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WO1997033899A1 - Molecule i induisant l'apoptose - Google Patents

Molecule i induisant l'apoptose Download PDF

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Publication number
WO1997033899A1
WO1997033899A1 PCT/US1996/003773 US9603773W WO9733899A1 WO 1997033899 A1 WO1997033899 A1 WO 1997033899A1 US 9603773 W US9603773 W US 9603773W WO 9733899 A1 WO9733899 A1 WO 9733899A1
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WIPO (PCT)
Prior art keywords
polypeptide
aim
polynucleotide
leu
ser
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PCT/US1996/003773
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English (en)
Inventor
Steven M. Ruben
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Human Genome Sciences, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by Human Genome Sciences, Inc. filed Critical Human Genome Sciences, Inc.
Priority to PCT/US1996/003773 priority Critical patent/WO1997033899A1/fr
Priority to AU57111/96A priority patent/AU5711196A/en
Publication of WO1997033899A1 publication Critical patent/WO1997033899A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention relates, in part, to newly identified polynucleotides and polypeptides; variants and derivatives of the polynucleotides and polypeptides; processes for making the polynucleotides and the polypeptides, and their variants and derivatives; agonists and antagonists of the polypeptides; and uses of the polynucleotides, polypeptides, variants, derivatives, agonists and antagonists.
  • the invention relates to polynucleotides and polypeptides of human Apoptosis Inducing Molecule I (AIM-I).
  • TNF- ⁇ and ⁇ are related members of a broad class of polypeptide mediators, which includes the interferons. interieukins and growth factors, collectively called cytokines (Beutler, B. and Cerami, A.. Annu. Rev. Immunol. 7:625-655, 1989).
  • Tumor necrosis factor (TNF- ⁇ and TNF-3) 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 retaliation and inflammation.
  • TNF- ⁇ tumor necrosis factor
  • TNF-/3 lymphotoxin-c.
  • LT- 3. OX40L
  • Fas ligand CD30L
  • CD27L CD40L
  • 4- 1BBL 4- 1BBL.
  • 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: 192. 1991 )
  • 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. Immunolo. 148: 1274, 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 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 11 transmembrane protein belonging to the TNF family. Fas ligand is expressed in splenocytes and thymocytes, consistent with T-cell mediated cytotoxicity. 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, 57J.441 , 1995).
  • Activation of T-cells induces expression of 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 interaciion 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.
  • AIM-I novel Apoptosis Inducing Molecule I
  • polynucleotides that encode AIM-I pa ⁇ icularly polynucleotides that encode the polypeptide herein designated AIM-I.
  • the polynucleotide comprises the region encoding human AIM-I in the sequence set out in Figure 1.
  • an isolated nucleic acid molecule encoding a mature polypeptide expressed by the human cDNA contained in ATCC Deposit No. 97448.
  • this aspect of the invention there are provided isolated nucleic acid molecules encoding human AIM-I. including mRNAs, cDNAs, genomic DNAs and, in further embodiments of this aspect of the invention, biologically, diagnostically, clinically or therapeuticallv useful variants, analogs, derivatives and/or fragments thereof, including fragments ot the ⁇ ar ⁇ ants. analogs and derivatives.
  • isolated nucleic acid molecules encoding human AIM-I. including mRNAs, cDNAs, genomic DNAs and, in further embodiments of this aspect of the invention, biologically, diagnostically, clinically or therapeuticallv useful variants, analogs, derivatives and/or fragments thereof, including fragments ot the ⁇ ar ⁇ ants. analogs and derivatives.
  • particularly preferred embodiments of this aspect of the invention are naturally occurring allelic variants of human AIM-I r
  • AIM-I polypeptides particularly human AIM-I polypeptides.
  • w hich may be employed to treat lymphadenopathy, autoimmune disease, graft versus host disease; which may be used to stimulate peripheral tolerance, destroy pathologic transformed cell lines, mediate cell activation and proliferation; and are functionally linked as primary mediators of immune regulation and inflammatory response.
  • This aspect of the invention provides novel polypeptides of human origin referred to herein as AIM-I as well as biologically, diagnostically or therapeutically useful fragments, variants and derivatives thereof, variants and derivatives of the fragments, and analogs of all of the foregoing.
  • AIM-I novel polypeptides of human origin referred to herein as AIM-I as well as biologically, diagnostically or therapeutically useful fragments, variants and derivatives thereof, variants and derivatives of the fragments, and analogs of all of the foregoing.
  • variants of human AIM-I encoded by naturally occurring alleles of the human AIM-I gene are particularly preferred embodiments of human AIM-I encoded by naturally occurring alleles of the human AIM-I gene.
  • methods for producing the aforementioned AIM-I polypeptides comprising culturing host cells having expressibly incorporated therein an exogenously-derived human AIM-I-encoding polynucleotide under conditions for expression of human AIM-I in the host and then recovering the expressed polypeptide.
  • products, compositions and methods for, among other things: assessing AIM-I expression in cells by determining AIM-I polypeptides or AIM-I-encoding mRN A; treaiinc disease or disorder caused by under-expression of the AIM-I in vitro, ex vivo or in vivo by exposing cells to AIM-I polypeptides or polynucleotides as disclosed herein; assaying genetic variation and aberrations, such as defects, in AIM-I genes; and administering a AIM-I polypeptide or polynucleotide to an organism to augment AIM-I function or remediate AIM-I dysfunction.
  • Ce ⁇ ain additional preferred aspects related to the above aspects of the invention provides antibodies against AIM-I polypeptides.
  • the antibodies are highly selective for human AIM-I, and may be employed, inter alia, to treat autoimmune diseases.
  • AIM-I agonists are provided.
  • preferred agonists are molecules that mimic AIM-I, that bind to AIM-I-binding molecules or receptor molecules, and that elicit or augment AIM-I- induced responses.
  • AIM-I antagonists are provided.
  • preferred antagonists are those which mimic AIM-I so as to bind to AIM-I receptor or binding molecules but not elicit an AIM-I-induced response or more than one AIM-I-induced response.
  • molecules that bind to or interact with AIM-I so as to inhibit an effect of AIM-I or more than one effect of AIM-I or which prevent expression of AIM-I.
  • 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.
  • compositions comprising an AIM-I polynucleotide or an AIM-I 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-I polynucleotide for expression of an AIM-I polypeptide in a host organism for treatment of disease.
  • Pa ⁇ icularly preferred in this regard is expression in a human patient for treatment of a dysfunction associated with aberrant endogenous activity of AIM-I.
  • Figure 1 shows the nucleotide and deduced amino acid sequence of human AIM-I.
  • Figure 2 shows the regioas of similarity and identity between amino acid sequence of AIM-I of the present invention and human Fas ligand polypeptide (SEQ ID NO:3).
  • Figure 3 shows regions of similarity and identity between amino acid sequences of AIM-I of the present invention and human Fas ligand polypeptide, TNF ⁇ and TNF / 3.
  • Figure 4 shows structural and functional features of AIM-I deduced by the indicated techniques, as a function of amino acid sequence.
  • the term "digestion of DNA” refers to catalytic cleavage of DNA with a restriction enzyme that acts only upon ce ⁇ ain sequences in the DNA.
  • the various restriction enzymes referred to herein are all commercially available and their reaction conditions, cofactors and other requirements for use are known and routine to the skilled a ⁇ isan.
  • plasmid or DNA fragment is digested with about 2 units of enzyme in about 20 ⁇ l of reaction buffer.
  • isolating DNA fragments for plasmid construction typically 5 to 50 ⁇ g of DNA are digested with 20 to 250 units of enzyme in propo ⁇ ionately larger volumes.
  • Incubation times ol about 1 hour at 37 "C are ordinarily used, but conditions may vary in accordance with standard procedures, the supplier's instructions and the pa ⁇ iculars of the reaction.
  • reactions may be analyzed, and fragments may be purified by electrophoresis through an agarose or polyacrylamide gel, using well known methods that are routine for those skilled in the an.
  • genetic element generally means a polynucleotide comprising a region that encodes a polypeptide or a region that regulates transcription or translation or other processes impo ⁇ ant to expression of the polypeptide in a host cell, or a polynucleotide comprising both a region that encodes a polypeptide and a region operably linked thereto that regulates expression.
  • Genetic elements may be comprised within a vector that replicates as an episomal element; that is, as a molecule physically independent of the host cell genome. They may be comprised within mini-chromosomes, such as those that arise during amplification of transfected DNA by methotrexate selection in eukaryotic cells. Genetic elements also may be comprised within a host cell genome; not in their natural state but, rather, following manipulation such as isolation, cloning and introduction into a host cell in the form of purified DNA or in a vector, among others.
  • isolated means altered “by the hand of man” from its natural state; i.e. , that, if it occurs in nature, it has been changed or removed from its original environment, or both.
  • a naturally occurring polynucleotide or a polypeptide naturally present in a living animal in its natural state is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated”, as the term is employed herein.
  • isolated means that it is separated from the chromosome and cell in which it naturally occurs
  • polynucleotides can be joined to other polynucleotides, such as DNAs. for mutagenesis, to form fusion proteins, and for propagation or expression in a host, for instance.
  • the isolated polynucleotides, alone or joined to other polynucleotides such as vectors, can be introduced into host cells, in culture or in whole organisms. Introduced into host cells in culture or in whole organisms, such DNAs still would be isolated, as the term is used herein, because they would not be in their naturally occurring form or environment.
