WO2011103667A1 - Agonistic antibodies to trkb receptors and uses thereof - Google Patents

Abstract

The present invention relates to the production of monoclonal antibodies (mAbs) that selectively bind and activate Tropomyosin-related kinase B (TrkB) receptors. An anti-TrkB receptor mAb that specifically binds the D2-D3 domain of TrkB is isolated, produced by the hybridoma cell line having ATCC patent deposit designation 090310-01, and does not bind or activate TrkA, TrkC or p75NTR or interfere with BDNF binding. There are also provided herein pharmaceutical compositions thereof and the use thereof for treating or preventing conditions which require activation of TrkB, such as glaucoma and other ocular neurodegenerative conditions, and for inhibiting neurodegeneration.

Description

AGONISTIC ANTIBODIES TO TRKB RECEPTORS AND USES THEREOF

FIELD OF THE INVENTION

This invention relates to novel monoclonal antibodies that selectively bind and activate TrkB receptors, pharmaceutical compositions thereof and use thereof for treating or preventing conditions which require activation of TrkB and for inhibiting

neurodegeneration.

BACKGROUND OF THE INVENTION

Trk tyrosine kinase receptors are multi-domain single-transmembrane receptors that play an important role in a wide spectrum of neuronal responses including survival, differentiation, growth and regeneration. They are high affinity receptors for neurotrophins, a family of protein growth factors which includes nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophins-4/5 (NT-4/5). The role of TrkB in the central nervous system has been well-characterized. TrkB receptors are widely distributed in the brain and play a key role in neuronal survival, differentiation and neuroregeneration, which has been demonstrated in a number of neurodegenerative models, including stroke, spinal cord injury, optic nerve axotomy, glaucoma and ALS.

BDNF and NT-4/5 are preferred ligands for the TrkB receptor. The receptors for BDNF include TrkB and p75NTR. In adult neurons these two receptors are usually expressed at varying ratios. BDNF binding to the TrkB receptor mediates pro-survival signals, whereas BDNF binding to p75NTR generally mediates pro-death signals, depending on the biological context.

The neuronal cells of the retina, the retinal ganglion cells (RGCs), normally express TrkB but not the p75NTR receptor, which is expressed preferentially on glial cells in normal retina. The expression levels of TrkB and p75NTR can be up-regulated in disease states of the retina, such as optic nerve (ON) axotomy or glaucoma. Experimental ON axotomy is a model of acute injury where the optic nerve is completely severed, causing the RGCs to die and their cell bodies to degenerate very rapidly. The episcleral vein cautery (EVC) experimental glaucoma model is a chronic and progressive neuropathy associated with high intraocular pressure (IOP). The experimental glaucoma model causes a slow and progressive RGC death.

The selective cellular distribution of TrkB and p75NTR receptors on developmentally and functionally different cell types in the retina, prompted the use of BDNF to study neurotrophic mechanisms in experimental animal models of neurodegeneration. Administration of exogenous BDNF can delay RGC death during ON axotomy or glaucoma, however, the pharmacological use of BDNF affords shortlived neuroprotection and requires administration at high doses or high frequency. BDNF has therefore not proven useful in the clinic. Furthermore, as sustained Trk activation leads to long-lived physiological effects in neurons whereas transient Trk activation leads to incomplete or limited physiological effects, the failure of BDNF in vivo may be due to poor activation of retinal TrkB in vivo, to poor pharmacokinetics, or also because BDNF leads to p75NTR activation in glia with release of neurotoxic factors such as pro-neurotrophins or TNF-a.

TrkB agonist antibodies have been described (Qian, M.D. et al, J. Neurosci 2006, 26: 9394-9403; WO 2006/133164) and shown to have neuroprotective and neurotrophic effects on cultured neurons in vitro and to stimulate survival of RGCs in vitro and in vivo (Qian, M.D. et al, J. Neurosci 2006, 26: 9394-9403; Hu, Y. et al, Invest Ophthalmol Vis Sci. 2009 [Epub ahead of print]). However, there is a need for antibodies with greater efficacy, for antibodies which do not compete with BDNF and/or for antibodies which bind or activate TrkB more strongly than BDNF.

There is a need for agents that selectively target TrkB, without binding to p75NTR, and for potent and selective agonists of TrkB.

SUMMARY OF THE INVENTION

The present invention relates to monoclonal antibodies (mAbs) that selectively target TrkB, without binding to p75NTR, including agonist monoclonal antibodies that activate TrkB, pharmaceutical compositions thereof, and use thereof for treating or preventing conditions (including symptoms, disorders, or diseases) which require activation of TrkB, such as diseases involving neurodegeneration.

In accordance with one embodiment of the present invention, there are provided monoclonal antibodies that specifically bind the D2-D3 domain of the TrkB receptor, or a peptide sequence within the D2-D3 domain of TrkB. Fragments, portions, variants or derivatives of the monoclonal antibodies which retain the binding specificity or agonist activity of the full-length antibodies are also provided herein. In an embodiment, the antibodies provided herein specifically bind TrkB, and do not bind the same site on TrkB that BDNF binds or block the binding between BDNF and TrkB .

In another embodiment, the antibodies provided herein can activate TrkB, i.e. can act as agonists of the TrkB receptor. In one aspect, the antibodies provided herein are specific for TrkB and do not bind and/or activate TrkA, TrkC and/or p75NTR receptors. In a further aspect, the antibodies provided herein bind or activate TrkB more strongly than BDNF. The TrkB may be any mammalian TrkB, including but not limited to human TrkB, murine TrkB and rat TrkB.

In yet another embodiment, the antibodies provided herein specifically bind an epitope of TrkB with a sequence comprising the D2-D3 domain of TrkB. The TrkB may be any mammalian TrkB, including but not limited to human TrkB, murine TrkB and rat TrkB.

There is also provided herein a monoclonal antibody that is produced from the hybridoma deposited with the International Depositary Authority of Canada on May 26, 2010 (originally deposited on March 9, 2010) and having accession no. 090310-01 (also referred to herein as 1D7) or from a progenitor cell thereof. In another aspect, antibodies (or fragments, portions, variants or derivatives thereof) binding to the same epitope as the monoclonal antibody produced from the hybridoma having accession no. 090310-01 are provided.

The antibodies of the invention may be humanized or modified in any way which provides benefit without altering the binding properties or the biological activity of the antibodies. Non-limiting examples of fragments, portions, variants or derivatives of the antibodies include single chain antibodies and Fab fragments thereof. A hybridoma that produces a monoclonal antibody according to the invention is also encompassed herein.

There is further provided herein an antibody which comprises complementarity- determining regions (CDRs) of an antibody produced by a hybridoma having ATCC patent deposit designation 090310-01.

In a further embodiment, pharmaceutical compositions comprising the antibodies of the invention or the fragments, portions, variants or derivatives thereof, and a pharmaceutically acceptable carrier, are provided.

In accordance with another embodiment of the invention, there is provided a method of activating TrkB in a subject, comprising administering a therapeutically effective amount of a monoclonal antibody of the invention or a fragment, portion, variant or derivative thereof to the subject, such that TrkB is activated in the subject. In an aspect, the subject is human and the TrkB is human TrkB. In another aspect, the subject suffers from a neurological or neurodegenerative condition which requires activation of TrkB. For example, the subject may have been injured by a wound, surgery, ischemia, infection, a metabolic disease, malnutrition, a malignant tumor or a toxic drug, or may have suffered a stroke, spinal cord injury or an axotomy. In one aspect, the subject suffers from a neurodegenerative disease which is amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease or Huntington's chorea or is an ocular disease, or the subject has suffered an axotomy of the optical nerve. In one embodiment, the subject has glaucoma.

In an aspect, the antibodies of the invention may be administered to a subject parenterally, intravenously, subcutaneously or interperitoneally. In another aspect, the antibodies of the invention may be administered in combination with a second therapeutic agent, such as an agent for lowering intraocular pressure.

In other embodiments, methods for treating glaucoma or for treating or preventing a neurodegenerative condition in a subject, comprising administering a therapeutically effective amount of a monoclonal antibody of the invention, or a fragment, portion, variant or derivative thereof, are provided.

In further embodiments, fragments, portions, variants or derivatives of the monoclonal antibody produced by the hybridoma having ATCC patent deposit designation 090310-01, said fragments, portions, variants or derivatives binding specifically to the same epitope as the monoclonal antibody, are provided herein. The monoclonal antibody produced by the hybridoma having ATCC patent deposit designation 090310-01 or antigen-binding fragments, portions, variants or derivatives thereof may also be humanized, veneered, or chimeric.

In some embodiments, the monoclonal antibody produced by the hybridoma having ATCC patent deposit designation 090310-01 or antigen-binding fragments, portions, variants or derivatives thereof specifically bind TrkB receptor, or may specifically bind TrkB receptor domain D2-D3. In additional embodiments, the monoclonal antibody produced by the hybridoma having ATCC patent deposit designation 090310-01 or antigen-binding fragments, portions, variants or derivatives thereof activate TrkB receptor.

There are also provided herein methods of in vitro screening for an agent which binds to TrkB receptor and can thereby affect TrkB receptor biological activity, comprising combining the antibodies or the fragments, portions, variants or derivatives of the invention with TrkB receptor, in the presence or absence of a candidate agent, and determining whether binding of the antiobodies to TrkB receptor is reduced in the presence of the candidate agent, wherein a reduction in antibody binding in the presence of the candidate agent indicates that said candidate agent binds to TrkB receptor, and can thereby affect TrkB receptor biological activity.

