDR2, and the heavy chain CDR2 comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 4, 10, 39, 43, 70, and 84.

In some embodiments, the heavy chain or a fragment thereof comprises a heavy chain CDR3, and the heavy chain CDR3 comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 11, and 95.

In some embodiments, the heavy chain or a fragment thereof comprises a heavy chain CDR3, and the heavy chain CDR3 comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 5, 11, 73, 74, 75, 85, 86, 87, 88, 89, and 90.

In some embodiments, the heavy chain or a fragment thereof comprises a heavy chain variable region, and the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 100 or SEQ ID NO: 102.

In some embodiments, the heavy chain or a fragment thereof comprises a heavy chain variable region, and the heavy chain variable region comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 15-22, 28-30, and 77-82.

In some embodiments, the heavy chain or a fragment thereof comprises a heavy chain constant region, and the heavy chain constant region comprises a human IgG constant region.

In some embodiments, the antibody comprises a light chain or a fragment thereof.

In some embodiments, the light chain or a fragment thereof comprises a light chain CDR1, and the light chain CDR1 comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 96-97.

In some embodiments, the light chain or a fragment thereof comprises a light chain CDR1, and the light chain CDR1 comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 6, 12, 53, and 64.

In some embodiments, the light chain or a fragment thereof comprises a light chain CDR2, and the light chain CDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 98 (X ₁X ₂SX ₃RX ₄S, wherein X ₁ is K or W, X ₂ is A or V, X ₃ is N or T, X ₄ is E, F, or L) .

In some embodiments, the light chain or a fragment thereof comprises a light chain CDR2, and the light chain CDR2 comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 7, 13, and 62.

In some embodiments, the light chain or a fragment thereof comprises a light chain CDR3, and the light chain CDR3 comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 14, and 99.

In some embodiments, the light chain or a fragment thereof comprises a light chain CDR3, and the light chain CDR3 comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 8, 14, 61, and 63.

In some embodiments, the heavy chain or a fragment thereof comprises a light chain variable region, and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 101 or SEQ ID NO: 103.

In some embodiments, the light chain or a fragment thereof comprises a light chain variable region, and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NOs: 23-27, and 31-33.

In some embodiments, the light chain or a fragment thereof comprises a light chain constant region, and the light chain constant region comprises a human Igκ constant region or a human Igλconstant region.

In some embodiments, the antibody or the antigen binding fragment thereof comprises:

1) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 6, 7 and 8 respectively, and heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 3, 4 and 5 respectively;

2) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 6, 7 and 8 respectively, and heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 3, 70 and 5 respectively;

3) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 6, 7 and 8 respectively, and heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NO: 3, 70 and 85 respectively;

4) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 6, 7 and 8 respectively, and heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 3, 70 and 86 respectively;

5) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 6, 7 and 8 respectively, and heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 3, 70 and 87 respectively;

6) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 6, 7 and 8 respectively, and heavy chain CDR 1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 3, 70 and 88 respectively;

7) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 6, 7 and 8 respectively, and heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 3, 70 and 89 respectively;

8) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NO: 6, 7 and 8 respectively, and heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 3, 70 and 90 respectively;

9) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 6, 7 and 8 respectively, and heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 3, 70 and 50 respectively;

10) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 6, 62 and 63 respectively, and heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 67, 84 and 5 respectively;

11) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 6, 7 and 61 respectively, and heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 67, 70 and 5 respectively;

12) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 6, 7 and 61 respectively, and heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 3, 70 and 73 respectively;

13) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NO: s64, 7 and 61 respectively, and heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 3, 84 and 74 respectively;

14) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 64, 7 and 61 respectively, and heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 3, 84 and 75 respectively;

15) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 6, 7 and 61 respectively, and heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 3, 84 and 75 respectively;

16) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 12, 13 and 14 respectively, and heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NO: 9, 10 and 11 respectively;

17) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NO: 12, 13 and 14 respectively, and heavy chain CDR 1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 9, 39 and 11 respectively; or

18) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 53, 13 and 14 respectively, and heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 9, 43 and 11 respectively.

1) a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 23, and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 15;

2) a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 24, and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 16;

3) a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 24, and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 77;

4) a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 24, and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 78;

5) a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 24, and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 79;

6) a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 24, and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 80;

7) a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 24, and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 81;

8) a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 24, and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 82;

9) a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 24, and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 17;

10) a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 26, and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 19;

11) a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 25, and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 18;

12) a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 25, and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 20;

13) a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 27, and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 21;

14) a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 27, and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 22;

15) a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 25, and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 22;

16) a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 31, and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 28;

17) a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 32, and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 29; or

18) a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 33, and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 30.

In another aspect, the present application provides a fusion protein comprising the antibody or the antigen binding fragment thereof of the present application.

In another aspect, the present application provides a protein conjugate comprising the antibody or the antigen binding fragment thereof of the present application

In another aspect, the present application provides an isolated nucleic acid molecule or molecules, encoding for the antibody or the antigen binding fragment thereof of the present application, or the fusion protein of the present application.

In another aspect, the present application provides a vector or vectors, comprises the isolated nucleic acid molecule or molecules of the present application.

In another aspect, the present application provides a cell, comprises the isolated nucleic acid molecule or molecules of the present application, or the vector or vectors of the present application.

In another aspect, the present application provides a method for producing the antibody or the antigen binding fragment thereof of the present application, or the fusion protein of the present application, comprises culturing the cell of the present application under conditions enabling expression of the antibody or the antigen binding fragment thereof of the present application, or the fusion protein of the present application.

In another aspect, the present application provides a composition, comprises the antibody or the antigen binding fragment thereof of the present application, the fusion protein of the present application, the protein conjugate of the present application, the isolated nucleic acid molecule or molecules of the present application, the vector or vectors of the present application, and/or the cell of the present application, and optionally a pharmaceutically acceptable excipient.

In another aspect, the present application provides a kit comprises the antibody or the antigen binding fragment thereof of the present application.

In another aspect, the present application provides a use of the antibody or the antigen binding fragment thereof of the present application, the fusion protein of the present application, the protein conjugate of the present application, the isolated nucleic acid molecule or molecules of the present application, the vector or vectors of the present application, the cell of the present application, and/or the composition of the present application in the manufacture of a medicament for enhancing cell survival, enhancing neural injury repairing, protecting neural cells from apoptosis and/or necroptosis in neural cells expressing TrkB or promoting sensorimotor function in a TrkB associated condition.

In another aspect, the present application provides a use of the antibody or the antigen binding fragment thereof of the present application, the fusion protein of the present application, the protein conjugate of the present application, the isolated nucleic acid molecule or molecules of the present application, the vector or vectors of the present application, the cell of the present application, and/or the composition of the present application in the manufacture of a medicament for neuroprotection, enhancing synaptic development, enhancing neurite branching, enhancing cell survival or protecting cells from apoptosis and/or necroptosis.

In another aspect, the present application provides a use of the antibody or the antigen binding fragment thereof of the present application, or the fusion protein of the present application in the manufacture of an agent for determining the presence and/or amount and/or activity of TrkB in a sample.

In another aspect, the present application provides a method for neuroprotection, enhancing cell survival, enhancing neural injury repairing, protecting neural cells from apoptosis and/or necroptosis in neural cells expressing TrkB or promoting sensorimotor function in a TrkB associated condition in a subject in need thereof, comprises administering to the subject an effective amount of the antibody or the antigen binding fragment thereof of the present application, the fusion protein of the present application, the protein conjugate of the present application, the isolated nucleic acid molecule or molecules of the present application, the vector or vectors of the present application, the cell of the present application and/or the composition of the present application.

In another aspect, the present application provides a method for determining the presence and/or amount and/or activity of TrkB in a sample, comprising: a) contacting the sample with the antibody or the antigen binding fragment thereof of the present application, or the fusion protein of the present application; and b) determining the presence and/or amount and/or activity of the antibody, the antigen binding fragment or variant thereof, or the fusion protein bound to the sample.

In another aspect, the present application provides the antibody or the antigen binding fragment thereof of the present application, the fusion protein of the present application, the isolated nucleic acid molecule or molecules of the present application, the vector or vectors of the present application, and/or the cell of the present application, for a) neuroprotection, enhancing cell survival, enhancing neural injury repairing, protecting neural cells from apoptosis and/or necroptosis in neural cells expressing TrkB or promoting sensorimotor function in a TrkB associated condition, and/or b) determining the presence and/or amount and/or activity of TrkB in a sample, wherein the disease or disorder is a disease or disorder associated with an inappropriate expression or function of TrkB.

In another aspect, the present application provides a use of the antibody or the antigen binding fragment thereof of the present application, the fusion protein of the present application, the protein conjugate of the present application, the isolated nucleic acid molecule or molecules of the present application, the vector or vectors of the present application, the cell of the present application and/or the composition of the present application in the manufacture of a medicament for preventing and/or treating a disease or disorder, wherein said disease or disorder is neurodegenerative disease, depression, anxiety, autism, schizophrenia, and post-traumatic stress disorder, CNS injuries, stroke and/or traumatic brain injury.

In another aspect, the present application provides a method of preventing and/or treating a disease or disorder, comprising administering the antibody or the antigen binding fragment thereof of the present application, the fusion protein of the present application, the protein conjugate of the present application, the isolated nucleic acid molecule or molecules of the present application, the vector or vectors of the present application, the cell of the present application and/or the composition of the present application.

In some embodiments, the neurodegenerative disease comprises Alzheimer's Disease and related dementias, Parkinson's Disease, Huntington's Disease, Lewy Body Disease and related movement disorders, Amyotrophic lateral sclerosis, glaucoma and/or Friedrich's Ataxia and related Spinocerebellar Ataxia.

Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWING

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are employed, and the accompanying drawings (also “figure” and “FIG. ” herein) , of which:

FIG. 1 illustrates the results of ELISA analysis for exemplary TrkB antibodies of the present disclosure.

FIG. 2 illustrates the results of FACS analysis for exemplary TrkB antibodies of the present disclosure.

FIG. 3 illustrate the results of p-ERK HTRF assay for exemplary TrkB antibodies of the present disclosure.

FIG. 4 illustrates the results of p-ERK HTRF assay for exemplary TrkB antibodies of the present disclosure.

FIGs. 5A-5C illustrate the results of p-ERK HTRF assay for exemplary TrkB hybridoma (5A) , chimeric (5B) , and humanized (5C) antibody of the present disclosure.

FIGs. 6A-6B illustrate the results of ELISA for exemplary TrkB affinity maturation antibodies to human TrkB (6A) and mouse TrkB (6B) .

FIG. 7 illustrates the results of FACS for exemplary TrkB antibodies of the present disclosure to CHO-human TrkB.

FIG. 8 illustrates the results of p-ERK HTRF assay for exemplary TrkB affinity maturation antibodies in vtiro.

FIG. 9 illustrates the results of the effect of exemplary TrkB antibodies in BDNF deprivation assay using CellTiter-Glo detection on neurons at Day 16.

FIG. 10 illustrates the results of p-ERK HTRF assay for exemplary TrkB antibody of the present disclosure.

FIG. 11 illustrates the results of the effect of TrkB antibodies in BDNF deprivation assay using CellTiter-Glo detection on neurons.

FIG. 12 illustrates the pro-survival effect of BDNF, Ab419 and 6C10B4 in neuronal differentiation.

FIG. 13 illustrates diagram of the electrode fixed to the skull.

FIGs. 14A-14B illustrates the results of epileptiform discharge events and epileptiform discharge duration after administrating the exemplary TrkB antibody in the present disclosure.

FIGs. 15A-15B illustrate the results of the dark-adapted full-field electroretinography (ERG) examination.

FIG. 16 illustrates the body weight change of SOD1 mice after administrating the exemplary TrkB antibody. Compared with anti-TNP control. **p≤0.01, ***p≤0.001 , ****p≤0.0001 (N=13/12, M=7 , F=6/5)

FIG. 17 illustrates the results of hanging wire test in SOD1 mice. Compared with anti-TNP control. p≤0.05, **p≤0.01 (N=13/12, M=7, F=6/5)

FIG. 18 illustrates the body weight change of SOD1 mice after administrating the exemplary TrkB antibody of different dosages. Compared with PBS control. *p≤0.05, **p≤0.01 ***p≤0.001, ****p≤0.0001 (N=7)

FIG. 19 illustrates the results of hanging wire test in SOD1 mice under different dosage of TrkB antibody. Compared with PBS control. *p≤0.05 (N=7)

FIG. 20 illustrates the results of rotarod test in SOD1 mice under different dosage of TrkB antibody. Compared with PBS control. *p≤0.05 (N=7)

DETAILED DESCRIPTION

While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

The term “antibody” , as used herein, generally refers to an immunoglobulin or an immunoglobulin-like molecule capable of specifically recognizing or binding to an antigen. An antibody may comprise a light chain (L) and a heavy chain (H) . The light chains of an antibody can be classified as к and λ light chains. The heavy chains can be classified as μ, δ, γ, α or ε, and the isotypes of an antibody are defined as IgM, IgD, IgG (e.g., IgGl, IgG2, IgG3 or IgG4 subtype) , IgA and IgE, respectively. Each heavy chain may comprise a heavy chain variable region (VH) and a heavy chain constant region (CH) . The heavy chain constant region may comprise three domains (CH1, CH2 and CH3) . Each light chain may comprise a light chain variable region (VL) and a light chain constant region (CL) . The light chain constant region may comprise a CL domain. The VH and VL regions can also be subdivided into regions with high variability known as complementarity determining regions (CDRs) interspersed with more conserved regions known as framework regions (FRs) . Each VH and VL consists of 3 CDRs and 4 FRs arranged from N-terminal to C-terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy/light chain pair form the antibody binding site, respectively. Distribution of amino acids to regions or domains follows the definition of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991) ) , or Chothia &Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883. The term “antibody” is not limited by any antibody-producing method. For example, it includes recombinant antibodies, monoclonal antibodies, and other forms of antibodies. In some cases, an antibody of the present disclosure is an isolated antibody.

