Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4747—Apoptosis related proteins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates generally to molecules capable of modulating apoptosis of animal cells. More particularly, the present invention provides cell homologues of viral-derived, apoptotic-inhibiting molecules which are useful in modulating apoptosis of animal cells.
• the molecules contemplated by the present invention may be used to promote or inhibit cell apoptosis depending on the exigencies of the therapeutic situation.
• Apoptosis is a cellular death program that may be initiated by a variety of stimuli and typically leads to characteristic changes in cells, frequently resulting in activation of a endonuclease which catalyses chromatin fragmentation and condensation, membrane blebbing and collapse of the nucleus.
• proteins resembling Bcl-2 can protect nematode, insect and vertebrate cells from apoptosis (Vaux etal, 1992; Alnemri etal, 1992), and cysteine proteases resembling interleukin- l ⁇ converting enzyme (ICE) are required for apoptosis in both C. elgans and mammals (Ellis and Horvitz, 1986; Miura et al, 1993; Kuida et al, 1995).
• ICE interleukin- l ⁇ converting enzyme
• apoptosis effector proteases exist as precursors in many cells, but must be cleaved and assembled into tetramers before they are active (Thornberry et al, 1992; Wilson et al, 1994; Walker et al, 1994; Munday e/ ⁇ /, 1995).
• the cytoplasmic domain of CD95 which bears a motif termed the "death domain" is known to associate with other death domain bearing proteins such as FADD/MORT1 (Boldin et al, 1995; Chinnaiyan et al, 1995) and RTP (Stager et al, 1995) which are believed to participate in signalling from CD95.
• FADD/MORT1 Bosset et al, 1995; Chinnaiyan et al, 1995
• RTP Ster et al, 1995
• Tumour Necrosis Factor can also trigger apoptosis, and its receptor resemble CD95
• TNF-receptor 1 (p55) is known to bind to TRADD (Hsu et al, 1995) and the TNF-receptor 2 (p75) is known to bind to TNF receptors associated factors (TRAFs) 1 and 2 (Rothe et al, 1994), but how these proteins work has not yet been determined.
• the inventors studied viral anti-apoptosis proteins and the intermediate steps of apoptosis signalling.
• Apoptosis can be used as a defence against viruses, but many viruses carry genes for anti-apoptosis proteins, presumably to keep the host cell alive while they replicate.
• Some viral anti-apoptosis proteins resemble known cellular proteins such as Bcl-2 (reviewed in Vaux, 1993).
• baculovirus p35 proteins have unknown cellular counterparts, but can function in heterologous systems such as nematodes and mammals where they are thought to act as competitive inhibitors of ICE-like cysteine proteases (Clem et al, 1991; Hay et al, 1994; Rabizadeh et al, 1993; Xue and Horvitz, 1995; Bump et al, 1995). Miller and coworkers identified a family of proteins in baculoviruses they designated "IAPs" because they could inhibit the apoptotic response of insect cells to viral infection (Birnbaum et al, 1994; Crook et al, 1993).
• Viral IAP proteins typically have two terminal repeats designated baculovirus LAP repeats (BLRs), and a carboxy terminal RING finger domain.
• BLRs baculovirus LAP repeats
• AcNPW Autographa californica nuclear polyhedrosis virus encodes a IAP protein that does not inhibit apoptosis, so it is possible that IAP proteins also have other functions, for example, regulatory cytokine function.
• the present inventors sought cellular IAP homologues and chemical analogues including derivatives which function in cell death pathways by screening for gene segments potentially encoding novel IAP proteins. Full length cDNA clones were then obtained and tested for their ability to mediate apoptosis by ICE and by FADD.
• one aspect of the present invention is directed to a homologue or chemical analogue of a viral derived peptide, polypeptide or protein or a derivative thereof which viral- derived molecule is capable of inhibiting an apoptotic response of cells to viral infection.
• the present invention provides an isolated proteinaceous molecule or derivative of chemical analogue thereof capable of inhibiting an apoptotic response in cells to viral infection, said proteinaceous molecule comprising a cell-derived homologue of a viral inhibitor of apoptosis (IAP).
• IAP apoptosis
• Viral derived IAPSs are polypeptides or proteins.
• the IAPs represent a family of proteinaceous molecules which inhibit the apoptotic response of cells to viral infection. More particularly, the IAPs inhibit the apoptotic response of insect cells to infection by baculoviruses.
• the present invention provides, therefore, homologues or chemical analogues of baculovirus IAPs which are useful in modulating apoptosis of animal cells.
• the apoptosis is mediated by ICE or by FADD.
• another aspect of the present invention contemplates a homologue or chemical analogue of a baculovirus IAP peptide, polypeptide or protein or a derivative thereof.
• the homologue is a animal, cell derived molecule.
• animal cells are those from humans, livestock animals (e.g. sheep, pigs, horses, donkeys, cows), companion animals (e.g. dogs, cats), laboratory test animals (e.g. rabbits, mice, rats, guinea pigs), captive wild animals (e.g. foxes, deer, kangaroos), insects (e.g. mosquitoes) and nematodes.
• Homologous may also be from yeast or fungi.
• Particularly preferred homologues are for human, murine, insect, yeast or nematode cells.
• An example of a nematode is Caenorhabditis elegans.
• the mammalian homologues are derived from murine cells and are designated herein MLHA, MLHB, MLHC and MLHE.
• the homologue is an insect homologue from Drosophila designated herein DLHA.
