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WO1998047919A1 - Matieres et procedes lies a la stabilisation de substances dans des cellules - Google Patents

Matieres et procedes lies a la stabilisation de substances dans des cellules Download PDF

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Publication number
WO1998047919A1
WO1998047919A1 PCT/GB1998/001140 GB9801140W WO9847919A1 WO 1998047919 A1 WO1998047919 A1 WO 1998047919A1 GB 9801140 W GB9801140 W GB 9801140W WO 9847919 A1 WO9847919 A1 WO 9847919A1
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Prior art keywords
mdm2
cells
substance
binding domain
peptide
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PCT/GB1998/001140
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English (en)
Inventor
David Philip Lane
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University Of Dundee
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Priority to AU70642/98A priority Critical patent/AU7064298A/en
Publication of WO1998047919A1 publication Critical patent/WO1998047919A1/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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates materials and methods relating to stabilising substances in cells, and in particular in cells in which a mdm2 mediated degradation pathway does not operate efficiently, using substances comprising a mdm2 binding domain linked to a coupling partner.
  • tumour suppressor protein p53 is activated upon genotoxic insult to cells and acts as a transcription factor to induce cell cycle arrest or apoptosis in cells after DNA damage.
  • Previous reports have suggested that cell proliferation might depend on a fine balance between expression of the oncogene mdm2 and the tumour suppressor p53 (Chen et al , 1994; Finlay, 1993; Otto and Deppert, 1993) .
  • This is due to an autoregulatory feedback loop for p53 activity involving mdm2 , as mdm2 is transcriptionally activated by binding of p53 to an internal promoter within the mdm2 gene (Juven et al, 1993; u et al , 1993) . It then binds the N-terminus of p53 thereby preventing p53 from interacting with the transcriptional machinery (Momand et al, 1992; Oliner et al , 1993).
  • WO 93/20238 discloses that excess levels of mdm2 are present in some tumour cells such as certain types of sarcoma.
  • This application discloses that the overexpression of mdm2 interferes with the normal feedback loop between mdm2 and p53 , allowing cells overexpressing mdm2 to escape from p53 -regulated growth control by binding p53.
  • WO 93/20238 therefore suggests that molecules that inhibit the binding of p53 to mdm2 could be used as therapeutics for conditions in which mdm2 is overexpressed by alleviating this sequestration of p53, and thereby reestablishing normal p53 control.
  • WO 93/20238 maps the domains of p53 that are necessary for mdm2 binding to amino acid residues 13-41, as well as additional residues on either the carboxy or the amino terminal side of this peptide.
  • WO 96/02642 describes experiments to refine the peptide motif of p53 responsible for binding to mdm2 , and shows that the motif is less extensive than disclosed in WO 93/20238.
  • WO 96/02642 discloses that a FxxLW motif between amino acid residues 18-23 of p53 (where x is any amino acid) is sufficient to bind to mdm2. This motif can be used to screen for therapeutic compounds capable of disrupting the interaction so that the transcriptional activity of p53 in cells overexpressing mdm2 can be restored.
  • the present invention is based on the finding that mdm2 binds to p53 in cells in which mdm2 is not overexpressed, i.e. in cells in which mdm2 is expressed at normal or low levels, and that in these cells, this interaction targets p53 for degradation. More particularly, the present invention concerns materials and methods that exploit this mechanism of p53 degradation to stabilise a substance comprising a mdm2 binding domain linked to a coupling partner in cells in which this mdm2 mediated degradation pathway does not operate efficiently.
  • Examples of this are cells which are p53 null, or contain lower than normal amounts of endogenous p53, or cells in which mutant p53 accumulates and is not marked for degradation by mdm2 , e.g. because the p53 is not active for DNA specific binding and so does not induce mdm2 production.
  • the substance in normal cells expressing functional mdm2 , the substance will tend to be unstable as it will be marked for degradation through the interaction of the endogenous mdm2 with the mdm2 binding domain of the substance.
  • TIP 12/1 The most potent aptamer, TIP 12/1, showed a similar binding affinity for mdm2 as bacterial full length wt p53. This made it a powerful inhibitor which could be expressed in mammalian cells.
