EP1169449A2 - Clasp-2 transmembranproteine - Google Patents

Clasp-2 transmembranproteine

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Publication number
EP1169449A2
EP1169449A2 EP00923388A EP00923388A EP1169449A2 EP 1169449 A2 EP1169449 A2 EP 1169449A2 EP 00923388 A EP00923388 A EP 00923388A EP 00923388 A EP00923388 A EP 00923388A EP 1169449 A2 EP1169449 A2 EP 1169449A2
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EP
European Patent Office
Prior art keywords
clasp
polypeptide
sequence
polynucleotide
cell
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EP00923388A
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English (en)
French (fr)
Inventor
Peter S. Lu
Jonathan David Garman
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Arbor Vita Corp
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Arbor Vita Corp
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Publication of EP1169449A2 publication Critical patent/EP1169449A2/de
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    • 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
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates

Definitions

  • the present invention relates to molecules expressed in cells ofthe immune system.
  • the invention relates to a transmembrane protein that contains certain classical cadherin characteristics.
  • T H The helper T cell
  • APC antigen-presenting cell
  • TCR T-cell antigen receptor
  • Costimulatory molecules such as CD4, ICAM-1, LFA-1, CD28, CD2 have been proposed to stabilize the cell-cell contact (Dustin, et al, 1999, Science 283: 649). However, since these molecules are recruited to the synapse after activation they cannot account for the high specificity and avidity during the early phases of T-cell antigen recognition. Recent work demonstrated that a portion of the T cell surface at the leading edge is specialized to mediate the early phases of synapse formation (Negulescu, et al. , 1996, Immunity 4: 421-430).
  • Such a specialization must be a pre-formed structure containing cell surface adhesion proteins (ectodomains) to augment TCR engagement and corresponding cytoplasmic portions (endodomains) to transduce signals and bind cytoskeleton to maintain structural/functional polarity.
  • ectodomains cell surface adhesion proteins
  • endodomains cytoplasmic portions
  • CLASP-1 The ectodomain ofthe pre-formed synapse or "immune gateway" was recently discovered and is created in part by CLASP-1 (U.S.S.N. 09/411,328, filed October 1, 1999; PCT/US99/22996).
  • CLASP-1 also contains a CRK-SH3 binding domain, tyrosine phosphorylation sites, and coiled/coil domains suggesting direct interaction with cytoskeleton and regulation by adaptor molecules such as CRK.
  • the CLASP-1 transcript is present in lymphoid organs and neural tissue, and the protein is expressed by T and B lymphocytes and macrophages in the MOMA-1 subregion of the marginal zone ofthe spleen, an area known to be important in T: B cell interaction.
  • CLASP- 1 staining of individual T and B cells exhibits a preactivation structural polarity, being organized as a "ball” or “cap” structure in B cells, and forming a "ring", "ball” or “cap” structure in T cells. The placement of these structures is adjacent to the microtubule- organizing center ("MTOC").
  • MTOC microtubule- organizing center
  • CLASP-1 antibody staining indicates that CLASP-1 is at the interface of T-B cell conjugates that are fully committed to differentiation. Antibodies to the extracellular domain of CLASP-1 also block T-B cell conjugate formation and T cell activation.
  • the present invention relates to a cell surface molecule, a member of a new multigene family designated cadherin-like asymmetry protein(s) ("CLASP(s)").
  • CLASP(s) cadherin-like asymmetry protein
  • a polynucleotide comprising a coding sequence for CLASP-2, a polynucleotide that selectively hybridizes to the complement of a CLASP-2 coding sequence, expression vectors containing such polynucleotides, genetically-engineered host cells containing such polynucleotides, CLASP-2 polypeptides, CLASP-2 fusion proteins, therapeutic compositions, CLASP-2 domain mutants, antibodies specific for CLASP-2 polypeptides, methods for detecting the expression of CLASP-2, and methods of inhibiting an immune response by interfering with CLASP-2 function.
  • a wide variety of uses are encompassed by the invention, including but not limited to, treatment of autoimmune diseases and hypersensitivities, prevention of transplantation rejection responses,
  • the invention provides an isolated CLASP-2 polynucleotide that is: (a) a polynucleotide that has the sequence of SEQ ID NO: 1, 3, 5 or 9; (b) a polynucleotide that hybridizes under stringent hybridization conditions to (a) and encodes a polypeptide having the sequence of SEQ ID NO: 2, 4, 6 or 10 or an allelic variant or homologue of a polypeptide having the sequence of SEQ ID NO: 2, 4, 6 or 10; or (c) a polynucleotide that hybridizes under stringent hybridization conditions to (a) and encodes a polypeptide with at least 25 contiguous residues ofthe polypeptide of SEQ ID NO: 2, 4, 6 or 10; or (d) a polynucleotide that hybridizes under stringent hybridization conditions to (a) and has at least 12 contiguous bases identical to or exactly complementary to SEQ ID NO: 1, 3, 5, or 9.
  • the invention provides a CLASP-2 polynucleotide wherein the polynucleotide encodes a polypeptide that binds to the PDZ domain of PSD95, DLG1 or neDLG. 2. In another related aspect, the invention provides a CLASP-2 polynucleotide wherein the polynucleotide encodes a polypeptide that has a binding affinity of at least 10 4 M "1 for binding PSD95, DLG1 or neDLG.
  • the invention provides a CLASP-2 polynucleotide that encodes a polypeptide having the full-length sequence of SEQ ID NO: 2, 4, 6, or 10 or the cDNA coding sequence of ATCC Deposit Nos: PTA-1562 and PTA-1563.
  • the invention further provides an isolated CLASP-2 polynucleotide comprising a nucleotide sequence that has at least 90% percent identity to SEQ ID NO: 1, 3, 5 or 9 as calculated using FAST A wherein said sequences are aligned so that highest order match between said sequences is obtained.
  • the invention further provides an isolated polypeptide comprising a nucleotide sequence that has at least 90% sequence identity to SEQ ID NO: 2, 4, 6 or 10 and is immunologically crossreactive with SEQ ID NO: 2, 4, 6 or 10 or shares a biological function with native CLASP-2.
  • the invention also provides vectors, such as expression vectors, comprising a polynucleotide sequence ofthe invention.
  • the invention provides host cells or progeny of the host cells comprising a vector of the invention.
  • the host cell is a eukaryote.
  • the expression vector comprises a CLASP-2 polynucleotide in which the nucleotide sequence ofthe polynucleotide is operatively linked with a regulatory sequence that controls expression of the polynucleotide in a host cell.
  • the invention provides a host cell comprising a CLASP-2 polynucleotide, wherein the nucleotide sequence of the polynucleotide is operatively linked with a regulatory sequence that controls expression of the polynucleotide in a host cell, or progeny ofthe cell.
  • the invention further provides a CLASP-2 polynucleotide that is an antisense polynucleotide.
  • the antisense polynucleotide is less than about 200 bases in length.
  • the invention provides an antisense oligonucleotide complementary to a messenger RNA comprising SEQ ID NO: 1, 3, 5 or 9 and encoding CLASP-2, wherein the oligonucleotide inhibits the expression of CLASP-2.
  • the invention provides an isolated DNA that encodes a CLASP-2 protein as shown in SEQ ID NO: 2, 4, 6 or 10.
  • the CLASP-2 polynucleotide is RNA.
  • the invention provides a method for producing a polypeptide comprising: (a) culturing the host cell containing a CLASP-2 polynucleotide under conditions such that the polypeptide is expressed; and (b) recovering the polypeptide from the cultured host cell or its cultured medium.
  • the invention further provides an isolated CLASP-2 polypeptide encoded by a CLASP-2 polynucleotide.
  • the CLASP-2 polypeptide has the amino acid sequence of SEQ ID NO: 2, 4, 6 or 10, or a fragment thereof.
  • the isolated CLASP-2 polypeptide is cell-membrane associated.
  • the isolated CLASP -2 polypeptide is soluble.
  • the soluble CLASP-2 polypeptide is fused with a heterologous polypeptide.
  • the invention further provides an isolated CLASP-2 protein having the sequence as shown in SEQ ID NO: 2, 4, 6 or 10.
  • the invention provides a CLASP-2 protein comprising the sequence as shown in SEQ. ID.
  • the invention further provides an isolated antibody that specifically binds to a polypeptide having the amino acid sequence as shown in SEQ ID NO: 2, 4, 6 or 10, or a binding fragment thereof. In some embodiments the antibody is monoclonal. In other embodiments, the invention provides a hybridoma capable of secreting the antibody.
  • the invention further provides a method of identifying a compound or agent that binds a CLASP-2 polypeptide comprising: i) contacting a CLASP-2 polypeptide with the compovmd or agent under conditions which allow binding of the compound to the CLASP-2 polypeptide to form a complex and ii) detecting the presence ofthe complex.
  • the invention further provides a method of detecting a CLASP-2 polypeptide in a sample, comprising: (a) contacting the sample with a CLASP-2 antibody or binding fragment and (b) determining whether a complex has been formed between the antibody and with CLASP-2 polypeptide.
  • the invention further provides a method of detecting a CLASP-2 polypeptide in a sample, comprising: (a) contacting the sample with a CLASP-2 polynucleotide or a polynucleotide that comprises a sequence of at least 12 nucleotides and is complementary to a contiguous sequence of the CLASP-2 polynucleotide and (b) determining whether a hybridization complex has been formed.
  • the invention further provides a method of detecting a CLASP-2 nucleotide in a sample, comprising: (a) using a polynucleotide that comprises a sequence of at least 12 nucleotides and is complementary to a contiguous sequence of a CLASP-2 polynucleotide in an amplification process; and (b) determining whether a specific amplification product has been formed.
  • the invention further provides pharmaceutical compositions comprising a CLASP-2 polynucleotide, a CLASP-2 polypeptide, or a CLASP-2 antibody and a pharmaceutically acceptable carrier.
  • the invention provides a method of inhibiting an immune response in a cell comprising: (a) interfering with the expression of a CLASP-2 gene in the cell; (b) interfering with the ability of a CLASP-2 protein to mediate cell-cell interaction (e.g., interfering with a heterotypic and/or homotypic interaction) between CLASP-2 and an extracellular protein; (c) interfering with the ability of a CLASP-2 protein to bind to another protein.
  • the cell is a T cell or a B cell.
  • Some such methods comprise contacting the cell with an effective amount of a polypeptide which comprises the amino acid sequence of SEQ ID NO: 2, 4, 6 or 10 or a fragment thereof.
  • the invention provides a method of inhibiting an immune response in a subject, comprising administering to the subject a therapeutically effective amount of an antibody which specifically binds a polypeptide having the sequence of SEQ ID NO: 2, 4, 6 or 10.
  • the invention provides a method of preventing or treating a CLASP-2-mediated disease comprising administering to a subject in need thereof a therapeutically effective amount of a CLASP-2 pharmaceutical composition.
  • the CLASP-2-mediated disease is an autoimmune disease.
  • the invention further provides a method of treating an autoimmune disease in a subject caused or exacerbated by increased activity of T H 1 cells consisting of administering a therapeutically effective amount of a CLASP-2 pharmaceutical composition to the subject.
  • FIG. 1 Nucleotide and predicted amino acid sequence of CLASP-2A cDNA. Notable protein motifs are indicated above the nucleotide sequence in bold. Potential initiator methionines are underscored. The notable, predicted protein motifs are: a cadherin cleavage site encoded by nucleotides 854-868, a cadherin ectodomain (EC) encoded by nucleotides 1253-1264, a transmembrane domain encoded by nucleotides 2861-2917, a coiled coil domain encoded by nucleotides 3579-3682, a second coiled coil domain encoded by nucleotides 3827-3937, and a PDZ binding motif (PBM) encoded by nucleotides 4046-4057.
  • EC cadherin ectodomain
  • PBM PDZ binding motif
  • FIG. 1 A. Schematic of CLASP-2 splice variants. Splice variants are compared to Human (h) CLASP-2A. Numbers above hCLASP-2A line diagram indicate where splice variations comprising deletions and insertions relative to hCLASP-2A are found. Abbreviations: "KIAA” KIAA1058 sequence (Genbank Accession No. AB028981).
  • B Nucleotide and predicted amino acid sequence of CLASP-2 A cDNA. Notable protein motifs are indicated above the nucleotide sequence in bold. Exact position of insertions and deletions are indicated above the CLASP-2A sequence with arrows and "x", respectively.
  • nucleotide sequence of insertions schematized in FIG.2 A are indicated above the arrow.
  • the insertions and deletions are as follows (numeration refers to Human CLASP-2A nucleotide sequence): Nucleotides 1966-2034 are deleted in CLASP-2D. Nucleotides 2219- 2224 are deleted in CLASP-2B. There is an insertion of 69 amino acids at nucleotide 2927 found in CLASP-2D.
  • the nucleotide sequence for this insertion is: AAGCAGTCCAGTGGGAGCCGCCCCTTCTCCCCCACAGCCATAGCGCCTGCCTGA GGAGGAGCCGGGGAG and encodes amino acids ANQWEPPLLPHSHSACLRRSRG (one letter amino acid abbreviation).
  • This amino acid sequence encodes a putative immunoreceptor tyrosine-based activation motif (ITAM).
  • ITAM immunoreceptor tyrosine-based activation motif
  • FIG. 1 Alignment of nucleotide sequences ofthe CLASP-2 isoforms. Sequences were aligned using ClustalW
  • B Alignment of amino acid sequence ofthe CLASP-2 isoforms. Sequences were aligned using ClustalW. One letter amino acid abbreviation is used.
  • FIG. 4 Expression of CLASP-2 in human cell lines and human tissues as determined by Northern hybridization.
  • a CLASP-2-specific DNA fragment was generated by PCR from a CLASP-2 cDNA clone (HC2-5'), using primers HC2AS2 and HC2S1. The fragment was labeled by incorporation of radioactive 32 P dCTP.
  • a Northern with RNA from multiple cells lines was hybridized with the same hCLASP-2 probe.
  • a similarly migrating band is detected in Jurkat (T-cell derived), 9D10 (B-cell derived) and 293 (human kidney derived) cell lines.
  • 9D10 B-cell derived
  • 293 human kidney derived
  • There are multiple weaker bands in the 9D10 lane indicating possible splice variants of hCLASP-2.
  • Weak expression is also detected in the mouse cell lines CH27 (B cell lymphoma) and 3 A9 (T-cell hybridoma). Since hybridization and washing were carried out at high stringency, this indicates that the human CLASP-2 probe may cross-react with mouse CLASP mRNA.
  • FIG. 5 A. Amino acid sequence of human and rat CLASP proteins. Sequences were aligned using ClustalW. One letter amino acid abbreviation used. Protein motifs are found within the labeled boxes. A “-” indicates gaps that are placed to acquire a best overall alignment. Other abbreviations: “HC2A” Human CLASP-2 sequence, “KIAA” KIAA1058 sequence (Genbank Accession No. AB028981), “rat” TRG gene (Genbank Accession No. X68101), “HC4" Human CLASP-4 sequence, “HCl” Human CLASP-1 sequence, “HC3” Human CLASP-3 sequence, "HC5" Human CLASP-5 sequence. B. Alignment of DOCK motifs found within the human CLASPs and compared to canonical DOCK motifs. Consensus amino acids found within all DOCK motifs are also indicated.
  • FIG. 6 A. Nucleotide and predicted amino acid sequence of CLASP-2AcDNA. Notable protein motifs are indicated (see FIG. 1 legend for details). Additionally, boundaries between exons and introns are indicated by arrows. These boundaries were defined by sequencing Bacterial Artificial Chromosomes (BACs) containing genomic DNA corresponding to CLASP-2. BACs were sequenced using primers derived from exon sequences corresponding to the CLASP-2 cDNA. Each exon/intron boundary is noted (as "Ref with an appropriate reference number) above the cDNA sequence. The references contain exact nucleotide location of introns. The names and nucleotide numbers ofthe primers that were used in sequence reactions are also indicated.
  • BACs Bacterial Artificial Chromosomes
  • FIG. 7 Southern hybridization analysis of CLASP-2. Genomic DNA from HeLa cells or a BAC DNA clone was digested with EcoRI or HinDm (genomic DNA) or Pst I (BAC DNA) and eletrophoresed and transferred to nylon membrane by standard methods. For a probe, a CLASP-2-specific DNA fragment was generated by PCR from a CLASP-2 cDNA clone (HC2-5'), using primers HC2AS2 and HC2S1. The fragment was labeled by incorporation of radioactive P dCTP. Probe HC2.1 is 800 bp long and it recognizes two fragments (-4.5 kb and 1.85 kb) on Eco RI digested genomic DNA. Three fragments are revealed by this probe when hybridized to digested DNA of BACs 4 and 6, with the two major ones identical in size to those detected on genomic DNA.
  • FIG. 8 Expression of human CLASP-1 (hCLASP-1) CLASP-1 and CLASP-2 Glutathion- S-Transferase (GST) fusion proteins. Nucleotides encoding a portion of the hCLASP-2A intracellular domain (nucleotides 3230-4065) were subcloned into pGEX vectors (Pharmacia). Recombinant plasmids were transformed into E. coli (strain DH5 ⁇ ), and transformed strains were grown by standard conditions. While in log phase cells were either induced (I) with IPTG (0.1 mM final concentration) or left uninduced (U). After several additional hours of growth cells were harvested and soluble protein lysates generated by standard methods.
  • I induced
  • U left uninduced
  • CLASP-2 Inhibitor refers to a synthetic peptide composed of the eight C-terminal amino acids of CLASP-2.
  • KN1.3 Inhibitor refers to a synthetic peptide composed of the 19 C- terminal amino acids of KN1.3, a lymphocyte potassium channel. The amino acid sequence of the KV1.3 inhibitor is TT ⁇ P ⁇ SAN ⁇ IKKIFTDN. D. Inhibition of KV1.3 - PDZ binding.
  • patient or “subject” are used interchangeably and refer to mammals such as human patients and non-human primates, as well as experimental animals such as rabbits, rats, and mice, and other animals.
  • biological sample is a sample of biological tissue, fluid, or cells that contains hCLASP-2 or nucleic acid encoding hCLASP-2 protein. Such samples include, but are not limited to, tissue isolated from humans. Biological samples may also include sections of tissues such as frozen sections taken for histologic purposes.
  • a biological sample is typically obtained from a eukaryotic organism, preferably eukaryotes such as fungi, plants, insects, protozoa, birds, fish, reptiles, and preferably a mammal such as rat, mice, cow, dog, guinea pig, or rabbit, and most preferably a primate such as chimpanzees or humans.
  • treating includes the administration ofthe compounds or agents of the present invention to prevent or delay the onset of the symptoms, complications, or biochemical indicia of a disease, alleviating the symptoms or arresting or inhibiting further development of the disease, condition, or disorder (e.g., autoimmune disease).
  • Treatment may be prophylactic (to prevent or delay the onset of the disease, or to prevent the manifestation of clinical or subclinical symptoms thereof) or therapeutic suppression or alleviation of symptoms after the manifestation ofthe disease.
  • lymphocyte as used herein has the normal meaning in the art, and refers to any ofthe mononuclear, nonphagocytic leukocytes, found in the blood, lymph, and lymphoid tissues, i. e. , B and T lymphocytes.
  • isolated refers to material that is substantially free from components that normally accompany it as found in its native state (e.g., recombinantly produced or purified away from other cell components with which it is naturally associated). Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography.
  • purified denotes that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. Particularly, it means that the nucleic acid or protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure.
  • nucleic acid and “polynucleotide” are used interchangeably" and refer to refers to DNA, RNA and nucleic acid polymers containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non- naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides.
  • analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).
  • polypeptide peptide
  • protein protein
  • amino acids may be natural amino acids, or include an artificial chemical mimetic of a corresponding naturally occurring amino acid.
  • nucleic acid probe is defined as a nucleic acid capable of specifically binding to a target nucleic acid of complementary sequence (e.g., through complementary base pairing).
  • a probe may include natural (i.e., A, G, C, or T) or modified bases (7-deazaguanosine, inosine, and the like).
  • the bases in a probe may be joined by a linkage other than a phosphodiester bond, so long as it does not interfere with hybridization (e.g., probes may be peptide nucleic acids).
  • the probes can be directly labeled as with isotopes, chromophores, lumiphores, chromogens, or indirectly labeled such as with biotin to which a streptavidin complex may later bind.
  • recombinant when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or, in the case of cells, to progeny of a cell so modified.
  • recombinant cells express genes that are not found within the native (non- recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.
  • heterologous when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not fovmd in the same relationship to each other in nature.
  • the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source.
  • a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).
  • sequence identity refers to a measure of similarity between amino acid or nucleotide sequences, and can be measured using methods known in the art, such as those described below:
  • substantially identical in the context of two nucleic acids or polypeptides, refers to two or more sequences or subsequences that have at least of at least 60%, often at least 70%, preferably at least 80%, most preferably at least 90% or at least 95% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.
  • the substantial identity exists over a region ofthe sequences that is at least about 50 bases or residues in length, more preferably over a region of at least about 100 bases or residues, and most preferably the sequences are substantially identical over at least about 150 bases or residues.
  • the sequences are substantially identical over the entire length ofthe coding regions.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • sequence comparison of nucleic acids and proteins to CLASP-2 nucleic acids and proteins the BLAST and BLAST 2.0 algorithms and the default parameters discussed below are used.
  • a “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, 1981, Adv. Appl. Math.2: 482), by the homology alignment algorithm of Needleman & Wunsch, 1970, J. Mol.
  • a preferred example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the FASTA algorithm, which is described in Pearson, W.R. & Lipman, D.J., 1988, Proc. Natl. Acad. Sci. U.S, A. 85 : 2444. See also W. R. Pearson, 1996, Methods Enzymol.266: 227-258.
  • BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: //www.ncbi.nlm.nih.gov/).
  • This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive- valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased.
  • HSPs high scoring sequence pairs
  • Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always ⁇ 0).
  • M forward score for a pair of matching residues; always > 0
  • N penalty score for mismatching residues; always ⁇ 0.
  • a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative- scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed ofthe alignment.
  • W wordlength
  • E expectation
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 90: 5873-5787).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication ofthe probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison ofthe test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
  • PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show relationship and percent sequence identity. It also plots a tree or dendogram showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, 1987, J. Mol. Evol.35: 351-360. The method used is similar to the method described by Higgins & Sharp, 1989, CABIOS 5 : 151- 153. The program can align up to 300 sequences, each of a maximum length of 5,000 nucleotides or amino acids.
  • the multiple alignment procedure begins with the pairwise alignment ofthe two most similar sequences, producing a cluster of two aligned sequences. This cluster is then aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences are aligned by a simple extension ofthe pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive, pairwise alignments.
  • the program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison and by designating the program parameters. Using PILEUP, a reference sequence is compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps.
  • PILEUP can be obtained from the GCG sequence analysis software package, e.g., version 7.0 (Devereaux et al, 1984, Nuc. Acids Res. 12: 387-395.
  • Another preferred example of an algorithm that is suitable for multiple DNA and amino acid sequence alignments is the CLUSTALW program (Thompson, J. D. et al. , 1994, Nucl. Acids. Res. 22: 4673-4680).
  • ClustalW performs multiple pairwise comparisons between groups of sequences and assembles them into a multiple alignment based on homology. Gap open and Gap extension penalties were 10 and 0.05 respectively.
  • the BLOSUM algorithm can be used as a protein weight matrix (Henikoff and Henikoff, 1992, Proc. Natl. Acad. Sci. U.S.A. 89: 10915-10919).
  • a “label” is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means.
  • useful labels include 32 P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins for which antisera or monoclonal antibodies are available (e.g., the polypeptide of SEQ ID NO: 1 can be made detectable, e.g., by incorporating a radiolabel into the peptide, and used to detect antibodies specifically reactive with the peptide).
  • sorting in the context of cells as used herein to refers to both physical sorting ofthe cells, as can be accomplished using, e.g., a fluorescence activated cell sorter, as well as to analysis of cells based on expression of cell surface markers, e.g., FACS analysis in the absence of sorting.
  • the phrase "specifically (or selectively) binds" to an antibody refers to a binding reaction that is determinative of the presence of the protein in a heterogeneous population of proteins and other biologies.
  • the specified antibodies bind to a particular protein at least two times the background and do not substantially bind in a significant amount to other proteins present in the sample.
  • the phrase "specifically bind(s)" or “bind(s) specifically” when referring to a peptide refers to a peptide molecule which has intermediate or high binding affinity, exclusively or predominately, to a target molecule.
  • the phrases “specifically binds to” refers to a binding reaction which is determinative ofthe presence of a target protein in the presence of a heterogeneous population of proteins and other biologies.
  • the specified binding moieties bind preferentially to a particular target protein and do not bind in a significant amount to other components present in a test sample. Specific binding to a target protein under such conditions may require a binding moiety that is selected for its specificity for a particular target antigen.
  • a variety of assay formats may be used to select ligands that are specifically reactive with a particular protein.
  • solid-phase ELISA immunoassays, immunoprecipitation,Biacore and Western blot are used to identify peptides that specifically react with PDZ domain-containing proteins.
  • a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 times background.
  • Specific binding between a monovalent peptide and a PDZ-containing protein means a binding affinity of at least 10 4 M "1 , and preferably 10 5 or 10* NT 1 .
  • homotypic interaction refers to the binding of a given protein to another molecule ofthe same protein (e.g., the binding of hCLASP-2 to hCLASP-2).
  • heterotypic interaction refers to the binding of a given protein to a different protein or other molecule (e. g. , the binding of hCL ASP-2 to a PDZ domain-containing protein or the binding of a transcription factor to DNA).
  • immune cell response refers to the response of immune system cells to external or internal stimuli (e.g. , antigen, cytokines, chemokines, and other cells) producing biochemical changes in the immune cells that result in immune cell migration, killing of target cells, phagocytosis, production of antibodies, other soluble effectors ofthe immune response, and the like.
  • external or internal stimuli e.g. , antigen, cytokines, chemokines, and other cells
  • B lymphocyte response and “B lymphocyte activity” are used interchangeably to refer to the component of immune response carried out by B lymphocytes (i.e. the proliferation and maturation of B lymphocytes, the binding of antigen to cell surface immunogobulin, the intemalization of antigen and presentation of that antigen via MHC molecules to T lymphocytes, and the synthesis and secretion of antibodies).
  • T lymphocyte response and "T lymphocyte activity” are used here interchangeably to refer to the component of immune response dependent on T lymphocytes (/.-.., the proliferation and/or differentiation of T lymphocytes into helper, cytotoxic killer, or suppressor T lymphocytes, the provision of signals by helper T lymphocytes to B lymphocytes that cause or prevent antibody production, the killing of specific target cells by cytotoxic T lymphocytes, and the release of soluble factors such as cytokines that modulate the function of other immune cells).
  • immune response refers to the concerted action of lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the above cells or the liver (including antibodies, cytokines, and complement) that results in selective damage to, destruction of, or elimination from the human body of invading pathogens, cells or tissues infected with pathogens, cancerous cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues.
  • cytotoxic T lymphocytes can be incubated with radioactively labeled target cells and the lysis of these target cells detected by the release of radioactivity
  • helper T lymphocytes can be incubated with antigens and antigen presenting cells and the synthesis and secretion of cytokines measured by standard methods (Windhagen A; et al, 1995, Immunity 2(4): 373- 80)
  • antigen presenting cells can be incubated with whole protein antigen and the presentation of that antigen on MHC detected by either T lymphocyte activation assays or biophysical methods (Harding et al, 1989, Proc.
  • mast cells can be incubated with reagents that cross-link their Fc-epsilon receptors and histamine release measured by enzyme immunoassay (Siraganian, et al, 1983, TIPS 4: 432-437).
  • enzyme immunoassay enzyme immunoassay
  • products of an immune response in either a model organism (e.g., mouse) or a human patient can also be detected by various methods that are well known to those of ordinary skill in the art.
  • the production of antibodies in response to vaccination can be readily detected by standard methods currently used in clinical laboratories, e.g., an ELISA;
  • the migration of immune cells to sites of inflammation can be detected by scratching the surface of skin and placing a sterile container to capture the migrating cells over scratch site (Peters et al, 1988, Blood 72: 1310-5);
  • the proliferation of peripheral blood mononuclear cells in response to mitogens or mixed lymphocyte reaction can be measured using 3 H-thymidine;
  • the phagocitic capacity of granulocytes, macrophages, and other phagocytes in PBMCs can be measured by placing PMBCs in wells together with labeled particles (Peters et al, 1988);
  • the differentation of immune system cells can be measured by labeling PBMCs with antibodies to CD molecules such as CD4 and CD8 and measuring the fraction ofthe PBMCs expressing these markers.
  • signal transduction pathway or “signal transduction event” refers to at least one biochemical reaction, but more commonly a series of biochemical reactions, which result from interaction of a cell with a stimulatory compound or agent.
  • the interaction of a stimulatory compound with a cell generates a "signal” that is transmitted through the signal transduction pathway, ultimately resulting in a cellular response, e.g., an immune response described above.
  • a signal transduction pathway refers to the biochemical relationship between a variety of signal transduction molecules that play a role in the transmission of a signal from one portion of a cell to another portion of a cell.
  • Signal transduction molecules ofthe present invention include, for example, extracellular and intracellular domains of CLASP-2.
  • the phrase "cell surface receptor” includes molecules and complexes of molecules capable of receiving a signal and the transmission of such a signal across the plasma membrane of a cell.
  • An example of a "cell surface receptor" ofthe present invention is the T cell receptor (TCR).
  • intracellular signal transduction molecule includes those molecules or complexes of molecules involved in transmitting a signal from the plasma membrane of a cell through the cytoplasm ofthe cell, and in some instances, into the cell's nucleus.
  • CLASP-2 can be referred to as an "intracellular signal transduction molecule”, but can also be referred to as a “signal transduction molecule”.
  • a signal transduction pathway in a cell can be initiated by interaction of a cell with a stimulator that is inside or outside ofthe cell. If an exterior (i.e., outside ofthe cell) stimulator (e.g., an MHC-antigen complex on an antigen presenting cell) interacts with a cell surface receptor (e.g., a T cell receptor), a signal transduction pathway can transmit a signal across the cell's membrane, through the cytoplasm ofthe cell, and in some instances into the nucleus. If an interior (e.g., inside the cell) stimulator interacts with an intracellular signal transduction molecule, a signal transduction pathway can result in transmission of a signal through the cell's cytoplasm, and in some instances into the cell's nucleus.
  • an exterior stimulator e.g., an MHC-antigen complex on an antigen presenting cell
  • a cell surface receptor e.g., a T cell receptor
  • a signal transduction pathway can transmit a signal across the cell's membrane, through
  • Signal transduction can occur through, e.g., the phosphorylation of a molecule; non-covalent allosteric interactions; complexing of molecules; the conformational change of a molecule; calcium release; inositol phosphate production; proteolytic cleavage; cyclic nucleotide production and diacylglyceride production.
  • signal transduction occurs through phosphorylating a signal transduction molecule.
  • a CLASP-2 signal transduction pathway refers generally to a pathway in which CLASP-2 protein regulates a pathway that includes engaged-receptors, PKC-substrates, G proteins, and other molecules.
  • the present invention relates to a novel transmembrane protein, CLASP-2, a new member of the CLASP family that contains an endodomain that displays the appropriate properties to organize the cytoskeleton and signal transduction apparatus ofthe immune gateway.
  • CLASP-2 functions in cells ofthe immune system, e.g., T cells and B cells, as well as non-immune cells.
  • the CLASP-2 protein functions in a variety of cellular processes, particularly related to immune function, regulation of T cell and B cell interactions, T cell activation, and in the organization, establishment and maintenance ofthe "immunological synapse" (see Dustin et al, 1999, Science 283: 680-682; Paul et al, 1994, Cell 76: 241-251; Dustin et al, 1996, J. Immunol. 157: 2014; Dustin et al, 1998, Cell 94: 667), including signal transduction, cytoskeletal interactions, and membrane organization.
  • the CLASP-2 protein is believed to be a component ofthe lymphocyte organelle called the "immune gateway" that creates a docking site or portal for cell-cell contact during antigen-presentation. It is believed the cytoplasmic domains of CLASP-2 proteins organize it into a patch at the leading edge of T cells.
  • the carboxy-terminus encoded sequences mediate interaction with PDZ domain proteins and with cytoskeletal proteins (e.g., spectrin or ankyrin) to connect CLASP-2 to the microtubule network and hold the receptors at a polarized configuration just above the microtubule-organizing center (“MTOC").
  • MTOC microtubule-organizing center
  • the CLASP-2 molecules engage one another to dock the two cells and organize the immune synapse.
  • Modulating the expression of the CLASP-2 protein, and interference with, or enhancement of, CLASP-2 protein interactions with other proteins has a number of beneficial physiological effects, e.g., altered signaling in response to antigen, altered T and B cell response to antigen, and modulation of T cell activation.
  • the CLASP-2 extracellular domain is targeted (e.g., using anti-CLASP-2 antibody, soluble CLASP-2 fragments, and the like) to regulate T cell activation (and thus regulate immune responses).
  • disorders that can be treated by disrupting CLASP-2 function include without limitation, multiple sclerosis, juvenile diabetes, rheumatoid arthritis, pemphigus, pemphigoid, epidermolysis bullosa acquista, lupus, endometriosis, toxemia or pregnancy induced hypertension, pruritic urticarial papules and plaques of pregnancy (PUPPP), herpes gestationis, impetigo herpetiformis, pruritus gravidarum, placenta-related disorders, and Rh incompatibility.
  • the present invention provides methods and reagents for detection of CLASP-2 expression and CLASP-2-expressing cells. Abnormal expression patterns or expression levels are diagnostic for immune and other disorders.
  • diseases characterized by overproduction or depletion of lymphocytes in blood or other organs may be detected or monitored by monitoring the level of CLASP-2 polypeptide or mRNA in a biological sample (e.g., peripheral blood), e.g., the number or percentage of CLASP-2 expressing cells.
  • a biological sample e.g., peripheral blood
  • T cells Diseases characterized by overproduction of T cells include, e.g., leukemia (both ALL and CLL), lymphoma (including non-Hodgkins lymphoma, Burkitt's lymphoma, mycosis fungoides, and sezary syndrome), EBV, CMV, toxoplasmosis, syphilis, typhoid, brucellosis, tuberculosis, influenza, hepatitis, serum sickness, and thyrotoxicosis.
  • Diseases associated with the depletion of T cells include, e.g., HIV and myelodysplasia.
  • B cells Diseases associated with the overproduction of B cells include, e.g., leukemia (both ALL and CLL), non-Hodgkins lymphoma, Burkitt's lymphoma, myeloma, EBV, CMV, toxoplasmosis, syphilis, typhoid, brucellosis, tuberculosis, influenze, hepatitis, serum sickness, and thyrotoxicosis.