• the homologue is a C elegans homologue designated herem CLA-1 and CIA-2.
• the homologue is a yeast homologue designated herein YIA-1.
• the present invention extends to human and other mammalian homologues of baculovirus IAPs or homologues or chemical analogues of MLHA, MLHB, MLHC and/or DLHA.
• an isolated molecule having an amino acid sequence comprising:
• Xaa is an amino acid residue
• [Xaa] m is a series of at least 5 and preferably at least 9 amino acids;
• [Xaa] n is Met or Leu;
• [Xaa] 0 is a series of at least 3 and preferably at least 5 amino acids;
• [Xaa] p is Val or Ala; [Xaa]q is Phe or Tyr; [Xaa] r is Leu or Val;
• MLHA having a amino acid sequence substantially as set forth in SEQ LD NO: 2 or having at least 40% similarity to all or part thereof or a homologue, chemical analogue or derivative thereof.
• a related aspect of the present invention is directed to a isolated molecule designated MLHB having an amino acid sequence substantially as set forth in SEQ LD NO:4 or having at least 40% similarity to all or part thereof or a homologue, chemical analogue or derivative thereof.
• MLHC isolated molecule designated MLHC having an amino acid sequence substantially as set forth in SEQ LD NO: 6 or having at least 40% similarity to all or part thereof or a homologue, chemical analogue or derivative thereof.
• Still a further related aspect of the present invention provides a isolated molecule designated DLHA having an amino acid sequence substantially as set forth in SEQ LD NO: 8 or having at least 40% similarity to all or part thereof or a homologue, chemical analogue or derivative thereof.
• Another related aspect of the present invention provides a isolated molecule designated DLHA having an amino acid sequence substantially as set forth in SEQ LD NO:l 1 or having at least 40% similarity to all or part thereof or a homologue, chemical analogue or derivative thereof.
• Still another related aspect of the present invention provides a isolated molecule designated DLHA having an amino acid sequence substantially as set forth in SEQ LD NO: 13 or having at least 40% similarity to all or part thereof or a homologue, chemical analogue or derivative thereof.
• a further related aspect ofthe present invention provides a isolated molecule designated DLHA having an amino acid sequence substantially as set forth in SEQ LD NO: 15 or having at least
• Yet another related aspect of the present invention provides a isolated molecule designated DLHA having an amino acid sequence substantially as set forth in SEQ LD NO: 17 or having at least 40% similarity to all or part thereof or a homologue, chemical analogue or derivative thereof.
• the present invention also encompasses nucleic acid molecules encoding IAP homologues such as MLHA, MLHB, MLHC, MLHE, CIA-1, CIA-2, YLA-1 and DLHA.
• the LAP homologues may or may not bear RING finger domains, for example, MLHA, MHB and MLHC bear the domain whereas MLHE, CIA-1, CIA-2 or YIAY do not bear the domain.
• the present invention further contemplates a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding the amino acid sequence comprising:
• Xaa is an amino acid residue
• [Xaa] m is a series of at least 5 and preferably at least 9 amino acids
• n is Met or Leu
• [Xaa] 0 is a series of at least 3 and preferably at least 5 amino acids; [Xaa] p is Val or Ala; [Xaa]q is Phe or Tyr; [Xaa] r is Leu or Val;
• the present invention contemplates a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding the amino acid sequence substantially set forth in SEQ LD NO:2 or having at least 40% similarity to a nucleotide sequence encoding the amino acid sequence substantially set forth in SEQ LD NO:2.
• nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding the amino acid sequence substantially set forth in SEQ LD NO:4 or having at least 40% similarity to a nucleotide sequence encoding the amino acid sequence substantially set forth in SEQ LD NO:4.
• nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding the amino acid sequence substantially set forth in SEQ LD NO:6 or having at least 40% similarity to a nucleotide sequence encoding the amino acid sequence substantially set forth in SEQ LD NO:6.
• a further embodiment is directed to a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding the amino acid sequence substantially set forth in SEQ LD NO: 8 or having at least 40% similarity to a nucleotide sequence encoding the amino acid sequence substantially set forth in SEQ LD NO: 8.
• nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding the amino acid sequence substantially set forth in SEQ LD NO: 11 or having at least 40% similarity to a nucleotide sequence encoding the amino acid sequence substantially set forth in SEQ LD NO: 11.
• Still another related aspect is directed to a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding the amino acid sequence substantially set forth in SEQ LD NO: 13 or having at least 40% similarity to a nucleotide sequence encoding the amino acid sequence substantially set forth in SEQ LD NO: 13.
• a further related aspect is directed to a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding the amino acid sequence substantially set forth in SEQ LD NO: 15 or having at least 40% similarity to a nucleotide sequence encoding the amino acid sequence substantially set forth in SEQ LD NO: 15.
• nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding the amino acid sequence substantially set forth in SEQ LD NO: 17 or having at least 40% similarity to a nucleotide sequence encoding the amino acid sequence substantially set forth in SEQ LD NO: 17.
• nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ LD NO:l or having at least 40% similarity thereto or is a nucleic acid molecule capable of hybridising to SEQ LD NO:l under low stringency conditions.
• nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ LD NO:3 or having at least 40% similarity thereto or is a nucleic acid molecule capable of hybridising to SEQ LD NO: 3 under low stringency conditions.
• nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ LD NO: 5 or having at least 40% similarity thereto or is a nucleic acid molecule capable of hybridising to SEQ LD NO: 5 under low stringency conditions.
• nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ LD NO: 7 or having at least 40% similarity thereto or is a nucleic acid molecule capable of hybridising to SEQ LD NO: 7 under low stringency conditions.
• nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ LD NO: 10 or having at least 40% similarity thereto or is a nucleic acid molecule capable of hybridising to SEQ LD NO: 10 under low stringency conditions.
• nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ LD NO: 12 or having at least 40% similarity thereto or is a nucleic acid molecule capable of hybridising to SEQ LD NO: 12 under low stringency conditions.
• nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ LD NO: 14 or having at least 40% similarity thereto or is a nucleic acid molecule capable of hybridising to SEQ LD NO: 14 under low stringency conditions.
• nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ LD NO: 16 or having at least 40% similarity thereto or is a nucleic acid molecule capable of hybridising to SEQ LD NO: 16 under low stringency conditions.
• Reference herein to a low stringency at 42 °C includes and encompasses from at least about 1% v/v to at least about 15% v/v formamide and from at least about IM to at least about 2M salt for hybridisation, and at least about IM to at least about 2M salt for washing conditions.
• Alternative stringency conditions may be applied where necessary, such as medium stringency, which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5M to at least about 0.9M salt for hybridisation, and at least about 0.5M to at least about 0.9M salt for washing conditions, or high stringency, which includes and encompasses from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.0 IM to at least about 0.15M salt for hybridisation, and at least about 0.0 IM to at least about 0.15M salt for washing conditions.
• medium stringency which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5M to at least about 0.9M salt for hybridisation, and at least about 0.5M to at least about 0.9M salt for washing conditions
• high stringency which includes and encompasses from at least about 31% v/v to at least about 50% v/
• the nucleic acid molecules are preferably in isolated form and/or carried by a vector molecule such as an expression vector.
• the LAP homologues and their derivatives and chemical analogues of the present invention are useful in inhibiting apoptosis and are useful in the treatment of diseases including but not limited to those characterised by apoptosis such as degenerative diseases including Alzheimer's disease, motor neuron disease, neuropathies; ischemic vascular disease including stroke and myocardial infarction; infectious diseases including Acquired Immuno-Deficiency Syndrome caused by HIV.
• Certain derivatives of the homologues contemplated herein may promote contemplated herein may promote apoptosis or inhibit anti-apoptotic processes and are potentially useful in the treatment of diseases characterised by failure of apoptosis of certain cells including but not limited to cancer or autoimmune disease, alzheimer's and motor neuron diseases.
• Derivatives of the homologues contemplated herein many promote apoptosis or inhibit anti-apoptotic processes and are useful in the treatment of, for example, certain cancers or in the promotion of the death of malignant cells.
• Derivatives of the homologues of the present invention include mutants, parts and fragments of the homologue molecules as well as single or multiple amino acid substitutions, deletions and/or additions to the homologue molecules.
• Nucleic acid derivatives including single or multiple nucleotide substitutions, deletions and/or additions to the nucleic acid molecules as defined in SEQ LD NO: 1 or SEQ LD NO:3 or SEQ LD NO:5 or SEQ LD NO:7 or SEQ LD NO: 10 or SEQ LD NO: 12 or SEQ LD NO: 14 or SEQ LD NO.16.
• Chemical analogues ofthe baculovirus IAPs or of their homologues include, but are not limited to, modifications to side chains, incorporation of unnatural amino acids and/or their derivatives during peptide synthesis and the use of crosslinkers and other methods which impose conformational constraints on the peptides or their analogues.
• side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with a aldehyde followed by reduction with BBH4; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; triitrobezylation of amino groups with 2, 4, 6, -trinitrobezene sulphonic acid (TBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5 '- phosphate followed by reduction with ⁇ BH ⁇
• the guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.
• the carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitisation, for example, to a corresponding amide.
• Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuribezoate, 4- chloromercuripheylsulphonic acid, phenylmercury chloride, 2-chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.
• Tryptophan residues may be modified by, for example, oxidation with -bromosuccinimide or alkylation of the idole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides.
• Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.
• Modification of the imidazole ring of a hisitidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.
• Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5- phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, norithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D- isomers of amino acids.
• a list of unnatural amino acids is provided in Table 1.
• Non-conventional Code Non-conventional Code amino acid amino acid
• peptides can be conformationally constrained by, for example, inco ⁇ oration of C ⁇ and N ⁇ -methylamine acids, introduction of double bonds between C ⁇ and C p atoms of amino acids and the formation of cyclic peptides or analogues by introducing covalent bonds such as forming a amide bond between the and C termini, between two side chains or between a side chain and the N or C terminus.
• the present invention further contemplates a method for modulating cell apoptosis in a animal, said method comprising administering to said animal a cell apoptosis modulating effective amount of a homologue or chemical analogue of a baculovirus IAP or a derivative thereof for a time and under conditions sufficient for said cell apoptosis to be modulated.
• modulating refers to the promotion or enhancement of cell apoptosis or the deminuation, inhibition or reduction of cell apoptosis. Enhancing cell apoptosis may be important in treating certain cancers and cell malignancies; reducing cell apoptosis may be important in the treatment of degenerative disorders such as neuropathies and alzheimer's and motor neuron diseases.
• the present invention is also directed to therapeutic and pharmaceutical compositions useful for modulating cell apoptosis in a animal.
