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WO1996016989A1 - PROTEINES p53 A DOMAINES DE TETRAMERISATION MODIFIES - Google Patents

PROTEINES p53 A DOMAINES DE TETRAMERISATION MODIFIES Download PDF

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WO1996016989A1
WO1996016989A1 PCT/US1995/015353 US9515353W WO9616989A1 WO 1996016989 A1 WO1996016989 A1 WO 1996016989A1 US 9515353 W US9515353 W US 9515353W WO 9616989 A1 WO9616989 A1 WO 9616989A1
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seq
sequence
gly
pro
residues
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PCT/US1995/015353
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Thanos D. Halazonetis
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The Wistar Institute Of Anatomy And Biology
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Priority claimed from US08/347,792 external-priority patent/US5573925A/en
Priority claimed from US08/431,357 external-priority patent/US5721340A/en
Application filed by The Wistar Institute Of Anatomy And Biology filed Critical The Wistar Institute Of Anatomy And Biology
Priority to AU42884/96A priority Critical patent/AU4288496A/en
Priority to EP95941474A priority patent/EP0799243A4/fr
Publication of WO1996016989A1 publication Critical patent/WO1996016989A1/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
    • C07K14/4746—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used p53
    • 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

  • the present invention relates to the field of p53 proteins with altered oligo erization domains, polynucleotide sequences encoding them, and their use in therapy.
  • Wild-type (wt) p53 is a sequence-specific DNA binding protein found in humans and other mammals, which has tumor suppressor function [See, e.g., Harris (1993), Science, 262: 1980-1981].
  • the wild-type p53 protein functions to regulate cell proliferation and cell death (also known as apoptosis) . It also participates in the response of the cell to DNA damaging agents [Harris (1993), cited above].
  • DNA damaging agents such as radiation and chemotherapeutics commonly used for cancer treatment.
  • nucleotide and amino acid sequences of human p53 are reported below as SEQ ID NOS: 1 and 2, respectively [Zakut-Houri et al, (1985), EMBO J., 4: 1251-1255; GenBank Code Hsp53].
  • the amino acid sequence of p53 is conserved across evolution [Soussi et al, (1990) , Oncogene, 5: 945-952], suggesting that its function is also conserved.
  • p53 is a tetrameric DNA sequence-specific transcription factor. Its DNA binding and transcriptional activities are required for p53 to suppress tumor growth [Pietenpol et al, (1994) , Proc. Natl. Acad. Sci. USA, 91: 1998-2002]. p53 forms homotetramers in the absence of DNA and maintains its tetrameric stoichiometry when bound to DNA [Kraiss et al, (1988), J. Virol., 62: 4737-4744; Stenger et al, (1992), Mol.
  • p53 activates gene transcription from neighboring promoters.
  • the ability of p53 to activate gene transcription has been mapped to within amino acid residues 1-90 of SEQ ID NO: 2 [Fields et al, (1990), Science, 249: 1046-1049].
  • the C-terminus of the human p53 tumor suppressor protein (i.e., amino acids 290-393 of human p53, SEQ ID NO: 2) has two functions. It induces p53 oligomerization and it regulates p53 DNA binding by controlling the conformation of p53 tetramers. These two functions map to independent regions. Oligomerization maps to amino acid residues 322-355 of SEQ ID NO: 2 [Wang et al,
  • Such hetero-tetra ers are biochemically inactive or characterized by considerably reduced activity compared to wild-type p53 tetramers [Milner and Medcalf (1991), Cell, 65: 765-774; Bargonetti et al, (1992), cited above; Farmer et al, (1992), Nature, 358: 83-86; Kern et al, (1992), Science, 256: 827-830].
  • Wild-type p53 tetramers
  • compositions which are not inhibited by endogenous p53, as well as for methods for the uses of such compositions for therapeutic purposes.
  • the present invention provides novel modified p53 proteins, including preferably chimeric proteins formed by the association of sequences of p53 and sequences of other selected proteins, which novel proteins have desirable functional characteristics.
  • the present invention provides p53 proteins with altered tetramerization domains characterized by the ability to form tetramers, bind DNA and activate transcription indistinguishably from wild-type p53, but incapable of forming hetero-tetramers with p53 proteins that have an intact tetramerization domain, such as wild-type p53 or tumor-derived p53 mutants.
  • These p53 proteins of the invention are preferably chimeric proteins, characterized by disruption of the native p53 tetramerization domain and insertion of a heterologous oligomerization domain in a way that preserves tetramerization.
  • the invention provides p53 proteins characterized by restricted DNA binding specificity from an alteration in the way the tetramerization domain orients the DNA binding domains of a p53 tetramer relative to one another.
  • These proteins are characterized by deletion of all or a significant portion of, or disruption of, the region between the DNA binding domain (amino acid residues 90-289 of human p53 of SEQ ID NO: 2) and tetramerization domain (amino acid residues 322-355 of human p53 of SEQ ID NO: 2) .
  • This region (spanning residues 290-321 of human p53 of SEQ ID NO: 2) is considered an extension of the p53 tetramerization domain.
  • the invention provides p53 proteins with both of the characteristics described above, namely: (1) ability to form tetramers, but inability to hetero-tetramerize with p53 proteins having an intact tetramerization domain, such as wild-type p53 or tumor-derived p53 mutants; (2) restricted DNA binding specificity from an alteration in the way that the tetramerization domain orients the DNA binding domains of a p53 tetramer relative to one another.
  • the invention provides further modifications of the p53 proteins provided above. These modifications include: (l) altered transcription activation sequences (amino acid residues 1-90 of human p53 of SEQ ID NO: 2) ; (2) insertion of one or more nuclear localization signals; and (3) replacement of selected regions of the p53 proteins with homologous regions of non-human p53 protein, as well as conventional modifications such as insertion or deletion or substitution of individual amino acid residues throughout the sequence, and the use of linkers between portions of the chimeric proteins.
  • Still another aspect of this invention provides p53 proteins having two or more of the above-described modifications.
  • the present invention provides a nucleic acid sequence encoding a protein of the invention.
  • These nucleic acids may be inserted into an appropriate vector for delivery to patients for gene therapy.
  • the nucleic acids may be inserted into a vector for in vitro expression of a protein of the invention, which is then introduced into patients.
  • the invention provides a method of treating an individual having a condition characterized by abnormal cell proliferation by delivering a protein or, preferably, a nucleic acid sequence, of the invention to the patient.
  • Fig. IA schematically illustrates wild-type p53.
  • the amino acid numbering which is also maintained throughout Figs. 1A-1K, refers to the residues of human p53 as indicated in SEQ ID NO: 2.
  • the entire length of human p53 is 393 amino acids.
  • Symbols for the DNA binding domain (residues 90-290 of SEQ ID NO: 2) (checkerboard bar) and the oligomerization domain (residues 322-355 of SEQ ID NO: 2) (solid bar) are maintained throughout Figs. 1A-1K.
  • Fig. IB schematically illustrates a heterologous dimerization domain p53 chimeric protein containing residues 1-346 of p53 [SEQ ID NO: 2], a glutamic acid for cloning convenience and a GCN4 dimerization domain corresponding to residues 253-281 of GCN4 of SEQ ID NO: 4 (hatched bar) .
  • Fig. IC schematically illustrates a heterologous dimerization domain p53 chimeric protein containing residues 1-347 of p53 [SEQ ID NO: 2] and residues 253-281 of GCN4 of SEQ ID NO: 4 (hatched bar).
  • Fig. ID schematically illustrates a heterologous dimerization domain chimeric p53 protein containing an insertion of a glutamic acid and residues 253-281 of GCN4 of SEQ ID NO: 4 (hatched bar) between residues 346 and 347 Of p53 [SEQ ID NO: 2].
  • Fig. IE schematically illustrates a heterologous dimerization domain chimeric p53 protein containing residues 1-356 of human p53 [SEQ ID NO: 2] with a mutation within the native p53 oligomerization domain (leucine 344 to alanine) linked to the dipeptide glycine-asparagine and residues 253-281 of GCN4 of SEQ ID NO: 4 (hatched bar) .
  • Fig. IF schematically illustrates a heterologous tetramerization domain chimeric p53 protein which contains residues 1-334 of human p53 [SEQ ID NO: 2] linked to an asparagine and then to a tetrameric variant of GCN4 residues 249-281 of SEQ ID NO: 6 (hatched bar) .
  • Fig. IG schematically illustrates a heterologous tetramerization domain chimeric p53 protein which contains residues 1-325 of human p53 [SEQ ID NO: 2] linked to the tripeptide arginine-glycine-asparagine [SEQ ID NO: 7] and then to a tetrameric variant of GCN4 residues 249-281 of SEQ ID NO: 6 (hatched bar) .
  • Fig. 1H schematically illustrates a heterologous tetramerization domain chimeric p53 protein which contains residues 1-323 of human p53 [SEQ ID NO: 2] linked to the hexapeptide arginine-glycine-glycine- asparagine-proline-glutamic acid [SEQ ID NO: 8] and then to a tetrameric variant of GCN4 residues 250-281 of SEQ ID NO: 6 (hatched bar) .
  • Fig. 1J schematically illustrates a heterologous tetramerization domain chimeric p53 protein which contains residues 1-300 of human p53 [SEQ ID NO: 2] linked to the pentapeptide glycine-glycine-asparagine- glutamine-alanine [SEQ ID NO: 9] and then to a tetrameric variant of GCN4 residues 250-281 of SEQ ID NO: 6 (hatched bar) .
  • Fig. IK schematically illustrates a heterologous tetramerization domain chimeric p53 protein which contains residues 1-325 of human p53 [SEQ ID NO: 2] linked to the tripeptide arginine-glycine-asparagine [SEQ ID NO: 7], a tetrameric variant of GCN4 residues 249-281 of SEQ ID NO: 6 and an isoleucine (hatched bar) , and then followed by residues 352-393 of human p53 [SEQ ID NO: 2].
  • Fig. 2A schematically illustrates the chimeric p53 protein of Fig. IF, which serves as a paradigm to indicate the various modifications that can be introduced into any of the p53 proteins of this invention (Figs.
  • FIG. 2B-2F schematically illustrates a deletion of residues 300-327 of human p53 [SEQ ID NO: 2], that confers novel DNA binding specificities.
  • Fig. 2C schematically illustrates the substitution of the transcription activation domain of p53 with that of the herpes simplex virus protein VP16 (reverse hatched bar) , also known as ⁇ trans-inducing factor.
  • Fig. 2D schematically illustrates the insertion of a nuclear localization signal (NLS) between amino acid residues 80 and 81 of p53 [SEQ ID NO: 2] (horizontal lined bar) .
  • the abbreviation a.a. represents amino acids.
  • Fig. 2E schematically illustrates the substitution of human p53 residues 3-80 of SEQ ID NO: 2 with the corresponding xenopus sequences (cross-hatched bar) .
  • Fig. 2F schematically illustrates two mutations that enhance function of the p53 proteins [SEQ ID NO: 2] of this invention, such as substitution of Arg 174 with Gin, or Arg 175 with Leu.
  • Fig. 3A schematically illustrates a wild-type p53 tetramer bound to a DNA site containing four contiguous pentanucleotides repeats.
