WO2007008547A2 - Sp35 antibodies and uses thereof - Google Patents
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- WO2007008547A2 WO2007008547A2 PCT/US2006/026271 US2006026271W WO2007008547A2 WO 2007008547 A2 WO2007008547 A2 WO 2007008547A2 US 2006026271 W US2006026271 W US 2006026271W WO 2007008547 A2 WO2007008547 A2 WO 2007008547A2
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Definitions
- This invention relates to neurology, neurobiology and molecular biology. More particularly, this invention relates to molecules and methods for treatment of neurological diseases, disorders and injuries such as spinal cord injury.
- Axons and dendrites extend from neurons.
- the distal tip of an extending axon or neurite includes a specialized region, known as the growth cone.
- Growth cones sense the local environment and guide axonal growth toward a neuron's target cell. Growth cones respond to environmental cues, for example, surface adhesiveness, growth factors, neurotransmitters and electric fields.
- the growth cones generally advance at a rate of one to two millimeters per day.
- the growth cone explores the area ahead of it and on either side, by means of elongations classified as lamellipodia and f ⁇ lopodia. When an elongation contacts an unfavorable surface, it withdraws. When an elongation contacts a favorable growth surface, it continues to extend and guides the growth cone in that direction. When the growth cone reaches an appropriate target cell a synaptic connection is created.
- myelin inhibitory proteins include NogoA (Chen et al, Nature, 2000, 403, 434- 439; Grandpre et al., Nature 2000, 403, 439-444), myelin associated glycoprotein (MAG) (McKerracher et al, 1994, Neuron 13:805-811; Mukhopadhyay et al, 1994, Neuron 13:757-767) and oligodendrocyte glycoprotein (OM-gp), Mikol et al, 1988, J. Cell. Biol.106: 1273-1279).
- NogoA Choen et al, Nature, 2000, 403, 434- 439; Grandpre et al., Nature 2000, 403, 439-444
- MAG myelin associated glycoprotein
- OM-gp oligodendrocyte glycoprotein
- Nogo receptor- 1 Nogo receptor-1 (NgRl) is a GPI-anchored membrane protein that contains 8 leucine rich repeats (Fournier et al, 2001, Nature 409:341-346).
- inhibitory proteins ⁇ e.g., NogoA, MAG and OM-gp
- Such diseases, disorders or injuries include, but are not limited to, multiple sclerosis (MS), progressive multifocal leukoencephalopathy (PML), encephalomyelitis (EPL), central pontine myelolysis (CPM), adrenoleukodystrophy, Alexander's disease, Pelizaeus Merzbacher disease (PMZ), Globoid cell Leucodystrophy (Krabbe's disease) and Wallerian Degeneration, optic neuritis, transverse myelitis, amyotrophic lateral sclerosis (ALS), Huntington's disease, Alzheimer's disease, Parkinson's disease, spinal cord injury, traumatic brain injury, post radiation injury, neurologic complications of chemotherapy, stroke, acute ischemic optic neuropathy, vitamin E deficiency, isolated vitamin E deficiency syndrome, AR, Bassen-Kornzweig syndrome, Marchiafava-Bignami syndrome, metachromatic leukodystrophy, trigeminal neuralgia, and Bell's palsy
- MS generally begins with a relapsing-remitting pattern of neurologic involvement, which then progresses to a chronic phase with increasing neurological damage.
- MS is associated with the destruction of myelin, oligodendrocytes and axons localized to chronic lesions.
- the demyelination observed in MS is not always permanent and remyelination has been documented in early stages of the disease. Remyelination of neurons requires oligodendrocytes.
- the present invention is based on the discovery that Sp35 (Sp35 is also designated in the literature as LINGO-I and LRRN6) is expressed in oligodendrocytes and neuronal cells and negatively regulates oligodendrocyte/neuronal differentiation, survival and axon myelination. Furthermore, certain antagonists of Sp35 promote survival, proliferation and differentiation of oligodendrocytes and neuronal cells, as well as myelination of neurons. Based on these discoveries, the invention relates generally to antibodies, antigen binding fragment or derivatives thereof which can be used as an antagonist of Sp35.
- the invention generally relates to methods for treating various disease, disorders or injuries associated with demyelination, dysmyelination, oligodendrocyte/neuronal cell death or axonal injury by the administration of an Sp35 antagonist antibody or antigen binding fragment.
- the invention includes an isolated antibody or antigen binding fragment thereof which specifically binds to the same Sp35 epitope as a reference monoclonal antibody selected from the group consisting of 201', 3A3, 3A6, 1A7, 1G7, 2B10, 2Cl 1, 2F3, 3P1D10.2C3, 3P1E11.3B7, 3P2C6.3G10.2H7, 3P2C9.2G4, 3P4A6.1D9, 3P4A1.2B9, 3P4C2.2D2, 3P4C5.1D8, 3P4C8.2G9, 30-C12 (LiOl), 38-D01 (LiO2), 35-E04 (Li03), 36-C09 (LiO4), 30-Al 1 (LiOS), 34-F02 (LiO6), 29-E07 (LiO7), 34-G04 (Li08), 36-A12 (LiO9), 28-D02 (LiIO), 30-B01 (L
- Certain embodiments of the invention include an isolated polypeptide comprising an immunoglobulin heavy chain variable region (VH) wherein the CDRl, CDR2 and CDR3 regions are selected from the polypeptide sequences shown in Table 4 or at least 80%, 85%, 90 or 95% identical to the polypeptide sequences shown in Table 4.
- VH immunoglobulin heavy chain variable region
- Certain embodiments of the invention include an isolated polypeptide comprising an immunoglobulin light chain variable region (VL) wherein the CDRl, CDR2 and CDR3 regions are selected from the polypeptide sequences shown in Table 5 or at least 80%, 85%, 90% or 95% identical to the polypeptide sequences shown in Table 5.
- VL immunoglobulin light chain variable region
- Certain embodiments of the invention include an isolated polypeptide comprising an immunoglobulin heavy chain variable region (VH) selected from the group consisting of SEQ ID NOs: 158 to 172, 372, 376, 380 and 384, as shown in Table 6, or at least 80%, 85%, 90% or 95% identical to said SEQ ID NOs: 158 to 172, 372, 376, 380 and 384 as shown in Table 6.
- VH immunoglobulin heavy chain variable region
- Certain embodiments of the invention include an isolated polypeptide comprising an immunoglobulin light chain variable region (VL) selected from the group consisting of SEQ ID NOs: 273 to 286, 373, 377, 381 and 385, as shown in Table 8, or at least 80%, 85%, 90% or 95% identical to said SEQ ID NOs: 273 to 286, 373, 377, 381 and 385, as shown in Table 8.
- VL immunoglobulin light chain variable region
- the invention includes an isolated polynucleotide comprising a nucleic acid encoding an immunoglobulin heavy chain variable region (VH) wherein the CDRl, CDR2 and CDR3 regions are selected from the group selected from the polynucleotide sequences shown in Table 4 or at least 80%, 85%, 90 or 95% identical to the polynucleotide sequences shown in Table 4.
- VH immunoglobulin heavy chain variable region
- the invention includes an isolated polynucleotide comprising a nucleic acid encoding an immunoglobulin light chain variable region (VL) wherein the CDRl, CDR2 and CDR3 regions are selected from the the polynucleotide sequences shown in Table 5 or at least 80%,
- VL immunoglobulin light chain variable region
- the invention includes compositions comprising the antibodies or antigen binding fragments described herein.
- Huntington's disease, Alzheimer's disease, Parkinson's disease, diabetic neuropathy and stroke comprising administering to an animal in need of said treatment an effective amount of an agent selected from the group consisting of an isolated Sp35 antibody or fragment thereof or compositions comprising said antibody or fragment thereof.
- Additional embodiments of the present invention include a method of decreasing inhibition of axonal growth of a central nervous system (CNS) neuron, comprising contacting the neuron with an effective amount of an agent selected from the group consisting of the isolated Sp35 antibody or fragment thereof of or compositions comprising said antibody or fragment thereof.
- CNS central nervous system
- Other embodiments of the present invention include a method of inhibiting growth cone collapse of a CNS neuron, comprising contacting the neuron with an effective amount of an agent selected from the group consisting of the isolated Sp35 antibody or fragment thereof or compositions comprising said antibody or fragment thereof.
- FIG.3 MAbs 1A7 and 2F3 protected DRG neurons from myelin-mediated inhibition of neurite outgrowth.
- FIG.4A-G Immunohistochemical staining ("IHC") of cocultures of DRG neurons and oligodendrocytes treated with monoclonal antibodies 1A7 and 2F3, or control antibody. Panels D and
- E are enlargements of panels B and C, respectively. Staining with anti- ⁇ ffl-tubulin antibody to identify axons, or anti-MBP antibody to identify oligodendrocytes.
- F Quantitation of MBP+ myelinating cells upon treatment of cocultures with 1A7 or 2F3.
- G Western blot analysis to quantify the MBP produced from cocultures of DRG neurons and oligodendrocytes treated with monoclonal antibodies 1A7 and
- Anti-MBP protein antibody or luxol fast blue staining of mouse neurons in cuprizone model C:
- FIG. 6 Surviving RGCs. Treatment with monoclonal antibody 1A7 Anti-Sp35 antibody
- FIG. 8 Western blot of co-cultured oligodendrocytes and DRGs after incubation with anti-
- FIG. 10 Graph of the number of regenerative neuronal fibers per section counted in animals receiving an intravitreal injection of the Sp35 antibody 1A7 after optic nerve crush.
- an "isolated" polypeptide or a fragment, variant, or derivative thereof is intended a polypeptide that is not in its natural milieu. No particular level of purification is required.
- an isolated polypeptide can be removed from its native or natural environment.
- Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for purposed of the invention, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique.
- variant polypeptides may also be referred to herein as "polypeptide analogs.”
- a “derivative" of an Sp35 antibody or antibody polypeptide refers to a subject polypeptide having one or more residues chemically derivatized by reaction of a functional side group.
- derivatives are those peptides which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. For example, 4-hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine; and ornithine may be substituted for lysine.
- isolated nucleic acid or polynucleotide is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment.
- a recombinant polynucleotide encoding an Sp35 antibody contained in a vector is considered isolated for the purposes of the present invention.
- Further examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution.
- Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides of the present invention.
- a "coding region” is a portion of nucleic acid which consists of codons translated into amino acids. Although a “stop codon” (TAG, TGA, or TAA) is not translated into an amino acid, it may be considered to be part of a coding region, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, and the like, are not part of a coding region. Two or more coding regions of the present invention can be present in a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate (different) vectors.
- Two DNA fragments are "operably associated" if induction of promoter function results in the transcription of mRNA encoding the desired gene product and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed.
- a promoter region would be operably associated with a nucleic acid encoding a polypeptide if the promoter was capable of effecting transcription of that nucleic acid.
- the promoter may be a cell-specific promoter that directs substantial transcription of the DNA only in predetermined cells.
- transcription control elements besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can be operably associated with the polynucleotide to direct cell-specific transcription.
- Suitable promoters and other transcription control regions are disclosed herein.
- transcription control regions are known to those skilled in the art. These include, without limitation, transcription control regions which function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (the immediate early promoter, in conjunction with intron-A), simian virus 40 (the early promoter), and retroviruses (such as Rous sarcoma virus).
- Other transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone and rabbit ⁇ -globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers as well as lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins).
- translation control elements include, but are not limited to ribosome binding sites, translation initiation and termination codons, and elements derived from picornaviruses (particularly an internal ribosome entry site, or IRES, also referred to as a CITE sequence).
- a polynucleotide of the present invention is RNA, for example, in the form of messenger RNA (mRNA).
- mRNA messenger RNA
- Polynucleotide and nucleic acid coding regions of the present invention may be associated with additional coding regions which encode secretory or signal peptides, which direct the secretion of a polypeptide encoded by a polynucleotide of the present invention.
- proteins secreted by mammalian cells have a signal peptide or secretory leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated.
- polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the complete or "full length" polypeptide to produce a secreted or "mature” form of the polypeptide.
- the native signal peptide e.g., an immunoglobulin heavy chain or light chain signal peptide is used, or a functional derivative of that sequence that retains the ability to direct the secretion of the polypeptide that is operably associated with it.
- a heterologous mammalian signal peptide, or a functional derivative thereof may be used.
- the wild-type leader sequence may be substituted with the leader sequence of human tissue plasminogen activator (TPA) or mouse ⁇ -glucuronidase.
- the present invention is directed to certain Sp35 antibodies, or antigen-binding fragments, variants, or derivatives thereof.
- Sp35 antibodies encompasses full-sized antibodies as well as antigen- binding fragments, variants, analogs, or derivatives of such antibodies, e.g., naturally occurring antibody or immunoglobulin molecules or engineered antibody molecules or fragments that bind antigen in a manner similar to antibody molecules.
- an antibody or immunoglobulin comprises at least the variable domain of a heavy chain, and normally comprises at least the variable domains of a heavy chain and a light chain.
- Basic immunoglobulin structures in vertebrate systems are relatively well understood. See, e.g., Harlow et al, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988).
- immunoglobulin comprises various broad classes of polypeptides that can be distinguished biochemically.
- heavy chains are classified as gamma, mu, alpha, delta, or epsilon, ( ⁇ , ⁇ , ⁇ , ⁇ , ⁇ ) with some subclasses among them ⁇ e.g., ⁇ l- ⁇ 4). It is the nature of this chain that determines the "class" of the antibody as IgG, IgM, IgA IgG, or IgE, respectively.
- immunoglobulin subclasses e.g., IgGi, IgQ -5 IgG 3 , IgG 4 , IgA 1 , etc. are well characterized and are known to confer functional specialization. Modified versions of each of these classes and isotypes are readily discernable to the skilled artisan in view of the instant disclosure and, accordingly, are within the scope of the instant invention. All immunoglobulin classes are clearly within the scope of the present invention, the following discussion will generally be directed to the IgG class of immunoglobulin molecules.
- a standard immunoglobulin molecule comprises two identical light chain polypeptides of molecular weight approximately 23,000 Daltons, and two identical heavy chain polypeptides of molecular weight 53,000-70,000.
- the four chains are typically joined by disulfide bonds in a "Y" configuration wherein the light chains bracket the heavy chains starting at the mouth of the "Y” and continuing through the variable region.
- Light chains are classified as either kappa or lambda (K, ⁇ ). Each heavy chain class may be bound with either a kappa or lambda light chain.
- the light and heavy chains are covalently bonded to each other, and the "tail" portions of the two heavy chains are bonded to each other by covalent disulfide linkages or non-covalent linkages when the immunoglobulins are generated either by hybridomas, B cells or genetically engineered host cells.
- the amino acid sequences run from an N-terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain.
- Both the light and heavy chains are divided into regions of structural and functional homology.
- the terms "constant” and “variable” are used functionally.
- the variable domains of both the light (V L ) and heavy (V H ) chain portions determine antigen recognition and specificity.
- the constant domains of the light chain (C L ) and the heavy chain (C H 1, C H 2 or C H 3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like.
- the N-terminal portion is a variable region and at the C-terminal portion is a constant region; the C H 3 and C L domains actually comprise the carboxy-terminus of the heavy and light chain, respectively.
- variable region allows the antibody to selectively recognize and specifically bind epitopes on antigens. That is, the V L domain and V H domain, or subset of the complementarity determining regions (CDRs), of an antibody combine to form the variable region that defines a three dimensional antigen binding site.
- This quaternary antibody structure forms the antigen binding site present at the end of each arm of the Y. More specifically, the antigen binding site is defined by three CDRs on each of the V H and V L chains.
- a complete immunoglobulin molecule may consist of heavy chains only, with no light chains. See, e.g., Hamers-Casterman et al, Nature 363:446-448 (1993).
- CDR complementarity determining region
- V H H the heavy chain variable region
- the main differences between camelid V H H variable regions and those derived from conventional antibodies (V H ) include (a) more hydrophobic amino acids in the light chain contact surface of V H as compared to the corresponding region in V H H, (b) a longer CDR3 in V H H, and (c) the frequent occurrence of a disulfide bond between CDRl and CDR3 in V H H.
- Antibodies or antigen-binding fragments, variants, or derivatives thereof of the invention include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized, or chimeric antibodies, single chain antibodies, epitope-binding fragments, e.g., Fab, Fab' and F(ab') 2 , Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising either a V L or V H domain, fragments produced by a Fab expression library, and anti- idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to Sp35 antibodies disclosed herein).
- anti-Id antigen-binding fragments, variants, or derivatives thereof of the invention
- Immunoglobulin or antibody molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.
- Antibody fragments, including single-chain antibodies may comprise the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region, C H 1, C H 2, and C H 3 domains.
- a binding polypeptide for use in the invention may lack at least a portion of a C H 2 domain (e.g., all or part of a C H 2 domain).
- a C H 2 domain e.g., all or part of a C H 2 domain.
- the heavy chain portions of one polypeptide chain of a multimer are identical to those on a second polypeptide chain of the multimer.
- heavy chain portion-containing monomers of the invention are not identical.
- each monomer may comprise a different target binding site, forming, for example, abispecific antibody.
- the heavy chain portions of a binding polypeptide for use in the diagnostic and treatment methods disclosed herein may be derived from different immunoglobulin molecules.
- a heavy chain portion of a polypeptide may comprise a C H I domain derived from an IgGl molecule and a hinge region derived from an IgG3 molecule.
- a heavy chain portion can comprise a hinge region derived, in part, from an IgGl molecule and, in part, from an IgG3 molecule.
- a heavy chain portion can comprise a chimeric hinge derived, in part, from an IgGl molecule and, in part, from an IgG4 molecule.
- the term "light chain portion" includes amino acid sequences derived from an immunoglobulin light chain.
- the light chain portion comprises at least one of a V L or C L domain.
- an "epitope" on a target polypeptide may be or include non-polypeptide elements, e.g., an "epitope may include a carbohydrate side chain.
- the minimum size of a peptide or polypeptide epitope for an antibody is thought to be about four to five amino acids.
- Peptide or polypeptide epitopes preferably contain at least seven, more preferably at least nine and most preferably between at least about 15 to about 30 amino acids. Since a CDR can recognize an antigenic peptide or polypeptide in its tertiary form, the amino acids comprising an epitope need not be contiguous, and in some cases, may not even be on the same peptide chain.
- antibody “A” may be deemed to have a higher specificity for a given epitope than antibody "B,” or antibody “A” may be said to bind to epitope “C” with a higher specificity than it has for related epitope “D.”
- an antibody of the invention may be said to bind a target polypeptide disclosed herein or a fragment or variant thereof with an off rate (k(off)) less than or equal to 5 X 10 "4 sec “1 , 10 "4 sec “1 , 5 X 10 "5 sec “1 , or 10 "5 sec “1 5 X 10 "6 sec “1 , 10 “6 sec “1 , 5 X 10 "7 sec “1 or 10 "7 sec “1 .
- Table 3B describes the ability of the named monoclonal antibodies or Fab fragments to detect Sp35 in various assays such as: Fluorescent Activated Cell Sorting (FACS), Immunoprecipitation (IP), Western blot analysis, Immunohistochemistry (IHC) and Enzyme Linked Immunosorbent Assay (ELISA). Detailed protocols for performing these assays are described herein or are well known and understood by those of ordinary skill in the art.
- Hybridoma-derived monoclonal antibodies listed in Table 3B were produced by injection of soluble Sp35 into mice and then isolated using hybridoma technology which is well known in the art and described herein.
- Monoclonal antibodies and antibody Fab fragments listed in Table 3B were isolated from two different phage display libraries using techniques known in the art.
- huSp35 human Sp35 protein
- mSp35 mouse Sp35 protein
- FACS Fluorescent Activated Cell Sorting
- the term "antigen binding domain” includes a site that specifically binds an epitope on an antigen (e.g., an epitope of Sp35).
- the antigen binding domain of an antibody typically includes at least a portion of an immunoglobulin heavy chain variable region and at least a portion of an immunoglobulin light chain variable region. The binding site formed by these variable regions determines the specificity of the antibody.