• compositions are generally known in the art and reference can conveniently be made to Remington's Pharmaceutical Sciences, 17th end., Mack Publishing Co., Easton, Pennsylvania, USA.
• the present invention contemplates a pharmaceutical composition comprising an apoptopic modulating effective amount of an IAP homologue as hereinbefore defined or chemical analogues or derivatives thereof and optionally including one or more other active molecules and one or more pharmaceutically acceptable carriers and/or diluents.
• the active ingredients of a pharmaceutical composition comprising the IAP homologues or their derivatives are contemplated herein to exhibit excellent therapeutic activity, for example, in modulating apoptosis of animal cells when administered in an amount which depends on the particular case. For example, from about 0.5 ⁇ g to about 20 mg per kilogram of body weight per day may be administered. Dosage regime may be adjusted to provide the optimum therapeutic response.
• the active compounds may be administered in any convenient manner such as by the oral, intravenous (where water soluble), intramuscular, subcutaneous, intranasal, intradermal or suppository routes or by implanting (eg using slow release molecules), topical administration or following or during surgery or biopsy or other invasive procedure.
• the active ingredients which comprise the LAP homologues or chemical analogues or derivatives may be required to be coated in a material to protect said ingredients from the action of enzymes, acids and other natural conditions which may inactivate said ingredients.
• homologues may be administered in an adjuvant, co ⁇ administered with enzyme inhibitors or in liposomes.
• adjuvants contemplated by the present invention include, but are not limited to, cytokines (e.g. interferons) as well as resorcinols, non-ionic surfactants such as polyoxyethelene oleyl ether and n-hexadecyl polyethylene ether.
• the active compounds may also be administered parenterally or intraperitoneally.
• Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
• the pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
• the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
• the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
• the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of superfactants.
• the preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thirmerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.
• Sterile injectable solutions are prepared by inco ⁇ orating the active compounds in the required amount in the appropriate solvent with various ofthe other ingredients enumerated above, as required, followed by filtered sterilisation.
• dispersions are prepared by inco ⁇ orating the various sterilised active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
• the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.
• the active, compound may be orally administered, for example, with an inert diluent or with a assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be inco ⁇ orated directly with the food of the diet.
• the active compound may be inco ⁇ orated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
• Such compositions and preparations should contain at least 1 % by weight of active compound.
• compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit.
• the amount of active compound in such therapeutically useful compositions in such that a suitable dosage will be obtained.
• Preferred compositions or preparations according to the present invention are prepared so that a oral dosage unit form contains between about 0.1 ⁇ g and 2000 mg of active compound.
• the tablets, troches, pills, capsules and the like may also contain the following: A binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such a sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring.
• a binder such as gum tragacanth, acacia, corn starch or gelatin
• excipients such as dicalcium phosphate
• a disintegrating agent such as corn starch, potato starch, alginic acid and the like
• a lubricant such as magnesium stearate
• a sweetening agent such as sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint
• tablets, pills, or capsules may be coated with shellac, sugar or both.
• a syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour.
• any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
• the active compound may be inco ⁇ orated into sustained-release preparations and formulations.
• a pharmaceutically acceptable carrier and/or diluent includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and abso ⁇ tion delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
• Dosage unit form refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit coating a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
• the specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such a active material for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired as herein disclosed in detail.
• the principal active ingredient is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form as hereinbefore disclosed.
• a unit dosage form can, for example, contain the principal active compound in amounts ranging from 0.5 ⁇ g to about 2000 mg. Expressed in proportions, the active compound is generally present in from about 0.5 ⁇ g to about 2000 mg/ml of carrier.
• the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.
• the pharmaceutical composition may also comprise genetic molecules such as a vector capable of transfecting target cells where the vector carries a nucleic acid molecule capable of modulating IAP homologue expression or IAP homologue activity.
• the vector may, for example, be a viral vector.
• Another aspect ofthe present invention contemplates the use of an animal cell homologue of an baculovirus IAP to modulate apoptosis in animals suffering from a degenerative disease, an infectious disease, cancer or an autoimmune disease.
• the cell homologue is as defined by one of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:ll, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO:17 or is a derivative or chemical analogue thereof.
• the present invention further contemplates antibodies to the IAP homologues and the derivatives of the present invention.
• Antibodies are useful in diagnostic assays for the homologues as well as purifying the homologues or isolating the homologues from biological fluid or culture medium.
• Such antibodies may be monoclonal or polyclonal and may be selected from naturally occurring antibodies to an IAP homologue or may be specifically raised to an IAP homologue or a derivative thereof. In the case of the latter, an IAP homologue or its derivatives may first need to be associated with a carrier molecule.
• the antibodies and/or recombinant IAP homologue or its derivatives ofthe present invention are particularly useful as therapeutic or diagnostic agents.
• an LAP homologue and its derivatives can be used to screen for naturally occurring antibodies to the homologue. These may occur, for example in some autoimmune diseases.
• specific antibodies can be used to screen for an IAP homologue.
• Techniques for such assays are well known in the art and include, for example, sandwich assays and ELISA. Knowledge of IAP homologue levels may be important for diagnosis of certain cancers or a predisposition to cancers or for monitoring certain therapeutic protocols.
• Antibodies to an IAP homologue of the present invention may be monoclonal or polyclonal.
• fragments of antibodies may be used such as Fab fragments.
