EP1007662A1 - METHODE PERMETTANT DE PROVOQUER UNE MORT CELLULAIRE PROGRAMMEE DANS DES CELLULES MALIGNES PAR REDUCTION DU RAPPORT PROTEINES Rb/PROTEINES INDUISANT L'APOPTOSE - Google Patents

METHODE PERMETTANT DE PROVOQUER UNE MORT CELLULAIRE PROGRAMMEE DANS DES CELLULES MALIGNES PAR REDUCTION DU RAPPORT PROTEINES Rb/PROTEINES INDUISANT L'APOPTOSE

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Publication number
EP1007662A1
EP1007662A1 EP98902970A EP98902970A EP1007662A1 EP 1007662 A1 EP1007662 A1 EP 1007662A1 EP 98902970 A EP98902970 A EP 98902970A EP 98902970 A EP98902970 A EP 98902970A EP 1007662 A1 EP1007662 A1 EP 1007662A1
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EP
European Patent Office
Prior art keywords
apoptosis
prb
ink4
gene
use according
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP98902970A
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German (de)
English (en)
Inventor
Michael Strauss
Volker Sandig
Jiri Bartek
Jiri Lukas
Ag Fur Gentherapie Hepavec
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Develogen AG
Original Assignee
Sandig Volker
HepaVec AG fur Gentherapie
Bartek Jiri
Lukas Jiri
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Filing date
Publication date
Application filed by Sandig Volker, HepaVec AG fur Gentherapie, Bartek Jiri, Lukas Jiri filed Critical Sandig Volker
Publication of EP1007662A1 publication Critical patent/EP1007662A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4747—Apoptosis related proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C07K14/4736—Retinoblastoma protein
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4746—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used p53
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/022—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from an adenovirus

Definitions

  • the present invention relates to a method of inducing cell death, such as apoptosis, in malignant cells in a mammal such as a human.
  • the invention further relates to the use of vectors for transfer of genetic material with the purpose of achieving growth arrest and subsequent induction of apoptosis in malignant cells.
  • the invention also relates to vectors for transfer of genetic material into malignant cells with the purpose of inducing cell death by apoptosis.
  • cytokine-gene aided tumour vaccination and selective prodrug activation.
  • the first strategy relies on the strong immunostimulatory effect of a relatively small number of genetically modified cyto- toxic T cells or tumour cells
  • the second one is based on conversion of a nontoxic prodrug into a toxic product by an enzyme-encoding gene where the toxic effect is exerted also on non-transduced dividing tumour cells due to a so-called bystander effect.
  • strategies can be envisaged where the malignant phenotype of a cell is reversed by either inactivating an oncogene or reestablishing an inactivated tumour suppressor gene.
  • tumour suppressors were isolated by isolation of the retino- blastoma susceptibility gene (Rbl ) . This discovery stimulated the search for tumour suppressors enormously. Since then several other tumour suppressor genes were isolated.
  • the function of the Rbl gene product pRb is strongly related to the regulation of cell division [Goodrich and Lee (1993) , Weinberg (1995), Bartek et al . (1996), Herwig and Strauss (1997)] . It is non-phosphorylated in the early G x phase.
  • Non- phosphorylated pRb forms complexes with the transcription factor E2F which is released from this complex by phosphory- lation in late G suggesting that its growth inhibiting function is based on binding of E2F which could either result in formation of an active repressor of E2F-dependent genes in GO/G-L or in sequestration of normally activating E2F.
  • the current view is that both alternative mechanisms may function with different genes or even with one and the same gene.
  • Sequential activation of certain genes is required to promote the progression from the quiescent state (G 0 ) through G into the phase of DNA replication (S) and subsequently through a second gap phase (G 2 ) into mitosis (M) [Pardee (1989)] . It seems obvious that every phase of the cell cycle requires the activity of a defined set of genes which are active only for a certain period of time.
  • One mechanism to ensure this ordered cascade of events is phase-specific phosphorylation and dephosphorylation of transcriptional regulators. These phase-specific phosphorylations are carried out by enzymes called cyclin-dependent kinases (cdks) .
  • cdk catalytic subunits form complexes with regulatory subunits called cyclins [Sherr (1993)] .
  • the cyclins turn the cdk into an active enzyme and determine the substrate specificity of the kinase by binding specifically to certain target proteins.
  • the cyclins are short-lived proteins whose mRNA and/or protein levels accumulate in a certain phase of the cell cycle.
  • CKI cyclin-dependent kinases
  • KIP kinase-inhibiting proteins
  • INK4 proteins members of the second group including pl5 INK4 , pl6 INK4 , pl8 INK4 , and pl9 INK4 , are specific for cdk4/cdk6 [Sherr and Roberts (1996)].
  • tumour suppressor p53
  • p53 Another tumour suppressor, p53, seems to exert at least one of its functions by inhibiting phosphorylation of pRb via induction of p21 INK4 [el Deiry et al . (1993), Harper et al . (1993), Dulic et al . (1994)].
  • This well-characterized response to DNA-damaging agents is charac- teristic for the so-called check point in late G-,_ .
  • the major target of p53 -mediated growth arrest is the growth control mechanism governed by pRb.