  • polynucleotides and polypeptides may occur in a composition, such as a media formulations, solutions for introduction of polynucleotides or polypeptides, for example, into cells, compositions or solutions for chemical or enzymatic reactions, for instance, which are not naturally occurring compositions, and, therein remain isolated polynucleotides or polypeptides within the meaning of that term as it is employed herein.
  • a composition such as a media formulations, solutions for introduction of polynucleotides or polypeptides, for example, into cells, compositions or solutions for chemical or enzymatic reactions, for instance, which are not naturally occurring compositions, and, therein remain isolated polynucleotides or polypeptides within the meaning of that term as it is employed herein.
  • ligation refers to the process of forming phosphodiester bonds between two or more polynucleotides, which most often are double stranded DNAs. Techniques for ligation are well known to the an and protocols for ligation are described in standard laboratory manuals and references, such as, for instance, Sambrook et al. Molecular Cloning. A Laboratory Manual. 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989) and Maniatis et al, pg. 146, as cited below.
  • oligonucleotide(s) refers to relatively short polynucleotides. Often the term refers to single-stranded deoxyribonucleotides, but it can refer as well to single- or double-stranded ribonucleotides, RNA:DNA hybrids and double-stranded DNAs, among others.
  • Oligonucleotides such as single-stranded DNA probe oligonucleotides. often are synthesized by chemical makersxls. such as those implemented on automated oligonucleotide synthesizers. However, oligonucleotides can be made by a variety of other methods, including in vitro recombinant DNA-mediated techniques and by expression of DNAs in cells and organisms.
  • oligonucleotides typically are obtained without a 5' phosphate.
  • the 5 ' ends of such oligonucleotides are not substrates for phosphodiester bond formation by ligation reactions that employ DNA ligases typically used to form recombinant DNA molecules.
  • a phosphate can be added by standard techniques, such as those that employ a kinase and ATP.
  • the 3' end of a chemically synthesized oligonucleotide generally has a free hydroxyl group and, in the presence of a ligase, such as T4 DNA ligase, readily will form a phosphodiester bond with a 5' phosphate of another polynucleotide, such as another oligonucleotide. As is well known, this reaction can be prevented selectively, where desired, by removing the 5' phosphates of the other polynucleotide(s) prior to ligation.
  • a ligase such as T4 DNA ligase
  • Plasmids generally are designated herein by a lower case p preceded and/or followed by capital letters and/or numbers, in accordance with standard naming conventions that are familiar to those of skill in the art.
  • plasmids disclosed herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids by routine application of well known, published procedures. Many plasmids and other cloning and expression vectors that can be used in accordance with the present invention are well known and readily available to those of skill in the art. Moreover, those of skill readily may construct any number of other plasmids suitable for use in the invention. The properties, construction and use of such plasmids, as well as other vectors, in the present invention w ill be readily apparent to those of skill from the present disclosure.
  • polynucleoiidcisr generally refers to any polyribonucleotide or polydeoxribonucleotide. which mj> he unmodified RNA or DNA or modified RNA or DNA.
  • polynucleotides as used herein refers to, among others, single-and double-stranded DNA.
  • polynucleotide as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • the strands in such regions may be from the same molecule or from different molecules.
  • the regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules.
  • One of the molecules of a triple-helical region often is an oligonucleotide.
  • polynucleotide includes DNAs or RNAs as described above that contain one or more modified bases.
  • DNAs or RNAs with backbones modified for stability or for other reasons are “polynucleotides” as that term is intended herein.
  • DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples are polynucleotides as the term is used herein.
  • polynucleotide as it is employed herein embraces such chemically, enzymatically or metabolicallv modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells, inter alia.
  • polypeptides as used herein, includes all polypeptides as described below.
  • the basic structure of polypeptides is well known and has been described in innumerable textbooks and other publications in the art.
  • the term is used herein to refer to any peptide or protein comprising two or more amino acids joined to each other in a linear chain by peptide bonds.
  • the term refers to both sho ⁇ chains, which also commonly are referred to in the an as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the an as proteins, of which there are many types.
  • polypeptides often contain amino acids other than the 20 amino acids commonly referred to as the 20 naturally occurring amino acids, and that many amino acids, including the terminal amino acids, may be modified in a given polypeptide, either by natural processes, such as processing and other post-translational modifications, but also by chemical modification techniques which are well known to the an. Even the common modifications that occur naturally in polypeptides are too numerous to list exhaustively here, but they are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature, and they are well known to those of skill in the an.
  • polypeptides of the present are, to name an illustrative few, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation. oxidation, proteolytic processing, phosphorylation. prenylation. racemization. selenoylation. sulfation. transfer-RNA mediated addition of amino acids to proteins such as
  • polypeptides are not always entirely linear.
  • polypeptides may be branched as a result of ubiquitination, and they may be circular, with or without branching, generally as a result of posttranslation events, including natural processing event and events brought about by human manipulation which do not occur naturally.
  • Circular, branched and branched circular polypeptides may be synthesized by non-translation natural process and by entirely synthetic methods, as well.
  • Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini.
  • blockage of the amino or carboxyl group in a polypeptide, or both, by a covalent modification is common in naturally occurring and synthetic polypeptides and such modifications may be present in polypeptides of the present invention, as well.
  • the amino terminal residue of polypeptides made in E. coli, prior to proteolytic processing almost invariably w ill be N-formylmethionine.
  • modifications that t ⁇ cur in a polypeptide often will be a function of how it is made.
  • polypeptides made b ⁇ expressing a cloned gene in a host for instance, the nature and extent of the modifications in large part will be determined by the host cell posttranslational modification capacity and the modification signals present in the polypeptide amino acid sequence
  • glycosylation often does not occur in bacterial hosts such as E. coli. Accordingly, when glycosylation is desired, a polypeptide should be expressed in a glycosylating host, generally a eukaryotic cell.
  • Insect cell often carry out the same posttranslational glycosylations as mammalian cells and, for this reason, insect cell expression systems have been developed to express efficiently mammalian proteins having native patterns of glycosylation, inter alia. Similar considerations apply to other modifications.
  • polypeptide encompasses all such modifications, particularly those that are present in polypeptides synthesized by expressing a polynucleotide in a host cell.
  • variant(s) of polynucleotides or polypeptides, as the term is used herein, are polynucleotides or polypeptides that differ from a reference polynucleotide or polypeptide, respectively. Variants in this sense are described below and elsewhere in the present disclosure in greater detail.
  • a polynucleotide variant can be, for example, a polynucleotide that differs in nucleotide sequence from another, reference polynucleotide. Generally, differences are limited so that the nucleotide sequences of the reference and the variant are closely similar overall and. in many regions, identical.
  • changes m the nucleotide sequence of the variant may be silent. That is, they may not alter the ammo acids encoded by the polynucleotide. Where alterations are limited to silent changes of this type a variant will encode a polypeptide with the same amino acid sequence as the reference. Also as noted below, changes in t.' ⁇ nucleotide sequence of the variant may alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Such nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below.
  • a polynucleotide variant can be, for example, a polypeptide that differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference and the variant are closely similar overall and, in many region, identical.
  • a variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions, fusions and truncations, which may be present in any combination.
  • receptor molecule refers to molecules which bind or interact specifically with AIM-I polypeptides of the present invention, including not only classic receptors, which are preferred, but also other molecules that specifically bind to or interact with polypeptides of the invention (which also may be referred to as “binding molecules” and “interaction molecules. " respectively and as "AIM-I binding molecules” and "AIM-I interaction molecules.
  • Binding between polypeptides of the invention and such molecules, including receptor or binding or interaction molecules may be exclusive to polypeptides of the invention, which is very highly preferred, or it may be highly specific for polypeptides of the invention, which is highly preferred, or it may be highly specific to a group of proteins that includes polypeptides of the invention, which is preferred, or it may be specific to several groups of proteins at least one of which includes polypeptides of the invention.
  • Receptors also may be non-naturallv occurring, such as antibodies and antibody- derived reagents that bind specifically to polypeptides of the invention.
  • the present invention relates to novel AIM-I polypeptides and polynucleotides, among other things, as described in greater detail below.
  • the invention relates to polypeptides and polv nucleotides of a novel human AIM-I, which is related by amino acid sequence homology to known human AIM-I.
  • the invention relates especially to AIM-I having the nucleotide and amino acid sequences set out in Figure 1 , and to the AIM-I nucleotide and amino acid sequences of the cDNA in ATCC Deposit No. 97448, which is herein referred to as "the deposited clone" or as the "cDNA of the deposited clone.
  • nucleotide and amino acid sequences set out in Figure 1 were obtained by sequencing the cDNA of the deposited clone.
  • sequence of the deposited clone is controlling as to any discrepancies between the two and any reference to the sequences of Figure 1 include reference to the sequence of the human cDNA of the deposited clone.
  • isolated polynucleotides which encode the AIM-I polypeptide having the deduced amino acid sequence of Figure 1 or the AIM-I polypeptide encoded by the cDNA in the deposited clone.
  • a polynucleotide of the present invention encoding human AIM-I polypeptide may be obtained using standard cloning and screening procedures, such as those for cloning cDNAs using human tissue as starting material.
