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WO2011103667A1 - Anticorps agonistes des récepteurs trkb et leurs utilisations - Google Patents

Anticorps agonistes des récepteurs trkb et leurs utilisations Download PDF

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Publication number
WO2011103667A1
WO2011103667A1 PCT/CA2011/000215 CA2011000215W WO2011103667A1 WO 2011103667 A1 WO2011103667 A1 WO 2011103667A1 CA 2011000215 W CA2011000215 W CA 2011000215W WO 2011103667 A1 WO2011103667 A1 WO 2011103667A1
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Prior art keywords
trkb
fragment
variant
derivative
monoclonal antibody
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Horacio Uri Saragovi
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6452728 CANADA Inc
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6452728 CANADA Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/06—Antiglaucoma agents or miotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/75—Agonist effect on antigen
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)

Definitions

  • 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
  • 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.
  • NGF nerve growth factor
  • BDNF brain derived neurotrophic factor
  • NT-3 neurotrophin-3
  • NT-4/5 neurotrophins-4/5
  • 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 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).
  • IOP intraocular pressure
  • the experimental glaucoma model causes a slow and progressive RGC death.
  • TrkB and p75NTR receptors 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.
  • BDNF BDNF-induced Trk activation
  • 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]).
  • 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
  • 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.
  • mAbs monoclonal antibodies
  • 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.
  • 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 .
  • the antibodies provided herein can activate TrkB, i.e. can act as agonists of the TrkB receptor.
  • the antibodies provided herein are specific for TrkB and do not bind and/or activate TrkA, TrkC and/or p75NTR receptors.
  • 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.
  • 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.
  • 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.
  • 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.
  • an antibody which comprises complementarity- determining regions (CDRs) of an antibody produced by a hybridoma having ATCC patent deposit designation 090310-01.
  • compositions comprising the antibodies of the invention or the fragments, portions, variants or derivatives thereof, and a pharmaceutically acceptable carrier, are 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.
  • the subject is human and the TrkB is human TrkB.
  • the subject suffers from a neurological or neurodegenerative condition which requires activation of TrkB.
  • 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.
  • 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.
  • 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.
  • the subject has glaucoma.
  • the antibodies of the invention may be administered to a subject parenterally, intravenously, subcutaneously or interperitoneally.
  • the antibodies of the invention may be administered in combination with a second therapeutic agent, such as an agent for lowering intraocular pressure.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • MAb 21G3 does not bind in western blots;
  • 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).
  • 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.
  • 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).
  • NTI NFL-GCL-IPL
  • 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. ⁇ _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).
  • A shows the nucleot
  • the present invention provides monoclonal antibodies (mAbs) that selectively target TrkB, without binding to p75NTR.
  • mAbs monoclonal antibodies 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.
  • the present invention provides monoclonal antibodies that bind specifically to TrkB.
  • the antibodies bind to human TrkB.
  • the antibodies bind to rat and/or mouse TrkB.
  • the antibodies bind preferentially to human TrkB, and do not bind to TrkA, TrkC and/or p75NTR receptors.
  • the antibodies provided herein are also agonists of TrkB.
  • the antibodies provided herein bind to the D2-D3 domain of TrkB, as shown in Fig. 7B.
  • 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.
  • the receptor site for antibody binding does not include the ENLVGED peptide sequence.
  • 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).
  • the present invention provides any monoclonal antibodies with the properties described herein that bind and/or activate human, rat or mouse TrkB.
  • 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.
  • 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.
  • the present invention provides hybridomas that produce any of the monoclonal antibodies of the invention.
  • the hybridoma that was deposited with the International Depositary Authority of Canada on May 26, 2010 and given accession no. 090310-01 is provided.
  • 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.
  • the monoclonal antibodies of the invention can be prepared by any known method.
  • 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).
  • 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.
  • 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).
  • 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).
  • 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.