BRIEF DESCRIPTION OF THE DRAWINGS

Particular embodiments of the present invention will now be explained by way of example and with reference to the accompanying drawings, in which:

FIG. 1 shows a FACScan binding profile of mAbs 1D7 (accession no. 090310-01) and 21G3 binding to wild type SY5Y cells or human TrkB-transfected SY5Y cells. Mouse IgG is the negative control. It can be seen that MAb B3 does not bind to native cell surface TrkB whereas mAbs 1D7 and 21G3 bind to cell surface human TrkB. FIG. 2 shows that MAb 1D7 binds to the D2-D3 domain of rat TrkB, wherein: (a) Scheme of the chimeric receptors is shown. TrkB (black) domains were replaced with TrkA (white) domains. These chimeras were expressed in HEK293 cells (Zaccaro MC, et al., J Biol Chem 2001 ;276:31023-31029; Perez P, et al., Mol Cell Neurosci 1995;6:97- 105); (b) Western blots of mAb 1D7 and B3 binding to samples prepared from cells expressing the chimeric receptors are shown. Actin is shown as loading control. MAb 21G3 does not bind in western blots; (c) Cells express functional chimeric receptors, as previously reported (Zaccaro MC, et al., J Biol Chem 2001 ;276:31023-31029; Perez P, et al., Mol Cell Neurosci 1995;6:97-105). Anti-phosphotyrosine western blot of samples prepared from cells expressing the chimeric receptors, after cells were treated or not treated for 15 minutes with 2 nM BDNF, as indicated, is shown.

FIG. 3 shows that mAb 1D7 is a TrkB agonist, wherein: (a) Representative 4G10 anti- phosphotyrosine western blot of whole cell lysates from SY5Y-TrkB cells treated with 2 nM BDNF, 10 nM mAb 1D7, 10 nM mAb 21G3, or 10 nM mlgG is shown. A protein with the Mr of TrkB is recognized, containing phosphotyrosine; (b) Representative 4G10 anti-phosphotyrosine western blot of whole cell lysates from SY5Y or SY5Y-TrkB cells is shown. Cells were treated with 10 nM mlgG, 2 nM BDNF, or 10 nM mAb 1D7. A heterogeneous protein containing phosphotyrosine, consistent with the Mr of TrkB, was recognized only for SY5Y-TrkB cells; (c) Cell survival was studied by MTT assays. SY5Y or SY5Y-TrkB cells were cultured in serum free media to induce cell death. The indicated concentrations of either BDNF, mAb 1D7, or 21G3 were added and further incubated for 3 days. 10% fetal bovine serum was used as positive control 10% (set to 100% survival). Average Optical Density ± sem, n=4.

FIG. 4 shows quantification of surviving RGCs in axotomy and glaucomatous retina, wherein: (a, b) Representative pictures of fluorogold-labeled RGCs after the indicated disease (14 days of axotomy, 42 days of glaucoma) and treatment are shown. Areas denote standardized distances form optic nerve (see Methods); (c,d) summary of RGC survival in ON axotomy and in glaucoma is shown. For each treatment RGC survival was quantified versus normal contralateral control from the indicated number of eyes ± sem (see Methods); (e) Averages of the IOPs measured weekly in rats used for glaucoma experiments are shown, n=18 ± sd. OD = cauterized, OS = naive.

FIG. 5 shows quantification of surviving RGCs in axotomy and glaucomatous retina, wherein: (a) Representative phospho-TrkB western blots of protein extracts from retinas (normal, day 1 axotomy, day 14 glaucoma) collected 6 hours or 18 hours after the indicated treatments, are shown; (b) Quantification of the phospho-TrkB, relative to β- tubulin is shown. Average ± sem, n=4 independent retinas each group. Control IgG- treated samples are standardized as 1.

FIG. 6 shows quantification of retinal structures in vivo, wherein: (a) 1. 3D reconstruction of a rat retina with 600 B-scans taken every 4 mm in the horizontal x and y axis (2.4 mm) is shown. A single isolated B-scan (rectangle) shows the z-axis with the inner segment at the top. 2. Fundus (top view) of the image in (al) is shown. Three volumes were randomly selected in different sectors of the retina at a distance of -1.5 mm from the optic nerve head. 3. From each volume six B-scans were randomly selected. 4. In each B-scan four measurements of the NFL-GCL-IPL (NGI) thickness were completed (yellow arrows). A total of seventy-two measurements per eye were thus performed and averaged. Each group consists of three rats (axotomy or glaucoma ± mAb 1D7 treatment). The normal contralateral eye of each animal serves as control; (b) Representative sections of B-scan images for normal, axotomy ± mAb 1D7 are shown. The images are unmodified and are not enhanced. Note the progressive loss of NGI thickness over time, particularly in the untreated axotomy sample; (c) Axotomy, time- dependent (days) changes to average NGI thickness ± sd, n =3 eyes. Treatment is significant versus untreated control at day 7 (p<0.05), day 10 (p<0.005), day 14 (p<0.001) by t-test; (d) Shows glaucoma, time-dependent (weeks) changes to average NGI thickness ± sd, n =3 eyes. Treatment is significant versus untreated control at week 3 (p<0.02), week 4 (p<0.05), week 5 (p<0.05), week 6 (p<0.02) by t-test. FIG. 7 shows the sequence of the D2-D3 domain of the rat TrkB receptor and of the human TrkB receptor, wherein (A) shows the nucleotide sequence encoding the D2-D3 domain, which includes nucleotides 920-1243 of the rat TrkB cDNA (Accession no. ΝΜ_001163169.1) (SEQ ID NO:l), and (B) shows the peptide sequence of the D2-D3 domain, which possesses amino acids 87-194 of the ratTrkB receptor (SEQ ID NO:2), and (C) shows the nucleotide sequence encoding the D2-D3 domain, which includes nucleotides 1 192-1515 of the human TrkB cDNA (Accession no. AB2091 18.1) (SEQ ID NO: 3), and (D) shows the peptide sequence of the D2-D3 domain, which possesses amino acids 87-194 of the human TrkB receptor (SEQ ID NO:4).

DETAILED DESCRIPTION OF THE INVENTION The present invention provides monoclonal antibodies (mAbs) that selectively target TrkB, without binding to p75NTR. Agonist monoclonal antibodies that activate TrkB, pharmaceutical compositions thereof, and use thereof for treating diseases involving neurodegeneration and or for providing neuroprotection are provided herein.

The present invention is based, at least in part, on the principle that monoclonal antibodies that specifically bind to the TrkB receptor can dimerize TrkB and be sufficient to induce the activation of the receptor, and therefore induce biological responses similar to those mediated by, for example, BDNF. Monoclonal antibodies such as those provided herein can act as agonists that mimic the biological effects of receptor-ligand interactions.

In one aspect, the present invention provides monoclonal antibodies that bind specifically to TrkB. In certain embodiments the antibodies bind to human TrkB. In another embodiment the antibodies bind to rat and/or mouse TrkB. In certain embodiments, the antibodies bind preferentially to human TrkB, and do not bind to TrkA, TrkC and/or p75NTR receptors.

In one embodiment the antibodies provided herein are also agonists of TrkB.

In another embodiment the antibodies provided herein bind to the D2-D3 domain of TrkB, as shown in Fig. 7B. In an embodiment, the receptor site for antibody binding does not overlap with the receptor sites for BDNF binding, that is, the antibodies provided herein and BDNF bind to distinct regions of TrkB and/or do not block each other's binding. In another embodiment, the receptor site for antibody binding does not include the ENLVGED peptide sequence. In yet another embodiment, the receptor site for antibody binding does not include any of the amino acids that are found outside of the D2-D3 domain of TrkB (e.g., the epitopes for the antibodies do not include the amino acids that are found in transmembrane and/or intracellular domains of TrkB, in other regions of the extracellular domain or in the BDNF binding site). In another aspect, the present invention provides any monoclonal antibodies with the properties described herein that bind and/or activate human, rat or mouse TrkB.

In certain embodiments, these antibodies bind and/or activate TrkB with an ED50 in the range of about 10 pM to about 500 nM, for example in the range of about 10 pM to about 1 nM, including in the range of about 10 pM to about 500 pM and the range of about 10 pM to about 100 pM.

In another aspect, the present invention provides monoclonal antibodies with any of the properties described herein that bind one or more specific epitopes within the D2- D3 domain of human TrkB. In yet another aspect, the present provides monoclonal antibodies with any of the properties described herein that bind specifically to the D2-D3 domain of human, rat or mouse TrkB.

In yet another aspect, the present invention provides hybridomas that produce any of the monoclonal antibodies of the invention. For example, the hybridoma that was deposited with the International Depositary Authority of Canada on May 26, 2010 and given accession no. 090310-01 is provided. In still another aspect, the present invention provides the monoclonal antibody produced by the hybridoma that was deposited with the ID AC on May 26, 2010 and given accession no. 090310-01, or antigen-binding fragments thereof. The present invention also provides antibodies that block the binding of this antibody and therefore share the same binding epitope on human, rat or mouse TrkB.

It is to be understood that the monoclonal antibodies of the invention can be prepared by any known method. For example, they can be prepared using synthetic, recombinant or hybridoma technology (e.g., as described in Antibodies: A Laboratory Manual, Ed. by E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1988 or Monoclonal Antibodies: Principles and Practice by J. W. Goding, Academic Press, 1996). In particular it will be appreciated that the antibodies provided herein can be prepared by initially immunizing an animal with human TrkB or a derivative thereof (e.g., a recombinant protein that includes the desired domain of human TrkB, such as recombinant human TrkB D2-D3 domain) and then preparing monoclonals from suitably prepared hybridomas. Those skilled in the art will appreciate that suitable immunogens can be prepared using standard recombinant technology (e.g., see Protocols in Molecular Biology Ed. by Ausubel et al, John Wiley & Sons, New York, NY, 1989 and Molecular Cloning: A Laboratory Manual Ed. by Sambrook et al., Cold Spring Harbor Press, Plainview, NY, 1989, the contents of which are incorporated herein by reference).

