WO1998058541A1 - Modulation de l'apoptose - Google Patents

Modulation de l'apoptose Download PDF

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Publication number
WO1998058541A1
WO1998058541A1 PCT/US1998/012595 US9812595W WO9858541A1 WO 1998058541 A1 WO1998058541 A1 WO 1998058541A1 US 9812595 W US9812595 W US 9812595W WO 9858541 A1 WO9858541 A1 WO 9858541A1
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Prior art keywords
bcl
polypeptide
peptide
cytochrome
seq
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PCT/US1998/012595
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English (en)
Inventor
Donald W. Kufe
Surender M. Kharbanda
Carlo M. Nalin
Sushil K. Sharma
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Dana-Farber Cancer Institute, Inc.
Novartis Ag
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Publication of WO1998058541A1 publication Critical patent/WO1998058541A1/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
    • C07K14/46—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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/795—Porphyrin- or corrin-ring-containing peptides

Definitions

  • the invention relates to compounds and methods useful in regulating apoptosis.
  • apoptosis An active mechanism of programmed cell death known as apoptosis has been described, whereby large numbers of cells die during normal development and tissue homeostasis [Kerr et al . (1972) Br. J Cancer ⁇ . :239] .
  • the cascade of intrinsic biochemical reactions resulting in the eventual death of the relevant cell may be initiated by external or internal stimuli.
  • apoptosis include cell membrane "blebbing", cell shrinkage, nuclear condensation and degradation of DNA at nucleosomal intervals [ yllie et al. (1980) Int. Rev. Cytol . 68:2511.
  • IR ionizing radiation
  • other genotoxic agents include cell cycle arrest and activation of DNA repair.
  • cells respond with induction of apoptosis.
  • intracellular signals that control the induction of apoptosis are unclear.
  • p53 has been implicated in promoting apoptosis induced by IR exposure [Lowe et al . (1993) Nature 3_62:847; Clarke et al . (1993) Nature 362 : 849]
  • Bcl-2 and Bcl-x L proteins inhibit IR-induced apoptosis [Sentman et al . (1991) Cell 67 . : 879; Strasser et al . (1991) Cell 6_7:889; Datta et al . (1995) Cell Growth and
  • Cytochrome C also induces DNA fragmentation in isolated nuclei incubated with cytosolic lysates [Liu et al . cited supra] .
  • the finding that intact cells undergo apoptosis following release of cytochrome C into the cytosol has provided further support for an apoptotic function of this mitochondrial protein [Liu et al . cited supra] .
  • the invention is based on the discovery that Bcl- x L blocks release of cytochrome C into cellular cytosol by binding to cytochrome C, thereby inhibiting apoptosis.
  • Cytochrome C has been found to bind directly to the anti- apoptotic, full-length Bcl-x L , and not the pro-apoptotic alteratively spliced form of Bcl-x (designated Bcl-x s ) .
  • certain peptide fragments of the region of Bclx L that is deleted in Bcl-x s block this binding.
  • the invention features a peptide or peptidomimetic that inhibits binding of cytochrome C to an anti- apoptotic member of the Bcl-2-related family.
  • the anti- apoptotic member of the Bel -2 -related family is preferably Bcl-x L or Bcl-2.
  • the peptide may be a fragment of a Bel -2 -related family member and is preferably three to seventy amino acids long, is more preferably a fragment of Bcl-x L or Bel -2, even more preferably contains the amino acid sequence Ala-Leu-Cys-Val-Glu (SEQ ID NO: 3) or Val-Met-Cys-Val-Glu (SEQ ID N0:4) , and most preferably includes the amino acid sequence of SEQ ID NO:l or SEQ ID NO: 2. Also included in the invention is a peptide that is five to seventy amino acids long and contains the amino acid sequence of SEQ ID NO : 3 or SEQ ID NO:4.
  • the invention encompasses methods for screening for compounds that promote or inhibit apoptosis.
  • the screening methods involve: (a) providing cytochrome C (or a derivative of cytochrome C such as apo-cytochrome C or porphyrin-cytochrome C) bound to a solid support; (b) contacting the cytochrome C (or a derivative of cytochrome C such as apo-cytochrome C or porphyrin-cytochrome C) with a polypeptide comprising the sequence of SEQ ID NO: 3 or SEQ ID NO: 4 or a peptide that inhibits binding of cytochrome C to an anti-apoptotic member of the Bel -2 -related family, in the presence of a test compound; and (c) determining whether the test compound inhibits or enhances binding of the polypeptide or peptide to cytochrome C (or a derivative of cytochrome C such as apo-cytochrome C or porphyrin-cytochrome C) .
  • the screening methods involve: (a) providing a polypeptide comprising the sequence of SEQ ID NO: 3 or SEQ ID NO : 4 or a peptide that inhibits binding of cytochrome C to an anti-apoptotic member of the Bcl-2- related family, the polypeptide or peptide being bound to a solid support; (b) contacting the polypeptide or peptide with cytochrome C (or a derivative of cytochrome C such as apo-cytochrome C or porphyrin-cytochrome C) in the presence of a test compound; and (c) determining whether the test compound inhibits or enhances binding of cytochrome C (or a derivative of cytochrome C such as apo-cytochrome C or porphyrin-cytochrome C) to the polypeptide or peptide.
  • the polypeptide containing the sequence of SEQ ID NO : 3 or SEQ ID NO: 4 can be (a) Bcl-x L ; (b) a truncated Bcl-x L lacking twenty one amino acid residues of the carboxy-terminus of naturally occurring Bcl-x L ; (c) Bel -2; (d) a truncated Bel -2 lacking twenty one amino acid residues of the carboxy terminus of naturally occurring Bel-2; or (e) a fusion protein containing any one of (a) - (d) . Truncation at the carboxy terminus increases solubility of these proteins .
  • Another polypeptide comprising the sequence of SEQ ID NO: 3 or SEQ ID NO : 4 and which may also be used in both screening methods is a peptide that is five to seventy amino acids long and is preferably a fragment of Bcl-x L or Bel-2.
  • Another embodiment of the invention is an antibody which binds specifically to an epitope within the amino acid sequence of SEQ ID NO : 3 or SEQ ID NO: 4.
  • the antibody is preferably a monoclonal antibody.
  • the invention features an expression vector comprising a polynucleotide that encodes a peptide that inhibits binding of cytochrome C to an anti-apoptotic member of the Bcl-2-related family. Also within the invention is a method for inhibiting binding of cytochrome C to Bcl-x L .
  • the method includes the steps of introducing into a cell a peptide or a peptidomimetic that inhibits binding of cytochrome C to an anti- apoptotic member of the Bel -2 -related family.
  • the cell is preferably in a mammal such as a human, non-human primate, mouse, rat, rabbit, guinea pig, hamster, cow, horse, sheep, goat, dog or cat.
  • the peptide or its peptidomimetic may be introduced into the cell of the mammal by directly administering the peptide or peptidomimetic to the mammal.
  • the peptide may alternatively be introduced to the cell of the mammal by administering an expression vector containing a polynucleotide that encodes the peptide.
  • the mammal is one having a condition wherein apoptosis in at least one cell type is desirable for the health and homeostasis of the individual, for example, an individual having an autoimmune disease or cancer, or a transplant recipient. Where the patient has cancer, the method may be carried out in conjunction with chemotherapy, radiation therapy or immunotherapy.
  • the invention includes a nucleic acid that encodes a polypeptide containing multiple peptide domains, each of which includes the amino acid sequence of SEQ ID NO: 3.
  • a method for inhibiting release of cytochrome C into cell cytosol is a also a component of the invention.
  • the method may involve introducing into appropriate cells of a patient (e.g., a human suffering from an immunodeficiency disease) the nucleic acid that encodes a polypeptide comprising multiple peptide domains, each of which contains the amino acid sequence of SEQ ID NO : 3.
  • the nucleic acid can be introduced into the patient's cells by administering the nucleic acid to the patient.
  • Bcl-2-related family is meant the group of homologous polypeptides that includes Bel -2, Bcl-x L , Bcl- w, Bfl-1, Brag-1, Mcl-1, Al , Bax, Bak, Bcl-x s , Bad, Bid, Bik and Hrk [Kroemer (1997) Nature Medicine 2:614].
  • Bcl-x L is meant the full-length, mature Bcl- x L polypeptide [Boise et al . (1993) Cell 74 :597] .
