WO2000068384A2 - Nouveaux acides nucleiques et proteines ayant une activite p53 et comportant des domaines de tetramerisation modifies - Google Patents

Nouveaux acides nucleiques et proteines ayant une activite p53 et comportant des domaines de tetramerisation modifies Download PDF

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WO2000068384A2
WO2000068384A2 PCT/US2000/013248 US0013248W WO0068384A2 WO 2000068384 A2 WO2000068384 A2 WO 2000068384A2 US 0013248 W US0013248 W US 0013248W WO 0068384 A2 WO0068384 A2 WO 0068384A2
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tet
protein
sequence
proteins
residues
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PCT/US2000/013248
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WO2000068384A3 (fr
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Klaus Fiebig
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Xencor, Inc.
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Priority to JP2000616350A priority Critical patent/JP2003520023A/ja
Priority to EP00930723A priority patent/EP1179062A2/fr
Priority to CA002372881A priority patent/CA2372881A1/fr
Priority to AU48492/00A priority patent/AU4849200A/en
Publication of WO2000068384A2 publication Critical patent/WO2000068384A2/fr
Publication of WO2000068384A3 publication Critical patent/WO2000068384A3/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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the invention relates to novel proteins with p53 activity and altered tetrame ⁇ zation domains, and nucleic acids encoding these proteins
  • the invention further relates to the use of the novel proteins in the treatment of p53 related disorders such as cancer
  • the wild-type human protein p53 is a sequence specific transcription factor that induces cell cycle arrest or programmed cell death in response to DNA damage It is a homotetramer that contains at least five functional regions an N-terminal transactivation region, a DNA binding domain, a nuclear localization signal, a tetrame ⁇ zation domain, and a C-terminal regulatory region
  • the tetrame ⁇ zation domain mediates the oligome ⁇ zation that is responsible for the high affinity, sequence * specific DNA binding activity that results in tumor suppression activity See Stav ⁇ di et al, Protein Science 8 1773 (1999) and Mateu et al , EMBO J 17 2748 (1998), and references cited within, all of which are incorporated by reference herein
  • the present invention provides non-naturally occurring tet-p53 proteins (e g the proteins are not found in nature) comprising ammo acid sequences that are less than about 97% identical to human p53 in the tetramenzation domain
  • the tet-p53 proteins will preferentially tetramerize with itself to form homotetramers rather than tetramerize with the tetramenzation domain of wild-type p53 to form heterotetramers
  • Preferred embodiments utilize tet- p53 proteins with at least about 3, 4, 5 and 7 ammo acid changes as compared to wild type In a preferred embodiment, these changes are at one or more positions selected from positions 328, 330,
  • non-naturally occurring tet-p53 proteins have substitutions selected from the group of substitutions consisting of F328Y, F328W, F328L, L330I, I332V, I332L, R337L, F338Y, M340L, M340I, F341 I, F341 L, F341V, E343R, E343T, E343V, E343K, E343Q, E343W, E343F, E343N, L344M, N345Y, N345F, N345L, N345V, N345W, L348F, L348M, L348W, E349R, E349L,
  • E349Q E349W, E349I, E349N, E349L, L350I, L350Y, I350F, I350W and 1350V
  • the invention provides recombinant nucleic acids encoding the non-naturally occurring tet-p53 proteins, expression vectors, and host cells
  • the invention provides methods of producing a non-naturally occur ⁇ ng tet-p53 protein comprising cultu ⁇ ng the host cell of the invention under conditions suitable for expression of the nucleic acid
  • the invention provides pharmaceutical compositions comprising a tet-p53 protein of the invention and a pharmaceutical carrier
  • the invention provides pharmaceutical compositions comprising a nucleic acid encoding a tet-p53 protein of the invention and a pharmaceutical carrier
  • the invention provides methods for treating an p53 responsive condition comprising administering a tet-p53 nucleic acid encoding a tet-p53 protein of the invention to a patient BRIEF DESCRIPTION OF THE DRAWINGS
  • Figure 1 depicts the tetramenzation domain of human p53, spanning ammo acid numbers 326 to 356
  • the human wild-type p53 sequence has accession number P04637 in the SwissProtem protein sequence database, and the residue numbering follows this entry
  • Figure 2 depicts the sequence alignment of a number of p53 tetramenzation domains, and the characterization of residues as core (c), surface (s) and boundary (b)
  • Figure 3 depicts a table of acceptable residues (similar to a probability table except that there are no probabilities listed) for alterations in the tetramenzation domain
  • Figure 4 depicts the synthesis of a full-length gene and all possible mutations by PCR Overlapping oligonucleotides corresponding to the full-length gene (black bar, Step 1 ) and comprising one or more desired mutations are synthesized, heated and annealed Addition of DNA polymerase to the annealed oligonucleotides results in the 5' to 3' synthesis of DNA (Step 2) to produce longer DNA fragments (Step 3) Repeated cycles of heating, annealing, and DNA synthesis (Step 4) result in the production of longer DNA, including some full-length molecules These can be selected by a second round of PCR using primers (indicated by arrows) corresponding to the end of the full-length gene
  • Figure 5 depicts a preferred method for synthesizing a library of the p53 proteins of the invention using
  • Figure 6 depicts an overlapping extension method At the top of Figure 6 is the template DNA showing the locations of the regions to be mutated (black boxes) and the binding sites of the relevant primers
  • the primers R1 and R2 represent a pool of primers, each containing a different mutation, as described herein, this may be done using different ratios of primers if desired
  • the variant position is flanked by regions of homology sufficient to get hybridization
  • three separate PCR reactions are done for step 1
  • the first reaction contains the template plus o gos F1 and R1
  • the second reaction contains template plus F2 and R2, and the third contains the template and F3 and R3
  • Step 2 The reaction products are shown In Step 2, the products from Step 1 tube 1 and Step 1 tube 2 are taken After purification away from the primers, these are added to a fresh PCR reaction together with F1 and R4 During the denaturation phase of the PCR, the overlapping regions anneal and the second strand is synthesized The product is then amphfed by the outside primers In Step 3, the purified product from Step 2 is used in a third PCR reaction, together with the product of Step 1 ,, tube 3 and the primers F1 and R3 The final product corresponds to the full length gene and contains the required mutations
  • Figure 7 depicts a ligation of PCR reaction products to synthesize the libraries of the invention
  • the primers also contain an endonuclease restriction site (RE), either blunt, 5' overhanging or 3' overhanging
  • RE endonuclease restriction site
  • Figure 8 depicts blunt end ligation of PCR products
  • the primers such as F1 and R1 do not overlap, but they abut Again three separate PCR reactions are performed The products from tube 1 and tube 2 are ligated, and then amplified with outside primers F1 and R4 This product is then
  • the present invention is directed to novel proteins and nucleic acids possessing p53 activity with novel tetramenzation domains (sometimes referred to herein as "tetrame ⁇ zation p53 proteins” or “tet-p53” proteins)
  • novel proteins are generated using a system previously described in WO98/47089 and U S S Nos 09/058,459, 09/127,926, 60/104,612, 60/158,700, 09/419,351 , 60/181 ,630, 60/186,904, and an application entitled “Protein Design Automation for Protein Libraries” filed April 14, 2000 (no U S serial number received yet) all of which are expressly incorporated by reference in their entirety, that is a computational modeling system that allows the generation of extremely stable proteins without necessarily disturbing the biological functions of the protein itself In this way, novel tet-p53 proteins and nucleic acids are generated, that can have a plurality of mutations in comparison to the wild-type protein yet retain significant biological activity
  • sequence based methods are used
  • molecular dynamics calculations can be used to computationally screen sequences by individually calculating mutant sequence scores and compiling a rank ordered list
  • residue pair potentials can be used to score sequences (Miyazawa et al , Macromolecules 18(3) 534-552 (1985), expressly incorporated by reference) during computational screening
  • sequence profile scores Bowie et al , Science 253(5016) 164-70 (1991 ), incorporated by reference
  • potentials of mean force Hertz et al , J Mol Biol 216(1 ) 167-
  • scoring functions can be used to screen for sequences that would create metal or co- factor binding sites in the protein (Hellmga, Fold Des 3(1 ) R1-8 (1998), hereby expressly incorporated by reference) Similarly, scoring functions can be used to screen for sequences that would create disulfide bonds in the protein These potentials attempt to specifically modify a protein structure to introduce a new structural motif
  • sequence and/or structural alignment programs can be used to generate the tet-p53 proteins of the invention
  • sequence-based alignment programs including for example, Smith-Waterman searches, Needleman-Wunsch, Double Affine Smith-Waterman, frame search, G ⁇ bskov/GCG profile search, G ⁇ bskov/GCG profile scan, profile frame search, Bucher generalized profiles, Hidden Markov models, Hframe, Double Frame, Blast, Psi-Blast, Clustal, and GeneWise
  • sequence alignment methodologies can be used to create sequence alignments of proteins related to the target structure (Altschul et al , J Mol Biol 215(3) 403-410 (1990), Altschul et al , Nucleic Acids Res 25 3389-3402 (1997), both incorporated by reference) These sequence alignments are then examined to determine the observed sequence variations
  • Sequence based alignments can be used in a variety of ways For example, a number of related proteins can be aligned, as is known in the art, and the "variable" and “conserved” residues defined, that is, the residues that vary or remain identical between the family members can be defined These results can be used to generate a probability table, as outlined below Similarly, these sequence variations can be tabulated and a secondary library defined from them as defined below Alternatively, the allowed sequence variations can be used to define the ammo acids considered at each position during the computational screening Another variation is to bias the score for ammo acids that occur in the sequence alignment, thereby increasing the likelihood that they are found du ⁇ ng computational screening but still allowing consideration of other ammo acids This bias would result in a focused library of tet-p53 proteins but would not eliminate from consideration ammo acids not found in the alignment In addition, a number of other types of bias may be introduced For example, diversity may be forced, that is, a "conserved" residue is chosen and altered to force diversity on
  • structural alignment of structurally related proteins can be done to generate sequence alignments (Orengo et al , Structure 5(8) 1093-108 (1997), Holm et al , Nucleic Acids Res 26(1 ) 316-9
  • sequence alignments can then be examined to determine the observed sequence variations Libraries can be generated by predicting secondary structure from sequence, and then selecting sequences that are compatible with the predicted secondary structure There are a number of secondary structure prediction methods such as helix-coil transition theory (Munoz and Serrano, Biopolymers 41 495, 1997), neural networks, local structure alignment and others (e g , see in Selbig et al , Biomformatics 15 1039-46, 1999)
  • the computational method used to generate the set or library of tet-p53 proteins is Protein Design Automation (PDA), as is described in U S S N s 60/061 ,097, 60/043,464, 60/054,678, 09/127,926, 60/104,612, 60/158,700, 09/419,351 , 60/181630, 60/186,904, and an application entitled "Protein Design Automation for Protein Libraries” filed April 14, 2000 (no U S serial number received yet) and PCT US98/07254, all of which are expressly incorporated herein by reference
  • PDA Protein Design Automation
  • a known protein structure is used as the starting point
  • the residues to be optimized are then identified, which may be the entire sequence or subset(s) thereof
  • the side chains of any positions to be varied are then removed
  • the resulting structure consisting of the protein backbone and the remaining sidechains is called the template
  • Each variable residue position is then preferably classified as a core residue, a surface residue, or a boundary residue, each classification defines a subset of possible ammo acid residues for the position (for example, core residues generally will be selected from the set of hydrophobic residues, surface residues generally will be selected from the hydrophilic residues, and boundary residues may be either)
  • Each ammo acid can be represented by a discrete set of all allowed conformers of each side chain, called rotamers
  • all possible sequences of rotamers must be screened, where each backbone position can be occupied either by each ammo acid in all its possible rotame ⁇ c states, or a subset of ammo acids, and thus a subset of rotamers
  • Two sets of interactions are then calculated for each rotamer at every position the interaction of the rotamer side chain with all or part of the backbone (the "singles" energy, also called the rotamer/template or rotamer/back
  • a Monte Carlo search may be done to generate a rank- ordered list of sequences in the neighborhood of the DEE solution
  • Starting at the DEE solution random positions are changed to other rotamers, and the new sequence energy is calculated If the new sequence meets the criteria for acceptance, it is used as a starting point for another jump
