EP1648488A2 - Proteine membranaire exprimee dans des mastocytes d'origine humaine - Google Patents

Proteine membranaire exprimee dans des mastocytes d'origine humaine

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Publication number
EP1648488A2
EP1648488A2 EP04777035A EP04777035A EP1648488A2 EP 1648488 A2 EP1648488 A2 EP 1648488A2 EP 04777035 A EP04777035 A EP 04777035A EP 04777035 A EP04777035 A EP 04777035A EP 1648488 A2 EP1648488 A2 EP 1648488A2
Authority
EP
European Patent Office
Prior art keywords
gpr91
antibody
cells
antibodies
cell
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP04777035A
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German (de)
English (en)
Other versions
EP1648488A4 (fr
Inventor
Kang Li
Shen-Wu Wang
Guanghui Hu
Zhengbin Yao
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Tanox Inc
Original Assignee
Tanox Inc
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Publication of EP1648488A2 publication Critical patent/EP1648488A2/fr
Publication of EP1648488A4 publication Critical patent/EP1648488A4/fr
Withdrawn legal-status Critical Current

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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies

Definitions

  • Combinatorial libraries include, without limitation, peptide libraries (e.g. U.S. Pat. No. 5,010,175; Furka, 1991, Int. J. Pept. Prot. Res., 37:487-493; and Houghton et al., 1991, Nature, 354:84-88).
  • Other chemistries for generating chemical diversity libraries can also be used.
  • Nonlimiting examples of chemical diversity library chemistries include, peptides (PCT Publication No. WO 91/019735), encoded peptides (PCT Publication No. WO 93/20242), random bio-oligomers (PCT Publication No.
  • a functional assay is not typically required. All that is needed is a target protein, preferably substantially purified, and a library or panel of compounds (e.g., ligands, drugs, small molecules) or biological entities to be screened or assayed for binding to the protein target. Preferably, most small molecules that bind to the target protein will modulate activity in some manner, due to preferential, higher affinity binding to functional areas or sites on the protein.
  • compounds e.g., ligands, drugs, small molecules
  • the source may be a whole cell lysate that can be prepared by successive freeze-thaw cycles (e.g., one to three) in the presence of standard protease inhibitors.
  • the GPR91 polypeptide may be partially or completely purified by standard protein purification methods, e.g., affinity chromatography using specific antibody described infra, or by ligands specific for an epitope tag engineered into the recombinant GPR91 polypeptide molecule, also as described herein. Binding activity can then be measured as described.
  • Another embodiment of the present invention includes antibodies which immunospecifically bind a polypeptide, polypeptide fragment, or variant of SEQ ID NO:2, and/or an epitope of GPR91, or an extracellular domain fragment of GPR91.
  • Antibodies of the invention include, but are not limited to, polyclonal, monoclonal, monovalent, bispecific, heteroconjugate, multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), and epitope-binding fragments of any of the above.
  • antibody or “monoclonal antibody” (Mab) is meant to include intact molecules, as well as, antibody fragments (such as, for example, Fab and F(ab')2 fragments) which are capable of specifically binding to protein.
  • Fab and F(ab')2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation of the animal or plant, and may have less non-specific tissue binding than an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)). Thus, these fragments are preferred, as well as the products of a FAB or other immunoglobuhn expression library.
  • antibodies of the present invention include chimeric, single chain, and humanized antibodies.
  • the antibodies may be human antigen-binding antibody fragments of the present invention and include, but are not limited to, Fab, Fab' and F(ab')2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide- linked Fvs (sdFv) and fragments comprising either a VL or VH domain.
  • Antigen-binding 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, CHI, CH2, and CH3 domains. Also included in the invention are antigen-binding fragments also comprising any combination of variable region(s) with a hinge region, CHI, CH2, and CH3 domains.
  • the antibodies of the invention may be from any animal origin including birds and mammals.
  • the antibodies are human, murine (e.g., mouse and rat), donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken.
  • "human” antibodies include antibodies having the amino acid sequence of a human immunoglobuhn and include antibodies isolated from human immunoglobuhn libraries or from animals transgenic for one or more human immunoglobuhn and that do not express endogenous immunoglobulins, as described infra and, for example in, U.S. Pat. No. 5,939,598 by Kucherlapati et al.
  • Antibodies of the present invention may be described or specified in terms of the epitope(s) or portion(s) of a polypeptide of the present invention which they recognize or specifically bind.
  • the epitope(s) or polypeptide portion(s) may be specified as described herein, e.g., by N-terminal and C-terminal positions, by size in contiguous amino acid residues, or listed in the Tables and Figures.
  • Antibodies which specifically bind any epitope or polypeptide of the present invention may also be excluded. Therefore, the present invention includes antibodies that specifically bind polypeptides of the present invention, and allows for the exclusion of the same.
  • antibodies of the present invention cross-react with murine, rat and/or rabbit homologues of human proteins and the corresponding epitopes thereof.
  • Antibodies that do not bind polypeptides with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention.
  • the above-described cross-reactivity is with respect to any single specific antigenic or immunogenic polypeptide, or combination(s) of 2, 3, 4, 5, or more of the specific antigenic and/or immunogenic polypeptides disclosed herein.
  • antibodies which bind polypeptides encoded by polynucleotides which hybridize to a polynucleotide of the present invention under stringent hybridization conditions as described herein.
  • Antibodies of the present invention may also be described or specified in terms of their binding affinity to a polypeptide of the invention.
  • Preferred binding affinities include those with a dissociation constant or Kd less than 5.times.l0-2 M, 10-2 M, 5.times.l0-3 M, 10-3 M, 5.times.10-4 M, 10-4 M, 5. times.10-5 M, 10-5 M, 5.times.l0-6 M, 10- 6M, 5.times.10-7 M, 107 M, 5.times.10-8 M, 10-8 M, 5.times.l0-9 M, 10-9 M, 5.times.l0-10 M, 10-10 M, 5.times.l0-l l M, 10-11 M, 5.times.10-12 M, 10-12 M, 5.times.l0-13 M, 10-13 M, 5.times.l0-14 M, 10-14 M, 5.times.l0-15 M, or 10-15 M.
  • Antibodies of the present invention may act as agonists or antagonists of the polypeptides of the present invention.
  • the present invention includes antibodies which disrupt the receptor/ligand interactions with the polypeptides of the invention either partially or fully.
  • antibodies of the present invention bind an antigenic epitope disclosed herein, or a portion thereof.
  • the invention features both receptor-specific antibodies and ligand-specific antibodies.
  • the invention also features receptor-specific antibodies which do not prevent ligand binding but prevent receptor activation.