  • humanized or veneered versions of the antibody of interest 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.
  • 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.
  • 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.
  • CDRs complementarity- determining regions
  • FR human supporting framework region
  • 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).
  • 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.
  • 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.
  • antibody 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).
  • 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).
  • 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).
  • CDR complementarity determining regions
  • FR framework regions
  • 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.
  • 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.
  • 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).
  • CDR complementarity determining region
  • 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).
  • single chain Fv single chain Fv
  • 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).
  • 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).
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • any given monoclonal antibody may use a competition assay (e.g., an ELISA) to determine whether the antibodies block the interaction of TrkB and BDNF.
  • a competition assay e.g., an ELISA
  • 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.
  • 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.
  • TrkB 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.
  • the monoclonal antibodies provided herein are administered to a subject in order to activate TrkB, in accordance with the present invention.
  • 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.
  • 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.
  • the term refers to that ingredient alone.
  • the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.
  • 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.).
  • one or more higher doses e.g., 2, 3 or 4 fold higher
  • the higher dose(s) may be administered at the onset of treatment only or at the beginning of each treatment cycle.
  • 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.
  • 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.
  • 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).
  • 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.
  • the carrier is preferably a finely divided solid which is in admixture with the finely divided active ingredient.
  • 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.
  • compositions are provided in unit dosage form, such as tablets or capsules.
  • 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.
  • 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.
  • 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.
  • 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.
  • the unit dose is in tablet or capsule form. In some cases, each unit dosage is a therapeutically effective amount.
  • monoclonal antibodies of the invention may be administered alone to modulate TrkB activity.
  • 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.
  • 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.
  • 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.
  • 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
  • the monoclonal antibodies are combined with IOP-lowering agents, such as those used for the treatment of glaucoma.
  • 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.
  • the antibodies may be formulated for immediate, delayed, modified, sustained, pulsed, or controlled-release delivery.
  • the antibodies are formulated for delivery by injection.
  • 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.
  • 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.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil can be employed including synthetic mono- or diglycerides.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a suitable propellant e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, a hydrofluoroalkane, carbon dioxide or other suitable gas.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the route of administration may be intravenous, intraocular, intrasynovial, intramuscular, transdermal and/or oral.
  • 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.
  • the invention provides methods for treating neurological conditions, neurodegenerative diseases and/or for providing neuroprotection.
  • 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.
  • 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).
  • 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.
  • ADHD attention-deficit hyperactivity disorder
  • ALS amyotrophic lateral sclerosis
  • 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.
  • ocular neurodegenerative diseases including but not limited to glaucoma, retinal degeneration, retinitis pigmentosa, axotomy, axotomy of the optic nerve, diabetic retinopathy and macular degeneration.
  • RGCs retinal ganglion cells
  • BDNF neurotrophic factor
  • 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.
  • IOP intraocular pressure
  • 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).
  • TrkB signaling includes 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.
  • the term "subject” may include animals, such as mammals, such as dogs, cats, cows, pigs, sheep and horses, and human.
  • the subject is a human.
  • the subject is an adult human.
  • mice 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 (LeSterrorism 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).
  • ELISA enzyme-linked immunosorbent assay
  • mAbs 1D7 Accession no. 090310-01
