EP1328549A1 - Für die humane prostata spezifischer g-protein-rezeptor hpraj70 - Google Patents

Für die humane prostata spezifischer g-protein-rezeptor hpraj70

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Publication number
EP1328549A1
EP1328549A1 EP01977303A EP01977303A EP1328549A1 EP 1328549 A1 EP1328549 A1 EP 1328549A1 EP 01977303 A EP01977303 A EP 01977303A EP 01977303 A EP01977303 A EP 01977303A EP 1328549 A1 EP1328549 A1 EP 1328549A1
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EP
European Patent Office
Prior art keywords
psgr
replaced
polypeptide
amino acid
seq
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Withdrawn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
EP01977303A
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English (en)
French (fr)
Other versions
EP1328549A4 (de
Inventor
Daniel R. Soppet
Yi Li
Craig A. Rosen
Steven M. Ruben
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Human Genome Sciences Inc
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Human Genome Sciences Inc
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Publication of EP1328549A1 publication Critical patent/EP1328549A1/de
Publication of EP1328549A4 publication Critical patent/EP1328549A4/de
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)

Definitions

  • the present invention relates to PSGR, a novel prostate specific gene with homology to a G-protein coupled receptor overexpressed in prostate cancer. More specifically, the invention relates to PSGR polynucleotides and the polypeptides encoded by these polynucleotides, and the use of PSGR polynucleotides and polypeptides for detecting disorders of the reproductive system, including disorders of the prostate, particularly the presence of cancer. This invention relates to PSGR polynucleotides and polypeptides as well as vectors, host cells, antibodies directed to PSGR polynucleotides and polypeptides and recombinant and synthetic methods for producing the same.
  • diagnostic methods for diagnosing and treating, preventing and/or prognosing disorders related to the prostate including cancer, and therapeutic methods for treating such disorders.
  • the invention further relates to screening methods for identifying agonists and antagonists of PSGR polynucleotides and polypeptides of the invention.
  • the present invention further relates to methods and/or compositions for inhibiting or enhancing the production and/or function of the PSGR polypeptides of the present invention.
  • Prostate cancer is the most common form of cancer among males, with an estimated incidence of 30% in men over the age of 50. Overwhelming clinical evidence shows that human prostate cancer has the propensity to metastasize to bone, and the disease appears to progress inevitably from androgen dependent to androgen refractory status, leading to increased patient mortality. This prevalent disease is currently the second leading cause of cancer death among men in the U.S.
  • PSA prostate specific antigen
  • PAP prostatic acid phosphatase
  • G-protein coupled receptors constitute a major class of proteins responsible for transducing a signal within a cell.
  • GPCRs have three structural domains: an amino terminal extracellular domain; a transmembrane domain containing seven transmembrane segments, three extracellular loops, and three intracellular loops; and a carboxy terminal intracellular domain.
  • a signal is transduced within the cell that results in a change in a biological or physiological property of the cell.
  • GPCRs, along with G-proteins and effectors are the components of a modular signalling system that connects the state of intracellular second messangers to extracellular inputs.
  • GPCRs are a major target for drug action and development. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown GPCRs.
  • the present invention advances the state of the art by providing a previously unidentified prostate specific seven transmembrane GPCR.
  • the present invention provides isolated nucleic acid molecules comprising, or alternatively, consisting of, a polynucleotide encoding at least a portion of PSGR.
  • the present invention provides isolated nucleic acid molecules comprising, or alternatively, consisting of, a polynucleotide encoding PSGR having the amino acid sequence shown in
  • Figures 1A-B SEQ ID NO:2; or the amino acid sequence encoded by the cDNA clone
  • the present invention further provides isolated nucleic acid molecules comprising, or alternatively consisting of, a polynucleotide encoding PSGR having the amino acid sequence shown in Figures 2A-B (SEQ ID NO:4).
  • the present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them for production of PSGR polypeptides or peptides by recombinant techniques.
  • the invention further provides an isolated PSGR polypeptide having an amino acid sequence encoded by a polynucleotide described herein.
  • the present invention also provides diagnostic assays such as quantitative and diagnostic assays for detecting levels of PSGR protein.
  • diagnostic assays such as quantitative and diagnostic assays for detecting levels of PSGR protein.
  • a diagnostic assay in accordance with the invention for detecting over-expression of PSGR, or soluble form thereof, compared to normal control tissue samples may be used to detect the presence of tumors.
  • GPCR genes and gene products are potential causative agents of disease (Spiegel et al, J. Clin. Invest. 92:1119-1125 (1993); McKusick et al., J. Med. Genet. 30:1-26 (1993)).
  • the invention further provides cells which express the PSGR polypeptide with a candidate compound and its ligand, assaying for inhibiting PSGR mediated signalling induced by said ligand which involves administering to a cell which expresses the PSGR polypeptide an effective amount of a PSGR agonist capable of decreasing PSGR mediated signalling.
  • the present invention is directed to a method for enhancing PSGR mediated signalling induced by its ligand which involves administering to a cell which expresses the PSGR polypeptide an effective amount of a PSGR agonist or antagonist capable of increasing PSGR mediated signalling.
  • any candidate "agonist” or “antagonist” of the present mvention can enhance or inhibit PSGR mediated signalling can be determined using art-known G-protein coupled ligand/receptor cellular response assays, including those well known in the art.
  • a screening method is provided for determining whether a candidate agonist or antagonist is capable of enhancing or inhibiting a PSGR mediated cellular response to a ligand.
  • the method involves contacting cellular response, and comparing the cellular response to a standard cellular response, the standard being assayed when contact is made with the ligand in absence of the candidate compound, whereby an increased cellular response over the standard indicates that the candidate compound is an agonist of the ligand/receptor signaling pathway and a decreased cellular response compared to the standard indicates that the candidate compound is an antagonist of the ligand/receptor signaling pathway.
  • a cell expressing the PSGR polypeptide can be contacted with either an endogenous or exogenously administered ligand.
  • Figures 1A-B shows the nucleotide (SEQ ID NO:l) and deduced amino acid sequence (SEQ ID NO:2) of the PSGR protein. It is predicted that amino acids 1 to 21 constitute the amino terminal extracellular domain and amino acids 294 to 320 constitute the carboxy terminal intracellular domain. The region spanning the entire transmembrane domain is from about amino acids 22 to about amino acid 293.
  • the seven transmembrane segments are as follows: from about amino acid 22 to about amino acid 42, from about amino acid 56 to about amino acid 76, from about amino acid 99 to about amino acid 119, from about amino acid 142 to about amino acid 162, from about amino acid 198 to about amino acid 218, from about amino acid 241 to about amino acid 261, and from about amino acid 273 to about amino acid 293.
  • the amino acids corresponding to the three extracellular loops are as follows: from about amino acid 77 to about 98, from about amino acid 163 to about amino acid 197, and from about amino acid 262 to about amino acid 272.
  • amino acids corresponding to the three intracellular loops are as follows: from about amino acid 43 to about amino acid 55, from about amino acid 120 to about amino acid 141, and from about amino acid 219 to about amino acid 240.
  • the conserved intracellular G protein signature sequence, "DRY" is found at amino acids 120 to 122. This sequence is implicated in signal transduction.
  • Figures 2A-B shows the nucleotide (SEQ ID NO: 3) and deduced amino acid sequence (SEQ ID NO:4) of a variant PSGR protein. It is predicted that amino acids 1 to 21 constitute the amino terminal extracellular domain and amino acids 294 to 320 constitute the carboxy terminal intracellular domain. The region spanning the entire transmembrane domain is from about amino acids 22 to about amino acid 293.
  • the seven transmembrane segments are as follows: from about amino acid 22 to about amino acid 42, from about amino acid 56 to about amino acid 76, from about amino acid 99 to about amino acid 119, from about amino acid 142 to about amino acid 162, from about amino acid 198 to about amino acid 218, from about amino acid 241 to about amino acid 261, and from about amino acid
  • the amino acids corresponding to the three extracellular loops are as follows: from about amino acid 77 to about 98, from about amino acid 163 to about amino acid 197, and from about amino acid 262 to about amino acid 272.
  • the amino acids corresponding to the three intracellular loops are as follows: from about amino acid 43 to about amino acid 55, from about amino acid 120 to about amino acid 141, and from about amino acid 219 to about amino acid 240.
  • the conserved intracellular G protein signature sequence, "DRY" is found at amino acids 120 to 122. This sequence is implicated in signal transduction.
  • Figure 3 shows the regions of similarity between the amino acid sequences of the PSGR (SEQ ID NO:2), and the human HGMP071 olfactory receptor (SEQ ID NO:5). Regions of identity are shaded.
  • Figure 4 shows an analysis of the PSGR amino acid sequence.
  • Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown, and all were generated using the default settings.
  • the positive peaks indicate locations of the highly antigenic regions of the PSGR protein, i.e., regions from which epitope-bearing peptides of the invention can be obtained.
  • the domains defined by these graphs are contemplated by the present invention.
  • amino acid residues T-50 to H-55, D-87 to E-90, L-227 to R-234, and S-309 to D-313 in Figures 1A-B correspond to the shown highly antigenic regions of the PSGR protein.
  • the present invention provides isolated nucleic acid molecules comprising, or alternatively consisting of, a polynucleotide encoding a PSGR polypeptide having the amino acid sequence shown in Figures 1A-B (SEQ ID ⁇ O:2).
  • the PSGR polypeptides of the present invention share sequence homology with G protein coupled odorant receptor family members ( Figure 3). Specifically, PSGR shows the closest similarity to a human OR gene, HPFHIOR.
  • the nucleotide sequence shown in Figures 1A-B (SEQ ID NO:l) was obtained by sequencing the cDNA clone HPRAJ70, which was deposited at the American Type Culture Collection, and given Accession Number 97131. The deposited HPRAJ70 clone is inserted in the UniZAP XR plasmid (Stratagene Cloning Systems, Inc.) using the EcoRI and Xhol restriction endonuclease cleavage sites.
  • the present invention further provides nucleic acid molecules comprising, or alternatively consisting of, a polynucleotide encoding a PSGR polypeptide having the amino acid sequence shown in Figures 2A-B (SEQ ID NO:4).
  • NO:l contains an open reading frame of 963 nucleotide base pairs encoding a protein of about 320 amino acid residues, and a deduced molecular weight of about 35.4 kDa.
  • PSGR polypeptides of the invention share the greatest degree of homology with human G protein coupled odorant family. Specifically, PSGR showed the closest similarity to a human
  • HPFHIOR SEQ ID NO:5 (see, Figure 3), which was mapped to the beta-globin gene cluster on chromosome llpl5.5.
  • the PSGR protein contains seven trans-membrane domains between amino acid residues 24 to 293 in SEQ ID NO:2 that are characteristic of G- protein coupled receptors.
  • PSGR polynucleotides as used herein is meant to encompass both of the above described PSGR polynucleotide variants and fragments thereof.
  • PSGR polypeptides as used herein is meant to encompass polypeptides encoded by the above described variants, and fragments thereof.
  • PSGR predominantly localized to epithelial cells of gland (data not shown).
  • the invention further provides polypeptides having various residues deleted from the N-terminus and/or C-terminus of the complete
  • PSGR including polypeptides lacking one or more amino acids from the N-termini of the
  • PSGR extracellular domains described herein which constitute soluble forms of the extracellular domain of the PSGR polypeptides respectively.
  • nucleic acid molecules of the present invention may be in the form of
  • RNA such as mRNA
  • DNA in the form of DNA, including, for instance, cDNA and genomic
  • DNA obtained by cloning or produced synthetically.
  • the DNA may be double-stranded or single-stranded.
  • Single-stranded DNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti-sense strand.
  • isolated nucleic acid molecule(s) is intended a nucleic acid molecule, DNA or
  • RNA which has been removed from its native environment.
  • recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention.
  • Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution.
  • Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules of the present invention.
  • Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.
  • a nucleic acid molecule contained in a clone that is a member of a mixed clone library e.g., a genomic or cDNA library
  • a chromosome isolated or removed from a cell or a cell lysate e.g., a cell lysate
  • chromosome spread as in a karyotype
  • Isolated nucleic acid molecules of the present invention include DNA molecules comprising an open reading frame (ORF) shown in Figures 1A-B (SEQ ID NO:l); DNA molecules comprising the coding sequence for the complete (full-length) PSGR protein shown in Figures 1A-B (SEQ ID NO:2); DNA molecules comprising an open reading frame
  • the genetic code is well known in the art. Thus, it would be routine for one skilled in the art to generate such degenerate variants.
  • the invention provides isolated nucleic acid molecules having a polynucleotide sequence encoding the PSGR polypeptide having an amino acid sequence as encoded by a cDNA clone contained in the plasmid deposited as ATCC Deposit No. 97131.
  • the invention further provides an isolated nucleic acid molecule having the nucleotide sequence shown in Figures 1A-B (SEQ ID NO:l), an isolated nucleic acid molecule having the nucleotide sequence shown in Figures 2A-B (SEQ ID NO:3), or a nucleic acid molecule having a sequence complementary to one of the above sequences.
  • isolated molecules particularly DNA molecules, are useful, for example, as probes for gene mapping by in situ hybridization with chromosomes, and for detecting expression of the PSGR gene in human tissue (e.g., in prostate tissue or prostate cancer tissue), for instance, by hybridization (e.g.,
  • the present invention is further directed to fragments of the isolated nucleic acid molecules described herein.
  • a fragment of an isolated DNA molecule having the nucleotide sequence of the deposited cDNA or the nucleotide sequence shown in Figures 1 A- B (SEQ ID NO:l) or the nucleotide sequence shown in Figures 2A-B (SEQ ID NO:3) is intended DNA fragments at least about 15nt, and more preferably at least about 20 nt, at least about 24 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt, at least about 50 nt, at least about 100 nt, at least about 150 nt, at least about 200 nt, at least about 250 nt, at least about 300 nt in length which are useful, for example, as diagnostic probes and primers as discussed herein.
  • fragments 350-1500 nt in length are also useful according to the present invention, as are fragments corresponding to most, if not all, of the nucleotide sequence of the deposited cDNA plasmids, or as shown in Figures 1A-B (SEQ ID NO:l), or as shown in Figures 2A-B (SEQ ID NO:3), or the complementary strand thereto.
  • fragments which include 20 or more contiguous bases from the nucleotide sequence of the deposited cDNA or the nucleotide sequence as shown in Figures 1A-B (SEQ ID NO:l) or the nucleotide sequence as shown in Figures 2A-B (SEQ ID ,NO:3).
  • “about” includes the particularly recited size, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini.
  • PSGR polynucleotide fragments of the invention include, for example, fragments that comprise, or alternatively, consist of, a sequence from about nucleotide 274 to 336, 337 to 399, 400 to 438, 439 to 501, 502 to 567, 568 to 630, 631 to 696, 697 to 759, 760 to 864, 865 to 927, 928 to 993, 994 to 1056, 1057 to 1089, 1090 to 1152, 1153 to 1233 of Figures 1 A-B (SEQ ID NO: 1), or Figures 2A-B (SEQ ID NO:3) or the complementary strand thereto, or the cDNA contained in one of the deposited cDNA clones.
