+

WO1999037777A2 - Sequences associees a une differentiation et methodes d'utilisation - Google Patents

Sequences associees a une differentiation et methodes d'utilisation Download PDF

Info

Publication number
WO1999037777A2
WO1999037777A2 PCT/US1999/001624 US9901624W WO9937777A2 WO 1999037777 A2 WO1999037777 A2 WO 1999037777A2 US 9901624 W US9901624 W US 9901624W WO 9937777 A2 WO9937777 A2 WO 9937777A2
Authority
WO
WIPO (PCT)
Prior art keywords
polypeptide
differentiation
cancer
patient
polynucleotide
Prior art date
Application number
PCT/US1999/001624
Other languages
English (en)
Other versions
WO1999037777A3 (fr
Inventor
Fei Huang
Paul B. Fisher
Neil I. Goldstein
Original Assignee
Genquest, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Genquest, Inc. filed Critical Genquest, Inc.
Priority to AU23433/99A priority Critical patent/AU2343399A/en
Publication of WO1999037777A2 publication Critical patent/WO1999037777A2/fr
Publication of WO1999037777A3 publication Critical patent/WO1999037777A3/fr

Links

Classifications

    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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

Definitions

  • the present invention relates generally to compounds and methods for inhibiting cell growth and for cancer therapy.
  • the invention is more specifically related to polypeptides and polynucleotides associated with terminal differentiation and growth arrest. Such molecules may be used, for example, to identify agents that may be used in vaccines and pharmaceutical compositions for cancer therapy.
  • the polypeptides and polynucleotides may also be used as markers for diagnosing and monitoring the progression of a cancer in a patient.
  • Cancer is a significant health problem in the United States and throughout the world. Although advances have been made in detection and treatment of cancer, no vaccine or other universally successful method for cancer prevention or treatment is currently available. Management of the disease currently relies on a combination of early diagnosis and aggressive treatment, which may include one or more of a variety of treatments such as surgery, radiotherapy, chemotherapy and hormone therapy. The course of treatment for a particular cancer is often selected based on a variety of prognostic parameters, including an analysis of specific tumor markers. However, the use of established markers often leads to a result that is difficult to interpret, and the high mortality continues to be observed in many cancer patients.
  • Malignant melanoma is one cancer that is increasingly common in North American populations. It is estimated that 1 in 100 children currently born may eventually develop superficial spreading-type melanoma. Although readily curable at early stages, surgical and chemotherapeutic intervention are virtually ineffective in preventing metastatic disease and death in patients with advanced states of malignant melanoma. Improved therapeutic approaches are needed to effectively treat metastatic melanoma. A potentially useful therapy for this and other malignancies could involve the use of agents capable of inducing an irreversible loss in proliferative capacity in tumor cells without the requirement for direct cytotoxicity (i.e., differentiation therapy).
  • this invention provides polypeptides and polynucleotides associated with terminal differentiation and growth arrest, and methods of using such compounds.
  • the present invention provides isolated polypeptides comprising at least a portion of a differentiation-associated protein, or a variant thereof, wherein: (a) the differentiation-associated protein comprises a sequence encoded by a polynucleotide sequence recited in Figure 1 (SEQ ID NO:l) or Figure 2 (SEQ ID NO:2), or a complement thereof; and (b) the portion retains at least one immunological and/or biological activity characteristic of the differentiation-associated protein.
  • isolated polynucleotides encoding such polypeptides are provided.
  • Such polynucleotides may comprise a sequence recited in Figure 1 (SEQ ID NO:l) or Figure 2 (SEQ ID NO:2).
  • Antisense polynucleotides comprising a sequence complementary to such a polynucleotide are also provided, along with expression vectors comprising such a polynucleotide and host cells transformed or transfected with such an expression vector.
  • monoclonal antibodies, or antigen-binding fragments thereof, that specifically bind to a polypeptide as provided above are provided.
  • the present invention provides pharmaceutical compositions, comprising a polypeptide, polynucleotide or antibody as described above in combination with a physiologically acceptable carrier.
  • the present invention further provides, within other aspects, vaccines comprising a polypeptide, polynucleotide or antibody as described above and an immune response enhancer.
  • the present invention provides methods for inhibiting the development of a cancer in a patient, comprising administering to a patient a pharmaceutical composition or vaccine as described above.
  • the present invention further provides, within other aspects, methods for determining whether a tumor in a patient is malignant, comprising determining the level of a polypeptide or polynucleotide as described above in a tumor sample obtained from a patient, and therefrom determining whether the tumor is malignant.
  • the present invention provides methods for monitoring the progression of a cancer in a patient, comprising: (a) detecting, in a biological sample obtained from a patient, an amount of a polypeptide or RNA molecule as provided above at a first point in time; (b) repeating step (a) at a subsequent point in time; and (c) comparing the amounts of polypeptide detected in steps (a) and (b), and therefrom monitoring the progression of a cancer in the patient.
  • the present invention further provides methods for preparing a polypeptide as described above, comprising the steps of: (a) culturing a host cell as described above under conditions suitable for the expression of the polypeptide; and (b) recovering the polypeptide from the host cell culture.
  • diagnostic kits comprising: (a) a monoclonal antibody or fragment thereof as described above; and (b) a second monoclonal antibody or fragment thereof that binds to (i) a monoclonal antibody recited in step (a); or (ii) a polypeptide as described above; wherein the second monoclonal antibody is conjugated to a reporter group.
  • the present invention further provides, within other aspects, methods for identifying a compound that modulates cell growth and/or differentiation, comprising the steps of: (a) contacting a candidate compound with a polypeptide as described above under conditions and for a time sufficient to allow the candidate compound to bind to the polypeptide; and (b) detecting binding of the candidate compound to the polypeptide, and therefrom identifying a compound that modulates cell growth and/or differentiation.
  • the present invention provides methods for identifying an agent that modulates cell growth and/or differentiation, comprising the steps of: (a) contacting a candidate agent with a cell capable of expressing a polypeptide as described above, under conditions and for a time sufficient to allow the candidate agent and the cell to interact; and (b) determining the effect of the candidate agent on the level of the polypeptide, and therefrom identifying an agent that modulates cell growth and/or differentiation.
  • polynucleotides comprising an endogenous promoter or regulatory element of a differentiation-associated protein, wherein the protein comprises a sequence encoded by a polynucleotide sequence recited in Figure 1 (SEQ ID NO:l) or Figure 2 (SEQ ID NO:2), or a complement thereof.
  • the present invention provides polynucleotides comprising a reporter gene under the control of an endogenous promoter or regulatory element of a differentiation-associated protein, wherein the protein comprises a sequence encoded by a polynucleotide sequence recited in Figure 1 (SEQ ID NO:l) or Figure 2, or a complement thereof, as well as cells transformed or transfected with such a polynucleotide.
  • methods for identifying an agent that modulates the expression of a differentiation-associated protein, comprising the steps of: (a) contacting a candidate agent with a cell as described above under conditions and for a time sufficient to allow the candidate agent to interact with the promoter or regulatory element thereof; and (b) determining the effect of the candidate agent on the level of reporter protein produced by the cell, and therefrom identifying an agent that modulates expression of a differentiation-associated protein.
  • Figure 1 depicts the sequence of a differentiation-associated sequence referred to herein as mda8-A (SEQ ID NO:l).
  • Figure 2 depicts the sequence of a differentiation-associated sequence referred to herein as mda8-B (SEQ ID NO:2).
  • Figure 3 is a Northern blot illustrating the tissue distribution of mda8-A and mda8-B mRNA. Tissues are indicated at the top of the blot.
  • Figure 4 is a histogram illustrating the effect of transfection with DNA encoding mda8-A on a colony formation assay in tumor and non-tumor cell lines.
  • Colony number is shown + standard deviation for T47D tumor cell lines, as well as
  • HBL100 and CREF non-tumor cell lines transfected with mda8-A or vector alone, as indicated.
  • the present invention is generally directed to compounds and methods for inhibiting cell growth and for cancer therapy.
  • the present invention is based, in part, on the identification of "differentiation-associated sequences," which are polypeptides and polynucleotides associated with terminal differentiation and growth arrest (i.e., expression of such sequences increases in cells committed to terminal differentiation).
  • a differentiation-associated mRNA is a mRNA that is present at a greater level in such committed cells than in the corresponding cells not committed to terminal differentiation (i.e., the level of RNA is at least 2-fold higher in tumor tissue).
  • a differentiation-associated cDNA molecule comprises the sequence of a differentiation-associated mRNA (and/or a complementary sequence).
  • Such cDNA molecules may be prepared from RNA or mRNA preparations using standard techniques, such as reverse transcription.
  • a differentiation-associated protein or polypeptide comprises a sequence encoded by a differentiation-associated mRNA.
  • Such polypeptides are generally present at a greater level in cells committed to terminal differentiation than in the corresponding uncommitted cells (i.e., the level of protein is at least 2-fold higher in committed cells).
  • compositions described herein may include one or more polypeptides, nucleic acid sequences and/or antibodies.
  • Polypeptides of the present invention generally comprise at least a portion of a differentiation-associated protein, or a variant thereof.
  • Nucleic acid sequences of the subject invention generally comprise a DNA or RNA sequence that encodes such a polypeptide, or that is complementary to such a coding sequence.
  • Antibodies are generally immune system proteins, or antigen- binding fragments thereof, that are capable of binding to a portion of a polypeptide as described above.
  • a composition may comprise one or more agents that modulate expression of a differentiation-associated gene. Such agents may generally be identified as described herein.
  • any polynucleotide that encodes a differentiation-associated polypeptide, or a portion or variant thereof as described herein, is encompassed by the present invention.
  • Such polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA or synthetic) or RNA molecules. Additional non-coding sequences may, but need not, be present within a polynucleotide of the present invention, and a polynucleotide may, but need not, be linked to other molecules and/or support materials.
  • Differentiation-associated polynucleotides may be prepared using any of a variety of techniques.
  • such a polynucleotide may be amplified from cD A prepared from certain human melanoma cells or from a melanoma cell line (e.g., HO-1 or GMB) induced to terminally differentiate.
  • Such polynucleotides may be amplified via polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • sequence-specific primers may be designed based on the sequences provided in Figures 1-2 (SEQ ID NOs:l and 2), and may be purchased or synthesized.
  • An amplified portion may then be used to isolate a full length gene from a human genomic DNA library or from a melanoma cDNA library, using well known techniques, as described below.
  • a full length gene can be constructed from multiple PCR fragments.
