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
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154
• • 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
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
  • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P33/00—Antiparasitic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P33/00—Antiparasitic agents
  • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
  • A61P33/06—Antimalarials
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
• • 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
  • C12N2799/00—Uses of viruses
  • C12N2799/02—Uses of viruses as vector
  • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
  • C12N2799/026—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus

Definitions

• the present invention relates to a novel member of the TNF-Ligand superfamily. More specifically, isolated nucleic acid molecules are provided encoding a human Apoptosis Inducing Molecule II (AIM II). AIM II polypeptides are also provided, as are vectors, host cells and recombinant methods for producing the same. The invention further relates to screening methods for identifying agonists and antagonists of AIM II activity. Also provided are therapeutic methods for treating lymphadenopathy, autoimmune disease, graft versus host disease, and to inhibit neoplasia, such as tumor cell growth.
• AIM II Apoptosis Inducing Molecule II
• TNF- ⁇ and ⁇ are related members of a broad class of polypeptide mediators, which includes the interferons, interleukins and growth factors, collectively called cytokines (Beutler, B. and Cerami. A., Annu. Ret,. Immunol, 7:625-655 (1989)).
• Tumor necrosis factor (TNF- ⁇ and TNF- ⁇ ) was originally discovered as a result of its anti-tumor activity, however, now it is recognized as a pleiotropic cytokine capable of numerous biological activities including apoptosis of some transformed cell lines, mediation of cell activation and proliferation and also as playing important roles in immune regulation and inflammation.
• TNF-ligand superfamily known members of the TNF-ligand superfamily include TNF- ⁇ , TNF- ⁇ (lymphotoxin- ⁇ ). LT- ⁇ , OX40L, Fas ligand, CD30L, CD27L, CD40L and 4-IBBL.
• the ligands of the TNF ligand superfamily are acidic, TNF-like molecules with approximately 20% sequence homology in the extracellular domains (range, 12%-36%) and exist mainly as membrane-bound forms with the biologically active form being a trimeric/multimeric complex. Soluble forms of the TNF ligand superfamily have only been identified so far for TNF, LT ⁇ , and Fas ligand (for a general review, see Gruss, H. and Dower, S.K., Blood, 85(12):3378-3404 (1995)), which is hereby incorporated by reference in its entirety.
• Apoptosis plays a critical role in the destruction of immune thymocytes that recognize self antigens. Failure of this normal elimination process may play a role in autoimmune diseases (Gammon et al, Immunology Today 12:193 (1991)).
• Fas/CD95 a cell surface antigen that mediates apoptosis and is involved in clonal deletion of T-cells. Fas is expressed in activated T-cells, B-cells, neutrophils and in thymus, liver, heart and lung and ovary in adult mice (Watanabe-Fukunaga et al., J. Immunolo. 148:1274 (1992)) in addition to activated T-cells, B-cells, neutorophils. In experiments where a monoclonal Ab to Fas is cross-linked to Fas, apoptosis is induced
• Fas antigen is a cell surface protein of relative MW of 45 Kd.
• Both human and murine genes for Fas have been cloned by Watanabe-Fukunaga et al., (J. Immunol. 148:1214 (1992)) and Itoh et al. (Cell 66:233 (1991)).
• the proteins encoded by these genes are both transmembrane proteins with structural homology to the Nerve Growth Factor/Tumor Necrosis Factor receptor superfamily, which includes two TNF receptors, the low affinity Nerve Growth Factor receptor and the LT ⁇ receptor CD40, CD27, CD30, and OX40.
• Fas ligand has been described (Suda et al., Cell 75:1169 (1993)).
• the amino acid sequence indicates that Fas ligand is a type II transmembrane protein belonging to the TNF family. Fas ligand is expressed in splenocytes and thymocytes.
• the purified Fas ligand has a MW of 40 kd.
• Fas/Fas ligand interactions are required for apoptosis following the activation of T-cells (Ju et al., Nature 373:444 (1995); Brunner et al., Nature 373:441 (1995)).
• Activation of T-cells induces both proteins on the cell surface.
• Subsequent interaction between the ligand and receptor results in apoptosis of the cells. This supports the possible regulatory role for apoptosis induced by Fas/Fas ligand interaction during normal immune responses.
• polypeptide of the present invention has been identified as a novel member of the TNF ligand super-family based on structural and biological similarities.
• the present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding the AIM II polypeptide having the amino acid sequence shown in Figures 1A-C (SEQ ID NO:2) or the amino acid sequence encoded by the cDNA clone deposited in a bacterial host as ATCC Deposit Number 97689 on August 22, 1996.
• the present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them for production of AIM II polypeptides or peptides by recombinant techniques.
• the invention further provides an isolated AIM II polypeptide having an amino acid sequence encoded by a polynucleotide described herein.
• AIM II polypeptide includes membrane-bound proteins (comprising a cytoplasmic domain, a transmembrane domain, and an extracellular domain) as well as truncated proteins that retain the AIM II polypeptide activity.
• soluble AIM II polypeptides comprise all or part of the extracellular domain of an AIM II protein, but lack the transmsmbrane region that would cause retention of the polypeptide on a cell membrane.
• Soluble AIM II may also include part of the transmembrane region or part of the cytoplasmic domain or other sequences, provided that the soluble AIM II protein is capable of being secreted.
• a heterologous signal peptide can be fused to the N-terminus of the soluble AIM II polypeptide such that the soluble AIM II polypeptide is secreted upon expression.
• the invention also provides for AIM II polypeptides, particularly human AIM-II polypeptides, which may be employed to treat lymphadenopathy, autoimmune disease, graft versus host disease, which may be used to stimulate peripheral tolerance, destroy some transformed cell lines, mediate cell activation and proliferation and are functionally linked as primary mediators of immune regulation and inflammatory response.
• AIM II polypeptides particularly human AIM-II polypeptides, which may be employed to treat lymphadenopathy, autoimmune disease, graft versus host disease, which may be used to stimulate peripheral tolerance, destroy some transformed cell lines, mediate cell activation and proliferation and are functionally linked as primary mediators of immune regulation and inflammatory response.
• compositions comprising an AIM II polynucleotide or an AIM II polypeptide for administration to cells in vitro, to cells ex vivo and to cells in vivo, or to a multicellular organism.
• the compositions comprise an AIM II polynucleotide for expression of an AIM II polypeptide in a host organism for treatment of disease.
• Particularly preferred in this regard is expression in a human patient for treatment of a dysfunction associated with aberrant endogenous activity of an AIM II.
• the present invention also provides a screening method for identifying compounds capable of enhancing or inhibiting a cellular response induced by AIM II, which involves contacting cells which express AIM II with the candidate compound, assaying a cellular response, and comparing the cellular response to a standard cellular response, the standard being assyed when contact is made in absence of the candidate compound; whereby, an increased cellular response over the standard indicates that the compound is an agonist and a decreased cellular response over the standard indicates that the compound is an antagonist.
• a screening assay for AIM II agonists and antagonists is provided.
• the antagonists may be employed to prevent septic shock, inflammation, cerebral malaria, activation of the HIV virus, graft-host rejection, bone resorption, rheumatoid arthritis and cachexia (wasting or malnutrition).
• An additional aspect of the invention is related to a method for treating an individual in need of an increased level of AIM II activity in the body comprising administering to such an indivdual a composition comprising a therapeutically effective amount of an isolated AIM II polypeptide of the invention or an agonist thereof.
• a still further aspect of the invention is related to a method for treating an individual in need of a decreased level of AIM II activity in the body comprising, administering to such an individual a composition comprising a therapeutically effective amount of an AIM II antagonist.
• Figures 1A-C show the nucleotide (SEQ ID NO:l) and deduced amino acid (SEQ ID NO:2) sequences of AIM II.
• the protein has a deduced molecular weight of about 26.4 kDa.
• the predicted Transmembrane Domain of the AIM II protein is underlined.
• Figures 2A-F show the regions of similarity between the amino acid sequences of the AIM II protein and human TNF- ⁇ (SEQ ID NO: 3), human TNF- ⁇ (SEQ ID NO:4), human lymphotoxin (SEQ ID NO:5) and human Fas
• Figures 3A-F show an analysis of the AIM II amino acid sequence. Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown.
• “Antigenic Index - Jameson- Wolf ' graph about amino acid residues 13-20, 23-36, 69-79, 85-94, 167-178, 184-196, 221-233 in Figures 1A-C correspond to the shown highly antigenic regions of the AIM II protein.
• the present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding an AIM II polypeptide having the amino acid sequence shown in Figures 1A-C (SEQ ID NO:2), which was determined by sequencing a cloned cDNA.
