WO1998037190A1 - Method for induction of apoptotic cell death in malignant cells, through reduction of the rb to apoptosis inducing proteins ratio - Google Patents

Abstract

The present invention relates to use of a vector containing 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 phosphorylation of the pRb protein. The invention further relates to specific vectors for efficient transfer.

Description

METHOD FOR INDUCTION OF APOPTOTIC CELL DEATH IN MALIGNANT CELLS, THROUGH REDUCTION OF THE RB TO APOPTOSIS INDUCING PROTEINS RATIO

FIELD OF THE INVENTION

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.

GENERAL BACKGROUND

There is only a limited number of basic strategies for cancer gene therapy which show some promise in preclinical models so far. The two major strategies are cytokine-gene aided tumour vaccination and selective prodrug activation. Whereas 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. Alternatively, 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. In both cases, highly efficient gene transfer to the cells in a tumour is required. Although high efficiencies of gene transfer can be obtained in vi tro and even in vivo under certain circumstances, correction of the malignant phenotype by reversing the major oncogenic change in the tumour cells is unlikely to result in normal cells. Thus, selective induction of tumour cell death would be preferable, and the development of methods enabling such induction will be of great importance. Several genetic changes in different oncogenes or tumour suppressor genes have to occur before a normal cell turns malignant. Most of the oncogenes or tumour suppressors are involved in a certain percentage of tumours but none of them are involved in the development of every single tumour. There is now growing evidence for the idea that only a very limited number of regulatory pathways exist which are involved in the key decisions about the fate of cells including control of the cell cycle. The common feature of multistep carcinogene- sis is the abrogation of at least one of the components of each pathway.

The idea of the existence of recessive cancer genes or tumour suppressors was first confirmed 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 (G₀) through G into the phase of DNA replication (S) and subsequently through a second gap phase (G₂) 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) . These 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.

Recently, a new class of growth regulators has been identified which are inhibitors of cyclin-dependent kinases (CKI) [Sherr and Roberts (1996)] . Whereas one group of CKI, the kinase-inhibiting proteins (KIP) including p21^KIP, p27^KIP, and p57^KIP, can inhibit several cdks, members of the second group (INK4 proteins) including pl5^INK4, pl6^INK4, pl8^INK4, and pl9^INK4, are specific for cdk4/cdk6 [Sherr and Roberts (1996)]. Positive regulators of the kinases (cyclins) and negative regulators (CKIs) seem to compete stoichiometrically for binding to cdks which is at least true for the

fie complexes of cdk4/cdk6 with either D cyclins or pl6^INK4 [Parry et al . (1995)] . This suggests that the balance of growth factors in the environment of the cell would decide about the proportion of cdk4/cdk6 which is in complex with either cyclin D or pl6^INK4 thereby determining the extent of phosphorylation of pRb. 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-,_ . Thus, it appears that 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)].

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) ] .

Most differentiated tissues are continuously regenerating with a certain percentage of their cells. In order to keep the number of cells in a solid tissue constant, simultaneous death of cells is required. This programmed cell death or apoptosis has first been detected in the liver and was later extensively studied in lymphoid cells. A number of genes have been identified which are involved in the induction or prevention of apoptosis [White (1996)] . One of the most extensively studied gene products in this context is the tumour suppressor p53. This protein was originally identified as a partner in complexes with SV40 T-antigen which also binds pRb. Simultaneous inactivation of pRb and p53 by SV40 and other DNA tumour viruses was suggested to be the sole basis for virus-induced malignant transformation [Hinds and Wein- berg (1994)] . The expression of p53 is strongly induced by irradiation and other DNA-damaging treatments. In turn p53 induces the expression of a number of other genes including p21^KIP [el Deiry et al . (1993)] which was shown to be a general inhibitor of cyclin-dependent kinases. Thus, it appears that p53 exerts cell-cycle inhibition in the G_x phase by inhibiting phosphorylation of pRb through p21^KIP-mediated blocking of cdk4 activity. However, p53 was also shown to induce apoptosis in late Gl which probably occurs when DNA damages cannot be repaired properly. On the other hand, 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.

