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WO1999016787A9 - Agonistes de mort cellulaire - Google Patents

Agonistes de mort cellulaire

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Publication number
WO1999016787A9
WO1999016787A9 PCT/US1998/019765 US9819765W WO9916787A9 WO 1999016787 A9 WO1999016787 A9 WO 1999016787A9 US 9819765 W US9819765 W US 9819765W WO 9916787 A9 WO9916787 A9 WO 9916787A9
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WO
WIPO (PCT)
Prior art keywords
seq
leu
bcl
polypeptide
domain
Prior art date
Application number
PCT/US1998/019765
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English (en)
Other versions
WO1999016787A1 (fr
Inventor
Stanley J Korsmeyer
Original Assignee
Univ Washington
Stanley J Korsmeyer
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Publication date
Application filed by Univ Washington, Stanley J Korsmeyer filed Critical Univ Washington
Priority to AU94028/98A priority Critical patent/AU9402898A/en
Publication of WO1999016787A1 publication Critical patent/WO1999016787A1/fr
Publication of WO1999016787A9 publication Critical patent/WO1999016787A9/fr

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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
    • 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

Definitions

  • This invention relates generally to the regulation of apoptosis and to compounds which regulate apoptosis, and more particularly, to a novel cell death agonist.
  • apoptosis plays an indispensable role in the development and maintenance of homeostasis within all multicellular organisms (Raff, Nature 356:397-400, 1992). Genetic and molecular analysis from nematodes to humans has indicated that the apoptotic pathway of cellular suicide is highly conserved (Hengartner and Horvitz, Cell 76:1107-1114, 1994) In addition to being essential for normal development and maintenance, apoptosis is important in the defense against viral infection and in preventing the emergence of cancer.
  • the BCL-2 family of proteins constitutes an intracellular checkpoint of apoptosis.
  • the founding member of this family is the apoptosis-inhibiting protein encoded by the bcl-2 protooncogene which was initially isolated from a follicular lymphoma (Bakhshi et al., Cell 41:889-906, 1985; Tsujimoto et al, Science 229:1390-1393, 1985; Cleary and Sklar, Proc Natl Acad Sci USA 82:7439- 7443, 1985).
  • the BCL-2 protein is a 25 kD, integral membrane protein localized to intracellular membranes including mitochondria.
  • BCL-2-related proteins is comprised of both anti-apoptotic and pro-apoptotic members that function in a distal apoptotic pathway common to all multi-cellular organisms. It has been suggested that the ratio of anti-apoptotic (BCL-2, BCL-X L , MCL-1 and Al ) to pro-apoptotic (BAX, BAK, BCL-X S , BAD, BIK and BID) molecules dictates whether a cell will respond to a proximal apoptotic stimulus.
  • BCL-Homology domains 1 and 2 (BH1 and BH2 ) have been shown to be important in dimerization and in modulating apoptosis (Yin et al., supra).
  • a third homology region, BH3, has been found in some family members and shown to be important in dimerization as well as promoting apoptosis (Boyd et al., Oncogene 12:1921- 1928; Chittenden et al., Embo J 14:5589-5596, 1995).
  • BH4 the most recently identified homology domain, is present near the amino terminal end of some pro-apoptotic family members ( Farrow et al. , supra) .
  • the BH3 domain may play a role in the promotion of death by full-length pro-apoptotic family members, although BAD was not heretofore known to contain a BH3 domain.
  • the pro-apoptotic family member may play a role in the promotion of death by full-length pro-apoptotic family members, although BAD was not heretofore known to contain a BH3 domain.
  • the pro-apoptotic family member may play a role in the promotion of death by full-length pro-apoptotic family members, although BAD was not heretofore known to contain a BH3 domain.
  • the pro-apoptotic family member may play a role in the promotion of death by full-length pro-apoptotic family members, although BAD was not heretofore known to contain a BH3 domain.
  • the pro-apoptotic family member may play a role in the promotion of death by full-length pro-apoptotic family members, although BAD was not heretofore known to contain a
  • BCL-X S which is translated from an alternatively spliced version of the mR ⁇ A encoding BCL-X L , contains BH3 and BH4 domains, but lacks BH1 and BH2 domains.
  • BCL-X S inhibits the ability of BCL-2 to enhance the survival of growth- factor deprived cells (Boise et al. Cell 74:597-608, 1993).
  • BIK and BID are other death promoting BCL-2 family members having a BH3 but not BH1 or BH2 domains and which also lack a BH4 domain (Boyd et al. , Oncogene 11:1921-1928, 1995; Wang et al., Nature 379:554-556, 1996).
  • pro- apoptotic BID also interacts with BCL-2, BCL-X L , and BAX through its BH3 domain and indicated that the corresponding binding site on these partner proteins is the BH1 domain, and perhaps also the BH2 domain (Wang et al. , supra.
  • the BAK peptide which is a random coil in solution, forms an ⁇ helix upon binding in a hydrophobic cleft formed by the BH1, BH2, and BH3 regions of BCL-X L , with certain hydrophobic side chains of the BAK peptide (Val 74 , Leu 78 , and lie 81 ) pointing into the cleft and certain charged side chains of the peptide (Arg 76 , Asp 83 , and Asp 84 ) being close to oppositely charged residues of BCL-X L . Smaller BAK peptides from this region, including an llmer peptide corresponding to BAK residues 77 to 87, reportedly did not bind to BCL-X, .
  • BH3-BH1 binding may not be involved in all interactions between BCL-2 related proteins.
  • pro-apoptotic BIK and BCL-X S both of which lack the BH1 and BH2 domains, have been shown to interact (Boyd et al., supra ) .
  • BAX does not require BH1 or BH2 to homodimerize ( Zha et al., supra ) .
  • Some disease conditions are believed to be related to the development of a defective down-regulation of apoptosis in the affected cells.
  • neoplasias may result, at least in part, from an apoptosis-resistant state in which cell proliferation signals inappropriately exceed cell death signals.
  • DNA viruses such as Epstein-Barr virus, African swine fever virus and adenovirus, parasitize the host cellular machinery to drive their own replication and at the same time modulate apoptosis to repress cell death and allow the target cell to reproduce the virus.
  • certain disease conditions such as lymphoproliferative conditions, cancer including drug resistant cancer, arthritis, inflammation, autoimmune diseases and the like may result from a down regulation of cell death regulation.
  • polypeptides including a BH3 domain derived from a pro-apoptotic member of the BCL-2 family can promote apoptosis.
  • Such polypeptides are shorter than the full length of the family member from which it is derived.
  • pro- apoptotic BCL-2 family member refers to any polypeptide having a BH3 domain as defined herein and having the ability to promote cell death in one or more of the assays described herein.
  • Pro-apoptotic family members include BAD, BAK, BAX, BID, and BIK.
  • the present invention is based on the discovery reported herein (1) that BAD (Bcl-2 Associated cell Death promoter) has a BH3 domain which is essential for apoptotic function and (2) that the BH3 domain of any pro-apoptotic member of the BCL-2 family is sufficient to promote apoptosis.