  • Receptor activation i.e., signaling
  • receptor activation may be determined by techniques described herein or otherwise known in the art. For example, receptor activation can be determined by detecting calcium flux, for example, as described herein.
  • antibodies are provided that inhibit ligand activity or receptor activity by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50% of the activity in absence of the antibody.
  • Antibodies of the present invention may be used, for example, but not limited to, to purify, detect, and target GPR91, including both in vitro and in vivo diagnostic and therapeutic methods.
  • the antibodies have use in immunoassays for qualitatively and quantitatively measuring levels of GPR91 in biological samples. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) (incorporated by reference herein in its entirety).
  • antibodies of the present invention may be used either alone or in combination with other compositions.
  • the antibodies may be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalently and non-covalently conjugations) to polypeptides or other compositions.
  • antibodies of the present invention may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionucleotides, or toxins. See, e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP 396,387.
  • the antibodies 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 from generating an anti-idiotypic response.
  • 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.
  • the antibodies of the present invention may be generated by any suitable method known in the art.
  • the antibodies of the present invention may comprise polyclonal antibodies. Methods of preparing polyclonal antibodies are known to the skilled artisan (Harlow, et al., Antibodies: A Laboratory Manual, (Cold spring Harbor Laboratory Press, 2.sup.nd ed. (1988), which is hereby incorporated herein by reference in its entirety).
  • the method comprises using isolated epitope-bearing polypeptides of GPR91 or antigenic fragments thereof as an immunogen for producing antibodies that bind to the GPR91 in a known protocol for producing antibodies.
  • the method comprises using host cells that express recombinant GPR91 as an antigen.
  • the method comprises using DNA expression vectors containing the GPR91 gene to express the receptor as an antigen for producing the antibodies.
  • Methods well known in the art include, but not limited to, in vivo immunization, in vitro immunization, and phage display methods. See, e.g., Sutcliffe et al., supra; Wilson et al., supra, and Bittle et al, J. Gen. Virol., 66:2347-2354 (1985). If in vivo immunization is used, animals may be immunized with free peptide; however, anti-peptide antibody titer may be boosted by coupling the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid.
  • KLH keyhole limpet hemacyanin
  • peptides containing cysteine residues may be coupled to a carrier using a linker such as maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to carriers using a more general linking agent such as glutaraldehyde.
  • a linker such as maleimidobenzoyl-N-hydroxysuccinimide ester (MBS)
  • MBS maleimidobenzoyl-N-hydroxysuccinimide ester
  • the immunogen can be administered to various host animals including, but not limited to, rabbits, mice, rats, etc. to induce the production of sera containing polyclonal antibodies specific for the antigen.
  • the administration of an immunogenic polypeptide of GPR91 may entail one or more injections and, if desired, may include an adjuvant.
  • immunizing agent may be defined as a polypeptide of encoding GPR91, including fragments, variants, and/or derivatives thereof, in addition to fusions with heterologous polypeptides and other forms of the polypeptides described herein.
  • Such conjugation includes either chemical conjugation by derivitizing active chemical functional groups to both the polypeptide of the present invention and the immunogenic protein such that a covalent bond is formed, or through fusion- protein based methodology, or other methods known to the skilled artisan.
  • immunogenic proteins include, but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor.
  • adjuvants may be used to increase the immunological response, depending on the host species, including but 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 hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Additional examples of adjuvants which may be employed includes the MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalose dicorynomycolate). The immunization protocol may be selected by one skilled in the art without undue experimentation.
  • the antibodies of the present invention may comprise monoclonal antibodies.
  • Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975) and U.S. Pat. No. 4,376,110, by Harlow, et al., Antibodies: A Laboratory Manual, (Cold spring Harbor Laboratory Press, 2.sup.nd ed. (1988), by Hammerling, et al, Monoclonal Antibodies and T-Cell Hybridomas (Elsevier, N.Y., (1981)), or other methods known to the artisan.
  • Such antibodies may be of any immunoglobuhn class including IgG, IgM, IgE, IgA, IgD and any subclass thereof.
  • the hybridoma producing the mAb of this invention may be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo makes this the presently preferred method of production.
  • a hybridoma method a mouse, a humanized mouse, a mouse with a human immune system, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro.
  • PBLs peripheral blood lymphocytes
  • spleen cells or lymph node cells are used if non-human mammalian sources are desired.
  • the lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986), pp. 59-103).
  • Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed.
  • the hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells.
  • Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, Calif, and the American Type Culture Collection, Manassas, Va. As inferred throughout the specification, human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al, Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63).
  • the culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the GPR91 receptor.
  • the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoadsorbant assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunoadsorbant assay
  • the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollart, Anal. Biochem., 107:220 (1980).
  • the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, supra). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal.
  • the monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobuhn purification procedures such as, for example, protein A- sepharose, hydroxyapatite chromatography, gel exclusion chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • immunoglobuhn purification procedures such as, for example, protein A- sepharose, hydroxyapatite chromatography, gel exclusion chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • the skilled artisan would acknowledge that a variety of methods exist in the art for the production of monoclonal antibodies and thus, the invention is not limited to their sole production in hydridomas.
  • the monoclonal antibodies may be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567.
  • the term "monoclonal antibody” refers to an antibody derived from a single eukaryotic, phage, or prokaryotic clone.
  • the DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies, or such chains from human, humanized, or other sources).
  • the hydridoma cells of the invention serve as a preferred source of such DNA.
  • the DNA may be subcloned into expression vectors, which are then transformed into host cells such as Simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobuhn protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • the DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (U.S. Pat. No.4,816,567; Morrison et al, supra) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.
  • non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.
  • the antibodies of the present invention can also be generated using various phage display methods known in the art.
  • phage display methods functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them.
  • phage can be utilized to display antigen binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine).
  • Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead.
  • Phage used in these methods are typically filamentous phage including fd and Ml 3 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein.
  • Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol.
  • the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below.
  • a chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region.
  • Methods for producing chimeric antibodies are known in the art. See e.g., Morrison, Science 229: 1202 (1985); Oi et al, BioTechniques 4:214 (1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; U.S. Pat. Nos.
  • Humanized antibodies are antibody molecules from non-human species antibody that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and a 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.
  • 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.
  • Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos.
  • a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain.
  • Humanization can be essentially performed following the methods of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
  • rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
  • such "humanized" antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • humanized antibodies are typically human antibodies in which some CDR residues and possible some FR residues are substituted from analogous sites in rodent antibodies.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., Nature, 321:522-525 (1986); Riechmann et al, Nature 332:323-329 (1988)1 and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992).
  • Fc immunoglobulin constant region
  • 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.