The term “antigen binding fragment” , as used herein, generally refers to an antibody fragment formed from a fragment of an antibody comprising one or more CDRs, or any other antibody portion that binds to an antigen but does not comprise an intact native antibody structure. In certain embodiments, the antibody provided herein is an antigen-binding fragment. Examples of antigen-binding fragment include, without limitation, a diabody, a Fab, a Fab', a F (ab') 2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv) , a (dsFv) 2, a bispecific dsFv (dsFv-dsFv′) , a disulfide stabilized diabody (ds diabody) , a single-chain antibody molecule (scFv) , an scFv dimer (bivalent diabody) , a multispecific antibody, a single domain antibody, a camelid single domain antibody, a VNAR, a nanobody, a domain antibody, an isolated CDR and a bivalent domain antibody. An antigen-binding fragment is capable of binding to the same antigen to which the parent antibody binds. In certain embodiments, an antigen-binding fragment may comprise one or more CDRs from a particular human antibody.

The term “TrkB” , as used herein, generally refers to the tropomyosin receptor kinase B (TrkB) , also known as tyrosine receptor kinase B, or BDNF/NT-3 growth factors receptor or neurotrophic tyrosine kinase receptor type 2, which is a protein that in humans is encoded by the NTRK2 gene. TrkB is located at the cellular membrane and is activated by binding of a ligand to the receptor's extracellular domain. TrkB has three isoforms in mammalian central nervous system (CNS) : the full-length isoform is a typical tyrosine kinase receptor, while the two C-terminal TrkB isoforms having the same extracellular domain, transmembrane domain and first 12 intracellular amino acid sequences as the full-length isoform but differing at the C-terminal sequences with 11 (T1 isoform) and 9 (T2 isoform) amino acids, respectively. In the present disclosure, the TrkB protein refers to either of the above mentioned three isoforms. TrkB is a receptor for brain-derived neurotrophic factor (BDNF) and binds BDNF in a ligand-specific manner. BDNF binding to TrkB triggers its dimerization through conformational changes and autophosphorylation of tyrosine residues in its intracellular domain, resulting in activation of the three major signaling pathways involving mitogen-activated protein kinase (MAPK) , phosphatidylinositol 3-kinase (PI3K) and phospholipase C-gamma 1 (PLC-gamma 1) . Several neurotrophins that are reported to activate TrkB, such as BDNF, neurotrophin-4 (NT-4) and neurotrophin-3 (NT-3) , thereby activating the multiple effects includes neuronal differentiation, neuronal repair, neuronal plasticity, proliferation and survival. Both BDNF and NT-4 can bind to TrkB and can activate many common signaling pathways. In certain embodiments, the TrkB as used herein is a human TrkB with the gene sequence with accession No. S76473.1, or a non-human animal TrkB, such as a mouse TrkB, a rat TrkB, a rabbit TrkB.

The term “BDNF” , as used herein, generally refers to a member of the neurotrophin family of growth factors, which are related to the canonical Nerve Growth Factor. BDNF are found in the brain such as the hippocampus, cortex, and basal forebrain, mediating survival and differentiative activities on neurons and modulating the synaptic function of neurons by binding and activating TrkB, as well as in the periphery nervous system, such as the retina, motor neurons, the kidneys, saliva, and the prostate. BDNF binds at least two receptors on the surface of cells, such as TrkB and p75 (also known as LNGFR) . All neurotrophins can interact with the p75 receptor. When the p75 is activated, it triggers apoptosis rather than survival pathways in cells expressing p75 receptor but lacking Trk receptors. BDNF also binds TrkA and TrkC, which together with TrkB belong to a sub-family of protein kinases.

The term “binding specificity” , as used herein, generally refers to an ability of one substance to bind another substance specifically, and not substantially bind to any other substance at random. For example, one protein may bind to another protein specifically due to their specific structures. Binding specificity may be measured by, e.g., cross-competing assays or other binding assays known in the art.

The term “K _D” , as used herein, generally refers to the dissociation constant, a specific type of equilibrium constant that measures the propensity of a larger object to separate (dissociate) reversibly into smaller components, as when a complex falls apart into its component molecules. The dissociation constant is the inverse of the association constant. In the specific case of antibodies (Ab) binding to antigen (Ag) , usually the term affinity constant refers to the association constant.

The term “specific binding” , as used herein, generally refers to a non-random binding reaction between two molecules, such as for example between an antibody and an antigen. For example, the antibodies or antigen binding fragments thereof of the present application may specifically bind human and/or non-human TrkB with a binding affinity (K _D) of about 0.01 nM to about 100 nM.

The term “block binding” , as used herein, generally refers to the ability of an antibody or antigen-binding fragment to inhibit the binding interaction between two molecules (e.g. human TrkB and a TrkB agonist antibody) to any detectable degree.

The term “monoclonal antibody” , as used herein, generally refers to antibodies that are made by identical immune cells that are all clones of a unique parent cell. Monoclonal antibodies can have a monovalent affinity, in that they bind to the same epitope (the part of an antigen that is recognized by the antibody) . It has become an important tool in biochemistry, molecular biology, and medicine. Several monoclonal antibody technologies had been developed recently, such as phage display, single B cell culture, single cell amplification from various B cell populations and single plasma cell interrogation technologies.

The term “chimeric antibody” , as used herein, generally refers to an antibody or antigen-binding fragment that has a portion of heavy and/or light chain derived from one species, and the rest of the heavy and/or light chain derived from a different species. In an illustrative example, a chimeric antibody may comprise a constant region derived from human and a variable region derived from a non-human species, such as from mouse.

The term “humanized antibody” , as used herein, generally refers to antibody or antigen-binding fragment, refers to the antibody or the antigen-binding fragment comprises CDRs derived from non-human animals (e.g. a rodent, rabbit, dog, goat, horse, or chicken) , FR regions derived from human, and when applicable, the constant regions derived from human. In certain embodiments, the constant regions from a human antibody are fused to the non-human variable regions. A humanized antibody or antigen-binding fragment is useful as human therapeutics. In certain embodiments because it has reduced immunogenicity or is less likely to induce an immune response in human, as compared to the non-human species antibody. In some embodiments, the non-human animal is a mammal, for example, a mouse, a rat, a rabbit, a goat, a sheep, a cattle, a horse, a guinea pig, a hamster, or a non-human primate (for example, a monkey (e.g., cynomolgus or rhesus monkey) or ape (e.g., chimpanzee, gorilla, simian or affen) ) . In some embodiments, the humanized antibody or antigen-binding fragment is composed of substantially all human sequences except for the CDR sequences which are non-human. In some embodiments, the humanized antibody or antigen-binding fragment is modified to improve the antibody performance, such as binding or binding affinity. For example, one or more amino acid residues in one or more non-human CDRs are altered to reduce potential immunogenicity in human, wherein the altered amino acid residues either are not critical for immunospecific binding or the alterations are conservative changes, such that the binding of the humanized antibody to the antigen is not significantly affected. In some embodiments, the FR regions derived from human may comprise the same amino acid sequence as the human antibody from which it is derived, or it may comprise some amino acid changes, for example, no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 changes of amino acid. In some embodiments, such change in amino acid could be present in heavy chain FR regions only, in light chain FR regions only, or in both chains. In some preferable embodiments, the humanized antibodies comprise human FR1-3 and human JH and Jκ.

The term “fully human antibody” and “human antibody” are used interchangeably herein, and generally refers to an antibody that comprises a human variable region and, most preferably a human constant region. In specific embodiments, the terms refer to an antibody that comprises a variable region and constant region of human origin. The term “fully human antibody” includes antibodies having variable and constant regions corresponding to human germline immunoglobulin sequences as described by Kabat et al. (See Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242) .

The term “Fab fragment” , as used herein, generally refers to a portion (such as an antigen-binding fragment) of an immunoglobulin molecule. A Fab fragment may comprise one light chain and part of a heavy chain with a single antigen-binding site. A Fab fragment may be obtained by papain digestion of an immunoglobulin molecule. For example, a Fab fragment may be composed of one constant and one variable domain of each of the heavy and the light chain. The variable domain may contain the paratope (the antigen-binding site) , comprising a set of the complementarity determining regions, at the amino-terminal end of the immunoglobulin molecule. The enzyme papain may be used to cleave an immunoglobulin molecule into two Fab fragments and one Fc fragment. The enzyme pepsin cleaves below the hinge region, so a F (ab') 2 fragment and a pFc'fragment is formed. Divalent F (ab) 2 or F (ab') 2 fragments have two antigen binding regions that are linked by disulfide bonds. Reduction of F (ab) 2 or F (ab') 2 fragments produces 2 monovalent Fab or Fab'fragments, which have a free sulfhydryl group that is useful for conjugation to other molecules.

The term “Fc” , as used herein, generally refers to portion of the antibody consisting of the second and third constant regions of a first heavy chain bound to the second and third constant regions of a second heavy chain via disulfide bond. IgG and IgM Fc regions contain three heavy chain constant regions (second, third and fourth heavy chain constant regions in each chain) . It can be obtained by papain digestion of an antibody. The Fc portion of the antibody is responsible for various effector functions such as ADCC, and CDC, but does not function in antigen binding.

The term “F (ab') 2” , as used herein, generally refers to a dimer of Fab'that comprises two light chains and part of two heavy chains.

The term “Fv fragment” , as used herein, generally refers to the smallest fragment of the antibody to bear the complete antigen binding site. A Fv fragment consists of the variable region of a single light chain bound to the variable region of a single heavy chain. A “dsFv” refers to a disulfide-stabilized Fv fragment that the linkage between the variable region of a single light chain and the variable region of a single heavy chain is a disulfide bond.

The term “ScFv” , as used herein, generally refers to a single-chain antibody fragment. An ScFv may be a recombinant single chain polypeptide molecule in which light and heavy chain variable regions of an antibody are connected, either directly or via a peptide linker. Single chain antibodies (ScFv) generally do not include portions of the Fc region of antibody, although methods are known for adding such regions to known ScFv molecules if desired. See Helfrich et al., A rapid and versatile method for harnessing ScFv antibody fragments with various biological functions. J Immunol Methods 237: 131-145 (2000) and de Haard et al., Creating and engineering human antibodies for immunotherapy. Advanced Drug Delivery Reviews 31: 5-31 (1998) .

The term “fusion protein” , as used herein, generally refers to a polypeptide that comprises, or alternatively consists of, an amino acid sequence of a polypeptide fused directly or indirectly (e.g., via a linker) to an amino acid sequence of a heterologous polypeptide (i.e., a polypeptide of a different origin, sequence or structure) .

The term “protein conjugate” , as used herein, generally refers to a conjugate comprising a protein (e.g., an antibody or a functional fragment thereof) conjugated to one or more additional moieties, such as cytotoxic agents, e.g., a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof) , a label (e.g., a fluorescent label) and/or a radioactive isotope (i.e., a radioconjugate) .

A number of CDR definitions are in use and are encompassed herein. The Kabat definition is based on sequence variability and is the most commonly used (Kabat EA et al., ibid. ) . Chothia refers instead to the location of the structural loops (Chothia &Lesk J. (1987) Mol. Biol. 196: 901-917) . The AbM definition is a compromise between the Kabat and the Chothia definitions and is used by Oxford Molecular's AbM antibody modelling software (Martin ACR et al., (1989) PNAS USA 86: 9268-9272; Martin ACR et al., (1991) Methods Enzymol. 203: 121-153; Pedersen JT et al., (1992) Immunomethods 1: 126-136; Rees AR et al., (1996) In Sternberg M.J.E. (ed. ) , Protein Structure Prediction. Oxford University Press, Oxford, 141-172) . The contact definition has been recently introduced (MacCallum RM et al., (1996) J. Mol. Biol. 262: 732-745) and is based on an analysis of the available complex structures available in the Protein Databank. The definition of the CDR by

the international ImMunoGeneTics information

(http: //www. imgt. org) is based on the IMGT numbering for all immunoglobulin and T cell receptor V-REGIONs of all species (

the international ImMunoGeneTics information

Lefranc MP et al., (1999) Nucleic Acids Res. 27 (1) : 209-12; Ruiz M et al., (2000) Nucleic Acids Res. 28 (1) : 219-21; Lefranc MP (2001) Nucleic Acids Res. 29 (1) : 207-9; Lefranc MP (2003) Nucleic Acids Res. 31 (1) : 307-10; Lefranc MP et al., (2005) Dev. Comp. Immunol. 29 (3) : 185-203; Kaas Q et al., (2007) Briefings in Functional Genomics &Proteomics, 6 (4) : 253-64) .