  • BAD Bcl-2 Associated cell Death promoter
  • small polypeptides of 50 or fewer amino acids comprising the 9 amino acid BH3 domain have significant death agonist activity when administered to cells. This discovery was unexpected because it was not previously known that all BCL-2 pro-apoptotic family members contain a BH3 domain, nor was it known that a polypeptide containing the BH3 domain of any pro- apoptotic member is sufficient to promote apoptosis.
  • one aspect of the present invention provides a polypeptide containing a bcl-homology domain 3_ (BH3 polypeptide) of from about 9 to about 50 contiguous amino acids having cell death agonist activity and comprising a BH3 domain of a pro-apoptotic BCL-2 family member.
  • the BH3 domain comprises a nine amino acid sequence as set forth in SEQ ID NO:40 ( Leu-Xaa 1 -Xaa 2 -Xaa 3 - Xaa 4 -Asp-Xaa 5 -Xaa 6 -Xaa 7 , wherein Xaa !
  • Xaa 2 is Arg, lie, Leu, Lys, Gin or Cys
  • Xaa 3 is Met, lie or Val
  • Xaa 4 is Ser or Gly
  • Xaa 5 is Glu, Asp or Ser
  • Xaa 6 is Phe, lie, Leu or Met
  • Xaa 7 is Val, Glu, Asn or Asp), or a conservatively substituted variant thereof, and which is identified more particularly by homology to the sequences shown in FIG. 1 (SEQ ID NO: 1-9).
  • the BH3 domain is identical to or is a conservatively substituted variant of a BH3 domain from a human or murine BAD, BAK, BAX, BID, or BIK polypeptide.
  • the BH3 polypeptide is operably linked to a cell penetrating agent.
  • Another aspect of the invention provides a BH3 domain peptide having death agonist activity which comprises between about five to eight contiguous amino acids from the BH3 domain as set forth in SEQ ID NO: 40, or a conservatively substituted variant thereof.
  • Yet another aspect of the invention provides polynucleotides encoding a BH3 polypeptide of no more than 50 amino acids having cell death agonist activity and comprising a BH3 domain of a pro-apoptotic BCL-2 family member.
  • the invention also provides polynucleotides encoding BH3 domain peptides of about five to eight contiguous amino acids from SEQ ID NO:40, or a conservatively substituted variant thereof. These polynucleotide may be used to transfect a target cell for expression of the BH3 polypeptide to promote death of the target cell.
  • the present invention provides a method for promoting apoptosis in a target cell comprising administering to the cell a death- promoting amount of a BH3 polypeptide or a BH3 domain peptide.
  • the BH3 polypeptide comprises no more than 50 contiguous amino acids having cell death agonist activity and comprising a BH3 domain of a pro-apoptotic BCL-2 family member, while the BH3 domain peptide has cell death agonist activity and comprises five to eight contiguous amino acids of the BH3 domain.
  • the BH3 polypeptide or BH3 domain peptide is operably linked to a cell-penetrating agent which improves entry of the BH3 polypeptide into the cell.
  • the BH3 polypeptide or BH3 domain peptide can be administered to the target cell by transfeeting the cell with an expression vector which comprises a polynucleotide encoding the BH3 polypeptide or BH3 domain peptide.
  • BH3 polypeptides which are relatively short in length and which possess cell death agonist activity
  • provision of peptides from the BH3 domain the provision of polynucleotides encoding these polypeptides and peptides
  • provision of BH3 polypeptide compositions and peptide compositions having cell death agonist activity and which can be readily delivered intracellularly to produce a death agonist activity and the provision of a method for promoting death of a target cell with these compositions.
  • Figure 1 illustrates the amino acid sequences of the BH3 domains from human (h) and murine (m) BAD, BAK, BAX, BIK, and BID (SEQ ID NO: 1-9);
  • Figure 2 illustrates the structures of BCL-2 family members showing the locations of the homology domains relative to the N-terminus as BH4, BH3, BHl, and BH2, with TM representing the hydrophobic transmembrane C-terminal tail present in most members;
  • FIG. 3 illustrates that BAD has a BH1/BH3 region that is required for cell death and heterodimerization with BCL-2 showing (A) a map of a nested set of BAD deletion mutants indicating retained amino acids and the position of the BH1/BH3 and BH2 domains and (B) the binding of P 32 -labeled GST-BCL-2 to these BAD deletion mutants transferred to nitrocellulose (upper panel) from a SDS-PAGE gel ( lower panel ) ;
  • Figure 4 illustrates aligned partial sequences of human and murine BAD, BAK, BAX, BID, and BIK (SEQ ID NO: 10-18) showing the sequence homology within BH3 domains (underlined) with identical amino acids boxed;
  • Figure 5 illustrates the predicted three- dimensional amphipathic ⁇ -helix structure of the BAD BH3 domain showing views of the hydrophobic surface (left) and polar surface (right ) with the locations of the hydrophobic and polar amino acids forming each surface identified;
  • FIG. 6 illustrates that the BAD BH1/BH3 domain is essential for pro-apoptotic function showing (A) the structure of BAD deletion mutants indicating retained amino acids and positions of the BH1/BH3 and BH2 domains, (B) the apoptosis-promoting activity of these BAD deletion mutants as measured by transient co-transfection with a luciferase reporter vector into BAD-deficient murine embryonic fibroblasts, and (C) the BCL-2 or BCL-X L binding ability of these BAD deletion mutants in an In vitro binding assay;
  • Figure 7 illustrates the effect of BAD BH3 mutations on heterodimerization of BAD with BCL-2 or BCL- X L showing (A) 35 S-labeled wild-type (WT) and mutant BAD proteins substituted with alanine at positions Gly 148 (G148A), Arg 149 (R149A), or Leul51 (L151A) produced by in vitro transcription-translation ( IVT ) and the amount of these 35 S-labeled BAD proteins that were captured by GST-BCL-2 or GST-BCL-X L bound to GSH-agarose beads in an In vitro binding assay, (B) a Western blot of lysates from FL5.12 BCL-X L cells stably expressing wild-type or mutant forms of BAD probed with an anti-BAD antibody (upper panel) or an anti-BCL-X L antibody (lower panel), and (C) a western blot analysis of levels of wild-type and mutant BAD proteins in total cell lysates ( lysates
  • Figure 9 illustrates the effect of BCL-2 BHl, BH2, and BH3 mutations on heterodimerization of BCL-2 with BAD showing 35 S-labeled wild-type (WT) and mutant BCL-2 proteins substituted with alanine at positions Gly 145 (G145A), Trp 188 (W188A), or Leu97 (L97A) produced by In vitro transcription-translation ( IVTT ) and the amount of these 35 S-labeled BCL-2 proteins that were captured by