  • standard cloning and screening procedures such as those for cloning cDNAs using human tissue as starting material.
  • the polynucleotide set out in Figure 1 was discovered in a cDNA library derived from cells of a human pancreas rumor.
  • Human AIM-I of the is structurally related to other proteins of the TNF family, as shown by the results ot sequencing the human cDNA encoding human AIM-I in the deposited clone
  • the cl)V ⁇ sequence thus obtained is set out in Figure 1 . It contains an open readinj. frame encodini! a protein of about 281 amino acid residues with a deduced molecular weight of about 31 kDa. The protein exhibits greatest homology to known human AIM I . among known proteins.
  • the AIM-I of Figure 1 has about 22.892 % identity and about 48.594 % similarity with the amino acid sequence of human Fas ligand.
  • Polynucleotides of the present invention may be in the form of RNA, such as mRNA, or in the form cDNA and genomic DNA obtained by cloning or produced by chemical synthetic techniques or by a combination thereof.
  • the DNA may be double- stranded or single-stranded.
  • Single-stranded DNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti-sense strand.
  • the coding sequence which encodes the polypeptide may be identical to the coding sequence of the polynucleotide shown in Figure 1. It also may be a polynucleotide with a different sequence, which, as a result of the redundancy (degeneracy) of the genetic code, encodes the polypeptide of the DNA of Figure 1.
  • Polynucleotides of the present invention which encode the polypeptide of Figure 1 may include, but are not limited to the coding sequence for the mature polypeptide, by itself; the coding sequence for the mature polypeptide and additional coding sequences, such as those encoding a leader or secretory sequence, such as a pre-, or pro- or prepro- protein sequence: the coding sequence of the mature polypeptide, with or without the aforementioned additional coding sequences, together with additional, non-coding sequences, including for example, but not limited to introns and non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription.
  • the polypeptide may be fused to a marker sequence, such as a peptide. which facilitates purification of the fused polypeptide.
  • the marker sequence is a hexa-histidine peptide, such as the tag provided in the pQE vector (Qiagen, Inc. , Chatsworth, CA), : -long others, many of which are commercially available. As described in Gentz et al , Proc. Natl. Acad. Sci..
  • hexa-histidine provides for convenient purification of the fusion protein.
  • the HA tag corresponds to an epitope derived of influenza hemagglutinin protein, which has been described by Wilson et al , Cell 37:161 (1984), for instance.
  • polynucleotide encoding a polypeptide encompasses polynucleotides which include a sequence encoding a polypeptide of the present invention, particularly the human AIM-I having the amino acid sequence set out in Figure 1.
  • the term encompasses polynucleotides that include a single continuous region or discontinuous regions encoding the polypeptide (for example, interrupted by introns) together with additional regions, that also may contain coding and/or non-coding sequences.
  • the present invention further relates to variants of the herein above described polynucleotides which encode for fragments, analogs and derivatives of the polypeptide having the deduced amino acid sequence of Figure 1.
  • a variant of the polynucleotide may be a namrally occurring variant such as a naturally occurring allelic variant, or it may be a variant that is not known to occur naturally.
  • Such non-narurally occurring variants of the polynucleotide may be made by mutagenesis techniques, including those applied to polynucleotides, cells or organisms.
  • variants in this regard are variants that differ from the aforementioned polynucleotides by nucleotide substimtions. deletions or additions. The substitutions, deletions or additions may involve one or more nucleotides. The variants may be altered in coding or non-coding regions or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions.
  • polynucleotides encoding polypeptides having the amino acid sequence of AIM-I set out in Figure 1 ; variants, analogs, derivatives and fragments thereof, and fragments of the variants, analogs and derivatives.
  • polynucleotides encoding AIM-I variants, analogs, derivatives and fragments, and variants, analogs and derivatives of the fragments which have the amino acid sequence of the AIM-I polypeptide of Figure 1 in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, deleted or added, in any combination.
  • Fu ⁇ her preferred embodiments of the invention are polynucleotides that are at least 70% identical to a polynucleotide encoding the AIM-I polypeptide having the amino acid sequence set out in Figure 1 , and polynucleotides which are complementary to such polynucleotides.
  • most highly preferred are polynucleotides that comprise a region that is at least 80% identical to a polynucleotide encoding the AIM-I polypeptide of the cDNA of the deposited clone and polynucleotides complementary thereto.
  • polynucleotides at least 90% identical to the same are particularly preferred, and among these pa ⁇ icularly preferred polynucleotides, those with at least 95 % are especially preferred. Furthermore, those with at least 97% are highly preferred among those with at least 95%. and among these those with at least 98% and at least 99 c i are particularly highly preferred, with at least 99% being the more preferred.
  • polynucleotides which encode polypeptides which retain substantially the same biological function or activity as the mature pol ⁇ peptide encoded by the cDNA of Figure 1.
  • the present invention further relates to polynucleotides that hybridize to the herein above-described sequences
  • the present invention especially relates to polynucleotides which hybridize under stringent conditions to the herein above- described polynucleotides.
  • stringent conditions means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences.
  • polynucleotides of the invention may be used as a hybridization probe for cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding human AIM-I and to isolate cDNA and genomic clones of other genes that have a high sequence similarity to the human AIM-I gene.
  • Such probes generally will comprise at least 15 bases.
  • such probes will have at least 30 bases and may have at least 50 bases.
  • Particularly preferred probes will have at least 30 bases and will have 50 bases or less.
  • the coding region of the AIM-I gene may be isolated by screening using the known DNA sequence to synthesize an oligonucleotide probe.
  • a labeled oligonucleotide having a sequence complementary to that of a gene of the present invention is then used to screen a library of human cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.
  • polynucleotides and polypeptides of the present invention may be employed as research reagents and materials for discovery of treatments and diagnostics to human disease, as further discussed herein relating to polynucleotide assays, inter alia.
  • the polynucleotides ma> encode a polypeptide which is the mature protein plus additional amino or carboxyl-terminal amino acids, or amino acids interior to the mature polypeptide (when the mature l rm has more than one polypeptide chain, for instance).
  • Such sequences may play a role in processing of a protein from precursor to a mature form, may facilitate protein trafficking, may prolong or shorten protein half-life or may facilitate manipulation of a protein for assay or production, among other things.
  • the additional amino acids may be processed away from the mamre protein by cellular enzymes.
  • a precursor protein, having the mamre form of the polypeptide fused to one or more prosequences may be an inactive form of the polypeptide.
  • inactive precursors When prosequences are removed such inactive precursors generally are activated. Some or all of the prosequences may be removed before activation. Generally, such precursors are called proproteins.
  • a polynucleotide of the present invention may encode a mamre protein, a mamre protein plus a leader sequence (which may be referred to as a preprotein), a precursor of a mature protein having one or more prosequences which are not the leader sequences of a preprotein. or a preproprotein. which is a precursor to a proprotein, having a leader sequence and one or more prosequences, which generally are removed during processing steps that produce active and mamre forms of the polypeptide.
  • a leader sequence which may be referred to as a preprotein
  • a precursor of a mature protein having one or more prosequences which are not the leader sequences of a preprotein.
  • a preproprotein which is a precursor to a proprotein, having a leader sequence and one or more prosequences, which generally are removed during processing steps that produce active and mamre forms of the polypeptide.
  • a deposit containing a human AIM-I cDNA has been deposited with the American Type Culture Collection, as noted above. Also as noted above, the human cDNA deposit is referred to herein as “the deposited clone” or as “the cDNA of the deposited clone. "
  • the deposited clone was deposited with the American Type Culture Collection, 12301 Park Lawn Dme. Rockv ille. Mar land 20852, USA. on February 20, 1996, and assigned ATCC Deposit No. 97448.
  • the deposited material is a pBluescript SK (-) plasmid (Stratagene, La Jolla, CA) that contains the full length AIM-I cDNA. referred to as "PF261 " upon deposit.
  • the deposit has been made under the terms of the Budapest Treaty on the international recognition of the deposit of micro-organisms for purposes of patent procedure. The strain will be irrevocably and without restriction or condition released to the public upon the issuance of a patent.
  • the deposit is provided merely as convenience to those of skill in the art and is not an admission that a deposit is required for enablement, such as that required under 35 U.S.C. ⁇ 112.
  • sequence of the polynucleotides contained in the deposited material, as well as the amino acid sequence of the polypeptide encoded thereby, are controlling in the event of any conflict with any description of sequences herein.
  • a license may be required to make, use or sell the deposited materials, and no such license is hereby granted.
  • the present invention further relates to a human AIM-I polypeptide which has the deduced amino acid sequence of Figure 1.
  • the invention also relates to fragments, analogs and derivatives of these polypeptides.
  • fragment when referring to the polypeptide of Figure 1 means a polypeptide which retains essentially the same biological function or activity as such polypeptide.
  • an analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mamre polypeptide.
  • polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic In ce ⁇ ain preferred embodiments it is a recombinant polypeptide.
  • the fragmeni. derivative or analog of the polypeptide of Figure 1 may be (i) one in which one or more of the ammo acid residues are substituted with a conserved or non-conserved ammo 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 mamre 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 mamre polypeptide.
  • particularly preferred embodiments of the invention in this regard are polypeptides having the amino acid sequence of AIM-I set out in Figure 1 , variants, analogs, derivatives and fragments thereof, and variants, analogs and derivatives of the fragments.