The term “isolated nucleic acid molecule or molecules” as used herein, generally refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, isolated from its native environment, or that is artificially synthesized.

The term “vector or vectors” as used herein, generally refers to a nucleic acid vehicle into which a polynucleotide encoding a protein can be inserted and expressed. The genetic material elements carried in the vector can be expressed in a host cell by transforming, transducing, or transfecting the host cell with the vector. A vector may contain a variety of elements that control expression, including promoter sequences, transcriptional initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain an origin of replication. It is also possible that the vector may include components that assist its entry into the cell, such as viral particles, liposomes or protein shells, but not only these substances.

The term “cell” as used herein, generally refers to a cell that may be used to carry the vector or vectors of the present disclosure, or be used to express or produce the antibody, the antigen binding fragment of the present disclosure. A cell of the present disclosure may be a host cell.

The terms “disease” and “disorder” may be used interchangeably herein, and generally refer to any condition that impairs the normal functioning of the body. Disease is often construed as a medical condition associated with specific symptoms and signs. It may be caused by external factors such as pathogens or by internal dysfunctions, particularly of the immune system, such as an immunodeficiency, or by a hypersensitivity, including allergies and autoimmunity.

As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. For example, the subject may be human.

The term “effective amount” , as used herein, generally refers to the designated carrier, vehicle, diluent, excipient (s) , and/or salt are generally chemically and/or physically compatible with the other ingredients comprising the formulation, and physiologically compatible with the recipient thereof. An “excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

The term “pharmaceutically acceptable excipient” , as used herein, generally refers to any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, etc., that are compatible with pharmaceutical administration.

The term “TrkB associated condition” , as used herein, generally refers to any condition that is caused by, exacerbated by, or otherwise linked to increased or decreased expression or activities of TrkB (e.g. a human TrkB) , or a disorder that is caused or exacerbated by a decrease in BDNF signaling, or any other intracellular signaling cascade that is activated through TrkB. Examples of neurological and psychiatric disorders may comprise neurodegenerative diseases (includes but are not limited to Alzheimer's Disease and related dementias, Parkinson's Disease, Huntington's Disease, Lewy Body Disease and related movement disorders, Amyotrophic lateral sclerosis, glaucoma and Friedrich's Ataxia and related Spinocerebellar Ataxia's ) , depression, anxiety, autism, schizophrenia, and post- traumatic stress disorder, CNS injuries, stroke and traumatic brain injury, and the like. Examples of metabolic disorders include obesity and hyperphagia.

The term “about” , as used herein, generally refers to an approximation to a given value that would reasonably be inferred based on the ordinary skill in the art, including equivalents and approximations due to the experimental and/or measurement conditions for such given value. For example, it may refer to a value that is no more than 10%above or below the value being modified by the term.

The terms “polypeptide” or “protein” , as used herein, generally refers to macromolecule having the amino acid sequence of a native protein, that is, a protein produced by a naturally-occurring and non-recombinant cell; or it is produced by a genetically-engineered or recombinant cell, and comprise molecules having the amino acid sequence of the native protein, or molecules having deletions from, additions to, and/or substitutions of one or more amino acids of the native sequence. The term also includes amino acid polymers in which one or more amino acids are chemical analogs of a corresponding naturally occurring amino acid and polymers. The terms “polypeptide” and “protein” specifically encompass TrkB antigen binding proteins, antibodies, or sequences that have deletions from, additions to, and/or substitutions of one or more amino acid of antigen-binding protein. The term “polypeptide fragment” refers to a polypeptide that has an amino-terminal deletion, a carboxyl-terminal deletion, and/or an internal deletion as compared with the full-length native protein. Such fragments can also contain modified amino acids as compared with the native protein. In certain embodiments, fragments are about five to 500 amino acids long. For example, fragments can be at least 5, 6, 8, 10, 14, 20, 50, 70, 100, 1 10, 150, 200, 250, 300, 350, 400, or 450 amino acids long. Useful polypeptide fragments include immunologically functional fragments of antibodies, including binding domains. In the case of a TrkB-binding antibody, useful fragments include but are not limited to a CDR region, a variable domain of a heavy and/or light chain, a portion of an antibody chain or just its variable region including two CDRs, and the like.

The term “isolated protein” (such as isolated antibody) , as used herein, generally refers to a subject protein (1) is free of at least some other proteins with which it would normally be found, (2) is essentially free of other proteins from the same source, e.g., from the same species, (3) is expressed by a cell from a different species, (4) has been separated from at least about 50 percent of polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature, (5) is operably associated (by covalent or noncovalent interaction) with a polypeptide with which it is not associated in nature, or (6) does not occur in nature. Typically, an “isolated protein” constitutes at least about 5%, at least about 10%, at least about 25%, or at least about 50%of a given sample. Genomic DNA, cDNA, mRNA or other RNA, of synthetic origin, or any combination thereof can encode such an isolated protein. Preferably, the isolated protein is substantially free from proteins or polypeptides or other contaminants that are found in its natural environment that would interfere with its therapeutic, diagnostic, prophylactic, research or other use.

A “variant” of a polypeptide (e.g., an antigen binding protein, or an antibody) comprises an amino acid sequence wherein one or more amino acid residues are inserted into, deleted from and/or substituted into the amino acid sequence relative to another polypeptide sequence. Variants include fusion proteins.

The term “identity” , as used herein, generally refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. “Percent identity” means the percent of identical residues between the amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest of the molecules being compared. For these calculations, gaps in alignments (if any) are preferably addressed by a particular mathematical model or computer program (i.e., an “algorithm” ) . Methods that can be used to calculate the identity of the aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, A.M., ed. ) , 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, D.W., ed. ) , 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, A.M., and Griffin, H.G., eds. ) , 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds. ) , 1991, New York: M. Stockton Press; and Carillo et al, 1988, SIAMJ. Applied Math. 48: 1073.

In calculating percent identity, the sequences being compared are typically aligned in a way that gives the largest match between the sequences. One example of a computer program that can be used to determine percent identity is the GCG program package, which includes GAP (Devereux et al., 1984, Nucl. Acid Res. 12: 387; Genetics Computer Group, University of Wisconsin, Madison, WI) . The computer algorithm GAP is used to align the two polypeptides or polynucleotides for which the percent sequence identity is to be determined. The sequences are aligned for optimal matching of their respective amino acid or nucleotide (the "matched span" , as determined by the algorithm) . A gap opening penalty (which is calculated as 3x the average diagonal, wherein the “average diagonal” is the average of the diagonal of the comparison matrix being used; the “diagonal” is the score or number assigned to each perfect amino acid match by the particular comparison matrix) and a gap extension penalty (which is usually 1/10 times the gap opening penalty) , as well as a comparison matrix such as PAM 250 or BLOSUM 62 are used in conjunction with the algorithm. In certain embodiments, a standard comparison matrix (see, Dayhoff et al, 1978, Atlas of Protein Sequence and Structure 5: 345-352 for the PAM 250 comparison matrix; Henikoff et ah, 1992, Proc. Natl. Acad. ScL U.S.A. 89: 10915-10919 for the BLOSUM 62 comparison matrix) is also used by the algorithm.

Conservative amino acid substitutions can encompass non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics and other reversed or inverted forms of amino acid moieties.

A “multispecific antigen binding protein” or “multispecific antibody” is one that targets more than one antigen or epitope.

A “bispecific” , “dual-specific” or “bifunctional” antigen binding protein or antibody is a hybrid antigen binding protein or antibody, respectively, having two different antigen binding sites. Bispecific antigen binding proteins and antibodies are a species of multispecific antigen binding protein antibody and can be produced by a variety of methods including, but not limited to, fusion of hybridomas or linking of Fab’ fragments. See, e.g., Songsivilai and Lachmann, 1990, Clin. Exp. Immunol. 79: 315-321; Kostelny et al, 1992, J. Immunol. 148: 1547-1553. The two binding sites of a bispecific antigen binding protein or antibody will bind to two different epitopes, which can reside on the same or different protein targets.

Unless otherwise indicated, the term “antibody” includes, in addition to antibodies comprising two full-length heavy chains and two full-length light chains, derivatives, variants, fragments, and muteins thereof, examples of which are described below. Furthermore, unless explicitly excluded, antibodies include monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as “antibody mimetics” ) , chimeric antibodies, humanized antibodies, human antibodies, antibody fusions (sometimes referred to herein as “antibody conjugates” ) , and fragments thereof, respectively. In some embodiments, the term also encompasses peptibodies.

In certain embodiments, an antibody heavy chain binds to an antigen in the absence of an antibody light chain. In certain embodiments, an antibody light chain binds to an antigen in the absence of an antibody heavy chain. In certain embodiments, an antibody binding region binds to an antigen in the absence of an antibody light chain. In certain embodiments, an antibody binding region binds to an antigen in the absence of an antibody heavy chain. In certain embodiments, an individual variable region specifically binds to an antigen in the absence of other variable regions.

In certain embodiments, definitive delineation of a CDR and identification of residues comprising the binding site of an antibody is accomplished by solving the structure of the antibody and/or solving the structure of the antibody-ligand complex. In certain embodiments, that can be accomplished by any of a variety of techniques known to those skilled in the art, such as X-ray crystallography. In certain embodiments, various methods of analysis can be employed to identify or approximate the CDR regions. Examples of such methods include, but are not limited to, the Kabat definition, the Chothia definition, the AbM definition and the contact definition.

As used herein, “substantially” or “substantial” generally means to a great or significant extent (e.g., to an extent of at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%or more) .

The term “treat” and “treatment” includes therapeutic treatments, prophylactic treatments, and applications in which one reduces the risk that a subject will develop a disorder or other risk factor. Treatment does not require the complete curing of a disorder and encompasses embodiments in which one reduces symptoms or underlying risk factors.

The term “prevent” does not require the 100%elimination of the possibility of an event. Rather, it denotes that the likelihood of the occurrence of the event has been reduced in the presence of the protein or method.

Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection) . Enzymatic reactions and purification techniques can be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures can be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (1989) ) , which is incorporated herein by reference for any purpose. Unless specific definitions are provided, the nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques can be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

Antibody, or the antigen binding fragment thereof

In one aspect, the present application provides an antibody or an antigen binding fragment thereof, which binds to TrkB and exhibits at least one of the following properties: 1) is capable of binding to TrkB with a K _D of less than 2.7E-06 M, as measured by Octet; 2) does not bind and/or does not activate human TrkA; 3) does not bind and/or does not activate human TrkC; 4) does not bind or and/or does not activate human p75; 5) is capable of activating TrkB; 5) is capable of activating ERK signaling pathway; 6) does not induce the reduction of body weight; 7) is capable of reducing the epileptiform discharge events and epileptiform discharge duration; and 8) has the ability of neuroprotection.

In the present application, the antibody or the antigen binding fragment has the ability of neuroprotection.

In the present application, the TrkB may be human TrkB. As described herein, TrkB proteins can also include fragments of the full length TrkB protein, such as the extracellular domain (ECD) thereof. An exemplary human TrkB ECD amino acid sequence is as set forth in SEQ ID NO: 1.

In some embodiments, the antibody, or the antigen binding fragment thereof comprises one or more CDRs (e.g., 1, 2, 3, 4, 5 or 6 CDRs) . In some embodiments, the antibody, or the antigen binding fragment thereof comprises (a) a polypeptide structure and (b) one or more CDRs that are inserted into and/or joined to the polypeptide structure. The polypeptide structure can take a variety of different forms. For example, it can be, or comprise, the framework of a naturally occurring antibody, or fragment or variant thereof, or can be completely synthetic in nature.

In certain embodiments, the polypeptide structure of the antibody, or the antigen binding fragment thereof is an antibody or is derived from an antibody, including, but not limited to, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as “antibody mimetics” ) , chimeric antibodies, humanized antibodies, antibody fusions (sometimes referred to as “antibody conjugates” ) , and portions or fragments of each, respectively. In some instances, the antibody, or the antigen binding fragment thereof is an immunological fragment of an antibody (e.g., a Fab, a Fab’ , a F (ab’ ) ₂, a scFv, or a VHH) .

The antibody, or the antigen binding fragment thereof may comprise a light chain constant region. The light chain constant region may comprise a Igκ constant region or a Igλ constant region.

The antibody, or the antigen binding fragment thereof may comprise a heavy chain constant region. The heavy chain constant region may comprise a IgG constant region (such as a IgG1, IgG2, or IgG4 constant region) .