  • cole et al. and Boerder et al., are also available for the preparation of human monoclonal antibodies (cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Riss, (1985); and Boerner et al., J. Immunol., 147(l):86-95, (1991)).
  • Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes.
  • the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells.
  • the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes.
  • the mouse heavy and light chain immunoglobulin genes may be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production.
  • the modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice.
  • the chimeric mice are then bred to produce homozygous offspring which express human antibodies.
  • the transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention.
  • Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology.
  • the human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation.
  • human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and creation of an antibody repertoire. This approach is described, for example, in U.S. Pat. Nos.
  • Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as "guided selection.”
  • a selected non-human monoclonal antibody e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. (Jespers et al., Bio/technology 12:899-903 (1988)).
  • antibodies to the polypeptides of the invention can, in turn, be utilized to generate anti-idiotype antibodies that "mimic" polypeptides of the invention using techniques well known to those skilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444; (1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)).
  • antibodies which bind to and competitively inhibit polypeptide multimerization and/or binding of a polypeptide of the invention to a ligand can be used to generate anti-idiotypes that "mimic" the polypeptide multimerization and/or binding domain and, as a consequence, bind to and neutralize polypeptide and or its ligand.
  • Such neutralizing anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize polypeptide ligand.
  • anti-idiotypic antibodies can be used to bind a polypeptide of the invention and/or to bind its ligands/receptors, and thereby block its biological activity.
  • bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobuhn heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture often different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published May 13, 1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).
  • Suitable reagents for this purpose include iminothiolate and methyl-4- mercaptobutyrimidate and those disclosed, for example, in U.S. Pat. No. 4,676,980. POLYNUCLEOTIDES ENCODING ANTIBODIES
  • the invention further provides polynucleotides comprising a nucleotide sequence encoding an antibody of the invention and fragments thereof.
  • the invention also encompasses polynucleotides that hybridize under stringent or lower stringency hybridization conditions, e.g., as defined supra, to polynucleotides that encode an antibody, preferably, that specifically binds to GPR91, preferably, an antibody that binds to a polypeptide having the amino acid sequence of SEQ ID NO:2.
  • the polynucleotides may be obtained, and the nucleotide sequence of the polynucleotides determined, 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 al., 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.
  • a polynucleotide encoding an antibody may be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin may be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably poly A+ RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody of the invention) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by a suitable source (e.
  • nucleotide sequence and corresponding amino acid sequence of the antibody 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 al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
  • 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, as described supra.
  • the framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions (see, e.g., Chothia et al., J. Mol. Biol.
  • the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds a polypeptide of the invention.
  • one or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions improve binding of the antibody to its antigen. Additionally, such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds.
  • Other alterations to the polynucleotide are encompassed by the present invention and within the skill of the art.
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region, e.g., humanized antibodies.
  • the present invention provides a vector comprising a nucleotide sequence encoding anti- GPR91 antibodies of the present invention and a host cell comprising such a vector.
  • a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of an antibody molecule, 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 ⁇ UR278 (Ruther et al., EMBO J.
  • pGEX vectors may be used to express foreign polypeptides as fusion proteins of glutathione S-transferase (GST).
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to 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.
  • AcNPV Autographa californica nuclear polyhedrosis virus
  • the virus grows in Spodoptera frugiperda 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).
  • a number of viral-based expression systems may be utilized.
  • the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence.
  • This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts, (e.g., see Logan & Shenk, Proc.
  • Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., Methods in Enzymol. 153:51-544 (1987)).
  • 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 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. Sci. 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.
  • the expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol.3. (Academic Press, New York, 1987)).
  • vector amplification for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol.3. (Academic Press, New York, 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., Mol. Cell. Biol. 3:257 (1983)).
  • sequences encoding appropriate signal peptides that are not naturally associated with GPR91 can be incorporated into expression vectors.
  • a nucleotide sequence for a signal peptide secretory leader
  • a signal peptide that is functional in the intended host cells enhances extracellular secretion of the appropriate polypeptide.
  • the signal peptide may be cleaved from the polypeptide upon secretion from the cell.
  • Yeasts useful as host cells in the present invention include those from the genus Saccharomyces, Pichia, K. Actinomycetes and Kluyveromyces.
  • Yeast vectors will often contain an origin of replication sequence from a 2 ⁇ yeast plasmid, an autonomously replicating sequence (ARS), a promoter region, sequences for polyadenylation, sequences for transcription termination, and a selectable marker gene.
  • ARS autonomously replicating sequence
  • Suitable promoter sequences for yeast vectors include, among others, promoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman et al, J. Biol. Chem. 255:2073, (1980)) or other glycolytic enzymes.
  • Other suitable promoters and vectors for yeast and yeast transformation protocols are well known in the art.
  • 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.
  • Such mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, WI38, and in particular, breast cancer cell lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary gland cell line such as, for example, CRL7030 and Hs578Bst.
  • Recombinant expression of an antibody of the invention, or fragment, derivative or analog thereof, involves the construction of an expression vector containing a polynucleotide that encodes the antibody.
  • an expression vector containing a polynucleotide that encodes the antibody Once 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.
  • 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 antibody of the invention.
  • the invention includes host cells containing a polynucleotide encoding an antibody of the invention, or a heavy or light chain thereof, or a single chain antibody of the invention, 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-expression vector systems may be utilized to express the antibody molecules of the invention.
  • 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, Pichia) 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, BHK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mamm
  • 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
  • the antibodies of the present invention or fragments thereof can be fused to heterologous polypeptide sequences described herein or otherwise known in the art, to facilitate purification.
  • the present invention further includes compositions comprising the polypeptides of the present invention fused or conjugated to antibody domains other than the variable regions.
  • the polypeptides of the present invention may be fused or conjugated to an antibody Fc region, or portion thereof.
  • the antibody portion fused to a polypeptide of the present invention may comprise the constant region, hinge region, CHI domain, CH2 domain, and CH3 domain or any combination of whole domains or portions thereof.
  • the polypeptides may also be fused or conjugated to the above antibody portions to form multimers.
  • Fc portions fused to the polypeptides of the present invention can form dimers through disulfide bonding between the Fc portions.
  • polypeptides corresponding to a polypeptide, polypeptide fragment, or a variant of SEQ ID NO:2 may be fused or conjugated to the above antibody portions to increase the in vivo half life of the polypeptides or for use in immunoassays using methods known in the art. Further, the polypeptides corresponding to SEQ ID NO:2 may be fused or conjugated to the above antibody portions to facilitate purification.
  • One reported example describes chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. (EP 394,827; Traunecker et al, Nature 331:84-86 (1988).
