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  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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Definitions

• This invention relates to neurobiology and molecular biology. More particularly, this invention relates to immunogenic Nogo receptor-1 polypeptides, Nogo receptor-1 antibodies, antigen-binding fragments thereof, soluble Nogo receptors and fusion proteins thereof and nucleic acids encoding the same. This invention further relates to compositions comprising, and methods for making and using, such Nogo receptor antibodies, antigen-binding fragments thereof, immunogenic Nogo receptor-1 polypeptides, soluble Nogo receptors and fusion proteins thereof and nucleic acids encoding the same.
• Axons and dendrites of neurons are long cellular extensions from neurons.
• the distal tip of an extending axon or neurite comprises a specialized region, known as the growth cone .
• Growth cones sense the local environment and guide axonal growth toward the neuron's target cell. Growth cones respond to several environmental cues, for example, surface adhesiveness, growth factors, neurotransmitters and electric fields.
• the guidance of growth at the cone involves various classes of adhesion molecules, intercellular signals, as well as factors that stimulate and inhibit growth cones .
• the growth cone of a growing neurite advances at various rates, but typically at the speed of one to two millimeters per day.
• Growth cones are hand shaped, with broad flat expansion (microspikes or filopodia) that differentially adhere to surfaces in the embryo.
• the filopodia are continually active, some filopodia retract back into the growth cone, while others continue to elongate through the substratum.
• the elongations between different filopodia form lamellipodia.
• the growth cone explores the area that is ahead of it and on either side with its lamellipodia and filopodia. When an elongation contacts a surface that is unfavorable to growth, it withdraws. When an elongation contacts a favorable growth surface, it continues to extend and guides the growth cone in that direction.
• the growth cone can be guided by small variations in surface properties of the substrata.
• Nerve cell function is greatly influenced by the contact between the neuron and other cells in its immediate environment (U. Rutishauser, T. M. Jessell, Physiol . Rev. 1988, 68, p. 819). These cells include specialized glial cells, oligodendrocytes in the central nervous system (CNS) , and Schwann cells in the peripheral nervous system (PNS) , which ensheathe the neuronal axon with myelin (an insulating structure of multi-layered membranes) (G. Lemke, in An Introduction to Molecular Neurobiology, Z. Hall, Ed. [Sinauer, Sunderland, Mass., 1992], p. 281).
• CNS neurons While CNS neurons have the capacity to regenerate after injury, they are inhibited from doing so because of the presence of inhibitory proteins present in myelin and possibly also by other types of molecules normally found in their local environment (Brittis and Flanagan, Neuron 2001, 30, pp. 11-14; Jones et al . , J. Neurosci . 2002, 22, pp. 2792-2803; Grimpe et al . , J. Neurosci. 2002, 22, pp. 3144- 3160) .
• myelin inhibitory proteins that are found on oligodendrocytes have been characterized, e . g. , NogoA (Chen et al .
• Nogo receptor-1 is a GPI-anchored membrane protein that contains 8 leucine rich repeats (Fournier et al . , Nature 2001, 409, 341-346) .
• an inhibitory protein e.g., NogoA, MAG and OM-gp
• the Nogo receptor-1 complex transduces signals that lead to growth cone collapse and inhibition of neurite outgrowth.
• the invention relates to soluble Nogo receptor-1 polypeptides and fusion proteins comprising them, and antibodies and antigenic fragments thereof directed against specific immunogenic regions of Nogo receptor-1.
• the invention also relates to immunogenic Nogo receptor-1 polypeptides that bind to the antibodies of the invention.
• the invention also relates to Nogo receptor-1 polypeptides that are bound by a monoclonal antibody that binds to Nogo receptor-1.
• Such polypeptides may be used, inter alia, as immunogens or to screen antibodies to identify those with similar specificity to an antibody of the invention.
• the invention further relates to nucleic acids encoding the polypeptides of this invention, vectors and host cells comprising such nucleic acids and methods of making the peptides.
• the antibodies, soluble receptors and receptor fusion proteins of this invention antagonize or block Nogo receptor-1 and are useful for inhibiting binding of Nogo receptor-1 to its ligands, inhibiting growth cone collapse in a neuron and decreasing the inhibition of neurite outgrowth or sprouting in a neuron.
• the invention provides a polypeptide selected from the group consisting of AAAFGLTLLEQLDLSDNAQLR (SEQ ID NO: 26); LDLSDNAQLR (SEQ ID NO: 27); LDLSDDAELR (SEQ ID NO: 29); LDLASDNAQLR (SEQ ID NO;
• LDLASDDAELR SEQ ID NO 31
• LDALSDNAQLR SEQ ID NO: 32
• LDALSDDAELR SEQ ID NO 33
• LDLSSDNAQLR SEQ ID NO: 34
• LDLSSDEAELR SEQ ID NO 35
• DNAQLRWDPTT SEQ ID NO: 36
• DNAQLR DNAQLR
• ADLSDNAQLRWDPTT SEQ ID NO: 41
• LALSDNAQLR ⁇ /VDPTT SEQ ID NO: 42
• LDLSDNAALRWDPTT SEQ ID NO: 43
• LDLSDNAQLHWDPTT SEQ ID NO: 44
• LDLSDNAQLAWDPTT SEQ ID NO : 45
• the invention provides a nucleic acid encoding a polypeptide of the invention.
• the nucleic acid is operably linked to an expression control sequence.
• the invention provides a vector comprising a nucleic acid of the invention.
• the invention provides a host cell comprising a nucleic acid or comprising the vector of the invention.
• the invention provides a method of producing a polypeptide of the invention comprising culturing a host cell comprising a nucleic acid or vector of the invention and recovering the polypeptide from the host cell or culture medium.
• the invention provides a method of producing an antibody comprising the steps of immunizing a host with a polypeptide of the invention or a host cell comprising a nucleic acid or comprising the vector of the invention and recovering the antibody.
• the invention also provides an antibody produced by the method or an antigen-binding fragment thereof.
• the invention provides an antibody or an antigen-binding fragment thereof that specifically binds to a polypeptide of the invention, wherein the antibody is not the monoclonal antibody produced by hybridoma cell line HB 7E11 (ATCC ® accession No. PTA- 4587) .
• the antibody or antigen-binding fragment (a) inhibits growth cone collapse of a neuron; (b) decreases the inhibition of neurite outgrowth and sprouting in a neuron; and (c) inhibits Nogo receptor-1 binding to a ligand.
• the neurite outgrowth and sprouting is axonal growth.
• the neuron is a central nervous system neuron.
• an antibody of the invention is monoclonal.
• an antibody of the invention is a murine antibody.
• an antibody of the invention is selected from the group consisting of a humanized antibody, a chimeric antibody and a single chain antibody.
• the invention provides a method of inhibiting Nogo receptor-1 binding to a ligand, comprising the step of contacting Nogo receptor-1 with an antibody of the invention or antigen-binding fragment thereof.
• the ligand is selected from the group consisting of NogoA, NogoB, NogoC, MAG and OM-gp.
• the invention provides a method for inhibiting growth cone collapse in a neuron, comprising the step of contacting the neuron with an antibody of the invention or antigen-binding fragment thereof .
• the invention provides a method for decreasing the inhibition of neurite outgrowth or sprouting in a neuron, comprising the step of contacting the neuron with an antibody of the invention or antigen-binding fragment thereof.
• the neurite outgrowth or sprouting is axonal growth.
• the neuron is a central nervous system neuron.
• the invention provides a composition comprising a pharmaceutically acceptable carrier and an antibody of the invention or an antigen-binding fragment thereof. In some embodiments, the composition further comprises one or more additional therapeutic agents.
• the invention provides a method Of promoting survival of a neuron at risk of dying, comprising contacting the neuron with an effective amount of an anti-Nogo receptor-1 antibody of the invention or antigen-binding fragment thereof.
• the neuron is in vi tro .
• the neuron is in a mammal.
• the mammal displays signs or symptoms of multiple sclerosis, ALS, Huntington' s disease, Alzheimer's disease, Parkinson's disease, diabetic neuropathy, stroke, traumatic brain injuries or spinal cord injury.
• the invention provides a method of promoting survival of a neuron in a mamm l, which neuron is at risk of dying, comprising (a) providing a cultured host cell expressing an anti-Nogo receptor-1 antibody of the invention or antigen-binding fragment thereof; and (b) introducing the host cell into the mammal at or near the site of the neuron.
• the invention provides a gene therapy method of promoting survival of a neuron at risk of dying, which neuron is in a mammal, comprising administering at or near the site of the neuron a viral vector comprising a nucleotide sequence that encodes an anti-Nogo receptor-1 antibody of the invention or an antigen-binding fragment thereof, wherein the anti-Nogo receptor-1 antibody or antigen-binding fragment is expressed from the nucleotide sequence in the mammal in an amount sufficient to promote survival of the neuron.
• Figure 1 is a schematic representation of the structure of Nogo receptor-1.
• Human sNogoR310 contains residues 26-310 and sNogoR344 contains residues 26-344.
• Rat sNogoR310 contains residues 27-310 and sNogoR344 contains residues 27-344
• Figure 2 depicts an antigenicity plot for the Nogo receptor-1 protein using the Vector Nti software.
• Rat P- 617 is SEQ ID NO: 10
• rat P-618 is SEQ ID NO: 11.
• Figure 3A is a graph depicting the binding activity of anti-Nogo receptor-1 antibody, 7E11. The graph presents the effect of 7E11 concentration on the binding of Nogo66 to Nogo receptor-1.
• Figure 3B depicts the binding activity of anti-Nogo receptor-1 antibody, 1H2.
• the graph presents the effect of 1H2 concentration on the binding of Nogo66 to sNogoR344-Fc (also referred to herein and in United States patent application 60/402,866 as Fc-sNogoR344 or Ig-sNogoR344) .
• Fc-MAG did not compete with Nogo66 for binding to sNogoR3 4-Fc .
• Figure 4 depicts the results of an ELISA for anti- Nogo-R-1 antibodies 1H2 , 3G5 and 2F7. The effect of the antibodies on OD 450 in the presence of immobilized antigens was determined.
• FIG. 5 is a graph depicting the effects of monoclonal antibody, 7E11, on rat DRG neurite outgrowth in the presence of varying amounts of myelin.
• Figure 6A is a graph depicting the effect of binding of sNogoR310 to 12 ⁇ I-Nogo66 and 125 I-Nogo40 in the presence of the following competitors: Nogo66, Nogo40 and anti-Nogo receptor-1 monoclonal antibody supernatant.
• Figure 6B depicts the binding activity of 1 5 I-Nogo66 to sNogoR310.
• Figure 7 is a graph depicting the effect of sNogoR310-Fc on 125 I-Nogo40 binding to sNogoR310.
• Figure 8 is a graph depicting the binding activity of sNogoR310-Fc to 125 I-Nogo40.
• Figure 9A is a graph of the effect of sNogoR310 on neurite outgrowth/cell in the presence or absence of myelin.
• Figure 9B is a graph of the effect of sNogoR310 on neurite outgrowth in the presence or absence of myelin.
• Figure 10A is a graph depicting the effect of sNogoR310-Fc on P4 rat DRG neurite outgrowth in the presence or absence of increasing amounts of myelin.