  • Bcl-x s is meant the full-length, mature Bcl- x s polypeptide [Boise et al . , cited supra] .
  • Bel -2 is meant the full length, mature Bel -2 polypeptide [Cleary et al . (1986) Cell 47 . : 19].
  • peptide is meant a polypeptide of at least 2 amino acids and not more than one hundred amino acids in length.
  • encodes a given peptide is meant that the coding sequence consists solely of codons defining the amino acid sequence of the given peptide.
  • Fig. 1A is a line graph illustrating the binding of Gst-Bcl-x LT to cytochrome C in an ELISA.
  • Fig. IB is a bar graph illustrating the inhibition of binding of GST-Bcl-x LT to cytochrome C by GST-Bcl-x s ⁇ in an ELISA.
  • Fig. 2 is a diagram depicting linear representations of Bcl-x L and Bcl-x s polypeptides . The region of the Bcl-x L molecule not present in the Bcl-x s molecule, designated ⁇ X L/S , is also shown. Below the ⁇ X L/S structure is illustrated the panel of decamer peptides that were synthesized and are described in greater detail in Fig. 3.
  • Fig. 3 is a diagram depicting the ⁇ X L/S region which spans amino acids 126 to 189 of the Bcl-x L molecule.
  • the diagram also illustrates a panel of twelve overlapping decamer peptides which together span the whole ⁇ X L/S region.
  • the peptides are appropriately aligned relative to the ⁇ X L/S region and the numbers assigned to each peptide are shown. All the peptides except peptide (12) contain a 5 amino acid overlap with the peptide immediately preceding it (see also Table 1) .
  • Peptide (12) contains seven amino acids of peptide (11) .
  • the amino acid sequences of peptide (7) (SEQ ID N0:1) and peptide (8) (SEQ ID NO: 2) are given, as is the amino acid sequence of their region of overlap (SEQ ID NO: 3) .
  • Fig. 4 is a line graph illustrating the inhibition of binding of Bcl-x L to cytochrome C by ⁇ X L/S peptides (7) (SEQ ID N0:1) and (8) (SEQ ID N0:2) in an ELISA.
  • cytochrome C When cells are exposed to apoptosis-inducing agents, cytochrome C is released from mitochondria into the cell cytosol. This released cytochrome C initiates the cascade of biochemical reactions that culminate in apoptosis of the cell. It has now been discovered that Bcl-x L inhibits both cytochrome C release into the cytosol and the sequelae that lead to apoptosis of the cell. Bcl- x L inhibits cytochrome C release, and hence also apoptosis, by binding to it and thereby preventing it from exiting mitochondria and entering the cytosol. Also of great interest is the observation that certain peptide fragments of Bcl-x L inhibit binding of Bcl-x L to cytochrome C.
  • U-937 cells overexpressing a gene that encodes Bcl-x L U-937/Bcl-x L
  • accumulation of cytosolic cytochrome C in response to IR or CDDP treatment was prevented.
  • treatment of U-937/Bcl-x L cells with IR or CDDP did not result in PARP cleavage.
  • U-937 cells stably overexpressing the cysteine protease inhibitor p35 were irradiated and their cytosolic proteins analyzed.
  • Lysates from control unirradiated and irradiated U-937/Bcl-x L cells were subjected to immunoprecipitation with antibody specific for cytochrome C, and the resulting immunoprecipitates were analyzed with antibody specific for Bcl-x (i.e., both Bcl-x L and Bcl-x s ) .
  • the data showed that the cytochrome C-specific antibody co-precipitated Bcl-x L but not Bcl-x s , whether or not the cells had been irradiated. This finding was the first to demonstrate a physical association between a Bcl-2- related family member and cytochrome C .
  • a reco binant truncated fusion protein consisting of Bcl-x L lacking 21 carboxy-terminal acids of naturally occurring Bcl-x L (designated Bcl-x LT ) and fused to hexahistidine (H6) was resolved by gel electrophoresis, transferred to a nitrocellulose filter and renatured in aqueous buffer.
  • Consecutive exposure to cytochrome C and cytochrome C-specific antibody demonstrated a direct interaction of cytochrome C and Bcl-x L , but no such interaction between cytochrome C and Bcl-x s ⁇ (recombinant , truncated Bcl-x s lacking 21 carboxy- terminal amino acids of natural Bcl-x s ) or a control protein, MAP kinase (MAPK) .
  • MAPK MAP kinase
  • cytochrome C was resolved by electrophoresis and transferred to nitrocellulose filters, which were incubated with purified H6 , H6-Bcl-x LT or Bcl-x s ⁇ and analyzed by immunoblotting with anti-Bcl-x antibody. Only the filters exposed to H6-Bcl-x LT showed reactivity with cytochrome C, thereby again demonstrating a specific interaction between cytochrome C and Bcl-x L .
  • This model was supported by an ELISA in which cytochrome C adsorbed to wells of a plastic ELISA plate was incubated with glutathione-S-transferase (GST) (as a control) , or a fusion protein of GST and either Bcl-x LT (GST-Bcl-x LT ) or Bcl-x s ⁇ (GST-Bcl-x s ⁇ ) . Unbound proteins were removed by washing. Rabbit antibody specific for GST, enzyme- conjugated anti-rabbit Ig (immunoglobulin) and a fluorogenic substrate for the enzyme were consecutively added to the wells of the ELISA plate. (Intermediate washing steps were included.) The results showed that GST-Bcl-x LT , but not GST or GST-Bcl-X s ⁇ , bound to the adsorbed cytochrome C.
  • Bcl-x s itself failed to bind to cytochrome C, an ELISA indicated that it inhibited binding of Bcl-x L to cytochrome C.
  • These ELISA experiments confirmed that the binding between cytochrome C and Bcl-x L shown by co- immunoprecipitation of cellular lysates is due to a specific interaction which can be blocked by Bcl-x s .
  • Bcl- x s is known to form a heteroduplex with Bcl-x L [Sato et al. (1994) Proc. Natl . Acad. Sci .
  • Bcl-x L binds to cytochrome C and Bcl-x s does not bind to cytochrome C.
  • Bcl-x s lacks an internal 63 amino acid domain ( ⁇ X L/S ) of Bcl-x L [Boise et al . , cited supra] . Therefore, the possibility that residues within ⁇ X LS are involved in binding of Bcl-x L to cytochrome C was tested.
  • a panel of twelve overlapping decamer peptides spanning ⁇ X L/S were synthesized for use in bindng inhibition experiments.
  • peptides were tested for their ability to inhibit binding of Bcl-x L to cytochrome C in an ELISA similar to that described for inhibition of binding to chromosome C of Bcl-x L by Bcl-x s (see supra) . Inhibition was measured at a range of peptide concentrations (1-100 ⁇ M) .
  • Peptide (7) (SEQ ID N0:1) and peptide (8) (SEQ ID NO:2) decreased binding of Bcl-x L to cytochrome C to a level approximately 20% of the control without peptide (i.e. 80% inhibition) .
  • the carboxy-terminus of peptide (8) (Phe-Ser-Phe-Gly-Gly- Ala-Leu-Cys-Val-Glu) (SEQ ID N0:2) overlaps the amino- terminus of peptide (7) (Ala-Leu-Cys-Val-Glu-Ser-Val-Asp- Lys-Glu) (SEQ ID NO:l) by 5 amino acids.
  • the amino acid sequence of this region of overlap is Ala-Leu-Cys-Val-Glu (SEQ ID NO: 3) .
  • a pentameric peptide with this sequence inhibited binding of Bcl-x L to cytochrome C almost as well (approximately 3 -fold less avidly) as peptide (7) (SEQ ID N0:1) or peptide (8) (SEQ ID N0:8).
  • Peptides containing the amino acid sequences of either or both of SEQ ID NO : 3 and SEQ ID NO : 4 may be used in the methods of invention. As described infra, the peptides and nucleic acids encoding them can be used for therapy of a variety of disease conditions, including human diseases. The peptides can also be used to generate and screen for antibodies specific for epitopes within the amino acid sequence of SEQ ID NOS:l, 2, 3 or 4. The resulting antibodies could be used in screening assays analogous to those described infra.
  • Such antibodies are another aspect of the invention. While the results of the binding inhibition experiments indicate that peptides (7) (SEQ ID N0:1) and (8) (SEQ ID NO:2) define a dominant site on Bcl-x L for binding of cytochrome C, they also suggest that this site is not the only site involved in binding. The latter possibility is shown by the fact that peptides other than (7) (SEQ ID NO:l) and (8) (SEQ ID NO : 2 ) showed less but still significant ability to inhibit binding of Bcl-x L to cytochrome C.
  • Peptides of the invention can be chemically synthesized by techniques familiar to those skilled in the art [e.g. see Creighton (1983) Proteins: Structures and Molecular Principles, W.H. Freeman and Co., N.Y.]. Larger peptides (longer than 50 amino acids) can be produced by a number of methods including recombinant DNA technology (see infra) .
  • a peptide of the invention is a polypeptide of 2 to 100 residues that inhibits binding of cytochrome C to an anti-apoptotic member of the Bcl-2 -related family, such as Bcl-x L , Bcl-2, Bcl-w, Bfl-1, Brag-1, Mcl-1 or Al .