  • polypeptides of the present invention fused or conjugated to an antibody having disulfide-linked dimeric structures may also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone.
  • the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties.
  • EP A 232,262 Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired.
  • the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations.
  • human proteins such as hIL-5
  • Fc portions for the purpose of high- throughput screening assays to identify antagonists of hIL-5.
  • the antibodies or fragments thereof of the present invention can be fused to marker sequences, such as a peptide to facilitate purification.
  • 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.
  • the present invention further encompasses antibodies or fragments thereof conjugated to a diagnostic or therapeutic agent.
  • the antibodies can be used diagnostically to, for example, monitor the development or progression of a tumor as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of 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.
  • the detectable substance may be coupled or conjugated either directly to the antibody (or fragment thereof) or indirectly, through an intermediate (such as, for example, a linker known in the art) using techniques known in the art. 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, beta-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 1251, 1311, l llln or 99Tc
  • an antibody or fragment thereof may be conjugated to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters such as, for example, 213Bi.
  • a cytotoxin or cytotoxic agent includes any agent that is detrimental to cells.
  • Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologues thereof.
  • Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5- fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g.
  • the conjugates of the invention can be used for modifying a given biological response, the therapeutic agent or drug moiety is not to be construed as limited to classical chemical therapeutic agents.
  • the drug moiety may be a protein or polypeptide possessing a desired biological activity.
  • Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, a-interferon, .beta.-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (See, International Publication No. WO 97/33899), AIM II (See, International Publication No. WO 97/34911), Fas Ligand (Takahashi et al, Int.
  • a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin
  • a protein such as tumor necrosis factor, a-interferon, .beta.-interferon, nerve growth factor, platelet derived growth factor, tissue plasm
  • VEGI See, International Publication No. WO 99/23105
  • a thrombotic agent or an anti-angiogenic agent e.g., angiostatin or endostatin
  • biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-1"), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.
  • IL-1 interleukin-1
  • IL-2 interleukin-2
  • IL-6 interleukin-6
  • GM-CSF granulocyte macrophage colony stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • Antibodies of the invention 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 al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, et al., J. Cell. Biol. 105:3087-3096 (1987)).
  • Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
  • Suitable antibody assay labels include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine (1251, 1211), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.
  • enzyme labels such as, glucose oxidase
  • radioisotopes such as iodine (1251, 1211), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc)
  • luminescent labels such as luminol
  • fluorescent labels such as fluorescein and rhodamine, and biotin.
  • diagnosis comprises: a) administering (for example, parenterally, subcutaneously, or intraperitoneally) to a subject an effective amount of a labeled molecule which specifically binds to GPR91, b) waiting for a time interval following the administering for permitting the labeled molecule to preferentially concentrate at sites in the subject where the polypeptide is expressed (and for unbound labeled molecule to be cleared to background level), c) determining background level; and d) detecting the labeled antibody in the subject, such that detection of labeled antibody above the background level indicates that the subject has a particular disease or disorder associated with mast cells Background level can be determined by various methods including, comparing the amount of labeled molecule detected to a standard value previously determined for a particular system
  • the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images
  • the quantity of radioactivity injected will normally range from about 5 to 20 milhcuries of 99 mTc
  • the labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the specific protein
  • In vivo tumor imaging is described in S W Burchiel et al , "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments " (Chapter 13 m Tumor Imaging The Radiochemical Detection of Cancer, S W Burchiel and B A Rhodes, eds , Masson Publishing Inc (1982)
  • the time interval following the administration for permitting the labeled molecule to preferentially concentrate at sites in the subject and for unbound labeled molecule to be cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours In another embodiment the time interval following administration is 5 to 20 days or 5 to 10 days
  • monitoring of the disease or disorder is carried out by repeating the method for diagnosing the disease or disease, for example, one month after initial diagnosis, six months after initial diagnosis, one year after initial diagnosis, etc
  • Presence of the labeled molecule can be detected in the patient using methods known in the art for in vivo scanning These methods depend upon the type of label used Skilled artisans will be able to determine the appropriate method for detecting a particular label
  • Methods and devices that may be used m the diagnostic methods of the invention include, but are not limited to, computed tomography (CT), whole body scan such as position emission tomography (PET), magnetic resonance imaging (MRI), and sonography
  • the method comprises collecting a cell, tissue, or body fluid sample known to contain a defined level of GPR91 receptor from a patient, analyzing the tissue or body fluid for the amount of GPR91 receptor in the tissue, and predicting the predisposition of the patient to certain immune diseases based upon the change in the amount of GPR91 receptor in the tissue or body fluid compared to a defined or tested level established for normal cell, tissue, or bodily fluid.
  • the defined level of GPR91 receptor may be a known amount based upon literature values or may be determined in advance by measuring the amount in normal cell, tissue, or body fluids. Specifically, determination of GPR91 receptor levels in certain tissues or body fluids permits specific and early, preferably before disease occurs, detection of immune diseases in the patient.
  • the tissue or body fluid is peripheral blood, peripheral blood leukocytes, biopsy tissues such as lung or skin biopsies, and synovial fluid and tissue.
  • the present invention provides a method for blocking or modulating the expression of a cellular GPR91 receptor by interfering with the transcription or translation of a DNA or RNA polynucleotide encoding the GPR91 activating receptor.
  • the method comprises exposing a cell capable of expressing a GPR91 receptor to a molecule that interferes with the proper transcription or translation of a DNA or RNA polynucleotide encoding the GPR91 activating receptor.
  • the molecule can be an organic molecule, a bioorganic molecule, an antisense nucleotide, an RNAi nucleotide, or a ribozyme.
  • the present invention provides a method for blocking or modulating expression of GPR91 receptor in an animal by administering to the animal a polynucleotide that is antisense to or forms a triple helix with GPR91 activating receptor-encoding DNA or with DNA regulating expression of GPR91 activating receptor- encoding DNA.
  • the animal is administered antisense polynucleotide or triple helix-forming polynucleotide in an amount sufficient to inhibit or regulate expression of GPR91 receptor in the animal.
  • the antisense polynucleotide or triple helix-forming polynucleotide is a DNA or RNA polynucleotide.
  • the present invention provides a method for preventing or treating GPR91 protein mediated diseases in a mammal.
  • the method comprises administering a disease preventing or treating amount of a GPR91 receptor agonist or antagonist to the mammal.
  • the agonist or antagonist binds to the GPR91 receptor and regulates cytokine and cellular receptor expression to produce cytokine levels characteristic of non-disease states.
  • the disease is an allergy, asthma, autoimmune, or other inflammatory disease.
  • the disease is an allergy or asthma.