• Figure 10B depicts the number of neurites/cell following treatment with
• Figure 11 is a graph depicting the effect of monoclonal antibody 5B10 on DRG neurite outgrowth/cell in the presence of increasing amounts of myelin.
• Figure 12 is a graph depicting the effect of sNogoR310-Fc on the BBB score up to 30 days following induction of injury in a rat spinal cord transection model.
• Figures 13A and 13B report the locomotor BBB score as a function of time after dorsal hemisection in the WT or transgenic mice from Line 08 or Line 01.
• Figure 13C graphs the maximal tolerated inclined plane angle as a function of time after injury for WT and transgenic mice.
• Figure 13D shows hindlimb errors during inclined grid climbing as a function of post-injury time. In all the graphs, means ⁇ s.e.m. from 7-9 mice in each group are reported. The values from transgenic group are statistically different from the WT mice, (double asterisks, P ⁇ 0.01; Student's t-test) .
• Figure 14A shows the locomotor BBB score as a function of time after dorsal hemisection in vehicle or sNogoR310-Fc treated animals.
• Figure 14B shows hindlimb errors during grid walking as a function of time after injury.
• Figure 14C shows footprint analysis revealing a shorter stride length and a greater stride width in control mice than uninjured or injured + sNogoR310-Fc rats.
• the values of sNogoR310-Fc group are statistically different from the control ( Figures 14A-B) .
• the control values are statistically different from no-SCI or SCI + sNogoR310-Fc rats in Figure 14C. (asterisk, p ⁇ 0.05; double asterisks, p ⁇ 0.01; Student's t-test) .
• antibody means an intact immunoglobulin, or an antigen-binding fragment thereof.
• Antibodies of this invention can be of any isotype or class (e.g., M, D, G, E and A) or any subclass ( e . g.
• Fc means a portion of the heavy chain constant region of an antibody that is obtainable by papain digestion.
• NogoR fusion protein means a protein comprising a soluble Nogo receptor-1 moiety fused to a heterologous polypeptide.
• humanized antibody means an antibody in which at least a portion of the non-human sequences are replaced with human sequences. Examples of how to make humanized antibodies may be found in United States Patent Nos. 6,054,297, 5,886,152 and 5,877,293.
• chimeric antibody means an antibody that contains one or more regions from a first antibody and one or more regions from at least one other antibody.
• the first antibody and the additional antibodies can be from the same or different species.
• "Nogo receptor,” “NogoR,” “NogoR-1,” “NgR,” and “NgR-1” each means Nogo receptor-1.
• the present invention relates to Nogo receptor-1 polypeptides that are immunogenic.
• the immunogenic polypeptide consists essentially of an amino acid sequence selected from the group consisting of: LDLSDNAQLRWDPTT (rat) (SEQ ID NO: 1); LDLSDNAQLRSVDPAT (human) (SEQ ID NO : 2); AVASGPFRPFQTNQLTDEELLGLPKCCQPDAADKA (rat) (SEQ ID NO: 3); AVATGPYHPIWTGRATDEEPLGLPKCCQPDAADKA (human) (SEQ ID NO: 4); and CRLGQAGSGA (mouse) (SEQ ID NO : 5) .
• the invention relates to Nogo receptor 1 polypeptides that are bound by a monoclonal antibody that binds to Nogo receptor-1.
• the polypeptide is recognized by the 7E11 monoclonal antibody.
• the polypeptide is selected from the group consisting of: LDLSDNAQLR (SEQ ID NO: 28); LDLSDDAELR (SEQ ID NO : 30); LDLASDNAQLR (SEQ ID NO:
• LDLASDDAELR SEQ ID NO 32
• LDALSDNAQLR SEQ ID NO: 33
• LDALSDDAELR SEQ ID NO 34
• LDLSSDNAQLR SEQ ID NO: 35
• LDLSSDEAELR SEQ ID NO 36
• DNAQLRWDPTT SEQ ID NO: 37
• DNAQLR SEQ ID NO: 38
• ADLSDNAQLRWDPTT SEQ ID NO: 42
• LALSDNAQLRWDPTT SEQ ID NO: 43
• LDLSDNAALRWDPTT SEQ ID NO: 44
• LDLSDNAQLHWDPTT SEQ ID NO : 45
• LDLSDNAQLAWDPTT SEQ ID NO : 46
• the invention relates to a nucleic acid encoding a polypeptide of SEQ ID NOs : 1-5, 26- 27, 29-37 and 41-45.
• the nucleic acid molecule is linked to an expression control sequence (e . g. , pCDNA(I)).
• the present invention also relates to a vector comprising a nucleic acid coding for a polypeptide of the invention.
• the vector is a cloning vector.
• the vector is an expression vector.
• the vector contains at least one selectable marker .
• the present invention also relates to host cells comprising the above-described nucleic acid or vector.
• the present invention also relates to a method of producing an immunogenic polypeptide of the invention comprising the step of culturing a host cell.
• the host cell is prokaryotic.
• the host cell is eukaryotic.
• the host cell is yeast.
• Antibodies [0055]
• the present invention further relates to an antibody or an antigen-binding fragment thereof that specifically binds a Nogo receptor-1 polypeptide of the invention.
• the antibody or antigen- binding fragment binds a polypeptide consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-5, 26-27, 29-37 and 41-45.
• the antibody or antigen-binding fragment of the present invention may be produced in vivo or in vi tro . Production of the antibody or antigen-binding fragment is discussed below.
• An antibody or an antigen-binding fragment thereof of the invention inhibits the binding of Nogo receptor-1 to a ligand ( e . g. , NogoA, NogoB, NogoC, MAG, OM-gp) and decreases myelin-mediated inhibition of neurite outgrowth and sprouting, particularly axonal growth, and attenuates myelin mediated growth cone collapse.
• the anti-Nogo receptor-1 antibody or antigen-binding fragment thereof is murine.
• the Nogo receptor-1 is from rat.
• the Nogo receptor-1 is human.
• the anti-Nogo receptor-1 antibody or antigen- binding fragment thereof is recombinant, engineered, humanized and/or chimeric.
• the antibody is selected from the group consisting of: monoclonal 7E11 (ATCC accession No. PTA-4587) ; monoclonal 1H2 (ATCc " accession No. PTA-
• the antibody is polyclonal antibody 46.
• Exemplary antigen-binding fragments are, Fab,
• CDR complementarity determining region
• Fd means a fragment that consists of the V H and C H ⁇ domains
• Fv means a fragment that consists of the V L and V H domains of a single arm of an antibody
• dAb means a fragment that consists of a V H domain (Ward et al . , Nature 341:544-546, 1989).
• single- chain antibody means an antibody in which a V L region and a V region are paired to form a monovalent molecules via a synthetic linker that enables them to be made as a single protein chain (Bird et al . , Science 242:423-426, 1988 and Huston et al . , Proc.
• diabody means a bispecific antibody in which V H and V L 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 . , Proc. Natl. Acad. Sci. USA 90:6444-6448, 1993, and Poljak, R. J., et al . , Structure 2:1121-1123, 1994).
• immunoadhesin that specifically binds an antigen of interest means a molecule in which one or more CDRs may be incorporated, either covalently or noncovalently.
• the invention provides a subunit polypeptide of a Nogo receptor-1 antibody of the invention, wherein the subunit polypeptide is selected from the group consisting of: (a) a heavy chain or a variable region thereof; and (b) a light chain or a variable region thereof .
• the invention provides a nucleic acid encoding the heavy chain or the variable region thereof, or the light chain and the variable region thereof of a subunit polypeptide of a Nogo receptor-1 antibody of the invention.
• the invention provides a hypervariable region (CDR) of a Nogo receptor-1 antibody of the invention or a nucleic acid encoding a CDR.
• CDR hypervariable region
• Antibodies of the invention can be generated by immunization of a suitable host (e . g. , vertebrates, including humans, mice, rats, sheep, goats, pigs, cattle, horses, reptiles, fishes, amphibians, and in eggs of birds, reptiles and fish) . Such antibodies may be polyclonal or monoclonal.
• a suitable host e . g. , vertebrates, including humans, mice, rats, sheep, goats, pigs, cattle, horses, reptiles, fishes, amphibians, and in eggs of birds, reptiles and fish.
• the host is immunized with an immunogenic Nogo receptor-1 polypeptide of the invention.
• the host is immunized with Nogo receptor-1 associated with the cell membrane of an intact or disrupted cell and antibodies of the invention are identified by binding to a Nogo receptor-1 polypeptide of the invention.
• the Nogo receptor-1 antigen is administered with an adjuvant to stimulate the immune response.
• Adjuvants often need to be administered in addition to antigen in order to elicit an immune response to the antigen.
• These adjuvants are usually insoluble or undegradable substances that promote nonspecific inflammation, with recruitment of mononuclear phagocytes at the site of immunization.
• Examples of adjuvants include, but are not limited to, Freund ' s adjuvant, RIBI (muramyl dipeptides) , ISCOM (immunostimulating complexes) or fragments thereof .
• Determination of immunoreactivity with an immunogenic Nogo receptor-1 polypeptide of the invention may be made by any of several methods well known in the art, including, e . g. , immunoblot assay and ELISA.
• Monoclonal antibodies of the invention can made by standard procedures as described, e . g. , in Harlow and Lane (1988) , supra. [0069] Briefly, at an appropriate period of time the animal is sacrificed and lymph node and/or splenic B-cells are immortalized by any one of several techniques that are well-known in the art, including but not limited to transformation, such as with EBV or fusion with an immortalized cell line, such as myeloma cells. Thereafter, the cells are clonally separated and the supernatants of each clone tested for production of an antibody specific for an immunogenic Nogo receptor-1 polypeptide of the invention.
• Antigens in this case Nogo receptor-1 or an immunogenic polypeptide of the invention
• antibodies can be labeled by joining, either covalently or non-covalently, a substance that provides for a detectable signal .
• Suitable labels include, but are not limited to, radionucleotides, enzymes, substrates, cofactors, inhibitors, fluorescent agents, chemiluminescent agents, magnetic particles and the like. Patents teaching the use of such labels include U.S. Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241.
• an antibody has multiple binding specificities, such as a bifunctional antibody prepared by any one of a number of techniques known to those of skill in the art including the production of hybrid hybridomas, disulfide exchange, chemical cross- linking, addition of peptide linkers between two monoclonal antibodies, the introduction of two sets of immunoglobulin heavy and light chains into a particular cell line, and so forth (see below for more detailed discussion) .
• the antibodies of this invention may also be human monoclonal antibodies, for example those produced by immortalized human cells, by SCID-hu mice or other non-human animals capable of producing "human" antibodies.
• Anti-Nogo receptor-1 antibodies of this invention can be isolated by screening a recombinant combinatorial antibody library.
• exemplary combinatorial libraries are for binding to an immunogenic Nogo receptor-1 polypeptide of the invention, such as a scFv phage display library, prepared using V L and V H cDNAs prepared from mRNA derived an animal immunized with an immunogenic Nogo receptor-1 polypeptide of the invention.