  • the present invention provides a substance which comprises a mdm2 binding domain linked to a coupling partner for use in a method of treatment, wherein the substance is stabilised in cells in which a mdm2 mediated degradation pathway does not operate efficiently, e.g cells which do not express normal levels of mdm2.
  • the present invention provides the use of a substance which comprises a mdm2 binding domain linked to a coupling partner in the preparation of a medicament wherein the substance is stabilised in cells in which a mdm2 mediated degradation pathway does not operate efficiently.
  • the present invention provides a method of selectively stabilising a substance in cells in which a mdm2 mediated degradation pathway does not operate efficiently, the method comprising exposing the cells a substance which comprises a mdm2 binding domain linked to a coupling partner so that the substance is marked for degradation in cells containing functional mdm2.
  • the method can be used to treat cells in vi tro or to treat patients having conditions that respond to the coupling partner or a product derivable from the coupling partner.
  • the present invention provides a pharmaceutical composition comprising one or more of the above substances in combination with a pharmaceutically acceptable carrier.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an expression vector comprising a nucleic acid sequence encoding a substance which comprises a mdm.2 binding domain linked to a coupling partner, the nucleic acid being under the control of sequences to direct its expression, in combination with a pharmaceutically acceptable carrier.
  • These compositions can be used in methods of gene therapy to deliver the substance to cells, wherein the substance is selectively stabilised in those cells in the population in which the mdm2 mediated degradation pathway does not operate efficiently.
  • the present invention provides an expression vector comprising a nucleic acid sequence encoding a substance which comprises a mdm2 binding domain linked to an effector, wherein the effector is a drug, a prodrug, a toxin, a label and/or a transport molecule.
  • the mdm2 binding domain is or is based on one or more of the mdm2 binding domains of p53 disclosed in WO 93/20238 or WO 96/02642, or variants of these peptides which retain at least some binding affinity for mdm2.
  • the binding affinity of the mdm2 binding domain is substantially the same as or greater than the binding affinity of wild type p53 for mdm2. Examples of variant mdm2 binding sequences are the TIP peptides described below and in B ⁇ ttger et al (1996) .
  • the present invention allows a wide variety of coupling partners to be linked to the mdm2 binding domain including effectors, drugs, prodrugs, toxins, peptides, labels, and transport molecules.
  • the synthesis and use of coupling partners are discussed further below.
  • the above methods can be used to obtain preferential stabilisation of the substance in populations of cells in which the mdm2 mediated degradation pathway does not operate efficiently.
  • This is the case in p53 null cells in which mdm2 is not present or present at very low levels as mdm2 production is induced by p53.
  • a further example is cells in which endogenous p53 tends to accumulate, e.g. because the p53 is mutated and is not active for DNA specific binding, and which therefore do not induce mdm2 production, or because those cells express a viral or cellular function that inactivates the transcription function of p53.
  • the mdm2 binding domain of p53 in the substance does not include a domain of p53 associated with the DNA specific binding property of p53, so that the substance does not induce mdm2 production in the cells in which it is stable.
  • FIG. 1 Schematic representation of the aptamers TIP and TIP 12/1 showing the peptide sequences inserted between G 33 and P 34 of E. coli thioredoxin. Deviations from the p53 wt sequence in TIP 12/1 are in bold with the non exchangeable amino acids underlined.
  • the 3D structure for thioredoxin was obtained from the Protein Data Bank (PDB) , Brookhaven National Laboratory and displayed using the public domain program RasMol .
  • PDB Protein Data Bank
  • FIG. 2 Immunoprecipitation from cellular lysates of U2-OS, MCF-7 and OSA cells using anti-p53 Pab 421 (lanes 2, 5 and 7) , anti-mdm2 Mab 4B2 (lanes 3, 6 and 8) or no antibody for controls (lanes 1 and 4) .
  • Precipitated proteins were separated by SDS PAGE and Western blotted.
  • Western blots were stained with a mixture of anti- mdm2 monoclonal antibodies (3G5, 4B2 and SMP 14) .
