WO2005092378A2

WO2005092378A2 - Anti-vglut glutamate transporter antibodies for use in pain therapy

Info

Publication number: WO2005092378A2
Application number: PCT/EP2005/002695
Authority: WO
Inventors: Heribert Bohlen; Silke Schwengberg; Andreas Ehlich
Original assignee: Axiogenesis Ag
Priority date: 2004-03-12
Filing date: 2005-03-14
Publication date: 2005-10-06
Also published as: WO2005092378A3

Abstract

Pharmaceutical compositions as well as methods of alleviating pain, such as central pain, pain arising from cancer and phantom limb pain, are provided comprising administration of an antibody or antigen binding fragment thereof that specifically binds a VGLUT glutamate transporter.

Description

Novel pain therapy

Field of the invention

The present invention pertains to a method of alleviating pain, such as chronic, neuropathic and central pain, pain arising from cancer and phantom limb pain, by administration of an antibody or antigen binding fragment thereof that specifically binds a NGLUT glutamate transporter.

Background of the invention

Pain is a sensation that hurts. It may cause discomfort or distress or agony. It may be steady or throbbing. It may be stabbing, aching, or pinching. However pain is felt, only the person experiencing pain can describe it or define it. Because pain is so individual, pain cannot be truly evaluated by any third person. The World Health Organization (WHO) recognizes a "Three-step Analgesic Ladder" for pharmacologic management of pain. The ladder begins with relatively low doses of low-potency analgesics and progresses to higher doses of more potent compounds. The three steps involve use of nonopioid analgesics with or without coanalgesics, such as Νonsteroid Anti-inflammatory Drugs (ΝSAIDs); lower-potency opioids with or without coanalgesics as pain persists or increases to moderate levels; and high- potency opioids with or without nonopioid coanalgesics as pain persists or increases to severe levels. Use of opioid analgesics, even for treatment of severe pain, is controversial in the medical community, due to the possibility of addiction; see, e.g. Weitz et al., New Jersey Medicine 97 (2000), 63-67.

Thus, there is a constant need for pharmaceutical compositions for the treatment of pain. The solution to said technical problem is achieved by providing the embodiments characterized in the claims, and described further below.

Description of the invention

The present invention relates to a pharmaceutical composition useful in the treatment or amelioration of pain comprising an antibody or antigen binding fragment thereof that specifically binds a vesicular glutamate transporter (NGLUT), and optionally a pharmaceutically acceptable carrier. A "NGLUT transporter" refers to a member of a glutamate transporter family of vesicular glutamate transporters characterized by NGLUT1, NGLUT2, and NGLUT3. The NGLUT glutamate transporters belong to a larger family known as the type I phosphate transporters. However, members of this family transport organic anions (such as sialic acid and glutamate) rather than inorganic phosphate. Within this family, the NGLUTs show much stronger sequence similarity (>50% amino acid identity to each other from C. elegans to mammals and >80% within mammals) than to other type I phosphate transporters such as sialin and ΝaPi-1 (35-45% amino acid identity). Thus, preferred NGLUT glutamate transporters of this invention show 50% or greater amino acid sequence identity, preferably 65% or greater amino acid sequence identity, more preferably 80%) or greater amino acid sequence identity, still more preferably 90%> or greater amino acid sequence identity, and most preferably 95% or greater amino acid sequence identity, to VGLUT1 and/or to NGLUT2 and/or to NGLUT3. For review of vesicular glutamate transporters in the brain see Hisano, Anatomical Science International 78 (2003), 191-204.