• the present invention extends to recombinant and synthetic antibodies and to antibody hybrids.
• a "synthetic antibody” is considered herein to include fragments and hybrids of antibodies.
• the antibodies of this aspect of the present invention are particularly useful for immunotherapy and may also be used as a diagnostic tool for assessing apoptosis or monitoring the program of a therapeutic regimen.
• LAP homologues For example, specific antibodies can be used to screen for an LAP homologues.
• the latter would be important, for example, as a means for screening for levels of an IAP homologue in a cell extract or other biological fluid or purifying LAP homologue made by recombinant means from culture supernatant fluid.
• Techniques for the assays contemplated herein are known in the art and include, for example, sandwich assays and ELISA.
• second antibodies (monoclonal, polyclonal or fragments of antibodies or synthetic antibodies) directed to the first mentioned antibodies discussed above. Both the first and second antibodies may be used in detecuon assays or a first antibody may be used with a commercially available anti-immunoglobulin antibody.
• An antibody as contemplated herein includes any antibody specific to any region of an LAP homologue. An antibody may also be directed to an amino acid sequence such as set forth in SEQ LD NO:9.
• Both polyclonal and monoclonal antibodies are obtainable by immunization with the enzyme or protein and either type is utilizable for immunoassays.
• the methods of obtaining both types of sera are well known in the art.
• Polyclonal sera are less preferred but are relatively easily prepared by injection of a suitable laboratory animal with an effective amount of LAP homologue, or antigenic parts thereof, collecting serum from the animal, and isolating specific sera by any ofthe known immunoadsorbent techniques.
• antibodies produced by this method are utilizable in virtually any type of immunoassay, they are generally less favoured because ofthe potential heterogeneity of the product.
• the use of monoclonal antibodies in an immunoassay is particularly preferred because ofthe ability to produce them in large quantities and the homogeneity of the product.
• the preparation of hybridoma cell lines for monoclonal antibody production derived by fusing an immortal cell line and lymphocytes sensitized against the immunogenic preparation can be done by techniques which are well known to those who are skilled in the art.
• Another aspect of the present invention contemplates a method for detecting an LAP homologue in a biological sample from a subject said method comprising contacting said biological sample with an antibody specific for the IAP homologue or its derivatives or homologues for a time and under conditions sufficient for an antibody-IAP homologue complex to form, and then detecting said complex.
• IAP homologue may be accomplished in a number of ways such as by Western blotting and ELISA procedures.
• a wide range of immunoassay techniques are available as can be seen by reference to US Patent Nos. 4,016,043, 4, 424,279 and 4,018,653. These, of course, includes both single-site and two-site or "sandwich" assays of the non- competitive types, as well as in the traditional competitive binding assays. These assays also include direct binding of a labelled antibody to a target.
• Sandwich assays are among the most useful and commonly used assays and are favoured for use in the present invention.
• an unlabelled antibody is immobilized on a solid substrate and the sample to be tested brought into contact with the bound molecule.
• a second antibody specific to the antigen is then added and incubated, allowing time sufficient for the formation of another complex of antibody-antigen-labelled antibody.
• the sample is one which might contain an LAP homologue including a cell extract, tissue biopsy or possibly serum, saliva, mucosal secretions, lymph, tissue fluid and respiratory fluid.
• the sample is, therefore, generally a biological sample comprising biological fluid but also extends to fermentation fluid and supernatant fluid such as from a cell culture.
• a first antibody having specificity for the IAP homologue or antigenic parts thereof is either covalently or passively bound to a solid surface.
• the solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
• the solid supports may be in the form of tubes, beads, discs of microplates, or any other surface suitable for conducting an immunoassay.
• the binding processes are well-known in the art and generally consist of cross-linking covalently binding or physically adsorbing, the polymer- antibody complex is washed in preparation for the test sample.
• an aliquot ofthe sample to be tested is then added to the solid phase complex and incubated for a period of time sufficient (e.g. 2-40 minutes) and under suitable conditions (e.g. 25 °C) to allow binding of any subunit present in the antibody.
• the antibody subunit solid phase is washed and dried and incubated with a second antibody specific for a portion of the hapten.
• the second antibody is linked to a reporter molecule which is used to indicate the binding of the second antibody to the hapten.
• An alternative method involves immobilizing the target molecules in the biological sample and then exposing the immobilized target to specific antibody which may or may not be labelled with a reporter molecule. Depending on the amount of target and the strength of the reporter molecule signal, a bound target may be detectable by direct labelling with the antibody. Alternatively, a second labelled antibody, specific to the first antibody is exposed to the target- first antibody complex to form a target-first antibody-second antibody tertiary complex. The complex is detected by the signal emitted by the reporter molecule.
• reporter molecule as used in the present specification, is meant a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of antigen-bound antibody. Detection may be either qualitative or quantitative.
• reporter molecules in this type of assay are either enzymes, fiuorophores or radionuclide containing molecules (i.e. radioisotopes) and chemiluminescent molecules.
• an enzyme immunoassay an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate.
• glutaraldehyde or periodate As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan.
• Commonly used enzymes include horseradish peroxidase, glucose oxidase, beta-galactosidase and alkaline phosphatase, amongst others.
• the substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, ofa detectable colour change.
• suitable enzymes include alkaline phosphatase and peroxidase.
• fluorogenic substrates which yield a fluorescent product rather than the chromogenic substrates noted above.