  • “about” includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini.
  • the polynucleotide fragments of the invention encode a polypeptide which demonstrates a PSGR functional activity.
  • a polypeptide demonstrating a PSGR “functional activity” is meant, a polypeptide capable of displaying one or more known functional activities associated with a full-length (complete) PSGR protein.
  • Such functional activities include, but are not limited to, biological activity, antigenicity (ability to bind (or compete with a PSGR polypeptide for binding) to an anti-PSGR antibody), immunogenicity (ability to generate antibody which binds to a PSGR polypeptide), ability to form multimers with PSGR polypeptides of the invention, and ability to bind to a receptor or ligand for a PSGR polypeptide.
  • PSGR polypeptides and fragments, variants derivatives, and analogs thereof, can be assayed by various methods.
  • immunoassays known in the art can be used, including but not limited to, competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc.
  • ELISA enzyme linked immunosorbent assay
  • sandwich immunoradiometric assays immunoradiometric assays
  • gel diffusion precipitation reactions immunodiffusion assays
  • in situ immunoassays using colloidal gold,
  • antibody binding is detected by detecting a label on the primary antibody.
  • the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody.
  • the secondary antibody is labeled.
  • binding can be assayed, e.g., by means well-known in the art, such as, for example, reducing and non-reducing gel chromatography, protein affinity chromatography, and affinity blotting. See generally, Phizicky, E., et ah, Microbiol. Rev. 59:94-123 (1995).
  • physiological correlates of PSGR binding to its substrates can be assayed.
  • assays described herein may routinely be applied to measure the ability of PSGR polypeptides and fragments, variants derivatives and analogs thereof to elicit PSGR related biological activity.
  • techniques described herein and otherwise known in the art may be applied or routinely modified to assay for the ability of the compositions of the invention to bind to the PSGR ligand or couple to G protein.
  • Preferred nucleic acid fragments of the present invention include nucleic acid molecules encoding a member selected from the group: a polypeptide comprising or alternatively, consisting of, the PSGR transmembrane domain I (amino acid residues from about 22 to about 42 in Figures 1A-B (SEQ ID NO:2) or Figures 2A-B (SEQ ID NO:4); a polypeptide comprising, or alternatively consisting of, the PSGR transmembrane domain II (amino acid residues from about 56 to about 76 in Figures 1A-B (SEQ ID NO:2) or Figures 2A-B (SEQ ID NO:4); a polypeptide comprising, or alternatively consisting of the PSGR transmembrane domain III (amino acid residues from about 99 to about 119 in Figures 1A-B (SEQ ID NO:2) or Figures 2A-B (SEQ ID NO:
  • Preferred nucleic acid fragments of the invention encode a full-length PSGR polypeptide lacking the nucleotides encoding the amino terminal methionine in Figures 1 A-B
  • nucleic acid fragments of the present invention further include nucleic acid molecules encoding epitope-bearing portions of the PSGR receptor proteins.
  • nucleic acid fragments of the present invention include nucleic acid molecules encoding: a polypeptide comprising amino acid residues from about 50 to about 55 in Figures
  • Figures 1A-B SEQ ID NO:2 or Figures 2A-B (SEQ ID NO:4); and a polypeptide comprising amino acid residues from about 309 to about 313 in Figures 1A-B (SEQ ID NO:2) or Figures 2A-B (SEQ ID NO:4); and a polypeptide comprising amino acid residues from about 309 to about 313 in Figures 1A-B (SEQ ID NO:2) or Figures 2A-B (SEQ ID NO:4); and a polypeptide comprising amino acid residues from about 309 to about 313 in Figures 1A-B (SEQ ID NO:2) or Figures 2A-B (SEQ ID NO:4); and a polypeptide comprising amino acid residues from about 309 to about 313 in Figures 1A-B (SEQ ID NO:2) or Figures 2A-B (SEQ ID NO:4); and a polypeptide comprising amino acid residues from about 309 to about 313 in Figures 1A-B (SEQ ID
  • the polynucleotides of the invention encode functional attributes of PSGR.
  • Preferred embodiments of the invention in this regard include fragments that comprise alpha-helix and alpha-helix forming regions ("alpha-regions"), beta-sheet and beta-sheet forming regions ("beta-regions"), turn and turn-forming regions ("turn-regions”), coil and coil-forming regions ("coil-regions”), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index regions of PSGR.
  • Figure 4 and/or Table I was generated using the various modules and algorithms of the DNA* STAR set on default parameters.
  • the data presented in columns VIII, XII, and XIII of Table I can be used to determine regions of
  • Regions of high antigenicity are determined from the data presented in columns VIII, XII, and/or XIII by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.
  • Table I be represented or identified by using tabular representations of the data presented in
  • Figure 4 The DNA*STAR computer algorithm used to generate Figure 4 (set on the original default parameters) was used to present the data in Figure 4 in a tabular format (See Table I).
  • the tabular format of the data in Figures 4 may be used to easily determine specific boundaries of a preferred region.
  • the above-mentioned preferred regions set out in Figure 4 and in Table I include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequences set out in Figures 1A-B. As set out in Figure 4 and in Table I, such preferred regions include Garnier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions,
  • the invention provides an isolated nucleic acid molecule comprising a polynucleotide which hybridizes under stringent hybridization conditions to a portion of the polynucleotide in a nucleic acid molecule of the invention described above, for instance, the cDNA clone contained in ATCC Deposit No. 97131, or the complementary strand of nucleotides of SEQ ID NO:l, or the complementary strand of nucleotides of SEQ
  • stringent hybridization conditions is intended overnight incubation at 42°C in a solution comprising: 50% formamide, 5x SSC (750 mM NaCl, 75mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 g/ml denatured, sheared salmon sperm DNA, followed by washing the filters in O.lx SSC at about
  • Polypeptides encoded by these nucleic acids are also encompassed by the invention.
  • a polynucleotide which hybridizes to a "portion" of a polynucleotide is intended a polynucleotide (either DNA or RNA) hybridizing to at least about 15 nucleotides (nt), and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably about 30-70 nt of the reference polynucleotide.
  • nt nucleotides
  • nucleotide sequence of the reference polynucleotide e.g., the deposited cDNA or the nucleotide sequence as shown in Figures 1 A-
  • a polynucleotide which hybridizes only to a poly A sequence (such as the
  • Figures 2A-B (SEQ ID NO:3), or to a complementary stretch of T (or U) resides, would not be included in a polynucleotide of the invention used to hybridize to a portion of a nucleic acid of the invention, since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone generated using oligo dT as a primer).
  • the polynucleotides of the invention are less than 110000 kb, 50000 kb, 10000 kb, 1000 kb, 500 kb, 400 kb, 350 kb, 300 kb, 250 kb, 200 kb, 175 kb,
  • polynucleotides of the invention comprise at least 15, at least 30, at least 50, at least 100, or at least 250, at least 500, or at least 1000 contiguous nucleotides of PSGR coding sequence, but consist of less than or equal to 107 kb, 75 kb, 50 kb, 30 kb, 25 kb, 20 kb, 15 kb, 10 kb, or 5 kb of genomic DNA that flanks the 5' or 3' coding nucleotide set forth in Figures 1A-B (SEQ ID NO:l) or Figures 2A-B (SEQ ID NO:3).
  • polynucleotides of the invention comprise at least 15, at least 30, at least 50, at least 100, or at least 250, at least 500, or at least 1000 contiguous nucleotides of
  • the nucleic acid comprising PSGR coding sequence does not contain coding sequences of a genomic flanking gene (i.e., 5 ' or 3' to the PSGR gene in the genome).
  • the polynucleotides of the invention do not contain the coding sequence of more than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1 genomic flanking gene(s).
  • nucleic acid molecules of the present invention which encode a PSGR polypeptide may include, but are not limited to, the coding sequence for the mature polypeptide, by itself; the coding sequence for the mature polypeptide and additional sequences, such as those encoding a.leader or secretory sequence, such as a pre-, or pro- or prepro- protein sequence; the coding sequence of the mature polypeptide, with or without the aforementioned additional coding sequences, together with additional, non-coding sequences, including for example, but not limited to introns and non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA processing - including splicing and polyadenylation signals, for example - ribosome binding and stability of mRNA; additional coding sequence which codes for additional amino acids, such as those which provide additional functionalities.
  • the polypeptide may be fused to a marker sequence, such as a peptide
  • the marker sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (Qiagen, Inc.), among others, many of which are commercially available. As described in Gentz et al, Proc. Natl.
  • hexa-histidine provides for convenient purification of the fusion protein.
  • the "HA” tag is another peptide useful for purification which corresponds to an epitope derived from the influenza hemagglutinin protein, which has been described by Wilson et al, Cell 37:767-778 (1984).
  • other such fusion proteins include the PSGR receptor fused to Fc at the N- or C-terminus.
  • the present invention further relates to variants of the nucleic acid molecules of the present invention, which encode portions, analogs, or derivatives of the PSGR receptor.
  • Variants may occur naturally, such as a natural allelic variant.
  • allelic variant is intended one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985). Non- naturally occurring variants may be produced using art-known mutagenesis techniques.
  • variants include those produced by nucleotide substitutions, deletions or additions which may involve one or more nucleotides.
  • the variants may be altered in coding or non-coding regions or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions, or additions. Especially preferred among these are silent substitutions, additions, and deletions, which do not alter the properties and activities of the PSGR receptor or portions thereof. Also especially preferred in this regard are conservative substitutions.
  • nucleic acid molecules comprising, or alternatively consisting of, a polynucleotide having a nucleotide sequence at least 90% identical, and more preferably at least 95%, 96%, 97%, 98%, or 99% identical to:
  • Figures 1A-B SEQ ID NO:2 or Figures 2A-B (SEQ ID NO:4); (b) a nucleotide sequence encoding the polypeptide having the amino acid sequence in Figures 1 A-B (SEQ ID NO:2) or
  • Figures 2A-B (SEQ ID NO:4), but lacking the amino terminal methionine; (c) a nucleotide sequence encoding the polypeptide having the amino acid sequence encoded by a cDNA clone contained in ATCC Deposit No.
  • PSGR with one, two, three, four, five, six, or all seven of the transmembrane domains deleted; (m) a nucleotide sequence encoding PSGR extracellular loop I; (n) a nucleotide sequence encoding PSGR extracellular loop II; (o) a nucleotide sequence encoding PSGR extracellular loop DI; (p) a nucleotide sequence encoding one, two, or all three of the PSGR extracellular loops; (q) a nucleotide sequence encoding PSGR intracellular loop I; (r) a nucleotide sequence encoding PSGR intracellular loop D; (s) a nucleotide sequence encoding
  • PSGR intracellular loop DI and (t) a nucleotide sequence encoding any combination of thenucleotide sequences in (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (1), (m), (n), (o), (p),
  • the invention also encompasses a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), (h), (i), (j),
  • nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five mismatches per each 100 nucleotides of the reference nucleotide sequence encoding the PSGR polypeptide.
  • a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.
  • These mismatches of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
  • the reference (query) sequence may be the entire
  • PSGR polynucleotide fragment e.g., a polynucleotide encoding the amino acid sequence of any of the PSGR N- and/or C- terminal deletions described herein, variant, derivative or analog, as described herein.
  • Figures 1A-B SEQ ID NO:2 or Figures 2A-B (SEQ ID NO:4) or to the nucleotide sequence of the deposited cDNA clone can be determined conventionally using known computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for
  • the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.
  • the identity between a reference (query) sequence (a sequence of the present invention) and a subject sequence is determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)).
  • Preferred parameters used in a FASTDB alignment of DNA sequences to calculate percent identity are:
  • the percent identity is corrected by calculating the number of bases of the query sequence that are 5' and 3' of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. A determination of whether a nucleotide is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This corrected score is what is used for the purposes of this embodiment. Only bases outside the 5' and 3' bases of the subject sequence, as displayed by the FASTDB alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the percent identity score.
  • a 90 base subject sequence is aligned to a 100 base query sequence to determine percent identity.
  • the deletions occur at the 5' end of the subject sequence and therefore, the FASTDB alignment does not show a matched/alignment of the first 10 bases at
  • the 10 unpaired bases represent 10% of the sequence (number of bases at the 5' and
  • FASTDB is not manually corrected. Once again, only bases 5' and 3' of the subject sequence which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are made for the purposes of this embodiment.
  • nucleic acid molecules comprising, or alternatively consisting of a nucleotide sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence for example, shown in Figures 1 A-B (SEQ ID NO:2) or
  • Figures 2 A-B (SEQ ID NO:4), or to the nucleic acid sequence of a deposited cDNA, irrespective of whether they encode a polypeptide having PSGR receptor activity. This is because even where a particular nucleic acid molecule does not encode a polypeptide having PSGR receptor activity. This is because even where a particular nucleic acid molecule does not encode a polypeptide having PSGR receptor activity.
  • PSGR functional activity one of skill in the art would still know how to use the nucleic acid molecule, for instance, as a hybridization probe or a polymerase chain reaction (PCR) primer.
  • PCR polymerase chain reaction
  • nucleic acid molecules of the present invention that do not encode a polypeptide having PSGR activity include, inter alia: (1) isolating the PSGR gene or allelic variants thereof in a cDNA library; (2) in situ hybridization (e.g., "FISH") to metaphase chromosomal spreads to provide precise chromosomal location of the PSGR gene, as described in Verma et al, Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York
  • PSGR mRNA expression in specific tissues e.g., normal prostate or prostate cancer tissues.
  • nucleic acid molecules comprising, or alternatively consisting of, a nucleotide sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to for example, the nucleic acid sequence shown in Figures 1 A-B (SEQ ID NO:2) or Figures 2A-B
  • a polypeptide having PSGR functional activity is intended polypeptides exhibiting activity similar, but not necessarily identical, to an activity of the PSGR of the invention, as measured in a particular biological assay.
  • Figures 1A-B (SEQ ID NO:2) or Figures 2A-B (SEQ ID NO:4), will encode a polypeptide
  • nucleic acid molecules having a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to, for example, a nucleic acid sequence centered in the deposited cDNA or the nucleic acid sequence shown in
  • Figures 1A-B (SEQ ID NO:l) or Figures 2A-B (SEQ ID NO:3) will encode a polypeptide
  • This invention is also related to the use of PSGR polynucleotides to detect complementary polynucleotides such as, for example, as a diagnostic reagent. Detection of a normal and mutated form of PSGR associated with a dysfunction will provide a diagnostic tool that can add or define a diagnosis of a disease or susceptibility to a disease which results from under-expression over-expression or altered expression of PSGR (or a soluble form thereof), such as, for example, tumors or autoimmune disease.