  • diseases associated with the depletion of B cells include, e.g., myelodysplasia.
  • CLASP-2 protein is type I transmembrane glycoprotein, characterized by multiple forms produced by alternative exon usage (i.e., production of splice variants). In one naturally occurring form, CLASP-2 has the structure shown in FIG. 1. However, as discussed in detail infra, the CLASP-2 gene encodes a variety of gene product due to alternative splicing of mRNA. FIG. 1
  • hCLASP-2A cDNA SEQ ID NO: 1
  • hCLASP-2A polypeptide SEQ ID NO: 2
  • hCLASP-2B cDNA SEQ ID NO: 3
  • hCLASP-2B polypeptide SEQ ID NO: 4
  • hCLASP-2C cDNA SEQ ID NO: 5
  • hCLASP-2C polypeptide SEQ ID NO: 6
  • hCLASP-2D cDNA SEQ ID NO: 7
  • hCLASP-2D polypeptide SEQ ID NO: 8
  • hCLASP-2E cDNA SEQ ID NO: 9
  • hCLASP-2E polypeptide SEQ ID NO: 10
  • hCLASP-2 human CLASP-2 (hCLASP-2)
  • hCLASP-2A hCLASP-2A
  • hCLASP-2B hCLASP-2C
  • hCLASP-2E hCLASP-2D
  • CLASP-2 polypeptides typically include an approximately 120 residue leader sequence, followed by a cadherin proteolytic cleavage signal RXXR, an extracellular domain, a transmembrane domain, and an intracellular domain.
  • the present invention provides a polynucleotide having the sequence of SEQ. ID. NO: 1, or a fragment thereof, and a polypeptide having the sequence of SEQ. ID NO: 2, or a fragment thereof.
  • the invention provides polynucleotides comprising hCLASP-2 genomic sequences, CLASP-2 homologs from other species, naturally occurring alleles of hCLASP-2, and hCLASP-2 variants as described herein, and methods for using CLASP-2 polynucleotide, polypeptides, antibodies and other reagents.
  • CLASP-2 Polypeptide Domains As is shown in FIG. 1, one naturally occurring CLASP-2 cDNA encodes a polypeptide characterized by several structural and functional domains and defined sequence motifs. To provide guidance to the practitioner, the structural features are described infra. However, it will be understood that the present invention is not limited to polypeptides that include all, or any particular one of these domains or motifs. For example, a CLASP-2 fusion protein of the invention contains only the extracellular domain of CLASP-2. Similarly, the CLASP-2A polypeptide of SEQ ID NO: 2 does not have the ITAM motifs (discussed infra) found in the CLASP-2B and 2C polypeptides.
  • the structurally (and functionally) different domains of CLASP-2 polypeptides are of interest, in part, because they may be separately targeted or modified (e.g., deleted or mutated) to affect the activity or expression of a CLASP-2 gene product (in order to, for example, modulate an immune response).
  • the extracellular domain of a CLASP-2 protein can be targeted (e.g., using an anti-CLASP monoclonal antibody to (a) block the interaction of a CLASP-2-expressingcell (e.g., a T cell) and a second cell (e.g., a B cell) displaying a protein that is bound by CLASP-2 (i. e.
  • a CLASP-2 ligand a CLASP-2 ligand
  • an intracellular domain e.g., ITAM or DOCK, see infra
  • an intracellular domain can be targeted to interfere with signal transduction without interfering with extracellular ligand binding.
  • inhibiting CLASP-2 expression or CLASP-2 polypeptide function will result in modulation of immune function including, for example, changing the threshold for T cell activation by affecting formation ofthe immune synapse. Modulation of immune function can be screened and quantitated by a number of assays known in the art and described herein (see also ⁇ 5.14).
  • the human CLASP-2 sequence presented in FIG. 1 encodes two potential start sites for translation.
  • the first predicted methionine appears at nucleotide 278 (ATG).
  • the second methionine appears at nucleotide 476.
  • Both have an acceptable consensus sequence for a translational start (A/GxxATGG; Kozak, M., 1996, Mamm. Genome 7(8): 563-74 ).
  • a polypeptide beginning at the second methionine is also predicted to encode a signal peptide capable of localizing the protein to the secretory pathway by SignalP, a signal sequence prediction program (Nielsen, H. et al, 1997, Protein Eng. 10(1): 1-6).
  • Polypeptides beginning at the first methionine are not predicted to contain a signal sequence; however, the consensus for signal prediction is only 80-90% accurate for known signal sequences.
  • a third possibility for a translational start is that the cDNA listed in FIG. 1 is incomplete and another methionine is encoded in frame and upstream ofthe sequence shown in FIG.1 .
  • the CLASP-2 extracellular domain is characterized by one cadherin EC-like motif (Pigott, R. and Power, C, 1993, The Adhesion Molecule Factbook. Academic Press, pg. 6; Jackson, R. M. and Russell, R. B., 2000, J. Mol. Biol. 296: 325-34).
  • Several highly conserved cysteines are found in the extracellular domain, as well as various glycosylation signals.
  • CLASP-2 may interact with ligands in a homotypic and/or heterotypic manner to establish the immunological synapse in conjunction with molecules such as TCR, MHC class I, MHC class II, CD3 complex and accessory molecules such as CD4, CD3, ICAM-1, LFA-1, and others.
  • Many cadherins contain a pro- domain of approximately 50 to 150 amino acids that is removed before localization to the plasma membrane. This cleavage is presumed to be carried out by Furin (Posthaus, H. et al, 1998, FEBS Let 438: 306-1010) at a consensus sequence of RKQR.
  • Furin is a protease that is at least partially responsible for the maturation of certain cadherins.
  • CLASP-2 has the sequence RNQR at nucleotides 945 through 957. By homology, this region is around 120 amino acids into the predicted protein start site for hCLASP-2A. This region may be a pro- domain and cleavage may be required for CLASP-2 function, or aspects of CLASP-2 function.
  • Antibodies raised against the extracellular domain can be added to cells expressing CLASP-2. These antibodies can either block the interaction of CLASP-2 with potential ligands or stabilize these interactions. Any immunoassay known in the art, e.g., listed and described herein, may be used to assess the modulation of immune function brought about by this approach. Similarly, portions of the extracellular domain of CLASP-2 can be expressed as soluble protein.
  • This soluble protein can then be added to cells expressing CLASP-2. These proteins may interact with potential ligands to competitively inhibit their binding to endogenous CLASP-2. This could modulate CLASP-2 function via the immunoassays described herein. Recombinant proteins could interfere in a positive or negative fashion with CLASP-2 interactions.
  • CLASP-2 predicted amino acid sequence was analyzed using the PHDhtm analysis software for prediction of transmembrane helices (Rost, B., et al, 1996, Prot.
  • transmembrane domain is located from nucleotides 2861-2917 (see FIG. 1 ), as well as three other potential transmembrane domains located near the amino terminal end.
  • the CLASP-2 intracellular domains contain motifs corresponding to several types of protein domains. Depending on the specific CLASP-2 (i.e., specific family member or splice variant) all or only some of the domains can be present. Listed from amino terminus to carboxy terminus, the domains include: (1) ITAM (Chan et al. 1994, Annual Review of Immunology 12: 555-592), (2) a newly discovered DOCK CLASP-2 motif, (3) a coiled-coil motif, and (4) a C-terminal PDZ binding motif (PBM) (also referred to as PDZ ligand or "PL"). 5.2.1.5. ITAM
  • Immunoreceptor Tyrosine-based Activation Motifs are motifs contained within antigen receptors for T and B cells, and Fc receptors on other leukocytes, and are necessary for proper activation and signal transduction in these cells. They are characterized by the consensus sequence YXXL/I - X7/8- YXXL/I (Grucza etal, 1999, Biochemistry 38: 5024- 5033), usually separated by 6-8 amino acids (Watson et al. , 1998, Immunol. Today 19: 260- 264; Isakov, J. Leukoc. Biol. 61: 6-16).
  • ITAM is used as an intracellular regulatory motif through its ability to be tyrosine phosphorylated by src-family tyrosine kinases such as Lyn that are involved in leukocyte signal transduction. Once phosphorylated, the ITAM acts as a high affinity binding site for SH2 containing proteins. Signal transduction components including ZAP-70, Syk, Lyn, She, PI3 kinase, and Grb2 contain SH2 domains and have been shown to bind ITAMs (Clements et al, 1999, Annu. Rev. Immunol. 17: 89-108). This places ITAM-containing molecules in a central role of intracellular signal regulation in leukocytes.
  • ITAM motifs in leukocyte signaling can facilitate signal transduction (e.g., tyrosine kinase signaling) by acting as temporal scaffolds where other transduction components could bind and be properly positioned to mediate transduction.
  • ITAM motifs often appear in multiples in a protein, however, it is known that one set of YXXL/I alone can transduce signals ofthe PTK pathway, though weakly.
  • CLASP-2 proteins typically have ITAM YXXL/I motifs (where X is any amino acid) separated by 3 or 13 amino acids.
  • the CLASP-2 polypeptide ofthe invention is characterized by one or more ofthe motifs shown in Table 1.
  • ITAM motifs in CLASPs proteins indicate that they may be engaged by multiple signal transduction components (e.g., ZAP-70/Syk, She, PI3 kinase, and Grb2).
  • the ITAM motif in CLASP proteins match identically to the canonical ITAM motif with some motifs containing a conservative amino acid change (i.e. valine instead of isoleucine or leucine).
  • the ITAMs within CLASPs can bind SH2-containing proteins including ZAP-70, Syk, She, PI3 kinase, and Grb2. Since CLASPs have an extracellular domain, CLASPs protein can independently initiate a signal transduction cascade through engagement of its extracellular domain. Otherwise CLASPs may cooperate with an antigen receptor signaling complex (e.g., with CD3/TCR, BCR, FcR), to facilitate tyrosine kinase signal transduction
  • an antigen receptor signaling complex e.g., with CD3/TCR, BCR, FcR
  • the ITAMs have demonstrated different binding specificity and affinities for SH2 domains (Clements, etal, 1999, Ann. Rev. Immunol. 17: 89-108). For example, She, PI3 kinase, and Grb2 bind to dual and mono phosphorylated ITAMs with different affinities. Thus the ITAMs in CLASPs are believed to provide quantitative as well as qualitative differences in signal transduction depending up their phosphorylation state, as well as to inhibit or augment specific protein interactions and hence specific tyrosine kinase-mediated signaling pathways in leukocytes. Antagonizing the PTK-CLASP-2 interaction (e.g., phosphorylation of
  • DOCK CLASP-2 polypeptides contain a new "DOCK” motif, not previously described in the scientific literature.
  • the CLASP DOCK motif includes a series of five tyrosines surrounded by conserved sequences in regions A, B, C, D, and G (see FIG. 5B). There are also two highly conserved non-tyrosine containing regions (E and G) separated by nine amino acids (P+EXAI+XM) and (LXMXL+GXVXXXVNXG) (where X is any amino acid).
  • the cytoplasmic region of CLASP-2 immediately following the ITAM domains exhibits sequence similarity to the C-terminal third of the so-called "DOCK” proteins.
  • the DOCK gene family includes three molecules that are the human homologues ofthe C. elegans CED proteins known to be involved in apoptosis.
  • CED-5 (DOCK180), a major CRK-binding protein, alters cell morphology upon translocation to the membrane ( mediates the membrane motion that scavenger cells exhibit as they surround and engulf dying cells; its function can be partially rescued by the human DOCK180 (Wu et al. , 1998, Nature 392: 501-504).
  • MSC Drosophila
  • DOCK1 when transfected into spindle cells, can make them flattened and polygonal (Takai, et al, 1996, Genomics 35: 403-303). DOCK1 expression is ubiquitous except in hematopoetic cells. DOCK2 is expressed in hematopoetic cells and when transfected into spindle cells can make them round up (Nishihara, H., 1999, Hokkaido Igaku Zasshi 74: 157-66). DOCK2 is expressed in peripheral blood lymphocytes, thymus, spleen, and liver.
  • CLASP-2s have the two coiled-coil domains (Lupas et al. , 1991 , Science 252: 1162-64; Lupas, A., 1996, Meth. Enzymology 266: 513-525). Coiled-coil domains are known to interact directly with cytoskeleton, indicating that that CLASP-2 proteins interact directly with the cytoskeleton.
  • CLASP-2 binds cytoskeletal proteins, e.g., spectrin, ankyrin, hsp70, talin, ezrin, tropomyosin, myosin, plectin, syndecans, paralemmin, Band 3 protein, Cytoskeletal protein 4.1 , Tyrosine phosphatase PTP36 and other molecules.
  • cytoskeletal proteins e.g., spectrin, ankyrin, hsp70, talin, ezrin, tropomyosin, myosin, plectin, syndecans, paralemmin, Band 3 protein, Cytoskeletal protein 4.1 , Tyrosine phosphatase PTP36 and other molecules.
  • CLASP-2 proteins contain a PDZ-ligand motif ("PBM” or "PL”) at the C- terminus ofthe protein.
  • PBM PDZ-ligand motif
  • PL PDZ-ligand motif
  • This short (3 - 8 amino acid) motif mediates the binding of proteins terminating at their carboxyl terminus in the motif (most commonly S/T - X - V — free carboxyl-terminus) to other proteins containing one or more specific PDZ domains (See Songyang et al. , 1997, Science 275 : 72 and Doyle et al , 1996, Cell 85 : 1067 for a discussion of PDZ-ligand structures).
  • PDZ domain-containing proteins are involved in the organization of ion channels and receptors at the neurological synapse and in establishing and maintaining polarity in epithelial cells via their binding to the C-termini of transmembrane receptors. It has been shown that PDZ-domain containing proteins can mediate protein-protein interactions in immune system cells (e.g., DLG1 binds to the lymphocyte potassium channel KVl.3 in human T lymphocytes, (Hanada et al, 1997, J. Biol. Chem. 272: 26899).
  • immune system cells e.g., DLG1 binds to the lymphocyte potassium channel KVl.3 in human T lymphocytes, (Hanada et al, 1997, J. Biol. Chem. 272: 26899).
  • FIG. 9A Biochemical evidence that CLASP-2 interacts with the PDZ domains of three closely related proteins is shown in FIG 9A-D.
  • FIG. 9A demonstrates the specificity ofthe interaction, as the C-terminal 20 amino acids of CLASP-2 bind PSD-95, NeDLG, and DLG1 , but not to the PDZ domains of the ⁇ AM- 1 protein.
  • FIG.9B demonstrates the affinity of the interaction. Notably, the highest affinity interaction occurs between CLASP-2 and NeDLG, with a specific binding affinity of at least 10 4 M '1 . Affinities in the micromolar range have been found for other biologically important PDZ-ligand interactions.
  • FIG.9C demonstrates the ability to inhibit CLASP-2 PDZ interactions using either a short fragment of CLASP-2 (the eight C-terminal amino acids) or the C-terminus of KVl .3.
  • KVl .3 is known to bind to DLG1 in live lymphocytes.
  • FIG. 9D demonstrates that CLASP-2 and KV1.3 compete for PDZ binding; i.e., not only does KV1.3 block CLASP-2 binding but CLASP-2 also blocks KVl.3 binding.
  • the results indicate that the CLASP-2 C-terminal sequence can slightly increase basal intracellular calcium concentrations and can slightly decrease the proportional increase in calcium upon activation ofthe cells with anti-CD3 antibody. Such changes would be expected for a compound that disrupts localization ofthe T cell activation- associated CLASP-2 protein and the KVl.3 potassium channel. Small changes in T cell calcium flux can result in large changes in the functional activity ofthe cells (Wulfing et al. , 1997, J. Exp. Med. 185: 1815).
  • CLASP-2 proteins modulate immune function in a variety of ways and through a variety of mechanisms (i.e., changing the threshold for T cell activation) by affecting formation of the immunological synapse.
  • Establishment and maintenance ofthe immunological synapse can involve: (A) signal transduction, (B) cell-cell interactions, and (C) membrane organization.
  • Human CLASP proteins as discussed above, contain SH3 domains and tyrosine phosphorylation sites. These regions have been shown to be involved in signal transduction in a variety of cells including lymphocytes. Thus, human CLASP proteins are believed to interact with these regions during signal transduction events which lead to modulation of immune responses.
  • CLASP proteins can interact with Tec sub-family of nonreceptor tyrosine kinases.
  • the Tec sub-family of nonreceptor tyrosine kinases consists of Tec, Btk, Tsk/Itk/Emt Itk, and Bmx, and is defined by the presence of SH3 and SH2 domains adjacent to the catalytic domain and an amino-terminal region containing a pleckstrin homology (PH) domain, a Tec homology (TH) domain, and a proline-rich region (Mano, H.; 1999, Cytokine Growth Factor Rev 10: 267-80).
  • the T cell specific Tsk/Itk/Emt, and Btk expressed in most hematopoietic cells other than T cells are important components of antigen receptor signaling pathways in hematopoietic cells.
  • Btk has been identified as the gene defective in murine X-linked immunodeficiency (xid) and human X-linked agammaglobulinemia (XL A) (Nisitani, S., 2000, Proc Natl Acad Sci U.S.A. 97: 2737-42).
  • xid mice B cell numbers are reduced to one-half of normal and the titers of specific immunoglobulin isotypes are significantly reduced; in addition, xid B cells are insensitive to a number of mitogenic stimuli.
  • the human disorder is much more severe, resulting in nearly complete elimination of the B cell compartment and dramatically reduced immunoglobulin levels. Biochemical studies have supported multiple roles for Btk in B cell activation.
  • Btk kinase activity and tyrosine phosphorylation are increased after cross-linking either the B cell receptor on B cells or the high affinity IgE receptor, FcRI, on mast cells.
  • Interleukin-5 and interleukin-6 treatment have also been shown to lead to the activation of Btk.
  • Itk like Btk, is tyrosine-phosphorylated upon antigen receptor cross-linking (Mano, H., 1999, Cytokine Growth Factor Rev, 10: 267-80).
  • peripheral T cells from mice lacking functional Itk are refractory to stimulation by antibodies to CD3 plus antigen presenting cells.
  • These Itk-deficient T cells can be stimulated by phorbol ester and calcium ionophore, demonstrating that Itk acts in signaling pathways proximal to the TCR.
  • the Tec family kinases lack the amino-terminal myristylation site crucial for the membrane localization of Src family kinases, suggesting that some adaptor proteins are required for the their membrane localization (Mano, H., 1999, Cytokine Growth Factor Rev 10: 267-80). Since all the Tec family kinases contain a proline-rich region which could be bound by a SH3 domain, and since all the human CLASPs contain a SH3 domain, it is believed that human CLASPs could serve as adaptors for the members in the Tec family in different hematopoietic cells.
  • GTP-binding proteins play an important role in immune response (Mach, B., 1999, Science 285: 1367). A number of biochemical events triggered by TCR/CD3-induced T cell activation are ablated by agents that modulate the action of G proteins. Pertinent to this is the ability of cholera toxin to inhibit the cellular proliferation and intracellular Ca 2+ mobilization that is mediated by anti-CD3 antibody treatment of T cells.
  • the G protein competitive inhibitor GDPS can impede the extent of inositol phosphates generated upon stimulation in peripheral T lymphocytes.
  • Nonhydrolyzable analogs of GTP such as GTPS, or other agents such as ALF that activate G proteins by circumventing the need for receptor engagement, can result in T cell activation.
  • adaptor proteins such as NCK, CBL (Bachmaier, K., 2000, Nature 403: 211-6), SHC, LAT, LNK, SLP-76 (Krause M et al, 2000, J Cell Biol 149: 181-94), HSl, SIT, VAV, GrB2 (Zhang W. and Samelson, L.E., 2000, Semin Immunol 12: 35-41), and BRDG1 , kinases such as SYK and LCK, and tyrosine phosphatases such as SHP-1 and SHP-2.
  • adaptor proteins such as NCK, CBL (Bachmaier, K., 2000, Nature 403: 211-6), SHC, LAT, LNK, SLP-76 (Krause M et al, 2000, J Cell Biol 149: 181-94), HSl, SIT, VAV, GrB2 (Zhang W. and Samelson, L.E., 2000, Semin Immunol 12: 35-
  • interactions can be defined by a number of different biochemical or cell biological methods including in vitro binding assays, co-immunoprecipitation assays, co-immunostaining (Harlow, E. and Lane, D., 1999, Using Antibodies: A laboratory Manual. Cold Spring Harbor Press) or genetic assays such as yeast the yeast two hybrid system, in which a CLASP-2 protein or fragment can be used as "bait” (Zervos et al, 1993, Cell 72: 223-232; Madura et al, 1993, J. Biol. Chem 268: 12046-12054).
  • assays include in vitro binding assays, co-immunoprecipitationassays, co-immunostaining assays, and yeast two hybrid system screening assays in which a CL ASP- 2 domain or fragment can be used as "bait” or "trap” protein (Zervos et al. (1993), Cell 72: 223-232; Madura et al. (1993) J. Biol. Chem. 268: 12046-12054).
  • CLASP polypeptides are transfected into lymphocytes.
  • a variety of standard assays can be used to evaluate, for example, CLASP modulation of T cell activation. These assays include calcium influx assays, NF-AT nuclear translocation assays (e.g., Cell, 1998, 93: 851-61), NF-AT/luciferase reporter assays (e.g., MCB 1996 16: 7151-7160), tyrosine phosphorylation of early response proteins such as HS 1 , PLC- , ZAP-76, and Vav (e.g., J. Biol. Chem. 1997, 272: 14562-14570).
  • NF-AT nuclear translocation assays e.g., Cell, 1998, 93: 851-61
  • NF-AT/luciferase reporter assays e.g., MCB 1996 16: 7151-7160
  • tyrosine phosphorylation of early response proteins such as HS 1 ,
  • CLASP-2 contains both a cadherin cleavage domain and a cadherin ectodomain. Therefore CLASP-2 proteins may interact with cadherins through these domains.
  • the cadherins constitute a family of cell surface adhesion molecules that are involved in calcium-dependent cell to cell adhesion.
  • Human cadherins, E-, P- N- and VE- cadherin have a restricted tissue distribution: E- and P-cadherin are expressed in epithelial tissues, N-cadherin is found mainly on neural cells, and VE-cadherin is fovmd on vascular endothelium.
  • E-cadherin is required for the formation of adherens junctions between mature epithelial cells and is involved in Langerhans cell adhesion to keratinocytes
  • VE-cadherin is needed for the maintenance of lateral association between endothelial cells.
  • the extracellular regions of mature mammalian cadherins are comprised of five "CAD" modules of approximately 1110 amino acids. Crystallographic and biochemical studies indicate that cadherins can form dimers on the cell surface, and that interaction with dimeric cadherins on opposing cell surfaces can lead to the formation of "zipper-like" cell junctions.
  • the integrins are a second family of transmembrane adhesion molecules that are involved in both cell to cell and cell to matrix interactions. At least 15 chains associate with 8 chains to form a large number of heterodimeric integrins that can be classified into several major subfamilies based on their shared use of a particular chain. Members of three subfamilies, the 1, 2, and 7 integrins, are commonly found on leukocytes. The expression of 1 integrins is widespread (for example, 51, CD49e/CD29, is found on T cells, granulocytes, platelets, fibroblasts, endothelium, and epithelium), whereas the 2 and 7 integrins have a restricted pattern of expression.
  • E-cadherin on human epithelial cells has been found to be a ligand for the mucosal lymphocyte integrin, E7, and a similar interaction has been indicated in the mouse.
  • Monoclonal antibodies to E-cadherin or to E7 block IEL adherence to epithelial cells, and transfection of cells with E7 confers upon them the ability to adhere to cells transfected with E-cadherin.
  • L929 cells can be transfected with CLASP-2 and Neomycin. G418-resistant clones can be screened for CLASP-expressionwith anti-CLASP peptide-specific antibodies. CLASP-expressing clones can be used to test for homotypic and/or heterotypic calcium dependent cell adhesion using the "cell aggregation assay" described for cadherin molecules (Murphy-Erdosh, C. et al, 1995, J. Cell Biol. 129: 1379-1390). Several approaches can be used to identify the amino acids involved in the binding domains.
  • Soluble fusion molecules e.g., EC12-IgG, ECC-IgG, ECM-IgG, and GST- EC 12
  • peptides e.g., EC12-IgG, ECC-IgG, ECM-IgG, and GST- EC 12
  • Transfectants generated by site-directed mutagenesis can also be used.
  • WASP a GTPase-binding protein
  • N-WASP Another WASP family protein, N-WASP, has also been shown to play important roles in filopodium formation.
  • WASP mainly localizes at perinuclear areas and causes actin clustering, but most N-WASP is present at plasma membranes and induces filopodium formation (Miki, H.; 1998, Nature 391 : 93-6).
  • Both WASP and N-WASP contain a proline-rich domain which could interact with the SH3 domain present in all the human CLASPs.
  • CLASP-2 may interact with F-actin filament through CLASP-2 binding to WASP or WASP-like proteins.
  • Standard assays can be used for detecting CLASP protein interaction with cytoskeletal proteins. These assays include co-sedimentation assays, far western blot analysis (Ohba, T., 1998, Anal. Biochem.262: 185-192), surface pasman resonance, F-actin staining withphalloidinin CLASP-transfectedlymphocytes(e.g., Small, J. etal 1999, Microsc. Res. Tech.
  • Alternative splice variants affecting the untranslated regions of an RNA can be a way of regulating RNA stability.
  • alternative splicing is likely to represent a regulatory switch that governs different functions of CLASP-2 in immune responses.
  • CLASP-2 gene expression is characterized by alternative exon usage. Intron exon structure can be predicted by computer analysis of genomic DNA, however, splice junctions and alternative splicing can only be elucidated by comparison of genomic clones to cDNA clones.
  • Alternative splicing and RNA editing are mechanisms generate a variety of proteins from the same gene that are closely related to each other but that can differ in structure and thus can exert distinct functions.
  • Numerous diseases are caused or are thought to be caused by splice site mutations that can cause exon skipping or otherwise result in a truncated protein product
  • Some of these diseases include, e.g., Marfan Syndrome (Liu W, et al, 1997, Nat. Genet. 16: 328-9), Hunter disease (Bonucelli G, et al, 2000, Hum. Mutat. (Online) 2000 15(4): 389, Duchenne muscular dystrophy (Wibawa T, et al, 2000, Brain Dev. 22(2): 107-112), Myelomonocytic leukemia (Wutz D, et al, 1999, Leuk.
  • Lymphoma 35: 491-9. Lymphoma 35: 491-9.
  • Isovaleric acidemia Vockley J, et al, 2000, Am. J. Hum. Genet. 66: 356-67. This is especially true for genes composed of many exons (such as CLASP-2).
  • CLASP-2 The genomic sequence around CLASP-2 exon/intron boundaries is useful for diagnostic approaches towards the identification of diseases caused by splice site mutations.
  • One of skill in the art can correlate the abundance or presence of CLASP-2 isoforms in cell populations (e.g., hematopoietic cells, lymphocytes) and a disease state by comparing the abundance of CLASP-2 in cells from subjects suffering from the disease with the level of CLASP-2 in cells from healthy subjects.
  • RNA editing is mechanisms generate a variety of proteins from the same gene that are closely related to each other but that can differ in structure and thus can exert distinct functions.
  • An example for how alternative splicing is used to generate thousands of different proteins from only a few genes is represented by the Neurexin gene family (for review of Neurexins, see Missler M. and Sudhof, T., 1998, Trends in Genetics, 14: 20-25).
  • CLASP-2 alternative transcripts identified in this study are summarized in FIG. 3. Briefly, one alternative exon missing in CLASP-2 A is present in CLASP-2B and CLASP-2D. This exon contains the DNA portion encoding the ITAM motif and DOCK motif 1.
  • the CLASP-2D protein product does not contain the C-terminal 38 amino acids of CLASP-2A and CLASP-2B: Thus, a PDZ binding motif (SSVV; amino acid residue 1286 through 1289) that is only present in the CLASP-2 A/B-specific C-terminal end is missing in the CLASP-2D gene product. The presence or absence of this PDZ binding motif can be attributed to alternative RNA processing.
  • CLASP-2 is a member of a superfamily of immune-cell associated proteins with similar motifs.
  • CLASP-1 was described in U.S.S.N. 09/411,328, filed October 1, 1999.
  • CLASP-1 uniquely among the known CLASPs contains SH3 binding domain motifs.
  • CLASP-2A, -B, -C, and -E polypeptides have no adaptor binding sites or SH3 binding domains found in CLASP-1.
  • CLASP-3, CLASP-4, CLASP-5 and CLASP-7 are described in copending U.S.S.N.60/182,296, filed February 14, 2000, and which is inco ⁇ orated by reference herein in its entirely for all purposes.
  • Jurkat human T cell line
  • MV4-M B myelomonocyte
  • 9D10 B cell line
  • THP-1 monocyte
  • 3A9 mouse T cell
  • CH27 mouse B cell line
  • HL60 human promyelocyte
  • 293 embryonic kidney epithelial cells (293)
  • a CLASP-2 EST (EST 815795) was identified from a bone marrow cDNA library.
  • the probe used did not distinguish between CLASP-2A, -2B, -2C and 2D.. This probe encompasses nucleotides 3920 to 4650 (731 bp long) from CLASP-2A cDNA.
  • RNA from 9D10 the major transcript runs substantially shorter than the major transcripts seen in Jurkat and 293 cells; however, the longer transcript is also present in 9D10.
  • Hybridization of probe HC2.2 with 9D10 total RNA reveals at least 3 different transcripts. See FIG. 4B
  • CLASP-2 is expressed most strongly in placenta followed by lung, kidney and heart;
  • CLASP-3 is expressed strongly in kidney and heart, and less strongly in placenta and skeletal muscle ;
  • CLASP-4 is expressed exclusively in peripheral blood lymphocytes;
  • CLASP-5 is expressed strongly in peripheral blood leukocytes, present in placenta, kidney, spleen and thymus, and weakly in lung, small intestine and liver. It is not expressed in brain, heart, skeletal muscle and large intestine;
  • CLASP-7 is expressed strongly in lung, heart, liver and kidney, but not in PBL, brain or thymus.
  • the present invention provides a variety of CLASP-2 polynucleotides and methods for using them.
  • the polynucleotide of the invention encodes a polypeptide comprising at least a fragment (e.g., an immunogenic fragment) of a CLASP-2 protein (e.g., at least a fragment of SEQ. ID. NO: 2, 4, 6 or 10) or variant thereof.
  • the molecules that comprise a CLASP-2 polynucleotide that, while not necessarily encoding a CLASP-2 protein or fragment is useful as a probe or primer for detecting CLASP-2 expression, for inhibition of CLASP-2 expression (e.g., antisense or ribozyme- mediated inhibition), for gene knockout, and the like.
  • the invention also provides isolated or purified nucleic acids having at least 8 nucleotides (i.e., a hybridizable portion) of a CLASP-2 sequence or its complement; in other embodiments, the nucleic acids consist of at least about 25 (continuous) nucleotides, about 50 nucleotides, about 100 nucleotides, about 150 nucleotides, about 200 nucleotides, about 250 nucleotides, about 500 nucleotides, about 550 nucleotides, about 600 nucleotides, or about 650 nucleotides or more of a CLASP-2 sequence, or a full-length CLASP-2 coding sequence. In another embodiment, the nucleic acids are smaller than about 35, about 200 or about 500 nucleotides in length. Polynucleotides can be single or double stranded, and may be DNA, RNA, PNA or a hybrid molecule.
  • nucleic acids are provided which comprise a sequence complementary to at least about 10, 25, 50, 100, 150, 200, 250, 500, 550, 600, or 650 nucleotides or the entire coding region of a CLASP-2 coding sequence.
  • the isolated polynucleotide is less than about 100 kbp, generally less than about 50 kbp, and often less than about 20 kbp, less than about 10 kbp, less than about 5 kbp, or less than about 1000 nucleotides in length.
  • a nucleic acid that is hybridizable to a CLASP-2 nucleic acid or its complement, or to a nucleic acid encoding a CLASP-2 derivative, under conditions of low stringency is provided.
  • Derivatives of CLASP-2 contemplated include, but are not limited to, splice variants of a gene encoding a CLASP-2, other members of a CLASP-2 gene family which differ from one of the CLASP-2 nucleotide or amino acid sequences disclosed herein by the insertion or deletion of one or several domains, and the like.
  • the CLASP-2 polynucleotide is identical or exactly complementary to SEQ. ID NO: 1, 3, 5 or 9 or selectively hybridizes to an aforementioned sequence.
  • the polynucleotide is identical or exactly complementary to, or selectively hybridizes to, the nucleotide sequence encoding a particular protein domain or region, or a particular gene exon ofthe CLASP-2 mRNA or genomic sequence.
  • Such polynucleotides are particularly useful as probes, because they can be selected to identify a defined species of CLASP-2.
  • the invention contemplates CLASP-2 homologues from other species, allelic and splice variants, and other variants disclosed herein.
  • the CLASP-2 gene exhibits evidence of alternative splicing of transcripts.
  • CLASP-2A and CLASP-2C are related to each other as apparent splice variants, with CLASP-2C containing an exon not found in CLASP-2A.
  • the exon sequence is 5'-AGG GAT TTT GAG AGG CTG GCC CAT CTG TAT GAC ACG CTG CAC CGG GCC TAC AGC AAA GTG ACC GAG GTC ATG CAC TCG GGC CGC AGT TNC TGG GGA CCT ACT TCC GGG TAG CCT TCT TCG GGC AG-3' (encoding the peptide sequence: RDFERLAHLYDTLHRAYSKVTEVMHSGRRLLGTYFRVAFFGQGF).
  • the CLASP-2 polynucleotides of the invention are substantially identical to SEQ ID NOs: 1, 3, 5, or 9, or to a fragment thereof.
  • nucleic acid sequences are substantially identical is that the two polynucleotides have a specified percentage sequence identity e.g., usually at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 98 identity over a specified region when optimally aligned.
  • a specified percentage sequence identity e.g., usually at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 98 identity over a specified region when optimally aligned.
  • nucleic acid sequences are substantially identical is that a polypeptide encoded by the first nucleic acid is immimologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below.
  • a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions.
  • Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below.
  • CLASP-2 polynucleotides can be PCR amplified from cDNA derived from human lymphocytes using the primer pairs shown in Table 3.
  • the primers of Table 3 are also useful for amplification of CLASP-2 splice variants. Another indication that two nucleic acid sequences are substantially identical is that they selective hybridize under stringent conditions (i.e., one sequence hybridizes to the complement ofthe second sequence), as described infra.
  • the invention also relates to nucleic acids that selectively hybridize to exemplified CLASP-2 sequences (including hybridizing to the exact complements of these sequences). Selective hybridization can occur under conditions of high stringency (also called “stringent hybridization conditions”), moderate stringency, or low stringency.
  • Stringent hybridization conditions are conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acid, but not to other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is fovmd in Tijssen, Techniques in
  • T m thermal melting point
  • the T m is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T m , 50% of the probes are occupied at equilibrium).
  • Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For high stringency hybridization, a positive signal is at least two times background, preferably 10 times background hybridization.
  • Exemplary high stringency or stringent hybridization conditions include: 50% formamide, 5x SSC and 1% SDS incubated at 42° C or 5x SSC and 1% SDS incubated at 65° C, with a wash in 0.2x SSC and 0.1% SDS at 65° C.
  • a nucleic acid which is hybridizable to a CLASP-2 nucleic acid under the following conditions of high stringency is provided: Prehybridization of filters containing DNA is carried out for 8 h to overnight at 65°C in buffer composed of 6X SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 ⁇ g/ml denatured salmon sperm DNA. Filters are hybridized for 8-16 h at 65°C in prehybridization mixture containing 100 ⁇ g/ml denatured salmon sperm DNA and 5-20 X 10 6 cpm of 32 P-labeled probe. Washing of filters is done at 65°C for 15-30 h in a solution containing 2X SSC, 0.1% SDS. This is followed by a wash in 0.2X SSC and 0.1% at 50°C for 15-30 min before autoradiography.
  • a nucleic acid which is hybridizable to a CLASP-2 nucleic acid under conditions of moderate stringency.
  • procedures using such conditions of moderate stringency are as follows: Filters containing
  • DNA are pretreated for 6 h at 55°C in a solution containing 6X SSC, 5X Denhart's solution,
  • Hybridizations are carried out in the same solution with the following modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 g/ml salmon sperm DNA, 10% (wt/vol) dextran sulfate, and 5-20 X 10 6 cpm
  • P-labeled probe is used. Filters are incubated in hybridization mixture for 18-20 h at 40 C, and then washed for 1.5 h at 55 C in a solution containing 2X SSC and 0.1% SDS. The wash solution is replaced with fresh solution and incubated an additional 30 minutes at 50-55°C.
  • Filters are blotted dry and exposed for autoradiography. If necessary, filters are washed for a third time at 60-65°C and reexposed to film. Other conditions of low stringency that can be used are well known in the art (e.g., as employed for cross-species hybridizations).
  • the CLASP-2 variants ofthe invention can contain alterations in the coding regions, non-coding regions, or both. Especially preferred are polynucleotide variants containing alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities ofthe encoded polypeptide. Nucleotide variants produced by silent substitutions due to the degeneracy of the genetic code are preferred. CLASP-2 polynucleotide variants can be produced for a variety of reasons, e.g., to optimize codon expression for a particular host (change codons in the human mRNA to those preferred by a bacterial host such as E. coli).
  • Exemplary CLASP-2 polynucleotide fragments are preferably at least about 15 nucleotides, and more preferably at least about 20 nucleotides, still more preferably at least about 30 nucleotides, and even more preferably, at least about 40 nucleotides in length, or larger 50, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650 nucleotides.
  • Exemplary fragments include fragments having at least a sequence from about nucleotide number 1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451-500, 501- 550, 551-600 to the end of SEQ ID NO: 1 or comprising the cDNA coding sequence in the deposited clone.
  • “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.
  • these fragments encode a polypeptide which has biological activity. More preferably, these polynucleotides can be used as probes or primers as discussed herein.
  • the CLASP-2 variants differ from SEQ ID NOs 1, 3, 5, 7 or 9 by virtue of incorporating a different combination of exons than found in the exemplified sequences.
  • 81 gOl Genbank Accession Number AF85864; Locus HUMYN81g01; 526 bp; EST sequence submitted August 29, 1998 by Washington University at St. Louis; see FIG. 3 A and FIG. 3B
  • hCLASP-2 is a variant of hCLASP-2 on the basis of CLASP-2 identity along certain stretches ofthe sequence. From 5' to 3', it begins with a 315 nucleotide stretch which is identical to CLASP-2A.
  • variants can be generated to improve or alter the characteristics of the CLASP-2 polypeptides. For instance, one or more amino acids can be deleted from the N-terminus or C-terminus ofthe CLASP-2 protein without substantial loss of biological function.
  • the invention further includes CLASP-2 polypeptide variants which show biological activity.
  • Such variants include deletions, insertions, inversions, repeats, and substitutions selected according to general rules known in the art so as have little effect on activity.
  • Guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie, J. U. et al, Science 247: 1306-1310 (1990), wherein the authors indicate that there are two main strategies for studying the tolerance of an amino acid sequence to change.
  • 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 ofthe protein.
  • the second strategy uses genetic engineering to introduce amino acid changes at 30 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, 1989, Science 244: 1081-1085) The resulting mutant molecules can then be tested for biological activity.
  • CLASP-2 polynucleotide fragments include coding regions for, or regions hybridizable to, the CLASP-2 structural or functional domains described supra. As set out in the Figures, such preferred regions include the following domains/motifs: ITAM, DOCK, COILED/COILED, and PBM. Thus, for example, polypeptide fragments of SEQ ID NO: 2, 4, 6 or 10 falling within conserved domains are specifically contemplated by the present invention (see FIG. 3). Moreover, polynucleotide fragments encoding these domains are also contemplated. Such polypeptide fragments find use, for example, as inhibitors of CLASP-2 function in CLASP-2-expressing cells.
  • CLASP-2 polynucleotides of the invention are useful in a variety of applications.
  • the polypeptide-encoding CLASP-2 polynucleotides of the invention are used to express CLASP-2 polypeptides (e.g., as described herein) for example to produce anti-CLASP-antibodies or for use as therapeutic polypeptides.
  • the CLASP-2 polynucleotide or fragments thereof can be used for diagnostic purposes (e.g., as probes for CLASP-2 expression).
  • a CLASP-2 polynucleotide can be used to detect the expression of CLASP-2 as a lymphocyte marker.
  • a CLASP-2 polynucleotide can be used to detect CLASP-2 gene expression or aberrant CLASP-2 gene expression in disease states.
  • the CLASP-2 polynucleotide or fragments are used for therapeutic purposes.
  • included in the scope ofthe invention are methods for inhibiting CLASP-2 expression, e.g., using oligonucleotide sequences, such as antisense RNA and DNA molecules and ribozymes, that function to inhibit expression of CLASP-2.
  • CLASP-2 polynucleotides can be used to construct transgenic and knockout animals, e.g., for screening of CLASP-2 agonists and antagonists.
  • CLASP-2 polynucleotides can be used for screening of CLASP-2 agonists and antagonists.
  • CLASP-2 Polynucleotides are useful for detection of
  • Aberrant expression of CLASP-2 mRNA or protein means expression in lymphocytes (e.g., T lymphocytes or B lymphocytes) or other CLASP-2 expressing cells of at least 2-fold, preferably at least 5 -fold greater or less than expression in control lymphocytes obtained from a healthy subject.
  • CLASP-2 polypeptide expression is easily measured by ELISA using anti- CLASP-2 antibodies ofthe invention.
  • CLASP-2 mRNA expression (including expression of specific species or splice variants of CLASP-2) can be measured by quantitative Northern analysis or quantitative PCR, LCR, or other methods, using the probes and primers of the invention.
  • the assays of the present invention are amplification- based assays for detection of an CLASP-2 gene product.
  • an amplification based assay all or part of a CLASP-2 mRNA or cDNA (hereinafter also referred to as "target") is amplified, and the amplification product is then detected directly or indirectly.
  • target a CLASP-2 mRNA or cDNA
  • no amplification product is produced (e.g., of the expected size), or amplification is non-specific and typically there is no single amplification product.
  • the target sequence is amplified, providing an indication ofthe presence and/or quantity ofthe underlying gene or mRNA.
  • Target amplification-based assays are well known to those of skill in the art.
  • the present invention provides a wide variety of primers and probes for detecting CLASP-2 genes and gene products.
  • primers and probes are sufficiently complementary to the CLASP-2 gene or gene product to hybridize to the target nucleic acid.
  • Primers are typically at least 6 bases in length, usually between about 10 and about 100 bases, typically between about 12 and about 50 bases, and often between about 14 and about 25 bases in length, often PCR primers of 15-30 (e.g., 18-22 nucleotides) are used. However, the length of primers can be adjusted by one skilled in the art.
  • probes and primers can be selected to distinguish between species and splice variants based on the guidance of this disclosure, by targeting primers or probes to differentially used exons (or exon-exon junctions that differ between variants).
  • Methods can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells ofthe sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to an CLASP- 2 gene under conditions such that hybridization and amplification ofthe CLASP-2-gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample.
  • nucleic acid e.g., genomic, mRNA or both
  • CLASP-2 gene products are expressed in the immvme system (e.g., T lymphocytes, B lymphocytes and macrophages), expression will be typically assayed in these cells.
  • Methods which are well known to those skilled in the art can be used to isolate lymphocytes, macrophages, and alike (See, e.g., Coligan, J. E., et al. (eds.), 1991, Current Protocols in Immunology, John Wiley & Sons, NY; this reference is incorporated by reference for all purposes).
  • assays are carried out on biopsy or autopsy- derived tissue.
  • CLASP-2 gene expression is detected by hybridization of a detectable probe to mRNA or cDNA obtained from cells (e.g., lymphocytes).
  • mRNA or cDNA obtained from cells (e.g., lymphocytes).
  • hybridization based assays refer to assays in which a probe nucleic acid is hybridized to a target nucleic acid, forming a hybridization complex.
  • nucleic acid hybridization probes of the invention are entirely or substantially identical to a contiguous sequence of the CLASP-2 gene or RNA sequence.
  • nucleic acid probes are at least about 50 bases, often at least about 20 bases, and sometimes at least about 200 bases, at least about 300-500 nucleotides or more in length.
  • Various hybridization techniques are well known in the art, and are in fact the basis of many commercially available diagnostic kits.
  • nucleic acid probe sequences for use in nucleic acid hybridization are discussed in Sambrook et al, supra.
  • at least one ofthe target and probe is immobilized.
  • the immobilized nucleic acid can be DNA, RNA, or another oligo- or poly-nucleotide, and can comprise natural or non-naturally occurring nucleotides, nucleotide analogs, or backbones.
  • Such assays can be in any of several formats including: Southern, Northern, dot and slot blots, high-density polynucleotide or oligonucleotide arrays (e.g., GeneChipsTM Affymetrix), dip sticks, pins, chips, or beads.
  • Hybridization techniques are generally described in Hames et al. , ed., 1985, Nucleic Acid Hybridization, A Practical Approach IRL Press; Gall and Pardue, 1969, Proc. Natl. Acad. Sci. U.S.A., 63: 378-383; and John et al, 1969, Nature, 223: 582- 587.
  • nucleic acid hybridization formats are known to those skilled in the art.
  • one common format is direct hybridization, in which a target nucleic acid is hybridized to a labeled, complementary probe.
  • labeled nucleic acids are used for hybridization, with the label providing the detectable signal.
  • One method for evaluating the presence, absence, or quantity of CLASP-2 mRNA is carrying out a Northern transfer of RNA from a sample and hybridization of a labeled CLASP-2 specific nucleic acid probe.
  • a useful method for evaluating the presence, absence, or quantity of DNA encoding CLASP-2 proteins in a sample involves a Southern transfer of DNA from a sample and hybridization of a labeled CLASP-2 specific nucleic acid probe.
  • Sandwich assays are commercially useful hybridization assays for detecting or isolating nucleic acid sequences. Such assays utilize a "capture" nucleic acid covalently immobilized to a solid support and a labeled "signal" nucleic acid in solution. The biological or clinical sample will provide the target nucleic acid. The "capture” nucleic acid and “signal” nucleic acid probe hybridize with the target nucleic acid to form a "sandwich” hybridization complex. To be effective, the signal nucleic acid cannot hybridize with the capture nucleic acid.
  • CLASP-2 polypeptides or polynucleotides are useful in treating deficiencies or disorders of the immune system, by activating or inhibiting the activation, differentiation of immune cells.
  • Immune cells develop through a process called hematopoiesis, producing myeloid (platelets, red blood cells, neutrophils, and macrophages) and lymphoid (B and T lymphocytes) cells from pluripotentstem cells.
  • myeloid platelets, red blood cells, neutrophils, and macrophages
  • lymphoid cells lymphoid cells from pluripotentstem cells.
  • the etiology of these immune deficiencies or disorders can be genetic, somatic, such as cancer or some autoimmune disorders, acquired (e.g., by chemotherapy or toxins), or infectious.
  • CLASP-2 polynucleotides or polypeptides are useful in treating or detecting deficiencies or disorders of hematopoietic cells.
  • CLASP-2 polypeptides or polynucleotides could be used to increase differentiation and proliferation of hematopoietic cells, including the pluripotent stem cells, in an effort to treat those disorders associated with a decrease in certain (or many) types hematopoietic cells.
  • immunologic deficiency syndromes include, but are not limited to: blood protein disorders (e.g., agammaglobulinemia, dysgammaglobulinemia), ataxia telangiectasia, common variable immunodeficiency, Digeorge Syndrome, HIV infection, HTLV-BLV infection, leukocyte adhesion deficiency syndrome, lymphopenia, phagocyte bactericidal dysfunction, severe combined immunodeficiency (SCIDs), Wiskott-Aldrich Disorder, anemia, thrombocytopenia, or hemoglobinuria.
  • blood protein disorders e.g., agammaglobulinemia, dysgammaglobulinemia
  • ataxia telangiectasia common variable immunodeficiency
  • common variable immunodeficiency e.g., Digeorge Syndrome, HIV infection, HTLV-BLV infection, leukocyte adhesion deficiency syndrome, lymphopenia, phagocyte bactericidal dysfunction, severe combined
  • CLASP-2 polynucleotides or polypeptides are useful in treating or detecting autoimmune diseases.
  • autoimmune disease has the normal meaning in the art and refers to a spontaneous or induced malfunction ofthe immune system of mammals in which the immune system fails to distinguish between foreign immunogenic substances within the mammal and/or autologous ("self) substances and, as a result, treats autologous ("self) tissues and substances as if they were foreign and mounts an immune response against them.
  • Autoimmune disease is characterized by production of either antibodies that react with self tissue, and/or the activation of immvme effector T cells that are autoreactive to endogenous self antigens.
  • autoantibodies are directed against functional cellular receptors or other cell surface molecules, and either stimulate or inhibit specialized cellular function with or without destruction of cells or tissues; 2) autoantigen—autoantibody immune complexes form in intercellular fluids or in the general circulation and ultimately mediate tissue damage; and 3) lymphocytes produce tissue lesions by release of cytokines or by attracting other destructive inflammatory cell types to the lesions. These inflammatory cells in turn lead to production of lipid mediators and cytokines with associated inflammatory disease.
  • CLASP-2 polypeptides or polynucleotides that can inhibit an immune response, particularly the proliferation, or differentiation of T-cells, can be an effective therapy in preventing autoimmune disorders.
  • autoimmune disorders that can be treated or detected by CLASP- 2 include, but are not limited to: Addison's Disease, hemolytic anemia, antiphospholipid syndrome, rheumatoid arthritis, dermatitis, allergic encephalomyelitis, glomerulonephritis, Goodpasture's Syndrome, Graves' Disease, Multiple Sclerosis, Myasthenia Gravis, Neuritis, Ophthalmia, Bullous Pemphigoid, Pemphigus, Polyendocrinopathies, Purpura, Reiter's Disease, Stiff-Man Syndrome, Autoimmune Thyroiditis, Systemic Lupus Erythematosus, Autoimmune Pulmonary Inflammation, Guillain-Barre Syndrome, insulin dependent diabetes mellitis, and autoimmune inflammatory eye disease.
  • CLASP-2 polypeptides or polynucleotides can be used to treat anaphylaxis or hypersensitivity to an antigenic molecules.
  • CLASP-2 polynucleotides or polypeptides are used to treat and/or prevent organ rejection or graft-versus-host disease (GVHD).
  • Organ rejection occurs by host immune cell destruction of the transplanted tissue through an immune response.
  • an immvme response is also involved in GVHD, but, in this case, the foreign transplanted immune cells destroy the host tissues.
  • the administration of CLASP-2 polypeptides or polynucleotides that inhibits an immune response, particularly the proliferation, differentiation of T-cells can be an effective therapy in preventing organ rejection or GVHD.
  • CLASP-2 polypeptides or polynucleotides are used to modulate inflammation.
  • inflammation refers to both acute responses (i.e., responses in which the inflammatory processes are active) and chronic responses (i.e., responses marked by slow progression and formation of new connective tissue).
  • Acute and chronic inflammation can be distinguished by the cell types involved. Acute inflammation often involves polymorphonuclear neutrophils; whereas chronic inflammation is normally characterized by a lymphohistiocytic and/or granulomatous response.
  • Inflammation includes reactions of both the specific and non-specific defense systems.
  • a specific defense system reaction is a specific immune system reaction response to an antigen (possibly including an autoantigen).
  • a non-specific defense system reaction is an inflammatory response mediated by leukocytes incapable of immunological memory. Such cells include granulocytes, macrophages, neutrophils and eosinophils.
  • CLASP-2 polypeptides or polynucleotides can inhibit the proliferation and differentiation of cells involved in an inflammatory response.
  • These molecules can be used to treat inflammatory conditions, both chronic and acute conditions, including inflammation associated with infection (e.g., septic shock, sepsis, or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine induced lung injury, inflammatory bowel disease, Crohn's disease, or resulting from over production of cytokines (e.g., TNF or IL-1.).
  • cytokines e.g., TNF or IL-1.
  • Examples of specific types of inflammation are diffuse inflammation, focal inflammation, croupous inflammation, interstitial inflammation, obliterative inflammation, parenchymatous inflammation, reactive inflammation, specific inflammation, toxic inflammation and traumatic inflammation.
  • CLASP-2 polypeptides or polynucleotides are used to treat or detect infectious agents.
  • infectious diseases can be treated.
  • the immune response can be increased by either enhancing an existing immune response, or by initiating a new immune response.
  • CLASP-2 polypeptides or polynucleotides can be used to treat or detect any of these symptoms or diseases.
  • hCLASP-2-based diagnostics involves screening assays for vaginal bleeding of unknown cause.
  • the cause ofthe bleeding can be in part differentiated by knowledge of whether the vaginal bleeding contains placental components (Hart FD, Ed., 1985, French's Index of Differential Diagnosis, 12th Ed. John Wright & Sons, pp. 561-63). In these cases, the high expression of hCLASP-2 nucleotide sequences in placenta relative to its low expression in blood (FIG.
  • hCLASP-2 can be used in the following screening assays:
  • hCLASP-2-based diagnostics involve screening assays to determine injury to vital tissues that express hCLASP-2 at high levels.
  • tissues include kidney, heart, and lung (Fig 4A).
  • Injury to these tissues can result in leakage of cells and cellular constituents including hCLASP-2 into surrounding fluids (specified below).
  • Detection of abnormally high levels of hCLASP-2 protein in these surrounding fluids by Western analysis or ELISA, or detection of abnormally high levels of hCLASP-2 RNA in these fluids by RT-PCR or Northern analysis is expected to aid in the diagnosis of tissue injury.
  • hCLASP-2 nucleotide or amino acid sequences or fragments thereof would be expected to appear in the urine. Detection of abnormally high levels of hCLASP-2 can aid in the diagnosis of both nephritis and tubular necrosis, and differentiate from non-renal causes of proteinuria. Early diagnosis of nephritis is of particular value in patients with clinical signs and symptoms suggestive of systemic lupus erythematosis in whom early diagnosis and treatment of lupus nephritis can prevent irreversible kidney damage (Cameron J.S., 1999, J Nephrol 12 Suppl 2: S29-41).
  • tubular necrosis currently cannot be reversed by pharmacotherapy, differentiation of tubular necrosis from pre-renal failure is critical in formulating a treatment plan for oligouric hospitalized patients (Bidani A. and Churchill P.C, 1989, Dis Mon 35: 57-132).
  • hCLASP-2 nucleic or amino acid sequence or fragments thereof are expected to appear in the blood.
  • myocardial proteins e.g., creatine kinase, troponin
  • electrophoretic methodologies Fauci et al, (eds.), 1998, Harrison's Principles of Internal Medicine, 14th Ed., McGraw Hill, pp. 1352-1375.
  • the presence of hCLASP-2 in cardiac muscle and its absence in skeletal muscle and blood makes hCLASP-2 an ideal marker to diagnose and monitor myocardial injury.
  • pulmonary injury is not routinely diagnosed by assaying serum for lung-specific proteins.
  • pulmonary infarction also releases lung-specific proteins and cells into systemic circulation.
  • Pulmonary infarction due to pulmonary embolism (PE) or pneumonia is expected to release hCLASP-2- bearing cells or protein/peptides into systemic circulation. Detection of hCLASP-2 protein in serum or RNA in blood can aid in the diagnosis of pulmonary infarction in the appropriate clinical setting.
  • Current methods to diagnose PE are not only expensive but lack specificity and sensitivity, and the misdiagnosis of this condition is a leading cause of preventable death in hospitalized patients (Raskob G.E. and Hull R.D., 1999, Curr Opin Hematol.6(5): 280-4).
  • hCLASP-2-based diagnostics involve screening assays for identifying disorders of cells of hematopoietic lineage.
  • hCLASP-2 is expressed in human T cells, B cells but not cells from the myeloid lineage. Different hCLASP-2 isoforms in T and B cells permit further discrimination between malignancies of T and B lineage (FIG. 4B).
  • Precise identification of hematopoietic cell types is vital to guide chemotherapy and radiation therapy of patients with leukemia and lymphoma (Fauci et al Eds., 1998, Harrison's
  • hCLASP-2 expression differences can be detected by using FACS, immvmofluorescence, immunoperoxidase staining, RT-PCR, in situ hybridization or RNA blot analysis (Sambrook, Fritsch and Maniatas, Molecular Cloning, 2nd Ed. Cold Spring Harbor Lab. Press, 1989; Ward MS,
  • hCLASP-2-based diagnostics involve screening assays for identifying activated immune system cells.
  • hCLASP-2 is generally expressed at quite low levels in PBMCs (which is critical for some ofthe above applications), it is known that the surface expression of the closely related mouse CLASP-1 protein is altered during the process of lymphocyte activation. An analogous change in expression is expected for the hCLASP-2 protein.
  • Subtyping lymphocytes specific for a particular antigen for example, using MHC-based multimeric staining reagents (Airman et. al., 1996, Science
  • hCLASP-2 can also serve as a potential therapeutic agent for Wilms' tumor.
  • Wilms tumor is the most common primary renal tumor of childhood (Cotran, Kumar, and
  • hCLASP-2 is highly expressed in 293 cells, embryonic kidney epithelial cells. Therefore, hCLASP-2 nucleic or amino acid sequence or fragments can serve as tumor markers for Wilms' tumor.
  • Antibodies directed against a hCLASP-2 variant that is expressed only in Wilms' tumor can serve as novel therapeutic agents for Wilms' tumor, and can also function as delivery vehicles for other targeted therapeutics that may be attached to the anti- hCLASP-2 antibody (e.g., chemotherapeutics or radiolabeling).
  • Oligonucleotide sequences that include anti-sense RNA and DNA molecules and ribozymes that function to inhibit the translation of a CLASP-2 mRNA are within the scope ofthe invention. Such molecules are useful in cases where downregulation of CLASP-2 expression is desired.
  • Anti-sense RNA and DNA molecules act to directly block the translation of mRNA by binding to targeted mRNA and preventing protein translation.
  • the invention provides methods and antisense oligonucleotide or polynucleotide reagents which can be used to reduce expression of CLASP-2 gene products in vitro or in vivo. Administration of the antisense reagents of the invention to a target cell results in reduced CLASP activity.
  • antisense oligonucleotides bind to, and interfere with the translation of, the sense CLASP-2 mRNA.
  • the antisense molecule can render the CLASP-2 mRNA susceptible to nuclease digestion, interfere with transcription, interfere with processing, localization or otherwise with RNA precursors ("pre-mRNA"), repress transcription of mRNA from the CLASP-2 gene, or act through some other mechanism.
  • pre-mRNA RNA precursors
  • the antisense polynucleotides of the invention comprise an antisense sequence of at least 7 to 10 to typically 20 or more nucleotides that specifically hybridize to a sequence from mRNA encoding CLASP-2 or mRNA transcribed from the CLASP-2 gene. More often, the antisense polynucleotide of the invention is from about 10 to about 50 nucleotides in length or from about 14 to about 35 nucleotides in length. In other embodiments, antisense polynucleotides are polynucleotides of less than about 100 nucleotides or less than about 200 nucleotides.
  • the antisense polynucleotide should be long enough to form a stable duplex but short enough, depending on the mode of delivery, to administer in vivo, if desired.
  • the minimum length of a polynucleotide required for specific hybridization to a target sequence depends on several factors, such as G/C content, positioning of mismatched bases (if any), degree of uniqueness ofthe sequence as compared to the population of target polynucleotides, and chemical nature of the polynucleotide (e.g., methylphosphonate backbone, peptide nucleic acid, phosphorothioate), among other factors.
  • the antisense sequence is substantially complementary to the target CLASP-2 mRNA sequence.
  • the antisense sequence is exactly complementary to the target sequence.
  • the antisense polynucleotides can also include, however, nucleotide substitutions, additions, deletions, transitions, transpositions, or modifications, or other nucleic acid sequences or non-nucleic acid moieties so long as specific binding to the relevant target sequence corresponding to CLASP-2 RNA or its gene is retained as a functional property of the polynucleotide.
  • CLASP-2 polynucleotides and oligonucleotides of the invention can be made using nonstandard bases (e.g., other than adenine, cytidine, guanine, thymine, and uridine) or nonstandard backbone structures to provides desirable properties (e.g., increased nuclease-resistance, tighter-binding, stability or a desired TM).
  • nonstandard bases e.g., other than adenine, cytidine, guanine, thymine, and uridine
  • nonstandard backbone structures e.g., other than adenine, cytidine, guanine, thymine, and uridine
  • desirable properties e.g., increased nuclease-resistance, tighter-binding, stability or a desired TM.
  • oligonucleotides having a peptide-nucleic acid (PNA) backbone (Nielsen et al, 1991, Science 254: 1497) or incorporating 2'-O-methyl ribonucleotides, phosphorothioate nucleotides, methyl phosphonate nucleotides, phosphotriester nucleotides, phosphorothioate nucleotides, phosphoramidates.
  • PNA peptide-nucleic acid
  • Still other useful oligonucleotides may contain alkyl and halogen-substituted sugar moieties comprising one ofthe following at the 2' position: OH, SH, SCH3, F, OCN, OCH3OCH3, OCH3O(CH2)nCH3, 0(CH2)nNH2 or O(CH2)nCH3, where n is from 1 to about l0; Cl to CIO lower alkyl, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF3; OCF3; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH3 ; SO2CH3; ONO2; NO2; N3; NH2; heterocycloalkyl;heterocycloalkaryl;aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a cholesteryl group; a folate group; a
  • Folate, cholesterol or other groups that facilitate oligonucleotide uptake may be conjugated directly or via a linker at the 2' position of any nucleoside or at the 3' or 5' position ofthe 3'-terminal or 5'-terminal nucleoside, respectively.
  • One or more such conjugates may be used.
  • Oligonucleotides may also have sugar mimetics such as cyclobutyls in place of the pentofuranosyl group.
  • inventions may include at least one modified base form or "universal base” such as inosine, or inclusion of other nonstandard bases such as queosine and wybutosine as well as acetyl-, methyl-, thio- and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases.
  • modified base form or "universal base” such as inosine, or inclusion of other nonstandard bases such as queosine and wybutosine as well as acetyl-, methyl-, thio- and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases.
  • the antisense oligonucleotide can comprise at least one modified base moiety which is selected from the group including, but not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine,
  • the invention further provides oligonucleotides having backbone analogues such as phosphodiester, phosphorothioate, phosphorodithioate, methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate, 3'-thioacetal, methylene(methylimino), 3 '-N-carbamate, morpholino carbamate, chiral-methyl phosphonates, nucleotides with short chain alkyl or cycloalkyl intersugar linkages, short chain heteroatomic or heterocyclic intersugar ("backbone") linkages, or CH2-NH-O-CH2, CH2-N(CH3)-OCH2, CH2-O- N(CH3)-CH2, CH2-N(CH3)-N(CH3)-CH2 and O-N(CH3)-CH2-CH2 backbones (where phosphodiester is O-P-O-CH2), or mixtures ofthe same. Also useful are oligonucleotides having morph
  • the antisense sequence is complementary to relatively accessible sequences ofthe CLASP-2 mRNA (e.g., relatively devoid of secondary structure). This can be determined by analyzing predicted RNA secondary structures using, for example, the MFOLD program (Genetics Computer Group, Madison WI) and testing in vitro or in vivo as is known in the art.
  • Another useful method for identifying effective antisense compositions uses combinatorial arrays of oligonucleotides (see, e.g., Milner et al, 1997, Nature Biotechnology 15: 537). Examples of oligonucleotides that can be tested in cells for antisense suppression of CLASP-2 function are those capable of hybridizing to (i.e., substantially complementary to) the following positions from SEQUENCE ID NO: 1 :
  • administration of antisense oligonucleotides will result in reduction of hCLASP-mRNA expression by at least about 50%, as assessed by Northern analysis after administration of an antisense phosphorothioate oligonucleotide at a concentration of 1 ⁇ M, 5 ⁇ M, 10 ⁇ M or 20 ⁇ M.
  • the invention also provides an antisense polynucleotide that has sequences in addition to the antisense sequence (/ ' . e. , in addition to anti-CLASP-2-sense sequence).
  • the antisense sequence is contained within a polynucleotide of longer sequence.
  • the sequence of the polynucleotide consists essentially of, or is, the antisense sequence.
  • antisense nucleic acids can be made using any suitable method for producing a nucleic acid, such as the chemical synthesis and recombinant methods disclosed herein.
  • antisense RNA molecules ofthe invention can be prepared by de novo chemical synthesis or by cloning.
  • an antisense RNA that hybridizes to CLASP-2 mRNA can be made by inserting (ligating) an CLASP-2 DNA sequence (e.g., SEQUENCE ID No: 1 , or fragment thereof) in reverse orientation operably linked to a promoter in a vector (e.g., plasmid).
  • operably linked refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter or enhancer) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
  • antisense DNA oligodeoxyribonucleotidesderived from the translation initiation site e.g., between -10 and 410 regions of a CLASP-2 nucleotide sequence
  • antisense polynucleotides see ANTISENSE RNA AND DNA, 1988, D.A. Melton, Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). See also, Dagle ef ⁇ /., 1991, Nucleic Acids Research, 19: 1805.
  • antisense therapy see, e.g., Uhlmann et al, 1990, Chem. Reviews, 90: 543-584.
  • Ribozyme Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
  • engineered hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of CLASP-2 RNA sequences.
  • Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences, GUA, GUU and GUC.
  • RNA sequences of between 15 and 20 ribonucleotides corresponding to the region ofthe target gene containing the cleavage site can be evaluated for predicted structural features such as secondary structure that can render the oligonucleotide sequence unsuitable.
  • the suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using ribonuclease protection assays.
  • endogenous target gene expression can be reduced by targeting deoxyribonucleotide sequences complementary to the regulatory region of the target gene (i.e., the target gene promoter and/or enhancers) to form triple helical structures that prevent transcription of the target gene in target cells in the body.
  • deoxyribonucleotide sequences complementary to the regulatory region of the target gene i.e., the target gene promoter and/or enhancers
  • triple helical structures that prevent transcription of the target gene in target cells in the body.
  • Nucleic acid molecules to be used in triplex helix formation for the inhibition of transcription should be single stranded and composed of deoxynucleotides.
  • the base composition of these oligonucleotides must be designed to promote triple helix formation via Hoogsteen base pairing rules, which generally require sizable stretches of either purines or pyrimidines to be present on one strand of a duplex.
  • Nucleotide sequences can be pyrimidine-based, which will result in TAT and CGC + triplets across the three associated strands of the resulting triple helix.
  • the pyrimidine-rich molecules provide base complementarily to a purine-rich region of a single strand of the duplex in a parallel orientation to that strand.
  • nucleic acid molecules can be chosen that are purine- rich, for example, contain a stretch of G residues. These molecules will form a triple helix with a DNA duplex that is rich in GC pairs, in which the majority ofthe purine residues are located on a single strand ofthe targeted duplex, resulting in GGC triplets across the three strands in the triplex.
  • Switchback molecules are synthesized in an alternating 5 '-3', 3 '-5' manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizable stretch of either purines or pyrimidines to be present on one strand of a duplex.
  • RNA molecules can be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule.
  • DNA sequences can be incorporated into a wide variety of vectors which contain suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters.
  • antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.
  • DNA molecules can be introduced as a means of increasing intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences of ribo- or deoxy- nucleotides to the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the oligodeoxyribonucleotide backbone.
  • Methods for introducing polynucleotides into such cells or tissue include methods for in vitro introduction of polynucleotides such as the insertion of naked polynucleotide, i.e., by injection into tissue, the introduction of a CLASP-2 polynucleotide in a cell ex vivo, the use of a vector such as a virus, (e.g., a retrovirus, adenovirus, adeno- associated virus, and the like), phage or plasmid, and the like or techniques such as electroporation or calcium phosphate precipitation.
  • a virus e.g., a retrovirus, adenovirus, adeno- associated virus, and the like
  • phage or plasmid e.g., a retrovirus, adenovirus, adeno- associated virus, and the like
  • techniques such as electroporation or calcium phosphate precipitation.
  • gene therapy can be used to treat conditions in which the cells do not express normal CLASP-2 or express abnormal/inactive CLASP-2.
  • the polynucleotide encoding a CLASP-2 is intended to replace or act in the place of a functionally deficient endogenous gene.
  • abnormal conditions characterized by overexpression can be treated using the gene therapy techniques described below.
  • nucleic acids comprising a sequence encoding a
  • CLASP-2 protein or functional derivative thereof are administered to promote CLASP-2 function, by way of gene therapy.
  • Gene therapy refers to therapy performed by the administration of a nucleic acid to a subj ect.
  • the nucleic acid produces its encoded protein that mediates a therapeutic effect by promoting CLASP-2 function.
  • the therapeutic composition comprises a CLASP-2 nucleic acid that is part of an expression vector that encodes a CLASP-2 protein or fragment or chimeric protein thereof in a suitable host.
  • a nucleic acid has a promoter operably linked to the CLASP-2 coding region, said promoter being inducible or constitutive, and, optionally, tissue-specific.
  • a nucleic acid molecule is used in which the CLASP-2 coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the CLASP-2 nucleic acid (Koller and
  • nucleic acid into a patient can be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid-carrying vector, or indirect, in which case, cells are first transformed with the nucleic acid in vitro, then transplanted into the patient. These two approaches are known, respectively, as in vivo or ex vivo gene therapy.
  • the nucleic acid is directly administered in vivo, where it is expressed to produce the encoded product.