• Methodologies for preparing and screening such libraries are known in the art. There are commercially available methods and materials for generating phage display libraries ( e . g. , the Pharmacia Recombinant Phage Antibody
• nucleic acid encoding the selected antibody can be recovered from the display package (e . g. , from the phage genome) and subcloned into other expression vectors by standard recombinant DNA techniques. If desired, the nucleic acid can be further manipulated to create other antibody forms of the invention, as described below.
• DNA encoding the antibody heavy chain and light chain or the variable regions thereof is cloned into a recombinant expression vector and introduced into a mammalian host cell, as described above.
• Anti-Nogo receptor-1 antibodies of the invention can be of any isotype.
• An antibody of any desired isotype can be produced by class switching.
• nucleic acids encoding V L or V H that do not include any nucleotide sequences encoding C L or C H , are isolated using methods well known in the art.
• the nucleic acids encoding V L or V H are then operatively linked to a nucleotide sequence encoding a C L or C H from a desired class of immunoglobulin molecule. This may be achieved using a vector or nucleic acid that comprises a C L or C H chain, as described above.
• an anti-Nogo receptor-1 antibody of the invention that was originally IgM may be class switched to an IgG. Further, the class switching may be used to convert one IgG subclass to another, e . g. , from IgGl to IgG2.
• antibodies or antigen- binding fragments of the invention may be mutated in the variable domains of the heavy and/or light chains to alter a binding property of the antibody.
• a mutation may be made in one or more of the CDR regions to increase or decrease the K d of the antibody for Nogo receptor-1, to increase or decrease K 0 f, or to alter the binding specificity of the antibody.
• Techniques in site-directed mutagenesis are well known in the art. See e . g. , Sambrook et al . and Ausubel et al . , supra .
• mutations are made at an amino acid residue that is known to be changed compared to germline in a variable region of an anti-Nogo receptor-1 antibody of the invention. In some embodiments, mutations are made at one or more amino acid residues that are known to be changed compared to the germline in a variable region of an anti-Nogo receptor-1 antibody of the invention. In another embodiment, a nucleic acid encoding an antibody heavy chain or light chain variable region is mutated in one or more of the framework regions . A mutation may be made in a framework region or constant domain to increase the half-life.
• a mutation in a framework region or constant domain also may be made to alter the immunogenicity of the antibody, to provide a site for covalent or non-covalent binding to another molecule, or to alter such properties as complement fixation. Mutations may be made in each of the framework regions, the constant domain and the variable regions in a single mutated antibody. Alternatively, mutations may be made in only one of the framework regions, the variable regions or the constant domain in a single mutated antibody.
• a fusion antibody or immunoadhesin may be made which comprises all or a portion of an anti-Nogo receptor-1 antibody of the invention linked to another polypeptide.
• only the variable region of the anti-Nogo receptor-1 antibody is linked to the polypeptide.
• the V H domain of an anti-Nogo receptor-1 antibody of this invention is linked to a first polypeptide, while the V L domain of the antibody is linked to a second polypeptide that associates with the first polypeptide in a manner that permits the V H and V L domains to interact with one another to form an antibody binding site.
• the V H domain is separated from the V L domain by a linker that permits the V H and V L domains to interact with one another (see below under Single Chain Antibodies) .
• the V H -linker- V L antibody is then linked to a polypeptide of interest.
• the fusion antibody is useful to directing a polypeptide to a cell or tissue that expresses a Nogo receptor-1 ligand.
• the polypeptide of interest may be a therapeutic agent, such as a toxin, or may be a diagnostic agent, such as an enzyme that may be easily visualized, such as horseradish peroxidase .
• 'fusion antibodies can be created in which two (or more) single-chain antibodies are linked to one another. This is useful if one wants to create a divalent or polyvalent antibody on a single polypeptide chain, or if one wants to create a bispecific antibody.
• the present invention includes a single chain antibody (scFv) that binds a Nogo receptor-1 polypeptide of the invention.
• scFv single chain antibody
• V H ⁇ and V L -encoding DNA is operatively linked to DNA encoding a flexible linker, e . g. , encoding the amino acid sequence (Gly 4 -Ser) 3 (SEQ ID NO: 10), such that the V H and V L sequences can be expressed as a contiguous single-chain protein, with the V L and V H regions joined by the flexible linker (see e . g. , Bird et al . (1988) Science 242:423-426; Huston et al . (1988) Proc.
• the single chain antibody may be monovalent, if only a single V H and V L are used, bivalent, if two V and V L are used, or polyvalent, if more than two V H and VL are used.
• Chimeric Antibodies [0080]
• the present invention further includes a bispecific antibody or antigen-binding fragment thereof in which one specificity is for a Nogo receptor-1 polypeptide of the invention.
• a chimeric antibody can be generated that specifically binds to a Nogo receptor- 1 polypeptide of the invention through one binding domain and to a second molecule through a second binding domain.
• the chimeric antibody can be produced through recombinant molecular biological techniques, or may be physically conjugated together.
• a single chain antibody containing more than one V H and V L may be generated that binds specifically to a polypeptide of the invention and to another molecule that is associated with attenuating myelin mediated growth cone collapse and inhibition of neurite outgrowth and sprouting.
• Such bispecific antibodies can be generated using techniques that are well known for example, Fanger et al .
• the chimeric antibodies are prepared using one or more of the variable regions from an antibody of the invention. In another embodiment, the chimeric antibody is prepared using one or more CDR regions from said antibody.
• An antibody or an antigen-binding fragment of the invention can be derivatized or linked to another molecule (e . g. , another peptide or protein) .
• the antibody or antigen-binding fragment is derivatized such that binding to a polypeptide of the invention is not affected adversely by the derivatization or labeling.
• an antibody or antibody portion of the invention can be functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody ( e . g.
• a derivatized antibody is produced by crosslinking two or more antibodies (of the same type or of different types, e . g. , to create bispecific antibodies) .
• Suitable crosslinkers include those that are heterobifunctional , having two distinctly reactive groups separated by an appropriate spacer ( e . g.
• the derivatized antibody is a labeled antibody.
• detection agents with which an antibody or antibody portion of the invention may be derivatized are fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-l-napthalenesulfonyl chloride, phycoer thrin, lanthanide phosphors and the like.
• An antibody also may be labeled with enzymes that are useful for detection, such as horseradish peroxidase, /3-galactosidase, luciferase, alkaline phosphatase, glucose oxidase and the like.
• enzymes that are useful for detection
• the antibody is detected by adding additional reagents that the enzyme uses to produce a detectable reaction product.
• horseradish peroxidase with hydrogen peroxide and diaminobenzidine for example, horseradish peroxidase with hydrogen peroxide and diaminobenzidine .
• An antibody also may be labeled with biotin, and detected through indirect measurement of avidin or streptavidin binding.
• An antibody may also be labeled with a predetermined polypeptide epitopes recognized by a secondary reporter ( e . g.
• An anti-Nogo receptor-1 antibody or an antigen- fragment thereof also may be labeled with a radio-labeled amino acid.
• the radiolabel may be used for both diagnostic and therapeutic purposes.
• the radio-labeled anti-Nogo receptor-1 antibody may be used diagnostically, for example, for determining Nogo receptor-1 levels in a subject. Further, the radio-labeled anti-Nogo receptor-1 antibody may be used therapeutically for treating spinal cord injury.
• labels for polypeptides include, but are not limited to, the following radioisotopes or radionucleotides - 3 H, 14 C, 15 N, 35 S, 90 Y, "Tc, U1 ln 125 I, 131 I .
• An anti-Nogo receptor-1 antibody or an antigen- fragment thereof may also be derivatized with a chemical group such as polyethylene glycol (PEG) , a methyl or ethyl group, or a carbohydrate group. These groups may be useful to improve the biological characteristics of the antibody, e . g. , to increase serum half-life or to increase tissue binding .
• The..class and subclass of anti-Nogo receptor-1 antibodies may be determined by any method known in the art.
• the class and subclass of an antibody may be determined using antibodies that are specific for a particular class and subclass of antibody. Such antibodies are available commercially.
• the class and subclass can be determined by ELISA, Western Blot as well as other techniques.
• the class and subclass may be determined by sequencing all or a portion of the constant domains of the heavy and/or light chains of the antibodies, comparing their amino acid sequences to the known amino acid sequences of various class and subclasses of immunoglobulins, and determining the class and subclass of the antibodies .
• binding affinity and dissociation rate of an anti-Nogo receptor-1 antibody of the invention to a Nogo receptor-1 polypeptide of the invention may be determined by any method known in the art.
• the binding affinity can be measured by competitive ELISAs, RIAs, BIAcore or KinExA technology.
• the dissociation rate also can be measured by BIAcore or KinExA technology.
• the binding affinity and dissociation rate are measured by surface plasmon resonance using, e . g. , a BIAcore.
• the K d of 7E11 and 1H2 were determined to be 1 x 10 "7 M and 2 x 10 "8 M, respectively.
• an anti-Nogo receptor-1 antibody or an antigen-binding fragment of the invention thereof inhibits the binding of Nogo receptor-1 to a ligand.
• the IC 50 of such inhibition can be measured by any method known in the art, e . g. , by ELISA, RIA, or Functional Antagonism.
• the IC 50 is between 0.1 and 500 nM.
• the IC 50 is between 10 and 400 nM.
• the antibody or portion thereof has an IC 50 of between 60 nM and 400 nM.
• the IC 50 of 7E11 and 1H2 were determined to be 400 nM and 60 nM, respectively, in a binding assay. See also Table 3, infra .
• Table 3, infra The IC 50 of 7E11 and 1H2 were determined to be 400 nM and 60 nM, respectively, in a binding assay. See also Table 3, infra .
• one of skill in the art provided with the teachings of this invention, has available a variety of methods which may be used to alter the biological properties of the antibodies of this invention including methods which would increase or decrease the stability or half-life, immunogenicity, toxicity, affinity or yield of a given antibody molecule, or to alter it in any other way that may render it more suitable for a particular application.
• Compositions comprising, and uses of, the antibodies of the present invention are described below.
• Nogo Receptor-1 Polypeptides Protein [0093] Full-length Nogo receptor-1 consists of a signal sequence, a N-terminus region (NT) , eight leucine rich repeats (LRR) , a LRRCT region (a leucine rich repeat domain C-terminal of the eight leucine rich repeats) , a C-terminus region (CT) and a GPI anchor (see Fig. 1) . [0094] Some embodiments of the invention provide a soluble Nogo receptor-1 polypeptide. Soluble Nogo receptor-
• polypeptides of the invention comprise an NT domain; 8 LRRs and an LRRCT domain and lack a signal sequence and a functional GPI anchor (i.e., no GPI anchor or a GPI anchor that lacks the ability to efficiently associate to a cell membrane) .
• a soluble Nogo receptor-1 polypeptide comprises a heterologous LRR.
• a soluble Nogo receptor-1 polypeptide comprises 2, 3, 4, 5, 6, 7, or 8 heterologous LRR ' s .
• a heterologous LRR means an LRR obtained from a protein other than Nogo receptor-1.
• Exemplary proteins from which a heterologous LRR can be obtained are toll-like receptor (TLR1.2); T-cell activation leucine repeat rich protein; deceorin; OM-gp; insulin-like growth factor binding protein acidic labile subunit slit and robo; and toll-like receptor 4.