  • PSGR polynucleotides of the invention are used to detect complementary polynucleotides as a diagnostic reagent for the detection of prostate cancer.
  • Individuals carrying mutations in the PSGR gene may be detected at the DNA level by a variety of techniques. Nucleic acids for diagnosis may be obtained from a biological sample from a patient (e.g., a patient's cells, such as from urine, semen, blood, saliva, tissue biopsy and autopsy material). The genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR prior to analysis. (Saiki et al., Nature 324:163-166 (1986)). RNA or cDNA may also be used in the same ways.
  • PCR primers complementary to the nucleic acid encoding PSGR can be used to identify and analyze PSGR expression and mutations. For example, deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to radiolabeled PSGR RNA or alternatively, radiolabeled PSGR antisense DNA sequences. Perfectly matched sequences can routinely be distinguished from mismatched duplexes by techniques known in the art, such as, for example, RNase A digestion or by differences in melting temperatures.
  • Sequence differences between a reference gene and genes having mutations also may be revealed by direct DNA sequencing.
  • cloned DNA segments may be employed as probes to detect specific DNA segments.
  • the sensitivity of such methods can be greatly enhanced by appropriate use of PCR or another amplification method.
  • a sequencing primer is used with double-stranded PCR product or a single-stranded template molecule generated by a modified PCR.
  • the sequence determination is performed by conventional procedures with radiolabeled nucleotide or by automatic sequencing procedures with fluorescent-tags.
  • DNA sequence differences may be achieved by detection of alteration in electrophoretic mobility of DNA fragments in gels, with or without denaturing agents. Small sequence deletions and insertions can be visualized by high resolution gel electrophoresis using techniques known in the art. DNA fragments of different sequences may be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e.g., Myers et al, Science 230:1242
  • Sequence changes at specific locations also may be revealed by nuclease protection assays, such as RNase and SI protection or the chemical cleavage method (e.g., Cotton et al,
  • the detection of a specific DNA sequence may be achieved by methods which include, but are not limited to, hybridization, RNase protection, chemical cleavage, direct
  • restriction enzymes e.g., restriction fragment length polymorphisms ("RFLP") and Southern blotting of genomic DNA.
  • RFLP restriction fragment length polymorphisms
  • the present invention also relates to vectors which include the isolated DNA molecules of the present invention, host cells which are genetically engineered with the recombinant vectors and/or nucleic acids of the invention, and the production of PSGR polypeptides or fragments thereof by recombinant and synthetic techniques.
  • Host cells can be genetically engineered to incorporate nucleic acid molecules and express polypeptides of the present invention.
  • the polynucleotides may be introduced alone or with other polynucleotides. Such other polynucleotides may be introduced independently, co-introduced or introduced joined to the polynucleotides of the invention.
  • the vector may be, for example, a plasmid vector, a single or double-stranded phage vector, a single or double-stranded RNA or DNA viral vector.
  • Such vectors may be introduced into cells as polynucleotides, preferably DNA, by well known techniques for introducing DNA and RNA into cells.
  • Viral vectors may be replication competent or replication defective. In the latter case viral propagation generally will occur only in complementing host cells.
  • vectors are those for expression of polynucleotides and polypeptides of the present invention.
  • such vectors comprise cis-acting control regions effective for expression in a host operatively linked to the polynucleotide to be expressed.
  • Appropriate trans-acting factors either are supplied by the host, supplied by a complementing vector or supplied by the vector itself upon introduction into the host.
  • the polynucleotides may be joined to a vector containing a selectable marker for propagation in a host.
  • a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
  • the DNA insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp, phoA and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters will be known to the skilled artisan.
  • the expression constructs will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation.
  • the coding portion of the mature transcripts expressed by the constructs will preferably include a translation initiating at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.
  • the expression vectors will preferably include at least one selectable marker.
  • markers include dihydrofolate reductase, G418 or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria.
  • Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession
  • insect cells such as Drosophila S2 and Spodoptera Sf9 cells
  • animal cells such as CHO, COS and Bowes melanoma cells
  • plant cells Appropriate culture mediums and conditions for the above-described host cells are known in the art.
  • vectors preferred for use in bacteria include pQ ⁇ 70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia.
  • preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.
  • Preferred expression vectors for use in yeast systems include, but are not limited to ⁇ YES2, pYDl, pTEFl/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalph, pPIC9, pPIC3.5, pHIL-D2, pHIL-Sl, pPIC3.5K, pPIC9K, and
  • PAO815 (all available from Invitrogen, Carlbad, CA). Other suitable vectors will be readily apparent to the skilled artisan.
  • the present invention also relates to host cells containing the above-described vector constructs described herein, and additionally encompasses host cells containing nucleotide' sequences of the invention that are operably associated with one or more heterologous control regions (e.g., promoter and/or enhancer) using techniques known of in the art.
  • the host cell can be a higher eukaryotic cell, such as a mammalian cell (e.g., a human derived cell), or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell.
  • the host strain may be chosen which modulates the expression of the inserted gene sequences, or modifies and processes the gene product in the specific fashion desired. Expression from certain promoters can be elevated in the presence of certain inducers; thus expression of the genetically engineered polypeptide may be controlled. Furthermore, different host cells have characteristics and specific mechanisms for the translational and post-translational processing and modification (e.g., phosphorylation, cleavage) of proteins. Appropriate cell lines can be chosen to ensure the desired modifications and processing of the foreign protein expressed.
  • Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al, Basic Methods In Molecular Biology
  • the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., PSGR coding sequence), and/or to include genetic material (e.g., heterologous polynucleotide sequences) that is operably associated with PSGR polynucleotides of the invention, and which activates, alters, and/or amplifies endogenous genetic material (e.g., PSGR coding sequence), and/or to include genetic material (e.g., heterologous polynucleotide sequences) that is operably associated with PSGR polynucleotides of the invention, and which activates, alters, and/or amplifies endogenous genetic material (e.g., PSGR coding sequence), and/or to include genetic material (e.g., heterologous polynucleotide sequences) that is operably associated with PSGR polynucleotides of the invention, and which activate
  • PSGR polynucleotides may be used to operably associate heterologous control regions (e.g., promoter and/or enhancer) and endogenous
  • PSGR polynucleotide sequences via homologous recombination see, e.g., US Patent Number
  • the PSGR polypeptide may be expressed in a modified form, such as a fusion protein (comprising the polypeptide joined via a peptide bond to a heterologous protein sequence (of a different protein)), and may include not only secretion signals but also additional heterologous functional regions.
  • a fusion protein can be made by protein synthetic techniques, e.g., by use of a peptide synthesizer.
  • a region of additional amino acids, particularly charged amino acids may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification or during subsequent handling and storage.
  • peptide moieties may be added to the polypeptide to facilitate purification.
  • polypeptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art.
  • PSGR polypeptides of the invention may be fused to the pelB pectate lyase signal sequence to increase the efficiency to expression and purification of such polypeptides in Gram- negative bacteria. See, US Patent Nos. 5,576,195 and 5,846,818, the contents of which are herein incorporated by reference in their entireties.
  • a preferred fusion protein comprises a heterologous region from immunoglobulin that is useful to solubilize proteins.
  • EP-A-O 464 533 (Canadian counterpart
  • Polypeptides of the present invention include: products purified from natural sources, including bodily fluids, tissues and cells, whether directly isolated or cultured; products of chemical synthetic procedures; and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or non- glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.
  • N-terminal methionine encoded by the translation initiation codon generally is removed with high efficiency from any protein after translation in all eukaryotic cells. While the N-terminal methionine on most proteins also is efficiently removed in most prokaryotes, for some proteins this prokaryotic removal process is inefficient, depending on the nature of the amino acid to which the N-terminal methionine is covalently linked.
  • the yeast Pichia pastoris is used to express PSGR in a eukaryotic system.
  • Pichia pastoris is a methylotrophic yeast which can metabolize methanol as its sole carbon source.
  • a main step in the methanol metabolization pathway is the oxidation of methanol to formaldehyde using O 2 . This reaction is catalyzed by the enzyme alcohol oxidase.
  • Pichia pastoris In order to metabolize methanol as its sole carbon source, Pichia pastoris must generate high levels of alcohol oxidase due, in part, to the relatively low affinity of alcohol oxidase for O 2 .
  • alcohol oxidase gene in a growth medium depending on methanol as a main carbon source, the promoter region of one of the two alcohol oxidase genes (AOX1) is highly active.
  • alcohol oxidase produced from the AOX1 gene comprises up to approximately 30% of the total soluble protein in Pichia pastoris. See, Ellis,
  • a heterologous coding sequence such as, for example, a PSGR polynucleotide of the present invention, under the transcriptional regulation of all or part of the AOX1 regulatory sequence may be expressed at exceptionally high levels in Pichia yeast grown in the presence of methanol.
  • the plasmid vector pPIC9K is used to express DNA encoding a
  • PSGR polypeptide of the invention as set forth herein, in a Pichea yeast system essentially as described in "Pichia Protocols: Methods in Molecular Biology," D.R. Higgins and J.
  • This expression vector is used to express and secrete a PSGR protein of the invention by virtue of the strong AOX1 promoter linked to the yeast alpha factor prepro peptide signal sequence (i.e., leader) located upstream of a multiple cloning site.
  • yeast vectors could be used in place of pPIC9K, such as, pYES2, pYDl, pTEFl/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, pHEL-Sl, pPIC3.5K, and PAO815, as one skilled in the art would readily appreciate, as long as the proposed expression construct provides appropriately located signals for transcription, translation, secretion (if desired), and the like, including an in-frame AUG as required.
  • high-level expression of a heterologous coding sequence such as, for example, a PSGR polynucleotide of the present invention
  • a heterologous coding sequence such as, for example, a PSGR polynucleotide of the present invention
  • an expression vector such as, for example, pGAPZ or pGAPZalpha
  • the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., PSGR coding sequence), and/or to include genetic material (e.g., heterologous polynucleotide sequences) that is operably associated with PSGR polynucleotides of the invention, and which activates, alters, and/or amplifies endogenous genetic material (e.g., PSGR coding sequence), and/or to include genetic material (e.g., heterologous polynucleotide sequences) that is operably associated with PSGR polynucleotides of the invention, and which activates, alters, and/or amplifies endogenous genetic material (e.g., PSGR coding sequence), and/or to include genetic material (e.g., heterologous polynucleotide sequences) that is operably associated with PSGR polynucleotides of the invention, and which activate
  • PSGR polynucleotides may be used to operably associate heterologous control regions (e.g., promoter and/or enhancer) and endogenous
  • proteins of the invention can be chemically synthesized using techniques known in the art (e.g., see Creighton, Proteins: Structures and Molecular Principles, W.H.
  • a polypeptide corresponding to a fragment of the PSGR polypeptides of the invention can be synthesized by use of a peptide synthesizer.
  • nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the PSGR polypeptide sequence.
  • Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, a- amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx,
  • the invention additionally, encompasses PSGR polypeptides which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, including but not limited to, specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease,
  • Additional post-translational modifications encompassed by the invention include, for example, e.g., N-linked or O-linked carbohydrate chains, processing of N-terminal or
  • polypeptides may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the protein.
  • chemically modified derivatives of PSGR which may provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immunogenicity (see U. S. Patent No. 4,179,337).
  • the chemical moieties for derivitization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like.
  • the polypeptides may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.
  • the polymer may be of any molecular weight, and may be branched or unbranched.
  • the preferred molecular weight is between about 1 kDa and about 100 kDa (the term "about” indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing.
  • Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog).
  • the polyethylene glycol may have an average molecular weight of about 200, 500, 1000, 1500, 2000, 2500
  • the polyethylene glycol may have a branched structure.
  • Branched polyethylene glycols are described, for example, in U.S. Patent No. 5,643,575; Morpurgo et al, Appl. Biochem. Biotechnol. 56:59-12 (1996); Vorobjev et al, Nucleosides Nucleotides
  • polyethylene glycol molecules (or other chemical moieties) should be attached to the protein with consideration of effects on functional or antigenic domains of the protein.
  • polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as, a free amino or carboxyl group.
  • Reactive groups are those to which an activated polyethylene glycol molecule may be bound.
  • the amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues glutamic acid residues and the C-terminal amino acid residue.
  • Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecules. Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group.
  • polyethylene glycol may be attached to proteins via linkage to any of a number of amino acid residues.
  • polyethylene glycol can be linked to a proteins via covalent bonds to lysine, histidine, aspartic acid, glutamic acid, or cysteine residues.
  • One or more reaction chemistries may be employed to attach polyethylene glycol to specific amino acid residues (e.g., lysine, histidine, aspartic acid, glutamic acid, or cysteine) of the protein or to more than one type of amino acid residue (e.g., lysine, histidine, aspartic acid, glutamic acid, cysteine and combinations thereof) of the protein.
  • polyethylene glycol as an illustration of the present composition, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to protein (or peptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein.
  • the method of obtaining the N-terminally pegylated preparation i.e., separating this moiety from other monopegylated moieties if necessary
  • Selective proteins chemically modified at the N-terminus modification may be accomplished by reductive alkylation which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminal) available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved.
  • pegylation of the proteins of the invention may be accomplished by any number of means.
  • polyethylene glycol may be attached to the protein either directly or by an intervening linker.
  • Linkerless systems for attaching polyethylene glycol to proteins are described in Delgado et al, Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992); Francis et al, Intern. J. of Hematol. 68:1-1$ (1998); U.S. Patent No. 4,002,531; U.S. Patent No. 5,349,052; WO 95/06058; and WO 98/32466, the disclosures of each of which are incorporated herein by reference.
  • One system for attaching polyethylene glycol directly to amino acid residues of proteins without an intervening linker employs tresylated MPEG, which is produced by the modification of monmethoxy polyethylene glycol (MPEG) using tresylchloride (ClSO 2 CH 2 CF 3 ).
  • MPEG monmethoxy polyethylene glycol
  • ClSO 2 CH 2 CF 3 tresylchloride
  • polyethylene glycol is directly attached to amine groups of the protein.
  • the invention includes protein- polyethylene glycol conjugates produced by reacting proteins of the invention with a polyethylene glycol molecule having a 2,2,2-trifluoreothane sulphonyl group.
  • Polyethylene glycol can also be attached to proteins using a number of different intervening linkers. For example, U.S.
  • Patent No. 5,612,460 discloses urethane linkers for connecting polyethylene glycol to proteins.
  • Protein-polyethylene glycol conjugates wherein the polyethylene glycol is attached to the protein by a linker can also be produced by reaction of proteins with compounds such as MPEG-succinimidylsuccinate, MPEG activated with 1 , 1 -carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate, MPEG-p- nitrophenolcarbonate, and various MPEG-succinate derivatives.
  • the number of polyethylene glycol moieties attached to each protein of the invention may also vary.