  • pRb functions as the major known repressor of Gl phase progression [Pardee (1989), Sherr (1993), Sherr and Roberts (1996)].
  • pl6 INK4 The gene coding for pl6 INK4 was shown to be a tumour suppressor which is lost at a high rate in certain tumours and even more frequently in tumour-derived cell lines [Kamb et al . (1994), Nobori et al . (1994)]. Loss of pl6 INK4 function can occur by various mechanisms including point mutation, deletion and also by silencing through hypermethylation [Merlo et al . (1995)] . Most recent studies have demonstrated that the loss of Rb and pl6 INK4 occurs in a mutually exclusive manner and the function of one of the two tumour suppressor genes is lost in almost every tumour cell line [Otterson et al . (1994), Okamoto et al . (1994), Tarn et al . (1994), Lukas et al. (1995) ] .
  • p53 was also shown to induce apoptosis in late Gl which probably occurs when DNA damages cannot be repaired properly.
  • apoptosis in the absence of p53 has been shown suggesting that p53 is an inducer of apoptosis but might not be required for apoptosis and, therefore, not part of the apoptotic machinery [White (1996)] .
  • the apoptotic role of p53 may be its most important contribution to the suppression of tumour cell growth. Where the response of normal cells to p53 seems to be cell cycle arrest, malignant cells are probably more sensitive to p53 -induced apoptosis. Thus, p53 positive tumour cells might generally be susceptible to apoptosis induced by radiation or chemotherapy.
  • a first-generation El-deficient vector with the p53 gene under control of the CMV promoter was introduced into human non-small cell lung carcinoma cells deficient in p53 [Zhang et al . (1994)] . Almost 100% gene transfer was achieved at multiplicities of infection (m.o.i.) of 50 pfu/cell and high level p53 protein expression was detected. Whereas growth of p53 -deficient tumour cells was significantly inhibited, almost no effect was found in the cells which had no defect in the p53 gene. Tumour-suppressing effects of p53 vectors were demonstrated in various animal models [Wills et al . (1994), Liu et al . (1994)].
  • pl6 INK4 is a bona fide tumour suppressor.
  • the function of the pl6 INK4 gene is lost in a variety of tumour types amounting to about 60% of all tumours and reaching 80-90% loss of function in pancreatic tumours and melanomas [Kamb et al. (1994), Nobori et al . (1994), Merlo et al . (1995), Otter- son et al . (1994), Okamoto et al . (1994), Tarn et al . (1994), Hannon and Beach (1994), Marx (1994), Zhang et al . (1994), Jen et al . (1994), Washimi et al . (1995), Hussussian et al .
  • the pl6 INK4 gene product specifically inhibits cdk4 and, thereby, the phosphorylation of pRb [Serrano et al . (1993), Serrano et al . (1995)]. Thus, it functions directly upstream of pRb [Lukas et al . (1995), Koh et al (1995)] . Therefore, loss of pRb and loss of pl6 INK4 are alternative events.
  • Rb-deficient tumours are generally positive for pl6 INK4
  • pl6 INK4 -deficient tumours have normal Rb [Strauss et al . (1995), White et al . (1996)].
  • the present invention aims at gene therapy of cancer based on the combined use of tumour suppressors for repression of cell proliferation and induction of programmed cell death (apoptosis) .
  • various tumour suppressors can inhibit proliferation of cells in general without specificity for tumour cells.
  • Apoptosis can be induced by various agents in normal cells but is more difficult to induce in tumour cells.
  • the ability to respond to inducers of apoptosis is largely dependent on the presence of a normal p53 gene.
  • the product of this gene is the main endogenous inducer of apoptosis.
  • the p53 gene is functionally inactivated in the majority of cancers .
  • the speed of tumour growth depends mainly on the balance between increased cell proliferation and apoptosis.
  • Transfer of the pl6 INK4 gene into different types of tumour cells by various means leads to inhibition of cell proliferation by inhibition of phosphorylation of the Rb protein which lasts as long as the pl6 INK4 protein is synthesised from the transferred gene. Later, cells can recover from the block and resume division. Thus, transient inhibition of cell division would not be sufficient for efficient block of tumour growth and for tumour regression.
  • a very important aspect of the present invention is that not only the absolute levels of pRb and an apoptosis-inducing protein such as p53 are important for induction of apoptosis but also the ratio of the two proteins (Example 5) .
  • the ratio pRb/apoptosis-inducing protein which differs between various cell types (when the apoptosis-inducing protein is p53 between 1:1 and 1:20) has to be reduced in two ways after cell cycle arrest.
  • the level of pRb has to be reduced by at least 5 fold (below 20% of the original level) , preferably by at least 10 fold (below 10%) or even by 20 fold (below 5%) .
  • the p53 level should preferably be increased to more than two fold of the original level.
  • a quantification of the changes in the level of pRb and p53 can be made by extracting tissue from a diagnosed tumour as well as tissue from the same area subsequent to treatment according to the invention, and comparing the levels of pRb and apoptosis-inducing protein in the tissue samples by employing standard immunological techniques, such as immunoblotting, ELISA or other techniques using antibodies specific for pRb and the apoptosis-inducing protein.
  • the total amount of pRb will be measured including phosphorylated pRb as well as non-phosphorylated pRb.