• the AIM II protein of the present invention shares sequence homology with human TNF- ⁇ (SEQ ID NO: 3), human TNF- ⁇ (SEQ ID NO: 4), human TNF- ⁇ (SEQ ID NO:
• nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer (such as the Model 373 from Applied Biosystems, Inc.), and all amino acid sequences of polypeptides encoded by DNA molecules determined herein were predicted by translation of a DNA sequence determined as above. Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about
• the actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art.
• a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion.
• nucleic acid molecule of the present invention encoding an AIM II polypeptide may be obtained using standard cloning and screening procedures, such as those for cloning cDNAs using mRNA as starting material.
• nucleic acid molecule described in Figures 1A-C SEQ ID NO:1 was discovered in a cDNA library derived from human macrophage ox LDL (HMCCB64). The gene was also identified in cDNA libraries from activated T-cells (HT4CC72).
• the determined nucleotide sequence of the AIM II cDNA of Figures 1 A-C contains an open reading frame encoding a protein of 240 amino acid residues, with an initiation codon at positions 49-51 of the nucleotide sequence in Figures 1A-C (SEQ ID NO:1), an extracellular domain comprising amino acid residues from about 60 to about 240 in Figures 1 A-C (SEQ ID NO:2), a transmembrane domain comprising amino acid residues from about 37 to about 59 in Figures 1A-C (SEQ ID NO:2), a intracellular domain comprising amino acid residues from about 1 to about 36 in
• Figures 1A-C (SEQ ID NO:2) and a deduced molecular weight of about 26.4 kDa.
• the AIM II protein shown in Figures 1 A-C (SEQ ID NO:2) is about 27% identical and about 51% similar to the amino acid sequence of human Fas Ligand ( Figures 2A-F) and is about 26% identical and about 47% similar to the amino acid sequence of human TNF- ⁇ ( Figures 2A-F).
• the predicted AIM II polypeptide encoded by the deposited cDNA comprises about 240 amino acids, but may be anywhere in the range of 230-250 amino acids. It will further be appreciated that, depending on the criteria used, concerning the exact "address" of the extracelluar, intracelluar and transmembrane domains of the AIMII polypeptide differ slightly. For example, the exact location of the AIM II extracellular domain in Figures 1 A-C [SEQ ID NO:2] may vary slightly (e.g., the address may "shift" by about 1 to 5 residues) depending on the criteria used to define the domain.
• nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA obtained by cloning or produced synthetically.
• the DNA may be double-stranded or single-stranded.
• Single-stranded DNA or RNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti-sense strand.
• isolated nucleic acid molecule(s) is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment
• recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention.
• Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution.
• Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.
• Isolated nucleic acid molecules of the present invention include DNA molecules comprising an open reading frame (ORF) shown in Figures 1A-C (SEQ ID NO:1); DNA molecules comprising the coding sequence for the AIM II protein shown in Figures 1A-C (SEQ ID NO:2); and DNA molecules which comprise a sequence substantially different from those described above but which, due to the degeneracy of the genetic code, still encode the AIM II protein.
• ORF open reading frame
• the invention provides isolated nucleic acid molecules encoding the AIM II polypeptide having an amino acid sequence encoded by the cDNA clone contained in the plasmid deposited as ATCC Deposit No. 97689 on
• this nucleic acid molecule will encode the polypeptide encoded by the above-described deposited cDNA clone.
• the invention further provides an isolated nucleic acid molecule having the nucleotide sequence shown in Figures 1 A-C (SEQ ID NO: 1) or the nucleotide sequence of the AIM II cDNA contained in the above-described deposited clone, or a nucleic acid molecule having a sequence complementary to one of the above sequences.
• Such isolated molecules, particularly DNA molecules are useful as probes for gene mapping, by in situ hybridization with chromosomes, and for detecting expression of the AIM II gene in human tissue, for instance, by Northern blot analysis.
• the present invention is further directed to fragments of the isolated nucleic acid molecules described herein.
• a fragment of an isolated nucleic acid molecule having the nucleotide sequence of the deposited cDNA or the nucleotide sequence shown in Figures 1A-C is intended fragments at least about 15 nt. and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length which are useful as diagnostic probes and primers as discussed herein.
• fragments 50-1500 nt in length are also useful according to the present invention as are fragments corresponding to most, if not all, of the nucleotide sequence of the deposited cDNA or as shown in Figures 1 A-C (SEQ
• fragments at least 20 nt in length are intended fragments which include 20 or more contiguous bases from the nucleotide sequence of the deposited cDNA or the nucleotide sequence as shown in Figures 1A-C (SEQ ID NO:1).
• nucleic acid fragments of the present invention include nucleic acid molecules encoding epitope-bearing portions of the AIM II protein.
• nucleic acid fragments of the present invention include nucleic acid molecules encoding: a polypeptide comprising amino acid residues from about 13 to about 20 in Figures 1 A-C (SEQ ID NO:2); a polypeptide comprising amino acid residues from about 23 to about 36 in Figures 1A-C (SEQ ID NO:2); a polypeptide comprising amino acid residues from about 69 to about 79 in Figures 1 A-C (SEQ ID NO:2); a polypeptide comprising amino acid residues from about 85 to about 94 in Figures 1 A-C (SEQ ID NO:2);a polypeptide comprising amino acid residues from about 167 to about 178 in Figures 1A-C (SEQ ID NO:2);a polypeptide comprising amino acid residues from about 184 to about 196 in
• Figures 1 A-C SEQ ID NO:2; and a polypeptide comprising amino acid residues from about 221 to about 233 in Figures 1 A-C (SEQ ID NO:2).
• the inventors have determined that the above polypeptide fragments are antigenic regions of the AIM II protein. Methods for determining other such epitope-bearing portions of the AIM II protein are described in detail below.
• AIM-II polynucleotides may be used in accordance with the present invention for a variety of applications, particularly those that make use of the chemical and biological properties of the AIM-II. Among these applications in autoimmune disease and aberrant cellular proliferation. Additional applications relate to diagonsis and to treatment of disorders of cells, tissues, and organisms.
• This invention is also related to the use of the AIM-II polynucleotides to detect complementary polynucleotides such as, for example, as a diagnostic reagent. Detection of a mutated form of an AIM-II associated with a dysfunction will provide a diagnostic tool that can add or define a diagnosis of a disease or susceptibility to disease which reults from under-expression, over-expression or altered expression of AIM-II, such as, for example, autoimmune diseases.
• the polynucleotide encoding the AIM-II may also be employed as a diagnostic marker for expression of the polypeptide of the present invention since the gene is found in many tumor cell lines including pancreatic tumor, testcs tumor, endometrial tumor and T-cell lymphoma.
• the invention provides an isolated nucleic acid molecule comprising a polynucleotide which hybridizes under stringent hybridization conditions to a portion of the polynucleotide in a nucleic acid molecule of the invention described above, for instance, the cDNA clone contained in ATCC Deposit 97689.
• stringent hybridization conditions is intended overnight incubation at 42°C in a solution comprising: 50% formamide, 5x SSC (150 mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 g/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1 x SSC at about 65 °C.
• a polynucleotide which hybridizes to a "portion" of a polynucleotide is intended a polynucleotide (either DNA or RNA) hybridizing to at least about 15 nucleotides (nt), and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably about 30-70 nt of the reference polynucleotide. These are useful as diagnostic probes and primers as discussed above and in more detail below.
• a portion of a polynucleotide of "at least 20 nt in length,” for example, is intended 20 or more contiguous nucleotides from the nucleotide sequence of the reference polynucleotide (e.g., the deposited cDNA or the nucleotide sequence as shown in Figures 1A-C (SEQ ID NO:1)).
• a polynucleotide which hybridizes only to a poly A sequence such as the 3' terminal poly(A) tract of the AIM II cDNA shown in Figures 1 A-C (SEQ ID NO:1)), or to a complementary stretch of T (or U) resides, would not be included in a polynucleotide of the invention used to hybridize to a portion of a nucleic acid of the invention, since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone).
• nucleic acid molecules of the present invention which encode an AIM II polypeptide may include, but are not limited to those encoding the amino acid sequence of the polypeptide, by itself; the coding sequence for the polypeptide and additional sequences, such as those encoding a leader or secretory sequence, such as a pre-, or pro- or prepro- protein sequence; the coding sequence of the polypeptide, with or without the aforementioned additional coding sequences, together with additional, non-coding sequences, including for example, but not limited to introns and non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA processing, including splicing and polyadenylation signals, for example - ribosome binding and stability of mRNA; an additional coding sequence which codes for additional amino acids, such as those which provide additional functionalities.
• the sequence encoding the polypeptide may be fused to a marker sequence, such as a sequence encoding a peptide which facilitates purification of the fused polypeptide.
• the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (Qiagen, Inc.), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein.
• the "HA” tag is another peptide useful for purification which corresponds to an epitope derived from the influenza hemagglutinin protein, which has been described by Wilson et al., Cell 37: 161 (1984).
• other such fusion proteins include the AIM II fused to Fc at the N- or C-terminus.