  • the dosages of GPR91 receptor agonist or antagonist vary according to the age, size, and character of the particular mammal and the disease. Skilled artisans can determine the dosages based upon these factors.
  • the agonist or antagonist can be administered in treatment regimes consistent with the disease, e.g., a single or a few doses over one to several days to ameliorate a disease state or periodic doses over an extended time to prevent allergy or asthma.
  • the agonists and antagonists can be administered to the mammal in an injectable formulation containing any biocompatible agent and agonists and antagonists compatible carrier such as various vehicles, adjuvants, additives, and diluents.
  • Aqueous vehicles such as water having no nonvolatile pyrogens, sterile water, and bacteriostatic water are also suitable to form injectable solutions.
  • aqueous vehicles can be used. These include isotonic injection compositions that can be sterilized such as sodium chloride, Ringer's, dextrose, dextrose and sodium chloride, and lactated Ringer's.
  • the compounds or pharmaceutical compositions of the invention are preferably tested in vitro, and then in vivo for the desired therapeutic or prophylactic activity, prior to use in humans.
  • in vitro assays to demonstrate the therapeutic or prophylactic utility of a compound or pharmaceutical composition include, the effect of a compound on a cell line or a patient tissue sample.
  • the effect of the compound or composition on the cell line and/or tissue sample can be determined utilizing techniques known to those of skill in the art including, but not limited to, rosette formation assays and cell lysis assays.
  • in vitro assays which can be used to determine whether administration of a specific compound 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.
  • Various delivery systems are known and can be used to administer a compound of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem.. 262:4429-4432 (1987)), construction of a nucleic acid as part of a retroviral or other vector, etc.
  • a compound of the invention e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem.. 262:4429-4432 (1987)), construction of a nucleic acid as part of a retroviral or other vector, etc.
  • Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.
  • the compounds or compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.
  • the pharmaceutical compounds or compositions of the invention may be desirable to administer the pharmaceutical compounds or compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.
  • a protein including an antibody
  • care must be taken to use materials to which the protein does not absorb.
  • polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190 (1985); During et al., Ann.
  • compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.
  • the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin.
  • Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.
  • the formulation should suit the mode of administration.
  • the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • the amount of the compound of the invention which will be effective in the treatment, inhibition and prevention of a disease or disorder associated with aberrant expression and or activity of a polypeptide of the invention can be determined by standard clinical techniques.
  • in vitro assays may optionally be employed to help identify optimal dosage ranges.
  • the precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • the compound comprises nucleic acid sequences encoding an antibody, said nucleic acid sequences being part of expression vectors that express the antibody or fragments or chimeric proteins or heavy or light chains thereof in a suitable host.
  • nucleic acid sequences have promoters operably linked to the antibody coding region, said promoter being inducible or constitutive, and, optionally, tissue-specific
  • nucleic acid molecules are used in which the antibody coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the antibody encoding nucleic acids (Koller and Smithies, Proc Natl.
  • the expressed antibody molecule is a single chain antibody; alternatively, the nucleic acid sequences include sequences encoding both the heavy and light chains, or fragments thereof, of the antibody.
  • Delivery of the nucleic acids into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid-carrying vectors, or indirect, in which case, cells are first transformed with the nucleic acids in vitro, then transplanted into the patient. These two approaches are known, respectively, as in vivo or ex vivo gene therapy.
  • the nucleic acid sequences are directly administered in vivo, where it is expressed to produce the encoded product. This can be accomplished by any of numerous methods known in the art, e.g., by constructing them as part of an appropriate nucleic acid expression vector and administering it so that they become intracellular, e.g., by infection using defective or attenuated retrovirals or other viral vectors (see U.S. Pat. No.
  • nucleic acid- ligand complexes can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation.
  • the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publications WO 92/06180; WO 92/22635; WO92/20316; W093/14188, WO 93/20221).
  • the nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989)).
  • viral vectors that contains nucleic acid sequences encoding an antibody of the invention are used.
  • a retroviral vector can be used (see Miller et al., Meth. Enzymol. 217:581- 599 (1993)). These retroviral vectors contain the components necessary for the correct packaging of the viral genome and integration into the host cell DNA.
  • the nucleic acid sequences encoding the antibody to be used in gene therapy are cloned into one or more vectors, which facilitates delivery of the gene into a patient.
  • retroviral vectors More detail about retroviral vectors can be found in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of a retroviral vector to deliver the mdrl gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy.
  • Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); . Salmons and Gunzberg, Human Gene Therapy 4: 129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3: 110-114 (1993).
  • Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499- 503 (1993) present a review of adenovirus-based gene therapy.
  • adenovirus vectors are used.
  • Adeno-associated virus has also been proposed for use in gene therapy (Walsh et al, Proc. Soc. Exp. • Biol. Med. 204:289-300 (1993); U.S. Pat. No. 5,436,146).
  • Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated tiansfection, or viral infection.
  • the method of transfer includes the transfer of a selectable marker to the cells. The cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene. Those cells are then delivered to a patient.
  • the nucleic acid is introduced into a cell prior to administration in vivo of the resulting recombinant cell.
  • introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc.
  • Numerous techniques are known in the art for the introduction of foreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol. 217:599-618 (1993); Cohen et al, Meth. Enzymol.
  • the technique should provide for the stable transfer of the nucleic acid to the cell, so that the nucleic acid is expressible by the cell and preferably heritable and expressible by its cell progeny.
  • the resulting recombinant cells can be delivered to a patient by various methods known in the art.
  • Recombinant blood cells e.g., hematopoietic stem or progenitor cells
  • the amount of cells envisioned for use depends on the desired effect, patient state, etc., and can be determined by one skilled in the art.
  • Cells into which a nucleic acid can be introduced for purposes of gene therapy encompass any desired, available cell type, and include but are not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, Blymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, e.g., as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc
  • the cell used for gene therapy is autologous to the patient.
  • nucleic acid sequences encoding an antibody are introduced into the cells such that they are expressible by the cells or their progeny, and the recombinant cells are then administered in vivo for therapeutic effect.
  • stem or progenitor cells are used. Any stem and/or progenitor cells which can be isolated and maintained in vitro can potentially be used in accordance with this embodiment of the present invention (see e.g. PCT Publication WO 94/08598; Stemple and Anderson, Cell 71:973-985 (1992); Rheinwald, Meth. Cell Bio. 21A:229 (1980); and Pittelkow and Scott, Mayo Clinic Proc. 61:771 (1986)).
  • the nucleic acid to be introduced for purposes of gene therapy comprises an inducible promoter operably linked to the coding region, such that expression of the nucleic acid is controllable by controlling the presence or absence of the appropriate inducer of transcription.