  • the p53 DNA binding domains are shown as circles, the oligomerization domain as a thin rectangle, the linker between the DNA binding and oligomerization domains as curved lines, the DNA as a thick rectangle and the specific pentanucleotides as arrows.
  • Fig. 3B schematically illustrates a wild-type p53 tetramer bound to a DNA site containing two pentanucleotide pairs separated by a 20-30 nucleotide insert.
  • Figs. 3C and 3D schematically illustrate a p53 tetramer with antiparallel alignment of its oligomerization domains and a short linker between the DNA binding and oligomerization domains.
  • Such a p53 tetramer cannot bind to a DNA site containing four contiguous pentanucleotides repeats (Fig. 3C) , but can bind to a DNA site containing two pentanucleotide pairs separated by a 20-30 nucleotide insert (Fig. 3D) .
  • Fig. 3E schematically illustrates a chimeric p53 tetramer with parallel alignment of its oligomerization domains bound to a DNA site containing four contiguous pentanucleotides repeats.
  • Fig. 3F schematically illustrates a chimeric p53 tetramer with parallel alignment of its oligomerization domains bound to a DNA site containing two pentanucleotide pairs separated by a 20-30 nucleotide insert.
  • Figs. 3G and 3H schematically illustrate a chimeric p53 tetramer with parallel alignment of its oligomerization domains and a short linker between the DNA binding and oligomerization domains.
  • a p53 tetramer can bind to a DNA site containing four contiguous pentanucleotides repeats (Fig. 3G) , but not to a DNA site containing two pentanucleotide pairs separated by a 20-30 nucleotide insert (Fig. 3H) .
  • Fig. 4 is a bar graph demonstrating the tumor suppressing activities of the proteins encoded by the listed expression plasmids, presented as means + l standard error of G418 resistant colonies per plate.
  • FIG. 5 is a graph charting tumor suppressor activities of p53 proteins in the presence of the p53 tumor-derived mutant tryptophan 248 (W248) . The results are presented relative to activity in the absence of p53W248.
  • Fig. 6A schematically illustrates how a c-Jun modified leucine zipper directs parallel assembly of p53 - c-Jun chimeric proteins.
  • the p53 segment is indicated as a rectangle with rounded edges
  • the c-Jun zipper as a rectangle with sharp edges
  • the leucine (Leu) and isoleucine (lie) residues which mediate oligomerization are indicated.
  • the p53 - c-Jun chimera forms tetramers, but for simplicity only two of the subunits are indicated.
  • Fig. 6B schematically illustrates how a c-Jun modified leucine zipper can direct antiparallel assembly of p53 - c-Jun chimeric proteins.
  • the number of hydrophobic interactions are the same whether the zippers assemble parallel (Fig. 6A) or antiparallel (Fig. 6B) .
  • Fig. 6C schematically illustrates how substitution of one isoleucine (lie) of the c-Jun modified leucine zipper with asparagine (Asn) compromises one hydrophobic interaction when the p53 - c-Jun chimeric proteins are assembled parallel.
  • Fig. 6D schematically illustrates how substitution of one isoleucine (lie) of the c-Jun modified leucine zipper with asparagine (Asn) compromises two hydrophobic interactions when the p53 - c-Jun chimeric proteins are assembled antiparallel.
  • Fig. 7 is a graph charting the tumor suppressor activities of p53-zipper chimera in Saos-2 cells using the colony-forming assay.
  • the present invention provides p53 proteins with modifications in the native p53 sequence. These modifications, which do not interfere with its native tumor-suppressor function, provide the protein with at least one of the following functional characteristics: (1) the ability to bind DNA and activate transcription like wild-type p53, but to not hetero-oligo erize with wild-type p53 or tumor-derived p53 mutants; and (2) restricted DNA binding specificity from an alteration in the way that the tetramerization domain orients the DNA binding domains of a p53 tetramer relative to one another.
  • nucleic acids encoding such proteins and methods of using such proteins or nucleic acid sequences therapeutically are provided.
  • a dimerization domain is defined as a domain that allows formation of dimers, while a tetramerization domain is defined as a domain that allows formation of tetramers.
  • An oligomerization domain allows formation of oligomers, which can be of any subunit stoichiometry (of course greater than one).
  • the term oligomerization domain is more general and encompasses both dimerization and tetramerization domains (which direct formation of oligomers of subunit stoichiometries 2 and 4, respectively) .
  • the term chimeric protein refers to a protein containing sequences from two different proteins, for example from p53 and GCN4.
  • a protein of this invention is comprised of a p53 protein bearing a partial functional inactivation of its tetramerization domain and a heterologous dimerization domain.
  • certain regions of the p53 tetramerization domain must be maintained (so that the chimeric protein can form tetramers, in spite of containing a heterologous dimerization domain) , while other regions are inactivated (so that tetramerization is dependent on the heterologous dimerization domain) .
  • the p53 tetramerization domain maps to residues 322-355 of SEQ ID NO: 2 [Wang et al, (1994), cited above; Clore et al, (1994), cited above].
  • a disruption of the p53 tetramerization domain involving residues 335-348 of SEQ ID NO: 2 or a subset of these residues, sufficiently disrupts the function of this domain, so that it can no longer drive tetramerization with wild-type p53 or tumor-derived p53 mutants.
  • introduction of a heterologous dimerization domain reestablishes the ability to form tetramers, which is mediated both by the heterologous dimerization domain and by the residual tetramerization domain of p53.
  • a heterologous dimerization domain is defined herein as a sequence of amino acids heterologous to p53 and capable of forming homodi ers.
  • One example of a dimerization domain is the leucine zipper (LZ) element.
  • LZ leucine zipper
  • a leucine zipper has been defined as stretch of about 35 amino acids containing 4-5 leucine residues separated from each other by six amino acids [Maniatis and Abel (1989), Nature, 341: 24-25].
  • the leucine zipper occurs in a variety of evikaryotic DNA binding proteins, such as GCN4, C/EBP, c-Fos, c-Jun, c-Myc and c-Max.
  • Heterologous dimerization domains may also be selected from other proteins, such as the retinoic acid receptor, the thyroid hormone receptor or other nuclear hormone receptors [Kurokawa et al, (1993), Genes Dev. , 7:1423-1435] or from the yeast transcription factors Gal4 and HAPl [Marmonstein et al, (1992), Nature, 356:408-414; Zhang et al, (1993), Proc. Natl. Acad. Sci. USA, 90:2851-2855].
  • proteins such as the retinoic acid receptor, the thyroid hormone receptor or other nuclear hormone receptors [Kurokawa et al, (1993), Genes Dev. , 7:1423-1435] or from the yeast transcription factors Gal4 and HAPl [Marmonstein et al, (1992), Nature, 356:408-414; Zhang et al, (1993), Proc. Natl. Acad. Sci. USA, 90:2851-2855].
  • dimerization domains including artificial dimerization domains [O'Shea et al, (1992), Cell, 68:699-708; Krylov et al, (1994), EMBO J., 13: 2849-2861].
  • the leucine zipper of the yeast transcription factor GCN4 is used herein as the exemplary dimerization domain.
  • the nucleotide and amino acid sequences of GCN4 are presented as SEQ ID NO: 3 and NO: 4, respectively. The numbering of the GCN4 nucleotide and amino acid residues follows Hinnenbusch (1984) Proc. Natl. Acad. Sci.
  • GCN4 The coding region of GCN4 is encompassed by nucleotide 778-1623 of SEQ ID NO: 3.
  • the nucleotide and amino acid sequence are found in GenBank under the Code Yscgcn4.
  • Partial functional inactivation of the p53 tetramerization domain can be accomplished by deletions, insertions and/or amino acid substitutions targeting part of this domain. Such mutations should involve residues 335-348 of SEQ ID NO: 2 or a subset of these residues, but need not be confined within the p53 tetramerization domain. For example, a deletion whose N-terminal boundary is within residues 335-348 of SEQ ID NO: 2 may extend as far as the p53 C-terminus. The precise boundaries of the mutations will depend on the nature of the heterologous dimerization domain and the presence, if any, of amino acid sequences introduced for cloning or other purposes between p53 and the heterologous dimerization domain.
  • residues 1-346 of human p53 [SEQ ID NO: 2] are juxtaposed to the dimerization domain of GCN4 (residues 253-281 of GCN4 SEQ ID NO: 4) through a glutamic acid linker (Fig. IB) .
  • residues 1-347 of human p53 [SEQ ID NO: 2] are juxtaposed to residues 253-281 of GCN4 [SEQ ID NO: 4] (Fig. IC) .
  • the function of the p53 tetramerization domain may be partially disrupted by insertion of the heterologous dimerization domain within the p53 tetramerization domain and preferably between residues 335 and 348 of human p53 [SEQ ID NO: 2].
  • a glutamic acid and residues 253-281 of GCN4 [SEQ ID NO: 4] are inserted between residues 346 and 347 of human p53 [SEQ ID NO:2].
  • the function of the p53 tetramerization domain may be partially disrupted by insertions, deletions or amino acid substitutions, while the heterologous dimerization domain is inserted outside the boundaries of the p53 tetramerization domain.
  • the mutations should again target residues 335-348 of human p53 [SEQ ID NO: 2], or a subset thereof.
  • the function of the p53 [SEQ ID NO: 2] tetramerization domain is inactivated by substitution of residue 344 by alanine. This mutation only partially disrupts the function of the p53 tetramerization domain (see Examples section) .
  • a heterologous dimerization domain can then be inserted even outside the p53 tetramerization domain, for example following residue 356 of human p53 [SEQ ID NO: 2], to reestablish tetramer formation (Fig. IE) .
  • Fig. IE human p53 [SEQ ID NO: 2]
  • At least two novel features characterize the class of proteins described here. First these chimeric proteins form tetramers. This was unexpected because the disruption in the p53 tetramerization domain is of sufficient magnitude to disrupt p53 tetramers into monomers. Yet, when the heterologous dimerization domain is introduced, the chimeric protein forms tetramers, rather than dimers, as would be expected.
  • a second novel feature of these chimeric proteins is that their ability to form tetramers with wild-type p53 or with tumor-derived p53 mutants is greatly reduced. This is surprising, because these proteins must utilize p53 structural determinants to form tetramers (recall that in the invention a heterologous dimerization domain is juxtaposed to the p53 sequence) .
  • the p53 protein bears a partial or preferably a complete functional inactivation of its tetramerization domain and contains a heterologous tetramerization domain.
  • a heterologous tetramerization domain is defined as a sequence of amino acids heterologous to p53 and capable of forming stable homo-tetramers.
  • exemplary suitable tetramerization domains include that of the lac repressor, or an artificial tetramerization domain, such as variants of the GCN4 leucine zipper that form tetramers [Alberti et al, (1993), EMBO J., 12: 3227-3236; Harbury et al, (1993), Science, 262: 1401-1407; Krylov et al, (1994), cited above].
  • One of skill in the art could readily select alternate tetramerization domains.
  • the tetrameric variant of the GCN4 leucine zipper [Harbury et al, (1993) , cited above] is used herein as the exemplary tetramerization domain.
  • This variant has isoleucines at positions d of the coiled coil and leucines at positions a, in contrast to the original zipper which has leucines and valines, respectively [Harbury et al, (1993), cited above].