  • Figure 10 illustrates (A) the BH3 domain of murine BID, represented with two upstream and two downstream amino acids (SEQ ID NOS: 19) and a schematic representation of mutations introduced into BID (SEQ ID NOS: 20-23) and (B) In vitro binding of BCL-2 or BAX with GST-BID or BID mutants;
  • Figure 11 illustrates (A) the viability of FL5.12- Bcl-2 clones expressing wild type or BH3-domain mutant BID, (B) Western blot showing BID expression and (C) Western blot showing association of wild type or BH3- domain mutant BID with BCL-2 and BAX (Lane 1: FL5.12-Bcl- 2/Hygro.l; Lane 2: FL5.12-Bcl-2/Bid-8; Lane 3: FL5.12- Bcl-2/BidmIII-1.15; Lane 4: FL5.12-Bcl-2/BidmIII-2.10; Lane 5: FL5.12-Bcl-2/BidmIII-3.1; Lane 6: FL5.12-Bcl- 2/BidmIII-4.1);
  • Figure 12 illustrates (A) the viability of Jurkat cells expressing wild type and BH3-domain mutant BID; (B) Western blot showing levels of BID polypeptides; and (C) viability measured in luciferase activity in Rat-1 fibroblasts co-transfected with the luciferase reporter gene and with 2>cI-2, bcl-2 along with bid, and with wild type and BH3-domain mutant ibid;
  • Figure 13 illustrates the death-promoting activity of full-length BAX BH3-domain mutants showing (A) the location of substitution mutations made in or near the BH3 domain (SEQ ID NOS: 24-29), (B) the luciferase activity in Rat-1 cells co-transfected with a luciferase reporter gene and a recombinant pcDNA3 vector encoding wild-type BAX, a BAX BH3-domain mutant or wild-type BCL- 2, and (C) the amount of luciferase activity in
  • Figure 15 illustrates the death-promoting ability of various BAX and BID regions showing (A) and (B) the amount of luciferase expression in Rat-1 cells at 20 hours after co-transfection with or without a pcDNA3 vector encoding BCL-2 and with recombinant pcDNA3 vectors encoding the (A) BAX regions or (B) BID regions, and (C) the amount of luciferase expression in Rat-1 cells grown in the presence or absence of the caspase inhibitor z- VAD-fmk at 20 hrs following transfection with recombinant pcDNA3 vectors encoding the indicated BAX and BID regions;
  • Figure 16 illustrates the effect of BH3 polypeptides on nuclear morphology of cells showing photographs of Rat-1 cells transfected with (A) BAX WT, (B) BAX 53-104, (C) BID WT, or (D) BID 74-128 and stained with the DNA dye Hoechest 33342;
  • Figure 17 illustrates the death-promoting ability of Tat-BH3 peptides showing (A) the sequences of synthetic peptides consisting of an 11 amino acid sequence from the HIV I Tat protein (SEQ ID NO: 55) linked to BAX or BID amino acid sequences containing a wild-type or mutant (m) BH3 domain and varying lengths of wild-type flanking region (SEQ ID NOS: 30-39) and (B) the viability of 2B4 cells determined by trypan blue dye exclusion at four hours after no treatment or treatment with 100 ⁇ M of the Tat peptide or one of the Tat-BH3 peptides shown in (A);
  • Figure 18 illustrates the kinetics and dose- response relationship of cell death induced by Tat-BH3 peptides containing a wild-type or mutant BH3 domain from BAX or BID showing the viability of 2B4 cells determined by trypan blue dye exclusion (A) at different times following no treatment or treatment with 100 ⁇ M of the designated Tat-BH3 peptide and (B) at
  • Figure 19 illustrates the effect of BCL-2 and z- VAD-fmk on cell death induced by Tat-BH3 peptides showing (A) the viability of 2B4 cells overexpressing BCL-2 or the vector alone (neo) determined by trypan blue dye exclusion at two hours after no treatment or treatment with Tat-BAX( 57-71) or Tat-BID( 81-100 ) at 100 ⁇ M concentration in the presence or absence of 200 ⁇ M z-VAD- fmk and (B) the percentage of these cells with subdiploid DNA ( ⁇ 2n) as determined by PI staining followed by flow cytometry;
  • Figure 20 illustrates the effect of Tat-BH3 peptides on cell morphology showing photographs of Jurkat cells treated for two hours with 100 ⁇ M of (A, B) Tat- BAX( 57-71) or (C, D) Tat-BID( 81-120 ) , stained with the DNA dye Hoescht 33342 and examined by (A, C) phase contrast light microscopy or (B, D) fluorescent microscopy;
  • Figure 21 illustrates the amino acid sequences for murine and human pro-apoptotic family members showing (A) full-length murine BAD and partial human BAD sequences (SEQ ID NOS: 41 and 42), with conservative amino acid substitutions indicated by a dot (.), (B) full-length murine and human BAK sequences (SEQ ID NOS: 43 and 44 ) , (C) full-length murine and human BAX sequences (SEQ ID NOS: 45 and 46), (D) full-length murine and human BID sequences ( SEQ ID NOS : 47 and 48 )
  • Figure 22 illustrates the nucleotide sequences of human cDNAs showing (A) a partial bad cDNA (SEQ ID NO: 50) which encodes a BH3-containing BAD polypeptide, (B) a bak cDNA (SEQ ID NO: 51) encoding full-length BAK, (C) a bax cDNA (SEQ ID NO: 52) encoding full-length BAX, (D) a bid cDNA (SEQ ID NO: 53) encoding full-length BID, and (E) a bik cDNA (SEQ ID NO: 54) encoding full-length BIK.
  • SEQ ID NO: 50 a partial bad cDNA
  • BAK a bak cDNA
  • Bax cDNA SEQ ID NO: 52
  • BAX full-length BAX
  • BID bid cDNA
  • E a bik cDNA
  • the present invention is based, in part, upon the unexpected discovery that BAD, like all other known pro- apoptotic members of the BCL-2 family, has a BH3 domain and that this domain is necessary for BAD's death agonist activity.
  • This discovery was unexpected because BAD has been previously reported as containing only BHl and BH2 domains in common with BCL-2 family members. Yang et al., Cell 80:285-291, 1995, incorporated herein by reference.
  • the BH3 domain is located N-terminal to the BHl domain (Fig.
  • the BH3 domain of BAD is located between the BHl and BH2 domains and indeed partially overlaps the C-terminal portion of the BHl domain (Fig. 2).
  • the heretofore unrecognized presence of a BH3 domain in all known pro-apoptotic members of the BCL-2 family along with the herein described death inducing activity of short BH3-containing polypeptides establishes for the first time that the BH3 domain is sufficient for inducing cell death. It is also believed that peptides as short as five amino acids from the BH3 domain will also have death agonist activity.
  • the present invention provides a BH3 polypeptide of at least 9 and no more than 50 amino acids comprising a BH3 domain of a pro-apoptotic BCL-2 family member.