  • cDNA molecules encoding a native differentiation-associated protein, or a portion thereof may also be prepared by screening a cDNA library prepared from, for example, melanoma mRNA (e.g., the HO-1 melanoma cell treated with IFN- ⁇ and MEZ to induce terminal differentiation).
  • melanoma mRNA e.g., the HO-1 melanoma cell treated with IFN- ⁇ and MEZ to induce terminal differentiation.
  • Such libraries may be commercially available, or may be prepared using standard techniques (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 1989, and references cited therein).
  • a library may be a cDNA expression library and may, but need not, be subtracted using well known subtractive hybridization techniques.
  • a screen for differentiated-associated cDNAs may be performed as described in WO 95/11986.
  • cDNA libraries may be screened in microarrays using, for example, chips available from Synteni (Palo Alto, CA).
  • a differentiation-associated cDNA molecule may be sequenced using well known techniques employing such enzymes as Klenow fragment of DNA polymerase I, Sequenase® (US Biochemical Corp., Cleveland OH) Taq polymerase (Perkin Elmer, Foster City CA), thermostable T7 polymerase (Amersham, Chicago, IL) or combinations of recombinant polymerases and proofreading exonucleases such as the ELONGASE Amplification System (Gibco BRL, Gaithersburg, MD).
  • An automated sequencing system may be used, using instruments available from commercial suppliers such as Perkin Elmer and Pharmacia.
  • the sequence of a partial cDNA may be used to identify a polynucleotide sequence that encodes a full length differentiation-associated protein using any of a variety of standard techniques.
  • a library cDNA or genomic
  • a library is screened using one or more polynucleotide probes or primers suitable for amplification.
  • a library is size-selected to include larger molecules. Random primed libraries may also be preferred for identifying 5' and upstream regions of genes. Genomic libraries are preferred for obtaining introns and extending 5' sequence.
  • a partial sequence may be labeled (e.g., by nick-translation or end-labeling with 32 P) using well known techniques.
  • a bacterial or bacteriophage library is then screened by hybridizing filters containing denatured bacterial colonies (or lawns containing phage plaques) with the labeled probe (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 1989). Hybridizing colonies or plaques are selected and expanded, and the DNA is isolated for further analysis.
  • cDNA clones may be analyzed to determine the amount of additional sequence by, for example, PCR using a primer from the partial sequence and a primer from the vector.
  • Restriction maps and partial sequenced may be generated to identify one or more overlapping clones.
  • the complete sequence may then be determined using standard techniques, which may involve generating a series of deletion clones.
  • the resulting overlapping sequences are then assembled into a single contiguous sequence.
  • a full length cDNA molecule can be generated by ligating suitable fragments, using well known techniques.
  • Primers are preferably 22-30 nucleotides in length, have a GC content of at least 50% and anneal to the target sequence at temperatures of about 68°C to 72°C.
  • the amplified region may be sequenced as described above, and overlapping sequences assembled into a contiguous sequence.
  • amplification technique is inverse PCR (see Triglia et al., Nucl. Acids Res. 7(5:8186, 1988), which uses restriction enzymes to generate a fragment in the known region of the gene. The fragment is then circularized by intramolecular ligation and used as a template for PCR with divergent primers derived from the known region.
  • sequences adjacent to a partial sequence may be retrieved by amplification with a primer to a linker sequence and a primer specific to a known region. The amplified sequences are typically subjected to a second round of amplification with the same linker primer and a second primer specific to the known region.
  • EST expressed sequence tag
  • Nucleic acid sequences of partial differentiation-associated cDNA molecules are provided in Figures 1 and 2 (SEQ ID NOs:l and 2). These polynucleotides were isolated based on similarity to the differentiation-associated cDNA mda-8 (see PCT Application WO 95/11986), and are referred to herein as members of the mda-8 gene family. One such polynucleotide is referred to herein as mda8-A (SEQ ID NO:l) and another is mda8-B (SEQ ID NO:2).
  • polynucleotides recited herein, as well as full length polynucleotides comprising such sequences, other portions of full length polynucleotides, and sequences complementary to all or a portion of such full length molecules, are specifically encompassed by the present invention. Additional differentiation associated sequences may be identified based on similarity to any of the above sequences.
  • Polynucleotide variants may contain one or more substitutions, deletions, insertions and/or modifications such that the antigenic, immunogenic and/or biological properties of the encoded polypeptide are not diminished. The effect on the properties of the encoded polypeptide may generally be assessed as described herein.
  • Preferred variants contain nucleotide substitutions, deletions, insertions and/or modifications at no more than 20%, preferably at no more than 10%, of the nucleotide positions. Certain variants are substantially homologous to a native gene, or a potion or complement thereof.
  • Such polynucleotide variants are capable of hybridizing under moderately stringent conditions to a naturally occurring DNA sequence encoding a differentiation- associated protein (or a complementary sequence).
  • Suitable moderately stringent conditions include prewashing in a solution of 5 X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50°C-65°C, 5 X SSC, overnight; followed by washing twice at 65° C for 20 minutes with each of 2X, 0.5X and 0.2X SSC containing 0.1% SDS).
  • Such hybridizing DNA sequences are also within the scope of this invention.
  • polynucleotide sequences that encode a polypeptide as described herein. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any native gene. Nonetheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present invention. As noted above, portions of any of the above sequences are also contemplated by the present invention. Such polynucleotides may generally be prepared by any method known in the art, including chemical synthesis by, for example, solid phase phosphoramidite chemical synthesis.
  • RNA molecules may be generated by in vitro or in vivo transcription of DNA sequences encoding a differentiation-associated protein, or a portion thereof, provided that the DNA is incorporated into a vector with a suitable RNA polymerase promoter (such as T7 or SP6). Certain portions may be used to prepare an encoded polypeptide, as described herein. In addition, or alternatively, a portion may function as a probe (e.g., for diagnostic purposes), and may be labeled by a variety of reporter groups, such as radionuclides and enzymes. Such portions are preferably at least 10 nucleotides in length, more preferably at least 20 nucleotides in length and still more preferably at least 30 nucleotides in length.
  • a portion of a sequence complementary to a coding sequence may also be used as a probe or to modulate gene expression.
  • cDNA constructs that can be transcribed into antisense RNA may also be introduced into cells of tissues to facilitate the production of antisense RNA.
  • An antisense polynucleotide may be used, as described herein, to inhibit expression of a differentiated-associated gene.
  • Antisense technology can be used to control gene expression through triple-helix formation, which compromises the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors or regulatory molecules (see Gee et al., In Huber and Carr, Molecular and Immunologic Approaches, Futura Publishing Co.
  • an antisense molecule may be designed to hybridize with a control region of a gene (e.g., promoter, enhancer or transcription initiation site), and block transcription of the gene; or to block translation by inhibiting binding of a transcript to ribosomes.
  • a control region of a gene e.g., promoter, enhancer or transcription initiation site
  • Any polynucleotide may be further modified to increase stability in vivo. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends; the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages in the backbone; and/or the inclusion of nontraditional bases such as inosine, queosine and wybutosine, as well as acetyl- methyl-, thio- and other modified forms of adenine, cytidine, guanine, thymine and uridine.
  • Nucleotide sequences as described herein may be joined to a variety of other nucleotide sequences using established recombinant DNA techniques.
  • a polynucleotide may be cloned into any of a variety of cloning vectors, including plasmids, phagemids, lambda phage derivatives and cosmids.
  • Vectors of particular interest include expression vectors, replication vectors, probe generation vectors and sequencing vectors.
  • a vector will contain an origin of replication functional in at least one organism, convenient restriction endonuclease sites and one or more selectable markers. Other elements will depend upon the desired use, and will be apparent to those of ordinary skill in the art.
  • polynucleotides may be formulated so as to permit entry into a cell of a mammal, and expression therein. Such formulations are particularly useful for therapeutic purposes, as described below.
  • a polynucleotide may be incorporated into a viral vector such as, but not limited to, adenovirus, adeno-associated virus, retrovirus, or vaccinia or other pox virus (e.g., avian pox virus). Techniques for incorporating DNA into such vectors are well known to those of ordinary skill in the art.
  • a retroviral vector may additionally transfer or incorporate a gene for a selectable marker (to aid in the identification or selection of transduced cells) and/or a targeting moiety, such as a gene that encodes a ligand for a receptor on a specific target cell, to render the vector target specific. Targeting may also be accomplished using an antibody, by methods known to those of ordinary skill in the art.
  • Other therapeutic formulations for polynucleotides include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • a preferred colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (i.e., an artificial membrane vesicle). The preparation and use of such systems is well known in the art.
  • a promoter of a differentiation-associated protein may be isolated using standard techniques.
  • the present invention provides nucleic acid molecules comprising such a promoter or one or more cis- or trans-acting regulatory elements thereof. Such regulatory elements may activate or suppress expression of the differentiation-associated protein.
  • One method for identifying a promoter region uses a PCR-based method to clone unknown genomic DNA sequences adjacent to a known cDNA sequence (e.g., a human PromoterFinderTMDNA Walking Kit, available from Clontech). This approach may generate a 5' flanking region, which may be subcloned and sequenced using standard methods. Primer extension and/or RNase protection analyses may be used to verify the transcriptional start site deduced from the cDNA.
  • putative promoter inserts of varying sizes may be subcloned into a heterologous expression system containing a suitable reporter gene without a promoter or enhancer may be employed.
  • suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase or the Green Fluorescent Protein gene, and may be generated using well known techniques Internal deletion constructs may be generated using unique internal restriction sites or by partial digestion of non-unique restriction sites. Constructs may then be transfected into cells that display high levels of differentiation-associated protein expression In general, the construct with the minimal 5' flanking region showing the highest level of expression of reporter gene is identified as the promoter.
  • Cis-acting sequences may generally be identified based on homology to previously characterized transcriptional motifs. Point mutations may then be generated within the identified sequences to evaluate the regulatory role of such sequences. Such mutations may be generated using site-specific mutagenesis techniques or a PCR-based strategy. The altered promoter is then cloned into a reporter gene expression vector, as described above, and the effect of the mutation on reporter gene expression is evaluated. Trans-acting factors that bind to cis-acting sequences may be identified using assays such as gel shift assays.