• the invention provides a soluble Nogo receptor-1 polypeptide of 319 amino acids (soluble Nogo receptor-1 344, sNogoRl-344, or sNogoR344) (residues 26-344 of SEQ ID NOs : 6 and 8 or residues 27-344 of SEQ ID NO: 8) .
• the invention provides a soluble Nogo receptor-1 polypeptide of 285 amino acids (soluble Nogo receptor-1 310, sNogoRl-310, or sNogoR310) (residues 26-310 of SEQ ID NOs : 7 and 9 or residues 27-310 of SEQ ID NO: 9) . See Fig. 1.
• the soluble Nogo receptor-1 polypeptides of the invention are used to inhibit the binding of a ligand to Nogo receptor-1 and act as an antagonist of Nogo receptor-1 ligands.
• the soluble Nogo receptor-1 polypeptides of the invention are used to decrease inhibition of neurite outgrowth and sprouting in a neuron, such as axonal growth and to inhibit myelin mediated growth cone collapse in a neuron.
• the neuron is a CNS neuron.
• the soluble Nogo receptor-1 polypeptide of the invention is a component of a fusion protein that further comprises a heterologous polypeptide.
• the heterologous polypeptide is an immunoglobulin constant domain.
• the immunoglobulin constant domain is a heavy chain constant domain.
• the heterologous polypeptide is an Fc fragment.
• the Fc is joined to the C-terminal end of the soluble Nogo receptor-1 polypeptide of the invention.
• the fusion Nogo receptor-1 protein is a dimer.
• nucleic Acid Molecules of the Present Invention provide a nucleic acid that encodes a polypeptide of the invention, including the polypeptides of any one of SEQ ID NOs: 1-9, 26-27, 29-37 and 41-45.
• the nucleic acid encodes a polypeptide selected from the group consisting of amino acid residues 26-344 of Nogo receptor-1 as shown in SEQ ID NOs: 6 and 8 or amino acid residues 27-344 of Nogo receptor-1 as shown in SEQ ID NO: 8.
• the nucleic acid molecule encodes a polypeptide selected from the group consisting of amino acid residues 26-310 of Nogo receptor-1 as shown in SEQ ID NOs : 7 and 9 or amino acid residues 27- 310 of Nogo receptor-1 as shown in SEQ ID NO: 9.
• nucleic acid means genomic DNA, cDNA, mRNA and antisense molecules, as well as nucleic acids based on alternative backbones or including alternative bases whether derived from natural sources or synthesized.
• the nucleic acid further comprises a transcriptional promoter and optionally a signal sequence each of which is operably linked to the nucleotide sequence encoding the polypeptides of the invention.
• the invention provides a nucleic acid encoding a Nogo receptor-1 fusion protein of the invention, including a fusion protein comprising a polypeptide selected from the group consisting of amino acid residues 26-344 of Nogo receptor-1 as shown in SEQ ID NOs : 6 and 8 or amino acid residues 27-344 of SEQ ID NO : 8 and amino acid residues 26-310 of Nogo receptor-1 as shown in SEQ ID NOs: 7 and 9 or amino acid residues 27-310 of SEQ ID NO: 9.
• the nucleic acid encodes a Nogo receptor-1 fusion protein comprising a polypeptides selected from the group consisting of SEQ ID NOs: 26-27, 29-37 and 41-45.
• the nucleic acid encoding a Nogo receptor-1 fusion protein further comprises a transcriptional promoter and optionally a signal sequence.
• the nucleotide sequence further encodes an immunoglobulin constant region.
• the immunoglobulin constant region is a heavy chain constant region.
• the nucleotide sequence further encodes an immunoglobulin heavy chain constant region joined to a hinge region.
• the nucleic acid further encodes Fc .
• the Nogo receptor-1 fusion proteins comprise an Fc fragment.
• labels include, but are not limited to, biotin, radiolabeled nucleotides and the like. A skilled artisan can employ any of the art known labels to obtain a labeled encoding nucleic acid molecule.
• compositions comprising a polypeptide selected from the group consisting of SEQ ID NOs: 1-5, 26-27, 29-37 and 41-45.
• the invention provides compositions comprising an anti-Nogo receptor-1 antibody or an antigen-binding fragment thereof, or a soluble Nogo receptor-1 polypeptide or fusion protein of the present invention.
• the present invention may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically for delivery to the site of action.
• Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water- soluble form, for example, water-soluble salts.
• suspensions of the active compounds as appropriate oily injection suspensions may be administered.
• Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides .
• Aqueous injection suspensions may contain substances which increase the viscosity of the suspension include, for example, sodium carboxymethyl cellulose, sorbitol and dextran.
• the suspension may also contain stabilizers. Liposomes can also be used to encapsulate the molecules of this invention for delivery into the cell.
• compositions comprise isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride.
• compositions comprise pharmaceutically acceptable substances such as wetting or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibodies, antigen- binding fragments, soluble Nogo receptors or fusion proteins of the invention.
• auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibodies, antigen- binding fragments, soluble Nogo receptors or fusion proteins of the invention.
• Compositions of the invention may be in a variety of forms, including, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions ( e . g. , injectable and infusible solutions) , dispersions or suspensions . The preferred form depends on the intended mode of administration and therapeutic application.
• compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for passive immunization of humans with other antibodies .
• the composition can be formulated as a solution, micro emulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration.
• Sterile injectable solutions can be prepared by incorporating an anti-Nogo receptor-1 antibody in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
• dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
• the preferred methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
• the proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
• Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
• the active compound may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems.
• a controlled release formulation including implants, transdermal patches, and microencapsulated delivery systems.
• Biodegradable, , biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See e . g. , Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
• Supplementary active compounds also can be incorporated into the compositions.
• a Nogo receptor-1 antibody or an antigen-binding fragments thereof, or soluble Nogo receptor-1 polypeptides or fusion proteins of the invention are coformulated with and/or coadministered with one or more additional therapeutic agents .
• the pharmaceutical compositions of the invention may include a "therapeutically effective amount” or a “prophylactically effective amount” of an antibody, antigen- binding fragment, polypeptide (s) , or fusion protein of the invention.
• a “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result.
• a therapeutically effective amount of the Nogo receptor-1 antibody or antigen-binding fragment thereof, soluble Nogo receptor-1 polypeptide or Nogo receptor fusion protein may vary according to factors such as the disease state, age, sex, and weight of the individual.
• a therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody, antigen-binding fragment, soluble Nogo receptor-1 polypeptide or Nogo receptor fusion protein are outweighed by the therapeutically beneficial effects.
• a “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.
• Dosage regimens may be adjusted to provide the optimum desired response (e . g. , a therapeutic or prophylactic response) .
• a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.
• parenteral compositions in dosage unit form for ease of administration and uniformity of dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated, each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
• a therapeutically effective dose range for Nogo receptor-1 antibodies or antigen-binding fragments thereof is 0.1 - 4 mg/Kg per day. In some embodiments a therapeutically effective dose range for Nogo receptor-1 antibodies or antigen-binding fragments thereof is 0.2 - 4 mg/Kg per day. In some embodiments a therapeutically effective dose range for Nogo receptor-1 antibodies or antigen-binding fragments thereof is 0.2 mg/Kg per day.
• the invention provides methods for inhibiting Nogo receptor-1 activity by administering anti-Nogo receptor-1 antibodies, antigen- binding fragments of such antibodies, soluble Nogo receptor- 1 polypeptides, or fusion proteins comprising such polypeptides to a mammal in need thereof.
• the invention provides a method of inhibiting Nogo receptor-1 binding to a ligand, comprising the step of contacting Nogo receptor-1 with an antibody or antigen-binding fragment of this invention.
• the ligand is selected from the group consisting of NogoA, NogoB, NogoC, MAG and OM-gp.
• the invention provides a method for inhibiting growth cone collapse in a neuron, comprising the step of contacting the neuron with the antibody or antigen-binding fragment thereof of this invention.
• the invention provides a method for decreasing the inhibition of neurite outgrowth or sprouting in a neuron, comprising the step of contacting the neuron with the antibody or antigen-binding fragment of this invention.
• the neuron is a CNS neuron. In some of these methods, the neurite outgrowth or sprouting is axonal growth.
• the invention provides a method of promoting survival of a neuron in a mammal, which neuron is at risk of dying, comprising (a) providing a cultured host cell expressing (i) an anti-Nogo receptor-1 antibody or antigen-binding fragment thereof; or (ii) a soluble Nogo receptor-1 polypeptide; and (b) introducing the host cell into the mammal at or near the site of the neuron.
• a cultured host cell expressing (i) an anti-Nogo receptor-1 antibody or antigen-binding fragment thereof; or (ii) a soluble Nogo receptor-1 polypeptide
• introducing the host cell into the mammal at or near the site of the neuron comprising (a) providing a cultured host cell expressing (i) an anti-Nogo receptor-1 antibody or antigen-binding fragment thereof; or (ii) a soluble Nogo receptor-1 polypeptide; and (b) introducing the host cell into the mammal at or near the site of the neuron.
• the invention provides a gene therapy method of promoting survival of a neuron at risk of dying, which neuron is in a mammal, comprising administering at or near the site of the neuron a viral vector comprising a nucleotide sequence that encodes (a) an anti-Nogo receptor-l antibody or antigen-binding fragment thereof; or (b) a soluble Nogo receptor-1 polypeptide, wherein the anti- Nogo receptor-1 antibody, antigen-binding fragment, or soluble Nogo receptor-1 polypeptide is expressed from the nucleotide sequence in the mammal in an amount sufficient to promote survival of the neuron.
• a viral vector comprising a nucleotide sequence that encodes (a) an anti-Nogo receptor-l antibody or antigen-binding fragment thereof; or (b) a soluble Nogo receptor-1 polypeptide, wherein the anti- Nogo receptor-1 antibody, antigen-binding fragment, or soluble Nogo receptor-1 polypeptide is expressed from the nucleotide sequence in the mammal
• the invention provides a method of inhibiting Nogo receptor-1 binding to a ligand, comprising the step of contacting the ligand with the soluble Nogo receptor-1 polypeptide or the Nogo receptor-1 fusion protein of this invention.
• the invention provides a method of modulating an activity of a Nogo receptor-1 ligand, comprising the step of contacting the Nogo receptor- 1 ligand with a soluble Nogo receptor-1 polypeptide or a Nogo receptor-1 fusion protein of the invention.
• the invention provides a method for inhibiting growth cone collapse in a neuron, comprising the step of contacting a Nogo receptor-1 ligand with a soluble Nogo receptor-1 polypeptide or a Nogo receptor-1 fusion protein of this invention.
• the invention provides a method for decreasing the inhibition of neurite outgrowth or sprouting in a neuron, comprising the step of contacting a Nogo receptor-1 ligand with the soluble Nogo receptor-1 polypeptide or the Nogo receptor-1 fusion protein of this invention.
• the neuron is a CNS neuron.
• the ligand is selected from the group consisting of NogoA, NogoB, NogoC, MAG and OM-gp.
• the neurite outgrowth or sprouting is axonal growth .