  • the pegylated proteins of the invention may be linked, on average, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or more polyethylene glycol molecules.
  • the average degree of substitution within ranges such as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17,
  • PSGR proteins of the invention may be modified by either natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given PSGR polypeptide.
  • PSGR polypeptides may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic
  • PSGR polypeptides may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to
  • the PSGR polypeptides of the mvention can be recovered and purified from chemical synthesis and recombinant cell cultures by standard methods which include, but are not limited to, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification. Well known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured during isolation and/or purification.
  • HPLC high performance liquid chromatography
  • PSGR polynucleotides and polypeptides may be used in accordance with the present invention for a variety of applications, particularly those that make use of the chemical and biological properties of PSGR. Among these are applications in the detection of prostate cancer, treatment of tumors, resistance to parasites, bacteria and viruses, to inhibit proliferation of B cells, to induce proliferation of T-cells, endothelial cells and certain hematopoietic cells, to treat restenosis, graft vs. host disease, to regulate anti-viral responses and to prevent certain autoimmune diseases after stimulation of PSGR by an agonist.
  • Additional applications relate to diagnosis and to treatment of disorders of cells, tissues and organisms. These aspects of the invention are discussed further below.
  • PSGR proteins (polypeptides) of the invention may be in monomers or multimers
  • the present invention relates to monomers and multimers of the PSGR proteins (polypeptides) of the invention, their preparation, and compositions (preferably, pharmaceutical compositions) containing them.
  • the polypeptides of the invention are monomers, dimers, trimers or tetramers.
  • the multimers of the invention are at least dimers, at least trimers, or at least tetramers.
  • Multimers encompassed by the invention may be homomers or heteromers.
  • the term homomer refers to a multimer containing only PSGR proteins of the invention (including PSGR fragments, variants, and fusion proteins, as described herein).
  • homomers may contain PSGR proteins having identical or different polypeptide sequences.
  • a homomer of the invention is a multimer containing only PSGR proteins having an identical polypeptide sequence.
  • a homomer of the invention is a multimer containing PSGR proteins having different polypeptide sequences (e.g., containing PSGR proteins having identical or different polypetide sequences..
  • the multimer of the invention is a homodimer (e.g., containing PSGR proteins having identical or different polypeptide sequences) or a homotrimer (e.g., containing PSGR proteins having identical or different polypeptide sequences).
  • the homomeric multimer of the invention is at least a homodimer, at least a homotrimer, or at least a homotetramer.
  • heteromer refers to a multimer containing heterologous proteins (i.e., proteins containing only polypeptide sequences that do not correspond to a polypeptide sequences encoded by the PSGR gene) in addition to the PSGR proteins of the invention.
  • the multimer of the invention is a heterodimer, a heterotrimer, or a heterotetramer.
  • the heteromeric multimer of the invention is at least a heterodimer, at least a heterotrimer, or at least a heterotetramer.
  • Multimers of the invention may be the result of hydrophobic, hydrophilic, ionic and/or covalent associations and/or may be indirectly linked, by for example, liposome formation.
  • multimers of the invention such as, for example, homodimers or homotrimers
  • heteromultimers of the invention such as, for example, heterotrimers or heterotetramers
  • proteins of the invention contact antibodies to the polypeptides of the invention (including antibodies to the heterologous polypeptide sequence in a fusion protein of the invention) in solution.
  • multimers of the invention are formed by covalent associations with and/or between the
  • covalent associations may involve one or more amino acid residues contained in the polypeptide sequence of the protein (e.g., the polypeptide sequence shown in Figures 1A-B (SEQ ID NO:2) or Figures 2A-B (SEQ ID NO:4), or a polypeptide encoded by the deposited cDNA clone).
  • the covalent associations are cross-linking between cysteine residues located within the polypeptide sequences of the proteins which interact in the native (i.e., naturally occurring) polypeptide.
  • covalent associations are the consequence of chemical or recombinant manipulation.
  • covalent associations may involve one or more amino acid residues contained in the heterologous polypeptide sequence in a PSGR fusion protein.
  • covalent associations are between the heterologous sequence contained in a fusion protein of the invention (see, e.g., US Patent Number 5,478,925).
  • the covalent associations are between the heterologous sequence contained in a PSGR-Fc fusion protein of the invention (as described herein).
  • covalent associations of fusion proteins of the invention are between heterologous polypeptide sequences from another PSGR family ligand/receptor member that is capable of forming covalently associated multimers, such as for example, oseteoprotegerin (see, e.g.,
  • two or more PSGR polypeptides of the invention are joined through synthetic linkers (e.g., peptide, carbohydrate or soluble polymer linkers). Examples include those peptide linkers described in U.S. Pat. No. 5,073,627
  • Proteins comprising multiple PSGR polypeptides separated by peptide linkers may be produced using conventional recombinant DNA technology.
  • Another method for preparing multimer PSGR polypeptides of the invention involves use of PSGR polypeptides fused to a leucine zipper or isoleucine polypeptide sequence.
  • Leucine zipper domains and isoleucine zipper domains are polypeptides that promote multimerization of the proteins in which they are found. Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al., Science 240:1759,
  • leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize.
  • PSGR proteins are those described in PCT application WO 94/10308, hereby incorporated by reference.
  • Recombinant fusion proteins comprising a soluble PSGR polypeptide fused to a peptide that dimerizes or trimerizes in solution are expressed in suitable host cells, and the resulting soluble multimeric PSGR is recovered from the culture supernatant using techniques known in the art.
  • trimeric PSGR may offer the advantage of enhanced biological activity.
  • Preferred leucine zipper moieties are those that preferentially form trimers.
  • One example is a leucine zipper derived from lung surfactant protein D (SPD), as described in Hoppe et al. (FEBS Letters
  • proteins of the invention are associated by interactions between
  • Flag® polypeptide sequence contained in Flag®-PSGR fusion proteins of the invention are associated by interactions between heterologous polypeptide sequence contained in Flag®-PSGR fusion proteins of the invention and anti-Flag® antibody.
  • the multimers of the invention may be generated using chemical techniques known in the art.
  • proteins desired to be contained in the multimers of the invention may be chemically cross-linked using linker molecules and linker molecule length optimization techniques known in the art (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
  • multimers of the invention may be generated using techniques known in the art to form one or more inter-molecule cross-links between the cysteine residues located within the polypeptide sequence of the proteins desired to be contained in the multimer (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
  • proteins of the invention may be routinely modified by the addition of cysteine or biotin to the C terminus or N-terminus of the polypeptide sequence of the protein and techniques known in the art may be applied to generate multimers containing one or more of these modified proteins (see, e.g., US Patent
  • multimers of the invention may be generated using genetic engineering techniques known in the art.
  • proteins contained in multimers of the invention are produced recombinantly using fusion protein technology described herein or otherwise known in the art (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
  • polynucleotides coding for a homodimer of the invention are generated by ligating a polynucleotide sequence encoding a polypeptide of the invention to a sequence encoding a linker polypeptide and then further to a synthetic polynucleotide encoding the translated product of the polypeptide in the reverse orientation from the original C-terminus to the N-terminus (lacking the leader sequence) (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
  • recombinant techniques described herein or otherwise known in the art are applied to generate recombinant polypeptides of the invention which contain a transmembrane domain and which can be incorporated by membrane reconstitution techniques into liposomes (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
  • the PSGR proteins of the invention can also be expressed in transgenic animals.
  • Animals of any species including, but not limited to, mice, rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep, cows and non-human primates, e.g., baboons, monkeys, and chimpanzees may be used to generate transgenic animals.
  • techniques described herein or otherwise known in the art are used to express polypeptides of the invention in humans, as part of a gene therapy protocol.
  • transgene i.e., nucleic acids of the invention
  • transgene i.e., nucleic acids of the invention
  • Such techniques include, but are not limited to, pronuclear microinjection (Paterson et al, Appl. Microbiol. Biotechnol 40:691-698 (1994); Carver et al, Biotechnology (NY)
  • transgenic clones containing polynucleotides of the invention for example, nuclear transfer into enucleated oocytes of nuclei from cultured embryonic, fetal, or adult cells induced to quiescence (Campell et al,
  • the present invention provides for transgenic animals that carry the transgene in all their cells, as well as animals which carry the transgene in some, but not all their cells, i.e., mosaic animals or chimeric animals.
  • the transgene may be integrated as a single transgene or as multiple copies such as in concatamers, e.g., head-to-head tandems or head-to-tail tandems.
  • the transgene may also be selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et al. (Proc. Natl.
  • the transgene may also be selectively introduced into a particular cell type, thus inactivating the endogenous gene in only that cell type, by following, for example, the teaching of Gu et al. (Science 265:103-106 (1994)).
  • the regulatory sequences required for such a cell-type specific inactivation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art. The contents of each of the documents recited in this paragraph is herein incorporated by reference in its entirety.
  • the expression of the recombinant gene may be assayed utilizing standard techniques. Initial screening may be accomplished by Southern blot analysis or PCR techniques to analyze animal tissues to verify that integration of the transgene has taken place. The level of mRNA expression of the transgene in the tissues of the transgenic animals may also be assessed using techniques which include, but are not limited to, Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and reverse transcriptase-PCR (rt-PCR). Samples of transgenic gene- expressing tissue may also be evaluated immunocytochemically or immunohistochemically using antibodies specific for the transgene product.
  • founder animals may be bred, inbred, outbred, or crossbred to produce colonies of the particular animal.
  • breeding strategies include, but are not limited to: outbreeding of founder animals with more than one integration site in order to establish separate lines; inbreeding of separate lines in order to produce compound transgenics that express the transgene at higher levels because of the effects of additive expression of each transgene; crossing of heterozygous transgenic animals to produce animals homozygous for a given integration site in order to both augment expression and eliminate the need for screening of animals by DNA analysis; crossing of separate homozygous lines to produce compound heterozygous or homozygous lines; and breeding to place the transgene on a distinct background that is appropriate for an experimental model of interest.
  • Transgenic and "knock-out" animals of the invention have uses which include, but are not limited to, animal model systems useful in elaborating the biological function of PSGR polypeptides, studying conditions and/or disorders associated with aberrant PSGR expression, and in screening for compounds effective in ameliorating such conditions and/or disorders.
  • cells that are genetically engineered to express the proteins of the invention, or alternatively, that are genetically engineered not to express the proteins of the invention are administered to a patient in vivo.
  • Such cells may be obtained from the patient (i.e., animal, including human) or an MHC compatible donor and can include, but are not limited to fibroblasts, bone marrow cells, blood cells (e.g., lymphocytes), adipocytes, muscle cells, endothelial cells, etc.
  • the cells are genetically engineered in vitro using recombinant DNA techniques to introduce the coding sequence of polypeptides of the invention into the cells, or alternatively, to disrupt the coding sequence and/or endogenous regulatory sequence associated with the polypeptides of the invention, e.g., by transduction (using viral vectors, and preferably vectors that integrate the transgene into the cell genome) or transfection procedures, including, but not limited to, the use of plasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc.
  • the coding sequence of the polypeptides of the invention can be placed under the control of a strong constitutive or inducible promoter or promoter/enhancer to achieve expression, and preferably secretion, of the polypeptides of the invention.
  • the engineered cells which express and preferably secrete the polypeptides of the invention can be introduced into the patient systemically, e.g., in the circulation, or intraperitoneally. Alternatively, the cells can be incorporated into a matrix and implanted in the body, e.g., genetically engineered fibroblasts can be implanted as part of a skin graft; genetically engineered endothelial cells can be implanted as part of a lymphatic or vascular graft. (See, for example, Anderson et al.
  • the cells to be administered are non-autologous or non-MHC compatible cells, they can be administered using well known techniques which prevent the development of a host immune response against the introduced cells.
  • the cells may be introduced in an encapsulated form which, while allowing for an exchange of components with the immediate extracellular environment, does not allow the introduced cells to be recognized by the host immune system.
  • polypeptides of the present invention are preferably provided in an isolated form.
  • isolated polypeptide is intended a polypeptide removed from its native environment.
  • a polypeptide produced and/or contained within a recombinant host cell is considered isolated for purposes of the present invention.
  • polypeptides that have been purified, partially or substantially, from a recombinant host cell are polypeptides that have been purified, partially or substantially, from a recombinant host cell.
  • a recombinantly produced version of the PSGR polypeptide can be substantially purified by the one-step method described in Smith and Johnson, Gene 67:31-40 (1988).
  • the invention provides an isolated PSGR polypeptide having the amino acid sequence encoded by encoded by the cDNA contained in ATCC Deposit No. 97131, or the amino acid sequence in Figures 1A-B (SEQ ID NO:2), or the amino acid sequence in Figures 2A-B (SEQ ID NO:4), or a polypeptide comprising a portion of the above polypeptides, such as for example, the PSGR extracellular loop I comprising, or alternatively consisting of, amino acids 77 to 98 of Figures 1A-B (SEQ ID NO:2) or Figures 2A-B (SEQ ID NO:4); the PSGR extracellular loop II comprising, or alternatively consisting of, amino acids 163 to 197 of Figures 1A-B (SEQ ID NO:2) or Figures 2A-B (SEQ ID NO:4); the PSGR extracellular loop DI comprising, or alternatively consisting of, amino acids 262 to 272 of Figures 1A-B (SEQ ID NO:2)
  • nucleotide sequence contained in the deposited clone or an amino acid sequence encoded by a nucleic acid containing a polynucleotide sequence which hybridizes to the complementary strand of the nucleotide sequence shown in Figures 1A-B (SEQ ID NO:l) or Figures 2A-B (SEQ ID NO:3).
  • Polynucleotides encoding these polypeptides are also encompassed by the invention.
  • Protein fragments may be "free-standing,” or comprised within a larger polypeptide of which the fragment forms a part or region, most preferably as a single continuous region.
  • polypeptide fragments of the invention include, for example, fragments that comprise or alternatively, consist of from about amino acid residues: 1 to 21,
  • polypeptide fragments can be at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110,
  • polypeptides are also encompassed by the invention.
  • “about” includes the particularly recited ranges, larger or smaller by several (5, 4, 3,
  • polypeptide fragments of the invention comprise, or alternatively consist of, one or more PSGR domains.