  • a rank-ordered list of sequences is generated Monte Carlo searching is a sampling technique to explore sequence space around the global minimum or to find new local minima distant in sequence space.
  • Monte Carlo searching is a sampling technique to explore sequence space around the global minimum or to find new local minima distant in sequence space.
  • the kinds of jumps allowed can be altered (e g random jumps to random residues, biased jumps (to or away from wild-type, for example), jumps to biased residues (to or away from similar residues, for example), etc )
  • the acceptance criteria of whether a sampling jump is accepted can be altered
  • the protein backbone (comprising (for a naturally occunng protein) the nitrogen, the carbonyl carbon, the ⁇ -carbon, and the carbonyl oxygen, along with the direction of the vector from the ⁇ -carbon to the ⁇ -carbon) may be altered prior to the computational analysis, by varying a set of parameters called supersecondary structure parameters
  • the protein backbone structure contains at least one variable residue position
  • the residues, or ammo acids, of proteins are generally sequentially numbered starting with the N- termmus of the protein
  • a protein having a methionine at it's N-termmus is said to have a methionme at residue or ammo acid position 1 , with the next residues as 2, 3, 4, etc
  • the wild type (i e naturally occunng) protein may have one of at least 20 ammo acids, in any number of rotamers
  • variable residue position herein is meant an ammo acid position of the protein to be designed that is not fixed in the design method as a specific residue or rotamer, generally the wild-type residue or rotamer
  • all of the residue positions of the protein are variable That is, every ammo acid side chain may be altered in the methods of the present invention This is particularly desirable for smaller proteins, although the present methods allow the design of larger proteins as well While there is no theoretical limit to the length of the protein which may be designed this way, there is a practical computational limit
  • residue positions of the protein are variable, and the remainder are "fixed' , that is, they are identified in the three dimensional structure as being in a set conformation
  • a fixed position is left in its original conformation (which may or may not correlate to a specific rotamer of the rotamer library being used)
  • residues may be fixed as a non-wild type residue, for example, when known site-directed mutagenesis techniques have shown that a particular residue is desirable (for example, to eliminate a proteolytic site or alter the substrate specificity of an enzyme), the residue may be fixed as a particular ammo acid
  • the methods of the present invention may be used to evaluate mutations de novo, as is discussed below
  • a fixed position may be "floated", the ammo acid at that position is fixed, but different rotamers of that ammo acid are tested
  • the variable residues may be at least one, or anywhere from 0 1% to 99 9% of the
  • residues which can be fixed include, but are not limited to, structurally or biologically functional residues, alternatively, biologically functional residues may specifically not be fixed
  • residues which are known to be important for biological activity such as the residues which the binding site for a binding partner ( gand/receptor, antigen/antibody, etc ), phosphorylation or glycosylation sites which are crucial to biological function, or structurally important residues, such as disulfide bridges, metal binding sites, critical hydrogen bonding residues, residues critical for backbone conformation such as prolme or glycme, residues critical for packing interactions, etc may all be fixed in a conformation or as a single rotamer, or "floated"
  • residues which may be chosen as variable residues may be those that confer undesirable biological attributes, such as susceptibility to proteolytic degradation, dimerization or aggregation sites, glycosylation sites which may lead to immune responses, unwanted binding activity, unwanted allostery, undesirable enzyme activity but with a preservation of binding, etc
  • undesirable biological attributes such as susceptibility to proteolytic degradation, dimerization or aggregation sites, glycosylation sites which may lead to immune responses, unwanted binding activity, unwanted allostery, undesirable enzyme activity but with a preservation of binding, etc
  • each variable position is classified as either a core, surface or boundary residue position, although in some cases, as explained below, the variable position may be set to glycme to minimize backbone strain
  • residues need not be classified, they can be chosen as variable and any set of ammo acids may be used Any combination of core, surface and boundary positions can be utilized core, surface and boundary residues, core and surface residues, core and boundary residues, and surface and boundary residues, as well as core residues alone, surface residues alone, or boundary residues alone
  • the classification of residue positions as core, surface or boundary may be done in several ways, as will be appreciated by those in the art
  • the classification is done via a visual scan of the original protein backbone structure, including the side chains, and assigning a classification based on a subjective evaluation of one skilled in the art of protein modelling
  • a preferred embodiment utilizes an assessment of the orientation of the C ⁇ -C ⁇ vectors relative to a solvent accessible surface computed using only the template C ⁇ atoms, as outlined in U S S N s 60/061 ,097, 60/043,464, 60/054,678, 09/127,926 60/104,612, 60/158,700, 09/419,351 , 60/181630, 60/186,904, and an application entitled "Protein Design Automation for Protein Libraries ' filed April 14, 2000 (no U S serial number received yet) and PCT US98/07254
  • a surface area calculation can be done
  • Suitable core and boundary positions for tet-p53 proteins are outlined below Once each variable position is classified as either core, surface or boundary, a set of ammo acid side chains, and thus a set of rotamers, is assigned to each position That is, the set of possible ammo acid side chains that the program will allow to be considered at any particular position is chosen Subsequently, once the possible ammo acid side chains are chosen, the set of rotamers that will be evaluated at a particular position can be determined Thus, a core residue will generally be selected from the group of hydrophobic residues consisting of alanine, valme, isoleucme, leucme, phenylalanine, tyrosine, tryptophan, and methionine (in some embodiments, when the ⁇ scaling factor of the van der Waals scoring function, described below, is low, methionine is removed from the set), and the rotamer set for each core position potentially includes rotamers for these eight ammo acid side chains (
  • prolme, cysteine and glycme are not included in the list of possible ammo acid side chains, and thus the rotamers for these side chains are not used
  • the position is set to glycme to minimize backbone strain
  • Equation 1 the total energy is the sum of the energy of the van der Waals potential (E vdw ), the energy of atomic solvation (E as ), the energy of hydrogen bonding (E h bond ⁇ ng ), the energy of secondary structure (E ss ) and the energy of electrostatic interaction (E elec )
  • E vdw van der Waals potential
  • E as the energy of atomic solvation
  • E h bond ⁇ ng the energy of hydrogen bonding
  • E ss the energy of secondary structure
  • E elec the energy of electrostatic interaction
  • 25 computational analysis comprises the determination of the interaction of each possible rotamer with all or part of the remainder of the protein That is, the energy of interaction, as measured by one or more of the scoring functions, of each possible rotamer at each variable residue position with either the backbone or other rotamers, is calculated
  • the interaction of each rotamer with the entire remainder of the protein, i e both the entire template and all other rotamers is done
  • portion' or similar grammatical equivalents thereof, as used herein, with regard to a protein refers to a fragment of that protein This fragment may range in size from 6-10 amino acid residues to the entire ammo acid sequence minus one ammo acid Accordingly, the term "portion", as used herein, 35 with regard to a nucleic refers to a fragment of that nucleic acid This fragment may range in size from
  • the first step of the computational processing is done by calculating two sets of interactions for each rotamer at every position the interaction of the rotamer side chain with the template or backbone (the “singles” energy), and the interaction of the rotamer side chain with all other possible rotamers at every other position (the “doubles” energy), whether that position is varied or floated
  • the backbone in this case includes both the atoms of the protein structure backbone, as well as the atoms of any fixed residues, wherein the fixed residues are defined as a particular conformation of an am o acid
  • DEE Dead End Elimination
  • PDA viewed broadly, has three components that may be varied to alter the output (e g the primary library) the scoring functions used in the process, the filtering technique, and the sampling technique
  • the scoring functions may be altered
  • the scoring functions outlined above may be biased or weighted in a variety of ways
  • a bias towards or away from a reference sequence or family of sequences can be done, for example, a bias towards wild-type or homolog residues may be used
  • the entire protein or a fragment of it may be biased, for example, the active site may be biased towards wild-type residues, or domain residues towards a particular desired physical property can be done
  • a bias towards or against increased energy can be generated
  • Additional scoring function biases include, but are not limited to applying electrostatic potential gradients or hydrophobicity gradients, adding a substrate or binding partner to the calculation, or biasing towards a desired charge or hydrophobicity
  • there are a variety of additional scoring functions that may be used Additional scoring functions include, but are not limited to torsional potentials, or residue pair potentials, or residue entropy potentials Such additional scoring functions can be used alone, or as functions for processing the library
  • filtering techniques can be done, including, but not limited to, DEE and its related counterparts Additional filtering techniques include, but are not limited to branch- and-bound techniques for finding optimal sequences (Gordon and Mayo, Structure Fold Des 7 1089-
  • sequence space sampling methods can be done, either in addition to the preferred Monte Carlo methods, or instead of a Monte Carlo search That is, once a sequence or set of sequences is generated, preferred methods utilize sampling techniques to allow the generation of additional, related sequences for testing
  • sampling methods can include the use of amino acid substitutions, insertions or deletions, or recombinations of one or more sequences
  • a preferred embodiment utilizes a Monte Carlo search, which is a series of biased, systematic, or random jumps
  • Monte Carlo search is a series of biased, systematic, or random jumps
  • the kinds of jumps allowed can be altered (e g random jumps to random residues, biased jumps (to or away from wild- type, for example), jumps to biased residues (to or away from similar residues, for example, etc ) Jumps where multiple residue positions are coupled (two residues always change together, or never change together), jumps where whole sets of residues change to other sequences (e g , recombination)
  • the acceptance criteria of whether a sampling jump is accepted can be altered
  • the preferred methods of the invention result in a rank ordered list of sequences, that is, the sequences are ranked on the basis of some objective criteria
  • it is possible to create a set of non-ordered sequences for example by generating a probability table directly (for example using SCMF analysis or sequence alignment techniques) that lists sequences without ranking them
  • the sampling techniques outlined herein can be used in either situation
  • Boltzman sampling is done as will be appreciated by those in the art, the temperature criteria for Boltzman sampling can be altered to allow broad searches at high temperature and narrow searches close to local optima at low temperatures (see e g , Metropolis et al , J Chem Phys 21 1087, 1953)
  • the sampling technique utilizes genetic algorithms, e g , such as those described by Holland (Adaptation in Natural and Artifical Systems, 1975, Ann Arbor, U Michigan Press) Genetic algorithm analysis generally takes generated sequences and recombines them computationally, similar to a nucleic acid recombination event, in a manner similar to "gene shuffling'
  • Genetic algorithm analysis generally takes generated sequences and recombines them computationally, similar to a nucleic acid recombination event, in a manner similar to "gene shuffling'
  • the "jumps" of genetic algorithm analysis generally are multiple position jumps
  • correlated multiple jumps may also be done Such jumps can occur withdifferent crossover positions and more than one recombination at a time, and can involve recombination of two or more sequences
  • deletions or insertions random or biased
  • genetic algorithm analysis may also be used after the secondary library has been generated
  • the sampling technique utilizes simulated annealing, e g , such as described by Kirkpat ⁇ ck et al [Science, 220 671-680 (1983)] Simulated annealing alters the cutoff for accepting good or bad jumps by altering the temperature That is, the stringency of the cutoff is altered by altering the temperature This allows broad searches at high temperature to new areas of sequence space, altering with narrow searches at low temperature to explore regions in detail
  • sampling methods can be used to further process a first set to generate additional sets of tet-p53 proteins
  • each optimized tet-p53 protein sequence, within the tetramenzation domain preferably comprises at least about 3-10% variant amino acids from the starting or wild type sequence, with at least about 10-15% being preferred, with at least about 15- 20% changes being more preferred and at least 25% being particularly preferred