- the present invention is more specifically directed to an Sp35 antibody, or antigen-binding fragment, variant or derivatives thereof, where the Sp35 antibody binds to the same epitope as a monoclonal antibody selected from the group consisting of 201', 3A3, 3A6, 1A7, 1G7, 2B10, 2Cl 1, 2F3, 3P1D10.2C3, 3P1E11.3B7, 3P2C6.3G10.2H7, 3P2C9.2G4, 3P4A6.1D9, 3P4A1.2B9, 3P4C2.2D2, 3P4C5.1D8, 3P4C8.2G9, 30-C12 (LiOl), 38-D01 (LiO2), 35-E04 (Li03), 36-C09 (Li04), 30-Al 1 (LiO5), 34-F02 (LiO6), 29-E07 (LiO7), 34-G04 (Li08), 36-A12 (LiO9), 28-D02
- the invention is further drawn to an Sp35 antibody, or antigen-binding fragment, variant or derivatives thereof, where the Sp35 antibody competitively inhibits a monoclonal antibody selected from the group consisting of 201', 3A3, 3A6, 1A7, 1G7, 2B10, 2C11, 2F3, 3P1D10.2C3, 3P1E11.3B7, 3P2C6.3G10.2H7, 3P2C9.2G4, 3P4A6.1D9, 3P4A1.2B9, 3P4C2.2D2, 3P4C5.1D8, 3P4C8.2G9, 30- C12 (LiOl), 38-D01 (LiO2), 35-E04 (Li03), 36-C09 (LiO4), 30-Al 1 (Li05), 34-F02 (LiO6), 29-E07 (LiO7), 34-G04 (LiO8), 36-A12 (LiO9), 28-D02 (LiIO), 30-B
- the invention is also drawn to an Sp35 antibody, or antigen-binding fragment, variant or derivatives thereof, where the Sp35 antibody comprises at least the antigen binding region of a monoclonal antibody selected from the group consisting of 201', 3A3, 3A6, 1A7, 1G7, 2B10, 2Cl 1, 2F3, 3P1D10.2C3, 3P1E11.3B7, 3P2C6.3G10.2H7, 3P2C9.2G4, 3P4A6.1D9, 3P4A1.2B9, 3P4C2.2D2, 3P4C5.1D8, 3P4C8.2G9, 30-C12 (LiOl), 38-D01 (LiO2), 35-E04 (LiO3), 36-C09 (LiO4), 30-Al 1 (LiO5), 34-F02 (LiO6), 29-E07 (LiO7), 34-G04 (LiO8), 36-A12 (LiO9), 28-D02
- the present invention is directed to an antibody, or antigen-binding fragment, variant, or derivative thereof which specifically or preferentially binds to a particular Sp35 polypeptide fragment or domain.
- Sp35 polypeptide fragments include, but are not limited to, an Sp35 polypeptide comprising, consisting essentially of, or consisting of amino acids 34 to 532; 34 to 417; 34 to 425; 34 to 493; 66 to 532; 66 to 417; 66 to 426; 66 to 493;66 to 532;417 to 532;417 to 532; 417 to 425 (the Sp35 basic region) ; 417 to 493; 417 to 532;, 419 to 493 (the Sp35 Ig region) ;, or 425 to 532 of SEQ ID NO:2; or an Sp35 variant polypeptide at least 70%, 75%, 80%, 85%, 90%, or 95% identical to amino acids 34 to 532; 34 to 417; 34 to 425; 34 to 493; 66 to
- Sp35 peptide fragments to which certain antibodies, or antigen-binding fragments, variants, or derivatives thereof of the present invention bind include, but are not limited to those fragments comprising, consisting essentially of, or consisting of one or more leucine-rich-repeats (LRR) of Sp35.
- LRR leucine-rich-repeats
- Such fragments include, for example, fragments comprising, consisting essentially of, or consisting of amino acids 66 to 89; 66 to 113; 66 to 137; 90 to 113; 114 to 137; 138 to 161; 162 to 185; 186 to 209; 210 to 233;, 234 to 257; 258 to 281; 282 to 305; 306 to 329; or 330 to 353 of SEQ ID NO:2.
- Corresponding fragments of a variant Sp35 polypeptide at least 70%, 75%, 80%, 85%, 90%, or 95% identical to amino acids 66 to 89; 66 to 113; 90 to 113; 114 to 137; 138 to 161; 162 to 185; 186 to 209; 210 to 233; 234 to 257; 258 to 281; 282 to 305; 306 to 329; or 330 to 353 of SEQ ID NO:2 are also contemplated.
- Additional Sp35 peptide fragments to which certain antibodies, or antigen-binding fragments, variants, or derivatives thereof of the present invention bind include, but are not limited to those fragments comprising, consisting essentially of, or consisting of one or more cysteine rich regions flanking the LRR of Sp35.
- Such fragments include, for example, a fragment comprising, consisting essentially of, or consisting of amino acids 34 to 64 of SEQ ID NO:2 (the N-terminal LRR flanking region (LRRNT)), or a fragment comprising, consisting essentially of, or consisting of amino acids 363 to 416 of SEQ ID NO:2 (the C-terminal LRR flanking region (LRRCT)), amino acids
- LRRNT N-terminal LRR flanking region
- LRRNT the N-terminal LRR flanking region
- LRRNT the N-terminal LRR flanking region
- sequence identity between two polypeptides is determined by comparing the amino acid sequence of one polypeptide to the sequence of a second polypeptide.
- sequence identity is determined by comparing the amino acid sequence of one polypeptide to the sequence of a second polypeptide.
- whether any particular polypeptide is at least about 70%, 75%, 80%, 85%, 90% or 95% identical to another polypeptide can be determined using methods and computer programs/software known in the art such as, but not limited to, the BESTFIT program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711).
- BESTFIT uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981), to find the best segment of homology between two sequences.
- the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference polypeptide sequence and that gaps in homology of up to 5% of the total number of amino acids in the reference sequence are allowed.
- Additional Sp35 peptide fragments to which certain antibodies, or antigen-binding fragments, variants, or derivatives thereof of the present invention bind include, but are not limited to those fragments comprising, consisting essentially of, or consisting of amino acids 41 to 525 of SEQ ID NO:2; 40 to 526 of SEQ ID NO:2; 39 to 527 of SEQ ID NO:2; 38 to 528 of SEQ ID NO:2; 37 to 529 of SEQ ID NO:2; 36 to 530 of SEQ ID NO:2; 35 to 531 of SEQ ID NO:2; 34 to 531 of SEQ ID NO:2; 46 to 520 of SEQ ID NO:2; 45 to 521 of SEQ ID NO:2; 44 to 522 of SEQ ID NO:2; 43 to 523 of SEQ ID NO:2; and 42 to 524 of SEQ ID NO:2.
- Sp35 peptide fragments to which certain antibodies, or antigen-binding fragments, variants, or derivatives thereof of the present invention bind include, but are not limited to those fragments comprising, consisting essentially of, or consisting of amino acids 1 to 33 of SEQ ID NO:2; 1 to 35 of SEQ ID NO:2; 34 to 64 of SEQ ID NO:2; 36 to 64 of SEQ ID NO:2; 66 to 89 of SEQ ID NO:2; 90 to 113 of SEQ ID NO:2; 114 to 137 of SEQ ID NO:2; 138 to 161 of SEQ ID NO:2; 162 to 185 of SEQ ID NO:2; 186 to 209 of SEQ ID NO:2; 210 to 233 of SEQ ID NO:2; 234 to 257 of SEQ ID NO:2; 258 to 281 of SEQ ID NO:2; 282 to 305 of SEQ ID NO:2; 306 to 329 of SEQ ID NO:2; 330 to 3
- Sp35 peptide fragments to which certain antibodies, or antigen-binding fragments, variants, or derivatives thereof of the present invention bind include, but are not limited to those fragments comprising, consisting essentially of, or consisting of amino acids 1 to 33 of SEQ ID NO:2; 1 to 35 of SEQ ID NO:2; 1 to 64 of SEQ ID NO:2; 1 to 89 of SEQ ID NO:2; 1 to 113 of SEQ ID NO:2; 1 to 137 of SEQ ID NO:2; 1 to 161 of SEQ ID NO:2; 1 to 185 of SEQ ID NO:2; 1 to 209 of SEQ ID NO:2; 1 to 233 of SEQ ID NO:2; 1 to 257 of SEQ ID NO:2; 1 to 281 of SEQ ID NO:2; 1 to 305 of SEQ ID NO:2; 1 to 329 of SEQ ID NO:2; 1 to 353 of SEQ ID NO:2; 1 to 416 of SEQ ID NO:2;
- Additional Sp35 peptide fragments to which certain antibodies, or antigen-binding fragments, variants, or derivatives thereof of the present invention bind include, but are not limited to those fragments comprising, consisting essentially of, or consisting of amino acids 34 to 64 of SEQ ID NO:2; 34 to 89 of SEQ ID NO:2; 34 to 113 of SEQ ID NO:2; 34 to 137 of SEQ ID NO:2; 34 to 161 of SEQ ID NO:2; 34 to 185 of SEQ ID NO:2; 34 to 209 of SEQ ID NO:2; 34 to 233 of SEQ ID NO:2; 34 to 257 of SEQ ID NO:2; 34 to 281 of SEQ ID NO:2; 34 to 305 of SEQ ID NO:2; 34 to 329 of SEQ ID NO:2; 34 to 353 of SEQ ID NO:2; 34 to 416 of SEQ ID NO:2; 34 to 424 of SEQ ID NO:2; 34 to 493 of SEQ ID NO:2;
- Sp35 peptide fragments to which certain antibodies, or antigen-binding fragments, variants, or derivatives thereof of the present invention bind include, but are not limited to those fragments comprising, consisting essentially of, or consisting of amino acids 34 to 530 of SEQ ID NO:2; 34 to 531 of SEQ ID NO:2; 34 to 532 of SEQ ID NO:2; 34 to 533 of SEQ ID NO:2; 34 to 534 of SEQ ID NO:2; 34 to 535 of SEQ ID NO:2; 34 to 536 of SEQ ID NO:2; 34 to 537 of SEQ ID NO:2; 34 to 538 of SEQ ID NO:2; 34 to 539 of SEQ ID NO:2; 30 to 532 of SEQ ID NO:2; 31 to 532 of SEQ ID NO:2; 32 to 532 of SEQ ID NO:2; 33 to 532 of SEQ ID NO:2; 34 to 532 of SEQ ID NO:2; 35 to 532 of SEQ ID NO:
- Sp35 peptide fragments to which certain antibodies, or antigen-binding fragments, variants, or derivatives thereof of the present invention bind include, but are not limited to those fragments comprising, consisting essentially of, or consisting of amino acids 36 to 64 of SEQ DD NO:2; 36 to 89 of SEQ DD NO:2; 36 to 113 of SEQ DD NO:2; 36 to 137 of SEQ DD NO:2; 36 to 161 of SEQ DD NO:2; 36 to 185 of SEQ DD NO:2; 36 to 209 of SEQ DD NO:2; 36 to 233 of SEQ DD NO:2; 36 to 257 of SEQ DD NO:2; 36 to 281 of SEQ DD NO:2; 36 to 305 of SEQ DD NO:2; 36 to 329 of SEQ DD NO:2; 36 to 353 of SEQ DD NO:2; 36 to 416 of SEQ DD NO:2; 36 to 424 of SEQ
- Additional Sp35 peptide fragments to which certain antibodies, or antigen-binding fragments, variants, or derivatives thereof of the present invention bind include, but are not limited to those fragments comprising, consisting essentially of, or consisting of amino acids 36 to 530 of SEQ DD NO:2; 36 to 531 of SEQ DD NO:2; 36 to 532 of SEQ DD NO:2; 36 to 533 of SEQ DD NO:2; 36 to 534 of SEQ DD NO:2; 36 to 535 of SEQ DD NO:2; 36 to 536 of SEQ DD NO:2; 36 to 537 of SEQ DD NO:2; 36 to 538 of SEQ DD NO:2; and 36 to 539 of SEQ DD NO:2.
- More Sp35 peptide fragments to which certain antibodies, or antigen-binding fragments, variants, or derivatives thereof of the present invention bind include, but are not limited to those fragments comprising, consisting essentially of, or consisting of amino acids 417 to 493 of SEQ DD NO:2; 417 to 494 of SEQ DD NO:2; 417 to 495 of SEQ DD NO:2; 417 to 496 of SEQ ID NO:2; 417 to 497 of SEQ DD NO:2; 417 to 498 of SEQ DD NO:2; 417 to 499 of SEQ DD NO:2; 417 to 500 of SEQ DD NO:2; 417 to 492 of SEQ ID NO:2; 417 to 491 of SEQ DD NO:2; 412 to 493 of SEQ DD NO:2; 413 to 493 of SEQ ID NO:2; 414 to 493 of SEQ ID NO:2; 415 to 493 of SEQ TD NO:2;
- Sp35 peptide fragments to which certain antibodies, or antigen-binding fragments, variants, or derivatives thereof of the present invention bind include, an Sp35 polypeptide comprising, consisting essentially of, or consisting of peptides of the Ig domain of Sp35 or fragments, variants, or derivatives of such polypeptides.
- polypeptides comprising, consisting essentially of, or consisting of the following polypeptide sequences: ITX 1 X 2 X3 (SEQ E) NO:287), ACXiX 2 X 3 (SEQ E) NO:288), VCX 1 X 2 X 3 (SEQ E) NO:289) and SPX 1 X 2 X 3 (SEQ E ) NO:290) where Xi is lysine, arginine, histidine, glutamine, or asparagine, X 2 is lysine, arginine, histidine, glutamine, or asparagine and X 3 is lysine, arginine, histidine, glutamine, or asparagine.
- Sp35 peptide fragments to which certain antibodies, or antigen-binding fragments, variants, or derivatives thereof of the present invention bind include, those fragments comprising, consisting essentially of, or consisting of the following polypeptide sequences: SPRKH (SEQ E ) NO:291), SPRKK (SEQ E) NO:292), SPRKR (SEQ E) NO:293), SPKKH (SEQ E) NO:294), SPHKH (SEQ E) NO:295), SPRRH (SEQ E) NO:296), SPRHH (SEQ E) NO:297), SPRRR (SEQ E ) NO:298), SPHHH (SEQ E) NO:299) SPKKK (SEQ E) NO:300), LSPRKH (SEQ E) NO:301), LSPRKK (SEQ E) NO:302), LSPRKR (SEQ E) NO:303), LSPKKH (SEQ E) NO:304), LSPHKH (SEQ
- Sp35 polypeptides include the basic "RKH loop” (Arginine-Lysine-Histidine amino acids 456-458) in the Ig domain of Sp35.
- Additional Sp35 peptides which include a basic tripeptide are ITPKRR (SEQ TD NO:321), ACHHK (SEQ TD NO:322) and VCHHK (SEQ TD NO:323).
- Sp35 peptide fragments to which certain antibodies, or antigen-binding fragments, variants, or derivatives thereof of the present invention bind include, an Sp35 polypeptide comprising, consisting essentially of, or consisting of peptides of the Ig domain of Sp35 or fragments, variants, or derivatives of such polypeptides.
- peptides comprising, consisting essentially of, or consisting of the following polypeptide sequences: X 4 X 5 RKH (SEQ TD NO:324), X 4 X 5 RRR (SEQ TD NO:325), X 4 X 5 KKK (SEQ TD NO:326), X 4 X 5 HHH (SEQ TD NO:327), X 4 X 5 RKK (SEQ TD NO:328), X 4 X 5 RKR (SEQ TD NO:329), X 4 X 5 KKH (SEQ TD NO:330), X 4 X 5 HKH (SEQ TD NO:331), X 4 X 5 RRH (SEQ ID NO:332) and X 4 X 5 RHH (SEQ ID NO:333) where X 4 is any amino acid and X 5 is any amino acid.
- Sp35 peptide fragments to which certain antibodies, or antigen- binding fragments, variants, or derivatives thereof of the present invention bind include, an Sp35 polypeptide comprising, consisting essentially of, or consisting of peptides of the Ig domain of Sp35 or fragments, variants, or derivatives of such polypeptides.
- polypeptides comprising, consisting essentially of, or consisting of the following polypeptide sequences: ITX 6 X 7 X 8 (SEQ ID NO:334), ACX 6 X 7 X 8 (SEQ ID NO:335), VCX 6 X 7 X 8 (SEQ ID NO:336) and SPX 6 X 7 X 8 (SEQ ID NO:337) where X 6 is lysine, arginine, histidine, glutamine, or asparagine, X 7 is any amino acid and X 8 is lysine, arginine, histidine, glutamine, or asparagine.
- a polypeptide comprising, consisting essentially of, or consisting of the following polypeptide sequence: SPRLH (SEQ ID NO:338).
- Sp35 peptide fragments to which certain antibodies, or antigen-binding fragments, variants, or derivatives thereof of the present invention bind include, an Sp35 polypeptide comprising, consisting essentially of, or consisting of peptides which contain amino acids 452-458 in the Ig domain of Sp35,or derivatives thereof, wherein amino acid 452 is a tryptophan or phenylalanine residue.
- Sp35 polypeptide comprising, consisting essentially of, or consisting of peptides of the basic domain of Sp35.
- peptides comprising, consisting essentially of, or consisting of the following polypeptide sequences: RRARIRDRK (SEQ ID NO:339), KKVKVKEKR (SEQ ID NO:340), RRLRLRDRK (SEQ ID NO:341), RRGRGRDRK (SEQ ID NO:342) and RRIRARDRK (SEQ ID NO:343).
- Epitope Mapping Protocols New Jersey: Humana Press (1996), which are both incorporated herein by reference in their entireties. Epitope mapping can also be performed by commercially available means (i.e. ProtoPROBE, Inc. (Milwaukee, Wisconsin)).
- antibodies produced which bind to any portion of Sp35 can then be screened for their ability to act as an antagonist of Sp35 and thus promote neurite outgrowth, neuronal and oligodendrocyte survival, proliferation and differentiation as well as promote myelination.
- Antibodies can be screened for oligodendrocyte/neuronal survival by using the method as described in Examples 10 and 11. Additionally, antibodies can be screened for their ability to promote myelination by using the method of Example 9. Finally, antibodies can be screened for their ability to promote oligodendrocyte proliferation and differentiation, as well as neurite outgrowth by using the method as described in Example 7.
- Other antagonist functions of antibodies of the present invention can be tested using other assays as described in the Examples herein.
- the present invention includes an antibody, or antigen-binding fragment, variant, or derivative thereof which specifically or preferentially binds to at least one epitope of Sp35, where the epitope comprises, consists essentially of, or consists of at least about four to five amino acids of SEQ ID NO:2, at least seven, at least nine, or between at least about 15 to about 30 amino acids of SEQ ID NO:2.
- the amino acids of a given epitope of SEQ ID NO:2 as described may be, but need not be contiguous or linear.
- the at least one epitope of Sp35 comprises, consists essentially of, or consists of a non-linear epitope formed by the extracellular domain of Sp35 as expressed on the surface of a cell or as a soluble fragment, e.g., fused to an IgG Fc region.
- the at least one epitope of Sp35 comprises, consists essentially of, or consists of at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, between about 15 to about 30, or at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 contiguous or non-contiguous amino acids of SEQ ID NO:2, where the non-contiguous amino acids form an epitope through protein folding.
- the present invention includes an antibody, or antigen-binding fragment, variant, or derivative thereof which specifically or preferentially binds to at least one epitope of Sp35, where the epitope comprises, consists essentially of, or consists of, in addition to one, two, three, four, five, six or more contiguous or non-contiguous amino acids of SEQ ID NO:2 as described above, and an additional moiety which modifies the protein, e.g., a carbohydrate moiety may be included such that the Sp35 antibody binds with higher affinity to modified target protein than it does to an unmodified version of the protein. Alternatively, the Sp35 antibody does not bind the unmodified version of the target protein at all.
- the present invention is directed to an antibody, or antigen-binding fragment, variant, or derivative thereof which specifically binds to a Sp35 polypeptide or fragment thereof, or an Sp35 variant polypeptide, with an affinity characterized by a dissociation constant (K D ) which is less than the K D for said reference monoclonal antibody.
- K D dissociation constant
- an antibody, or antigen-binding fragment, variant, or derivative thereof of the invention binds specifically to at least one epitope of Sp35 or fragment or variant described above, i.e., binds to such an epitope more readily than it would bind to an unrelated, or random epitope; binds preferentially to at least one epitope of Sp35 or fragment or variant described above, i.e., binds to such an epitope more readily than it would bind to a related, similar, homologous, or analogous epitope; competitively inhibits binding of a reference antibody which itself binds specifically or preferentially to a certain epitope of Sp35 or fragment or variant described above; or binds to at least one epitope of Sp35 or fragment or variant described above with an affinity characterized by a dissociation constant K D of less than about 5 x 10 "2 M, about 10 "2 M, about 5 x 10 '3 M, about 10 "3 M, about 5 x 10
- the term "about” allows for the degree of variation inherent in the methods utilized for measuring antibody affinity. For example, depending on the level of precision of the instrumentation used, standard error based on the number of samples measured, and rounding error, the term “about 10 "2 M” might include, for example, from 0.05 M to 0.005 M.
- an antibody, or antigen-binding fragment, variant, or derivative thereof of the invention binds Sp35 polypeptides or fragments or variants thereof with an off rate (k(off)) of less than or equal to 5 X 10 "2 sec “1 , 10 "2 sec “1 , 5 X 10 "3 sec “1 or 10 "3 sec “1 .