  • Preferred polypeptide fragments of the present invention include a member selected from the group: (a) a polypeptide comprising or alternatively, consisting of, the PSGR transmembrane domain I (predicted to constitute amino acid residues from about 22 to about 42 in Figures 1A-B (SEQ DD NO:2) or Figures
  • PSGR transmembrane domain D (predicted to constitute amino acid residues from about 56 to about 76 in Figures 1A-B (SEQ DD NO:2) or Figures 2A-B (SEQ DD NO:4)); (c) a polypeptide comprising, or alternatively consisting of the PSGR transmembrane domain DI
  • PSGR transmembrane domain V (predicted to constitute amino acid residues from about 198 to about 218 in Figures 1A-B (SEQ DD NO:2) or Figures 2A-B (SEQ DD NO:4)); (f) a polypeptide comprising, or alternatively consisting of, the PSGR transmembrane domain VI
  • polypeptide comprising, or alternatively consisting of, the PSGR extracellular loop DI (predicted to constitute amino acid residues from about 262 to about 272 in Figures
  • a polypeptide comprising, or alternatively consisting of, the PSGR intracellular loop D (predicted to constitute amino acid residues from about 120 to about 141 in Figures 1A-B (SEQ DD NO:2) or Figures 2A-B (SEQ DD NO:4));
  • a polypeptide comprising, or alternatively consisting of, the PSGR intracellular loop DI (predicted to constitute amino acid residues from about 219 to about 240 in Figures 1A-B (SEQ DD NO:2) or Figures 2A-B (SEQ DD NO:4)); or (n) any combination of polypeptides (a)-(m).
  • Polynucleotides encoding these polypeptides are also encompassed by the invention.
  • fragments of the invention are fragments characterized by structural or functional attributes of PSGR.
  • Such fragments include amino acid residues that comprise alpha-helix and alpha-helix forming regions ("alpha-regions”), beta-sheet and beta-sheet-forming regions (“beta-regions”), turn and turn-forming regions
  • turn-regions coil and coil-forming regions
  • hydrophilic regions alpha amphipathic regions, beta amphipathic regions, surface forming regions, and high antigenic index regions (i.e., containing four or more contiguous amino acids having an antigenic index of greater than or equal to 1, as identified using the default parameters of the Jameson-Wolf program) of complete (i.e., full-length) PSGR ( Figures 1A-B (SEQ DD NO:2)).
  • Certain preferred regions are those set out in Figure 4 and include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence depicted in Figures 1A-B (SEQ DD NO:2), such preferred regions include; Garnier-Robson predicted alpha-regions, beta-regions, turn-regions, and coil-regions; Chou-Fasman predicted alpha- regions, beta-regions, and turn-regions; Kyte-Doolittle predicted hydrophilic; Eisenberg alpha and beta amphipathic regions; Karplus-Schulz predicted flexible regions; Jameson- Wolf high antigenic index regions; and Emini surface-forming regions, as predicted using the default parameters of these computer programs. Polynucleotides encoding these polypeptides are also encompassed by the invention.
  • the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the PSGR amino acid sequence shown in Figures 1A-B, up to the glutamine residue at position number 315 and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides comprising the amino acid sequence of residues n ! -320 of Figures 1A-B, where n 1 is an integer from 2 to 315 corresponding to the position of the amino acid residue in Figures 1 A-B (SEQ DD NO:2).
  • the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues: S-2 to K-320;
  • K-320 P-157 to K-320; L-158 to K-320; P-159 to K-320; L-160 to K-320; L-161 to K-320;
  • K-320 M-211 to K-320; F-212 to K-320; 1-213 to K-320; S-214 to K-320; L-215 to K-320;
  • K-320 V-260 to K-320; H-261 to K-320; R-262 to K-320; F-263 to K-320; G-264 to K-320;
  • K-320 V-271 to K-320; R-272 to K-320; V-273 to K-320; V-274 to K-320; M-275 to K-320;
  • K-320 A-293 to K-320; K-294 to K-320; T-295 to K-320; K-296 to K-320; Q-297 to K-320;
  • K-320 A-304 to K-320; M-305 to K-320; F-306 to K-320; K-307 to K-320; 1-308 to K-320;
  • Polypeptides encoded by these polynucleotides are also encompassed by the invention.
  • C-terminus of a protein results in modification of loss of one or more biological functions of the protein, other functional activities (e.g., biological activities, ability to multimerize, ability to bind PSGR ligand may still be retained).
  • other functional activities e.g., biological activities, ability to multimerize, ability to bind PSGR ligand may still be retained.
  • PSGR mutein to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking C-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a PSGR mutein with a large number of deleted C-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six PSGR amino acid residues may often evoke an immune response.
  • the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the PSGR polypeptide shown in Figures 1A-B, up to the phenylalanine residue at position number 6, and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides comprising the amino acid sequence of residues 1-m 1 of Figures 1A-B, where m 1 is an integer from 6 to 319 corresponding to the position of the amino acid residue in Figures 1A-B (SEQ DD NO:2).
  • the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues: M-l to G-
  • M-l to K-312 M-l to D-311; M-l to C-310; M-l to S-309; M-l to 1-308; M-l to K-
  • M-l to F-306 M-l to M-305; M-l to A-304; M-l to L-303; M-l to V-302; M-l to R-
  • G-276 M-l to M-275; M-l to V-274; M-l to V-273; M-l to R-272; M-l to V-271; M-l to I-
  • T-239 M-l to G-238; M-l to F-237; M-l to A-236; M-l to K-235; M-l to A-234; M-l to R-
  • M-l to A-201 M-l to T-200
  • M-l to L-199 M-l to G-198
  • M-l to Y-197 M-l to V-
  • M-l to V-195 M-l to ⁇ -194; M-l to P-193; M-l to L-192; M-l to T-191; M-l to D-
  • M-l to V-146 M-l to 1-145; M-l to G-144; M-l to 1-143; M-l to Q-142; M-l to A-
  • M-l to T-140; M-l to V-139 ) ;; M-l to T-138; M-l to N-137; M-l to N-136; M-l to L- 135; M-l to V-134; M-l to A-1335;; M-l to A-132; M-l to H-131; M-l to R-130; M-l to L- 129; M-l to P-128; M-l to H-1271;; M-l to C-126; M-l to 1-125; M-l to A-124; M-l to V- 123; M-l to Y-122; M-l to R-121L;; M-l to D-120; M-l to F-119; M-l to A-118; M-l to M- 117; M-l to A-116; M-l to L-115; M-l to L-114; M-l to 1-113; M-l to T-112; M-l to S-lll;
  • the invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini, which may be described generally as having residues n ⁇ m 1 of Figures 1A-B (i.e., SEQ DD NO:2), where n 1 and m 1 are integers as described above.
  • SEQ DD NO:2 residues n ⁇ m 1 of Figures 1A-B
  • n 1 and m 1 are integers as described above.
  • the invention provides the above described polypeptide deletions (e.g., n 1 -320, 1-m 1 , or n 1 - m 1 , where n 1 and m 1 are integers as described above) containing one, two or all three of the following substitutions: a Fll replaced with C; F39 replaced with C; and F93 replaced with I of the PSGR amino acid sequence shown in Figures 1A-B (SEQ ID NO:
  • polypeptides are also encompassed by the invention.
  • the present invention is also directed to nucleic acid molecules comprising, or alternatively, consisting of, a polynucleotide sequence at least 80%, 85%, 90%, 92%, 95%,
  • polypeptides encoded by these nucleic acids and/or polynucleotide sequences are also encompassed by the invention, as are polypeptides comprising, or alternatively consisting of, an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence described above, and polynucleotides that encode such polypeptides.
  • Additional preferred polynucleotides encode polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues: M-l to G-15; S-2 to 1-16; S-
  • V-36 to T-50; A-37 to E-51; M-38 to R-52; C-39 to S-53; G-40 to L-54; N-41 to H-55;
  • T-140 H-127 to A-141; P-128 to Q-142; L-129 to 1-143; R-130 to G-144; H-131 to 1-145; A-
  • V-149 to K-163 R-150 to R-164; G-151 to L-165; S-152 to A-166; L-153 to F-167; F-
  • M-206 P-193 to G-207; N-194 to V-208; V-195 to D-209; V-196 to V-210; Y-197 to M-
  • R-272 V-259 to V-273; V-260 to V-274; H-261 to M-275; R-262 to G-276; F-263 to D-277;
  • polypeptide fragments may retain the biological activity of PSGR polypeptides of the invention and/or may be useful to generate or screen for antibodies, as described further below.
  • Polynucleotides encoding these polypeptide fragments are also encompassed by the invention.
  • the invention provides the above described polypeptide fragments containing one, two or all three of the following substitutions: a FI 1 replaced with C; F39 replaced with C; and F93 replaced with I of the PSGR amino acid sequence shown in Figures
  • nucleic acid molecules comprising, or alternatively, consisting of, a polynucleotide sequence at least 90%, 92%, 95%, 96%, 97%,
  • the present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence. Additionally, the present application is also directed to proteins containing polypeptides at least 90%, 92%, 93%, 94%,
  • Polypeptides encoded by these polynucleotides are also encompassed by the invention.
  • the invention further includes variations of the PSGR receptor, which show substantial PSGR receptor activity or which include regions of PSGR proteins, such as the protein portions discussed herein. Such mutants include deletions, insertions, inversions, repeats, and type substitutions.
  • the first strategy exploits the tolerance of amino acid substitutions by natural selection during the process of evolution. By comparing amino acid sequences in different species, conserved amino acids can be identified. These conserved amino acids are likely important for protein function. In contrast, the amino acid positions where substitutions have been tolerated by natural selection indicates that these positions are not critical for protein function. Thus, positions tolerating amino acid substitution could be modified while still maintaining biological activity of the protein.
  • the second strategy uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene to identify regions critical for protein function. For example, site directed mutagenesis or alanine-scanning mutagenesis (introduction of single alanine mutations at every residue in the molecule) can be used. (Cunningham and Wells,
  • tolerated conservative amino acid substitutions involve replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu and He; replacement of the hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and Glu; replacement of the amide residues Asn and Gin, replacement of the basic residues Lys, Arg, and His; replacement of the aromatic residues Phe, Tyr, and Trp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly.
  • site directed changes at the amino acid level of PSGR can be made by replacing a particular amino acid with a conservative amino acid.
  • Preferred conservative mutations include: Ml replaced with A, G, I, L, S, T, or V; S2 replaced with A, G, I, L, T, M, or V; S3 replaced with A, G, I, L, T, M, or V; N5 replaced with Q; F6 replaced with W, or Y;
  • T7 replaced with A, G, I, L, S, M, or V
  • H8 replaced with K, or R
  • A9 replaced with G, I, L
  • T10 replaced with A, G, I, L, S, M, or V
  • V12 replaced with A, G, I, L, S, T, or M
  • L13 replaced with A, G, I, S, T, M, or V
  • 114 replaced with A, G, L, S, T, M, or V;
  • G15 replaced with A, I, L, S, T, M, or V
  • 116 replaced with A, G, L, S, T, M, or V
  • G18 replaced with A, I, L, S, T, M, or V
  • L19 replaced with A, G, I, S, T, M, or V
  • E20 replaced with D
  • K21 replaced with H, or R
  • A22 replaced with G, I, L, S, T, M, or V
  • H23 replaced with K, or R
  • F24 replaced with W, or Y
  • W25 replaced with F, or Y
  • V26 replaced with A
  • G, I, L, S, T, or M G27 replaced with A, I, L, S, T, M, or V; F28 replaced with W, or Y; L30 replaced with A, G, I, S, T, M, or V; L31 replaced with A, G, I, S, T, M, or V; S32 replaced with A, G, I, L, T, M, or V; M33 replaced with A, G, I, L, S, T, or V; Y34 replaced with F, or
  • V35 replaced with A, G, I, L, S, T, or M
  • V36 replaced with A, G, I, L, S, T, or M
  • A37 replaced with G, I, L, S, T, M, or V
  • M38 replaced with A, G, I, L, S, T, or V
  • G40 replaced with A, I, L, S, T, M, or V
  • N41 replaced with Q
  • 143 replaced with A, G, L, S, T, M, or V;
  • V44 replaced with A, G, I, L, S, T, or M
  • V45 replaced with A, G, I, L, S, T, or M
  • F46 replaced with W, or Y
  • 147 replaced with A, G, L, S, T, M, or V
  • V48 replaced with A, G, I,
  • M58 replaced with A, G, I, L, S, T, or V
  • Y59 replaced with F, or W
  • L60 replaced with A, G, I, S, T, M, or V
  • F61 replaced with W, or Y
  • L62 replaced with A, G, I,
  • A71 replaced with G, I, L, S, T, M, or V
  • L72 replaced with A, G, I, S, T, M, or V
  • F86 replaced with W, or Y; D87 replaced with E; S88 replaced with A, G, I, L, T, M, or V
  • R89 replaced with H, or K
  • E90 replaced with D
  • 191 replaced with A, G, L, S, T, M, or V
  • S92 replaced with A, G, I, L, T, M, or V
  • 193 replaced with A, G, L, S, T, M, or V
  • E94 replaced with D
  • A95 replaced with G, I, L, S, T, M, or N
  • L97 replaced with A, G, I, S, T,
  • L, S, T, M, or V L106 replaced with A, G, I, S, T, M, or V; S107 replaced with A, G, I, L, T,
  • G, I, L, S, M, or V 1113 replaced with A, G, L, S, T, M, or V; LI 14 replaced with A, G, I, S,
  • T, M, or V T, M, or V
  • LI 15 replaced with A, G, I, S, T, M, or V
  • A116 replaced with G, I, L, S, T, M, or V
  • Ml 17 replaced with A, G, I, L, S, T, or V
  • A118 replaced with G, I, L, S, T, M, or V;
  • F119 replaced with W, or Y; D120 replaced with E; R121 replaced with H, or K; Y122 replaced with F, or W; V123 replaced with A, G, I, L, S, T, or M; A124 replaced with G, I, L,
  • A132 replaced with G, I, L, S, T, M, or V
  • A133 replaced with G, I, L, S, T, M, or V
  • V134 replaced with A, G, I, L, S, T, or M
  • L135 replaced with A, G, I, S, T, M, or V
  • ⁇ 136 replaced with Q
  • N137 replaced with Q
  • T138 replaced with A, G, I, L, S, M, or V
  • V139 replaced with A, G, I, L, S, T, or M
  • T140 replaced with A, G, I, L, S, M, or V
  • A141 replaced with G, I, L, S, T, M, or V
  • Q142 replaced with N; 1143 replaced with A, G, L, S, T,
  • G144 replaced with A, I, L, S, T, M, or V
  • 1145 replaced with A, G, L, S, T, M, or
  • V replaced with A, G, I, L, S, T, or M
  • A147 replaced with G, I, L, S, T, M, or V
  • V148 replaced with A, G, I, L, S, T, or M
  • V149 replaced with A, G, I, L, S, T, or M
  • R150 replaced with H, or K
  • G151 replaced with A, I, L, S, T, M, or V
  • S152 replaced with A, G, I,
  • L, T, M, or V L153 replaced with A, G, I, S, T, M, or V; F154 replaced with W, or Y; F155 replaced with W, or Y; F156 replaced with W, or Y; L158 replaced with A, G, I, S, T, M, or
  • L160 replaced with A, G, I, S, T, M, or V
  • L161 replaced with A, G, I, S, T, M, or V
  • 1162 replaced with A, G, L, S, T, M, or V
  • K163 replaced with H, or R
  • R164 replaced with H, or
  • L165 replaced with A, G, I, S, T, M, or V
  • A166 replaced with G, I, L, S, T, M, or V
  • F167 replaced with W, or Y; H169 replaced with K, or R; S170 replaced with A, G, I, L, T,
  • K185 replaced with H, or R
  • L186 replaced with A, G, I, S, T, M, or V
  • A187 replaced with G, I, L, S, T, M, or V
  • Y188 replaced with F, or W
  • A189 replaced with G, I, L
  • V196 replaced with A, G, I, L, S, T, or M
  • Y197 replaced with F, or W
  • G198 replaced with A, I, L, S, T, M, or V
  • L199 replaced with A, G, I, S, T, M, or V
  • T200 replaced with A, G, I, L, S, M, or V
  • A201 replaced with G, I, L, S, T, M, or V
  • 1202 replaced with A, G, L, S, T, M, or V
  • L203 replaced with A, G, I, S, T, M, or V
  • L204 replaced with
  • V205 replaced with A, G, I, L, S, T, or M
  • M206 replaced with A, G
  • G207 replaced with A, I, L, S, T, M, or V
  • V208 replaced with A, G, I, L, S,
  • D209 replaced with E
  • V210 replaced with A, G, I, L, S, T, or M
  • M211 replaced with A, G, I, L, S, T, or V
  • F212 replaced with W, or Y
  • 1213 replaced with A, G, L, S, T, M, or V
  • S214 replaced with A, G, I, L, T, M, or V
  • L215 replaced with A, G, I, S, T, M, or V;
  • L219 replaced with A, G, I, S, T, M, or V
  • 1220 replaced with A, G, L, S, T, M, or
  • V 1221 replaced with A, G, L, S, T, M, or V
  • R222 replaced with H, or K
  • T223 replaced with A, G, I, L, S, M, or V
  • V224 replaced with A, G, I, L, S, T, or M
  • L225 replaced with A
  • G, I, S, T, M, or V G, I, S, T, M, or V
  • Q226 replaced with N
  • L227 replaced with A, G, I, S, T, M, or V
  • S229 replaced with A, G, I, L, T, M, or V
  • K230 replaced with H, or R
  • S231 replaced with A, G, I,
  • K235 replaced with H, or R
  • A236 replaced with G, I, L, S, T, M, or V
  • F237 replaced with W, or Y; G238 replaced with A, I, L, S, T, M, or V; T239 replaced with
  • V241 replaced with A, G, I, L, S, T, or M
  • S242 replaced with A, G, I,
  • L, T, M, or V H243 replaced with K, or R; 1244 replaced with A, G, L, S, T, M, or V; G245 replaced with A, I, L, S, T, M, or V; V246 replaced with A, G, I, L, S, T, or M; V247 replaced with A, G, I, L, S, T, or M; L248 replaced with A, G, I, S, T, M, or V; A249 replaced with G, I, L, S, T, M, or V; F250 replaced with W, or Y; Y251 replaced with F, or
  • V252 replaced with A, G, I, L, S, T, or M
  • L254 replaced with A, G, I, S, T, M, or V
  • L267 replaced with A, G, I, S, T, M, or V
  • H268 replaced with K, or R
  • 1270 replaced with A, G, L, S, T, M, or V
  • V271 replaced with A, G, I, L, S, T, or M
  • R272 replaced with H, or K
  • V273 replaced with A, G, I, L, S, T, or M
  • V274 replaced with A, G,
  • I, L, S, T, or M replaced with A, G, I, L, S, T, or V; G276 replaced with A, I, L, S, T,
  • G292 replaced with A, I, L, S, T, M, or V
  • A293 replaced with G, I, L, S, T, M, or V
  • K294 replaced with H, or R
  • T295 replaced with A, G, I, L, S, M, or V
  • K296 replaced with H, or
  • K307 replaced with H, or R; 1308 replaced with A, G, L, S, T, M, or V; S309 replaced with
  • A, G, I, L, T, M, or V D311 replaced with E; K312 replaced with H, or R; D313 replaced with E; L314 replaced with A, G, I, S, T, M, or V; Q315 replaced with N; A316 replaced with G, I, L, S, T, M, or V; V317 replaced with A, G, I, L, S, T, or M; G318 replaced with A,
  • the resulting constructs can be routinely screened for activities or functions described throughout the specification and known in the art.