  • the BH3 domain comprises a nine amino acid sequence as set forth in SEQ ID NO: 40: Leu-Xaa 1 -Xaa 2 -Xaa 3 - Xaa 4 -Asp-Xaa 5 -Xaa 6 -Xaa 7 , wherein Xaa x is Arg or Ala, Xaa 2 is Arg, lie, Leu, Lys, Gin or Cys, Xaa 3 is Met, lie or Val, Xaa 4 is Ser or Gly, Xaa 5 is Glu, Asp or Ser, Xaa 6 is Phe, lie, Leu or Met, and Xaa 7 is Val, Glu, Asn or Asp; or a conservatively substituted variant thereof.
  • a conservatively substituted variant of SEQ ID NO:40 is an amino acid sequence having identity to or conservative amino acid substitutions at any of the nine positions of SEQ ID NO:42.
  • Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains.
  • Conservatively substituted amino acids can be grouped according to the chemical properties of their side chains.
  • one grouping of amino acids includes those amino acids which have neutral and hydrophobic side chains (A, V, L, I, P, W, F, and M); another grouping is those amino acids having neutral and polar side chains (G, S, T, Y, C, N, and Q); another grouping is those amino acids having basic side chains (K, R, and H); another grouping is those amino acids having acidic side chains (D and E); another grouping is those amino acids having aliphatic side chains (G, A, V, L, and I); another grouping is those amino acids having aliphatic-hydroxyl side chains ( S and T ) ; another grouping is those amino acids having amine-containing side chains (N, Q, K, R, and H); another grouping is those amino acids having aromatic side chains (F, Y, and W); and another grouping is those amino acids having sulfur-containing side chains (C and M).
  • A, V, L, I, P, W, F, and M amino acids having neutral and polar side chains
  • a conservatively substituted variant of SEQ ID NO:40 also includes the amino acid sequence of a BH3 domain identified in any subsequently discovered BCL-2 family member which has cell death agonist activity.
  • the BH3 domain is from a mammalian pro-apoptotic BCL-2 family member. More preferably, the BH3 domain is from murine or human BAD, (FIG. 21A) BAK (FIG. 21B), BAX (FIG. 21C), BID (FIG. 21D), or human BIK (FIG. 21E) and comprises an amino acid sequence as set forth in any of SEQ ID NO: 1-9 (FIG 1). Most preferably, the BH3 domain is a human amino acid sequence as set forth in any of SEQ ID N0:1, SEQ IN NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9.
  • the BH3 polypeptide can comprise at least one and up to 41 additional amino acids which flank the BH3 domain or which are contiguous to the N-terminal or C-terminal amino acids of the BH3 domain.
  • the BH3 polypeptide comprises between at least about 9 and about 50 contiguous amino acids and can have a length of any number between 9 and 50. More preferably, the BH3 polypeptide comprises at least 11 amino acids and even more preferably, the BH3 polypeptide is between at least 15 and 24 contiguous amino acids in length.
  • the amino acid sequence of the BH3 polypeptide can be any sequence provided that it includes a BH3 domain as defined above and that the polypeptide has cell death agonist activity.
  • the term "cell death agonist activity" is intended to mean that the BH3 polypeptide is capable of inducing cell death in a similar fashion, although not necessarily to the same degree, as the polypeptides particularly exemplified herein.
  • the cell death agonist activity of a polypeptide can be readily examined using one of the cell assays described herein. It is believed that the amino acid sequence of the BH3 polypeptide should be one which folds in such a manner that the BH3 domain is exposed on the surface of the surface of the polypeptide.
  • the BH3 polypeptide comprises a BH3- containing sequence of between at least 9 and 50 contiguous amino acids from a pro-apoptotic BCL-2 family member.
  • the BH3-containing sequence is from one of the human polypeptide sequences shown in Figure 21: BAD (SEQ ID NO: 41), BAK (SEQ ID NO:42), BAX (SEQ ID NO:43), BID (SEQ ID N0:44) or BIK (SEQ ID NO: 45), or a conservatively substituted variant thereof.
  • a conservatively substituted variant of a BH3- containing sequence means the sequence contains conservative amino acid substitutions of one or more of the amino acids in the naturally occurring sequence.
  • the BH3 polypeptides of the invention can also include unusual amino acids and/or amino acids containing modifications such as glycosylations.
  • Preferred BH3 polypeptides are human BAX polypeptides BAX 53-76 (SEQ ID N0:31), BAX 57-71 (SEQ ID NO:33), BAX 61-71 (SEQ ID NO:35), and a human BID polypeptide, BID 81-100 (SEQ ID NO: 37), which are defined by reference to the full-length BAX and BID sequences (FIGS. 21C and 21D).
  • the BH3 polypeptide comprises human BAX 57-71 which consists of the sequence Lys-Lys-Leu-Ser-Glu-Cys-Leu-Lys-Arg-Ile-Gly- Asp-Glu-Leu-Asp (SEQ ID NO: 33).
  • the invention also provides BH3 domain peptides having cell death agonist activity.
  • a BH3 domain peptide comprises five to eight contiguous amino acids from a BH3 domain as defined by SEQ ID NO: 40, or a conservatively substituted variant thereof.
  • Methods for preparation of the BH3 polypeptides and BH3 domain peptides of the invention include, but are not limited to, chemical synthesis, recombinant DNA techniques or isolation from biological samples.
  • Chemical synthesis of a peptide can be performed, for example, by the classical Merrifeld method of solid phase peptide synthesis (Merrifeld, J Am Chem Soc 85:2149, 1963 which is incorporated by reference) or the FMOC strategy on a Rapid Automated Multiple Peptide Synthesis system (DuPont Company, Wilmington, DE) (Caprino and Han, J Org Chem 37:3404, 1972 which is incorporated by reference).
  • polypeptides and peptides of the present invention are also intended to include non-peptidal substances such as peptide mimetics which possess the death-inducing activity of BH3 polypeptides or BH3 domain peptides.
  • non-peptidal substances such as peptide mimetics which possess the death-inducing activity of BH3 polypeptides or BH3 domain peptides.
  • the techniques for development of peptide mimetics are well known in the art. (See for example, Navia and Peattie, Trends Pharm Sci 14:189-195, 1993; Olson et al, J Med Chem 36:3039-3049 which are incorporated by reference ) . Typically this involves identification and characterization of the interaction between a protein target and its peptide ligand using X- ray crystallography and nuclear magnetic resonance technology.
  • the BH3 polypeptide or BH3 domain peptide is operably linked to a cell penetrating agent.
  • a cell penetrating agent is the 11 amino acid Tat peptide of HIV-I (SEQ ID NO:55).
  • the Tat peptide may be directly fused to the BH3 polypeptide or it may contain a short spacer sequence.
  • the cell penetrating agent can also be a conservatively substituted variant of SEQ ID NO: 55.
  • the present invention also includes therapeutic or pharmaceutical compositions comprising the BH3 polypeptide or BH3 domain peptide in an amount effective to promote death. Also encompassed within the present invention are methods for promoting apoptosis in a target cell comprising administering to the cell a death- promoting effective amount of the BH3 polypeptide.