• the enzyme-labelled antibody is added to the first antibody hapten complex, allowed to bind, and then the excess reagent is washed away. A solution containing the appropriate substrate is then added to the complex of antibody-antigen-antibody.
• the substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication ofthe amount of hapten which was present in the sample.
• Reporter molecule also extends to use of cell agglutination or inhibition of agglutination such as red blood cells on latex beads, and the like.
• fluorescent compounds such as fluorescein and rhodamine
• fluorescein and rhodamine may be chemically coupled to antibodies without altering their binding capacity.
• the fluorochrome-labelled antibody When activated by illumination with light of a particular wavelength, the fluorochrome-labelled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic colour visually detectable with a light microscope.
• the fluorescent labelled antibody is allowed to bind to the first antibody-hapten complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to the light ofthe appropriate wavelength the fluorescence observed indicates the presence ofthe hapten of interest.
• Immunofluorescene and EIA techniques are both very well established in the art and are particularly preferred for the present method. However, other reporter molecules, such as radioisotope, chemiluminescent or bioluminescent molecules, may also be employed.
• the present invention also contemplates genetic assays such as involving PCR analysis to detect IAP homolgoue gene or its derivatives.
• Alternative methods or methods used in conjunction include direct nucleotide sequencing or mutation scanning such as single stranded conformation polymo ⁇ homs analysis (SSCP) as specific oligonucleotide hybridisation, as methods such as direct protein truncation tests.
• SSCP single stranded conformation polymo ⁇ homs analysis
• Figure 1 is a comparison of deduced peptide sequences of LAP proteins.
• CPI derives from Cydia pomonella granulosis virus
• OpIAP derives from Orgyia pseudotsugata PV
• CilAP derives from Chilo iridescent virus
• ALP is a candidate gene for SMA
• AcIAP derives from Autographa Califoraica PV
• ASV derives from IAP- like sequences from African swine fever virus.
• CRAF Comparison of RLNG finger motifs of IAPs and other proteins. Strongly conserved residues are highlighted.
• CRAF (TRAF3) and TRAF2 are TRAF family members; BCRAl and RAG1 are two mammalian proteins with RING finger motifs; c-CBL is a cellular oncogene and SLI-1 is its C.
• elgans homologue cpi derives from Cydia pomonella granulosis virus
• OpIAP derives from Orgyia pseudotsugata PV
• CilAP derives from Chilo iridescent virus
• AcIAP derives from Autographa californica PV
• UKMT derives from the Drosophila Unkempt gene
• EctV derives from the p28 protein of Ectromelia Virus.
• Figure 2 is a photographic representation showing mammalian LAP homologues are expressed in a variety of tissues.
• RNA Northern blot was probed with the /wMLHA cDNA coding region at high stringency. This Northern was also probed with GAPDH as a indicator of each lane's loading.
• B-D A adult mouse tissue poly (A)+ RNA Northern blot (CLONTECH) was probed with the ⁇ MLHB CDNA coding region (B) and the ⁇ MLHC CDNA coding region (C) at low stringency.
• Figure 3 is a graphical representation showing that MLHA and MLHB protect against death induced by overexpression of ICE, but not FADD.
• Figure 4 is a schematic representation showing a hypothetical and non-limiting cell death model showing the proposed site of action of IAP proteins. Direct interactions are indicated by solid lines, and indirect actions are show as dotted lines. Uncertain interactions are indicated by a question mark. Spontaneous self-association of molecules such as TRAFs and death-domain bearing proteins may generate a intrinsic activation signal that in normal circumstances is insufficient to activate enough cell death proteases to cause apoptosis. Increased amounts of ICE precursor due to transfection could allow activation of enough enzyme to induce apoptosis. IAP proteins may act to decrease the spontaneous activation signals to various extents depending on their affinity for their target(s).
• OpIAP may be able to offer greater protection against FADD than the mammalian LAP homologues because it has greater affinity for its target, but it is not as effective against FADD as it is against pro-ICE because it is required to block a larger intrinsic activation signal in FADD transfected cells.
• CrmA and p35 which directly inhibit active ICE, are effective inhibitors of ICE mediated apoptosis on matter how ICE is activated.
• Figure 5 is a representation of the nucleotide sequence and corresponding amino acid sequence of MLHA.
• Figure 6 is a representation of the nucleotide sequence and correspondmg amino acid sequence of MLHB.
• Figure 7 is a representation of the nucleotide sequence and corresponding amino acid sequence of MLHC.
• Figure 8 is a representation of the nucleotide sequence and corresponding amino acid sequence of DLHA.
• Figure 9A and 9B show protection by MLHA, MLHB and MLHC against death induced by over expression of ICH-1 protease.
• Figure 10 is a photographic representation of a Western blot of 293T cells transfected with MLHA constructs. The following single and three letter abbreviations are used for amino acid residues:
• a fragment isolated from this library was used to probe a mouse liver cDNA library (Stratagene) at low stringency yielding three murine cDNA clones that were designated mammalian LAP homologueA (MLHA).
• MLHA mammalian LAP homologueA
• Translations ofthe human EST sequences GenBank:R19628 and GenBank: T96284 were found to resemble the BLR repeats of OpIAP. These sequences were used to design PCR primers within their putative BLR domains which were used to generate probes that were used to scree a human fetal liver cDNA library (Stratagene).
• the hybridising cDNA clones were designated MLHB and MLHC, respectively.