  • Proteins displaying binding activity within such assays may be partially digested, and the resulting peptides separated and sequenced. Peptide sequences may be used to design degenerate primers for use within RT-PCR to identify cDNAs encoding the trans-acting factors. DIFFERENTIATION-ASSOCIATED POLYPEPTIDES
  • Polypeptides within the scope of the present invention comprise at least a portion of a differentiation-associated protein or variant thereof, such that the portion is immunologically and/or biologically active.
  • Such polypeptides may be of any length, including a full length protein, an oligopeptide (i.e., consisting of a relatively small number of amino acid residues, such as 8-10 residues, joined by peptide bonds), or a peptide of intermediate length.
  • a polypeptide may further comprise additional sequences, which may or may not be derived from a native differentiation-associated protein. Such sequences may (but need not) possess immunogenic properties and/or a biological activity.
  • a polypeptide is "immunologically active," within the context of the present invention if it is recognized (i.e., specifically bound) by a B-cell and/or T-cell surface antigen receptor. Immunological activity may generally be assessed using well known techniques, such as those summarized in Paul, Fundamental Immunology, 3rd ed., 243-247 (Raven Press, 1993) and references cited therein. Such techniques include screening polypeptides derived from the native polypeptide for the ability to react with antigen-specific antisera and/or T-cell lines or clones, which may be prepared using well known techniques.
  • An immunologically active portion of a differentiation- associated protein reacts with such antisera and/or T-cells at a level that is not substantially lower than the reactivity of the full length polypeptide (e.g., in an ELISA and/or T-cell reactivity assay).
  • Such screens may generally be performed using methods well known to those of ordinary skill in the art, such as those described in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988.
  • B-cell and T-cell epitopes may also be predicted via computer analysis.
  • immunogenic portions may be identified using computer analysis, such as the Tsites program (see Rothbard and Taylor, EMBO J. 7:93-100, 1988; Deavin et al., Mol. Immunol.
  • CTL peptides with motifs appropriate for binding to murine and human class I or class II MHC may be identified according to BIMAS (Parker et al., J. Immunol. 752:163, 1994) and other HLA peptide binding prediction analyses.
  • a peptide may be tested using an HLA A2 transgenic mouse model and/or an in vitro stimulation assay using dendritic cells, fibroblasts or peripheral blood cells.
  • a polypeptide is "biologically active" if it possesses one or more structural, regulatory and/or biochemical functions of the native differentiation- associated protein.
  • a biological activity may be assessed using well known methods. For example, sequence comparisons may indicate a particular biological activity for the protein. Appropriate assays designed to evaluate the activity may then be designed based on existing assays known in the art.
  • a differentiation-associated protein may inhibit growth and induce terminal differentiation in a human melanoma or glioblastoma multiforme tumor. Certain portions and other variants of such proteins should also exhibit this property, within an in vivo or in vitro assay.
  • polypeptides may comprise one or more portions of a variant of an endogenous protein, where the portion is immunologically and/or biologically active (i.e., the portion exhibits one or more antigenic, immunogenic and/or biological properties characteristic of the full length protein).
  • a portion is at least as active as the full length protein within one or more assays to detect such properties.
  • a polypeptide "variant,” as used herein, is a polypeptide that differs from a native protein in substitutions, insertions, deletions and/or amino acid modifications, such that the antigenic, immunogenic and/or biological properties of the native protein are not substantially diminished.
  • a variant preferably retains at least 80% sequence identity to a native sequence, more preferably at least 90% identity, and even more preferably at least 95% identity.
  • Guidance in determining which and how many amino acid residues may be substituted, inserted, deleted and/or modified without diminishing immunological and/or biological activity may be found using any of a variety of computer programs known in the art, such as DNAStar software.
  • Properties of a variant may generally be evaluated by assaying the reactivity of the variant with antisera and/or T-cells as described above and/or evaluating a biological property characteristic of the native protein.
  • a variant contains conservative substitutions.
  • a “conservative substitution” is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged.
  • Amino acid substitutions may generally be made on the basis of similarity on polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues.
  • negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine.
  • amino acids that may represent conservative changes include: (1) ala, pro, gly, glu, asp, gin, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his.
  • a variant may also, or alternatively, contain nonconservative changes.
  • Variants within the scope of this invention also include polypeptides in which the primary amino acid structure of a native protein is modified by forming covalent or aggregative conjugates with other polypeptides or chemical moieties such as glycosyl groups, lipids, phosphate, acetyl groups and the like. Covalent derivatives may be prepared, for example, by linking particular functional groups to amino acid side chains or at the N- or C-termini.
  • the present invention also includes polypeptides with or without associated native-pattern glycosylation.
  • Polypeptides expressed in yeast or mammalian expression systems may be similar to or slightly different in molecular weight and glycosylation pattern than the native molecules, depending upon the expression system.
  • Expression of DNA in bacteria such as E. coli provides non-glycosylated molecules.
  • N- glycosylation sites of eukaryotic proteins are characterized by the amino acid triplet Asn-A j -Z, where A> is any amino acid except Pro, and Z is Ser or Thr.
  • Variants having inactivated N-glycosylation sites can be produced by techniques known to those of ordinary skill in the art, such as oligonucleotide synthesis and ligation or site-specific mutagenesis techniques, and are within the scope of this invention.
  • N- linked glycosylation sites can be added to a polypeptide.
  • polypeptides may comprise sequences that are not related to an endogenous differentiation-associated protein.
  • an N-terminal signal (or leader) sequence may be present, which co-translationally or post- translationally directs transfer of the polypeptide from its site of synthesis to a site inside or outside of the cell membrane or wall (e.g., the yeast ⁇ -factor leader).
  • the polypeptide may also comprise a linker or other sequence for ease of synthesis, purification or identification of the polypeptide (e.g., poly-His or hemagglutinin), or to enhance binding of the polypeptide to a solid support.
  • the peptide described by Hopp et al., Bio/Technology 6:1204 (1988) is a highly antigenic peptide that can be used to facilitate identification.
  • Such a peptide provides an epitope reversibly bound by a specific monoclonal antibody, enabling rapid assay and facile purification of expressed recombinant protein.
  • the sequence of Hopp et al. is also specifically cleaved by bovine mucosal enterokinase, allowing removal of the peptide from the purified protein. Fusion proteins capped with such peptides may also be resistant to intracellular degradation in E. coli.
  • Protein fusions encompassed by this invention further include, for example, polypeptides conjugated to an immunoglobulin Fc region or a leucine zipper domain as described, for example, in published PCT Application WO 94/10308.
  • Polypeptides comprising leucine zippers may, for example, be oligomeric, dimeric or trimeric. All of the above protein fusions may be prepared by chemical linkage or as fusion proteins, as described below.
  • Alleles are alternative forms of a native protein resulting from one or more genetic mutations (which may be amino acid deletions, additions and/or substitutions), resulting in an altered mRNA. Allelic proteins may differ in sequence, but overall structure and function are substantially similar.
  • Differentiation-associated polypeptides, variants and portions thereof may generally be prepared from nucleic acid encoding the desired polypeptide using well known techniques.
  • an isolated cDNA may be used.
  • standard mutagenesis techniques such as oligonucleotide-directed site-specific mutagenesis may be used, and sections of the DNA sequence may be removed to permit preparation of truncated polypeptides.
  • any of a variety of expression vectors known to those of ordinary skill in the art may be employed to express recombinant polypeptides of this invention.
  • Expression may be achieved in any appropriate host cell that has been transformed or transfected with an expression vector containing a DNA sequence that encodes a recombinant polypeptide.
  • Suitable host cells include prokaryotes, yeast and higher eukaryotic cells.
  • the host cells employed are E. coli, yeast or a mammalian cell line such as COS or CHO.
  • supematants from host/vector systems which secrete recombinant protein or polypeptide into culture media may be first concentrated using a commercially available filter. Following concentration, the concentrate may be applied to a suitable purification matrix such as an affinity matrix or an ion exchange resin.
  • a suitable purification matrix such as an affinity matrix or an ion exchange resin.
  • One or more reverse phase HPLC steps can be employed to further purify a recombinant polypeptide.
  • portions and other variants may also be generated by synthetic means, using techniques well known to those of ordinary skill in the art. For example, portions and other variants having fewer than about 500 amino acids, preferably fewer than about 100 amino acids, and more preferably fewer than about 50 amino acids, may be synthesized.
  • Polypeptides may be synthesized using any of the commercially available solid-phase techniques, such as the Merrifield solid-phase synthesis method, where amino acids are sequentially added to a growing amino acid chain. See Merrifield, J. Am. Chem. Soc. ⁇ 5:2149-2146, 1963.
  • Various modified solid phase techniques are also available (e.g., the method of Roberge et al., Science 269:202-204, 1995).
  • Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Applied BioSystems, Inc. (Foster City, CA), and may be operated according to the manufacturer's instructions.
  • polypeptides and polynucleotides as described herein are isolated.
  • An "isolated" polypeptide or polynucleotide is one that is removed from its original environment.
  • a naturally-occurring protein is isolated if it is separated from some or all of the coexisting materials in the natural system.
  • a polynucleotide is considered to be isolated if, for example, it is cloned into a vector that is not a part of the natural environment.
  • the present invention further provides binding agents, such as antibodies, and antigen-binding fragments thereof, that specifically bind to a differentiation-associated protein.
  • a differentiation-associated protein such an agent is said to "specifically bind" to a differentiation-associated protein if it reacts at a detectable level (within, for example, an ELISA) with a differentiation-associated protein or a portion or variant thereof, and does not react detectably with unrelated proteins.
  • binding refers to a noncovalent association between two separate molecules, such that a complex is formed. The ability to bind may be evaluated by, for example, determining a binding constant for the formation of the complex. The binding constant is the value obtained when the concentration of the complex is divided by the product of the component concentrations. In general, two compounds are said to "bind,” when the binding constant for complex formation exceeds about 10 3 L/mol. The binding constant maybe determined using methods well known in the art.
  • a binding agent is an antibody or antigen-binding fragment thereof.
  • Antibodies may be prepared by any of a variety of techniques known to those of ordinary skill in the art. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In general, antibodies can be produced by cell culture techniques, including the generation of monoclonal antibodies as described herein, or via transfection of antibody genes into suitable bacterial or mammalian cell hosts, in order to allow for the production of recombinant antibodies.