• the anti-Nogo receptor-1 antibody is a human antibody.
• the mammal is a human patient.
• the antibody or antigen- binding fragment thereof is administered to a non-human mammal expressing a Nogo receptor-1 with which the antibody cross-reacts (e . g. , a primate, cynomologous or rhesus monkey) for veterinary purposes or as an animal model of human disease.
• a non-human mammal expressing a Nogo receptor-1 with which the antibody cross-reacts e . g. , a primate, cynomologous or rhesus monkey
• Such animal models may be useful for evaluating the therapeutic efficacy of antibodies of this invention.
• administration of 'anti-Nogo receptor-1 antibody or antigen-binding fragment, or soluble Nogo receptor-1 polypeptide or fusion protein is used to treat a spinal cord injury to facilitate axonal growth throughout the injured site.
• the anti-Nogo receptor-1 antibodies or antigen- binding fragments, or soluble Nogo receptor-1 polypeptides or fusion proteins of the present invention can be provided alone, or in combination, or in sequential combination with other agents that modulate a particular pathological process.
• anti-inflammatory agents may be co- administered following stroke as a means for blocking further neuronal damage and inhibition of axonal regeneration.
• the Nogo receptor-1 antibodies, antigen-binding fragments, soluble Nogo receptor-1 and Nogo receptor fusion proteins are said to be administered in combination with one or more additional therapeutic agents when the two are administered simultaneously, consecutively or independently.
• the anti-Nogo receptor-1 antibodies, antigen- binding fragments, soluble Nogo receptor-1 polypeptides, Nogo receptor-1 fusion proteins of the present invention can be administered via parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal , transdermal, inhalational or buccal routes.
• an agent may be administered locally to a site of injury via microinfusion. Typical sites include, but are not limited to, damaged areas of the r spinal cord resulting from injury.
• the dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
• the compounds of this invention can be utilized in vivo, ordinarily in mammals, such as humans, sheep, horses, cattle, pigs, dogs, cats, rats and mice, or in vi tro .
• the invention provides recombinant DNA molecules (rDNA) that contain a coding sequence.
• a rDNA molecule is a DNA molecule that has been subjected to molecular manipulation. Methods for generating rDNA molecules are well known in the art, for example, see Sambrook et al . , (1989) Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory Press.
• a coding DNA sequence is operably linked to expression control sequences and vector sequences.
• the invention provides vectors comprising the nucleic acids encoding the polypeptides of the invention.
• vector and expression control sequences to which the nucleic acids of this invention is operably linked depends directly, as is well known in the art, on the functional properties desired (e . g. , protein expression, and the host cell to be transformed) .
• a vector of the present invention may be at least capable of directing the replication or insertion into the host chromosome, and preferably also expression, of the structural gene included in the rDNA molecule.
• Expression control elements that are used for regulating the expression of an operably linked protein encoding sequence are known in the art and include, but are not limited to, inducible promoters, constitutive promoters, secretion signals, and other regulatory elements.
• the inducible promoter is readily controlled, such as being responsive to a nutrient in the host cell's medium.
• the vector containing a coding nucleic acid molecule will include a prokaryotic replicon, i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extra-chromosomally in a prokaryotic host cell, such as a bacterial host cell, transformed therewith.
• a prokaryotic replicon i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extra-chromosomally in a prokaryotic host cell, such as a bacterial host cell, transformed therewith.
• replicons are well known in the art.
• vectors that include a prokaryotic replicon may also include a gene whose expression confers a detectable or selectable marker such as a drug resistance.
• Vectors that include a prokaryotic replicon can further include a prokaryotic or bacteriophage promoter capable of directing the expression (transcription and translation) of the coding gene sequences in a bacterial host cell, such as E. coli .
• a promoter is an expression control element formed by a DNA sequence that permits binding of RNA polymerase and transcription to occur. Promoter sequences compatible with bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a DNA segment of the present invention.
• vector plasmids examples include pUC8, pUC9, pBR322 and pBR329 (Bio-Rad " Laboratories), pPL and pKK223 (Pharmacia) .
• Any suitable prokaryotic host can be used to express a recombinant DNA molecule encoding a protein of the invention.
• Expression vectors compatible with eukaryotic cells can also be used to form a rDNA molecules that contains a coding sequence.
• Eukaryotic cell expression vectors are well known in the art and are available from several commercial sources. Typically, such vectors are provided containing convenient restriction sites for insertion of the desired DNA segment . Examples of such vectors are pSVL and pKSV-10 (Pharmacia) , pBPV-1, pML2d (International
• Eukaryotic cell expression vectors used to construct the rDNA molecules of the present invention may further include a selectable marker that is effective in an eukaryotic cell, preferably a drug resistance selection marker.
• a preferred drug resistance marker is the gene whose expression results in neomycin resistance, i.e., the neomycin phosphotransferase (neo) gene. (Southern et al . , (1982) J. Mol. Anal. Genet. 1, 327-341).
• the selectable marker can be present on a separate plasmid, the two vectors introduced by co-transfection of the host cell, and transfectants selected by culturing in the appropriate drug for the selectable marker.
• DNAs encoding partial or full-length light and heavy chains are inserted into expression vectors such that the genes are operatively linked to transcriptional and translational control sequences.
• Expression vectors include plasmids, retroviruses, cosmids, YACs, EBV-derived episomes, and the like.
• the antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene.
• the expression vector and expression control sequences are chosen to be compatible with the expression host cell used.
• the antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors. In some embodiments, both genes are inserted into the same expression vector.
• the antibody genes are inserted into the expression vector by standard methods (e . g. , ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present) .
• a convenient vector is one that encodes a functionally complete human C H or C L immunoglobulin sequence, with appropriate restriction sites engineered so that any V H or V L sequence can be easily inserted and expressed, as described above.
• splicing usually occurs between the splice donor site in the inserted J region and the splice acceptor site preceding the human C region, and also at the splice regions that occur within the human C H exons .
• Polyadenylation and transcription termination occur at native chromosomal sites downstream of the coding regions.
• the recombinant expression vector can also encode a signal peptide that facilitates secretion of the antibody chain from a host cell .
• the antibody chain gene may be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene.
• the signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein) .
• the recombinant expression vectors of the invention carry regulatory sequences that control their expression in a host cell. It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.
• Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from retroviral LTRs, cytomegalovirus (CMV) (such as the CMV promoter/enhancer) , Simian Virus 40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, ( e .g. , the adenovirus major late promoter (AdMLP) ) , polyoma and strong mammalian promoters such as native immunoglobulin and actin promoters.
• CMV cytomegalovirus
• SV40 Simian Virus 40
• AdMLP adenovirus major late promoter
• polyoma and strong mammalian promoters such as native immunoglobulin and actin promoters.
• the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e . g. , origins of replication) and selectable marker genes.
• the selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e . g. , U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017, all by Axel et al .
• the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced.
• drugs such as G418, hygromycin or methotrexate
• Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr " host cells with methotrexate selection/amplification) and the neo gene (for G418 selection) .
• DHFR dihydrofolate reductase
• neo gene for G418 selection
• Nucleic acid molecules encoding anti-Nogo receptor-1 antibodies, immunogenic peptides, soluble Nogo receptor-1 polypeptides, soluble Nogo receptor-1 fusion proteins of this invention and vectors comprising these nucleic acid molecules can be used for transformation of a suitable host cell. Transformation can be by any known method for introducing polynucleotides into a host cell .
• Methods for introduction of heterologous polynucleotides into mammalian cells include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide (s) in liposomes, and direct microinjection of the DNA into nuclei.
• nucleic acid molecules may be introduced into mammalian cells by viral vectors.
• Transformation of appropriate cell hosts with a rDNA molecule of the present invention is accomplished by well known methods that typically depend on the type of vector used and host system employed. With regard to transformation of prokaryotic host cells, electroporation and salt treatment methods can be employed (see, for example, Sambrook et al . , (1989) Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory Press;
• cells resulting from the introduction of an rDNA of the present invention can be cloned to produce single colonies.
• Cells from those colonies can be harvested, lysed and their DNA content examined for the presence of the rDNA using a method such as that described by Southern, (1975) J. Mol. Biol. 98, 503-517 or the proteins produced from the cell may be assayed by an immunological method.
• Mammalian cell lines available as hosts for expression are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC ®) . These include, inter alia,
• CHO Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e . g. , Hep G2), A549 cells, and a number of other cell lines.
• Cell lines of particular preference are selected through determining which cell lines have high expression levels.
• Other cell lines that may be used are insect cell lines, such as Sf9 cells.
• recombinant expression vectors encoding the immunogenic polypeptides, Nogo receptor-1 antibodies or antigen-binding fragments, soluble Nogo receptor-1 polypeptides and soluble Nogo receptor-1 fusion proteins of the invention are introduced into mammalian host cells, they are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody, polypeptide and fusion polypeptide in the host cells or, more preferably, secretion of the immunogenic polypeptides, Nogo receptor-1 antibodies or antigen-binding fragments, soluble Nogo receptor-1 polypeptides and soluble Nogo receptor-1 fusion proteins of the invention into the culture medium in which the host cells are grown.
• Immunogenic polypeptides, Nogo receptor-1 antibodies or antigen-binding fragments, soluble Nogo receptor-1 polypeptides and soluble Nogo receptor-1 fusion proteins of the invention can be recovered from the culture medium using standard protein purification methods.
• expression of immunogenic polypeptides, Nogo receptor-1 antibodies or antigen-binding fragments, soluble Nogo receptor-1 polypeptides and soluble Nogo receptor-1 fusion proteins of the invention of the invention (or other moieties therefrom) from production cell lines can be enhanced using a number of known techniques .
• the glutamine synthetase gene expression system (the GS system) is a common approach for enhancing expression under certain conditions. The GS system is discussed in whole or part in connection with European Patent Nos. 0 216 846, 0 256 055, and 0 323 997 and European Patent Application No. 89303964.4.
• the present invention further provides host cells transformed with a nucleic acid molecule that encodes a Nogo receptor-1 antibody, antigen-binding fragment, soluble Nogo receptor-1 polypeptide and/or soluble Nogo receptor-1 fusion protein of the invention.
• the host cell can be either prokaryotic or eukaryotic.
• Eukaryotic cells useful for expression of a protein of the invention are not limited, so long as the cell line is compatible with cell culture methods and compatible with the propagation of the expression vector and expression of the gene product.
• Preferred eukaryotic host cells include, but are not limited to, yeast, insect and mammalian cells, preferably vertebrate cells such as those from a mouse, rat, monkey or human cell line.
• useful eukaryotic host cells include ® Chinese hamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swiss mouse embryo cells NIH-3T3 available from the ATCC as CRL1658, baby hamster kidney cells (BHK) , and the like eukaryotic tissue culture cell lines.
• the present invention further provides methods for producing an a Nogo receptor-1 antibody or antigen-binding fragment, soluble Nogo receptor-1 polypeptide and/or soluble Nogo receptor-1 fusion protein of the invention using nucleic acid molecules herein described.
• the production of a recombinant form of a protein typically involves the following steps: [0143] First, a nucleic acid molecule is obtained that encodes a protein of the invention. If the encoding sequence is uninterrupted by introns, it is directly suitable for expression in any host.