  • the peptide may be a fragment of a Bel -2 -related family member.
  • the peptide is no more than 70 amino acids long and more preferably no more than 50 (e.g., less than 40 or even 30) . Examples of such peptides would be three, four, five, six, seven, eight, nine, ten, twelve, fifteen, twenty or twenty-five amino acids in length.
  • peptides may be fragments of Bcl-x L or Bcl-2, and can contain the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4. Examples include peptides with the amino acid sequence of SEQ ID NO : 1 or SEQ ID NO: 2 or peptides containing such a sequence.
  • Another embodiment of the invention is a peptide three to seventy amino acids long containing at least three (e.g., Cys-Val-Glu) and preferably four residues of SEQ ID NO: 3 or SEQ ID NO : 4.
  • Peptides may also include variants of the peptides described supra . These variants would contain different amino acids (preferably conservative changes) from the parental peptides but retain the ability to inhibit binding of cytochrome C to Bel -2 -related family members. Such variants can be synthesized by standard means, and are readily tested in the binding assays described above and below.
  • Peptides of the invention will also include those described above but modified for in vivo use by:
  • blocking agent in order to facilitate survival of the relevant peptide in vivo .
  • This can be useful in those situations in which the peptide termini tend to be degraded ("nibbled") by proteases prior to cellular or mitochondrial uptake.
  • blocking agents can include, without limitation, additional related or unrelated peptide sequences that can be attached to the amino and/or carboxy terminal residues of the peptide to be administered. This can be done either chemically during the synthesis of the peptide or by recombinant DNA technology (see Section C.l infra) .
  • blocking agents such as pyroglutamic acid or other molecules known to those of average skill in the art may be attached to the amino and/or carboxy terminal residues, or the amino group at the amino terminus or carboxyl group at the carboxy terminus replaced with a different moiety.
  • the peptides can be covalently or noncovalently coupled to pharmaceutically acceptable "carrier” proteins prior to administration.
  • peptidomimetic compounds based upon the amino acid sequence of the peptides of the invention.
  • Peptidomimetic compounds are synthetic compounds having a three-dimensional structure (i.e. a "peptide motif") based upon the three-dimensional structure of a selected peptide.
  • the peptide motif provides the peptidomimetic compound with the activity of inhibiting or enhancing the binding of cytochrome C to a member of the Bel -2 -related family that is the same or greater than the activity of the peptide from which the peptidomimetic was derived.
  • Peptidomimetic compounds can have additional characteristics that enhance their therapeutic application such as increased cell permeability, greater affinity and/or avidity and prolonged biological half-life.
  • the peptidomimetics of the invention typically have a backbone that is partially or completely non-peptide, but with side groups identical to the side groups of the amino acid residues that occur in the peptide on which the peptidomimetic is based.
  • Several types of chemical bonds e.g.
  • the truncated polypeptides described in (b) supra typically lack 21 carboxy-terminal amino acids. It is understood that they may lack no more than fifty, preferably forty and more preferably thirty (e.g. twenty seven or twenty three) carboxy-terminal amino acids. Methods to test for the utility of these truncated polypeptides in the screening assays of the invention are described infra .
  • test compound may be a defined, isolated and purified candidate compound (e.g., a synthetic small molecule) or may be present in a biological sample such as a biological fluid, tissue extract, subcellular fraction or cellular lysate .
  • cytochrome C is provided bound to a solid support.
  • solid supports are given infra.
  • Unbound cytochrome C is removed from the solid support (e.g., by washing) and the bound cytochrome C is contacted with detectably labeled "Polypeptide B" in the presence of the test compound.
  • detectably labeling polypeptides are described infra. After co- incubation, unbound substances are removed and any "Polypeptide B" that remains bound is detected by any number of techniques well-known in the art, examples of which are described infra. Instead of using detectably labeled "Polypeptide B", an unlabeled "Polypeptide B" can be used.
  • Bound "Polypeptide B” can then be detected by addition of a detectably labeled antibody that binds specifically to "Polypeptide B” . After incubation, unbound antibody is removed and any antibody that remains bound is detected by any one of the methods described infra.
  • the detectably labeled antibodies include those that react specifically with Bcl-x L , Bcl-2, any component of a fusion protein containing Bcl-x L or Bcl-2, or an epitope within the amino acid sequence of SEQ ID NO : 3 or SEQ ID:NO: 4.
  • an unlabeled antibody can be used instead of using a detectably labeled antibody that specifically binds to "Polypeptide B". After incubation, unbound antibody is removed and a detectably labeled "secondary" antibody that specifically recognizes the first antibody is added. After incubation and removal of unbound detectably labeled secondary antibody, the presence of the bound detectably labeled secondary antibody is detected by one of the methods described infra.
  • Polypeptide B may be conjugated to biotin by methods known to those of average skill in the art.
  • bound biotinylated “Polypeptide B” is contacted with detectably labeled avidin and avidin which remains bound to the biotin and thus to the solid support after washing is detected by one of the methods described infra.
  • antibody that binds specifically to "Polypeptide B” may be conjugated to biotin.
  • bound biotinylated antibody is contacted with detectably labeled avidin and avidin which remains bound to the biotin and thus to the solid support is detected by one of the methods described infra.
  • inhibition of binding of "Polypeptide B" to cytochrome C by a test compound is an indication that the test compound will permit release of cytochrome C from the mitochondria into the cytosol, and thus act as a promoter of apoptosis.
  • enhancement of binding of "Polypeptide B" to cytochrome C by a test compound is an indication that the test compound is an inhibitor of apoptosis.
  • Polypeptide B is provided fixed to the solid support. This "Polypeptide B” is contacted with soluble cytochrome C in the presence of a test compound. Cytochrome C bound to "Polypeptide B” is detected, for example, by either: (a) the cytochrome C being detectably labeled; (b) unlabeled cytochrome C and a detectably labeled anti-cytochrome C antibody; (c) unlabeled cytochrome C, unlabeled anti-cytochrome C antibody and a detectably labeled "secondary" antibody that binds specifically to the anti -cytochrome C antibody; (d) the cytochrome C being biotinylated and detectably labeled avidin; or (e) unlabeled, unconjugated cytochrome C, biotinylated anti-cytochrome C antibody and detectably labeled avidin, including the same steps for removal of unbound material described infra.
  • inhibition of binding of cytochrome C to "Polypeptide B" by a test compound is again an indication that the test compound is a promoter of apoptosis.
  • enhancement of binding of "Polypeptide B" by a test compound is an indication that the test compound is an inhibitor of apoptosis.
  • Sources include but are not restricted to serum, plasma, tissue extracts, cell lysates, cytosolic extracts or mitochondrial extracts.
  • Sources include but are not restricted to serum, plasma, tissue extracts, cell lysates, cytosolic extracts or mitochondrial extracts.
  • Methods to test for the utility of truncated polypeptides lacking more than twenty one carboxy-terminal amino acids (see supra on page 18) in the screening assays of the invention include, but are not restricted to, performing the screening assays in the absence of the test compound. "Polypeptide B" in such assays would be:
  • test truncated polypeptide lacking twenty one carboxy- terminal amino acids.
  • the test truncated polypeptide may be used in the screening assays of the invention.
  • Test compounds showing either inhibitory or enhancing activity in these screening assays can be further tested for their ability to regulate cytosolic cytochrome C levels, cleavage of PARP into apoptotic fragments and apoptosis in cells upon exposure to an appropriate stimulus, e.g. exposure to U-937 cells overexpressing Bcl-X L to IR.