  • particularly preferred embodiments of the invention in this regard are polypeptides having the amino acid sequence of the AIM-I of the cDNA in the deposited clone, variants, analogs, derivatives and fragments thereof, and variants, analogs and derivatives of the fragments.
  • Such substimtions are those that substitute a given amino acid in a polypeptide by another amino acid of like characteristics. Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala. Val. Leu and He; interchange of the hydroxyl residues Ser and Thr. exchange of the acidic residues Asp and Glu. substimtion between the amide residues Asn and Gin, exchange of the basic residues Lys and Arg and replacements among the aromatic residues Phe. Tyr.
  • variants, analogs, derivatives and fragments, and variants, analogs and derivatives of the fragments having the amino acid sequence of the AIM-I polypeptide of Figure 1 in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, deleted or added, in any combination.
  • silent substimtions, additions and deletions which do not alter the properties and activities of the AIM-I.
  • conservative substimtions are also especially preferred in this regard.
  • polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.
  • polypeptides of the present invention include the polypeptide of SEQ ID NO: 2 (in particular the mamre polypeptide) as well as polypeptides which have at least 70% similarity (preferably at least 70% identity) to the polypeptide of SEQ ID NO:2 and more preferably at least 90% similarity (more preferably at least 90% identity) to the polypeptide of SEQ ID NO:2 and still more preferably at least 95% similarity (still more preferably at least 95 % identity) to the polypeptide of SEQ ID NO:2 and also include portions of such polypeptides with such portion of the polypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids.
  • Fragments or portions of the polypeptides of the present invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, the fragments may be employed as intermediates for producing the full-length polypeptides. Fragments or portions of the polynucleotides of the present invention may be used to synthesize full-length polynucleotides of the present invention.
  • polypeptides comprising fragments of AIM-I, most particularly fragments of the AIM-I having the amino acid set out in Figure 1 , and fragments of variants and derivatives of the AIM-I of Figure 1.
  • a fragment is a polypeptide having an amino acid sequence that entirely is the same as part but not all of the amino acid sequence of the aforementioned AIM-I polypeptides and variants or derivatives thereof.
  • fragments may be "free-standing, " i.e. , not part of or fused to other amino acids or polypeptides, or they may be comprised within a larger polypeptide of which they form a part or region. When comprised within a larger polypeptide. the presently discussed fragments most preferably form a single continuous region. However, several fragments may be comprised within a single larger polypeptide. For instance, certain preferred embodiments relate to a fragment of an AIM-I polypeptide of the present comprised within a precursor polypeptide designed for expression in a host and having heterologous pre and pro-polypeptide regions fused to the amino terminus of the AIM-I fragment and an additional region fused to the carboxyl terminus of the fragment. Therefore, fragments in one aspect of the meaning intended herein, refers to the portion or portions of a fusion polypeptide or fusion protein derived from AIM-I.
  • polypeptide fragments of the invention there may be mentioned those w hich have from about 100 to about 281 amino acids.
  • “about” includes the particularly recited range and ranges larger or smaller by several, a few. 5.
  • about 100-281 amino acids in this context means a polypeptide fragment of 100 plus or minus several, a few . 5, 4, 3, 2 or 1 amino acids to 281 plus or minus several a few . 5.
  • 4. 3. 2 or I amino acid residues, i.e. ranges as broad as 100 minus several amino acids 10 281 plus several amino acids to as narrow as 100 plus several amino acids to 281 minus several amino acids.
  • Truncation mutants include AIM-I polypeptides having the amino acid sequence of Figure 1, or of variants or derivatives thereof, except for deletion of a continuous series of residues (that is, a continuous region, pan or po ⁇ ion) that includes the amino terminus, or a continuous series of residues that includes the carboxyl terminus or, as in double truncation mutants, deletion of two continuous series of residues, one including the amino terminus and one including the carboxyl terminus. Fragments having the size ranges set out about also are preferred embodiments of truncation fragments, which are especially preferred among fragments generally.
  • fragments characterized by structural or functional attributes of AIM-I are also preferred in this aspect of the invention.
  • Preferred embodiments of the invention in this regard include fragments that comprise alpha-helix and alpha-helix forming regions ("alpha-regions"), beta-sheet and beta-sheet-forming regions ("beta-regions"), turn and turn-forming regions ("turn- regions”), coil and coil-forming regions ("coil-regions”), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index regions of AIM-I.
  • Ce ⁇ ain preferred regions in these regards are set out in Figure 3, and include, but are not limited to. regions of the aforementioned types identified by analysis of the amino acid sequence set out in Figure 1 .
  • such preferred regions include Gamier- Robson alpha-regions, beta-regions, turn-regions and coil-regions, Chou-Fasman alpha-regions, beta-regions and turn-regions, Kyte-Doolittle hydrophilic n. _ions and hydrophilic regions. Eisenberg alpha and beta amphipathic regions, Karplus- Schulz flexible regions. Emini surface-forming regions and Jameson-Wolf high antigenic index regions.
  • highly preferred fragments in this regard are those that comprise regions of AIM-I that combine several structural features, such as several of the features set out above.
  • the regions defined by the residues about 1 to about 281 of Figure 1 which all are characterized by amino acid compositions highly characteristic of turn-regions, hydrophilic regions, flexible-regions, surface-forming regions, and high antigenic index-regions, are especially highly preferred regions.
  • Such regions may be comprised within a larger polypeptide or may be by themselves a preferred fragment of the present invention, as discussed above. It will be appreciated that the term "about” as used in this paragraph has the meaning set out above regarding fragments in general.
  • a highly preferred fragment comprises amino acids 39 through 281 which constitute a soluble po ⁇ ion of the overall AIM-I polypeptide sequence of Figure 1.
  • Fu ⁇ her preferred regions are those that mediate activities of AIM-I.
  • Most highly preferred in this regard are fragments that have a chemical, biological or other activity of AIM-I, including those with a similar activity or an improved activity, or with a decreased undesirable activity.
  • Highly preferred in this regard are fragments that contain regions that are homologs in sequence, or in position, or in both sequence and to active regions of related polypeptides. such as the related polypeptides set out in Figure 2. which include members of the TNF family.
  • particularly preferred fragments in these regards are truncation mutants, as discussed above.
  • the invention also relates to. among others, polynucleotides encoding the aforementioned fragments, polynucleotides that hybridize to polynucleotides encoding the tragments. particularly those that hybridize under stringent conditions, and po I nucleotides. such as PCR primers, for amplifying polynucleotides that encode the tragments
  • preferred polynucleotides are those that correspondent to the preferred fragments, as discussed above.
  • the present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.
  • Host cells can be genetically engineered to incorporate polynucleotides and express polypeptides of the present invention.
  • polynucleotides may be introduced into host cells using well known techniques of infection, transduction, transfection, transvection and transformation.
  • the polynucleotides may be introduced alone or with other polynucleotides.
  • Such other polynucleotides may be introduced independently, co-introduced or introduced joined to the polynucleotides of the invention.
  • polynucleotides of the invention may be transfected into host cells with another, separate, polynucleotide encoding a selectable marker, using standard techniques for co-transfection and selection in, for instance, mammalian cells.
  • polynucleotides generallv w ill be stably incorporated into the host cell genome.
  • the polynucleotides may be joined to a vector containing a selectable marker for propagation in a host.
  • the vector construct may be introduced into host cells by the aforementioned techniques.
  • a plasmid vector is introduced as DNA in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid tlectroporation also may be used to introduce polynucleotides into a host.
  • the vector is a virus, it may be packaged in vitro or introduced into a packaging cell and the packaged virus may be transduced into cells.
  • the vector may be, for example, a plasmid vector, a single or double-stranded phage vector, a single or double-stranded RNA or DNA viral vector.
  • Such vectors may be introduced into cells as polynucleotides, preferably DNA, by well known techniques for introducing DNA and RNA into cells.
  • the vectors in the case of phage and viral vectors also may be and preferably are introduced into cells as packaged or encapsidated virus by well known techniques for infection and transduction.
  • Viral vectors may be replication competent or replication defective. In the latter case viral propagation generally will occur only in complementing host cells.
  • vectors are those for expression of polynucleotides and polypeptides of the present invention.
  • such vectors comprise -acting control regions effective for expression in a host operatively linked to the polynucleotide to be expressed.
  • Appropriate trans-acting factors either are supplied by the host, supplied by a complementing vector or supplied by the vector itself upon introduction into the host.
  • the vectors provide for specific expression.
  • Such specific expression may be inducible expression or expression only in ce ⁇ ain types of cells or both inducible and cell-specific.
  • Pa ⁇ icularly preferred among inducible vectors are vectors that can be induced for expression by environmental factors that are easy to manipulate, such as temperamre and nutrient additives.
  • a variety of vectors suitable to this aspect of the invention, including constimtive and inducible expression vectors for use in prokaryotic and eukaryotic hosts, are well known and employed routinely bv those of skill in the an.
  • the engineered host cells can be cultured in conventional nutrient media, which may be modified as appropriate for. inter alia, activating promoters, selecting transformants or amplifying genes. Culture conditions, such as temperamre, pH and the 1 > '. previously used with the host cell selected for expression generally will be suitable for expression of polypeptides of the present invention as will be apparent to those of skill in the an.