• the nucleic acid molecule is then optionally placed in operable linkage with suitable control sequences, as described above, to form an expression unit containing the protein open reading frame.
• the expression unit is used to transform a suitable host and the transformed host is cultured under conditions that allow the production of the recombinant protein.
• the recombinant protein is isolated from the medium or from the cells; recovery and purification of the protein may not be necessary in some instances where some impurities may be tolerated.
• Each of the foregoing steps can be done in a variety of ways.
• the desired coding sequences may be obtained from genomic fragments and used directly in appropriate hosts.
• expression vectors that are operable in a variety of hosts is accomplished using appropriate replicons and control sequences, as set forth above.
• the control sequences, expression vectors, and transformation methods are dependent on the type of host cell used to express the gene and were discussed in detail earlier.
• Suitable restriction sites can, if not normally available, be added to the ends of the coding sequence so as to provide an excisable gene to insert into these vectors.
• Nogo receptor-1 As the Immunogen [0149]
• the rat Nogo receptor-1 gene (GenBank No. AF 462390) was subcloned into the mammalian expression vector ® pEAG1256 (Biogen ) that contained the CMV promotor and geneticin resistance gene for drug selection.
• the recombinant plasmid was transfected into COS-7 cells using ® Superfect (Qiagen ) . Transfectants were selected using geneticin (Gibco , 2 mg/ml) , cloned and verified for surface expression of Nogo receptor-1 protein by FACS .
• COS-7 membranes were prepared from these cells according to procedures as described [Wang et al . , J. Neuroch em . , 75 : 1155- 1161 (2000)] with two washings, and stored at 1 mg/ml [protein concentration] in 10% glycerol at -70 °C.
• Nogo receptor-1 Peptides as the Immunogen
• the rat Nogo receptor-1 gene sequence was subjected to antigenicity analyses using Vector NTi software (Fig. 2) .
• Antigenic peptides identified in the analyses were conjugated to Keyhole Limpet Hemocyanin (KLH) using standard glutaldehyde procedures .
• KLH Keyhole Limpet Hemocyanin
• Eight-week-old female RBF mice (Jackson Labs, Bar Harbor, ME) were immunized intraperitoneally with an emulsion containing 50 ⁇ g KLH-conjugated peptides and 50 ⁇ l ® complete Freund ' s adjuvant (Sigma Chemical Co., St. Louis, MO) once every two weeks.
• Serum from the immunized mice was collected before the first immunization and 1 week after the second and third immunizations and anti-Nogo receptor-1 antibody titers were measured. A booster dose was given after the third immunization. Three days after this booster dose immunization, fusion experiments were initiated.
• Splenocytes were isolated from the mice and fused to the FL653 myeloma (an APRT- derivative of a Ig-/HGPRT- Balb/c mouse myeloma, maintained in DMEM containing 10% FBS, 4500 mg/L glucose, 4 mM L-glutamine, and 20 mg/ml 8-azaguanine) as described (Kennett et al . , 1993. Monoclonal Antibodies: A New Dimension in Biological Analysis. Plenum Press, New York) . Fused cells were plated into 24- or 48-well plates (Corning Glass Works, Corning, NY), and fed with adenine, aminopterin and thymidine containing culture media.
• FL653 myeloma an APRT- derivative of a Ig-/HGPRT- Balb/c mouse myeloma, maintained in DMEM containing 10% FBS, 4500 mg/L glucose, 4 mM L-glutamine, and 20
• AAT resistant cultures were screened by ELISA or flow cytometry for binding to either Nogo receptor-l-COS-7 cells or to a Nogo receptor-1 antigenic peptide as described below. Cells in the positive wells were further subcloned by limiting dilution. [0154] To screen for antibody binding to a Nogo receptor- 1 antigenic peptide, the peptides that were used as immunogens were conjugated to BSA. 0.5 ⁇ g of the conjugated peptide in 50 ⁇ l of 0.1 M sodium bicarbonate buffer, pH 9.0 was added to each well of a 96-well MaxiSorp plate (Nunc ) . The plate was then incubated at 37°C for 1 hour or 4°C for
• Antibodies were screened for binding to full length Nogo receptor-1 as follows. COS-7 cells were labeled with 0.1 uM CellTracker Green CMFDA (Molecular Probes, Eugene, OR) as described by the vendor. Equal volumes of
• the cells were washed and incubated with 50 ⁇ l of R- phycoerythrin-conjugated affinity pure F(ab')2 fragment goat anti-mouse IgG Fc gamma specific second antibody (1:200, Jackson ImmunoResearch Laboratory, West Grove, PA) in PBS.
• the cells were washed twice with PBS and suspended in 200 ⁇ l of PBS containing 1% FBS, and subjected to FACS analyses.
• Nogo receptor- l-COS-7 cells were mixed with hybridoma supernatant and then treated with R-phycoerythrin-conjugated goat anti-mouse secondary antibody and directly subjected to standard FACS analyses.
• 25 anti-Nogo receptor-1 antibodies using a variety of immunogens.
• primers comprise degenerate nucleotides as follows: S represents G or C; M represents A or C, R represents G or A; W represents A or T; K represents G or T; and Y represents T or C .
• S represents G or C
• M represents A or C
• R represents G or A
• W represents A or T
• K represents G or T
• Y represents T or C .
• the light chains of 7E11 and 5B10 are have 94% amino acid sequence identity and the heavy chains have 91% amino acid sequence identity.
• mAbs 7E11, 5B10, and 1H2 are of the IgGl isotype and mAbs 3G5 and 2F7 are of the IgG2a isotype. Each of these five mAbs has a light chain of the kappa isotype. We analyze the sequence of the other monoclonal antibodies by this approach. Table 2. Amino Acid Sequence of mAbs 7E11 and 5B10
• Mab 7E11 binds both rat and human NgRl. To determine the epitope responsible for 7E11 binding, we generated fragments and synthetic peptides of rat NgRl and tested them for 7E11 binding. [0159] A recombinant fragment of the rat NgRl that contains all 8 LRR domains and the N- and C- terminal caps (sNgR310) was treated with either acid or cyanogen bromide (CNBr) and separated the fragments by gel electrophoresis. Untreated sNgR310 migrates with an apparent molecular weight of 42 kDa.
• CNBr cyanogen bromide
• Acid treatment of sNgR310 produced two major cleavage products of 15 kDa (aa 27-aa 122) and 30 kDa (aa 123-aa 310) .
• CNBr treatment generated three fragments, a 33/35 kDa doublet (aa 27-aa 229) , which may represent fragments with heterogeneous glycosylation, a 10 kDa product (aa 241-aa 310) , and an 11-amino-acid fragment (aa 230-aa 240), which is not retained on the gel.
• the 7E11 binding site was further analyzed by testing trypsic peptide digests of sNgR310. HPLC analyses showed several fragments, indicating that there were several trypsin-sensitive lysine and arginine residues in the NgRl sequence. 7E11 bound only a single tryptic digest peptide, providing additional evidence that 7E11 binds to a single epitope on NgRl. Subsequent mass spectroscopy (MS) and sequence analyses identified the bound peptide to be AAAFGLTLLEQLDLSDNAQLR (SEQ ID NO : 26) . [0161] The LDLSDNAQLRWDPTT peptide (SEQ ID NO: 1) was subjected to further mapping analysis.
• the peptide was digested with trypsin, which yielded two major fragments, LDLSDNAQLR (SEQ ID NO : 27) and WDPTT (SEQ ID NO : 28), and the ability of 7E11 to bind them was tested.
• MS analysis revealed that the antibody bound peptide LDLSDNAQLR (SEQ ID NO: 27), and therefore this peptide contains the binding epitope for 7E11.
• detailed MS analysis identified several scrambled peptides that also bound 7E11, including peptides with deamination at Asnll5 and Glnll7, addition of Alanine at 112 or 113, or addition of Serine at 114 (Table 3) . These data indicate that several amino acid residues located in this peptide fragment may not be critical for 7E11 binding.
• Table 3 Mutant peptides bound by 7E11
• the LDLSDNAQLRWDPTT peptide was also digested with the endoprotease Asp-N and 7E11 binding was tested. Endoprotease Asp-N cleaved the peptide into 3 peptide fragments, L, DLS and DNAQLRWDPTT (SEQ ID NO: 36) . Of these products, 7E11 bound the DNAQLRWDPTT peptide. Taken together, the trypsin and Asp-N cleavage data further localize the 7E11 binding epitope to the sequence shared between them, DNAQLR (SEQ ID NO : 37) .
• NgRl, NgR2 , and NgR3 were analyzed to predict critical residues in the 7E11 binding epitope based on the observation that 7E11 bound rat and human NgRl but not mouse NgRl, human NgR2 or mouse NgR3. Sequence alignment revealed that amino acids 110-125 of rat NgRl and the corresponding sequence of human NgRl are identical and that the mouse NgRl sequence differs only by one amino acid at position 119 (Argil9 in rat and human NgRl, and Hisll9 in mouse NgRl; Table 4) . Table 4. Sequence alignment of NgRs from different species
• Argll9 on NgRl contributes to 7E11 binding because it binds well to rat and human NgRl but poorly to mouse NgRl.
• 7E11 does not bind well to NgR3
• Alall ⁇ is involved in the epitope because within the DNAQLR sequence (SEQ ID NO: 38) NgR3 only differs from NgRl by an Arginine at the corresponding sequence .
• SEQ ID NO: 38 DNAQLR sequence
• 4 out of 6 of the residues in NgR2 are identical to rat NgRl. Alall6 and Glnll7 are replaced with Arginine and Histidine, respectively.
• the position of the 7E11 binding epitope was also determined in the recently resolved crystal structure of sNgR310. As expected, the structure shows that the 7E11 epitope is exposed on the surface of the molecule. Residues Argll9, Glnll7, Alall6, and Aspll4 protrude outward from the structure while Leull8 and Asnll5 are located inward. The epitope falls on top of an acidic patch within the concave surface of the structure and a basic surface that faces one of the sides.
• SPA beads coupled with Fc-sNogoR-1, anti- Nogo receptor-1 mAb and I ⁇ l 125 I-Nogo66 (Amersham, 2000 Ci/mmol, InM) in 50 ⁇ l of the HEPES-buffered incubation medium (10 mM HEPES, pH 7.4, 0.1% bovine serum albumin, 0.1% ovalbumin, 2 mM MgCl 2 , 2 mM CaCl 2 and protease inhibitors) was added to each sample well. After 16 hours, radioactivity was measured in quadruplicate samples using a ® TopCount (Packard) . IC 50 values were calculated from a curve-fit analysis (Fig. 3) (PRISM, GraphPad Software, NJ) .
• AP-ligand conjugates e.g. AP-Nogo66
• AP-Nogo66 AP-ligand conjugates
• Monoclonal antibodies 7E11, 5B10, 1H2 , 3G5 and 2F7 all inhibited binding of Nogo66, MAG and OM-gp to sNogoR344- Fc .
• the calculated IC 50 for Nogo66 for 7E11 and 1H2 were 400 nM and 60 nM, respectively.
• the data from ELISAs monitoring mAb-mediated inhibition of binding of the three ligands to Nogo receptor-1 are summarized in Table 6.