In certain embodiments, the immunogen used does not include any of the amino acids that are found outside of the D2-D3 domain of TrkB (e.g., they do not include the amino acids that are found in transmembrane and/or intracellular domains of TrkB, in other regions of the extracellular domain or in the BDNF binding site). Once suitable immunogens have been prepared, the immunogens are injected into any of a wide variety of animals (e.g., mice, rats, rabbits, etc.) and antibodies are prepared using standard, art- recognized techniques.

When using the antibodies provided herein for therapeutic purposes it may prove advantageous to use a humanized or veneered version of the antibody of interest to reduce any potential immunogenic reaction. In general, humanized or veneered antibodies minimize unwanted immunological responses that limit the duration and effectiveness of therapeutic applications of non-human antibodies in human recipients.

A number of methods for preparing humanized antibodies comprising an antigen binding portion derived from a non-human antibody have been described in the art. In particular, antibodies with rodent variable regions and their associated complementarity- determining regions (CDRs) fused to human constant domains have been described, as have rodent CDRs grafted into a human supporting framework region (FR) prior to fusion with an appropriate human antibody constant domain. Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Such antibodies can be produced using transgenic mice that are incapable of expressing endogenous immunoglobulin heavy and light chain genes, but which can express human heavy and light chain genes. It should be understood that partially or completely humanized versions of the antibodies provided herein are encompassed by the present invention. Veneered versions of the antibodies provided herein may also be used in the methods of the present invention. The process of veneering involves selectively replacing FR residues from, e.g., a murine heavy or light chain variable region, with human FR residues in order to provide an antibody that comprises an antigen binding portion which retains substantially all of the native FR protein folding structure. Veneering techniques are based on the understanding that the antigen binding characteristics of an antigen binding portion are determined primarily by the structure and relative disposition of the heavy and light chain CDR sets within the antigen- association surface (e.g., see Davies et al., Ann. Rev. Biochem. 59:439, 1990). Thus, antigen association specificity can be preserved in a humanized antibody only wherein the CDR structures, their interaction with each other and their interaction with the rest of the variable region domains are carefully maintained. By using veneering techniques, exterior (e.g., solvent-accessible) FR residues which are readily encountered by the immune system are selectively replaced with human residues to provide a hybrid molecule that comprises either a weakly immunogenic, or substantially non- immunogenic veneered surface. It should be understood that veneered versions of the antibodies provided herein are encompassed by the present invention.The term "antibody", as used herein, broadly refers to any immunoglobulin (Ig) molecule comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivative thereof, which retains the essential epitope binding features of an Ig molecule. Such mutant, variant, or derivative antibody formats are known in the art. A "monoclonal antibody" as used herein is intended to refer to a preparation of antibody molecules, which share a common heavy chain and common light chain amino acid sequence, in contrast with "polyclonal" antibody preparations that contain a mixture of different antibodies. Monoclonal antibodies can be generated by several novel technologies like phage, bacteria, yeast or ribosomal display, as well as classical methods exemplified by hybridoma-derived antibodies (e.g., an antibody secreted by a hybridoma prepared by hybridoma technology, such as the standard Kohler and Milstein hybridoma methodology ((1975) Nature 256:495-497). In a full-length antibody, each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHI, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy- terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass.

The term "antigen-binding portion" or "antigen-binding fragment" of an antibody (or simply "antibody portion" or "antibody fragment"), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., D2-D3 domain of TrkB). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Such antibody embodiments may also be bispecific, dual specific, or multi-specific formats; specifically binding to two or more different antigens. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')₂ fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341 :544-546, Winter et al., PCT publication WO 90/05144 Al, herein incorporated by reference), which comprises a single variable domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879- 5883). Such single chain antibodies are also intended to be encompassed within the present invention. Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444- 6448; Poljak, R. J., et al. (1994) Structure 2:1121-1 123). Such antibody binding portions are known in the art (Kontermann and Dubel eds., Antibody Engineering (2001) Springer- Verlag. New York. 790 pp. (ISBN 3-540-41354-5).

It should be understood that the antibodies of the invention include fragments, portions, variants or derivatives thereof, such as single-chain antibodies or Fab fragments, that retain the same binding properties (e.g. specificity or affinity) of the full- length antibodies.

The antibodies of the invention also include functional equivalents that include polypeptides with amino acid sequences substantially the same as the amino acid sequence of the variable or hypervariable regions of the antibodies of the present invention. "Substantially the same" amino acid sequence includes an amino acid sequence with at least 70%, preferably at least 80%, and more preferably at least 90% identity to another amino acid sequence when the amino acids of the two sequences are optimally aligned and compared to determine exact matches of amino acids between the two sequences. "Substantially the same" amino acid sequence also includes an amino acid sequence with at least 70%, preferably at least 80%, and more preferably at least 90% homology to another amino acid sequence, as determined by the FASTA search method in accordance with Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85, 2444-8 (1988).

In addition, proteins and non-protein agents may be conjugated to the antibodies by methods that are known in the art. Conjugation methods include direct linkage, linkage via covalently attached linkers, and specific binding pair members (e.g., avidin- biotin). For example, an antibody of the invention may include modifications that retain specificity for the D2-D3 domain of TrkB. Such modifications include, but are not limited to, conjugation to an effector molecule such as another therapeutic agent or conjugation to detectable reporter moieties. Modifications that extend antibody half-life (e.g., pegylation) are also included.

In an embodiment, the antibodies of the invention and/or fragments, portions, varants or derivatives thereof do not bind to the BDNF binding domain in TrkB and/or do not compete with BDNF for binding to TrkB receptor. In another embodiment, the antibodies of the invention and/or fragments, portions, variants or derivatives thereof show greater efficacy than BDNF at activating TrkB receptor, for example the antibody may be more effective than BDNF at activating TrkB and/or may bind the TrkB receptor more strongly than BDNF.

In certain embodiments, the antibodies presented herein are characterized for their binding activities to human TrkB protein (e.g., using ELISA, FACS and/or other methods known in the art). In certain embodiments binding to human TrkB proteins that are expressed on a cell surface may also be assessed (e.g., using cell lines, as described herein and as known in the art). Antibodies may also be tested for their cross-species binding activity; this allows monoclonal antibodies that bind TrkB from more than one species to be identified. In an embodiment, the mAbs bind to both human TrkB and rat TrkB. These antibodies are of interest since they can be tested in animal models with the knowledge that they can also be applied in human clinical trials.

In certain embodiments it may prove advantageous to further characterize the binding properties of any given monoclonal antibody. In particular, one may use a competition assay (e.g., an ELISA) to determine whether the antibodies block the interaction of TrkB and BDNF. One may also assess whether the antibodies bind non- human TrkB and/or human TrkA, TrkC or p75NTR. Mapping of the relative antibody binding epitopes on TrkB (human or other) may also be conducted, e.g., by examining the activity of each individual antibody in blocking the binding of other antibodies to TrkB. For example, the observation that two antibodies block each other's binding suggests these antibodies may bind to the same epitope or overlapping epitopes on TrkB. Methods for mapping epitopes are well-known in the art. In certain embodiments, the antibodies presented herein are characterized for their functional ability to activate TrkB which may be human or non-human TrkB (e.g. murine, rat, chicken, etc). Any agonist assay may be used. The Examples describe MTT- based survival/proliferation assays in cell lines and experiments conducted using rat retinal degeneration models. Other useful assays are known in the art and will be recognized by those skilled in the art.

Pharmaceutical compositions

In one aspect, the monoclonal antibodies provided herein are administered to a subject in order to activate TrkB, in accordance with the present invention. In another aspect, the monoclonal antibodies provided herein are administered in the context of a pharmaceutical composition, that contains a therapeutically effective amount of one or more antibodies together with one or more other ingredients known to those skilled in the art for formulating pharmaceutical compositions. As used herein, the terms "pharmaceutically effective amount" or "therapeutically effective amount" mean the total amount of each active ingredient of the pharmaceutical composition or method that is sufficient to show a meaningful patient benefit, e.g., treatment, prevention or amelioration of a condition which requires TrkB activation. When applied to an individual active ingredient that is administered alone, the term refers to that ingredient alone. When applied to a combination of active ingredients, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.

In certain embodiments of the invention, inventive antibodies are administered with a weekly dose in the range of about 0.1 to about 1000 mg/kg body weight, or about 1 to about 500 mg/kg body weight, in certain embodiments about 10 to about 300 mg/kg body weight. Doses may be administered as a single regimen or as a continuous regimen divided by two or more doses over the course of a day or week. Delivery may be as a bolus or in certain embodiments as a gradual infusion (e.g., by injection over 30 min.). In certain embodiments one or more higher doses (e.g., 2, 3 or 4 fold higher) may be administered initially followed by one or more, lower maintenance doses. The higher dose(s) may be administered at the onset of treatment only or at the beginning of each treatment cycle. These dosage levels and other dosage levels herein are for intravenous or intraperitoneal administration. The skilled person will readily be able to determine the dosage levels required for a different route of administration. It will be appreciated that, in general, the precise dose used will be as determined by the prescribing physician and will depend not only on the weight of the subject and the route of administration, but also on the age of the subject and the severity of the symptoms.

Additional ingredients useful in preparing pharmaceutical compositions in accordance with the present invention include, for example, carriers (e.g., in liquid or solid form), flavoring agents, lubricants, solubilizers, suspending agents, fillers, glidants, compression aids, binders, tablet-disintegrating agents, encapsulating materials, emulsifiers, buffers, preservatives, sweeteners, thickening agents, coloring agents, viscosity regulators, stabilizers or osmo-regulators, or combinations thereof. Liquid pharmaceutical compositions preferably contain one or more monoclonal antibodies of the invention and one or more liquid carriers to form solutions, suspensions, emulsions, syrups, elixirs, or pressurized compositions. Pharmaceutically acceptable liquid carriers include, for example water, organic solvents, pharmaceutically acceptable oils or fat, or combinations thereof. The liquid carrier can contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, colors, viscosity regulators, stabilizers or osmo-regulators, or combinations thereof. If the liquid formulation is intended for pediatric use, it is generally desirable to avoid inclusion of alcohol.