  • the nucleotide and amino acid sequences of this tetrameric leucine zipper variant are presented in the context of the full-length sequences, as SEQ ID NO: 5 and NO: 6, respectively. The numbering of the amino acid residues follows Ellenberger et al, (1992) [cited above].
  • the insertion of the tetramerization domain in the p53 chimeric protein can be quite liberal, provided the functions of the transcription activation (also known as transactivation) and DNA binding domains are not disrupted.
  • the heterologous tetramerization domain would be inserted C-terminally to residue 290 of human p53 [SEQ ID NO: 2], since this maintains the integrity of both the transactivation and DNA binding domains.
  • Functional inactivation of the p53 tetramerization domain can be accomplished by deletions, insertions and/or amino acid substitutions.
  • Such mutations should involve residues 322-355 of SEQ ID NO: 2, or a subset of these residues, since the p53 tetramerization domain maps to these residues [Wang et al, (1994), cited above; Clore et al, (1994), cited above].
  • selected mutations target residues 328-348 of human p53 [SEQ ID NO: 2], or a subset thereof. Within this region the most critical residues for tetramer formation are residues 337, 341 and 344 of SEQ ID NO: 2. However, mutation of other residues within the regions indicated above can disrupt tetramer formation.
  • mutations should involve residues 322-355 [SEQ ID NO: 2], or a subset thereof, they need not be confined within the p53 tetramerization domain. Thus, they can extend as far N-terminally as residue 290 of human p53 [SEQ ID NO: 2] or as far as the p53 C-terminus (residue 393 of SEQ ID NO: 2) .
  • functional inactivation of the p53 tetramerization domain can be accomplished by inserting the heterologous tetramerization domain within residues 322-355 of human p53 [SEQ ID NO: 2], and preferably within residues 328-348 of SEQ ID NO: 2.
  • the chimeric protein comprises a p53 sequence spanning amino acids 1 to 334 of human p53 [SEQ ID NO:2] fused to an asparagine linker and then to a tetrameric variant of GCN4 residues 249-281 [SEQ ID NO:6] (Fig. IF) .
  • the chimeric protein comprises a p53 sequence spanning amino acids 1 to 325 of human p53 [SEQ ID NO:2] fused to an arginine-glycine-asparagine linker [SEQ ID NO: 7] and then to a tetrameric variant of GCN4 residues 249-281 [SEQ ID NO:6] (Fig. IG) .
  • the chimeric protein comprises a p53 sequence spanning amino acids 1 to 323 of human p53 [SEQ ID NO:2] fused to an arginine-glycine-glycine-asparagine-proline-glutamic acid linker [SEQ ID NO: 8] and then to a tetrameric variant of GCN4 residues 250-281 [SEQ ID NO:6] (Fig. 1H) .
  • the chimeric protein comprises a p53 sequence spanning from amino acid 1 to 300 of human p53 [SEQ ID NO:2] fused to a glycine-glycine-asparagine- glutamine-alanine linker [SEQ ID NO: 9] and then to a tetrameric variant of GCN4 residues 250-281 [SEQ ID NO:6] (Fig. 1J) .
  • the chimeric protein comprises a p53 sequence spanning amino acids 1 to 325 of human p53 [SEQ ID NO:2] fused to an arginine-glycine- asparagine linker [SEQ ID NO: 7], a tetrameric variant of GCN4 residues 249-281 [SEQ ID NO:6], an isoleucine linker and then to residues 352-393 of human p53 [SEQ ID NO:2] (Fig. IK) .
  • a heterologous tetramerization domain would not be able to substitute for the native p53 tetramerization domain, because the function of the tetramerization domain is not only to drive tetramerization, but also to position the subunits appropriately relative to one another, so that the p53 tetramer can align to the DNA site.
  • the tetrameric variant of the GCN4 leucine zipper would be expected to be a particularly unsuitable choice for a heterologous tetramerization domain, since it drives parallel subunit assembly [Harbury et al, (1993) , cited above] , while the native p53 tetramerization domain drives antiparallel assembly [Clore et al, (1994), cited above; Sakamoto et al, (1994), Proc. Natl. Acad. Sci. USA, 91: 8974-8978]. Nevertheless, the inventor observed that such chimeric proteins bound DNA as homotetramers with very high efficiency.
  • p53 subunits align antiparallel in the absence of DNA, they adopt a parallel orientation upon DNA binding.
  • a heterologous tetramerization domain that drives parallel assembly of p53 subunits such as the tetrameric variant of the GCN4 leucine zipper, is compatible with DNA binding.
  • the proteins described in this section form homotetramers and maintain high affinity for the specific p53 DNA sites, but do not maintain the integrity of the native p53 oligomerization domain, they do not form hetero-tetramers with wild-type p53 or tumor-derived p53 mutants, and thus will display tumor suppressing activity even in cancer cells expressing high amounts of mutant p53. Additional p53 proteins of this invention can be generated by one of skill in the art following the teachings herein.
  • the p53 proteins described herein contain modifications. These modifications can be trivial (defined as having no effect on function) or beneficial (i.e. they improve upon some aspect of the protein) , and can include deletions, insertions, amino acid substitutions and/or replacement of functional domains or regions of functional domains by functionally equivalent domains or regions of other proteins.
  • modifications of the p53 proteins encompassed by the invention are illustrated in Figs. 2A through 2F. It is understood that the proteins of the invention may contain more than one of the modifications described below.
  • the following modification may be made in the context of wild-type p53 or in the context of the p53 proteins described in sections Al and A2 above.
  • This modification restricts the DNA binding specificities of the above mentioned proteins and involves a change in the length of the sequence between the p53 DNA binding and tetramerization domains.
  • This modification does not affect the ability of p53 to tetramerize, rather it affects the positioning of the DNA binding domains relative to one another in a p53 tetramer.
  • this modification is considered an alteration of the p53 tetramerization domain, as it affects a function of the tetramerization domain and involves sequences that are extensions of the p53 tetramerization domain.
  • Changing the length of the sequences between the DNA binding and tetramerization domains can affect the DNA binding properties of wild-type p53 or of a chimeric p53 protein of this invention both in terms of sequence specificity and affinity for DNA. Such changes can therefore confer desired properties.
  • the inventor realizes that the tetramerization domain of p53 is the site at which four p53 subunits contact each other.
  • the positioning of the four p53 DNA binding domains relative to each other is dependent on the length of the sequence between the C-terminal boundary of the DNA binding domain (residue 289 of human p53, [SEQ ID NO: 2]) and the N-terminal boundary of the tetramerization domain (residue 322 for human wild-type p53, [SEQ ID N0:2]).
  • a long linker such as the linker present in wild-type p53 (i.e., residues 289-322 of SEQ ID NO: 2) provides freedom in positioning the DNA binding domains relative to one another, which in turn allows p53 to bind to different types of DNA sites.
  • a long linker reduces the affinity for DNA, since it allows p53 to adopt multiple conformations, only one of which is compatible with a specific DNA site.
  • a short linker allows p53 to bind only to specific types of DNA sites, but the affinity for these sites is increased because p53 can adopt few alternate conformations.
  • Figs. 3A-3H illustrate the effect of deletions between these two domains.
  • Fig. 3A shows a schematic of a wild-type p53 tetramer bound to a DNA site with contiguous pentanucleotides.
  • Figure 3B shows the same p53 tetramer bound to a DNA site with a 20-30 nucleotide insert between the 2 pentanucleotide pairs. From Figs. 3A and 3B it is apparent that the naturally-occurring sequence between the tetramerization and DNA binding domains provides the flexibility for wild-type p53 to recognize both types of DNA sites.
  • Figs. 3C and 3D the sequences (linkers) between the tetramerization and DNA binding domains are shortened. This is performed by deletions within the region spanning residues 290-327 of human p53 [SEQ ID NO: 2], preferably involving more than 22 amino acids. Such deletions in the context of wild-type p53 limit the ability to position one pair of DNA binding domains close enough to the other pair.
  • Fig. 3C DNA sites with contiguous pentanucleotides cannot be recognized.
  • the same deletions do not limit the ability to recognize DNA sites with a 20-30 nucleotide insert between the two pentanucleotide pairs (Fig. 3D) .
  • wild-type p53, and p53 mutants lacking residues 290-297 of SEQ ID NO: 2 or 300-308 of SEQ ID NO: 2 or 300-317 of SEQ ID NO: 2 or 300-321 of SEQ ID NO: 2 bind to both types of DNA sites.
  • This chimeric protein can bind to both types of DNA sites via flexibility in positioning its DNA binding domains. Deletions within the sequences between the DNA binding and tetramerization domains create the opposite effect than the one observed for wild-type p53.
  • a short linker preferably by the deletion of 22 or more amino acids between residues 290 and 334 of SEQ ID NO: 2, allows p53 chimeras with parallel tetramerization domains to recognize only the DNA sites with contiguous pentanucleotides (Figs. 3G and 3H) .
  • the inserted sequences are p53 or non-p53 sequences. It is most meaningful to introduce insertions in the context of p53 proteins of this invention with very short sequences (i.e., 0 to about 12 amino acid residues) between the DNA binding and tetramerization domains, for example the protein of Fig. 1J, to expand the range of DNA sites they can recognize. Finally the length of the sequences between the DNA binding and tetramerization domains can be altered by changing the site of insertion of the heterologous oligomerization domain.
  • wild-type p53 recognizes DNA sites with 20 or more nucleotides between the two pairs of contiguous pentanucleotides, as well as to the observation that changes in the length of the sequences between the p53 DNA binding and tetramerization domains of p53 modulate its ability to bind to the different types of DNA sites. It has not been appreciated before that wild-type p53 can bind DNA sites, where the pairs of contiguous pentanucleotide repeats are separated by as many as 20 or more nucleotides. While it had been established that wild-type p53 can bind to the mdm2 site, it was thought that this site actually contains two DNA sites (each comprising four contiguous pentanucleotides) , as indicated below:
  • the therapeutic significance of altering the DNA binding properties of the p53 chimeric proteins, or of wild-type p53 relates to the biological consequences of activation of the different p53 target genes. More specifically, induction of the wafl gene which contains four contiguous pentanucleotides leads to tumor suppression [El-Deiry et al, (1993), cited above], and is thus desirable for cancer therapy.
  • the nucleotide sequence of this wafl site is: -GAACA-TGTCC-CAACA-TGTTG- [SEQ ID NO: 10].
  • mdm2 induction of the mdm2 gene leads to expression of the Mdm2 protein, which in turn downregulates the activity of p53 by masking its transactivation domain [Oliner et al, (1992), Nature, 358: 80-83; Momand et al, (1992), Cell, 69: 1237-1245; Oliner et al, (1993), Nature, 362: 857-860; Wu et al, (1993), cited above], and is thus undesirable for cancer therapy.
  • the mdm2 site contains two pairs of contiguous pentanucleotides separated by more than thirty nucleotides [Wu et al, (1993), cited above]. This site is as follows:
  • p53 proteins of this invention are constructed as described above that recognize only DNA sites with contiguous pentanucleotides or only DNA sites with 20 to 30 nucleotide inserts between the two pentanucleotide pairs.
  • Such a p53 protein of this invention that recognizes the wafl DNA site, but not the mdm2 DNA site, has the ability to suppress tumor growth, but is not subject to negative regulatory feedback by Mdm2.