• Nucleic acid molecules according to the present invention further include those encoding the full-length AIM-II polypeptide lacking the N-terminal methonine.
• the present invention further relates to variants of the nucleic acid molecules of the present invention, which encode portions, analogs or derivatives of the AIM II protein.
• Variants may occur naturally, such as a natural allelic variant.
• allelic variant is intended one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985). Non-naturally occurring variants may be produced using art-known mutagenesis techniques.
• variants include those produced by nucleotide substitutions, deletions or additions which may involve one or more nucleotides.
• the variants may be altered in coding regoins, non-coding regions, or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions. Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of the AIM II protein or portions thereof. Also especially preferred in this regard are conservative substitutions.
• nucleic acid molecules comprising a polynucleotide having a nucleotide sequence at least 90% identical, and more preferably at least 95%, 96%, 97%, 98% or 99% identical to (a) a nucleotide sequence encoding the AIM II polypeptide having the complete amino acid sequence in Figures 1 A-C (SEQ ID NO:2); (b) a nucleotide sequence encoding the AIM II polypeptide having the complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No.
• nucleotide sequence encoding a soluble AIM II polypeptide having the extracellular and intracellular domains but lacking the transmembrane domain; and (g) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d), (e) or (f) above.
• polynucleotide having a nucleotide sequence at least, for example, 95% "identical" to a reference nucleotide sequence encoding an AIM II polypeptide is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the AIM II polypeptide.
• a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.
• These mutations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
• nucleic acid molecule is at least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the nucleotide sequence shown in Figures 1 A-C or to the nucleotides sequence of the deposited cDNA clone can be determined conventionally using known computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711. Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2: 482-489 (1981), to find the best segment of homology between two sequences.
• Bestfit program Wiconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711. Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2: 482-489 (1981), to find the best segment of homology between two sequences.
• the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.
• the present application is directed to nucleic acid molecules at least 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence shown in Figures 1A-C (SEQ ID NO: 1) or to the nucleic acid sequence of the deposited cDNA, irrespective of whether they encode a polypeptide having AIM II activity. This is because even where a particular nucleic acid molecule does not encode a polypeptide having AIM II activity, one of skill in the art would still know how to use the nucleic acid molecule, for instance, as a hybridization probe or a polymerase chain reaction (PCR) primer.
• PCR polymerase chain reaction
• nucleic acid molecules of the present invention that do not encode a polypeptide having AIM II activity include, inter alia, (1) isolating the AIM II gene or allelic variants thereof in a cDNA library; (2) in situ hybridization (e.g., "FISH") to metaphase chromosomal spreads to provide precise chromosomal location of the AIM II gene, as described in Verma et al., Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York (1988); and Northern Blot analysis for detecting AIM II mRNA expression in specific tissues.
• FISH in situ hybridization
• nucleic acid molecules having sequences at least 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence shown in Figures 1A-C (SEQ ID NO:1) or to the nucleic acid sequence of the deposited cDNA which do, in fact, encode a polypeptide having AIM II protein activity.
• a polypeptide having AIM II activity is intended polypeptides exhibiting activity similar, but not necessarily identical, to an activity of the AIM II protein of the invention, as measured in a particular biological assay.
• AIM II protein cytotoxic activity can be measured using propidium iodide staining to demonstrate apoptosis as described by Zarres et al., Cell 70: 31-46 (1992).
• AIMII induced apoptosis can also be measured using TU NEL staining as described by Gavierli et al., J. Cell. Biol. 119: 493-501 (1992).
• the propidium iodide staining is performed as follows. Cells either from tissue or culture are fixed in formaldehyde, cut into frozen sections and stained with propidium iodide. The cell nuclei are visualized by propidium iodide using confocal fluorescent microscopy. Cell death is indicated by pyknotic nuclei ( chromosome clumping, shrinking and/or fragmentation of nuclei).
• nucleic acid molecules having a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of the deposited cDNA or the nucleic acid sequence shown in Figures 1A-C (SEQ ID NO:1) will encode a polypeptide "having AIM II protein activity.”
• degenerate variants of these nucleotide sequences all encode the same polypeptide, this will be clear to the skilled artisan even without performing the above described comparison assay.
• nucleic acid molecules that are not degenerate variants, a reasonable number will also encode a polypeptide having AIM II protein activity. This is because the skilled artisan is fully aware of amino acid substitutions that are either less likely or not likely to significantly effect protein function (e.g., replacing one aliphatic amino acid with a second aliphatic amino acid).
• the present invention also relates to vectors which include the isolated DNA molecules of the present invention, host cells which are genetically engineered with the recombinant vectors, and the production of AIM II polypeptides or fragments thereof by recombinant techniques.
• the polynucleotides may be joined to a vector containing a selectable marker for propagation in a host.
• a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
• the DNA insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few.
• an appropriate promoter such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few.
• Other suitable promoters will be known to the skilled artisan.
• the expression constructs will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation.
• the coding portion of the mature transcripts expressed by the constructs will preferably include a translation initiating at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.
• the expression vectors will preferably include at least one selectable marker.
• markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria.
• Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli,
• Streptomyces and Salmonella typhimurium cells
• fungal cells such as yeast cells
• insect cells such as Drosophila S2 and Spodoptera Sf9 cells
• animal cells such as CHO, COS and Bowes melanoma cells
• plant cells Appropriate culture mediums and conditions for the above-described host cells are known in the art.
• vectors preferred for use in bacteria include pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, ⁇ KK233-3, pDR540, pRIT5 available from Pharmacia.
• preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.
• Other suitable vectors will be readily apparent to the skilled artisan.
• Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986).
• the polypeptide may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art.
• a preferred fusion protein comprises a heterologous region from immunoglobulin that is useful to solubilize proteins. For example, EP-A-O 464
• fusion proteins comprising various portions of constant region of immunoglobin molecules together with another human protein or part thereof.
• the Fc part in a fusion protein is thoroughly advantageous for use in therapy and diagnosis and thus results, for example, in improved pharmacokinetic properties (EP-A 0232 262).
• EP-A 0232 262 it would be desirable to be able to delete the Fc part after the fusion protein has been expressed, detected and purified in the advantageous manner described. This is the case when Fc portion proves to be a hindrance to use in therapy and diagnosis, for example when the fusion protein is to be used as antigen for immunizations.
• human proteins such as, hIL5- has been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5.
• Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5.
• the AIM II protein can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction, chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography
• Polypeptides of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes. AIMII Polypeptides and Fragments
• the invention further provides an isolated AIM II polypeptide having the amino acid sequence encoded by the deposited cDNA, or the amino acid sequence in Figures 1A-C (SEQ ID NO:2), or a peptide or polypeptide comprising a protion of the above polypeptides. It will be recognized in the art that some amino acid sequences of the AIM II polypeptide can be varied without significant effect of the structure or function of the protein. If such differences in sequence are contemplated, it should be remembered that there will be critical areas on the protein which determine activity.
• the invention further includes variations of the AIM II polypeptide which show substantial AIM II polypeptide activity or which include regions of AIM II protein such as the protein portions discussed below.
• Such mutants include deletions, insertions, inversions, repeats, and type substitutions.
• guidance concerning which amino acid changes are likely to be phenotypically silent can be found in Bowie, J.U., et al., "Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions," Science 247:1306-1310 (1990).
• amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as an IgG Fc fusion region peptide of leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence.
• a conserved or non-conserved amino acid residue preferably a conserved amino acid residue
• substituted amino acid residue may or may not be one encoded by the genetic code
• amino acid residues includes a substituent group
• the mature polypeptide is fused
• the replacement of amino acids can also change the selectivity of binding to cell surface receptors. Ostade et al., Nature 361:266-268 (1993) describes certain mutations resulting in selective binding of TNF- ⁇ to only one of the two known types of TNF receptors.
• the AIM II receptor of the present invention may include one or more amino acid substitutions, deletions or additions, either from natural mutations or human manipulation.
• changes are preferably of a minor nature, such as conservative amino acid substitutions that do not significantly affect the folding or activity of the protein (see Table 1).
• Amino acids in the AIM II protein of the present invention that are essential for function can be identified by methods known in the art, such as site- directed mutagenesis or alanine-scanning mutagenesis (Cunmngham and Wells, Science 244: 1081-1085 (1989)). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity such as receptor binding or in vitro, or in vitro proliferative activity. Sites that are critical for ligand-receptor binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al., J. Mol Biol 224:899-904 (1992) and de Vos et al. Science 255:306-312 (1992)).
• polypeptides of the present invention are preferably provided in an isolated form.
• isolated polypeptide is intended a poypeptide removed from its native environment.
• a polypeptide produced and contained within a recombinant host cell is considered “isolated” for purposes of the present invention.
• polypeptides that have been purified, partially or substantially, from a recombinant host are polypeptides that have been purified, partially or substantially, from a recombinant host.