Examples of liquid carriers suitable for oral or parenteral administration include water (preferably containing additives such as cellulose derivatives such as sodium carboxymethyl cellulose), alcohols or their derivatives (including monohydric alcohols or polyhydric alcohols such as glycols) or oils (e.g., fractionated coconut oil and arachis oil). For parenteral administration the carrier can also be an oily ester such as ethyl oleate and isopropyl myristate. The liquid carrier for pressurized compositions can be halogenated hydrocarbons or other pharmaceutically acceptable propellant.

Solid pharmaceutical compositions preferably contain one or more solid carriers, and optionally one or more other additives such as flavoring agents, lubricants, solubilizers, suspending agents, fillers, glidants, compression aids, binders or tablet- disintegrating agents or an encapsulating material. Suitable solid carriers include, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, methyl cellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidine, low melting waxes or ion exchange resins, or combinations thereof. In powder pharmaceutical compositions, the carrier is preferably a finely divided solid which is in admixture with the finely divided active ingredient. In tablets, the active ingredient(s) are generally mixed with a carrier having the necessary compression properties in suitable proportions, and optionally, other additives, and compacted into the desired shape and size.

In some embodiments of the invention, pharmaceutical compositions are provided in unit dosage form, such as tablets or capsules. In such form, the composition is sub-divided in unit dose containing appropriate quantities of the active ingredient(s). The unit dosage forms can be packaged compositions, for example packeted powders, vials, ampoules, pre-filled syringes or sachets containing liquids. The unit dosage form can be, for example, a capsule or tablet itself, or it can be an appropriate number of any such compositions in package form. Thus, the present invention also provides a pharmaceutical composition in unit dosage form for activating TrkB, where the composition contains a therapeutically effective unit dosage of at least one monoclonal antibody of the invention. As one skilled in the art will recognize, the certain therapeutically effective unit dosage will depend on the method of administration. The present invention also provides a therapeutic package for dispensing the monoclonal antibodies of the invention to an individual being treated for a condition which requires TrkB activation. In some embodiments, the therapeutic package contains one or more unit dosages of at least one inventive monoclonal antibody, a container containing the one or more unit dosages, and labeling directing the use of the package for treatment. In certain embodiments, the unit dose is in tablet or capsule form. In some cases, each unit dosage is a therapeutically effective amount.

According to the present invention, monoclonal antibodies of the invention may be administered alone to modulate TrkB activity. Alternatively the antibodies may be administered in combination with (whether simultaneously or sequentially) one or more other pharmaceutical agents useful in the treatment, prevention or amelioration of one or more other conditions (including symptoms, disorders, or diseases) which require TrkB activity. For example, other pharmaceutical agents that can modulate TrkB activity may be used in combination with the monoclonal antibodies of the invention, including other activators of TrkB, including but not limited to BDNF derivatives and compositions.

Additionally or alternatively, the monoclonal antibodies may be used in conjunction with other pharmaceutical agents that are useful in the treatment, prevention or amelioration of neurological disorders and diseases. In certain embodiments, the monoclonal antibodies are combined with agents that are useful in the treatment, prevention or amelioration of disorders and diseases caused by injuries to the nervous system (e.g., by wound, surgery, ischemia, infection, metabolic diseases, malnutrition, malignant tumor, toxic drugs, etc.). It is to be understood that any suitable agent known

th in the art may be used, including those listed in the Physicians' Desk Reference, 55 Edition, 2001, published by Medical Economics Company, Inc. at Monvale, NJ, the relevant portions of which are incorporated herein by reference. In an embodiment, the monoclonal antibodies are combined with IOP-lowering agents, such as those used for the treatment of glaucoma.

In the therapeutic methods provided herein, the monoclonal antibodies may be delivered to a subject using any appropriate route of administration including, for example, parenteral, intravenous, topical, nasal, oral (including buccal or sublingual), rectal or other modes. In general, the antibodies may be formulated for immediate, delayed, modified, sustained, pulsed, or controlled-release delivery.

In certain embodiments, the antibodies are formulated for delivery by injection. In such embodiments, administration may be, for example, intracavernous, intravenous, intra-arterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular or subcutaneous, or via infusion or needle-less injection techniques. For such parenteral administration, the antibodies of the invention may be prepared and maintained in conventional lyophilized formulations and reconstituted prior to administration with a pharmaceutically acceptable saline solution, such as a 0.9% saline solution. The pH of the injectable formulation can be adjusted, as is known in the art, with a pharmaceutically acceptable acid, such as methanesulfonic acid. Other acceptable vehicles and solvents that may be employed include Ringer's solution and U.S. P. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

In order to prolong the effect of the inventive antibody, it may be desirable to slow its absorption from an intramuscular or subcutaneous injection. Delayed absorption of such an administered antibody may be accomplished by dissolving or suspending the agent in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the antibody in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of antibody to polymer and the nature of the particular polymer employed, the rate of antibody release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the antibodies in liposomes or microemulsions which are compatible with body tissues.

For application topically to the skin, the antibodies can be formulated as a suitable ointment containing the active ingredient suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water. Alternatively, they can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. The inventive antibodies can also be administered intranasally or by inhalation and are conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurized container, pump, spray, atomiser or nebuliser, with or without the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, a hydrofluoroalkane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The pressurized container, pump, spray, atomiser or nebuliser may contain a solution or suspension of the antibody, e.g., using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e.g., sorbitan trioleate. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of the antibodies of the invention and a suitable powder base such as lactose or starch.

For oral delivery, such delivery may be accomplished using solid or liquid formulations, for example in the form of tablets, capsules, multiparticulates, gels, films, ovules, elixirs, solutions or suspensions. In certain embodiments, the monoclonal antibodies are administered as oral tablets or capsules. Such preparations may be mixed chewable or liquid formulations or food materials or liquids if desirable, for example to facilitate administration to children, to individuals whose ability to swallow tablets is compromised, or to animals. Compositions for rectal administration are preferably suppositories which can be prepared by mixing the inventive antibodies with suitable non- irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectal vault and release the antibodies. Retention enemas and rectal catheters can also be used as is known in the art. Viscosity-enhancing carriers such as hydroxypropyl cellulose are also certain carriers of the invention for rectal administration since they facilitate retention of the pharmaceutical composition within the rectum. Generally, the volume of carrier that is added to the pharmaceutical composition is selected in order to maximize retention of the composition. In particular, the volume should not be so large as to jeopardize retention of the administered composition in the rectal vault.

A number of techniques have been disclosed for administration of drugs to the eye and are known to the skilled artisan. The compositions of this invention can be administered to the eye, for example by intravenous delivery to the eye, by implantation of a depot comprising a composition of the invention, by injection into the eye or into tissues proximal to the eye, or using any route or means of administration which is suitable. In one embodiment, the route of administration may be intravenous, intraocular, intrasynovial, intramuscular, transdermal and/or oral. Therapeutic Uses

In one aspect, inventive antibodies are useful for treating or preventing conditions (including symptoms, disorders, or diseases) which require activation of TrkB. Such methods involve administering a therapeutically effective amount of one or more of the antibodies provided herein to a subject. In certain embodiments, the invention provides methods for treating neurological conditions, neurodegenerative diseases and/or for providing neuroprotection. For example, and without limitation, the antibodies provided herein may be used to treat individuals with a nervous system that has been injured by wound, surgery, ischemia, infection, metabolic diseases, malnutrition, malignant tumor, toxic drug, etc. Specific examples include stroke, spinal cord injury, traumatic brain injury, retinal degeneration and axotomy. The inventive antibodies may also be used to treat disorders such as attention-deficit hyperactivity disorder (ADHD), depression and age-associated mental impairment (i.e., by providing cognitive enhancement). The inventive antibodies may also be used to treat congenital or neurodegenerative conditions including Alzheimer's disease, Parkinson's disease, Huntington's chorea, amyotrophic lateral sclerosis (ALS) and conditions related to these. The benefits of TrkB activation in treating non- neurological diseases such as cancer and diabetes has also been described and the antibodies provided herein may therefore find utility in such contexts.

In certain embodiments, the antibodies provided herein may be used to prevent or treat ocular neurodegenerative diseases, including but not limited to glaucoma, retinal degeneration, retinitis pigmentosa, axotomy, axotomy of the optic nerve, diabetic retinopathy and macular degeneration.

Virtually all optic neuropathies, such as glaucoma, involve death of retinal ganglion cells (RGCs). In degenerative diseases such as glaucoma, or after optic nerve injury, RGCs fail to regenerate their injured axons and eventually die. The failure of survival and axon regeneration is due in part to a lack of adequate trophic support after injury. For example, death of RGCs can be delayed by application of neurotrophic factors such as BDNF.

Glaucoma refers to a group of eye diseases that can gradually cause an individual to lose his or her sight. It is a relatively common retinal disease characterized by progressive neurodegenerative death of RGCs. This disease can lead to slowly progressive vision loss and, eventually, blindness. Glaucoma is a frequent condition affecting 2% of people over age 40 worldwide. Vision loss is clinically evident when >30% of RGCs have died. High intraocular pressure (IOP) is a risk factor for glaucoma. Current therapeutic options for glaucoma aim to normalize IOP, however the disease remains chronic with continuing RGC death. The most effective IOP lowering drugs only delay vision loss in patients. In addition, some patients have normal tension glaucoma. Preventing neurodegeneration is therefore attractive as a therapy for glaucoma, alone or in combination with IOP-lowering agents. Indeed, a recent study in humans (Pasutto F, et al. American Journal of Human Genetics 2009;85:447-456) indicates a correlation between reduced TrkB activation and disease progression in glaucoma, due to a mutation in the NT-4 gene (which encodes for a TrkB-activating ligand).