  • the target cell can be treated ex vivo or it can be present in a patient.
  • compositions and methods are useful for treating diseases or disease conditions in which the cell death signal is down .
  • regulated and the affected cell has an inappropriately diminished propensity for cell death, which is referenced herein as being a decreased apoptotic state.
  • diseases include, for example, cancer, other lymphoproliferative conditions, arthritis, inflammation, autoimmune diseases and the like which may result from a down regulation of cell death regulation.
  • the compositions and methods of the invention are also useful in treating diseases or disease conditions in which it is desirable to kill certain types of cells, such as virus- infected or autoantibody-expressing cells.
  • compositions of the present invention can be administered by any suitable route known in the art including, for example, intravenous, subcutaneous, intramuscular, transdermal, intrathecal or intracerebral or administration to cells in ex vivo treatment protocols. Administration can be either rapid as by injection or over a period of time as by slow infusion or administration of slow release formulation. For treating tissues in the central nervous system, administration can be by injection or infusion into the cerebrospinal fluid (CSF). When it is intended that a BH3 polypeptide be administered to cells in the central nervous system, administration can be with one or more agents capable of promoting penetration of the BH3 polypeptide across the blood-brain barrier.
  • CSF cerebrospinal fluid
  • the polypeptide can also be linked or conjugated with agents that provide desirable pharmaceutical or pharmacodynamic properties.
  • the BH3 polypeptide can be coupled to any substance known in the art to promote penetration or transport across the blood- brain barrier such as an antibody to the transferrin receptor, and administered by intravenous injection.
  • the BH3 polypeptide can be stably linked to a polymer such as polyethylene glycol to obtain desirable properties of solubility, stability, half-life and other pharmaceutically advantageous properties.
  • a polymer such as polyethylene glycol
  • compositions of the invention can also comprise agents which aid in targeting the BH3 polypeptide to a particular cell type and/or delivery into the cytosol of a cell.
  • the BH3 polypeptide can be encapsulated in liposomes that have various targeting ligands on their surface such as monoclonal antibodies that recognize antigens specifically expressed by the target cell or ligands which bind to receptors specific for the target cell.
  • targeting ligands on their surface
  • Such methods are well known in the art (see e.g., Amselem et al., Chem Phys Lipids 64:219-237, 1993 which is incorporated by reference).
  • the BH3 polypeptide can also be administered in a capsule comprised of a biocampatible polymer.
  • compositions can also include absorption enhancers which increase the pore size of the mucosal membrane.
  • absorption enhancers which have been used to enable peptides the size of insulin to be transported across the mucosal membrane, include sodium deoxycholate, sodium glycocholate, dimethyl- ⁇ -cyclodextrin, lauroyl-1- lysophosphatidylcholine and other substances having structural similarities to the phospholipid domains of the mucosal membrane.
  • compositions are usually employed in the form of pharmaceutical preparations. Such preparations are made in a manner well known in the pharmaceutical art.
  • One preferred preparation utilizes a vehicle of physiological saline solution, but it is contemplated that other pharmaceutically acceptable carriers such as physiological concentrations of other non-toxic salts, five percent aqueous glucose solution, sterile water or the like may also be used. It may also be desirable that a suitable buffer be present in the composition.
  • Such solutions can, if desired, be lyophilized and stored in a sterile ampoule ready for reconstitution by the addition of sterile water for ready injection.
  • the primary solvent can be aqueous or alternatively non-aqueous.
  • BID can also be incorporated into a solid or semi-solid biologically compatible matrix which can be implanted into tissues requiring treatment.
  • the carrier can also contain other pharmaceutically-acceptable excipients for modifying or maintaining the pH, osmolarity, viscosity, clarity, color, sterility, stability, rate of dissolution, or odor of the formulation.
  • the carrier may contain still other pharmaceutically-acceptable excipients for modifying or maintaining release or absorption or penetration across the blood-brain barrier.
  • excipients are those substances usually and customarily employed to formulate dosages for parenteral administration in either unit dosage or multi-dose form or for direct infusion by continuous or periodic infusion.
  • formulations containing the BH3 polypeptide are to be administered orally.
  • Such formulations are preferably encapsulated and formulated with suitable carriers in solid dosage forms.
  • suitable carriers, excipients, and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, gelatin, syrup, methyl cellulose, methyl- and propylhydroxybenzoates, talc, magnesium, stearate, water, mineral oil, and the like.
  • the formulations can additionally include lubricating agents, wetting agents, emulsifying and suspending agents, preserving agents, sweetening agents or flavoring agents.
  • the compositions may be formulated so as to provide rapid, sustained, or delayed release of the active ingredients after administration to the patient by employing procedures well known in the art.
  • the formulations can also contain substances that diminish proteolytic degradation and/or substances which promote absorption such as, for example, surface active agents.
  • the specific dose is calculated according to the approximate body weight or body surface area of the patient or the volume of body space to be occupied.
  • the dose will also be calculated dependent upon the particular route of administration selected. Further refinement of the calculations necessary to determine the appropriate dosage for treatment is routinely made by those of ordinary skill in the art. Such calculations can be made without undue experimentation by one skilled in the art in light of the activity disclosed herein in cell death assays. Exact dosages are determined in conjunction with standard dose-response studies.
  • a BH3 polypeptide may be therapeutically administered by implanting into patients vectors or cells capable of producing a biologically-active form of the polypeptide or a precursor thereof, i.e. a molecule that can be readily converted to a biologically-active form of the BH3 polypeptide by the body.
  • cells transformed to express and secrete the BH3 polypeptide may be encapsulated into semipermeable membranes for implantation into a patient. It is preferred that the cell be of human origin and that the BH3 polypeptide have a human amino acid sequence when the patient is human.
  • the formulations and methods herein can be used for veterinary as well as human applications and the term "patient” as used herein is intended to include human and veterinary patients.
  • the BH3 polypeptide can be administered to a target cell by transfecting the cell with a polynucleotide encoding for expression the BH3 polypeptide.
  • the encoding polynucleotide can be targeted to the cell using methods known in the art, such as encapsulating the polynucleotide in liposomes bearing targeting ligands or by non-covalently binding the polynucleotide to a ligand conjugate which directs the polynucleotide to the target cell. See, e.g., Wu et al., U.S. 5,635,383 and WO 95/25809.
  • the invention also provide polynucleotides encoding the BH3 polypeptides described herein.
  • the polynucleotide comprises a nucleotide sequence encoding a BH3 domain consisting of the amino acid sequence set forth in SEQ ID NO:40.
  • Preferred polynucleotides comprise a nucleotide sequence from one of the human cDNA sequences shown in Figure 22: bad (SEQ ID NO:47), bax (SEQ ID N0:48), bak (SEQ ID N0:49), bid (SEQ ID NO: 50), or bik (SEQ ID NO: 51). Preferred embodiments of the invention are described in the following examples.