  • the present invention is directed to tet-p53 proteins that have p53 activity
  • p53 activity herein is meant that the tet-p53 protein exhibits at least one, and preferably more, of the biological functions of a wild-type p53 protein
  • the biological function of a tet- p53 protein is altered, preferably improved, over the corresponding activity of a wild-type p53
  • protein herein is meant at least two covalently attached ammo acids, which includes proteins, polypeptides, o gopeptides and peptides
  • the protein may be made up of naturally occurring ammo acids and peptide bonds, or synthetic peptidomimetic structures, i e , "analogs” such as peptoids [see Simon et al , Proc Natl Acd Sci U S A 89(20 9367-71 (1992)], generally depending on the method of synthesis
  • “ammo acid”, or “peptide residue”, as used herein means both naturally occurring and synthetic ammo acids
  • homo-phenylalanme, citrulline, and noreleucine are considered ammo acids for the purposes of the invention
  • Ammo acid also includes imino acid residues such as prolme and hydroxyproline
  • any ammo acid representing a component of the tet-p53 proteins can be replaced by the same ammo acid but of the opposite chira ty
  • Aromatic am o acids may be replaced with D- or L-naphylalanme, D- or L-Phenylglycme, D- or L-2- thieneylalanine, D- or L-1-, 2-, 3- or 4-pyreneylalan ⁇ ne, D- or L-3-th ⁇ eneylalan ⁇ ne, D- or L-(2-py ⁇ d ⁇ nyl)- alanme, D- or L-(3-py ⁇ d ⁇ nyl)-alan ⁇ ne, D- or L-(2-pyraz ⁇ nyl)-alan ⁇ ne, D- or L-(4- ⁇ sopropyl)-phenylglyc ⁇ ne, D-(t ⁇ fluoromethyl)-phenylglyc ⁇ ne, D-(t ⁇ fluoromethyl)-phenylalan ⁇ ne, D-p-fluorophenylalanine, D- or L-p- biphenylphenylalanme, D- or
  • alkyl may be substituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl, sec-isotyl, iso-pentyl, non-acidic ammo acids, of C1-C20
  • Acidic ammo acids can be substituted with non-carboxylate ammo acids while maintaining a negative charge, and derivatives or analogs thereof, such as the non-limiting examples of (phosphono)alan ⁇ ne, glycme, leucme, isoleucme, threonme, or se ⁇ ne, or sulfated (e g , -SO sub 3 H) threonme, se ⁇ ne, tyrosine
  • alkyl refers to a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isoptopyl, n- butyl, isobutyl, t-butyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracisyl and the like
  • Alkyl includes heteroalkyl, with atoms of nitrogen, oxygen and sulfur Preferred alkyl groups herein contain 1 to 12 carbon atoms
  • Basic ammo acids may be substituted with alkyl groups at any position of the naturally occurring ammo acids lysine, arginme, ornithine, citrullme, or (guan ⁇ d ⁇ no)-acet
  • any amide linkage in any of the tet-p53 polypeptides can be replaced by a ketomethylene moiety
  • Such derivatives are expected to have the property of increased stability to degradation by enzymes, and therefore possess advantages for the formulation of compounds which may have increased in vivo half lives, as administered by oral, intravenous, intramuscular, intrape ⁇ toneal, topical, rectal, intraocular, or other routes
  • Additional ammo acid modifications of amino acids of tet-p53 polypeptides of to the present invention may include the following Cystemyl residues may be reacted with alpha-haloacetates (and corresponding amines), such as 2-chloroacet ⁇ c acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives Cystemyl residues may also be de ⁇ vatized by reaction with compounds such as bromot ⁇ fluoroacetone, alpha-bromo-beta-(5- ⁇ m ⁇ dozoyl)prop ⁇ on ⁇ c acid, chloroacetyl phosphate, N-alkylmaleimides, 3-n ⁇ tro-2-py ⁇ dyl disulfide, methyl 2-py ⁇ dyl disulfide, p- chloromercu ⁇ benzoate, 2-chloromercu ⁇ -4-n ⁇ trophenol, or chloro-7-n ⁇ trobenzo-2-oxa-1 ,3-d ⁇ azole
  • Histidyl residues may be de ⁇ vatized by reaction with compounds such as diethylprocarbonate e g , at pH 5 5-7 0 because this agent is relatively specific for the histidyl side chain, and para-bromophenacyl bromide may also be used, e g , where the reaction is preferably performed in 0 1 M sodium cacodylate at pH 6 0
  • Lysmyl and ammo terminal residues may be reacted with compounds such as succinic or other carboxylic acid anhydrides De ⁇ vatization with these agents is expected to have the effect of reversing the charge of the lysmyl residues
  • suitable reagents for de ⁇ vatizing alpha-amino-contaming residues include compounds such as imidoesters/e g , as methyl picolmimidate, py ⁇ doxal phosphate, py ⁇ doxal chloroborohyd ⁇ de, t ⁇ nitrobenzenesulfonic acid O-methylisourea, 2,4 pentanedione, and transaminase-catalyzed reaction with glyoxylate
  • Argmyl residues may be modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butaned ⁇ one, 1 ,2-cyclohexaned ⁇ one, and ninhyd ⁇ n according to known method steps De ⁇ vatization of arginme residues requires that the reaction be performed in alkaline conditions because of the high pKa of the guanidme functional group Furthermore, these reagents may react with the groups of lysine as well as the arginme epsilon-amino group
  • the specific modification of tyrosyl residues per se is well-known, such as for introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane N- acetylimidizol and tetranitromethane may be used to form O-acetyl tyrosyl species and 3-n ⁇ tro derivatives, respectively
  • Carboxyl side groups (aspartyl or glutamyl) may be selectively modified by reaction with carbodiimides
  • R'-N-C-N-R' such as 1-cyclohexyl-3-(2-morphol ⁇ nyl- (4-ethyl) carbodiimide or 1-ethyl-3-(4-azon ⁇ a-4,4- dimethylpentyl) carbodiimide
  • aspartyl and glutamyl residues may be converted to asparagmyl and glutammyl residues by reaction with ammonium ions
  • Glutammyl and asparagmyl residues may be frequently deamidated to the corresponding glutamyl and aspartyl residues Alternatively, these residues may be deamidated under mildly acidic conditions
  • the p53 may be from any number of organisms, with p53 proteins from mammals being particularly preferred Suitable mammals include, but are not limited to, rodents (rats, mice, hamsters, guinea pigs, etc ), primates, farm animals (including sheep, goats, pigs, cows, horses, etc) and in the most preferred embodiment, from humans (this is sometimes referred to herein as hp53, the tetramenzation sequence of which is depicted in Figure 1 ) As will be appreciated by those in the art, p53s based on p53s from mammals other than humans may find use in animal models of human disease
  • tet-p53 proteins of the invention exhibit at least one biological function of a p53
  • p53' herein is meant a wild type p53 or an allelic variant thereof
  • p53 refers to all forms of p53 that are active in accepted p53 assays
  • the tet-p53 proteins of the invention exhibit at least one biological function of a p53
  • biological function or “biological property” herein is meant any one of the properties or functions of a p53, including, but not limited to, activities of the transactivation domain, DNA binding domain, tetramenzation domain, and regulatory domain, the ability to effect cellular growth, in particular inhibition of cell proliferation, the ability to induce growth arrest and/or apoptosis
  • tet-p53 proteins will exhibit at least 25-50% of the wild-type activity More preferred are tet-p53 proteins that exhibit at least 75%, even more preferred are tet-p53 proteins that exhibit at least 90%, and most preferred are tet-p53 proteins that exhibit more than 100% of a biological activity of the wild type p53 Suitable assays include, but are not limited to, DNA binding assays, transcription assays (using reporter constructs, see Stav ⁇ di, supra), tumor suppression assays (using transfection assays and cell counting see Stav ⁇ di supra), tetramenzation assays (gel electrophoresis assays see Mateu, supra, size exclusion chromatography assays and radiolabeling/immunoprecipitation, see Stav ⁇ di, supra), and stability assays (including the use of circular dichroism (CD) assays and equilibrium studies, see Mateu, supra), all of which are expressly incorporated by reference
  • At least one biological property of the tet-p53 protein is altered when compared to the same property of p53, and in particular, tet-p53 proteins will altered tetramenzation domains and properties are preferred Particularly preferred are tet-p53 proteins with altered tetramenzation domains and substantially no alterations in any other p53 biological activity
  • the invention provides tet-p53 proteins with altered tetramenzation domains such that the tet- p53 proteins will preferentially oligome ⁇ ze with each other, but will not substantially oligome ⁇ ze with naturally occunng mutant p53 forms, which generally exhibit wild-type tetramenzation domains That is, under physiological conditions, the tet-p53 proteins will form homotetramers with themselves preferentially over heterotetramers with wild-type tetramenzation sequences "Preferentially” in this case means that given equal amounts of tet-p53 monomers and p53 monomers containing wild-type tetramenzation sequences (which, as will be appreciated by those in the art, can include mutant p53 proteins that have altered DNA binding properties leading to disease, but exhibit normal tetramenzation domains), at least 25% of the resulting tetramers are homotetramers of tet-p53, with at least about 50% being preferred
  • the invention provides tet-p53 nucleic acids encoding tet-p53 polypeptides
  • the tet-p53 polypeptide preferably has at least one property, which is substantially different from the same property of the corresponding naturally occurring p53 polypeptide
  • the property of the tet-p53 polypeptide is the result the PDA analysis of the present invention
  • altered property or grammatical equivalents thereof in the context of a polypeptide refer to any characteristic or attribute of a polypeptide that can be selected or detected and compared to the corresponding property of a naturally occurring protein
  • properties include, but are not limited to tetramenzation with wild-type or naturally occurring mutant p53 forms, oxidative stability, substrate specificity, substrate binding or catalytic activity, thermal stability, alkaline stability, pH activity profile, resistance to proteolytic degradation, kinetic association (K on ) and dissociation (K of( ) rate, protein folding, inducing an immune response, ability to bind to a ligand, ability to bind to a receptor, ability to be secreted, ability to be displayed on the surface of a cell, ability to oligome ⁇ ze, ability to signal, ability to stimulate cell proliferation, ability to inhibit cell proliferation, ability to induce apoptosis, ability to be modified by phosphorylation or glycosylation, ability to treat disease
  • a substantial change in any of the above-listed properties, when comparing the property of a tet-p53 polypeptide to the property of a naturally occurring p53 protein is preferably at least a 20%, more preferably, 50%, more preferably at least a 2-fold increase or decrease
  • a change in oxidative stability is evidenced by at least about 20%, more preferably at least 50% increase of activity of a tet-p53 protein when exposed to various oxidizing conditions as compared to that of p53 Oxidative stability is measured by known procedures
  • alkaline stability is evidenced by at least about a 5% or greater increase or decrease (preferably increase) in the half life of the activity of a tet-p53 protein when exposed to increasing or decreasing pH conditions as compared to that of p53 Generally, alkaline stability is measured by known procedures
  • thermal stability is evidenced by at least about a 5% or greater increase or decrease (preferably increase) in the half life of the activity of a tet-p53 protein when exposed to a relatively high temperature and neutral pH as compared to that of p53 Generally, thermal stability is measured by known procedures
  • tet-p53 proteins for example are experimentally tested and validated in in vivo and in in vitro assays Suitable assays include, but are not limited to, e g , examining their binding affinity to natural occurring or variant p53 tetramenzation domains, and can include quantitative comparisons comparing kinetic and equilibrium binding constants The kinetic association rate (K on ) and dissociation rate (K off ), and the equilibrium binding constants (K d ) can be determined using surface plasmon resonance on a BIAcore instrument following the standard procedure in the literature [Pearce et al , Biochemistry 38 81-89 (1999)] Again, as outlined herein, tet-p53 proteins that will auto- oligome ⁇ ze but will not oligome ⁇ ze with the wild-type p53 tetramenzation domain are preferred
  • the antigenic profile in the host animal of the tet-p53 protein is similar, and preferably identical, to the antigenic profile of the host p53, that is, the tet-p53 protein does not significantly stimulate the host organism (e g the patient) to an immune response, that is, any immune response is not clinically relevant and there is no allergic response or neutralization of the protein by an antibody That is, in a preferred embodiment, the tet-p53 protein does not contain additional or different epitopes from the p53
  • 'epitope" or "determinant” herein is meant a portion of a protein which will generate and/or bind an antibody
  • no significant amount of antibodies are generated to a tet-p53 protein In general, this is accomplished by not significantly altering surface residues, as outlined below nor by adding any ammo acid residues on the surface which can become glycosylated, as novel glycosylation can result in an immune response
  • tet-p53 proteins and nucleic acids of the invention are distinguishable from naturally occurring p53s
  • naturally occurring or wild type or grammatical equivalents
  • allelic variations that is, an ammo acid sequence or a nucleotide sequence that usually has not been intentionally modified
  • non-naturally occurring or “synthetic” or “recombinant” or grammatical equivalents thereof herein is meant an ammo acid sequence or a nucleotide sequence that is not found in nature that is, an ammo acid sequence or a nucleotide sequence that usually has been intentionally modified