Other conditions where it would be desirable to increase TrkB signaling include without limitation regulation of food intake, Rett Syndrome, Huntington's disease and depression. Other such conditions include other neuropathies and neurodegenerative conditions of the eye, such as Stargardt's disease or fundus flavimaculatus, hypertensive retinopathy, occlusive retinopathy or retinal vein occlusion.

As used herein the term "subject" may include animals, such as mammals, such as dogs, cats, cows, pigs, sheep and horses, and human. In a particular embodiment, the subject is a human. In yet another embodiment, the subject is an adult human.

EXAMPLES

The present invention will be more readily understood by referring to the following examples, which are provided to illustrate the invention and are not to be construed as limiting the scope thereof in any manner.

EXPERIMENTAL PROCEDURES Production and characterization of monoclonal antibodies (mAbs) The mouse mAbs were produced through standard hybridoma techniques, as described previously for other agonistic anti-Trk receptor mAbs (LeSauteur L, et al., J Neurosci 1996;16: 1308-1316; Guillemard V, et al.. Dev Neurobiol 2009). Female Balb/c mice (8 weeks old) were immunized with baculovirus recombinant human TrkB ectodomain. Splenocytes were fused to SP2/0 myelomas, and hybridomas were screened by differential binding in an enzyme-linked immunosorbent assay (ELISA) using the original TrkB immunogen (data not shown). Several mAbs were identified in ELISA, and mAbs 1D7 (Accession no. 090310-01), B3, and 21G3 were selected for further study. All mAbs were purified by affinity column chromatography to >95%.

Cell lines

HEK293 stably transfected with rat TrkB were used. For specificity wild type HEK293 cells, or HEK293 stably transfected with either rat p75NTR, human TrkC, human TrkA, or rat TrkA were used (Zaccaro MC, et al., J Biol Chem 2001;276:31023- 31029). The neuronal cell line SY5Y stably transfected with human TrkB cDNA (SY5Y- TrkB) was also used. For specificity wild type SY5Y cells were used (Yan C, et al., Mol Pharmacol 2002;61 :710-719).

Antibody Binding FACScan Assays

Characterization of mAbs 1D7, B3, and 21G3 binding to the cell surface was done by FACScan assays using live cells, as described (LeSauteur L, et al., J Neurosci 1996;16:1308-1316). Cells (2.5 x 105) in 0.1 ml of binding buffer (Hanks' Balanced Salt Solution (HBSS), 0.1% bovine serum albumin (BSA), and 0.1% NaN3) were incubated with primary mAbs (7nM) for 30 min at 4°C and washed in binding buffer, followed by incubation with FITC-conjugated goat anti-mouse secondary antibody for 30 min at 4°C. As negative controls, no primary (background fluorescence), or irrelevant mouse IgG primary (Sigma) were used. Cells were acquired and analyzed on a Fluorescent Activated Cell Scanner (FACScan) (Becton Dickinson, San Jose, CA) using the Cell Quest program. FACscan reveals specific binding to native cell surface receptors. MAbs 1D7 and 21G3 bound cell surface TrkB and were thus used in biological assays, but mAb B3 did not bind to native cell surface receptors.

Functional Assays for TrkB Agonist Activity The biological properties of the ligands were measured by quantification of TrkB tyrosine phosphorylation by western blot after treatment of cell lines with ligands, and by effects on MTT-based survival/proliferation assays.

The tyrosine phosphorylation of TrkB was studied after treatment of cells in culture with TrkB ligands mAb 1D7, mAb 21G3, control mouse IgG, or control BDNF (each at 10 nM) for 12 minutes at 37°C. Then cells were solubilized and protein concentrations were determined with Bio-Rad Detergent Compatible Protein Assay (Bio- Rad). Western blot analysis was performed as described (Maliartchouk S, et al., J Biol Chem 2000;275:9946-9956) with anti-phosphotyrosine mAb 4G10, or with anti- phospho-TrkB serum (a kind gift of Dr. Moses Chao), and the enhanced chemiluminescence system (PerkinElmer Life Sciences). Anti-actin antibody (Sigma) confirmed equal protein loading. Tyrosine phosphorylation of TrkB provides a direct measure of TrkB activation (Atwal JK, et al., Neuron 2000;27:265-277).

For survival assays, SY5Y-wt or SY5Y-TrkB cells (10,000 cells/well) in serum- free media (PFHM-II; Gibco; supplemented with 0.2% BSA) were added to 96-well plates (Falcon, Lincoln Park, NJ) ± mAb 1D7, mAb 21G3, mouse IgG as negative control, or BDNF as positive control (each at 10 nM), or serum (final 10% FBS) as normal culture conditions. Cell survival was quantified spectrophotometrically at 595 nm with 4-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT; Sigma) 48-72 hours after plating, as described (Zaccaro MC, et al., J Biol Chem 2001 ;276:31023- 31029).

Animals and Anesthesia

All animal work respected the guidelines set out by the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and institutional IACUC protocols. Wistar female rats (250-300 g, Charles River) were housed in a 12 hour dark-light cycle with food and water ad libitum. Deep anesthesia (ketamine, xylazine, acepromazine injected intraperitoneally; 50/5/1 mg/kg, as per IACUC recommendations) was used for ECV (glaucoma), ON axotomy, fiuorogold labeling, intraocular injection procedures, FD-OCT measurements, and euthanasia. For measuring IOP light anesthesia was used (a gas mixture of Oxygen, 2% isofluorane mixture, at a rate of 2.5 liter/min, as per IACUC recommendations) . Rat Retinal Degeneration Models

Optic nerve transaction model. The procedure was as described (Lebrun-Julien F, et al., Mol Cell Neurosci 2009;40:410-420; Shi Z, et al., J Biol Chem 2008;283:29156-29165). In brief, a 1.5-2.0 cm skin incision was made along the edge of the right orbit bone; lachrymal glands, orbital fats were excised and extraocular muscles were separated to expose the optic nerve. An 18G needle was used to lacerate the sheath longitudinally in order to not disturb the ophthalmic artery; the ON parenchyma was then separated out and lifted by a homemade hook, and then completely transected 0.5-1.0 mm posterior to the eye ball with the micro-tweezers. At seven days and fourteen days post-axotomy, respectively, -50% and -90% RGCs die (Lebrun-Julien F, et al., Mol Cell Neurosci 2009;40:410-420).

Glaucoma model. The episcleral vein cauterization (EVC) model of glaucoma (Shi Z, et al., J Biol Chem 2008;283:29156-29165) is validated in comparative studies (Urcola JH et al., Exp Eye Res 2006;83:429-437). Radial incisions were made in conjunctiva and three of the episcleral veins (two dorsal episcleral veins located near the superior rectus muscle and one temporal episcleral vein located near the lateral rectus muscle) were cauterized with a 30" cautery tip. The contralateral control eyes had sham-surgery to only isolate the three veins but without cauterization.

Intraocular pressure (IOP) was measured immediately after the EVC surgery and every week until the endpoint of each experiment. The mean normal IOP of rats under light anesthesia was measured by Tonopen XL applanation tonometer immediately after the EVC surgery and every week until the endpoint of each experiment. Normal IOP was 10-14 mm Hg, while after cauterization the IOP was elevated to 18-23 mm Hg (-1.7 fold in -90% of the rats) throughout the duration of the experiment. The high IOP is chronic and causes progressive disease, and after six weeks of disease -70% RGCs remain alive (Shi Z, et al, Dev Neurobiol 2007;67:884-894; Shi Z, et al., J Biol Chem 2008;283:29156-29165).

IOP measurements. The mean normal IOP of rats under light anesthesia was 10-14 mm Hg, while in glaucoma model the IOP was elevated -1.7 fold in -90% of the rats (range 18 - 23 mm Hg) throughout the duration of the experiment. Stable and chronic high IOP in this model has been published (Shi Z, et al., Dev Neurobiol 2007;67:884-894; Shi Z, et al., J Biol Chem 2008;283:29156-29165). Intraocular pressure (IOP) was measured by Tonopen XL applanation tonometer (Buckingham BP et al., J Neurosci 2008;28:2735- 2744; Danias J, et al., Invest Ophthalmol Vis Sci 2003;44:1138-1141) immediately after the EVC surgery and every week until the endpoint of each experiment. In our experiments, 4 consecutive readings obtained from each eye with a coefficient of variation <5%, and the average number was taken as the IOP for the day. In this high IOP animal model of glaucoma, a chronic and stable elevated IOP of ~1.7-fold over normal is achieved. IOP data was reported previously in many similar studies (Shi Z, et al., Dev Neurobiol 2007;67:884-894; Shi Z, et al., J Biol Chem 2008;283:29156-29165). Approximately 90% of the cauterized eyes experienced sustained elevation of IOP. Animals (and data) were discarded in the rare cases in which the retinal vasculature showed signs of ischemia, when an average IOP of < 1.4-fold (too low) or >2.8-fold (too high) in the cauterized eyes occurred at any point during the study, and in the rare cases in which the animals had cataracts.

Intraocular Injections and Drug treatment

A 30G needle was used for intraocular injections. The needle was injected at a 45° angle 2 mm behind the cornea-scleral limbal until all the bevel of the needle was inserted into the vitreous body, without damaging the lens. The whole procedure was finished in 2 minutes. After the injection, the needle was left in place for another minute to allow dispersion of the compound into the vitreous. The experimental eyes were injected with test agent or control vehicle while the contralateral eyes served as normal uninjected controls. Our previous publications showed that normal contralateral eyes are not different from each other when they remain uninjected or receive control PBS injections (Shi Z, et al., Dev Neurobiol 2007;67:884-894; Shi Z, et al., J Biol Chem 2008;283:29156-29165).