• the percent displacement is shown at 30 nM antibody and the EC 50 for certain mAbs determined from curve-fit analysis as described. "—” indicates no detectable activity and “ND” indicates not determined.
• Anti-Nogo receptor-1 Fab-phage antibodies that specifically bind an immunogenic Nogo receptor-1 polypeptide of the invention were also made by screening a Fab-phage library as follows. ® [0171] The MorphoSys Fab-phage library HuCAL GOLD was screened against recombinant rat soluble sNogoR310-Fc protein and COS7 cells expressing rat Nogo receptor-1. Fab- phages that specifically bound to Nogo receptor-1 were purified and characterized. The heavy chain of 14D5 is derived from the V H 2 gene and the light chain is derived from the V ⁇ l gene. The amino acid sequences of the CDRs of the heavy chain and light chain of one of these Fab-phages, 14D5, are shown in Table 7.
• 14D5 binds to rat Nogo receptor-1 in both monovalent and bivalent forms. In addition, 14D5 binds to mouse and human Nogo receptor-1 and human Nogo receptor-2 but not mouse Nogo receptor-3.
• polypeptides p-4 and p-5 polypeptides from the LRR5 and LRRCT regions of Nogo receptor-1, respectively. Ovalbumin and BSA were used as controls.
• mAbs 1H2 , 3G5 and 2F7 all specifically bound to sNogoR344-Fc .
• those antibodies also specifically bound a polypeptide consisting of amino acids 310-344 of rat Nogo receptor-1 (SEQ ID NO: 3) and mAbs 7E11 and 5B10 specifically bound polypeptide p-617 (SEQ ID NO: 1) .
• CNS myelin or PBS was spotted as 3 ⁇ l drops. Fluorescent microspheres (Polysciences) were added to the myelin/PBS to allow later identification of the drops (Grandpre et al , ® Nature 403, 2000) . Lab-Tek slides were then rinsed and coated with 10 ⁇ g/ml laminin (Gibco ) .
• DRG's Dorsal root ganglions (DRG's) from P3-4 Sprague Dawley rat pups were dissociated with 1 mg/ml collagenase type 1 (Worthington) , triturated with fire-polished Pasteur pipettes pre-plated to enrich in neuronal cells and finally plated at 23,000 ® cells/well on the pre-coated Lab-Tek culture slides.
• the culture medium was F12 containing 5% heat inactivated donor horse serum, 5% heat inactivated fetal bovine serum and 50 ng/ml NGF and incubated at 37°C and 5% C0 2 for 6 hours. Fifteen ⁇ g/ml of mAb 7E11 was added immediately after plating.
• P7 DRG neurons were cultured on a CNS myelin substrate, bivalent 14D5 also efficiently promoted neurite outgrowth.
• Nogo receptor-1 transfected and non-transfected ® cells were plated in 8-well Lab-Tek culture slides, fixed with 4% paraformaldehyde for 15 minutes, blocked with 10% normal goat serum, 0.1% Triton X-100 in PBS for 1 hour. Mab
• 7E11 was added at 15 ⁇ g/ml and 1.5 ⁇ g/ml in blocking solution and incubated for 2 hours at room temperature; ® Alexa -conjugated secondary antibody anti-mouse (Molecular
• Probes was incubated at a 1:300 dilution in blocking solution for 1 hour; DAPI was added at 5 ⁇ g/ml to the secondary antibody to label all nuclei.
• EXAMPLE 7 Mouse Model of Spinal Cord Contusive Injury
• Female mice (18-22 g) are treated prophylactically with analgesic and antibiotic agents. Mice are anesthetized and placed in a stereotaxic apparatus with vertebral column fixation under a stereomicroscope . Trauma to the spinal cord is introduced by a modified version of the weight-drop method (M. Li et al . , Functional role and therapeutic implications of neuronal caspase-1 and -3 in a mouse model of traumatic spinal cord injury. Neuroscience Vol. 99, pp. 333-342, 2000) .
• a T9 and T10 laminectomy is made and the vertebral column is stabilized using a pair of mouse transverse clamps supporting the T9-T10 transverse processes bilaterally.
• a stainless steel impact rod with a diameter of 1.4 mm and weight of 2 g, is raised 2.5 cm above the dura and dropped onto the spinal cord at the T10 level.
• mice are kept on a 37°C warming blanket and 1 ml of warmed sterile saline is administered subcutaneously to each mouse after surgery to avoid dehydration.
• the bladder is manually expressed once daily until reflexive bladder control is regained.
• soluble Nogo receptors (sNogoR310-Fc and sNogoR344-Fc) were immobilized on 250 ⁇ g WGA-SPA beads and received 0.5 ⁇ L of radioactive ligand (final concentration 0.5 nM) in a final volume of 100 ⁇ L of binding buffer (20 mM HEPES, pH 7.4, 2 mM Ca, 2 mM Mg, 0.1% BSA, 0.1% ovalubmin and protease inhibitors).
• Ligands included 10 ⁇ M Nogo66, 10 ⁇ M 125 I-Nogo40 (amino acids 1-40 of NogoA) and 10 ⁇ L of anti-Nogo receptor-1 antibody supernatant for each ligand set.
• Nogo40-A,-B and -C The three tyrosines on Nogo40 were separately iodinated and designated as Nogo40-A,-B and -C respectively. Mean values of triplicates are presented as normalized % bound radioactivity (Figs. 6, 7 and 8) . Error bars indicate SEM. Bound radioactivity in the absence of inhibitors was taken as 100% and the lowest bound radioactivity in the presence of 10 ⁇ M Nogo40 was taken as the 0% for data normalization.
• EXAMPLE 9 Inhibition of Ligand Binding to Soluble Nogo receptor-1 Fusion Protein [0193] A binding assay similar to the binding assay of Example 8 was used to test the ability of two mAbs produced in Example 1 to inhibit 125 I-Nogo66 binding to sNogoR344-Fc . Mabs 2F7 and 3G5 inhibited ⁇ 25 I-Nogo66 binding to sNogoR344- Fc.
• EXAMPLE 10 Neurite Outgrowth Assay ® [0194] Lab-Tek culture slides (4 wells) were coated with 0.1 mg/ml poly-D-lysme (Sigma ®) . CNS myelin alone or mixed with sNogoR310, sNogoR310-Fc fusion protein, mAb 5B10 or control PBS were separately spotted as 3 ⁇ l drops.
• the culture medium was F12 containing 5% heat inactivated donor horse serum, 5% heat inactivated fetal bovine serum and 50 ng/ml mNGF and incubated at 37 °C and 5% C0 2 for 6 hours .
• Slides were fixed for 20 minutes with 4% paraformaldehyde containing 20% sucrose and stained for the 1 neuronal marker anti beta-III-tubulin (Covance TUJl) diluted 1:500.
• As secondary antibody anti-mouse Alexa ® Fluor 594 (Molecular Probes) was diluted 1:300 and slides were coverslipped with Gel/Mount (Bi ⁇ meda ) . 5x digital images were acquired with OpenLab software and analyzed by ® using the MetaMorph software for quantification of neurite outgrowth.
• sNogoR310, sNogoR310-Fc and mAb 5B10 all protected DRG neurons from myelin-mediated inhibition of neurite outgrowth (Figs. 9-11) .
• sNogoR310 was used in a similar assay using chick neurons and was found to be protective.
• DRG's were dissected from post-natal day 6-7 rat pups (P6-7) , dissociated into single cells and plated on 96- well plates pre-coated with poly-D-lysine as described above. In some wells 2 ⁇ g/ml laminin was added for 2-3 hours and rinsed before the cells were plated. After an 18- 20 h incubation the plates were fixed with 4% paraformaldehyde, stained with rabbit anti-Beta-III-tubulin ® antibody diluted 1:500 (Covance ) and anti-HuC/D diluted
• Neuroscreen was used (Cellomics ) .
• the Neuroscreen cells were pre-differentiated for 7 days with 200 ng/ml NGF, detached and replated on 96-well plates pre-coated with poly-D-lysine. In some wells 5 ⁇ g/ml laminin was added for 2-3 hours and rinsed before the cells were plated. After 2 days incubation the plates were fixed with 4% paraformaldehyde, stained with rabbit anti-Beta-III-tubulin ® antibody diluted 1:500 (Covance ) and Hoechst (nuclear stain) . The ArrayScan ® II was used to quantify neurite outgrowth as in the DRG cells. [0201] sNogoR344-Fc or rat IgG were added in solution to
• a cDNA construct encoding amino acids 1-310 of rat Nogo receptor-1 was fused to rat IgGl Fc contained in a mammalian expression vector and this vector was electroporated into Chinese hamster ovary (CHO) (DG44) cells.
• CHO Chinese hamster ovary
• Cells were maintained in alpha-MEM, supplemented with 10% dialyzed fetal bovine serum, 2 mM glutamine and antibiotic-antimycotic reagents. Two days after transfection, the conditioned media was collected and analyzed by Western blot under reducing conditions. A protein band about 60 kDa was detected using a polyclonal rabbit anti-Nogo receptor-1 antibody. Cells were expanded and sorted using a R-PE conjugated goat anti-rat IgG antibody.
• CHO cells expressing the sNogoR310-Fc fusion protein were cultured in large scale. 1.7 L of concentrated conditioned media was obtained from a 10 L bioreactor run. The pH was raised by addition of one-tenth volume 1.0 M Tris-HCl, pH 8.9. Solid sodium chloride and glycine were added to 3.0 M and 1.5 M respectively. A 60 mL protein A-
• Sepharose column equilibrated with 10 mM Tris-HCl, 3 M sodium chloride, 1.5 M glycine, pH 8.9 was prepared. Concentrated conditioned media was applied to the column at 1.5 mL/min using a peristaltic pump. The column was washed with 300 mL of 10 mM Tris-HCl, 3 M sodium chloride, 1.5 M glycine, pH 8.9 followed with 120 mL 5 mM Tris-HCl, 3 M sodium chloride, pH 8.9. Protein was eluted with 25 mM sodium phosphate, 100 mM sodium chloride, pH 2.8. 10 mL fractions were collected in tubes containing 1.0 mL of 1.0 M HEPES, pH 8.5. Protein fractions were pooled and dialyzed against 3 x 2 L of 5 mM sodium phosphate, 300 mM NaCl , pH 7.4.
• sNogoR-1 fusion protein was tested in a rat spinal cord transection assay.
• ® Alzet osmotic pumps were loaded with test solution (sNogoR310-Fc in PBS) made up freshly on the day of use. The loading concentration was calculated to be 5 and 50 ⁇ M. Pumps were primed for >40 hours at 37 °C prior to implantation into animals. Female Long Evans rats were given pre-operative analgesia and tranquilizer and anesthetized using isoflurane (3% in 0 2 ) .
• rat behavioral testing BBB locomotor scale, grid walking and footprint analysis were performed.
• grid walking the rats were trained to walk on a wire grid (70 cm long with 2.54 cm squares), and the number of instances in which the hindpaw dropped below the grid plane was counted.