  • the tet-p53 protein has an am o acid sequence that differs from a wild-type p53 sequence by at least 1-3% of the residues in the tetramenzation domain (as outlined herein, additional residues (e g outside the tetramenzation domain) can be changed as well) That is, the tet-p53 proteins of the invention are less than about 97-99% identical to an p53 ammo acid sequence in the tetramenzation domain Accordingly, a protein is an "tet-p53 protein" if the overall homology of the protein sequence to the ammo acid sequence shown in Figure 1 is preferably less than about 97%, more preferably less than about 95%, even more preferably less than about 90% and most preferably less than 85% In some embodiments the homology will be as low as about 75 to 80% Stated differently, based on the human p53 sequence o Figure 1 , tet-p53 proteins have at least about 1 residue in
  • sequence similarity means sequence similarity or identity, with identity being preferred
  • a number of different programs can be used to identify whether a protein (or nucleic acid as discussed below) has sequence identity or similarity to a known sequence
  • Sequence identity and/or similarity is determined using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith & Waterman, Adv Appl Math , 2 482 (1981 ), by the sequence identity alignment algorithm of Needleman & Wunsch, J Mol Biol , 48 443 (1970), by the search for similarity method of Pearson & Lipman, Proc Natl Acad Sci U S A , 85 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wl), the Best Fit sequence program described by Devereux et al , Nucl Acid Res , 12 387-395 (1984), preferably using the default settings,
  • PILEUP PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments It can also plot a tree showing the clustering relationships used to create the alignment PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J Mol Evol 35 351-360 (1987), the method is similar to that described by Higgms & Sharp CABIOS 5 151-153 (1989)
  • Useful PILEUP parameters including a default gap weight of 3 00, a default gap length weight of 0 10, and weighted end gaps
  • a particularly useful BLAST program is the WU- BLAST-2 program which was obtained from Altschul et al , Methods in Enzymology, 266 460-480 (1996) http //blast wustl/edu/blast/ README html]
  • WU-BLAST-2 uses several search parameters, most of which are set to the default values
  • the HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched however the values may be adjusted to increase sensitivity
  • Gapped BLAST uses BLOSUM-62 substitution scores, threshold T parameter set to 9 the two-hit method to trigger ungapped extensions charges gap lengths of k a cost of ⁇ 0+k, X u set to 16 and X g set to 40 for database search stage and to 67 for the output stage of the algorithms Gapped alignments are triggered by a score corresponding to -22 bits
  • a % amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the "longer" sequence in the aligned region
  • the "longer” sequence is the one having the most actual residues in the aligned region (gaps introduced by WU- Blast-2 to maximize the alignment score are ignored)
  • percent (%) nucleic acid sequence identity with respect to the coding sequence of the polypeptides identified herein is defined as the percentage of nucleotide residues in a candidate sequence that are identical with the nucleotide residues in the coding sequence of the cell cycle protein
  • a preferred method utilizes the BLASTN module of WU-BLAST-2 set to the default parameters, with overlap span and overlap fraction set to 1 and 0 125, respectively
  • the alignment may include the introduction of gaps in the sequences to be aligned
  • the percentage of sequence identity will be determined based on the number of identical am o acids in relation to the total number of ammo acids
  • sequence identity of sequences shorter than that shown in Figure 1 will be determined using the number of ammo acids in the shorter sequence, in one embodiment In percent identity calculations relative weight is not assigned to various manifestations of sequence
  • identities are scored positively (+1) and all forms of sequence variation including gaps are assigned a value of "0", which obviates the need for a weighted scale or parameters as described below for sequence similarity calculations
  • Percent sequence identity can be calculated, for example, by dividing the number of matching identical residues by the total number of residues of the "shorter" sequence in the aligned region and multiplying by 100 The "longer" sequence is the one having the most actual residues in the aligned region
  • tet-p53 proteins of the present invention may be shorter or longer than the ammo acid sequence shown in Figure 1A
  • tet-p53 proteins included within the definition of tet-p53 proteins are portions or fragments of the sequences depicted herein Fragments of tet-p53 proteins are considered tet-p53 proteins if a) they share at least one antigenic epitope, b) have at least the indicated homology, c) and preferably have tet-p53 biological activity as defined herein
  • the tet-p53 proteins include further ammo acid variations, as compared to a wild type p53, than those outlined herein.
  • any of the variations depicted herein may be combined in any way to form additional novel tet- p53 proteins
  • tet-p53 proteins can be made that are longer than those depicted in the figures, for example, by the addition of epitope or purification tags, as outlined herein, the addition of other fusion sequences, etc
  • the tet-p53 proteins of the invention may be fused to other therapeutic proteins or to other proteins such as Fc or serum albumin for pharmacokmetic purposes See for example U S Patent No 5,766,883 and 5,876,969, both of which are expressly incorporated by reference
  • the tet-p53 proteins comprise variable residues in core and boundary residues Human p53 core residues of the tetramenzation domain are as follows positions 330, 332, 340, 341 , 344, 347, 348 and 350 (see Figure 3) Accordingly, in a preferred embodiment, tet-p53 proteins have variable positions selected from these positions
  • tet-p53 proteins have variable positions selected solely from core residues of human p53 Alternatively, at least a majority (51 %) of the variable positions are selected from core residues with at least about 75% of the va ⁇ able positions being preferably selected from core residue positions, and at least about 90% of the variable positions being particularly preferred A specifically preferred embodiment has only core variable positions altered as compared to human p53
  • variable core positions are altered to any of the other 19 ammo acids
  • variable core residues are chosen from Ala, Val, Phe, He, Leu, Tyr, Trp and Met
  • human p53 surface residues of the tetramenzation domain are as follows positions 326, 327, 329, 331 , 333, 334, 335, 336, 339, 342, 346, 351 , 352, 353, 354 and 355 (see
  • tet-p53 proteins have variable positions selected from these positions
  • variable surface positions are altered to any of the other 19 ammo acids
  • vartable surface residues are chosen from Ser, Thr, Asp, Asn, Glu, Gin,
  • human p53 boundary residues of the tetramenzation domain are as follows positions 328, 337, 338, 343, 345 and 349 (see Figure 3) Accordingly, in a preferred embodiment, tet-p53 proteins have variable positions selected from these positions
  • variable boundary positions are altered to any of the other 19 ammo acids
  • variable boundary residues are chosen from Ala, Val, Phe, He, Leu, Tyr,
  • Trp Met, Ser, Thr, Asp, Asn, Glu, Gin, Lys, Arg, His and Ala
  • Preferred ammo acids for each position including the human p53 residues, are shown in Figures 2-3
  • preferred ammo acids are Tyr, Trp, Phe and Leu
  • at position 330 He and Leu are preferred
  • position 332 He, Val or Leu are preferred
  • Preferred changes are as follows.
  • Residues in the tetramer interface are 343, 344, 348, 350, 346 and 351
  • Residues in the dimer interface are 328, 330, 332, 337, 338 and 345
  • preferred changes are at these residues
  • sequences are [E343I, L344M, I350Y, E346(K or H), K351 E, F328(Y or W or L), L330I, I332(V or L), R337L, F338Y and N345(Y or F)] (24 sequences), [M340A, L344(W or M), L348(Y, M, F or W), L350 (L or I)] (16 different sequences), [F341 A, M340(L or M), L344M, L348 (W or F), L350(L or I or Y)] (12 sequences), [L344A, M340 (M or L), F341 (F or M or Y), L348(F or M or W) and L350(L or I or Y or W] (108 sequences) These latter sequences were designed by forcing a mutation to occur and then finding the adjustments in
  • the tet-p53 proteins of the invention are human p53 conformers
  • 'conformer' herein is meant a protein that has a protein backbone 3D structure that is virtually the same but has significant differences in the ammo acid side chains.
  • the tet-p53 proteins of the invention define a conformer set, wherein all of the proteins of the set share a backbone structure and yet have sequences that differ by at least 1-3-5%
  • the three dimensional backbone structure of a tet- p53 protein thus substantially corresponds to the three dimensional backbone structure of human p53
  • "Backbone' in this context means the non-side chain atoms the nitrogen, carbonyl carbon and oxygen, and the ⁇ -carbon, and the hydrogens attached to the nitrogen and ⁇ -carbon
  • a protein must have backbone atoms that are no more than 2 A from the human p53 structure, with no more than 1 5 ⁇ being preferred, and no more than 1 A being particularly preferred In general, these distances may be determined in two ways In one embodiment, each potential conformer is crystallized and its three dimensional structure determined Alternatively, as the former is quite tedious, the sequence of each potential conformer is run in the
  • tet-p53 proteins may also be identified as being encoded by tet-p53 nucleic acids
  • nucleic acid the overall homology of the nucleic acid sequence is commensurate with ammo acid homology but takes into account the degeneracy in the genetic code and codon bias of different organisms Accordingly, the nucleic acid sequence homology may be either lower or higher than that of the protein sequence, with lower homology being preferred
  • a tet-p53 nucleic acid encodes a tet-p53 protein
  • an extremely large number of nucleic acids may be made, all of which encode the tet-p53 proteins of the present invention
  • those skilled in the art could make any number of different nucleic acids, by simply modifying the sequence of one or more codons in a way which does not change the ammo acid sequence of the t
  • nucleic acid homology is determined through hybridization studies
  • nucleic acids which hybridize under high stringency to the nucleic acid sequence shown in Figure 1 or its complement and encode a tet-p53 protein is considered a tet-p53 gene
  • stringent conditions are selected to be about 5-10 ° C lower than the thermal melting point (TJ for the specific sequence at a defined ionic strength and pH
  • TJ thermal melting point
  • the T m is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T m , 50% of the probes are occupied at equilibrium)
  • Stringent conditions will be those in which the salt concentration is less than about
  • less stringent hybridization conditions are used, for example, moderate or low stringency conditions may be used, as are known in the art, see Maniatis and Ausubel, supra, and Tijssen, supra
  • nucleic acid may refer to either DNA or RNA, or molecules which contain both deoxy- and ⁇ bonucleotides
  • the nucleic acids include genomic DNA, cDNA and oligonucleotides including sense and anti-sense nucleic acids
  • Such nucleic acids may also contain modifications in the ⁇ bose- phosphate backbone to increase stability and half life of such molecules in physiological environments
  • the nucleic acid may be double stranded, single stranded, or contain portions of both double stranded or single stranded sequence
  • the depiction of a single strand also defines the sequence of the other strand ("Crick"), thus the sequence depicted in Figure 1 also includes the complement of the sequence
  • recombinant nucleic acid herein is meant nucleic acid, originally formed in vitro, in general, by the
  • a "recombinant protein” is a protein made using recombinant techniques, i e through the expression of a recombinant nucleic acid as depicted above
  • a recombinant protein is distinguished from naturally occurring protein by at least one or more characteristics
  • the protein may be isolated or purified away from some or all of the proteins and compounds with which it is normally associated in its wild type host and thus may be substantially pure
  • an isolated protein is unaccompanied by at least some of the material with which it is normally associated in its natural state, preferably constituting at least about 0 5%, more preferably at least about 5% by weight of the total protein in a given sample
  • a substantially pure protein comprises at least about 75% by weight of the total protein, with at least about 80% being preferred, and at least about 90% being particularly preferred
  • the definition includes the production of a tet-p53 protein from one organism in a different organism or host cell Alternatively, the protein may be made at a significantly higher concentration than is normally seen, through the use of a m
  • tet-p53 proteins of the present invention are ammo acid sequence variants of the tet-p53 sequences outlined herein and shown in the Figures That is, the tet-p53 proteins may contain additional variable positions as compared to human p53 These variants fall into one or more of three classes substitutional, insertional or deletional variants These variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding a tet-p53 protein, using cassette or PCR mutagenesis or other techniques well known in the art, to produce DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture as outlined above
  • variant tet-p53 protein fragments having up to about 100-150 residues may be prepared by in vitro synthesis using established techniques Ammo acid sequence variants are characterized by the predetermined nature of the variation, a feature that sets them apart from naturally occurring allelic or mterspecies variation of the tet
  • the mutation per se need not be predetermined
  • random mutagenesis may be conducted at the target codon or region and the expressed tet-p53 variants screened for the optimal combination of desired activity
  • Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example, M13 primer mutagenesis and PCR mutagenesis Screening of the mutants is done using assays of tet-p53 protein activities
  • Ammo acid substitutions are typically of single residues, insertions usually will be on the order of from about 1 to 20 ammo acids, although considerably larger insertions may be tolerated Deletions range from about 1 to about 20 residues, although in some cases deletions may be much larger
  • substitutions may be made which more significantly affect the structure of the polypeptide backbone in the area of the alteration, for example the alpha-helical or beta-sheet structure, the charge or hydrophobicity of the molecule at the target site, or the bulk of the side chain
  • substitutions which in general are expected to produce the greatest changes in the polypeptide's properties are those in which (a) a hydrophilic residue, e g seryl or threonyl, is substituted for (or by) a hydrophobic residue, e g leucyl, isoleucyl, phenylalanyl valyl or alanyl, (b) a cysteine or prolme is substituted for (or by) any other residue, (c) a residue having an electropositive side chain, e g lysyl
  • the variants typically exhibit the same qualitative biological activity and will elicit the same immune response as the original tet-p53 protein, although variants also are selected to modify the characteristics of the tet-p53 proteins as needed Alternatively, the variant may be designed such that the biological activity of the tet-p53 protein is altered For example, glycosylation sites may be altered or removed Similarly, the biological function may be altered, for example, in some instances it may be desirable to have more or less potent p53 activity
  • tet-p53 proteins and nucleic acids of the invention can be made in a number of ways Individual nucleic acids and proteins can be made as known in the art and outlined below Alternatively, libraries of tet-p53 proteins can be made for testing
  • sets or libraries of tet-p53 proteins are generated from a probability distribution table
  • a probability distribution table there are a variety of methods of generating a probability distribution table, including using PDA, sequence alignments, forcefield calculations such as SCMF calculations, etc
  • the probability distribution can be used to generate information entropy scores for each position, as a measure of the mutational frequency observed in the library
  • the frequency of each ammo acid residue at each variable position in the list is identified Frequencies can be thresholded, wherein any variant frequency lower than a cutoff is set to zero This cutoff is preferably 1 %, 2%, 5%, 10% or 20%, with 10% being particularly preferred These frequencies are then built into the tet-p53 library That is, as above, these variable positions are collected and all possible combinations are generated, but the ammo acid residues that "fill" the library are utilized on a frequency basis Thus, in a non-frequency based library, a variable position that has 5 possible residues will have 20% of the proteins comprising that variable position with the first possible residue, 20% with the second, etc However, in a frequency based library, a variable position that has 5 possible residues with frequencies of 10%, 15%, 25%, 30% and 20%, respectively, will have 10% of the proteins comprising that variable position with the first possible residue, 15% of the proteins with the second residue, 25% with the third, etc As will be appreciated by those in the art, the actual frequency may depend on the method
  • SCMF self-consistent mean field
  • a probability table generated in this way can be used to create libraries as described herein
  • SCMF can be used in three ways the frequencies of am o acids and rotamers for each ammo acid are listed at each position, the probabilities are determined directly from SCMF (see Delarue et la Pac Symp Biocomput 109-21 (1997), expressly incorporated by reference)
  • highly variable positions and non-variable positions can be identified Alternatively, another method is used to determine what sequence is jumped to during a search of sequence space, SCMF is used to obtain an accurate energy for that sequence this energy is then used to rank it and create a rank-ordered list of sequences (similar to a Monte Carlo sequence list)
  • Similar methods include, but are not limited to, OPLS-AA (Jorgensen, et al , J Am Chem Soc (1996), v 118, pp 11225-11236, Jorgensen, W L , BOSS, Version 4 1 , Yale University New Haven, CT (1999)), OPLS (Jorgensen, et al , J Am Chem Soc (1988), v 110, pp 1657ff, Jorgensen, et al , J Am Chem Soc (1990), v 112, pp 4768ff), UNRES (United Residue Forcefield, Liwo, et al ,
  • a tet-p53 library created by recombming variable positions and/or residues at the variable position may not be in a rank-ordered list In some embodiments, the entire list may just be made and tested Alternatively, in a preferred embodiment, the tet-p53 library is also in the form of a rank ordered list This may be done for several reasons, including the size of the library is still too big to generate experimentally, or for predictive purposes This may be done in several ways In one embodiment, the library is ranked using the scoring functions of PDA to rank the library members Alternatively, statistical methods could be used For example, the library may be ranked by frequency score, that is, proteins containing the most of high frequency residues could be ranked higher, etc This may be done by adding or multiplying the frequency at each variable position to generate a numerical score Similarly, the library different positions could be weighted and then the proteins scored for example, those containing certain residues could be arbitrarily ranked
  • the different protein members of the tet-p53 library may be chemically synthesized This is particularly useful when the designed proteins are short, preferably less than 150 ammo acids in length, with less than 100 ammo acids being preferred, and less than 50 ammo acids being particularly preferred, although as is known in the art, longer proteins can be made chemically or enzymatically See for example Wilken et al, Curr 0pm Biotechnol 9 412-26 (1998), hereby expressly incorporated by reference
  • the library sequences are used to create nucleic acids such as DNA which encode the member sequences and which can then be cloned into host cells, expressed and assayed, if desired
  • nucleic acids, and particularly DNA can be made which encodes each member protein sequence This is done using well known procedures
  • codons, suitable expression vectors and suitable host cells will vary depending on a number of factors, and can be easily optimized as needed
  • multiple PCR reactions with pooled oligonucleotides is done, as is generally depicted in the Figures
  • overlapping oligonucleotides are synthesized which correspond to the full length gene
  • these oligonucleotides may represent all of the different ammo acids at each variant position or subsets
  • these oligonucleotides are pooled in equal proportions and multiple PCR reactions are performed to create full length sequences containing the combinations of mutations defined by the library In addition, this may be done using error-prone PCR methods
  • the different oligonucleotides are added in relative amounts corresponding to the probability distribution table
  • the multiple PCR reactions thus result in full length sequences with the desired combinations of mutaions in the desired proportions
  • the total number of oligonucleotides needed is a function of the number of positions being mutated and the number of mutations being considered at these positions
  • each overlapping oligonucleotide comprises only one position to be varied in alternate embodiments, the variant positions are too close together to allow this and multiple variants per oligonucleotide are used to allow complete recombination of all the possibilities That is, each oligo can contain the codon for a single position being mutated, or for more than one position being mutated The multiple positions being mutated must be close in sequence to prevent the oligo length from being impractical
  • particular combinations of mutations can be included or excluded in the library by including or excluding the oligonucleotide encoding that combination
  • there may be correlations between variable regions that is, when position X is a certain residue, position Y must (or must not) be a particular residue
  • correlations and shuffling can be fixed or optimized by altering the design of the oligonucleotides, that is, by deciding where the oligonucleotides (primers) start and stop (e g where the sequences are "cut")
  • the start and stop sites of ohgos can be set to maximize the number of clusters that appear in single oligonucleotides, thereby enriching the library with higher scoring sequences
  • Different oligonucleotide start and stop site options can be computationally modeled and ranked according to number of clusters that are represented on single ohgos, or the percentage of the resulting sequences consistent with the predicted library of sequences
  • the total number of oligonucleotides required increases when multiple mutable positions are encoded by a single oligonucleotide
  • the annealed regions are the ones that remain constant, i e have the sequence of the reference sequence
  • Oligonucleotides with insertions or deletions of codons can be used to create a library expressing different length proteins
  • computational sequence screening for insertions or deletions can result in secondary libraries defining different length proteins, which can be expressed by a library of pooled oligonucleotide of different lengths
  • the tet-p53 library is done by shuffling the family (e g a set of variants), that is, some set of the top sequences (if a rank-ordered list is used) can be shuffled, either with or without error-prone PCR
  • shuffling in this context means a recombination of related sequences generally in a random way It can include “shuffling” as defined and exemplified in U S Patent Nos 5,830,721 , 5,811 ,238, 5,605,793, 5,837,458 and PCT US/19256, all of which are expressly incorporated by reference in their entirety
  • This set of sequences can also be an artificial set, for example, from a probability table (for example generated using SCMF) or a Monte Carlo set
  • the "family" can be the top 10 and the bottom 10 sequences, the top 100 sequence, etc This may also be done using error-prone PCR
  • sihco shuffling is
  • error-prone PCR is done to generate the tet-p53 library See U S Patent Nos 5,605,793, 5,811 ,238, and 5,830,721 , all of which are hereby incorporated by reference This can be done on the optimal sequence or on top members of the library, or some other artificial set or family
  • the gene for the optimal sequence found in the computational screen of the primary library can be synthesized
  • Error prone PCR is then performed on the optimal sequence gene in the presence of oligonucleotides that code for the mutations at the variant positions of the library (bias oligonucleotides)
  • bias oligonucleotides bias oligonucleotides
  • gene shuffling with error prone PCR can be performed on the gene for the optimal sequence, in the presence of bias oligonucleotides, to create a DNA sequence library that reflects the proportion of the mutations found in the tet-p53 library
  • bias oligonucleotides can be done in a variety of ways, they can chosen on the basis of their frequency, i e oligonucleotides encoding high mutational frequency positions can be used, alternatively, oligonucleotides containing the most variable positions can be used, such that the diversity is increased, if the secondary library is ranked, some number of top scoring positions can be used to generate bias oligonucleotides, random positions may be chosen, a few top scoring and a few low scoring ones may be chosen, etc What is important is to generate new sequences based on preferred variable positions and sequences
  • PCR using a w d type gene or other gene can be used, as is schematically depicted in the Figures
  • a starting gene is used, generally, although this is not required, the gene is usually the wild type gene In some cases it may be the gene encoding the global optimized sequence, or any other sequence of the list, or a consensus sequence obtained e g from aligning homologous sequences from different organisms
  • oligonucleotides are used that correspond to the variant positions and contain the different ammo acids of the library PCR is done using PCR primers at the termini, as is known in the art This provides two benefits, the first is that this generally requires fewer oligonucleotides and can result in fewer errors
  • it has experimental advantages in that if the wild type gene is used, it need not be synthesized
  • tet-p53 library may be computationally remanipulated to form an additional tet-p53 library (sometimes referred to herein as "tertiary libraries")
  • additional tet-p53 library (sometimes referred to herein as "tertiary libraries)
  • any of the tet-p53 library sequences may be chosen for a second round of PDA, by freezing or fixing some or all of the changed positions in the first library Alternatively, only changes seen in the last probability distribution table are allowed Alternatively, the stringency of the probability table may be altered, either by increasing or decreasing the cutoff for inclusion Similarly, the tet-p53 library may be recombmed experimentally after the first round, for
  • a tertiary library can be generated from combining different tet-p53 libraries
  • a probability distribution table from a first tet-p53 library can be generated and recombmed, either computationally or experimentally, as outlined herein
  • a PDA tet-p53 library may be combined with a sequence alignment tet-p53 library, and either recombmed (again, computationally or experimentally) or just the cutoffs from each joined to make a new tertiary library
  • the top sequences from several libraries can be recombmed Sequences from the top of a library can be combined with sequences from the bottom of the library to more broadly sample sequence space, or only sequences distant from the top of the library can be combined tet-p53 libraries that analyzed different parts of a protein can be combined to a tertiary library that treats the combined parts of the protein