  • RNA interference RNA interference
  • siRNAs short interference RNAs
  • siRNA-expressing DNA construct of the invention an animal, preferably a mammal, and most preferably a human, patient for treating one or more of the disclosed diseases, disorders, or conditions (Nature 2001, 411:494; Target 2003, 2:42;FEBS 2002, 527:274).
  • Therapeutic compounds of the invention include, but are not limited to, siRNAs of the invention (including fragments, analogs and derivatives thereof as described herein) and nucleic acids homologous to siRNAs of the invention (including fragments, analogs and derivatives thereof and anti-idiotypic antibodies as described herein).
  • siRNAs of the invention can be used to treat, inhibit or prevent diseases, disorders or conditions associated with aberrant expression and/or activity of a polypeptide of the invention, including, but not limited to, any one or more of the diseases, disorders, or conditions described herein.
  • the treatment and/or prevention of diseases, disorders, or conditions associated with aberrant expression and/or activity of a polypeptide of the invention includes, but is not limited to, alleviating symptoms associated with those diseases, disorders or conditions.
  • Antibodies of the invention may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.
  • siRNAs of the present invention may be used therapeutically includes binding polynucleotides of the present invention locally or systemically in the body or by direct cytotoxicity of the siRNA, e.g. as mediated by complement (CDC) or by effector cells (ADCC). Some of these approaches are described in more detail below.
  • siRNAs of this invention may be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3 and IL- 7), for example, which serve to increase the number or activity of effector cells which interact with the antibodies.
  • lymphokines or hematopoietic growth factors such as, e.g., IL-2, IL-3 and IL- 7
  • siRNAs of the invention may be administered alone or in combination with other types of treatments (e.g., radiation therapy, chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents). Generally, administration of products of a species origin or species reactivity (in the case of siRNAs) that is the same species as that of the patient is preferred. Thus, in a preferred embodiment, human antibodies, fragments derivatives, analogs, or nucleic acids, are administered to a human patient for therapy or prophylaxis.
  • siRNAs directed against polypeptides of the present invention are useful for inhibiting allergic reactions in animals.
  • a therapeutically acceptable dose of an siRNA, or siRNAs, of the present invention, or a cocktail of the present siRNAs, or in combination with other siRNAs of varying sources the animal may not elicit an allergic response to antigens.
  • the polypeptide of the present invention is responsible for modulating the immune response to auto-antigens
  • transforming the organism and/or individual with a construct comprising any of the promoters disclosed herein or otherwise known in the art in addition, to a polynucleotide encoding the siRNA directed against the polypeptide of the present invention could effective inhibit the organisms immune system from eliciting an immune response to the a ⁇ to-antigen(s).
  • a polynucleotide encoding the siRNA directed against the polypeptide of the present invention could effective inhibit the organisms immune system from eliciting an immune response to the a ⁇ to-antigen(s).
  • Detailed descriptions of therapeutic and/or gene therapy applications of the present invention are provided elsewhere herein.
  • the compound comprises nucleic acid sequences encoding a siRNA, said nucleic acid sequences being part of expression vectors that express the antibody or fragments or chimeric proteins or heavy or light chains thereof in a suitable host.
  • nucleic acid sequences have promoters operably linked to the antibody coding region, said promoter being inducible or constitutive, and, optionally, tissue-specific
  • nucleic acid molecules are used in which the antibody coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the antibody encoding nucleic acids (Koller and Smithies, Proc. Natl.
  • the expressed antibody molecule is a single chain antibody; alternatively, the nucleic acid sequences include sequences encoding both the heavy and light chains, or fragments thereof, of the antibody.
  • microparticle bombardment e.g., a gene gun; Biolistic, Dupont
  • coating lipids or cell-surface receptors or transfecting agents, encapsulation in liposomes, microparticles, or microcapsules, or by administering them in linkage to a peptide which is known to enter the nucleus, by administering it in linkage to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem . . . 262:4429-4432 (1987)) (which can be used to target cell types specifically expressing the receptors), etc.
  • Adeno-associated virus has also been proposed for use in gene therapy (Walsh et al, Proc. Soc. Exp. Biol Med. 204:289-300 (1993); U.S. Pat. No. 5,436,146).
  • Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection.
  • the method of transfer includes the transfer of a selectable marker to the cells. The cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene. Those cells are then delivered to a patient.
  • the cell used for gene therapy is autologous to the patient.
  • nucleic acid sequences encoding a siRNA are introduced into the cells such that they are expressible by the cells or their progeny, and the recombinant cells are then administered in vivo for therapeutic effect.
  • stem or progenitor cells are used. Any stem and/or progenitor cells which can be isolated and maintained in vitro can potentially be used in accordance with this embodiment of the present invention (see e.g. PCT Publication WO 94/08598; Stemple and Anderson, Cell 71:973-985 (1992); Rheinwald, Meth. Cell Bio. 21A:229 (1980); and Pittelkow and Scott, Mayo Clinic Proc. 61:771 (1986)).
  • KNOCKOUT ANIMALS KNOCKOUT ANIMALS
  • the present invention provides a knockout animal comprising a genome having a heterozygous or homozygous disruption in its endogenous GPR91 receptor gene that suppresses or prevents the expression of biologically functional GPR91 receptor proteins.
  • the knockout animal of the present invention has a homozygous disruption in its endogenous GPR91 receptor gene.
  • the knockout animal of the present invention is a mouse. The knockout animal can be made easily using techniques known to skilled artisans.
  • mice or other animals derived from these oocytes will be, at a frequency of about 10 to 20%, the transgenic founders that through breeding will give rise to the different transgenic mouse lines.
  • Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art, e.g., U.S. Pat. Nos. 4,736,866, 4,870,009, and 4,873,191 and in Hogan, B., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used for production of other transgenic animals.
  • Embryonic stem cell (“ES cell”) technology can be used to create knockout mice (and other animals) with specifically deleted genes.
  • Totipotent embryonic stem cells which can be cultured in vitro- and genetically modified, are aggregated with or microinjected into mouse embryos to produce a chimeric mouse that can transmit this genetic modification to its offspring. Through directed breeding, a mouse can thus be obtained that lacks this gene.
  • ICSI intracytoplasmic sperm injection technique
  • Microarray technology was used to compare gene expression quantitatively between Mast Cells, THPl and PBMC, and identify those genes preferentially expressed (>2.5 fold) in Mast Cells versus THPl and PBMC.