  • a great variety of expression vectors can be used to express a polypeptide of the invention.
  • Such vectors include chromosomal, episomal and virus-derived vectors e.g.
  • any vector suitable to maintain, propagate or express polynucleotides to express a polypeptide in a host may be used for expression in this regard.
  • the appropriate DNA sequence may be inse ⁇ ed into the vector by any of a variety of well-known and routine techniques.
  • a DNA sequence for expression is joined to an expression vector by cleaving the DNA sequence and the expression vector with one or more restriction endonucleases and then joining the restriction fragments together using T4 DNA ligase.
  • Procedures for restriction and ligation that can be used to this end are well known and routine to those of skill. Suitable procedures in this regard, and for constmcting expression vectors using alternative techniques, which also are well known and routine to those skill, are set forth in great detail in Sambrook et al cited elsewhere herein.
  • the DNA sequence in the expression vector is operatively linked to appropriate expression control sequence! s».
  • appropriate expression control sequence! s» including, for instance, a promoter to direct mRNA transcription.
  • promoters include the phage lambda PL promoter, the E. coli lac. tip and tae promoters, the SV40 early and late promoters and promoters of retroviral LTRs. to name just a few of the well-known promoters. It will be understood that numerous promoters not mentioned are suitable for use in this aspect of the invention are well known and readily may be employed by those of skill in the manner illustrated by the discussion and the examples herein.
  • expression constructs will contain sites for transcription initiation and termination, and, in the transcribed region, a ribosome binding site for translation.
  • the coding po ⁇ ion of the mamre transcripts expressed by the constructs will include a translation initiating AUG at the beginning and a termination codon appropriately positioned at the end of the polypeptide to be translated.
  • constmcts may contain control regions that regulate as well as engender expression.
  • control regions that regulate as well as engender expression.
  • such regions will operate by controlling transcription, such as repressor binding sites and enhancers, among others.
  • Vectors for propagation and expression generally will include selectable markers. Such markers also may be suitable for amplification or the vectors may contain additional markers for this purpose.
  • the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells.
  • Preferred markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, and tetracycline or ampicillin resistance genes for culruring £ coli and other bacteria.
  • the vector containing the appropriate DNA sequence as described elsewhere herein, as well as an appropriate promoter, and other appropriate control sequences, may be introduced into an appropriate host using a variety of well known techniques suitable to expression therein of a desired polypeptide.
  • appropriate hosts include bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as east cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells, animal cells such as CHO, COS and Bowes melanoma cells; and plant cells.
  • the present invention also includes recombinant constmcts, such as expression constmcts, comprising one or more of the sequences described above.
  • the constmcts comprise a vector, such as a plasmid or viral vector, into which such a sequence of the invention has been inse ⁇ ed.
  • the sequence may be inse ⁇ ed in a forward or reverse orientation.
  • the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence.
  • regulatory sequences including, for example, a promoter, operably linked to the sequence.
  • vectors preferred for use in bacteria are pQE70, pQE60 and pQE- 9, available from Qiagen: pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A. pNHl ⁇ a, pNH18A, pNH46A. available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540. pRIT5 available from Pharmacia.
  • preferred eukaryotic vectors are pWLNEO.
  • pMSG and pSVL available from Pharmacia. These vectors are listed solely by way of illustration of the many commercially available and well known vectors that are available to those of skill in the art for use in accordance with this aspect of the present invention. It will be appreciated that any other plasmid or vector suitable for. for example, introduction, maintenance, propagation or expression of a polynucleotide or polypeptide of the invention in a host may be used in this aspect of the invention.
  • Promoter regions can be selected from any desired gene using vectors that contain a reporter transcript ion unit lacking a promoter region, such as a chloramphenicol acetyl transferase ( " cat" ) transcription unit, downstream of restriction site or sites for introducing a candidate promoter fragment; i.e. , a fragment that may contain a promoter.
  • a reporter transcript ion unit lacking a promoter region such as a chloramphenicol acetyl transferase ( " cat” ) transcription unit, downstream of restriction site or sites for introducing a candidate promoter fragment; i.e. , a fragment that may contain a promoter.
  • introduction into the vector of a promoter- containing fragment at the restriction site upstream of the cat gene engenders production of CAT activity, which can be detected by standard CAT assays.
  • Vectors suitable to this end are well known and readily available. Two such vectors are pKK232-8 and pCM7.
  • bacterial promoters suitable for expression of polynucleotides and polypeptides in accordance with the present invention are the £. coli lad and lacZ and promoters, the T3 and T7 promoters, the T5 tae promoter, the lambda PR, PL promoters and the trp promoter.
  • known eukaryotic promoters suitable in this regard are the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous sarcoma vims ("RSV"). and metallothionein promoters, such as the mouse metallothionein-I promoter.
  • the present invention also relates to host cells containing the above-described constmcts discussed above.
  • the host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukar otic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell.
  • constructs into the host cell can be effected by calcium phosphate transfection. DE ⁇ F.-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 Biolo ⁇ v. ( 1986). Constmcts in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively, the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.
  • Mamre proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constmcts of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook et al. Molecular Cloning: A Laboratory Manual. 2nd Ed.. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).
  • recombinant expression vectors will include origins of replication, a promoter derived from a highly -expressed gene to direct transcription of a downstream structural sequence, and a selectable marker to permit isolation of vector containing cells after exposure to the vector.
  • Enhancers are cis-acting elements of DNA. usually about from 10 to 300 bp that act to increase transcriptional activity of a promoter in a given host cell-type.
  • enhancers include the SV4O enhancer, w hich is located on the late side of the replication origin at bp 100 to 270. the cv iomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovims enhancers.
  • Polynucleotides of the invention encoding the heterologous structural sequence of a polypeptide of the invention generally w ill be inse ⁇ ed into the vector using standard techniques so that it is onerablv linked to the promoter for expression.
  • the polynucleotide will he positioned so that the transcription sta ⁇ site is located appropriately 5' to a ribosome binding site.
  • the ribosome binding site will be 5' to the AUG that initiates translation of the polypeptide to be expressed.
  • secretion signals may be incorporated into the expressed polypeptide.
  • the signals may be endogenous to the polypeptide or they may be heterologous signals.
  • 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.
  • 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.
  • region also 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 an.
  • the selected promoter is inducible it is induced by appropriate means (e.g.. temperature shift or exposure to chemical inducer) and cells are cultured for an additional period. Cells typically then are harvested by centrifugation. dismpted by physical or chemical means, and the resulting cmde extract retained for further purification.
  • Microbial cells employed in expression of proteins can be dismpted by any convenient method, including freeze-thaw cycling, sonication, mechanical dismption. or use of cell lysing agents, such methods are well know to those skilled in the art.
  • mammalian cell cul re systems can be employed for expression, as well.
  • mammalian expression systems include the COS-7 lines of monkey kidney fibroblast, described in Gluzman et al, Cell, 23: 175 (1981).
  • Other cell lines capable of expressing a compatible vector include for example, the C127, 3T3, CHO, HeLa, human kidney 293 and BHK cell lines.
  • the AIM-I polypeptide 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, hydroxy lapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC") is employed for purification. Well known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purification.
  • HPLC high performance liquid chromatography
  • 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-glycosv lated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host- mediated processes.
  • AIM-I polynucleotides and polypeptides may be used in accordance with the present invention for a variety of applications, particularly those that make use of the chemical and biological properties AIM-I. Among these are applications in autoimmune disease and aberrant cellular proliferation. Additional applications relate to diagnosis and to treatment of disorders of cells, tissues and organisms. These aspects of the invention are illustrated further by the following discussion.
  • This invention is also related to the use of the AIM-I polynucleotides to detect complementary polynucleotides such as, for example, as a diagnostic reagent. Detection of a mutated form of AIM-I associated with a dysfunction will provide a diagnostic tool that can add or define a diagnosis of a disease or susceptibility to diseases which results from under-expression over-expression or altered expression of AIM-I, such as, for example, autoimmune diseases.
  • Nucleic acids for diagnosis may be obtained from a patient's cells, such as from blood, urine, saliva, tissue biopsy and autopsy material.
  • the genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR prior to analysis.
  • PCR Saiki et al, Nature, 324: 163-166, ( 1986).
  • RNA or cDNA may also be used in the same ways.
  • PCR primers complementary to the nucleic acid encoding AIM-I can be used to identify and analyze AIM-1 expression and mutations.
  • deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype.
  • Point mutations can be identified by hybridizing amplified DNA to radiolabeled AIM-I RS ⁇ or alterrutivel .
  • radiolabeled AIM-I antisense DNA sequences Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melung lemperatures.
  • Sequence differences between a reference gene and genes having mutations also may be revealed by direct DNA sequencing.
  • cloned DNA segments may be employed as probes to detect specific DNA segments.
  • the sensitivity of such methods can be great Iv enhanced bv appropriate use of PCR or another amplification method.
  • a sequencing primer is used with double-stranded PCR product or a single-stranded template molecule generated by a modified PCR.
  • the sequence determination is performed by conventional procedures with radiolabeled nucleotide or by automatic sequencing procedures with fluorescent- tags.
  • DNA sequence differences may be achieved by detection of alteration in electrophoretic mobility of DNA fragments in gels, with or without denaturing agents. Small sequence deletions and insertions can be visualized by high resolution gel electrophoresis. DNA fragments of different sequences may be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e.g. , Myers et al, Science, 230: 1242, 1985).