  • One exemplary p53 protein bearing a modification in the length of the sequences between the DNA binding and tetramerization domains is shown in Fig. 2B, i.e., deletion of residues 300-327 of SEQ ID NO: 2.
  • the p53 transactivation (also known as transcription activation) domain contained within amino acids 1-90 of human p53 [SEQ ID NO: 2] is substituted with that of another protein, e.g., the herpes simplex virus protein VP16, also known as ⁇ trans-inducing factor [Pellett et al,
  • nucleotide sequence spanning VP16 nucleotides 2074 - 2307 [Pellett et al, (1985) cited above] is reported as nucleotides 1 - 234 in SEQ ID NO: 12.
  • amino acid sequence of the VP16 HSV fragment from amino acid 402 through 479 [Pellett et al, (1985) cited above] is reported as amino acid 1 - 78 in SEQ ID NO: 13. See, also, GenBank Code Helcg.
  • Fig. 2C One exemplary modified p53 protein of the invention is illustrated in Fig. 2C, in which amino acid residues 402-479 of VP16 [aa 1-78 of SEQ ID NO: 13] have replaced amino acid residues 3-80 of human p53 [SEQ ID NO:2].
  • An equivalent substitution has been reported to maintain p53 tumor suppressor function [Pietenpol et al, (1994), cited above] .
  • the advantage of this substitution is that in certain tumors, overexpression of the Mdm2 protein suppresses p53-mediated transcription by masking its transactivation domain [Oliner et al, (1992) , cited above; Momand et al, (1992), cited above; Oliner et al, (1993), cited above].
  • Wild-type p53 contains three nuclear localization signals (NLS) , all of which map to the C-terminus of wild-type p53 and specifically to residues 316-325, 369-375 and 379-384 of p53 [SEQ ID NO: 2] [Shaulsky et al, (1990), Mol. Cell. Biol., 10: 6565-6577].
  • NLS nuclear localization signals
  • an analog of the p53 proteins described above may contain a NLS fused to its N-terminus, or its C-terminus, or at the junction of the transactivation and DNA binding domains or at the junction of the DNA binding and tetramerization domains or elsewhere in the protein, as long as the function of the p53 protein is not disrupted by insertion of the NLS.
  • Fig. 2D demonstrates the insertion of a NLS at the boundary of the transactivation and DNA binding domains.
  • the NLS may be that of p53 or of any other nuclear protein, such as the NLS of SV40 large T antigen which is comprised of amino acids proline-lysine-lysine-lysine- arginine-lysine-valine [SEQ ID NO: 14] [Kalderon et al, (1984), Cell, 39: 499-509]. Additional heterologous NLS are described by Shaulsky et al, (1990); (1991) [cited above] .
  • human p53 [SEQ ID NO: 2] sequences with equivalent non-human sequences relates to the realization that interactions of p53 with specific cellular or viral proteins are species-specific.
  • human p53 is inactivated by the human Mdm2 protein [Oliner et al, (1993), cited above; Momand et al, (1992), cited above; Wu et al, (1993), cited above].
  • Non-human p53 sequences have lower or no affinity for the human Mdm2.
  • p53 proteins of this invention that contain non-human p53 sequences are not susceptible to inhibition by Mdm2.
  • the species of p53 that can be used to substitute for the human p53 sequences can readily be selected by one of skill in the art. Species, such as xenopus and trout, that diverge most from human p53 [Soussi et al, (1990), cited above] are preferred, although other species may also be selected. As an exemplary modification of this type, residues 3-80 of human p53 [SEQ ID NO: 2] are substituted by the homologous xenopus sequence (Fig. 2E) to produce a modified p53 protein incapable of interacting with Mdm2. B5. Amino Acid Substitutions, Deletions and Insertions
  • modifications of the p53 proteins described in this invention include amino acid substitutions, small deletions and small insertions. (Deletions and insertions within the sequences between the DNA binding and tetramerization domains are discussed in section Bl above.) These modifications involve either the p53 sequences or the heterologous oligomerization domain sequences or both. The modifications may enhance function or introduce a useful property. For example a modification may introduce a tag to optimize protein purification [Scopes (1994) , Protein Purification, Principles and Practice, third edition, Springer-Verlag, New York] , or may enhance expression and/or stability of a p53 protein of the invention when expressed in vitro or in a patient.
  • Modifications in the p53 fragment may enhance DNA binding and growth suppressing activities. Two such modifications have already been described: substitution of arginine 174 with glutamine or of arginine 175 with leucine (the numbering refers to human p53 [SEQ ID NO: 2]; in mouse p53 the corresponding residues are 171 and 172 of SEQ ID NO: 15, respectively) [Halazonetis and Kandil (1993), cited above; Li et al, (1994), Cell Growth Differentiation, 5: 711-721].
  • Modifications in p53 may also affect interaction with cellular or viral proteins, for example, substitution of leucine 14 of SEQ ID NO: 2 with glutamine and phenylalanine 19 of SEQ ID NO: 2 with serine abolish the p53-Mdm2 interaction [Lin et al, (1994), cited above].
  • Modifications in the heterologous oligomerization domain may increase the stability of tetramer formation, for example, substitutions that stabilize oligomerization driven by leucine zippers are known [Krylov et al, (1994), cited above; O'Shea et al, (1992), cited above].
  • residues 174 or 175 of human p53 [SEQ ID NO: 2] are substituted by glutamine or leucine, respectively (Fig. 2F) in a p53 chimeric protein of this invention.
  • FIG. IC amino acid or peptide linker between the p53 fragment and the heterologous oligomerization domain.
  • FIG. IC there is no linker between p53 and the GCN4 leucine zipper.
  • Figs. IB, IF and IK there are glutamic acid or asparagine or isoleucine linkers, respectively.
  • Linkers may be present for cloning convenience or to confer some useful property. For example, residues that stabilize specific secondary structure elements, such as ⁇ -helices, are known [Richardson and Richardson (1988), Science 240: 1648-1652].
  • Such residues can be introduced in the linkers to stabilize the heterologous oligomerization domains.
  • the linkers glycine-asparagine, arginine-glycine-asparagine [SEQ ID NO: 7], arginine- glycine-glycine-asparagine-proline-glutamic acid [SEQ ID NO: 8], glycine-glycine-asparagine-glutamine-alanine [SEQ ID NO: 9] present in the examples shown in Figs. IE, IG and IK, 1H and 1J, respectively, are all designed to stabilize the N-terminus of the ⁇ -helical heterologous oligomerization domain.
  • a variety of other amino acid or peptide linkers may be used for the reasons discussed above, provided they do not interfere with the function of the p53 chimeric protein.
  • the present invention further provides nucleic acid sequences encoding the proteins of this invention, which includes the proteins described in sections A and B above.
  • the nucleic acid sequences of the invention include the complementary DNA sequence representing the non-coding strand, the messenger RNA sequence, the corresponding cDNA sequence and the RNA sequence complementary to the messenger RNA sequence.
  • Variants of these nucleic acids of the invention include variations due to the degeneracy of the genetic code and are encompassed by this invention. Such variants may be readily identified and/or constructed by one of skill in the art. In certain cases specific codon usage may be employed to optimize expression.
  • the above nucleotide sequences can be included within larger DNA or RNA fragments, or may be interrupted by introns.
  • nucleic acids encoding the p53 proteins of the invention are present in the context of vectors suitable for amplification in prokaryotic or eukaryotic cells.
  • vectors suitable for amplification in prokaryotic or eukaryotic cells Many such vectors are known [Ausubel et al, (1994), cited above] and many of these are commercially available.
  • plasmids with bacterial or yeast replication origins allow amplification in bacteria or yeast, respectively.
  • Such vectors allow the production of large quantities of nucleic acids encoding the proteins of the invention, which nucleic acids can be used for gene therapy or for expression of the p53 proteins of the invention.
  • the nucleic acids encoding the proteins of the invention are present in the context of vectors suitable for expression in cell-free extracts or lysates or in prokaryotic or eukaryotic cells.
  • vectors suitable for expression in cell-free extracts or lysates or in prokaryotic or eukaryotic cells.
  • Many such vectors are known [Ausubel et al, (1994) , cited above] and many of these are commercially available.
  • the vector pGEM4 Promega, Madison, WI
  • the vector pSV2 [Mulligan et al, (1992), cited above] is suitable for expression in mammalian cells.
  • Such vectors allow the production of the proteins of the invention in vitro for analysis of their functional properties or for delivery to patients. D.
  • the nucleic acid sequences of the invention may be inserted into a vector capable of targeting and infecting a desired cell, either in vivo or ex vivo for gene therapy, and causing the encoded p53 protein of this invention to be expressed by that cell.
  • viral vectors are useful for this purpose, e.g., adenoviruses, retroviruses and adeno-associated viruses (AAV) [Schreiber et al, (1993), Biotechniques, 14: 818-823; Davidson et al, (1993), Nature Genetics, 3: 219-223; Roessler et al, (1993), J. Clin.
  • a recombinant viral vector e.g. an adenovirus
  • a recombinant viral vector comprises DNA of at least that portion of the viral genome which is capable of infecting the target cells operatively linked to the nucleic acid sequences of the invention.
  • infection is generally meant the process by which a virus transfers genetic material to its host or target cell.
  • the virus used in the construction of a vector of the invention is rendered replication-defective to remove the effects of viral replication on the target cells.
  • the replication-defective viral genome can be packaged by a helper virus in association with conventional techniques.
  • the vector(s) containing the nucleic acids encoding a protein of the invention is suspended in a pharmaceutically acceptable carrier, such as saline, and administered parenterally (or by other suitable means) in sufficient amounts to infect the desired cells and provide sufficient levels of p53 activity to arrest abnormal cellular proliferation.
  • a pharmaceutically acceptable carrier such as saline
  • Other pharmaceutically acceptable carriers are well known to those of skill in the art.
  • a suitable amount of the vector containing the chimeric nucleic acid sequences is between about 10 6 to 10 9 infectious particles per mL carrier. The delivery of the vector may be repeated as needed to sustain satisfactory levels of p53 activity, as determined by monitoring clinical symptoms.
  • this therapy may be combined with other therapies for the disease or condition being treated.
  • therapy involving the administration of a vector capable of expressing a p53 protein of the invention is well suited for use in conjunction with conventional cancer therapies, including surgery, radiation and chemotherapy.
  • Nucleic acid sequences driving expression of a p53 protein of the invention may also be introduced by "carriers" other than viral vectors, such as liposomes, nucleic acid-coated gold beads or can simply be injected in situ [Fujiwara et al (1994b) , cited above; Fynan et al, (1993), Proc. Natl. Acad. Sci. USA, 90: 11478-11482; Cohen (1993), Science, 259: 1691-1692; Wolff et al, (1991), Biotechniques, 11: 474-485].
  • the proteins of this invention may also be formulated into pharmaceutical compositions and administered using a therapeutic regimen compatible with the particular formulation.
  • Pharmaceutical compositions within the scope of the present invention include compositions containing a protein of the invention in an effective amount to have the desired physiological effect, e.g. to arrest the growth of cancer cells without causing unacceptable toxicity for the patient.
  • Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble or water-dispersible form, e.g. saline. Alternatively, suspensions of the active compounds may be administered in suitable conventional lipophilic carriers or in liposomes.
  • the compositions may be supplemented by active pharmaceutical ingredients, where desired.