Nucleic and amino acid sequences of NGLUT transporter are well known in the art and comprise NGLUT1 (GenBank Accession No: AB032436) and homologs and orthologues thereof, NGLUT2 (GenBank Accession Νos: rat NGLUT2: AF271235; human VGLUT2: AB032435) and homologs and orthologues thereof, and NGLUT3 (GenBank Accession No: ALl 57942) and homologs and orthologues thereof; as well as "BNPI" which refers to a brain- specific inorganic phosphate transporter (see, e.g., Rosteck et al., Proc. Natl. Acad. Sci. USA 91 (1994), 5607-5611; Glinn and Paul, J. Neurochem 65 (1995), 2358-2365; and Glinn et al., J. Neurochem. 70 (1998), 1850-1858); see also, GenBank accession number AB032436. BNPI is used herein synonymously with VGLUT1. For review of NGLUT glutamate transporters see also US application US2002/0098473.

The present invention is based on the observation that an antibody that is capable of specifically binding a NGLUT transporter, in particular NGLUT2, is capable of alleviating pain, probably by modulating glutamate uptake into synaptic vesicles. A better understanding of the theory underlying the present invention may be had from the review articles by Nenkatesh et al., Annu. Rev. Νeurosci. 26 (2003), 701-28 and Matthew et al., Annu. Rev. Cell Dev. Biol. 15 (1999), 733-98. In principle, the antibody or a corresponding antibody-like molecule may modulate NGLUT activity either by up- or down-regulation of the transporter or by complete blocking the transporter activity. Preferably, however, but without intending to be bound by a particular theory, it is believed that the release of too much glutamate causes excessive excitation in the nervous system and leads to or is involved in the origin, maintenance, severity or otherwise persistence of pain. Accordingly, in accordance with the present invention it is expected that by using antibodies specific for NGLUT transporters and interfering with transport glutamate into synaptic vesicles the generation of an anesthetic/analgesic effect in a subject experiencing pain can be achieved.

Thus, the present invention provides a pharmaceutical composition useful in the treatment or amelioration of pain comprising an antibody or antigen binding fragment thereof that specifically binds a NGLUT glutamate transporter, and optionally a pharmaceutically acceptable carrier.

Hitherto antibodies and antibody derived molecules have only been considered for diagnostic uses. Hence, methods for the generation of antibodies to NGLUT transporter polypeptides, which may be used in accordance with the present invention are described in the prior art; see, e.g., in US application US2002/0098473 and international application WO00/071709.

The pharmaceutical compositions of the present invention comprise the NGLUT antibody, or a binding fragment, or derivative, or corresponding receptor molecule thereof, or a chemical derivative of any one thereof. The composition of the present invention preferably further comprise a pharmaceutically acceptable carrier. The term "chemical derivative" describes a molecule that contains additional chemical moieties that are not normally a part of the base molecule. Such moieties may improve the solubility, half-life, absorption, etc. of the base molecule. Alternatively, the moieties may attenuate undesirable side effects of the base molecule or decrease the toxicity of the base molecule. Examples of such moieties are described in a variety of texts, such as Remington's Pharmaceutical Sciences. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Compositions comprising such carriers can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose. Administration of the suitable compositions may be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. Aerosol formulations such as nasal spray formulations include purified aqueous or other solutions of the active agent with preservative agents and isotonic agents. Such formulations are preferably adjusted to a pH and isotonic state compatible with the nasal mucous membranes. Formulations for rectal or vaginal administration may be presented as a suppository with a suitable carrier.

The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs , being administered concurrently. A typical dose can be, for example, in the range of 0.001 to 1000 μg (or of nucleic acid for expression in this range); however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors. Generally, the regimen as a regular administration of the pharmaceutical composition should be in the range of 1 μg to 10 mg units per day. If the regimen is a continuous infusion, it should also be in the range of 1 μg to 10 mg units per kilogram of body weight per minute, respectively. Progress can be monitored by periodic assessment. Dosages will vary but a preferred dosage for intravenous administration of DNA is from approximately 10 to 10 copies of the DNA molecule. The compositions of the invention may be administered locally or systemically. Administration will generally be parenterally, e.g., intravenously; DNA may also be administered directly to the target site, e.g., by biolistic delivery to an internal or external target site or by catheter to a site in an artery. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of 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. Parenteral vehicles include sodium chloride solution, 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, anti-oxidants, chelating agents, and inert gases and the like. Furthermore, the pharmaceutical composition of the invention may comprise further agents such as interleukins or interferons depending on the intended use of the pharmaceutical composition.