  • Figure 3 Soluble ⁇ -galactosidase assays of cell lysates transfected with RGC ⁇ lacz and TIP 12/1 (black bars) or Trx (white bars) encoding DNA. The highest activity was measured in MCF-7 cells transfected with TIP 12/1 and set 100%.
  • FIG. 4 Western blot of SAOS 2 cell lysates 48 hrs after transfecting of control plasmid (lane 1) , wt p 53 alone (land 2) or together with mdm2 (lane 3) and F 19 ⁇ A mutant p53 alone (lane 4) and in combination with mdm2
  • the above methods can be used to obtain preferential stabilisation of the substance in populations of cells in which the mdm2 mediated degradation pathway does not operate efficiently.
  • This is the case in p53 null cells in which mdm2 is not present or present at very low levels as mdm2 production is induced by p53.
  • Another example is cells in which endogenous p53 tends to accumulate, e.g. because the p53 is mutated and is not active for DNA specific binding, and which therefore do not induce mdm2 production or because those cells express a viral or cellular function that inactivates the transcription function of p53.
  • the mdm2 binding domain of p53 in the substance does not include a domain of p53 associated with the DNA specific binding property of p53 , so that the substance does not induce mdm2 production in the cells in which it is stable.
  • the substances mentioned above comprise a mdm2 binding domain and one or more coupling partners.
  • a mdm2 binding domain means a substance that bind to mdm2 so that it is marked for degradation.
  • the mdm2 binding domain a peptide that is or is based on one or more p53 domains that interact with mdm2. Examples of mdm2 binding domains of p53 are disclosed in WO 93/20238 and WO 96/02642.
  • Such peptides tend to be small molecules, preferably less than 25 amino acids, more preferably less than 20 amino acids, more preferably less than 15 amino acids, and more preferably less than 10 amino acids in length.
  • it may be preferable to employ larger polypeptides e.g. to try and ensure that the mdm2 binding domain is displayed in a suitable conformation.
  • the present invention also encompasses peptides which are sequence variants of mdm2 binding domains based on wild type p53 sequence.
  • Variant peptides have an amino acid sequence which differs from wt p53 sequence, e.g. in the mdm2 binding motif between amino acids 13-41 described in WO96/02642, by one or more of addition, substitution, deletion and insertion of one or more amino acids, but which retains the activity of binding to mdm2.
  • Such variants preferably include the motif FxxxW, where x is any amino acid, and will typically share at least about 70%, more preferably at least about 80%, more preferably at least about 90%, or more preferably at least about 95% amino acid sequence identity with the corresponding portion of human p53.
  • variant mdm2 binding domains are the thioredoxin insert peptides (TIPs) disclosed in B ⁇ ttger et al, 1996, and in the examples below, see especially peptide TIP 12/1.
  • the coupling partner can be one or more compound (s) whose delivery to the target population of cells is desired for direct or indirect diagnostic or therapeutic effect.
  • Preferred coupling partners include (a) therapeutic reagents, such as drugs, toxins (e.g. ricin) or prodrugs which are converted to active form by reaction with a second reagent such as an enzyme, (b) diagnostic reagents such as labels or precursors of compounds that can produce a detectable result by reaction with a second reagent, and/or (c) transport molecules such as the Penetratin peptide described in WO 91/19981 to provide delivery of the substance to cells.
  • therapeutic reagents such as drugs, toxins (e.g. ricin) or prodrugs which are converted to active form by reaction with a second reagent such as an enzyme
  • diagnostic reagents such as labels or precursors of compounds that can produce a detectable result by reaction with a second reagent
  • transport molecules such as the Penetratin peptide described in WO
  • the substances are peptides and can be produced using recombinant techniques and/or chemically synthesised.
  • a convenient way of producing it is to express nucleic acid encoding it in a suitable expression system.
  • the use of expression system has reached an advanced degree of sophistication today.
  • the present invention also encompasses a method of making the substances disclosed herein, the method including expression from nucleic acid encoding the substance. This can conveniently be achieved by growing a host cell in culture, containing a vector comprising the nucleic acid under the control of sequences to direct its expression, under appropriate conditions which cause or allow expression of the peptide. Peptides may also be expressed in in vi tro systems, such as reticulocyte lysate .