- an antibody, or antigen-binding fragment, variant, or derivative thereof of the invention binds binds Sp35 polypeptides or fragments or variants thereof with an off rate (k(off)) of less than or equal to 5 X 10 "4 sec “1 , 10 "4 sec “1 , 5 X 10 "5 sec “1 , or 10 "5 sec “1 5 X 10 "6 sec “1 , 10 “6 sec “1 , 5 X 10 "7 sec “1 or 10 "7 sec “1 .
- an antibody, or antigen-binding fragment, variant, or derivative thereof of the invention binds Sp35 polypeptides or fragments or variants thereof with an on rate (k(on)) of greater than or equal to 10 3 M "1 sec “1 , 5 X 10 3 M “1 sec “1 , 10 4 M “1 sec “1 or 5 X 10 4 M “1 sec “1 .
- an antibody, or antigen-binding fragment, variant, or derivative thereof of the invention binds Sp35 polypeptides or fragments or variants thereof with an on rate (k(on)) greater than or equal to 10 5 M "1 sec “1 , 5 X 10 5 M “1 sec “1 , 10 6 M “1 sec “1 , or 5 X 106 M “1 sec “1 or 10 7 M “1 sec “1 .
- an Sp35 antibody, or antigen-binding fragment, variant, or derivative thereof as described herein is an antagonist of Sp35 activity.
- binding of an antagonist Sp35 antibody to Sp35, as expressed on neurons blocks myelin- associated neurite outgrowth inhibition or neuronal cell death.
- binding of the Sp35 antibody to Sp35, as expressed on oligodendrocytes blocks inhibition of oligodendrocyte growth or differentiation, or blocks demyelination or dysmyelination of CNS neurons.
- an Sp35 antibody e.g., an antibody of the invention is a bispecific Sp35 antibody, binding polypeptide, or antibody, e.g., a bispecific antibody, minibody, domain deleted antibody, or fusion protein having binding specificity for more than one epitope, e.g., more than one antigen or more than one epitope on the same antigen.
- a bispecific Sp35 antibody, binding polypeptide, or antibody has at least one binding domain specific for at least one epitope on a target polypeptide disclosed herein, e.g., Sp35.
- a bispecific Sp35 antibody, binding polypeptide, or antibody has at least one binding domain specific for an epitope on a target polypeptide and at least one target binding domain specific for a drug or toxin.
- a bispecific Sp35 antibody, binding polypeptide, or antibody has at least one binding domain specific for an epitope on a target polypeptide disclosed herein, and at least one binding domain specific for a prodrug.
- a bispecific Sp35 antibody, binding polypeptide, or antibody may be a tetravalent antibody that has two target binding domains specific for an epitope of a target polypeptide disclosed herein and two target binding domains specific for a second target.
- a tetravalent bispecific Sp35 antibody, binding polypeptide, or antibody may be bivalent for each specificity.
- Sp35 antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention can comprise a constant region which mediates one or more effector functions.
- binding of the Cl component of complement to an antibody constant region may activate the complement system.
- Activation of complement is important in the opsonisation and lysis of cell pathogens.
- the activation of complement also stimulates the inflammatory response and may also be involved in autoimmune hypersensitivity.
- antibodies bind to receptors on various cells via the Fc region, with a Fc receptor binding site on the antibody Fc region binding to a Fc receptor (FcR) on a cell.
- FcR Fc receptor
- Fc receptors which are specific for different classes of antibody, including IgG (gamma receptors), IgE (epsilon receptors), IgA (alpha receptors) and IgM (mu receptors). Binding of antibody to Fc receptors on cell surfaces triggers a number of important and diverse biological responses including engulfment and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (called antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental transfer and control of immunoglobulin production.
- ADCC antibody-dependent cell-mediated cytotoxicity
- certain embodiments of the invention include an Sp35 antibody, or antigen- binding fragment, variant, or derivative thereof, in which at least a fraction of one or more of the constant region domains has been deleted or otherwise altered so as to provide desired biochemical characteristics such as reduced effector functions, the ability to non-covalently dimerize, increased ability to localize at the site of a tumor, reduced serum half-life, or increased serum half-life when compared with a whole, unaltered antibody of approximately the same immunogenicity.
- certain antibodies for use in the diagnostic and treatment methods described herein are domain deleted antibodies which comprise a polypeptide chain similar to an immunoglobulin heavy chain, but which lack at least a portion of one or more heavy chain domains. For instance, in certain antibodies, one entire domain of the constant region of the modified antibody will be deleted, for example, all or part of the C H 2 domain will be deleted.
- the Fc portion may be mutated to decrease effector function using techniques known in the art.
- the deletion or inactivation (through point mutations or other means) of a constant region domain may reduce Fc receptor binding of the circulating modified antibody thereby increasing tumor localization.
- constant region modifications consistent with the instant invention moderate complement binding and thus reduce the serum half life and nonspecific association of a conjugated cytotoxin.
- modifications of the constant region may be used to modify disulfide linkages or oligosaccharide moieties that allow for enhanced localization due to increased antigen specificity or antibody flexibility.
- the resulting physiological profile, bioavailability and other biochemical effects of the modifications such as tumor localization, biodistribution and serum half-life, may easily be measured and quantified using well know immunological techniques without undue experimentation.
- Modified forms of Sp35 antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention can be made from whole precursor or parent antibodies using techniques known in the art. Exemplary techniques are discussed in more detail herein.
- both the variable and constant regions of Sp35 antibodies, or antigen-binding fragments, variants, or derivatives thereof are fully human.
- Fully human antibodies can be made using techniques that are known in the art and as described herein. For example, fully human antibodies against a specific antigen can be prepared by administering the antigen to a transgenic animal which has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled. Exemplary techniques that can be used to make such antibodies are described in US patents: 6,150,584; 6,458,592; 6,420,140 which are incorporated by reference in their entireties. Other techniques are known in the art. Fully human antibodies can likewise be produced by various display technologies, e.g., phage display or other viral display systems, as described in more detail elsewhere herein.
- Sp35 antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention can be made or manufactured using techniques that are known in the art.
- antibody molecules or fragments thereof are "recombinantly produced," i.e., are produced using recombinant DNA technology. Exemplary techniques for making antibody molecules or fragments thereof are discussed in more detail elsewhere herein.
- S ⁇ 35 antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention also include derivatives that are modified, e.g., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from specifically binding to its cognate epitope.
- the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.
- Sp35 antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention will not elicit a deleterious immune response in the animal to be treated, e.g., in a human.
- Sp35 antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention are modified to reduce their immunogenicity using art- recognized techniques.
- antibodies can be humanized, primatized, deimmunized, or chimeric antibodies can be made. These types of antibodies are derived from a non-human antibody, typically a murine or primate antibody, that retains or substantially retains the antigen-binding properties of the parent antibody, but which is less immunogenic in humans.
- CDRs complementarity determining regions
- De-immunization can also be used to decrease the immunogenicity of an antibody.
- the term "de-immunization” includes alteration of an antibody to modify T cell epitopes (see, e.g., WO9852976A1, WO0034317A2).
- V H and V L sequences from the starting antibody are analyzed and a human T cell epitope "map" from each V region showing the location of epitopes in relation to complementarity-determining regions (CDRs) and other key residues within the sequence.
- Individual T cell epitopes from the T cell epitope map are analyzed in order to identify alternative amino acid substitutions with a low risk of altering activity of the final antibody.
- a range of alternative V H and V L sequences are designed comprising combinations of amino acid substitutions and these sequences are subsequently incorporated into a range of binding polypeptides, e.g., Sp35 -specific antibodies or immunospecif ⁇ c fragments thereof for use in the diagnostic and treatment methods disclosed herein, which are then tested for function.
- variant antibodies typically, between 12 and 24 variant antibodies are generated and tested. Complete heavy and light chain genes comprising modified V and human C regions are then cloned into expression vectors and the subsequent plasmids introduced into cell lines for the production of whole antibody. The antibodies are then compared in appropriate biochemical and biological assays, and the optimal variant is identified.
- Sp35 antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention may be generated by any suitable method known in the art.
- Polyclonal antibodies to an antigen of interest can be produced by various procedures well known in the art.
- an Sp35 antibody e.g., a binding polypeptide, e.g., an Sp35-specific antibody or immunospecif ⁇ c fragment thereof can be administered to various host animals including, but not limited to, rabbits, mice, rats, chickens, hamsters, goats, donkeys, etc., to induce the production of sera containing polyclonal antibodies specific for the antigen.
- adjuvants may be used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Such adjuvants are also well known in the art.
- Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.
- monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et ah, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. (1988); Hammerling et al, in: Monoclonal Antibodies and T- CeIl Hybridomas Elsevier, N. Y., 563-681 (1981) (said references incorporated by reference in their entireties).
- the term "monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology.
- the term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. Thus, the term “monoclonal antibody” is not limited to antibodies produced through hybridoma technology.
- Monoclonal antibodies can be prepared using Sp35 knockout mice to increase the regions of epitope recognition.
- Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma and recombinant and phage display technology as described elsewhere herein.
- lymphocytes from a mammal which has been injected with antigen are fused with an immortal tumor cell line (e.g. a myeloma cell line), thus, producing hybrid cells or "hybridomas" which are both immortal and capable of producing the genetically coded antibody of the B cell.
- an immortal tumor cell line e.g. a myeloma cell line
- hybrid cells or "hybridomas" which are both immortal and capable of producing the genetically coded antibody of the B cell.
- the resulting hybrids are segregated into single genetic strains by selection, dilution, and regrowth with each individual strain comprising specific genes for the formation of a single antibody. They produce antibodies which are homogeneous against a desired antigen and, in reference to their pure genetic parentage, are termed "monoclonal.”
- Hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
- suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
- reagents, cell lines and media for the formation, selection and growth of hybridomas are commercially available from a number of sources and standardized protocols are well established.
- culture medium in which the hybridoma cells are growing is assayed for production of monoclonal antibodies against the desired antigen.
- the monoclonal antibodies secreted by the subclones may be separated from culture medium, ascites fluid or serum by conventional purification procedures such as, for example, protein-A, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.
- Antibody fragments that recognize specific epitopes may be generated by known techniques.
- Fab and F(ab') 2 fragments may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab') 2 fragments).
- F(ab') 2 fragments contain the variable region, the light chain constant region and the C H I domain of the heavy chain.
- Ribosomal display can be used to replace bacteriophage as the display platform (see, e.g., Hanes et al, Nat. Biotechnol 75:1287 (2000); Wilson et al, Proc. Natl. Acad.
- Humanized antibodies are antibody molecules derived from a non-human species antibody that bind the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule.
- CDRs complementarity determining regions
- framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding.
- These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Pat. No.
- Human antibodies are particularly desirable for therapeutic treatment of human patients.
- Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated herein by reference in its entirety.
- the amino acid sequence of the heavy and/or light chain variable domains may be inspected to identify the sequences of the complementarity determining regions (CDRs) by methods that are well know in the art, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability.
- CDRs complementarity determining regions
- one or more of the CDRs may be inserted within framework regions, e.g., into human framework regions to humanize a non-human antibody.
- the framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions ⁇ see, e.g., Chothia et al., J. MoI. Biol.
- Yet other embodiments of the present invention comprise the generation of human or substantially human antibodies in transgenic animals ⁇ e.g., mice) that are incapable of endogenous immunoglobulin production (see e.g., U.S. Pat. Nos. 6,075,181, 5,939,598, 5,591,669 and 5,589,369 each of which is incorporated herein by reference).
- transgenic animals ⁇ e.g., mice
- U.S. Pat. Nos. 6,075,181, 5,939,598, 5,591,669 and 5,589,369 each of which is incorporated herein by reference.
- the homozygous deletion of the antibody heavy-chain joining region in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of a human immunoglobulin gene array to such germ line mutant mice will result in the production of human antibodies upon antigen challenge.
- Another preferred means of generating human antibodies using SCID mice is disclosed in U.S. Pat. No. 5,811,524 which is incorporated herein
- lymphocytes can be selected by micromanipulation and the variable genes isolated.
- peripheral blood mononuclear cells can be isolated from an immunized mammal and cultured for about 7 days in vitro. The cultures can be screened for specific IgGs that meet the screening criteria. Cells from positive wells can be isolated.
- Individual Ig- producing B cells can be isolated by FACS or by identifying them in a complement-mediated hemolytic plaque assay.
- Ig-producing B cells can be micromanipulated into a tube and the V H and V L genes can be amplified using, e.g., RT-PCR.
- the V H and V L genes can be cloned into an antibody expression vector and transfected into cells (e.g., eukaryotic or prokaryotic cells) for expression.
- Domain deleted constructs can be derived using a vector (e.g., from Biogen IDEC Incorporated) encoding an IgG 1 human constant domain (see, e.g., WO 02/060955 A2 and WO02/096948A2, which are incorporated by reference in their entireties).
- This exemplary vector was engineered to delete the C H 2 domain and provide a synthetic vector expressing a domain deleted IgGj constant region.
- an Sp35 antibody, or antigen-binding fragment, variant, or derivative thereof of the invention comprises an immunoglobulin heavy chain having deletion or substitution of a few or even a single amino acid as long as it permits association between the monomelic subunits.
- the mutation of a single amino acid in selected areas of the C H 2 domain may be enough to substantially reduce Fc binding and thereby increase tumor localization.
- Such partial deletions of the constant regions may improve selected characteristics of the antibody (serum half-life) while leaving other desirable functions associated with the subject constant region domain intact.
- the variants encode less than 50 amino acid substitutions, less than 40 amino acid subsitutions, less than 30 amino acid substitutions, less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the reference V H region, V H CDR1, V H CDR2, V H CDR3, V L region, V L CDR1, V L CDR2, or V L CDR3.
- a "conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a side chain with a similar charge.
- mutations only in framework regions or only in CDR regions of an antibody molecule.
- Introduced mutations may be silent or neutral missense mutations, i.e., have no, or little, effect on an antibody's ability to bind antigen. These types of mutations may be useful to optimize codon usage, or improve a hybridoma's antibody production.
- non-neutral missense mutations may alter an antibody's ability to bind antigen. The location of most silent and neutral missense mutations is likely to be in the framework regions, while the location of most non-neutral missense mutations is likely to be in CDR, though this is not an absolute requirement.
- the present invention also provides for nucleic acid molecules encoding Sp35 antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention.
- the CDRl, CDR2, and CDR3 regions of the VH are at least 80%, 85%, 90% or 95% identical to reference heavy chain CDRl, CDR2, and CDR3 amino acid sequences from monoclonal Sp35 antibodies disclosed herein.
- a heavy chain variable region of the invention has CDRl, CDR2, or CDR3 polypeptide sequences related to the polypeptide sequences shown in Table 4:
- N nucleotide sequence
- P polypeptide sequence
- the present invention provides an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding an immunoglobulin heavy chain variable region (VH) in which the CDRl, CDR2, and CDR3 regions are encoded by nucleotide sequences which are identical to the nucleotide sequences which encode the CDRl, CDR2, and CDR3 groups shown in Table 4.
- VH immunoglobulin heavy chain variable region
- an antibody or antigen-binding fragment comprising the VH encoded by the polynucleotide specifically or preferentially binds to Sp35.
- an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of a VH encoded by one or more of the polynucleotides described above specifically or preferentially binds to the same epitope as a monoclonal antibody selected from the group consisting of: 201', 3A3, 3A6, 1A7, 1G7, 2B10, 2Cl 1, 2F3, 3P1D10.2C3, 3P1E11.3B7, 3P2C6.3G10.2H7, 3P2C9.2G4, 3P4A6.1D9, 3P4A1.2B9, 3P4C2.2D2, 3P4C5.1D8, 3P4C8.2G9, 30-C12 (LiOl), 38-D01 (LiO22), 35-E04 (LiO33), 36-C09 (LiO4), 30-Al 1 (LiO5), 34-F02 (LiO6), 29-E07 (LiO
- the present invention provides an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding an immunoglobulin light chain variable region (VL), where at least one of the CDRs of the light chain variable region or at least two of the CDRs of the light chain variable region are at least 80%, 85%, 90% or 95% identical to reference light chain CDRl, CDR2, or CDR3 amino acid sequences from monoclonal Sp35 antibodies disclosed herein.
- VL immunoglobulin light chain variable region
- the CDRl, CDR2, and CDR3 regions of the VL are at least 80%, 85%, 90% or 95% identical to reference light chain CDRl, CDR2, and CDR3 amino acid sequences from monoclonal Sp35 antibodies disclosed herein.
- a light chain variable region of the invention has CDRl, CDR2, or CDR3 polypeptide sequences related to the polypeptide sequences shown in Table 5:
- N nucleotide sequence
- P polypeptide sequence
- an antibody or antigen-binding fragment comprising the VL encoded by the polynucleotide specifically or preferentially binds to Sp35.
- an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of a VL encoded by one or more of the polynucleotides described above specifically or preferentially binds to an Sp35 polypeptide or fragment thereof, or a Sp35 variant polypeptide, with an affinity characterized by a dissociation constant (K D ) no greater than 5 x 10 "2 M, 10 "2 M, 5 x 10 "3 M, 10 "3 M, 5 x 10 "4 M, 10 "4 M, 5 x 10 '5 M, 10 "5 M, 5 x 10 "6 M, 10 “6 M, 5 x 10- 7 M, 1(T 7 M, 5 x 10- 8 M, 10 "s M, 5 x IO ⁇ 9 M, IO ⁇ 9 M, 5 x 1(T 10 M, 10 "10 M, 5 x 1(T 11 M, 10 " " M, 5 x 10 " 12 M, 10 "12 M, 5 x 10
- the present invention includes an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding a VH at least 80%, 85%, 90% or 95% identical to a reference VH polypeptide sequence selected from the group consisting of SEQ ID NOs: 158 to 172, 372, 376, 380, and 384 shown in Table 6.
- an antibody or antigen-binding fragment comprising the VH encoded by the polynucleotide specifically or preferentially binds to Sp35.
- the present invention includes an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence encoding a VH having a polypeptide sequence selected from the group consisting of SEQ ID NOs: 158 to 172, 372, 376, 380 and 384 shown in Table 6.
- an antibody or antigen-binding fragment comprising the VH encoded by the polynucleotide specifically or preferentially binds to Sp35.
- the present invention includes an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding a VH at least 80%, 85%, 90% or 95% identical to a reference VH polypeptide sequence selected from the group consisting of SEQ ID NOs: 158-172, 372, 376, 380, and 384.
- an antibody or antigen- binding fragment comprising the VH encoded by the polynucleotide specifically or preferentially binds to Sp35.
- the present invention includes an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence encoding a VH of the invention, selected from the group consisting of SEQ ID NOs: 158-172, 372, 376, 380, and 384.
- an antibody or antigen-binding fragment comprising the VH encoded by the polynucleotide specifically or preferentially binds to Sp35.
- an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of a VH encoded by one or more of the polynucleotides described above specifically or preferentially binds to the same epitope as a monoclonal antibody selected from the group consisting of,(201') 3A3, 3A6, 1A7, 1G7, 2B10, 2Cl 1, 2F3, 3P1D10.2C3, 3P1E11.3B7, 3P2C6.3G10.2H7, 3P2C9.2G4, 3P4A6.1D9, 3P4A1.2B9, 3P4C2.2D2, 3P4C5.1D8, 3P4C8.2G9, 30-C12 (LiOl), 38-D01 (LiO2), 35-E04 (Li03), 36-C09 (LiO4), 30-Al 1 (LiO5), 34-F02 (LiO6), 29-E07 (LiO7)
- the present invention includes an isolated polynucleotide which encodes a heavy chain variable region (V H ), where the polynucleotide comprises a V H nucleic acid sequence selected from the group consisting of SEQ ID NOs 173 to 184, 370, 374, 378 and 382, as shown in Table 7.
- V H heavy chain variable region
- an antibody or antigen-binding fragment comprising the VH encoded by the polynucleotide specifically or preferentially binds to Sp35.
- the present invention includes an isolated polynucleotide comprising, consisting essentially of, or consisting of a VH-encoding nucleic acid at least 80%, 85%, 90% or 95% identical to a reference nucleic acid sequence selected from the group consisting of SEQ ID NOs: 173-184, 370, 374, 378 and 382 of Table 7.
- the polynucleotide encodes a VH polypeptide which specifically or preferentially binds to Sp35.