  • BAD contains a BH3 domain that is required for heterodimerization and cell death.
  • BAD was initially identified by its interaction with BCL-2 and BCL-X L .
  • BCL-2 BCL-X L
  • a nested set of deletion mutants was generated (Fig. 3A) and tested for their ability to interact with BCL-2 protein.
  • the deletion mutants were prepared by inserting fragments of a murine Jbad cDNA with engineered Hindlll and EcoRI sites into the pET17b expression vector in frame with the T7-gene-10 promoter and the resulting recombinant expression vectors were transformed into BL21 cells (Novagen). One hour after inducing expression of the truncated BAD proteins by IPTG (0.1 mM), total cell lysates were prepared. Lysates (40 ⁇ g) were size fractionated by SDS-PAGE and transferred to a nitrocellulose membrane.
  • the resulting blot was hybridized with a 32 P-labeled glutathione s-transferase - BCL-2 (GST-BCL-2) fusion protein according to the protocol of Blanar and Rutter, Science 256:1014-1018, 1992, and the results are shown in Figure 2B.
  • BAD proteins 141-181, 141-172, 141-183, and 141-194 exhibited binding to GST-BCL-2 while the truncated BAD proteins 152-204, 163-204, and 173-204 did not bind to GST-BCL-2. Therefore, a small 31-amino acid region (BAD 141-172) is both sufficient and essential for BAD to heterodimerize with BCL-2.
  • Example 2 This example demonstrates that the BH3 domain is required for BAD's apoptotsis-promoting activity and that BAD deletion mutants lacking the BH3 domain do not bind to BCL-2 or BCL-X L in vitro.
  • BAD- deficient murine embryonic fibroblasts DNA fragments encoding for full-length BAD or truncated BAD proteins (1-181, 1-141, 127-204, and full-length with a deletion from 142 to 165) (Fig. 6A) and engineered to contain BamHI and EcoRI restriction sites were inserted into pcDNA3 ( Invitrogen ) , downstream of T7 and CMV promoters. MEF cells were allowed to grow to about 80% confluence in 12-well plates before transfection.
  • a luciferase reporter plasmid (0.1 mg) was mixed with 0.05 mg of a pcDNA3 recombinant construct or the pcDNA3 vector as a control and 3 ml of lipofectAMINETM (Gibco BRL) and 0.5 ml of the mixture was added to MEF cells for 5 hrs.
  • the transfected cells were lysed 18-20 hrs later and luciferase assays were performed using a standard substrate (Promega). Luciferase activities were quantified by a luminometer (OptocompII, MGM Instruments Inc. ) and the relative luciferase activity for cells co- transfected with a recombinant pcDNA3 construct compared to luciferase activity in cells co-transfected with the control were determined. The means ⁇ ISD of 3 experiments are shown in Fig. 6B.
  • the effect of recombinantly expressed full-length or truncated BAD on cell viability of the BAD-deficient MEF cells can be estimated by its effect on the activity of the co-transfected luciferase gene, with a low relative luciferase activity indicating low cell viability and high activity indicating good cell viability. As expected, lysates of cells co-transfected with full- length BAD (1-204) showed very little cell viability.
  • BAD constructs lacking the BH1/BH3 region (1- 141 and ⁇ 142-165) had substantially diminished death- promoting activity.
  • Example 3 This example demonstrates that binding of BAD to BCL-2 and BCL-X L is affected by single amino acid changes in the BAD BH3 domain.
  • BAD mutant proteins were prepared with the following single- amino acid changes: Gly at position 148 to Ala (G148A); Arg at position 149 to Ala (R149A); and Leu at position 151 to Ala (BADL151A).
  • G148A Gly at position 148 to Ala
  • R149A Arg at position 149 to Ala
  • BADL151A Leu at position 151 to Ala
  • the resulting recombinants were used in an n vitro transcription-translation system (IVTT, Promega) to generate 35 S-labeled wild-type (WT) and mutant BAD proteins, which are shown in the upper panel of FIG. 7A (IVTT). Binding of the 35 S-labeled wild-type and BH1/BH3 mutant BAD proteins to GST-BCL-2 and GST-BCL-X L fusion proteins was assessed by an In vitro binding assay, which was performed as described in Example 2. The amount of radioactively labeled heterodimers captured on GSH agarose beads are shown in the middle and lower panels of FIG. 7A.
  • Example 4 This example demonstrates the ability of BAD BH1/BH3 mutants to bind to BCL-X L in vivo .
  • the recombinant pSFFV expression vectors encoding the wild-type BAD and the BAD mutants described in Example 3 were electroporated into the murine hematopoietic cell line FL5.12 BCL-X L , which overexpresses BCL-X L .
  • Clones expressing similar levels of WT and mutant BAD proteins as well as BCL-X L were identified by probing Western blots of cell lysates with either a rabbit polyclonal anti-BAD antibody (#10929, described in Yang et al., Cell 80: 285-291, 1995) (Fig. 7B, upper panel) or a rabbit polyclonal anti-BCL-XL antibody (13.6, described in Boise et al., Immunity 3 : 87-98, 1995) (Fig. 7B, lower panel ) .
  • BAD/BCL-X L heterodimers were immunoprecipitated from cell lysates using 7B2, a murine monoclonal Ab against human BCL-X L (Boise et al., supra).
  • NP-40 isotonic lysis buffer with freshly added protease inhibitors (142.5 mM KC1, 5 mM MgCl 2 , 10 mM HEPES [pH 7.2], 1 mM EDTA, 0.25% NP-40, 0.2 mM PMSF, 0.1% aprotinin, 1 ⁇ g/ml pepstatin, and 1 ⁇ g/ml leupeptin), incubated on ice for 30 min, and centrifuged at 15,000 X g for 10 min to precipitate nuclei and non-lysed cells.
  • protease inhibitors 142.5 mM KC1, 5 mM MgCl 2 , 10 mM HEPES [pH 7.2], 1 mM EDTA, 0.25% NP-40, 0.2 mM PMSF, 0.1% aprotinin, 1 ⁇ g/ml pepstatin, and 1 ⁇ g/ml leupeptin
  • the mutants BAD G148A and BAD R149A were co- precipitated with BCL-X L in amounts similar to that seen for wild-type BAD (FIG. 7C, compare lanes 2 and 5 with lane 11).
  • 7B2 mAb co-precipitated greatly reduced amounts of BAD L151A with BCL-X L as compared to wild-type BAD (FIG. 7C, compare lanes 8 and 11). Consistent with this, a markedly increased amount of BAD L151A was present in the supernatant (Sup) of this immunoprecipitate compared to the supernatants of the other mutants and wild-type (Sup, compare lane 9 with lanes 3, 6 and 12.
  • Example 5 This example demonstrates the effect of the BH1/BH3 mutations on intracellular distribution of BAD and apoptotic activity.