  • Suitable host cells include bacteria, eukaryotic cells such as mammalian and yeast, and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous peptide include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, COS cells and many others. A common, preferred bacterial host is E. coli .
  • Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Vectors may be plasmids, viral e.g.
  • a still further aspect provides a method which includes introducing the nucleic acid into a host cell .
  • the introduction which may (particularly for in vi tro introduction) be generally referred to without limitation as "transformation", may employ any available technique.
  • suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g. vaccinia or, for insect cells, baculovirus.
  • suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage .
  • direct injection of the nucleic acid could be employed.
  • Marker genes such as antibiotic resistance or sensitivity genes may be used in identifying clones containing nucleic acid of interest, as is well known in the art.
  • the introduction may be followed by causing or allowing expression from the nucleic acid, e.g. by culturing host cells (which may include cells actually transformed although more likely the cells will be descendants of the transformed cells) under conditions for expression of the gene, so that the encoded peptide is produced. If the peptide is expressed coupled to an appropriate signal leader peptide it may be secreted from the cell into the culture medium.
  • a polypeptide may be isolated and/or purified from the host cell and/or culture medium, as the case may be, and subsequently used as desired, e.g. in the formulation of a composition which may include one or more additional components, such as a pharmaceutical composition which includes one or more pharmaceutically acceptable excipients, vehicles or carriers (e.g. see below).
  • nucleic acid may take place in vivo by way of gene therapy, as discussed below.
  • the peptides can also be generated wholly or partly by chemical synthesis.
  • the compounds of the present invention can be readily prepared according to well-established, standard liquid or, preferably, solid-phase peptide synthesis methods, general descriptions of which are broadly available (see, for example, in J.M. Stewart and J.D. Young, Solid Phase Peptide Synthesis, 2nd edition, Pierce Chemical Company, Rockford, Illinois (1984) , in M. Bodanzsky and A.
  • Bodanzsky The Practice of Peptide Synthesis, Springer Verlag, New York (1984) ; and Applied Biosystems 430A Users Manual, ABI Inc., Foster City, California
  • they may be prepared in solution, by the liquid phase method or by any combination of solid-phase, liquid phase and solution chemistry, e.g. by first completing the respective peptide portion and then, if desired and appropriate, after removal of any protecting groups being present, by introduction of the residue X by reaction of the respective carbonic or sulfonic acid or a reactive derivative thereof.
  • the agents of the invention can be formulated in pharmaceutical compositions, e.g. as medicaments for activating p53 in cells that do not overexpress mdm2.
  • These compositions may comprise, in addition to one of the above substances, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non- toxic and should not interfere with the efficacy of the active ingredient.
  • the precise nature of the carrier or other material may depend on the route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes.
  • Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form.
  • a tablet may include a solid carrier such as gelatin or an adjuvant.
  • Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil.
  • a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil.
  • Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
  • the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection.
  • Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.
  • administration is preferably in a "prophylactically effective amount” or a "therapeutically effective amount” (as the case may be, although prophylaxis may be considered therapy) , this being sufficient to show benefit to the individual.
  • a prophylaxis may be considered therapy
  • the actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol , A. (ed) , 1980.
  • targeting therapies may be used to deliver the active agent more specifically to certain types of cell, by the use of targeting systems such as antibody or cell specific ligands. Targeting may be desirable for a variety of reasons; for example if the agent is unacceptably toxic, or if it would otherwise require too high a dosage, or if it would not otherwise be able to enter the target cells.
  • these agents could be produced in the target cells by expression from an encoding gene introduced into the cells, eg in a viral vector (a variant of the VDEPT technique - see below) .
  • the vector could be targeted to the specific cells to be treated, or it could contain regulatory elements which are switched on more or less selectively by the target cells.