• the Drosophila genomic sequence (GenBank:DROCCAAT) was used to design primers to amplify a 900 bp product from Drosophila CDNA. This fragment was subcloned and used to screen an oligo(dT) - primed Drosophila larval cDNA library constructed in lambda ZAP (Stratagene). A 2kb cDNA (DLHA) clone encoding all but the 8 -terminal amino acids was isolated. For peptide sequence comparisons (Figure 1) these residues deduced from the genomic sequence GenBank:DROCCAAT were added.
• RNA ANALYSIS Radiolabelled mMLHA and GAPDH were hybridised at high stringency to a mouse tissue Northern blot bearing 5 ⁇ g/lane of total RNA.
• MLHA MLHB and MLHC were amplified by PCR using primers incorporating an EcoRI site at the 5 ' end and a BamHl site at the 3 ' end (or a Bglll site in the case of MLHB), such that the proteins would be expressed as in frame fusions with the GAL4 DNA binding domain.
• the PCR products were the ligated into the pGBT9 vector (CLONTECH).
• the OpIAP gene from the HindTTl site 17 codons upstream of the initiating ATG was cloned into the pGBT9 vector such that a in frame fusion would result.
• PCR- derived inserts were sequenced to check for misinco ⁇ orations by Taq polymerase.
• TRAFl, TRAF2 and TRAF3 expression vectors have been previously described (Rothe et al, 1994; Hu et al, 1994b). Vectors with the coding regions of c-jun in GBT9 and fos were used as controls for the detection of interacting proteins.
• the yeast strain HF7c was transformed with these plasmids using the lithium acetate protocol (Gietz et ⁇ l, 1992).
• the p32-/ ⁇ cZ fusion plasmid p ⁇ actMl 1Z was as described by Miura et ⁇ l (1993).
• the FADD expression construct FADD-AUl was as described by Chinnaiyan et ⁇ l (1995).
• the coding regions of bcl-2, crmA, p35 from AcPV, and IAP from OpPV were inserted into the pEF vector.
• the truncated OpIAP plasmid was constructed by digestion of the pEF vector coating full length IAP with ru ⁇ and Sm ⁇ l, and re-ligating. This deleted sequences 3' of the nil site in the OpIAP gene which encode the RLNG finger domain.
• GenBank CLONING OF DLHA, MLHA MLHB and MLHC Searches of GenBank revealed a Drosophila genomic sequence (GenBank:DROCCAAT) that resembled baculoviral IAP genes.
• a Drosophila cDNA library was screened using PCR generated probes and clones isolated which encodes this protein, designated herein Drosophila IAP homologue A (DLHA).
• the GenBank searches also revealed a number of mammalian sequences that resembled either the BLRs or the RING finger domains of viral IAPs.
• the first mammalian IAP homologue identified was cloned using a PCR generated probe corresponding to a STS sequence on the X chromosome, HUMSWX595, which when translated encoded a peptide resembUng a baculoviral IAP RING finger motif.
• the inventors isolated partial length human genomic clones and mouse MLHA cDNA clones compassing the entire coding region.
• a further pair of closely related human LAP homologues (MLHB and MLHC) were isolated using PCR generated probes corresponding to GenBank EST sequences R19628 and T96284, which encode BLR-like motifs.
• Figure IA compares the predicted amino acid sequences for DLHA (predicted molecular weight 55 kD), MLHA (56 kD), MLHB (70 kD) and MLHC (68 kD). Start codons were chose as the most 5' methionine with upstream, in frame stop codons. All four proteins bear three BLR repeats in the amino terminal half and a single RING finger domain close to the carboxy terminus. MLHB and MLHC are the most closely related, with 73% amino acid identity. MLHA shares 43% identity and 62% similarity with MLHB and MLHC. DLHA has 36% identity and 57% similarity to MLHC.
• the BLR repeats of these proteins are compared to each other, the BLR repeats of ALP and the viral IAPs in Figure IB.
• some of the BLRs resemble the analogous BLR from another IAP more closely than they resemble the remaining BLRs in the same protein, indicating that duplication of a primordial BLR was a early event.
• Four ofthe most highly conserved residues in the BLRs are three cysteines and a histidine in the pattern CX 2 CX, 6 HX 6 C. These may bind metal ions (Birnbaum et al, 1994).
• Figure IC shows a comparison ofthe C3HC4 RLNG finger domains ofthe IAP molecules with RING fingers from other selected proteins.
• the RING fingers from IAP proteins resemble each other more than they resemble the RLNG fingers from other proteins, such as TRAF2 (Rothe et /, 1994) and CRAF (Hu et ⁇ /, 1994b; Cheng et al, 1995; Sato et al, 1995), that do not bear BLRs.
• TRAF2 Rothe et /, 1994
• CRAF Hu et ⁇ /, 1994b; Cheng et al, 1995; Sato et al, 1995
• the most similar RING fingers in o-BLR bearing proteins are in the mammalian oncogene, c-Cbl (Blake et al, 1991) and its C. elgans homologue, Sli-1 (Yoon et al, 1995).
• the RING finger bearing protein p28 of Ectromelia virus is not required for growth, but is essential for virulence (Senkevich et al, 1994). However, p28 does not have BLRs and its function is unknown.
• the message for murine MLHA is about 7.5 kb, and is expressed in most mouse tissues with the except of skeletal and cardiac muscle.
• the highest level detected was in the lung, with intermediate levels detected in the brain and kidney, and lower levels in the thymus, liver, bone marrow, skin and testes (Figure 2A).