  • the total amount of pRb When reducing the total amount of pRb according to the concept of the present invention, it is the non-phosphorylated pRb which is reduced because reduction occurs only if almost no phosphorylated pRb is present anymore.
  • Treatment would typically comprise transfection of tumorous tissue with a vector containing a preferred version of the p 1 g iNK4 g en ⁇ / optionally combined with an apoptosis-inducing gene, and tissue for quantification of pRb and the apoptosis- inducing protein level would typically be extracted one week or two weeks after initiation of treatment .
  • the present invention relates to the use of a vector containing genetic material for the preparation of a compo- sition for the treatment of malignant disease, said vector, upon administration in a subject suffering from malignant disease, inducing apoptosis in malignant cells by reducing the ratio of the level of pRb protein to the level of an apoptosis-inducing protein in malignant cells, by reducing the level of pRb and increasing the level of the apoptosis- inducing protein, after having first achieved growth arrest of said malignant cells by inducing herein an inhibition of phosphorylation of the pRb protein.
  • the present invention also relates to vectors for the transfer of genetic material.
  • the present invention provides several events which generally take place over a period of hours or days in a sequential manner starting with overexpression of a gene which causes block of cell division and subsequent reduction of pRb levels (for which gene pl6 INK4 is a prototype) and later induction of apoptosis by overexpression of an apoptosis-inducing protein such as p53.
  • the latter can be omitted in the tumours expressing well detectable levels of wild-type p53.
  • the reduction of the ratio of pRb/apoptosis-inducing protein is the crucial point .
  • Overexpression is preferably achieved by the use of viral vectors, preferably adenoviral vectors. Care must be taken with regard to selection of high expressers (more than 5 -fold of normal levels) . Most favourable are vectors which express both genes at the same time.
  • the preferred tools of the invention are therefore adenoviral (or other viral) vectors allowing for simultaneous expression of pl6 INK4 or one of its relatives and p53 or one of its downstream effectors. Since pRb and p53 are master regulators of certain pathways, there are several potential replacements for pl6 INK4 and p53.
  • the pl6 INK4 gene can be replaced with good success by pl5 INK4 , pl8 INK4 or pl9 INK4 , or alternatively by p21 KIP , p27 KIP or p57 KIP .
  • the p53 gene induces a number of pro-apoptotic genes such as bax, bak and bcl-X which could be used as part of a therapeutic vector instead of p53. When the term "apoptosis-inducing protein" is used, reference is made to the protein products of such genes.
  • the method of the invention was used to cause inhibition of tumour development in nude mice from pre-transduced cells (Example 6) and to cause tumour growth inhibition by in vivo gene transfer into existing tumours (Example 7) .
  • the data clearly demonstrate that single administration of pl6 INK4 or p53 has little or no effect whereas the combined effect of the two genes is very strong. Whereas tumour growth is inhibited by the combined treatment in most animals, some others show even complete regression of the tumours.
  • the outcome of in vivo application of the method of the invention largely depends on the proper delivery of the vectors to the tumour tissue.
  • a new strategy for tumour gene therapy which is based on the combinatorial effects of growth inhibition by pl6 INK4 and induction of apoptosis by p53 is employed.
  • pl6 INK4 induces down-modulation of pRb protein synthesis resulting in increased sensitivity of tumour cells to apoptotic stimuli.
  • efficient block of the cyclin D/cdk-pRb pathway in tumour cells is a crucial step towards growth arrest and regression of tumours by apoptotic cell death as long as it can be induced in the majority of tumour cells.
  • the present invention teaches, for the first time, a method of inducing apoptosis in the large majority of malignant cells in a living organism.
  • the present invention provides an answer to the long felt need for effective anti-tumour treatment.
  • WO 96/27008 discloses transfer of pl6 INK4 encoding genetic material contained in an adenoviral vector into cells for the treatment of tumours and other hyperplasias .
  • the document does not disclose or discuss whether certain levels of cellu- lar expression of pl6 K4 are necessary or preferred.
  • the present invention discloses that only significant overexpression of pl6 INK4 protein employed in the presence of p53 protein will result in efficient and wide- spread cell death.
  • WO 95/11301 concerns methods for reducing the viability of proliferating mammalian cells, such as cancer cells, by increasing the cellular levels of p53 protein or by increasing the activity of p53.
  • the role of other cellular proteins in relation to tumour suppression is not discussed or mentioned.
  • the use of p53 alone is in direct contrast to the approach used in relation to the present invention which shows that it is not sufficient to express or overexpress p53 in malignant cells in order to induce cell death by apoptosis (see Example 3) .
  • WO 96/20207 discloses mutants of the pRb and p53 proteins, which are used therapeutically for treating a variety of pathophysiological cell proliferative diseases. Cancer cells are treated with nucleic acids encoding the above two pro- teins, either concurrently or consecutively. However, nothing is mentioned with respect to the levels of expression that are necessary to achieve cell death. The significance of the cellular ratio of the two proteins is not discussed. Furthermore, a positive effect of treatment is only shown to occur in cell lines deficient in either p53 or pRb. This does not suggest that the method of WO 96/20207 is suitable for cancer treatment in general .
  • WO 96/27008 From WO 96/27008 is it known that ectopic expression of pl6 INK4 in various tumour cell lines can inhibit prolifera- tion of these cells.