  • Well known solid supports that may be used for screening assays of the invention include, but are not restricted to, glass, polystyrene, polypropylene, polyethylene, dextran, nylon, natural and modified celluloses, and polyacrylamides .
  • microtiter plates may conveniently be utilized as the solid phase.
  • the anchored component can be immobilized by non-covalent or covalent attachments. Non-covalent attachment can be accomplished by simply coating the solid surface with a solution of the protein and drying.
  • an immobilized antibody preferably a monoclonal antibody, specific for the protein to be immobilized can be used to anchor the protein to the solid surface.
  • the surfaces can be prepared in advance and stored.
  • a protein e.g., "Polypeptide B", cytochrome C, an antibody or some other "secondary” reagent such as avidin
  • EIA enzyme immunoassay
  • Enzymes that can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5 -steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta- galactosidase, ribonuclease, urease, catalase, glucose-6- phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
  • the detection can be accomplished by colorimetric methods that employ a chromogenic substrate for the enzyme. Detection can also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards .
  • Detection can also be accomplished using any of a variety of other immunoassays .
  • a radioimmunoassay RIA
  • the radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter, or by autoradiography .
  • fluorescent labeling compound When the fluorescently labeled material is exposed to light of the proper wave length, its presence can ⁇ then be detected due to fluorescence .
  • fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine .
  • Proteins can also be detectably labeled using fluorescence emitting metals such as 152 Eu, or others of the lanthanide series . These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA) .
  • DTPA diethylenetriaminepentacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • the polypeptides also can be detectably labeled by coupling to a chemiluminescent compound.
  • the presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
  • particularly useful chemiluminescent labeling compounds are luminal, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • Bioluminescence is a type of chemiluminescence found in biological systems, in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence .
  • Important bioluminescent compounds for purposes of labeling are luciferin, luciferase, and aequorin.
  • Peptides and polypeptides used in the screening assays of the invention may be obtained by a variety of means. Smaller peptides (less than 50 amino acids long) may be conveniently synthesized by standard chemical methods. Some polypeptides (e.g. cytochrome C antibodies) may be purchased from commercial sources. Modified forms of cytochrome C, which may be used in the assays of the invention (e.g., apocytochrome C and porphyrin cytochrome C) , can be either purchased from commercial sources or chemically derived by methods familiar to those of average skill in the art. Where otherwise unavailable, antibodies can be generated as described in Section D infra. Detectably labeled antibodies either can be purchased from commercial sources or are readily prepared by those of ordinary skill in the art.
  • Polypeptides such as Bcl-x L and Bel -2 may be purified from biological sources by methods well-known to those skilled in the art (Protein Purification, Principles and Practice, second edition (1987) Scopes, Springer Verlag, N.Y.] . They may also be produced in their naturally occurring, truncated, or fusion protein forms by recombinant DNA technology using techniques well known in the art. These methods include, for example, in vi tro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. See, for example, the techniques described in Sambrook et al . (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, N.Y. , ; and Ausubel et al .
  • RNA encoding the proteins may be chemically synthesized. See, for example, the techniques described in Oligonucleotide Synthesis, (1984) Gait, M.J. ed., IRL Press, Oxford, which is incorporated by reference herein in its entirety.
  • a variety of host-expression vector systems may be utilized to express the nucleotide sequences. Where the peptide or polypeptide is soluble, it can be recovered from: (a) the culture, i.e., from the host cell in cases where the peptide or polypeptide is not secreted; or (b) from the culture medium in cases where the peptide or polypeptide is secreted by the cells.
  • the expression systems also encompass engineered host cells that express the polypeptide in si tu, i.e., anchored in the cell membrane. Purification or enrichment of the polypeptide from such an expression system can be accomplished using appropriate detergents and lipid micelles and methods well known to those skilled in the art. Alternatively, such engineered host cells themselves may be used in situations where it is important not only to retain the structural and functional characteristics of the protein, but also to assess biological activity.
  • the expression systems that may be used for purposes of the invention include but are not limited to microorganisms such as bacteria (for example, E. coli and B . subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing the nucleotide sequences; yeast transformed with recombinant yeast expression vectors; insect cells infected with recombinant viral expression vectors (baculovirus) ; plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors; or mammalian cells (e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g. metallothionein promoter) or from mammalian viruses .
  • microorganisms such as bacteria (for
  • a number of expression vectors may be advantageously selected depending upon the use intended for the gene product being expressed. For example, when a large quantity of such a protein is to be produced, e.g. for raising antibodies to the protein, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable.
  • Such vectors include, but are not limited to, the E. coli expression vector pUR278 [Ruther et al . (1983) EMBO J. 2:1791], in which the coding sequence may be ligated individually into the vector in frame with the lacZ coding region so that a fusion protein is produced; pIN vectors [Inouye & Inouye (1985) Nucleic Acids Res.
  • pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST) .
  • GST glutathione S-transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione.
  • the PGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
  • a number of viral -based expression systems may be utilized.
  • the nucleotide sequence of interest may be ligated to an adenovirus transcription/translation control complex, e . g. , the late promoter and tripartite leader sequence.
  • This chimeric gene may then be inserted in the adenovirus genome by in vi tro or in vivo recombination. Insertion in a non-essential region of the viral genome ⁇ e . g. , region El or E3) will result in a recombinant virus that is viable and capable of expressing the gene product in infected hosts [e.g.
  • Specific initiation signals may also be required for efficient translation of inserted nucleotide sequences. These signals include the ATG initiation codon and adjacent sequences. In cases where an entire gene or cDNA, including its own initiation codon and adjacent sequences, is inserted into the appropriate expression vector, no additional translational control signals may be needed. However, in cases where only a portion of the coding sequence is inserted, exogenous translational control signals, including, perhaps, the ATG initiation codon, must be provided. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert.
  • exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic.
  • the efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. [Bittner et al . (1987) Methods in Enzymol. 153:516] .
  • a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e . g. , glycosylation) and processing ( e . g. , cleavage) of protein products may be important for the function of the protein.
  • Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.
  • Mammalian host cells include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, and WI38. For long-term, high-yield production of recombinant proteins, stable expression is preferred.
  • cell lines which stably express the sequences described above may be engineered.