  • Optional antibacterial, antiseptic, and antioxidant agents in the compositions can perform their ordinary functions.
  • the pharmaceutical compositions of the invention may further contain any of a number of suitable viscosity enhancers, stabilizers, excipients and auxiliaries which facilitate processing of the active compounds into preparations that can be used pharmaceutically. Preferably, these preparations, as well as those preparations discussed below, are designed for parenteral administration. However, compositions designed for oral or rectal administration are also considered to fall within the scope of the present invention.
  • suitable amount or “effective amount” means an amount which is effective to treat the conditions referred to below.
  • a preferred dose of a pharmaceutical composition containing a protein of this invention is generally effective above about 0.1 mg p53 protein per kg of body weight (mg/kg), and preferably from about 1 mg/kg to about 100 mg/kg. These doses may be administered with a frequency necessary to achieve and maintain satisfactory p53 activity levels. Although a preferred range has been described above, determination of the effective amounts for treatment of each type of tumor or other condition may be determined by those of skill in the art.
  • Dosage units of such pharmaceutical compositions containing the proteins of this invention preferably contain about 1 mg to 5 g of the protein.
  • nucleic acids and proteins of the invention can be introduced into human patients for therapeutic benefits in conditions characterized by insufficient wild-type p53 activity.
  • the nucleic acids of the invention may be introduced into the patient in the form of a suitable viral vectors (or by direct DNA delivery) to harness the patient's cellular machinery to express the proteins of the invention in vivo.
  • proteins of the invention may be introduced into the patient in appropriate pharmaceutical formulations as described above.
  • the pharmaceutical compositions of this invention may be employed to induce the cellular defence to DNA damaging agents.
  • DNA damaging agents include sunlight UV irradiation, as well as radiation and chemotherapeutics used for cancer treatment.
  • compositions of this invention are in inducing apoptosis of specific cells, such as proliferating lymphocytes.
  • a suitable amount of an appropriate pharmaceutical composition of this invention is administered to a subject to enhance the development of immune tolerance.
  • This method may employ both in vivo and ex vivo modes of administration.
  • this therapy is useful as the sole treatment or as an accessory treatment to prevent transplant rejection, or to treat autoimmune diseases, e.g., systemic lupus erythrematosis, rheumatoid arthritis and the like.
  • compositions of this invention may also be employed to restore p53 function in tumor cells.
  • Introduction of p53 function in tumor cells leads to arrest of cell proliferation or to cell death [Finlay et al, (1989), cited above; Eliyahu et al, (1989), cited above; Baker et al, (1990) , cited above; Mercer et al, (1990), cited above; Diller et al, (1990), cited above; Isaacs et al, (1991), cited above; Yonish-Rouach et al, (1993), cited above; Fujiwara et al, (1993), cited above] .
  • p53 function primes tumor cells to undergo cell death in response to DNA damaging agents currently used in cancer therapy [Lowe et al, (1993), cited above; Fujiwara et al, (1994) , cited above; Fisher (1994), Cell, 78: 539-542].
  • a suitable amount of the composition of this invention is administered systemically, or locally to the site of the tumor with or without concurrent administration of conventional cancer therapy (i.e. DNA damaging agents).
  • compositions of this invention may be administered in methods to suppress cell proliferation in diseases other than cancers, which are characterized by aberrant cell proliferation.
  • diseases include psoriasis, atherosclerosis and arterial restenosis. This method is conducted by administering a suitable amount of the selected composition systemically or locally to the patient.
  • the present invention provides p53 proteins with modifications in the native p53 sequence. These modifications, which do not interfere with its native tumor-suppressor function, provide the protein with at least one of the following functional characteristics: (1) the ability to bind DNA and activate transcription like wild-type p53, but to not hetero-oligomerize with wild-type p53 or tumor-derived p53 mutants; and (2) restricted DNA binding specificity from an alteration in the way that the tetramerization domain orients the DNA binding domains of a p53 tetramer relative to one another.
  • Exemplary p53 proteins of this invention which demonstrate the aforementioned functional characteristics, are described in sections A and B, above.
  • Plasmid pGEMhump53wt encodes full-length human wild-type p53 [SEQ ID NOS: 1 and 2].
  • This plasmid was prepared by PCR [Innis et al, (1990), cited above] using a human p53 cDNA, which is readily available to those practicing the art.
  • the PCR procedure was designed to incorporate unique restriction sites within the coding sequence of human p53 [SEQ ID NO: 1]: Kpn I at codon 218, Sst I at codon 299, Sst II at codon 333, Bst BI at codon 338 and Sal I immediately following the termination codon.
  • An Msc I site at codon 138 was eliminated.
  • Plasmid, pGEMhump53wt, was used to generate all the p53 mutants described below, as well as for expression of wild-type p53 by in vitro translation.
  • the sequence of the 5' PCR primer GCAGAGGAGC AAAAGCTTGA AGACAAGGTT [SEQ ID NO: 21] incorporates a HindiII site, while the sequence of the 3' primer CTTCAGGTCG ACTCAGCGTT CGCCAACTAA TTTC [SEQ ID NO: 22] incorporates a termination codon and a Sal I restriction site.
  • pGEMhump53LZ346E encodes amino acids 1-346 of human p53 [SEQ ID NO: 2], a glutamic acid, and then amino acids 253-281 of GCN4 [SEQ ID NO: 4].
  • Plasmi GEHhvwp53 Z347 A fragment encoding amino acids 253-281 [SEQ ID NO: 4] of the yeast transcription factor GCN4 [SEQ ID NO: 4] [Hinnenbusch et al, (1984), cited above] was prepared by PCR of plasmid pSP64-GCN4 [Halazonetis et al, (1988), cited above].
  • the PCR fragment was cloned into pGEMhump53wt linearized with Stu I and Sal I.
  • the resultant plasmid, pGEMhump53LZ347 encodes amino acids 1-347 of human p53 [SEQ ID NO: 2] and amino acids 253-281 Of GCN4 [SEQ ID NO: 4].
  • the sequence of the 5' PCR primer GCAGAGGAGC AAAAGCTTGA AGACAAGGTT [SEQ ID NO: 21] incorporates a Hind III site, while the sequence of the 3• primer CTTCAGGTCG ACTCAGCGTT CGCCAACTAA TTTC [SEQ ID NO: 22] incorporates a termination codon and a Sal I restriction site.
  • the PCR fragment was blunt-ended at the Hind III site and cloned into pGEMhump53wt linearized with Sst II and Sal I.
  • the Sst II site of the vector and the blunt-ended Hind III site of the PCR product were bridged by annealed synthetic oligonucleotides GGGCGTC [SEQ ID NO: 24] and GACGCCCGC [SEQ ID NO: 25].
  • the resultant plasmid, pGEMhump53LZ335Q encodes amino acids 1-335 of human p53 [SEQ ID NO: 2], a glutamine, and then amino acids 253-281 Of GCN4 [SEQ ID NO: 4].
  • AGACAAGGTT [SEQ ID NO: 21] incorporates a Hind III site, while the sequence of the 3' primer CTTCAGGTCG ACTCAGCGTT CGCCAACTAA TTTC [SEQ ID NO: 22] incorporates a termination codon and a Sal I restriction site.
  • the PCR fragment was blunt-ended at the Hind III site and cloned into pGEMhump53wt linearized with Bst BI and Sal I.
  • the Bst BI site of the vector and the blunt-ended Hind III site of the PCR product were bridged by annealed synthetic oligonucleotides CGAAATGTTC CGAGAGCGAA TGAAAC and GTTTCATTCG CTCTCGGAAC ATTT [SEQ ID NO: 26 and 27].
  • the resultant plasmid, pGEMhump53LZ343RMKQ encodes amino acids 1-343 of human p53 [SEQ ID NO: 2] and then amino acids 249-281 of GCN4 [SEQ ID NO: 4].
  • B5. Plasmid pGEMhump53TZ334NR Synthetic oligonucleotides were used to generate a tetrameric variant of the GCN4 leucine zipper.
  • oligonucleotides TATCCGCGGT AATCGTCTGA AACAGATCGA AGACAAGTTA GAAGAAATCC TTTCGAAGCT CTATCACATC GAG and TTTGTCGACT CAACGTTCAC CCAATAATTT TTTGATGCGC GCTAACTCAT TCTCGATGTG ATAGAGCTTC G [SEQ ID NO: 28 and 29] were subjected to a PCR cycle in the absence of any additional DNA.
  • the PCR product was digested with restriction endonucleases Sst II and Sal I and cloned into pGEMhump53wt linearized with Sst II and Sal I.
  • the resultant plasmid, pGEMhump53TZ334NR encodes amino acids 1-334 of human p53 [SEQ ID NO: 2], an asparagine, and then the tetrameric zipper variant corresponding to amino acids 249-281 [SEQ ID NO: 6] of GCN4.
  • the resultant plasmid, pGEMhump53TZ323RGN encodes amino acids 1-323 of human p53 [SEQ ID NO: 2], an arginine-glycine-asparagine tripeptide [SEQ ID NO: 7], and then the tetrameric zipper variant corresponding to amino acids 249-281 [SEQ ID NO: 6] of GCN4.
  • SEQ ID NO: 2 human p53
  • SEQ ID NO: 7 an arginine-glycine-asparagine tripeptide
  • tetrameric zipper variant corresponding to amino acids 249-281 [SEQ ID NO: 6] of GCN4.
  • This plasmid is a modification of plasmid pGEMhump53TZ334NR.
  • the Sst II-Sal I fragment of pGEMhump53TZ334NR containing the tetrameric zipper was modified by PCR using the primer TATCCGCGGT GGAAATCCTG AACTGaAAACA GATCGAAGAC AAG [SEQ ID NO: 31].
  • the PCR fragment was cloned using the Sst II-Sal I sites into pGEMhump53TZ334NR, replacing the original Sst II-Sal I fragment.
  • the resultant plasmid, pGEMhump53TZ334GNPE encodes amino acids 1-334 of human p53 [SEQ ID NO: 2], a glycine-asparagine-proline-glutamic acid tetrapeptide [SEQ ID NO: 32] and the tetrameric zipper variant corresponding to amino acids 250-281 [SEQ ID NO: 6] of GCN4.
  • Plasmid pGEMhumP53LZ346E352I This plasmid is a modification of plasmid pGEMhump53LZ346E.
  • a Cla I restriction site was introduced just after the last codon of pGEMhump53LZ346E by PCR with the primer GTCATCGATG CGTTCGCCAA CTAATTTCTT [SEQ ID NO: 33].
  • a PCR fragment encoding residues 352-393 of human p53 [SEQ ID NO:2] containing a Cla I site at its 5' end was also generated using the primer ATGAGGCCTT GGAACTCATC GATGCCCAGG CTGGG of SEQ ID NO: 34.
  • the latter fragment was cloned using the Cla I-Sal I sites into the modified (using the primer of SEQ ID NO: 33) pGEMhump53LZ346E vector.
  • the resultant plasmid, pGEMhump53LZ346E352I encodes amino acids 1-346 of human p53 [SEQ ID NO: 2], a glutamic acid, the leucine zipper corresponding to amino acids 253-281 of GCN4 [SEQ ID NO: 4], an isoleucine and then residues 352-393 of human p53 [SEQ ID NO: 2].