  • This can be accomplished by any of numerous methods known in the art, e.g., by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by infection using a defective or attenuated retroviral or other viral vector (see, U.S. Patent No.
  • microparticle bombardment e.g., a gene gun; Biolistic, Dupont
  • coating lipids or cell-surface receptors or transfecting agents, encapsulation in liposomes, microparticles, or microcapsules, or by administering it in linkage to a peptide which is known to enter the nucleus, by administering it in linkage to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262: 4429-4432) (which can be used to target cell types specifically expressing the receptors), and the like.
  • a nucleic acid-ligand complex can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation.
  • the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publications WO 92/06180 dated April 16, 1992; WO 92/22635 dated December 23, 1992; WO 92/20316 dated November 26, 1992; WO 93/14188 dated July 22, 1993; WO 93/20221 dated October 14, 1993).
  • the nucleic acid can be introduced intracellularly and inco ⁇ orated within host cell DNA for expression, by homologous recombination (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. U.S.A. 86: 8932-8935; Zijlstra et al, 1989, Nature 342: 435-438).
  • a viral vector that contains the CLASP-2 nucleic acid is used.
  • a retroviral vector can be used (see, Miller et al, 1993, Meth. Enzymol. 217: 581-599). These retroviral vectors have been modified to delete retroviral sequences that are not necessary for packaging ofthe viral genome and integration into host cell DNA.
  • the CLASP-2 nucleic acid to be used in gene therapy is cloned into the vector, which facilitates delivery ofthe gene into a patient.
  • retroviral vectors can be fovmd in Boesen et al, 1994, Biotherapy 6: 291-302, which describes the use of a retroviral vector to deliver the mdrl gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy.
  • Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al, 1994, J. Clin. Invest. 93: 644-651; Kiem et al, 1994, Blood 83: 1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy 4: 129- 141; and Grossman and Wilson, 1993, Curr. Opin. in Genetics and Devel . 3 : 110-114.
  • Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson 1993, Current Opinion in Genetics and Development 3: 499-503) present a review of adenovirus-based gene therapy.
  • Adeno-associated virus has also been proposed for use in gene therapy (Walsh et al, 1993, Proc. Soc. Exp. Biol. Med. 204: 289-300.
  • Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection.
  • the method of transfer includes the transfer of a selectable marker to the cells. The cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene. Those cells are then delivered to a patient.
  • the nucleic acid is introduced into a cell prior to administration in vivo ofthe resulting recombinant cell.
  • introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, and the like.
  • Numerous techniques are known in the art for the introduction of foreign genes into cells (see, e.g., Loeffler and Behr, 1993, Meth. Enzymol. 217: 599-618; Cohen et al, 1993, Meth. Enzymol.
  • the technique should provide for the stable transfer ofthe nucleic acid to the cell, so that the nucleic acid is expressible by the cell and preferably heritable and expressible by its cell progeny.
  • the resulting recombinant cells can be delivered to a patient by various methods known in the art.
  • epithelial cells are injected, e.g., subcutaneously.
  • recombinant skin cells can be applied as a skin graft onto the patient.
  • Recombinant blood cells e.g., hematopoietic stem or progenitor cells
  • the amount of cells envisioned for use depends on the desired effect, patient state, and the like., and can be determined by one skilled in the art.
  • Cells into which a nucleic acid can be introduced for purposes of gene therapy encompass any desired, available cell type, and include but are not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, e.g., as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, and the like.
  • the cell used for gene therapy is autologous to the patient.
  • the nucleic acid to be introduced for purposes of gene therapy comprises an inducible promoter operably linked to the coding region, such that expression ofthe nucleic acid is controllable by controlling the presence or absence ofthe appropriate inducer of transcription.
  • endogenous target gene expression can also be reduced by inactivating or "knocking out” the target gene or its promoter using targeted homologous recombination (see, e.g., Smithies et al, 1985, Nature 317: 230-234; Thomas and Capecchi, 1987, Cell 51: 503-512; Thompson et al, 1989, Cell 5: 313-321; each of which is incorporated by reference herein in its entirety).
  • targeted homologous recombination see, e.g., Smithies et al, 1985, Nature 317: 230-234; Thomas and Capecchi, 1987, Cell 51: 503-512; Thompson et al, 1989, Cell 5: 313-321; each of which is incorporated by reference herein in its entirety).
  • a mutant, nonfunctional target gene flanked by DNA homologous to the endogenous target gene (either the coding regions or regulatory regions of the target gene) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express the target gene in vivo. Insertion ofthe DNA construct, via targeted homologous recombination, results in inactivation ofthe target gene.
  • embryonic stem cells can be used to generate animal offspring with an inactive target gene
  • the CLASP-2 gene product can also be expressed in transgenic animals.
  • Animals of any species including, but not limited to, mice, rats, rabbits, guinea pigs, pigs, micro-pigs, goats, sheep, and non-human primates, e.g., baboons, monkeys, and chimpanzees can be used to generate CLASP-2 transgenic animals.
  • transgenic refers to animals expressing CLASP-2 gene sequences from a different species (e.g., mice expressing human CLASP-2 gene sequences), as well as animals that have been genetically engineered to overexpress endogenous (i.e., same species) CLASP-2 sequences or animals that have been genetically engineered to no longer express endogenous CLASP-2 gene sequences (i. e. , "knock-out” animals), and their progeny.
  • Any technique known in the art can be used to introduce a CLASP-2 transgene into animals to produce the founder lines of transgenic animals.
  • Such techniques include, but are not limited to pronuclearmicroinjection(Hoppe and Wagner, 1989, U.S. Pat. No. 4,873,191); retrovirus mediated gene transfer into germ lines (Van der Putten et al, 1985, Proc. Natl. Acad. Sci., U.S.A. 82: 6148-6152); gene targeting in embryonic stem cells (Thompson etal, 1989, Cell 56: 313-321); electroporation of embryos (Lo, 1983, Mol. Cell.
  • the present invention provides for transgenic animals that carry a CLASP-2 transgene in all their cells, as well as animals that carry the transgene in some, but not all their cells, i.e., mosaic animals.
  • the transgene can be integrated as a single transgene or in concatamers, e.g., head-to-head tandems or head-to-tail tandems.
  • the transgene can also be selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et al (1992, Proc. Natl. Acad. Sci. U.S.A. 89: 6232-6236).
  • the regulatory sequences required for such a cell-type specific activation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.
  • gene targeting is preferred.
  • vectors containing some nucleotide sequences homologous to the endogenous CLASP-2 gene are designed for the purpose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function ofthe nucleotide sequence of the endogenous CLASP-2 gene.
  • the transgene can also be selectively introduced into a particular cell type, thus inactivating the endogenous CLASP-2 gene in only that cell type, by following, for example, the teaching of Gu et al. (1994, Science 265: 103-106).
  • 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 level of mRNA expression ofthe transgene in the tissues ofthe transgenic animals can also be assessed using techniques that include, but are not limited to, Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and RT-PCR (reverse transcriptase PCR). Samples of CLASP-2 gene-expressing tissue, can also be evaluated immunocytochemically using antibodies specific for the CLASP-2 transgene product.
  • Sequences can be mapped to chromosomes by preparing PCR primers from SEQ ID NO: 1 , 3, 5, or 9. These primers can be can be less than 50 nucleotides in length, generally less than 46 nucleotides, more generally less than 41 nucleotides, most generally less than 36 nucleotides, preferably less than 31 nucleotides, more preferably less than 26 nucleotides, and most preferably less than 21 nucleotides in length.
  • the probes can also be less than 16 nucleotides, less than 13 nucleotides in length, less than 9 nucleotides in length and less than 7 nucleotides in length.
  • Primers can be selected so that the primers do not span more than one predicted exon in the genomic DNA. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes (i.e., chromosome 13). Only those hybrids containing the human CLASP-2 gene corresponding to SEQ ID NO: 1, 3, 5, or 9 will yield an amplified fragment. Similarly, somatic hybrids provide a rapid method of PCR mapping the polynucleotides to particular chromosomes. Precise chromosomal location ofthe CLASP-2 polynucleotides can also be achieved using fluorescence in situ hybridization (FISH) of a metaphase chromosomal spread.
  • FISH fluorescence in situ hybridization
  • the CLASP-2 polynucleotides can be used for identifying individuals from minute biological samples as DNA markers for restriction fragment length polymo ⁇ hism (RFLP).
  • RFLP restriction fragment length polymo ⁇ hism
  • An individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot with CLASP-2 DNA markers to yield unique bands for identifying the individual.
  • CLASP-2 polynucleotides can be also be used as polymo ⁇ hic markers for forensic analysis. See generally National Research Council, The Evaluation of Forensic DNA Evidence (Eds. 1996, Pollard et al, National Academy Press, Washington D.C.). The capacity to identify a distinguishing or unique set of forensic markers in an individual is useful for forensic analysis. For example, one can determine whether a blood sample from a suspect matches a blood or other tissue sample from a crime scene by determining whether the set of polymo ⁇ hic forms occupying selected polymo ⁇ hic sites is the same in the suspect and the sample.
  • the set of polymo ⁇ hic markers does not match between a suspect and a sample, it can be concluded (barring experimental error) that the suspect was not the source ofthe sample. If the set of markers does match, one can conclude that the DNA from the suspect is consistent with that found at the crime scene. If frequencies ofthe polymo ⁇ hic forms at the loci tested have been determined (e.g., by analysis of a suitable population of individuals), one can perform a statistical analysis to determine the probability that a match of suspect and crime scene sample would occur by chance.
  • PCR technology can be used to amplify DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, or semen found at a crime scene. The amplified sequence can then be compared to a standard, thereby allowing identification of the origin of the biological sample.
  • the CLASP-2 polynucleotide sequences of the present invention can be used to provide polynucleotide reagents, e.g., PCR primers, targeted to specific loci in the human genome, which can enhance the reliability of DNA-based forensic identifications by, for example, providing another "identification marker" (/. e. another DNA sequence that is unique to a particular individual).
  • polynucleotide reagents include the CLASP-2 nucleotide sequences or portions thereof, e.g., fragments derived from the noncoding regions of SEQ ID NO: 1, 3, 5, or 9 having a length of at least 20 bases, preferably at least 25 bases, and more preferably at least 30 bases. CLASP-2 polynucleotides can also be used as reagents for paternity testing.
  • the obj ect of paternity testing is usually to determine whether a male is the father of a child. In most cases, the mother ofthe child is known and thus, the mother's contribution to the child's genotype can be traced. Paternity testing investigates whether the part ofthe child's genotype not attributable to the mother is consistent with that ofthe putative father. Paternity testing can be performed by analyzing sets of polymo ⁇ hisms in the putative father and the child. Of course, the present invention can be expanded to the use of this procedure to determine if one individual is related to another. Even more broadly, the present invention can be employed to determine how related one individual is to another, for example, between races or species.
  • a CLASP-2 polynucleotide which encodes the CLASP-2 polypeptides, mutant polypeptides, peptide fragments, CLASP-2 fusion proteins or functional equivalents thereof, can be used to express CLASP-2 proteins in appropriate host cells.
  • the CLASP-2 polypeptides expressed will be identical or substantially similar to SEQ ID NOs: 2, 4, 6 or 10 or a fragment thereof.
  • altered DNA sequences which can be used in accordance with the invention include deletions, additions or substitutions of different nucleotide residues resulting in a sequence that encodes the same or a functionally equivalent gene product.
  • other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence can be used in the practice of the invention for the expression of the CLASP-2 protein.
  • a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
  • nucleic acid variations are "silent variations," which are one species of conservatively modified variations.
  • each codon in a nucleic acid sequence such SEQ ID NO: 1 except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.
  • a polypeptide having the sequence of SEQ ID NO: 2 or a fragment thereof can be encoded by numerous polynucleotides other than SEQ ID NO: 1.
  • the degenerate sequence will hybridize with SEQ ID NO: 1 under high or moderate stringency conditions, but this is not strictly required (e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cased, the nucleic acids typically hybridize under moderately stringent hybridization conditions.
  • the gene product itself can contain deletions, additions or substitutions of amino acid residues within a CLASP-2 sequence, which result in a silent change thus producing a functionally equivalent CLASP-2 protein.
  • Such conservative amino acid substitutions can be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature ofthe residues involved.
  • negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine, histidine and arginine; amino acids with uncharged polar head groups having similar hydrophilicity values include the following: glycine, asparagine, glutamine, serine, threonine, tyrosine; and amino acids with nonpolar head groups include alanine, valine, isoleucine, leucine, phenylalanme, proline, methionine, tryptophan.
  • the DNA sequences of the invention can be engineered in order to alter a
  • CLASP-2 coding sequence for a variety of ends, including but not limited to, alterations which modify processing and expression ofthe gene product.
  • mutations can be introduced using techniques which are well known in the art, e.g., site-directed mutagenesis, to insert new restriction sites, to alter glycosylation patterns, phosphorylation, and the like.
  • site-directed mutagenesis e.g., site-directed mutagenesis
  • new restriction sites e.g., to insert new restriction sites, to alter glycosylation patterns, phosphorylation, and the like.
  • CLASP-2 mutant polypeptides can be constructed by modifying or rearranging the nucleotide sequences that encode the CLASP-2 extracellular, transmembrane and cytoplasmic domains.
  • the present invention provides homologues ofthe
  • CLASP-2 polypeptides which function as either an CLASP-2 agonists or an CLASP-2 antagonist.
  • the CLASP-2 agonists and antagonists stimulate or inhibit, respectively, a subset ofthe biological activities ofthe naturally occurring form of the CLASP-2 polypeptide.
  • specific biological effects can be elicited by treatment with a homologue of limited function.
  • treatment of a subject with a homologue having a subset ofthe biological activities ofthe naturally occurring form ofthe polypeptide has fewer side effects in a subject relative to treatment with the naturally occurring form ofthe CLASP-2 polypeptide.
  • the invention contemplates both full-length CLASP-2 polypeptides and fragments, e.g., fragments having a length of at least about 10, often 20, frequently 50 or 100 residues substantially identical to the exemplified CLASP-2 polypeptide sequences of the invention.
  • Protein fragments can 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.
  • Representative examples of polypeptide fragments of the invention include, for example, fragments from about amino acid number 1-20, 2 1-40, 4 1-60, 61-80, 81-100, 102-120, 121-140, 141-160, 161-180, 181-200, or 201 to the end of the coding region.
  • polypeptide fragments can be about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200 amino acids in length.
  • “about” includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes.
  • Preferred polypeptide fragments include the CLASP-2 protein.
  • Further preferred polypeptide fragments include the CLASP-2 protein having a continuous series of deleted residues from the amino or the carboxy terminus, or both. For example, any number of amino acids, ranging from 1-X, can be deleted from the amino terminus of either the CLASP-2 polypeptide. Furthermore, any combination of the above amino and carboxy terminus deletions are preferred.
  • polynucleotide fragments encoding these CLASP- 2 polypeptide fragments are also preferred. Even if deletion of one or more amino acids from the N-terminus of a protein results in modification of loss of one or more biological functions of the protein, other biological activities can still be retained.
  • the ability of shortened CLASP-2 muteins to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptides generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the N-terminus. Whether a particular polypeptide lacking N-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 CLASP-2 mutein with a large number of deleted N- terminal amino acid residues can retain some biological or immunogenic activities. In fact, peptides composed of as few as four CLASP-2 amino acid residues can often evoke an immune response.
  • Homologues ofthe CLASP-2 polypeptide can be generated by mutagenesis, e.g., discrete point mutation or truncation ofthe CLASP-2 polypeptide.
  • the term "homologue” refers to a variant form of the CLASP-2 polypeptide which acts as an agonist or antagonist ofthe activity ofthe CLASP-2 polypeptide.
  • An agonist ofthe CLASP-2 polypeptide can retain substantially the same, or a subset, ofthe biological activities ofthe CLASP-2 polypeptide.
  • An antagonist ofthe CLASP-2 polypeptide can inhibit one or more ofthe activities ofthe naturally occurring form ofthe CLASP-2 polypeptide, by, for example, competitively binding to a downstream or upstream member of the CLASP-2 molecular pathway which includes the CLASP-2 polypeptide.
  • Modulation can be assayed by determining any parameter that is indirectly or directly affected by the expression ofthe target gene.
  • parameters include, e.g., changes in RNA or protein levels, changes in protein activity, changes in product levels, changes in downstream gene expression, changes in reporter gene transcription (luciferase, CAT, - galactosidase, -glucuronidase, GFP ⁇ ee, e.g., Mistili & Spector, 1997, Nature Biotechnology 15: 961-964); changes in signal transduction, phosphorylation and dephosphorylation, receptor-ligand interactions, second messenger concentrations (e.g., cGMP, cAMP, IP 3 , and Ca 2+ ), and cell growth.
  • reporter gene transcription e.g., luciferase, CAT, - galactosidase, -glucuronidase, GFP ⁇ ee, e.g., Mistili & Spector, 1997, Nature Biotechnology 15: 961-964
  • RNA or protein levels can be measured by any means known to those skilled in the art, e.g., measurement of RNA or protein levels, measurement of RNA stability, identification of downstream or reporter gene expression, e.g., via chemiluminescence, fluorescence, colorimetric reactions, antibody binding, inducible markers, ligand binding assays; changes in intracellular second messengers such as cGMP and inositol triphosphate (IP 3 ); changes in intracellular calcium levels; cytokine release, and the like.
  • chemiluminescence, fluorescence, colorimetric reactions e.g., via chemiluminescence, fluorescence, colorimetric reactions, antibody binding, inducible markers, ligand binding assays
  • changes in intracellular second messengers such as cGMP and inositol triphosphate (IP 3 )
  • changes in intracellular calcium levels cytokine release, and the like.
  • CLASP-2 Polypeptide Expression Systems In order to express a biologically active CLASP-2, the nucleotide sequence coding for CLASP-2, or a functional equivalent, is inserted into an appropriate expression vector.
  • the CLASP-2 gene product as well as host cells or cell lines transfected or transformed with recombinant CLASP-2 expression vectors can be used for a variety of p uposes. These include, but are not limited to, generating antibodies (i.e., monoclonal or polyclonal) that competitively inhibit activity of CLASP-2 protein and neutralize its activity; antibodies that activate CLASP-2 function and antibodies that detect its presence on the cell surface or in solution.
  • Anti-CLASP-2 antibodies can be used in detecting and quantifying expression of CLASP-2 levels in cells and tissues such as lymphocytes and macrophages, as well as isolating CLASP-2-positive cells from a cell mixture.
  • Methods which are well known to those skilled in the art can be used to construct recombinant expression vectors containing the CLASP-2 coding sequence and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination/genetic recombination. (See, e.g., the techniques described in Sambrook et al, 1989, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y. and Ausubel et al, supra).
  • the recombinant expression vectors ofthe invention comprise a nucleic acid ofthe invention in a form suitable for expression ofthe nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis ofthe host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of polypeptide desired, and the like.
  • the expression vectors ofthe invention can be introduced into host cells to thereby produce polypeptides or peptides, including fusion polypeptides or peptides, encoded by nucleic acids as described herein (e.g., CLASP-2 polypeptides, mutant forms of CLASP-2, fusion polypeptides, and the like).
  • fusion polypeptides or peptides encoded by nucleic acids as described herein (e.g., CLASP-2 polypeptides, mutant forms of CLASP-2, fusion polypeptides, and the like).
  • a variety of host-expression vector systems can be utilized to express a variety of host-expression vector systems can be utilized to express a
  • CLASP-2 coding sequence include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the CLASP-2 coding sequence; yeast transformed with recombinant yeast expression vectors containing the CLASP-2 coding sequence; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the CLASP-2 coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the CLASP-2 coding sequence; or animal cell systems.
  • microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the CLASP-2 coding sequence
  • any of a number of suitable transcription and translation elements can be used in the expression vector.
  • inducible promoters such as pL of bacteriophage ⁇ , plac, pt ⁇ , ptac (ptip- lac hybrid promoter; cvtomegalovirus promoter) and the like can be used;
  • promoters such as the baculovirus polyhedron promoter can be used;
  • promoters derived from the genome of plant cells e.g., heat shock promoters; the promoter for the small subunit of RUBISCO; the promoter for the chlorophyll ⁇ / ⁇ binding protein
  • plant viruses e.g., the 35 S RNA promoter of CaMV; the coat protein promoter of TMV
  • a number of expression vectors can be advantageously selected depending upon the use intended for the expressed CLASP-2 product.
  • vectors which direct the expression of high levels of fusion protein products that are readily purified can be desirable.
  • Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al. , 1983 , EMBO J. 2 : 1791 ), in which the CLASP-2 coding sequence can be ligated into the vector in frame with the lacZ coding region so that a hybrid protein is produced; pI ⁇ vectors (Inouye & Inouye, 1985, Nucleic acids Res.
  • pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
  • GST glutathione S-transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by adso ⁇ tionto glutathione-agarose beads followed by elution in the presence of free glutathione.
  • Proteins made in such systems may be designed to include heparin, thrombin, or factor XA protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.
  • yeast a number of vectors containing constitutive or inducible promoters can be used.
  • the expression of the CLASP-2 coding sequence can be driven by any of a number of promoters.
  • viral promoters such as the 35S RNA and 19S RNA promoters of CaMV (Brisson et al, 1984, Nature 310: 511-514), or the coat protein promoter of TMV (Takamatsu e/ ⁇ /., 1987, EMBO J. 6: 307-311) can be used; alternatively, plant promoters such as the small subunit of RUBISCO (Coruzzi et al, 1984, EMBO J.
  • An alternative expression system which could be used to express CLASP-2 is an insect system.
  • a utographa calif ornica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes.
  • the virus grows in Spodoptera frugiperda cells.
  • the CLASP-2 coding sequence can be cloned into non-essential regions (e.g., the polyhedron gene) of the virus and placed under control of an AcNPV promoter (e.g., the polyhedron promoter).
  • Successful insertion ofthe CLASP-2 coding sequence will result in inactivation ofthe polyhedron gene and production of non-occluded recombinant virus (i.e., virus lacking the proteinaceous coat coded for by the polyhedron gene).
  • the CLASP-2 coding sequence can be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence.
  • This chimeric gene can then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region ofthe viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing CLASP-2 in infected hosts.
  • a non-essential region ofthe viral genome e.g., region El or E3
  • the vaccinia 7.5K promoter can be used.
  • Regulatable expression vectors such as the tetracycline repressible vectors can also be used to express a coding sequence in a controlled fashion.
  • Specific initiation signals can also be required for efficient translation of inserted CLASP-2 coding sequences. These signals include the ATG initiation codon and adjacent sequences. In cases where the entire CLASP-2 gene, including its own initiation codon and adjacent sequences, is inserted into the appropriate expression vector, no additional translational control signals can be needed. However, in cases where only a portion ofthe CLASP-2 coding sequence is inserted, exogenous translational control signals, including the ATG initiation codon, must be provided. Furthermore, the initiation codon must be in phase with the reading frame of the CLASP-2 coding sequence to ensure translation ofthe entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, and the like, (see Bittner et al, 1987, Methods in Enzymol. 153: 516-544).
  • a host cell strain can be chosen which modulates the expression ofthe inserted sequences, or modifies and processes the gene product in a specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for the function ofthe protein.
  • modifications e.g., glycosylation
  • processing e.g., cleavage
  • the presence of several consensus N-glycosylation sites in CLASP-2 extracellular domains support the possibility that proper modification can play a role in CLASP-2 function.
  • Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins.
  • Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing ofthe foreign protein expressed.
  • eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation ofthe gene product can be used.
  • mammalian host cells include, but are not limited to, CHO, VERO, BHK, HeLa, COS, MDCK, 293, WI38, and the like.
  • Host cells transformed with nucleotide sequences encoding CLASP-2 may be cultured under conditions suitable for the expression and recovery ofthe soluble protein from cell culture.
  • the protein produced by a transformed cell may be secreted or contained intracellularly depending on the sequence and/or the vector used.
  • expression vectors containing polynucleotides which encode CL ASP- 2 may be designed to contain signal sequences which direct secretion of CLASP-2 through a prokaryotic or eukaryotic cell membrane.
  • Other constructions may be used to join sequences encoding CLASP-2 to nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins.
  • purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin,
  • cell lines which stably express CLASP-2 proteins can be engineered.
  • host cells can be transformed with the CLASP-2 DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, and the like.), and a selectable marker.
  • expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, and the like.
  • engineered cells can be allowed to grow for 1-2 days in an enriched medium, and then switched to a selective medium.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
  • This method can advantageously be used to engineer cell lines which express the CLASP-2 protein(s) on the cell surface. Such engineered cell lines are particularly useful in screening for molecules or drugs that affect CLASP-2 function.
  • a number of selection systems can be used, including but not limited to, the he ⁇ es simplex virus thymidine kinase (Wigler et al, 1977, Cell 11 : 223), hypoxanthine- guanine phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc. Natl. Acad. Sci. U.S.A. 48: 2026), and adenine phosphoribosyltransferase (Lowy et al, 1980, Cell 22: 817) genes which can be employed in tk " , hgprt " or aprt " cells, respectively.
  • antimetabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler et al, 1980, Natl. Acad. Sci. U.S.A. 77: 3567; O'Hare et al, 1981, Proc. Natl. Acad. Sci. U.S.A. 78: 1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981), Proc. Natl. Acad. Sci. U.S.A. 78: 2072); neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapine/ al, 1981 , J. Mol. Biol.
  • hygro which confers resistance to hygromycin
  • Additional selectable genes have been described, namely trpB, which allows cells to utilize indole in place of tryptophan; hisD, which allows cells to utilize histinol in place of histidine (Hartman & Mulligan, 1988, Proc. Natl. Acad. Sci. U.S.A.
  • ODC ornithine decarboxylase
  • 2- (difluoromethyl)-DL-ornithineJ)FMO McConlogueL., 1987, In: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory ed.
  • glutamine synthetase Bebbington et al, 1992, Biotech 10: 169.
  • the coding sequence of CLASP-2 could be synthesized in whole or in part, using chemical methods well known in the art.
  • chemical methods See, e.g., Caruthers et al, 1980, Nuc. Acids Res. Symp. Ser. 7: 215-233; Crea and Horn, 180, Nuc. Acids Res. 9(10): 2331; Matteucci and Caruthers, 1980, Tetrahedron Letter 21 : 719; and Chow and Kempe, 1981, Nuc. Acids Res. 9(12): 2807-2817.
  • the protein itself could be produced using chemical methods to synthesize a CLASP-2 amino acid sequence in whole or in part.
  • peptides can be synthesized by solid phase techniques, cleaved from the resin, and purified by preparative high performance liquid chromatography. (See Creighton, 1983, Proteins Structures And Molecular Principles, W.H. Freeman and Co., N.Y. pp. 50-60). The composition ofthe synthetic polypeptides can be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; see Creighton, 1983, Proteins, Structures and Molecular Principles, W.H. Freeman and Co., N.Y., pp. 34-49).
  • the CLASP-2 polypeptide contains non-naturally occurring amino acids or amino acid analogs (i.e., compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an ⁇ carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium).
  • non-naturally occurring amino acids or amino acid analogs i.e., compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an ⁇ carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium).
  • the recombinant host cells which contain the coding sequence and which express a CLASP-2 gene product or fragments thereof can be identified by at least four general approaches; (a) DNA-DNA or DNA-RNA hybridization; (b) the presence or absence of "marker" gene functions; (c) assessing the level of transcription as measured by the expression of CLASP-2 mRNA transcripts in the host cell; and (d) detection of the gene product as measured by immunoassay or by its biological activity. Prior to the identification of gene expression, the host cells can be first mutagenized in an effort to increase the level of expression of CLASP-2, especially in cell lines that produce low amounts of CLASP-2.
  • the presence ofthe CLASP-2 coding sequence inserted in the expression vector can be detected by DNA-DNA or DNA-RNA hybridization using probes comprising nucleotide sequences that are homologous to the CLASP-2 coding sequence, respectively, or portions or derivatives thereof.
  • the recombinant expression vector/host system can be identified and selected based upon the presence or absence of certain "marker" gene functions (e.g., thymidine kinase activity, resistance to antibiotics, resistance to methotrexate, transformation phenotype, occlusion body formation in baculovirus, and the like).
  • certain "marker" gene functions e.g., thymidine kinase activity, resistance to antibiotics, resistance to methotrexate, transformation phenotype, occlusion body formation in baculovirus, and the like.
  • certain "marker” gene functions e.g., thymidine kinase activity, resistance to antibiotics, resistance to methotrexate, transformation phenotype, occlusion body formation in baculovirus, and the like.
  • a marker gene can be placed in tandem with the CLASP-2 sequence under the control of the same or different promoter used to control the expression of the CLASP-2 coding sequence. Expression of
  • transcriptional activity for the CLASP-2 coding region can be assessed by hybridization assays.
  • RNA can be isolated and analyzed by Northern blot using a probe homologous to the CLASP-2 coding sequence or particular portions thereof.
  • total nucleic acids of the host cell can be extracted and assayed for hybridization to such probes.
  • reverse transcription-polymerase chain reactions can be used to detect low levels of gene expression.
  • the expression ofthe CLASP-2 protein product can be assessed immunologically, for example by Western blots, immunoassays such as radioimmuno-precipitation, enzyme-linked immunoassays, fluorescent activated cell sorting ("FACS"), and the like. This can be achieved by using an anti-CLASP-2 antibody.
  • CLASP-2 protein can be expressed as a fusion protein with green-fluorescent protein to facilitate its detection in cells (United States Patent Nos. 5,491,084; 5,804,387; 5,777,079).
  • Identification of cells or tissues expressing CLASP protein or mRNA, especially CLASP-2 isoforms, can be useful for determining normal and abnormal CLASP expression in a given cell or tissue.
  • CLASP-2 isoforms a number of CLASP-2 isoforms have been identified, e.g., in Jurkat cells, peripheral blood, and brain.
  • the identification of mRNA or protein expression in various cell types and tissues can allow for identification of isoforms improperly expressed in either a spatial or temporal manner.
  • Expression of hCLASP-2D isoform in hematopoietic cells may cause problems due to the presence ofthe SH3 domain not seen in the Jurkat and peripheral blood isoforms.
  • the CLASP-2 protein and/or cell lines that express CLASP-2 can be used to screen for antibodies, peptides, small molecules, natural and synthetic compounds or other cell bound or soluble molecules that bind to the CLASP-2 protein resulting in stimulation or inhibition of CLASP-2 function.
  • anti- CLASP-2 antibodies can be used to inhibit or stimulate CLASP-2 function and to detect its presence.
  • screening of peptide libraries with recombinantly expressed soluble CLASP-2 protein or cell lines expressing CLASP-2 protein can be useful for identification of therapeutic molecules that function by inhibiting or stimulating the biological activity of CLASP-2.
  • the uses of the CLASP-2 protein and engineered cell lines, described in the subsections below, can be employed equally well for homologous CLASP-2 genes in various species.
  • cell lines may be engineered to express the extracellular or intracellular domain of CLASP fused to another molecule such as GST.
  • CLASP its extracellular domain or its intracellular domain may be fused to an immvmoglobulin constant region (Hollenbaugh and Aruffo, 1992, Current Protocols in Immunology, Unit 10.19; Aruffo et al, 1990, Cell 61 : 1303) to produce a soluble molecule with increased half life.
  • the soluble protein or fusion protein can be used in binding assays, affinity chromatography, immunoprecipitation, Western blot, and the like. Synthetic compounds, natural products, and other sources of potentially biologically active materials can be screened in assays that are well known in the art.
  • Random peptide libraries consisting of all possible combinations of amino acids attached to a solid phase support can be used to identify peptides that are able to bind to a specific domain of CLASP-2 (Lam, K.S. etal, 1991, Nature 354: 82-84).
  • the screening of peptide libraries can have therapeutic value in the discovery of pharmaceutical agents that stimulate or inhibit the biological activity of CLASP-2.
  • Identification of molecules that are able to bind to the CLASP-2 protein can be accomplished by screening a peptide library with recombinant soluble CLASP-2 protein. Methods for expression and purification of CLASP-2 are described in Section 5.7, supra, and can be used to express recombinant full length CLASP-2 or fragments of CLASP-2 depending on the functional domains of interest. Such domains include CLASP-2 extracellular domain, transmembrane domain, CLASP-2 intracellular domain, ITAM containing domain, tyrosine phosphorylation site containing domain, cysteine cluster containing domain, cadherin motif containing domain, and coil/coil domain.
  • CLASP-2 protein can be conjugated to enzymes such as alkaline phosphatase or horseradish peroxidase or to other reagents such as fluorescent labels which can include fluorescein isothiocyanate (FITC), phycoerythrin (PE) or rhodamine. Conjugation of any given label to CLASP-2 can be performed using techniques that are well known in the art.
  • CLASP-2 expression vectors can be engineered to express a chimeric CLASP-2 protein containing an epitope for which a commercially available antibody exist. The epitope- specific antibody can be tagged with a detectable label using methods well known in the art including an enzyme, a fluorescent dye or colored or magnetic beads.
  • the "tagged" CLASP-2 conjugate is incubated with the random peptide library for 30 minutes to one hour at 22°C to allow complex formation between CLASP-2 and peptide species within the library. The library is then washed to remove any unbound protein. If CLASP-2 has been conjugated to alkaline phosphatase or horseradish peroxidase the whole library is poured into a petri dish containing substrates for either alkaline phosphatase or peroxidase, for example, 5-bromo-4-chloro-3-indoyl phosphate (BOP) or 3,3',4,4"-diaminobenzidine (DAB), respectively.
  • BOP 5-bromo-4-chloro-3-indoyl phosphate
  • DAB 3,3',4,4"-diaminobenzidine
  • the peptide/solid phase- CLASP-2 complex changes color, and can be easily identified and isolated physically under a dissecting microscope with a micromanipulator. If a fluorescent tagged CLASP-2 molecule has been used, complexes can be isolated by fluorescence activated sorting. If a chimeric CLASP-2 protein expressing a heterologous epitope has been used, detection of the peptide/CLASP-2 complex can be accomplished by using a labeled epitope-specific antibody. Once isolated, the identity of the peptide attached to the solid phase support can be determined by peptide sequencing.
  • soluble CLASP-2 molecules in another embodiment, it is possible to detect peptides that bind to cell-associated CLASP-2 using intact cells.
  • the use of intact cells is preferred for use with cell surface molecules.
  • Methods for generating cell lines expressing CLASP-2 are described in Section 5.8.
  • the cells used in this technique can be either live or fixed cells.
  • the cells can be incubated with the random peptide library and bind to certain peptides in the library to form a "rosette" between the target cells and the relevant solid phase support/peptide.
  • the rosette can thereafter be isolated by differential centrifugation or removed physically under a dissecting microscope. Techniques for screening combinatorial libraries are known in the art (Gallop et al, 1994, J. Med. Chem., 37: 1233; Gordon, 1994, J. Med. Chem., 37: 1385).