  • the agent could be administered in a precursor form, for conversion to the active form by an activating agent produced in, or targeted to, the cells to be treated. This type of approach is sometimes known as
  • ADEPT or VDEPT the former involving targeting the activating agent to the cells by conjugation to a cell-specific antibody, while the latter involves producing the activating agent, e.g. an enzyme, in a vector by expression from encoding DNA in a viral vector (see for example, EP-A-
  • a composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
  • the composition can be used to treat conditions such as cancer, virus infections or any other condition in which p53 or mdm2 is not functioning.
  • the nucleic acid encoding the substance can be used in a method of gene therapy, to treat a patient with the coupling partner or a compound derivable from the coupling partner.
  • This is particularly useful in the present invention for the stabilisation of the coupling partner in cells which a mdm2 mediated degradation pathway does not operate efficiently, as is the case in many tumour cells.
  • this feature of the invention makes it particularly useful to improve selective delivery to these classes of cells; the agent being unstable in normal cells but stable in cells lacking p53 or mdm2 function.
  • Vectors such as viral vectors have been used in the prior art to introduce genes into a wide variety of different target cells. Typically, the vectors are exposed to the target cells so that transfection can take place in a sufficient proportion of the cells to provide a useful therapeutic or prophylactic effect from the expression of the desired polypeptide.
  • the transfected nucleic acid may be permanently incorporated into the genome of each of the targeted tumour cells, providing long lasting effect, or alternatively the treatment may have to be repeated periodically.
  • vectors both viral vectors and plasmid vectors
  • a number of viruses have been used as gene transfer vectors, including papovaviruses , such as SV40, vaccinia virus, herpesviruses, including HSV and EBV, and retroviruses .
  • papovaviruses such as SV40
  • vaccinia virus such as SV40
  • herpesviruses including HSV and EBV
  • retroviruses including vaccinia virus
  • retroviruses including HSV and EBV
  • retroviruses include vaccinia virus, cowpoviruses, cowpoviruses, and vaccinia virus, and retroviruses .
  • Many gene therapy protocols in the prior art have used disabled murine retroviruses .
  • nucleic acid into cells includes electroporation, calcium phosphate co- precipitation, mechanical techniques such as microinjection, transfer mediated by liposomes and direct DNA uptake and receptor-mediated DNA transfer.
  • E. coli 1724 cells were transformed with the resulting plasmids. They were grown in RM medium at 30°C overnight, then inoculated into fresh induction medium and grown to OD 0.5. The cultures were transferred to 37°C, induced with L-tryptophan at a final concentration of 100 ⁇ g/ml and grown for three to four hours .
  • Soluble extracts were obtained by resuspending pellets in ice cold 20 mM Tris/HCl, pH 8, 2.5 mM EDTA with protease inhibitors (1 mM PMSF, ImM benzamidine, leupeptin, approtinin and pepstatin at 10 ⁇ g/ml each) and three times shock freezing, thawing and sonicating, followed by centrifugation for 20 min at 10,000 g. Heat shock lysates were obtained by resuspending pellets to an OD of 100 and then treating at 80°C for 10 min followed by centrifugation at 10,000g for 20 min.
  • the thioredoxin coding region complete with the peptide insertions, was amplified from pTrx, pTrx 12/1 and pTrx wt using the following primers :
  • the resulting PCR products were cleaved with BamHI and Eco RI and ligated into BamHI, EcoRI cleaved pcDNA3.
  • the TIP 12/1 sequence in pcDNA3 was verified by sequencing.
  • ELISAs were carried out as previously described (B ⁇ ttger et al , 1996). Briefly, plates were coated with l ⁇ g/ml p53 or dilutions of p53 overnight at 4°C. They were blocked and a preincubated mixture of GST-hdm2 (1.3 ⁇ g/ml) and synthetic peptides inhibition ELISA) or hdm2 alone
  • DMEM Dulbecco's modified Eagle medium
  • microinjection cells were seeded onto tissue culture dishes and grown to 60-70% confluence. Microinjection was performed using an Eppendorf microinjection system (Microinjector 5242, Micromanipulator
  • TIP 12/1, TIP and Trx was purified using Quiagen purification system or phenol/chloroform precipitation and injected at a concentration of 0.25 mg/ml in water. Following microinjection fresh medium was added to the cell cultures and they were incubated for 24 hours.