  • Sequence changes at specific locations also may be revealed by nuclease protection assays, such as RNase and SI protection or the chemical cleavage method (e.g. , Cotton er a/.. Proc. Natl. Acad. Sci., USA, 55:4397-4401 , 1985).
  • nuclease protection assays such as RNase and SI protection or the chemical cleavage method (e.g. , Cotton er a/.. Proc. Natl. Acad. Sci., USA, 55:4397-4401 , 1985).
  • the detection of a specific DNA sequence may be achieved by methods such as hybridization. RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes, (e.g.. restriction fragment length polymorphisms ("RFLP”) and Southern blotting of genomic DNA.
  • restriction enzymes e.g.. restriction fragment length polymorphisms ("RFLP")
  • RFLP restriction fragment length polymorphisms
  • mutations also can be detected h ⁇ in situ analysis.
  • sequences 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.
  • Few chromosome marking reagents based on actual sequence data (repeat polymorphisms) are presently available for marking chromosomal location.
  • 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-I gene. This can be accomplished using a variety of well known techniques and libraries, which generally are available commercially.
  • the genomic DNA the is used for in situ chromosome mapping using well known techniques for this purpose. Typically, in accordance with routine procedures for chromosome mapping, some trial and error may be necessary to identify a genomic probe that gives a good in situ hybridization signal.
  • 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. Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment.
  • PCR mapping of somatic cell hybrids is a rapid procedure for assigning a pa ⁇ icular DNA to a particular chromosome.
  • suhlocalization can be achieved with panels of fragments from specific chromosomes or pools of large genomic clones in an analogous manner.
  • Other mapping strategies that can similarly be used to map to its chromosome include in situ hybridization, prescreemng w ith labeled flow-sorted chromosomes and preselection by hybridization to construct chromosome specific-cDNA libraries.
  • 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 cDNA as short as 50 or 60.
  • Verma et al Human Chromosomes: A Manual Of Basic Techniques, Pergamon Press, New York (1988).
  • a cDNA precisely localized to a chromosomal region associated with the disease could be one of between 50 jnu 500 potential causative genes. (This assumes 1 megabase mapping resolution and one gene per 20 kb).
  • the present invention alv> relates to a diagnostic assays such as quantitative and diagnostic assays for detecting levels of AIM-I protein in cells and tissues, including determination of normal and abnormal levels.
  • a diagnostic assay in accordance with the invention tor detecting over-expression of AIM-I protein compared to normal control tissue samples may be used to detect the presence of aberrant cellular proliferation such as cancer, for example.
  • Assay techniques that can be used to determine levels of a protein, such as an AIM-I protein of the present invention, in a sample derived from a host are well-known to those of skill in the an. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays.
  • An ELISA assay initially comprises preparing an antibody specific to AIM-I, preferably a monoclonal antibody.
  • a reponer antibody generally is prepared which binds to the monoclonal antibody.
  • the reponer antibody is attached a detectable reagent such as radioactive, fluorescent or enzymatic reagent, in this example horseradish peroxidase enzyme.
  • a sample is removed from a host and incubated on a solid suppo ⁇ . e.g. a polystyrene dish, that binds the proteins in the sample. Any free protein binding sites on the dish are then covered by incubating with a non-specific protein such as bovine semm albumin.
  • the monoclonal antibody is incubated in the dish during which time the monoclonal antibodies attach to any AIM-I proteins attached to the polystyrene dish. Unbound monoclonal antibody is washed out with buffer.
  • the reponer antibody linked to horseradish peroxidase is placed in the dish resulting in binding of the reponer antibody to any monoclonal antibody bound to AIM-I.
  • Unattached reponer antibody is then washed out.
  • Reagents for peroxidase activity including a colorimetric substrate are then added to the dish.
  • Immobilized peroxidase, linked to AIM-I through the primary and secondary antibodies, produces a colored reaction product.
  • the amount of color developed in a given time period indicates the amount of AIM-I protein present in the sample.
  • Quantitative results typically are obtained by reference to a standard curve.
  • a competition assay may be employed wherein antibodies specific to AIM-I attached to a solid suppo ⁇ and labeled AIM-I and a sample derived from the host are passed over the solid suppo ⁇ and the amount of label detected attached to the solid suppo ⁇ can be correlated to a quantity of AIM-I in the sample.
  • the polypeptides, their fragments or other derivatives, or analogs thereof, or cells expressing them can be used as an immunogen to produce antibodies thereto. These antibodies can be, for example, polyclonal or monoclonal antibodies.
  • the present invention also includes chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library. Various procedures known in the an may be used for the production of such antibodies and fragments.
  • Antibodies generated against the polypeptides corresponding to a sequence of the present invention can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptides to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptides itself. In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptides. Such antibodies can then be used to isolate the polypeptide from tissue expressing that polypeptide.
  • any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler. G. and Milstein, C., Nature, 256:495-497 (1975), the trioma technique, the human B-cell hybridoma technique (Kozbor et al. Immunology Today, 4:12 (1983) and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al . pg. 77-96 in Monoclonal Antibodies And Cancer Therapy, Alan R. Liss. Inc. ( 1985 ).
  • the AIM-I of the present invention may be employed to treat lymphoproliferative disease which results in lymphadenopathy, the AIM-I mediates apoptosis by stimulating clonal deletion of T-cells and may therefore, by employed to treat autoimmune disease, to stimulate peripheral tolerance and cytotoxic T-cell mediated apoptosis.
  • the AIM-1 may also be employed as a research tool in elucidating the biology of autoimmune disorders including systemic lupus erythematosus, immunoproliferative disease lymphadenopathy (IPL), angioimmunoproliferative lymphadenopathy (AIL), rheumatoid a ⁇ hritis. diabetes, and multiple sclerosis, and to treat graft versus host disease.
  • the AIM-I may of the present invention may also be employed to inhibit neoplasia, such as tumor cell growth.
  • the AIM-I polypeptide may be responsible for tumor destmction through apoptosis and cytotoxicity to ce ⁇ ain cells.
  • AIM-I may also be employed to treat diseases which require growth promotion activity, for example, restenosis, since AIM-I has proliferation effects on cells of endothelial origin.
  • AIM-I may. therefore, also be employed to regulate hematopoiesis in endothelial cell development.
  • the polynucleotide encoding the AlM-1 may be employed as a diagnostic marker for determining expression of the polypeptide of the present invention since the gene is found in many tumor cell lines including pancreatic tumor, testes mmor, endometrial rumor and T-cell lymphoma.
  • This invention also provides a method for identification of molecules, such as receptor molecules, that bind AIM-I.
  • Genes encoding proteins that bind AIM-I, 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.
  • polyadenylated RNA is prepared from a cell responsive to AIM-I
  • 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-I.
  • the transfected cells then are exposed to labeled AIM-I.
  • AIM-I 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-I is determined. These procedures conveniently are carried out on glass slides.
  • Pools are identified of cDNA that produced AIM-I-binding cells. 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 photoaffinity 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.
  • 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-I binding capacity of AIM-I binding molecules, such as receptor molecules, in cells or in cell-free preparations.
  • the invention also provides a method of screening compounds to identify those which enhance or block the action of AIM-I on cells, such as its interaction with AIM-I- binding molecules such as receptor molecules.
  • An agonist is a compound which increases the namral biological functions of AIM-I or which functions in a manner similar to AIM-I, while antagonists decrease or eliminate such functions.
  • a cellular compartment such as a membrane or a preparation thereof, such as a membrane-preparation, may be prepared from a cell that expresses a molecule that binds AIM-I.
  • AIM-I a molecule of a signaling or regulatory pathway modulated by AIM-I.
  • the preparation is incubated with labeled AIM-I in the absence or the presence of a candidate molecule which may be an AIM-I agonist or antagonist.
  • the ability of the candidate molecule to bind the binding molecule is reflected in decreased binding of the labeled ligand.
  • AlM-I-like effects of potential agonists and antagonists may by measured, for instance, by determining activ ity 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-I or molecules that elicit the same effects as AIM-I.
  • 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.
  • Another example of an assay for AIM-I antagonists is a competitive assay that combines AIM-I and a potential antagonist with membrane-bound AIM-I receptor molecules or recombinant AIM-I receptor molecules under appropriate conditions for a competitive inhibition assay.
  • AIM-I can be labeled, such as by radioactivity, such that the number of AIM-I 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-I-induced activities, thereby preventing the action of AIM-I by excluding AIM-I from binding.
  • Antisense molecules 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 : Oligodeoxvnucleotides 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, 257: 1360 1991. The methods are based on binding of a polynucleotide to a complementary DNA or RNA.
  • the 5' coding portion of a pol ucleotide that encodes the mamre polypeptide of the present invention may be used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length ⁇ DS ⁇ oligonucleotide is designed to be complementary to a region of the gene inv lved in transcription thereby preventing transcription and the production of AIM-l
  • the antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into AIM-I 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-I.
  • 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 suppressed by AIM-I.
  • the AIM-I antagonists may also be employed to treat cerebral malaria in which AIM-I appears to play a pathogenic role.
  • the antagonists may also be employed to treat rheumatoid arthritis by inhibiting AIM-I induced production of inflammatory cytokines, such as IL1 in the synovial cells. When treating arthritis, AIM-I is preferably injected intra-articularly.