As mentioned before, it is believed that antibodies and antibody-like molecules are capable of modulating NGLUT transporter activity, preferably by inhibiting glutamate transport. Hence, inhibition of glutamate is expected to be useful in anesthesia and the management of pain. Thus, in a preferred embodiment of the pharmaceutical composition, said NGLUT antibody or antigen binding fragment thereof is capable of modulating the transporter activity of the NGLUT glutamate transporter, most preferably said antibody or antibody-like molecule is capable of down-regulating or blocking the activity of the NGLUT transporter.

In a particular preferred embodiment of the pharmaceutical composition of the present invention, said NGLUT transporter is the NGLUT2-Transporter also termed "Differentiation- Associated Sodium-Dependent Inorganic Phosphate Transporter" (DΝPI). Identification of NGLUT2 as the differentiation-associated Νa+/PI transporter expressed in a distinct set of glutamatergic synapses is described by Varoquil et al. in J. Neuroscience 22 (2002), 142- 155, see also Takamori et al. and Herzog et al., both in J. Neuroscience 21 (2001). Antibodies directed against DNPI that may be used and adapted in accordance with the present invention are also described; see, e.g., international application WO02/101394. This international application describes the nucleic and amino acid sequences of NGLUT 1 (BΝPI) and NGLUT2 (DΝPI).

In a preferred embodiment of the present invention, the antibody or antibody-like molecule binds to a domain of the NGLUT transporter, which is accessible to the antibody in vivo. Methods of generating such antibodies for example by selection in cell based assays and/or computer predicted selection of appropriate peptides are well known in the art; see also the references cited herein. For example, antigenic regions of NGLUT transporters can be identified using PSORT and PC-GEΝE and used to design short peptide sequences for immunization or screening of compound libraries.

The antibody in the pharmaceutical composition of the present invention can be a monoclonal antibody, a single chain antibody, chimeric antibody, humanized antibody, diabody, xenogeneic antibody, or a fragment and/or a chemically modified derivative of any one thereof that specifically binds a NGLUT transporter as defined above also including bispecific antibody, synthetic antibody, antibody fragment, such as Fab, Fv or scFv fragments etc., or a chemically modified derivative of any of these. Antibodies or fragments thereof can be obtained by using methods which are described, e.g., in Harlow and Lane "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988. When derivatives of said antibodies are obtained by the phage display technique, surface plasmon resonance as employed in the BIAcore system can be used to increase the efficiency of phage antibodies which bind to the same epitope as that of a given antibody (Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13). The production of chimeric antibodies is described, for example, in international application WO89/09622. Methods for the production of humanized antibodies are described in, e.g., European patent application EP-A1 0 239 400 and international application WO90/07861. A further source of antibodies to be utilized in accordance with the present invention are so- called xenogeneic antibodies. The general principle for the production of xenogeneic antibodies such as human antibodies in mice is described in, e.g., international applications WO91/10741, WO94/02602, WO96/34096 and WO96/33735. As discussed above, the antibody in the pharmaceutical composition of the invention may exist in a variety of forms besides complete antibodies; including, for example, Fv, Fab and F(ab)2, as well as in single chains; see e.g., international application WO88/09344. Furthermore, the production of diabody molecules that are heterodimerized by creating a fusion protein with the CL an CHI immunoglobulin constant domains is described in international application WO02/02781. In addition, the construction of bispecific diabody libraries form scFv libraries is described in European patent application EP 1 500 665 A.