  • the present invention shows that significant levels of overexpression of pl6 INK4 , leading to growth arrest, are crucial to the induction of apoptosis in p53 -expressing tumour cells, through the down-modulation of pRb level and concomitant increase of p53 level leading to a lowering of the ratio of pRb protein to p53 protein (pRb/p53) .
  • the actual magnitude of the change in level of pRb and p53, respectively has been determined for several kinds of cells in relation to the present invention.
  • the present invention relates to the use of a vector contai- ning genetic material for the preparation of a composition for the treatment of malignant disease, said vector, upon administration in a subject suffering from malignant disease, inducing apoptosis in malignant cells by reducing the ratio of the level of pRb protein to the level of an apoptosis- inducing protein in malignant cells, by reducing the level of pRb and increasing the level of the apoptosis-inducing protein, after having first achieved growth arrest of said malignant cells by inducing herein an inhibition of phosp- horylation of the pRb protein.
  • growth arrest means a substantial block of cell division, which is understood as an almost complete block, typically at least 90%.
  • the ratio of the level of pRb protein to the level of apoptosis- inducing protein is reduced in malignant cells by reducing the level of pRb by at least 5 fold (to less than 20% of the original level) , by at least 10 fold (to less than 10% of the original level) , or by at least 20 fold (to less than 5% of the original level) and preferably increasing the level of the apoptosis-inducing protein such as p53 by at least 2 fold.
  • the ratio pRb/apoptosis-inducing protein in various types of malignant cells determines the possibility of in- ducing cell death by apoptosis after growth arrest has first been achieved.
  • the actual ratio has been seen to vary according to the type of cell in question.
  • the inventors have been able to induce apoptosis in at least 90% of malignant cells in a tumour. This percentage of cell killing is unprecedentedly high and thus offers a significant improvement in cancer treatment.
  • the ratio of the level of pRb protein to the level of apoptosis-inducing protein will preferably be quantified by using immu- noblotting of the apoptosis-inducing protein (e.g. p53) and pRb proteins on the same gel using specific antibodies.
  • the amounts of detectable primary antibody which is measured, e.g., by the common secondary antibody and streptavidin-POD reaction correspond roughly to the amount (molecules) of the respective target protein.
  • the ratio does not give very exact molar ratios but relative measures of the detected immunocomplexes . Therefore, measurement of the reduction of pRb level and increase of apoptosis-inducing protein level is more reproducible and reliable than measurement of molar ratios of the two proteins.
  • a quantification of the changes in the level of pRb and apoptosis-inducing protein will preferably be carried out by extracting tissue from a diagnosed tumour as well as tissue from the same area subsequent to treatment according to the invention, and comparing the level of pRb and apotosis-in- ducing protein in these tissue samples by employing standard immunological techniques, such as immunoblotting or ELISA or other techniques using antibodies specific for pRb and the apoptosis-inducing protein. It is also preferred to use tissue sections from a tumour immunologically stained for pRb and the apoptosis-inducing protein, respectively, for comparison with tissue sections from the same area subsequent to treatment according to the invention.
  • Treatment according to the invention would preferably comprise transfection of tumorous tissue with an effective amount of a vector containing a preferred version of the pl6 INK4 gene, optionally combined with the p53 gene or a p53 pro-apoptotic downstream gene.
  • Tissue or tissue sections for quantification of pRb and apoptosis-inducing protein level would preferably be extracted or cut, respectively, at least one week, preferably at least two weeks, after initiation of treatment. If the ratio of the level of pRb/apoptosis-inducing protein is not sufficiently reduced, the treatment may be repeated.
  • the malignant cells are p53 positive.
  • p53 positive is meant that the normal (wild type) protein is expressed and can be detected by standard techniques such as immunohistochemistry or immunoblotting.
  • the malignant cells are p53 deficient.
  • p53 deficient is meant the lack of expression due to gross rearrangement or deletion of the gene .
  • the level of pRb protein in malignant cells is reduced.
  • the ratio pRb/apoptosis-inducing protein (e.g. pRb/p53) is crucial to the induction of apoptosis in malignant cells.
  • the inventors have shown that the presence of a normal wild type level of pRb is necessary as a starting point for the reduction of the ratio.
  • the presence of p53 is necessary.
  • the ratio of level of pRb to level of apoptosis-inducing protein is reduced in malignant cells by reducing the level of pRb and increasing the level of apoptosis-in- ducing protein e.g. p53, by transfer of genetic material into said cells.
  • genetic material is meant sequences of nucleic acids.
  • the genetic material is one or more genes selected from the group consisting of pl5 INK4 , pl8 INK4 , p21 KIP , p27 KIP , p57 KIP , and p53.
  • a modified version of the pl6 INK4 gene is preferred, in particular a modified version of the pl6 INK4 gene which only encodes the cdk-binding domain of the pl6 INK4 protein or a mutant version of this domain.
  • modified version genetic material derived from the original gene by deleting, adding or substituting specific nucleic acids in the genetic sequence.
  • pl6 INK4 It is the interaction of pl6 INK4 with cdk4 that brings about the growth arresting effect according to the invention, and consequently it is desirable to achieve as strong and efficient an interaction as possible.