  • CLASP-2 molecules can be reconstituted into liposomes where label or "tag" can be attached.
  • a CLASP-2 or a modified CLASP-2 sequence can be ligated to a heterologous sequence to encode a fusion protein.
  • a fusion protein can also be engineered to contain a cleavage site located between a CLASP-2 sequence and the heterologous protein sequence, so that the CLASP-2 can be cleaved away from the heterologous moiety.
  • fusion proteins ofthe invention can contain the CLASP-2 extracellular domain comprising at least about residues 1 through 816 or fragment thereof. In another embodiment, fusion proteins can contain the CLASP-2 intracellular domain comprising at least about residue 843 through the end ofthe CLASP-2 sequence or fragment thereof. 5.6. Cloning Alleles, Variants, and Species Homologs of CLASP-2
  • labeled DNA probes made from nucleic acid fragments corresponding to any partial cDNA disclosed herein can be used to screen a cDNA library derived from lymphoid cells or brain cells. More specifically, oligonucleotides corresponding to either the 5 ' or 3' terminus ofthe cDNA sequence can be used to obtain longer nucleotide sequences. Briefly, the library can be plated out to yield a maximum of 30,000 pfu for each 150 mm plate. Approximately 40 plates can be screened.
  • Nylon filters are placed onto the soft top agarose and after 60 seconds, the filters are peeled off and floated on a DNA denaturing solution consisting of 0.4N sodium hydroxide. The filters are then immersed in neutralizing solution consisting of 1 M Tris-HCl, pH 7.5, before being allowed to air dry.
  • the filters are prehybridized in hybridization buffer such as casein buffer containing 10% dextran sulfate, 0.5M NaCl, 50mM Tris-HCl, pH 7.5, 0.1% sodium pyrophosphate, 1% casein, 1% SDS, and denatured salmon sperm DNA at 0.5 mg/ml for 6 hours at 60°C.
  • hybridization buffer such as casein buffer containing 10% dextran sulfate, 0.5M NaCl, 50mM Tris-HCl, pH 7.5, 0.1% sodium pyrophosphate, 1% casein, 1% SDS, and denatured salmon sperm DNA at 0.5 mg/ml for 6 hours at 60°C.
  • the radiolabeled probe is then denatured by heating to 95 °C for 2 minutes and then added to the prehybridization solution containing the filters.
  • the filters are hybridized at 60°C for 16 hours.
  • the filters are then washed in IX wash mix (1 OX wash mix contains 3M NaCl, 0.6M Tris base, and 0.02M EDTA) twice for 5 minutes each at room temperature, then in 1 X wash mix containing 1% SDS at 60°C for 30 minutes, and finally in 0.3X wash mix containing 0.1% SDS at 60°C for 30 minutes.
  • IX wash mix (1 OX wash mix contains 3M NaCl, 0.6M Tris base, and 0.02M EDTA) twice for 5 minutes each at room temperature, then in 1 X wash mix containing 1% SDS at 60°C for 30 minutes, and finally in 0.3X wash mix containing 0.1% SDS at 60°C for 30 minutes.
  • the filters are then air dried and exposed to x-ray film for autoradiography. After developing, the film is aligned with the filters to select a positive plaque.
  • the agar plug containing the plaques will be removed and placed in lambda dilution buffer containing 0.1M NaCl, 0.01M magnesium sulfate, 0.035M Tris HCl, pH 7.5, 0.01 % gelatin.
  • the phage can then be replated and rescreened to obtain single, well isolated positive plaques.
  • Positive plaques can be isolated and the cDNA clones sequenced using primers based on the known cDNA sequence. This step can be repeated until a full length cDNA is obtained. It can be necessary to screen multiple cDNA libraries from different tissues to obtain a full length cDNA.
  • RACE Rapid Amplification of cDNA Ends
  • RACE Rapid Amplification of cDNA Ends
  • 5'-RACE- Ready RNA synthesized from human tissues containing a unique anchor sequence is commercially available (Clontech).
  • PCR is carried out on 5 ' -RACE-Ready cDN A using the provided anchor primer and the 3 ' primer.
  • a secondary PCR reaction is then carried out using the anchored primer and a nested 3' primer according to the manufacturer's instructions.
  • the full length cDNA sequence can be translated into amino acid sequence and examined for certain landmarks such as a continuous open reading frame flanked by translation initiation and termination sites, a cadherin-like domain, an ITAM domain, a tyrosine phosphorylation site, a cysteine cluster, a transmembrane domain, and finally overall structural similarity to the CLASP-2 genes disclosed herein. See, Ponassi et al, 1999, Mech. Dev.
  • an endogenous CLASP-2 gene within a cell population can be modified by inserting a heterologous DNA regulatory element into the genome of the cell line such that the inserted regulatory element is operatively linked with the endogenous CLASP-2 gene.
  • a heterologous DNA regulatory element for example, an endogenous CLASP-2 gene which is normally "transcriptionally silent", i.e., an CLASP-2 gene which is normally not expressed, or is expressed only at very low levels in a cell population, can be activated by inserting a regulatory element which is capable of promoting the expression of a normally expressed gene product in the cells.
  • a transcriptionally silent, endogenous CLASP-2 gene can be activated by insertion of a promiscuous regulatory element that works across cell types.
  • a heterologous regulatory element can be inserted into a cell line population, such that it is operatively linked with an endogenous CLASP-2 gene, using techniques, such as targeted homologous recombination, which are well known to those of skill in the art, (see e.g., in Chappel, U.S. Patent No. 5,272,071; PCT publication No. WO 91/06667, published Jan 16, 1991). 5.8. Anti-CLASP-2 Antibodies
  • Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, human or humanized, IgG, IgM, IgA, IgD or IgE, a complementarity determining region, Fab fragments, F(ab') 2 and fragments produced by an Fab expression library as well as anti- idiotypic antibodies.
  • Antibodies which compete for CLASP-2 binding are especially preferred for diagnostics and therapeutics.
  • Monoclonal antibodies that bind CLASP-2 can be radioactively labeled allowing one to follow their location and distribution in the body after injection. Radioisotope tagged antibodies can be used as a non-invasive diagnostic tool for imaging de novo lymphoid tumors and metastases that express CLASP-2.
  • Immunotoxins can also be designed which target cytotoxic agents to specific sites in the body.
  • high affinity CLASP-2 specific monoclonal antibodies can be covalently complexed to bacterial or plant toxins, such as diphtheria toxin or ricin.
  • a general method of preparation of antibody/hybrid molecules can involve use of thiol- crosslinking reagents such as SPDP, which attack the primary amino groups on the antibody and by disulfide exchange, attach the toxin to the antibody.
  • SPDP thiol- crosslinking reagents
  • the hybrid antibodies can be used to specifically eliminate CLASP-2 expressing lymphocytes.
  • various host animals can be immunized by injection with the recombinant or naturally purified CLASP-2 protein, fusion protein or peptides, including but not limited to goats, rabbits, mice, rats, hamsters, and the like
  • Various adjuvants can be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacilli Calmette-Guerin) and Corynebacterium parvum.
  • BCG Bacilli Calmette-Guerin
  • Corynebacterium parvum bacilli Calmette-Guerin
  • Monoclonal antibodies to CLASP-2 can be prepared by using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique originally described by Kohler and Milstein, (Nature, 1975, 256: 495-497), the human B-cell hybridoma technique (Kosbor et al, 1983, Immunology Today, 4: 72; Cote et al, 1983, Proc. Natl. Acad. Sci. U.S.A., 80: 2026-2030) and the EBV-hybridoma technique (Cole et al, 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
  • phage display technology is used to identify antibodies and heteromeric Fab fragments that specifically bind to selected antigens (see, e.g., McCafferty et al, Nature 348: 552-554 (1990); Marks et al, Biotechnology 10: 779-783 (1992)).
  • Hybridomas can be screened using enzyme-linked immunosorbent assays (ELISA) in order to detect cultures secreting antibodies specific for refolded recombinant CLASP-2.
  • Cultures can also be screened by ELISA to identify those cultures secreting antibodies specific for mammalian-produced CLASP-2. Confirmation of antibody specificity can be obtained by western blot using the same antigens. Subsequent ELISA testing can use recombinant CLASP-2 fragments to identify the specific portion ofthe CLASP-2 molecule with which a monoclonal antibody binds.
  • Additional testing can be used to identify monoclonal antibodies with desired functional characteristics such as staining of histological sections, immunoprecipitation of CLASP-2, inhibition of CLASP-2 binding or stimulation of CLASP-2 to transmit an intracellular signal. Determination ofthe monoclonal antibody isotype can be accomplished by ELISA, thus providing additional information concerning purification or function.
  • Some anti-CLASP-2 monoclonal antibodies of the present invention are humanized, human or chimeric, in order to reduce their potential antigenicity, without reducing their affinity for their target.
  • Humanized antibodies have been described in the art. See, e.g., Queen, et al, 1989, Proc. Natl Acad. Sci. U.S.A. 86: 10029; U.S. Patent Nos. 5,563,762; 5,693,761 ; 5,585,089 and 5,530,101.
  • the human antibody sequences used for humanization can be the sequences of naturally occurring human antibodies or can be consensus sequences of several human antibodies. See Kettleborough et al, 1991, Protein Engineering 4: 773; Kolbinger et al, 1993, Protein Engineering 6: 971.
  • Humanized monoclonal antibodies against CLASP-2 peptides can also be produced using transgenic animals having elements of a human immune system (see, e.g., U.S. Patent Nos. 5,569,825; 5,545,806; 5,693,762; 5,693,761; and 5,7124,350).
  • an anti-CLASP-2 polypeptide monoclonal or polyclonal antiserum is produced that is specifically immvmoreactive with a particular CLASP-2 polypeptide and is selected to have low cross-reactivity against other molecules (e.g., other CLASP polypeptides) and any such cross-reactivity is removed by immunoabsorbtion prior to use in the immunoassay.
  • polyclonal antibodies raised to hCLASP-2A as shown in SEQ ID NO: 1, or splice variants, or immunogenic portions thereof, can be selected to obtain only those polyclonal or monoclonal antibodies that are specifically immunoreactive with the target protein not with other proteins. This selection may be achieved by subtracting out antibodies that cross-react with molecules.
  • a variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein.
  • solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).
  • a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background.
  • antibodies that cross-react with a selected set of polypeptides may be prepared.
  • Antibody fragments which contain specific binding sites of V can be generated by known techniques.
  • fragments include, but are not limited to, the F(ab') 2 fragments which can be produced by pepsin digestion ofthe antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges ofthe F(ab') 2 fragments.
  • Fab expression libraries can be constructed (Huse et al, 1989, Science, 246: 1275-1281 ) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity to CLASP-2.
  • Anti-CLASP-2 antibodies can also be used to identify, isolate, inhibit or eliminate CLASP-2-expressing cells.
  • the present invention includes a method of identifying an abnormal T cell profile of an immunocompromised subject relative to the T cell profile of a non-immunocompromised subject.
  • the method includes (i) sorting a sample of peripheral blood mononuclear cells (PBMC) isolated from the immunocompromised subject into sets of T cell types, (ii) determining the ratio of CLASP-2 + cells relative to the total number of cells (CLASP-2 + : total) in each set, and identifying an abnormal T cell profile in the immunocompromised subject by comparing the CLASP-2 + : total ratios of sets from the immunocompromised subject with the CLASP-2 + : total ratios of analogous sets from a non-immunocompromised subject.
  • PBMC peripheral blood mononuclear cells
  • anti-CLASP-2 antibodies can be used for detection of hCLASP-2 protein in assays such as fluorescent activated cell sorting (FACS), ELISA, fluorescent or electron immunomicroscopy, Western blots, gel shift analyses.
  • FACS fluorescent activated cell sorting
  • ELISA ELISA
  • fluorescent or electron immunomicroscopy Western blots
  • gel shift analyses Western blots
  • CLASP-2 expression in various cells, localization within cells, interactions with other proteins, and differentiation between CLASP-2 isoform expression can be determined by use of the techniques listed herein.
  • the invention provides methods for identifying compounds or agents that modulate ( . e. , inhibit or enhance) CLASP-2 expression or activity.
  • CLASP-2 expression or activity modulators are useful for treatment of disorders characterized by (or associated with) aberrant or abnormal CLASP-2 expression or activity.
  • Aberrant expression of CLASP-2 mRNA or protein means expression in lymphocytes (e.g., T lymphocytes or B lymphocytes) or other CLASP-2 expressing cells of at least 2-fold, preferably at least 5-fold greater than expression in control lymphocytes obtained from a healthy subject.
  • the CLASP-2 expression assays can include the steps of contacting a cell expressing CLASP-2 with a compound or agent and assaying CLASP-2 expression.
  • CLASP- 2 polypeptide expression is easily measured by ELI S A using anti-CLASP-2 antibodies of the invention.
  • CLASP-2 mRNA expression (including expression of specific species or splice variants of CLASP-2) can be measured by quantitative Northern analysis or quantitative PCR.
  • CLASP-2 activities include, for example, the CLASP-2 polypeptide binding to PDZ-domain containing molecules and CLASP-2 polypeptide involvement in signal transduction (e.g., leading to T cell activation).
  • Compounds or agents that modulate the interaction of a CLASP-2 polypeptide and a target molecule, modulate CLASP-2 nucleic acid expression, or modulate CLASP-2 polypeptide activity are all contemplated by the methods ofthe present invention.
  • Test compounds include, for example, 1) peptides (e.g., soluble peptides, including Ig-tailed fusion peptides and members of random peptide libraries (see, e.g., Lam, K. S. et al, 1991, Nature 354: 82-84; Houghten, R. et al, 1991, Nature 354: 84-86) and combinatorial chemistry-derived molecular libraries made of D- and/or L-configuration amino acids; 2) phosphopeptides (e.g., members of random and partially degenerate, directed phosphopeptide libraries, see, e.g., Songyang, Z. et al.
  • peptides e.g., soluble peptides, including Ig-tailed fusion peptides and members of random peptide libraries (see, e.g., Lam, K. S. et al, 1991, Nature 354: 82-84; Houghten, R. et al, 1991
  • CLASP-2 antibodies as described above
  • small organic and inorganic molecules e.g., molecules obtained from combinatorial and natural product libraries
  • antisense RNA and DNA molecules and ribozymes as described above.
  • the CLASP modulators can be any of a large variety of compounds, both naturally occurring and synthetic, organic and inorganic, and including polymers (e.g., oligopeptides, polypeptides, oligonucleotides, and polynucleotides), small molecules, antibodies, sugars, fatty acids, nucleotides and nucleotide analogs, analogs of naturally occurring structures (e.g., peptide mimetics, nucleic acid analogs, and the like), and numerous other compounds.
  • polymers e.g., oligopeptides, polypeptides, oligonucleotides, and polynucleotides
  • small molecules e.g., antibodies, sugars, fatty acids, nucleotides and nucleotide analogs, analogs of naturally occurring structures (e.g., peptide mimetics, nucleic acid analogs, and the like), and numerous other compounds.
  • the invention provides assays for screening test compounds which bind to CLASP-2 polypeptides.
  • the assays can be recombinant cell based or cell-free assays. These assays can include the steps of combining a cell expressing a CLASP-2 polypeptide or a binding fragment thereof, and a compound or agent under conditions which allow binding ofthe compound or agent to the CLASP-2 polypeptide to form a complex. Complex formation can then be determined. The ability ofthe candidate compound or agent to bind to the CLASP-2 polypeptide or fragment thereof is indicated by the presence ofthe candidate compound in the complex. Formation of complexes between the CLASP-2 polypeptide and the candidate compound can be quantitated, for example, using standard immunoassays.
  • the invention provides screening assays to identify test compounds which modulate the interaction (and most likely CLASP-2 activity as well) between a CLASP-2 polypeptide and a molecule (target molecule with which the CLASP-2 polypeptide normally interacts.
  • these CLASP-2 target molecules can be tyrosine kinases (e.g., lyn, lck, fyn, ZAP-70m SyK, and CSK).
  • these CLASP-2 target molecules can be tyrosine phosphatases (e.g., EZRIN, SHP-1 , SHP-2 and PTP36).
  • these CLASP-2 target molecules can be adaptor proteins (e.g., NCK, CBL, SHC, LNK, SLP-76, HS 1 , SIT, VAV, GrB2, and BRDG1 ).
  • these CLASP-2 target molecules can be cytoskeletal associated proteins such as ankyrin, spectrin, talin, ezrin, tropomyosin, myosin, plectin, syndecans, paralemmin, Band 3 protein, cytoskeletal protein 4.1, and PTP36.
  • CLASP-2 target molecules can be members ofthe integrin family.
  • the assays are recombinant cell based or cell-free assay.
  • These assays can include the steps of combining a cell expressing a CLASP-2 polypeptide or a binding fragment thereof, a CLASP-2 target molecule (e.g., a CLASP-2 ligand) and a test compound, under conditions where but for the presence of the candidate compovmd, the CLASP-2 polypeptide or biologically active portion thereof binds to the target molecule. Detecting complex formation between the CLASP-2 polypeptide or the binding fragment thereof, the CLASP-2 target molecule and a test compound detecting the formation of a complex which includes the CLASP-2 polypeptide and the target molecule can be accomplished.
  • a CLASP-2 target molecule e.g., a CLASP-2 ligand
  • Detection of complex formation can include direct quantitation of the complex by, for example, measuring inductive effects, such as T cell activation, of the CLASP-2 polypeptide.
  • a significant change, such as a decrease, in the interaction of the CLASP-2 and target molecule (e.g., in the formation of a complex between the CLASP-2 and the target molecule) in the presence of a candidate compound (relative to what is detected in the absence of the candidate compound) is indicative of a modulation of the interaction between the CLASP-2 polypeptide and the target molecule.
  • Modulation ofthe formation of complexes between the CLASP-2 polypeptide and the target molecule can be quantitated using, for example, an immunoassay.
  • CLASP-2 binding to a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and microcentrifuge tubes.
  • a fusion polypeptide can be provided which adds a domain that allows the polypeptide to be bound to a matrix.
  • the complexes can be dissociated from the matrix, separated by SDS-PAGE, and the level of CLASP-2-binding polypeptide found in the bead fraction quantitated from the gel using standard electrophoretic techniques.
  • CLASP-2 or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated CLASP-2 molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, 111.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
  • antibodies reactive with CLASP-2 but which do not interfere with binding of the polypeptide to its target molecule can be derivatized to the wells of the plate, and CLASP-2 trapped in the wells by antibody conjugation.
  • preparations of a CLASP-2 -binding polypeptide and a candidate compound are incubated in the CLASP-2 -presenting wells of the plate, and the amount of complex trapped in the well can be quantitated.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies reactive with the CLASP-2 target molecule, or which are reactive with CLASP-2 polypeptide and compete with the target molecule; as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the target molecule.
  • One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant DNA molecules expressing the CLASP-2, e.g., the protein having the sequence of SEQ ID NO: 2.
  • Such cells either in viable or fixed form, can be used for standard ligand/receptor binding assays (see, e.g., Parce et al (1989) Science 246: 243-247; and Owicki et al (1990) Proc. Natl Acad. Sci. U.S.A.87: 4007-4011 , which describe sensitive methods to detect cellular responses.
  • a test compound, often labeled can be assayed for binding or for competition with another ligand for binding.
  • the invention provides a method for identifying a compound (e.g., a screening assay) capable of use in the treatment of a disorder characterized by (or associated with) aberrant or abnormal CLASP-2 nucleic acid expression or CLASP-2 polypeptide activity.
  • This method typically includes the step of assaying the ability of the compound or agent to modulate the expression ofthe CLASP-2 nucleic acid or the activity of the CLASP-2 polypeptide thereby identifying a compound for treating a disorder characterized by aberrant or abnormal CLASP-2 nucleic acid expression or CLASP-2 polypeptide activity.
  • Methods for assaying the ability ofthe compound or agent to modulate the expression of the CLASP-2 nucleic acid or activity ofthe CLASP-2 polypeptide are typically cell-based assays. For example, cells which are sensitive to ligands which transduce signals via a pathway involving CLASP-2 can be induced to overexpress a CLASP-2 polypeptide in the presence and absence of a candidate compound. Candidate compounds which produce a change in CLASP-2-dependent responses can be identified. In one embodiment, expression ofthe CLASP-2 nucleic acid or activity of a CLASP-2 polypeptide is modulated in cells and the effects of candidate compounds on the readout of interest (such as T cell activation) are measured. For example, the expression of genes which are up- or down-regulated in response to a CLASP-2-dependent signal cascade can be assayed.
  • modulators of CLASP-2 expression can be identified in a method where a cell is contacted with a candidate compound and the expression of CLASP-2 mRNA or polypeptide in the cell is determined. The level of expression of CLASP-2 mRNA or polypeptide in the presence of the candidate compound is compared to the level of expression of CLASP-2 mRNA or polypeptide in the absence ofthe candidate compound. The candidate compound can then be identified as a modulator of CLASP-2 nucleic acid expression based on this comparison. For example, when expression of CLASP-2 mRNA or polypeptide is greater in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of CLASP-2 nucleic acid expression.
  • the candidate compound when CLASP-2 nucleic acid expression is less in the presence ofthe candidate compound than in its absence, the candidate compound is identified as an inhibitor of CLASP-2 nucleic acid expression.
  • the level of CLASP-2 nucleic acid expression in the cells can be determined by methods described herein for detecting CLASP-2 mRNA or polypeptide. Modulators of CLASP-2 polypeptide activity and CLASP-2 nucleic acid expression identified according to these drug screening assays can be used to treat, for example, immune disorders.
  • These methods of treatment include the steps of administering the modulators of CLASP-2 polypeptide activity or nucleic acid expression, e.g., in a pharmaceutical composition as described in ⁇ 5.10.1 below, to a subject in need of such treatment, e.g., a subject with a disorder described herein.
  • CLASP-2 is expressed in lymphocytes and, as noted supra, play a role in regulating T cell and B cell interactions, thus making CLASP-2 activity (e.g., CLASP-2 binding of regulatory proteins) a target for diagnostic and treatment of immune disorders and for modulation of immvme function (e.g., T cell activation). Additionally, since CLASP-2 contains domains capable of transducing an intracellular signal, cell surface CLASP-2 can be triggered by an anti- CLASP-2 antibody or soluble CLASP-2 or a fragment thereof in order to enhance the activation state of a lymphocyte.
  • CLASP-2 activity e.g., CLASP-2 binding of regulatory proteins
  • immvme function e.g., T cell activation
  • a CLASP-2 polypeptide, a fragment thereof, anti-CLASP-2 antibody, CLASP-2 polynucleotide (e.g., antisense or ribozyme), or small molecule agonists or antagonists can be administered to a subject er se or in the form of a pharmaceutical or therapeutic composition.
  • Pharmaceutical compositions comprising the proteins of the invention can be manufactured by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or ly ophilizing processes.
  • compositions can be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries which facilitate processing of the protein or active peptides into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • the CLASP-2 protein or fragment (encoding a functional domain of CLASP-2) can be introduced into the cell as a fusion protein tied to a transporter protein derived from homeoprotein transcription factors such as ANTP.
  • the CLASP-2 protein or fragment (encoding a functional domain of CLASP-2) can be introduced into the cell as a fusion protein tied to other transcription factors such as the HIV Tat protein and the he ⁇ es simplex virus type 1 (HSV-1) VP22 protein.
  • the CLASP-2 protein or fragment (encoding a functional domain of CLASP-2) can be introduced into the cell as a fusion protein tied to peptides derived from signal- sequences present in several proteins such as HIV-1 gp41.
  • there are several synthetic and/or chemeric cell-penetrating peptides such as transportan and Amphiphiloc model peptide (Lindgren, M.; et al, 2000, Trends Pharmacol Sci.21 : 99-103) that can be used.
  • the CLASP-2 protein or fragment can be introduced by using anti-DNA antibodies (see, e.g., Zack, D. J., et al, 1996, J. Immunol. 157: 2082-8
  • the proteins ofthe invention can be formulated as solutions, gels, ointments, creams, suspensions, and the like, as are well-known in the art.
  • Systemic formulations include those designed for administration by injection, e.g., subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal injection, as well as those designed for transdermal, transmucosal, oral or pulmonary administration.
  • the proteins of the invention can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
  • physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
  • the solution can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the proteins can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art.
  • compositions can be readily formulated by combining the proteins with pharmaceutically acceptable carriers well known in the art.
  • pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the proteins to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
  • suitable excipients include fillers such as sugars, such as lactose, sucrose, mannitol and sorbitol; cellulose preparations such as maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose,and/or polyvinylpyrrolidone (PVP); granulating agents; and binding agents.
  • disintegrating agents can be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • solid dosage forms can be sugar-coated or enteric-coated using standard techniques.
  • suitable carriers, excipients or diluents include water, glycols, oils, alcohols, and the like. Additionally, flavoring agents, preservatives, coloring agents and the like can be added.
  • the proteins can take the form of tablets, lozenges, and the like, formulated in conventional manner.
  • the proteins for use according to the present invention are conveniently delivered in the form of an aerosol spray from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit can be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • the proteins can also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
  • the proteins can also be formulated as a depot preparation. Such long acting formulations can be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
  • the proteins can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • Liposomes and emulsions are well known examples of delivery vehicles that can be used to deliver the proteins or peptides of the invention.
  • Certain organic solvents such as dimethylsulfoxide also can be employed, although usually at the cost of greater toxicity.
  • the proteins can be delivered using a sustained-release system, such as semipermeable matrices of solid polymers containing the therapeutic agent.
  • sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules can, depending on their chemical nature, release the proteins for a few weeks up to over 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization can be employed.
  • proteins and peptides ofthe invention can contain charged side chains or termini, they can be included in any ofthe above-described formulations as the free acids or bases or as pharmaceutically acceptable salts.
  • Pharmaceutically acceptable salts are those salts which substantially retain the biologic activity of the free bases and which are prepared by reaction with inorganic acids. Pharmaceutical salts tend to be more soluble in aqueous and other protic solvents than are the conesponding free base forms.
  • CLASP-2 polypeptides, CLASP-2 fragments and anti-CLASP-2 antibodies will generally be used in an amount effective to achieve the intended pu ⁇ ose.
  • the proteins ofthe invention, or pharmaceutical compositions thereof are administered or applied in a therapeutically effective amount.
  • therapeutically effective amount is meant an amovmt effective ameliorate or prevent the symptoms, or prolong the survival of, the patient being treated. Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.
  • a therapeutically effective dose can be estimated initially from in vitro assays.
  • a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC 50 as determined in cell culture ( . e. , the concentration of test compound that inhibits 50% of CLASP-2 binding interactions). Such information can be used to more accurately determine useful doses in humans.
  • Initial dosages can also be estimated from in vivo data, e.g., animal models, using techniques that are well known in the art. One having ordinary skill in the art could readily optimize administration to humans based on animal data. Dosage amount and interval can be adjusted individually to provide plasma levels of the proteins which are sufficient to maintain therapeutic effect. Usual patient dosages for administration by injection range from about 0.1 to 5 mg/kg/day, preferably from about 0.5 to 1 mg/kg/day. Therapeutically effective serum levels can be achieved by administering multiple doses each day.
  • the effective local concentration ofthe proteins can not be related to plasma concentration.
  • One having skill in the art will be able to optimize therapeutically effective local dosages without undue experimentation.
  • the amount of CLASP-2 administered will, of course, be dependent on the subject being treated, on the subject's weight, the severity ofthe affliction, the manner of administration and the judgment ofthe prescribing physician.
  • the therapy can be repeated intermittently while symptoms detectable or even when they are not detectable.
  • the therapy can be provided alone or in combination with other drugs.
  • the drugs that can be used in combination with CLASP-2 or fragments thereof include, but are not limited to, steroid and non-steroid immunosuppressive agents.
  • a therapeutically effective dose ofthe proteins described herein will provide therapeutic benefit without causing substantial toxicity.
  • Toxicity of the proteins described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD 50 (the dose lethal to 50% ofthe population) or the LD 100 (the dose lethal to 100% ofthe population). The dose ratio between toxic and therapeutic effect is the therapeutic index.
  • the data obtained from these cell culture assays and animal studies can be used in formulating a dosage range that is not toxic for use in human.
  • the dosage ofthe proteins described herein lies preferably within a range of circulating concentrations that include the effective dose with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view ofthe patient's condition. (See, e.g., Fingl et al, 1975, In: The Pharmacological Basis of Therapeutics. Ch.l, p.l). 5.11. Binding Assays
  • CLASP-2 polypeptides can be used to screen for molecules that bind to CLASP-2 or for molecules to which CLASP-2 binds.
  • the binding of CLASP-2 by the molecule can activate (agonist), increase, inhibit (antagonist), or decrease activity of the CLASP-2 or the molecule bound.
  • Examples of such molecules include antibodies, oligonucleotides, proteins (e.g., receptors), or small molecules.
  • the molecule is closely related to the natural ligand of CLASP-2, e.g., a fragment ofthe ligand, or a natural substrate, a ligand, a structural or functional mimetic.
  • the molecule can be closely -related to the natural receptor to which CLASP-2 binds, or at least, a fragment ofthe receptor capable of being bound by CLASP-2 (e.g., active site). In either case, the molecule can be rationally designed using known techniques.
  • the screening for these molecules involves producing appropriate cells which express CLASP-2, either as a secreted protein or on the cell membrane.
  • Preferred cells include cells from mammals, yeast, Drosophila, or E. coli.
  • Cells expressing CLASP-2 (or cell membrane containing the expressed polypeptide) are then preferably contacted with a test compound potentially containing the molecule to observe binding, stimulation, or inhibition of activity of either CLASP-2 or the molecule.
  • the assay can simply test binding of a candidate compound to CLASP-2, where binding is detected by a label, or in an assay involving competition with a labeled competitor. Further, the assay can test whether the candidate compound results in a signal generated by binding to CLASP-2.
  • the assay can be carried out using cell-free preparations, polypeptide affixed to a solid support, chemical libraries, or natural product mixtures.
  • the assay can also simply comprise the steps of mixing a candidate compound with a solution containing CLASP-2, measuring CLASP-2 activity or binding, and comparing the CLASP-2 activity or binding to a standard.
  • an ELISA assay can measure CLASP-2 level or activity in a sample (e.g., biological sample) using a monoclonal or polyclonal antibody.
  • the antibody can measure CLASP-2 level or activity by either binding, directly or indirectly, to CLASP-2 or by competing with CLASP-2 for a substrate.
  • the CLASP-2 polypeptides, or fragments thereof can be used as "bait proteins" in a two-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al, 1993, Cell 72: 223-232; Madura et al, 1993, J. Biol. Chem.
  • CLASP-2-binding proteins or "CLASP-2-bp"
  • CLASP-2-binding proteins are also likely to be involved in the propagation of signals by the CLASP-2 polypeptides as, for example, upstream or downstream elements ofthe CLASP-2 pathway.
  • All of these above assays can be used as diagnostic or prognostic markers.
  • the molecules discovered using these assays can be used to treat disease or to bring about a particular result in a patient by activating or inhibiting the CLASP-2 molecule.
  • the assays can discover agents which can inhibit or enhance the production of CLASP-2 from suitably manipulated cells or tissues.
  • the invention includes a method of identifying compounds or agents that bind to CLASP-2 polypeptides comprising the steps of: (a) contacting a CLASP-2 polypeptide with a compound or agent under conditions which allow binding of the compound to the CLASP-2 polypeptide to form a complex and (b) determining if binding has occurred.
  • the invention includes a method of identifying agonists or antagonists comprising the steps of: (a) incubating a candidate compound with CLASP-2, (b) assaying a biological activity, and (b) determining if a biological activity of CLASP-2 has been altered.
  • the polynucleotides, polypeptides, polypeptide homologues, modulators, and antibodies described herein can be used in one or more ofthe following methods: a) drug screening assays; b) diagnostic assays particularly in disease identification, allelic screening and pharmocogenetic testing; and c) pharmacogenomics.
  • a CLASP-2 polypeptide of the invention has one or more of the activities described herein and can thus be used to, for example, modulate an immune response in an immvme cell, for example by binding to a CLASP-2 binding partner making it unavailable for binding to the naturally present CLASP- 2 polypeptide.
  • these CLASP-2 binding partners can be tyrosine kinases (e.g., lyn, lck, fyn, ZAP-70m SyK, and CSK).
  • these CLASP-2 binding partners can be tyrosine phosphatases (e.g., EZRIN, SHP- 1 , SHP-2 and PTP36).
  • these CLASP-2 target molecules can be adaptor proteins (e.g., NCK, CBL, SHC, LNK, SLP-76, HSl, SIT, VAV, GrB2, and BRDG1.
  • CLASP-2 binding partners can be cytoskeletal associated proteins such as ankyrin, spectrin, talin, ezrin, tropomyosin, myosin, plectin, syndecans, paralemmin, Band 3 protein, cytoskeletal protein 4.1, and PTP36.
  • CLASP-2 binding partners can be members of the integrin family.
  • the isolated nucleic acid molecules ofthe invention can be used to express CLASP-2 polypeptide (e.g., via a recombinant expression vector in a host cell or in gene therapy applications), to detect CLASP-2 mRNA (e.g., in a biological sample) or a naturally occurring or recombinantly generated genetic mutation in an CLASP-2 gene, and to modulate CLASP-2 activity, as described further below.
  • the CLASP-2 polypeptides can be used to screen drugs or compounds which modulate CLASP-2 polypeptide activity as well as to treat disorders characterized by insufficient production of CLASP-2 polypeptide or production of CLASP-2 polypeptide forms which have decreased activity compared to wild type CLASP-2.
  • the anti-CLASP-2 antibodies of the invention can be used to detect and isolate an CLASP-2 polypeptide, particularly fragments of CLASP-2 present in a biological sample, and to modulate CLASP-2 polypeptide activity.
  • the invention further provides a method for detecting the presence of CLASP-2, or fragment thereof, in a biological sample.
  • the biological sample contains lymphocytes (e.g., from blood).
  • the method involves contacting the biological sample with a compound or an agent capable of detecting CLASP-2 polypeptide or mRNA such that the presence of CLASP-2 is detected in the biological sample.
  • a preferred agent for detecting CLASP-2 mRNA is a directly or indirectly labeled nucleic acid probe capable of hybridizing to CLASP-2 mRNA.
  • the nucleic acid probe can be, for example, the full-length CLASP-2 cDNA of SEQ ID NO: 1 , or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to CLASP-2 mRNA.
  • a preferred agent for detecting CLASP-2 polypeptide is a directly or indirectly labeled antibody capable of binding to a CLASP-2 polypeptide.
  • Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab) 2 ) can be used.
  • the term "directly or indirectly", with regard to the probe or antibody, is intended to encompass direct labeling ofthe probe or antibody by coupling ( . e. , physically linking) a detectable substance to the probe or antibody, as well as indirect labeling ofthe probe or antibody by reactivity with another reagent that is directly labeled.
  • Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.
  • the detection method ofthe invention can be used to detect CLASP-2 mRNA or polypeptide in a biological sample in vitro as well as in vivo.
  • in vitro techniques for detection of CLASP-2 mRNA include Northern hybridizations and in situ hybridizations.
  • In vitro techniques for detection of CLASP-2 polypeptide include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence.
  • CLASP-2 polypeptide can be detected in vivo in a subject by introducing into the subject a labeled anti-CLASP-2 antibody.
  • the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
  • Particularly useful are methods which detect the allelic variant of CLASP-2 expressed in a subject and methods which detect fragments of an CLASP-2 polypeptide in a sample.
  • kits for detecting the presence of CLASP-2 in a biological sample can comprise a directly or indirectly labeled compound or agent capable of detecting CLASP-2 polypeptide or mRNA in a biological sample; means for determining the amount of CLASP-2 in the sample; and means for comparing the amount of CLASP-2 in the sample with a standard.
  • the compound or agent can be packaged in a suitable container.
  • the kit can further comprise instructions for using the kit to detect CLASP-2 mRNA or polypeptide.
  • the methods ofthe invention can also be used to detect naturally occurring genetic mutations in an CLASP-2 gene, thereby determining if a subject with the mutated gene is at risk for a disorder characterized by aberrant or abnormal CLASP-2 nucleic acid expression or CLASP-2 polypeptide activity as described herein.
  • the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic mutation characterized by at least one of an alteration affecting the integrity of a gene encoding an CLASP-2 polypeptide, or the misexpression ofthe CLASP-2 gene.
  • CLASP-2 mediates a variety of cell functions in lymphocytes and other cells.
  • assays are useful for detecting or quantitating CLASP-2 activity, or for identifying agents (including polynucleotides, polypeptides, and antibodies of the invention) that modulate CLASP-2 activity (i.e., biological activity, e.g., binding) or expression.
  • agents include polynucleotides, polypeptides, and antibodies of the invention
  • Such agents are useful for treatment of diseases and conditions associated with aberrant CLASP-2 expression or activity.
  • other CLASP-2 -mediated activities can be identified by those of skill using routine assays, such as those described below.
  • Exemplary assays for CLASP-2 function include assays for modulation of an in vitro or in vivo cell response (e.g., an immune response such as lymphocyte activation, antibody production, inflammation) by detecting a change in an activity (e.g., cytokine production, calcium flux, tyrosine phosphorylation, regulation of early activation markers, cell metabolism, proliferation, and the like, as described below) of cells in vitro or in vivo.
  • the cells are lymphocytes.
  • recombinant CLASP-2 protein, peptides, or antibodies corresponding to the CLASP-2 extracellular domain can be mixed directly with T and B cells.
  • Cytokine production by these cells can then be measured and the degree of modulation of the immune response quantitated.
  • antigen-presenting B cells are mixed with untransfected T cells or T cells that have been transfected with CLASP-2 isoforms. Cytokine production (or calcium flux or other assays in ⁇ 5.14.3) is be measured at the appropriate time to determine the effect of CLASP-2 on such an immune response.
  • B cells transfected with CLASP-2 constructs are tested for their ability to stimulate a T cell to generate an immune response.
  • Transfected constructs in any of these cases could encode, for example, full or partial length CLASP-2 sequences, or antisense constructs to inhibit translation of endogenous CLASP-2 gene. Any of the examples described herein can be used to stimulate an immune response in the presence or absence of CLASP-2 isoforms or antibodies and assay the resulting effects on immune response by the methods listed in ⁇ 5.14.3.
  • an effect of an agent on immune cells is detected using an in vitro assay.
  • the degree of an immune response can be measured or quantitated by a number of standard assays including those described below.
  • human peripheral blood mononuclear cells PBMC
  • human T cell clones e.g., Jurkat E6, ATCC TIB-152
  • EBV-transformed B cell clones e.g., 9D10, ATCC CRL-8752
  • antigen-specific T cell clones or lines can be used to examine immune responses in vitro. Activation, enhanced activation or inhibition of activation of these cells or cell lines can be used for the evaluation of potential CLASP therapeutics.
  • Standard methods by which hematopoietic cells are stimulated to undergo activation characteristic of an immune response are, for example:
  • A) Antigen specific stimulation of immune responses Either pre-immunized or naive mouse splenocytes can be generated by standard procedures. In addition, antigen-specific T cell clones and hybridomas (e.g., MBP-specific), and numerous B cell lymphoma cell lines (e.g., CH27), have been previously characterized are available for the assays discussed below. Antigen specific splenocytes or B-cells can be mixed with specific T-cells in the presence of antigen to generate an immune response. This can be performed in the presence or absence of CLASP-2 to assay whether CLASP-2 modulates the immune response as measured by any ofthe assays in section 5.14.2.
  • MBP-specific antigen-specific T cell clones and hybridomas
  • B cell lymphoma cell lines e.g., CH27
  • TCR cross-linking T cell receptor
  • receptor activation molecules e.g., TCR, CD3, or CD2
  • co-stimulator molecules for example anti-CD28
  • activation molecules e.g., TCR, CD3, or CD2
  • co-stimulator molecules for example anti-CD28
  • PHA phytohemagglutinin
  • pharmacological agents that activate protein kinase C e.g., phorbol esters
  • increase cytoplasmic Ca 2+ e.g., ionomycin
  • Non-specific B cell activation 1) application of antibodies against cell surface molecules such as IgM, CD20, or CD21. 2) Lipopolysaccharide (LPS), phorbol esters, calcium ionophores and ionomycin can also be used to by-pass receptor triggering.
  • LPS Lipopolysaccharide
  • phorbol esters phorbol esters
  • calcium ionophores phorbol esters
  • ionomycin can also be used to by-pass receptor triggering.
  • MLR Mixed lymphocyte reaction
  • a standard approach is to generate tetanus toxin-specific T cells from a donor that has recently been boosted with tetanus toxin.
  • Major histocompatability complex- (MHC-) matched antigen presenting cells and a source of tetanus toxin are used to maintain antigen specificity of the cell line or T cell clone (Lanzavecchia, A., et al, 1983, Eur. J. Immun. 13: 733-738).
  • MHC- Major histocompatability complex-
  • the anticipated mechanism of action of a CLASP-2 polypeptide or polynucleotide should define the appropriate assay to use to investigate its potential enhancement or inhibition of lymphocyte activation.
  • soluble proteins containing the CLASP extracellular domain may interfere with the interaction between T cells and antigen presenting cells. Such interaction plays a role in the MLR and in antigen- specific T cell activation, but not in non-specific T or B cell activation.
  • the assays described above have the advantage of several possible detection methods for quantitation.
  • an effect of an agent on immune cells is detected using an in vivo assay.
  • the degree of an immune response can be measured or quantitated by a number of standard assays including those described below.
  • a standard animal model for graft versus host rejection is ectopic heart transplantation (Fulmer et al, 1963, Am. J. Anat. 113: 273-281).
  • This method involves using BALB/C mice (either sex, and range from 1-9 months) for transplanting cardiac tissue into a surgically-created pocket on the dorsum for both ears made by slitting the skin over the auricular artery at the base ofthe ear. Small curved forceps are forced into the slit, bluntly dissecting between the skin and the cartilage plate. Donor tissue is eased into the base ofthe pocket near the distal edge of the ear. The auricular artery is used to seal off the opening of the pocket.
  • CIA Collagen Induced Arthritis
  • DBA/a mice can be used as an assay for the in vivo relevance of CLASP-2 in vitro testing potential immvme therapeutics. In vivo experiments will be performed to examine the ability of potential therapeutics to prevent CIA. We will use 3-5 mice per group to statistically justify our results.
  • mice will be immunized with three different concentrations of CII 50, 200, and 400 ⁇ g per animal (Nabozny et al, 1996, J. Exp. Med., 183: 27-37).
  • animals can be immunized with an appropriate concentration of CH, determined as described above.
  • One half of a 1:1 ratio of antigen:CFA can be injected at the base ofthe tail and the remainder equally divided in each hind footpad.
  • Mice can be carefully monitored every day for the onset and progression of CIA thoughout the experiment until its termination 12 weeks post-immunization with CII.
  • the pieces of heart transplanted can be approximately 3 X 3 mm in size. The severity of arthritis can be assessed following standard procedures known to one of skill in the art.
  • Tyrosine phosphorylation of early response proteins such as HSl, PLC-r, ZAP-76, and Vav is an early biochemical event following T cell activation.
  • the tyrosine phosphorylated proteins can be detected by Western blot using antibodies against phosphorylated tyrosine residues. Tyrosine phosphorylation of these early response proteins can be used as a standard assay for T cell activation (J. Biol. Chem., 1997, 272(23): 14562- 14570). Any change in the phosphorylation pattern of these or related proteins when immune responses are generated in the presence of CLASP-2 is indicative of a CLASP-2 modulation of this response.
  • B Intracellular Calcium Flux
  • MTS [5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazolyl)- 3 (4-sulfopheny l)tetrazolium,inner salt] is a colorimetric method for determining the number of viable cells in proliferation or cytotoxicity assays (Barltrop, J.A. et al, 1991, Bioorg. & Med. Chem. Lett. 1 : 611). 1-5 days after lymphocyte activation, MTS tetrazolium compound, Owen's reagent, is bioreduced by cells into a colored formazan product that is soluble in tissue culture media. Color intensity is read at 490 nm minus 650 nm using a microplate reader.
  • BrdU-pulsed cells are labeled with an enzyme-conjugated anti-BrdU antibody (Gratzner, H.G., 1982, Science 218: 474-475.).
  • a colorimetric, soluble substrate is used to visualize proliferating cells that have inco ⁇ orated BrdU. Reaction is stopped with sulfuric acid and plate is read at 450 nm using a microplate reader. Any statistically significant increase or decrease in color intensity of CLASP-2- treated sample, as compared to control sample (no treatment), suggest an effect of CLASP-2 on biological function.
  • apoptosis is an early event in a cascade of catabolic reactions leading to cell death. A lose in the integrity ofthe cell membrane allows for the binding of fluorescently conjugated phosphatidylserine. Stained cells can be measured by fluorescence microscopy and flow cytometry (Vermes, I., 1995, J. Immunol. Methods. 180: 39-52). In one embodiment, any statistically significant increase or decrease in apoptotic cell number of CLASP-2-treated sample, as compared to control sample (no treatment), suggest an effect of CLASP-2 on biological function. For evaluating apoptosis in situ, assays for evaluating cell death in tissue samples can also be used in vivo studies.
  • cytokine production Cell supematants harvested after cell stimulation for 16-48 hrs are stored at -80°C until assayed or directly tested for cytokine production. Multiple cytokine assays can be performed on each sample. IL-2, IL-3, IFN- and other cytokine ELISA Assays are available for mouse, rat, and human (Endogen, Inc. and BioSource). Cytokine production is measured using a standard two-antibody sandwich ELISA protocol as described by the manufacturer. The presence of horseradish peroxidase is detected with 3, 3'5, 5' tertamethyl benziidine (TMB) substrate and the reaction is stopped with sulfuric acid. The absorbency at 450 nm is measured using a microplate reader. Any statistically significant increase or decrease in color intensity of CLASP-2-treated sample, as compared to control sample (no treatment), suggest an effect of CLASP-2 on biological function.
  • TMB 3, 3'5, 5' tertamethyl benziidine
  • T cell activation requires the import of nuclear factor of activated T cells (NFAT) to the nucleus.
  • NFAT nuclear factor of activated T cells
  • This translocation of NF-AT can be visualized by immunostaining with anti- NF-AT antibody (Cell 1998, 93: 851-861). Therefore, NF-AT nuclear translocation has been used to assay T cell activation.
  • NF-AT/luciferase reporter assays have been used as a standard measurement of T cell activation (MCB 1996, 12: 7151-7160).
  • C(II) ELISA for collagen type II (C ⁇ I)-specific antibodies see above for related in vivo assay
  • C(II) titers from serum of animals immunized with CLASP-2 can be measured and compared.
  • Both TH1 -dependent IgG2a and TH2-dependent IgGl and IgE CII- specific antibody isotypes will be measured by ELISA.
  • Mouse blood will be obtained by orbital bleed one and two months post-immunization with CII. Samples will be allowed to coagulate and centrifuge to obtain sera, and stored at -80°C until assayed by ELISA. Coat ELISA plates with CII and dilute sera. HRP conjugated goat, isotype specific antibody.
  • ELISPOT enzyme-linked immunospot
  • FC Rl is the high affinity receptor for IgE complexes, which when coupled to biotin can be cross-linked with avidin to induce degranulation and histamine release of lymphocytes.
  • Histamine is quantified with an enzyme immunoassay competition assay (Immunotech). Histamine release.
  • Immunotech enzyme immunoassay competition assay
  • Determining the tissue distribution of lymphocytes following a pathological disorder can aid in identifying specific organ, tissue and lymphocyte involved in an immune response.
  • Cellular phenotyping of lymphocyte trafficking is generally performed with by flow cytometry and Immunocytochemistry.
  • L929 cells can be transfected with CLASP-2 and Neomycin. G418-resistant clones can be screened for CL ASP-expression with anti-CL ASP peptide-specific antibodies.
  • CLASP-expressing clones can then be used to test for homotypic and/or heterotypic calcium dependent cell adhesion using the "cell aggregation assay" described for cadherin molecules (Mvuphy-Erdosh, C. et al, 1995, J. Cell Biol. 129: 1379-1390). Any change in the levels of cellular aggregation when immune responses are generated in the presence of
  • CLASP-2 is indicative of a CLASP-2 modulation of this response.
  • hCLASP-2A 3' clone (AVC-PD1) ATCC accession number PTA-1563 hCL ASP-2A 5 ' clone (AVC-PD2) ATCC accession number PTA- 1562 hCLASP-2B clone (AVC-PD12) ATCC accession number
  • CLASP-2 was cloned in the following manner: an expressed sequence tag or EST clone (IMAGE clone 815795, derived from human germinal B cells) was identified based on a BLAST search of human GenBank human EST database using CLASP-1 sequences. IMAGE clone 815795 was sequenced completely. A polynucleotide probe prepared from 815975 sequence was labeled with 32 P-dCTP and used to screen human cDNA libraries including Jurkat (Stratagene) and Ramos B cell cDNA library (James Boulter, UCLA). The screening methods employed were as described in Maniatis et al, 1989, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, New York.
  • the PCR fragment was cloned using the pGEM-T system (Promega). The sequence corresponded to encompassing the 5' end of CLASP-2 including an additional 399 bp of 5' UTR.
  • Preliminary CLASP-2 nucleotide sequence is shown in FIG. 10.
  • the sequence ofthe CLASP-2 cDNA is shown in FIG. 1.
  • RNA samples were run over night on a 1.1% agarose gel containing 1.5% formaldehyde (both gel and running buffer were 20 mM sodium phosphate, pH 7.5). To visualize RNA after gel migration, approx. 0.5 ⁇ g ethidium bromide was added to each sample prior to the run together with RNA loading buffer. RNA in the gel was then visualized by 260nm wavelength light.
  • Probe HC2.2 encompasses to nucleotides 3920 to 4650 (731 bp long) of CLASP- 2A.
  • the HC2.2 probe was prepared using standard labeling kits and desalted using pasteur pipette G-50 Sephadex column in TEN (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, and 100 mM NaCl).
  • Hybridizations of 32 P dCTP labeled DNA probes to the membrane bound RNAs were carried out in CLONTECH EXPRESSHYB solution, at 68°C and for 1-2 hours. Blots were washed 2 times in 2x SSC 0.1% SDS for 10' each at 50°C and then twice in 0.2 x SSC 0.1% SDS for 10' each at 50°C, followed by a 5' wash in 2xSSC at 50°C. Exposure to KODAK BIOMAX MS film was carried out at minus 80°C using amplifying screens. Typical exposure times were 10 to 36 hours.
  • Genomic DNA was prepared from HeLa cells (ATCC #CCL-17) using the methods described by Sambrook, Fritsch and Maniatis (1989); DNA concentrations were determined by the 260nm light abso ⁇ tion of the DNA solution, and aliquots corresponding to 20 microgram ( ⁇ g) genomic DNA or 2 ⁇ g for BAC DNA were used for restriction enzyme digests with Eco Rl or HinD HI (genomic DNA) or Eco Rl and Pst I (BAC DNA). Digests were carried out in 150 microtiter volume for 4 hours at 37°C.
  • Digested DNA was ethanol precipitated and the pellet was resuspended in 20 microtiter deionized water prior to migration over a 1.2 % agarose gel at 35 V over night.
  • Running buffer was TAE, and the gel contained 0.1 ⁇ g ethidiumbromide/ml to visualize DNA.
  • DNA was visualized by 260 nm wavelength light.
  • the gel was then washed twice for 20' in denaturing buffer (0.5M NaCl, 0.4 N NaOH) and twice in neutralization buffer (1.5 M NaCl, 0.5 M TRIS pH 8.0).
  • DNA was transferred from the gel onto AMERSHAM HYBOND N membrane by capillary blotting in 20 x SSC for 5 hours.
  • the DNA was crosslinked to the membrane by UV light using a Stratagene Stratalinker.
  • Probe HC2.1 encompasses nucleotides 325 to 1126 (802 bp long) of CLASP-2A.
  • the HC2.1 probe was prepared using standard labeling kits and desalted using pasteur pipette G-50 Sephadex column in TEN (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, and 100 mM Nacl).
  • Hybridizations of P dCTP labeled DNA against DNA immobilized onto the membrane were carried out at 65°C overnight in modified CHURCH hybridization solution (7% SDS, 0.5 M sodiumphosphate, ImM EDTA).
  • Membranes were then exposed to KODAK BIOMAX MS film at minus 80°C.
  • genomic DNA southern analysis revealed two fragments (-4.5 kb and 1.85 kb) in the Eco Rl digested DNA but three fragments in BACs 4 and 6 DNA. The two major bands are identical in both genomic and BAC DNA (FIG. 7).
  • Genomic clones of human CLASP-2 were obtained using the Release I high density filters from Genome Systems Ine (cat # FBAC-4434). Two rounds of screening were completed. The first round of screening was carried out using a probe corresponding to nucleotides 3830 to 4558 ofthe human CLASP-2 cDNA by standard protocols specific by Genome Systems. This screen identified two genomic clones, referred to as AVC BAC4 and 7. A second round of screening using a probe that corresponded to nucleotides 1208 to 1604 of human CLASP-2 cDNA identified clone AVC BAC26. All the clones were partially sequenced to authenticate that they were indeed CLASP-2 genomic clones, to verify exon sequences, and to identify exon/intron boundaries.
  • Oligonucleotides for sequencing the BACs were based upon human CLASP-2 cDNA sequence. Sense and antisense sequencing oligonucleotides were designed along the length of the human CLASP-2 cDNA spaced approximately every 200 nucleotides to ensure a high density of coverage of the corresponding genomic regions. Sequencing reactions with primers and BAC DNA were carried out by standard PCR sequencing using Big Dye termination sequencing mix (ABI). Results from sequence reactions were analyzed using Sequencher software (Genecodes). The results are summarized in FIG. 6.
  • Amplified DNA sequences were digested with restriction enzymes for cloning in-frame into GST expression vectors. Fusion proteins were expressed by IPTG induction in DH5 ⁇ and purified according to instructions from Pharmacia using glutathione-Sepharose (Pharmacia). SDS-PAGE gel stained with Coomassie Blue showing induced and uninduced expression of the GST-CLASP-2-cyto construct is shown in FIG. 8. These recombinant proteins were expressed in DH5 ⁇ and purified according to instructions from Pharmacia using glutathione- Sepharose. Such recombinant proteins were used to generate antibodies (Josman laboratories) using a AVC Rapid Immunization Protocol.
  • the full length CLASP can easily be expressed from either the beginning of the hCLASP-2 sequence (in frame with nucleotide 2) or from the first or second methionine (nucleotide 278 or nucleotide 476, underlined in Figure 1) through to the stop codon (nucleotide 4058). Assuming that the GST moiety has a weight of 26 kD, the total predicted sizes are 180, 168, and 164.5 kD respectively. Alternatively, other bacterial expression systems such as 6CLASP HIS tags, Calmodulin binding protein, maltose binding protein can also be used in a similar manner.
  • hlgG fusions using the CD5gamma-l expression vector (kindly provided by B. Seed, Harvard University). Polypeptides were cloned into this vector in frame with a CD5 leader sequence that directs the fusion protein into the secretory pathway and in frame with a C- terminal hIgG(Fc) protein. This fusion can be secreted from cell lines such as 293 (Hsieh, J-C, 1999, Nature 398: 431-436).
  • Sense primers with hCLASP-2 sequences beginning at nucleotide 866 and antisense primers at nucleotide 1459 (EC12-IgG), nucleotide 2389 (ECC- IgG) and nucleotide 2857 (ECM-IgG) were used to amplify portions of the extracellular domain for insertion into this vector.
  • Recombinant vectors were purified by Maxiprep (Qiagen) and transfected into 293 EBNA- T cells (kindly provided by B. Seed, Harvard University) by calcium phosphate techniques (Sambrook and Maniatis).
  • Example 6B Intracellular fusions Similar methods have been used to construct fusions for expression of full length hCLASP-2 isoforms as well as truncated C-terminal forms in other cell lines such as Jurkat. Recombinant hCLASP-2 fragments were either isolated by digestion of cDNA clones or amplified by primers flanking specific regions (Please provide some specific regions). These can be cloned into expression vectors such as pBJl-neo (Mark Davis, Stanford University), Peakl2 (B. Seed, Harvard University), and pDsRedl-Nl (Clontech).
  • inhibition of CLASP-2 expression is examined using an in vitro cell-free expression system.
  • PS-ODNs antisense phosphorothioate oligonucleotides
  • a CLASP-2 transcription/expressionplasmid can be used according to standard methodology for in vitro transcription and translation of sense CLASP-2 RNA.
  • Coupled transcription-translation reactions can be performed with a reticulocyte lysate system (Promega TNTTM) according to standard conditions.
  • Each coupled transcription/translationreaction can include CLASP-2 RNA transcribed from the expression plasmid, and a test antisense polynucleotide at a range of standard test concentrations, as well as the luciferase transcription/translation internal control to normalize each reaction (see, e.g., Sambrook et al. , supra, Ausubel et al. , supra).
  • the translation reaction can also be performed with sense CLASP-2 RNA that is synthesized in vitro in a separate reaction and then added to the translation reaction. 35 S-Met is included in the reaction to label the translation products.
  • the negative control is performed without added PS-ODN or a sense PS-ODN.
  • the labeled translation products can be separated by gel electrophoresis and quantitated after exposing the gel to a phosphorimager screen.
  • the amovmt of CLASP-2 protein expressed in the presence of CLASP-2 specific PS-ODNs can be normalized to the co-expressed luciferase control.
  • RPMI 1640 medium BioWhitaker; DMEM/M199 medium, BioWhitaker; EMEM, BioWhitaker; Fetal Bovine Serum, Summit (stored frozen at -20°C, stored thawed at 4°C); Trypsin-EDTA, GIBCO (catalogue #25300-054) (stored frozen at -20°C, stored thawed 4°C; Isoton II (stored at RT); DMSO (stored at RT); oligonucleotides (see Table 1 and Figure 3, stored in solution at -20°C); PBS (Ca 2+ Mg 2+ free); TE; 10 mM Tris-HCL, pH 8.0; 1 mM EDTA.
  • Oligonucleotide nucleotides can dissolved in the appropriate amount of TE to make a concentrated stock solution (1 - 20 mM).
  • 1.1 x 10 5 cells total per well, 2 ml/well is added.
  • the amount of cells can be scaled up or down proportionally for 12-well, 100 mm, or 150 mm dishes.
  • 12-well dishes use 4.6 x 10 4 cells in 2 ml media; for 100 mm dishes use 6 x 10 5 cells in 10 ml media; for 150 mm dishes use 1.7 x 10 6 cells in 35 ml media.
  • An appropriate number of cells (as described in step 2 above) are collected, centrifuged and resuspended in media containing a range of ODN concentrations.
  • the cells are treated in single, duplicate, or triplicate wells. Control wells are treated with TE or sense ODNs diluted in media.
  • the suspension cultures are washed and resuspended daily with PS-ODN media.
  • Suspension cultures are grown for 2-4 days. Cells are washed with PBS and density measured using a Coulter counter or a hemocytometer. If necessary, the cells are replated at 1.1 x 10 5 cells per well, 2 ml media per well, and fed with PS-ODN as described above. Samples ofthe cells can also be harvested for analysis to determine the effects of CLASP-2 antisense ODNs. Samples are harvested for RNA and analyzed by either Northern analysis or RT-PCR for the presence of CLASP-2 mRNA. Functional consequences of CLASP-2 antisense ODNs can be analyzed by measuring the ability of Jurkat and 9D 10 cells to be activated.
  • Jurkat cells are activated by exposure to anti-CD3 and anti-CD28 crosslinking antibodies, and 9D10 cells are activated by exposure to anti-IgM crosslinking antibody or P. aeruginosalipopolysaccharide.A hallmark of activation, calcium influx, can be measured by flow cytometry. Additionally, ELISA assays can be used to measure Interleukin-2 production from Jurkat cells and secreted IgM can be measured using standard assays from 9D10.
  • the GST-PDZ fusion proteins are made following standard procedures.
  • An exemplary GST-PDZ fusion protein was constructed as follows: A 572 bp fragment encoding two PDZ domains of the human neDLG gene (Genbank Accession No. U49089.1) was amplified from total Jurkat RNA by RT-PCR according to standard protocols (Sambrook, Fritsch, and Maniatis, 1989, Molecular Cloning - A Laboratory Manual. Cold Spring Harbor Press.) using primers flanked by restriction endonuclease sites for cloning. Fragments were purified by Sephaglas (Pharmacia), digested with the appropriate enzymes, and ligated into the GST expression vector pGEX-3X (Pharmacia) cut with similar enzymes.
  • Synthetic peptides corresponding to the carboxyl-terminus of a protein of interest are synthesized by standard resin-based chemistry (e.g., FMOC), labeled with biotin at the amino-terminus when indicated, and cleaved from the resin using a halide containing acid (e.g., trifluoroacetic acid).
  • the synthetic peptides are then purified by reverse phase high performance liquid chromatography (HPLC) and the identity ofthe peptides are confirmed by mass spectrometry.
  • Example 8B Measurement of CLASP-2 peptide binding to PDZ Domain-containing proteins
  • GST fusion protein containing one or more PDZ domain(s) is coated onto a protein- binding surface.
  • the protein-binding surface is the surface of a polystyrene plate, which in some cases has been pre-treated by coating with 5 ⁇ g/ml of goat-anti-GST polyclonal antibody followed by blocking with excess bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • the concentration of GST fusion protein used is 5-10 ⁇ g/ml and the reaction of the GST fusion protein with the plate is carried out in PBS for 1 - 16 hours at 4°C. If not already blocked, the plate is then blocked with BSA (2% in PBS, 2 hours, 4°C)
  • the plate is washed with PBS.
  • the biotinylated peptide (generally 0.2-20 ⁇ M) is then added to the plate and allowed to react in PBS/2% BSA buffer with the GST fusion protein for 10 minutes at 4°C followed by 20 minutes at 25°C. In cases where competition between a labeled (biotinylated) and unlabeled (non-biotinylated) peptide is performed, the unlabeled peptide is added immediately prior to adding the labeled peptide.
  • the plate is washed with PBS.
  • steptavidin-HRP conjugate 0.5 ⁇ g/ml steptavidin-HRP conjugate is added to the plate in PBS/2 % BSA buffer and allowed to react for 20 minutes at 4°C.
  • the plate is washed 5 X with detergent (tween 20) containing solution.
  • the plate is developed by addition of HRP-substrate solution for 20 minutes at room temperature.
  • Binding [peptide] Saturation Binding x ([peptide] / ([peptide] + Kd))
  • Binding [peptide] is the binding of a given concentration of peptide to the GST-PDZ fusion protein minus binding to the GST alone control
  • Kd is the apparent affinity ofthe binding reaction
  • Saturation Binding is computed to allow the best fit ofthe data to the above equation.
  • apparent affinity is used because the reaction may not reach equilibrium during the duration ofthe binding reaction in which case the apparent affinity would underestimate the actual affinity (i.e., actual Kd ⁇ observed Kd).
  • the present invention relates to molecules involved in cell-cell interactions in the immune system.
  • the invention relates to a cell surface protein that contains certain classical cadherin characteristics, but it exhibits an apical distribution pattern on the surface of lymphocytes.
  • the membrane location of this molecule correlates with the contact interface between T and B cells, and antibodies against an extracellular domain of this protein disrupt T cell/B cell interactions.
  • An antigen introduced into the immune system first encounters an antigen presenting cell.
  • An antigen presenting cell processes the antigen and presents antigenic fragments to helper T cells (TH), which, in turn, stimulate two types of immune responses; i.e., cellular and humoral immune responses.
  • TH respond to antigen stimulation by producing lymphokines which "help" or activate other effector cell types in the immune system.
  • TH activate B cells to secrete antibodies which function as the major effector molecule in humoral immune responses.
  • Antibodies neutralize foreign antigens and cooperate with other effector cells in mediating antibody-dependent cellular cytotoxicity.
  • TH regulate cellular immune responses by stimulating another T cell subset to develop into antigen-specific cytotoxic effector cells, which directly kill antigen-expressing target cells.
  • TH are distinguished from cytotoxic T lymphocytes (CTL) and B cells by their expression of a cell surface glycoprotein marker termed CD4.
  • CTL cytotoxic T lymphocytes
  • CD4 a cell surface glycoprotein marker
  • type 1 helper T cells THI
  • IL-2 interleukin-2
  • ⁇ -IFN ⁇ -interferon
  • TH2 type 2 helper T cells
  • Tm Based on the profile of lymphokine production, Tm appear to be involved in promoting the activation and proliferation of other T cell subsets such as CTL, whereas TH2 specifically regulate B cell proliferation and differentiation, antibody synthesis, and antibody class switching.
  • CTL express the CD8 surface marker. Unlike most TH, these cells display cytolytic activity by direct contact with target cells, although they are also capable of producing certain lymphokines. In vivo, these cells are particularly important in situations where an antibody response alone is inadequate. There is a preponderance of experimental evidence that cellular immune responses play a principal role in the defense against viral infections and cancer.
  • immune cells communicate with each other by direct contact via surface proteins and by secretory cytokines which bind surface receptors.
  • TCR T cell receptor
  • T lymphocytes exhibit functional polarity (Negulescu et al, 1996, Immunity 4: 421-30). Cells that encounter antigen at their leading edge readily activate, whereas those that encounter antigen at the uropod do so much more poorly. Since TCR density does not seem to be greater at the cell's leading edge prior to antigen activation, other molecule(s) may be responsible for this intrinsic polarity.
  • cytoplasmic molecules display polar distribution in lymphocytes before antigen activation.
  • spectrin, ankyrin, and the microtubule-organizing center demarcate a structural pole in T cells that has been suggested to be important in the directional delivery of signaling molecules after cell-cell coupling (Geiger etal, 1982, J. Cell Biol. 95: 137-43; Gregorio etal, 1994, J. Cell Biol. 125: 345- 58; Kupfer ef a/., 1986, J. Exp. Med. 163: 489-98; Kupfer ef a/., 1994, J. Exp. Med. 179: 1507-15; Lee et al, 1988, Cell 55: 807-16).
  • MTOC microtubule-organizing center
  • a cell surface molecule had not been identified to have a polar distribution on lymphocytes before antigen activation.
  • a cell surface molecule had not been identified to have a polar distribution on lymphocytes before antigen activation.
  • Casp-1 cadherin-like asymmetry protein-1
  • polynucleotides comprising coding sequences for Clasp-1, polynucleotides that selectively hybridize to Clasp-1 coding sequences, expression vectors containing such polynucleotides, genetically-engineered host cells containing such polynucleotides, Clasp-1 polypeptides, Clasp-1 fusion proteins, therapeutic compositions, Clasp-1 domain mutants, antibodies specific for Clasp-1 , methods for detecting the expression of Clasp-1, and methods of inhibiting an immune response by interfering with Clasp-1 function.
  • a wide variety of uses are encompassed by the invention, including but not limited to, treatment of autoimmune diseases and hypersensitivities, prevention of transplantation rejection responses, and augmentation of immune responsiveness in immunodeficiency states.
  • Clasp-1 is expressed in lymphoid tissues and the brain, but is undetectable in most major adult organs. In particular, Clasp-1 is expressed in both T and B cells, as well as macrophages.
  • the cell surface distribution pattern of Clasp-1 in lymphocytes is apical, and it is localized at the pole associated with the leading edge of the cell. More importantly, Clasp-1 is concentrated at the interface between T cell/B cell clusters, and antibodies directed to its extracellular domain inhibit T cell/B cell interactions.
  • Figure la Clasp-1 amino acid sequence (SEQ ID NO:1).
  • the 3.9 kb open reading frame (ORF) is flanked by multiple stop codons in all three reading frames and a polyadenylation signal (AATAAA) located 620 bp downstream of the translation termination codon.
  • AATAAA polyadenylation signal located 620 bp downstream of the translation termination codon.
  • the sequences corresponding to the original degenerate PCR primers are marked by arrows.
  • the propeptide extends from amino acid residues 1-120 ending in the putative cadherin processing signal RAQR (Pigott and Power, 1993, The Adhesion Molecule Facts Book, Academic Press Limited) (triangle).
  • the extracellular domain contains four potential N-glycosylation sites (hexagons) and a cluster of cysteines (double underlines) before the 20 amino acid residue transmembrane domain (shade and double-underline) typical of classical cadherins (Hofman and Stoffel, 1993, Biol. Chem. Hoppe-Seyler 374: 166).
  • the cytoplasmic domain contains a CRK-SH3 binding domain (Knudsen et al, 1994, J. Biol. Chem.
  • FIG. 1b Schematic domain structure of Clasp-1.
  • Clasp-I contains a signal peptide (S) terminating in the cadherin proteolytic processing signal at amino acid residue #120.
  • the extracellular domain has four glycosylation sites (hexagons) and a cluster of cysteines ("C's") typical of cadherins.
  • the transmembrane domain (TM) is followed by a cadherin-like domain (CAD) which contains the CRK-SH3 binding domain (pentagon) and tyrosine phosphorylation sites (star), and a coiled/coil domain (“C/C”).
  • FIG. 2 Cadherin sequence motifs.
  • the cadherin sequence motifs are composed of four stretches of conserved cadherin amino acid sequences (A-D) which are separated by nearly identical numbers of amino acids (in parentheses). Motif A is also the CRK-SH3 binding domain and is similar to the E-cadherin sequence.