• a recombinantly produced version of the AIM II polypeptide can be substantially purified by the one-step method described in Smith and Johnson, Gene 67:31-40 (1988).
• polypeptides of the present invention include the polypeptide encoded by the deposited cDNA, the polypeptide of Figures 1A-C (SEQ ID NO:2), the polypeptide of Figures 1 A-C (SEQ ID NO:2) lacking the N-terminal methinone, the extracellular domain, the transmembrane domain, the intracellular domain, soluble polypeptides comprising all or part of the extracellular and intracelluar domains but lacking the transmembrane domain, as well as polypeptides which are at least 80% identical, more preferably at least 90% or 95% identical, still more preferably at least 96%, 97%, 98% or 99% identical to the polypeptide encoded by the deposited cDNA, to the polypeptide of Figures 1A-C (SEQ ID NO:2), and also include portions of such polypeptides with at least 30 amino acids and more preferably at least 50 amino acids.
• polypeptide having an amino acid sequence at least, for example, 95% "identical" to a reference amino acid sequence of an AIM II polypeptide is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of the AIM II polypeptide.
• up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence.
• These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
• any particular polypeptide is at least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid sequence shown in Figures 1A-C (SEQ ID NO:2) or to the amino acid sequence encoded by deposited cDNA clone can be determined conventionally using known computer programs such the Bestfit program (Wisconsin Sequence Analysis
• AIM II polypeptide includes membrane-bound proteins (comprising a cytoplasmic domain, a transmembrane domain, and an extracellular domain) as well as truncated proteins that retain the AIM II polypeptide activity.
• soluble AIM II polypeptides comprise all or part of the extracellular domain of an AIM II protein, but lack the transmsmbrane region that would cause retention of the polypeptide on a cell membrane.
• Soluble AIM II may also include part of the transmembrane region or part of the cytoplasmic domain or other sequences, provided that the soluble AIM II protein is capable of being secreted.
• a heterologous signal peptide can be fused to the N-terminus of the soluble AIM II polypeptide such that the soluble AIM II polypeptide is secreted upon expression.
• polypeptide of the present invention could be used as a molecular weight marker on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those of skill in the art.
• the invention provides a peptide or polypeptide comprising an epitope-bearing portion of a polypeptide of the invention.
• the epitope of this polypeptide portion is an immunogenic or antigenic epitope of a polypeptide described herein.
• An "immunogenic epitope” is defined as a part of a protein that elicits an antibody response when the whole protein is the immunogen.
• a region of a protein molecule to which an antibody can bind is defined as an "antigenic epitope.”
• the number of immunogenic epitopes of a protein generally is less than the number of antigenic epitopes. See, for instance, Geysen et al., Proc. Natl. Acad. Sei. USA 81:3998- 4002 (1983).
• peptides or polypeptides bearing an antigenic epitope i.e., that contain a region of a protein molecule to which an antibody can bind
• relatively short synthetic peptides that mimic part of a protein sequence are routinely capable of eliciting an antiserum that reacts with the partially mimicked protein. See, for instance, Sutcliffe, J. G., Shinnick, T. M., Green, N. and Learner, R.A. (1983) Antibodies that react with predetermined sites on proteins. Science 219:660-666.
• Peptides capable of eliciting protein-reactive sera are frequently represented in the primary sequence of a protein, can be characterized by a set of simple chemical rules, and are confined neither to immunodominant regions of intact proteins (i.e., immunogenic epitopes) nor to the amino or carboxyl terminals.
• Antigenic epitope-bearing peptides and polypeptides of the invention are therefore useful to raise antibodies, including monoclonal antibodies, that bind specifically to a polypeptide of the invention. See, for instance, Wilson et al. Cell 37: 767-778 (1984) at 777.
• Antigenic epitope-bearing peptides and polypeptides of the invention preferably contain a sequence of at least seven, more preferably at least nine and most preferably between about at least about 15 to about 30 amino acids contained within the amino acid sequence of a polypeptide of the invention.
• Non-limiting examples of antigenic polypeptides or peptides that can be used to generate AIM Il-specific antibodies include: a polypeptide comprising amino acid residues from about 13 to about 20 in Figures 1 A-C (SEQ ID NO:2); a polypeptide comprising amino acid residues from about 23 to about 36 in
• Figures 1A-C (SEQ ID NO:2); a polypeptide comprising amino acid residues from about 69 to about 79 in Figures 1A-C (SEQ ID NO:2); a polypeptide comprising amino acid residues from about 85 to about 94 in Figures 1 A-C (SEQ ID NO:2); a polypeptide comprising amino acid residues from about 167 to about 178 in Figures 1A-C (SEQ ID NO:2); a polypeptide comprising amino acid residues from about 184 to about 196 in Figures 1A-C (SEQ ID NO:2); and a polypeptide comprising amino acid residues from about 221 to about 233 in Figures 1A-C (SEQ ID NO:2).
• the inventors have determined that the above polypeptide fragments are antigenic regions of the AIM II protein.
• the epitope-bearing peptides and polypeptides of the invention may be produced by any conventional means. Houghten, R. A. (1985) General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen-antibody interaction at the level of individual amino acids. Proc. Natl. Acad. Sci. USA 82:5131-5135. This "Simultaneous Multiple Peptide Synthesis (SMPS)" process is further described in U.S. Patent No. 4,631,21 1 to Houghten et al. (1986).
• SMPS Simultaneous Multiple Peptide Synthesis
• the AIM II polypeptide of the present invention may be employed to treat lymphoproliferative disease which results in lymphadenopathy, the AIM II mediates apoptosis by stimulating clonal deletion of T-cells and may therefore, be employed to treat autoimmune disease, to stimulate peripheral tolerance and cytotoxic T-cell mediated apoptosis.
• the AIM II may also be employed as a research tool in elucidating the biology of autoimmune disorders including systemic lupus erythematosus (SLE), immunoproliferative disease lymphadenopathy (IPL), angioimmunoproliferative lymphadenopathy (AIL), immunoblastive lymphadenopathy (IBL), rheumatoid arthritis, diabetes, and multiple sclerosis, allergies and to treat graft versus host disease.
• SLE systemic lupus erythematosus
• IPL immunoproliferative disease lymphadenopathy
• AIL angioimmunoproliferative lymphadenopathy
• IBL immunoblastive lymphadenopathy
• rheumatoid arthritis diabetes
• multiple sclerosis allergies and to treat graft versus host disease.
• the AIM II polypeptide of the present invention may also be employed to inhibit neoplasia, such as tumor cell growth.
• the AIM II polypeptide may be responsible for tumor destruction through apoptosis and cytotoxicity to certain cells.
• AIM II may also be employed to treat diseases which require growth promotion activity, for example, restenosis, since AIM II has proliferation effects on cells of endothelial origin.
• AIM II may, therefore, also be employed to regulate hematopoiesis in endothelial cell development.
• This invention also provides a method for identification of molecules, such as receptor molecules, that bind AIM II.
• Genes encoding proteins that bind AIM II, such as receptor proteins can be identified by numerous methods known to those of skill in the art, for example, ligand panning and FACS sorting. Such methods are described in many laboratory manuals such as, for instance, Coligan et al, Current Protocols in Immunology, 1 (2): Chapter 5 (1991).
• expression cloning may be employed for this purpose.
• polyadenyiated RNA is prepared from a cell responsive to AIM II, a cDNA library is created from this RNA, the library is divided into pools and the pools are transfected individually into cells that are not responsive to AIM II. The transfected cells then are exposed to labeled AIM II.
• AIM II can be labeled by a variety of well-known techniques including standard methods of radio-iodination or inclusion of a recognition site for a site-specific protein kinase.
• the cells are fixed and binding of AIM II is determined. These procedures conveniently are carried out on glass slides.
• Sub-pools are prepared from these positives, transfected into host cells and screened as described above. Using an iterative sub-pooling and re-screening process, one or more single clones that encode the putative binding molecule, such as a receptor molecule, can be isolated.
• a labeled ligand can be photo affinity linked to a cell extract, such as a membrane or a membrane extract, prepared from cells that express a molecule that it binds, such as a receptor molecule.
• Cross-linked material is resolved by polyacrylamide gel electrophoresis ("PAGE") and exposed to X-ray film.
• PAGE polyacrylamide gel electrophoresis
• the labeled complex containing the ligand-receptor can be excised, resolved into peptide fragments, and subjected to protein microsequencing.
• the amino acid sequence obtained from microsequencing can be used to design unique or degenerate oligonucleotide probes to screen cDNA libraries to identify genes encoding the putative receptor molecule.
• Polypeptides of the invention also can be used to assess AIM II binding capacity of AIM II binding molecules, such as receptor molecules, in cells or in cell-free preparations.