• footprint analysis the walking patterns of rat hindpaws were recorded with ink during a continuous locomotion across a 90 cm runway, and stride length on each side and stride width were calculated (Metz et al . , 2000, Brain Res., 883, 165-177) . All of these behavioral tests were performed by at least two individuals. Throughout the surgery, behavioral testing and histologic analysis, researchers were blind to the identity of the compound in the minipump . [0210] sNogoR310-Fc promoted functional recovery (Fig. 12) .
• EXAMPLE 13 Rat Spinal Cord Contusion Assay The effect of soluble Nogo receptor-1 polypeptides and fusion proteins on neurons in vivo are tested in a rat spinal cord contusion assay.
• Female hooded Long Evans rats (170-190 g) are treated prophylactically with analgesic and antibiotic agents. Ten minutes before surgery, animals are tranquilized with 2.5 mg/kg Midazolam i.p. and anesthetized in 2-3% isoflurane in 0 2 . Rats are then shaved, wiped down with alcohol and betadine, and ocular lubricant applied to their eyes. Next, an incision is made down the midline and the T7 to T12 vertebrae exposed. [0213] A dorsal laminectomy is performed at T9 1/2 and T10 to expose the cord. The rat is mounted on the Impactor.
• T7 and T8 segments are first clamped and then the Til and T12 segments are attached to the caudal clamp.
• a soft material is placed underneath the chest of the rat.
• the Impactor rod is set to the zero position and the electrical ground clip is attached to the wound edge.
• the Impactor rod is then raised to 25.0 mm and appropriately adjusted to a position directly above the exposed spinal cord.
• the Impactor rod is released to hit the exposed cord and the Impactor rod is immediately lifted.
• ® [0214] The rat is then dismounted, and Gelfoam placed on the wound.
• the muscle over the wound is sutured, and the incision is surgically stapled. Animals are placed in an incubator until they recover from anesthesia. Rats are given antibiotics, analgesics, and saline as required.
• Soluble Nogo receptor-1 fusion protein e.g., sNogoR310-Fc
• BBB scoring is performed one-day after surgery, then every week thereafter until 4 to 6 weeks.
• EXAMPLE 14 Expression of sNogoR310 in Transgenic Mice
• mice expressing soluble Nogo receptor-1 protein to test its effect when expressed in vivo.
• mice cloned the mouse sNogoR310 cDNA (corresponding to amino acids 1-310 of the Nogo receptor-1) into the NotI site of the C-3123 vector.
• s ⁇ ogoR310 expression is under the control of the glial fibrillary acidic protein (gfap) gene regulatory elements, which allow high level expression with enhanced secretion from reactive astrocytes at site of injury.
• gfap glial fibrillary acidic protein
• EXAMPLE 15 Expression of sNogoR310 in Transgenic Mice After Injury
• mice were perfused transcardially with PBS, followed by 4% paraformaldehyde. Mice used for sNogoR310 expression experiments did not receive any tracer injection.
• the spinal cord at a level between T3 and L3 was collected without perfusion 14 days after injury. Mice used for Nogo receptor-1 immunohistochemical staining were perfused with 4% paraformaldehyde 10 days after hemisection, and the injured spinal cord was removed for sectioning.
• sNogoR310 expression in the injured brain of transgenic and WT mice was performed with a number 11 scalpel blade held in a stereotaxic apparatus (David Kopf, Tujunga, CA) . A 4 mm parasagittal cut was made, 0.5 mm posterior to Bregma, 1.5 mm laterally from midline and 3.5 mm deep . [0223] We detected increased levels of sNogoR310 in soluble extracts of spinal cords ten days after the injury in transgenic mice but not in WT mice, consistent with the upregulation after injury of GFAP around the lesion.
• Nogo receptor-1 protein is co-localized with astrocytic marker GFAP only in the transgenic mice. There is also a greatly enhanced diffuse non-cellular staining in the transgenic samples, consistent with sNogoR310in the extracellular space. Neuronal cell body Nogo receptor-1 staining is detected in both WT and transgenic mice. EXAMPLE 16 Secreted sNogoR310 Induces CST Sprouting in Transgenic Mice [0225] We tested whether increased expression of sNogoR310 around the lesion in transgenic mice results in the regeneration of injured axons .
• CST corticospinal tracts
• the sections rostral to dorsal hemisection from injured sNogoR310 transgenic mice indicate a quite different BDA labeling pattern.
• a high density of BDA-labeled CST fibers are observed outside of prominent dCST in all the transgenic mice from line Tg08 or line TgOl.
• Ectopic fibers extend throughout the gray matter area, and some fibers reach into lateral and dorsolateral white matter.
• Several fibers (4-12 sprouts per transverse section) are seen on the opposite side of the spinal cord (ipsilateral to the tracer injection site) .
• Micro densitometric measurement of the collateral sprouts indicates approximately a tenfold increase in sprouting density in sNogoR310 transgenic mice.
• EXAMPLE 17 Regenerating CST Axons Bypass the Lesion Site into Distal Spinal Cord in sNogoR310 Transgenic Mice
• Collaterals and arborized fibers are most frequently seen in gray matter area of distal spinal cord.
• the reconstructions demonstrate 5-15 BDA-labeled regenerating fibers coursing in the rostral -caudal axis at any level 1-4 mm caudal to the lesion in each transgenic mouse.
• BDA-labeled CST axons are seen in both the gray matter and white matter areas in each transgenic mouse.
• the fiber counts for the transgenic mice indicate approximately a similar number of BDA-labeled CST fibers to the proximal levels in the sagittal sections.
• the other descending tracts such as raphespinal fibers
• the transection injures a majority of the serotonergic fibers, decreasing the density of these fibers by approximately 80% in the ventral horn.
• Analysis of total length of serotonin fibers in the ventral horn of caudal spinal cord indicates a much greater number of these fibers in transgenic mice than WT group, indicating that the growth-promoting effects of sNogoR310 in transgenic mice are not limited to one axon descending pathway.
• EXAMPLE 18 Transgenic Expression of sNogoR310 Improves Locomotor Recovery
• the CST axon tracing and serotonergic fiber analysis indicate that the sNogoR310 released from astrocytes in transgenic mice stimulates extensive anatomical regeneration of injured descending axons in the spinal cord. We performed several behavioral tests as described in Example 12 to determine whether these regenerated fibers benefit functional recovery. [0233] As assessed by the BBB test, the WT mice partially recover locomotor function during a 4 -week period of survival.
• mice can sustain their posture on board angled at 55 degrees. On days 7-28 after injury, the sustainable angle is reduced in all mice, but the angles sustainable by the transgenic mice are significantly greater than those for the control group (Fig. 13C) .
• mice climbed a grid placed at a 45 degree angle to vertical and excursions of the hmdlimbs below the plane of the grid were counted (Metz et al . , 2000) . No mice made errors on this test during the pre-injury training.
• transgenic mice secreting sNogoR310 from astrocytes exhibit CST regeneration, raphespinal sprouting and improved motor function after thoracic spinal hemisection.
• sNogoR310-Fc protein intrathecally to rats with a mid-thoracic dorsal hemisection injury through an osmotic minipump .
• 1.2 mg sNogoR310-Fc protein was locally administered in each rat.
• rats receiving the vehicle treatment 1.2 mg rat IgG
• sections rostral to hemisection display the tightly bundled prominent dorsal CST and very few ectopic BDA- labeled CST fibers above the lesion site. Sections rostral to lesion from injured rats receiving sNogoR310-Fc protein exhibit a quite different pattern of labeling.
• ectopic fibers sprouting from the BDA-labeled CST are observed from transverse and parasagittal sections. In some cases, projections cross from the dCST area near the midline to the circumference of the cord, becoming intermingled with the dorsolateral CST. The sprouting axons extend through gray matter to a greater extent than white matter.
• a measure of ectopic sprouting fibers (>100 ⁇ m in transverse sections, ⁇ 200 ⁇ m in sagittal sections) adjacent to the dCST reveals a greater increase in the sNogoR310-Fc-treated rats.
• a measure of the sprouts from distal spinal cord demonstrates that the total collateral length of each sprout in sNogoR310-Fc-treated rats is twice as great as that from NEP 1-40-treated animals.
• Example 21 Local sNogoR310-Fc Induces Sprouting of Rubropinal and Serotonergic Axons in Injured Rat Spinal Cord [0241]
• a burr hole was made on each side of the skull overlying the sensorimotor cortex of the lower limbs to trace CST fibers.
• the anterograde neuronal tracer BDA (10% in PBS, 3.5 ⁇ l per cortex) was applied at seven injection sites at a depth of 1.5 mm from dura on each side (Grandpre, 2002) .
• Sections at same level treated with sNogoR310- Fc exhibit many linking fibers between the main RST and dorsal horn gray matter.
• Transverse sections 11-15 mm distal to SCI no BDA-labeled RST fibers in vehicle-treated rats.
• sections at the same level receiving sNogoR310- Fc treatment display many BDA-labeled RST fibers in both gray and white matter contralateral to tracer injection. Some sprouts with a branching pattern are seen in the gray matter ipsilateral to BDA injection.
• Ruphespinal spinal fibers were also examined in sNogoR310-Fc treated spinal injured rats.
• Immunostaining demonstrates the density of serotonergic fibers 11-15 mm rostral to lesion that is similar between vehicle and sNogoR310-Fc treated groups. In the sections 11-15 mm below the lesion, the seroton fibers in sNogoR310-Fc treated rats are twice as numerous as those in the control group. These results demonstrate that the responsiveness to Nogo receptor-1 inhibition by sNogoR310-Fc protein is not limited to CST fibers, and that the other descending tracts, such as rubrospinal and serotonergic axons, are also responsive to Nogo receptor-1 antagonism.
• Intrathecal administration of sNogoR310-Fc protein stimulates axon regeneration in several descending pathways after traumatic spinal cord injury. We tested whether the protein also improves functional recovery in the injured spinal cord.
• the locomotor BBB score in vehicle-treated rats reaches a stable level of 12 (Fig. 14A) .
• most of controls (6 out of 7) have frequent-consistent weight-supported plantar steps and frequent-consistent forelimb-hindlimb coordination, but they have a rotation of predominant paw position when making initial contact with surface.
• the locomotor score continues to improve between 2-4 weeks post-trauma.
• all 9 of the sNogoR310-Fc treated animals had consistent forelimb- hindlimb coordination and a parallel paw position at initial contact with the testing surface.
• Grid walking has been used to assess the deficits in descending fine motor control after spinal cord injury (Metz et al . , 2000) . This performance requires forelimb- hindlimb coordination and voluntary movement control mediated by ventrolateral, corticospinal and rubrospinal fibers. During the pre-injury training, all the rats accurately place their hmdlimbs on the grid bars. At 2-4 weeks post-injury, control rats make 8-9 errors per session with only minimal improvement over time. In contrast, the rats treated with sNogoR310-Fc exhibit a progressive improvement on grid walking and make significant fewer errors (4-7/session on average) . The majority of the improvement occurs at 2-3 weeks after injury.
• Hybridomas HB 7E11 (ATCC ® accession No. PTA-4587) , HB 1H2 (ATCC ® accession No. PTA-4584) , HB 3G5 (ATCC * accession No. PTA-4586), HB 5B10 (ATCC " accession No.

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