The antibodies in the pharmaceutical compositions of the present invention or their corresponding immunoglobulin chain(s) can be further modified using conventional techniques known in the art, for example, by using amino acid deletion(s), insertions), substitution(s), addition(s), and/or recombination(s) and/or any other modification(s) known in the art either alone or in combination. Methods for introducing such modifications in the DNA sequence underlying the amino acid sequence of an immunoglobulin chain are well known to the person skilled in the art; see, e.g., Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. Modifications of the antibody of the invention include chemical and/or enzymatic derivatizations at one or more constituent amino acid, including side chain modifications, backbone modifications, and N- and C-terminal modifications including acetylation, hydroxylation, methylation, amidation, and the attachment of carbohydrate or lipid moieties, cofactors, and the like. Likewise, the present invention encompasses chimeric proteins which comprise the described NGLUT antibody or some fragment thereof at the amino terminus fused to heterologous molecule such as an immunostimulatory ligand at the carboxyl terminus; see, e.g., international application WO00/30680 for corresponding technical details. Hence, the present invention relates to any antibody and similar binding molecules which recognize the same epitope and with substantially the same affinity, or at least 1/10 of the affinity as the NGLUT antibody for the medical use of the invention exemplified herein. Such antibodies and binding molecules can be tested for their binding specificity and affinity for example by the methods described in US application US2002/0098473.

In a preferred embodiment, the antibody in the pharmaceutical composition of the invention is a chimeric or a humanized antibody. Chimeric antibodies are antibodies whose light and heavy chain genes have been constructed, typically by genetic engineering, from immunoglobulin gene segments belonging to different species. For example, the variable (N) segments of the genes from a mouse monoclonal antibody may be joined to human constant (C) segments, such as γl and γ3. A typical therapeutic chimeric antibody is thus a hybrid protein consisting of the N or antigen-binding domain from a mouse antibody and the C or effector domain from a human antibody, although other mammalian species may be used as well if for example veterinary application is envisaged. Human constant region DΝA sequences can be isolated in accordance with well known procedures from a variety of human cells, but preferably immortalized B-cells (see, Kabat, infra and international application WO87/02671). For example, the human kappa immunoglobulin constant and J region genes and sequences are described in Heiter, Cell 22 (1980), 197-207 and the nucleotide sequence of a human immunoglobulin C gene is described in Ellison, Νucl. Acids Res. 10 (1982), 4071, both of which are incorporated herein by reference.

In accordance with the present invention, in one preferred embodiment the term antibody also comprises the polynucleotides encoding the NGLUT antibody or a binding fragment or derivative thereof such as intrabodies described for example in US application US2002/0098473. A review on targeting scFv, i.e. intrabody expression to eukaryotic intracellular compartments is given in Cohen, Methods Mol. Biol. 178 (2002), 367-378. Direct in vivo screening of intrabody libraries constructed on a highly stable single-chain framework is described in der Maur et al., J. Biol. Chem. 277 (2002), 45075-45085. As a further example, the intrabody-based strategy for inhibition of vascular endothelial growth factor receptor-2: effects on apoptosis, cell growth, and angiogenesis is described in Wheeler et al, FASEB J. 17 (2003), 1733-1735. In accordance with the present invention these polynucleotides can be used alone or as part of a vector to express the (poly)peptide of the invention in cells, for, e.g., gene therapy of pain. The polynucleotides or vectors are introduced into the cells which in turn produce the antibody. Gene therapy, which is based on introducing therapeutic genes into cells by ex-vivo or in-vivo techniques is one of the most important applications of gene transfer. Suitable vectors and methods for in-vitro or in-vivo gene therapy are described in the literature and are known to the person skilled in the art; see, e.g., Giordano, Nature Medicine 2 (1996), 534-539; Schaper, Circ. Res. 79 (1996), 911-919; Anderson, Science 256 (1992), 808-813; Isner, Lancet 348 (1996), 370-374; Muhlhauser, Circ. Res. 77 (1995), 1077-1086; Wang, Nature Medicine 2 (1996), 714-716; WO94/29469; WO 97/00957 or Schaper, Current Opinion in Biotechnology 7 (1996), 635-640, and references cited therein. The polynucleotides and vectors may be designed for direct introduction or for introduction via liposomes, or viral vectors (e.g. adenoviral, retroviral) into the cell.