  • host cells can be transformed with DNA controlled by appropriate expression control elements (e . g. , promoter, enhancer sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched medium, and then are switched to a selective medium.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
  • This method may advantageously be used to engineer cell lines which express the gene product.
  • Such engineered cell lines may be particularly useful in screening and evaluation of compounds that affect the endogenous activity of the gene product.
  • a fusion protein may be readily purified by utilizing an antibody or a ligand that specifically binds to the fusion protein being expressed.
  • a system described by Janknecht et al . [(1991) Proc. Natl. Acad. Sci. USA 28:8972] allows for the ready purification of non- denatured fusion proteins expressed in human cell lines.
  • the gene of interest is subcloned into a vaccinia recombination plasmid such that the gene's open reading frame is translationally fused to an amino-terminal tag consisting of six histidine residues.
  • Extracts from cells infected with recombinant vaccinia virus are loaded onto Ni 2+ nitriloacetic acid-agarose columns and histidine- tagged proteins are selectively eluted with imidazole- containing buffers. If desired, the histidine tag can be selectively cleaved with an appropriate enzyme.
  • Antibodies that specifically recognize epitopes within the amino acid sequence of SEQ. ID NOS:l, 2, 3 and 4 are also encompassed by the invention.
  • Such antibodies include but are not limited to polyclonal antibodies, monoclonal antibodies (mAbs) , humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') 2 fragments, and epitope-binding fragments of any of the above .
  • the antibodies of the invention may be used, for example, in the detection of the Bcl-x L or Bcl-2 in a biological sample, and may therefore be utilized as part of a diagnostic or prognostic technique whereby patients are tested for abnormal amounts of Bcl-x L or Bcl-2. Such antibodies may also be utilized in the screening assays of the invention.
  • a host animal is immunized by injection with a peptide containing the amino acid sequence of SEQ. ID N0S:1, 2, 3 and 4.
  • Such host animals may include but are not limited to rabbits, mice, and rats, to name but a few.
  • Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete) adjuvant, mineral gels such as aluminum hydroxide, lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, BCG (bacille Calmette-Guerin) and Corynebacterium parvum.
  • Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of the immunized animals .
  • the immunogen may be coupled to a carrier.
  • carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) .
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers.
  • Methods of coupling a peptide to a carrier are well known in the art and include the use of glutaraldehyde, carbodiimide and m-maleimidobenzoyl-N-hydroxysuccinimide ester.
  • epitopes within SEQ ID NOS : 1 , 2 and 3 and SEQ ID NO: 4 are predicted to interact with cytochrome C, they would be exposed on the surface of Bcl-x L and Bel -2, respectively. Therefore, they would not be predicted to be "hidden” epitopes when immunizing with intact Bcl-x L and Bcl-2.
  • Bcl-x L and Bcl-2 are intracellular proteins and thus normally sequestered from the immune system, neither CD4 + helper T-cells nor B-cells of the host animal will likely be tolerant of the epitopes within SEQ ID N0S:1, 2, 3 and 4. Therefore, the host animal is unlikely to be immunologically tolerant and thus unresponsive to the immunogen.
  • the amount of antigen to be used can be determined readily by those with average skill in the art without undue experimentation.
  • the antigen can be administered by a number of routes (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal) .
  • routes subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal
  • polyclonal antibodies is monitored by sampling blood of the immunized animal at various time points after administration. When the desired level of antibody is obtained, the animal is bled and the serum is stored.
  • Monoclonal antibodies which are homogeneous populations of antibodies to a particular antigen, may be obtained by any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique [Kohler and Milstein (1975) Nature 256:495-497; U.S. Patent No. 4,376,110; Howell and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Press, N.Y.], the human B-cell hybridoma technique [Kosbor et al . (1983) Immunology Today 4 . : 72; Cole et al . (1983) Proc. Natl. Acad. Sci.
  • Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof.
  • chimeric antibodies In addition, techniques developed for the production of "chimeric antibodies” can be used [Morrison et al . (1984) Proc. Natl. Acad. Sci. USA 81:6851; Neuberger et al . (1984) Nature 312 :604; Takeda et al . (1985) Nature 314 :452l . These involve splicing a portion of a gene encoding a mouse antibody of appropriate antigen specificity to a portion of a gene encoding a human antibody of appropriate biological activity.
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region.
  • single chain antibodies can be adapted to produce single chain antibodies against the epitopes of SEQ ID N0S:1, 2, 3 and 4.
  • Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. They are conveniently produced by recombinant DNA techniques. Antibody fragments which recognize specific epitopes may be generated by known techniques.
  • such fragments include but are not limited to the F(ab') 2 fragments which can be produced by pepsin digestion of the antibody molecule, and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab') 2 fragments.
  • Fab expression libraries may be constructed [Huse et al . (1989) Science 246 :1275l to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. Methods for screening antibodies for binding specificity are well known.
  • the invention includes methods for producing a medically desirable effect in a patient by either enhancing apoptosis or inhibiting apoptosis.
  • Patients that would benefit from promotion of apoptosis include but are not limited to those with, or at least suspected of having:
  • autoimmune diseases in which it is desirable to enhance the death of the specifically activated, apoptosis- susceptible lymphocytes (T and/or B) which are the effectors of the tissue destruction resulting in the symptoms of the disease;
  • Patients that would benefit from inhibition of apoptosis include but are not limited to those with, or at least suspected of having:
  • autoimmune diseases in which affected tissues are protected from the destructive activities of the effector cells of the immune system, these activities substantially involving apoptosis of affected tissue cells;
  • xeno- and allograft transplant patients in which, similar to the situation described in (d) supra , it is desirable to protect the cells of the grafted tissue from apoptosis-inducing action of the immunological effector cells;
  • Therapeutic methods of the invention that result in enhancement of apoptosis involve the use of peptides of the invention. These include all those described in Section B supra . Therapeutic methods of the invention that result in inhibition of apoptosis utilize a polypeptide (or a nucleic acid that encodes a polypeptide) that includes multiple peptide domains, each of which contains the amino acid sequence of SEQ ID NO: 3. E.l Methods of enhancing apoptosis
  • a peptide or peptidomimetic that inhibits binding of cytochrome C to an anti-apoptotic Bcl-2-related family member (such as a polypeptide containing the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4) is delivered to relevant cells. Delivery involves administering to a patient either the peptide or peptidomimetic itself, a nucleic acid encoding the peptide, an expression vector encoding the peptide, or cells transfected or transduced with the vector.
  • Peptides and peptidomimetics may be delivered directly to cells in vi tro . Methods of achieving this are well known to those of average skill in the art. Furthermore, peptides may be delivered to a cell of a mammal using techniques substantially the same as those described infra for delivery to human patients. Examples of appropriate mammals include but are not restricted to humans, non-human primates, horses, cattle, sheep, dogs, cats, mice, rats, guinea pigs, hamsters, rabbits and goats. A peptide of the invention may be delivered to cells of a patient in its unmodified state, dissolved in an appropriate physiological solution, e.g. physiological saline.