  • Plasmid pGEMhumP53TZ334NR/I352 This plasmid is a modification of plasmid pGEMhump53TZ334NR.
  • a Cla I restriction site was introduced just after the last codon of pGEMhump53TZ334NR by PCR with the primer TTTGTCGACT CAATCGATAC GTTCACCCAA TAATTTTTTG [SEQ ID NO: 35].
  • a PCR fragment encoding residues 352-393 of human p53 [SEQ ID NO:2] containing a Cla I site at its 5' end was also generated using the primer ATGAGGCCTT GGAACTCATC GATGCCCAGG CTGGG of SEQ ID NO: 34.
  • the latter fragment was cloned using the Cla I-Sal I sites into the modified (using the primer of SEQ ID NO: 35) pGEMhump53TZ334NR vector.
  • the resultant plasmid, pGEMhump53TZ334NR/I352 encodes amino acids 1-334 of human p53 [SEQ ID NO: 2], an asparagine, the tetrameric zipper variant corresponding to amino acids 249-281 [SEQ ID NO: 6] of GCN4, an isoleucine and then residues 352-393 of human p53 [SEQ ID NO: 2].
  • pGEMhump53D290-297 Plasmids pGEMhump53D290-297, pGEMhump53D290-297D300-321, pGEMhump53D300-308, pGEMhump53D300-317, pGEMhump53D300-321, pGEMhump53D300-327, and pGEMhump53D364-393 encode proteins that contain deletions within wild-type human p53 [SEQ ID NO: 2]. These deletions involve residues
  • Plasmid pSV2hump53wt encodes full-length human wild-type p53 [SEQ ID NO: 2], and directs transcription of this protein in mammalian cells.
  • the pSV2 vector has been previously described [Mulligan et al, (1981), Proc. Natl. Acad. Sci. USA, 78: 2072-2076].
  • a pSV2 vector containing a human c-jun insert has also been described [Zhang et al, (1990), Proc. Natl. Acad. Sci. USA, 87: 6281-6285].
  • the c-jun insert was removed from the latter plasmid using Sal I and Bgl II restriction endonucleases, and the ends of the vector were blunted.
  • Into this vector a blunted Eco RI-Hind III p53 insert from pGEMhump53wt was cloned.
  • plasmids pGEMhump53wt and pSV2hump53wt contain the same p53 insert, it is possible to use restriction sites that are common within the inserts of these plasmids, to transfer p53 subfragments from plasmids of the pGEMhump53 series to pSV2hump53wt.
  • pSV2hump53 vector it is possible to transfer, for example, Sst II-Sal I fragments encoding altered tetramerization domains into the pSV2hump53 vector, and thus allow expression of p53 proteins of the invention in mammalian cells.
  • pSV2 vectors expressing most of the proteins described above have been constructed.
  • the name of the p53 protein with altered tetramerization domain is retained from the pGEM to the pSV2 series.
  • pSV2hump53TZ334NR transfer of the Sst II-Sal I fragment of pGEMhump53TZ334NR to pSV2hump53wt, yields pSV2hump53TZ334NR, which allows expression in mammalian cells of a p53 protein containing amino acids 1-334 of human p53 [SEQ ID NO: 2], an asparagine, and then the tetrameric zipper variant corresponding to amino acids 249-281 [SEQ ID NO: 6] of GCN4.
  • Plasmids pEwafl-TK-SEAP, pBC.V4A-TK-SEAP and pBC-TK-SEAP have one copy each of double-stranded oligonucleotides Ewafl [SEQ ID NO: 16], BC.V4A [SEQ ID NO: 17] and BC [SEQ ID NO: 18] , respectively cloned into the unique Eco RV site of pTK-SEAP. These oligonucleotides contain p53 binding sites of different affinities.
  • oligonucleotide Ewafl (top strand) is: CCC-GAACA-TGTCC-CAACA-TGTTG-GGG [SEQ ID NO: 16]. This oligonucleotide corresponds to the enhancer that drives p53-dependent transcription of the wafl gene
  • oligonucleotide BC.V4A top strand
  • TC-GAGCA-TGTTC- GAGCA-TGTTC-GAGCATGT sequence of oligonucleotide BC (top strand)
  • sequence of oligonucleotide BC (top strand) is: CC-GGGCA-TGTCC- GGGCA-TGTCC-GGGCATGT [SEQ ID NO: 18].
  • Oligonucleotides BC.V4A [SEQ ID NO: 17] and BC [SEQ ID NO: 18] contain artificial sites recognized by p53.
  • Plasmid pTK-SEAP drives expression of a secreted form of alkaline phosphatase under the control of a minimal thymidine kinase promoter [Halazonetis (1992) , Anticancer Res., 12: 285-292]. It contains no p53 binding site, and thus serves as a control.
  • Plasmid pSV2crot Plasmid pSV2crot
  • Plasmid pSV2gpt [Mulligan et al, (1981) , cited above] drives expression of gpt in mammalian cells. In these studies it only serves to bring the total amount of transfected DNA to 30 ⁇ g, when necessary. Expression of gpt does not interfere with p53 function.
  • Example 2 In Vitro Translation and DNA Binding Assay
  • Plasmids of the pGEMhump53 series of Example 1 were used to produce in vitro transcribed mRNA according to standard procedures [Halazonetis et al, (1988) , cited above].
  • the mRNA is subsequently translated in vitro using preferably rabbit reticulocyte lysate (Promega, Madison, WI) [Halazonetis et al (1988) , cited above] .
  • In vitro translated p53 can be used directly for DNA binding, without further purification.
  • Alternate strategies for expression of p53 for DNA binding assays include expression in E. coli or in Sf9 insect cells using appropriate vectors (many are commercially available) for expression in bacterial cells or baculovirus vectors, respectively. Lysates or extracts prepared from bacterial or insect cells are used without purification, or optimally, following partial or complete purification using standard protein purification techniques [Scopes (1994), cited above].
  • the in vitro translated protein is incubated with a radioactively labeled oligonucleotide containing a p53 binding site in the presence of non-specific competitor DNA.
  • the reaction mixture is incubated 20 min. at room temperature and directly loaded on a native 5% polyacrylamide electrophoresis gel.
  • free DNA migrates to the bottom of the gel, whereas p53/DNA complexes migrate more slowly.
  • p53 DNA binding which can be detected by autoradiography, indicates p53 DNA binding [Halazonetis et al (1993), cited above; Halazonetis and Kandil (1993), cited above].
  • non-specific competitor DNAs the following were used: 0.1 ⁇ g single-stranded oligonucleotide MI7 [GAGAGCCCCAGTTACCATAACTACTCT, SEQ ID NO: 36] and 0.05 ⁇ g double-stranded oligonucleotide TF3 [ATCACGTGATATCACGTGATATCACGTGAT, SEQ ID NO: 37] per reaction.
  • oligonucleotides containing p53 binding sites were radioactively labeled for these experiments. These included oligonucleotides Ewafl, BC.V4A and BC [SEQ ID NOS: 16, 17 and 18, respectively], and oligonucleotide BC.S21.
  • the sequence Of BC.S21 is: TAT-GGGCA-TGTCC-TATATATATGCGTATATATAT- GGGCA-TGTCC-TAT [SEQ ID NO: 19].
  • the pentanucleotide repeats, which are recognized by p53, are indicated by hyphens. These DNAs were radioactively labeled using
  • A. DNA Binding Activities of Wild-type Human P53 The ability of wild-type human p53 to recognize the DNA sites present in oligonucleotides Ewafl, BC.V4A and BC [SEQ ID NOS: 16, 17 and 18, respectively] has been previously demonstrated [El-Deiry et al, (1993), cited above; Halazonetis et al, (1993), cited above]. Using the assay described in Example 2, wild-type p53 recognized all these DNAs. The highest signal was obtained using the BC oligonucleotide [SEQ ID NO: 18], while oligonucleotide Ewafl [SEQ ID NO: 16] gave the weakest signal.
  • the intensity of the signal in this assay reflects the affinity of p53 for the different DNA sites.
  • the intensity of the signal using oligonucleotides BC.V4A [SEQ ID NO: 17] or Ewafl [SEQ ID NO: 16] was enhanced in the presence of 0.1 ⁇ g anti-p53 antibody PAb421 [Oncogene Science, Uniondale, NY].
  • This antibody activates DNA binding of wild-type p53, by switching the conformation of the protein [Halazonetis et al, (1993), cited above; Halazonetis and Kandil (1993), cited above].
  • oligonucleotide BC [SEQ ID NO: 18] is quite potent, and very little further enhancement is observed following incubation with antibody PAb421.
  • the conformation of p53 can be switched by a C-terminal truncation that removes residues 364-393 of human p53 [SEQ ID NO: 2] [Halazonetis and Kandil (1993), cited above; Hupp et al (1992), Cell, 71:875-886; Hupp and Lane (1994), Current Biology, 4: 865-875].
  • the C-terminally truncated p53 protein, p53D364-393 bound all three oligonucleotides with high affinity, comparable to wild-type p53 in the presence of PAb421.
  • oligonucleotide BC.S21 contains two pairs of contiguous pentanucleotide repeats separated by 21 nucleotides. Wild-type p53 bound efficiently to this DNA, as indicated by a strong signal in the DNA binding assay described in Example 2, above. The signal was as strong as with oligonucleotide BC [SEQ ID NO: 18] (which represents the optimal p53 DNA site) and was not further enhanced by antibody PAb421. As discussed in the Section V.B1, oligonucleotide BC.S21 [SEQ ID NO: 19] does not match the consensus p53 DNA site. Thus, the ability of wild-type p53 to bind to oligonucleotide BC.S21 [SEQ ID NO: 19] is a novel finding.
  • Proteins p53LZ346E, p53LZ347, p53TZ334NR and p53TZ323RGN represent chimeric proteins of the invention, which are encoded by plasmids pGEMhump53LZ346E, pGEMhump53LZ347, pGEMhump53TZ334NR and pGEMhump53TZ323RGN, respectively, described in Example 1.
  • the DNA complexes of proteins p53LZ346E, p53LZ347, P53TZ334NR and p53TZ323RGN comigrated with the DNA complex of wild-type p53 or the DNA complex of p53D364-393. Since migration on acrylamide gels depends on the molecular size of the migrating species [Hope and Struhl (1987), EMBO J. , 6: 2781-2784] this indicates that the complexes of wild-type p53, p53D364-393, p53LZ346E, P53LZ347, p53TZ334NR and p53TZ323RGN with DNA have similar molecular sizes.
  • Proteins p53LZ335Q and p53LZ343RMKQ are chimeric proteins of p53 with the GCN4 leucine zipper, encoded by plasmids pGEMhump53LZ335Q and pGEMhump53LZ343RMKQ, respectively.
  • Protein p53LZ343RMKQ was first described by Pietenpol et al, (1994) [cited above]. The ability of these proteins to bind oligonucleotides BC.V4A and BC [SEQ ID NOS: 17 and 18, respectively] was examined using the assay described in Example 2.
  • the molecular sizes of the complexes of p53LZ335Q and p53LZ343RMKQ are smaller than those of wild-type p53 or p53D364-393. Since wild-type p53 and p53D364-393 are tetramers
  • p53LZ335Q and P53LZ343RMKQ are dimers. They cannot be monomers, because monomeric p53 does not bind DNA [Halazonetis and Kandil (1993), cited above].