  • an immunogen comprising the polypeptide is initially injected into any of a wide variety of mammals (e.g., mice, rats, rabbits, sheep or goats).
  • the polypeptides of this invention may serve as the immunogen without modification.
  • a superior immune response may be elicited if the polypeptide is joined to a carrier protein, such as bovine serum albumin or keyhole limpet hemocyanin.
  • the immunogen is injected into the animal host, preferably according to a predetermined schedule incorporating one or more booster immunizations, and the animals are bled periodically.
  • Polyclonal antibodies specific for the polypeptide may then be purified from such antisera by, for example, affinity chromatography using the polypeptide coupled to a suitable solid support.
  • Monoclonal antibodies specific for the antigenic polypeptide of interest may be prepared, for example, using the technique of Kohler and Milstein, Eur. J. Immunol. (5:511-519, 1976, and improvements thereto. Briefly, these methods involve the preparation of immortal cell lines capable of producing antibodies having the desired specificity (i.e., reactivity with the polypeptide of interest). Such cell lines may be produced, for example, from spleen cells obtained from an animal immunized as described above. The spleen cells are then immortalized by, for example, fusion with a myeloma cell fusion partner, preferably one that is syngeneic with the immunized animal. A variety of fusion techniques may be employed.
  • the spleen cells and myeloma cells may be combined with a nonionic detergent for a few minutes and then plated at low density on a selective medium that supports the growth of hybrid cells, but not myeloma cells.
  • a preferred selection technique uses HAT (hypoxanthine, aminopterin, thymidine) selection. After a sufficient time, usually about 1 to 2 weeks, colonies of hybrids are observed. Single colonies are selected and their culture supematants tested for binding activity against the polypeptide. Hybridomas having high reactivity and specificity are preferred.
  • Monoclonal antibodies may be isolated from the supematants of growing hybridoma colonies.
  • various techniques may be employed to enhance the yield, such as injection of the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host, such as a mouse.
  • Monoclonal antibodies may then be harvested from the ascites fluid or the blood.
  • Contaminants may be removed from the antibodies by conventional techniques, such as chromatography, gel filtration, precipitation, and extraction.
  • the polypeptides of this invention may be used in the purification process in, for example, an affinity chromatography step.
  • antigen-binding fragments of antibodies may be preferred.
  • Such fragments include Fab fragments, which may be prepared using standard techniques. Briefly, immunoglobulins may be purified from rabbit serum by affinity chromatography on Protein A bead columns (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988) and digested by papain to yield Fab and Fc fragments. The Fab and Fc fragments may be separated by affinity chromatography on protein A bead columns. Monoclonal antibodies and fragments thereof may be coupled to one or more diagnostic agents, such as radioactive agents to facilitate tracing of differentiated and undifferentiated cells.
  • diagnostic agents such as radioactive agents to facilitate tracing of differentiated and undifferentiated cells.
  • a diagnostic agent may be coupled (e.g., covalently bonded) to a suitable monoclonal antibody either directly or indirectly (e.g. , via a linker group).
  • a direct reaction between an agent and an antibody is possible when each possesses a substituent capable of reacting with the other.
  • a nucleophilic group such as an amino or sulfhydryl group
  • on one may be capable of reacting with a carbonyl- containing group, such as an anhydride or an acid halide, or with an alkyl group containing a good leaving group (e.g., a halide) on the other.
  • a linker group can function as a spacer to distance an antibody from an agent in order to avoid interference with binding capabilities.
  • a linker group can also serve to increase the chemical reactivity of a substituent on an agent or an antibody, and thus increase the coupling efficiency. An increase in chemical reactivity may also facilitate the use of agents, or functional groups on agents, which otherwise would not be possible.
  • linker group may be employed as the linker group. Coupling may be effected, for example, through amino groups, carboxyl groups, sulfhydryl groups or oxidized carbohydrate residues, using any of a variety of well known techniques. It may be desirable to couple multiple agents (of the same type or of different types) to an antibody. Immunoconjugates with multiple agents may be prepared in a variety of ways. For example, more than one agent may be coupled directly to an antibody molecule, or linkers which provide multiple sites for attachment can be used. Alternatively, a carrier can be used.
  • Certain carriers bear the agents via covalent bonding (directly or via a linker group).
  • Such carriers include proteins such as albumins (e.g., U.S. Patent No. 4,507,234, to Kato et al.), peptides and polysaccharides such as aminodextran (e.g., U.S. Patent No. 4,699,784, to Shih et al.).
  • a carrier may also bear an agent by noncovalent bonding or by encapsulation, such as within a liposome vesicle (e.g., U.S. Patent Nos. 4,429,008 and 4,873,088).
  • Carriers specific for radionuclide agents include radiohalogenated small molecules and chelating compounds that contain, for example, nitrogen and sulfur atoms as the donor atoms for binding the metal, or metal oxide, radionuclide.
  • anti-idiotypic antibodies that mimic an immunogenic portion of a native protein. Such antibodies may be raised against an antibody, or antigen-binding fragment thereof, that specifically binds to an immunogenic portion of a differentiation-associated protein, using well known techniques. Anti-idiotypic antibodies that mimic an immunogenic portion are those antibodies that bind to an antibody, or antigen-binding fragment thereof, that specifically binds to an immunogenic portion of a differentiation-associated protein, as described herein.
  • compositions comprise one or more such compounds and a physiologically acceptable carrier.
  • Vaccines may comprise one or more polypeptides and an immune response enhancer, such as an adjuvant or a liposome (into which the compound is incorporated).
  • Pharmaceutical compositions and vaccines may additionally contain a delivery system, such as biodegradable microspheres which are disclosed, for example, in U.S. Patent Nos. 4,897,268 and 5,075,109.
  • Pharmaceutical compositions and vaccines within the scope of the present invention may also contain other compounds, which may be biologically active or inactive.
  • a pharmaceutical composition or vaccine may contain DNA encoding one or more of the polypeptides as described above, such that the polypeptide is generated in situ.
  • the DNA may be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems, bacteria and viral expression systems. Appropriate nucleic acid expression systems contain the necessary DNA sequences for expression in the patient (such as a suitable promoter and terminating signal).
  • Bacterial delivery systems involve the administration of a bacterium (such as Bacillus-Calmette-Guerri ⁇ ) that expresses an immunogenic portion of the polypeptide on its cell surface.
  • the DNA may be introduced using a viral expression system (e.g., vaccinia or other pox vims, retrovims, or adenovirus), which may involve the use of a non-pathogenic (defective), replication competent vims.
  • a viral expression system e.g., vaccinia or other pox vims, retrovims, or adenovirus
  • vaccinia or other pox vims, retrovims, or adenovirus e.g., vaccinia or other pox vims, retrovims, or adenovirus
  • a viral expression system e.g., vaccinia or other pox vims, retrovims, or adenovirus
  • compositions of the present invention may be formulated for any appropriate manner of administration, including for example, topical, oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous or intramuscular administration.
  • parenteral administration such as subcutaneous injection
  • the carrier preferably comprises water, saline, alcohol, a fat, a wax or a buffer.
  • any of the above carriers or a solid carrier such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate, may be employed.
  • Biodegradable microspheres e.g., polylactate polyglycolate
  • compositions may also comprise buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide) and/or preservatives.
  • buffers e.g., neutral buffered saline or phosphate buffered saline
  • carbohydrates e.g., glucose, mannose, sucrose or dextrans
  • mannitol proteins
  • proteins polypeptides or amino acids
  • proteins e.glycine
  • antioxidants e.g., antioxidants, chelating agents such as EDTA or glutathione
  • adjuvants e.g., aluminum hydroxide
  • preservatives e.g., aluminum hydroxide
  • adjuvants may be employed in the vaccines of this invention to nonspecifically enhance the immune response.
  • Most adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as lipid A, Bortadella pertussis or Mycobacterium tuberculosis derived proteins.
  • Suitable adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, MI), Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ), alum, biodegradable microspheres, monophosphoryl lipid A and quil A.
  • Cytokines such as GM-CSF or interleukin-2, -7, or -12, may also be used as adjuvants.
  • compositions described herein may be administered as part of a sustained release formulation (i.e., a formulation such as a capsule or sponge that effects a slow release of compound following administration).
  • a sustained release formulation i.e., a formulation such as a capsule or sponge that effects a slow release of compound following administration.
  • Such formulations may generally be prepared using well known technology and administered by, for example, oral, rectal or subcutaneous implantation, or by implantation at the desired target site.
  • Sustained-release formulations may contain a polypeptide, polynucleotide or antibody dispersed in a carrier matrix and/or contained within a reservoir surrounded by a rate controlling membrane.
  • Carriers for use within such formulations are biocompatible, and may also be biodegradable; preferably the formulation provides a relatively constant level of cyclic peptide release.
  • the amount of active compound contained within a sustained release formulation depends upon the site of implantation, the rate and expected duration of release and the nature of the condition to be treated or prevented.
  • the compounds described herein may be used for cancer therapy.
  • differentiation-associated polypeptides and polynucleotides may be used to inhibit growth and induce terminal differentiation in specific human melanomas and glioblastoma multiforme tumors.
  • Agents that enhance the expression of such polypeptides and polynucleotides may also be employed within such therapeutic techniques.
  • the compounds are preferably incorporated into pharmaceutical compositions or vaccines, as described above.
  • Suitable patients for therapy may be any warm-blooded animal, preferably a human.
  • a patient may or may not be afflicted with cancer, as determined by standard diagnostic methods.
  • the above pharmaceutical compositions and vaccines may be used to inhibit the development of a cancer at various stages of the disease (e.g., to prevent the development of cancer or to treat a patient afflicted with cancer).
  • Pharmaceutical compositions of the present invention may be administered in a manner appropriate to the specific cancer to be treated (or prevented).
  • the route, duration and frequency of administration will be determined by such factors as the condition of the patient, the type and severity of the patient's disease and the method of administration. Routes and frequency of administration may vary from individual to individual, and may be readily established using standard techniques.
  • the pharmaceutical compositions and vaccines may be administered by injection (e.g., intracutaneous, intramuscular, intravenous or subcutaneous), intranasally (e.g., by aspiration) or orally.
  • injection e.g., intracutaneous, intramuscular, intravenous or subcutaneous
  • intranasally e.g., by aspiration
  • Preferably, between 1 and 10 doses may be administered over a 52 week period.
  • 6 doses are administered, at intervals of 1 month, and booster vaccinations may be given periodically thereafter. Alternate protocols may be appropriate for individual patients.