Variable regions of immunoglobulin chains generally exhibit the same overall structure, comprising relatively conserved framework regions (FR) joined by three hypervariable regions, more often called “complementarity determining regions” or CDRs. The CDRs from the two chains of each heavy chain/light chain pair mentioned above typically are aligned by the framework regions to form a structure that binds specifically with a specific epitope on the target protein (e.g., TrkB) . From N-terminal to C-terminal, naturally occurring light and heavy chain variable regions both typically conform with the following order of these elements: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. A numbering system has been devised for assigning numbers to amino acids that occupy positions in each of these domains. This numbering system is defined in Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, MD) , or Chothia &Lesk, 1987, J. MoL Biol. 196: 901-917; Chothia et al., 1989, Nature 342: 878-883.

Various heavy chain and light chain variable regions are provided herein. In some embodiments, each of these variable regions can be attached to a heavy and light chain constant region to form a complete antibody heavy and light chain, respectively. Further, each of the so generated heavy and light chain sequences can be combined to form a complete antibody structure.

Specific examples of some of the variable regions of the light (VL) and heavy (VH) chains of the antibodies are provided and their corresponding amino acid sequences are summarized in Table 1 below.

Table 1

Name	VH	VL
6C10B4	SEQ ID NO: 15	SEQ ID NO: 23
6C10B4-chi	SEQ ID NO: 15	SEQ ID NO: 23
hu6C10B4-V1	SEQ ID NO: 16	SEQ ID NO: 24
hu6C10B4-V1-DG1	SEQ ID NO: 77	SEQ ID NO: 24
hu6C10B4-V1-DG3	SEQ ID NO: 78	SEQ ID NO: 24
hu6C10B4-V1-NG1	SEQ ID NO: 79	SEQ ID NO: 24
hu6C10B4-V1-NG2	SEQ ID NO: 80	SEQ ID NO: 24
hu6C10B4-V1-NG3	SEQ ID NO: 81	SEQ ID NO: 24
hu6C10B4-V1-NG4	SEQ ID NO: 82	SEQ ID NO: 24
hu6C10B4-AM1	SEQ ID NO: 17	SEQ ID NO: 24

hu6C10B4-AM2	SEQ ID NO: 19	SEQ ID NO: 26
hu6C10B4-AM6	SEQ ID NO: 18	SEQ ID NO: 25
hu6C10B4-AM8	SEQ ID NO: 20	SEQ ID NO: 25
hu6C10B4-AM22	SEQ ID NO: 21	SEQ ID NO: 27
hu6C10B4-AM23	SEQ ID NO: 22	SEQ ID NO: 27
hu6C10B4-AM27	SEQ ID NO: 22	SEQ ID NO: 25
24B5D6	SEQ ID NO: 28	SEQ ID NO: 31
24B5D6-chi	SEQ ID NO: 28	SEQ ID NO: 31
hu24B5D6-V1	SEQ ID NO: 29	SEQ ID NO: 32
hu24B5D6-V7	SEQ ID NO: 30	SEQ ID NO: 33

Each of the exemplary variable heavy chains listed in Table 1 can be combined with any of the exemplary variable light chains shown in Table 1 to form an antibody. Table 1 shows exemplary light and heavy chain pairings found in several of the antibodies disclosed herein. In some instances, the antibodies include at least one variable heavy chain and one variable light chain from those listed in Table 1. In other instances, the antibodies contain two identical light chains and two identical heavy chains. As an example, an antibody or antigen binding fragment thereof can include a heavy chain and a light chain, two heavy chains, or two light chains. In some embodiments, the antibody or the antigen binding fragment thereof comprises (and/or consists of) 1, 2, and/or 3 heavy and/or light CDRs from at least one of the sequences listed in Table 1. In some embodiments, all 6 CDRs (CDRl-3 from the light (LCDR1, LCDR2, LCDR3) and CDR 1-3 from the heavy (HCDR1, HCDR2, and HCDR3) ) are part of the antibody or the antigen binding fragment thereof. In some embodiments, 1, 2, 3, 4, 5, or more CDRs are included in the antibody or the antigen binding fragment thereof. In some embodiments, one heavy and one light CDR from the CDRs in the sequences in Table 1 is included in the antibody or the antigen binding fragment thereof. In some embodiments, additional sections are also included in the antibody or the antigen binding fragment thereof. Optional light chain variable sequences (including CDR1, CDR2, and CDR3) can be selected from the following: SEQ ID NO: 15-22; 28-30; 77-82. Optional heavy chain variable sequences (including CDR1, CDR2 and CDR3) can be selected from the following amino acid sequences: SEQ ID NOs: 23-27, and 31-33.

Some of the exemplary antibodies provided in the present disclosure are considered alternatives or variants to each other. For example, 6C10B4, hu6C10B4-V1, hu6C10B4-V1-DG1, hu6C10B4-V1-DG3, hu6C10B4-V1-NG1, hu6C10B4-V1-NG2, hu6C10B4-V1-NG3, hu6C10B4-V1-NG4, hu6C10B4-AM1, hu6C10B4-AM2, hu6C10B4-AM6, hu6C10B4-AM8, hu6C10B4-AM22, hu6C10B4-AM23 and hu6C10B4-AM27 may be considered variants or alternatives to each other. As another example, 24B5D6, hu24B5D6-V1, and hu24B5D6-V7 may be considered variants or alternatives to each other.

As described herein, the TrkB antibody or the antigen binding fragment thereof can comprise a humanized antibody and/or part thereof. An important practical application of such a strategy is the “humanization” of the mouse humoral immune system. In certain embodiments, a humanized antibody is substantially non-immunogenic in humans. In certain embodiments, a humanized antibody has substantially the same affinity for a target as an antibody from another species from which the humanized antibody is derived.

In certain embodiments, amino acids of an antibody variable domain that can be modified without diminishing the native affinity of the antigen binding domain while reducing its immunogenicity are identified.

In certain embodiments, modification of an antibody by methods known in the art is typically designed to achieve increased binding affinity for a target and/or to reduce immunogenicity of the antibody in the recipient. In certain embodiments, humanized antibodies are modified to eliminate glycosylation sites in order to increase affinity of the antibody for its cognate antigen. See, e.g., Co et al., MoI. Immunol., 30: 1361-1367 (1993) . In certain embodiments, techniques such as “reshaping” , “hyperchimerization” or “veneering/resurfacing” are used to produce humanized antibodies. See, e.g., Vaswami et al., Annals of Allergy, Asthma, &Immunol. 81: 105 (1998) ; Roguska et al, Prot. Engineer., 9: 895-904 (1996) ; and U.S. Patent No. 6,072,035. In certain such embodiments, such techniques typically reduce antibody immunogenicity by reducing the number of foreign residues, but do not prevent anti-idiotypic and anti-allotypic responses following repeated administration of the antibodies.

In certain instances, humanizing antibodies results in a loss of antigen binding capacity. In certain embodiments, humanized antibodies are “back mutated” . In certain such embodiments, the humanized antibody is mutated to include one or more of the amino acid residues found in the donor antibody. See, e.g., Saldanha et ai, MoI Immunol 36: 709-19 (1999) .

In certain embodiments the complementarity determining regions (CDRs) of the light and heavy chain variable regions of an antibody to TrkB can be grafted to framework regions (FRs) from the same, or another, species. In certain embodiments, the CDRs of the light and heavy chain variable regions of an antibody to TrkB can be grafted to consensus human FRs. To create consensus human FRs, in certain embodiments, FRs from several human heavy chain or light chain amino acid sequences are aligned to identify a consensus amino acid sequence. In certain embodiments, the FRs of an antibody to TrkB heavy chain or light chain are replaced with the FRs from a different heavy chain or light chain. In certain embodiments, rare amino acids in the FRs of the heavy and light chains of an antibody to TrkB are not replaced, while the rest of the FR amino acids are replaced. Rare amino acids are specific amino acids that are in positions in which they are not usually found in FRs. In certain embodiments, the grafted variable regions from an antibody to TrkB can be used with a constant region that is different from the constant region of an antibody to TrkB. In certain embodiments, the grafted variable regions are part of a single chain Fv antibody. CDR grafting is described, e.g., in U.S. Patent Nos. 6,180,370, 6,054,297, 5,693,762, 5,859,205, 5,693,761, 5,565,332, 5,585,089, and 5,530,101, and in Jones et al, Nature, 321: 522-525 (1986) ; Riechmann et al, Nature, 332: 323-327 (1988) ; Verhoeyen et al, Science, 239: 1534-1536 (1988) , Winter, FEBS Letts., 430: 92-94 (1998) , which are hereby incorporated by reference for any purpose.

In the present application, the antibody or the antigen binding fragment may comprise at least one Complementarity Determining Region (CDR) of a heavy chain variable region (VH) , the VH may comprise the amino acid sequence as set forth in SEQ ID NO: 100 or SEQ ID NO: 102.

In the present application, the antibody or the antigen binding fragment may comprise at least one Complementarity Determining Region (CDR) of a heavy chain variable region (VH) , the VH may comprise the amino acid sequence as set forth in any one of SEQ ID NOs: 15-22, 28-30, and 77-82.

In the present application, the antibody or the antigen binding fragment may comprise at least one Complementarity Determining Region (CDR) of a light chain variable region (VL) , the VL may comprise the amino acid sequence as set forth in SEQ ID NO: 101 or SEQ ID NO: 103.

In the present application, the antibody or the antigen binding fragment may comprise at least one Complementarity Determining Region (CDR) of a light chain variable region (VL) , the VL may comprise the amino acid sequence as set forth in any one of SEQ ID NOs: 23-27, and 31-33.

In the present application, the antibody may comprise a heavy chain or a fragment thereof.

In the present application, the heavy chain or a fragment thereof may comprise a heavy chain CDR1, and the heavy chain CDR1 may comprise the amino acid sequence as set forth in SEQ ID NO: 91 (X ₁YX ₂MH, wherein X ₁ is G, S, or T, X ₂ is T or W) .

For example, the heavy chain or a fragment thereof may comprise a heavy chain CDR1, and the heavy chain CDR1 may comprise an amino acid sequence as set forth in any one of SEQ ID NOs: 3, 9, and 67.

For example, the heavy chain or a fragment thereof may comprise a heavy chain CDR2, and the heavy chain CDR2 may comprise the amino acid sequence as set forth in SEQ ID NO: 92.

In the present application, the heavy chain or a fragment thereof may comprise a heavy chain CDR2, and the heavy chain CDR2 may comprise an amino acid sequence as set forth in any one of SEQ ID NOs: 93-94.

For example, the heavy chain or a fragment thereof may comprise a heavy chain CDR2, and the heavy chain CDR2 may comprise an amino acid sequence as set forth in any one of SEQ ID NOs: 4, 10, 39, 43, 70, and 84.

In the present application, the heavy chain or a fragment thereof may comprise a heavy chain CDR3, and the heavy chain CDR3 may comprise an amino acid sequence as set forth in any one of SEQ ID NOs: 11, and 95.

For example, the heavy chain or a fragment thereof may comprise a heavy chain CDR3, and the heavy chain CDR3 may comprise an amino acid sequence as set forth in any one of SEQ ID NOs: 5, 11, 73, 74, 75, 85, 86, 87, 88, 89, and 90.

For example, the antibody or the antigen binding fragment thereof may comprise:

1) heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 3, 4 and 5 respectively;

2) heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 3, 70 and 5 respectively;

3) heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 3, 70 and 85 respectively;

4)heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 3, 70 and 86 respectively;

5)heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 3, 70 and 87 respectively;

6)heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 3, 70 and 88 respectively;

7) heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 3, 70 and 89 respectively;

8) heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 3, 70 and 90 respectively;

9) heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 3, 70 and 50 respectively;

10) heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 67, 84 and 5 respectively;

11) heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 67, 70 and 5 respectively;

12) heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 3, 70 and 73 respectively;

13) heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 3, 84 and 74 respectively;

14) heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 3, 84 and 75 respectively;

15) heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 3, 84 and 75 respectively;

16) heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 9, 10 and 11 respectively;

17) heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 9, 39 and 11 respectively; or

18) heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 9, 43 and 11 respectively.

In the present application, the heavy chain or a fragment thereof may comprise a heavy chain variable region, and the heavy chain variable region may comprise an amino acid sequence as set forth in SEQ ID NO: 100 or SEQ ID NO: 102.

In the present application, the heavy chain or a fragment thereof may comprise a heavy chain variable region, and the heavy chain variable region may comprise an amino acid sequence as set forth in any one of SEQ ID NOs: 15-22, 28-30, and 77-82.

In the present application, the heavy chain or a fragment thereof may comprise a heavy chain constant region, and the heavy chain constant region may comprise a human IgG constant region.

In the present application, the antibody may comprise a light chain or a fragment thereof.

In the present application, the light chain or a fragment thereof may comprise a light chain CDR1, and the light chain CDR1 may comprise an amino acid sequence as set forth in any one of SEQ ID NOs: 96-97.

For example, the light chain or a fragment thereof may comprise a light chain CDR1, and the light chain CDR1 may comprise an amino acid sequence as set forth in any one of SEQ ID NOs: 6, 12, 53, and 64.