  • a tertiary library can be generated using correlations in a tet-p53 library That is, a residue at a first variable position may be correlated to a residue at second variable position (or correlated to residues at additional positions as well) For example, two variable positions may ste ⁇ cally or electrostatically interact, such that if the first residue is X, the second residue must be Y This may be either a positive or negative correlation
  • the expression vectors may be either self-replicating extrachromosomal vectors or vectors which integrate into a host genome Generally, these expression vectors include transc ⁇ ptional and translational regulatory nucleic acid operably linked to the nucleic acid encoding the tet-p53 protein
  • control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism
  • the control sequences that are suitable for prokaryotes include a promoter, optionally an operator sequence, and a ⁇ bosome binding site
  • Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers
  • Nucleic acid is "operably linked” when it is placed into a functional relationship with another nucleic acid sequence For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if
  • a replacement of the naturally occurring secretory leader sequence is desired
  • an unrelated secretory leader sequence is operably linked to a tet-p53 encoding nucleic acid leading to increased protein secretion
  • any secretory leader sequence resulting in enhanced secretion of the tet-p53 protein, when compared to the secretion of p53 and its secretory sequence, is desired Suitable secretory leader sequences that lead to the secretion of a protein are know in the art
  • a secretory leader sequence of a naturally occurring protein or a protein is removed by techniques known in the art and subsequent expression results in mtracellular accumulation of the recombinant protein
  • transc ⁇ ptional and translational regulatory nucleic acid will generally be appropriate to the host cell used to express the fusion protein, for example, transc ⁇ ptional and translational regulatory nucleic acid sequences from Bacillus are preferably used to express the fusion protein in Bacillus Numerous types of appropriate expression vectors, and suitable regulatory sequences are known in the art for a variety of host cells
  • transc ⁇ ptional and translational regulatory sequences may include, but are not limited to, promoter sequences, ⁇ bosomal binding sites, transc ⁇ ptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences
  • the regulatory sequences include a promoter and transc ⁇ ptional start and stop sequences
  • Promoter sequences encode either constitutive or mducible promoters
  • the promoters may be either naturally occurring promoters or hybrid promoters Hybrid promoters, which combine elements of more than one promoter, are also known in the art, and are useful in the present invention
  • the promoters are strong promoters, allowing high expression in cells, particularly mammalian cells, such as the CMV promoter, particularly in combination with a Tet regulatory element
  • the expression vector may comprise additional elements
  • the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in mammalian or insect cells for expression and in a prokaryotic host for cloning and amplification
  • the expression vector contains at least one sequence homologous to the host cell genome, and preferably two homologous sequences which flank the expression construct
  • the integrating vector may be directed to a specific locus in the host cell by selecting the appropriate homologous sequence for inclusion in the vector Constructs for integrating vectors are well known in the art
  • the expression vector contains a selectable marker gene to allow the selection of transformed host cells Selection genes are well known in the art and will vary
  • a preferred expression vector system is a retroviral vector system such as is generally described in
  • the expression vector comprises the components described above and a gene encoding a tet-p53 protein
  • a vector composition a gene encoding a tet-p53 protein
  • the tet-p53 nucleic acids are introduced into the cells either alone or in combination with an expression vector
  • introduction into or grammatical equivalents herein is meant that the nucleic acids enter the cells in a manner suitable for subsequent expression of the nucleic acid
  • Exemplary methods include CaP0 4 precipitation, hposome fusion, lipofectm®, electroporation, viral infection, etc
  • the tet- p53 nucleic acids may stably integrate into the genome of the host cell (for example, with retroviral introduction, outlined below), or may exist either transiently or stably in the cytoplasm (i.e through the use of traditional plasmids, utilizing standard regulatory sequences, selection markers, etc )
  • the tet-p53 proteins of the present invention are produced by cultu ⁇ ng a host cell transformed with an expression vector containing nucleic acid encoding a tet-p53 protein, under the appropriate conditions to induce or cause expression of the tet-p53 protein
  • the conditions appropriate for tet-p53 protein expression will vary with the choice of the expression vector and the host cell, and will be easily ascertained by one skilled in the art through routine experimentation
  • the use of constitutive promoters in the expression vector will require optimizing the growth and proliferation of the host cell, while the use of an mducible promoter requires the appropriate growth conditions for induction
  • the timing of the harvest is important
  • the baculoviral systems used in insect cell expression are lytic viruses, and thus harvest time selection can be crucial for product yield
  • Appropriate host cells include yeast, bacteria, archebacte ⁇ a, fungi, and insect and animal cells, including mammalian cells Of particular interest are Drosophila melangaster cells, Saccharomyces cerevisiae and other yeasts, E coli, Bacillus subtilis, SF9 cells, C129 cells, 293 cells, Neurospora, BHK, CHO, COS, Pichia Pastons, etc.
  • the tet-p53 proteins are expressed in mammalian cells
  • Mammalian expression systems are also known in the art, and include retroviral systems
  • a mammalian promoter is any DNA sequence capable of binding mammalian RNA polymerase and initiating the downstream (3') transcription of a coding sequence for the fusion protein into mRNA
  • a promoter will have a transcription initiating region, which is usually placed proximal to the 5' end of the coding sequence, and a TATA box, using a located 25-30 base pairs upstream of the transcription initiation site The TATA box is thought to direct RNA polymerase II to begin RNA synthesis at the correct site
  • a mammalian promoter will also contain an upstream promoter element (enhancer element), typically located within 100 to 200 base pairs upstream of the TATA box An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation
  • An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation
  • transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3' to the translation stop codon and thus, together with the promoter elements, flank the coding sequence The 3' terminus of the mature mRNA is formed by site-specific post-translational cleavage and polyadenylation Examples of transcription terminator and polyadenlytion signals include those derived form SV40
  • suitable cell types include, but are not limited to, tumor cells of all types (particularly melanoma, myeloid leukemia, carcinomas of the lung, breast, ovaries, colon, kidney, prostate, pancreas and testes), cardiomyocytes, endothehal cells, epithelial cells, lymphocytes (T-cell and B cell) , mast cells, eosmophils, vascular intimal cells, hepatocytes, leukocytes including mononuclear leukocytes, stem cells such as haemopoetic, neural, skin, lung, kidney, liver and myocyte stem cells (for use in screening for differentiation and de-differentiation factors), osteoclasts, chondrocytes and other connective tissue cells, keratmocytes, melanocytes, liver cells, kidney cells, and adipocytes Suitable cells also include known research cells, including, but not limited to, Jurkat T cells, NIH3T3 cells, CHO, Cos, etc See the ATCC cell line
  • the cells may be additionally genetically engineered, that is, contain exogeneous nucleic acid other than the tet-p53 nucleic acid
  • the tet-p53 proteins are expressed in bacterial systems Bacterial expression systems are well known in the art
  • a suitable bacterial promoter is any nucleic acid sequence capable of binding bacterial RNA polymerase and initiating the downstream (3') transcription of the coding sequence of the tet-p53 protein into mRNA
  • a bacterial promoter has a transcription initiation region which is usually placed proximal to the 5' end of the coding sequence This transcription initiation region typically includes an RNA polymerase binding site and a transcription initiation site
  • Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences Examples include promoter sequences derived from sugar metabolizing enzymes, such as galactose, lactose and maltose, and sequences derived from biosynthetic enzymes such as tryptophan Promoters from bacte ⁇ ophage may also be used and are known in the art
  • synthetic promoters and hybrid promoters are also useful, for example, the tac promoter is a hybrid of the trp and lac promoter sequences
  • a bacterial promoter can include naturally occurring promoters of non-bacterial
  • the expression vector may also include a signal peptide sequence that provides for secretion of the tet-p53 protein in bacteria
  • the signal sequence typically encodes a signal peptide comprised of hydrophobic am o acids which direct the secretion of the protein from the cell, as is well known in the art
  • the protein is either secreted into the growth media (gram-positive bacteria) or into the pe ⁇ plasmic space, located between the inner and outer membrane of the cell (gram-negative bacteria)
  • bacterial secretory leader sequences operably linked to a tet-p53 encoding nucleic acid, are preferred
  • the bacterial expression vector may also include a selectable marker gene to allow for the selection of bacterial strains that have been transformed Suitable selection genes include genes which render the bacteria resistant to drugs such as ampicillin, chloramphenicol, erythromycm, kanamycm, neomycin and tetracyc ne Selectable markers also include biosynthetic genes, such as those in the histidme, tryptophan and leucme biosynthetic pathways
  • Expression vectors for bacteria are well known in the art, and include vectors for Bacillus subtilis, E coli, Streptococcus cremons, and Streptococcus lividans, among others
  • the bacterial expression vectors are transformed into bacterial host cells using techniques well known in the art, such as calcium chloride treatment, electroporation, and others
  • tet-p53 proteins are produced in insect cells
  • Expression vectors for the transformation of insect cells, and in particular, baculovirus-based expression vectors, are well known in the art
  • tet-p53 protein is produced in yeast cells
  • Yeast expression systems are well known in the art, and include expression vectors for Saccharomyces cerevisiae, Candida albicans and C maltosa, Hansenula polymorpha, Kluyveromyces fragilis and K lactis, Pichia guillenmondii and P pasto ⁇ s, Schizosaccharomyces pombe, and Yarrowia lipolytica
  • Preferred promoter sequences for expression in yeast include the mducible GAL1.10 promoter, the promoters from alcohol dehydrogenase, enolase, glucokmase, glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate- dehydrogenase, hexokmase, phosphofructokmase, 3-phosphoglycerate mutase, pyruvate kmase, and the acid phosphatase gene Yeast selectable markers include ADE
  • tet-p53 polypeptides of the invention may be further fused to other proteins, if desired, for example to increase expression or stabilize the protein
  • the tet-p53 nucleic acids, proteins and antibodies of the invention are labeled with a label other than the scaffold
  • label herein is meant that a compound has at least one element, isotope or chemical compound attached to enable the detection of the compound
  • labels fall into three classes a) isotopic labels, which may be radioactive or heavy isotopes b) immune labels, which may be antibodies or antigens, and c) colored or fluorescent dyes
  • the labels may be incorporated into the compound at any position
  • the tet-p53 proteins may be covalently modified Covalent and non-covalent modifications of the protein are thus included within the scope of the present invention
  • modifications may be introduced into a tet-p53 polypeptide by reacting targeted ammo acid residues of the polypeptide with an organic de ⁇ vatizing agent that is capable of reacting with selected side chains or terminal residues
  • One type of covalent modification includes reacting targeted ammo acid residues of a tet-p53 polypeptide with an organic de ⁇ vatizing agent that is capable of reacting with selected side chains or the N-or C-terminal residues of a tet-p53 polypeptide
  • De ⁇ vatization with bifunctional agents is useful, for instance, for crosshnking a tet-p53 protein to a water-insoluble support matrix or surface for use in the method for purifying ant ⁇ -tet-p53 antibodies or screening assays, as is more fully described below
  • Commonly used crosshnking agents include, e g , 1 ,1-b ⁇ s(d ⁇ azoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccmimide esters, for example, esters with 4-az ⁇ dosal ⁇ cyl ⁇ c acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'
  • Another type of covalent modification of the tet-p53 polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of the polypeptide "Altering the native glycosylation pattern" is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence tet-p53 polypeptide, and/or adding one or more glycosylation sites that are not present in the native sequence tet-p53 polypeptide
  • Addition of glycosylation sites to tet-p53 polypeptides may be accomplished by altering the ammo acid sequence thereof
  • the alteration may be made, for example, by the addition of, or substitution by one or more se ⁇ ne or threonme residues to the native sequence tet-p53 polypeptide (for 0-hnked glycosylation sites)
  • the tet-p53 ammo acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the tet-p53 polypeptide at preselected bases such that codons are generated that will translate into the desired ammo acids