  • Human cord blood CD34 + cells (Bio-Whittaker, Walkersville, MD) were cultured up to 9 weeks in culture media consisting of RP MI 1640 (Invitrogen) supplemented with 20% FBS (Sigma- Aldrich, St. Louis, MO), 2 mM L-glutamine, 50 ⁇ M 2-ME, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin, 1 ⁇ g/ml gentamicin, 80 ng/ml SCF, 50 ng/ml IL-6 and 5 ng/ml IL-10. Cells were stained with anti-tryptase mAb to determine the percentage of mast cells.
  • GeneChip expression analysis was done using the Affymetrix expression assay service.
  • Affymetric's standard array (GeneChip Human Genome U133 Set) is composed of two microarrays containing over 1 million unique oligonucleotides covering more than 39,000 transcript variants which in turn represent greater than 33,000 human genes.
  • RNA from each of Mast cells, THPl, and PBMC cultures was used to obtain biotin- labeled cRNA.
  • Single-stranded cDNA was synthesized by reverse transcription using poly(A) RNA present in the total RNA sample and then converted to double-strnaded cDNA.
  • An in vitro transcription reaction was performed in the presence of biotinylated UTP and CTP to produce biotin-labeled cRNA.
  • cRNA was then fragmented in the presence of heat and Mg 2 *. The fragmented cRNA from each cell type was hybridized to a test array to assess target quality and labeling efficiency.
  • the test array was then washed, stained with streptavidin-phycoerythrin, and scanned using a GeneArray scanner. Images were analyzed using quality control parameters such as 375' ratio of housekeeping genes, presence of spiked control cRNA sequences, noise (Q values), scaling factors, etc. After assessment, the cRNA was hybridized to the U133 standard array for 16 hours at 45 °C. The array was then washed, stained with streptavidin-phycoerythrin, and scanned using a GeneArray scanner. The expression data obtained was extracted from black and white images using Affymetrix MAS 5.0 software, and statistical algorithms were employed to calculate a quantitative value (Signal Intensity) and a qualitative value (presence or absence) for each transcript on the array.
  • GPCR GeneBank accession no. NM_348078, BC030948, AF348078, AC068647 and other homologous sequences
  • RNAs were isolated to measure the level of GPR91 mRNA in the following cells: Daudi (a B lymphoblast cell line derived from Burkitt's lymphoma, ATCC No. CCL-213), THP-1 (a monocytic leukemia cell line, ATCC No. TIB202), HMC-1, (a mastoma cell line); peripheral blood mononuclear cells (PBMC); primary monocytes; primary B cells; primary neutrophils; in vitro cultured mast cells at week 8-9.
  • Daudi a B lymphoblast cell line derived from Burkitt's lymphoma, ATCC No. CCL-213
  • THP-1 a monocytic leukemia cell line, ATCC No. TIB202
  • HMC-1 a mastoma cell line
  • PBMC peripheral blood mononuclear cells
  • primary monocytes primary B cells
  • primary neutrophils in vitro cultured mast cells at week 8-9.
  • RNAs from the cells indicated above were used in reverse transcriptase reaction to generate first strand cDNAs, which were used as templates in quantitative PCR reactions to obtain the threshold amplification cycle ( ).
  • the C t was normalized using the control from 18S RNAs to obtain ⁇ C t .
  • ⁇ Q values were calculated by using the lowest expression level as the base, which were then converted to the values of relative expression difference.
  • the quantitative RT-PCR analysis showed that GPR91 mRNA was expressed at highest level in human mast cells, and was expressed at moderate levels in kidney, spleen, THP-1, and PBMC (Table 1).
  • GPR91 The coding sequence of GPR91 (SEQ ID NO 1) was amplified by PCR from mast cell RNA and cloned into pcDNA3.1TOPO (Invitrogen, Carlsbad, CA). The sequence of GPR91 expression construct was verified to be identical to NM_348078, BC030948, AF348078, and AC068647 (GenBank Accession Number). A number of mutant GPR91 expression constructs were constructed by inserting epitope tag at various position of the coding region. One of the insertion mutations, GPR91-20Flag (SEQ ID NO 3), showed much stronger activity than the wild type GPR91 in GPR91-luciferase reporter assay (Table 2).
  • GPR91-20Flag was generated by PCR SOEing (Ho et al, 1989 Gene 77:51-59; Horton et al, 1990 Biotechniques 8:528-535), which contains a Flag tag sequence inserted after the first 20 amino acid residues (SEQ ID NO 3 and SEQUENCE 2). TABLE 2 Luciferase assay of the wild type and tagged GPR91 expression constructs co-transfected with Luc reporter plasmids
  • EXAMPLE 4 ACTIVATION OF NFAT AND API SIGNALING PATHWAYS BY GPR91
  • the intracellular signaling pathways activated by GPR91 were investigated using a luciferase reporter assay. Transient transfection was performed using Lipofectamine 2000 system (Invitrogen, Carlsbad, CA). For Western blot analysis, twenty micrograms of plasmid DNA was transfected into 293T cells in a 100 mm tissue culture dish; and 40 hours later, the cells were harvested in PBS-based, enzyme-free cell dissociation buffer (Invitrogen) and processed as described in the following sections.
  • GPR91 expression construct was cotransfected in HMC-1 cells with Mercury Pathway Profiling reporter plasmids, or with luciferase reporter plasmids for monitering mast cell activation (DB BioScience Clontech, Polo Alto, CA) and a control luciferase plasmid, pRL-SChA.
  • the cells were harvested 40 hours later, and assayed for luciferase activity using the dual luciferase assay kit following manufacturer's protocol (Promega, Madison, WI).
  • the firefly luciferase reporter constructs for monitering mast cell activation were generated by inserting the PCR-amplif ⁇ ed promoter sequences from human IL8, IL13, TNF- , tryptase jSl, tryptase ⁇ 2, FceRIc. and genes into the promoterless luciferase reporter plasmid vector, TA-Luc (DB Bioscience Clontech). Those promoters were chosen because of their pivotal role in immune responses.
  • GPR91 activates the signaling pathway mediated through the intracellular calcium flux to calcineurin and GPR91 activation.
  • the well-known calcineurin inhibitor, cyclosporin A could block the NFAT activation by GPR91.
  • Table 4 the stimulatory effect of GPR91 on NFAT-Luc reporter was reduced by 82-87% at micro molar or submicro molar concentrations of cyclosporin A.
  • GPR91 ENHANCES THE CYTOKINE AND TRYPTASE PROMOTER ACTIVITY
  • the cytokine secretion and tryptase release are the hallmarks of mast cell activation, which are accompanied with increased de novo synthesis of the released proteins.
  • Melanophores are Xenopus cells containing pigment granules (melanosomes), whose movement in the cytoplasm is affected by GPCR activity and second messenger levels of cAMP and diacylglycerol When a GPCR is activated by its ligand, it in turn may activate Gq or Gs, which causes melanosomes undergoing rapid dispersion throughout the cell and the cell appearing dark.