  • This can be done by constructing modified versions of the pl6 INK4 gene which, upon transfer into cells, will give rise to a pl6 INK4 protein species that will interact more strongly with the endogenous cdk4 protein than normal pl6 INK4 .
  • pl6 INK4 relatives means genes of the same recognized INK4 gene family, such as
  • the transferred genetic material is the pl6 INK4 gene in combination with the p53 gene. Also preferred is genetic transfer of a modified version of the pl6 INK4 gene in combi- nation with a p53 pro-apoptotic downstream gene.
  • p53 pro-apoptotic downstream gene a gene which acts downstream of p53 which is on the top of a regulatory cascade (like pRb) .
  • p53 activates and represses a number of genes, and all these genes are called downstream genes. Some of these genes are only cell cycle inhibitors (like p21 KIP ) and others are pro-apoptotic. Examples of p53 pro-apoptotic downstream genes are the bax, bak and bcl-X genes.
  • pl6 INK4 and p53 make it possible to achieve the desired effect of growth arrest followed by induction of apoptosis in malignant cells, in both p53 positive and p53 deficient cells.
  • Example 3 shows that there is a synergistic effect of the simultaneous transfer of the two genes.
  • the genetic material is transferred in a vector.
  • the vector can be a non-viral or a viral vector. If the vector is viral, it is preferred according to the invention that the vector is selected from the group consisting of an adenoviral vector, a retroviral vector, a herpes viral vector and a hybrid vector. It is particularly preferred that the viral vector is the Ad- pl6-9 vector (ECACC accession number V97021335) .
  • a high level of overexpression of the transferred ge- netic material is achieved.
  • Preferably more than 2 fold overexpression is achieved, more preferably more than 4 fold, still more preferably more than 6 fold, even more preferably more than 8 fold, most preferably more than 10 fold overexpression is achieved.
  • overexpression means expression at levels exceeding those present in normal cells.
  • the inventors show that cellular overexpression of the pl6 INK4 gene is crucial to achieving growth arrest and induction of apoptosis according to the invention.
  • the present invention provides the basis for induction of apoptosis in the vast majority of malignant cells by disclosing that over- expression, preferably a high level of overexpression, is necessary.
  • p53 deficient cells are made p53 positive.
  • Particularly preferred p53 deficient cells are made p53 positive by trans- fer of the p53 gene.
  • a preferred embodiment of the present invention is the use according to the invention for the treatment of a malignant disease.
  • the malignant disease is a solid tumour. More preferably the malignant disease is selected from the group consisting of colorectal cancer, mammary cancer, liver cancer, pancreatic cancer, prostate cancer, lung cancer, head and neck cancer, kidney cancer, and melanoma.
  • An important aspect of the present invention is the use of a vector containing genetic material for the preparation of a composition for the treatment of a malignant disease, said vector, upon administration in a subject suffering from a malignant disease, inducing cell death, such as apoptosis, in malignant cells by reducing the ratio of the level of pRb protein to the level of an apoptosis-inducing protein in malignant cells, by reducing the level of pRb and increasing the level of the apoptosis-inducing protein, after having first achieved growth arrest of said malignant cells by inducing therein an inhibition of phosphorylation of the pRb protein.
  • a preferred embodiment of the present invention is the use according to the invention, wherein the vector is a non-viral vector. Also preferred is the use of a viral vector, in particular an adenoviral vector.
  • an particularly preferred embodiment of the present invention is the use according to the invention, wherein the viral vector is selected from the group consisting of an adenoviral vector, a retroviral vector, a herpes viral vector and a hybrid vector.
  • the viral vector is selected from the group consisting of an adenoviral vector, a retroviral vector, a herpes viral vector and a hybrid vector.
  • Ad-pl6-9 ECACC accession number V97021335) .
  • hybrid vectors means vectors which are generated by combination of parts from different vectors. Different parts of two or more viruses can be combined but also parts of viruses with non-viral components .
  • Example 1 the inventors show that use of the vector Ad- pl6-9 (ECACC accession number V97021335) enables a very high level of cellular overexpression of genes that are transferred into cells by means of the vector. Since such levels of overexpression are preferred according to the present invention, the disclosed adenoviral vector represents an important advantage over hitherto disclosed vectors which have not been known to mediate significant overexpression of the genes they transfer.
  • a preferred embodiment of the present invention is the use according to the invention, wherein the genetic material is one or more genes selected from the group consisting of pl6 INK4 , pl8 INK4 , pl9 INK4 , p21 KIP , p27 KIP , p57 KIP , and p53.
  • the genetic material is one or more genes selected from the group consisting of pl6 INK4 , pl8 INK4 , pl9 INK4 , p21 KIP , p27 KIP , p57 KIP , and p53.
  • the use of a modified version of the pl6 INK4 gene or a number of derivatives thereof More preferred is the use of a modified version of the pl6 INK4 gene which only encodes the cdk-binding domain of the pl6 INK4 protein. Even more preferred is the use of the pl6 INK4 gene in combination with the p53 gene.