- an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of a VH encoded by one or more of the polynucleotides described above specifically or preferentially binds to the same epitope as a monoclonal antibody selected from the group consisting of,(201') 3A3, 3A6, 1A7, 1G7, 2B10, 2Cl 1, 2F3, 3P1D10.2C3, 3P1E11.3B7, 3P2C6.3G10.2H7, 3P2C9.2G4, 3P4A6.1D9, 3P4A1.2B9, 3P4C2.2D2, 3P4C5.1D8, 3P4C8.2G9, 30-C12 (LiOl), 38-D01 (LiO2), 35-E04 (LiO3), 36-C09 (LiO4), 30-Al 1 (Li05), 34-F02 (LiO6), 29-E07 (LiO7)
- an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of a VH encoded by one or more of the polynucleotides described above specifically or preferentially binds to an Sp35 polypeptide or fragment thereof, or a Sp35 variant polypeptide, with an affinity characterized by a dissociation constant (K D ) no greater than 5 x 10 "2 M, 10 "2 M, 5 x 10 "3 M, 10 "3 M, 5 x 10 "4 M, 10 "4 M, 5 x 1O -5 M, 10 "5 M, 5 x 10 "6 M, lO '6 M, 5 x 10 "7 M, 10 "7 M, 5 x 10 "8 M, 10 “8 M, 5 x 10 "9 M, 10 "9 M, 5 x 10 "10 M, 10 “10 M, 5 x 10 "11 M, 10 "11 M, 5 x 10 " 12 M, 10 "12 M, 5 x 10 "13
- the present invention includes an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding a VL at least 80%, 85%, 90% or 95% identical to a reference VL polypeptide sequence selected from the group consisting of SEQ ID NOs: 273 to 286, 373, 377, 381 and 385, shown in Table 8.
- an antibody or antigen-binding fragment comprising the VL encoded by the polynucleotide specifically or preferentially binds to Sp35.
- the present invention includes an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence encoding a VL having a polypeptide sequence selected from the group consisting of SEQ ID NOs: 273 to 286, 373, 377, 381 and 385, shown in Table 8.
- an antibody or antigen-binding fragment comprising the VL encoded by the polynucleotide specifically or preferentially binds to Sp35.
- the present invention includes an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding a VL at least 80%, 85%, 90% or 95% identical to a reference VL polypeptide sequence selected from the group consisting of SEQ ID NOs: 273 to 286, 373, 377, 381 and 385,.
- an antibody or antigen- binding fragment comprising the VL encoded by the polynucleotide specifically or preferentially binds to Sp35.
- the present invention includes an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence encoding a VL of the invention, selected from the group consisting of SEQ ID NOs: 273 to 286, 373, 377, 381 and 385.
- an antibody or antigen-binding fragment comprising the VL encoded by the polynucleotide specifically or preferentially binds to Sp35.
- an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of a VL encoded by one or more of the polynucleotides described above specifically or preferentially binds to the same epitope as a monoclonal antibody selected from the group consisting of 201', 3A3, 3A6, 1A7, 1G7, 2B10, 2Cl 1, 2F3, 3P1D10.2C3, 3P1E11.3B7, 3P2C6.3G10.2H7, 3P2C9.2G4, 3P4A6.1D9, 3P4A1.2B9, 3P4C2.2D2, 3P4C5.1D8, 3P4C8.2G9, 30-C12 (LiOl), 38-D01 (LiO2), 35-E04 (LiO3), 36-C09 (LiO4), 30-Al 1 (Li05), 34-F02 (LiO6), 29-E07 (LiO7),
- an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of a VL encoded by one or more of the polynucleotides described above specifically or preferentially binds to an Sp35 polypeptide or fragment thereof, or a Sp35 variant polypeptide, with an affinity characterized by a dissociation constant (K D ) no greater than 5 x 10 "2 M, 10 "2 M, 5 x 10 "3 M, 10 "3 M, 5 x 10 "4 M, 10 "4 M, 5 x 10 '5 M, 1(T 5 M, 5 x 10 "6 M, 10 "6 M, 5 x 10 "7 M 5 1(T 7 M, 5 x It) "8 M, 10 "s M, 5 x 10 "9 M, 10 “9 M, 5 x 1(T 10 M, 1(T 10 M, 5 x 10 "11 M, 10 "n M, 5 x 10 " 12 M, 1(T 12 M, 5 x
- the present invention includes an isolated polynucleotide which encodes a light chain variable region (V L ), where the polynucleotide comprises a V L nucleic acid sequence selected from the group consisting of SEQ ID NOs 185 to 194, 371, 375, 379 and 383, as shown in Table 9.
- V L light chain variable region
- an antibody or antigen-binding fragment comprising the VL encoded by the polynucleotide specifically or preferentially binds to Sp35.
- the present invention includes an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding a VL at least 80%, 85%, 90%, or 95% identical to a VL polynucleotide selected from the group consisting of SEQ E) NOs: 185- 194, 371, 375, 379 and 383 of Table 9.
- the polynucleotide encodes a VL polypeptide which specifically or preferentially binds to Sp35.
- an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of a VL encoded by one or more of the polynucleotides described above specifically or preferentially binds to the same epitope as a monoclonal antibody selected from the group consisting of 201', 3A3, 3A6, 1A7, 1G7, 2B10, 2Cl 1, 2F3, 3P1D10.2C3, 3P1E11.3B7, 3P2C6.3G10.2H7, 3P2C9.2G4, 3P4A6.1D9, 3P4A1.2B9, 3P4C2.2D2, 3P4C5.1D8, 3P4C8.2G9, 30-C12 (LiOl), 38-D01 (LiO2), 35-E04 (Li03), 36-C09 (LiO4), 30-Al 1 (Li05), 34-F02 (LiO6), 29-E07 (LiO7), 34
- an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of a VL encoded by one or more of the polynucleotides described above specifically or preferentially binds to an Sp35 polypeptide or fragment thereof, or a Sp35 variant polypeptide, with an affinity characterized by a dissociation constant (K D ) no greater than 5 x 10 "2 M, IQ- 2 M, 5 x 10 "3 M, 10 "3 M, 5 x 10 "4 M, 10 "4 M, 5 x 10 "5 M, 10 "5 M, 5 x 10 ⁇ 6 M, 10 "5 M, 5 x 10 "7 M, 10 "7 M, 5 x 1(T 8 M, 10 "8 M, 5 x 10 "9 M, 1(T 9 M, 5 x 1(T 10 M, 1(T 10 M, 5 x 10 "11 M, l(T n M, 5 x IQT 12 M, 1(T 12 M
- any of the polynucleotides described above may further include additional nucleic acids, encoding, e.g., a signal peptide to direct secretion of the encoded polypeptide, antibody constant regions as described herein, or other heterologous polypeptides as described herein.
- the present invention includes compositions comprising the polynucleotides comprising one or more of the polynucleotides described above.
- the invention includes compositions comprising a first polynucleotide and second polynucleotide wherein said first polynucleotide encodes a VH polypeptide as described herein and wherein said second polynucleotide encodes a VL polypeptide as described herein.
- composition which comprises, consists essentially of, or consists of a VH polynucleotide, as show in Table 7, and a VL polynucleotide, as shown in Table 9, wherein said VH polynucleotide and said VL polynucleotide are selected from the group consisting of: i) SEQ ID NO:173 and SEQ ID NO:185; ii) SEQ ID NO: 174 and SEQ ID NO: 186; iii) SEQ ID NO: 175 and SEQ ID NO: 187; iv) SEQ ID NO: 176 and SEQ ID NO: 188; v) SEQ ID NO: 178 and SEQ ID NO: 189; vi) SEQ ID NO: 179 and SEQ ID NO: 190; vii) SEQ ID NO:180 and SEQ ID NO:191; viii) SEQ ID NO:181 and SEQ ID NO:192; ix) SEQ ID NO:182 and SEQ ID NO:19
- the present invention also includes fragments of the polynucleotides of the invention, as described elsewhere. Additionally polynucleotides which encode fusion polynucleotides, Fab fragments, and other derivatives, as described herein, are also contemplated by the invention. [0210] The polynucleotides may be produced or manufactured by any method known in the art.
- a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et ah, BioTechniques 17:242 (1994)), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.
- chemically synthesized oligonucleotides e.g., as described in Kutmeier et ah, BioTechniques 17:242 (1994)
- nucleotide sequence and corresponding amino acid sequence of the Sp35 antibody, or antigen-binding fragment, variant, or derivative thereof may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et ah, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
- a polynucleotide encoding an Sp35 antibody, or antigen-binding fragment, variant, or derivative thereof can be composed of any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
- a polynucleotide encoding Sp35 antibody, or antigen-binding fragment, variant, or derivative thereof can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
- a polynucleotide encoding an Sp35 antibody, or antigen-binding fragment, variant, or derivative thereof can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA.
- a polynucleotide encoding an Sp35 antibody, or antigen-binding fragment, variant, or derivative thereof may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons.
- Modified bases include, for example, tritylated bases and unusual bases such as inosine.
- polynucleotide embraces chemically, enzymatically, or metabolically modified forms.
- An isolated polynucleotide encoding a non-natural variant of a polypeptide derived from an immunoglobulin can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of the immunoglobulin such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations may be introduced by standard techniques, such as site- directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more non-essential amino acid residues.
- the present invention is further directed to isolated polypeptides which make up Sp35 antibodies, antigen binding fragments, variants or derivatives thereof.
- Sp35 antibodies of the present invention comprise polypeptides, e.g., amino acid sequences encoding Sp35-specific antigen binding regions derived from immunoglobulin molecules.
- a polypeptide or amino acid sequence "derived from" a designated protein refers to the origin of the polypeptide.
- the present invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin heavy chain variable region (VH), where at least one of CDRs of the heavy chain variable region or at least two of the CDRs of the heavy chain variable region are at least 80%, 85%, 90% or 95% identical to reference heavy chain CDRl, CDR2 or CDR3 amino acid sequences from monoclonal Sp35 antibodies disclosed herein.
- VH immunoglobulin heavy chain variable region
- the CDRl, CDR2 and CDR3 regions of the VH are at least 80%, 85%, 90% or 95% identical to reference heavy chain CDRl, CDR2 and CDR3 amino acid sequences from monoclonal Sp35 antibodies disclosed herein.
- a heavy chain variable region of the invention has CDRl, CDR2, and CDR3 polypeptide sequences related to the groups shown in Table 4, supra.
- an antibody or antigen-binding fragment comprising the VH polypeptide specifically or preferentially binds to Sp35.
- the present invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin heavy chain variable region (VH) in which the CDRl, CDR2, and CDR3 regions have polypeptide sequences which are identical to the CDRl, CDR2, and CDR3 groups shown in Table 4.
- VH immunoglobulin heavy chain variable region
- an antibody or antigen- binding fragment comprising the VH polypeptide specifically or preferentially binds to Sp35.
- the present invention includes an isolated polypeptide comprising, consisting essentially of, or consisting of a VH polypeptide selected from the group consisting of SEQ ID NOs: 158 to 172, 372, 376, 380 and 384 as shown in Table 6.
- an antibody or antigen-binding fragment comprising the VH polypeptide specifically or preferentially binds to Sp35.
- an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of a one or more of the VH polypeptides described above specifically or preferentially binds to the same epitope as a monoclonal antibody selected from the group consisting of 201', 3A3, 3A6, 1A7, 1G7, 2B10, 2C11, 2F3, 3P1D10.2C3, 3P1E11.3B7, 3P2C6.3G10.2H7, 3P2C9.2G4, 3P4A6.1D9, 3P4A1.2B9, 3P4C2.2D2, 3P4C5.1D8, 3P4C8.2G9, 30- C12 (LiOl), 38-D01 (LiO2), 35-E04 (Li03), 36-C09 (LiO4), 30-Al 1 (Li05), 34-F02 (LiO6), 29-E07 (LiO7), 34-G04 (Li
- an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of one or more of the VH polypeptides described above specifically or preferentially binds to an Sp35 polypeptide or fragment thereof, or a Sp35 variant polypeptide, with an affinity characterized by a dissociation constant (K D ) no greater than 5 x 10 "2 M, 10 "2 M, 5 x 10 ⁇ 3 M, 10 "3 M, 5 x IO ⁇ 4 M, 1O -4 M, 5 x 10 "5 M, 10 "5 M, 5 x 10 "6 M, 10 "6 M, 5 x 10 '7 M, 10 "7 M, 5 x 10- 8 M, 10 "8 M, 5 x 10 "9 M, 10 "9 M, 5 x 10 "10 M, 10 “10 M, 5 x 10 "11 M, 10 '11 M, 5 x 10 "12 M, 10 "12 M, 5 x 10 ⁇ 13 M, 10 "13 M
- the present invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin light chain variable region (VL), where at least one of the CDRs of the light chain variable region or at least two of the CDRs of the light chain variable region are at least 80%, 85%, 90% or 95% identical to reference heavy chain CDRl, CDR2, or CDR3 amino acid sequences from monoclonal Sp35 antibodies disclosed herein.
- VL immunoglobulin light chain variable region
- the CDRl, CDR2 and CDR3 regions of the VL are at least 80%, 85%, 90% or 95% identical to reference light chain CDRl, CDR2, and CDR3 amino acid sequences from monoclonal Sp35 antibodies disclosed herein.
- a light chain variable region of the invention has CDRl, CDR2, and CDR3 polypeptide sequences related to the polypeptides shown in Table 5, supra.
- an antibody or antigen-binding fragment comprising the VL polypeptide specifically or preferentially binds to Sp35.
- the present invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin light chain variable region (VL) in which the CDRl, CDR2, and CDR3 regions have polypeptide sequences which are identical to the CDRl, CDR2, and CDR3 groups shown in Table 5.
- VL immunoglobulin light chain variable region
- an antibody or antigen-binding fragment comprising the VL polypeptide specifically or preferentially binds to Sp35.
- the present invention includes an isolated polypeptide comprising, consisting essentially of, or consisting of a VL polypeptide at least 80%, 85%, 90% or 95% identical to a reference VL polypeptide sequence selected from the group consisting of SEQ ID NOs:273 to 286, 373, 377, 381 and 385, shown in Table 8.
- an antibody or antigen-binding fragment comprising the VL polypeptide specifically or preferentially binds to Sp35.
- the present invention includes an isolated polypeptide comprising, consisting essentially of, or consisting of a VL polypeptide selected from the group consisting of SEQ ID NOs: 273 to 286, 373, 377, 381 and 385,shown in Table 8.
- an antibody or antigen-binding fragment comprising the VL polypeptide specifically or preferentially binds to Sp35.
- an antibody or antigen-binding fragment thereof comprising, consisting essentially of, one or more of the VL polypeptides described above specifically or preferentially binds to the same epitope as a monoclonal antibody selected from the group consisting of 20r, 3A3, 3A6, 1A7, 1G7, 2B10, 2Cl 1, 2F3, 3P1D10.2C3, 3P1E11.3B7, 3P2C6.3G10.2H7, 3P2C9.2G4, 3P4A6.1D9, 3P4A1.2B9, 3P4C2.2D2, 3P4C5.1D8, 3P4C8.2G9, 30-C12 (LiOl), 38-D01 (LiO2), 35-E04 (Li03), 36-C09 (LiO4), 30-Al 1 (LiO5), 34-F02 (LiO6), 29-E07 (LiO7), 34-G04 (LiO8), 36-Al 1 (LiO5),
- an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of a one or more of the VL polypeptides described above specifically or preferentially binds to an Sp35 polypeptide or fragment thereof, or a Sp35 variant polypeptide, with an affinity characterized by a dissociation constant (K D ) no greater than 5 x 10 "2 M, IQ- 2 M, 5 x 10 "3 M, IQ- 3 M, 5 x 10 "4 M, 10 "4 M, 5 x 10 "5 M, 10 "5 M, 5 x 10 '6 M, 10 "6 M, 5 x 10 "7 M, 10 "7 M, 5 x Kr 8 M, 10 "8 M, 5 x 10 "9 M, 10 "9 M, 5 x 10 "10 M, 1(T 10 M, 5 x 10 '11 M, 10 "11 M, 5 x 10 "12 M, 10 "12 M, 5 x 10 "13 M, 10 "13
- an antibody or antigen-binding fragment thereof comprises, consists essentially of or consists of a VH polypeptide, as shown in Table 6, and a VL polypeptide, as shown in Table 8 , selected from the group consisting of: i) SEQ ID NO: 170 and SEQ ID NO:283; ii) SEQ ID NO:171 and SEQ ID NO:284; iii) SEQ ID NO: 172 and SEQ ID NO:285; iv) SEQ ID NO: 172 and SEQ ID NO:286; v) SEQ TD NO: 158 and SEQ TD NO:273; vi) SEQ TD NO: 159 and SEQ TD NO:274; vii) SEQ ID NO: 160 and SEQ ID NO:275; viii) SEQ E) NO:161 and SEQ E) NO:276; ix) SEQ TD NO: 163 and SEQ TD NO:277; x) SEQ E)
- polypeptides described above may further include additional polypeptides, e.g., a signal peptide to direct secretion of the encoded polypeptide, antibody constant regions as described herein, or other heterologous polypeptides as described herein.
- polypeptides of the invention include polypeptide fragments as described elsewhere.
- polypeptides of the invention include fusion polypeptide, Fab fragments, and other derivatives, as described herein.
- the present invention includes compositions comprising the polypeptides described above.
- Sp35 antibody polypeptides as disclosed herein may be modified such that they vary in amino acid sequence from the naturally occurring binding polypeptide from which they were derived.
- a polypeptide or amino acid sequence derived from a designated protein may be similar, e.g., have a certain percent identity to the starting sequence, e.g., it may be 60%, 70%, 75%, 80%, 85%, 90%, or 95% identical to the starting sequence.
- nucleotide or amino acid substitutions, deletions, or insertions leading to conservative substitutions or changes at "non-essential" amino acid regions may be made.
- a polypeptide or amino acid sequence derived from a designated protein may be identical to the starting sequence except for one or more individual amino acid substitutions, insertions, or deletions, e.g., one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty or more individual amino acid substitutions, insertions, or deletions.
- a polypeptide or amino acid sequence derived from a designated protein has one to five, one to ten, one to fifteen, or one to twenty individual amino acid substitutions, insertions, or deletions relative to the starting sequence.
- Certain Sp35 antibody polypeptides of the present invention comprise, consist essentially of, or consist of an amino acid sequence derived from a human amino acid sequence.
- certain Sp35 antibody polypeptides comprise one or more contiguous amino acids derived from another mammalian species.
- an Sp35 antibody of the present invention may include a primate heavy chain portion, hinge portion, or antigen binding region.
- one or more murine- derived amino acids may be present in a non-murine antibody polypeptide, e.g., in an antigen binding site of an Sp35 antibody.
- Sp35 -specific antibodies, or antigen- binding fragments, variants, or analogs thereof are designed so as to not be immunogenic in the animal to which the antibody is administered.
- an Sp35 antibody polypeptide comprises an amino acid sequence or one or more moieties not normally associated with an antibody. Exemplary modifications are described in more detail below.
- a single-chain fv antibody fragment of the invention may comprise a flexible linker sequence, or may be modified to add a functional moiety (e.g., PEG, a drug, a toxin, or a label).
- An Sp35 antibody polypeptide of the invention may comprise, consist essentially of, or consist of a fusion protein.
- Fusion proteins are chimeric molecules which comprise, for example, an immunoglobulin antigen-binding domain with at least one target binding site, and at least one heterologous portion, i.e., a portion with which it is not naturally linked in nature.
- the amino acid sequences may normally exist in separate proteins that are brought together in the fusion polypeptide or they may normally exist in the same protein but are placed in a new arrangement in the fusion polypeptide. Fusion proteins may be created, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship.
- heterologous as applied to a polynucleotide or a polypeptide, means that the polynucleotide or polypeptide is derived from a distinct entity from that of the rest of the entity to which it is being compared.
- a “heterologous polypeptide” to be fused to an Sp35 antibody, or an antigen-binding fragment, variant, or analog thereof is derived from a non- immunoglobulin polypeptide of the same species, or an immunoglobulin or non-immunoglobulin polypeptide of a different species.
- a "conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
- Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
- basic side chains e
- a nonessential amino acid residue in an immunoglobulin polypeptide is preferably replaced with another amino acid residue from the same side chain family.
- a string of amino acids can be replaced with a structurally similar string that differs in order and/or composition of side chain family members.
- mutations may be introduced randomly along all or part of the immunoglobulin coding sequence, such as by saturation mutagenesis, and the resultant mutants can be incorporated into Sp35 antibodies for use in the diagnostic and treatment methods disclosed herein and screened for their ability to bind to the desired antigen, e.g., Sp35.
- Sp35 antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalent and non-covalent conjugations) to polypeptides or other compositions.
- Sp35-specific Sp35 antibodies may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionuclides, or toxins. See, e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Patent No. 5,314,995; and EP 396,387, which are incorporated herein by reference in their entireties.
- Sp35 antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention include derivatives that are modified, i.e., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody binding Sp35.