  • an appropriate dosage and treatment regimen provides the active compound(s) in an amount sufficient to provide therapeutic and/or prophylactic benefit. Such a response can be monitored by establishing an improved clinical outcome
  • a polypeptide may be administered at a dosage ranging from the amount of each polypeptide present in a dose ranges from about 100 ⁇ g to 5 mg.
  • DNA molecules encoding such polypeptides may generally be administered in amounts sufficient to generate comparable levels of polypeptide.
  • Appropriate dosages may generally be determined using experimental models and/or clinical trials. In general, the use of the minimum dosage that is sufficient to provide effective therapy is preferred.
  • Patients may generally be monitored for therapeutic effectiveness using assays suitable for the condition being treated or prevented, which will be familiar to those of ordinary skill in the art. Suitable dose sizes will vary with the size of the patient, but will typically range from about 0.1 mL to about 5 mL.
  • Polypeptides, polynucleotides and antibodies, as described herein, may be used within a variety of methods for detecting a cancer in a patient.
  • the presence of differentiation-associated polypeptides and/or polynucleotides as described herein in the cells of a host is indicative of terminal differentiation and growth arrest.
  • differentiation-associated sequences may be used as markers for distinguishing between normal and malignant cells (e.g., distinguishing between normal glial cells and malignant astrocytomas, such as glioblastoma multiforme tumors, or for diagnosing carcinomas such as prostate, breast, lung and colorectal carcinomas). Differentiation- associated sequences may also be used as markers to monitor treatment.
  • Methods involving the use of an antibody may detect the presence or absence of a polypeptide as described herein in any suitable biological sample.
  • suitable biological samples include tumor or normal tissue biopsy, melanomas, or other tissue, homogenate or extract thereof obtained from a patient.
  • assay formats known to those of ordinary skill in the art for using an antibody to detect polypeptide markers in a sample. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988.
  • the assay may be performed in a Western blot format, wherein a protein preparation from the biological sample is submitted to gel electrophoresis, transferred to a suitable membrane and allowed to react with the antibody. The presence of the antibody on the membrane may then be detected using a suitable detection reagent, as described below.
  • the assay involves the use of antibody immobilized on a solid support to bind to the polypeptide and remove it from the remainder of the sample.
  • the bound polypeptide may then be detected using a second antibody or reagent that contains a reporter group.
  • a competitive assay may be utilized, in which a polypeptide is labeled with a reporter group and allowed to bind to the immobilized antibody after incubation of the antibody with the sample. The extent to which components of the sample inhibit the binding of the labeled polypeptide to the antibody is indicative of the reactivity of the sample with the immobilized antibody, and as a result, indicative of the concentration of polypeptide in the sample.
  • the solid support may be any material known to those of ordinary skill in the art to which the antibody may be attached.
  • the solid support may be a test well in a microtiter plate or a nitrocellulose filter or other suitable membrane.
  • the support may be a bead or disc, such as glass, fiberglass, latex or a plastic material such as polystyrene or polyvinylchloride.
  • the support may also be a magnetic particle or a fiber optic sensor, such as those disclosed, for example, in U.S. Patent No. 5,359,681.
  • the antibody may be immobilized on the solid support using a variety of techniques known to those in the art, which are amply described in the patent and scientific literature.
  • immobilization refers to both noncovalent association, such as adsorption, and covalent attachment (which may be a direct linkage between the antigen and functional groups on the support or may be a linkage by way of a cross-linking agent). Immobilization by adsorption to a well in a microtiter plate or to a membrane is preferred. In such cases, adsorption may be achieved by contacting the antibody, in a suitable buffer, with the solid support for a suitable amount of time. The contact time varies with temperature, but is typically between about 1 hour and 1 day.
  • contacting a well of a plastic microtiter plate (such as polystyrene or polyvinylchloride) with an amount of antibody ranging from about 10 ng to about 1 ⁇ g, and preferably about 100-200 ng, is sufficient to immobilize an adequate amount of polypeptide.
  • a plastic microtiter plate such as polystyrene or polyvinylchloride
  • Covalent attachment of antibody to a solid support may also generally be achieved by first reacting the support with a bifunctional reagent that will react with both the support and a functional group, such as a hydroxyl or amino group, on the antibody.
  • a bifunctional reagent that will react with both the support and a functional group, such as a hydroxyl or amino group, on the antibody.
  • the antibody may be covalently attached to supports having an appropriate polymer coating using benzoquinone or by condensation of an aldehyde group on the support with an amine and an active hydrogen on the binding partner using well known techniques.
  • the assay for detection of polypeptide in a sample is a two-antibody sandwich assay.
  • This assay may be performed by first contacting an antibody that has been immobilized on a solid support, commonly the well of a microtiter plate, with the biological sample, such that the polypeptide within the sample are allowed to bind to the immobilized antibody. Unbound sample is then removed from the immobilized polypeptide-antibody complexes and a second antibody (containing a reporter group) capable of binding to a different site on the polypeptide is added. The amount of second antibody that remains bound to the solid support is then determined using a method appropriate for the specific reporter group.
  • the immobilized antibody is then incubated with the sample, and polypeptide is allowed to bind to the antibody.
  • the sample may be diluted with a suitable diluent, such as phosphate-buffered saline (PBS) prior to incubation.
  • PBS phosphate-buffered saline
  • an appropriate contact time i.e., incubation time is that period of time that is sufficient to detect the presence of polypeptide within a sample obtained from an individual without cancer.
  • the contact time is sufficient to achieve a level of binding that is at least 95% of that achieved at equilibrium between bound and unbound polypeptide.
  • the time necessary to achieve equilibrium may be readily determined by assaying the level of binding that occurs over a period of time. At room temperature, an incubation time of about 30 minutes is generally sufficient.
  • Unbound sample may then be removed by washing the solid support with an appropriate buffer, such as PBS containing 0.1% Tween 20TM.
  • the second antibody which contains a reporter group, may then be added to the solid support.
  • Preferred reporter groups include enzymes (such as horseradish peroxidase), substrates, cofactors, inhibitors, dyes, radionuclides, luminescent groups, fluorescent groups and biotin.
  • the conjugation of antibody to reporter group may be achieved using standard methods known to those of ordinary skill in the art.
  • the second antibody is then incubated with the immobilized antibody- polypeptide complex for an amount of time sufficient to detect the bound polypeptide.
  • An appropriate amount of time may generally be determined by assaying the level of binding that occurs over a period of time. Unbound second antibody is then removed and bound second antibody is detected using the reporter group.
  • the method employed for detecting the reporter group depends upon the nature of the reporter group. For radioactive groups, scintillation counting or autoradiographic methods are generally appropriate. Spectroscopic methods may be used to detect dyes, luminescent groups and fluorescent groups. Biotin may be detected using avidin, coupled to a different reporter group (commonly a radioactive or fluorescent group or an enzyme).
  • Enzyme reporter groups may generally be detected by the addition of substrate (generally for a specific period of time), followed by spectroscopic or other analysis of the reaction products.
  • the signal detected from the reporter group that remains bound to the solid support is generally compared to a signal that corresponds to a predetermined cut-off value established from non-tumor tissue.
  • the cut-off value is the average mean signal obtained when the immobilized antibody is incubated with samples from patients without cancer.
  • a sample generating a signal that is three standard deviations below the predetermined cut-off value may be considered positive for cancer.
  • the cut-off value is determined using a Receiver Operator Curve, according to the method of Sackett et al., Clinical Epidemiology: A Basic Science for Clinical Medicine, p. 106-7 (Little Brown and Co., 1985). Briefly, in this embodiment, the cut-off value may be determined from a plot of pairs of tme positive rates (i.e., sensitivity) and false positive rates (100%-specif ⁇ city) that correspond to each possible cut-off value for the diagnostic test result.
  • tme positive rates i.e., sensitivity
  • false positive rates (100%-specif ⁇ city
  • the cut-off value on the plot that is the closest to the upper left-hand comer is the most accurate cut-off value, and a sample generating a signal that is lower than the cut-off value determined by this method may be considered positive.
  • the cut-off value may be shifted to the left along the plot, to minimize the false positive rate, or to the right, to minimize the false negative rate.
  • a sample generating a signal that is lower than the cut-off value determined by this method is considered positive for cancer.
  • the assay is performed in a flow-through or strip test format, wherein the antibody is immobilized on a membrane, such as nitrocellulose.
  • a membrane such as nitrocellulose.
  • the polypeptide within the sample bind to the immobilized antibody as the sample passes through the membrane.
  • a second, labeled antibody then binds to the antibody-polypeptide complex as a solution containing the second antibody flows through the membrane.
  • the detection of bound second antibody may then be performed as described above.
  • the strip test format one end of the membrane to which antibody is bound is immersed in a solution containing the sample. The sample migrates along the membrane through a region containing second antibody and to the area of immobilized antibody. Concentration of second antibody at the area of immobilized antibody is negative for cancer.
  • the concentration of second antibody at that site generates a pattern, such as a line, that can be read visually.
  • a pattern such as a line
  • the amount of antibody immobilized on the membrane is selected to generate a visually discernible pattern when the biological sample contains a level of polypeptide that would be sufficient to generate a positive signal in the two-antibody sandwich assay, in the format discussed above.
  • the amount of antibody immobilized on the membrane ranges from about 25 ng to about 1 ⁇ g, and more preferably from about 50 ng to about 1 ⁇ g. Such tests can typically be performed with a very small amount of biological sample.
  • the presence or absence of a cancer in a patient may also be determined by evaluating the level of mRNA encoding a polypeptide of the present invention within the biological sample (e.g., a biopsy) relative to a predetermined cut-off value. Such an evaluation may be achieved using any of a variety of methods known to those of ordinary skill in the art such as, for example, in situ hybridization and amplification by polymerase chain reaction. Probes and primers for use within such assays may generally be designed based on the sequences recited in Figures 1-2, or on similar sequences identified in other individuals. Probes may be used within well known hybridization techniques, and may be labeled with a detection reagent to facilitate detection of the probe. Such reagents include, but are not limited to, radionuclides, fluorescent dyes and enzymes capable of catalyzing the formation of a detectable product.
  • Primers may generally be used within detection methods involving polymerase chain reaction (PCR), such as RT-PCR, in which PCR is applied in conjunction with reverse transcription.