  • Immunofluorescence VRn.6 cells were fixed with 4% paraformaldehyde in PBS for 10 min, washed and permeabilised with 1% NP40 in PBS.
  • Primary antibodies rabbit anti -galactosidase or monoclonal anti-thioredoxin antibody (Anti-ThioTM Antibody, Invitrogen) were applied 1/500, Texas red conjugated goat anti mouse Ig or FITC conjugated goat and rabbit Ig F(ab') 2 fragments (Jackson) were applied 1/500 as second antibodies. Incubations were carried out for 1 hour or 45 min at RT. Washes were carried out in PBS.
  • T22 cells were fixed with ice cold acetone/methanol (%) for 8 min at 4°C.
  • Primary antibodies, protein A purified CM5 at 2 ⁇ g/ml or anti-ThioTM 1/500 were added for 1 hour at RT, after PBS washes FITC conjugated anti mouse Ig at 1/80 or Texas red anti rabbit Ig at 1/400 were added for 30 min at RT.
  • Cells were seeded into 6 well plates at 1.5 x 10 6 cells per well. They were grown to a density of 80% confluence and transfected using different lipophilic reagents (lipofectin and lipofectamin, Promega, DOSPER and DOTAP,
  • Cells were grown to > 90% confluence on 14 cm dishes. They were scraped into PBS and lysed in 50 mM Tris/HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 0.5% NP 40 and protease inhibitors (Boehringer CompleteTM) . To 100 ⁇ l lysate 1 ⁇ g PAb 421 in 400 ⁇ l DMEM/10% FCS or 400 ⁇ l Mab 4B2 supernatant was added and the lysates incubated overnight at 4°C. Protein G sepharose beads were added and incubation carried out for 2 hours. Beads were washed 5 times with PBS, 0.2% Tween and then boiled in SDS sample buffer.
  • Proteins were separated on PAGE gels, western blotted and blots stained with rabbit polyclonal anti-p53 antibodies CM5 and CM1 followed by HRP anti rabbit IgG or a mixture of anti-mdm2 monoclonal antibodies 3G5, SMP14 and 4B2 followed by HRP anti-mouse IgG (DAKO) .
  • HRP activity was established by ECL (Amersham) .
  • SAOS 2 cells were seeded 24 hrs before transfection to 80% confluence in 10 cm dishes. Calcium phosphate mediated transfections were performed as previously described (Lin and Green, 1989) . 5 ⁇ g of p53 wt and mutant encoding plasmids (pcDNA3) or control vector and 5 ⁇ g mdm2 encoding plasmid (X2 , Haupt et al . , 1996) were cotransfected per 10 cm dish. 48 hrs after transfection cell lysates were fractionated by 12% SDS-PAGE and transferred to nitrocellulose membranes. Blots were stained with rabbit anti p53 antiserum CM5 (1/8000) and HRP conjugated anti rabbit Ig. Peroxidase activity was established using ECL.
  • F 19 ⁇ A was accomplished by site directed mutagenesis using the TransformerTM site directed mutagenesis kit (Clonetech) .
  • the sequence of the selection primer was: 5' -3' GACTCTGGGGATCGATATGACCGACC
  • the sequence of the mutagenic primer was: 5' -3'
  • Peptide inserts into Thioredoxin create potent inhibitors of the p53-md ⁇ r ⁇ 2 interaction.
  • FIG. 1 shows a schematic representation of the three aptamers we constructed by inserting additional peptide sequences into the active site of E.coli thioredoxin.
  • TIP 12/1 Thioredoxin Insert Protein
  • TIP wt contains the sequence corresponding to p53 wild type sequence P 13 to .
  • As controls we expressed thioredoxin lacking a peptide insertion (Trx) in bacteria.
  • TIP 12/1 inhibits the interaction between p53 and mdm2 in this assay with the same strength as full length p53. This should make it a suitable agent to be tested in cellular systems for effects on the interaction between p53 and mdm2 in vivo .