- the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc.
- Sp35 antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention can be composed of amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids.
- Sp35-specf ⁇ c antibodies may be modified by natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art.
- Cyclic, branched, and branched cyclic Sp35-specific antibodies may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, s
- the present invention also provides for fusion proteins comprising an Sp35 antibody, or antigen-binding fragment, variant, or derivative thereof, and a heterologous polypeptide.
- the heterologous polypeptide to which the antibody is fused may be useful for function or is useful to target the Sp35 polypeptide expressing cells.
- a fusion protein of the invention comprises, consists essentially of, or consists of, a polypeptide having the amino acid sequence of any one or more of the V H regions of an antibody of the invention or the amino acid sequence of any one or more of the V L regions of an antibody of the invention or fragments or variants thereof, and a heterologous polypeptide sequence.
- a fusion protein for use in the diagnostic and treatment methods disclosed herein comprises, consists essentially of, or consists of a polypeptide having the amino acid sequence of any one, two, three of the V H CDRs of an Sp35-specific antibody, or fragments, variants, or derivatives thereof, or the amino acid sequence of any one, two, three of the V L CDRs of an Sp35-specific antibody, or fragments, variants, or derivatives thereof, and a heterologous polypeptide sequence.
- the fusion protein comprises a polypeptide having the amino acid sequence of a V H CDR3 of an Sp35-specif ⁇ c antibody of the present invention, or fragment, derivative, or variant thereof, and a heterologous polypeptide sequence, which fusion protein specifically binds to at least one epitope of Sp35.
- a fusion protein comprises a polypeptide having the amino acid sequence of at least one V H region of an Sp35-specific antibody of the invention and the amino acid sequence of at least one V L region of an Sp35-specific antibody of the invention or fragments, derivatives or variants thereof, and a heterologous polypeptide sequence.
- the V H and V L regions of the fusion protein correspond to a single source antibody (or scFv or Fab fragment) which specifically binds at least one epitope of Sp35.
- a fusion protein for use in the diagnostic and treatment methods disclosed herein comprises a polypeptide having the amino acid sequence of any one, two, three or more of the V H CDRS of an Sp35-specif ⁇ c antibody and the amino acid sequence of any one, two, three or more of the V L CDRs of an Sp35-specif ⁇ c antibody, or fragments or variants thereof, and a heterologous polypeptide sequence.
- V H CDR(s) or V L CDR(s) correspond to single source antibody (or scFv or Fab fragment) of the invention.
- Nucleic acid molecules encoding these fusion proteins are also encompassed by the invention.
- Exemplary fusion proteins reported in the literature include fusions of the T cell receptor (Gascoigne et al, Proc. Natl. Acad. Sd. USA 54:2936-2940 (1987)); CD4 (Capon et ah, Nature 337:525-531 (1989); Traunecker et al, Nature 339:68-70 (1989); Zettmeissl et al, DNA Cell Biol. USA P:347-353 (1990); and Byrn et al, Nature 344:667-670 (1990)); L-selectin (homing receptor) (Watson et al, J. Cell. Biol.
- CD44 (Aruffo et al, Cell (57:1303-1313 (1990)); CD28 and B7 (Linsley et al, J. Exp. Med. 773:721-730 (1991)); CTLA-4 (Lisley et al, J. Exp. Med. 174:561-569 (1991)); CD22 (Stamenkovic et al, Cell 66: 1133-1144 (1991)); TNF receptor (Ashkenazi et al, Proc. Natl. Acad. ScL USA 55:10535-10539 (1991); Lesslauer et al, Eur. J.
- Sp35 antibodies, antibody fragments, derivatives and variants thereof further comprise a targeting moiety.
- Targeting moieties include a protein or a peptide which directs localization to a certain part of the body, for example, to the brain or compartments therein.
- Sp35 antibodies, antibody fragments, derivatives and variants thereof are attached or fused to a brain targeting moiety.
- the brain targeting moieties are attached covalently (e.g., direct, translational fusion, or by chemical linkage either directly or through a spacer molecule, which can be optionally cleavable) or non-covalently attached (e.g., through reversible interactions such as avidin, biotin, protein A, IgG, etc.).
- the Sp35 antibodies, antibody fragments, derivatives and variants thereof are attached to one more brain targeting moieties.
- the brain targeting moiety is attached to a plurality of Sp35 antibodies, antibody fragments, derivatives and variants thereof.
- a brain targeting moiety associated with an Sp35 antibody, antibody fragment, derivative or variant thereof enhances brain delivery of such an Sp35 antibodies, antibody fragments, dervatives and variants thereof.
- a number of polypeptides have been described which, when fused to a protein or therapeutic agent, delivers the protein or therapeutic agent through the blood brain barrier (BBB).
- BBB blood brain barrier
- Non-limiting examples include the single domain antibody FC5 (Abulrob et al. (2005) J. Neurochem. 95, 1201-1214); mAB 83-14, a monoclonal antibody to the human insulin receptor (Pardridge et al. (1995) Pharmacol. Res.
- administering to an animal a radioactively, enzymatically or fluorescently labeled Sp35 antibody, antibody fragment, derivative and variant thereof linked to a brain targeting moiety; determining brain localization; and comparing localization with an equivalent radioactively, enzymatically or fluorescently labeled Sp35 antibody, antibody fragment, deirvative or variant thereof that is not associated with a brain targeting moiety.
- Other means of determining enhanced targeting are described in the above references.
- Sp35 antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention may be fused to heterologous polypeptides to increase the in vivo half life of the polypeptides or for use in immunoassays using methods known in the art.
- PEG can be conjugated to the Sp35 antibodies of the invention to increase their half-life in vivo. Leong, S.R., et al, Cytokine 16:106 (2001); Adv. in Drug Deliv. Rev. 54:531 (2002); or Weir et al., Biochem. Soc. Transactions 30:512 (2002).
- Sp35 antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention can be fused to marker sequences, such as a peptide to facilitate their purification or detection.
- the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available.
- hexa-histidine provides for convenient purification of the fusion protein.
- peptide tags useful for purification include, but are not limited to, the "HA” tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:161 (1984)) and the "flag" tag.
- Fusion proteins can be prepared using methods that are well known in the art (see for example US Patent Nos. 5,116,964 and 5,225,538). The precise site at which the fusion is made may be selected empirically to optimize the secretion or binding characteristics of the fusion protein. DNA encoding the fusion protein is then transfected into a host cell for expression.
- Sp35 antibodies or antigen-binding fragments, variants, or derivatives thereof of the present invention may be used in non-conjugated form or may be conjugated to at least one of a variety of molecules, e.g., to improve the therapeutic properties of the molecule, to facilitate target detection, or for imaging or therapy of the patient.
- Sp35 antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention can be labeled or conjugated either before or after purification, when purification is performed.
- Sp35 antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention may be conjugated to therapeutic agents, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceutical agents, or PEG.
- conjugates may also be assembled using a variety of techniques depending on the selected agent to be conjugated.
- conjugates with biotin are prepared e.g. by reacting a binding polypeptide with an activated ester of biotin such as the biotin N-hydroxysuccinimide ester.
- conjugates with a fluorescent marker may be prepared in the presence of a coupling agent, e.g. those listed herein, or by reaction with an isothiocyanate, preferably fluorescein-isothiocyanate.
- Conjugates of the Sp35 antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention are prepared in an analogous manner.
- the present invention further encompasses Sp35 antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention conjugated to a diagnostic or therapeutic agent.
- the Sp35 antibodies can be used diagnostically to, for example, monitor the development or progression of a neurological disease as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment and/or prevention regimen. Detection can be facilitated by coupling the Sp35 antibody, or antigen-binding fragment, variant, or derivative thereof to a detectable substance.
- detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. See, for example, U.S. Pat. No. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention.
- suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase;
- suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
- suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
- an example of a luminescent material includes luminol;
- examples of bioluminescent materials include luciferase, luciferin, and aequorin;
- suitable radioactive material include 125 1, 131 1, 111 In or 99 Tc.
- An Sp35 antibody, or antigen-binding fragment, variant, or derivative thereof also can be detectably labeled by coupling it to a chemiluminescent compound.
- the presence of the chemiluminescent-tagged Sp35 antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
- particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
- an Sp35 antibody, or antigen-binding fragment, variant, or derivative thereof can be detectably labeled is by linking the same to an enzyme and using the linked product in an enzyme immunoassay (EIA) (Voller, A., "The Enzyme Linked Immunosorbent Assay (ELISA)” Microbiological Associates Quarterly Publication, Walkersville, Md., Diagnostic Horizons 2:1-7 (1978)); Voller et al., J. Clin. Pathol. 37:507-520 (1978); Butler, J. E., Meth. Enrymol 73:482- 523 (1981); Maggio, E.
- EIA enzyme immunoassay
- the enzyme which is bound to the Sp35 antibody will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorimetric or by visual means.
- Enzymes which can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5 -steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. Additionally, the detection can be accomplished by colorimetric methods which employ a chromogenic substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.
- Detection may also be accomplished using any of a variety of other immunoassays.
- a radioimmunoassay ⁇ see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, (March, 1986)), which is incorporated by reference herein).
- the radioactive isotope can be detected by means including, but not limited to, a gamma counter, a scintillation counter, or autoradiography.
- An Sp35 antibody, or antigen-binding fragment, variant, or derivative thereof can also be detectably labeled using fluorescence emitting metals such as 152Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
- DTPA diethylenetriaminepentacetic acid
- EDTA ethylenediaminetetraacetic acid
- RNA may be isolated from the original hybridoma cells or from other transformed cells by standard techniques, such as guanidinium isothiocyanate extraction and precipitation followed by centrifugation or chromatography. Where desirable, mRNA may be isolated from total RNA by standard techniques such as chromatography on oligo dT cellulose. Suitable techniques are familiar in the art.
- cDNAs that encode the light and the heavy chains of the antibody may be made, either simultaneously or separately, using reverse transcriptase and DNA polymerase in accordance with well known methods.
- PCR may be initiated by consensus constant region primers or by more specific primers based on the published heavy and light chain DNA and amino acid sequences.
- PCR also may be used to isolate DNA clones encoding the antibody light and heavy chains, m this case the libraries may be screened by consensus primers or larger homologous probes, such as mouse constant region probes.
- DNA typically plasmid DNA
- DNA may be isolated from the cells using techniques known in the art, restriction mapped and sequenced in accordance with standard, well known techniques set forth in detail, e.g., in the foregoing references relating to recombinant DNA techniques.
- the DNA may be synthetic according to the present invention at any point during the isolation process or subsequent analysis.
- the polynucleotides encoding the Sp35 antibodies are typically inserted in an expression vector for introduction into host cells that may be used to produce the desired quantity of Sp35 antibody.
- Recombinant expression of an antibody, or fragment, derivative or analog thereof, e.g., a heavy or light chain of an antibody which binds to a target molecule described herein, e.g., Sp35 requires construction of an expression vector containing a polynucleotide that encodes the antibody.
- a polynucleotide encoding an antibody molecule or a heavy or light chain of an antibody, or portion thereof (preferably containing the heavy or light chain variable domain), of the invention has been obtained, the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art.
- Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No. 5,122,464) and the variable domain of the antibody may be cloned into such a vector for expression of the entire heavy or light chain.
- the host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide.
- the two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides.
- a single vector may be used which encodes both heavy and light chain polypeptides.
- the light chain is advantageously placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc. Natl. Acad. Sd. USA 77:2197 (1980)).
- the coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.
- vectors used in accordance with the present invention as a vehicle for introducing into and expressing a desired gene in a host cell.
- vectors may easily be selected from the group consisting of plasmids, phages, viruses and retroviruses.
- vectors compatible with the instant invention will comprise a selection marker, appropriate restriction sites to facilitate cloning of the desired gene and the ability to enter and/or replicate in eukaryotic or prokaryotic cells.
- vectors For the purposes of this invention, numerous expression vector systems may be employed.
- one class of vector utilizes DNA elements which are derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses (RSV, MMTV or MOMLV) or SV40 virus.
- Others involve the use of polycistronic systems with internal ribosome binding sites.
- cells which have integrated the DNA into their chromosomes may be selected by introducing one or more markers which allow selection of transfected host cells. The marker may provide for prototrophy to an auxotrophic host, biocide resistance (e.g., antibiotics) or resistance to heavy metals such as copper.
- the selectable marker gene can either be directly linked to the DNA sequences to be expressed, or introduced into the same cell by cotransformation. Additional elements may also be needed for optimal synthesis of mRNA. These elements may include signal sequences, splice signals, as well as transcriptional promoters, enhancers, and termination signals.
- the cloned variable region genes are inserted into an expression vector along with the heavy and light chain constant region genes (preferably human) synthetic as discussed above.
- this is effected using a proprietary expression vector of Biogen IDEC, Inc., referred to as NEOSPLA (U.S. patent 6,159,730).
- NEOSPLA a proprietary expression vector of Biogen IDEC, Inc.
- This vector contains the cytomegalovirus promoter/enhancer, the mouse beta globin major promoter, the SV40 origin of replication, the bovine growth hormone polyadenylation sequence, neomycin phosphotransferase exon 1 and exon 2, the dihydrofolate reductase gene and leader sequence.
- This vector has been found to result in very high level expression of antibodies upon incorporation of variable and constant region genes, transfection in CHO cells, followed by selection in G418 containing medium and methotrexate amplification.
- any expression vector which is capable of eliciting expression in eukaryotic cells may be used in the present invention.
- Suitable vectors include, but are not limited to plasmids pcDNA3, pHCMV/Zeo, pCR3.1, pEF I/His, pEMD/GS, pRc/HCMV2, pSV40/Zeo2, pTRACER-HCMV, pUB6/V5-His, pVAXl, and pZeoSV2 (available from Invitrogen, San Diego, CA), and plasmid pCI (available from Promega, Madison, WI).
- screening large numbers of transformed cells for those which express suitably high levels if immunoglobulin heavy and light chains is routine experimentation which can be carried out, for example, by robotic systems. Vector systems are also taught in U.S. Pat. Nos.
- the Sp35 antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention may be expressed using polycistronic constructs such as those disclosed in United States Patent Application Publication No. 2003-0157641 Al, filed November 18, 2002 and incorporated herein in its entirety.
- polycistronic constructs such as those disclosed in United States Patent Application Publication No. 2003-0157641 Al, filed November 18, 2002 and incorporated herein in its entirety.
- multiple gene products of interest such as heavy and light chains of antibodies may be produced from a single polycistronic construct.
- These systems advantageously use an internal ribosome entry site (IRES) to provide relatively high levels of Sp35 antibodies, e.g., binding polypeptides, e.g., Sp35-specific antibodies or immunospecific fragments thereof in eukaryotic host cells.
- IVS internal ribosome entry site
- Compatible IEES sequences are disclosed in U.S. Pat. No. 6,193,980 which is also incorporated herein. Those skilled in the
- the expression vector may be introduced into an appropriate host cell.
- Introduction of the plasmid into the host cell can be accomplished by various techniques well known to those of skill in the art. These include, but are not limited to, transfection (including electrophoresis and electroporation), protoplast fusion, calcium phosphate precipitation, cell fusion with enveloped DNA, microinjection, and infection with intact virus. See, Ridgway, A. A. G. "Mammalian Expression Vectors" Vectors, Rodriguez and Denhardt, Eds., Butterworths, Boston, Mass., Chapter 24.2, pp. 470- 472 (1988).
- plasmid introduction into the host is via electroporation.
- the host cells harboring the expression construct are grown under conditions appropriate to the production of the light chains and heavy chains, and assayed for heavy and/or light chain protein synthesis.
- Exemplary assay techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or fluorescence-activated cell sorter analysis (FACS), immunohistochemistry and the like.
- the expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibodyfor use in the methods described herein.
- the invention includes host cells containing a polynucleotide encoding an antibody of the invention, or a heavy or light chain thereof, operably linked to a heterologous promoter.
- vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.
- host cells refers to cells which harbor vectors constructed using recombinant DNA techniques and encoding at least one heterologous gene.
- the terms “cell” and “cell culture” are used interchangeably to denote the source of antibody unless it is clearly specified otherwise.
- recovery of polypeptide from the “cells” may mean either from spun down whole cells, or from the cell culture containing both the medium and the suspended cells.
- host-expression vector systems may be utilized to express antibody molecules for use in the methods described herein.
- Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ.
- These include but are not limited to microorganisms such as bacteria ⁇ e.g., E. coli, B.
- subtilis transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichid) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors ⁇ e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors ⁇ e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors ⁇ e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems ⁇ e.g., COS, CHO, BLK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells ⁇ e.g., metallothionein promoter)
- bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule.
- mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., Gene ⁇ 5:101 (1986); Cockett et al., Bio/Technology 8:2 (1990)).
- the host cell line used for protein expression is often of mammalian origin; those skilled in the art are credited with ability to preferentially determine particular host cell lines which are best suited for the desired gene product to be expressed therein.
- Exemplary host cell lines include, but are not limited to, CHO (Chinese Hamster Ovary), DG44 and DUXBIl (Chinese Hamster Ovary lines, DHFR minus), HELA (human cervical carcinoma), CVI (monkey kidney line), COS (a derivative of CVI with SV40 T antigen), VERY, BHK (baby hamster kidney), MDCK, 293, WI38, R1610 (Chinese hamster fibroblast) BALBC/3T3 (mouse fibroblast), HAK (hamster kidney line), SP2/0 (mouse myeloma), P3x63-Ag3.653 (mouse myeloma), BFA-Ic IBPT (bovine endothelial cells), RAJI (human lymph
- a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications ⁇ e.g., glycosylation) and processing ⁇ e.g., cleavage) of protein products may be important for the function of the protein.
- Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.
- eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used.
- cell lines which stably express the antibody molecule may be engineered.
- host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
- appropriate expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
- engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
- the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
- This method may advantageously be used to engineer cell lines which stably express the antibody molecule.
- a number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al, Cell 11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. ScL USA 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817 1980) genes can be employed in tk-, hgprt- or aprt-cells, respectively.
- antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Natl. Acad. ScL USA 77:357 (1980); O'Hare et al, Proc. Natl. Acad. ScL USA 78:1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad.
- the expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, TJie use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Academic Press, New York, Vol. 3. (1987)).
- vector amplification for a review, see Bebbington and Hentschel, TJie use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Academic Press, New York, Vol. 3. (1987)).
- a marker in the vector system expressing antibody is amplifiable
- increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Grouse et al, MoI. Cell. Biol. 3:257 (1983)).
- the solutions of polypeptides can be purified by the customary chromatography methods, for example gel filtration, ion-exchange chromatography, chromatography over DEAE-cellulose or (immuno-)affinity chromatography, e.g., after preferential biosynthesis of a synthetic hinge region polypeptide or prior to or subsequent to the HIC chromatography step described herein.
- customary chromatography methods for example gel filtration, ion-exchange chromatography, chromatography over DEAE-cellulose or (immuno-)affinity chromatography, e.g., after preferential biosynthesis of a synthetic hinge region polypeptide or prior to or subsequent to the HIC chromatography step described herein.
- Genes encoding Sp35 antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention can also be expressed non-mammalian cells such as bacteria or yeast or plant cells.
- Bacteria which readily take up nucleic acids include members of the enterobacteriaceae, such as strains of Escherichia coli or Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus, and Haemophilus influenzae. It will further be appreciated that, when expressed in bacteria, the heterologous polypeptides typically become part of inclusion bodies. The heterologouspolypeptides must be isolated, purified and then assembled into functional molecules. Where tetravalent forms of antibodies are desired, the subunits will then self-assemble into tetravalent antibodies (WO02/096948A2).
- a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed.
- vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable.
- Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al, EMBO J. 2: 1791 (1983)), in which the antibody coding sequence may be ligated individually into the vector in frame with the lacZ coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res.
- pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
- GST glutathione S-transferase
- fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to a matrix glutathione-agarose beads followed by elution in the presence of free glutathione.
- the pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
- eukaryotic microbes may also be used. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among eukaryotic microorganisms although a number of other strains are commonly available, e.g., Pichia pastoris :
- the plasmid YRp7 for example, (Stinchcomb et ah, Nature 252:39 (1979); Kingsman et al., Gene 7:141 (1979); Tschemper et ah, Gene 10:157 (1980)) is commonly used.
- This plasmid already contains the TRPl gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4- 1 (Jones, Genetics 85:12 (1977)).
- the presence of the trpl lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.
- Autographa californica nuclear polyhidrosis virus (AcNPV) is typically used as a vector to express foreign genes.
- the virus grows in Spodoptera ⁇ rwgzperd ⁇ cells.