  • the resulting constructs have an increased and/or a decreased PSGR activity or function, while the remaining PSGR activities or functions are maintained. More preferably, the resulting constructs have more than one increased and/or decreased PSGR activity or function, while the remaining PSGR activities or functions are maintained.
  • variants of PSGR include (i) substitutions with one or more of the non-conserved amino acid residues, where the substituted amino acid residues may or may not be one encoded by the genetic code, or (ii) substitution with one or more of amino acid residues having a substituent group, or (iii) fusion of the mature polypeptide with another compound, such as a compound to increase the stability and/or solubility of the polypeptide (for example, polyethylene glycol), or (iv) fusion of the polypeptide with additional amino acids, such as, for example, an IgG Fc fusion region peptide, or leader or secretory sequence, or a sequence facilitating purification or (v) fusion of the the polypeptide with another compound, such as albumin (including but not limited to recombinant albumin (see, e.g., U.S.
  • PSGR polypeptide variants containing amino acid substitutions of charged amino acids with other charged or neutral amino acids may produce proteins with improved characteristics, such as less aggregation. Aggregation of pharmaceutical formulations both reduces activity and increases clearance due to the aggregate's immunogenic activity.
  • preferred non-conservative substitutions of PSGR include Ml replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S2 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S3 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C4 replaced with D,
  • V, P, or C replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; L30 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L31 replaced with D, E, H, K, R, N, Q,
  • V35 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V36 replaced with
  • E, H, K, R, N, Q, F, W, Y, P, or C V44 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V45 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F46 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; 147 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V48 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R49 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T50 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E51
  • K79 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; 180 replaced with
  • N, Q, F, W, Y, P, or C replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or
  • E, H, K, R, N, Q, F, W, Y, P, or C F101 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; F102 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; 1103 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; H104 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A105 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L106 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S107 replaced with D, E, H, K, R, N, Q, F, W,
  • H127 replaced with D, E, A, G, I, L, S, T, M, V, N, Q,
  • P128 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; P128 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or
  • L129 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R130 replaced with D, E, A, G,
  • H131 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; H131 replaced with D, E, A, G, I, L, S, T, M, V, N,
  • L135 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C
  • N136 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C
  • N137 replaced with D, E, H, K,
  • T138 replaced with D, E, H, K, R, N, Q, F, W, Y,
  • V139 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C
  • T140 replaced with D, E
  • Q142 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C
  • 1143 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C
  • G144 replaced with D, E, H, K, R, N, Q, F, W, Y,
  • V148 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
  • V149 replaced with D, E, H, K, R,
  • G151 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C
  • S152 replaced with D, E, H, K
  • L158 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P159 replaced with D,
  • L161 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C
  • 1162 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C
  • K163 replaced with D, E, A, G, I, L, S, T, M, V,
  • L165 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
  • A166 replaced with D, E, H, K,
  • N171 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,
  • V172 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L173 replaced with
  • E, H, K, R, N, Q, F, W, Y, P, or C K185 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L186 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A187 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y188 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; A189 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D190 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T191 replaced with D, E, H, K, R, N, Q,
  • V205 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C
  • M206 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C
  • G207 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C
  • V208 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C
  • D209 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C
  • V210 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C
  • M211 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C
  • F212 replaced with D, E, H, K, R, N, Q, A,
  • F, W, Y, P, or C F250 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; Y251 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; V252 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P253 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; L254 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; 1255 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G256 replaced with D, E, H, K, R, N, Q, F, W, Y, P
  • K312 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C
  • D313 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C
  • L314 replaced with D, E, H, K
  • A316 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
  • V317 replaced with D, E,
  • G319 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; and/or K320 replaced with D, E,
  • Figures 1A-B SEQ DD NO:2.
  • the resulting constructs can be routinely screened for activities or functions described throughout the specification and known in the art.
  • the resulting constructs have an increased and/or decreased PSGR activity or function, while the remaining PSGR activities or functions are maintained. More preferably, the resulting constructs have more than one increased and/or decreased PSGR activity or function, while the remaining PSGR activities or functions are maintained.
  • more than one amino acid e.g., 2, 3, 4, 5, 6, 7, 8, 9 and 10
  • substituted amino acids can occur in the full length, mature, or proprotein form of PSGR protein, as well as the N- and C- terminal deletion mutants, having the general formula n ⁇ m 1 , listed above.
  • a further embodiment of the invention relates to a polypeptide which comprises the amino acid sequence of a PSGR polypeptide having an amino acid sequence which contains at least one amino acid substitution, but not more than 50 amino acid substitutions, even more preferably, not more than 40 amino acid substitutions, still more preferably, not more than 30 amino acid substitutions, and still even more preferably, not more than 20 amino acid substitutions.
  • a polypeptide it is highly preferable for a polypeptide to have an amino acid sequence which comprises the amino acid sequence of a PSGR polypeptide, which contains at least one, but not more than 10, 9, 8, 7, 6,
  • the number of additions, substitutions, and/or deletions in the amino acid sequence of Figures 1A-B or fragments thereof is 1-5, 5-10, 5-25, 5-
  • substitutions include FI 1 replaced with C; F39 replaced with C; and F93 replaced with I of the PSGR amino acid sequence shown in Figures 1A-B (SEQ DD NO:2).
  • Polynucleotides encoding these polypeptides are also encompassed by the invention.
  • 97131 may be (i) one in which at least one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue(s), and more preferably at least one but less than ten conserved amino acid residues) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as an IgG Fc fusion region peptide or leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence or (v) one in which the mature polypeptide is fused with another compound, such as albumin (including but not limited
  • PSGR polypeptides of the present invention may include one or more amino acid substitutions, deletions, or additions, either from natural mutations or human manipulation.
  • the number of substitutions, additions or deletions in the amino acid sequence of Figures 1A-B and/or any of the polypeptide fragments described herein is 75, 70, 60, 50, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 30-20, 20-15, 20-10, 15-10, 10- 1, 5-10, 1-5, 1-3 or 1-2.
  • Amino acids in the PSGR proteins of the present invention that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)).
  • the resulting mutant molecules are then tested for biological activity such as receptor binding or in vitro prohferative activity.
  • Sites that are critical for ligand-receptor binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al, J. Mol. Biol. 224:899-904 (1992) and de Vos et al.
  • PSGR polypeptides may be employed.
  • Recombinant DNA technology known to those skilled in the art can be used to create novel mutant proteins or "muteins including single or multiple amino acid substitutions, deletions, additions or fusion proteins.
  • Such modified polypeptides can show, e.g., enhanced activity or increased stability.
  • they may be purified in higher yields and show better solubility than the corresponding natural polypeptide, at least under certain purification and storage conditions.
  • Non-naturally occurring variants may be produced using art-known mutagenesis techniques, which include, but are not limited to oligonucleotide mediated mutagenesis, alanine scanning, PCR mutagenesis, site directed mutagenesis (see e.g., Carter et al, Nucl.
  • the invention also encompasses PSGR derivatives and analogs that have one or more amino acid residues deleted, added, or substituted to generate PSGR polypeptides that are better suited for expression, scale up, etc., in the host cells chosen.
  • cysteine residues can be deleted or substituted with another amino acid residue in order to eliminate disulfide bridges; N-linked glycosylation sites can be altered or eliminated to achieve, for example, expression of a homogeneous product that is more easily recovered and purified from yeast hosts which are known to hyperglycosylate N-linked sites.
  • amino acid residues of the polypeptides of the invention may be deleted or substituted with another residue to eliminate undesired processing by proteases such as, for example, furins or kexins.
  • polypeptides of the present . invention include a polypeptide comprising, or alternatively, consisting of a polypeptide having the amino acid sequence encoded by the cDNA in ATCC Deposit No. 97131; a polypeptide comprising, or alternatively, consisting of a polypeptide having the amino acid sequence encoded by the cDNA in ATCC Deposit No.
  • polypeptides which are at least 80% identical, more preferably at least 90% or
  • polypeptide of Figures 1A-B SEQ DD NO:2
  • polypeptide of Figures 1A-B SEQ DD NO:2
  • Polypeptide encoding these polypeptides are also encompassed by the invention.
  • polypeptide having an amino acid sequence at least, for example, 95%
  • amino acid sequence of a PSGR polypeptide is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of PSGR.
  • up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence.
  • These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
  • Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence according to the present invention, the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference amino acid sequence and that gaps in homology of up to 5% of the total number of amino acid residues in the reference sequence are allowed.
  • the identity between a reference (query) sequence (a sequence of the present invention) and a subject sequence is determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)).
  • the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminal of the subject sequence, which are not matched aligned with a corresponding subject residue, as a percent of the total bases of the query sequence.
  • a determination of whether a residue is matched aligned is determined by results of the
  • N- and C-terminal residues of the subject sequence For example, a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to determine percent identity.
  • the deletion occurs at the N-terminus of the subject sequence and therefore, the FASTDB alignment does not show a matching/alignment of the first 10 residues at the N-terminus.
  • the 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C- termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%.
  • a 90 residue subject sequence is compared with a 100 residue query sequence. This time the deletions are internal deletions so there are no residues at the N- or C-termini of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected.
  • FASTDB alignment which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are made for the purposes of this embodiment.
  • the present application is also directed to proteins cotaining polypeptides at least
  • PSGR proteins of the invention comprise fusion proteins as described above wherein the PSGR polypeptides are those described as n ⁇ m 1 , herein.
  • the application is directed to nucleic acid molecules at least 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequences encoding polypeptides having the amino acid sequence of the specific N- and C-terminal deletions recited herein. Polynucleotides encoding these polypeptides are also encompassed by the invention.
  • the term "epitopes,” as used herein, refers to portions of a polypeptide having antigenic or immunogenic activity in an animal, preferably a mammal, and most preferably in a human.
  • the present invention encompasses a polypeptide comprising an epitope, as well as the polynucleotide encoding this polypeptide.
  • immunological epitope is defined as a portion of a protein that elicits an antibody response in an animal, as determined by any method known in the art, for example, by the methods for generating antibodies described infra. (See, for example, Geysen et al.,
  • antigenic epitope is defined as a portion of a protein to which an antibody can immunospecifically bind its antigen as determined by any method well known in the art, for example, by the immunoassays described herein. Immunospecific binding excludes non-specific binding but does not necessarily exclude cross- reactivity with other antigens. Antigenic epitopes need not necessarily be immunogenic.
  • Fragments which function as epitopes may be produced by any conventional means. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985), further described in U.S. Patent No. 4,631,211).