  • TIP wt inhibits the interaction 20 times less than TIP 12/1. This has to be attributed to the 50 times less potent inhibition achieved by the wt peptide when compared with peptide 12/1 in peptide competition assays (B ⁇ ttger et al, 1997) .
  • Trx does not show inhibition in our assay, making it a suitable negative control for in vivo experiments.
  • TIP 12/1 exhibits strong enough inhibitory potential to compete against endogenous levels of wt p53 in tumour cells for binding to mdm2. It therefore offers an agent that should be capable to function inside mammalian cells.
  • OSA cells a human osteosarcoma cell line (Florenes et al, 1994) as an example for a cell line with highly elevated mdm2 levels due to gene amplification.
  • U2-OS cells another osteosarcoma cell line, which has no gene amplification for mdm2 but elevated levels of mdm2-mRNA (Florenes et al,
  • MCF-7 cells a breast cancer cell line with heterogeneously expressed low levels of wt p53 and no reported mdm2 elevation.
  • Figure 2 shows the results.
  • 4B2 coprecipitates p53 in similar amounts to PAb 421 (Fig 2b, lane 7 for PAb 421 and lane 8 for 4B2) .
  • PAb 421 precipitates p53 (Fig 2b, lane 5) , but no coprecipitation of p53 by anti-mdm2 antibody 4B2 is observed (Fig 2b, lane 6, signal is the same as in control precipitation, lane 4) .
  • PAb 421 precipitates p53 ( Figure 2b, lane 3) and 4B2 precipitates a large percentage of p53 ( Figure 2b, lane 3), suggesting a high degree of hdm2-p53 complex formation.
  • the inhibitory potential of the 15 kD TIP 12/1 protein was the same as that of tetrameric wt p53 and greatly exceeded that achieved by the simple transplantation of the wild type p53 sequence into thioredoxin.
  • both aptamers TIP 12/1 and TIP wt were able to induce p53 dependent transcriptional activation of a reporter gene.
  • the intensity of the effect in microinjection experiments exerted by TIP 12/1 in comparison with TIP wt was proportional to their in vitro inhibitory potential. With these mdm2 binding aptamers we therefore have developed powerful tools to study the biological consequences of disrupting the interaction between mdm2 and p53 in tumour cells.
  • Mdm2 regulates p53 levels
  • F 19 ⁇ A mutation blocks mdm2 dependent reductions in p53 levels in transfection studies.
  • Marston et al found that an in frame deletion of conserved box 1 resulted in a mutant p53 that was very stable but retained transcriptional activity (Marston et al, 1994) whilst it is well established that larger N-terminal deletions also stabilise p53.
  • the model that p53 drives the transcription of the key regulator of its own degradation also provides an intensely attractive model for the stability of the p53 protein in cells in which the protein is inactive transcriptionally. This includes cells that contain only mutant p53 such as the majority of human tumour cells and cells infected or transformed by DNA tumour viruses whose products bind an inactivate the transcriptional function of p53.
  • wild type p53 can mediate sequence-specific transactivation of an internal promoter within the mdm2 gene.
  • Oncogene 8 3411-3416.
  • the mdm-2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation.

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Abstract

Mdm2 se lie à p53 dans des cellules dans lesquelles mdm2 n'est pas surexprimé c'est-à-dire dans des cellules dans lesquelles mdm2 est exprimé à des niveaux normal ou faible, cette intéraction ciblant p53 pour la dégradation. Cette invention exploite ce mécanisme de dégradation de p53 pour stabilise une substance comprenant un domaine de liaison mdm2 lié à un partenaire de couplage dans des cellules dans lesquelles ce processus de dégradation à médiateur mdm2 ne fonctionne pas de manière efficace. Par contre dans des cellules normales exprimant le mdm2 fonctionel la substance aura tendance à être instable une fois marquée pour la dégradation par l'interaction du mdm2 endogène avec le domaine de liaison mdm2 de la substance. On peut par conséquent utiliser ces substances pour apporter le partenaire de couplage à ces cellules c'est-à-dire pour les utiliser dans le diagnostic et/ou le traitement de cancers, d'infections virales ou d'autres pathologies associées à une p53 ou un mdm2 non fonctionnel.