- the antibody coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
- an antibody molecule of the invention may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography ⁇ e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
- chromatography e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography
- centrifugation e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography
- differential solubility e.g., differential solubility
- Sp35 antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention can relieve NgRl -mediated inhibition of axonal extension that normally takes place in CNS neurons. This is beneficial in situations where axonal extension or neurite sprouting is needed in the brain or spinal cord.
- Spinal cord injury, including partial or complete crush or severance, exemplifies a situation in which axonal extension is needed, but is normally inhibited through operation of the Nogo pathway.
- diseases or disorders in which axonal extension and/or neurite sprouting in the brain would be beneficial include stroke, multiple sclerosis, and other neurodegenerative diseases or disorders such as multiple sclerosis (MS), progressive multifocal leukoencephalopathy (PML), encephalomyelitis (EPL), central pontine myelolysis (CPM), adrenoleukodystrophy, Alexander's disease, Pelizaeus Merzbacher disease (PMZ), Globoid cell Leucodystrophy (Krabbe's disease) and Wallerian Degeneration, optic neuritis, transverse myelitis, amyotrophic lateral sclerosis (ALS), Huntington's disease, Alzheimer's disease, Parkinson's disease, spinal cord injury, traumatic brain injury, post radiation injury, neurologic complications of chemotherapy, stroke, neuropathy, acute ischemic optic neuropathy, vitamin E deficiency, isolated vitamin E deficiency syndrome, AR, Bassen-Kornzweig syndrome, Marchiafava-Bignami
- Sp35 is expressed in oligodendrocytes, and contributes to oligodendrocyte biology.
- Soluble derivatives of S ⁇ 35, certain polynucleotides ⁇ e.g. RNAi), as well as certain antibodies which specifically bind to Sp35, as described herein act as antagonists to Sp35 function in oligodendrocytes, promoting proliferation, differentiation and survival of oligodendrocytes and promoting myelination of neurons in vitro and in vivo. This is beneficial in for diseases, disorders or conditions involving demyelination and dysmyelination.
- Examples of diseases or disorders in which oligodendrocyte proliferation, differentiation and survival, and/or myelination or remyelination would be beneficial include multiple sclerosis (MS), progressive multifocal leukoencephalopathy (PML), encephalomyelitis (EPL), central pontine myelolysis (CPM), adrenoleukodystrophy, Alexander's disease, Pelizaeus Merzbacher disease (PMZ), Globoid cell Leucodystrophy (Krabbe's disease), Wallerian Degeneration, optic neuritis, transverse myelitis, amyotrophic lateral sclerosis (ALS), Huntington's disease, Alzheimer's disease, Parkinson's disease, spinal cord injury, traumatic brain injury, post radiation injury, neurologic complications of chemotherapy, stroke, acute ischemic optic neuropathy, vitamin E deficiency, isolated vitamin E deficiency syndrome, AR, Bassen-Kornzweig syndrome, Marchiafava-Bignami syndrome, metachromatic le
- one embodiment of the present invention provides methods for treating spinal cord injury, diseases or disorders associated with inhibition of neuronal growth in the CNS, diseases or disorders associated with inhibition of oligodendrocyte growth or differentiation, and diseases involving demyelination or dysmyelination of CNS neurons in an animal suffering from such injury or disease or predisposed to contract such disease, the method comprising, consisting essentially of, or consisting of administering to the animal an effective amount of an Sp35 antibody, or antigen-binding fragment, variant, or derivative thereof.
- Antibodies of the invention are described herein, and include the monoclonal antibodies listed in Table 3A and 3B, antibodies which specifically bind to the same epitope as the monoclonal antibodies listed in Table 3A and 3B, antibodies which competitively inhibit binding of the monoclonal antibodies listed in Table 3A and 3B to Sp35, and antibodies comprising polypeptides derived from the monoclonal antibodies listed in Table 3 A and 3B.
- a therapeutic Sp35 antibody to be used in treatment methods disclosed herein can be prepared and used as a therapeutic agent which promotes CNS neurite outgrowth, neuronal survival, axon guidance and axon regeneration, which promotes oligodendrocyte survival, growth, and/or differentiation, and which promotes myelination or remyelination of CNS neurons.
- Characteristics of suitable therapeutic Sp35 antibodies include: binding to Sp35 epitopes which result in blocking of Sp35 activity, binding to Sp35 with sufficient affinity to elicit a therapeutic effect, and binding to Sp35 preferentially to normal binding partners, e.g., Nogo Receptor.
- Therapeutic Sp35 antibodies may be monoclonal, chimeric or humanized antibodies, or fragments of antibodies that bind specifically to Sp35.
- the antibodies may be monovalent, bivalent, polyvalent, or bifunctional antibodies.
- Antibody fragments include without limitation Fab F(ab') 2 , and Fv fragments.
- Therapeutic Sp35 antibodies, or antigen-binding fragments, variants or derivatives thereof according to the invention can be used in unlabeled or unconjugated form, or can be coupled or linked to drugs, labels or stabilization agents which may or may not exert additional therapeutic effects.
- a specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the particular Sp35 antibody, or antigen-binding fragment, variant or derivative thereof used, the patient's age, body weight, general health, sex, and diet, and the time of administration, rate of excretion, drug combination, and the severity of the particular disease being treated. Judgment of such factors by medical caregivers is within the ordinary skill in the art.
- the amount will also depend on the individual patient to be treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The amount used can be determined by pharmacological and pharmacokinetic principles well known in the art.
- the Sp35 antibodies, or antigen-binding fragments, variants or derivatives thereof may be administered directly to the nervous system, intracerebroventricularly, or intrathecally, e.g. into a chronic lesion of MS, as discussed in more detail below.
- an Sp35 antibody as described above is an antagonist of Sp35 activity.
- binding of an antagonist Sp35 antibody to Sp35, as expressed on neurons blocks myelin-associated neurite outgrowth inhibition or neuronal cell death.
- binding of the Sp35 antibody to Sp35, as expressed on oligodendrocytes blocks inhibition of oligodendrocyte growth or differentiation, or blocks demyelination or dysmyelination of CNS neurons.
- an Sp35 antibody, or an antigen-binding fragment, variant, or derivative thereof, in particular the Sp35 antibodies described herein can be administered directly as a preformed polypeptide, or indirectly through a nucleic acid vector, to permit beneficial axonal outgrowth, promote oligodendrocyte proliferation, differentiation, and survival, and/or promote myelination or remyelination.
- a subject may be treated with a nucleic acid molecule encoding an Sp35 antibody, or antigen-binding fragment, variant, or analog thereof, e.g., in a vector.
- Doses for nucleic acids encoding polypeptides range from about 10 ng to 1 g, 100 ng to 100 mg, 1 ⁇ g to 10 mg, or 30-300 ⁇ g DNA per patient.
- Doses for infectious viral vectors vary from 10-100, or more, virions per dose.
- an Sp35 antibody, or an antigen-binding fragment, variant, or derivative thereof is administered in a treatment method that includes: (1) transforming or transfecting an implantable host cell with a nucleic acid, e.g., a vector, that expresses an Sp35 antibody, or an antigen-binding fragment, variant, or derivative thereof; and (2) implanting the transformed host cell into a mammal, at the site of a disease, disorder or injury.
- the transformed host cell can be implanted at the site of a spinal cord injury or at a site of dysmyelination.
- the implantable host cell is removed from a mammal, temporarily cultured, transformed or transfected with an isolated nucleic acid encoding a an Sp35 antibody, and implanted back into the same mammal from which it was removed.
- the cell can be, but is not required to be, removed from the same site at which it is implanted.
- Such embodiments sometimes known as ex vivo gene therapy, can provide a continuous supply of the Sp35 polypeptide, localized at the site of site of action, for a limited period of time.
- the methods for treating spinal cord injury, diseases or disorders associated with inhibition of neuronal growth in the CNS, diseases or disorders associated with inhibition of oligodendrocyte growth or differentiation, and diseases involving demyelination or dysmyelination of CNS neurons comprising administration of an Sp35 antibody, or antigen-binding fragment, variant, or derivative thereof of the invention are typically tested in vitro, and then in vivo in an acceptable animal model, for the desired therapeutic or prophylactic activity, prior to use in humans. Suitable animal models, including transgenic animals, are will known to those of ordinary skill in the art.
- in vitro assays to demonstrate the therapeutic utility of Sp35 antibody described herein include the effect of an Sp35 antibody on a cell line or a patient tissue sample.
- in vitro assays which can be used to determine whether administration of a specific Sp35 antibody is indicated, include in vitro cell culture assays in which a patient tissue sample is grown in culture, and exposed to or otherwise administered a compound, and the effect of such compound upon the tissue sample is observed.
- Supplementary active compounds also can be incorporated into the compositions of the invention.
- a Sp35 antibody, or antigen-binding fragment, variant, or derivative thereof of the invention may be coformulated with and/or coadministered with one or more additional therapeutic agents.
- the invention encompasses any suitable delivery method for a Sp35 antibody, or antigen- binding fragment, variant, or derivative thereof of the invention to a selected target tissue, including bolus injection of an aqueous solution or implantation of a controlled-release system. Use of a controlled-release implant reduces the need for repeat injections.
- Methods of preparing and administering Sp35 antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention to a subject in need thereof are well known to or are readily determined by those skilled in the art.
- the route of administration of the Sp35 antibody, or antigen-binding fragment, variant, or derivative thereof may be, for example, oral, parenteral, by inhalation or topical.
- parenteral as used herein includes, e.g., intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal administration.
- a form for administration would be a solution for injection, in particular for intravenous or intraarterial injection or drip.
- a suitable pharmaceutical composition for injection may comprise a buffer (e.g. acetate, phosphate or citrate buffer), a surfactant (e.g. polysorbate), optionally a stabilizer agent (e.g. human albumin), etc.
- a buffer e.g. acetate, phosphate or citrate buffer
- a surfactant e.g. polysorbate
- optionally a stabilizer agent e.g. human albumin
- Sp35 antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention can be delivered directly to the site of the adverse cellular population thereby increasing the exposure of the diseased tissue to the therapeutic agent.
- Sp35 antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention may be administered in a pharmaceutically effective amount for the in vivo treatment of mammalian spinal cord injury, diseases or disorders associated with inhibition of neuronal growth in the CNS, diseases or disorders associated with inhibition of oligodendrocyte growth or differentiation, and diseases involving demyelination or dysmyelination of CNS.
- the disclosed antibodies will be formulated so as to facilitate administration and promote stability of the active agent.
- pharmaceutical compositions in accordance with the present invention comprise a pharmaceutically acceptable, non-toxic, sterile carrier such as physiological saline, non-toxic buffers, preservatives and the like.
- a pharmaceutically effective amount of an Sp35 antibody, or antigen-binding fragment, variant, or derivative thereof, conjugated or unconjugated shall be held to mean an amount sufficient to achieve effective binding to a target and to achieve a benefit, e.g., to ameliorate symptoms of a disease or disorder or to detect a substance or a cell.
- compositions used in this invention comprise pharmaceutically acceptable carriers, including, e.g., ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose- based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.
- pharmaceutically acceptable carriers including, e.g., ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as
- Preparations for parenteral administration includes sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
- non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
- Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
- pharmaceutically acceptable carriers include, but are not limited to, 0.01-0. IM and preferably 0.05M phosphate buffer or 0.8% saline.
- Intravenous vehicles include sodium phosphate solutions, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
- Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present such as for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.
- compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
- the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and will preferably be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
- the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol ⁇ e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
- the proper fluidity 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.
- a coating such as lecithin
- surfactants Suitable formulations for use in the therapeutic methods disclosed herein are described in Remington's Pharmaceutical Sciences, Mack Publishing Co., 16th ed. (1980).
- Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like.
- isotonic agents for example, sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride in the composition.
- Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
- sterile injectable solutions can be prepared by incorporating an active compound (e.g., an Sp35 antibody, or antigen-binding fragment, variant, or derivative thereof, by itself or in combination with other active agents) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization.
- an active compound e.g., an Sp35 antibody, or antigen-binding fragment, variant, or derivative thereof, by itself or in combination with other active agents
- dispersions are prepared by incorporating the active compound into a sterile vehicle, which 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, which yields a powder of an active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
- the preparations for injections are processed, filled into containers such as ampoules, bags, bottles, syringes or vials, and sealed under aseptic conditions according to methods known in the art. Further, the preparations may be packaged and sold in the form of a kit such as those described in co-pending U.S. S.N. 09/259,337 (US-2002-0102208 Al), which is incorporated herein by reference in its entirety.
- Such articles of manufacture will preferably have labels or package inserts indicating that the associated compositions are useful for treating a subject suffering from, or predisposed to autoimmune or neoplastic disorders.
- Parenteral formulations may be a single bolus dose, an infusion or a loading bolus dose followed with a maintenance dose. These compositions may be administered at specific fixed or variable intervals, e.g., once a day, or on an "as needed" basis.
- compositions used in this invention may be orally administered in an acceptable dosage form including, e.g., capsules, tablets, aqueous suspensions or solutions. Certain pharmaceutical compositions also may be administered by nasal aerosol or inhalation. Such compositions may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, and/or other conventional solubilizing or dispersing agents.
- the amount of an Sp35 antibody, or fragment, variant, or derivative thereof that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration.
- the composition may be administered as a single dose, multiple doses or over an established period of time in an infusion. Dosage regimens also may be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response).
- Sp35 antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention may be administered to a human or other animal in accordance with the aforementioned methods of treatment in an amount sufficient to produce a therapeutic effect.
- the Sp35 antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention can be administered to such human or other animal in a conventional dosage form prepared by combining the antibody of the invention with a conventional pharmaceutically acceptable carrier or diluent according to known techniques. It will be recognized by one of skill in the art that the form and character of the pharmaceutically acceptable carrier or diluent is dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well-known variables. Those skilled in the art will further appreciate that a cocktail comprising one or more species of Sp35 antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention may prove to be particularly effective.
- Effective doses of the compositions of the present invention, for treatment of spinal cord injury, diseases or disorders associated with inhibition of neuronal growth in the CNS, diseases or disorders associated with inhibition of oligodendrocyte growth or differentiation, and diseases involving demyelination or dysmyelination of CNS vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Usually, the patient is a human but non-human mammals including transgenic mammals can also be treated. Treatment dosages may be titrated using routine methods known to those of skill in the art to optimize safety and efficacy.
- the dosage can range, e.g., from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg (e.g., 0.02 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, lmg/kg, 2 mg/kg, etc.), of the host body weight.
- dosages can be 1 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg, preferably at least 1 mg/kg. Doses intermediate in the above ranges are also intended to be within the scope of the invention.
- Subjects can be administered such doses daily, on alternative days, weekly or according to any other schedule determined by empirical analysis.
- An exemplary treatment entails administration in multiple dosages over a prolonged period, for example, of at least six months. Additional exemplary treatment regimes entail administration once per every two weeks or once a month or once every 3 to 6 months.
- Exemplary dosage schedules include 1-10 mg/kg or 15 mg/kg on consecutive days, 30 mg/kg on alternate days or 60 mg/kg weekly.
- two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dosage of each antibody administered falls within the ranges indicated.
- Sp35 antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention can be administered on multiple occasions. Intervals between single dosages can be daily, weekly, monthly or yearly. Intervals can also be irregular as indicated by measuring blood levels of target polypeptide or target molecule in the patient. In some methods, dosage is adjusted to achieve a plasma polypeptide concentration of 1-1000 ⁇ g/ml and in some methods 25-300 ⁇ g/ml. Alternatively, Sp35 antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the antibody in the patient.
- the half-life of an Sp35 antibody can also be prolonged via fusion to a stable polypeptide or moeity, e.g., albumin or PEG.
- a stable polypeptide or moeity e.g., albumin or PEG.
- humanized antibodies show the longest half-life, followed by chimeric antibodies and nonhuman antibodies.
- the Sp35 antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention can be administered in unconjugated form.
- the Sp35 antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention can be administered multiple times in conjugated form.
- Sp35 antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention can be administered in unconjugated form, then in conjugated form, or vice versa.
- compositions of the present invention may be administered by any suitable method, e.g., parenterally, intraventricularly, orally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.
- parenteral as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.
- Sp35 antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention act in the nervous system to promote survival, proliferation and differentiation of oligodendrocytes and myelination of neurons and neuronal survival, axon regeneration and axon guidance. Accordingly, in the methods of the invention, the Sp35 antibodies, or antigen-binding fragments, variants, or derivatives thereof are administered in such a way that they cross the blood-brain barrier. This crossing can result from the physico-chemical properties inherent in the Sp35 antibody molecule itself, from other components in a pharmaceutical formulation, or from the use of a mechanical device such as a needle, cannula or surgical instruments to breach the blood-brain barrier.
- the Sp35 antibody is a molecule that does not inherently cross the blood-brain barrier, e.g., a fusion to a moiety that facilitates the crossing
- suitable routes of administration are, e.g., intrathecal or intracranial, e.g., directly into a chronic lesion of MS.
- the route of administration may be by one or more of the various routes described below.
- antibodies are administered as a sustained release composition or device, such as a MedipadTM device. Delivery across the blood brain barrier can be enhanced by a carrying molecule, such as anti-Fc receptor, transferrin, anti-insulin receptor or a toxin conjugate or penetration enhancer.
- the Sp35 antibodies, or antigen-binding fragments, variants, or derivatives thereof used in the methods of the invention may be directly infused into the brain.
- Various implants for direct brain infusion of compounds are known and are effective in the delivery of therapeutic compounds to human patients suffering from neurological disorders. These include chronic infusion into the brain using a pump, stereotactically implanted, temporary interstitial catheters, permanent intracranial catheter implants, and surgically implanted biodegradable implants. See, e.g., Gill et al, "Direct brain infusion of glial cell line-derived neurotrophic factor in Parkinson disease," Nature Med.
- compositions may also comprise an Sp35 antibody dispersed in a biocompatible carrier material that functions as a suitable delivery or support system for the compounds.
- sustained release carriers include semipermeable polymer matrices in the form of shaped articles such as suppositories or capsules.
- Implantable or microcapsular sustained release matrices include polylactides (U.S. Patent No. 3,773,319; EP 58,481), copolymers of L-glutamic acid and gamma-ethyl- L-glutamate (Sidman et al, Biopolymers 22:547-56 (1985)); poly(2-hydroxyethyl-methacrylate), ethylene vinyl acetate (Langer et al, J. Biomed. Mater. Res. 15:167-277 (1981); Langer, Chem. Tech. 12:98-105 (1982)) or poly-D-(-)-3hydroxybutyric acid (EP 133,988).
- an Sp35 antibody, or antigen-binding fragment, variant, or derivative thereof of the invention is administered to a patient by direct infusion into an appropriate region of the brain. See, e.g., Gill et al, supra.
- Alternative techniques are available and may be applied to administer an Sp35 antibody according to the invention. For example, stereotactic placement of a catheter or implant can be accomplished using the Riechert-Mundinger unit and the ZD (Zamorano-Dujovny) multipurpose localizing unit.
- a contrast-enhanced computerized tomography (CT) scan injecting 120 ml of omnipaque, 350 mg iodine/ml, with 2 mm slice thickness can allow three-dimensional multiplanar treatment planning (STP, Fischer, Freiburg, Germany). This equipment permits planning on the basis of magnetic resonance imaging studies, merging the CT and MRI target information for clear target confirmation.
- CT computerized tomography
- the Leksell stereotactic system (Downs Surgical, Inc., Decatur, GA) modified for use with a GE CT scanner (General Electric Company, Milwaukee, WT) as well as the Brown-Roberts-Wells (BRW) stereotactic system (Radionics, Burlington, MA) can be used for this purpose.
- GE CT scanner General Electric Company, Milwaukee, WT
- BRW stereotactic system Radionics, Burlington, MA
- Serial CT sections can be obtained at 3 mm intervals though the (target tissue) region with a graphite rod localizer frame clamped to the base plate.
- a computerized treatment planning program can be run on a VAX 11/780 computer (Digital Equipment Corporation, Maynard, Mass.) using CT coordinates of the graphite rod images to map between CT space and BRW space.
- Sp35 antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention can optionally be administered in combination with other agents that are effective in treating the disorder or condition in need of treatment (e.g., prophylactic or therapeutic).
- the invention further provides a diagnostic method useful during diagnosis of neronal disorders or injuries, which involves measuring the expression level of Sp35 protein or transcript in tissue or other cells or body fluid from an individual and comparing the measured expression level with a standard Sp35 expression levels in normal tissue or body fluid, whereby an increase in the expression level compared to the standard is indicative of a disorder.
- Sp35-specific antibodies can be used to assay protein levels in a biological sample using classical immunohistological methods known to those of skill in the art (e.g., see Jalkanen, et ah, J. Cell. Biol. 101:976-985 (1985); Jalkanen, et al, J. Cell Biol. 705:3087-3096 (1987)).