Anti-NGLUT antibodies suitable for use/expression as intrabodies in the methods of this invention can be readily produced by a variety of methods. Such methods include, but are not limited to, traditional methods of raising "whole" polyclonal antibodies, which can be modified to form single chain antibodies, or screening of, e.g. phage display libraries to select for antibodies showing high specificity and/or avidity for NGLUT. Such screening methods are described in the references cited herein in some detail. The antibody cassette is delivered to the cell by any of the known means. This discloses the use of a fusion protein comprising a target moiety and a binding moiety. The target moiety brings the vector to the cell, while the binding moiety carries the antibody cassette. Other methods include those described in, for example, Miller, Nature 357 (1992), 455-460; Anderson, Science 256 (1992), 808-813; Wu et al., J. Biol. Chem. 263 (1988), 14621-14624. For example, a cassette containing these (anti- NGLUT) antibody genes, such as the sFv gene, can be targeted to a particular cell by a number of techniques including, but not limited to the use of tissue-specific promoters, the use of tissue specific vectors, and the like. Methods of making and using intrabodies are described in detail in US Patent 6,004,940.

In one embodiment, the pharmaceutical composition of the present invention may further comprise a pain-suppressive agent such as an opioid or ΝSAID, for example aspirin; see also the background section above.

Pain can originate for many reasons. A familiar cause is trauma, such as a sprain or muscle injury or broken bone, or from surgery. Pain due to inflammation, such as a toothache, is also familiar to many. Headache is a common experience and arises often for unknown reasons. Pain may be acute or chronic. Acute pain can be severe, but lasts a relatively short time. It is usually a signal that body tissue is being injured in some way, and the pain generally disappears when the injury heals. Chronic pain may range from mild to severe, and it is present to some degree for long periods of time. Chronic pain often arises without any detectable injury.

In one embodiment of the present invention, the pharmaceutical composition is designed for the treatment of acute, central or chronic pain. Since the it has been recently shown that VGLUT1 and NGLUT2 are probably the only two transporters in the mammalian central nervous system and thus the only two gene products for vesicular glutamate uptake (see, e.g., Kaneko and Fujiyama, Νeurosci. Res. 42 (2002), 243-250), the management of pain originating from the central nervous system including neuropathic pain is particularly envisaged.

The alleviation of chronic severe pain, such as that arising from cancer and "phantom limb pain" is an important topic in modern medicine. Cancer is highly pervasive in the human population. A person suffering from cancer frequently experiences severe pain. This pain can also be known as central pain or chronic pain. However, a patient need not be suffering from cancer to experience central pain or chronic pain. A related type of pain is phantom limb pain. These types of pain have been treated by opiates such as morphine. A drawback of the opiate analgesia is the addictive nature of the opiates. Acute local pain can arise, for example, from toothaches, eye irritation, inflammation in a nervous tissue region, canker sores, genital ulcers, and pain in epithelial tissues caused by burns, surgery or soreness. Perception of pain can also be divided into three areas; acute nociceptive processing, facilitated pain arising from persistent afferent input (as after tissue injury) and neuropathic pain that arises from altered processing after nerve injury.

All those types of pain may be treated with a pharmaceutical composition of the present invention. In one embodiment the pain is caused by cancer, surgery, mechanical stress or is phantom limb pain. Since it appears as if vesicular glutamate transporters are predominantly expressed and localized in pancreatic islets, upper gastrointestinal tract, and testis (see Hayashi et al, J. Histochem. Cytochem. 51 (2003), 1375-1390), the treatment of pain originating from those tissue and organs, for example due to cancer or injury, is a preferred embodiment of the present invention. Furthermore, vesicular glutamate transporter 2 appears to be expressed in the gut (Tong et al., Neuroreport 12 (2001), 3929-3934) and therefore this represents a preferred target for the pain therapy as well.