  • Xenopus melanophores may be transfected with GPR91, and subject to agonist compound treatment. The melanosome movement and cell color changes may be detected by a microplate reader or video image system. (References: J. Biol. Chem. 1993, 268: 5957- 5964; J. Biol. Chem. 1999, 274: 8597-8603). This screening technique has been described in PCT WO92/01810 and is incorporated by reference.
  • the c. subunit of G protein in non-active state bound with GDP is tightly associated with ⁇ subunits that are targeted to plasma membrane.
  • Ligand binding to GPR91 triggers activation of the heterotrimeric G protein by virtue of guanine nucleotide exchange, converting the a subunit into a GTP-bound state which is recruited to GPR91.
  • the reaction was well documented being used to assess GPR91 and ligand interaction. Briefly, the membrane fraction of the cells transfected with a specific GPCR was isolated and incubated with a labeled GTP such as 35S-GTP ⁇ S (guanosine 5'-3-0-(thio) triphosphate) or Eu-GTP (PerkinElmer Life Science).
  • a labeled GTP such as 35S-GTP ⁇ S (guanosine 5'-3-0-(thio) triphosphate) or Eu-GTP (PerkinElmer Life Science).
  • This feature may be utilized to screen ligand or agonist compound if NFAT is linked to a reporter system, such as luciferase (DB BioSciences Clontech, Palo Alto, CA), j ⁇ -lactamase or green fluorescent protein (DB BioSciences Clontech, Palo Alto, CA). (Reference: Yang J. et al.2003, J. Biol. Chem. 278, in press).
  • Xenopus oocytes may be micro-injected with RNAs that encode for GPCR and potassium channels (Kir3.1-3.4). If a ligand binds to the GPCR expressed on the oocyte surface, it will activate the GPCR, resulting in the release of G ⁇ y subunits from the heterotrimeric G protein and subsequent activation of the potassium channel. The current may be recorded by electrographic recorder. (References: Nature 2001, 409: 202-205; J. Biol. Chem. 2000, 275: 30531-30536; J. Biol. Chem. 1995, 270: 29059- 29062). G. TITLE
  • Another method involves co-transfecting HEK-293 cells with a mammalian expression plasmid encoding a GPR91, along with a mixture comprised of mammalian expression plasmids cDNAs encoding GUI 5 (Wilkie T. M. et al Proc Natl Acad Sci USA 1991 88: 10049-10053), GU16 (Amatruda T. T. et al Proc Natl Acad Sci USA 1991 8: 5587-5591, and three chimeric G-proteins refered to as Gqi5, Gqs5, and Gqo5 (Conklin B R et al Nature 1993 363: 274-276, Conklin B. R.
  • transfected HEK-293 cells are plated into poly-D-lysine coated 96 well black/clear plates (Becton Dickinson, Bedford, Mass.).
  • the cells are assayed on FLIPR (Fluorescent Imaging Plate Reader, Molecular Devices, Sunnyvale, Calif.) for a calcium mobilization response following addition of test ligands.
  • FLIPR Fluorescent Imaging Plate Reader, Molecular Devices, Sunnyvale, Calif.
  • subsequent experiments are performed to determine which, if any, G-protein is required for the functional response.
  • HEK-293 cells are then transfected with GPR91, or co-transfected with GPR91 and G015, GD16, GqiS, Gqs5, or Gqo5.
  • GPR91 requires the presence of one of the G-proteins for functional expression in HEK-293 cells, all subsequent experiments are performed with HEK-293 cell cotransfected with the G- protein which gives the best response.
  • the receptor can be expressed in a different cell line, for example RBL-2H3, without additional G proteins.
  • a calcium flux assay was performed using a commercially available Calcium 3 Assay kit and FlexStation II (Molecular Devices, Sunnyvale, CA). The results showed that succinate stimulated calcium flux in LAD2 cells in a dose-dependent manner, and the effective concentration at 50% activation level (EC50) was estimated at ⁇ 180 ⁇ M (Table 7). Similarly, succinate induced calcium flux in CBMC (data not shown). In contrast, succinate did not trigger any calcium flux in HMC-1 cells (Table 7), which was attributed to the lack of expression of GPR91 in these cells. These finding indicated that succinate activated calcium flux in mast cells through a GPR91 -mediated signaling pathway.

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Abstract

La présente invention se rapporte à GPR91, un polypeptide de type récepteur purinergique, à son expression dans des mastocytes, et à son utilisation pour le diagnostic et/ou le traitement de maladies médiées par des mastocytes, et notamment l'asthme allergique et non allergique, les broncho-pneumopathies chroniques obstructives (COPD), les rhinites allergiques, l'anaphylaxie, les maladies gastro-intestinales allergiques, les dermatites atopiques, la polyarthrite rhumatoïde et d'autres maladies allergiques, auto-immunes et inflammatoires. L'invention se rapporte également à des procédés de criblage d'agonistes et/ou d'antagonistes du récepteur GPR91.
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CN111781369A (zh) * 2019-04-03 2020-10-16 复旦大学 琥珀酸gpr-91受体作为分子靶点在制药中的用途

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WO2001098351A2 (fr) * 2000-06-16 2001-12-27 Incyte Genomics, Inc. Recepteurs couples a la proteine g
US20020137887A1 (en) * 2001-01-17 2002-09-26 Hedrick Joseph A. Adenosine receptor
WO2005050220A1 (fr) * 2003-10-31 2005-06-02 Bayer Healthcare Ag Diagnostics et traitements de maladies associees au recepteur 91 couple aux proteines g (gpr91)

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WO2001098351A2 (fr) * 2000-06-16 2001-12-27 Incyte Genomics, Inc. Recepteurs couples a la proteine g
US20020137887A1 (en) * 2001-01-17 2002-09-26 Hedrick Joseph A. Adenosine receptor
WO2005050220A1 (fr) * 2003-10-31 2005-06-02 Bayer Healthcare Ag Diagnostics et traitements de maladies associees au recepteur 91 couple aux proteines g (gpr91)

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YANG J ET AL: "Calcineurin/nuclear factors of activated T cells (NFAT)-activating and immunoreceptor tyrosine-based activation motif (ITAM)-containing protein (CNAIP), a novel ITAM-containing protein that activates the calcineurin/NFAT-signaling pathway" JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY OF BIOLOCHEMICAL BIOLOGISTS, BIRMINGHAM,, US, vol. 278, no. 19, 9 May 2003 (2003-05-09), pages 16797-16801, XP002273544 ISSN: 0021-9258 *

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