Drug treatments were done with the experimenters blinded to the treatment code. For the glaucoma model the intraocular injections were performed at days 14 and 21 after cauterization; and the endpoint was at day 42 of high IOP. Thus, in this paradigm, before treatment there is pre-existing damage for 14 days (causing -8% RGC death). For the axotomy model the intraocular injections were performed within 5 min of injury; and the endpoint was at day 7 or day 14 after ON transection. Recombinant BDNF and anti- TrkB mAbs 1D7 and 21G3 were prepared in PBS. Intraocular injections in retinas used for therapeutic assays (endpoints at 14 days in ON axotomy; and 42 days in glaucoma) delivered 3 μΐ with 3 μg of compound. Intraocular injections in retinas used for biochemical assays (endpoints at 6 hr or 18 hr) delivered 3 μΐ with 1 μg of compound. Fluorogold Retrograde Labeling

RGCs were retrogradely labeled with a 4% Fluorogold solution (Fluorochrome, Englewood, CO) applied bilaterally to the superior colliculous (SC) (Lebrun-Julien F, et al., Mol Cell Neurosci 2009;40:410-420; Shi Z, et al., Dev Neurobiol 2007;67:884-894; Shi Z, et al., J Biol Chem 2008;283:29156-29165). Briefly, rats were mounted on stereotactic apparatus (Kopf Instruments, Tujunga, CA.), holes were drilled at a position 1.3 mm lateral to the sagital suture and 2.5 mm anterior to the lambda suture on each side, and fluorogold (3 μΐ) was injected into the SC at the depth of 6.0 mm below the skull. Then, the holes were filled with gelfoam soaked in 4% Fluorogold. In the glaucoma model, retrograde labeling was performed at day 35 after ocular hypertension (7 days before the experimental endpoint), while in the axotomy model retrograde labeling was carried out 7 days before optic nerve transection (14 or 21 days before the experimental endpoint). These times afford excellent labeling efficacy and are practical and compatible with experimental procedures, including the long-lived glaucoma model (6 weeks).

In vivo TrkB phosphorylation

Rats (n= 4 per group) received 1 μg of the indicated treatment. In axotomy drugs were injected within 5 min after ON transection. In glaucoma, drugs were injected after fourteen days of high IOP. Six hours or eighteen hours after drug treatment retinas were dissected and lysed in 80 μΐ SDS-PAGE sample buffer containing 2% SDS. After SDS- PAGE and transfer, membranes were western blotted with rabbit antisera to TrkB-pTyr (a gift of Moses Chao), followed by goat anti-rabbit secondary antibodies conjugated to horseradish peroxidase (Sigma) at a 1 :10,000 dilution. Loading was controlled with antibodies to β-tubulin (Sigma). Detection was by ECL. For digital quantification, membranes were scanned and analyzed using ImageJ software.

RGCs Survival Quantification and Statistical Analysis Quantification of surviving RGCs was performed as reported previously (Lebrun- Julien F, et al, Mol Cell Neurosci 2009;40:410-420; Shi Z, et al., Dev Neurobiol 2007;67:884-894; Shi Z, et al., J Biol Chem 2008;283:29156-29165). At day fourteen of ON axotomy or at day forty-two of glaucoma both eyes were enucleated; the anterior parts were cut out, and the remaining part was fixed in 4% paraformaldehyde (PFA) for 30 minutes. Then, retinas were flat-mounted on a glass slide and dissected by four radial cuts to facilitate flattening of the retinas into a Maltese cross shape, with the vitreous side up. Pictures for each freshly flat-mounted retina were taken using a Zeiss fluorescence microscope (Carl Zeiss Meditec, Jena, Germany), with 12 pictures/retina at 20X magnification. For each quadrant there were 3 pictures at the distance of 1 mm, 2 mm, 3 mm radially from the optic nerve (indicated as areas 1, 2, and 3). Microglia and macrophages which incorporated Fluorogold after phagocytosis of dying RGCs were excluded according to their morphology, as previously reported (Lebrun-Julien F, et al., Mol Cell Neurosci 2009;40:410-420; Shi Z, et al., Dev Neurobiol 2007;67:884-894; Shi Z, et al., J Biol Chem 2008;283:29156-29165).

In all cases, manual RGC counting was performed by two independent persons. One person was the experimental performer, blinded to drug treatment code, the second person was unrelated to the experiment and was blinded to the whole protocol. Standardization of % RGCs survival in each rat were calculated as the ratio of the experimental right eye (OD) versus control left eye (OS) (RGC_eXperimentai/RGC_COntraiaterai)- The mean ± sem were calculated for all the % RGCs survival data, for each experimental group (untreated, PBS, BDNF, 1D7, 21G3). Data analysis was performed using GraphPad Prizm 5 software (GraphPad Software Inc., San Diego, CA). Comparison between the RGCs survival rate is using one-way ANOVA with Dunnett's multiple comparison test, and p<0.05 was considered statistically significant.

Fourier Domain Optical Coherence Tomography (FD-OCT)

A non-invasive prototype spectrometer based FD-OCT system was used to acquire the retinal images. FD-OCT is a non-invasive method that allows time-kinetic studies in the same animal, with axial resolution in tissue nominally better than 4 μιη, and repeatability of the measurements from B-scans better than 1 μπι. Data acquisition was performed using custom software written in C++ for rapid frame grabbing, processing, and display of two-dimensional images. Manual segmentations were used to measure the thickness of the rat retinas in glaucoma and axotomy experiments. After anesthesia, the rats were placed on a homemade rack, and the head was oriented to an angle where the eye was properly aligned to the optical beam. The pupils were dilated using a topical solution (Atropine sulphate 1%, Alcon). Refraction of light at the cornea was cancelled by placing over the eye a flat coverslip coated with a generic artificial tear gel. Alignment of the optical system to the rat retina required a few minutes, and was followed by rapid acquisition of data, -5 seconds per volume. During retinal scanning, three volumes were acquired in different sectors of the retina containing the ON head and retinal blood vessels as landmarks. This is sufficient to comprise most of the retina. The volumes can be rendered in 3D, or visualized en face as a fundus image. In post processing, six B-scans were randomly selected from each volume. The retinal thickness measurements were performed with ImageJ software, using the saved data. In each B- scan the thickness of the NFL-GCL-IPL (NGI) was measured at four adjacent points at a distance nominally 1.5 mm from the ON head.

EXAMPLE 1. Generation and Characterization of anti-TrkB mAbs 1D7 and 21G3

Initial screening of hybridomas was done by ELISA binding studies (data not shown). Clones mAb 1D7, mAb 21G3, and mAb B3 were selected for FACScan binding assays using live cells.

The mAb 1D7 and mAb 21 G3 bind to SY5Y cells stably transfected to express human TrkB (SY5Y-TrkB), but do not bind to wild type SY5Y cells above background control mouse immunoglobulin (FIG. 1). On fluorescent units represented on a logarithmic scale, the mean can be measured from bell-shaped histograms. The mean fluorescence of SY5Y-TrkB cells exposed to background mouse Ig is ~8 units, and when exposed to mAb 1D7 or mAb 21G3 the mean fluorescence increases to -40 units and -150 units, respectively. In controls, the mean of wild type SY5Y cells exposed to background mouse Ig is -4 units, and when exposed to mAb 1D7 or mAb 21G3 the mean is -8 units.

In further tests, the mAb 1D7 and mAb 21G3 also bind to HEK293 cells transfected with rat TrkB; but do not bind to HEK293 cells transfected with either rat p75 , human TrkC, human TrkA receptors, or rat TrkA receptors (Table 1). Because the cells are intact, the FACScan data indicate that mAb 1D7 and mAb 21G3 bind to native TrkB ectodomain expressed on the cell surface. The data also indicate that mAb 1D7 and mAb 21G3 can bind selectively to TrkB, and that they can bind both human TrkB as well as rat TrkB. In contrast, the mAb B3 does not bind to any of the cells in FACScan, indicating that it does not recognize native cell surface receptor.

Further work aimed to characterize the binding site of these mAbs on the TrkB ectodomain. MAb 1D7 and mAb B3 recognize denatured TrkB in western blots when samples are resolved in non-reduced SDS-PAGE. In contrast, mAb 21G3 does not recognize denatured TrkB under any condition and can not be studied using western blotting.

To identify the TrkB domain where mAb 1D7 and mAb B3 bind, we studied HEK293 cells expressing transfected chimeric receptors. In these transfectants a domain of rat TrkA was spliced in to replace the corresponding domain in rat TrkB (Zaccaro MC, et al., J Biol Chem 2001 ;276:31023-31029; Perez P, et al., Mol Cell Neurosci 1995;6:97-105)(FIG. 2a). MAb 1D7 does not bind chimeras 2.1 and 2.2, meaning that it recognizes the D2-D3 domain(s) of rat TrkB. MAb B3 does not bind chimeras 2.2 and 3.1, meaning that it recognizes the D4 domain of rat TrkB (FIG. 2b).

The expressed chimeric receptors are functional, as shown in phospho-tyrosine western blots after treatment of live cells with 2 nM BDNF (FIG. 2c). The 3.1 chimera is activated without ligand, as previously reported (Zaccaro MC, et al., J Biol Chem 2001 ;276:31023-31029; Perez P, et al., Mol Cell Neurosci 1995;6:97-105). The chimeras 2.1 and 2.2, not recognized by mAb 1D7, can be fully activated by BDNF.