• AIM II polypeptides of the present invention and the epitope-bearing fragments thereof described above can be combined with parts of the constant domain of immunoglobulins (IgG), resulting in chimeric polypeptides.
• IgG immunoglobulins
• These fusion proteins facilitate purification and show an increased half-life in vivo. This has been shown, e.g., for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins (EPA 394,827; Traunecker et al., Nature 331:84- 86 (1988)).
• Fusion proteins that have a disulfide-linked dimeric structure due to the IgG part can also be more efficient in binding and neutralizing other molecules than the monomeric AIM II protein or protein fragment alone (Fountoulakis et al., J Biochem 270:3958-3964 (1995)).
• AIM II is expressed in spleen, thymus and bone marrow tissue.
• disorders such as septic shock, inflammation, cerebral malaria, activation of the HIV virus, graft-host rejection, bone resorption, rheumatoid arthritis and cachexia
• bodily fluids e.g., serum, plasma, urine, synovial fluid or spinal fluid
• the invention provides a diagnostic method useful during diagnosis of a disorder, which involves: (a) assaying AIM II gene expression level in cells or body fluid of an individual; (b) comparing the
• AIM II gene expression level with a standard AIM II gene expression level, whereby an increase or decrease in the assayed AIM II gene expression level compared to the standard expression level is indicative of a disorder.
• AIM II Agonists and Antagonists The invention also provides a method of screening compounds to identify those which enhance or block the action of AIM II on cells, such as its interaction with AIM II-binding molecules such as receptor molecules.
• An agonist is a compound which increases the natural biological functions of AIM II or which functions in a manner similar to AIM II. while antagonists decrease or eliminate such functions.
• a cellular compartment such as a membrane preparation
• a cellular compartment may be prepared from a cell that expresses a molecule that binds AIM II, such as a molecule of a signaling or regulatory pathway modulated by AIM II.
• the preparation is incubated with labeled AIM II in the absence or the presence of a candidate molecule which may be an AIM II agonist or antagonist.
• AIM II a molecule of a signaling or regulatory pathway modulated by AIM II.
• the ability of the candidate molecule to bind the binding molecule or AIM II itself is reflected in decreased binding of the labeled ligand.
• AIM II-like effects of potential agonists and antagonists may by measured, for instance, by determining activity of a second messenger system following interaction of the candidate molecule with a cell or appropriate cell preparation, and comparing the effect with that of AIM II or molecules that elicit the same effects as AIM II.
• Second messenger systems that may be useful in this regard include but are not limited to AMP guanylate cyclase, ion channel or phosphoinositide hydrolysis second messenger systems.
• an assay for AIM II antagonists is a competitive assay that combines AIM II and a potential antagonist with membrane-bound AIM II receptor molecules or recombinant AIM II receptor molecules under appropriate conditions for a competitive inhibition assay.
• AIM II can be labeled, such as by radioactivity, such that the number of AIM II molecules bound to a receptor molecule can be determined accurately to assess the effectiveness of the potential antagonist.
• Potential antagonists include small organic molecules, peptides, polypeptides and antibodies that bind to a polypeptide of the invention, and thereby inhibit or extinguish its activity. Potential antagonists also may be small organic molecules, a peptide, a polypeptide such as a closely related protein or antibody that binds the same sites on a binding molecule, such as a receptor molecule, without inducing AIM II-induced activities, thereby preventing the action of AIM II by excluding AIM II from binding. Other potential antagonists include antisense molecules. Antisense technology can be used to control gene expression through antisense DNA or RNA or through triple-helix formation. Antisense techniques are discussed, for example, in Okano, J. Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988).
• Triple helix formation is discussed in, for instance, Lee et al., Nucleic Acids Research 3:173 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al, Science 251:1360 (1991).
• the methods are based on binding of a polynucleotide to a complementary DNA or RNA.
• the 5' coding portion of a polynucleotide that encodes the mature polypeptide of the present invention may be used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length.
• a DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription thereby preventing transcription and the production of AIM II.
• the antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into AIM II polypeptide.
• the oligonucleotides described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of AIM II.
• the antagonists may be employed in a composition with a pharmaceutically acceptable carrier, e.g., as hereinafter described.
• the antagonists may be employed for instance to treat cachexia which is a lipid clearing defect resulting from a systemic deficiency of lipoprotein lipase, which is believed to be suppressed by AIM II.
• the AIM II antagonists may also be employed to treat cerebral malaria in which AIM II may play a pathogenic role.
• the antagonists may also be employed to treat rheumatoid arthritis by inhibiting AIM-II induced production of inflammatory cytokines, such as IL1 in the synovial cells.
• AIM II antagonists are preferably injected intra-articularly.
• the AIM II antagonists may also be employed to prevent graft-host rejection by preventing the stimulation of the immune system in the presence of a graft.
• the AIM II antagonists may also be employed to inhibit bone resorption and, therefore, to treat and/or prevent osteoporosis.
• the antagonists may also be employed as anti-inflammatory agents, and to treat endotoxic shock. This critical condition results from an exaggerated response to bacterial and other types of infection.
• tissue in mammals with cancer express significantly reduced levels of the AIM II protein and mRNA encoding the AIM II protein when compared to a corresponding "standard" mammal, i.e., a mammal of the same species not having the cancer.
• reduced levels of the AIM II protein can be detected in certain body fluids (e.g., sera, plasma, urine, and spinal fluid) from mammals with cancer when compared to sera from mammals of the same species not having the cancer.
• the invention provides a diagnostic method useful during tumor diagnosis, which involves assaying the expression level of the gene encoding the AIM II protein in mammalian cells or body fluid and comparing the gene expression level with a standard AIM II gene expression level, whereby an decrease in the gene expression level over the standard is indicative of certain tumors.
• the present invention is useful as a prognostic indicator, whereby patients exhibiting enhanced AIM II gene expression may experience a better clinical outcome relative to patients expressing the gene at a lower level.
• saying the expression level of the gene encoding the AIM II protein is intended qualitatively or quantitatively measuring or estimating the level of the AIM II protein or the level of the mRNA encoding the AIM II protein in a first biological sample either directly (e.g., by determining or estimating absolute protein level or mRNA level) or relatively (e.g., by comparing to the AIM II protein level or mRNA level in a second biological sample).
• the AIM II protein level or mRNA level in the first biological sample is measured or estimated and compared to a standard AIM II protein level or mRNA level, the standard being taken from a second biological sample obtained from an individual not having the cancer.
• a standard AIM II protein level or mRNA level is known, it can be used repeatedly as a standard for comparison.
• biological sample any biological sample obtained from an individual, cell line, tissue culture, or other source which contains AIM II protein or mRNA.
• Biological samples include mammalian body fluids (such as sera, plasma, urine, synovial fluid and spinal fluid) which contain secreted mature AIM II protein, and ovarian, prostate, heart, placenta, pancreas liver, spleen, lung, breast and umbilical tissue.
• the present invention is useful for detecting cancer in mammals.
• the invention is useful during diagnosis of the of following types of cancers in mammals: breast, ovarian, prostate, bone, liver, lung, pancreatic, and spleenic.
• Preferred mammals include monkeys, apes, cats, dogs, cows, pigs, horses, rabbits and humans. Particularly preferred are humans.
• Total cellular RNA can be isolated from a biological sample using the single-step guanidinium-thiocyanate-phenol-chloroform method described in Chomczynski and Sacchi, Anal. Biochem. 162:156-159 (1987). Levels of mRNA encoding the AIM II protein are then assayed using any appropriate method.
• AIM II protein levels in a biological sample can occur using antibody-based techniques. For example, AIM II protein expression in tissues can be studied with classical immunohistological methods (Jalkanen, M., et al.,
• antibody-based methods useful for detecting AIM II protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
• immunoassays such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
• Suitable lables are known in the art and include enzyme lables, such as, Glucose oxidase, and radioisotopes, such as iodine ( 125 I, 121 I), carbon ( 14 C), sulpher ( 35 S), tritium ( 3 H), indium ( 112 In), and technetium ( 99m Tc), and fluorescent labels, such as fluorescein and rhodamine, and biotin.
• enzyme lables such as, Glucose oxidase, and radioisotopes, such as iodine ( 125 I, 121 I), carbon ( 14 C), sulpher ( 35 S), tritium ( 3 H), indium ( 112 In), and technetium ( 99m Tc)
• fluorescent labels such as fluorescein and rhodamine, and biotin.
• the AIM II polypeptides may be employed to treat neoplasia, lymphadenopathy, autoimmune disease, graft versus host disease.
• the AIM II polypeptide of the present invention may be employed to inhibit neoplasia, such as tumor cell growth.
• the AIM II polypeptide may be responsible for tumor destruction through apoptosis and cytotoxicity to certain cells.
• AIM II may also be employed to treat diseases which require growth promotion activity, for example, restenosis, since AIM II has proliferation effects on cells of endothelial origin. AIM II may, therefore, also be employed to regulate hematopoiesis in endothelial cell development.