  • Antigenic epitope-bearing peptides and polypeptides of the invention are therefore useful, for example, to raise antibodies, including monoclonal antibodies, that bind specifically to a polypeptide of the invention. See, for instance, Wilson et al, Cell 37:161-
  • Antigenic epitope-bearing peptides and polypeptides of the invention preferably contain a sequence of at least seven, more preferably at least nine, at least 20, at least 25, at least 30, at least 40, at least 50 and most preferably between at least about 55 to about 100 amino acids contained within the amino acid sequence of a polypeptide of the invention.
  • Non-limiting examples of antigenic polypeptides or peptides that can be used to generate PSGR-specific antibodies include: a polypeptide comprising or alternatively consisting of amino acid residues from about 50 to about 55 in Figures 1A-B (SEQ DD
  • polypeptide comprising or alternatively consisting of amino acid residues from about 87 to about 90 in Figures 1A-B (SEQ DD NO:2); a polypeptide comprising or alternatively consisting of amino acid residues from about 227 to about 234 in Figures 1 A-B
  • PSGR protein Polynucleotides encoding theses polypeptides are also encompassed by the invention.
  • the epitope-bearing peptides and polypeptides of the invention may be produced by any conventional means.
  • R.A. Houghten "General Method for the Rapid Solid-phase
  • SMPS Single Peptide Synthesis
  • PSGR polypeptides of the present invention and the epitope-bearing fragments thereof, described herein e.g., including, but not limited to, a portion of one, two, or all three of the the extracellular loops, such as, for example, amino acid residues 77 to 98, 163 to 197, or 262 to 272 of SEQ DD NO:2; or a portion of one, two, or all three of the intracellular loops, such as for example, amino acid residues 43 to 55,
  • polypeptides of the present invention can be combined with heterologous polypeptide sequences, for example, the polypeptides of the present invention may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM) or portions thereof (CHI, CH2,
  • albumin including, but not limited to, recombinant albumin (see, e.g., U.S. Patent No.
  • chimeric polypeptides resulting in chimeric polypeptides.
  • These fusion proteins facilitate purification and show an increased half-life in vivo. This has been shown, e.g., for 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 (EPA 394,827; Traunecker et al, Nature
  • Fusion proteins that have a disulfide-linked dimeric structure due to the IgG part can also be more efficient in binding and neutralizing other molecules than the monomeric PSGR protein or protein fragment alone (Fountoulakis et al, J. Biochem.
  • Preferred Fc fusions of the present invention include, but are not limited to constructs comprising, or alternatively consisting of, amino acid residues 1 to 98, 1 to 197, 1 to 272, 22 to 320, 77 to 320, 163 to 320, 262 to 320, 77 to 272, 77 to 197, and/or 22 to 98 of SEQ DD
  • polypeptides of the present invention have uses which include, but are not limited to, as sources for generating antibodies that bind the polypeptides of the invention, and as molecular weight markers on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those of skill in the art.
  • the compounds of the present invention are useful for diagnosis or treatment of various prostate related disorders in mammals, preferably humans.
  • Such disorders include but are not limited to including inflammatory disorders, such as chronic prostatitis, granulomatous prostatitis and malacoplakia, prostatic hyperplasia and prostate neoplastic disorders, including adenocarcinoma, transitional cell carcinomas, ductal carcinomas, squamous cell carcinomas, or as hormones or factors with systemic or reproductive functions.
  • PSGR is expressed in prostate, predominantly in the epithelial cells of gland, with an increased expression level in prostate cancer.
  • substantially altered (increased or decreased) levels of PSGR gene expression can be detected in prostate tissue or other cells or bodily fluids (e.g., sera, plasma, urine, semen, synovial fluid or spinal fluid) taken from an individual having such a disorder, relative to a "standard" PSGR gene expression level, that is, the PSGR expression level in prostate tissues or bodily fluids from an individual not having the prostate disorder.
  • bodily fluids e.g., sera, plasma, urine, semen, synovial fluid or spinal fluid
  • the invention provides a diagnostic method useful during diagnosis of an system disorder, which involves measuring the expression level of the gene encoding the PSGR polypeptide in prostate tissue or other cells or body fluid from an individual and comparing the measured gene expression level with a standard PSGR gene expression level, whereby an increase or decrease in the gene expression level(s) compared to the standard is indicative of an prostate disorder.
  • a diagnostic method useful during diagnosis of an system disorder which involves measuring the expression level of the gene encoding the PSGR polypeptide in prostate tissue or other cells or body fluid from an individual and comparing the measured gene expression level with a standard PSGR gene expression level, whereby an increase or decrease in the gene expression level(s) compared to the standard is indicative of an prostate disorder.
  • certain tissues in mammals with cancer of cells or tissue of the prostate express significantly enhanced or reduced levels of normal or altered PSGR polypeptide and mRNA encoding the PSGR polypeptide when compared to a corresponding "standard" level.
  • PSGR polypeptide can be detected in certain body fluids (e.g., sera, plasma, urine, and spinal fluid) or cells or tissue from mammals with such a cancer when compared to sera from mammals of the same species not having the cancer.
  • body fluids e.g., sera, plasma, urine, and spinal fluid
  • cells or tissue from mammals with such a cancer when compared to sera from mammals of the same species not having the cancer.
  • PSGR polypeptides of the invention are expressed in prostate. Accordingly, polynucleotides of the invention (e.g., polynucleotide sequences complementary to all or a portion of PSGR mRNA) and antibodies (and antibody fragments) directed against the polypeptides of the invention may be used to quantitate or qualitate concentrations of cells of the prostate expressing PSGR on their cell surfaces.
  • polynucleotides of the invention e.g., polynucleotide sequences complementary to all or a portion of PSGR mRNA
  • antibodies (and antibody fragments) directed against the polypeptides of the invention may be used to quantitate or qualitate concentrations of cells of the prostate expressing PSGR on their cell surfaces.
  • These antibodies additionally have diagnostic applications in detecting abnormalities in the level of PSGR gene expression, or abnormalities in the structure and/or temporal, tissue, cellular, or subcellular location of PSGR.
  • diagnostic assays may be performed in vivo or in vitro, such as, for example, on blood samples, biopsy tissue or autopsy tissue.
  • the invention provides a diagnostic method useful during diagnosis of a prostate disorder, including cancers, which involves measuring the expression level of the gene encoding the PSGR polypeptide in prostate tissue or other cells or body fluid from an individual and comparing the measured gene expression level with a standard PSGR gene expression level, whereby an increase or decrease in the gene expression level compared to the standard is indicative of a prostate disorder.
  • the present invention is useful as a prognostic indicator, whereby patients exhibiting enhanced or depressed PSGR gene expression will experience a worse clinical outcome relative to patients expressing the gene at a level nearer the standard level.
  • test the expression level of the gene encoding the PSGR polypeptide is intended qualitatively or quantitatively measuring or estimating the level of the PSGR polypeptide or the level of the mRNA encoding the PSGR polypeptide in a first biological sample either directly (e.g., by determining or estimating absolute protein level or mRNA level) or relatively (e.g., by comparing to the PSGR polypeptide level or mRNA level in a second biological sample).
  • the PSGR polypeptide level or mRNA level in the first biological sample is measured or estimated and compared to a standard PSGR polypeptide level or mRNA level, the standard being taken from a second biological sample obtained from an individual not having the disorder or being determined by averaging levels from a population of individuals not having a disorder of the prostate.
  • a standard PSGR polypeptide level or mRNA level it can be used repeatedly as a standard for comparison.
  • biological sample any biological sample obtained from an individual, cell line, tissue culture, or other source containing PSGR protein (including portions thereof) or mRNA.
  • biological samples include body fluids (such as sera, plasma, urine, synovial fluid and spinal fluid) which contain soluble domains of the
  • PSGR polypeptide cells expressing PSGR polypeptides, prostate tissue, and other tissue sources found to express the full length or soluble domains of PSGR.
  • Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. Where the biological sample is to include mRNA, a tissue biopsy is the preferred source.
  • Total cellular RNA can be isolated from a biological sample using any suitable technique such as the single-step guanidinium-thiocyanate-phenol-chloroform method described in Chomczynski and Sacchi, Anal. Biochem. 762:156-159 (1987). Levels of mRNA encoding the PSGR polypeptide are then assayed using any appropriate method.
  • PCR reverse transcription in combination with the polymerase chain reaction
  • RT-PCR reverse transcription in combination with the ligase chain reaction
  • the present invention also relates to diagnostic assays such as quantitative and diagnostic assays for detecting levels of PSGR protein, or the soluble form thereof, in a biological sample (e.g., cells and tissues), including determination of normal and abnormal levels of polypeptides.
  • a diagnostic assay in accordance with the invention for detecting over-expression of PSGR compared to normal control tissue samples may be used to detect the presence of tumors, for example.
  • Assay techniques that can be used to determine levels of a protein, such as a PSGR protein of the present invention in a sample derived from a host are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays. Assaying PSGR protein levels in a biological sample can occur using any art-known method.
  • Assaying PSGR polypeptide levels in a biological sample can occur using antibody-based techniques.
  • PSGR polypeptide expression in tissues can be studied with classical immunohistological methods (Jalkanen, M., et al, J. Cell. Biol. 707:976-985 (1985); Jalkanen, M., et al, J. Cell . Biol. 705:3087-3096 (1987)).
  • Other antibody-based methods useful for detecting PSGR polypeptide gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
  • ELISA enzyme linked immunosorbent assay
  • RIA radioimmunoassay
  • Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase, and radioisotopes, such as iodine ( 125 1, 121 I), carbon
  • fluorescent labels such as fluorescein and rhodamine, and biotin.
  • the tissue or cell type to be analyzed will generally include those which are known, or suspected, to express the PSGR gene (such as, for example, cells of the prostate or prostate cancer).
  • the protein isolation methods employed herein may, for example, be such as those described in Harlow and Lane (Harlow, E. and Lane, D., 1988, "Antibodies: A
  • the isolated cells can be derived from cell culture or from a .patient.
  • the analysis of cells taken from culture may be a necessary step in the assessment of cells that could be used as part of a cell-based gene therapy technique or, alternatively, to test the effect of compounds on the expression of the
  • antibodies, or fragments of antibodies, such as those described herein may be used to quantitatively or qualitatively detect the presence of PSGR gene products or conserved variants or peptide fragments thereof. This can be accomplished, for example, by immunofluorescence techniques employing a fluorescently labeled antibody coupled with light microscopic, flow cytometric, or fluorimetric detection.
  • antibodies, or fragments of antibodies directed to any one or all of the extracellular or intracellular domains of PSGR may be used to quantitatively or qualitatively detect the presence of PSGR gene products or conserved variants or peptide fragments thereof. This can be accomplished, for example, by immunofluorescence techniques employing a fluorescently labeled antibody coupled with light microscopic, flow cytometric, or fluorimetric detection.
  • antibodies, or fragments of antibodies directed to a conformational epitope of PSGR may be used to quantitatively or qualitatively detect the presence of PSGR gene products or conserved variants or peptide fragments thereof. This can be accomplished, for example, by immunofluorescence techniques employing a fluorescently labeled antibody coupled with light microscopic, flow cytometric, or fluorimetric detection.
  • the antibodies (or fragments thereof), and/or PSGR polypeptides of the present invention may, additionally, be employed histologically, as in immunofluorescence, immunoelectron microscopy or non-immunological assays, for in situ detection of PSGR gene products or conserved variants or peptide fragments thereof.
  • In situ detection may be accomplished by removing a histological specimen from a patient, and applying thereto a labeled antibody or PSGR polypeptide of the present invention.
  • the antibody (or fragment) or PSGR polypeptide is preferably applied by overlaying the labeled antibody (or fragment) onto a biological sample.
  • PSGR polypeptide binding but also its distribution in the examined tissue.
  • histological methods such as staining procedures
  • Immunoassays and non-immunoassays for PSGR gene products or conserved variants or peptide fragments thereof will typically comprise incubating a sample, such as a biological fluid, a tissue extract, freshly harvested cells, or lysates of cells which have been incubated in cell culture, in the presence of a detectably labeled antibody capable of binding
  • a sample such as a biological fluid, a tissue extract, freshly harvested cells, or lysates of cells which have been incubated in cell culture
  • PSGR gene products or conserved variants or peptide fragments thereof and detecting the bound antibody by any of a number of techniques well-known in the art.
  • the biological sample may be brought in contact with and immobilized onto a solid phase support or carrier such as nitrocellulose, or other solid support which is capable of immobilizing cells, cell particles or soluble proteins.
  • a solid phase support or carrier such as nitrocellulose, or other solid support which is capable of immobilizing cells, cell particles or soluble proteins.
  • the support may then be washed with suitable buffers followed by treatment with the detectably labeled anti-PSGR antibody or detectable PSGR polypeptide.
  • the solid phase support may then be washed with the buffer a second time to remove unbound antibody or polypeptide.
  • the antibody is subsequently labeled.
  • the amount of bound label on solid support may then be detected by conventional means.
  • solid phase support or carrier any support capable of binding an antigen or an antibody.
  • supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite.
  • the nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention.
  • the support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody.
  • the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod.
  • the surface may be flat such as a sheet, test strip, etc.
  • Preferred supports include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.
  • binding activity of a given lot of anti-PSGR antibody or PSGR polypeptide may be determined according to well known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.
  • PSGR polypeptide or polynucleotide can also be detected in vivo by imaging.
  • PSGR polypeptide and/or anti-PSGR antibodies are used to image prostate neoplasms.
  • PSGR polynucleotides of the invention e.g., polynucleotides complementary to all or a portion of PSGR mRNA
  • anti-PSGR antibodies e.g., antibodies directed to any one or a combination of the extracellular or intracellular domains of PSGR, antibodies directed to a conformational epitope of PSGR, antibodies directed to the full length polypeptide expressed on the cell surface of a mammalian cell
  • PSGR polynucleotides of the invention e.g., polynucleotides complementary to all or a portion of PSGR mRNA
  • anti-PSGR antibodies e.g., antibodies directed to any one or a combination of the extracellular or intracellular domains of PSGR, antibodies directed to a conformational epitope of PSGR, antibodies directed to the full length polypeptide expressed on the cell surface of a mammalian cell
  • Antibody labels or markers for in vivo imaging of PSGR polypeptide include those detectable by X-radiography, NMR, MRI, CAT-scans or ESR.
  • suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject.
  • suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the antibody by labeling of nutrients for the relevant hybridoma.
  • in vivo imaging is used to detect enhanced levels of PSGR polypeptide for diagnosis in humans, it may be preferable to use human antibodies or "humanized" chimeric monoclonal antibodies. Such antibodies can be produced using techniques described herein or otherwise known in the art. For example methods for producing chimeric antibodies are known in the art. See, for review,
  • any PSGR polypeptide whose presence can be detected can be administered.
  • PSGR polypeptides labeled with a radio-opaque or other appropriate compound can be administered and visualized in vivo, as discussed, above for labeled antibodies. Further such PSGR polypeptides can be utilized for in vitro diagnostic procedures.