  • Most preferred is the use of a modified version of the pl6 INK4 gene in combination with
  • a preferred embodiment of the present invention is the use according to the invention, wherein high levels of over- expression of the transferred genetic material is achieved. It is preferred that more than a 2 fold level of overexpres- sion of the selected gene is achieved. It is more preferred that more than a 4 fold level of overexpression of the selected gene is achieved. It is still more preferred that more than a 6 fold level of overexpression of the selected gene is achieved. It is even more preferred that more than an 8 fold level of overexpression of the selected gene is achieved. It is most preferred that more than a 10 fold level of over- expression of the selected gene is achieved.
  • An especially preferred embodiment of the present invention is the use according to the invention, wherein the ratio (pRb/apoptosis-inducing protein) is reduced by reducing the level of pRb by at least 5 fold (to below 20% of the original level) , preferably by at least 10 fold (to below 10% of the original level) or even by at least 20 fold (to below 5% of the original level) , while at the same time, the p53 level should be increased to at least two fold of the original level.
  • the ratio pRb/apoptosis-inducing protein
  • An important aspect of the present invention relates to a vector comprising the nucleic acid sequence encoding the pl6 INK4 protein or a functional equivalent thereof, and further optionally comprising a nucleic acid sequence encoding the p53 protein or a functional equivalent thereof, in combination with a genetic element inherent to the vector, said inherent element effecting significant overexpression of said gene sequences .
  • a further very important aspect of the invention relates to the specific adenoviral vector Ad-pl6-9 which was deposited on 13 February 1997 with the collection of European Collec- tion of Cell Cultures (ECACC) under the accession number
  • FIG. 1 The figure shows overexpression of pl6 INK4 in IMR- 90 cells after gene transfer with the vector Ad-pl6-9. Infection with an m.o.i. of 50 was done for 1 hour. Cells were washed and extracted 48 hours later, protein was separated by SDS-polyacrylamide gel electrophoresis and blotted onto nitrocellulose membrane. Detection of pl6 IN 4 was done using a monoclonal anti-pl6 INK4 antibody (DCS50) . The upper band in the right lane corresponds to the endogenous pl6 INK4 which is up-regulated in virus-infected cells. The lower band repre- sents the ectopic overexpressed pl6 INK4 derived from a gene with a short N-terminal truncation of the coding sequence.
  • DCS50 monoclonal anti-pl6 INK4 antibody
  • FIG. 1 The figure shows the effect of Ad-pl6 INK4 gene transfer on cell cycle regulation.
  • Two cell lines lacking endogenous pl6 INK4 expression HuH7, LOVO
  • two Rb negative cell lines taken for control (BT549, C33A) were treated with PBS (upper panel) or infected with either Ad-bg (middle panel) or Ad-pl6 INK4 adenoviruses at 50, 30, 50 and 50 m.o.i., respectively.
  • BrdU bromodeoxy- uridine
  • FIG. 3 The figure shows stimulation of apoptosis by simultaneous transfer of pl6 INK4 and p53 genes.
  • the DNA content of HuH7, LOVO and IMR-90 cells was analyzed by flow cytometry after propidium iodine staining.
  • Cells were either treated with PBS (-) or infected with Ad-p53 or Ad-pl6 INK4 adenoviruses at m.o.i. 25 (HuH7) or 15 (LOVO) 96 hours before analysis.
  • Ad ⁇ gal virus was added at m.o.i. 25 (HuH7) and 15 (LOVO) .
  • Double infection with both Ad-p53 and Ad-pl6 INK4 was carried out at m.o.i.
  • FIG. 5 The figure shows the detection of reduced pRb level and increased p53 level after transfer after overexpression of pl6 INK4 and p53.
  • the figure shows adenoviral infection of HuH7 cells with Ad-pl6 (15 m.o.i.) and Ad-p53 (15 m.o.i.), which was carried out for 1 hour in phosphate-buffered saline (PBS) in the presence of 1 mM MgCl 2 . Cells were incubated at 37°C for three days, washed and extracted in lysis buffer. Protein (50 ⁇ g) was separated on a 10% polyacrylamide-SDS gel, transferred to a nitrocellulose membrane by semidry blotting and immunodetection was performed.
  • PBS phosphate-buffered saline
  • FIG. 6 The figure shows tumour size over time as a function of treatment with p53 and pl6 INK4 .
  • 3xl0 6 HuH7 cells were injected into the left inguinal region of nude mice. After 15 days, when visible tumours had grown, adenoviral vectors (6xl0 9 , 150 ⁇ l ) were injected into the tumours (day 1) . Injection was repeated 4 days later. Tumour size was measured as described in Methods.
  • the pl6 INK4 cDNA [Lukas et al . (1995)] was fused to a CMV polyadenylation signal and cloned downstream of the CMV immediate early promoter.
  • the complete expression unit was inserted into the adenoviral shuttle vector PdElsplA [Bett et al . (1994)] .
  • Recombinant virus was generated by co-transfec- tion of the shuttle plasmid with pJM17 [McGrory et al . (1988)] in subconfluent cultures of late passage 293 cells using a modified Ca-P0 4 precipitation technique [Lieber et al . (1995)] .
  • the viruses were plaque purified once and analyzed by restriction analysis. One of the plaques gave Ad- pl6-9.
  • the adenovirus containing the p53 gene driven by the CMV promoter was a kind gift from Wei-Wei Zang (Houston) and the adenovirus harbouring the ⁇ -galactosidase gene under control of the RSV promoter was kindly provided by Michel Perricaudet (Paris) .