  • BAD is known to exist as a nonphosphorylated form that heterodimerizes with BCL-2 and BCL-X L at membrane sites and as a hyperphosphorylated form that does not bind to BCL-2 or BCL-X L but instead binds to the 14-3-3 protein in the cytosol ( Zha et al. , supra ) .
  • the inventors compared the intracellular distribution and 14-3-3 binding activity of wild-type BAD and the BH1/BH3 mutants.
  • the above-described FL5.12 cells co-expressing BCL-X L and wild-type or mutant BAD proteins were washed with PBS twice, resuspended in Buffer A ( 10 mM Tris pH 7.5, 25 mM NaF, 5 mM MgCl 2 , 1 mM EGTA, 1 mM DTT, aprotinin 0.15 U/ml, 20 mM leupeptin, 1 mM PMSF) and incubated on ice for fifteen minutes. Cells were then homogenized in a Dounce homogenizer with fifty strokes and nuclei were removed by centrifugation at 500g for ten minutes.
  • Buffer A 10 mM Tris pH 7.5, 25 mM NaF, 5 mM MgCl 2 , 1 mM EGTA, 1 mM DTT, aprotinin 0.15 U/ml, 20 mM leupeptin, 1 mM PMSF
  • the supernatant was further centrifuged at 315,000g for thirty minutes to separate cytosol from crude membranes.
  • Membrane fractions were solubilized in 1% SDS and centrifuged at 12,000g for five minutes at room temperature.
  • the resulting membrane fractions and cytosol fractions were diluted 1:10 in 1% Triton X-100, 100 mM NaCl in buffer A and analyzed by western blot using the 10929 anti-BAD Ab and the results are shown in FIG. 8A.
  • BAD L151A The majority of BAD L151A was present in the cytosoli ⁇ fraction (Cyt), with the more prominent upper band representing the hyperphosphorylated form and the lower band representing the nonphosphorylated form (Fig. 8A, lane 5). In contrast, the majority of wild-type BAD was detected as the nonphosphorylated form in the crude membrane fraction (CM, lane 8) as was the majority of BAD G148A (lane 2).
  • BAD R149A which bears a mutation closer to the BH3 domain than G148A, displayed an intracellular distribution pattern that was intermediate between that observed for BAD G148A and L151A.
  • Binding ability to 14-3-3 was assessed by immunoprecipitation of BAD/14-3-3 complexes from the cytosolic fraction using the anti-BAD mAb 2G11 ( Zha et al., supra ) .
  • the amount of 14-3-3 protein in the immunoprecipitates was analyzed by western blot using an anti-14-3-3 antibody from Upstate Biotechnology, Inc., and the results are shown in FIG 8B.
  • the anti-BAD mAb 2G11 co-precipitated significantly more 14-3-3 protein associated with BAD L151A than with WT BAD or the other mutants. These data indicate that BAD L151A, which is incapable of binding to BCL-X L , is also functionally inactive and localized to the cytosol where it is bound to 14-3-3.
  • Example 7 This example illustrates the effect of BH3 domain mutations on the death agonist activity of BID and the binding of BID to BCL-2 or BAX.
  • BH3-mutant Bid constructs were generated in two steps. First, the 5 ' portion of the molecule was PCR amplified. The 5' primer added an EcoRI site, while the 3' primer ended at the Wiiel site 324 bp into the open reading frame. Second, the amplified EcoRI/Nhel fragment plus the 3' Nhel/EcoRI fragment were ligated into the EcoRI site of pBTM.
  • the BH3 mutants of BID were tested for their binding to BCL-2 and BAX in vitro (Figure 10B). All four mutants tested disrupted BID's interaction with either BCL-2 or BAX. However, the mutants did display different specificities: BIDmIII-1 (M97A,D98A) bound to BAX but not to BCL-2, BIDmIII-3 (G94A) bound to BCL-2 but not BAX, whereas BIDmIII-2 and mlII-4 did not bind to either (Figure 10B).
  • each BID mutant into FL5.12-Bcl-2 cells and selected stable expressing clones.
  • the expression level of BID mutants was comparable to that of a wild-type BID transfectant ( Figure 11B).
  • the ability of each mutant to interact with BCL-2 or BAX was assessed by immunoprecipitation with an anti-BID Ab followed by an anti-BCL-2 or anti-BAX immunoblot ( Figure 11C).
  • Anti- human-BCL-2 monoclonal Ab 6C8 and biotinylated anti- murine-BAX polyclonal Ab 651 were used for blot analyses (1:2000 and 1:500, respectively).
  • Site specific mutagenesis of BID revealed that BH3 was required for death promoting activity. This included the capacity to counter protection by BCL-2 as well as induce a cysteine protease dependent apoptosis when expressed in Jurkat T cells or Rat-1 fibroblasts ( able 1 ) .
  • BIDmIII-3 which binds BCL-2 but not BAX lost its capacity to counter BCL-2 and induce apoptosis.
  • BIDmIII-1 M97A,D98A
  • BIDmIII-1 M97A,D98A
  • Example 8 This example illustrates the effect of mutations in the BH3 domain on the dimerizing and death agonist activities of BAX.
  • the polynucleotides encoding these mutants were subcloned into the mammalian expression vector pSFFV and introduced by electroporation into FL5.12 cells over- expressing BCL-2.
  • Clones expressing exogenous HA-tagged mutant BAX were screened by Western blot with a polyclonal anti-BAX Ab 651, and those with the highest amount of expression were retained.
  • BAX mutants were subcloned into the mammalian expression vector pcDNA3 under the control of a CMV promoter, and were co-transfected with a luciferase reporter into Rat-1 cells. Luciferase activity assays as described above were performed 16-18 hrs after transfection. Co-transfection of wild-type BAX with the luciferase reporter resulted in a 10-fold decrease in luciferase activity (Fig. 13B) reflecting its apoptosis activity. Mutants 1, 3 and 5 retained close to wild-type activity, while mutants 2 and 4 were 6- and 3-fold less potent then wild-type BAX, respectively (Fig. 13C).
  • Rat-1 cells were co- transfected with polynucleotides encoding BCL-2 and wild- type BAX or a BAX mutant.
  • FIG. 13C co- transfection of wild-type BAX and BCL-2 resulted in an intermediate luciferase activity confirming the capacity of BAX to counteract BCL-2.
  • Mutants 1 and 5 retained wild-type like activity, mutant 2 lost 90% of the activity, while mutants 3 and 4 lost 50-60% of the activity.
  • Example 9 This example demonstrates the death-promoting activity of BAX and BID BH3-containing fragments when expressed in cells.
  • the nuclear morphology of cells expressing BAX 53-104 or BID 74-128 was compared to that of cells expressing the respective full-length molecules by staining the cells with Hoechest 33342, which is a DNA-specific dye ( Figure 16).
  • Example 10 This example demonstrates that small BH3-containing BAX and BID fragments fused to a tat-peptide can promote cell death.