PCT/GB1998/001140 1997-04-22 1998-04-20 Matieres et procedes lies a la stabilisation de substances dans des cellules WO1998047919A1 (fr)

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Cited By (4)

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Publication number Priority date Publication date Assignee Title
US7531515B2 (en) 2000-04-05 2009-05-12 The Research Foundation Of State University Of New York Peptides selectively lethal to malignant and transformed mammalian cells
CN103974973A (zh) * 2011-11-09 2014-08-06 莫茨制药有限及两合公司 显示出缩短的生物学活性的神经毒素
US9539327B2 (en) 2007-11-26 2017-01-10 The Research Foundation For The State University Of New York Small molecule cancer treatments that cause necrosis in cancer cells but do not affect normal cells
US12239720B2 (en) 2018-04-06 2025-03-04 Oncolyze, Inc. Compositions for use in lysis of selective cancer cells

Citations (2)

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WO1993020238A2 (fr) * 1992-04-07 1993-10-14 The Johns Hopkins University Amplification du gene mdm2 dans des tumeurs chez l'homme
WO1996002642A1 (fr) * 1994-07-20 1996-02-01 University Of Dundee INTERRUPTION DE LA LIAISON DE LA PROTEINE MDM2 ET p53 ET SON APPLICATION THERAPEUTIQUE

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WO1993020238A2 (fr) * 1992-04-07 1993-10-14 The Johns Hopkins University Amplification du gene mdm2 dans des tumeurs chez l'homme
WO1996002642A1 (fr) * 1994-07-20 1996-02-01 University Of Dundee INTERRUPTION DE LA LIAISON DE LA PROTEINE MDM2 ET p53 ET SON APPLICATION THERAPEUTIQUE

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M. KUBBUTAT ET AL: "Regulation of p53 stability by mdm2", NATURE, vol. 387, 15 May 1997 (1997-05-15), pages 299 - 303, XP002075660 *
PICKSLEY S M ET AL: "IMMUNOCHEMICAL ANALYSIS OF THE INTERACTION OF P53 WITH MDM2;- FINEMAPPING OF THE MDM2 BINDING SITE ON P53 USING SYNTHETIC PEPTIDES", ONCOGENE, vol. 9, 1994, pages 2523 - 2529, XP002058480 *
WILLS K N ET AL: "DEVELOPMENT AND CHARACTERIZATION OF RECOMBINANT ADENOVIRUSES ENCODING HUMAN P53 FOR GENE THERAPY OF CANCER", HUMAN GENE THERAPY, vol. 5, no. 9, 1 September 1994 (1994-09-01), pages 1079 - 1088, XP000579605 *
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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7531515B2 (en) 2000-04-05 2009-05-12 The Research Foundation Of State University Of New York Peptides selectively lethal to malignant and transformed mammalian cells
US7745405B2 (en) 2000-04-05 2010-06-29 The Research Foundation Of State University Of New York Peptides selectively lethal to malignant and transformed mammalian cells
EP1608409A4 (fr) * 2003-03-12 2009-09-16 Univ New York State Res Found Peptides selectivement letaux pour les cellules malignes et transformees
AU2004220114B2 (en) * 2003-03-12 2010-03-04 The Research Foundation Of State University Of New York Peptides selectively lethal to malignant and transformed mammalian cells
US9539327B2 (en) 2007-11-26 2017-01-10 The Research Foundation For The State University Of New York Small molecule cancer treatments that cause necrosis in cancer cells but do not affect normal cells
CN103974973A (zh) * 2011-11-09 2014-08-06 莫茨制药有限及两合公司 显示出缩短的生物学活性的神经毒素
US9809809B2 (en) 2011-11-09 2017-11-07 Merz Pharma Gmbh & Co. Kgaa Neurotoxins exhibiting shortened biological activity
US12239720B2 (en) 2018-04-06 2025-03-04 Oncolyze, Inc. Compositions for use in lysis of selective cancer cells

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AU7064298A (en) 1998-11-13

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