- Other antibody- based methods useful for detecting protein expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA), immunoprecipitation, or western blotting. Suitable assays are described in more detail elsewhere herein.
- an assaying the expression level of Sp35 polypeptide is intended qualitatively or quantitatively measuring or estimating the level of Sp35 polypeptide in a first biological sample either directly (e.g., by determining or estimating absolute protein level) or relatively (e.g., by comparing to the cancer associated polypeptide level in a second biological sample).
- Sp35 polypeptide expression level in the first biological sample is measured or estimated and compared to a standard Sp35 polypeptide level, the standard being taken from a second biological sample obtained from an individual not having the disorder or being determined by averaging levels from a population of individuals not having the disorder.
- the standard Sp35 polypeptide level is known, it can be used repeatedly as a standard for comparison.
- biological sample any biological sample obtained from an individual, cell line, tissue culture, or other source of cells potentially expressing Sp35. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art.
- Sp35 antibodies for use in the diagnostic methods described above include any Sp35 antibody which specifically binds to an Sp35 gene product, as described elsewhere herein. XI. IMMUNOASSAYS
- Sp35 antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention may be assayed for immunospecific binding by any method known in the art.
- the immunoassays which can be used include but are not limited to competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few.
- Immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to the cell lysate, incubating for a period of time (e.g., 1-4 hours) at 4.degree.
- a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol
- protein phosphatase and/or protease inhibitors e.g.,
- Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washing the membrane in washing buffer (e.g., PBS-Tween 20), blocking the membrane with primary antibody (the antibody of interest) diluted in blocking buffer, washing the membrane in washing buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g., an anti-human antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., 32p or 1251) diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence of the antigen
- ELISAs comprise preparing antigen, coating the well of a 96 well microliter plate with the antigen, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence of the antigen.
- a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase)
- a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase)
- a second antibody conjugated to a detectable compound may be added following the addition of the antigen of interest to the coated well.
- ELISAs see, e.g., Ausubel et al., eds, Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, Vol. 1 (1994) at 11.2.1.
- the binding affinity of an antibody to an antigen and the off-rate of an antibody-antigen interaction can be determined by competitive binding assays.
- a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen ⁇ e.g., 3 H or 125 I) with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen.
- the affinity of the antibody of interest for a particular antigen and the binding off-rates can be determined from the data by scatchard plot analysis. Competition with a second antibody can also be determined using radioimmunoassays. In this case, the antigen is incubated with antibody of interest is conjugated to a labeled compound ⁇ e.g., 3 H or 125 I) in the presence of increasing amounts of an unlabeled second antibody.
- Sp35 antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention additionally, be employed histologically, as in immunofluorescence, immunoelectron microscopy or non-immunological assays, for in situ detection of cancer antigen gene products or conserved variants or peptide fragments thereof.
- In situ detection may be accomplished by removing a histological specimen from a patient, and applying thereto a labeled Sp35 antibody, or antigen-binding fragment, variant, or derivative thereof, preferably applied by overlaying the labeled antibody (or fragment) onto a biological sample.
- Immunoassays and non-immunoassays for Sp35 gene products or conserved variants or peptide fragments thereof will typically comprise incubating a sample, such as a biological fluid, a tissue extract, freshly harvested cells, or lysates of cells which have been incubated in cell culture, in the presence of a detectably labeled antibody capable of binding to Sp35 or conserved variants or peptide fragments thereof, and detecting the bound antibody by any of a number of techniques well- known in the art.
- the biological sample may be brought in contact with and immobilized onto a solid phase support or carrier such as nitrocellulose, or other solid support which is capable of immobilizing cells, cell particles or soluble proteins.
- a solid phase support or carrier such as nitrocellulose, or other solid support which is capable of immobilizing cells, cell particles or soluble proteins.
- the support may then be washed with suitable buffers followed by treatment with the detectably labeled Sp35 antibody, or antigen-binding fragment, variant, or derivative thereof.
- the solid phase support may then be washed with the buffer a second time to remove unbound antibody.
- the antibody is subsequently labeled.
- the amount of bound label on solid support may then be detected by conventional means.
- solid phase support or carrier any support capable of binding an antigen or an antibody.
- supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite.
- the nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention.
- the support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody.
- the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod.
- the surface may be flat such as a sheet, test strip, etc.
- Preferred supports include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.
- binding activity of a given lot of Sp35 antibody, or antigen-binding fragment, variant, or derivative thereof may be determined according to well known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.
- SPR Surface plasmon reasonance
- BIAcore offers a number of advantages over conventional methods of measuring the affinity of antibody-antigen interactions: (i) no requirement to label either antibody or antigen; (ii) antibodies do not need to be purified in advance, cell culture supernatant can be used directly; (iii) real-time measurements, allowing rapid semiquantitative comparison of different monoclonal antibody interactions, are enabled and are sufficient for many evaluation purposes; (iv) biospecif ⁇ c surface can be regenerated so that a series of different monoclonal antibodies can easily be compared under identical conditions; (v) analytical procedures are fully automated, and extensive series of measurements can be performed without user intervention.
- BIAapplications Handbook version AB (reprinted 1998), BIACORE code No. BR-1001-86; BIAtechnology Handbook, version AB (reprinted 1998), BIACORE code No. BR-1001-84.
- SPR based binding studies require that one member of a binding pair be immobilized on a sensor surface.
- the binding partner immobilized is referred to as the ligand.
- the binding partner in solution is referred to as the analyte.
- the ligand is attached indirectly to the surface through binding to another immobilized molecule, which is referred as the capturing molecule.
- SPR response reflects a change in mass concentration at the detector surface as analytes bind or dissociate.
- the resulting sensorgram can be divided into three essential phases: (i) Association of analyte with ligand during sample injection; (ii) Equilibrium or steady state during sample injection, where the rate of analyte binding is balanced by dissociation from the complex; (iii) Dissociation of analyte from the surface during buffer flow.
- the equilibrium phase provides information on the affinity of the analyte-ligand interaction (K D )-
- BIAevaluation software provides comprehensive facilities for curve fitting using both numerical integration and global fitting algorithms. With suitable analysis of the data, separate rate and affinity constants for interaction can be obtained from simple BIAcore investigations. The range of affinities measurable by this technique is very broad ranging from mM to pM.
- Epitope specificity is an important characteristic of a monoclonal antibody.
- Epitope mapping with BIAcore in contrast to conventional techniques using radioimmunoassay, ELISA or other surface adsorption methods, does not require labeling or purified antibodies, and allows multi-site specificity tests using a sequence of several monoclonal antibodies. Additionally, large numbers of analyses can be processed automatically.
- Pair-wise binding experiments test the ability of two MAbs to bind simultaneously to the same antigen.
- MAbs directed against separate epitopes will bind independently, whereas MAbs directed against identical or closely related epitopes will interfere with each other's binding.
- a capture molecule to bind the first Mab, followed by addition of antigen and second MAb sequentially.
- the sensorgrams will reveal: 1. how much of the antigen binds to first Mab, 2. to what extent the second MAb binds to the surface-attached antigen, 3. if the second MAb does not bind, whether reversing the order of the pair-wise test alters the results.
- Peptide inhibition is another technique used for epitope mapping. This method can complement pair-wise antibody binding studies, and can relate functional epitopes to structural features when the primary sequence of the antigen is known. Peptides or antigen fragments are tested for inhibition of binding of different MAbs to immobilized antigen. Peptides which interfere with binding of a given MAb are assumed to be structurally related to the epitope defined by that MAb.
- Sp35 is involved in oligodendrocyte biology
- Oligodendrocytes mature through several developmental stages from A2B5 progenitor cells (which express A2B5), differentiating into pre-myelinating oligodendrocytes (which express Ol and 04) and finally into mature myelinating oligodendrocytes (which express 01, 04 and MBP).
- A2B5 progenitor cells which express A2B5
- pre-myelinating oligodendrocytes which express Ol and 04
- mature myelinating oligodendrocytes which express 01, 04 and MBP.
- Monoclonal antibodies against 04, MBP and CNPase were from Sternberger Monoclonals; antibody to APC (clone CC-I; ref. 29) was from Calbiochem. Other antibodies were to ⁇ m tubulin (Covance), Sp35 (Biogen pout), Fyn (Santa Cruz Biotechnology) and phospho-Fyn (Biosource). Monoclonal antibodies against A2B5 are available from Chemicon.
- Sp35 is expressed in oligodendrocytes
- RT-PCR polymerase chain reaction after reverse transcription
- Sp35 is a component of the Nogo-66 receptor/p75 signaling complex
- Nat. Neurosci. 7: 221-28 (2004) and probed with digoxigenin-labeled Sp35 antisense or sense RNAs using the first 500 nucleotides of the Sp35 coding sequence.
- the sections were stained according to the manufacturers' instructions using a Tyramide Signal Amplification kit (Amersham Biosciences) and a fluorescent anti-digoxigenin conjugated antibody kit (Perkin Elmer).
- CCl antibody Calbiochem; a marker of mature oligodendrocytes
- anti-Sp35 antibody a marker of mature oligodendrocytes
- CCl antibody Calbiochem; a marker of mature oligodendrocytes
- anti-Sp35 antibody a marker of mature oligodendrocytes
- oligodendrocytes that hybridized to an antisense Sp35 probe also co- stained with an antibody to CCl (data not shown). No specific labeling was observed using a sense Sp35 probe.
- Sp35 expression in oligodendrocytes also was confirmed by immunohistochemistry studies of tissue sections from the lateral ventricle region of P7 rat cortex. A majority of cortical cells that labeled with CCl antibody also labeled with anti-Sp35 antibody. Data not shown. The specificity of the interaction was confirmed by preadsorption of the anti-Sp35 antibody with Sp35-Fc ⁇ see Example 2), which eliminated the signal.
- Sp35-specific RNAi knockdown of Sp35 expression promotes oligodendrocyte growth and differentiation
- Sp35-specific RNAi was used to ablate Sp35 expression in oligodendrocyte precursor cells to examine how Sp35 contributes to oligodendrocyte growth and differentiation.
- 50,000 A2B5 oligodendrocyte precursor cells were infected with lentivirus carrying Sp35-specific RNAi sequence or control RNAi prepared as follows.
- Murine and rat Sp35 DNA sequences were compared to find homologous regions to use for candidate small-hairpin RNAs (shRNA).
- CH324 for lentivirus expression of Sp35 RNAi, was constructed by annealing oligonucleotides LV1-035 and LV1-036 and ligating to Hpal and Xhol digested pLL3.7.
- the pLL3.7 vector, additional methodology and virus production were as described in Rubinson et ah, Nat. Genet. 33, 401-06 (2003).
- the Sp35 RNAi oligonucleotides were purchased from MWG and have the following sequences: LV1-035 (sense oligo) 5' - TGA TCG TCA TCC TGC TAG ACT TCA AGA GAG TCT AGC AGG ATG ACG ATC TTT TTT C - 3 ' (SEQ ID NO:346) and LV1-036 (antisense oligo) 5' - TCG AGA AAA AAG ATC GTC ATC CTG CTA GAC TCT CTT GAA GTC TAG CAG GAT GAC GAT CA - 3' (SEQ ED NO:347).
- Control RNAi was designed with the same oligonucleotide sequences except for the nucleotide changes indicated in lower-case letters: 5'-TGA TCc TCA TcC ttC Tat ACT TCA AGA GAG TgT AGC AGG ATG AcG ATC TTT TTT CTC GA-3' (SEQ ID NO:348) and 5'-TCG AGA AAA AAG ATC GTC ATC CTG CTA GAC TCT CTT GAA GTa TAG aAG GAT GAC GAT CA-3 '. (SEQ ID NO:349).
- DNA from pLL3.7 or candidate shRNA in pLL3.7 were cotransfected with murine Sp35-HA tagged plasmid at a ratio of 5 to 1 into CHO cells in a 6-well format. Knockdown was analyzed by western blot detection of Sp35-HA tag from transfected CHO cell lysates as well as by northern blot of total RNA prepared from duplicate wells. The blot was probed with a fragment of Sp35 cDNA. Assays were performed 48 hours post-transfection. As expected, there was a 10-fold reduction of Sp35 mRNA in CH324 RNAi-treated CHO cells relative to control-treated cells. Data not shown.
- RNAi lentiviruses carrying green fluorescent protein (GFP) were generated as described in Rubinson et al. In cultures treated with either control or Sp35 RNAi, approximately 80% of the oligodendrocytes were GFP positive. Total cell number was not altered by the RNAi treatments. To quantify the effects of RNAi on differentiation, only GFP-expressing oligodendrocytes were counted.
- GFP green fluorescent protein
- Enriched populations of oligodendrocytes were grown from female Long Evans P2 rats as described by Conn, Meth. Neurosci. 2:1-4 (Academic Press; 1990) with modifications as follows. Briefly, the forebrain was dissected and placed in Hank's buffered salt solution (HBSS; Invitrogen). The tissue was cut into 1-mm fragments and was incubated at 37 0 C for 15 min in 0.01% trypsin and 10 ⁇ g/ml DNase. Dissociated cells were plated on poly-L-lysine-coated T75 tissue culture flasks and were grown at 37°C for 10 d in DMEM medium with 20% fetal calf serum (Invitrogen).
- HBSS Hank's buffered salt solution
- Dissociated cells were plated on poly-L-lysine-coated T75 tissue culture flasks and were grown at 37°C for 10 d in DMEM medium with 20% fetal calf serum (Invitrog
- Oligodendrocyte precursors (A2B5 + ) were collected by shaking the flask overnight at 200 rpm at 37 0 C, resulting in a 95% pure population. Cultures were maintained in high-glucose Dulbecco's modified Eagle's medium (DMEM) with FGF/PDGF (10 ng/ml; Peprotech) for 1 week. Removal of FGF/PDGF allowed A2B5 + cells to differentiate into O4 + premyelinating oligodendrocytes after 3-7 d, and to differentiate into O4 + and MBP + mature oligodendrocytes after 7-10 d.
- DMEM high-glucose Dulbecco's modified Eagle's medium
- A2B5 + cells are bipolar in shape
- O4 + premyelinating oligodendrocytes have longer and more branched processes
- MBP + mature oligodendrocytes contain myelin sheet structures between processes.
- A2B5 oligodendrocyte precursor cells were infected with the lentivirus containing the CH324 RNAi. The resulting cells were cultured for 3 days and the number of 04-positive (a marker for oligodendrocyte differentiation) oligodendrocytes was counted. Endogenous S ⁇ 35 expression was reduced by infection with S ⁇ 35 RNAi lentivirus and was confirmed by RT-PCR. Reduction of Sp35 resulted in more highly differentiated, mature oligodendrocytes as compared with control infected cells, as was evident by increases in the length of cell processes and by the presence of abundant myelin sheet structures (data not shown). In cells that expressed Sp35 RNAi, there were three times as many mature (04-positive) oligodendrocytes as in control cultures. These data indicate that Sp35 may negatively regulate oligodendrocyte differentiation.
- Dominant-negative Sp35 promotes oligodendrocyte growth and differentiation
- Lentiviral vectors that express wild-type and a dominant-negative form of Sp35 were constructed.
- DNA sequence encoding mouse full length Sp35 (FL-Sp35, amino acid residues 34-614 of SEQ ID NO:2) was amplified by PCR using primers 5' - GAG GAT CTC GAC GCG GCC GCA TGG AGA CAG ACA CAC TCC TG - 3' (SEQ ID NO:350) and 5' - GGG GCG GAA TTG GAT CCT CAC AGA TCC TCT TCT GAG ATG AG-3' (SEQ ID NO:351) and inserted into the HRST- IRESeGFP lentiviral vector at the Not! and BamHI sites.
- DNA sequence encoding dominant negative Sp35 was amplified by PCT using primers 5' - GAG GAT CTC GAC GCG GCC GCA TGG AGA CAG ACA CAC TCC TG- 3' (SEQ ID NO:352) and 5' - GAT ACG GAT CCT CAG CCT TTG CCC CGG CTC CAT AGA AAC AGC - 3' (SEQ ID NO:353).
- the FL-Sp35 and DN-Sp35 plasmids were transfected into 293 cells to produce lentivirus as described by Rubinson et al., "A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference," Nat. Genet. 33: 401- 06 (2003). Oligodendrocytes were infected with lentivirus at 2 MOI per cell and confirmed expression of FL-Sp35 and DN-Sp35 by western blot.
- DN-Sp35 promoted oligodendrocyte differentiation, producing an increase in the number of mature oligodendrocytes.
- overexpression of full-length Sp35 had the opposite effect and inhibited differentiation, as was evident by a reduction in the number of mature oligodendrocytes as compared with the control (data not shown).
- a construct was made fusing the extra-cellular portion of human Sp35 (residues 1-532) to the hinge and Fc region of human IgGl to study the biological function of Sp35.
- a partial coding sequence for human Sp35 was obtained by PCR from clone 227.2 using the forward primer 5' - CAG CAG GTC GAC GCG GCC GCA TGC TGG CGG GGG GCG T - 3' (SEQ ID NO:354) and reverse primer 5' - CAG CAG GTC GAC CTC GCC CGG CTG GTT GGC CAA CCA GCC GGG CGA GGT CGA CCT CGA GG - 3' (SEQ ID NO:355).
- the blunt-end PCR product was subcloned into the Sr ⁇ site of the PCR SCRIPT AMP vector (Stratagene) to create PCR SCRIPT AMP-Sp35.
- a SaE fragment was isolated from PCR SCRIPT AMP-Sp35 and subcloned into the PCRCAMP Ig vector (derivative of Stratagene vector PCR SCRIPT AMP).
- the hinge and Fc gamma sequence is subcloned as a SaE(S 1 ) to NM(3') fragment.
- the SaE Sp35 fragment was subcloned into the SaE site of the PCRCAMP Ig vector thereby fusing the Sp35 signal sequence and extracellular domain (codons 1-532) in-frame with sequences encoding the hinge and Fc region of human IgI. Correct isolates were identified, and a Noil fragment encompassing the Sp35 Fc fragment was subcloned into the single Noil cloning site of the CHO expression vector, PV90 (Biogen pou). The resulting plasmid was confirmed by D ⁇ A sequencing aand designated GT 123.
- Stable cell lines expressing the Sp35-Fc fusion protein were generated by electroporation of CHO host cells DG44 with plasmid GT 123. Transfected CHO cells were cultured in alpha minus MEM in the presence of 10% dialyzed serum and 4 mM glutamine to select for nucleoside-independent growth. Fourteen days post-transfection, cells were fed fresh media. To screen for cells expressing Sp35-Fc, CHO cells were labeled with phycoerythrin (PE)-labeled goat anti-human IgG (Jackson Labs) and subjected to high speed flow cytometry sorting in a FACS Mo-Flo (Cytomation).
- PE phycoerythrin
- the cells that expressed the highest levels of Sp35-Fc were selected. These cells were expanded in culture for 7 days, then re-labeled and re-sorted. Cells expressing the highest levels of Sp35-Fc were isolated as individual clones in 96-well plates. These clones were grown for two weeks and then fed fresh media one day prior to FACS analysis to check for expression levels. Clones that expressed the highest levels of Sp35-Fc were expanded, and frozen cell banks were established. The cell lines were adapted to grow in suspension culture in the serum-free media BCMl 6.
- the titer of Sp35-Fc produced by these clones was determined by growing cell lines at 37°C for 4-5 passages, then growing the cells to 50% maximal cell density and culturing them for 10-15 days at 28°C until the viable cell density dropped to 75%. At this time, the culture media were harvested, cleared of cells and debris by centrifugation, and the culture supernatants titered for Sp35-Fc levels by Western blot analysis using an anti-human Ig antibody (Jackson Lab) as the probe.
- Sp35-Fc fusion protein was purified from the clarified culture medium as follows: 9 ml of IM HEPES pH 7.5 was added to 900 ml of conditioned medium.
- the medium was batch loaded for 3 hr at 4 0 C onto 3 ml of Protein A Sepharose (Amersham Bioscience).
- the resin was collected in a 1.5 cm (LD.) column, and washed four times with 3 ml PBS, two times with 4 ml of PBS containing 800 mM NaCl, and then again with 3 ml of PBS.
- the Sp35-Fc was eluted from the column with 25 mM NaH2PO4 5 pH 2.8 and 100 mM NaCl in 1.5 ml fractions and neutralized by adding 75 ⁇ l of 0.5 M NaH2PO4 j pH 8.6.
- Peak protein-containing fractions were identified by absorbance at 280 nm, pooled, and subjected to further purification on a 1 mL Protein A column.
- NaCl was added to 600 mM and HEPES, pH 7.5 to 50 mM.