  • Polypeptides containing an 11 amino acid sequence from the HIV-I Tat 1 protein (SEQ ID NO: 48) and a wild-type or mutated BH3 domain (m) of BAX or BID with different lengths of flanking region (FIG. 17A) were chemically synthesized.
  • the amino acid sequence in the mutated BH3 domains are scrambled versions of the sequential order of amino acids in wild-type BH3 from BAX of BID. It is believed the Tat sequence facilitates entry of the polypeptide into the cells.
  • These Tat-BH3 polypeptides were added to murine T cell hybridoma 2B4 cells at a concentration of 100 ⁇ M and cell viability was examined 4 hr. later by trypan blue dye exclusion.
  • Tat-BAX( 53-76 )M SEQ ID NO:32
  • Tat-BAX( 57-71 )M SEQ ID NO:34
  • Tat- BID(81-100)M SEQ ID NO:38
  • BAX53-86 displayed cell death agonist activity when expressed by cells (FIG. 15A) and Tat-BID(75- 106) reduced viability of 2B4 cells by more than 40% when trypan blue dye exclusion was determined 19 hours after polypeptide addition (data not shown).
  • Tat-BID(75- 106) reduced viability of 2B4 cells by more than 40% when trypan blue dye exclusion was determined 19 hours after polypeptide addition (data not shown).
  • This data suggests that therapeutic use of polypeptides longer than about 32 amino acids may require that they be administered with additional cell penetrating agents or expressed by polynucleotides transfected into the cell.
  • Example 11 This example demonstrates cell viability exposed illustrates the kinetics and dose-response relationship of cell death induced by Tat-BH3 polypeptides.
  • Tat-BAX( 53-76 ) Tat- BAX( 67-71)
  • Tat BID( 81-100) or their corresponding BH3 mutant derivatives were added at a concentration of 100 ⁇ M to multiple sets of 2B4 cultures and trypan blue dye exclusion was determined at various times after polypeptide addition.
  • Tat- BID( 81-100) achieved its maximum death promoting effect before the Tat-BAX fusion polypeptides, with more than 75% of the 2B4 cells losing viability by 1 hr. after addition of Tat-(BID)81-100 as compared to about 50% or 40% loss of viability in cells treated with Tat-BAX( 57-71 ) or Tat- BAX( 53-76), respectively.
  • Tat- BAX( 57-71) the greatest reduction in cell viability was displayed by Tat- BAX( 57-71), which killed almost all of the cells by that time, with about 15% and 35% of the cells treated with Tat- BID( 81-100) and Tat-BAX( 53-76) being viable.
  • the mutant Tat-BH3 fusion polypeptides did not display significant cell killing activity at early times in the assay.
  • Tat-BAX( 57-71 )m reduced cell viability about 35% by 16 hours, indicating the mutant BH3 domain in this polypeptide has a low level of cell death agonist activity.
  • Tat-BAX( 57-71), Tat-BAX( 57-71 )m, Tat-BID(81- 100), or Tat-BID(81-100)m was added to 2B4 cells at 25, 50, 75, 100, 125, or 150 ⁇ M and two hours later cell viability was determined by trypan blue dye exclusion. The results are shown in FIG. 18B.
  • Tat-BAX( 57-71 ) and Tat- BID( 81-100) were similar, with loss of cell viability increasing with increasing doses of these polypeptides. While the polypeptides were about equally potent at 75 and 100 ⁇ M doses, Tat-BAX( 57-71 ) killed a higher percentage of the 2B4 cells at 50 ⁇ M than a corresponding dose of Tat- BID( 81-100).
  • the Tat fusion polypeptides with mutant BH3 domains displayed no or very little effect on cell viability at all doses tested.
  • Example 12 This example illustrates that the cell death induced by Tat-BH3 fusion polypeptides is not inhibited by BCL-2 and z- VAD-fmk.
  • Duplicate cultures of 2B4 cells transfected with a recombinant vector encoding BCL-2 or control cells (neo) were treated with Tat-BAX( 57-71) or Tat-BID( 81-100) at 100 ⁇ M in the presence or absence of 100 ⁇ M of z-VAD-fmk. Two hours later, cell viability was measured by trypan blue dye exclusion (FIG. 19A) and the percentage of cells with subdiploid DNA ( ⁇ 2n) was determined by PI staining followed by flow cytometry (FIG. 19B).
  • the cell death induced by Tat-BH3 polypeptides added to the cells in culture was not significantly reversed by BCL-2, z-VAD-fmk, or when both BCL-2 and z-VAD-fmk were present (FIG. 19A).
  • the percentage of cells with subdiploid DNA was significantly increased in cultures treated with one of the TatBH3 peptides and this increase was not significantly alleviated by z-VAD-fmk (FIG. 19B).
  • the number of Tat- BID treated cells containing subdiploid DNA was reduced somewhat by BCL-2, but no significant reduction was seen for cells treated with Tat-BAX (FIG. 19B).
  • Example 13 This example demonstrates that cells treated with the Tat-BAX( 57-71) or Tat( BID)81-100 polypeptides are morphologically atypical for apoptotic cells.
  • Jurkat cells were treated for 2 hours with 100 ⁇ M of Tat-BAX(57-71) (FIG. 20A, 20B) or Tat(BID)81-100 (FIG. 20C, 20D).
  • the treated cells were stained with Hoechst 33342 and then examined by phase contrast light microscopy (FIG. 20A, 20C) or fluorescent microscopy (FIG. 20B, 20D).
  • Leu Ala lie lie Gly Asp Asp He Asn 1 5
  • MOLECULE TYPE protein
  • GAGAGCCCAT TCCCACCATT CTACCTGAGG CCAGGACGTC TGGGGTGTGG GGATTGGTGG 1260
  • GACTGACTCT GAAGAGGACC TGGACCCTAT
  • GGAGGACTTC GATTCTTTGG AATGCATGGA 180
  • CATCTACGAC CAGACTGAGG ACATCAGGGA TGTTCTTAGA AGTTTCATGG ACGGTTTCAC 360
  • GGTTTTTTGT TTTTTTTA TTCCAGTTTT CGTTTTTTCT AAAAGATGAA TTCCTATGGC 720 TCTGCAATTG TCACCGGTTA ACTGTGGCCT GTGCCCAGGA AGAGCCATTC ACTCCTGCCC 780

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Abstract

L'invention concerne de petits polypeptides et de petits peptides de 5 à 50 acides aminés présentant une activité agoniste de la mort cellulaire. Les polypeptides ont une longueur d'au moins 9 acides aminés et contiennent le domaine BH3 d'un membre de la famille BCL-2 pro-apoptotique. Les peptides contiennent de 5 à 8 acides aminés du domaine BH3. L'invention concerne également des méthodes favorisant l'apoptose avec ces polypeptides et peptides agonistes de la mort cellulaire, ainsi que les polynucléotides les codant.
PCT/US1998/019765 1997-09-26 1998-09-22 Agonistes de mort cellulaire WO1999016787A1 (fr)

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