In the present application, the light chain or a fragment thereof may comprise a light chain CDR2, and the light chain CDR2 may comprise the amino acid sequence as set forth in SEQ ID NO: 98 (X ₁X ₂SX ₃RX ₄S, wherein X ₁ is K or W, X ₂ is A or V, X ₃ is N or T, X ₄ is E, F, or L) .

For example, the light chain or a fragment thereof may comprise a light chain CDR2, and the light chain CDR2 may comprise an amino acid sequence as set forth in any one of SEQ ID NOs: 7, 13, and 62.

In the present application, the light chain or a fragment thereof may comprise a light chain CDR3, and the light chain CDR3 may comprise an amino acid sequence as set forth in any one of SEQ ID NOs: 14, and 99.

For example, the light chain or a fragment thereof may comprise a light chain CDR3, and the light chain CDR3 may comprise an amino acid sequence as set forth in any one of SEQ ID NOs: 8, 14, 61, and 63.

For example, In the present application, the antibody or the antigen binding fragment thereof may comprise:

1) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 6, 7 and 8 respectively;

2) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 6, 7 and 8 respectively;

3) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 6, 7 and 8 respectively;

4) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 6, 7 and 8 respectively;

5) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 6, 7 and 8 respectively;

6) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 6, 7 and 8 respectively;

7) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 6, 7 and 8 respectively;

8) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 6 , 7 and 8 respectively;

9) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 6, 7 and 8 respectively;

10) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 6, 62 and 63 respectively;

11) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 6, 7 and 61 respectively;

12) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 6, 7 and 61 respectively;

13) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 64, 7 and 61 respectively;

14) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 64, 7 and 61 respectively;

15) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 6, 7 and 61 respectively;

16) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 12, 13 and 14 respectively;

17) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 12, 13 and 14 respectively; or

18) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 53, 13 and 14 respectively.

In the present application, the heavy chain or a fragment thereof may comprise a light chain variable region, and the light chain variable region may comprise an amino acid sequence as set forth in SEQ ID NO: 101 or SEQ ID NO: 103.

In the present application, the light chain or a fragment thereof may comprise a light chain variable region, and the light chain variable region may comprise an amino acid sequence as set forth in any one of SEQ ID NOs: 23-27, and 31-33.

In the present application, the light chain or a fragment thereof may comprise a light chain constant region, and the light chain constant region may comprise a human Igκ constant region.

In the present application, the antibody or the antigen binding fragment thereof may comprise:

1) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 6, 7 and 8 respectively, and heavy chain CDR 1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 3, 4 and 5 respectively;

2) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 6, 7 and 8 respectively, and heavy chain CDR 1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 3, 70 and 5 respectively;

3) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 6, 7 and 8 respectively, and heavy chain CDR 1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 3, 70 and 85 respectively;

4) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 6, 7 and 8 respectively, and heavy chain CDR 1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 3, 70 and 86 respectively;

5) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 6, 7 and 8 respectively, and heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NO: 3, 70 and 87 respectively;

6) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 6, 7 and 8 respectively, and heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 3, 70 and 88 respectively;

8) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 6, 7 and 8 respectively, and heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 3, 70 and 90 respectively;

13) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 64, 7 and 61 respectively, and heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 3, 84 and 74 respectively;

16) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 12, 13 and 14 respectively, and heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 9, 10 and 11 respectively;

17) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 12, 13 and 14 respectively, and heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 9, 39 and 11 respectively; or

The antibody or antigen binding fragment may also encompass a homology or a variant thereof having substantially the same function/property thereto. In some cases, the homology or variant may be a polypeptide different from the antibody or antigen binding fragment thereof at least one amino acid. For example, the homology or variant may be a polypeptide different from the antibody or antigen binding fragment thereof by an addition, deletion or substitution of one or more amino acid, such as 1-50, 1-40, 1-30, 1-20, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 amino acids. In some cases, the homology or variant may be a polypeptide having a sequence identity of at least 80%with the antibody or antigen binding fragment thereof. For example, the homology or variant may be a polypeptide having a sequence identity of 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%or higher) to the antibody or antigen binding fragment thereof.

The term “percent (%) sequence identity, ” as used in the context of polypeptide sequences identified herein, generally refers to the percentage of amino acid residues or nucleotides in a query sequence that are identical with the amino acid residues or nucleotides of a second, reference polypeptide sequence or a portion thereof, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid/nucleotide sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Percent identity may be measured over the length of an entire defined polypeptide/polynucleotide sequence, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide/polynucleotide sequence. It is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.

In another aspect, the present application provides a fusion protein, comprising the antibody or the antigen binding fragment thereof of the present disclosure.

In another aspect, the present application provides a protein conjugate (such as an immunoconjugate) , comprising the antibody or the antigen binding fragment thereof of the present disclosure.

Nucleic acid, vector, cell and the method of preparation

In another aspect, the present disclosure provides isolated nucleic acid or molecules, encoding for the antibody or the antigen binding fragment thereof, or the fusion protein.

The isolated nucleic acids may comprise one or more nucleic acid molecules, with each encoding the antibody of the present disclosure or an antigen binding fragment thereof. For example, the isolated nucleic acids may comprise at least two nucleic acid molecules, with one encoding the antibody heavy chain or a fragment thereof, and one encoding the antibody light chain or a fragment thereof. In some cases, the isolated nucleic acids may encode for a fusion protein.

The isolated nucleic acid or isolated nucleic acids may be synthesized using recombinant techniques well known in the art. For example, the isolated nucleic acid or isolated nucleic acids may be synthesized with an automated DNA synthesizer. Standard recombinant DNA and molecular cloning techniques include those described by Sambrook, J., Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, (1989) (Maniatis) and by T.J. Silhavy, M.L. Bennan, and L.W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984) and by Ausubel, F.M. et al., Current Protocols in Molecular Biology, pub. by Greene Publishing Assoc. and Wiley-Interscience (1987) . Briefly, the subject nucleic acids may be prepared from genomic DNA fragments, cDNAs, and RNAs, all of which may be extracted directly from a cell or recombinantly produced by various amplification processes including but not limited to PCR and RT-PCR.

Direct chemical synthesis of nucleic acids typically involves sequential addition of 3’-blocked and 5’-blocked nucleotide monomers to the terminal 5’-hydroxyl group of a growing nucleotide polymer chain, wherein each addition is affected by nucleophilic attack of the terminal 5’-hydroxyl group of the growing chain on the 3’-position of the added monomer, which is typically a phosphorus derivative, such as a phosphotriester, phosphoramidite, or the like. See for example, Matteuci et al., Tet. Lett. 521: 719 (1980) ; U.S. Pat. No. 4,500,707 to Caruthers et al.; and U.S. Pat. Nos. 5,436,327 and 5,700,637 to Southern et al.

In another aspect, the present disclosure provides a vector or vectors, comprising the isolated nucleic acid molecule or molecules.

The vector may be any linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors and the like. Non-limiting examples of a viral vector may include a retrovirus, an adenovirus and an adeno-associated virus. In some embodiments, the vector is an expression vector, e.g. a phage display vector.

An expression vector may be suitable for use in particular types of host cells and not others. For example, the expression vector can be introduced into the host organism, which is then monitored for viability and expression of any genes/polynucleotides contained in the vector.

The expression vector may also contain one or more selectable marker genes that, upon expression, confer one or more phenotypic traits useful for selecting or otherwise identifying host cells that carry the expression vector. Non-limiting examples of suitable selectable markers for eukaryotic cells include dihydrofolate reductase and neomycin resistance.

The subject vectors can be introduced into a host cell stably or transiently by a variety of established techniques. For example, one method involves a calcium chloride treatment wherein the expression vector is introduced via a calcium precipitate. Other salts, for example calcium phosphate, may also be used following a similar procedure. In addition, electroporation (that is, the application of current to increase the permeability of cells to nucleic acids) may be used. Other examples of transformation methods include microinjection, DEAE dextran mediated transformation, and heat shock in the presence of lithium acetate. Lipid complexes, liposomes, and dendrimers may also be employed to transfect the host cells.

In another aspect, the present disclosure provides a cell (e.g., an isolated cell, such as a host cell) , comprising the isolated nucleic acid molecule or molecules of the present disclosure or the vector or vectors of the present disclosure.

The cell may express the antibody, or the antigen binding fragment thereof of the present disclosure, or the fusion protein of the present disclosure. The cell may be a eukaryotic cell or a prokaryotic cell. An appropriate cell may be transformed or transfected with the nucleic acid (s) or vector (s) of the present disclosure and utilized for the expression and/or secretion of the antibody, the antigen binding fragment thereof, or the fusion protein. For example, the cell may be E. coli cells, other bacterial host cells, yeast cells, or various higher eukaryotic cells.

In another aspect, the present disclosure provides a method for producing the antibody or the antigen binding fragment thereof, or the fusion protein of the present disclosure, comprising culturing the cell of the present disclosure under conditions enabling expression of the antibody, the antigen binding fragment thereof, or the fusion protein.

The method optionally may further comprise harvesting the antibody or the antigen binding fragment thereof, or the fusion protein of the present disclosure.

Compositions

In another aspect, the present disclosure provides a composition, comprising the antibody or the antigen binding fragment thereof, the fusion protein, the isolated nucleic acid molecule or molecules, the vector or vectors, and/or the cell of the present disclosure, and optionally a pharmaceutically acceptable excipient.

In some embodiments, the composition further comprises an effective amount of an additional therapeutically active component, for example, an additional therapeutically active component may be BDNF and/or NT-4.

Described below are non-limiting exemplary pharmaceutical compositions and methods for preparing the same. The pharmaceutical composition may, for example, be in a form suitable for oral administration as a tablet, capsule, pill, powder, sustained release formulations, solution, suspension, for parenteral injection as a sterile solution, suspension or emulsion, for topical administration as an ointment or cream or for rectal administration as a suppository. The pharmaceutical composition may be in unit dosage forms suitable for single administration of precise dosages. In some embodiments, the pharmaceutical composition may be a liquid pharmaceutical composition.

Pharmaceutical compositions of the disclosure can be presented as discrete dosage forms, with each dosage containing a predetermined amount of an active ingredient as a powder or in granules, a solution, or a suspension in an aqueous or non-aqueous liquid. Such dosage forms can be prepared by any of the methods known to a skilled person, for example, it may include the step of bringing the active ingredient into association with the carrier, which constitutes one or more other ingredients. In general, the compositions are prepared by uniformly and intimately mixing the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation.

The antibody, the antigen binding fragment thereof, or the fusion protein of the present disclosure can be combined in an intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier can take a wide variety of forms depending on the form of preparation desired for administration.

The composition can further include one or more pharmaceutically acceptable additives and excipients. Such additives and excipients include, without limitation, detackifiers, anti-foaming agents, buffering agents, polymers, antioxidants, preservatives, chelating agents, viscomodulators, tonicifiers, flavorants, colorants, odorants, opacifiers, suspending agents, binders, fillers, plasticizers, lubricants, and/or mixtures thereof.

The pharmaceutical compositions of the present disclosure may comprise a therapeutically effective amount of the active agent (e.g., the antibody, the antigen binding fragment thereof, or the fusion protein of the present disclosure) . A therapeutically effective amount is an amount of the subject pharmaceutical composition capable of preventing and/or curing (at least partially) a condition or disorder (e.g., an autoimmune diseases) and/or any complications thereof in a subject suffering from or having a risk of developing said condition or disorder. The specific amount/concentration of the active agent comprised may vary according to the method of administration and the need of a patient, and can be determined based on e.g., volume, viscosity, and/or body weight of a patient etc. For example, an appropriate dosage may be about 0.1mg or l mg/kg/day to about 50mg/kg/day; sometimes, the dosage can be even higher. It shall be understood that these specific doses may be conveniently adjusted by a skilled person in the art (e.g., a doctor or a pharmacist) based on conditions of a specific patient, formulation, and/or disease.

For example, the kit may be used for diagnosing, preventing, delaying or treating the TrkB associated conditions.

Medical Use and Methods of Treatment

In another aspect, the present application provides a use of the antibody or the antigen binding fragment thereof of the present application, the fusion protein of the present application, the isolated nucleic acid molecule or molecules of the present application, the vector or vectors of the present application, and/or the cell of the present application in the manufacture of a medicament for neuroprotection, enhancing cell survival, enhancing neural injury repairing, protecting neural cells from apoptosis and/or necroptosis in neural cells expressing TrkB or promoting sensorimotor function in a TrkB associated condition.

In another aspect, the present application provides a use of the antibody or the antigen binding fragment thereof of the present application, the fusion protein of the present application, the isolated nucleic acid molecule or molecules of the present application, the vector or vectors of the present application, and/or the cell of the present application in the manufacture of a medicament for neuroprotection, enhancing synaptic development, enhancing neurite branching, enhancing cell survival or protecting cells from apoptosis and/or necroptosis.

For example, the neurons may be neurons in the central nervous system (CNS) .