  • B3, and 21G3 were selected for further study. All mAbs were purified by affinity column chromatography to >95%.
  • HEK293 stably transfected with rat TrkB were used.
  • 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.
  • SY5Y- TrkB For specificity wild type SY5Y cells were used (Yan C, et al., Mol Pharmacol 2002;61 :710-719).
  • mAbs 1D7, B3, and 21G3 binding to the cell surface were done by FACScan assays using live cells, as described (LeSterrorism 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.
  • binding buffer Hanks' Balanced Salt Solution (HBSS), 0.1% bovine serum albumin (BSA), and 0.1% NaN3
  • FACScan Fluorescent Activated Cell Scanner
  • 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.
  • TrkB 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.
  • TrkB a direct measure of TrkB activation (Atwal JK, et al., Neuron 2000;27:265-277).
  • 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.
  • PFHM-II serum-free media
  • 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).
  • 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.
  • 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.
  • EMC episcleral vein cauterization
  • Intraocular pressure 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 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.
  • 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.
  • a chronic and stable elevated IOP of ⁇ 1.7-fold over normal is achieved.
  • 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.
  • Drug treatments were done with the experimenters blinded to the treatment code.
  • the intraocular injections were performed at days 14 and 21 after cauterization; and the endpoint was at day 42 of high IOP.
  • 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 ⁇ with 3 ⁇ g of compound.
  • Intraocular injections in retinas used for biochemical assays (endpoints at 6 hr or 18 hr) delivered 3 ⁇ with 1 ⁇ g of compound.
  • 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).
  • FC superior colliculous
  • 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 ⁇ ) 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.
  • 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).
  • Rats (n 4 per group) received 1 ⁇ g of the indicated treatment.
  • axotomy drugs were injected within 5 min after ON transection.
  • drugs were injected after fourteen days of high IOP.
  • Six hours or eighteen hours after drug treatment retinas were dissected and lysed in 80 ⁇ SDS-PAGE sample buffer containing 2% SDS.
  • 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 ⁇ -tubulin (Sigma). Detection was by ECL. For digital quantification, membranes were scanned and analyzed using ImageJ software.
  • PFA paraformaldehyde
  • 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).
  • 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 ⁇ , and repeatability of the measurements from B-scans better than 1 ⁇ .
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • MAb 1D7 and mAb B3 recognize denatured TrkB in western blots when samples are resolved in non-reduced SDS-PAGE.
  • mAb 21G3 does not recognize denatured TrkB under any condition and can not be studied using western blotting.
  • 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.
  • mAb 1D7 Treatment of SY5Y-TrkB cells with mAb 1D7 (10 nM) or BDNF (10 nM) induces tyrosine phosphorylation of TrkB.
  • 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.
  • 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.
  • 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.
  • mAb 21G3, and mouse Ig do not support SY5Y-TrkB cell survival.
  • TrkB is agonistic to TrkB
  • TrkB is biologically inert.
  • 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 .
  • Anti-TrkB mAb 1D7 supports long-lasting RGC survival in glaucoma and ON axotomy
  • 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).
  • 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.
  • IOP intraocular pressure
  • TrkB tyrosine phosphorylation 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).
  • BDNF and mAb 1D7 induce pTyr-TrkB
  • only mAb 1D7 can induce sustained pTyr-TrkB and can protect RGCs from death in retinal diseases.
  • NGI in normal retinas is 71 ⁇ 0.6 ⁇ .
  • the NGI thickness is 54.7 ⁇ 1.2 ⁇ , while in the mAb lD7-treated group it is 60.8 ⁇ 0.3 ⁇ (significant versus untreated axotomy, ⁇ 0.001) (representative data in FIG. 6b, summarized in FIG. 6c).
  • the NGI thickness is 51.2 ⁇ 2.6 ⁇ , while in the mAb lD7-treated group it is 60.4 ⁇ 0.1 ⁇ (significant versus untreated glaucoma, p ⁇ 0.02) (FIG. 6d).
  • mAb 1D7 treatment significantly protects the structure of the retinal neuronal layers in both acute (ON axotomy) and chronic (glaucoma) pathologies.
  • mAb 1D7 treatment does not protect the NGI fully.
  • 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.
  • TrkB agonist affords preservation of the retinal structure in both animal models, with maintenance of the layers comprising neurons and neuronal fibers.
  • TrkB agonist affords long-lasting neuroprotection, by causing sustained TrkB activation.
  • mAb 1D7 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.
  • TrkB ectodomain 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.
  • 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.
  • BDNF 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.
  • 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.
  • the quantitative structural data obtained with FD-OCT correlated well with RGC loss during glaucoma progression.
  • loss of structural integrity -30% loss of thickness
  • 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.
  • the use of FD-OCT to measure preservation of retinal structure may be a predictive endpoint for neuroprotective efficacy in glaucoma.