  • FIG. 3a Clasp-1 is predominantly expressed in lymphoid tissues and in the brain. Ten ⁇ g of total RNA were loaded and probed with the Clasp-1 cDNA sequence to reveal a 13 kb band, suggesting that the 5' untranslated region was very long, or that it was a polycistronic message. The initiation methionine is predicted from the Kozak consensus sequence. Lane: 1) thymus, 2) spleen, 3) small intestine, 4) skin, 5) muscle, 6) lymph node, 7) lung, 8) liver, 9) kidney, 10) heart, 11) colon, 12) bone marrow, 13) brain. Clasp-1 is found in the thymus, spleen,- lymph node and the brain.
  • FIG. 3b Clasp-1 is expressed in both T and B lymphocytes. Ten ⁇ g of total RNA were loaded in each lane. Lane: 1) S194 (IgA plasmacytoma), 2) NFS 40 (pre-B cell), 3) J558L (IgA plasmacytoma), 4) HSIC 5 (pre-B cell), 5) HAFTLJ (pro- granulocytemacrophage cell), 6) Bal 17 (mature B cell), 7) BAC 14 (pre-B cell), 8) 5CC7 (CD4 T cell). Clasp-1 is expressed in most cell lines tested; it is absent in J558 and in low levels in S194, both plasmacytomas.
  • Clasp-1 protein is about 130 kd molecular weight in both T and B cells.
  • Western blots of 2B4 cytoplasm/membrane (lane 1) or nuclei (lane 2) and CH27 cytoplasm/membrane (lane 3) or nuclei (lane 4) were probed with a goat antiserum to the cytoplasmic domain of Clasp-1.
  • a 130K Clasp-1 band was seen in both T and B cells in the cytosol/membrane fraction. The same 130 kd band was detected with antisera to the putative extracellular domain of Clasp-1.
  • a minor band of 55 kd may represent a degradation product of Clasp-1 or a cross-reactive protein.
  • Figure 4a-4f Clasp-1 localizes to the MOMA-1 marginal zone and appears to be focally distributed in T and B lymphocytes. Mice were perfusion-fixed. Their spleens were removed, cryoprotected, cryosectioned (7 micron) and probed with rabbit antiserum to Clasp-1 -cyto followed by rhodamine-conjugated goat anti-rabbit (red). A second FITC stain (green) was applied to CD3 ( Figures 4a, 4d), B220 ( Figures 4b, 4e), or MOMA-1 (Figures 4c, 4f). Figures 4a-4c are low power views (16X objective) and Figures 4d-4f are high power views (63X objective).
  • T cell zone The T cell zone, B cell zone, marginal zone, and central arterioles are labeled by T, B, M, and c respectively.
  • staining was mostly punctate except for a few scattered cells with dendritic morphology.
  • B cell zone the dominant staining was dendritic and heavily concentrated in the marginal zone ( Figure 4e, arrow).
  • Co-staining with macrophage markers showed localization to the MOMA-1 metallo-macrophage zone.
  • B cells Spleen cell suspensions were cytospun onto poly-L-lysine coated glass slides, fixed in periodate-lysine-paraformaldehyde (McLean and Nakane, 1974, J. Histochem. Cytochem. 22: 1077-83), stained with goat anti-Clasp-EC 12A (plus biotin conjugated mouse monoclonal anti-goat, followed by PE-conjugated strepavidin) and anti-B220- FITC. While most B cells were Clasp-1 negative, when Clasp-1 was present, it was organized into a membrane surface apical domain.
  • FIG. 4h Clasp-1 forms an apical cap or ring in CD3-positive splenic T cells.
  • Spleen cell suspensions were cytospun onto poly-L-lysine coated glass slides, fixed in periodate-lysine-paraformaldehyde, permeabilized in CSK (Greenberg and Edelman, 1983 Cell 33: 767-79), blocked, and stained with rabbit anti-Clasp-cyto (plus rhodamine conjugated anti-rabbit Fab'2) and anti-CD3-FITC.
  • Clasp-1 was organized into a cap or a ring.
  • Clasp-1 forms an apical cap on the surface of D10 T cells.
  • D10 T cells were prepared as described under Figure 3g above, and stained with goat anti-Clasp- EC 12A (plus biotin conjugated mouse monoclonal anti-goat, followed by PE-conjugated strepavidin) and anti-CD3-FITC. Clasp-1 formed a membrane apical domain.
  • FIG. 4j Clasp-1 is located on the same side of the cell as the MTOC.
  • D10 and 2B4 T cells were prepared as described under Figure 4h above, and stained with rabbit anti-Clasp-cyto (plus rhodamine conjugated anti-rabbit Fab'2), monoclonal rat anti- ⁇ - tubulin (YOL 1/34, plus FITC-conjugated mouse anti-rat Fab'2), and counterstained with DAPI.
  • the MTOC green was always located between the Clasp-1 surface and the nucleus.
  • FIG. 5a Clasp-1 in productive and non-productive T-B cell interactions. 3A9 and 5b HEL TCR transgenic splenocytes were cultured in the presence of HEL peptide for 10 hours. Cells were cytospun, fixed, permeabilized and stained for Clasp-1 (red) and CD3 (green). The corresponding phase-contrast (PC) picture is adjacent to each set.
  • Figure 5a Productive T-B cell couples were followed by T cell blast transformation (note the loss of condensed chromatin and nuclear border in the phase contrast picture of the T cells). All pairs show accumulation of Clasp-1 at the cell-cell interface.
  • Figure 5b In non-productive T-B interaction (T cell was not undergoing blast transformation), Clasp-1 was not facing the cell-cell interface.
  • Figure 6a Goat anti-Clasp-EC 12 blocks T-B cell coupling. 2B4 T cell hybridoma with specificity for moth cytochrome c (MCC) in the context of l-E k was mixed with CH27 B cell loaded with MCC peptide. Gamma-bind (Pharmacia, NJ) purified goat anti-Clasp- EC12 or preimmune serum was added at 0, 50, 150 and 300 ⁇ g/ml.
  • MCC moth cytochrome c
  • FIG. 6b Goat anti-Clasp-EC 12 blocks T cell activation.
  • 2B4 T cell hybridoma with specificity for moth cytochrome c (MCC) in the context of l-E k was mixed with CH27 B cell loaded with MCC peptide.
  • Gamma-bind (Pharmacia, NJ) purified goat anti-Clasp- EC12 or pre-immune serum was added at 0, 125, 500 and 1,000 ⁇ g/ml.
  • IL-2 levels were measured after 48 hours of co-incubation and found to diminish in a dose dependent fashion.
  • Pre-immune serum did not inhibit T cell activation as measured by IL-2 stimulation. Samples were performed in triplicate.
  • the amino acid sequence of mouse CLASP-1 is provided as SEQ ID NO:1.
  • the nucleotide sequence of the mouse CLASP-1 cDNA is provided in SEQ ID NO:2, which also shows the position of the start and stop of translation, and the encoded polypeptide.
  • the nucleotide sequence of human CLASP-1 is provided as SEQ ID NO:
  • Nucleic acid molecules that comprise mammalian Clasp-1 coding sequences and polypeptide encoded by the sequences are provided.
  • mouse and human Clasp-1 cDNA molecules were isolated, and their nucleotide and deduced amino acid sequences characterized. While Clasp-1 shares sequence homology with cadherin-encoding genes from different species, both the nucleotide coding sequences and the deduced amino acid sequences of Clasp-1 are unique.
  • any nucleotide sequence that encodes an amino acid sequence of a Clasp-1 gene product can be used to generate recombinant molecules that direct the expression of Clasp-1 polypeptides.
  • the invention also provides isolated or purified nucleic acids consisting of at least 8 nucleotides (i.e., a hybridizable portion) of a Clasp-1 sequence or its complement; in other embodiments, the nucleic acids consist of at least 25 (continuous) nucleotides, 50 nucleotides, 100 nucleotides, 150 nucleotides, or 200 nucleotides of a Clasp-1 sequence, or a full-length Clasp-1 coding sequence. In another embodiment, the nucleic acids are smaller than 35, 200 or 500 nucleotides in length. Nucleic acids can be single or double stranded. The invention also relates to nucleic acids that selectively hybridize to or complementary to the foregoing sequences.
  • nucleic acids are provided that comprise a sequence complementary to at least 10, 25, 50, 100, or 200 nucleotides or the entire coding region of a Clasp-1 coding sequence.
  • a nucleic acid is provided that hybridizes to a Clasp-1 nucleic acid (e.g., having SEQ ID NO:2, SEQ ID NO:3) or its complement, or to a nucleic acid encoding a Clasp-1 derivative.
  • hybridization may be performed under condition of low, moderate or high stringency.
  • the hybridization may utilize the 5' region of the provided sequences.
  • a nucleic acid may be determined to hybridize under stringent conditions to a probe selected from nucleotides 1 to approximately 3990 of SEQ ID NO:3.
  • Hybridizations are carried out in the same solution with the following modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 ⁇ g/ml salmon sperm DNA, 10% (wt/vol) dextran sulfate, and 5-20 X 10 6 cpm 32 P-labeled probe is used. Filters are incubated in hybridization mixture for 18-20 h at 40°C, and then washed for 1.5 h at 55°C in a solution containing 2X SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution is replaced with fresh solution and incubated an additional 1.5 h at 60°C.
  • Filters are blotted dry and exposed for autoradiography. If necessary, filters are washed for a third time at 65-68 °C and reexposed to film.
  • Other conditions of low stringency which may be used are well known in the art (e.g., as employed for cross-species hybridizations).
  • procedures using such conditions of high stringency are as follows: Prehybridization of filters containing DNA is carried out for 8 h to overnight at 65°C in buffer composed of 6X SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 ⁇ g/ml denatured salmon sperm DNA.
  • Filters are hybridized for 48 h at 65°C in prehybridization mixture containing 100 ⁇ g/ml denatured salmon sperm DNA and 5-20 X 10 ⁇ cpm of 32 P-labeled probe. Washing of filters is done at 37°C for 1 h in a solution containing 2X SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. This is followed by a wash in 0.1X SSC at 50°C for 45 min before autoradiography. Other conditions of high stringency which may be used are well known in the art.
  • Examples of procedures using such conditions of moderate stringency are as follows: Filters containing DNA are pretreated for 6 h at 55°C in a solution containing 6X SSC, 5X Denhart's solution, 0.5% SDS and 100 ⁇ g/ml denatured salmon sperm DNA. Hybridizations are carried out in the same solution and 5-20 X 10 6 cpm 3 P-labeled probe is used. Filters are incubated in hybridization mixture for 18-20 h at 55 °C, and then washed twice for 30 minutes at 60 °C in a solution containing 1X SSC and 0.1% SDS. Filters are blotted dry and exposed for autoradiography. Other conditions of moderate stringency which may be used are well-known in the art.
  • labeled DNA probes made from nucleic acid fragments corresponding to any partial cDNA disclosed herein may be used to screen a cDNA library derived from lymphoid cells or brain cells. More specifically, oligonucleotides corresponding to either the 5' or 3' terminus of the cDNA sequence may be used to obtain longer nucleotide sequences.
  • the library may be plated out to yield a maximum of 30,000 pfu for each 150 mm plate. Approximately 40 plates may be screened. The plates are incubated at 37 °C until the plaques reach a diameter of 0.25 mm or are just beginning to make contact with one another (3-8 hours). Nylon filters are placed onto the soft top agarose and after 60 seconds, the filters are peeled off and floated on a DNA denaturing solution consisting of 0.4N sodium hydroxide. The filters are then immersed in neutralizing solution consisting of 1 M Tris-HCl, pH 7.5, before being allowed to air dry.
  • the filters are prehybridized in hybridization buffer such as casein buffer containing 10% dextran sulfate, 0.5M NaCl, 50mM Tris-HCl, pH 7.5, 0.1% sodium pyrophosphate, 1% casein, 1% SDS, and denatured salmon sperm DNA at 0.5 mg/ml for 6 hours at 60°C.
  • hybridization buffer such as casein buffer containing 10% dextran sulfate, 0.5M NaCl, 50mM Tris-HCl, pH 7.5, 0.1% sodium pyrophosphate, 1% casein, 1% SDS, and denatured salmon sperm DNA at 0.5 mg/ml for 6 hours at 60°C.
  • the radiolabelled probe is then denatured by heating to 95°C for 2 minutes and then added to the prehybridization solution containing the filters.
  • the filters are hybridized at 60 °C for 16 hours.
  • the filters are then washed in 1X wash mix (10X wash mix contains 3M NaCl, 0.6M Tris base, and 0.02M EDTA) twice for 5 minutes each at room temperature, then in 1X wash mix containing 1% SDS at 60°C for 30 minutes, and finally in 0.3X wash mix containing 0.1 % SDS at 60 °C for 30 minutes.
  • 1X wash mix contains 3M NaCl, 0.6M Tris base, and 0.02M EDTA
  • the agar plug containing the plaques will be removed and placed in lambda dilution buffer containing 0.1M NaCl, 0.01 M magnesium sulfate, 0.035M Tris HCl, pH 7.5, 0.01% gelatin.
  • the phage may then be replated and rescreened to obtain single, well isolated positive plaques.
  • Positive plaques may be isolated and the cDNA clones sequenced using primers based on the known cDNA sequence. This step may be repeated until a full length cDNA is obtained.
  • RACE is a proven PCR-based strategy for amplifying the 5' end of incomplete cDNAs.
  • 5'-RACE-Ready RNA synthesized from human tissues containing a unique anchor sequence is commercially available (Clontech).
  • PCR is carried out on 5-RACE-Ready cDNA using the provided anchor primer and the 3' primer.
  • a secondary PCR reaction is then carried out using the anchored primer and a nested 3' primer according to the manufacturer's instructions.
  • the full length cDNA sequence may be translated into amino acid sequence and examined for certain landmarks such as a continuous open reading frame flanked by translation initiation and termination sites, a cadherin-like domain, an SH3 binding domain, and finally overall structural similarity to the Clasp-1 gene disclosed herein.
  • Clasp-1 polynucleotides encoding Clasp-1 polypeptides, mutant polypeptides, peptide fragments of Clasp-1 , Clasp-1 fusion proteins or functional equivalents thereof may be used to generate recombinant DNA molecules that direct the expression of Clasp-1 protein, Clasp-1 peptide fragments, fusion proteins or a functional equivalent thereof, in appropriate host cells.
  • Such Clasp- 1 polynucleotide sequences, as well as other polynucleotides that selectively hybridize to at least a part of such Clasp-1 polynucleotides or their complements may also be used in nucleic acid hybridization assays, Southern and Northern blot analyses, etc.
  • DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence, may be used in the practice ofthe invention for the expression of the Clasp-1 protein.
  • DNA sequences include those which are capable of hybridizing to the mouse Clasp-1 sequence or its complementary sequence under low, moderate or high stringent conditions as described above.
  • Altered DNA sequences which may be used in accordance with the invention include deletions, additions or substitutions of different nucleotide residues resulting in a sequence that encodes the same or a functionally equivalent gene product.
  • the gene product itself may contain deletions, additions or substitutions of amino acid residues within a Clasp-1 sequence, which result in a silent change thus producing a functionally equivalent Clasp-1 protein.
  • conservative amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.
  • negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine, histidine and arginine; amino acids with uncharged polar head groups having similar hydrophilicity values include the following: glycine, asparagine, glutamine, serine, threonine, tyrosine; and amino acids with nonpolar head groups include alanine, valine, isoleucine, leucine, phenylalanine, proline, methionine, tryptophan.
  • the DNA sequences of the invention may be engineered in order to alter a Clasp-1 coding sequence for a variety of ends, including but not limited to, alterations which modify processing and expression ofthe gene product.
  • mutations may be introduced using techniques which are well known in the art, e.g., site-directed mutagenesis, to insert new restriction sites, to alter glycosylation patterns, phosphorylation, etc.
  • a large number of Clasp-1 mutant polypeptides can be constructed by rearranging the nucleotide sequences that encode the Clasp-1 extracellular, transmembrane and cytoplasmic domains.
  • a Clasp-1 or a modified Clasp-1 sequence may be ligated to a heterologous sequence to encode a fusion protein.
  • a fusion protein may also be engineered to contain a cleavage site located between a Clasp-1 sequence and the heterologous protein sequence, so that the Clasp-1 may be cleaved away from the heterologous moiety.
  • an endogenous Clasp-1 gene within a cell population may be modified by inserting a heterologous DNA regulatory element into the genome of the cell line such that the inserted regulatory element is operatively linked with the endogenous Clasp-1 gene.
  • a heterologous DNA regulatory element for example, an endogenous Clasp-1 gene which is normally "transcriptionally silent", i.e., an Clasp-1 gene which is normally not expressed, or is expressed only at very low levels in a cell population, may be activated by inserting a regulatory element which is capable of promoting the expression of a normally expressed gene product in the cells.
  • a transcriptionally silent, endogenous Clasp-1 gene may be activated by insertion of a promiscuous regulatory element that works across cell types.
  • a heterologous regulatory element may be inserted into a cell line population, such that it is operatively linked with an endogenous Clasp-1 gene, using techniques, such as targeted homologous recombination, which are well known to those of skill in the art, (see e.g., in Chappel, U.S. Patent No. 5,272,071; PCT publication No. WO
  • the coding sequence of Clasp-1 could be synthesized in whole or in part, using chemical methods well known in the art.
  • chemical methods See, e.g., Caruthers et al, 1980, Nuc. Acids Res. Symp. Ser. 7:215-233; Crea and Horn, 180, Nuc. Acids Res. 9(10):2ZZV, Matteucci and Caruthers, 1980, Tetrahedron Letter 21:1 '19; and Chow and Kempe, 1981, Nuc. Acids Res. 9(12):2807-28M.
  • the protein itself could be produced using chemical methods to synthesize a Clasp-1 amino acid sequence in whole or in part.
  • peptides can be synthesized by solid phase techniques, cleaved from the resin, and purified by preparative high performance liquid chromatography. (See Creighton, 1983, Proteins Structures And Molecular Principles, W.H. Freeman and Co., N.Y. pp. 50-60). The composition ofthe synthetic polypeptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; see Creighton, 1983, Proteins, Structures and Molecular Principles, W.H. Freeman and Co., N.Y., pp. 34-49).
  • Clasp-1 gene products as well as host cells or cell lines transfected or transformed with recombinant Clasp-1 expression vectors can be used for a variety of purposes. These include, but are not limited to, generating antibodies (i.e., monoclonal or polyclonal) that competitively inhibit activity of Clasp-1 proteins and neutralize its activity; antibodies that activate Clasp-1 function and antibodies that detect its presence on the cell surface or in solution. Anti-Clasp-1 antibodies may be used in detecting and quantifying expression of Clasp-1 levels in cells and tissues such as lymphocytes and macrophages, as well as isolating Clasp-1 - positive cells from a cell mixture.
  • an appropriate expression vector i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
  • Clasp-1 gene products as well as host cells or cell lines transfected or transformed with recombinant Clasp-1 expression vectors can be used for a variety of purposes. These include, but are not limited to
  • a variety of host-expression vector systems may be utilized to express the Clasp- 1 coding sequence. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the Clasp-1 coding sequence; yeast transformed with recombinant yeast expression vectors containing the Clasp-1 coding sequence; insect cell systems infected with recombinant virus expression vectors (e.g.. baculovirus) containing the Clasp-1 coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g..).
  • microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the Clasp-1 coding sequence
  • yeast transformed with recombinant yeast expression vectors containing the Clasp-1 coding sequence yeast transformed with recombinant yeast expression vectors containing the Clasp
  • plasmid expression vectors e.g.. Ti plasmid
  • the expression elements of these systems vary in their strength and specificities.
  • any of a number of suitable transcription and translation elements including constitutive and inducible promoters, may be used in the expression vector.
  • inducible promoters such as pL of bacteriophage ⁇ , plac, ptrp, ptac (ptrp-lac hybrid promoter; cvtomegalovirus promoter) and the like may be used; when cloning in insect cell systems, promoters such as the baculovirus polyhedron promoter may be used; when cloning in plant cell systems, promoters derived from the genome of plant cells (e.g.. heat shock promoters; the promoter for the small subunit of RUBISCO; the promoter for the chlorophyll ⁇ / ⁇ binding protein) or from plant viruses (e.g..).
  • the 35S RNA promoter of CaMV; the coat protein promoter of TMV may be used; when cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (e.g.. metallothionein promoter) or from mammalian viruses (e.g.. the adenovirus late promoter; the vaccinia virus 7.5K promoter) may be used; when generating cell lines that contain multiple copies ofthe Clasp-1 DNA, SV40-, BPV- and EBV-based vectors may be used with an appropriate selectable marker.
  • promoters derived from the genome of mammalian cells e.g.. metallothionein promoter
  • mammalian viruses e.g. the adenovirus late promoter
  • the vaccinia virus 7.5K promoter e.g. the vaccinia virus 7.5K promoter
  • a number of expression vectors may be advantageously selected depending upon the use intended for the expressed Clasp-1 product.
  • vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable.
  • Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which the Clasp-1 coding sequence may be ligated into the vector in frame with the lacZ coding region so that a hybrid protein is produced; plN vectors (Inouye & Inouye, 1985, Nucleic acids Res.
  • pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S- transferase (GST).
  • GST glutathione S- transferase
  • fusion proteins are soluble and can be purified easily from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione.
  • the pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety.
  • yeast a number of vectors containing constitutive or inducible promoters may be used.
  • Current Protocols in Molecular Biology Vol. 2, 1988, Ed. Ausubel et al., Greene Publish. Assoc. & Wiley Interscience, Ch. 13; Grant et al., 1987, Expression and Secretion Vectors for Yeast, ]n Methods in Enzymology, Eds. Wu & Grossman, 1987, Acad. Press, N.Y., Vol. 153, pp. 516-544; Glover, 1986, DNA Cloning, Vol. II, IRL Press, Wash., D.C., Ch.
  • viral promoters such as the 35S RNA and 19S RNA promoters of CaMV (Brisson et al., 1984, Nature 310:511-514), or the coat protein promoter of TMV (Takamatsu et al., 1987, EMBO J. 3:1731) may be used; alternatively, plant promoters such as the small subunit of RUBISCO (Coruzzi et al., 1984, EMBO J. 3:1671-1680; Broglie et al., 1984, Science 224:838-843); or heat shock promoters, e.g.. soybean hsp17.5-E or hsp17.3-B (Gurley et al., 1986, Mol. Cell.
  • Biol. 6:559-565) may " be used. These constructs can be introduced into plant cells using Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, etc. (Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp.421-463; and Grierson & Corey, 1988, Plant Molecular Biology, 2d Ed., Blackie, London, Ch. 7-9.)
  • An alternative expression system which could be used to express Clasp-1 is an insect system.
  • Autographa califomica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes.
  • the virus grows in Spodoptera frugiperda cells.
  • the Clasp-1 coding sequence may be cloned into non-essential regions (e.g., the polyhedron gene) of the virus and placed under control of an AcNPV promoter (e.g., the polyhedron promoter).
  • Successful insertion of the Clasp-1 coding sequence will result in inactivation of the polyhedron gene and production of non- occluded recombinant virus (L , virus lacking the proteinaceous coat coded for by the polyhedron gene).
  • recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed, (e.g.. see Smith et al., 1983, J. Viol. 46:584; Smith, U.S. Patent No. 4,215,051).
  • a number of viral based expression systems may be utilized.
  • the Clasp-1 coding sequence may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination.
  • Insertion in a non-essential region of the viral genome will result in a recombinant virus that is viable and capable of expressing Clasp-1 in infected hosts, (e.g., See Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:3655-3659).
  • the vaccinia 7.5K promoter may be used. (See, e ⁇ g., Mackett et al., 1982, Proc. Natl. Acad. Sci. USA 79:7415-7419; Mackett et al., 1984, J. Virol. 49:857-864; Panicali et al., 1982, Proc. Natl. Acad. Sci. USA 79:4927-4931).
  • Regulatable expression vectors such as the tetracycline repressible vectors may also be used to express a coding sequence in a controlled fashion.
  • Specific initiation signals may also be required for efficient translation of inserted Clasp-1 coding sequences. These signals include the ATG initiation codon and adjacent sequences. In cases where an entire Clasp-1 gene, including its own initiation codon and adjacent sequences, is inserted into the appropriate expression vector, no additional translational control signals may be needed. However, in cases where only a portion of the Clasp-1 coding sequence is inserted, exogenous translational control signals, including the ATG initiation codon, must be provided. Furthermore, the initiation codon must be in phase with the reading frame of the Clasp-1 coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic.
  • telomeres may be shortened by the telomeres.
  • a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in a specific fashion desired. Such modifications (e.g.. glycosylation) and processing (e.g.. cleavage) of protein products may be important for the function of the protein.
  • modifications e.g.. glycosylation
  • processing e.g.. cleavage
  • the presence of several consensus N-glycosylation sites in the Clasp-1 extracellular domain support the possibility that proper modification may be important for Clasp-1 function.
  • Different host cells have characteristic and specific mechanisms for the post- translational processing and modification of proteins.
  • Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing ofthe foreign protein expressed.
  • eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation ofthe gene product may be used.
  • mammalian host cells include, but are not limited to, CHO, VERO, BHK, HeLa, COS, MDCK, 293, WI38, etc.
  • cell lines which stably express Clasp-1 may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with the Clasp-1 DNA controlled by appropriate expression control elements (e.g.. promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched medium, and then switched to a selective medium.
  • expression control elements e.g. promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
  • This method may advantageously be used to engineer cell lines which express the Clasp-1 protein on the cell surface. Such engineered cell lines are particularly useful in screening for molecules or drugs that affect Clasp-1 function.
  • a number of selection systems may be used, including but not limited to, the he ⁇ es simplex virus thymidine kinase (Wigler, et al., 1977, Cell 11:223), hypoxanthine- guanine phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adenine phosphoribosyltransferase (Lowy, et al., 1980, Cell 22:817) genes which can be employed in tk ' , hgprt ' or aprt ' cells, respectively.
  • antimetabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler, et al., 1980, Natl. Acad. Sci. USA 77:3567; O'Hare, et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981), Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin, et al., 1981, J. Mol. Biol.
  • trpB which allows cells to utilize indole in place of tryptophan
  • hisD which allows cells to utilize histinol in place of histidine
  • ODC ornithine decarboxylase
  • DFMO McConlogue L, 1987, In: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory ed.
  • glutamine synthetase Bebbington et al, 1992, Biotech 10:169.
  • the host cells which contain the coding sequence and which express a biologically active Clasp-1 gene product or fragments thereof may be identified by at least four general approaches; (a) DNA-DNA or DNA-RNA hybridization; (b) the presence or absence of "marker" gene functions; (c) assessing the level of transcription as measured by the expression of Clasp-1 mRNA transcripts in the host cell; and (d) detection ofthe gene product as measured by immunoassay or by its biological activity.
  • the host cells Prior to the identification of gene expression, the host cells may be first mutagenized in an effort to increase the level of expression of Clasp-1, especially in cell lines that produce low amounts of Clasp-1.
  • the presence ofthe Clasp-1 coding sequence inserted in the expression vector can be detected by DNA-DNA or DNA-RNA hybridization using probes comprising nucleotide sequences that are homologous to the Clasp-1 coding sequence, respectively, or portions or derivatives thereof.
  • the recombinant expression vector/host system can be identified and selected based upon the presence or absence of certain "marker" gene functions (e.g.. thymidine kinase activity, resistance to antibiotics, resistance to methotrexate, transformation phenotype, occlusion body formation in baculovirus, etc.).
  • certain "marker" gene functions e.g. thymidine kinase activity, resistance to antibiotics, resistance to methotrexate, transformation phenotype, occlusion body formation in baculovirus, etc.
  • recombinants containing the Clasp-1 coding sequence can be identified by the absence of the marker gene function.
  • a marker gene can be placed in tandem with the Clasp-1 sequence under the control of the same or different promoter used to control the expression of the Clasp-1 coding sequence. Expression of the marker in response to induction or selection indicates expression of the Clasp-1 coding sequence.
  • transcriptional activity for the Clasp-1 coding region can be assessed by hybridization assays. For example, RNA can be isolated and analyzed by Northern blot using a probe homologous to the Clasp-1 coding sequence or particular portions thereof. Alternatively, total nucleic acids of the host cell may be extracted and assayed for hybridization to such probes. Additionally, reverse transcription-polymerase chain reactions may be used to detect low levels of gene expression.
  • Clasp-1 protein product can be assessed immunologically, for example by Western blots, immunoassays such as radioimmuno-precipitation, enzyme-linked immunoassays and the like. This can be achieved by using an anti-Clasp-1 antibody.
  • Clasp-1 protein may be expressed as a fusion protein with green-fluorescent protein to facilitate its detection in cells (United States Patent Nos. 5,491,084; 5,804,387; 5,777,079).
  • the Clasp-1 protein and/or cell lines that express Clasp-1 may be used to screen for antibodies, peptides, small molecules, natural and synthetic compounds or other cell bound or soluble molecules that bind to the Clasp-1 protein resulting in stimulation or inhibition of Clasp-1 function.
  • anti-Clasp- 1 antibodies may be used to inhibit or stimulate Clasp-1 function and to detect its presence.
  • screening of peptide libraries with recombinantly expressed soluble Clasp-1 protein or cell lines expressing Clasp-1 protein may be useful for identification of therapeutic molecules that function by inhibiting or stimulating the biological activity of Clasp-1.
  • Clasp-1 protein and engineered cell lines may be employed equally well for homologous Clasp-1 genes in various species.
  • cell lines have been engineered to express the extracellular domain of Clasp-1 fused to another molecule such as GST.
  • Clasp-1 or its extracellular domain may be fused to an immunoglobulin constant region (Hollenbaugh and Aruffo, 1992, Current Protocols in Immunology, Unit
  • soluble protein or fusion protein may be used in binding assays, affinity chromatography, immunoprecipitation, Western blot, and the like. Synthetic compounds, natural products, and other sources of potentially biologically active materials can be screened in assays that are well known in the art.
  • Random peptide libraries consisting of all possible combinations of amino acids attached to a solid phase support may be used to identify peptides that are able to bind to a specific domain of Clasp-1 (Lam, K.S. et al., 1991, Nature 354: 82-84).
  • the screening of peptide libraries may have therapeutic value in the discovery of pharmaceutical agents that stimulate or inhibit the biological activity of Clasp-1.
  • Identification of molecules that are able to bind to the Clasp-1 protein may be accomplished by screening a peptide library with recombinant soluble Clasp-1 protein. Methods for expression and purification of Clasp-1 may be used to express recombinant full length Clasp-1 or fragments of Clasp-1 depending on the functional domains of interest. Such domains include Clasp-1 extracellular domain, transmembrane domain, cytoplasmic domain, SH2 domain, SH3 domain and coiled/coil domain. In a specific embodiment, a portion of the Clasp-1 extracellular domain corresponding to amino acid residues #131-327 is shown to contain a binding site that interacts with itself or other proteins.
  • Clasp-1 protein may be conjugated to enzymes such as alkaline phosphatase or horseradish peroxidase or to other reagents such as fluorescent labels which may include fluorescein isothiocyanate (FITC), phycoerythrin (PE) or rhodamine. Conjugation of any given label to Clasp-1 may be performed using techniques that are well known in the art.
  • Clasp-1 expression vectors may be engineered to express a chimeric Clasp-1 protein containing an epitope for which a commercially available antibody exist. The epitope-specific antibody may be tagged with a detectable label using methods well known in the art including an enzyme, a fluorescent dye or colored or magnetic beads.
  • the "tagged" Clasp-1 conjugate is incubated with the random peptide library for 30 minutes to one hour at 22 °C to allow complex formation between Clasp-1 and peptide species within the library. The library is then washed to remove any unbound protein. If Clasp-1 has been conjugated to alkaline phosphatase or horseradish peroxidase the whole library is poured into a petri dish containing substrates for either alkaline phosphatase or peroxidase, for example, 5-bromo-4-chloro-3-indoyl phosphate (BCIP) or 3,3',4,4''-diaminobenzidine (DAB), respectively.
  • BCIP 5-bromo-4-chloro-3-indoyl phosphate
  • DAB 3,3',4,4''-diaminobenzidine
  • the peptide/solid phase-Clasp-1 complex changes color, and can be easily identified and isolated physically under a dissecting microscope with a micromanipulator. If a fluorescent tagged Clasp-1 molecule has been used, complexes may be isolated by fluorescence activated sorting. If a chimeric Clasp-1 protein expressing a heterologous epitope has been used, detection of the peptide/Clasp-1 complex may be accomplished by using a labeled epitope-specific antibody. Once isolated, the identity of the peptide attached to the solid phase support may be determined by peptide sequencing.
  • soluble Clasp-1 molecules in another embodiment, it is possible to detect peptides that bind to cell-associated Clasp-1 using intact cells.
  • the use of intact cells is preferred for use with cell surface molecules.
  • Methods for generating cell lines expressing Clasp-1 are described above.
  • the cells used in this technique may be either live or fixed cells.
  • the cells may be incubated with the random peptide library and bind to certain peptides in the library to form a "rosette" between the target cells and the relevant solid phase support/peptide.
  • the rosette can thereafter be isolated by differential centrifugation or removed physically under a dissecting microscope. Techniques for screening combinatorial libraries are known in the art
  • Clasp-1 molecules can be reconstituted into liposomes where label or "tag" can be attached.
  • antibodies to epitopes ofthe natural and recombinantly produced Clasp-1 protein include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, humanized, a complementarity determining region, Fab fragments, F(ab') 2 and fragments produced by an Fab expression library as well as anti-id iotypic antibodies.
  • Antibodies which compete for Clasp-1 binding are especially preferred for diagnostics and therapeutics.
  • Monoclonal antibodies that bind Clasp-1 may be radioactively labeled allowing one to follow their location and distribution in the body after injection. Radioisotope tagged antibodies may be used as a non-invasive diagnostic tool for imaging de novo lymphoid tumors and metastases that express Clasp-1.
  • Immunotoxins may also be designed which target cytotoxic agents to specific sites in the body.
  • high affinity Clasp-1 specific monoclonal antibodies may be covalently complexed to bacterial or plant toxins, such as diphtheria toxin or ricin.
  • a general method of preparation of antibody/hybrid molecules may involve use of thiol-crosslinking reagents such as SPDP, which attack the primary amino groups on the antibody and by disulfide exchange, attach the toxin to the antibody.
  • SPDP thiol-crosslinking reagents
  • the hybrid antibodies may be used to specifically eliminate Clasp-1 expressing lymphocytes.
  • various host animals may be immunized by injection with the recombinant or naturally purified Clasp-1 protein, fusion protein or peptides, including but not limited to goats, rabbits, mice, rats, hamsters, etc.
  • adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacilli Calmette-Guerin) and Corynebacterium parvum.