For example, the sample may express TrkB. For example, the sample may be neural cells, including neural stem cells at various differentiation stages or terminally differentiated neural cells, such as neurons, astrocytes and oligodendrocytes. For example, the sample may be derived from a cell or tissue (e.g. biopsied tissue from an organ) , tumor cells, or bodily fluid (e.g. blood or serum) .

In the present application, the neural cell may comprise PC12 cells, hippocampal neurons, retinal ganglion cells, motor neurons, and dopaminergic neurons. In the present application, the regulated synaptic plasticity may comprise increased synapses, enhanced synaptic transmission, enhanced long term potentiation (LTP) , and enhanced γ oscillation.

In another aspect, the present application provides a use of the antibody or the antigen binding fragment thereof of the present application, the fusion protein of the present application, the isolated nucleic acid molecule or molecules of the present application, the vector or vectors of the present application, and/or the cell of the present application in the manufacture of a medicament for treating a condition. For example, the condition may comprise neurodegenerative diseases, psychiatric disorders, metabolic disorders and brain injury. For example, the neurodegenerative diseases may comprise Alzheimer's disease (AD) , Amyotrophic lateral sclerosis (ALS) , glaucoma, Huntington's disease (HD) , and Parkinson's disease (PD) . In certain embodiments, the psychiatric disorders comprise depression, autism, schizophrenia, and post-traumatic stress disorder (PTSD) . In certain embodiments, the psychiatric disorders comprise depression, autism, schizophrenia, and post-traumatic stress disorder (PTSD) .

In some cases, the antibody or the antigen binding fragment thereof, the fusion protein, the protein conjugate, the isolated nucleic acid molecule or molecules, the vector or vectors, the cell and/or the pharmaceutical composition of the present application may reduce the risk of epilepsy in a subject.

Examples

The following examples are set forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc. ) but some experimental errors and deviations should be accounted for.

Example 1. Development of TrkB agonist monoclonal antibodies

Identified antibodies were sub-cloned and examined, which bind to human TrkB (Q16620) with high specificity and strength, as well as show a strong agonistic effect in TrkB dependent HTRF assay, as shown in ELISA assay (FIG. 1) , and by FACS analysis (FIG. 2) , and by p-ERK TrkB HTRF assay (FIG. 3) . The procedures of the ELISA, FACS and HTRF analysis are briefly described below.

Binding to TrkB by ELISA

Hybridoma subclones were initially screened against human TrkB by indirect ELISA. Briefly, purified human TrkB extracellular domain protein was diluted to a final concentration of 1μg/mL in PBS buffer. Pipette 100 μL of the diluted antigen to each well of a 96-well plate, and incubate the plate at 4℃ overnight. The plates were washed with PBST (PBS comprising 0.05%Tween-20, pH 7.4) for three times, then pipetted 200μl/well of 2%bovine serum albumin (BSA) blocking solution diluted in PBST, and then incubated at room temperature for 2 hours for blocking. After removing blocking solution, the plate was washed 3 times with 300μL PBST. The diluted solutions of primary and secondary antibodies were prepared, along with substrate solution. Pipette 100μL of diluted primary antibody in each well, and plate was incubated for 1 hour at room temperature. The content in the wells was removed and wells were washed 3X with 300 μL PBST buffer. Flick and pat the plate to completely remove PBS buffer. Pipette 100μL of diluted Peroxidase-AffiniPure Goat Anti-Mouse IgG, Fcγ Fragment Specific (min X Hu, Bov, Hrs Sr Prot) in each well, and plate was incubated for 1 hour at 37℃. The content in the wells was removed and plate was washed 3X with 300μL PBST. 100μL of TMB substrate solution was pipetted to the wells. After sufficient color development, 100μL of stop solution was pipetted to the wells to terminate the reaction. Read the absorbance (OD: 450) of each well with a microplate reader and the data were analyzed.

Binding to TrkB by FACS

The ELISA-positive antibody producing clones were further verified by fluorescence-activated cell sorting (FACS) using a conventional method. Briefly,

TrkB-NFAT-bla CHO-K1 cell, the human TrkB overexpression cells were stained with anti-huTrkB antibody in U-bottom 96-well plates. The cells were resuspended to 2x10 ⁶ cells/ml in ice cold PBS. The diluted solutions of primary and secondary antibodies were prepared, and 100μL of diluted primary antibody was added in each well, and it was incubated for 1 hour at 4 ℃ in the dark. The cells were washed 2 times by centrifugation at 2500 rpm for 3 min and resuspended in ice cold PBS. The APC Goat anti-Mouse IgG was diluted in cold PBS at the 1μg/ml and then the cells were resuspended in 100μl of this solution. The cells were washed 2 times by centrifugation at 2500 rpm for 3 min and were resuspended in 100μl ice cold PBS. The cell suspension was stored immediately at 4℃ in the dark. The cells were analyzed on the flow cytometer as soon as possible.

Agonistic effect to TrkB activation using HTRF

To examine the effect of antibodies on the ERK pathway, the HTRF (Homogeneous Time-Resolved Fluorescence) technology were performed in

TrkB-NFAT-bla CHO-K1 cells. Cellsensor TrkB-NFAT-bla CHO-K1cells (K1435, Life Technologies) were seeded in 96-well plates with 100μl media on the day before experiment. The cells were pre-incubated with 3-fold serial diluted antibody in a humidified 37℃/5%CO ₂ incubator for 30 minutes. Cell supernatant was removed carefully either by aspirating supernatant or by flicking the plate. Immediately 50 μl of supplemented lysis buffer (1X) was added and incubated for 30 minutes at room temperature under shaking. After homogenization by pipetting up and down, 16 μl cell lysate was transferred from the 96-well plate to a 384-well plate. 16 μl of supplemented lysis buffer (1X) was dispensed to the Cell-free control wells. 16 μl of homogenized cell lysate was dispensed to the Test Sample wells, the Unstimulated Control wells, and Stimulated Control wells. It was incubated at least 4 hours at room temperature. The reader was set up for Eu ³⁺ Cryptate and read the fluorescence emission at two different wavelengths (665nm and 620nm) on a compatible

reader.

The selected clones 6C10B4 and 24B5D6 exhibited good human TrkB binding. Furthermore, as shown in the function assay (FIG. 3) , the selected antibodies could activate the TrkB-downstream ERK signal pathway.

Example 2. Cloning of the antibody variable region gene

Hybridoma cells were cultured in a 10cm dish, and were collected at logarithmic growth phase. Total cell RNA was extracted with Trizol (Invitrogen, 15596-018) following the manufacturer’s introduction. The RNA was resuspended in nuclease free water. The RNA concentration was determined by using absorbance at 260 nm on the bioTEK equipment.

To obtain the cDNA templates, 4 μg of each RNA was reverse transcribed using HiFiScript cDNA Synthesis Kit (CWBIO, CW2569) .

Then two rounds PCR reaction were performed to clone the variable gene of hybridoma mAbs. First round PCR were performed by using degenerate primers based on the sequences reported previously (Wang, Z et al, J Immunol Methods. 2000; 233 (1-2) : 167-177) . The PCR products were sent to GeneWiz for DNA sequencing. The sequencing results were analyzed using IMGT/V-QUEST web program (http: //www. imgt. org/IMGT_vquest/analysis) . The germline V gene of heavy chain (IGHV subgroups) and light chain (kappa and lambda subgroups) of each mAb was identified.

The second-round primers were designed to amplifying signal peptide and entire variable region, according to the germline information. The PCR product was directly sequenced.

To construct expression plasmid of the chimeric antibody, the VH gene were subcloned into pCNDA3.4-hIgG4 vector, and VL gene were subcloned into pCDNA3.4-hCK vector, further confirmed by DNA sequencing. Antibody complement determinant regions (CDRs) were identified based on Kabat by using ANARCI tools (Dunbar J et al Bioinformatics. 2016; 32 (2) : 298-300) .

Table 2. Antibody Kabat numbering

Example 3. Humanization

Humanization was carried out by CDR grafting, which was disclosed in many literatures. Briefly, the parental (murine antibody) variable region (VH and VL) frameworks were replaced with those of the selected human germline V and J genes of VH and VL.

After searching in database like IMGT, a template human germline is chosen on the basis of the homology between the parental antibody and the germline V and J genes.

For 6C10B4, IGHV1-46*01 and IGHJ6*01 were chosen as template heavy chain germline; IGKV4-1*01 and IGKJ4*01 were chosen as template light chain germline.

For 24B5D6, IGHV1-46*01 and IGHJ5*01 were chosen as template heavy chain germline; IGKV2-30*02 and IGKJ2*02 were chosen as template light chain germline.

After CDR grafting, some key residues in frameworks were back mutated. Gene synthesis was done by GeneWiz, and cloned into pCDNA3.4-hIgG4 or pCDNA3.4-hCK vectors. Some back mutations were done by site-direct mutagenesis PCR. All humanized versions were tested affinity by Octet (Table 3) , and some of them were selected for in-vitro function testing (FIGs. 4 and 5A-5C) . Results show that the TrkB antibody of the present application is capable of activating ERK signaling pathway.

The

TrkB-NFAT-bla CHO-K1 cells were used in conducting HTRF assays, and the experiments were conducted according to the protocols of

Affinity Determination by Octet

To determine the affinities of the antibodies, bio-layer interferometry (BLI) method was used with the Octet Red384 instrument. Briefly, anti-human Fc-coated biosensor AHQ tips (ForteBio) were pre-wet in PBS with 0.1%w/v bovine serum albumin and 0.05%Tween-20 for a minimum of 10 min in the pre-wetting plate. The purified antibodies at 100nM in PBS with 0.1%PBA and 0.05%Tween-20 (assay buffer) were captured on AHQ biosensors (ForteBio) to obtain capture levels of ～1 nm. The loaded biosensors were washed with assay buffer to remove any unbound protein. Then, association rates, dissociation rates and responses measurements were carried out with antigen prepared as 100 nM in PBS with 0.1%BSA and 0.05%Tween-20. All measurements were corrected for baseline drift by subtracting a control sensor exposed to assay buffer only. Association rate constants (k _on) and dissociation rate constants (k _off) for each antibody were calculated by applying a 1: 1 interaction model (fitting local, full) using the ForteBio data analysis software.

Table 3-1. CDR sequences of VH and VL

Table 3-2. Antibody VH/VL pairing

Table 4. Affinity by Octet

Name	Kon (1/Ms)	Koff (1/s)	KD (M)
6C10B4	4.21E+05	9.81E-03	2.33E-08
6C10B4-chi	3.57E+05	7.47E-03	2.09E-08
hu6C10B4-V1	5.08E+04	1.36E-01	2.67E-06
hu6C10B4-V1-DG1	9.87E+05	1.23E-02	1.25E-08
hu6C10B4-V1-DG3	2.91E+05	4.21E-02	1.45E-07
hu6C10B4-V1-NG1	3.98E+05	1.30E-02	3.27E-08
hu6C10B4-V1-NG2	2.99E+05	3.53E-02	1.18E-07
hu6C10B4-V1-NG3	6.14E+05	2.31E-02	3.75E-08
hu6C10B4-V1-NG4	5.08E+04	1.36E-01	2.67E-06
hu6C10B4-AM2	6.00E+05	1.90E-04	3.17E-10
hu6C10B4-AM6	5.78E+05	5.62E-04	9.72E-10
hu6C10B4-AM22	6.22E+05	7.24E-03	1.16E-08
hu6C10B4-AM23	5.99E+05	2.87E-03	4.79E-09
hu6C10B4-AM27	6.01E+05	4.32E-03	7.19E-09
24B5D6-chi	3.14E+05	1.07E-02	3.41E-08
hu24B5D6-V1	3.47E+05	1.79E-02	5.16E-08
hu24B5D6-V7	5.16E+05	2.02E-02	3.91E-08

Example 4. Post translational modification (PTM) removal and affinity maturation

According to the result of sequence analysis, several PTMs were found in heavy chain CDR3 (DG and NG) .

In order to reduce the risk of deamidation and isomerization, several mutations were done to the heavy chain CDR3 by quick change PCR.

The mutations were decided by the properties of amino acid and the steric collisions, the mutations are showed below:

DG->EG; DG->SG; DG->DA; NG->YG; NG->TG; NG->SG; NG->GG

The site-directed mutagenesis was done by following the standard protocol.

6C10B4 and its derived antibodies, like chimeric antibody (6C10B4-chi) , humanized antibody (hu6C10B4-V1) , PTM removal antibodies (hu6C10B4-DG1, hu6C10B4-DG3, hu6C10B4-NG1, hu6C10B4-NG2, hu6C10B4-NG3, hu6C10B4-NG4) are tested on Octet for their binding with human TrkB ECD protein (Table 4) .

With the aim of improving affinity, and getting cross species binding with mouse, affinity maturation libraries were generated and phage display was done.

First, humanized antibody was reformatted into single-chain fragment variable (scFv) , codon optimized in E. coli host, then gene was synthesized and inserted into a phage display plasmid.

The parsimonious mutagenesis library was generated as follow:

Primer design: forward primer should be between 25 and 45 bases in length and contain the desired mutation in degenerate code (NNS) in the center with correct sequences on both sides; the reverse primer is the reverse complement of this. For each amino acid site in 6 CDRs, a pair of primers were designed to generate mutations.