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Abstract

La présente invention concerne la production d'anticorps monoclonaux (mAb) qui se lient de manière sélective et activent les récepteurs kinase B apparentés à la tropomyosine. Un mAb anti-récepteur TrkB qui se lie spécifiquement au domaine D2-D3 de la TrkB est isolé, produit par la lignée cellulaire d'hybridome ayant le numéro de dépôt de brevet ATCC 090310-01, et qui ne se lie pas à ni n'active TrkA, TrkC ou p75NTR ni n'interfère avec la liaison à BDNF. L'invention concerne également des compositions pharmaceutiques de ceux-ci et leur utilisation pour le traitement et la prévention d'états nécessitant l'activation de TrkB, tels que le glaucome et d'autres états de neurodégénérescence oculaire, et pour l'inhibition de la neurodégénérescence.
PCT/CA2011/000215 2010-02-26 2011-02-25 Anticorps agonistes des récepteurs trkb et leurs utilisations Ceased WO2011103667A1 (fr)

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WO2018224630A1 (fr) * 2017-06-09 2018-12-13 Boehringer Ingelheim International Gmbh Anticorps anti-trkb
CN109414501A (zh) * 2016-05-03 2019-03-01 斯克利普斯研究院 用于治疗神经退行性疾病的TrkB激动剂抗体
CN110753702A (zh) * 2017-03-15 2020-02-04 清华大学 新型抗trkb抗体
US11066474B2 (en) 2017-11-30 2021-07-20 Regeneron Pharmaceuticals, Inc. Anti-TrkB monoclonal antibodies and methods of use
US11078287B2 (en) 2015-11-17 2021-08-03 Glaxosmithkline Intellectual Property Development Limited Binding agonist for treatment of neurological and other disorders

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EP3452088A4 (fr) * 2016-05-03 2020-04-15 The Scripps Research Institute Anticorps agonistes de trkb pour le traitement de troubles neurodégénératifs
CN109414501A (zh) * 2016-05-03 2019-03-01 斯克利普斯研究院 用于治疗神经退行性疾病的TrkB激动剂抗体
US9914781B1 (en) 2016-11-08 2018-03-13 Glaxosmithkline Intellectual Property Development Limited Binding agonist for treatment of neurological and other disorders
JP2020513806A (ja) * 2017-03-15 2020-05-21 チンファ ユニバーシティＴｓｉｎｇｈｕａ Ｕｎｉｖｅｒｓｉｔｙ 新規の抗ＴｒｋＢ抗体
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JP7216424B2 (ja) 2017-03-15 2023-02-01 チンファ ユニバーシティ 新規の抗ＴｒｋＢ抗体
EP3596126A4 (fr) * 2017-03-15 2021-03-03 Tsinghua University Nouveaux anticorps anti-trkb
US10941203B2 (en) 2017-03-15 2021-03-09 Tsinghua University Anti-TrkB agonist antibodies binding to D5 domain of TrkB and methods of promoting neuronal survival in motor neuron injury, stroke or glaucoma
CN110753702A (zh) * 2017-03-15 2020-02-04 清华大学 新型抗trkb抗体
CN110753702B (zh) * 2017-03-15 2022-09-20 清华大学 抗trkb抗体
CN114940713A (zh) * 2017-03-15 2022-08-26 清华大学 新型抗trkb抗体
US11866501B2 (en) 2017-06-09 2024-01-09 Boehringer Ingelheim International Gmbh Anti-TrkB antibodies
US10793634B2 (en) 2017-06-09 2020-10-06 Boehringer Ingelheim International Gmbh Anti-TrkB antibodies
JP7267208B2 (ja) 2017-06-09 2023-05-01 ベーリンガー インゲルハイム インターナショナル ゲゼルシャフト ミット ベシュレンクテル ハフツング 抗ｔｒｋｂ抗体
JP2023093621A (ja) * 2017-06-09 2023-07-04 ベーリンガー インゲルハイム インターナショナル ゲゼルシャフト ミット ベシュレンクテル ハフツング 抗ｔｒｋｂ抗体
TWI808086B (zh) * 2017-06-09 2023-07-11 德商百靈佳殷格翰國際股份有限公司 抗-trkb抗體
WO2018224630A1 (fr) * 2017-06-09 2018-12-13 Boehringer Ingelheim International Gmbh Anticorps anti-trkb
IL271067B1 (en) * 2017-06-09 2024-02-01 Boehringer Ingelheim Int Anti-TRKB antibodies
JP2020522270A (ja) * 2017-06-09 2020-07-30 ベーリンガー インゲルハイム インターナショナル ゲゼルシャフト ミット ベシュレンクテル ハフツング 抗ｔｒｋｂ抗体
IL271067B2 (en) * 2017-06-09 2024-06-01 Boehringer Ingelheim Int Anti-trkb antibodies
JP7505084B2 (ja) 2017-06-09 2024-06-24 ベーリンガー インゲルハイム インターナショナル ゲゼルシャフト ミット ベシュレンクテル ハフツング 抗ｔｒｋｂ抗体
US20180355046A1 (en) * 2017-06-09 2018-12-13 Boehringer Ingelheim International Gmbh Anti-trkb antibodies
US11066474B2 (en) 2017-11-30 2021-07-20 Regeneron Pharmaceuticals, Inc. Anti-TrkB monoclonal antibodies and methods of use
US12037401B2 (en) 2017-11-30 2024-07-16 Regeneron Pharmaceuticals, Inc. Anti-TrkB monoclonal antibodies and methods of use

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