• the invention further provides a method of treating an individual in need of an increased level of AIM II activity comprising administering to such an individual a pharmaceutical composition comprising an effective amount of an isolated AIM II polypeptide of the invention, particularly a mature form of the AIM II, effective to increase the AIM II activity level in such an individual.
• the total pharmaceutically effective amount of AIM II polypeptide administered parenterally per dose will be in the range of about 1 ⁇ g/kg/day to 10 mg/kg/day of patient body weight, although, as noted above, this will be subject to therapeutic discretion. More preferably, this dose is at least 0.01 mg/kg/day, and most preferably for humans between about 0.01 and 1 mg/kg/day for the hormone.
• the AIM II polypeptide is typically administered at a dose rate of about 1 ⁇ g/kg/hour to about 50 ⁇ g/kg/hour, either by 1-4 injections per day or by continuous subcutaneous infusions, for example, using a mini-pump. An intravenous bag solution may also be employed.
• compositions containing the AIM II of the invention may be administered orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (as by powders, ointments, drops or transdermal patch), bucally, or as an oral or nasal spray.
• pharmaceutically acceptable carrier is meant a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
• parenteral refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.
• AIM II polypeptides containing the transmembrane region can also be used when appropriately solubilized by including detergents, such as triton X-100, with buffer.
• the nucleic acid molecules of the present invention are also valuable for chromosome identification.
• the sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome.
• the mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease.
• the cDNA herein disclosed is used to clone genomic DNA of an AIM II protein gene. This can be accomplished using a variety of well known techniques and libraries, which generally are available commercially. The genomic DNA then is used for in situ chromosome mapping using well known techniques for this purpose.
• sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis of the 3' untranslated region of the gene is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes.
• Fluorescence in situ hybridization of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step.
• This technique can be used with probes from the cDNA as short as 50 or 60 bp.
• the DNA sequence encoding the AIM II protein in the deposited cDNA clone is amplified using PCR oligonucleotide primers specific to the amino terminal sequences of the AIM II protein and to vector sequences 3' to the gene.
• a 22 kDa AIM II protein fragment (lacking the N-terminus and transmembrane region) is expressed using the following primers:
• the 5' oligonucleotide primer has the sequence 5' GCGGGATCCGGAGAGATGGTCACC 3' (SEQ ID NO:7) containing the underlined BamHl restriction site, which encodes 244-258 nucleotides of the AIM II protein coding sequence in Figures 1A-C (SEQ ID NO:l).
• the 3 ' primer has the sequence 5 '
• CGCAAGCTTCCTTCACACCATGAAAGC 3' (SEQ ID NO:8) containing the underlined Hind III restriction site followed by 756-783 nucleotides as shown in Figures 1A-C.
• the entire AIM II protein can be expressed using the following primers:
• the 5' oligonucleotide primer has the sequence 5 ' GACC GGATCC ATG
• the 3' primer has the sequence 5' CGC AAGCTT CCT TCA CAC CAT GAA AGC 3' (SEQ ID NO: 10) containing the underlined Hindlll restriction site followed followed by 756-783 nucleotides as shown in Figures 1 A-C.
• restriction sites are convenient to restriction enzyme sites in the bacterial expression vector pQE9, which are used for bacterial expression in these examples. (Qiagen, Inc. 9259 Eton Avenue, Chatsworth, CA, 9131 1). pQE9 encodes ampicillin antibiotic resistance ("Amp r ”) and contains a bacterial origin of replication ("ori”), an IPTG inducible promoter, a ribosome binding site (“RBS”). a 6-His tag and restriction enzyme sites.
• amplified AIM II DNA and the vector pQE9 both are digested with
• Insertion of the AIM II protein DNA into the restricted pQE9 vector places the AIM II protein coding region downstream of and operably linked to the vector's IPTG- inducible promoter and in-frame with an initiating AUG appropriately positioned for translation of AIM II protein.
• the bacterial expression vector pQE60 is used for bacterial expression in this example. (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 9131 1). pQE60 encodes ampicillin antibiotic resistance ("Amp r ”) and contains a bacterial origin of replication ("ori”), an IPTG inducible promoter, a ribosome binding site
• RBS nickel-nitrilo-tri-acetic acid
• Ni-NTA nickel-nitrilo-tri-acetic acid
• suitable single restriction enzyme cleavage sites These elements are arranged such that an inserted DNA fragment encoding a polypeptide expresses that polypeptide with the six His residues (i.e., a "6 X His tag”) covalently linked to the carboxyl terminus of that polypeptide.
• the DNA sequence encoding the desired portion of the AIM II protein is amplified from the deposited cDNA clone using PCR oligonucleotide primers which anneal to the amino terminal sequences of the desired portion of the AIM II protein and to sequences in the deposited construct 3' to the cDNA coding sequence. Additional nucleotides containing restriction sites to facilitate cloning in the pQE60 vector are added to the 5' and 3' sequences, respectively.
• the 5' primer has the sequence 5' GACGC CCATGG AG GAG GAG AGT GTC GTA CGG C 3' (SEQ ID NO: 17) containing the underlined Ncol restriction site followed by nucleotides complementary to the amino terminal coding sequence of the AIM II sequence in Figures 1A-C.
• the 3' primer has the sequence 5' GACC GGATCC CAC
• the amplified AIM II DNA fragment and the vector pQE60 are digested with BamHI and Nco I and the digested DNAs are then ligated together. Insertion of the AIM II DNA into the restricted pQE60 vector places the AIM II protein coding region downstream from the IPTG-inducible promoter and in- frame with an initiating AUG and the six histidine codons.
• the ligation mixture from the HA tagged expression constructs made in A or B, above, is transformed into competent E. coli cells using standard procedures. Such procedures are described in Sambrook et al, Molecular Cloning: a Laboratory Manual, 2nd Ed.; Cold Spring Harbor Laboratory Press,
• E. coli strain M15/rep4 containing multiple copies of the plasmid pREP4, which expresses lac repressor and confers kanamycin resistance ("Kan r "), is used in carrying out the illustrative example described herein.
• This strain which is only one of many that are suitable for expressing AIM II protein, is available commercially from Qiagen. Transformants are identified by their ability to grow on LB plates in the presence of ampicillin and kanamycin. Plasmid DNA is isolated from resistant colonies and the identity of the cloned DNA confirmed by restriction analysis.
• Clones containing the desired constructs are grown overnight ("O/N") in liquid culture in LB media supplemented with both ampicillin (100 ⁇ g/ml) and kanamycin (25 ⁇ g/ml).
• the O/N culture is used to inoculate a large culture, at a dilution of approximately 1:100 to 1:250.
• the cells are grown to an optical density at 600nm
• IPTG Isopropyl-B-D-thiogalactopyranoside
• the 8M urea solution containing the solubilized protein is passed over a PD-10 column in 2X phosphate-buffered saline ("PBS"), thereby removing the urea, exchanging the buffer and refolding the protein.
• PBS 2X phosphate-buffered saline
• the protein is purified by a further step of chromatography to remove endotoxin. Then, it is sterile filtered.
• the sterile filtered protein preparation is stored in 2X PBS at a concentration of 95 ⁇ /ml.
• the bacterial expression vector pQE60 is used for bacterial expression in this example. (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 9131 1). pQE60 encodes ampicillin antibiotic resistance ("Amp r ”) and contains a bacterial origin of replication ("ori"). an IPTG inducible promoter, a ribosome binding site
• RBS nickel-nitrilo-tri-acetic acid
• Ni-NTA nickel-nitrilo-tri-acetic acid
• suitable single restriction enzyme cleavage sites These elements are arranged such that a DNA fragment encoding a polypeptide may be inserted in such as way as to produce that polypeptide with the six His residues (i.e., a "6 X His tag”) covalently linked to the carboxyl terminus of that polypeptide.
• the polypeptide coding sequence is inserted such that translation of the six His codons is prevented and, therefore, the polypeptide is produced with no 6 X His tag.
• the DNA sequence encoding the desired portion of the AIM II protein lacking the hydrophobic leader sequence is amplified from the deposited cDNA clone using PCR oligonucleotide primers which anneal to the amino terminal sequences of the desired portion of the AIM II protein and to sequences in the deposited construct 3 1 to the cDNA coding sequence. Additional nucleotides containing restriction sites to facilitate cloning in the pQE60 vector are added to the 5' and 3' sequences, respectively.
• the 5' primer has the sequence 5'GACGC CCATGG AG GAG GAG AGT GTC GTA CGG C 3' (SEQ ID NO: 17) containing the underlined Ncol restriction site followed by nucleotides complementary to the amino terminal coding sequence of the AIM II sequence in Figures 1A-C.