  • physiological solution e.g. physiological saline.
  • Section B.2 it may be modified as detailed in Section B.2 in order to facilitate transport across cell and/or intracellular membranes and to prevent extracellular or intracellular degradation. Its transport across biological membranes may also be enhanced by delivering it encapsulated in liposomes [Gabizon et al . (1990) Cancer Res. 5_0 . :6371; Ranade (1989) J. Clin. Pharmacol. 29:685] or an appropriate biodegradable polymeric microparticle (also referred to as a "microsphere” , “nanosphere” , “nanoparticle, or "microcapsule”) . Naturally, it is desirable that these peptides be selectively targeted to relevant tissues and cell types.
  • RA rheumatoid arthritis
  • IDDM insulin- dependent diabetes mellitus
  • the peptides could be introduced directly into affected joints or the pancreas, respectively, or, preferably, into draining lymphoid tissue in which the active autoimmune response occurs. The latter procedure would obviate the potential tissue damage caused by introducing pro-apoptotic peptides into the cells of the target organ.
  • peptides of the invention may be delivered in liposomes into which have been incorporated ligands for receptors on relevant cells (e.g., T cells or B cells) or antibodies to cell-surface markers expressed by these cells.
  • relevant cells e.g., T cells or B cells
  • an antibody specific for the CD4 T cell surface marker may direct liposomes containing both the anti-CD4 antibody and the relevant pro-apoptotic peptide to a CD4 + T cell.
  • an antibody specific for the relevant TCR component e.g. V/3 may be used.
  • the pro-apoptotic peptides are preferably directed to cancer cells.
  • the peptides could, for example, be injected directly into the tissues surrounding the tumor site after surgery to remove the tumor, in order to eliminate residual tumor cells.
  • the tumor could be treated by in si tu injection of the peptide into the tumor.
  • the treatment can be combined with radiotherapy, chemotherapy or immunotherapy to enhance tumor cell apoptosis.
  • the liposome methodology described supra could also be exploited. In this case antibodies specific for tumor-specific antigens (TSA) or tumor-associated antigens (TAA) would be exploited.
  • dosages for any one patient depend on many factors, as well as the particular compound to be administered, the time and route of administration and other drugs being administered concurrently.
  • Dosages for the peptides of the invention will vary, but can be, when administered intravenously, approximately 0.01 mg to 10 mg/ml blood volume. Routes and doses of administration are well known to skilled pharmacologists and physicians. Routes, in addition to those described supra , include, but are not restricted to: intraperitoneal , intramuscular, intrapulmonary, transmucosal , subcutaneous and intravenous . E. 1 .2 Administration of peptides utilizing expression vectors encoding the peptides .
  • An expression vector is composed of or contains a nucleic acid in which a polynucleotide sequence encoding a peptide or polypeptide of the invention is operatively linked to a promoter or enhancer-promoter combination.
  • a promoter is a trancriptional regulatory element composed of a region of a DNA molecule typically within 100 nucleotide pairs in front (uptream of) of the point at which transcription starts.
  • Another transcriptional regulatory element is an enhancer.
  • An enhancer provides specificity in terms of time, location and expression level. Unlike a promoter, an enhancer can function when located at variable distances from the transcription site, provided a promoter is present . An enhancer can also be located downstream of the transcription initiation site.
  • a coding sequence of an expression vector is operatively linked to a transcription terminating region.
  • To bring a coding sequence under control of a promoter it is necessary to position the translation initiation site of the translational reading frame of the peptide or polypeptide between one and about fifty nucleotides downstream (3') of the promoter. Examples of particular promoters are provided infra .
  • Expression vectors and methods for their construction are known to those familiar with the art.
  • Suitable vectors include plasmids, and viral vectors such as herpes viruses, retroviruses, canary pox viruses, adenoviruses and adeno-associated viruses, among others.
  • viral vectors such as herpes viruses, retroviruses, canary pox viruses, adenoviruses and adeno-associated viruses, among others.
  • the application of peptide-encoding genes to the treatment of cancer, autoimmune disease, or graft rejection in humans can utilize either in vivo ox ex vivo based therapeutic approaches.
  • the in vivo approach requires delivery of a genetic construct directly into the patient, preferably targeting it to the cells or tissue of interest.
  • a genetic construct directly into the patient, preferably targeting it to the cells or tissue of interest.
  • residual cells may be targeted by treating the vicinity of the tumor with a composition containing a retroviral vector encoding an immunostimulatory peptide.
  • the primary tumor could be treated by in si tu injection of the vector directly into the tumor.
  • Malignant cells distal to the primary tumor site may be reached by delivering the vector intravenously.
  • targeting of tissues under autoimmune attack may be achieved by direct injection of vectors.
  • Targeting of tumor cells or activated lymphocytes for example, can be accomplished by the use of a retrovirus, which stably transfects primarily proliferating cells.
  • Tissue specific targeting may also be achieved by the use of a molecular conjugate composed of a plasmid or other vector attached to poly-L-lysine by electrostatic or covalent forces.
  • Poly-L-lysine binds to a ligand that can bind to a receptor on tumor cells [Cristiano et al . (1995) J. Mol . Med 73:479].
  • tumor and cell specific antibodies of the type described supra in Section E.l.l can be bound to vectors and thereby target them to tumors or cells such as T-lymphocytes . The latter would be useful in both autoimmune diseases and organ transplantation.
  • a promoter inducing relatively tumor-specific expression can be used to achieve a further level of targeting: for example, the c ⁇ -fetoprotein promoter for hepatocellular carcinoma [Huber et al . (1991) Proc. Natl. Acad. Sci. USA 88:8039]; the DF3 tumor antigen promoter/enhancer for certain breast and lung carcinomas [Abe and Kufe (1993) Proc. Natl. Acad. Sci. USA 9J):282] or the tyrosinase promoter for melanoma [Hart et al . (1995) Br. Med. Bull. 5JL:647] .
  • Tissue-specific promoters for use in autoimmune or transplant patients include, for example, the inducible IL-2 [Thompson et al . (1992) Mol . Cell. Biol. 12: 1043], IL-4 [Todd et al . (1993) J. Exp . Med. 177:1663] and gamma- interferon [Penix et al . (1993) J. Exp. Med. 178 :4831 T-cell targeting promoters.
  • Such inducible promoters would have an invaluable additional advantage in that expression would occur selectively in activated T-cells. Included in this population of activated T-cells are the effector cells that an ideal immuno-therapeutic modality would selectively eliminate in autoimmune and transplant patients.
  • Vectors can also be delivered by incorporation into liposomes or other delivery vehicles either alone or co- incorporated with cell specific antibodies, as described supra in Section E.l.l for pro-apoptotic peptides.
  • CDDP see Example 1 infra
  • radiation therapy see Example 1 infra
  • immunotherapy all of which act substantially by inducing apoptosis in the tumor cells.
  • Such therapy combinations may result in synergistic or at least additive therapeutic effects.
  • DNA or transfected cells may be administered in a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers are biologically compatible vehicles which are suitable for administration to a human, e.g., physiological saline.
  • a therapeutically effective amount is an amount of the DNA of the invention which is capable of producing a medically desirable result in a treated animal .
  • the dosage for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Dosages will vary, but a preferred dosage for intravenous administration of DNA is from approximately 10 6 to 10 12 copies of the DNA molecule. This dose can be repeatedly administered, as needed. Routes of administration will be as for the peptides described supra in Section E.l.l. E.2 Methods of inhibiting apoptosis