  • Removal of carbohydrate moieties present on the tet-p53 polypeptide may be accomplished chemically or enzymatically or by mutational substitution of codons encoding for ammo acid residues that serve as targets for glycosylation
  • Chemical deglycosylation techniques are known in the art and described, for instance, by Hakimuddin et al , Arch Biochem Biophys , 259 52 (1987) and by Edge et al , Anal Biochem , 118 131 (1981 )
  • Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo-and exo-glycosidases as described by Thotakura et al Meth Enzymol , 138 350 (1987)
  • Such de ⁇ vatized moieties may improve the solubility, absorption, permeability across the blood brain barrier biological half life, and the like
  • Such moieties or modifications of tet-p53 polypeptides may alternatively eliminate or attenuate any possible undesirable side effect of the protein and the like
  • Moieties capable of mediating such effects are disclosed, for example, in Remington's Pharmaceutical Sciences, 16th ed , Mack Publishing Co , Easton, Pa (1980)
  • Another type of covalent modification of tet-p53 comprises linking the tet-p53 polypeptide to one of a variety of nonprotemaceous polymers, e g , polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U S Patent Nos 4,640,835, 4,496,689, 4,301 ,144, 4,670,417, 4,791 ,192 or 4,179,337
  • tet-p53 polypeptides of the present invention may also be modified in a way to form chime ⁇ c molecules comprising a tet-p53 polypeptide fused to another, heterologous polypeptide or am o acid sequence
  • a chime ⁇ c molecule comprises a fusion of a tet-p53 polypeptide with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind
  • the epitope tag is generally placed at the ammo-or carboxyl-termmus of the tet-p53 polypeptide
  • the presence of such epitope-tagged forms of a tet-p53 polypeptide can be detected using an antibody against the tag polypeptide
  • provision of the epitope tag enables the tet-p53 polypeptide to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag
  • the chime ⁇ c molecule may comprise
  • tag polypeptides and their respective antibodies are well known in the art Examples include poly-histidme (poly-his) or poly-histidine-glycme (poly-his-gly) tags, the flu HA tag polypeptide and its antibody 12CA5 [Field et al , Mol Cell Biol 8 2159-2165 (1988)], the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et al , Molecular and Cellular Biology, 5 3610-3616
  • Tag polypeptides include the Flag-peptide [Hopp et al , BioTechnology 6 1204-1210 (1988)], the KT3 epitope peptide [Martin et al , Science 255 192-194 (1992)], tubuhn epitope peptide [Skinner et al , J Biol Chem 266 15163-15166 (1991 )], and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al , Proc Natl Acad Sci U S A 87 6393-6397
  • the tet-p53 protein is purified or isolated after expression tet-p53 proteins may be isolated or purified in a variety of ways known to those skilled in the art depending on what other components are present in the sample Standard purification methods include electrophoretic, molecular, immunological and chromatographic techniques, including ion exchange, hydrophobic, affinity, and reverse-phase HPLC chromatography, and chromatofocusmg
  • the tet-p53 protein may be purified using a standard anti-library antibody column Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful For general guidance in suitable purification techniques, see Scopes, R , Protein Purification, Sp ⁇ nger-Verlag, NY (1982) The degree of purification necessary will vary depending on the use of the tet-p53 protein In some instances no purification will be necessary
  • the tet-p53 proteins and nucleic acids of the invention find use in a number of applications
  • the tet-p53 proteins are administered to a patient to treat an p53-assoc ⁇ ated disorder
  • p53 associated disorder or "p53 responsive disorder” or “condition” herein is meant a disorder that can be ameliorated by the administration of a pharamaceutical composition comprising a p53 or tet-p53 protein, including, but not limited to, cancer, particularly tumors and cancers known to have a mutant p53 relationship, including, but not limited to, breast, prostate, brain and lung cancer
  • a therapeutically effective dose of a tet-p53 protein is administered to a patient in need of treatment
  • therapeutically effective dose herein is meant a dose that produces the effects for which it is administered The exact dose will depend on the purpose of the treatment, and will be ascertamable by one skilled in the art using known techniques
  • dosages of about 5 ⁇ g/kg are used, administered either mtraveneously or subcutaneously
  • adjustments for tet-p53 protein degradation, systemic versus localized delivery, and rate of new protease synthesis are known in the art.
  • a "patient” for the purposes of the present invention includes both humans and other animals, particularly mammals, and organisms Thus the methods are applicable to both human therapy and veterinary applications
  • the patient is a mammal, and in the most preferred embodiment the patient is human
  • treatment in the instant invention is meant to include therapeutic treatment, as well as prophylactic, or suppressive measures for the disease or disorder
  • successful administration of a tet-p53 protein prior to onset of the disease results in “treatment” of the disease
  • successful administration of a tet-p53 protein after clinical manifestation of the disease to combat the symptoms of the disease comprises “treatment” of the disease
  • Treatment also encompasses administration of a tet-p53 protein after the appearance of the disease in order to eradicate the disease
  • Successful administration of an agent after onset and after clinical symptoms have developed, with possible abatement of clinical symptoms and perhaps amelioration of the disease comprises "treatment" of the disease
  • Those "in need of treatment” include mammals already having the disease or disorder, as well as those prone to having the disease or disorder, including those in which the disease or disorder is to be prevented
  • a therapeutically effective dose of a tet-p53 protein, a tet-p53 gene, or a tet- p53 antibody is administered to a patient having a disease involving inappropriate expression of p53
  • a "disease involving inappropriate expression of a p53" within the scope of the present invention is meant to include diseases or disorders characterized by aberrant p53, either by alterations in the amount of p53 present or due to the presence of mutant p53
  • An overabundance may be due to any cause, including, but not limited to, overexpression at the molecular level, prolonged or accumulated appearance at the site of action, or increased activity of p53 relative to normal Included within this definition are diseases or disorders characterized by a reduction of p53 This reduction may be due to any cause, including, but not limited to, reduced expression at the molecular level, shortened or reduced appearance at the site of action, mutant forms of p53, or decreased activity of p53 relative to normal Such an overabundance or
  • the administration of the tet-p53 proteins of the present invention can be done in a variety of ways, including, but not limited to, orally, subcutaneously, intravenously, intranasally, transdermally, intrape ⁇ toneally, intramuscularly, mtrapulmonary, vagmally, rectally, or intraocularly
  • the tet-p53 A protein may be directly applied as a solution or spray
  • the pharmaceutical composition may be formulated in a variety of ways
  • the concentration of the therapeutically active tet-p53 protein in the formulation may vary from about 0 1 to 100 weight %
  • the concentration of the tet-p53 protein is in the range of 0 003 to 1 0 molar, with dosages from 0 03, 0 05 0 1 , 0 2, and 0 3 mill
  • compositions of the present invention comprise a tet-p53 protein in a form suitable for administration to a patient
  • the pharmaceutical compositions are in a water soluble form, such as being present as pharmaceutically acceptable salts, which is meant to include both acid and base addition salts
  • “Pharmaceutically acceptable acid addition salt” refers to those salts that retain the biological effectiveness of the free bases and that are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfu ⁇ c acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glycohc acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succmic acid, fuma ⁇ c acid, tarta ⁇ c acid, citric acid, benzoic acid, cinnamic acid, mandehc acid, methanesulfonic acid, ethanesulfonic acid, p-toluen
  • compositions may also include one or more of the following carrier proteins such as serum albumin, buffers such as NaOAc, fillers such as microcrystalhne cellulose, lactose, corn and other starches, binding agents, sweeteners and other flavoring agents, coloring agents, and polyethylene glycol Additives are well known in the art, and are used in a variety of formulations
  • carrier proteins such as serum albumin, buffers such as NaOAc, fillers such as microcrystalhne cellulose, lactose, corn and other starches, binding agents, sweeteners and other flavoring agents, coloring agents, and polyethylene glycol Additives are well known in the art, and are used in a variety of formulations
  • the tet-p53 proteins are added in a micellular formulation, see U S Patent No 5,833,948, hereby expressly incorporated by reference in its entirety
  • compositions may be administered in combination with other therapeutics
  • antibodies including but not limited to monoclonal and polyclonal antibodies, are raised against tet-p53 proteins using methods known in the art In a preferred embodiment, these ant ⁇ -tet-p53 antibodies are used for immunotherapy.
  • methods of immunotherapy are provided by “immunotherapy” is meant treatment of an p53 related disorders with an antibody raised against a tet-p53 protein
  • immunotherapy can be passive or active Passive immunotherapy, as defined herein, is the passive transfer of antibody to a recipient
  • tet-p53 protein antigen may be provided by injecting a tet-p53 polypeptide against which antibodies are desired to be raised into a recipient, or contacting the recipient with a tet-p53 protein encoding nucleic acid, capable of expressing the tet-p53 protein antigen, under conditions for expression of the tet-p53 protein antigen
  • a therapeutic compound is conjugated to an antibody, preferably an ant ⁇ -tet-p53 protein antibody
  • the therapeutic compound may be a cytotoxic agent
  • targeting the cytotoxic agent to tumor tissue or cells results in a reduction in the number of afflicted cells, thereby reducing symptoms associated with cancer, and tet-p53 protein related disorders
  • Cytotoxic agents are numerous and varied and include, but are not limited to, cytotoxic drugs or toxins or active fragments of such toxins Suitable toxins and their corresponding fragments include dipthe ⁇ a A chain, exotoxm A chain, ⁇ cin A chain, ab ⁇ n A chain, curcm, crotin, phenomycm, enomyc and the like
  • Cytotoxic agents also include radiochemicals made by conjugating radioisotopes to antibodies raised against cell cycle proteins, or binding of a radionuchde to a chelatmg agent that has been covalently attached to the antibody
  • tet-p53 proteins are administered as therapeutic agents, and can be formulated as outlined above Similarly, tet-p53 genes (including both the full-length sequence, partial sequences, or regulatory sequences of the tet-p53 coding regions) can be administered in gene therapy applications, as is known in the art These tet-p53 genes can include antisense applications, either as gene therapy (i e for incorporation into the genome) or as antisense compositions, as will be appreciated by those in the art
  • the nucleic acid encoding the tet-p53 proteins may also be used in gene therapy In gene therapy applications, genes are introduced into cells in order to achieve in vivo synthesis of a therapeutically effective genetic product, for example for replacement of a defective gene "Gene therapy" includes both conventional gene therapy where a lasting effect is achieved by a single treatment, and the administration of gene therapeutic agents, which involves the one time or repeated administration of a therapeutically effective DNA or mRNA Antisense RNA
  • nucleic acid source there are a variety of techniques available for introducing nucleic acids into viable cells
  • the techniques vary depending upon whether the nucleic acid is transferred into cultured cells in vitro, or in vivo in the cells of the intended host
  • Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, the calcium phosphate precipitation method, etc
  • the currently preferred in vivo gene transfer techniques include transfection with viral (typically retroviral) vectors and viral coat protem- hposome mediated transfection [Dzau et al , Trends in Biotechnology 11 205-210 (1993)]
  • an agent that targets the target cells such as an antibody specific for a cell surface membrane protein or the target cell, a ligand for a receptor on the target cell, etc
  • proteins which bind to a cell surface membrane protein associated with endocytosis may be used
  • tet-p53 genes are administered as DNA vaccines, either single genes or combinations of tet-p53 genes
  • Naked DNA vaccines are generally known in the art Brower, Nature Biotechnology, 16 1304-1305 (1998)
  • Methods for the use of genes as DNA vaccines are well known to one of ordinary skill in the art, and include placing a tet-p53 gene or portion of a tet-p53 gene under the control of a promoter for expression in a patient in need of treatment
  • the tet-p53 gene used for DNA vaccines can encode full-length tet-p53 proteins, but more preferably encodes portions of the tet- p53 proteins including peptides derived from the tet-p53 protein
  • a patient is immunized with a DNA vaccine comprising a plurality of nucleotide sequences derived from a tet- p53 gene.
  • the DNA vaccines include a gene encoding an adjuvant molecule with the DNA vaccine.
  • adjuvant molecules include cytokines that increase the immunogenic response to the tet-p53 polypeptide encoded by the DNA vaccine. Additional or alternative adjuvants are known to those of ordinary skill in the art and find use in the invention.