These data indicate that the receptor sites for BDNF binding and activation are not overlapping with the receptor sites for mAb 1D7 binding. This notion is further supported by our observations that pre-binding of BDNF to cell surface TrkB does not inhibit mAb 1D7 binding to TrkB.

Table 1. Summary of FACScan data.

SY5Y 4 9 8 9

SY5Y-TrkB (human) 8 44 153 1 1

HEK293 10 14 15 15

HEK293-TrkB (rat) 9 93 186 13

HEK293-TrkA (rat) 6 9 12 11

HEK293-TrkA (human) 4 7 9 10

HEK293-TrkC (human) 8 9 5 1 1

HEK293-p75^{N ,K} (rat) 8 8 11 10

FACScan binding profile of the three anti-TrkB mAbs 1D7, 21G3, and B3 (or control mouse Ig) binding to the indicated cell lines. Specificity controls are the untransfected SY5Y parental cells, or untransfected HEK293 parental cells. Data shown are the mean channel fluorescence, from bell-shaped histograms as in Figure 1, with 5,000 cells analyzed. Numbers in bold indicate increases over background and over control cells. EXAMPLE 2. Anti-TrkB mAb 1D7 is an agonist

Functional assays were done with mAb 1D7 and mAb 21G3 because they recognize native cell surface receptors. These functional studies were not carried out with MAb B3 because it does not recognize native TrkB on the cell surface, and therefore cannot be studied functionally.

Treatment of SY5Y-TrkB cells with mAb 1D7 (10 nM) or BDNF (10 nM) induces tyrosine phosphorylation of TrkB. However, treatment with mAb 21G3 (10 nM) or with control mouse Ig does not induce TrkB tyrosine phosphorylation (FIG. 3a). Therefore, mAb 1D7 is a TrkB ligand that activates this receptor. In additional assays, treatment of control wild type SY5Y cells with mAb 1D7 (10 nM) or BDNF (10 nM) does not result in the detection of phospho-TrkB (FIG. 3 b), as expected because these cells do not express TrkB.

To further measure agonism, functional survival assays using wild type SY5Y cells or SY5Y-TrkB cells were performed. These cells die by apoptosis when placed in serum-free conditions, but they can be protected from death by supplementation with trophic support. MAb 1D7 and BDNF support SY5Y-TrkB cell survival, in a dose- dependent manner. In contrast, mAb 21G3, and mouse Ig do not support SY5Y-TrkB cell survival.

In specificity controls, neither the mAbs nor BDNF can support the survival of wild type SY5Y cells. As a further positive survival control, supplementing serum-free cultures with 10% serum supports the survival of both SY5Y-TrkB and wild type SY5Y cultures (FIG. 3c).

Together, the data indicate that mAb 1D7 and mAb 21G3 bind to native TrkB on the cell membrane. While mAb 1D7 is agonistic to TrkB, mAb 21G3 is biologically inert. This pair of TrkB ligands (one agonistic and one inert) were tested in vivo, and they were compared to BDNF which is an agonist of TrkB and p75^NTR.

EAMPLE 3. Anti-TrkB mAb 1D7 supports long-lasting RGC survival in glaucoma and ON axotomy

We compared in vivo the neuroprotective effect of the mAbs as TrkB ligands, versus BDNF or PBS controls. Equal protein concentrations of test agents were injected intravitreally (3 μg/3 μΐ), because little is known about pharmacokinetics, bioavailability, or biodistribution within retina. However, since mAbs have greater molecular weight they likely result in a lower retinal molarity than BDNF. The concentration of BDNF was selected from doses reported to be efficacious (Pease ME, et al., Invest Ophthalmol Vis Sci 2000;41 :764-774; Cheng L, et al., J Neurosci 2002;22:3977-3986; Pernet V and Di Polo A, Brain 2006;129: 1014-1026; Peinado-Ramon P, et al., Invest Ophthalmol Vis Sci 1996;37:489-500; Ko M, et al., Invest Ophtalmol Vis Sci 2000;41 :2967-2971 ; Martin KR, et al., Invest Ophthalmol Vis Sci 2003;44:4357-4365; Quigley HA, et al., Invest Ophthalmol Vis Sci 2000;41 :3460-3466). We quantified whether treatments promote RGC survival in vivo. In a rat model of ON axotomy a single intravitreal injection of test agents or PBS control was performed immediately after axotomy, and RGCs were quantified at the fourteen day endpoint. Representative micrographs of retinas show the labeled RGCs (FIG. 4a). From these pictures we quantified the % surviving RGCs after ON axotomy (FIG. 4c), versus the contralateral naive eye.

In the untreated axotomy group 1 1.3 ± 1.5% of the RGCs remain alive. Injection of PBS has no effect and 12.2 ± 1.1 of the RGCs remain alive. BDNF (14.6 ± 1.3% RGC survival) and mAb 21G3 (15.7 ± 1.4% RGC survival) did not prevent RGC death compared to the PBS control group. However, mAb 1D7 afforded statistically significant neuroprotection, with 27.0 ± 1.8% RGCs remaining alive (P<0.0001 versus PBS control group) (FIG. 4c). In a rat model of glaucoma, a total of two intravitreal injections of test agents or PBS control were performed at day fourteen and day twenty-one of glaucoma, and RGCs were quantified at the forty-two day endpoint glaucoma (i.e. twenty-one days after the last treatment). Representative micrographs of retinas show the labeled RGCs (FIG. 4b). From these pictures we quantified the % surviving RGCs after glaucoma (FIG. 4d), versus the contralateral naive eye.

The IOP measured in the rats used for the glaucoma studies show sustained IOP elevation in the cauterized eyes (FIG. 4e). Cauterization causes a ~ 1.7-fold stable increase in intraocular pressure (IOP), that remains chronic for many weeks. It is noteworthy that in this paradigm there is pre-existing RGC loss of -8-12% before treatment was applied at days fourteen and twenty-one of glaucoma. Moreover, the retina endured constant stress because the measured IOP remained high throughout the experiment.

In the untreated glaucoma group 73.6 ± 1.2% of the RGCs remain alive. Injection of PBS has no effect and 76.3 ± 1.3% of the RGCs remain alive. BDNF (76.5 ± 1.5% RGC survival) and mAb 21G3 (72.9 ± 1.8% RGC survival) did not prevent RGC death compared to the PBS control group. However, mAb 1D7 afforded statistically significant neuroprotection, with 86.0 ± 1.9% labeled RGCs remaining alive (P<0.0001 versus PBS control group) (FIG. 4d).

EXAMPLE 4. Long-lived RGC survival correlates with long-lasting TrkB activation

Functional activation of TrkB in vivo was tested by measuring receptor phosphorylation. Normal, axotomized or glaucomatous eyes were injected intravitreally with BDNF, mAb 1D7, or mlgG control. Retinal protein extracts were analyzed either six or eighteen hours later for TrkB tyrosine phosphorylation using a specific anti- phospho-TrkB rabbit antisera (a gift of Dr. Moses Chao).

At the six-hour point, treatment with BDNF and mAb 1D7 significantly increased TrkB tyrosine phosphorylation compared to mlgG control. However, at the eighteen- hour point only mAb 1D7 significantly increased TrkB tyrosine phosphorylation compared to mlgG control (FIG. 5a, data quantified in FIG. 5b). Comparable data were obtained by using a generic anti-phosphotyrosine mAb 4G10 (data not shown).

Thus, although both BDNF and mAb 1D7 induce pTyr-TrkB, only mAb 1D7 can induce sustained pTyr-TrkB and can protect RGCs from death in retinal diseases.

EXAMPLE 5. A selective TrkB agonist maintains retinal structures in Glaucoma and ON axotomy

We measured structural changes using FD-OCT, a non-invasive method that allows time-kinetic studies of retinas degenerating after axotomy or glaucoma (FIG. 6). In the normal rodent retina the RGCs form a single layer (the GCL), bracketed by the nerve fiber layer (NFL) and the inner plexiform layer (IPL). With FD-OCT it is possible to quantitatively measure the combination of the NFL+GCL+IPL (herein termed "NGI") layers. The NGI is relevant because it is where the RGC soma and the projecting RGC fibers are located.

The experimental protocol is described in FIG. 6a. The combined thickness of

NGI in normal retinas is 71 ± 0.6 μπι. Fourteen days after axotomy the NGI thickness is 54.7 ± 1.2 μιη, while in the mAb lD7-treated group it is 60.8 ± 0.3 μιη (significant versus untreated axotomy, <0.001) (representative data in FIG. 6b, summarized in FIG. 6c). Forty-two days after glaucoma the NGI thickness is 51.2 ± 2.6 μηι, while in the mAb lD7-treated group it is 60.4 ± 0.1 μπι (significant versus untreated glaucoma, p<0.02) (FIG. 6d).

Thus, mAb 1D7 treatment significantly protects the structure of the retinal neuronal layers in both acute (ON axotomy) and chronic (glaucoma) pathologies. However, mAb 1D7 treatment does not protect the NGI fully. At the experimental endpoints (day fourteen for ON axotomy, day 42 for glaucoma) the NGI of mAb 1D7 treated rats is significantly thinner than normal retinas ( <0.0001). This is not surprising given that these eyes endured continuous stress and had a low treatment frequency and dose. The above Examples show that a selective TrkB agonist affords long-lived TrkB activation, and delayed RGC death in models of acute and chronic retinal injury in vivo. Importantly, using non-invasive retinal imaging it is also shown that a selective TrkB agonist affords preservation of the retinal structure in both animal models, with maintenance of the layers comprising neurons and neuronal fibers. Thus in animal models of both acute and chronic neurodegeneration a TrkB agonist affords long-lasting neuroprotection, by causing sustained TrkB activation.

The results shown in the Examples indicate that the novel anti-TrkB monoclonal antibodies presented herein can be applied in vivo to the neurodegenerating retina and that the mAbs will be important tools in the clinic and for further research.