  • the AIM-I 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-I 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.
  • compositions comprising the polynucleotide or the polypeptides discussed above or the agonists or antagonists.
  • the polypeptides of the present invention may be employed in combination with a non-sterile or sterile carrier or carriers for use with cells, tissues or organisms, such as a pharmaceutical carrier suitable for administration to a subject.
  • a pharmaceutical carrier suitable for administration to a subject such as a pharmaceutical carrier suitable for administration to a subject.
  • Such compositions comprise, for instance, a media additive or a therapeutically effective amount of a polypeptide of the ;.' ention and a pharmaceutically acceptable carrier or excipient.
  • Such carriers may include, but are not limited to. saline, buffered saline, dextrose, water, glycerol, ethanol and combinations thereof. The formulation should suit the mode of administration.
  • the invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention.
  • Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacmre, use or sale of pharmaceuticals or biological products, reflecting approval by the agency of the manufacmre, use or sale of the product for human administration.
  • Polypeptides and other compounds of the present invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.
  • compositions may be administered in any effective, convenient manner including, for instance, administration by topical, oral, anal, vaginal, intravenous, intraperitoneal. intramuscular, subcutaneous, intranasal or intradermal routes among others.
  • compositions generally are administered in an amount effective for treatment or prophylaxis of a specific indication or indications.
  • the compositions are administered in an amount of at least about 10 ⁇ g/kg body weight. In most cases they will be administered in an amount not in excess of about 8 mg/kg body weight per day. Preferably, in most cases, dose is from about 10 ⁇ g/kg to about 1 mg/kg body weight, daily. It will be appreciated that optimum dosage will be determined by standard methods for each treatment modality and indication, taking into account the indication, us severity . route of administration, complicating conditions and the like.
  • polypeptides, agonists and antagonists that are polypeptides may be employed in accordance with the present invention by expression of such polypeptides in vivo, in treatment modalities often referred to as "gene therapy.
  • a polynucleotide such as a DNA or RNA
  • the engineered cells then can be provided to a patient to be treated with the polypeptide.
  • cells may be engineered ex vivo by the use of a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention.
  • cells may be engineered in vivo for expression of a polypeptide in vivo by procedures known in the an.
  • a polynucleotide of the invention may be engineered for expression in a replication defective retroviral vector, as discussed above.
  • the retroviral expression construct then may be isolated and introduced into a packaging cell is transduced with a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention such that the packaging cell now produces infectious viral panicles containing the gene of interest.
  • These producer cells may be administered to a patient for engineering cells in vivo and expression of the polypeptide in vivo.
  • Retrovimses from which the retroviral plasmid vectors herein above mentioned may be derived include, but are not limited to. Moloney Murine Leukemia Vims, spleen necrosis vims, retrov iruses such as Rous Sarcoma Vims, Harvey Sarcoma Vims, avian leukosis vims, gibbon ape leukemia v ims, human immunodeficiency vims, adenovims. Myeloproliferative Sarcoma Vims, and mammary tumor vims.
  • the retroviral plasmid vector is derived from Moloney Murine Leukemia Vims.
  • Such vectors well include one or more promoters for expressing the polypeptide.
  • Suitable promoters which may be employed include, but are not limited to, the retroviral LTR: the SV40 promoter: and the human cytomegalovirus (CMV) promoter described in Miller et al. Biotechntquei. 7 980-990 (1989), or any other promoter (e.g. , cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, RNA polymerase III, and ⁇ -actin promoters).
  • CMV human cytomegalovirus
  • viral promoters which may be employed include, but are not limited to, adenovims promoters, thymidine kinase (TK) promoters, and B19 parvovirus promoters. The selection of a suitable promoter will be apparent to those skilled in the an from the teachings contained herein.
  • Suitable promoters which may be employed include, but are not limited to, adenoviral promoters, such as the adenoviral major late promoter; or heterologous promoters, such as the cytomegalovims (CMV) promoter; the respiratory syncytial vims (RSV) promoter; inducible promoters, such as the MMT promoter, the metallothionein promoter; heat shock promoters; the albumin promoter; the ApoAI promoter: human globin promoters; viral thymidine kinase promoters, such as the Herpes Simplex thymidine kinase promoter; retroviral LTRs (including the modified retroviral LTRs herein above described); the ⁇ -actin promoter; and human growth hormone promoters.
  • the promoter also may be the native promoter which controls the gene encoding the
  • the retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines.
  • packaging cells which may be transfected include, but are not limited to. the PE501 . PA317, Y-2, Y-AM, PA12. T19-14X, VT- 19-17-H2. YCRE. YCRIP. GP + E-86. GP+envAml2. and DAN cell lines as described in Miller, A., Human Gene Therapv. / :5- 14 ( 1990).
  • the vector may be transduced into the packaging cells through any means known in the an. Such means include, but are not limited to. electroporation. the use of liposomes, and CaPO 4 precipitation.
  • the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid. and then administered to a host.
  • the producer cell line w ill generate infectious retroviral vector panicles, which include the nucleic acid sequence(s) encoding the polypeptides.
  • retroviral vector particles then may be employed to transduce eukaryotic cells, either in vitro or in vivo.
  • the transduced eukaryotic cells will express the nucleic acid sequence(s) encoding the polypeptide.
  • Eukaryotic cells which may be transduced include, but are not limited to, embryonic stem cells, embryonic carcinoma cells, as well as hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells, and bronchial epithelial cells.
  • ligations were accomplished using standard buffers, incubation temperatures and times, approximately equimolar amounts of the DNA fragments to be ligated and approximately 10 units of T4 DNA ligase ("ligase") per 0.5 ⁇ g of DNA.
  • ligase T4 DNA ligase
  • the DNA sequence encoding human AIM-I in the deposited polynucleotide was amplified using PCR oligonucleotide primers specific to the amino acid carboxyl terminal sequence of the human AIM-I protein and to vector sequences 3' to the gene. Additional nucleotides containing restriction sites to facilitate cloning were added to the 5' and 3' sequences respectively.
  • the 5' oligonucleotide primer had the sequence 5' GCG GCG GGA TCC ATG GCT ATG ATG GAG GTC CAG 3' containing the underlined BamHI restriction site, which encodes a start AUG. followed by 18 nucleotides of the human AIM-I coding sequence set out in Figure 1.
  • the 3' primer had the sequence 5 CGC GCG TCT AGA GCT TAG GCA ACT AAA AAG GCC 3 " containing the underlined Xbal restriction site followed by 21 nucleotides complementary to the last 21 nucleotides of the AIM-I coding sequence set out in Figure 1. including the stop codon.
  • the restrictions sites were convenient to restriction enzyme sites in the bacterial expression vectors pQi ⁇ which were used tor bacterial expression in these examples.
  • C ⁇ i 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.
  • the ligation mixture was transformed into competent £. 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, Cold Spring Harbor, N.Y. (1989).
  • This strain which is only one of many that are suitable for expressing AIM-I, is available commercially from Qiagen.
  • Transformants were identified by their ability to grow on LB plates in the presence of ampicillin. Plasmid DNA was isolated from resistant colonies and the identity of the cloned DNA was confirmed by restriction analysis.
  • Clones containing the desired constmcts were 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 was used to inoculate a large culture, at a dilution of approximately 1 : 100 to 1 :250.
  • the cells were grown to an optical density at 600 nm ("ODioo") of between 0.4 and 0.6.
  • Isopropyl-B-D-thiogalactopyranoside (“IPTG”) was then added to a final concentration of 1 mM to induce transcription from lac repressor sensitive promoters, by inactivating the lad repressor. Cells subsequently were incubated further for 3 to 4 hours. Cells then were harvested by centrifugation and dismpted, by standard methods.
  • the cDNA sequence encoding the full length human AIM-I 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' CCG CGC GGA TCC ATC ATG GCT ATG ATG GAG GTC C 3 ' containing the underlined restriction enzyme site followed by 22 bases of the sequence of AIM-I of Figure 1 .
  • 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 ' CGC GCG TCT AGA GCT TAG CCA ACT AAA AAG GCC 3' containing the underlined Xbal restriction followed by nucleotides complementary to the last 21 nucleotides of the AIM-I coding sequence set out in Figure 1 , including the stop codon.
  • 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 BamHI and Asp718 and again is purified on a 1 % agarose gel. This fragment is designated herein F2.
  • the vector pA2 is used to express the AIM-I protein in the baculovims expression system, using standard methods, such as those described in Summers et al, A Manual Of Methods For Baculovims 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 califomica nuclear polyhedrosis vims (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 vims 40 (“SV40") is used for efficient polyadenylation.
  • SV40 simian vims 40
  • the beta- galactosidase gene from £. 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 vims that express the cloned polynucleotide.
  • baculovims vectors could be used in place of pA2, such as pAc373, pVL941 and pAcIM l provided, as those of skill readily will appreciate, that constmction provides appropriately K»cated signals for transcription, translation, trafficking and the like, such as an in-frame Al G and a signal peptide, as required.
  • K K»cated signals for transcription, translation, trafficking and the like, such as an in-frame Al G and a signal peptide, as required.
  • Such vectors are described in Lucko et al . Vint ms . 170: 1 -39, among others.