- the column was washed twice with 600 ⁇ l of 10 mM HEPES pH 7.5 and 1 M NaCl , and then with 1 ml PBS.
- Sp35-Fc was eluted from the column with 25 mM NaH2PO4 ; pH 2.8 and 100 mM NaCl, collecting 0.5 mL fractions, and neutralized by adding 25 ⁇ l of 0.5 M NaH2P ⁇ 4 s pH 8.6.
- Peak protein-containing fractions were identified by absorbance at 280 nm and pooled.
- the Sp35-Fc protein migrated as a single band (>95% pure) with an apparent mass of 90 kDa.
- the protein ran as a dimer with an approximate mass of 180 kDa.
- the purified Sp35-Fc protein was aliquoted and stored at -7O 0 C.
- Anti-Sp35 Antibodies that specifically bind an Sp35 polypeptide of the invention were made using the following methods and procedures.
- Sp35-Fc (0.5 ⁇ g in 50 ⁇ l of 0.1 M sodium bicarbonate buffer, pH 9.0) was added to each well of 96-well MaxiSorpTM plates (NuncTM). The plates were then incubated at 37°C for 1 hour or 4°C for 16 hours. Non-specific binding sites on the plates were blocked using 25 mM HEPES, pH 7.4 containing 0.1% BSA, 0.1% ovalbumin, 0.1% (5% (w/v) nonfat dry milk in 15OmM NACE) and 0.001% azide. Dilutions of serum or hybridoma supernatants (for example, serial three-fold dilutions) were added across each row of the plate, and incubated at 25°C for 1 hour.
- COS-7 cells were labeled with 0.1 ⁇ M CellTrackerTM Green CMFDA (Molecular Probes, Eugene, OR) as described by the vendor. Equal volumes of CellTrackerTM labeled control cells were mixed with washed Sp35-COS-7 cells (produced by transient transfection of Sp35 expression vector) before incubation with anti-Sp35 test sera or hybridoma supernatants. Fifty microliters of the cell mixture was dispensed into each well of a 96-well V-bottom polystyrene plates (Costar® 3877, Corning, NY) and 100 ⁇ l of mouse serum, hybridoma supernatant, or a control anti-Sp35 antibody was added.
- CellTrackerTM Green CMFDA Molecular Probes, Eugene, OR
- the cells were washed and incubated with 50 ⁇ l of 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% fetal bovine serum (FBS), and subjected to FACS analyses.
- FBS fetal bovine serum
- Sp35-COS-7 cells were mixed with mouse serum or hybridoma supernatant and then treated with R-phycoerythrin-conjugated goat anti- mouse secondary antibody and directly subjected to standard FACS analyses.
- Sera from the immunized mice were collected before the first immunization and 1 week after the second and third immunizations, and anti-Sp35 antibody titers were measured by FACS assay on Sp35 -expressing COS- 7 cells as described above. A booster final dose was given after the third immunization and three days prior to when hybridoma fusions 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 in Monoclonal Antibodies.
- 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
- Hvbridomas A New Dimension in Biological Analyses, ed. Kennett, R.H., McKearn, T.J. and Bechtol, K.B. New York: Plenum Press (1982). Fused cells were plated into 24- or 48-well plates (Corning Glass Works, Corning, NY), and fed with adenine, aminopterin and thymidine (AAT, available from Sigma® Chemical Co., St. Louis, MO) containing culture medium. AAT resistant cultures were screened by ELISA or flow cytometry as described above for binding to either Sp35-COS-7 cells or to Sp35-Fc. Positive hybridomas were further subcloned by limiting dilution.
- V H and V L Polynucleotides encoding the variable domains (V H and V L ) of monoclonal antibodies 1A7, 2F3, 3P1D10.2C3 and 3P1E11.3B7 were isolated by PCR, cloned and were subjected to sequence analysis by the following method.
- Total RNA was extracted from hybridoma cells using Qiagen® RNeasy® mini kitand cDNA was generated from the isolated RNA by RT PCR, using standard conditions.
- a cocktail of primers were used for the RT-PCR.
- a prefered set of primers included a primer with the 5' of the primer hybridizing to the signal sequence and the 3' end of the primer hybridizing to the constant domain 3' of the FR4/constant domain junction.
- sequence data for designing more effective primers, can be obtained from the bulk RT-PCR products which have been gel purified and then sequenced.
- the PCR product can also be subcloned using, for example, the TOPO Cloning Kit (Invitrogen) then sequence. Sequence data is then obtained from multiple independent subclones or gel purified fragments to firmly establish the consensus sequence.
- the sequence of the light chain of the PlEl 1.3B7 was determined by using a cocktail of 5' murine kappa light chain signal sequence primers: (i) 5' GGG GAT ATC CAC CAT GGA TTT TCA GGT GCA GAT TTT CAG 3' (SEQ ID NO:356), (ii) 5' GGG GAT ATC CAC CAT GRA GTC ACA KAC YCA GGT CTT YRT A 3 1 (SEQ E) NO:357), (iii) 5' GGG GAT ATC CAC CAT GAA GTT GCC TGT TAG GCT GTT G 3' (SEQ E) NO:358), and (iv) 5' GGG GAT ATC CAC CAT GAG GKC CCC WGC TCA GYT YCT KGG A 3' (SEQ E) NO:359), with a single 3' murine kappa constant domain primer: 5' GCG TCT AGA ACT GGA TGG TGG GAG
- the resulting PCR product was subcloned and multiple - I l l - independent subclones were sequenced.
- the deduced consensus sequence was consistent with the Edman degradation sequencing data. Sequencing indicated that the degenerate signal sequence 5' primer 5' GGG GAT ATC CAC CAT GRA GTC ACA KAC YCA GGT CTT YRT A 3' (SEQ ID NO:357) was the one that had yielded the 3P1E11.3B7 light chain variable domain during the amplification.
- the design of the degenerate signal sequence primer was based upon signal sequences of the best hits derived from a TFASTA search of the Genbank rodent sequence database queried with the 3P1E11.3B7 consensus deduced FRl sequence from the PCR reaction with the "universal primer” described above. This PCR yielded a product with a complete murine heavy chain variable domain.
- the complete 3P1E11.3B7 murine variable domains were used (with silent mutagenesis as necessary to introduce restriction sites) in conjunction with human IgGl and kappa constant domain cDNAs to construct chimeric heavy and light chain cDNAs, respectively.
- the full-length immunoglobulin cDNAs were subcloned into an expression vector called pNEOOl, a derivative of the commercial EBV mammalian cell episomal expression vector pCEP4.
- the heavy and light chain expression vectors (called pXW372 and pXW363, respectively) were co-transfected into 293 -EBNA cells.
- Anti-Sp35 monoclonal antibody Fab fragments were identified and isolated from phage display libraries as described in Hoet et ah, Nat. Biotech. 23:344-348 (2005); Rauchenberger, et al., J. Biol. Chem. 278:194-205 (2003); and Knappik, et al., J. MoI. Biol. 296:57-86 (2000), all of which are incorporated herein by reference in their entireties.
- the MorphoSys Fab-phage display library HuCAL® GOLD (“Phage Display Library-2” in Table 3B), which comprises humanized synthetic antibody variable regions was screened against recombinant human soluble Sp35-Fc protein by standard ELISA AND IHC screening methods. See, e.g., Ostendorp, R., Frisch, C. and Urban M, "Generation, engineering and production of human antibodies using HuCAL®.” Antibodies, Volume 2 Novel Technologies and Therapeutic Use. New York: Kluwer Academic/Plenum 13-52 (2004).
- Fab-phages that specifically bound to Sp35 were purified and characterized. Properties of these phage display-derived monoclonal antibody Fab fragments are shown in Table 3B as "phage display library-2-derived monoclonal Fab fragments.” Isolated Fab-phage 1968 was selected for further analysis.
- COS-I cells expressing Sp35, fused to a hemaglutinin (HA) tag on the N-terminus were produced by transiently transfecting COS-I cells with a DNA construct which expresses the full-length Sp35 protein with an HA tag.
- Cells were harvested 48 hr after transfection and were lysed in 1 ml lysis buffer (5OmM HEPES, pH 7.5, 15OmM NaCl, 1.5mM MgCl2, ImM EGTA, 1% Triton X-IOO and 10% glycerol) for 30 min at 4 0 C. After centrifugation at 1 ml lysis buffer (5OmM HEPES, pH 7.5, 15OmM NaCl, 1.5mM MgCl2, ImM EGTA, 1% Triton X-IOO and 10% glycerol) for 30 min at 4 0 C. After centrifugation at 1 ml lysis buffer (5OmM HEPES, pH 7.5, 15
- the ELISA was performed as follows: The Mabs were coated onto ELISA plates, then incubated either with an Sp35-AP fusion protein at 4 0 C overnight followed by AP-linked anti-human (H+L) (1:5,000, Jackson ImmunoResearch) at RT for 1 hr, or with AP-fusion proteins at 4 0 C overnight. AP substrate was then developed by 10 mg/ml 4NPP in 0.1 M Glycin, 1 mM MgCl 2 , 1 mM ZnCl 2 , pH 10.5, and read at O.D. 405. EXAMPLE 6
- the cells were washed 3 times with 2% FBS/PBS, then incubated with PE labeled secondary antibody (1:100, JacksonlmmunoResearch) on ice for 30 min. After 2 washes with 2% FBS/PBS, cells were fixed in 2% PFA and subjected to FACS analysis by PE.) FACS result showed that MAbs 1A7 and 2F3 bound to COS-7 or 293 cells expressing Sp35, but not bind to control cells with no Sp35 expression (Fig 2).
- DRG's Dorsal root ganglions (DRG's) from P6-7 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 10,000 cells/well on the pre-coated Lab-Tek® culture slides. Ten ⁇ g/ml of mAb 1A7 or 2F3 were added immediately after plating of the DRGs.
- the culture medium was F12 (available from Gibco/Invitrogen) containing 5% heat inactivated donor horse serum, 5% heat inactivated fetal bovine serum and 50 ng/ml mouse nerve growth factor (mNGF) and incubated at 37°C and 5% CO2 for 6 hours. Following incubation, the slides were fixed in 4% paraformaldehyde/20% sucrose and stained with anti-BIII-tubulin TUJl antibody (Covance) after 16 hours.
- F12 available from Gibco/Invitrogen
- mNGF mouse nerve growth factor
- Monoclonal antibody 1 A7 promotes functional recovery in the rat Spinal Cord Injury model
- SCI Spinal cord injury
- CST corticospinal tract
- Hindlimb function was quantified using the Basso-Beattie-Bresnahan (BBB) open field scoring method (Eby, M.T. et al, J. Biol. Chem. 275, 15336-15342 (2000), incorporated herein by reference) and all animals sustained marked functional deficits after SCI, with almost complete hindlimb paralysis the day after surgery.
- BBB Basso-Beattie-Bresnahan
- an intrathecal catheter was inserted into the subarachnoid space at T7 and connected to a primed mini-osmotic pump (Alzet model 2004, Alza Corp) inserted into the subcutaneous space.
- Mini-osmotic pumps delivered Human IgG isotype control protein (5mg/ml) or monoclonal antibody 1A7 (4.8mg/ml) continuously at a rate of 0.25 ⁇ l/h over 5 weeks.
- Control (Human IgG-treated) animals recovered substantial function over the 5 week duration of the experiment, but plateaued at 3-4 weeks, ultimately attaining a mean BBB score of 9 ⁇ 0.45 (Fig. 7).
- continuous intrathecal infusion of 1A7 for 5 weeks after spinal cord transection resulted in significantly improved BBB scores over the control animals by 5 weeks with a continued improvement in function in the 2-5 week timeframe, reaching a mean BBB score of 11.1 ⁇ 0.7 (Fig. 4).
- A2B5-positive oligodendrocytes were collected by shaking the flasks overnight at 200 rpm at 37 0 C.
- the A2B5 oligodendrocytes were cultured for 7 days in DMEM (Gibco) containing 25 mM D-glucose, 4 mM L-glutamine, 1 mM sodium pyruvate, 50 ⁇ g/ml human apo-transferrin, 5 ⁇ g/ml bovine pancreatic insulin, 30 nM sodium selenate, 10 nM hydrocortisone, 10 nM D-biotin, 1 mg/ml BSA, 10ng/ml FGF and PDGF (Peprotech).
- the cells were then harvested by trypsinization.
- the cells then co-cultured with the DRG neurons in the presence or absence of 1, 3, 10, or 30 ⁇ g/ml of anti Sp35 monoclonal antibodies 1A7 or 2F3, or a negative control antibody in NLA medium containing 2% fetal bovine serum, 50 ⁇ g/ml ascorbic acid, 100 ng/ml NGF (Gibco).
- An effective antibody dose to administer in such an assay has been determined to be in the range of 0.1 ⁇ g/ml to 10 ⁇ g/ml, depending upon the antibody.
- One of skill in the art would be able to determine an effective dose using assays described herein.
- the culture medium was changed and the various monoclonal antibodies were replenished every three days. After 30 days at 37°C, the co-cultured cells were stained by immunohistochemical staining ("IHC") for neurofilaments with anti-BIII-tubulin antibody to identify axons, or anti-MBP antibody to identify oligodendrocytes (Fig. 4A-E). Co-cultured cells were also lysed and subjected to Western blot analysis to quantify the MBP (Fig. 4G).
- IHC immunohistochemical staining
- co- cultured cells treated with anti-Sp35 antibodies 1A7 and 2F3 showed increased survival of oligodendrocyte and neurons, increased numbers of bundled axons and increased numbers of MBP positive cells (Fig. 4F, 10-fold more MBP-positive cells when compared to control-antibody treated co- cultures.
- oligodendrocyte and DRG co-cultures were incubated in the presence or absence of anti-Sp35 antibodies Li05 and LiO6, or a negative control antibody.
- Co-cultured cells were lysed and subjected to Western blot analysis to quantify the MBP (Fig. 8). Based on Western blot analyses, co-cultured cells treated with anti-Sp35 antibodies LiO5 and LiO6 showed increased numbers of MBP positive cells, similar to co-cultured cells treated with 3, 10 and 30 ⁇ g of Sp35-Fc (LINGO-I-Fc).
- oligodendrocyte and DRG co-cultures were incubated in the presence or absence of anti-Sp35 antibodies 3P1D10.2C3, 3P1E11.3B7, 6P4F4.1D3, 6P4F4.1F9, 7P1D5.1G9, Li08, Lil3, Li28, and Li33 and also promoted myelination.
- full-length antibodies D05 and D08 also promoted myelination.
- Anti-Sp35 antibody 1A7 promotes oligodendrocyte survival and myelination in vivo
- the 1A7 and control monoclonal antibodies were delivered as follows.
- the cuprizone- treated mice were anesthetized with ketamine (80 mg/kg body weight) and xylazine (10 mg/kg body weight) and positioned in an immobilization apparatus designed for stereotactic surgery (David Kopf Instruments).
- the scalp was opened and the sterile compounds injected (1 ⁇ M in 1 ml of HBSS) unilaterally into the acutely demyelinated corpus callosum of the wild-type recipient mice with a 10 ⁇ l Hamilton syringe using stereotactic coordinates of 0.7 mm posterior and 0.3 mm lateral to bregma at a depth of 1.7 mm (Messier et al, Pharmacol. Biochem. Behav. 63: 313-18 (1999)). Additional control recipient mice were stereotactically injected with HBSS containing no compounds. The opening in the skull was filled with Gelfoam, and the area was swabbed with penicillin and streptomycin (Gibco) and the wound was sutured.
- mice were sacrificed every week of the experiment after injection and their brains removed and processed for molecular, biochemical and histological analysis.
- the animals receiving anti-Sp35 antibody 1A7 treatment showed increased mature oligodendrocyte survival (based on CCl antibody staining, Fig. 5A) and axon myelination by IHC using anti-MBP protein antibody or luxol fast blue (Fig. 5B).
- Fig. 5B CCl antibody-positive oligodendrocytes were quantitated at four weeks and 6 weeks (Fig. 5C).
- anti-Sp35 antibody 1A7 treatment promoted mature oligodendrocyte survival and axon myelination compared to control- antibody treated mice.
- animals receiving the 1A7 antibody in a lysolecithin model of demyelination also promoted axon myelination compared to control animals (data not shown).
- Anti-Sp35 antibody 1A7 promotes retinal ganglion cell (RGC) survival in the optic nerve transection model
- the right optic nerve receives complete crush by #5 forceps for 10 seconds around 1.5 mm behind the eyeball intraorbitally just before administration of 2 ⁇ l of monoclonal antibody 1A7, 2F3, Li05 and LiO6 in 2ml by intravitreal injection.
- the animals receive a second intravitreal injection of the same treatment one week after the surgery. Two weeks after the surgery, the animals are perfused with EM fixatives, postfixed and processed for semithin and ultrathin sections. The longitudinal optic nerve sections are stained and prepared for myelin observation. The myelination of the proximal and the distal parts of the crushed optic nerve are compared among different treatment groups. Sp35-Fc and 1A7, 2F3, Li05 and LiO6 treated animals, as well as appropriate controls, will be analyzed for remyelination in the distal part of the optic nerve compared to the controls.
- the right optic nerve was crushed by #5 forceps for 10 seconds around 1.5-2 mm behind the eyeball intraorbitally just before administration of 2 ⁇ g of monoclonal antibody 1A7 in PBS via intravitreal injection. 4 rats were tested with the 1A7 antibody and 8 rats were used as control animals. The animals received a second intravitreal injection of the same treatment one week after the surgery. Three days prior to sacrifice of the test animals (day 11 of the experiment), 2 ml of CTB-FITC was injected intravitreally to label, anterograde, the regenerative optic nerve axons. On the 14th day post geary, the animals were perfused and postfixed. The crushed optic nerve was processed for frozen longitudinal sections.
- Anti-Sp35 antibodies promote remyelination and repair in the optic nerve using the MOG induced EAE rat model.
- the Myelin Oligodendrocyte Glycoprotein (MOG) induced Experimental Autoimmune Encephalomyelitis (EAE) rat model was used. This is the animal model for human multiple sclerosis. 50 ⁇ l of 200 ng complete Freund's adjuvant (Chondrex Inc.) plus 50 ⁇ l of 50 ⁇ g MOG in saline was emulsified (1:1) and kept on ice before being injected intradermally at the base of the tail for each animal. Female brown Norway rats, 8-10 weeks old, were used for all experiments. General observation in the art indicates that the EAE model is induced around 15 days after MOG injection. Rats are scored for clinical signs of EAE.
- MOG Myelin Oligodendrocyte Glycoprotein
- the signs are scored as follows: grade 0.5, distal paresis of the tail; grade 1, complete tail paralysis; grade 1.5, paresis of the tail and mild hind leg paresis; grade 2.0, unilateral severe hind leg paresis; grade 2.5, bilateral severe hind limb paresis; grade 3.0, complete bilateral hind limb paralysis; grade 3.5, complete bilateral hind limb paralysis and paresis of one front limb; grade complete paralysis (tetraplegia), moribund state, or death.
- the animals receive treatment once the EAE model is induced.
- EAE is induced in the 129B6 mixed strain of mice by intradermal immunization (day 0) with 100 ⁇ g MOG1-125 protein emulsified with complete Freund's adjuvant (CFA).
- CFA complete Freund's adjuvant
- the injected volume is 100 ⁇ l per mouse and is distributed over 3 sites (pinnae, back and skin).
- the emulsion is prepared on the basis of a 1:1 volume ratio and contains 1 mg/ml MOGl -125 and 2 mg/ml M. tuberculosis (strain H37Ra, Chondrex).
- Pertussis toxin 200 ng/mouse is administered intra- peritoneally at the time of immunization and 2 days thereafter.
- Plasma is separated from PBMC by centrifugation and cell phenotyping performed by FACS staining.
- Profiling of the humoral anti-MOG antibody response is performed by ELISA using subclassVisotype-specific niAbs (Pharmingen).
- brain, spinal cord, optic nerves and sciatic nerves are harvested following perfusion.
- Sp35 knockout mice typically show lower EAE score (1.5), and no relapse compared to control (over a 45 day period), then wild type litter mates (EAE score 3.5).
- the His-tagged MOG 1 , 125 protein was expressed in Pichia pastoris using a Doxycycline inducible TetO-AOXl promoter (M. Levesque, D. Krushinskie and K. Strauch, manuscript in preparation).
- the extracellular coding sequence (Glyl through Glyl25 of the mature protein after removal of signal sequence) of rat MOG was PCR amplified using the following primers: 5'GGGGTATCTCTCGAGAAAAGAGAGCATCATCATCATCATCATATGGGACAGTTCAGAGT GATAGGG 3' (SEQ ID NO:368), and 5'TTCGCGGCCGCTATTAGCCAGGGTTG ATCCAGTAGAAGGG3' (SEQ E) NO:369).
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