  • a PSGR polypeptide-specific antibody or antibody fragment which has been labeled with an appropriate detectable imaging moiety such as a radioisotope (for example,
  • a radio-opaque substance or a material detectable by nuclear magnetic resonance
  • a radio-opaque substance or a material detectable by nuclear magnetic resonance
  • 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 millicuries of 99m Tc.
  • the labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain PSGR protein.
  • S.W. Burchiel et al. "Dnmunopharmacokinetics of Radiolabeled Antibodies and
  • one of the ways in which the anti-PSGR antibody can be detectably labeled is by linking the same to an enzyme and using the linked product in an enzyme immunoassay (EIA) (Voller, A., "The Enzyme Linked Immunosorbent Assay
  • the enzyme which is bound to the antibody will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorimetric or by visual means.
  • Enzymes which can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta- galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. Additionally, the detection can be accomplished by colorimetric methods which employ a chromogenic substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.
  • Detection may also be accomplished using any of a variety of other immunoassays.
  • a radioimmunoassay see, for example, Weintraub, B.,
  • the radioactive isotope can be detected by means including, but not limited to, a gamma counter, a scintillation counter, or autoradiography.
  • fluorescent labeling compounds fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, ophthaldehyde and fluorescamine.
  • the antibody can also be detectably labeled using fluorescence emitting metals such as 152 Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
  • DTPA diethylenetriaminepentacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • the antibody also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
  • chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • a bioluminescent compound may be used to label the antibody of the present invention.
  • Bioluminescence is a type of chemiluminescence found in biological systems in, which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence.
  • Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.
  • the present invention encompasses polypeptides comprising, or alternatively consisting of, an epitope of the polypeptide having an amino acid sequence of SEQ DD NO:2, or an eptiope of the polypeptide having an amino acid sequence of SEQ DD NO:4, or an epitope of the polypeptide sequence encoded by a polynucleotide sequence contained in ATCC deposit No. 97131, or encoded by a polynucleotide that hybridizes to the complement of the sequence of SEQ DD NO:l, or encoded by a polynucleotide that hybridizes to the complement of the sequence of SEQ DD NO:3, or contained in ATCC deposit No.
  • the present invention further encompasses polynucleotide sequences encoding an epitope of a polypeptide sequence of the invention (such as, for example, the sequence disclosed in SEQ DD ⁇ NO:l), polynucleotide sequences of the complementary strand of a polynucleotide sequence encoding an epitope of the invention, and polynucleotide sequences which hybridize to the complementary strand under stringent hybridization conditions or lower stringency hybridization conditions defined supra.
  • epitopes refers to portions of a polypeptide having antigenic or immunogenic activity in an animal, preferably a mammal, and most preferably in a human.
  • the present invention encompasses a polypeptide comprising an epitope, as well as the polynucleotide encoding this polypeptide.
  • An "immunogenic epitope,” as used herein, is defined as a portion of a protein that elicits an antibody response in an animal, as determined by any method known in the art, for example, by the methods for generating antibodies described infra. (See, for example, Geysen et al., Proc. Natl. Acad. Sci.
  • antigenic epitope is defined as a portion of a protein to which an antibody can immunospecifically bind its antigen as determined by any method well known in the art, for example, by the immunoassays described herein. Immunospecific binding excludes non-specific binding but does not necessarily exclude cross- reactivity with other antigens. Antigenic epitopes need not necessarily be immunogenic.
  • antigenic epitopes preferably contain a sequence of at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, and, most preferably, between about 15 to about 30 amino acids.
  • Preferred polypeptides comprising immunogenic or antigenic epitopes are at least 10, 15, 20,
  • Additional non-exclusive preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as portions thereof. Antigenic epitopes are useful, for example, to raise antibodies, including monoclonal antibodies, that specifically bind the epitope.
  • Preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these antigenic epitopes.
  • Antigenic epitopes can be used as the target molecules in immunoassays. (See, for instance, Wilson et al., Cell
  • immunogenic epitopes can be used, for example, to induce antibodies according to methods well known in the art. (See, for instance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al., J. Gen.
  • immunogenic epitopes include the immunogenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these immunogenic epitopes.
  • the polypeptides comprising one or more immunogenic epitopes may be presented for eliciting an antibody response together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse), or, if the polypeptide is of sufficient length (at least about 25 amino acids), the polypeptide may be presented without a carrier.
  • immunogenic epitopes comprising as few as 8 to 10 amino acids have been shown to be sufficient to raise antibodies capable of binding to, at the very least, linear epitopes in a denatured polypeptide (e.g., in Western blotting).
  • Epitope-bearing polypeptides of the present invention may be used to induce antibodies according to methods well known in the art including, 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).
  • 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.
  • Animals such as rabbits, rats and mice are immunized with either free or carrier- coupled peptides, for instance, by intraperitoneal and/or intradermal injection of emulsions containing about 100 ⁇ g of peptide or carrier protein and Freund's adjuvant or any other adjuvant known for stimulating an immune response.
  • booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody which can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface.
  • the titer of anti-peptide antibodies in serum from an immunized animal may be increased by selection of anti-peptide antibodies, for instance, by adsorption to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.
  • polypeptides of the present invention comprising an immunogenic or antigenic epitope can be fused to other polypeptide sequences.
  • the polypeptides of the present invention may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CHI, CH2, CH3, or any combination thereof and portions thereof) resulting in chimeric polypeptides.
  • immunoglobulins IgA, IgE, IgG, IgM
  • CHI constant domain of immunoglobulins
  • IgG Fusion proteins that have a disulfide-linked dimeric structure due to the IgG portion desulfide bonds have also been found to be more efficient in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone. See, e.g., Fountoulakis et al., J. Biochem., 270:3958-3964 (1995). Nucleic acids encoding the above epitopes can also be recombined with a gene of interest as an epitope tag (e.g., the hemagglutinin (“HA”) tag or flag tag) to aid in detection and purification of the expressed polypeptide.
  • an epitope tag e.g., the hemagglutinin (“HA") tag or flag tag
  • the gene of interest is subcloned into a vaccinia recombination plasmid such that the open reading frame of the gene is translationally fused to an amino-terminal tag consisting of six histidine residues.
  • the tag serves as a matrix binding domain for the fusion protein. Extracts from cells infected with the recombinant vaccinia virus are loaded onto Ni2+ nitriloacetic acid-agarose column and histidine-tagged proteins can be selectively eluted with imidazole-containing buffers.
  • Additional fusion proteins of the invention may be generated through the techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling
  • DNA shuffling may be employed to modulate the activities of polypeptides of the invention, such methods can be used to generate polypeptides with altered activity, as well as agonists and antagonists of the polypeptides.
  • alteration of polynucleotides corresponding to SEQ DD NO:l and the polypeptides encoded by these polynucleotides may be achieved by DNA shuffling.
  • DNA shuffling involves the assembly of two or more DNA segments by homologous or site-specific recombination to generate variation in the polynucleotide sequence.
  • polynucleotides of the invention, or the encoded polypeptides may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination.
  • one or more components, motifs, sections, parts, domains, fragments, etc., of a polynucleotide encoding a polypeptide of the invention may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.
  • polypeptides of the invention relate to antibodies and T-cell antigen receptors (TCR) which immunospecifically bind a PSGR polypeptide, polypeptide fragment, or variant of SEQ DD NO:2, and/or an epitope, of the present invention (as determined by immunoassays well known in the art for assaying specific antibody-antigen binding).
  • TCR T-cell antigen receptors
  • Antibodies of the invention include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') fragments, fragments produced by a Fab expression library, anti-idiotypic
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen.
  • the immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.
  • the immunoglobulin molecules of the invention are IgGl.
  • the immunoglobulin molecules of the invention are IgG4.
  • the antibodies are 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 NL 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 immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulin and that do not express endogenous immunoglobulins, as described infra and, for example in, U.S. Patent No. 5,939,598 by Kucherlapati et al.
  • the antibodies of the present invention may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of a polypeptide of the present invention or may be specific for both a polypeptide of the present invention as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material. See, e.g., PCT publications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J. Immunol. 147:60-69 (1991); U.S. Patent Nos.
  • 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 may also be described or specified in terms of their cross-reactivity. Antibodies that do not bind any other analog, ortholog, or homolog of a polypeptide of the present invention are included. Antibodies that bind polypeptides with at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 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. In specific embodiments, antibodies of the present invention cross-react with murine, rat and/or rabbit homologs 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 are also included in the present invention.
  • Preferred binding affinities include those with a dissociation constant or Kd less than 5 X IO "2 M, IO “2 M, 5 X 10 "3 M, 10 “3 M, 5 X IO “4 M, 10 " 4 M, 5 X 10 "5 M, IO “5 M, 5 X IO “6 M, 10 “6 M, 5 X IO “7 M, IO 7 M, 5 X 10 "8 M, 10 “8 M, 5 X IO “9 M, IO "9 M, 5 X 10 "10 M, 10 “10 M, 5 X 10 "11 M, IO “11 M, 5 X IO "12 M, 10"12 M, 5 X 10 "13 M, 10 " 13 M, 5 X IO “14 M, IO “14 M, 5 X 10 “15 M, or IO “15 M.
  • the invention also provides antibodies that competitively inhibit binding of an antibody to an epitope of the invention as determined by any method known in the art for determining competitive binding, for example, the immunoassays described herein.
  • the antibody competitively inhibits binding to the epitope 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%.
  • 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) may be determined by techniques described herein or otherwise known in the art.
  • receptor activation can be determined by detecting the phosphorylation (e.g., tyrosine or serine/threonine) of the receptor or its substrate by immunoprecipitation followed by western blot analysis (for example, as described supra).
  • phosphorylation e.g., tyrosine or serine/threonine
  • 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.
  • the invention also features receptor-specific antibodies which both prevent ligand binding and receptor activation as well as antibodies that recognize the receptor-ligand complex, and, preferably, do not specifically recognize the unbound receptor or the unbound ligand.
  • receptor-specific antibodies which both prevent ligand binding and receptor activation as well as antibodies that recognize the receptor-ligand complex, and, preferably, do not specifically recognize the unbound receptor or the unbound ligand.
  • neutralizing antibodies which bind the ligand and prevent binding of the ligand to the receptor, as well as antibodies which bind the ligand, thereby preventing receptor activation, but do not prevent the ligand from binding the receptor.
  • antibodies which activate the receptor are also act as receptor agonists, i.e., potentiate or activate either all or a subset of the biological activities of the ligand-mediated receptor activation, for example, by inducing dimerization of the receptor.
  • the antibodies may be specified as agonists, antagonists or inverse agonists for biological activities comprising the specific biological activities of the peptides of the invention disclosed herein.
  • the above antibody agonists can be made using methods known in the art. See, e.g., PCT publication WO 96/40281; U.S. Patent No. 5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chen et al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al., J. Immunol. 161(4): 1786-1794 (1998); Zhu et al., Cancer Res. 58(15):3209-3214 (1998); Yoon et al., J.
  • Antibodies of the present invention may be used, for example, but not limited to, to purify, detect, and target the polypeptides of the present invention, including both in vitro and in vivo diagnostic and therapeutic methods.
  • the antibodies have use in immunoassays for qualitatively and quantitatively measuring levels of the polypeptides of the present invention 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).
  • the antibodies of the present invention may be used either alone or in combination with other compositions.
  • the antibodies may further 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, radionuclides, or toxins. See, e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Patent No.
  • 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, phosphylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc.
  • the antibodies of the present invention may be generated by any suitable method known in the art.
  • Polyclonal antibodies to an antigen-of-interest can be produced by various procedures well known in the art.
  • a polypeptide of the invention 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.
  • adjuvants may be used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum. Such adjuvants are also well known in the art.
  • Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.
  • monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al.,
  • “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology.
  • the term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.
  • mice can be immunized with a polypeptide of the invention or a cell expressing such peptide.
  • an immune response e.g., antibodies specific for the antigen are detected in the mouse serum
  • the mouse spleen is harvested and splenocytes isolated.
  • the splenocytes are then fused by well known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC. Hybridomas are selected and cloned by limited dilution.
  • hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding a polypeptide of the invention.
  • Ascites fluid which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.
  • the present invention provides methods of generating monoclonal antibodies as well as antibodies produced by the method comprising culturing a hybridoma cell secreting an antibody of the invention wherein, preferably, the hybridoma is generated by fusing splenocytes isolated from a mouse immunized with an antigen of the invention with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind a polypeptide of the invention.
  • Antibody fragments which recognize specific epitopes may be generated by known techniques.
  • Fab and F(ab')2 fragments of the invention may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
  • F(ab ⁇ 2 fragments contain the variable region, the light chain constant region and the CHI domain of the heavy chain.
  • the antibodies of the present invention can also be generated using various phage display methods known in the art and as discussed in detail in the Examples
  • 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 ID or gene VID protein.
  • 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);
  • 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.
  • mammalian cells including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below.
  • F(ab')2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869
  • 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.,
  • 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. Often, framework residues in the human framework regions will be substituted with the conesponding residue from the CDR donor antibody to alter, preferably improve, antigen binding.
  • CDRs complementarity determining regions
  • 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. Patent Nos.
  • 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. Patent Nos. 4,444,887 and 4,716,111; and
  • 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.
  • 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)).

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US7361338B2 (en) 1999-10-05 2008-04-22 Agensys, Inc. Methods to inhibit growth of prostate cancer cells
US6790631B1 (en) 1999-10-05 2004-09-14 Agensys, Inc. G protein-coupled receptor up-regulated in prostate cancer and uses thereof

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WO1996039435A1 (en) * 1995-06-05 1996-12-12 Human Genome Sciences, Inc. Human g-protein receptor hpraj70
WO2001034802A2 (en) * 1999-11-12 2001-05-17 Corixa Corporation Compositions and methods for the therapy and diagnosis of prostate cancer
WO2001039798A1 (en) * 1999-12-06 2001-06-07 Diadexus, Inc. Method of diagnosing, monitoring, staging, imaging and treating prostate cancer

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Publication number Priority date Publication date Assignee Title
WO1996039435A1 (en) * 1995-06-05 1996-12-12 Human Genome Sciences, Inc. Human g-protein receptor hpraj70
WO2001034802A2 (en) * 1999-11-12 2001-05-17 Corixa Corporation Compositions and methods for the therapy and diagnosis of prostate cancer
WO2001039798A1 (en) * 1999-12-06 2001-06-07 Diadexus, Inc. Method of diagnosing, monitoring, staging, imaging and treating prostate cancer

Non-Patent Citations (2)

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Title
See also references of WO0228899A1 *
XU L L ET AL: "PSGR, a novel prostate-specific gene with homology to a G protein-coupled receptor, is overexpressed in prostate cancer." CANCER RESEARCH. 1 DEC 2000, vol. 60, no. 23, 1 December 2000 (2000-12-01), pages 6568-6572, XP002290228 ISSN: 0008-5472 *

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