The pharmaceutical compositions may be provided to the individual by a variety of routes such as by intracoronary, intraperitoneal, subcutaneous, intravenous, transdermal. intrasynovial, intramuscular or oral routes or as an aerosol. In addition, co-administration or sequential administration of other agents may be desirable. Formulation for each administration route is generally considered known in the art; see, e.g., "Remington, the Science and Practice of Pharmacy", 19th ed., A.R. Gennaro, ed., c. 1995 by The Philadelphia College of Pharmacy and Science. Administration of peripheral anti-Abeta antibody directed to the CNS is described for example by DeMattos et al., Proc. Natl. Acad. Sci. USA 98 (2001), 8850-8855.

A therapeutically effective dose refers to that amount of antibodies, polynucleotides and vectors of the invention ameliorate the symptoms or condition. Therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50.

Hence, the present invention generally relates a method of alleviating pain in a subject, comprising administering an effective amount of an VGLUT antibody as described above or a ligand binding molecule which has substantially the same binding specifitity to the subject. Most preferably, the subject is a human.

The person skilled in the art knows that each variable domain (the heavy chain NH and light chain VL) of an antibody comprises three hypervariable regions, sometimes called complementarity determining regions or "CDRs" flanked by four relatively conserved framework regions of "FRs". The CDRs contained in the variable regions of the antibody of the invention can be determined, for example, according to Kabat, Sequences of Proteins of Immunological Interest (U.S. Department of Health and Human Services, third edition, 1983, fourth edition, 1987, fifth edition 1990 and updates thereof). The person skilled in the art will readily appreciate that the variable domain of the antibody having the above-described variable domain can be used for the construction of other polypeptides or antibodies of desired specificity and biological function. Thus, the present invention also encompasses antibodies and ligand, i.e. NGLUT transporter binding molecules comprising at least one CDR of the variable domain of the above-described anti-NGLUT antibodies and which advantageously have substantially the same or similar binding properties as the original antibody. The person skilled in the art will readily appreciate that using the variable domains or CDRs described herein antibodies can be constructed according to methods known in the art, for example, as described in EP 0 451 216 Al and EP 0 549 581 Al. Furthermore, the person skilled in the art knows that binding affinity may be enhanced by making amino acid substitutions within the CDRs or within the hypervariable loops (Chothia and Lesk, J. Mol. Biol. 196 (1987), 901-917) which partially overlap with the CDRs as defined in Kabat. Thus, the present invention also relates to the use of antibodies and ligand binding molecules wherein one or more of the mentioned CDRs comprise one or more, preferably not more than two amino acid substitutions. Accordingly, the present invention encompasses the use of a ligand binding molecule comprising at least one CDR of a NGLUT antibody as defined above for treatment of pain. The appropriate concentration of the therapeutic antibody might be dependent on the particular agent. The therapeutically effective dose has to be compared 'with the toxic concentrations; the clearance rate as well as the metabolic products play a role as do the solubility and the formulation.

Likewise the present invention relates to the use of NGLUT, in particular NGLUT2 as a target for in antibody based therapy, especially in pain management.

These and other embodiments are disclosed and encompassed by the description of the present invention. Further literature concerning any one of the materials, methods, uses and compounds to be employed in accordance with the present invention may be retrieved from public libraries and databases, using for example electronic devices. For example the public database "Medline" may be utilized, which is hosted by the National Center for Biotechnology Information and/or the National Library of Medicine at the National Institutes of Health. Further databases and web addresses, such as those of the European Biomformatics Institute (EBI), which is part of the European Molecular Biology Laboratory (EMBL) are known to the person skilled in the art and can also be obtained using internet search engines. An overview of patent information in biotechnology and a survey of relevant sources of The above disclosure generally describes the present invention. Several documents are cited throughout the text of this specification. The contents of all cited references (including literature references, issued patents, published patent applications as cited throughout this application and manufacturer's specifications, instructions, etc) are hereby expressly incorporated by reference; however, there is no admission that any document cited is indeed prior art as to the present invention.