  • PCR polymerase chain reaction
  • RNA is extracted from a sample tissue and is reverse transcribed to produce cDNA molecules.
  • PCR amplification using specific primers generates a differentiation-associated cDNA molecule, which may be separated and visualized using, for example, gel electrophoresis.
  • Amplification is typically performed on samples obtained from matched pairs of tissue (tumor and non- tumor tissue from the same individual) or from unmatched pairs of tissue (tumor and non-tumor tissue from different individuals).
  • the amplification reaction may be performed on several dilutions of cDNA spanning two orders of magnitude. A two-fold or greater decrease in expression in several dilutions of the tumor sample as compared to the same dilutions of the non-tumor sample is typically considered positive.
  • Certain in vivo diagnostic assays may be performed directly on a tumor.
  • One such assay involves contacting tumor cells with an antibody or fragment thereof that binds to a differentiation-associated protein. The bound antibody or fragment may then be detected directly or indirectly via a reporter group.
  • Such antibodies may also be used in histological applications.
  • antisense polynucleotides may be used within such applications.
  • the progression and/or response to treatment of a cancer may be monitored by performing any of the above assays over a period of time, and evaluating the change in the level of the response (i.e., the amount of polypeptide or mRNA detected or, in the case of a skin test, the extent of the immune response detected).
  • the assays may be performed every month to every other month for a period of 1 to 2 years.
  • a cancer is progressing in those patients in whom the level of the response decreases over time.
  • a cancer is not progressing when the signal detected either remains constant or increases with time.
  • kits for use within any of the above diagnostic methods.
  • Such kits typically comprise two or more components necessary for performing the assay.
  • Such components may be compounds, reagents and/or containers or equipment.
  • one container within a kit may contain a monoclonal antibody or fragment thereof that specifically binds to a differentiation- associated polypeptide.
  • Such antibodies or fragments may be provided attached to a support material, as described above.
  • One or more additional containers may enclose elements, such as reagents or buffers, to be used in the assay.
  • Such kits may also contain a detection reagent (e.g., an antibody) that contains a reporter group suitable for direct or indirect detection of antibody binding.
  • the present invention further provides methods for identifying compounds that bind to and/or modulate the expression of a differentiation-associated protein.
  • Such agents may generally be identified by contacting a polypeptide as provided herein with a candidate compound or agent under conditions and for a time sufficient to allow interaction with the polypeptide. Any of a variety of well known binding assays may then be performed to assess the ability of the candidate compound to bind to the polypeptide.
  • agents may be screened using cells that are known to increase expression of a differentiation-associated gene when induced to differentiate. Such cells may be contacted with candidate agents and the expression of a differentiation-associated gene evaluated, relative to expression in the absence of candidate agent.
  • agents that increase expression of a differentiation- associated gene may have therapeutic potential for inducing terminal differentiation in cancers such as carcinomas (e.g., prostate, breast, lung and colorectal), melanomas, astrocytomas and glioblastoma multiforme tumors.
  • compounds may be screened for the ability to modulate expression (e.g., transcription) of a differentiation-associated protein.
  • a promoter or regulatory element thereof may be operatively linked to a reporter gene as described above. Such a constmct may be transfected into a suitable host cell, which is then contacted with a candidate agent.
  • cells may be used to screen a combinatorial small molecule library. Briefly, cells are incubated with the library (e.g., overnight). Cells are then lysed and the supernatant is analyzed for reporter gene activity according to standard protocols. Compounds that result in an increase in reporter gene activity are inducers of differentiation-associated gene transcription, and may be used to inhibit cancer progression.
  • This Example illustrates the cloning of differentiation-associated cDNAs.
  • treatment of melanoma cells with either IFN- ⁇ or MEZ alone results in a reversible alteration in differentiation phenotypes in the human melanoma cell line HO-1.
  • a subtraction hybridization protocol was used to identify melanoma differentiation associated (mda) genes displaying enhanced expression in cells treated with reversible- and terminal differentiation inducing compounds.
  • the human melanoma cell line HO-1 is a melanotic melanoma derived from a 49-year-old female and was used between passage 125 and 150.
  • HO-1 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine semm (DMEM-5) at 37°C in a 5% CO 2 -95% air humidified incubator.
  • Cells were either untreated (Ind) or treated (IncT) with a combination of IFN- ⁇ (2000 units per ml) and MEZ (10 ng per ml) for 2, 4, 8, 12 and 24 hr.
  • HO-1 cells were untreated or treated for 12 and 24 hr with IFN- ⁇ (2000 units per ml), MEZ (10 ng per ml) or IFN- ⁇ plus MEZ (2000 units per ml plus 10 ng per ml) prior to isolation of cellular RNA and Northern blotting analysis.
  • RNA from untreated (Ind) and IFN- ⁇ plus MEZ treated (2,4,8,12 and 24 hr) (Incf) samples was isolated by the guanidinium isothiocyanate/CsCl centrifugation procedure and poly(A + ) RNA was selected following oligo(dT) cellulose chromatography.
  • cDNA synthesis was performed using the ZAP-cDNATM synthesis kit from Stratagene ® (La Jolla, CA). A primer-adapter consisting of oligo(dT) next to a unique restriction site (Xhol was used for first strand synthesis. The double-stranded cDNAs were ligated to EcoRI adapters and then digested with the Xhol restriction endonuclease.
  • the resultant EcoRI and Xhol cohesive ends allowed the finished cDNAs to be inserted into the ⁇ ZAP II vector in a sense orientation with respect to the lac-Z promoter.
  • the ⁇ ZAP II vector contains pBluescript plasmid sequences flanked by bacteriophage-derived fl sequences that facilitate in vivo conversion of the phage clones into the phagemid.
  • Two cDNA libraries were constmcted: a differentiation inducer-treated cDNA library (Inct) (tester library); and a control uninduced cDNA library (Ind) (driver library).
  • the libraries were packaged with Gigapack II Gold Packaging Extract (Stratagene ® ) and amplified on PLK-F' bacterial cells (Stratagene ® ).
  • the Inct cDNA phagemid library was excised from ⁇ ZAP using the mass excision procedure described by Stratagene ® (La Jolla, CA). Briefly, 1 X 10 7 pfu of Inct cDNA library were mixed with 2 X 10 8 XL-1 Blue strain of Escherichia coli and 2 X 10 8 pfu of ExAssist helper phage in 10 mM MgSO 4 followed by absorption at 37°C for 15 min. After the addition of 10 ml of LB medium, the phage/bacteria mixture was incubated with shaking at 37°C for 2 hr followed by incubation at 70°C for 20 min to heat inactivate the bacteria and the ⁇ ZAP phage particles.
  • the supernatant was transferred to a sterile polystyrene tube, and stored at 4°C before use.
  • 5 X 10 7 pfu of the phagemids were combined with 1 X 10 9 SOLR strain of Escherichia coli, which are nonpermissive for the growth of the helper phages and therefore prevent coinfection by the helper phages, in 10 mM MgSO 4 followed by absorption at 37°C for 15 min.
  • the phagemids/bacteria were transferred to 250 ml LB medium containing 50 ⁇ g/ml ampicillin and incubated with shaking at 37°C overnight.
  • the bacteria were harvested by centrifugation, and the double-stranded phagemid DNA was isolated by the alkali lysis method and purified through a QIAGEN-tip 500 column (QIAGEN Inc., Chatsworth, CA).
  • Helper phage VCS Ml 3 (Stratagene ® , La Jolla, C A) was added to 2 X 10 7 pfu/ml, and after incubation for 1 hr, kanamycin sulfate (Sigma) was added to 70 ⁇ g/ml. The bacteria were grown overnight. The phagemids were harvested and single-stranded DNAs were prepared using standard protocols.
  • double-stranded DNA from the IncT cDNA library was digested with EcoRI and Xhol, and extracted with phenol and chloroform followed by ethanol precipitation. After centrifugation, the pellet was resuspended in distilled H 2 O.
  • Single-stranded DNA from Ind cDNA library was biotinylated using photoactivatable biotin (Photobiotin, Sigma, St. Louis, MO). In a 650 ⁇ l microcentrifuge tube, 50 ⁇ l of 1 ⁇ g/ ⁇ l single-stranded DNA was mixed with 50 ⁇ l of 1 ⁇ g/ ⁇ l photoactivatable biotin in H 2 O.
  • the solution was irradiated with the tube slanted on cmshed ice at a distance of 10 cm from a 300 watt sun lamp for 15 min.
  • the DNA was further biotinylated by adding 25 ⁇ l of photoactivatable biotin to the solution which was then exposed to an additional 15 min of irradiation as described above.
  • the reaction was diluted to 200 ⁇ l with 100 mM Tris-HCl, 1 mM EDTA, pH 9.0, and extracted 3X with 2-butanol. Sodium acetate, pH 6.5 was added to a concentration of 0.3 M, and the biotinylated DNA was precipitated with two volumes of ethanol.
  • the hybridization mixture was diluted to 400 ⁇ l with 0.5 M NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA and then 15 ⁇ g of streptavidin (BRL ® ) in H 2 O was added, followed by incubation at room temperature for 5 min.
  • the sample was extracted 2X with phenol/chloroform (1 :1), followed by back-extraction of the organic phase with 50 ⁇ l of 0.5 M NaCl in TE buffer, pH 8.0. An additional 10 ⁇ g of streptavidin was added and phenol/chloroform extraction was repeated.
  • the final solution was diluted to 2 ml with TE buffer, pH 8.0, and passed through a Centricon 100 filter (Amicon; Danvers, MA) 2X as recommended by the manufacturer.
  • the concentrated DNA solution (approximately 50 ⁇ l) was then lyophilized.
  • the subtracted cDNAs were ligated to EcoRI- and Xhol-digested and CIAP treated arms of the ⁇ ZAP II vector and packaged with Gigapack II Gold packaging extract (Stratagene ® , La Jolla, CA).
  • the library was then amplified using the PLK-F' bacterial cell.
  • the mass excision of the library was performed using ExAssist helper phage as described above.
  • the SOLR strain of Escherichia coli and cDNA phagemids were mixed at 37°C for 15 min and plated onto LB plates containing ampicillin and IPTG/X-gal. White colonies were chosen at random, isolated and grown in LB medium. Plasmid minipreps and restriction enzyme digestions were performed to confirm the presence of inserts. The inserts were isolated and used as robes for Northern blotting analysis (5,25).
  • Total cellular RNA was prepared from HO-1 cells treated with IFN- ⁇ (2000 units/ml), MEZ (10 ng/ml), and IFN- ⁇ plus MEZ (2000 units/ml plus 10 ng/ml), electrophoresed in 0.8% agarose gels and transferred to nylon membranes (Amersham, Arlington Heights, IL). Radiolabeled probes were generated by random oligonucleotide priming. Prehybridization, hybridization, posthybridization washes, and autoradiography were performed using standard protocols.
  • mda-8 is one novel cDNA identified as described above. The sequence and properties of mda-8 have been described in PCT application WO 95/11986.
  • the cDNA molecules designated mda8-A (SEQ ID NO:l) and mda8-B (SEQ ID NO:2) were identified from placental and skeletal libraries, respectively, based on sequence similarity to mda-8.
  • This Example illustrates the tissue distribution of mda8-A and mda8-B, as well as the effect of mda8-A on growth of normal and tumor cell lines.
  • a human tissue Northern blot (Clonetics) was probed mda8-A and mda8-B cDNA sequences. Probes were hybridized in 50 mM PIPES, pH 6.4, 100 mM NaCl, 1 mM EDTA, pH 8.0, 50 mM sodium phosphate and 5% SDS. The blot was then washed in 0.5x SSC, 1% SDS at room temperature for 5 minutes; at 50°C for ten minutes and at 65°C for 30 minutes. The resulting autoradiogram, shown in Figure 3, shows two bands at approximately 2 kb (believed to be mda8-A) and 2.8 kb (believed to be mda8-B). The highest expression of mda8-A and mda8-B was observed in heart and pancreas, with substantial expression also detected in placenta, skeletal muscle and kidney.
  • tumor T47D; available from American Type Culture Collection, Rockville, MD
  • normal HBLIOO and CREF
  • cDNA encoding mda8-A or vector alone
  • lipofectamine lipofectamine