  • 293 cells were infected at m.o.i. 5 and harvested after CPE became visible (48 hours) . Cells were lysed in PBS containing 0,2% NP40.
  • CsCl-step gradient centrifugation [Kanegae et al . (1994)].
  • CsCl was removed by gel filtration through Sephadex G25 columns (PD25, Pharmacia) and virus aliquots were stored at -70°C in storage buffer containing 150 mM NaCl, 3 mM KCl, 1 mM MgCl 2 , 10 mM Tris pH 7.4 and 10% glycerol . Titers were determined by plaque assay on 293 cells.
  • HuH7 human hepatocellular carcinoma
  • BT549 human breast carcinoma
  • MCF7 human breast carcinoma
  • IMR-90 normal human fibroblasts
  • 293 cells human primary embryonal kidney
  • DMEM Dulbecco's modified Eagle's medium
  • C33A human cervix carcinoma
  • LOVO cells colon carcinoma
  • Optimum concentrations of virus causing expression in 100% of the cells without visible toxic effect were determined by infection of cells with the ⁇ -galactosida- se expressing virus at different doses and subsequent staining with Xgal .
  • bromodeoxyuridine was applied to the cell culture medium at a final concentration of 10 mM, and cells were incubated at 37°C for 30 minutes. Cells were washed twice with PBS, trypsinised and fixed in 70% ethanol. DNA was denatured and RNA was degraded by incubation in 2M HCl/0,5% Triton X100 for 30 minutes. After neutralisation in 0. IM Na 2 B 4 0 7 , cells were exposed to a mouse monoclonal Anti-BrdU antibody (Becton Dickinson) followed by a FITC-conjugated goat anti-mouse antibody.
  • Becton Dickinson Becton Dickinson
  • the lower band corresponds to the protein encoded by the truncated transferred gene whereas the upper band shows that the endogenous pl6 INK4 gene was also activated by the gene transfer proce- dure.
  • the degree of overexpression calculated from the intensity of the lower band was 38 fold.
  • adenoviral vector carrying a truncated but functional pl6 INK4 gene under the control of the CMV promoter was generated and used to infect various carcinoma cell lines of different tissue origin.
  • the effect of pl6 INK4 overexpression on the cell cycle was then determined by flow cytometry analysis as described in Materials and Methods. Whereas infection of all cell lines with a control vector carrying the lacZ gene (Ad-bg) had no significant effect on the cell cycle on day 2, progression into S phase was blocked completely in the two Rb-positive carcinoma lines HuH7 and LOVO (ATCC).
  • Ad-p53 on its own did not show a significant effect on the cell cycle in all three cell types whereas Ad-pl6 INK4 induced Gl arrest in all cell lines under these conditions.
  • a combination of both viruses resulted in a complete shift of the DNA profile to a sub-Gl position in the two tumour cell lines suggesting that almost all cells entered apoptosis at this time point. No effect was detected in IMR-90 cells (Fig. 3) .
  • the facilitating function of p53 would best be explained by a mechanism in which the blocked Rb pathway (with subsequent reduction of pRb level) cooperates with p53, e.g. by synergistically acting towards induction of a downstream effector.
  • the bax gene product [Oltvai et al . (1993)] would be a good candidate for this effector since it has been shown to be induced by p53 [Oltvai et al . (1993)] and E2F might be involved in its up-regulation as well.
  • Some tumours, particularly those deficient in p53 and pRb, are obviously susceptible to apop- totic cell death by overexpression of p53 only [Yang et al .
  • pl6 INK4 close to normal levels which are sufficient to exert Gl arrest does not support p53-dependent apoptosis [Lukas et al . (1995) and unpublished results] . It is important to stress that this cooperative effect towards apoptosis was not observed in normal IMR-90 cells (Fig. 4) .
  • pi6 INK4 -dependent down-modulation of pRb level in the presence of overexpressed wild-type p53 causes apoptosis only in tumour cells whereas normal cells remain arrested in Gl .
  • tumour cell-specific effect of pRb down-modulation in the presence of p53 is unclear but it is most likely due to mutations in protooncogenes or tumour suppressor genes which have occurred in the genesis of the tumour in addition to abrogation of the Rb pathway.
  • down-modulation of pRb after Gl arrest is presumably not unusual and may serve as a differentiation-promoting event.
  • HuH7 cells (8xl0 5 cells/10 cm dish) were seeded the day before infection.
  • PBS phosphate-buffered saline
  • the virus suspension was replaced by fresh cell culture medium and the cells were incubated at 37°C for three days. After this period, cells were washed twice with PBS and extracted in lysis buffer. Protein concentration was measured and 50 ⁇ g of protein per lane were separated on a 10% poly- acrylamide-SDS gel.
  • Proteins were transferred to a nitrocellulose membrane by semidry blotting and immunodetection was performed.
  • Primary antibodies were G3245 (Phar- mingen) against pRb and Ab-2 (Oncogene Sciences) against p53.
  • Detection of specific immunocomplexes was done using biotiny- lated goat anti-mouse antibody and streptavidin-POD conjugate (ECL system, Amersham) .