The site-saturation mutagenesis was similar with site-directed mutagenesis.

The PCR process follows site-directed mutagenesis protocol with a system of 30μl/reaction.

Right after the PCR, pick 5μl PCR product of each tube and mix, DpnI digests for at least 2hrs.

Purified the products by PCR-clean kit (Axygen) .

Concentration was detected by BioTek C5.

The purified PCR product was transformed into TG1 competent cells with electroporation. After cultivation in 37℃ for 1 hour, spread the cells onto a pre-warmed selective plate and 37℃incubation overnight. The next day, phage library was generated following standard protocol (Thie H et.al Methods Mol Biol. 2009; 525: 309-xv, Bostrom J et al. Methods Mol Biol. 2009; 525: 353-xiii) . A traditional panning and screening process was carried out. After 3 rounds panning and screening with ELISA, top clones were picked up for sequencing. Then all the mutations were combined and expressed in expi293. Affinity and function were tested to aiding top leads selection (FIGs. 6A-6B) .

The parsimonious mutagenesis will scan all 6 CDRs for the potential “hot spots” . However, the affinity improvement might be induced by multiple mutated sites, which are not “hot spots” . So an oligonucleotide-directed library was also generated. The multi-site-directed mutagenesis was carried out in both VH-CDR3 and VL-CDR3. The processes of library construction and panning follow the traditional methods. Sequencing and expression in exp293 were done right after panning and screening. Binding was confirmed by FACS (FIG. 7) , affinity test was done on Octet (Table 4) and in vitro function test was performed (FIGs. 8 and 9) . Results show that compared with BDNF, the TrkB antibody of the present disclosure is more effective in maintaining neuronal cell survival, as illustrated by their significantly higher maximum neuroprotection level.

The

Assay was following a standard Promega protocol (

Luminescent Cell Viability Assay, G7570) .

Example 5 Function of the TrkB antibodies

The method of determining agonistic effect to TrkB activation using HTRF assay is the same as Example 1.

The BDNF deprived assay was performed as previously described (Fang Han, et al, Therapeutic potential of a TrkB agonistic antibody for ischemic brain injury, (2019) Neurobiology of Disease) . Briefly, the experimental procedure is as follows:

1. Prepare 1 X Borate Buffer by 20 X Borate Buffer (ThermoFisher #28341) diluted 20-fold in water.

2. Prepare a 0.1%PEI solution by diluting 50%PEI (Cat #P3143, Sigma) solution in 1 X Borate Buffer. Filter the 0.1%PEI solution through a 0.22μm filter.

3. Plate coating: Use 0.1%PEI to coat the cell culture plate, incubate 1 hours at room temperature. Rinse each well 4 times with sterile tissue culture grade water (UltraPure ^TM DNase/RNase-Free Distilled Water: Gibco #10977015) , aspirate last wash of water completely and air dry completely in the hood before use (until no water mark can be seen) . Air-dry the coated plate in a sterile biological safety cabinet overnight.

4. Medium preparation: Use sterile techniques to prepare complete HopCellTM Human Cortical Neuron Medium (Basal Medium + Supplement) . The following example is for preparing 500 mL complete medium. If preparing other volumes, adjust accordingly. Add 12.5 mL of Human Cortical Neuron Medium Supplement (HopCell-CNM-500B) to 488 mL Human Cortical Neuron Basal Medium (HopCell-CNM-500A) . Mix thoroughly. Filter the complete medium using 0.22 μm low protein binding polyethersulfone (PES) filter unit (e.g., Thermo Scientific, Cat #566-0020) .

5. Digestion: Save the conditioned medium in wells for later use. Wash the NPC cells once with D-PBS. Add 0.3 mL Accutase (Cat #A1110501, Gibco, 0.15 mL/cm ²) into one well of 24-well plate to digest NPC. Incubate the plate for 5-10 minutes at a 37℃ cell incubator or until cells start to come off. Add 1.5 mL of saved conditional medium into each well. Harvest the cells with the 1 mL culture tip to pipette up and down for several times until almost all cells are collected. Avoid creating bubbles or over pipetting. Transfer cells into a 15 mL centrifuge tube and centrifuge at 200g for 4 minutes.

6. After centrifugation, carefully aspirate the supernatant and gently resuspend the cell pellet with 2 mL fresh HopCellTM Human Cortical Neuron Medium. Count the live cells and add more medium to make the right seeding density cell suspension.

7. Seed cells at a density of 2.5-3 X 10 ⁴ live cells/cm ² (i.e., 3 X 10 ³ cells per well of 384-well plate, 1 X 10 ⁴ cells per well of 96-well plate and 5 X 10 ⁴ cells per well of 24-well plate with coverslip, 70 μL, 200 μL and 500 μL medium per well respectively) .

8. Medium change: half-change the medium every 3-7 days.

The

Assay was following a standard Promega protocol (

Luminescent Cell Viability Assay, G7570)

FIG. 10 shows that the TrkB antibody of the present disclosure could activate the TrkB-downstream ERK signal pathway. FIG. 11 shows that compared with BDNF and Ab419, the TrkB antibodies of the present disclosure are more effective in maintaining neuronal cell survival, as illustrated by their significantly higher maximum neuroprotection level. FIG. 12 shows that compared with BDNF and Ab419, the TrkB antibody of the present disclosure also maintains neuroprotective effect at high concentration.

Example 6 The risk of epilepsy induced by the TrkB antibody

On the day of surgery, under anesthesia with Sutai 50 (15mg/kg, i.p. ) + thirazine (8mg/kg, i.p. ) , the mice were fixed by stereotaxic instrument. The head of animal was sheared and disinfected, and the head skin was cut. The four corners were clamped with hemostatic forceps, with the skull fully exposed and the periosteum peeled. Drill according to the diameter of the implant submodel, and implant electrodes. Beside the dura mater, fix the electrodes on the skull with dental cement (Figure 13) . The dental cement dripping on the tissue and skin was cleaned up. The implant was then placed subcutaneously on the back, and then cefradine powder was applied locally to the surgical incision to suture the surgical wound. After surgery, the mice were placed in an insulated blanket in a lateral decubitus position. After the mice were awake, they were carefully placed in a clean recovery cage, reared in a single cage, placed in a shielded recovery room. The animals were given three days of nursing after surgery, and gentamicin (0.2mg/ml, 0.1ml/pc) and meloxicam (2mg/ml, 0.1ml/pc) were given subcutaneously daily. The experiment was carried out 10 days after postoperative recovery. Animals were randomized into groups according to the body weight, the experiment was designed blinded, divided into RB-5 (TAM-163, the heavy chain variable region of TAM-163 is as set forth in SEQ ID NO: 108, and the light chain variable region of TAM-163 is as set forth in SEQ ID NO: 109) group (30 mg/kg, i.v., N=8) , TA-001 (24B5D6) group (30 mg/kg, i.v., N=8) , and TA003 (control IgG) group (30 mg/kg, i.v., N=8) . After postoperative recovery, tail vein administration was started, and the day of the first administration was defined as Day 0. Baseline was recorded 1 h before administration on Day 0, and the second administration was given on Day 14. EEG was recorded continuously for 4 days from Day 14 to Day 17. Raw data on mouse EEG and real-time body temperature were collected by the DSI system Ponemah software and analyzed with NeuroScore software. EEG data were analyzed by comparison with baseline. If the spike amplitude is more than 2 times the baseline, the frequency is greater than 5Hz, and at least 3 spikes that meet this feature appear in a row, it is considered to produce epileptiform discharge. Data were analyzed by calculating the number of events and duration of epileptiform discharges with 1 house as the unit time. The experimental data are represented by mean ± standard error (Mean ± S.E.M) , which were statistically analyzed and plotted by GraphPad prism software using Two-way ANOVA and One-way ANOVA, *P < 0.05 indicates significant difference, **P < 0.01 and ***P < 0.001 indicate a very significant difference. The day on which the dose started is designated as Day 0.

Data FIGs. 14A-14B show that compared with TAM-163, the treatment of TrkB antibody of the present disclosure exhibited reduced epileptiform discharge events and epileptiform discharge duration to a comparable level as the negative IgG control, indicating that less risk of epilepsy would be induced by the TrkB antibody of the present disclosure.

Example 7 Neuroprotection by the TrkB antibodies

A rat retinal ischemia-reperfusion injury (RIRI) model was used to examine the protective effect of the TrkB antibody of the present disclosure on optic nerve injury.

The experimental procedures are briefly described below. To establish RIRI model, the right eye of each experimental animal was chosen as the modeling eye and the left eye was designated as its own control. After animals being immobilized, the right eye was epi-anesthetized with obvacaine eye drops. The infusion assembly consisted of a 27G needle and a saline infusion bottle, connected through an infusion set valve and connecting tubing. Raise the saline infusion bottle to a height of about 190 cm from the animal (higher than the animal's arterial systolic blood pressure) , drain all air bubbles in the tubing, use the 27G needle to pierce the anterior chamber along the temporal angle scleral limbus/cornea, open the infusion set valve, conduct the water pressure into the eye, and confirm the reliability of ischemia with ophthalmoscope. Apply carbomer to both eyes. After 60 minutes of ischemia in the experimental eye, lower the infusion bottle to the animal level position for 10 minutes (to restore the intraocular pressure to normal and restore the blood perfusion of the eye) , close the valve, and pull out the needle. After the operation, use ofloxacin eye ointment to coat both eyes.

According to the amplitude of ERG b wave before modeling, 16 animals that passed quarantine and had no abnormalities in ophthalmic examination were randomly divided into 2 groups. The first intravitreal administration was conducted 24 hrs before modeling by injecting 2μL of solvent or antibody 24B5 into the vitreal cavity of the right eye. 12 animals were enrolled according to the modeling situation, and the day of modeling was counted as D1. The second intravitreal administration was carried out in D15. The dark-adapted full-field electroretinography (ERG) examination was performed before intravitreal administration and 8, 15, 22, 29 and 36 days after modeling. The efficacy was judged by the downward trend of ERG values on days 15, 22, 29 and 36 compared with those on day 8.

Data (FIGs. 15A and 15B) show that the TrkB antibody of the present disclosure has neuroprotective effect in SD rat ocular ischemia-reperfusion model.

Example 8 The therapeutic effect of the TrkB antibodies on SOD1 mouse

SOD1*G93A mice were randomized into groups according to the baseline data of rotarod and hang wire test. After grouping, animals were administered with test articles or vehicle control biweekly via tail vein injection. After start of dose, the rotarod and hang wire test were recorded once per week.

Table 5 Grouping of SOD1*G93A Mice

Hang wire test

The mice were placed on a metal net, and after the mice grasped the grid, the grid was gently inverted to record the hanging time of the mice on the grid. If a mouse hang on the grid for more than 180 seconds, the hanging time was recorded as 180 seconds. This test was repeated for three times, and the average hanging time of three results was calculated as the evaluation value. Once a week. The heavy chain variable region of Anti-TNP is as set forth in SEQ ID NO: 106, and the light chain variable region of Anti-TNP is as set forth in SEQ ID NO: 107.

The body weight change of SOD1 mice is shown in FIG. 16 and the result of hanging wire test is shown in FIG. 17. Data show that compared with Ab419 (the heavy chain variable region of Ab419 is as set forth in SEQ ID NO: 104, and the light chain variable region of Ab419 is as set forth in SEQ ID NO: 105) , the TrkB antibody of the present disclosure does not induce reduction of body weight of the mice. And the TrkB antibody of the present disclosure delays the motor behavioral decline in SOD1 mice.

Example 9 The therapeutic effect on SOD1 mouse of the TrkB antibody

Table 6 Grouping of SOD1*G93A Mice

Accelerated rotarod test

Mice were placed in the behavioral room ahead of time to acclimate for more than 30 min, and then placed on a rotarod apparatus at 0 rpm for 10 s, followed by 4 rpm for 20 s, (if dropped during this period, repositioned. If dropped three consecutive times, presented as 0 s) , the acceleration program was set to gradually accelerate from a rotational speed of 4 rpm to 40 rpm within 5 min, and the time and speed at which the mouse dropped on the rotarod were recorded. Each mouse was tested three consecutive times at 15-min intervals, and the average of three residence times was measured once a week.

Hang wire test

The mice were placed on a metal net, and after the mice grasped the grid, the grid was gently inverted to record the hanging time of the mice on the grid. If a mouse hang on the grid for more than 180 seconds, the hanging time was recorded as 180 seconds. This test was repeated for three times, and the average hanging time of three results was calculated as the evaluation value. Once a week.

The body weight change of SOD1 mouse is shown in FIG. 18, the result of hanging wire test is shown in FIG. 19, and the result of Rotarod test is shown in FIG. 20. Data show that compared with Ab419, the TrkB antibody of the present disclosure does not induce reduction of body weight of the mouse. the TrkB antibody of the present disclosure delays the behavioral decline in SOD1 mice.