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Cette invention a trait à des compositions ainsi qu'aux méthodes afférentes visant à inhiber la croissance cellulaire et à provoquer une différentiation terminale. Ces compositions sont également destinées à intervenir dans le cadre d'une thérapie anticancéreuse. Ces composés sont, en l'occurrence, des polypeptides et des polynucléotides associés à une différentiation terminale et à un arrêt de la croissance, ainsi que des polynucléotides codant ces polypeptides. L'invention, qui concerne également des vaccins et des compositions pharmaceutiques renfermant ces composés, porte, de surcroît, sur leur utilisation en matière de thérapie anticancéreuse. On peut aussi utiliser ces molécules pour identifier, notamment, des agents susceptibles d'entrer dans la formulation de vaccins et de compositions pharmaceutiques aux fins d'une thérapie anticancéreuse. Les polypeptides et les polynucléotides de l'invention peuvent, en outre, être utilisés comme marqueurs permettant de diagnostiquer un cancer chez un patient et de surveiller son évolution.
PCT/US1999/001624 1998-01-26 1999-01-26 Sequences associees a une differentiation et methodes d'utilisation WO1999037777A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU23433/99A AU2343399A (en) 1998-01-26 1999-01-26 Differentiation-associated sequences and methods of use therefor

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US7252498P 1998-01-26 1998-01-26
US60/072,524 1998-01-26
US8465098P 1998-05-07 1998-05-07
US60/084,650 1998-05-07