The above disclosure generally describes the present invention. Detailed descriptions of conventional methods, such as those employed herein can be found in the cited literature. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Methods in molecular genetics and genetic engineering are described generally in the current editions of Molecular Cloning: A Laboratory Manual, (Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press); DNA Cloning, Volumes I and II (Glover ed., 1985); Oligonucleotide Synthesis (Gait ed., 1984); Nucleic Acid Hybridization (Hames and Higgins eds. 1984); Transcription And Translation (Hames and Higgins eds. 1984); Culture Of Animal Cells (Freshney and Alan, Liss, Inc., 1987); Gene Transfer Vectors for Mammalian Cells (Miller and Calos, eds.); Current Protocols in Molecular Biology and Short Protocols in Molecular Biology, 3rd Edition (Ausubel et al., eds.); and Recombinant DNA Methodology (Wu, ed., Academic Press). Gene Transfer Vectors For Mammalian Cells (Miller and Calos, eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al., eds.); Immobilized Cells And Enzymes (IRL Press, 1986); Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (Weir and Blackwell, eds., 1986). Reagents, cloning vectors, and kits for genetic manipulation referred to in this disclosure are available from commercial vendors such as BioRad, Stratagene, Invitrogen, and Clontech. General techniques in cell culture and media collection are outlined in Large Scale Mammalian Cell Culture (Hu et al., Curr. Opin. Biotechnol. 8 (1997), 148); Serum-free Media (Kitano, Biotechnology 17 (1991), 73); Large Scale Mammalian Cell Culture (Curr. Opin. Biotechnol. 2 (1991), 375); and Suspension Culture of Mammalian Cells (Birch et al., Bioprocess Technol. 19 (1990), 251); Extracting information from cDNA arrays, Herzel et al, CHAOS 11, (2001), 98-107.

Claims

1. A pharmaceutical composition useful in the treatment or amelioration of pain comprising an antibody or antigen binding fragment thereof that specifically binds a NGLUT glutamate transporter, and optionally a pharmaceutically acceptable carrier.

2. The pharmaceutical composition of claim 1, wherein said antibody or antigen binding fragment thereof is capable of modulating the transporter activity of the NGLUT glutamate transporter.

3. The pharmaceutical composition of claim 1 or 2, wherein said NGLUT glutamate transporter is the NGLU2-Transporter.

4. The pharmaceutical composition of any one of claims 1 to 3, wherein said antibody is a monoclonal antibody.

5. The pharmaceutical composition of any one claims 1 to 4, wherein said antibody is a chimeric or humanized antibody.

6. The pharmaceutical composition of any one of claims 1 to 5, further comprising a pain-suppressive agent.

7. The pharmaceutical composition of claim 6, wherein said pain-suppressive agent is an opioid or ΝSAID.

8. The pharmaceutical composition of claim 7, wherein said pain-suppressive agent is aspirin.

9. The pharmaceutical composition of any one of claims 1 to 8, wherein the pain is acute, central or chronic pain.

10. The pharmaceutical composition of any one of claims 1 to 9, wherein the pain is caused by cancer, surgery, mechanical stress or is phantom limb pain.

11. The pharmaceutical composition of any one of claims 1 to 10, wherein said pharmaceutical composition is designed to be administered intraveneous, intramuscular, subcutaneous, intraperitoneal, or as an aerosol.

12. A method of alleviating pain in a subject, comprising administering an effective amount of the antibody as defined in any one of claims 1 to 5 or a ligand binding molecule which has substantially the same binding specifitity to the subject.

13. Use of a ligand binding molecule comprising at least one CDR of an antibody as defined in any one of claims 1 to 5 for the preparation of a pharmaceutical composition for the treatment of pain.