  • a recombinant polypeptide containing multiple peptide domains each of which contains the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO : 4 , is introduced into the cells and preferably into the mitochondria of cells of a relevant tissue.
  • the polypeptide includes at least two copies of the domain, and preferably at least three (e.g., four, five, six, eight, ten, twelve, or twenty) .
  • Each domain can be represented by SEQ ID NO: 3 or 4 , or can be a larger segment or all of ⁇ X L/S or the corresponding portion of Bcl-2.
  • polypeptides in which the above described domains are either present in tandem on the polypeptide molecule or are separated by stretches of other polypeptide sequence (e.g. other Bclx or Bcl-2 sequence), would display an enhanced ability to bind cytochrome C, prevent its entry into the cytosol, and thereby render the cells relatively more resistant to apoptosis induction.
  • polypeptide sequence e.g. other Bclx or Bcl-2 sequence
  • the recombinant polypeptide is preferably introduced into the mitochondria of the patient by administering to the patient a nucleic acid of the invention that encodes the polypeptide.
  • Targeting to the mitochondria would be accomplished by: (a) employing an appropriate signal sequence in construction of the nucleic acid encoding the polypeptide (e.g., utilizing the same mechanism utilized by the cell to deliver other proteins such as Bcl-x L into the mitochondria) ; and (b) inserting nucleic segments encoding the amino acid sequences of SEQ ID NO : 3 or SEQ ID NO: 4 into a nucleic acid encoding Bcl-x L or Bcl-2 at positions predicted from the known structure of Bcl-x L [Muchmore et al .
  • Bcl-2 [Vance et al . , cited supra] to encode regions on the outer surface of the polypeptide and thereby be available for interaction with cytochrome C.
  • An example of an appropriate signal sequence as described in (a) would be a naturally-occuring Bcl-x L or Bcl-2 signal sequence.
  • Vehicles of delivery (vectors), methods of administration and methods of tissue-specific targeting are substantially the same as those used for in vivo administration of expression vectors encoding peptides that promote apoptosis (see Section E.1.2 supra) .
  • This method of the invention may be used to inhibit apoptosis in tissues undergoing autoimmune attack or transplanted organs undergoing rejection.
  • nucleic acid encoding the anti-apoptotic polypeptide to synovial cells in RA patients by conjugating it with an antibody that binds to a cell -surface molecule on such cells, and thereby protect the synovial cells from the lethal effects of the effector lymphocytes and inflammatory cytokines involved in the pathogenesis of RA.
  • a vector comprising an insulin promoter [Dandoy et al . (1991) Nucleic Acids Res. 19:4925; Selden et al . (1986) Nature 321:525] would target expression of the apoptosis inhibiting polypeptide to pancreatic islet cells and would thereby selectively protect them from the pathologic immunologic and inflammatory activities involved in autoimmune IDDM.
  • CD4 + T-cell specific antibodies and promoters could direct expression of the anti-apoptotic polypeptide to these T-cells in certain immunodeficiency patients, for example, patients that are either HIV-1 positive or have AIDS.
  • This therapeutic method would substantially preserve these CD4 + T-cells which are absolutely essential for a functional immune system and the loss of which leads to the devastating immunodeficiency observed in AIDS patients.
  • Targeting of the nucleic acid encoding the anti-apoptotic polypeptide to bone marrow with a pluripotent stem cell-specific antibody would also substantially preserve the hematological systems of cancer patients undergoing chemo- and/or radiation therapy. Bone marrow depletion and consequent hematological insufficiency is a frequent side- effect of these therapies. Directing expression of the anti- apoptotic polypeptide to cells of the neurological system would be useful in treatment of certain neurodegenerative conditions resulting from excessive neurological cell death. The following examples are meant to illustrate the invention and not to limit it.
  • Bcl-x I ⁇ T and Bcl- ⁇ g ⁇ proteins Full length Bcl-x L and Bcl-x s were cloned from a cDNA library of human Raj i lymphoma cells (Clontech) using the PCR primers: 5 '-primer: 5' -GGAATTCATATGTCTCAGAGCAACCG-3' (SEQ ID NO: 15); 3 '-primer: 5' -AGAATTCATTCATTTCCGACTGAAGAG-3' (SEQ ID NO: 16).
  • H 6 -Bcl-x LT , Bcl-x s ⁇ and H 6 polypeptides were resolved by SDS-PAGE and transferred to a nitrocellulose filter.
  • Purified MAP kinase (MAPK) protein was used as a negative control.
  • the filter was incubated with purified cytochrome C (bovine heart cytochrome C; Sigma) for 1 h at room temperature. The filters were then analyzed by immunoblotting with anti -cytochrome C antibody. In separate experiments, purified cytochrome C was separated by SDS-PAGE and transferred to three nitrocellulose filters.
  • the filters were incubated with purified, eluted H 6 , H 6 -Bcl-x LT or Bcl-x s ⁇ for 1 h at room temperature. The filters were then analyzed by immunoblotting with anti-Bcl-x antibody. ELISA immunoassay
  • Cytochrome C was adsorbed onto the plastic surface of a 96-well ELISA plate, followed by addition of blocking buffer (SuperblockTM, Pierce Chemical) to reduce non-specific binding.
  • Glutathione-S-transferase (GST) GST-Bcl-x LT or GST-Bcl-x s ⁇ in Tris-buffered saline containing 5% bovine serum albumin and 0.5% NP-40 were added to the wells. After incubation for 2 h at room temperature, the plates were washed and incubated with rabbit anti-GST, followed by washing and incubation with alkaline phosphatase-conjugated anti-rabbit IgG antibody. The plates were developed using a fluorogenic substrate and reactivity determined by measurement of fluorescence. Peptide synthesis
  • Peptides were synthesized on an Advanced Chemtech 396 MPS peptide synthesizer (Louisville, KY) using a 9- fluorenylmethyloxycarbonyl (Fmoc) protocol provided by the manufacturer.
  • 2- (lH-benzotriazole-yl) -1,1,3,3- tetramethyluronium hexafluorophosphate (HBTU) /N- hydroxybenzotriazole (HOBt) was used for amino acid couplings and Fmoc cleavages were accomplished with 25% piperidine in dimethylformamide (DMF) .
  • Fmoc-Aa-Wang resins (where Aa is the C-terminal amino acid of each sequence, 0.05 mmol, Midwest Biotech, IN) were employed for synthesis of each peptide. Side chains of functional amino acids were protected as Cys(Trt), Asn(Trt), Trp(Boc), Asp(t-Bu), Gin (Trt), Arg(Pbf), Glu(t-Bu), His (Trt), Lys (Boc) , Ser(t- Bu) , Thr(t-Bu), and Tyr(t-Bu) where tBu is a tert-butyl, Trt is a trityl, Boc is a tert-butyloxycarbonyl, and Pbf is a
  • This assay was a modification of the ELISA immunoassay described supra . All steps were the same except that the GST-Bcl-x LT was mixed with synthetic peptide in a separate plate prior to addition to the assay plate containing the adsorbed cytochrome C. The final concentration of peptide was in the range 1 - 100 ⁇ M
  • Example 1 Bcl-x LT blocks cytochrome C release from mitochondria of U- 937 cells and cytochrome C release occurs upstream to activation of cysteine proteases
  • U-937 cells that stably overexpress Bcl-x L are resistant to radiation-induced internucleosomal DNA fragmentation [Datta et al . (1995), cited supra] .
  • Immunoblotting of electrophoretically separated cytosolic proteins from U-937/Bcl-x L and control U-937 cells showed that Bcl-x L expression also prevented accumulation of cytosolic cytochrome C in response to IR or CDDP treatment.
  • IR-treated U-937/Bcl-x L cells failed to exhibit PARP cleavage as indicated by electrophoresis and immunoblotting of the same samples with anti-PARP antibody.
  • Cytochrome C associates with Bcl-x L but not with Bcl-x s , and the binding of cytochrome C to Bcl-x L is inhibited by Bcl-x s Since Bcl-x L blocks the increase of cytosolic cytochrome C, co-immunoprecipitation studies were carried out to determine whether these proteins form a specific complex in cells. Lysates from control and irradiated U- 937/Bcl-x L cells were subjected to immunoprecipitation with anti-cytochrome C antibody.
  • cytochrome C was also resolved by electrophoresis and transferred to nitrocellulose filters, which were then incubated with purified H 6 , H 6 -Bcl-x LT or Bcl-x s ⁇ proteins and analyzed by immunoblotting with anti- Bcl-x antibody. Only the blot incubated with H 6 -Bcl-x LT showed reactivity with cytochrome C.
  • Peptides (7) (SEQ ID N0:1) and (8) (SEQ ID NO: 8) inhibited binding by approximately 80%.
  • amino acid sequence SEQ ID NO: 3
  • amino acid sequence SEQ ID NO:l
  • peptide (8) SEQ ID NO: 2