Publications (2)

Publication Number Publication Date
WO1999037777A2 true WO1999037777A2 (fr) 1999-07-29
WO1999037777A3 WO1999037777A3 (fr) 1999-09-30

Family

ID=26753455

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1999/001624 WO1999037777A2 (fr) 1998-01-26 1999-01-26 Sequences associees a une differentiation et methodes d'utilisation

Country Status (2)

Country Link
AU (1) AU2343399A (fr)
WO (1) WO1999037777A2 (fr)

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100355111B1 (ko) * 1993-10-27 2003-03-03 더 트러스티스 오브 컬럼비아 유니버시티 인 더 시티 오브 뉴욕 발췌제거cDNA라이브러리제조법및제조된라이브러리의용도

Also Published As

Publication number Publication date
AU2343399A (en) 1999-08-09
WO1999037777A3 (fr) 1999-09-30

Similar Documents

Publication Publication Date Title
US6887660B2 (en) Compounds for immunodiagnosis of prostate cancer and methods for their use
US7202334B1 (en) Compositions and methods for the therapy and diagnosis of ovarian cancer
US5243041A (en) DNA vector with isolated CDNA gene encoding metallopanstimulin
EP1876241A2 (fr) Compositions et procédés pour le traitement et le diagnostic du cancer du sein
NZ578927A (en) Compositions and methods for the therapy and diagnosis of ovarian cancer
CA2742641A1 (fr) Composes permettant l'immunotherapie du cancer de la prostate et procedes concernant leur utilisation
JP4270523B2 (ja) 乳癌の処置および診断のための組成物および方法
CA2249742A1 (fr) Composes et methodes d'immunotherapie et d'immunodiagnostic pour le cancer de la prostate
WO1997008318A2 (fr) Composes et procedes de traitement et de diagnostic du cancer de la prostate
EP1650221B1 (fr) Nouveaux composés
WO2000052165A2 (fr) Compositions et methodes de therapie et de diagnostic du cancer du sein
WO1997025431A1 (fr) Compositions et procedes de traitement et de diagnostic du cancer
RU2307666C2 (ru) Полинуклеотид, модулирующий пролиферацию раковых клеток (варианты), полипептид, моноклональное антитело, вектор экспрессии, клетка-хозяин, лекарственное средство для лечения пролиферативного заболевания, фармацевтическая композиция, применение полипептида для получения лекарственного средства
WO1999037775A2 (fr) Compositions et procedes pour la detection et le traitement du cancer du sein
NZ567750A (en) Compositions and uses for cancer therapy
US6379951B1 (en) Compounds for immunotherapy of breast cancer and methods for their use
WO1999037774A2 (fr) Sequences associees a la differenciation cellulaire et procedes d'utilisation de ces sequences
WO1999037777A2 (fr) Sequences associees a une differentiation et methodes d'utilisation
WO1999054466A2 (fr) Antigenes associes au cancer du colon, et utilisations desdits antigenes a des fins diagnostiques ou therapeutiques
WO1999060124A2 (fr) Sequences liees a la differenciation et procede d'utilisation correspondant
US7008772B1 (en) Compounds for immunodiagnosis of prostate cancer and methods for their use
US7270980B2 (en) Compounds for immunodiagnosis of prostate cancer and methods for their use
CA2370482A1 (fr) Compositions et procedes utilises en therapie et dans le diagnostic de l'epithelioma malpighien spinocellulaire de la tete et du cou
WO1999037776A1 (fr) Techniques de modulation de l'angiogenese
WO1999037778A2 (fr) Compositions et procedes de detection et de traitement du cancer de la prostate

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG UZ VN YU ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW SD SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
AK Designated states

Kind code of ref document: A3

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG UZ VN YU ZW

AL Designated countries for regional patents

Kind code of ref document: A3

Designated state(s): GH GM KE LS MW SD SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
NENP Non-entry into the national phase

Ref country code: KR

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载