• the 3' primer has the sequence 5' CGC AAGCTT CCTT CAC ACC ATG AAA GC
• amplified AIM II DNA fragments and the vector pQE60 are digested with Ncol and Hind III and the digested DNAs are then ligated together.
• Insertion of the AIM II DNA into the restricted pQE60 vector places the AIM II protein coding region including its associated stop codon downstream from the IPTG-inducible promoter and in-frame with an initiating AUG.
• the associated stop codon prevents translation of the six histidine codons downstream of the insertion point.
• the ligation mixture is transformed into competent E. coli cells using standard procedures such as those described in Sambrook et al., Molecular Cloning: a Laboratory Manual, 2nd Ed; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).
• coli strain M15/rep4 containing multiple copies of the plasmid pREP4, which expresses the lac repressor and confers kanamycin resistance ("Kan r "), is used in carrying out the illustrative example described herein.
• This strain which is only one of many that are suitable for expressing AIM II protein, is available commercially from QIAGEN, Inc., supra. Transformants are identified by their ability to grow on LB plates in the presence of ampicillin and kanamycin. Plasmid DNA is isolated from resistant colonies and the identity of the cloned DNA confirmed by restriction analysis, PCR and DNA sequencing.
• Clones containing the desired constructs are grown overnight ("O/N") in liquid culture in LB media supplemented with both ampicillin (100 ⁇ g/ml) and kanamycin (25 ⁇ g/ml).
• the O/N culture is used to inoculate a large culture, at a dilution of approximately 1:25 to 1 :250.
• the cells are grown to an optical density at 600 nm ("OD600") of between 0.4 and 0.6.
• Isopropyl-b-D- thiogalactopyranoside (“IPTG”) is then added to a final concentration of 1 mM to induce transcription from the lac repressor sensitive promoter, by inactivating the lad repressor. Cells subsequently are incubated further for 3 to 4 hours.
• the cells are then stirred for 3-4 hours at 4°C in 6M guanidine-HCl, pH8.
• the cell debris is removed by centrifugation, and the supernatant containing the AIM II is dialyzed against 50 mM Na-acetate buffer pH6, supplemented with 200 mM NaCl.
• the protein can be successfully refolded by dialyzing it against 500 mM NaCl, 20% glycerol, 25 mM Tris/HCl pH7.4. containing protease inhibitors.
• the protein can be purified by ion exchange, hydrophobic interaction and size exclusion chromatography.
• an affinity chromatography step such as an antibody column can be used to obtain pure AIM II protein.
• the purified protein is stored at 4°C or frozen at -80°C.
• Example 2 Cloning and Expression of AIM II protein in a Baculovirus Expression System
• the cDNA sequence encoding the full length AIM II protein in the deposited clone is amplified using PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the gene:
• the 5' primer has the sequence 5'GCT CCA GGA TCC GCC ATC ATG GAG GAG AGT GTC GTA CGG C3' (SEQ ID NO: 1 1) containing the underlined Bam HI restriction enzyme site followed by 22 bases of the sequence of AIM II protein in Figures 1A-C. Inserted into an expression vector, as described below, the 5' end of the amplified fragment encoding AIM II provides an efficient signal peptide. An efficient signal for initiation of translation in eukaryotic cells, as described by Kozak, M., J. Mol Biol. 196:941-950 (1987) is appropriately located in the vector portion of the construct.
• the 3' primer has the sequence 5'GA CGC GGT ACC GTC CAA TGC ACC ACG CTC CTT CCT TC 3' (SEQ ID NO: 12) containing the underlined Asp 718 restriction site followed by nucleotides complementary to 769-795 nucleotides of the AIM II set out in Figures 1 A-C.
• the amplified fragment is isolated from a 1% agarose gel using a commercially available kit ("Geneclean,” BIO 101 Inc., La Jolla, Ca.). The fragment then is digested with Bam HI and Asp 718 and again is purified on a 1 % agarose gel. This fragment is designated herein F2.
• the vector pA2-GP is used to express the AIM II protein in the baculovirus expression system, using standard methods, as described in Summers et al , A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures, Texas Agricultural Experimental Station Bulletin No. 1555 (1987).
• This expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV) followed by convenient restriction sites.
• the signal peptide of AcMNPV gp67, including the N-terminal methionine, is located just upstream of a BamHI site.
• the polyadenylation site of the simian virus 40 (“SV40") is used for efficient polyadenylation.
• the beta- galactosidase gene from E. coli is inserted in the same orientation as the polyhedrin promoter and is followed by the polyadenylation signal of the polyhedrin gene.
• the polyhedrin sequences are flanked at both sides by viral sequences for cell-mediated homologous recombination with wild-type viral DNA to generate viable virus that express the cloned polynucleotide.
• baculovirus vectors could be used in place of pA2-GP, such as pAc373, pVL941 and pAcIMl provided, as those of skill readily will appreciate, that construction provides appropriately located signals for transcription, translation, trafficking and the like, such as an in-frame AUG and a signal peptide, as required.
• pA2-GP such as pAc373, pVL941 and pAcIMl
• the plasmid is digested with the restriction enzyme Bam HI and Asp 718 and then is dephosphorylated using calf intestinal phosphatase, using routine procedures known in the art.
• the DNA is then isolated from a 1 % agarose gel using a commercially available kit ("Geneclean" BIO 101 Inc., La Jolla, Ca.).
• This vector DNA is designated herein "V".
• Fragment F2 and the dephosphorylated plasmid V2 are ligated together with T4 DNA ligase.
• E. coli HB101 cells are transformed with ligation mix and spread on culture plates.
• Bacteria are identified that contain the plasmid with the human AIM II gene by digesting DNA from individual colonies using Xbal and then analyzing the digestion product by gel electrophoresis. The sequence of the cloned fragment is confirmed by DNA sequencing. This plasmid is designated herein pBac AIM II .
• 5 ⁇ g of the plasmid pBac AIM II is co-transfected with 1.0 ⁇ g of a commercially available linearized baculovirus DNA ("BaculoGoldTM baculovirus DNA", Pharmingen, San Diego, CA.), using the lipofection method described by Feigner et al. Proc. Natl. Acad. Sci. USA 84: 7413-7417 (1987).
• BaculoGoldTM virus DNA and 5 ⁇ g of the plasmid pBac AIM II are mixed in a sterile well of a microtiter plate containing 50 ⁇ l of serum-free Grace's medium (Life Technologies Inc., Gaithersburg, MD).
• plaque assay After four days the supernatant is collected and a plaque assay is performed, as described by Summers and Smith, cited above. An agarose gel with "Blue Gal” (Life Technologies Inc., Gaithersburg) is used to allow easy identification and isolation of gal -expressing clones, which produce blue-stained plaques. (A detailed description of a "plaque assay” of this type can also be found in the user's guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaithersburg, page 9-10).
• the virus is added to the cells. After appropriate incubation, blue stained plaques are picked with the tip of an Eppendorf pipette. The agar containing the recombinant viruses is then resuspended in an Eppendorf tube containing 200 ⁇ l of Grace's medium. The agar is removed by a brief centrifugation and the supernatant containing the recombinant baculovirus is used to infect Sf9 cells seeded in 35 mm dishes. Four days later the supematants of these culture dishes are harvested and then they are stored at 4°C. A clone containing properly inserted hESSB I, II and III is identified by DNA analysis including restriction mapping and sequencing.
• V-AIM II This is designated herein as V-AIM II .
• Sf9 cells are grown in Grace's medium supplemented with 10% heat- inactivated FBS.
• the cells are infected with the recombinant baculovirus V- AIM II at a multiplicity of infection ("MOI") of about 2 (about 1 to about 3).
• MOI multiplicity of infection
• vectors used for the transient expression of the AIM II protein gene sequence in mammalian cells should carry the SV40 origin of replication. This allows the replication of the vector to high copy numbers in cells (e.g., COS cells) which express the T antigen required for the initiation of viral DNA synthesis. Any other mammalian cell line can also be utilized for this purpose.
• a typical mammalian expression vector contains the promoter element, which mediates the initiation of transcription of mRNA, the protein coding sequence, and signals required for the termination of trancription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for
• RNA splicing Highly efficient transcription can be achieved with the early and late promoters from SV40, the long terminal repeats (LTRs) from Retroviruses, e.g., RSV, HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV).
• LTRs long terminal repeats
• CMV cytomegalovirus
• cellular signals can also be used (e.g., human actin promoter).
• Suitable expression vectors for use in practicing the present invention include, for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala. Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC 67109).
• Mammalian host cells that could be used include, human Hela, 283, H9 and Jurkart cells, mouse NlH3T3 and C127 cells, Cos 1, Cos 7 and CV1, African green monkey cells, quail QCl-3 cells, mouse L cells and Chinese hamster ovary cells.
• the gene can be expressed in stable cell lines that contain the gene integrated into a chromosome.