The potency of mAb 1D7 as a functional agonist of TrkB in ex vivo assays is equivalent to BDNF on a molar basis. However, mAb 1D7 binds to TrkB at a site distinct from BDNF. Because mAb 1D7 recognizes denatured TrkB in western blots, it is likely that it recognizes a linear or stable epitope on the receptor. The data indicates that the epitope is comprised within the D2-D3 domain.

Not all mAbs that are directed to the TrkB ectodomain are functionally active. This could be a consequence of each mAb having different binding sites and different domains. However, one property that all mAbs share is multivalency, hence the potential to induce receptor dimerization. In the case of functionally inert mAb 21G3 any putative TrkB dimerization it might induce is non-functional in terms of receptor pTyr, and in terms of neuronal survival ex vivo and in vivo. Testing an inert TrkB ligand such as mAb 21G3 in vivo is an important variable that controls for possible ligand- induced receptor dimerization. It also controls for non-specific effects because in vivo cell-bound antibodies can activate immune pathways such as opsonization of complement fixation.

Although both BDNF and mAb 1D7 induce pTyr-TrkB, only mAb 1D7 can protect RGCs from death in retinal disease. The pharmacological efficacy of mAb 1D7 may be due, at least in part, to its causing a long-lived TrkB activation, resulting in long-lived physiological effects. Trk receptors are activated with very long kinetics compared to other receptor tyrosine kinases. Sustained Trk activation leads to long-lived physiological effects in neurons even after the agonists and the activated receptors have been cleared. In contrast, the failure of BDNF may be due to its causing a short-lived TrkB activation, because it is known that transient Trk activation leads to incomplete physiological effects. It is noted that in the Examples above, the mAbs were administered at an 8-fold lower molar dose than BDNF.

An important difference between BDNF and the antibodies of the invention is that BDNF binds and activates p75^NTR, whereas mAb 1D7 does not. Activation of p75 may be undesirable given that p75 is up-regulated in disease states. BDNF activation of p75^NTR may cause glial release of neurotoxic pro-neurotrophins or TNF-a, and could compromise any benefits of TrkB activation.

In the above examples, the quantitative structural data obtained with FD-OCT correlated well with RGC loss during glaucoma progression. At forty-two days of glaucoma there is a close correlation between loss of structural integrity (-30% loss of thickness) and cellular loss (-30% RGC death). Neuroprotection with mAb 1D7 results in the survival of approximately half of the RGCs that would have died, and the maintenance of approximately half of the NGI thickness that would have been lost. Thus, the use of FD-OCT to measure preservation of retinal structure may be a predictive endpoint for neuroprotective efficacy in glaucoma.

The contents of all documents and references cited herein are hereby incorporated by reference in their entirety.

Unless defined otherwise or the context clearly dictates otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It should be understood that any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention.

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.

Claims

What is claimed is: 1. A monoclonal antibody that specifically binds the D2-D3 domain, a peptide within the D2-D3 domain, or a peptide comprising the D2-D3 domain, of TrkB.

2. A fragment, portion, variant or derivative of the monoclonal antibody of claim 1, wherein said fragment, portion, variant or derivative specifically binds the D2-D3 domain, a peptide within the D2-D3 domain, or a peptide comprising the D2-D3 domain, of TrkB.

3. A monoclonal antibody that specifically binds TrkB, wherein the antibody does not bind the same site on TrkB that BDNF binds or block the binding between BDNF and TrkB.

4. A fragment, portion, variant or derivative of the monoclonal antibody of claim 3, wherein said fragment, portion, variant or derivative of the monoclonal antibody specifically binds TrkB and does not bind the same site on TrkB that BDNF binds or block the binding between BDNF and TrkB.

5. The monoclonal antibody of claim 1 or 3, wherein the antibody comprises complementarity-determining regions (CDRs) of an antibody produced by a hybridoma having ATCC patent deposit designation 090310-01.

6. The monoclonal antibody or the fragment, portion, variant or derivative of any one of the preceding claims, wherein the antibody or fragment, portion, variant or derivative activates TrkB.

7. The monoclonal antibody of any one of claims 1, 3 and 5 to 6, or the fragment, portion, variant or derivative of any one claims 2, 4 and 6, wherein the antibody binds and/or activates human TrkB.

8. The monoclonal antibody of any one of claims 1, 3 and 5 to 6, or the fragment, portion, variant or derivative of any one claims 2, 4 and 6, wherein the antibody binds and/or activates murine or rat TrkB.

9. The monoclonal antibody or the fragment, portion, variant or derivative of any one of the preceding claims, wherein the antibody does not bind and/or activate TrkA,

TrkC and/or p75NTR.

10. A monoclonal antibody that is produced from the hybridoma deposited with the International Depositary Authority of Canada on May 26, 2010 and having accession no. 090310-01 , or from a progenitor cell thereof.

11. The monoclonal antibody or the fragment, portion, variant or derivative of any one of the preceding claims, wherein the antibody or fragment, portion, variant or derivative binds to the same epitope as the monoclonal antibody of claim 10.

12. The monoclonal antibody or the fragment, portion, variant or derivative of any one of the preceding claims, wherein the antibody is humanized.

13. A hybridoma deposited with the International Depositary Authority of Canada on May 26, 2010 and having accession no. 090310-01 or a progenitor cell thereof.

14. A pharmaceutical composition comprising the monoclonal antibody or the fragment, portion, variant or derivative of any one of the preceding claims and a pharmaceutically acceptable carrier.

15. A method of activating TrkB in a subject, comprising administering a therapeutically effective amount of the monoclonal antibody or the fragment, portion, variant or derivative of any one of claims 1 to 12 to the subject, such that TrkB is activated in the subject.

16. The method of claim 15, wherein the subject is a human and the TrkB is human TrkB.

17. The method of claim 15 or 16, wherein the subject suffers from a neurological or neurodegenerative condition which requires activation of TrkB.

18. The method of claim 17, wherein the subject has been injured by a wound, surgery, ischemia, infection, a metabolic disease, malnutrition, a malignant tumor or a toxic drug.

19. The method of claim 17, wherein the subject has suffered a stroke, spinal cord injury or an axotomy.

20. The method of claim 17, wherein the neurodegenerative disease is amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease or Huntington's chorea or is an ocular disease.

21. The method of claim 19, wherein the subject has suffered an axotomy of the optical nerve.

22. The method of claim 17, wherein the subject suffers from glaucoma.

23. The method of any one of claims 15 to 22, wherein the monoclonal antibody or the fragment, portion, variant or derivative is administered to the subject parenterally, intravenously, subcutaneously or interperitoneally.

24. The method of any one of claims 15 to 23, wherein the monoclonal antibody or the fragment, portion, variant or derivative is administered in combination with a second therapeutic agent.

25. The method of claim 24, wherein the second therapeutic agent is an agent for lowering intraocular pressure.

26. A method for treating glaucoma in a subject, comprising administering a therapeutically effective amount of the monoclonal antibody or the fragment, portion, variant or derivative of any one of claims 1 to 12 to the subject.

27. A method for treating or preventing a neurodegenerative condition in a subject, comprising administering a therapeutically effective amount of the monoclonal antibody or the fragment, portion, variant or derivative of any one of claims 1 to 12 to the subject.

28. Use of the monoclonal antibody or the fragment, portion, variant or derivative of any one of claims 1 to 12 for treating glaucoma in a subject.

29. The monoclonal antibody or fragment, portion, variant or derivative of any one of the preceding claims, comprising a single-chain antibody.

30. The monoclonal antibody or fragment, portion, variant or derivative of any one of the preceding claims, comprising a Fab fragment.

31. The monoclonal antibody or fragment, portion, variant or derivative of any one of the preceding claims, wherein the antibody or fragment, portion, variant or derivative binds and/or activates TrkB more strongly than BDNF.

32. The monoclonal antibody or fragment, portion, variant or derivative of any one of the preceding claims, wherein the antibody or fragment, portion, variant or derivative specifically binds an epitope of human TrkB with a sequence comprising the D2-D3 domain of human TrkB.

33. A hybridoma that produces a monoclonal antibody or fragment, portion, variant or derivative according to any one of the preceding claims.

34. A fragment, portion, variant or derivative of the monoclonal antibody of claim 10, wherein said fragment, portion, variant or derivative binds specifically to the same epitope as the monoclonal antibody of claim 10.

35. An antigen-binding fragment of the monoclonal antibody of claim 10.

36. The monoclonal antibody of claim 10 or the fragment, portion, variant or derivative of claim 34 or 35, wherein the antibody, fragment, portion, variant or derivative is humanized, veneered or chimeric.

37. The monoclonal antibody of claim 10 or 36 or an antigen-binding fragment thereof, wherein said antibody or fragment thereof specifically binds TrkB receptor.

38. The monoclonal antibody or fragment thereof of claim 37, wherein said antibody or fragment thereof specifically binds TrkB receptor under physiological conditions.

39. The monoclonal antibody or fragment thereof of claim 10, 36, 37 or 38, wherein said antibody or fragment thereof specifically binds TrkB receptor domain D2-D3.

40. The monoclonal antibody of any one of claims 10 and 36 to 39 or an antigen- binding fragment thereof, wherein said antibody or fragment thereof activates TrkB receptor.

41. A method of in vitro screening for an agent which binds to TrkB receptor and can thereby affect TrkB receptor biological activity, which comprises: combining the antibody or the fragment, portion, variant or derivative thereof of any one of the preceding claims with TrkB receptor, in the presence or absence of a candidate agent; and

determining whether binding of said antibody to TrkB receptor is reduced in the presence of the candidate agent;

wherein a reduction in antibody binding indicates that said candidate agent binds to TrkB receptor, and can thereby affect TrkB receptor biological activity.