  • p53LZ335Q and p53LZ343RMKQ are not proteins of this invention, since they fail to form tetramers.
  • Proteins p53Q334, p53L337, p53A341 and p53A344 are encoded by plasmids pGEMhump53Q334, pGEMhump53L337, pGEMhump53A34l and pGEMhump53A344, respectively, described in Example 1.
  • the ability of these proteins to bind oligonucleotide BC [SEQ ID NO: 18] was examined using the assay described in Example 2.
  • Proteins p53Q334, p53L337 and p53A341 bound DNA very weakly, if at all.
  • Proteins p53D290-297, p53D290-297D300-321, p53D300-308, p53D300-317, p53D300-321 and p53D300-327 are encoded by plasmids pGEMhump53D290-297, pGEMhump53D290-297D300-321, pGEMhump53D300-308, pGEMhump53D300-317, pGEMhump53D300-321 and pGEMhump53D300-327, respectively, described in Example 1.
  • the ability of these proteins to bind oligonucleotides BC and BC.S21 [SEQ ID NOS: 18 and 19, respectively] was examined using the assay described in Example 2.
  • Proteins p53D290-297, p53D300-308, p53D300-317, and p53D300-321 bound both oligonucleotides BC and BC.S21 [SEQ ID NOS: 18 and 19, respectively] with efficiencies paralleling that of wild-type p53.
  • the complexes of these proteins with DNA comigrated with the complexes of wild-type p53 with DNA.
  • p53D290-297, p53D300-308, p53D300-317 and p53D300-321 exhibit DNA binding properties similar to wild-type p53.
  • the p53 proteins were in vitro translated, as described in Example 2, in the presence of 35S-methionine, so that they would be radioactively labeled.
  • 3 ⁇ l of the lysate containing the translated protein(s) was incubated in 30 ⁇ l DNA binding buffer [Halazonetis et al, (1993) , cited above] for 20 minutes at room temperature.
  • Example 5 Hetero-Oligomerization Assay
  • p53LZ346E one of the proteins of the invention encoded by plasmid pGEMhump53LZ346E (Example 1) , does not hetero-oligomerize with p53 proteins having intact native p53 tetramerization domains, such as wild-type p53 and tumor-derived p53 mutants.
  • Wild-type p53 and p53LZ346E were cotranslated in the presence of 35S-methionine. Simultaneous translation of the two proteins provides opportunity for the two different subunit types to form hetero-oligomers. After cotranslation was completed, the mixture was immunoprecipitated using antibody PAb421, as described in Example 4 above. The epitope of antibody PAb421 maps to residues
  • PAb421 recognizes wild-type p53, but not p53LZ346E. Thus, if the two proteins hetero-oligomerize when cotranslated, then both will be precipitated by PAb421. If they do not hetero-oligomerize, then only wild-type p53 will be precipitated.
  • An assay for transcriptional activity entails introducing vectors expressing wild-type p53 or p53 proteins of the invention into cells together with a reporter plasmid that expresses a reporter marker in a p53-dependent manner.
  • the cells are human tumor cells that do not express endogenous p53, so that the transcriptional activity can be evaluated without interference from endogenous wild-type or mutant p53.
  • Transcriptional activity was assayed in Saos-2 human osteosarcoma cells [ATCC HTB 85] . These cells do not contain any endogenous p53, because both p53 alleles are deleted [Diller et al, (1990), Mol. Cell. Biol., 10: 5772-5781]. Thus, any transcriptional activity can be attributed to the transfected p53. Transcriptional activity was assayed as previously described [Halazonetis (1992), Anticancer Res., 12: 285-292].
  • plasmids expressing p53 in mammalian cells were cotransfected with reporter plasmids (of the pTK-SEAP series described in Example 1) using the calcium phosphate technique [Halazonetis (1992), cited above].
  • Alkaline phosphatase activity which reflects p53-mediated transcriptional activity, was assayed as previously described [Halazonetis (1992) , cited above] .
  • Example 7 Comparison of Transcriptional Activities of Wild-tVPe P53. P53LZ346E. P53LZ347. P53LZ335Q.
  • Wild-type p53, p53LZ346E, p53LZ347, p53LZ335Q, p53LZ343RMKQ and p53TZ334NR were expressed in Saos-2 cells [ATCC HTB 85] by transfecting plasmids pSV2hump53wt, pSV2hump53LZ346E, pSV2hump53LZ347, pSV2hump53LZ335Q, pSV2hump53LZ343RMKQ and pSV2hump53TZ334NR, respectively.
  • the transcriptional activities of the expressed proteins were assayed using one or more of the reporter plasmids pBC-TK-SEAP, pBC.V4A-TK-SEAP and pEwafl-TK-SEAP, described in Example 1.
  • wild-type p53 is able to activate transcription from all the reporter plasmids examined, including plasmid pEwaf1-TK-SEAP, which contains the weakest p53 binding site.
  • the tetrameric p53 proteins of the invention P53LZ346E, p53LZ347 and p53TZ334NR, all exhibit transcriptional activity.
  • the dimeric p53 chimeric proteins, such as p53LZ335Q and p53LZ343RMKQ do not exhibit transcriptional activity that is detectably above background in this assay, and are thus clearly inferior to the tetrameric proteins of this invention.
  • Tumor-derived p53 mutants are known to suppress the transcriptional activity of wild-type p53 by forming hetero-tetramers with wild-type p53 [Milner and Medcalf (1991), cited above; Bargonetti et al, (1992), cited above; Farmer et al, (1992), cited above; Kern et al, (1992), cited above].
  • the tetrameric p53 proteins of this invention do not hetero-tetramerize with tumor-derived p53 mutants, because the native p53 tetramerization domain is partially or completely disrupted (See Example 5) . Consequently, the transcriptional activities of the tetrameric p53 proteins should not be inhibited by tumor-derived p53 mutants.
  • the transcriptional activities of the p53 proteins of this invention in the presence of excess of a tumor-derived p53 mutant were compared to their transcriptional activities in the absence of the tumor-derived mutant.
  • the tumor-derived mutants p53Hisl75 and p53Ser249 have histidine at position 175 or serine at position 249 of human p53 [SEQ ID NO: 2], respectively.
  • Other tumor-derived p53 mutants [Caron de Fromentel and Soussi (1992), Genes Chrom. Cancer, 4: 1-15] can also be used, as long as they potently inhibit the transcriptional activity of wild-type p53.
  • results of relevant experiments are presented in Table 2 below. Suppression of transcriptional activity by the tumor-derived p53 mutant is presented as percent of residual transcriptional activity in the presence of the tumor-derived p53 mutant, as compared to the transcriptional activity in the absence of the mutant. For each transfection the amounts of transfected plasmids are indicated in ⁇ g. Where the total amount was less than 30 ⁇ g (as in the absence of the tumor-derived p53 mutant) , then plasmid pSV2gpt (described in Example 1) was used to bring the total to 30 ⁇ g. For these experiments the tumor-derived p53 mutant His 175 was used (described in Example 1) .
  • transcriptional activity of p53TZ334NR and p53TZ334NR/I352 were examined by transient transfection in Saos-2 osteosarcoma cells, which lack endogenous p53 [L. Diller et al, Mol. Cell. Biol.. 10:5772-5781 (1990)]. Briefly, as above. transcriptional activity was determined by transfecting in quadruplicated Saos-2 cells with 5 ⁇ g of p53 expression and 25 ⁇ g of reporter plasmids.
  • the reporter plasmids, Ep21/TK-seap and pEmdm2/TKseap have one copy of oligonucleotide Ep21 [SEQ ID NO: 45: CCC-GAACA-TGTCC-TGTTG-GGG] or Emdm2 [SEQ ID NO: 46: GGCT-GGTCA-AGTTG-GGACA-CGTCC- GGCGTCGGCTGTCGGAG-GAGCT-A-AGTCC-TGACA-CCAG] , respectively, cloned in the Eco RV site of pTKseap [Halazonetis, cited above] and express secreted alkaline phosphatase in a p53-responsive manner.
  • Both chimeric proteins of the invention activated transcription from reporter plasmids containing the p21 or mdm2 p53 sites.
  • the dimeric p53-leucine zipper hybrids were less potent transcriptional activators, especially with the reporter plasmid containing the mdm2 site.
  • Transcriptional activity for all p53 proteins examined was sequence-specific, since none of them activated transcription from a reporter plasmid that lacked a p53 site.
  • Example 9 Turcqr suppression Activities of p53 fusion proteins in Saos-2 cells.
  • Example 1 part B5 and p53TZ334NR/I352 of Example 1, part B9, as well as other expression plasmids, including p53wt (wild-type p53; see Example 1, part A), p53L2343 (Example 1, part B4), p53LZ335Q (Example 1, part B3) , p53W248 (wild-type p53 with a point mutation associated with human cancer at Trp 248), and p53W248TZ334N (p53T2334N containing the point mutation at Trp 248) were tested in a colony formation assay, by cotransfecting Saos-2 osteosarcoma cells in quadruplicate with 5 ⁇ g of expression plasmid directing p53 expression, and 1 ⁇ g of pSV7neo, a plasmid conferring neomycin resistance [Zhang et al, Proc.
  • Plasmid pGEMhump53 unTZ334N Plasmid pGEMhump53junTZ334N encodes a p53 - modified c-Jun chimeric protein consisting (in an N-terminal to C- erminal direction) of residues 1-334 of human p53 [SEQ ID NO: 2], an asparagine, a modified c-Jun leucine zipper corresponding to residues 276-313 of human c-Jun [SEQ ID NO: 41] and a tripeptide glycine-glutamic acid-arginine. Synthetic oligonucleotides were used to generate a tetrameric variant of the c-Jun leucine zipper.
  • oligonucleotides [SEQ ID NO: 42 and 43] were subjected to a PCR cycle in the absence of any additional DNA.
  • the PCR product was digested with restriction endonucleases Sst II and Sal I and cloned into pGEMhump53wt (described in Example IA) linearized with Sst II and Sal I.
  • Sst II and Sal I restriction endonucleases
  • Plasmid pGEMhump53junTZ334N was designed to resemble plasmid pGEMhump53TZ334NR as much as possible.
  • the latter plasmid encodes a p53 - modified GCN4 chimeric protein consisting in an N-terminal to C-terminal direction of residues 1-334 of human p53 [SEQ ID NO: 2], an asparagine, and a modified GCN4 leucine zipper corresponding to amino acids 249-281 of GCN4 [SEQ ID NO: 6].
  • plasmid pGEMhump53junTZ334N would resemble as much as possible pGEMhump53TZ334NR, the tripeptide glycine-glutamic acid-arginine was inserted C-terminal to the c-Jun modified leucine zipper.
  • the structures of wild-type p53 and of the protein encoded by plasmid pGEMhump53TZ334NR are represented schematically in Figs. IA and IF, respectively.
  • the protein encoded by plasmid pGEMhumpp53junTZ334N is substantially identical to the schematic diagram of Fig. IF, except that the tetrameric variant of c-Jun residues 276-313 and the above-identified tripeptide are substituted for tetrameric variant of GCN4.