• a additional transcript of -9.5 kb was detected in the testes but not see in other tissues.
• a full length human MLHC cDNA probe hybridised at low stringency to two messages of ⁇ 3.0 and -4.0 kb (Figure 2C). on 4.0 kb message was detectable in the spleen, high levels were detected in the testes and intermediate levels were detected in all other tissues. The -3.0 kb transcript could be see in the lung and skeletal muscle and very faintly in the spleen, liver, kidney and testes. As the AMLHB and ⁇ MLHC probes were hybridised at low stringency to mouse RNA, it is possible the upper (4.0 kb) transcript is the same as that detected by the MIHB probe. Some of the transcripts may also represent other closely related mammalian IAP homologues.
• HeLa cells were cotransfected with a plasmid bearing a ICE-lacZ fusion construct together with plasmids encoding the IAP homologues or controls. The cells were stained with X-gal to identify those that hand been transfected, and these were assessed visually for viability.
• MLHA, MLHB and OpIAP significantly reduced the amount of death caused by ICE, whereas MLHC did not provide detectable protection.
• Enforced expression ofthe Cd95 associated protein FADD also causes cell death (Chinnaiyan et al, 1995; Boldin et al, 1995). HeLa cells were cotransfected with three plasmids: a FADD expression construct, a plasmid carrying the lacZ gene and vectors encoding the mammalian LAP homologues or OpIAP.
• OpIAP provided partial protection against FADD, but it was not as effective as it was against ICE (compare Figure 3 A, lane 2 with Figure 3B, lane 2). The inventors could not detect any reduction in the amount of FADD induced cell death by MLHA, MLHB or MLHC.
• TRAF FAMILY MEMBERS Two ofthe mammalian LAP homologues, MLHB and MLHC, were isolated as part of a protein complex that binds to the cytoplasmic domain ofthe TNF-R2 (p75) together with the TNF-R2 associated molecules TRAFl and TRAF2 (Rothe et al, 1994).
• BLR baculovirus LAP repeats
• Xaa is an amino acid residue
• [Xaa] m is a series of at least 5 and preferably at least 9 amino acids; [Xaa] n is Met or Leu;
• [Xaa] 0 is a series of at least 3 and preferably at least 5 amino acids
• [Xaa] p is Val or Ala
• [Xaa]q is Phe or Tyr
• [Xaa] is Leu or Val; or a homologue, chemical analogue or derivative thereof.
• Figures 9A and B shows the protection from MLHA, MLHB and MLHC against death induced by over expression ofthe ICH-1 protease.
• a polyclonal rabbit antibody was prepared and affinity purified against the MLHA peptide KDLKKTMEEKIQTSG (SEQ LD NO: 18). This antibody was used in a western blot of 293T cells transfected inter alia MLHA. The results are shown in Figure 10.
• the yeast strain HF7c was cotransformed with constructs wliich express fusion proteins between the GAL4 DNA-binding domain and the LAP family members or controls, and vectors which encode fusions between the TRAF proteins or controls and the GAM activation domain. Expression from the his and lacZ reporter genes (which indicates interactions) was analysed by growth of double transformants on medium lacking histidine (Column 3), and blue staining of colonies, with X-gal (Column 4), c-jun and fos were used as control genes encoding interacting proteins in the DNA binding vector and activation vector, respectively.
• CAG TCT ACA AAT CCT GGT ATC CAA AAT GGC CAG TAC AAA TCT GAA AAC 568 Gin Ser Thr Asn Pro Gly Ile Gin Asn Gly Gin Tyr Lys Ser Glu Asn 105 110 115
• GAAGAAAGTC AACATGATGC TGCAGGAAAT GGAAACAAAT ACAAATGATA TTTAACAAAG 420
• GGT CCC TCG TAT CAA AAC ATT AAG AGT ATA ATG GAA GAT AGC ACG ATC 1240 Gly Pro Ser Tyr Gin Asn Ile Lys Ser Ile Met Glu Asp Ser Thr Ile 15 20 25
• CTTAATACAC ATCACTCTTC TGTGAAGGGT TTTAATTTTC AACACAGCTT ACTCTGTAGC 480
• AGCCATGCAC AAAACTACCT CCCTAGAGAA AGGCTAGTCC CTTTTCTTCC CCATTCATTT 720
• AGC GCC AAC ACG TTT GAA CTG AAA TAC GAC TTG TCA TGT GAA CTG TAC 817 Ser Ala Asn Thr Phe Glu Leu Lys Tyr Asp Leu Ser Cys Glu Leu Tyr 20 25 30
• GTC GCC AAT GGT TTC TTT GCC ACG GGA AAC TGG CTG GAG GCC GAG TGC 143 Val Ala Asn Gly Phe Phe Ala Thr Gly Asn Trp Leu Glu Ala Glu Cys 35 40 45
• CTGTAAACTT TTGCCTCCCT CCCCGACCTG TTTAACATCC GTAATTCATT CCAAAAATGT 1004
• Trp Leu Ile Asp Glu Leu Glu Lys Glu Asn Arg lie Asp Glu Ile Ile 275 280 285
• AGC AAT ATA ATG AGG CAA CAT CTG ACT GTT ACT GAT AAC AAA CAA GTT 336 Ser Asn Ile Met Arg Gin His Leu Thr Val Thr Asp Asn Lys Gin Val 100 105 110

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