  • BCG Bacilli Calmette-Guerin
  • Corynebacterium parvum bacilli Calmette-Guerin
  • Monoclonal antibodies to Clasp-1 may be prepared by using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique originally described by Kohler and Milstein, (Nature, 1975, 256:495-497), the human B-cell hybridoma technique (Kosbor et al., 1983, Immunology Today, 4:72; Cote et al., 1983, Proc. Natl. Acad. Sci. USA, 80:2026-2030) and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
  • Hybridomas may be screened using enzyme-linked immunosorbent assays (ELISA) in order to detect cultures secreting antibodies specific for refolded recombinant Clasp-1.
  • Cultures may also be screened by ELISA to identify those cultures secreting antibodies specific for mammalian-produced Clasp-1. Confirmation of antibody specificity may be obtained by western blot using the same antigens. Subsequent ELISA testing may use recombinant Clasp-1 fragments to identify the specific portion of the Clasp-1 molecule with which a monoclonal antibody binds.
  • Additional testing may be used to identify monoclonal antibodies with desired functional characteristics such as staining of histological sections, immunoprecipitation of Clasp-1, inhibition of Clasp-1 binding or stimulation of Clasp-1 to transmit an intracellular signal. Determination of the monoclonal antibody isotype may be accomplished by ELISA, thus providing additional information concerning purification or function.
  • Antibody fragments which contain specific binding sites of Clasp-1 may be generated by known techniques.
  • such fragments include, but are not limited to, the F(ab') 2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab') 2 fragments.
  • Fab expression libraries may be constructed (Huse et al., 1989, Science, 246:1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity to Clasp-1.
  • Anti- Clasp-1 antibodies may be used to identify, isolate, inhibit or eliminate Clasp-1- expressing cells.
  • a Clasp-1 polynucleotide or fragments thereof may be used for diagnostic and/or therapeutic purposes.
  • Clasp-1 since Clasp-1 is expressed in lymphocytes, a
  • Clasp-1 polynucleotide may be used to detect the expression of Clasp-1 as a lymphocyte marker.
  • a Clasp-1 polynucleotide may be used to detect Clasp-1 gene expression or aberrant Clasp-1 gene expression in disease states. Included in the scope ofthe invention are oligonucleotide sequences, such as antisense RNA and DNA molecules and ribozymes, that function to inhibit expression of Clasp-1.
  • Clasp-1 polynucleotide may be used to construct transgenic and knockout animals for screening of Clasp-1 agonists and antagonists.
  • the Clasp-1 gene products can also be expressed in transgenic animals.
  • Animals of any species including, but not limited to, mice, rats, rabbits, guinea pigs, pigs, micro-pigs, goats, sheep, and non-human primates, e.g., baboons, monkeys, and chimpanzees may be used to generate Clasp-1 transgenic animals.
  • transgenic refers to animals expressing Clasp-1 gene sequences from a different species (e.g., mice expressing human Clasp-1 gene sequences), as well as animals that have been genetically engineered to overexpress endogenous (i.e., same species) Clasp-1 sequences or animals that have been genetically engineered to no longer express endogenous Clasp-1 gene sequences (i.e., "knock-out” animals), and their progeny.
  • Any technique known in the art may be used to introduce a Clasp-1 transgene into animals to produce the founder lines of transgenic animals.
  • Such techniques include, but are not limited to pronuclear microinjection (Hoppe and Wagner, 1989, U.S. Pat. No. 4,873,191); retrovirus mediated gene transfer into germ lines (Van der Putten, et al, 1985, Proc. Natl. Acad. Sci., USA 82:6148-6152); gene targeting in embryonic stem cells (Thompson, et al, 1989, Cell 56:313-321); electroporation of embryos (Lo, 1983, Mol. Cell. Biol.
  • transgenic animal clones containing a Clasp-1 transgene for example, nuclear transfer into enucleated oocytes of nuclei from cultured embryonic, fetal or adult cells induced to quiescence (Campbell, et al, 1996, Nature 380:64-66; Wilmut, et al, Nature 385:810-813).
  • the present invention provides for transgenic animals that carry a Clasp-1 transgene in all their cells, as well as animals that carry the transgene in some, but not all their cells, i.e., mosaic animals.
  • the transgene may be integrated as a single transgene or 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 etal. (Lasko, et al, 1992, Proc. Natl. Acad. Sci. USA 89:6232-6236).
  • the regulatory sequences required for such a cell-type specific activation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.
  • gene targeting is preferred.
  • vectors containing some nucleotide sequences homologous to the endogenous Clasp-1 gene are designed for the purpose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of the nucleotide sequence of the endogenous Clasp-1 gene.
  • the transgene may also be selectively introduced into a particular cell type, thus inactivating the endogenous Clasp-1 gene in only that cell type, by following, for example, the teaching of Gu, et al. (1994, Science 265: 103-106).
  • 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 expression of the recombinant Clasp-1 gene may be assayed utilizing standard techniques. Initial screening may be accomplished by Southern blot analysis or PCR techniques to analyze animal tissues to assay whether 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 that include, but are not limited to, Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and RT-PCR (reverse transcriptase PCR). Samples of Clasp-1 gene-expressing tissue, may also be evaluated immunocytochemically using antibodies specific for the Clasp-1 transgene product. DIAGNOSTIC USES OF Clasp-1 POLYNUCLEOTIDE
  • a Clasp-1 polynucleotide may have a number of uses in the diagnosis of diseases or disorders resulting from aberrant expression of Clasp-1 such as immunodeficient states.
  • the Clasp-1 DNA sequence may be used in hybridization assays of biopsies or autopsies to detect abnormalities of Clasp-1 expression; e.g., Southern or Northern analysis, including in situ hybridization assays and PCR.
  • PCR primers of 15-30 nucleotides may be used.
  • a preferred length of a PCR primer is about 18-22 nucleotides. However, the length of primers may be adjusted by one skilled in the art.
  • a Clasp-1 probe a polynucleotide of 300-500 nucleotides is preferred.
  • Various hybridization techniques are well known in the art, and are in fact the basis of many commercially available diagnostic kits.
  • Clasp-1 polynucleotide may be useful in the treatment of various abnormal conditions.
  • gene therapy can be used to treat conditions in which the cells do not express normal Clasp-1 or express abnormal/inactive Clasp-1.
  • the polynucleotide encoding a Clasp-1 is intended to replace or act in the place of a functionally deficient endogenous gene.
  • abnormal conditions characterized by overexpression can be treated using the gene therapy techniques described below.
  • nucleic acids comprising a sequence encoding a
  • Clasp-1 protein or functional derivative thereof are administered to promote Clasp-1 function, by way of gene therapy.
  • Gene therapy refers to therapy performed by the administration of a nucleic acid to a subject.
  • the nucleic acid produces its encoded protein that mediates a therapeutic effect by promoting Clasp-1 function.
  • the therapeutic composition comprises a Clasp-1 nucleic acid that is part of an expression vector that encodes a Clasp-1 protein or fragment or chimeric protein thereof in a suitable host.
  • a nucleic acid has a promoter operably linked to the Clasp-1 coding region, said promoter being inducible or constitutive, and, optionally, tissue-specific.
  • a nucleic acid molecule is used in which the Clasp-1 coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the Clasp-1 nucleic acid (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932- 8935; Zijlstra et al., 1989, Nature 342:435-438).
  • nucleic acid into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid-carrying vector, or indirect, in which case, cells are first transformed with the nucleic acid in vitro, then transplanted into the patient. These two approaches are known, respectively, as in vivo or ex vivo gene therapy.
  • the nucleic acid is directly administered in vivo, where it is expressed to produce the encoded product.
  • This can be accomplished by any of numerous methods known in the art, e.g., by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by infection using a defective or attenuated retroviral or other viral vector (see U.S. Patent No.
  • microparticle bombardment e.g., a gene gun; Biolistic, Dupont
  • coating lipids or cell-surface receptors or transfectmg agents, encapsulation in liposomes, microparticles, or microcapsules, or by administering it in linkage to a peptide which is known to enter the nucleus, by administering it in linkage to a ligand subject to receptor-mediated endocytosis (see e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432) (which can be used to target cell types specifically expressing the receptors), etc.
  • a nucleic acid-ligand complex can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation.
  • the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publications WO 9206180 dated April 16, 1992; WO 92/22635 dated December 23, 1992; WO92/20316 dated November 26, 1992; WO93/14188 dated July 22, 1993; WO 93/20221 dated October 14, 1993).
  • the nucleic acid can be introduced intracellularly and inco ⁇ orated within host cell DNA for expression, by homologous recombination (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438).
  • a viral vector that contains the Clasp-1 nucleic acid is used.
  • a retroviral vector can be used (see Miller et al., 1993, Meth. Enzymol. 217:581-599). These retroviral vectors have been modified to delete retroviral sequences that are not necessary for packaging ofthe viral genome and integration into host cell DNA.
  • the Clasp-1 nucleic acid to be used in gene therapy is cloned into the vector, which facilitates delivery of the gene into a patient.
  • retroviral vectors More detail about retroviral vectors can be found in Boesen et al., 1994, Biotherapy 6:291-302, which describes the use of a retroviral vector to deliver the mdrl gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy.
  • Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., 1994, J. Clin. Invest. 93:644-651; Kiem et al., 1994, Blood 83:1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141; and Grossman and Wilson, 1993, Curr. Opin. in Genetics and Devel. 3:110-114.
  • Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson (1993, Current Opinion in Genetics and Development 3:499-503) present a review of adenovirus-based gene therapy.
  • Adeno-associated virus has also been proposed for use in gene therapy (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med. 204:289-300.
  • Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, iipofection, calcium phosphate mediated transfection, or viral infection.
  • the method of transfer includes the transfer of a selectable marker to the cells.
  • the cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene. Those cells are then delivered to a patient.
  • the nucleic acid is introduced into a cell prior to administration in vivo of the resulting recombinant cell.
  • Such introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcell- mediated gene transfer, spheroplast fusion, etc.
  • Numerous techniques are known in the art for the introduction of foreign genes into cells (see e.g., Loeffler and Behr, 1993, Meth. Enzymol. 217:599-618; Cohen et al., 1993, Meth. Enzymol. 217:618-644; Cline, 1985, Pharmac. Ther.
  • the technique should provide for the stable transfer of the nucleic acid to the cell, so that the nucleic acid is expressible by the cell and preferably heritable and expressible by its cell progeny.
  • the resulting recombinant cells can be delivered to a patient by various methods known in the art.
  • epithelial cells are injected, e.g., subcutaneously.
  • recombinant skin cells may be applied as a skin graft onto the patient.
  • Recombinant blood cells e.g., hematopoietic stem or progenitor cells
  • the amount of cells envisioned for use depends on the desired effect, patient state, etc., and can be determined by one skilled in the art.
  • Cells into which a nucleic acid can be introduced for pu ⁇ oses of gene therapy encompass any desired, available cell type, and include but are not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, e.g., as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc.
  • the cell used for gene therapy is autologous to the patient.
  • the nucleic acid to be introduced for pu ⁇ oses of gene therapy comprises an inducible promoter operably linked to the coding region, such that expression of the nucleic acid is controllable by controlling the presence or absence of the appropriate inducer of transcription.
  • Oligonucleotide sequences that include anti-sense RNA and DNA molecules and ribozymes that function to inhibit the translation of a Clasp-1 mRNA are within the scope of the invention. Such molecules are useful in cases where downregulation of
  • Anti-sense RNA and DNA molecules act to directly block the translation of mRNA by binding to targeted mRNA and preventing protein translation.
  • antisense DNA oligodeoxyribonucleotides derived from the translation initiation site, e.g., between -10 and +10 regions of a Clasp-1 nucleotide sequence, are preferred.
  • the antisense oligonucleotide may comprise at least one modified base moiety which is selected from the group including, but not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta- D-mannosyl
  • Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA.
  • ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
  • engineered hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of Clasp-1 RNA sequences.
  • ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences, GUA, GUU and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for predicted structural features such as secondary structure that may render the oligonucleotide sequence unsuitable. The suitability of candidate targets may also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using ribonuclease protection assays.
  • Endogenous target gene expression can also be reduced by inactivating or "knocking out” the target gene or its promoter using targeted homologous recombination (e.g., see Smithies, et al, 1985, Nature 317:230-234; Thomas and Capecchi, 1987, Cell 51:503-512; Thompson, et al, 1989, Cell 5:313-321; each of which is inco ⁇ orated by reference herein in its entirety).
  • targeted homologous recombination e.g., see Smithies, et al, 1985, Nature 317:230-234; Thomas and Capecchi, 1987, Cell 51:503-512; Thompson, et al, 1989, Cell 5:313-321; each of which is inco ⁇ orated by reference herein in its entirety.
  • a mutant, nonfunctional target gene flanked by DNA homologous to the endogenous target gene (either the coding regions or regulatory regions of the target gene) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express the target gene in vivo. Insertion of the DNA construct, via targeted homologous recombination, results in inactivation of the target gene.
  • ES embryonic stem
  • Such approaches are particularly suited in the agricultural field where modifications to ES (embryonic stem) cells can be used to generate animal offspring with an inactive target gene (e.g., see Thomas and Capecchi, 1987 and Thompson, 1989, supra).
  • this approach can be adapted for use in humans provided the recombinant DNA constructs are directly administered or targeted to the required site in vivo using appropriate viral vectors.
  • endogenous target gene expression can be reduced by targeting deoxyribonucleotide sequences complementary to the regulatory region of the target gene (i.e., the target gene promoter and/or enhancers) to form triple helical structures that prevent transcription of the target gene in target cells in the body.
  • deoxyribonucleotide sequences complementary to the regulatory region of the target gene i.e., the target gene promoter and/or enhancers
  • triple helical structures that prevent transcription of the target gene in target cells in the body.
  • Nucleic acid molecules to be used in triplex helix formation for the inhibition of transcription should be single stranded and composed of deoxynucleotides.
  • the base composition of these oligonucleotides must be designed to promote triple helix formation via Hoogsteen base pairing rules, which generally require sizeable stretches of either purines or pyrimidines to be present on one strand of a duplex.
  • Nucleotide sequences may be pyrimidine-based, which will result in TAT and CGC triplets across the three associated strands ofthe resulting triple helix.
  • the pyrimidine-rich molecules provide base complementarily to a purine-rich region of a single strand ofthe duplex in a parallel orientation to that strand.
  • nucleic acid molecules may be chosen that are purine-rich, for example, contain a stretch of G residues. These molecules will form a triple helix with a DNA duplex that is rich in GC pairs, in which the majority ofthe purine residues are located on a single strand ofthe targeted duplex, resulting in GGC triplets across the three strands in the triplex.
  • the potential sequences that can be targeted for triple helix formation may be increased by creating a so called "switchback" nucleic acid molecule.
  • Switchback molecules are synthesized in an alternating 5'-3 ⁇ 3-5' manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex.
  • the anti-sense RNA and DNA molecules, ribozymes and triple helix molecules ofthe invention may be prepared by any method known in the art for the synthesis of RNA molecules. These include techniques for chemically synthesizing oligodeoxyri- bonucleotides well known in the art such as for example solid phase phosphoramidite chemical synthesis.
  • RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule.
  • DNA sequences may be inco ⁇ orated into a wide variety of vectors which contain suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters.
  • antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.
  • DNA molecules may be introduced as a means of increasing intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences of ribo- or deoxy- nucleotides to the 5' and/or 3' ends ofthe molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the oligodeoxyribonucleotide backbone.
  • Methods for introducing polynucleotides into such cells or tissue include methods for in vitro introduction of polynucleotides such as the insertion of naked polynucleotide, i.e., by injection into tissue, the introduction of a Clasp-1 polynucleotide in a cell ex vivo, the use of a vector such as a virus, (retrovirus, adenovirus, adeno-associated virus, etc.), phage or plasmid, etc. or techniques such as electroporation or calcium phosphate precipitation.
  • the subject gene may be employed for producing all or portions of Clasp-1 polypeptides.
  • Fragments of interest include glycosylation sites, which may affect the stability and/or activity of the polypeptide, the protein interaction sites, etc.
  • Such domains will usually include at least about 20 amino acids of the provided sequence, more usually at least about 50 amino acids, and may include 100 amino acids or more, up to the complete domain.
  • Binding contacts may be comprised of non-contiguous sequences, which are brought into proximity by the tertiary structure ofthe protein. The sequence of such fragments may be modified through manipulation of the coding sequence, as described above. Truncations may be performed at the carboxy or amino terminus of the fragment, e.g. to determine the minimum sequence required for biological activity.
  • a polypeptide of particular interest comprises the mature portion of the Clasp-1 protein, i.e. the fragment that remain after cleavage of the signal peptide, or the propeptide sequence. Determination of this cleavage site may be determined experimentally, by producing the polypeptide in an expression system capable such cleavage, and then determining the terminus of the mature protein. Alternatively, the cleavage site may be determined by deduction, after comparison with known cleavage sites. For example, the cleavage site of the mouse Clasp-1 polypeptide is between residue 120 and 121. In the human homolog, a putative cleavage site (arg pro gin arg) is between residues 104 and 105.
  • Assays for the biological activity of the protein or fragments thereof may be determined as described in the art. Numerous in vitro assays for determining lymphocyte activation are known in the art, or as provided in the Examples. Inhibition of cellular adhesion and cell-cell contacts, is determined through in vivo or in vitro models (for reviews, see Fukuda (1995) Bioorg Med Chem 3(3):207-215; Zanetta et al. (1994) Histol Histopathol 9(2):385-412).
  • the protein may be isolated and purified in accordance with conventional ways.
  • a lysate may be prepared of the expression host and the lysate purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique.
  • the purified protein will generally be at least about 80% pure, preferably at least about 90% pure, and may be up to and including 100% pure. Pure is intended to mean free of other proteins, as well as cellular debris.
  • the Clasp-1 protein is expressed in lymphocytes, and is specifically localized at the interface of T cell-B cell interactions. Therefore, a soluble Clasp-1, a Clasp-1 fragment containing an extracellular domain or an anti-Clasp-1 antibody may be used to inhibit T cell-B cell interactions, thereby inhibiting an immune response. It is believed that the involvement of Clasp-1 in T cell-B cell contact occurs prior to B cell activation by the TH, thus inhibition of Clasp-1 binding can interfere with an early stage of the immune response.
  • Autoimmune disorders that may be treated by disrupting Clasp-1 function, include, but are not limited to, multiple sclerosis, juvenile diabetes, rheumatoid arthritis, pemphigus, pemphigoid, epidermolysis bullosa acquista, lupus, Rh incompatibility, etc.
  • Clasp-1 contains domains capable of transducing an intracellular signal
  • cell surface Clasp-1 may be triggered by an anti-Clasp- 1 antibody or soluble Clasp-1 or a fragment thereof in order to enhance the activation state of a lymphocyte.
  • a Clasp-1 polypeptide, a fragment thereof or an anti-Clasp-1 antibody may be administered to a subject per se or in the form of a pharmaceutical or therapeutic composition.
  • Pharmaceutical compositions comprising the proteins of the invention may be manufactured by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • Pharmaceutical compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries which facilitate processing ofthe protein or active peptides into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • proteins of the invention may be formulated as solutions, gels, ointments, creams, suspensions, etc. as are well-known in the art.
  • Systemic formulations include those designed for administration by injection, e.g. subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal injection, as well as those designed for transdermal, transmucosal, oral or pulmonary administration.
  • the proteins of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution,
  • Ringer's solution or physiological saline buffer.
  • the solution may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the proteins may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art.
  • a composition can be readily formulated by combining the proteins with pharmaceutically acceptable carriers well known in the art. Such carriers enable the proteins to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
  • suitable excipients include fillers such as sugars, such as lactose, sucrose, mannitol and sorbitol; cellulose preparations such as maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP); granulating agents; and binding agents.
  • disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • solid dosage forms may be sugar-coated or enteric-coated using standard techniques.
  • suitable carriers, excipients or diluents include water, glycols, oils, alcohols, etc. Additionally, flavoring agents, preservatives, coloring agents and the like may be added.
  • the proteins may take the form of tablets, lozenges, etc. formulated in conventional manner.
  • the proteins for use according to the present invention are conveniently delivered in the form of an aerosol spray from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • the proteins may also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, e.g, containing conventional suppository bases such as cocoa butter or other glycerides.
  • the proteins may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
  • the proteins may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • Liposomes and emulsions are well known examples of delivery vehicles that may be used to deliver the proteins or peptides ofthe invention.
  • Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity.
  • the proteins may be delivered using a sustained-release system, such as semipermeable matrices of solid polymers containing the therapeutic agent.
  • sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the proteins for a few weeks up to over 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed.
  • proteins and peptides ofthe invention may contain charged side chains or termini, they may be included in any ofthe above-described formulations as the free acids or bases or as pharmaceutically acceptable salts.
  • Pharmaceutically acceptable salts are those salts which substantially retain the biologic activity of the free bases and which are prepared by reaction with inorganic acids. Pharmaceutical salts tend to be more soluble in aqueous and other protic solvents than are the corresponding free base forms.
  • Clasp-1 polypeptides, Clasp-1 fragments and anti-Clasp-1 antibodies will generally be used in an amount effective to achieve the intended pu ⁇ ose.
  • the proteins of the invention, or pharmaceutical compositions thereof, are administered or applied in a therapeutically effective amount.
  • therapeutically effective amount is meant an amount effective ameliorate or prevent the symptoms, or prolong the survival of, the patient being treated. Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.
  • a therapeutically effective dose can be estimated initially from in vitro assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the C ⁇ as determined in cell culture (i.e., the concentration of test compound that inhibits 50% of Clasp-1 binding interactions). Such information can be used to more accurately determine useful doses in humans.
  • Initial dosages can also be estimated from in vivo data, e.g., animal models, using techniques that are well known in the art. One having ordinary skill in the art could readily optimize administration to humans based on animal data.
  • Dosage amount and interval may be adjusted individually to provide plasma levels of the proteins which are sufficient to maintain therapeutic effect.
  • Usual patient dosages for administration by injection range from about 0.1 to 5 mg/kg/day, preferably from about 0.5 to 1 mg/kg/day.
  • Therapeutically effective serum levels may be achieved by administering multiple doses each day.
  • the effective local concentration of the proteins may not be related to plasma concentration.
  • One having skill in the art will be able to optimize therapeutically effective local dosages without undue experimentation.
  • the amount of Clasp-1 administered will, of course, be dependent on the subject being treated, on the subject's weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.
  • the therapy may be repeated intermittently while symptoms detectable or even when they are not detectable.
  • the therapy may be provided alone or in combination with other drugs.
  • the drugs that may be used in combination with Clasp-1 or fragments thereof include, but are not limited to, steroid and non-steroid immunosuppressive agents.
  • a therapeutically effective dose of the proteins described herein will provide therapeutic benefit without causing substantial toxicity.
  • Toxicity of the proteins described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD ⁇ (the dose lethal to 50% of the population) or the LD 100 (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effect is the therapeutic index.
  • the data obtained from these cell culture assays and animal studies can be used in formulating a dosage range that is not toxic for use in human.
  • the dosage of the proteins described herein lies preferably within a range of circulating concentrations that include the effective dose with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view ofthe patient's condition. (See, e.g., Fingl et al, 1975, In: The Pharmacological Basis of Therapeutics. Ch.1, p.1).
  • ⁇ n vitro studies may use purified Clasp-1 macromolecules to screen large compound libraries for inhibitory drugs; or the purified target molecule may be used for a rational drug design program, which requires first determining the structure of the target or, the structure of the macromolecular target in association with its customary substrate or ligand. This information is then used to design inhibitory compounds which must be synthesized and tested further. Test results are used to refine the molecular models and drug design process in an iterative fashion until a lead compound emerges. Drug screening may be performed using an in vitro model, a genetically altered cell, or purified protein. One can identify ligands or substrates that bind to, modulate or mimic the action of the target genetic sequence or its product.
  • a wide variety of assays may be used for this pu ⁇ ose, including labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, and the like.
  • the purified protein may also be used for determination of three-dimensional crystal structure, which can be used for modeling intermolecular interactions.
  • agent as used herein describes any molecule, e.g. protein or pharmaceutical, with the capability of altering or mimicking the physiological function of RAB.
  • agent e.g. protein or pharmaceutical, with the capability of altering or mimicking the physiological function of RAB.
  • a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a differential response to the various concentrations.
  • one of these concentrations serves as a negative control, i.e. at zero concentration or below the level of detection.
  • Candidate agents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 50 and less than about 2,500 daltons.
  • Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups.
  • the candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
  • Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.
  • Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.
  • the screening assay is a binding assay
  • the label can directly or indirectly provide a detectable signal.
  • Various labels include radioisotopes, fluorescers, chemiluminescers, enzymes, specific binding molecules, particles, e.g. magnetic particles, and the like.
  • Specific binding molecules include pairs, such as biotin and streptavidin, digoxin and antidigoxin etc.
  • the complementary member would normally be labeled with a molecule that provides for detection, in accordance with known procedures.
  • reagents may be included in the screening assay. These include reagents like salts, neutral proteins, e.g. albumin, detergents, etc that are used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Reagents that improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc. may be used. The mixture of components are added in any order that provides for the requisite binding. Incubations are performed at any suitable temperature, typically between 4 and 40° C. Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high-throughput screening. Typically between 0.1 and 1 hours will be sufficient.
  • the compounds having the desired biological activity may be administered in a an acceptable carrier to a host for treatment of fungal infection, or prevention of infection, etc.
  • the inhibitory agents may be administered in a variety of ways. Depending upon the manner of introduction, the compounds may be formulated in a variety of ways.
  • the concentration of therapeutically active compound in the formulation may vary from about 0.01-100 wt.%.
  • RNA was primed with oligo dT and reverse transcribed with MMTV reverse transcriptase (BRL, NY) according to the method of Suzuki etal, (1988, Cell Regul. 2: 807-16).
  • the cDNA was then used for hot start (Ampli-wax, Perkin Elmer, CA) Polymerase Chain Reaction (PCR) in Promega PCR buffer (Promega, WI) containing Mg (1.5-3.0 mM), 1 ⁇ g primer each, 2.5 units of AmpliTaq (Perkin Elmer, CA).
  • the Perkin Elmer Thermocycler (Perkin Elmer, CA) was set to 94°C for a 30 second denaturation, 37°C for a 2 minute annealing, and ramped over 2 minutes to 65°C, which was maintained for a 3 minute extension reaction, for 35 cycles.
  • the PCR products were resolved in 3% NuSieve agarose (FMC, ME): 1% agarose (BRL, NY).
  • the band of predicted size was excised, purified using Sephaglas (Pharmacia, NJ), and reamplified for 20 rounds using the same program.
  • the final product was gel purified, digested with EcoRI and Xbal (New England Biolabs, MA), and cloned into pBluescript KS (Stratagene, La Jolla, CA) at the corresponding sites, and its nucleotide sequence determined. Half of the PCR clones sequenced were identical. A representative clone was used to screen a mouse neonatal thymus library to obtain a complete cDNA sequence designated Clasp-1.
  • the full length cDNA clone-(SEQ ID NO:2) contains an open reading frame that encodes a 1,289 amino acid type I transmembrahe protein which shares a number of cadherin features, including the cadherin proteolytic processing signal (Pigott and Power, 1993, The Adhesion Molecule Facts Book, Academic Press Limited), glycosylation sites, a cluster of cysteines proximal to the transmembrane domain (Hofman and Stoffel, 1993, Biol. Chem. Hoppe- Seyler 374: 166), and a cytoplasmic domain that exhibits several cadherin sequence motifs (Figure 2).
  • the processed polypeptide begins at amino acid residue #121 after the amino acid sequence RAQR ( Figure 1a). This gene was named Clasp-1 for cadherin-like asymmetry protein.
  • Clasp-1 may provide a direct interaction between signal transduction pathways and the cytoskeleton. FASTA searches of Clasp-1 (Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85: 2444-48; Ladunga et al., 1996, J. Mol. Biol. 259: 840-54) revealed similarities with two cDNAs of unknown function, the rat RNTRG (GenBank X68101) and a putative C.
  • Clasp-1 is a cell surface cadherin-like protein that contains domains known to be involved in signal transduction pathways.
  • Example 2 Expression Pattern of Clasp-1 And Cellular Localization of its Encoded Product Th1 cells (5CC7), pro-GMB cells (HAFTLJ), pre-B cells (NFS40, HSIC5, BAC14), mature B cells (BAL17), and plasmacytomas (S194, J598L) were maintained as cultured cell lines. RNA were extracted from these cells for Northern blot analysis.
  • Northern blot analysis Ten micrograms ( ⁇ g) total RNA from each cell sample were loaded onto a 1% agarose formaldehyde gel, transferred onto BioBlot nitrocellulose paper (Costar, MA) and crosslinked with Stratalink (Stratagene, La Jolla, CA). Prehybridization and hybridization were performed in 50% formamide, 25mM sodium phosphate (pH 6.5), 1X Denhardt's solution, 200 ⁇ g/ml herring sperm DNA, and 5X SSC, at 65°C.
  • Probes corresponding to the coding sequence of Clasp-1 were prepared using Ready-To-Go Labeling Kit (Pharmacia, NJ) according to the manufacturer's instructions, and desalted using pasteur pipet G-50 Sephadex column in TEN (10 mM Tris-HCl, pH 8, 1 mM EDTA, and 100 mM NaCl). The final wash ofthe blots was in 0.1X SCC at 60°C. Autoradiography was performed on Kodak XOMAT film at -80°C with an enhancing screen.
  • Cytospin and immunofluorescence Cells were cytospun (Cytospin 1 , Shandon]) onto poly-L-lysine (Sigma #P2636, MA) slides, fixed in periodate-lysine- paraformaldehyde (McLean and Nakane, 1974, J. Histochem. Cytochem. 22: 1077-83). Primary antibodies were added at 20-30 ⁇ g/ml in 10% normal donkey serum, TBS-C (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM CaCI 2 ), and 0.4% saponin, and incubated overnight at 4°C.
  • the first step was Gamma-bind purified goat antisera in 10% normal mouse serum, 10% normal rat serum, TBS-C for overnight at 4°C in a humidifying chamber; the second step was biotin-conjugated monoclonal anti-goat antibody (Sigma, MO) at 1/50 dilution for 2 hours at room temperature; and the last step was strepavidin-PE (Molecular Probes, OR) at 1/50 for 1 hour at room temperature. A FITC-conjugated anti-B200 or anti-CD3 antibody was also added at the last step.
  • Sections were incubated in 100 ⁇ l of the primary antibody in TBS-C + 25% normal goat serum overnight at 4°C and washed three times in TBS-C.
  • One hundred ⁇ l of a secondary antibody (Jackson Immunoresearch, PA) was added for two hours at room temperature. Stained sections were examined under Nikon Biophot or Zeiss Axiophot fluorescent microscope and photographs were taken using Kodak Elite ASA 100.
  • lymphoid tissues thymus, spleen, lymph nodes and bone marrow
  • a 13 kb Clasp-1 transcript was detected.
  • the same transcript was also observed in the brain, but was missing from the liver, lung, muscle, kidney, and skin ( Figure 3a).
  • Further analysis in lymphoid cell lines indicated that the Clasp-1 transcript was present in lymphocytes of both T and B cell lineage ( Figure 3b).
  • the transcript was either absent or minimally expressed in several plasmacytoma lines (S194 and J558L), suggesting that the gene may be turned off in terminally differentiated B cells.
  • T cells (D10) grown in the absence of antigen also exhibited the same surface polar distribution (Figure 4i), as well as B cells (CH27, Figure 4j), indicating that the apical grouping was not the result of antigen-induced crosslinking, but was an inherent property of Clasp-1 itself.
  • T cells (D10 and 2B4) were stained for both ⁇ -tubulin (green) and Clasp-I (red).
  • the apical Clasp-I structure was always observed on the same side of the nucleus (blue) as the microtubule- organizing center ( Figure 4k). For most cell types examined, the centrisome was always on the same side of the nucleus as the leading edge, indicating that Clasp-1 pole was associated with the leading edge.
  • splenic lymphocytes from 3A9 mice transgenic for TCR to hen egg lysozyme (HEL 46-61 peptide:l-A k ) (Ho et al, J. Exp. Med. 179: 1539-49), were cultured in the presence of the HEL peptide and allowed to form conjugate T-B cell pairs (Sagerstrom et al., 1993, Proc. Natl. Acad. Sci. USA 90: 8987-91). Cultured cells were removed at 4, 10 and 36 hours.
  • T-B cell pairs were observed by 4 hours, and by 10 hours more than 95% of the tight T-B cell pairs demonstrated early blast transformation (loss of nuclear membrane definition and heterochromatin).
  • Clasp-1 was always found at the cell-cell interface ( Figure 5a EXAMPLE 3 INHIBITION OF T CELL-B CELL COUPLING BY ANTI-Clasp-1 ANTIBODY
  • a coding sequence for amino acid residues 121- 327 of Clasp-1 was cloned into pGEX-4T-1 (Pharmacia, NJ) at the BamHI /Not 1 site and it was referred to as GST-Clasp-EC 12A.
  • GST-Clasp-cyto was a construct that contained DNA encoding amino acid residues 969-1289 of Clasp-1 cloned into pGEX-4T-3 (Pharmacia, N J) at the Notl/EcoRI sites.
  • Fusion proteins were expressed and purified according to instructions from Pharmacia using glutathione- Sepharose columns (Pharmacia, NJ) and used as immunogens for the generation of rabbit and goat antisera.
  • Antibodies MOMA-1 (Kraal and Janse, 1986, Immunology 58: 665-669) and ER-TR-9 (van Vliet et al., 1985, J. Histochem. Cytochem. 33: 40-44) were used as described.
  • Anti-CD4-FITC, CD8-FITC, CD3-FITC, CD45R (B220)-FITC antibodies were purchased from Caltag, CA.
  • YOL 1/34 was purchased from Sera-Tec, (NC).
  • Cell conjugation and inhibition assay 2B4 or D10 T cells and CH27 B cells (loaded overnight with 10 ⁇ M moth cytochrome c peptide 88-103 or with conalbumin) were resuspended at 3.4 x 10 5 cells/ml in RPMI + 10% FCS. 120 ⁇ l of each cell type were mixed in a 8 well coverslip slide chamber (Nunc, NY) and Gammabind (Pharmacia, NY) purified goat anti-Clasp-EC 12A or anti-Clasp-cyto antibodies were added to a final concentration of 0, 50, and 150 ⁇ g/ml (pre-immune serum up to 450 ⁇ g/ml).
  • Antibodies were generated against Clasp-1 fusion proteins.
  • Western blot analysis was performed using extracts from CH 27 (a mature B cell line) and 2B4 (a T cell hybridoma)
  • antibodies raised against GST-fusion proteins containing either the extracellular domain (Clasp-EC12A) or the cytoplasmic domain (Clasp-cyto) identified a band of about 130 kD molecular weight, which was consistent with the deduced molecular weight of 134 kD ( Figure 3c) of Clasp-1.

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