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  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Peptides et peptidomimétiques inhibant la liaison du cytochrome C à un membre de la famille apparentée à Bcl-2, à utiliser dans le traitement des maladies auto-immunes et du cancer ainsi que dans la prévention du rejet de greffon chez les patients transplantés, par l'administration du peptide, de son peptidomimétique ou d'un vecteur d'expression codant le peptide.
PCT/US1998/012595 1997-06-24 1998-06-16 Modulation de l'apoptose WO1998058541A1 (fr)

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US88164697A 1997-06-24 1997-06-24
US08/881,646 1997-06-24

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WO1998058541A1 true WO1998058541A1 (fr) 1998-12-30

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Cited By (5)

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Publication number Priority date Publication date Assignee Title
WO2005049073A2 (fr) * 2003-11-19 2005-06-02 Survac Aps Proteines appartenant a la famille bcl-2, fragments associes, et utilisation therapeutique de ces proteines et fragments sur des patients atteints du cancer
EP1573324A2 (fr) * 2002-12-13 2005-09-14 Elixir Pharmaceuticals, Inc. Acetylation du cytochrome c
US7790184B2 (en) 2006-08-01 2010-09-07 Washington University In St. Louis Cytochrome c synthesis inhibitors
EP2332964B1 (fr) * 2001-12-20 2016-04-20 CytoTools AG Peptides ayant un effet sur l'apoptose
US9528088B2 (en) 2002-06-28 2016-12-27 Life Technologies Corporation Methods for eliminating at least a substantial portion of a clonal antigen-specific memory T cell subpopulation

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US5656725A (en) * 1995-05-12 1997-08-12 Apoptosis Technology, Inc. Peptides and compositions which modulate apoptosis
US5702897A (en) * 1994-04-13 1997-12-30 The Burnham Institute Interaction of proteins involved in a cell death pathway

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US5702897A (en) * 1994-04-13 1997-12-30 The Burnham Institute Interaction of proteins involved in a cell death pathway
US5656725A (en) * 1995-05-12 1997-08-12 Apoptosis Technology, Inc. Peptides and compositions which modulate apoptosis

Non-Patent Citations (1)

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Title
PANDEY P., ET AL.: "ROLE FOR BCL-XL AS AN INHIBITOR OF CYTOSOLIC CYTOCHROME C ACCUMULATION IN DNA DAMAGE-INDUCED APOPTOSIS.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, US, vol. 94., 1 June 1997 (1997-06-01), US, pages 6939 - 6942., XP002910191, ISSN: 0027-8424, DOI: 10.1073/pnas.94.13.6939 *

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2332964B1 (fr) * 2001-12-20 2016-04-20 CytoTools AG Peptides ayant un effet sur l'apoptose
US9528088B2 (en) 2002-06-28 2016-12-27 Life Technologies Corporation Methods for eliminating at least a substantial portion of a clonal antigen-specific memory T cell subpopulation
US7771961B2 (en) 2002-12-13 2010-08-10 Elixir Pharmaceuticals, Inc Cytochrome c acetylation
EP1573324A4 (fr) * 2002-12-13 2007-08-01 Elixir Pharmaceuticals Inc Acetylation du cytochrome c
US7575883B2 (en) 2002-12-13 2009-08-18 Elixir Pharmaceuticals, Inc. Cytochrome c acetylation
EP1573324A2 (fr) * 2002-12-13 2005-09-14 Elixir Pharmaceuticals, Inc. Acetylation du cytochrome c
EP2087904A1 (fr) * 2003-11-19 2009-08-12 Survac ApS Utilisation thérapeutique de peptides dérivées du Bcl-XL protéine sur des patients atteints du cancer
WO2005049073A2 (fr) * 2003-11-19 2005-06-02 Survac Aps Proteines appartenant a la famille bcl-2, fragments associes, et utilisation therapeutique de ces proteines et fragments sur des patients atteints du cancer
AU2004290866B2 (en) * 2003-11-19 2010-09-02 Survac Aps Proteins belonging to the BcL-2 family and fragments thereof, and their use in cancer patients
US7842294B2 (en) 2003-11-19 2010-11-30 Survac Aps Proteins belonging to the Bcl-2 family and fragments thereof, and their use in cancer patients
KR101284237B1 (ko) * 2003-11-19 2013-07-09 수르벡 에이피에스 Ｂｃｌ-2 패밀리에 속한 단백질들, 그들의 단편들 및 암 환자에 대한 그들의 용도
WO2005049073A3 (fr) * 2003-11-19 2006-01-05 Survac Aps Proteines appartenant a la famille bcl-2, fragments associes, et utilisation therapeutique de ces proteines et fragments sur des patients atteints du cancer
US7790184B2 (en) 2006-08-01 2010-09-07 Washington University In St. Louis Cytochrome c synthesis inhibitors
US8889378B2 (en) 2006-08-01 2014-11-18 Washington University In St. Louis Cytochrome C synthesis inhibitors

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