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Abstract

L'invention concerne de nouvelles protéines p53 comportant des domaines de tétramérisation (tet-p53) modifiés et de nouveaux acides nucléiques. L'invention concerne en outre l'utilisation des protéines tet-p53 dans le traitement de troubles associés aux p53.
PCT/US2000/013248 1999-05-12 2000-05-12 Nouveaux acides nucleiques et proteines ayant une activite p53 et comportant des domaines de tetramerisation modifies WO2000068384A2 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
JP2000616350A JP2003520023A (ja) 1999-05-12 2000-05-12 ｐ５３活性および変更された四量体化ドメインを有する新規の核酸およびタンパク質
EP00930723A EP1179062A2 (fr) 1999-05-12 2000-05-12 Nouveaux acides nucleiques et proteines ayant une activite p53 et comportant des domaines de tetramerisation modifies
CA002372881A CA2372881A1 (fr) 1999-05-12 2000-05-12 Nouveaux acides nucleiques et proteines ayant une activite p53 et comportant des domaines de tetramerisation modifies
AU48492/00A AU4849200A (en) 1999-05-12 2000-05-12 Novel nucleic acids and proteins with p53 activity and altered tetramerization domains

Applications Claiming Priority (2)

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US13378399P 1999-05-12 1999-05-12
US60/133,783 1999-05-12

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WO2000068384A2 true WO2000068384A2 (fr) 2000-11-16
WO2000068384A3 WO2000068384A3 (fr) 2001-02-15

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JP (1) JP2003520023A (fr)
AU (1) AU4849200A (fr)
CA (1) CA2372881A1 (fr)
WO (1) WO2000068384A2 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001009325A2 (fr) * 1999-07-30 2001-02-08 The Government Of The United States Of America, Asrepresented By The Secretary, Dept. Of Health And Human Services Mutations du gene p53 humain, et systeme genetique de levures utilise pour l'identification fonctionnelle des mutations du gene p53 humain
WO2003045415A2 (fr) * 2001-11-26 2003-06-05 University Health Network Excipients d'administration de peptides auto-assembleurs
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

Citations (3)

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Publication number Priority date Publication date Assignee Title
WO1996016989A1 (fr) * 1994-11-28 1996-06-06 The Wistar Institute Of Anatomy And Biology PROTEINES p53 A DOMAINES DE TETRAMERISATION MODIFIES
WO1998031703A1 (fr) * 1997-01-17 1998-07-23 The Wistar Institute Of Anatomy & Biology Procedes de modification d'une structure proteique tridimensionnelle et compositions produites par un tel procede
WO2000022115A2 (fr) * 1998-10-13 2000-04-20 Board Of Regents, The University Of Texas System Dosages permettant d'identifier des alterations fonctionnelles dans le gene suppresseur de tumeur p53

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996016989A1 (fr) * 1994-11-28 1996-06-06 The Wistar Institute Of Anatomy And Biology PROTEINES p53 A DOMAINES DE TETRAMERISATION MODIFIES
WO1998031703A1 (fr) * 1997-01-17 1998-07-23 The Wistar Institute Of Anatomy & Biology Procedes de modification d'une structure proteique tridimensionnelle et compositions produites par un tel procede
WO2000022115A2 (fr) * 1998-10-13 2000-04-20 Board Of Regents, The University Of Texas System Dosages permettant d'identifier des alterations fonctionnelles dans le gene suppresseur de tumeur p53

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
MATEU MAURICIO G ET AL: "Mutually compensatory mutations during evolution of the tetramerization domain of tumor suppressor p53 lead to impaired hetero-oligomerization." PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES, vol. 96, no. 7, 30 March 1999 (1999-03-30), pages 3595-3599, XP002152303 March 30, 1999 ISSN: 0027-8424 *
MATEU MAURICIO G ET AL: "Nine hydrophobic side chains are key determinants of the thermodynamic stability and oligomerization status of tumour suppressor p53 tetramerization domain." EMBO (EUROPEAN MOLECULAR BIOLOGY ORGANIZATION) JOURNAL, vol. 17, no. 10, 15 May 1998 (1998-05-15), pages 2748-2758, XP002152304 ISSN: 0261-4189 cited in the application *
STAVRIDI ELENA S ET AL: "Change in oligomerization specificity of the p53 tetramerization domain by hydrophobic amino acid substitutions." PROTEIN SCIENCE, vol. 8, no. 9, September 1999 (1999-09), pages 1773-1779, XP002152305 ISSN: 0961-8368 cited in the application *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001009325A2 (fr) * 1999-07-30 2001-02-08 The Government Of The United States Of America, Asrepresented By The Secretary, Dept. Of Health And Human Services Mutations du gene p53 humain, et systeme genetique de levures utilise pour l'identification fonctionnelle des mutations du gene p53 humain
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
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
WO2003045415A2 (fr) * 2001-11-26 2003-06-05 University Health Network Excipients d'administration de peptides auto-assembleurs
WO2003045415A3 (fr) * 2001-11-26 2003-12-04 Univ Health Network Excipients d'administration de peptides auto-assembleurs

Also Published As

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WO2000068384A3 (fr) 2001-02-15
CA2372881A1 (fr) 2000-11-16
EP1179062A2 (fr) 2002-02-13
AU4849200A (en) 2000-11-21
JP2003520023A (ja) 2003-07-02

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