  • the plasmid is digested w ith the restriction enzymes BamHI and Xbal 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 "V2" .
  • Fragment F2 and the dephosphorylated plasmid V2 are ligated together with T4 DNA ligase.
  • £. coli HB101 cells are transformed with ligation mix and spread on culture plates.
  • Bacteria are identified that contain the plasmid with the human AIM-I gene by digesting DNA from individual colonies using BamHI and 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-I.
  • 5 ⁇ g of the plasmid pBacAIM-I is co-transfected with 1.0 ⁇ g of a commercially available linearized baculovims DNA ("BaculoGoldTM baculovims DNA”. Pharmingen, San Diego, CA.), using the lipofection method described by Feigner et al , Proc. Natl. Acad. Sci. USA, 54:7413-7417 ( 1987).
  • BaculoGoldTM vims DNA and 5 ⁇ g of the plasmid pBacAIM-I are mixed in a sterile well of a microtiter plate containing 50 ⁇ l of semm 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 p ⁇ xluce 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).
  • V- AIM-I A clone containing properly inserted AIM-I is identified by DNA analysis including restriction mapping and sequencing. This is designated herein as V- AIM-I.
  • Sf9 cells are grown in Grace's medium supplemented with 10% heat-inactivated FBS.
  • the cells are infected with the recombinant baculovims V- AIM-I at a multiplicity of infection ("MOD of about 2 (about 1 to about 3).
  • MOD multiplicity of infection
  • the medium is removed and is replaced with SF900 II medium minus methionine and cysteine (available from Life Technologies Inc. , Gaithersburg). 42 hours later, 5 ⁇ Ci of 35 S- methionine and 5 ⁇ Ci "S cysteine (available from Amersham) are added.
  • the cells are further incubated for 16 hours and then they are harvested by centrifugation, lysed and the labeled proteins are visualized by SDS-PAGE and autoradiography .
  • the expression plasmid. ⁇ IM-I HA. is made by cloning a cDNA encoding AIM-I into the expression vector pcDN Al/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 £.
  • coli and other prokaryotic cell (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 entire AIM-I precursor and a 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-I cDNA of the deposited clone is amplified using primers that contained convenient restriction sites, much as described above regarding the constmction of expression vectors for expression of AIM-I in £. coli and 5. fur ⁇ erda.
  • one of the primers contains a hemagglutinin tag ("HA tag") as described above.
  • Suitable primers include that following, which are used in this example.
  • the 5' primer containing the underlined restriction enzyme site, an AUG start codon and 22 codons thereafter, forming the hexapeptide haemagglutinin tag, has the sequence: 5' CCG CGC GGA ICC ATC ATG GCT ATG ATG GAG GTC C 3'
  • the 3' primer containing the underlined Xbal site and 21 nucleotides of 3' coding sequence (at the 3' end) has the sequence: 5' CGC GCG TCT AGA GCT TAG CCA ACT AAA AAG GCC 3'
  • the PCR amplified DNA fragment and the vector, pcDNAI/Amp, are digested with and then ligated.
  • the ligation mixmre is transformed into E. coli strain SURE (available from Stratagene Cloning Systems, 11099 North To ⁇ ey Pines Road, La Jolla, CA 92037) 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-I-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-l by the vector.
  • AIM-I 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. Ne 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. I S N P-40. 0. 1 % SDS, 1 % NP-40, 0.5% DOC, 50 mM TRIS. 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. .'., expression product of the expected size is seen in the cell lysate, which is not seen in negative controls.
  • Example 4 Tissue Distribution of AIM-I Expression
  • RNAzolTM B system Biotecx Laboratories, Inc. 6023 South Loop East, Houston, TX 77033.
  • RNA is isolated from tissue samples. The RNA is size resolved by electrophoresis through a 1 % agarose gel under strongly denaturing conditions. RNA is blotted from the gel onto a nylon filter, and the filter then is prepared for hybridization to a detectably labeled polynucleotide probe.
  • the antisense strand of the coding region of the cDNA inse ⁇ in the deposited clone is labeled to a high specific activity.
  • the cDNA is labeled by primer extension, using the Prime-It kit, available from Stratagene.
  • the reaction is carried out using 50 ng of the cDNA, following the standard reaction protocol as recommended by the supplier.
  • the labeled polynucleotide is purified away from other labeled reaction components by column chromatography using a Select-G-50 column, obtained from 5-Prime - 3-Prime, Inc. of 5603 Arapahoe Road. Boulder. CO 80303.
  • the labeled probe is hybridized to the filter, at a concentration of 1.000,000 cp /ml. in a small volume of 7 r ; SDS. 0.5 M NaPO 4 , pH 7.4 at 65 * C, overnight. Thereafter the probe solution is drained and the filter is washed twice at room temperamre and twice at 60 * C ith 0.5 x SSC, 0.1 % SDS. The filter then is dried and exposed to film at -70 overnight ith an intensifying screen. Autoradiography shows that mRNA for AIM-I is abundant in human hea ⁇ . bone marrow, CD4 + and CD19 * peripheral blood lymphocytes, and less so in lung and kidney tissue.
  • Example 5 Gene Therapeutic Expression of Human AIM-I
  • Fibroblasts are obtained from a subject by skin biopsy.
  • the resulting tissue is placed in tissue-culmre medium and separated into small pieces. Small chunks of the tissue are placed on a wet surface of a tissue culmre flask, approximately ten pieces are placed in each flask.
  • the flask is turned upside down, closed tight and left at room temperamre overnight. After 24 hours at room temperamre, the flask is inve ⁇ ed - the chunks of tissue remain fixed to the bottom of the flask - and fresh media is added (e.g. , Ham's F12 media, with 10% FBS, penicillin and streptomycin).
  • the tissue is then incubated at 37 'C for approximately one week. At this time, fresh media is added and subsequently changed every several days. After an additional two weeks in culmre. a monolayer of fibroblasts emerges. The monolayer is trypsinized and scaled into larger flasks.
  • a vector for gene therap is digested with restriction enzymes for cloning a fragment to be expressed.
  • the digested vector is treated with calf intestinal phosphatase to prevent self-ligation.
  • the dephosphorylated, linear vector is fractionated on an agarose gel and purified.
  • AIM-I cDNA capable of expressing active AIM-I is isolated.
  • the ends of the fragment are modified, if necessary , for cloning into the vector. For instance, 5" overhanging may be treated w ith DNA polymerase to create blunt ends. 3 ' overhanging ends may be removed using S I nuclease. Linkers may be ligated to blunt ends with T4 DNA ligase
  • Equal quantities of the Moloney murine leukemia vims linear backbone and the AIM-I fragment are mixed together and joined using T4 DNA ligase.
  • the ligation mixmre is used to transform £. coli and the bacteria are then plated onto agar-containing kanamycin. Kanamycin phenotype and restriction analysis confirm that the vector has the properly inse ⁇ ed gene.
  • Packaging cells are grown in tissue culmre to confluent density in Dulbecco's Modified Eagle's Medium (DMEM) with 10% calf semm (CS), penicillin and streptomycin.
  • DMEM Dulbecco's Modified Eagle's Medium
  • CS calf semm
  • penicillin and streptomycin The vector containing the AIM-I gene is introduced into the packaging cells by standard techniques. Infectious viral panicles containing the AIM-I gene are collected from the packaging cells, which now are called producer cells.
  • Fresh media is added to the producer cells, and after an appropriate incubation period media is harvested from the plates of confluent producer cells.
  • the media containing the infectious viral particles, is filtered through a Millipore filter to remove detached producer cells.
  • the filtered media then is used to infect fibroblast cells.
  • Media is removed from a sub-confluent plate of fibroblasts and quickly replaced with the filtered media.
  • Polybrene Aldrich
  • the media is removed and replaced with fresh media. If the titer of vims is high, then virtually all fibroblasts will be infected and no selection is required. If the titer is low, then it is necessary to use a retroviral vector that has a selectable marker, such as neo or his, to select out transduced cells for expansion.
  • Engineered fibroblasts then may be injected into rats, either alone or after having been grown to confluence on m ⁇ cn carr ⁇ er beads, such as cytodex 3 beads.
  • the injected fibroblasts produce AIM-I product, and the biological actions of the protein are conveyed to the host
  • ADDRESSEE Carella, Byrne, Bain, Gilfillan, Cecchi,
  • MOLECULE TYPE DNA (genomic)
  • CAAAACAAAC AAACAGAAAA CAGAAAACAA AAAAACCTCT ATGCAATCTG AGTAGAGCAG 1044
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Cette invention concerne des polypeptides AIM-I, des polynucléotides codant ces polypeptides, des procédés de production de ces polypeptides notamment par l'expression des polynucléotides, ainsi que des agonistes et des antagonistes de ces polypeptides. Cette invention concerne également des procédés d'utilisation de ces polynucléotides, polypeptides, agonistes et antagonistes dans des applications qui ont partiellement trait aux domaines diagnostique, clinique et de la recherche.
PCT/US1996/003773 1996-03-14 1996-03-14 Molecule i induisant l'apoptose WO1997033899A1 (fr)

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PCT/US1996/003773 WO1997033899A1 (fr) 1996-03-14 1996-03-14 Molecule i induisant l'apoptose
AU57111/96A AU5711196A (en) 1996-03-14 1996-03-14 Apoptosis inducing molecule i

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