  • Chemiluminescence was detected by exposure to X-ray film as shown in Fig. 5. Intensities of bands were recorded by densitometric scanning of different exposures to ensure linearity of the image.
  • the scanned relative intensities of the pRb- and p53 -specific bands of Fig. 5 are given in table 1. Table 1
  • the ratio of pRb to p53 was reduced by more than 20 fold by this treatment, as a result of a more than 5 fold reduction of pRb level and a concomitant 4 fold increase of p53 level.
  • Cells were analyzed for cell cycle profiles as described in Example 2, and it was found that almost 50% of the cells have entered apoptosis at that time as indicated by a sub-Gl peak in the flow cytometric scan (not shown) .
  • the experiment was repeated with higher amounts of Ad-pl6 INK4 and Ad-p53 (25 m.o.i. of each virus) which led to an even lower ratio of pRb/p53 (0.25) and to almost 100% apoptosis induction.
  • HuH7 cells were infected with either Ad- pl6 INK4 or Ad-p53, control Adtk virus (m.o.i. 100), or a mixture of Ad-pl6 INK4 and Ad-p53 (m.o.i. 50 for each) and were injected subcutaneously into nude mice.
  • Ad-pl6 INK4 and Ad-p53 half the amount of Ad-pl6 INK4 and Ad-p53 was used and Ad- ⁇ gal was added to a total m.o.i. of 100. Tumour size was measured after 6 weeks. The results are summarized in table 2.
  • tumours All control animals formed tumours whereas only two out of ten animals in the two groups injected either with Ad- pl6 INK4 infected cells or with doubly infected cells formed tumours at all. These tumours were less than 10% of the size of control tumours, independent of the titer. Results obtained with p53 -transduced cells were strongly titer dependent. High titers had some growth inhibiting effect which was not as pronounced as that of the pl6 INK4 virus.
  • Human hepatocellular carcinoma cells (HuH7) were transduced with adenoviruses containing the indicated genes and sub- sequently injected subcutaneously into nude mice. Tumour volumes were measured after 6 weeks. Figures are numbers of animals which developed tumours out of 5 animals treated in each group. Average sizes of the tumours and standard devi- ation are given.
  • Adtk, Ad-pl6 INK4 or Ad-p53 were each given at an m.o.i. of 100 if administered alone or at an m.o.i. of 50 if given together.
  • Ad- pl6 INK4 or Ad-p53 were each administered at an m.o.i. of 50 if given alone and Ad ⁇ gal was used to adjust the total viral m.o.i. to 100.
  • tumour cells Since pre-transduction of tumour cells by the viral vectors was expected to have a strong growth inhibiting effect, an in vivo experiment with preestablished tumours was carried out in which the viral vectors were directly injected into tumours developed from HuH7 hepatocellular carcinoma cells. The results of this experiment are given in Fig. 6. The data demonstrate the strength of the combinatorial approach. Whereas Ad-pl6 INK4 had some effect and Ad-p53 only a marginal growth-retarding effect, the combination of the two genes led to an almost complete block of tumour growth for at least 12 days. At that time one tumour was completely resolved.
  • tumour growth inhibition 3xl0 6 HuH7 cells were injected subcutaneously into the left inguinal region of nude mice. When visible tumours had grown, the skin was incised, tumours were exposed and injected with adenoviral vectors in a volume of 150 ⁇ l/animal. The procedure was repeated 4 days later. Development of tumour volumes was monitored over a period of 18 days. pl6 INK4 and p53 cooperate in tumour growth inhibition
  • the present example has shown that combinatorial transfer of pl6 INK4 and p53 genes by adenoviral vectors leads to efficient induction of apoptosis which could not be achieved by p53 on its own in Rb-positive tumour cells expressing either normal or mutant p53.
  • Cipl is a potent inhibitor of Gl cyclin-dependent kinases.
  • the retinoblastoma Protein a master regulator of cell cycle, differentiation and apoptosis.
  • Tbe indications made below relate to the microorganism referred to in the description on page 21 . line s 17 ⁇ 24
  • the applicants request that a sample of the deposited microorganisms only be made available to an expert nominated by the requester until the date on which the patent is granted or the date on which the application has been refused or withdrawn or is deemed to be withdrawn.

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Abstract

Cette invention porte sur l'utilisation d'un vecteur renfermant un matériel génétique aux fins de la préparation d'une composition de traitement d'affections malignes. Administré à un sujet atteint d'une affection maligne, ce vecteur provoque l'apoptose des cellules malignes par réduction du rapport teneur en protéine pRb/teneur en protéine induisant l'apoptose ainsi que par réduction de la teneur en protéine pRb et élévation de la teneur en protéine induisant l'apoptose et, une fois obtenue la cessation de la croissance desdites cellules malignes, il provoque une inhibition de la phosphorylisation de la protéine pRb. Cette invention concerne également des vecteurs spécifiques aux fins d'un transfert efficace.
EP98902970A 1997-02-20 1998-02-20 METHODE PERMETTANT DE PROVOQUER UNE MORT CELLULAIRE PROGRAMMEE DANS DES CELLULES MALIGNES PAR REDUCTION DU RAPPORT PROTEINES Rb/PROTEINES INDUISANT L'APOPTOSE Withdrawn EP1007662A1 (fr)

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