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

An antibody or an antigen binding fragment thereof, which specifically binds to TrkB and exhibits at least one of the following properties:

a) is capable of binding to TrkB with a K _D of less than 2.7E-06 M, as measured by Octet;

b) does not bind and/or does not activate human TrkA;

c) does not bind and/or does not activate human TrkC;

d) does not bind and/or does not activate human p75;

e) is capable of activating TrkB;

f) is capable of activating ERK signaling pathway;

g) does not induce the reduction of body weight;

h) is capable of reducing the epileptiform discharge events and epileptiform discharge duration; and

i) has the ability of neuroprotection.
The antibody or the antigen binding fragment thereof of claim 1, wherein said antibody is selected from the group consisting of: a monoclonal antibody, a chimeric antibody, a humanized antibody, a fully human antibody and a bispecific antibody.
The antibody or the antigen binding fragment thereof of any one of claims 1-2, wherein said antigen binding fragment is selected from the group consisting of: a Fab fragment, a Fab’ fragment, a F (ab) ₂ fragment, a Fv fragment, a ScFv and a VHH.
The antibody or the antigen binding fragment thereof of any one of claims 1-3, wherein said TrkB is human TrkB.
The antibody or the antigen binding fragment thereof of any one of claims 1-4, wherein said antibody or the antigen binding fragment comprises at least one Complementarity Determining Region (CDR) of a heavy chain variable region (VH) , said VH comprises the amino acid sequence as set forth in SEQ ID NO: 100 or SEQ ID NO: 102.
The antibody or the antigen binding fragment thereof of any one of claims 1-5, wherein said antibody or the antigen binding fragment comprises at least one CDR of a VH, said VH comprises the amino acid sequence as set forth in any one of SEQ ID NOs: 15-22, 28-30, and 77-82.
The antibody or the antigen binding fragment thereof of any one of claims 1-6, wherein said antibody or the antigen binding fragment comprises at least one CDR of a light chain variable region (VL) , said VL comprises the amino acid sequence as set forth in SEQ ID NO: 101 or SEQ ID NO: 103.
The antibody or the antigen binding fragment thereof of any one of claims 1-7, wherein said antibody or the antigen binding fragment comprises at least one CDR of a VL, said VL comprises the amino acid sequence as set forth in any one of SEQ ID NOs: 23-27 and 31-33.
The antibody or the antigen binding fragment thereof of any one of claims 1-8, which comprises a heavy chain CDR1, and said heavy chain CDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 91 (X ₁YX ₂MH, wherein X ₁ is G, S, or T, X ₂ is T or W) .
The antibody or the antigen binding fragment thereof of any one of claims 1-9, which comprises a heavy chain CDR1, and said heavy chain CDR1 comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 3, 9, and 67.
The antibody or the antigen binding fragment thereof of any one of claims 1-10, which comprises a heavy chain CDR2, and said heavy chain CDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 92.
The antibody or the antigen binding fragment thereof of any one of claims 1-11, which comprises a heavy chain CDR2, and said heavy chain CDR2 comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 93-94.
The antibody or the antigen binding fragment thereof of any one of claims 1-12, which comprises a heavy chain CDR2, and said heavy chain CDR2 comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 4, 10, 39, 43, 70, and 84.
The antibody or the antigen binding fragment thereof of any one of claims 1-13, which comprises a heavy chain CDR3, and said heavy chain CDR3 comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 11 and 95.
The antibody or the antigen binding fragment thereof of any one of claims 1-14, which comprises a heavy chain CDR3, and said heavy chain CDR3 comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 5, 11, 73, 74, 75, 85, 86, 87, 88, 89 and 90.
The antibody or the antigen binding fragment thereof of any one of claims 1-15, which comprises a heavy chain variable region, and said heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 100 or SEQ ID NO: 102.
The antibody or the antigen binding fragment thereof of any one of claims 1-16, which comprises a heavy chain variable region, and said heavy chain variable region comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 15-22, 28-30 and 77-82.
The antibody or the antigen binding fragment thereof of any one of claims 1-17, which comprises a heavy chain constant region, and said heavy chain constant region comprises a human IgG constant region.
The antibody or the antigen binding fragment thereof of any one of claims 1-18, which comprises a light chain CDR1, and said light chain CDR1 comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 96-97.
The antibody or the antigen binding fragment thereof of any one of claims 1-19, which comprises a light chain CDR1, and said light chain CDR1 comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 6, 12, 53, and 64.
The antibody or the antigen binding fragment thereof of any one of claims 1-20, which comprises a light chain CDR2, and said light chain CDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 98 (X ₁X ₂SX ₃RX ₄S, wherein X ₁ is K or W, X ₂ is A or V, X ₃ is N or T, X ₄ is E, F, or L) .
The antibody or the antigen binding fragment thereof of any one of claims 1-21, which comprises a light chain CDR2, and said light chain CDR2 comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 7, 13, and 62.
The antibody or the antigen binding fragment thereof of any one of claims 1-22, which comprises a light chain CDR3, and said light chain CDR3 comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 14, and 99.
The antibody or the antigen binding fragment thereof of any one of claims 1-23, which comprises a light chain CDR3, and said light chain CDR3 comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 8, 14, 61, and 63.
The antibody or the antigen binding fragment thereof of any one of claims 1-24, which comprises a light chain variable region, and said light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 101 or SEQ ID NO: 103.
The antibody or the antigen binding fragment thereof of any one of claims 1-25, which comprises a light chain variable region, and said light chain variable region comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 23-27; and 31-33.
The antibody or the antigen binding fragment thereof of any one of claims 1-26, which comprises a light chain constant region, and said light chain constant region comprises a human Igκ constant region or a human Igλ constant region.
The antibody or the antigen binding fragment thereof of any one of claims 1-27, comprising:

1) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 6, 7 and 8 respectively, and heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 3, 4 and 5 respectively;

2) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 6, 7 and 8 respectively, and heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 3, 70 and 5 respectively;

3) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 6, 7 and 8 respectively, and heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 3, 70 and 85 respectively;

4) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 6, 7 and 8 respectively, and heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 3, 70 and 86 respectively;

5) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 6, 7 and 8 respectively, and heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 3, 70 and 87 respectively;

6) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 6, 7 and 8 respectively, and heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 3, 70 and 88 respectively;

7) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 6, 7 and 8 respectively, and heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 3, 70 and 89 respectively;

8) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 6, 7 and 8 respectively, and heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 3, 70 and 90 respectively;

9) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 6, 7 and 8 respectively, and heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 3, 70 and 50 respectively;

10) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 6, 62 and 63 respectively, and heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 67, 84 and 5 respectively;

11) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 6, 7 and 61 respectively, and heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 67, 70 and 5 respectively;

12) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 6, 7 and 61 respectively, and heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 3, 70 and 73 respectively;

13) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 64, 7 and 61 respectively, and heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 3, 84 and 74 respectively;

14) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 64, 7 and 61 respectively, and heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 3, 84 and 75 respectively;

15) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 6, 7 and 61 respectively, and heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 3, 84 and 75 respectively;

16) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 12, 13 and 14 respectively, and heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 9, 10 and 11 respectively;

17) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 12, 13 and 14 respectively, and heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 9, 39 and 11 respectively; or

18) light chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 53, 13 and 14 respectively, and heavy chain CDR1-3 comprising an amino acid sequence as set forth in SEQ ID NOs: 9, 43 and 11 respectively.
The antibody or the antigen binding fragment thereof of any one of claims 1-28, comprising:

1) a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 23, and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 15;

2) a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 24, and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 16;

3) a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 24, and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 77;

4) a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 24, and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 78;

5) a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 24, and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 79;

6) a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 24, and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 80;

7) a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 24, and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 81;

8) a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 24, and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 82;

9) a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 24, and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 17;

10) a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 26, and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 19;

11) a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 25, and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 18;

12) a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 25, and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 20;

13) a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 27, and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 21;

14) a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 27, and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 22;

15) a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 25, and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 22;

16) a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 31, and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 28;

17) a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 32, and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 29; or

18) a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 33, and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 30.
A fusion protein comprising the antibody or the antigen binding fragment thereof of any one of claims 1-29.
A protein conjugate, comprising the antibody or the antigen binding fragment thereof of any one of claims 1-29, or the fusion protein of claim 30.
Isolated nucleic acid molecule or molecules, encoding for the antibody or the antigen binding fragment thereof of any one of claims 1-29, or the fusion protein of claim 30.
Vector or vectors, comprising the isolated nucleic acid molecule or molecules of claim 32.
A cell, comprising the isolated nucleic acid molecule or molecules of claim 32, or the vector or vectors of claim 33.
A method for producing the antibody or the antigen binding fragment thereof of any one of claims 1-29, or the fusion protein of claim 30, comprising culturing the cells of claim 34 under conditions enabling expression of the antibody or the antigen binding fragment thereof of any one of claims 1-29, or the fusion protein of claim 30.
A composition, comprising the antibody or the antigen binding fragment thereof of any one of claims 1-29, the fusion protein of claim 30, the protein conjugate of claim 31, the isolated nucleic acid molecule or molecules of claim 32, the vector or vectors of claim 33, and/or the cell of claim 34, and optionally a pharmaceutically acceptable excipient.
A kit comprising the antibody or the antigen binding fragment thereof of any of claims 1-29.
Use of the antibody or the antigen binding fragment thereof of any of claims 1-29, the fusion protein of claim 30, the protein conjugate of claim 31, the isolated nucleic acid molecule or molecules of claim 32, the vector or vectors of claim 33, the cell of claim 34, and/or the composition of claim 36 in the manufacture of a medicament for neuroprotection, enhancing cell survival, enhancing neural injury repairing, protecting neural cells from apoptosis and/or necroptosis in neural cells expressing TrkB or promoting sensorimotor function in a TrkB associated condition.
Use of the antibody or the antigen binding fragment thereof of any of claims 1-29, the fusion protein of claim 30, the protein conjugate of claim 31, the isolated nucleic acid molecule or molecules of claim 32, the vector or vectors of claim 33, the cell of claim 34, and/or the composition of claim 36 in the manufacture of a medicament for neuroprotection, enhancing synaptic development, enhancing neurite branching, enhancing cell survival or protecting cells from apoptosis and/or necroptosis.
A method for neuroprotection, enhancing cell survival, enhancing neural injury repairing, protecting neural cells from apoptosis and/or necroptosis in neural cells expressing TrkB or promoting sensorimotor function in a TrkB associated condition in a subject in need thereof, comprising administering to said subject an effective amount of the antibody or the antigen binding fragment thereof of any of claims 1-29, the fusion protein of claim 30, the protein conjugate of claim 31, the isolated nucleic acid molecule or molecules of claim 32, the vector or vectors of claim 33, the cell of claim 34, and/or the composition of claim 36.
The antibody or the antigen binding fragment thereof of any of claims 1-29, the fusion protein of claim 30, the protein conjugate of claim 31, the isolated nucleic acid molecule or molecules of claim 32, the vector or vectors of claim 33, the cell of claim 34, and/or the composition of claim 36, for a) neuroprotection, enhancing cell survival, enhancing neural injury repairing, protecting neural cells from apoptosis and/or necroptosis in neural cells expressing TrkB or promoting sensorimotor function in a TrkB associated condition, and/or b) determining the presence and/or amount and/or activity of TrkB in a sample, wherein said disease or disorder is a disease or disorder associated with an inappropriate expression or function of TrkB.
Use of the antibody or the antigen binding fragment thereof of any of claims 1-29, the fusion protein of claim 30, the protein conjugate of claim 31, the isolated nucleic acid molecule or molecules of claim 32, the vector or vectors of claim 33, the cell of claim 34, and/or the composition of claim 36 in the manufacture of a medicament for preventing and/or treating a disease or disorder, wherein said disease or disorder is neurodegenerative disease, depression, anxiety, autism, schizophrenia, and post-traumatic stress disorder, CNS injuries, stroke and/or traumatic brain injury.
The use of claim 42, wherein said neurodegenerative disease comprises Alzheimer's Disease and related dementias, Parkinson's Disease, Huntington's Disease, Lewy Body Disease and related movement disorders, Amyotrophic lateral sclerosis, glaucoma and/or Friedrich's Ataxia and related Spinocerebellar Ataxia.
A method of preventing and/or treating a disease or disorder, comprising administering the antibody or the antigen binding fragment thereof of any of claims 1-29, the fusion protein of claim 30, the protein conjugate of claim 31, the isolated nucleic acid molecule or molecules of claim 32, the vector or vectors of claim 33, the cell of claim 34, and/or the composition of claim 36 to a subject in need thereof, wherein said disease or disorder is neurodegenerative disease, depression, anxiety, autism, schizophrenia, and post-traumatic stress disorder, CNS injuries, stroke and/or traumatic brain injury.
The method of claim 44, wherein said neurodegenerative disease comprises Alzheimer's Disease and related dementias, Parkinson's Disease, Huntington's Disease, Lewy Body Disease and related movement disorders, Amyotrophic lateral sclerosis, glaucoma and/or Friedrich's Ataxia and related Spinocerebellar Ataxia.