  • Plasmid pGEMhump53junN287TZ334N encodes a protein that is identical to the protein encoded by pGEMhump53junTZ334N, except that one of the isoleucines at position a of the coiled-coil p53, corresponding to amino acid 287 of human c-Jun [SEQ ID NO: 39], was substituted with asparagine.
  • This plasmid was generated with PCR-directed mutagenesis [Innis et al, (1990)] using the oligonucleotide described in SEQ ID NO: 44 as the PCR primer. See, Figs. 6A through 6D.
  • Plasmid pSV2hump53junTZ334N directs expression of the p53 - c-Jun chimeric protein described above (Example 10A) in mammalian cells.
  • Plasmids pGEMhump53junTZ334N and pGEMhump53junN287TZ334N were used to generate in vitro translated proteins as described in Example 2. These proteins were subsequently tested for their ability to bind DNA, again as described in Example 2. Both proteins (hump53junTZ334N and hump53junN287TZ334N) bound DNA as efficiently as wild-type p53. Hump53junTZ334N bound DNA as a tetramer as determined by migration of its DNA complexes on native electrophoretic gels relative to the DNA complexes of wild-type p53. A second complex of hump53junTZ334N with DNA was also observed. This complex migrated more slowly.
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • AAAAATTTCC GACTTTAAAT ACGGAAGATA AATACTCCAA CCTTTTTTTC 100
  • GAAAACTGTC AGTTTTTTGA AGAGTTATTT GTTTTGTTAC CAATTGCTAT 600
  • GGT TCT AAA TCA ACC AAC GAA AAT GTA TCT GCT TCC ACT TCT 879 Gly Ser Lys Ser Thr Asn Glu Asn Val Ser Ala Ser Thr Ser
  • AAA CGT GCT AGA AAC ACT GAA GCC GCC AGG CGT TCT CGT GCG 1509 Lys Arg Ala Arg Asn Thr Glu Ala Ala Arg Arg Ser Arg Ala
  • MOLECULE TYPE DNA (genomic)
  • AAAAATTTCC GACTTTAAAT ACGGAAGATA AATACTCCAA CCTTTTTTTC 100
  • GAAAACTGTC AGTTTTTTGA AGAGTTATTT GTTTTGTTAC CAATTGCTAT 600
  • GGT TCT AAA TCA ACC AAC GAA AAT GTA TCT GCT TCC ACT TCT 879 Gly Ser Lys Ser Thr Asn Glu Asn Val Ser Ala Ser Thr Ser
  • AAA CGT GCT AGA AAC ACT GAA GCC GCC AGG CGT TCT CGT GCG 1509 Lys Arg Ala Arg Asn Thr Glu Ala Ala Arg Arg Ser Arg Ala
  • MOLECULE TYPE DNA (genomic)
  • Xi SEQUENCE DESCRIPTION: SEQ ID NO:10: GAACATGTCC CAACATGTTG 20 (2) INFORMATION FOR SEQ ID NO:11:
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:16: CCCGAACATG TCCCAACATG TTGGGG 26
  • MOLECULE TYPE DNA (genomic)
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:17: TCGAGCATGT TCGAGCATGT TCGAGCATGT 30
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • SEQUENCE DESCRIPTION SEQ ID NO:19: TATGGGCATG TCCTATATAT ATGCGTATAT ATATGGGCAT GTCCTAT 47
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • Xi SEQUENCE DESCRIPTION: SEQ ID NO:21: GCAGAGGAGC AAAAGCTTGA AGACAAGGTT 30
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • Xi SEQUENCE DESCRIPTION: SEQ ID NO:23: ATGAGGCCTT GGAAGACAAG GTTGAAGAAT TG 32
  • MOLECULE TYPE DNA (genomic)
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:24: GGGCGTC 7
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • SEQUENCE DESCRIPTION SEQ ID NO:26: CGAAATGTTC CGAGAGCGAA TGAAAC 26
  • MOLECULE TYPE DNA (genomic)
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:27: GTTTCATTCG CTCTCGGAAC ATTT 24
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • Xi SEQUENCE DESCRIPTION: SEQ ID NO:30: TTCTCCGCGG AGTGGTTTCT TCTTTGGCTG 30
  • MOLECULE TYPE DNA (genomic)
  • Xi SEQUENCE DESCRIPTION: SEQ ID NO:31: TATCCGCGGT GGAAATCCTG AACTGAAACA GATCGAAGAC AAG 43 96/16989 PC17US95/15353
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • Xi SEQUENCE DESCRIPTION: SEQ ID NO:35: TTTGTCGACT CAATCGATAC GTTCACCCAA TAATTTTTTG 40
  • MOLECULE TYPE DNA (genomic)
  • Xi SEQUENCE DESCRIPTION: SEQ ID NO:36: GAGAGCCCCA GTTACCATAA CTACTCT 27
  • MOLECULE TYPE DNA (genomic)
  • Xi SEQUENCE DESCRIPTION: SEQ ID NO:37: ATCACGTGAT ATCACGTGAT ATCACGTGAT 30 (2) INFORMATION FOR SEQ ID NO:38:
  • MOLECULE TYPE DNA (genomic)
  • GGC ATG GTG GCT CCC GCG GTA GCC TCG GTG GCA GGG
  • GGC AGC 462 Gly Met Val Ala Pro Ala Val Ala Ser Val Ala Gly Gly Ser
  • MOLECULE TYPE DNA (genomic)
  • FEATURE FEATURE:
  • GGC ATG GTG GCT CCC GCG GTA GCC TCG GTG GCA GGG
  • GGC AGC 462 Gly Met Val Ala Pro Ala Val Ala Ser Val Ala Gly Gly Ser

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Abstract

La présente invention concerne des protéines p53 à domaines de tétramérisation mofidiés qui conservent la fonction p53 du type sauvage ainsi que la possibilité de former des tétramères et qui possèdent au moins une des caractéristiques suivantes: (1) pas de formation d'hétéro-oligomères avec la protéine p53 du type sauvage ou avec des mutants de protéine p53 dérivés de tumeurs, et (2) spécificité de liasion ADN réduite à partir d'une modification de la manière de laquelle le domaine de tétramérisation oriente les domaines de liaisons ADN d'une tétramère p53 par rapport à un autre. Cette invention concerne également des acides nucléiques codant pour les protéines ci-dessus ainsi que des procédés permettant d'accroître la réponse cellulaire face à des agents provoquant un dommage à l'ADN, et elle concerne également des méthodes de traitement de maladies caractérisées par une prolifération anormale des cellules, et des méthodes de déclenchement de tolérance immunitaire pour faciliter les transplantations et le traitement des maladies autoimmunes, grâce à l'administration de protéines conformes à la présente invention ou de séquences d'acides nucléiques codant pour ces protéines.
PCT/US1995/015353 1994-11-28 1995-11-27 PROTEINES p53 A DOMAINES DE TETRAMERISATION MODIFIES WO1996016989A1 (fr)

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997004092A1 (fr) * 1995-07-19 1997-02-06 Rhone-Poulenc Rorer S.A. Variants de la proteine p53 et utilisations therapeutiques
WO1998006753A3 (fr) * 1996-08-13 1998-04-23 Univ Princeton Mutant de p53
US5847083A (en) * 1996-08-21 1998-12-08 The Wistar Institute Of Anatomy And Biology Modified p53 constructs which enhance DNA binding
WO2000068384A3 (fr) * 1999-05-12 2001-02-15 Xencor Inc Nouveaux acides nucleiques et proteines ayant une activite p53 et comportant des domaines de tetramerisation modifies
WO2001009325A3 (fr) * 1999-07-30 2001-08-30 Us Health Mutations du gene p53 humain, et systeme genetique de levures utilise pour l'identification fonctionnelle des mutations du gene p53 humain
US6388062B1 (en) 1998-05-08 2002-05-14 The Wistar Institute Of Anatomy And Biology Modified p53 tetramerization domains having hydrophobic amino acid substitutions
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Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6933373B2 (en) 1995-07-19 2005-08-23 Aventis Pharma S.A. P53 protein variants and therapeutic uses thereof
AU725841B2 (en) * 1995-07-19 2000-10-19 Aventis Pharma S.A. P53 protein variants and therapeutical uses thereof
WO1997004092A1 (fr) * 1995-07-19 1997-02-06 Rhone-Poulenc Rorer S.A. Variants de la proteine p53 et utilisations therapeutiques
US6326464B1 (en) 1995-07-19 2001-12-04 Aventis Pharma S.A. P53 protein variants and therapeutic uses thereof
WO1998006753A3 (fr) * 1996-08-13 1998-04-23 Univ Princeton Mutant de p53
US5847083A (en) * 1996-08-21 1998-12-08 The Wistar Institute Of Anatomy And Biology Modified p53 constructs which enhance DNA binding
US7071158B2 (en) 1997-07-01 2006-07-04 Atherogenics, Inc. Antioxidant enhancement of therapy for hyperproliferative conditions
US6388062B1 (en) 1998-05-08 2002-05-14 The Wistar Institute Of Anatomy And Biology Modified p53 tetramerization domains having hydrophobic amino acid substitutions
WO2000068384A3 (fr) * 1999-05-12 2001-02-15 Xencor Inc Nouveaux acides nucleiques et proteines ayant une activite p53 et comportant des domaines de tetramerisation modifies
WO2001009325A3 (fr) * 1999-07-30 2001-08-30 Us Health Mutations du gene p53 humain, et systeme genetique de levures utilise pour l'identification fonctionnelle des mutations du gene p53 humain
JP2003506041A (ja) * 1999-07-30 2003-02-18 アメリカ合衆国 ヒトｐ５３変異およびヒトｐ５３変異の機能的同定のための酵母遺伝子系
US7256260B1 (en) 1999-07-30 2007-08-14 The United States Of America, As Represented By The Secretary, Dept. Of Health And Human Services, Nih Human p53 mutations and a genetic system in yeast for functional identification of human p53 mutations
WO2003106501A1 (fr) * 2002-05-21 2003-12-24 Gou Young Koh Proteine de fusion comprenant un domaine se liant au recepteur de l'angiopoietine et un domaine de multimerisation
US7081443B2 (en) 2002-05-21 2006-07-25 Korea Advanced Institutes Of Science And Technology (Kaist) Chimeric comp-ang1 molecule
US7309586B2 (en) 2002-05-21 2007-12-18 Korea Advanced Institute Of Science And Technology (Kaist) Chimeric coiled coil molecules
US20120015884A1 (en) * 2009-01-19 2012-01-19 Alain Prochiantz Polypeptides for Specific Targeting to Otx2 Target Cells
US10842852B2 (en) 2009-01-19 2020-11-24 Centre National De La Recherche Scientifique Methods of delivering a polypeptide molecule to Otx2 target cells using an Otx2 targeting peptide
WO2014152878A2 (fr) 2013-03-14 2014-09-25 Agrivida, Inc. Utilisation de domaines de dimérisation à des fins de régulation par la température de l'activité enzymatique
WO2014152878A3 (fr) * 2013-03-14 2014-11-13 Agrivida, Inc. Utilisation de domaines de dimérisation à des fins de régulation par la température de l'activité enzymatique
US10240137B2 (en) 2013-03-14 2019-03-26 Agrivida, Inc. Use of dimerization domains for temperature regulation of enzyme activity

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EP0799243A1 (fr) 1997-10-08
AU4288496A (en) 1996-06-19

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