• a selectable marker such as dhfr, gpt, neomycin, hygromycin allows the identification and isolation of the transfected cells.
• the transfected gene can also be amplified to express large amounts of the encoded protein.
• the DHFR dihydrofolate reductase
• GS glutamine synthase
• Another useful selection marker is the enzyme glutamine synthase (GS) (Murphy et al., Biochem J. 227:277-279 (1991); Bebbington et al., Bio/Technology 10:169-175 (1992)).
• GS glutamine synthase
• the mammalian cells are grown in selective medium and the cells with the highest resistance are selected.
• These cell lines contain the amplified gene(s) integrated into a chromosome. Chinese hamster ovary (CHO) cells are often used for the production of proteins.
• the expression vectors pC1 and pC4 contain the strong promoter (LTR) of the Rous Sarcoma Virus (Cullen et al., Molecular and Cellular Biology, 438-447 (March, 1985)) plus a fragment of the CMV-enhancer (Boshart et al.,
• the expression plasmid, pAIM II HA is made by cloning a cDNA encoding AIM II into the expression vector pcDNAI/Amp (which can be obtained from Invitrogen, Inc.).
• the expression vector pcDNAI/amp contains: (1) an E.coli origin of replication effective for propagation in E.
• coli and other prokaryotic cells (2) an ampicillin resistance gene for selection of plasmid-containing prokaryotic cells; (3) an SV40 origin of replication for propagation in eukaryotic cells; (4) a CMV promoter, a polylinker, an SV40 intron, and a polyadenylation signal arranged so that a cDNA conveniently can be placed under expression control of the CMV promoter and operably linked to the SV40 intron and the polyadenylation signal by means of restriction sites in the polylinker.
• a DNA fragment encoding the AIM II protein and an HA tag fused in frame to its 3' end is cloned into the polylinker region of the vector so that recombinant protein expression is directed by the CMV promoter.
• the HA tag corresponds to an epitope derived from the influenza hemagglutinin protein described by Wilson et al., Cell 37: 767 (1984). The fusion of the HA tag to the target protein allows easy detection of the recombinant protein with an antibody that recognizes the HA epitope.
• the plasmid construction strategy is as follows.
• the AIM II cDNA of the deposited clone is amplified using primers that contain convenient restriction sites, much as described above regarding the construction of expression vectors for expression of AIM II in E. coli.
• primers that contain convenient restriction sites, much as described above regarding the construction of expression vectors for expression of AIM II in E. coli.
• one of the primers contains a hemagglutinin tag ("HA tag”) as described above.
• Suitable primers include the following, which are used in this example.
• the 5' primer, containing the underlined BamHI site, and an AUG start codon has the following sequence:
• the 3' primer containing the underlined Xba I site, a stop codon, 9 codons thereafter forming the hemagglutinin HA tag, and 31 bp of 3 ' coding sequence (at the 3' end) has the following sequence: 5'GATGTTCTAGAAAGC GTAGTC TGG GAC GTC GTA TGG GTA CAC CAT GAA AGC CCC GAA GTA AGA CCG GGT AC 3' (SEQ ID NO:14).
• the PCR amplified DNA fragment and the vector, pcDNAI/Amp are digested with Hindlll and Xhol and then ligated.
• the ligation mixture is transformed into E. coli strain SURE (available from Stratagene Cloning Systems, 1 1099 North Torrey Pines Road, La Jolla, CA 92037), and the transformed culture is plated on ampicillin media plates which then are incubated to allow growth of ampicillin resistant colonies. Plasmid DNA is isolated from resistant colonies and examined by restriction analysis and gel sizing for the presence of the AIM II -encoding fragment.
• COS cells are transfected with an expression vector, as described above, using DEAE-DEXTRAN, as described, for instance, in Sambrook et al., Molecular Cloning: a Laboratory Manual, Cold Spring Laboratory Press, Cold Spring Harbor, New York (1989). Cells are incubated under conditions for expression of AIM II by the vector.
• AIM II HA fusion protein is detected by radiolabelling and immunoprecipitation. using methods described in, for example Harlow et al., Antibodies: A Laboratory Manual, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1988). To this end, two days after transfection, the cells are labeled by incubation in media containing 35 S-cysteine for 8 hours. The cells and the media are collected, and the cells are washed and the lysed with detergent-containing RIPA buffer: 150 mM NaCl, 1% NP-40, 0.1% SDS, 1%NP-40, 0.5% DOC, 50 mM TR1S, pH 7.5, as described by Wilson et al. cited above.
• Proteins are precipitated from the cell lysate and from the culture media using an HA-specific monoclonal antibody. The precipitated proteins then are analyzed by SDS-PAGE gels and autoradiography. An expression product of the expected size is seen in the cell lysate, which is not seen in negative controls.
• Plasmid pC 1 is a derivative of the plasmid pSV2-dhfr [ATCC Accession No. 37146]. Both plasmids contain the mouse DHFR gene under control of the SV40 early promoter. Chinese hamster ovary- or other cells lacking dihydrofolate activity that are transfected with these plasmids can be selected by growing the cells in a selective medium (alpha minus MEM, Life. Technologies) supplemented with the chemotherapeutic agent methotrexate.
• a selective medium alpha minus MEM, Life. Technologies
• MTX methotrexate
• Plasmid pC4 contains for expressing the gene of interest the strong promoter of the long terminal repeat (LTR) of the Rous Sarcoma Virus (Cullen, et al. Molecular and Cellular Biology, March 1985:438-447) plus a fragment isolated from the enhancer of the immediate early gene of human cytomegalovirus (CMV) (Boshart et al., Cell 47:521-530 (1985)). Downstream of the promoter are Bam HI, Xbal, and Asp718 restriction enzyme cleavage sites that allow integration of the genes. Behind these cloning sites the plasmid contains the 3' intron and polyadenylation site of the rat preproinsulin gene.
• LTR long terminal repeat
• CMV cytomegalovirus
• high efficiency promoters can also be used for the expression, e.g., the human ⁇ -actin promoter, the SV40 early or late promoters or the long terminal repeats from other retroviruses, e.g., HIV and HTLVI.
• Clontech's Tet-Off and Tet-On gene expression systems and similar systems can be used to express the AIM II in a regulated way in mammalian cells (Gossen, M., & Bujard, H. 1992, Proc. Natl. Acad. Sci. USA 89: 5547-5551).
• Other signals e.g., from the human growth hormone or globin genes can be used as well.
• Stable cell lines carrying a gene of interest integrated into the chromosomes can also be selected upon co-transfection with a selectable marker such as gpt, G418 or hygromycin. It is advantageous to use more than one selectable marker in the beginning, e.g., G418 plus methotrexate.
• the plasmid pC4 is digested with the restriction enzymes Bam HI and
• the DNA sequence encoding the complete AIM II protein including its leader sequence is amplified using PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the gene.
• the 5' primer has the sequence 5'GCT
• the 3' primer has the sequence 5'GA CGC GGT ACC GTC CAA TGC ACC ACG CTC CTT CCT TC 3' (SEQ ID NO: 16) containing the underlined Asp 718 restriction site followed by nucleotides complementary to nucleotides 769-795 of the AIM II gene shown in Figures 1 A-C (SEQ ID NO:1).
• the amplified fragment is digested with the endonucleases BamHI and
• the plasmid pS V2neo contains a dominant selectable marker, the neo gene from Tn5 encoding an enzyme that confers resistance to a group of antibiotics including G418.
• the cells are seeded in alpha minus MEM supplemented with 1 mg/ml G418. After 2 days, the cells are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) in alpha minus MEM supplemented with 10, 25, or 50 ng/ml of metothrexate plus 1 mg/ml G418.
• single clones are trypsinized and then seeded in 6-well petri dishes or 10 ml flasks using different concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM). Clones growing at the highest concentrations of methotrexate are then transferred to new 6-well plates containing even higher concentrations of methotrexate (1 ⁇ M, 2 ⁇ M, 5 ⁇ M, 10 mM, 20 mM). The same procedure is repeated until clones are obtained which grow at a concentration of 100 - 200 ⁇ M. Expression of the desired gene product is analyzed, for instance, by SDS-PAGE and Western blot or by reverse phase HPLC analysis.
• Northern blot analysis is carried out to examine AIM II gene expression in human tissues, using methods described by, among others, Sambrook et al., cited above.
• a cDNA probe containing the entire nucleotide sequence of the AIM II protein (SEQ ID NO: 1) is labeled with 32 P using the rediprimeTM DNA labeling system (Amersham Life Science), according to manufacturer's instructions. After labelling, the probe is purified using a CHROMA SPIN- 100TM column (Clontech Laboratories, Inc.), according to manufacturer's protocol number PT1200-1. The purified labelled probe is then used to examine various human tissues for AIM II mRNA.
• MTN Multiple Tissue Northern
• H human tissues
• IM human immune system tissues

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