There is a strong tendency to lose p53 function in Rb-de- ficient cells. There is no known tumour cell line and prob- ably no tumour (besides retinoblastomas) which is Rb-defi- cient and still p53 positive. Loss of G phase control allows for cell multiplication for a few rounds but would probably be recognized as a serious deregulation by p53, may be due to accumulation of mutations, and would result in apoptosis. Since deregulation of the Rb pathway in cases of cyclin D overexpression or loss of pl6^INK4 is less pronounced than in the case of loss of Rb, inactivation of p53 is obviously not essential in the first two situations.

It was demonstrated that ectopic expression of the wild-type p53 gene inhibited proliferation in vi tro of human tumour cell lines either lacking p53 completely or expressing mutant p53 [Baker et al . (1990), Cheng et al . (1992), Takahashi et al. (1992)] . p53 gene-transduced tumour cells were less tumorigenic in nude mice. Intratracheal instillation of a retroviral vector carrying the p53 gene prevented the growth of orthotopic human lung cancer cells in nude mice [Cai et al. (1993)] . The use of a similar retroviral vector in a spheroid tumour model showed that the vector was able to penetrate into the inner tumour mass and also to induce significant apoptotic tumour cell death in lung cancer cells [Fujiwara et al . (1993)] . Lung cancers and also the malignant lesions of the epithelium of the oesophagus have frequently mutated p53 suggesting that these malignancies could be primary targets for p53 gene transfer by instillation of a suitable vector. Since retroviral vectors have several inherent difficulties in gene delivery in vivo, adenoviral vec- tors with the p53 gene were generated [Zhang et al . (1993)] . 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)]. Recently, the first clinical trial for application of p53 gene transfer was published [Roth et al . 1996)] . In this study, a retroviral vector was used to deliver the p53 gene into lung carcinomas of end- stage patients who all died within a few weeks after treatment. In some patients, clear signs of tumour regression correlating with apoptotic cell death were detectable. However, the extent of tumour cell death and regression of the tumour mass is difficult to assess from this study. Lung cancer cells are not only frequently deficient in p53 but also susceptible to the induction of apoptosis by overexpres- sed p53 making this tumour particularly suitable for gene therapy by p53.

pl6^INK4 (MTS1) 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 . (1994)] . As discussed above, 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, and pl6^INK4-deficient tumours have normal Rb [Strauss et al . (1995), White et al . (1996)]. Thus, reestab- lishment of pl6^INK4 function in tumour cells would restore normal growth control [Lukas et al . (1995)] but would not necessarily lead to inhibition of tumour growth. Recent application of an adenoviral vector with the pl6^INK4 in nude mice demonstrated some retardation of tumour growth. This result confirms the tumour suppressing function of pl6^INK4 but also indicates that no significant regression of tumours can be obtained by this tumour suppressor.

SUMMARY OF THE INVENTION

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) . Prior art has shown that 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. When p53 is lost, spontaneous apoptosis does not occur and the efficiency of chemotherapy-induced apoptosis is very low. Since the major goal for tumour therapy is not only inhibition of tumour growth but induction of tumour cell death, it would be particularly important to combine the two principles. The presence of a functional Rb has been related to prevention of apoptosis [Haas-Kogan et al . (1995), Herwig and Strauss (1997)] . Thus, it appears that cell cycle control and regulation of apoptosis are tightly coordinated by pRb and p53. Loss of both functions probably guarantees unrestricted growth without apoptotic cell death. The question arises whether reconstitution of one or the other tumour suppressor could result in reconstitution of growth control and/or apoptosis.

The present invention is based on the original finding that the product of the pl6^INK4 gene (also called MTS-1=CDKI4=CD- KN2) [Hannon et al . (1994)] inhibits cell proliferation in all cells which still have a functional Rbl gene, which is the case for about 85% of all tumours. 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. In relation to ectopic expression of the pl6^INK4 gene, some degree of spontaneous apoptosis in tumour cells which are p53 positive was observed when the pl6^INK4 gene was highly overexpressed using selected adenoviral vectors (Example 2) . Apoptosis was not observed in p53-defi- cient tumour cells and in normal cells. Combined overexpres- sion of pl6^INK4 (Ad-pl6-9) (ECACC accession number V97021335) and p53 leads to an unprecedented high degree (up to 100%) of apoptosis in tumour cells which are either p53 -positive or negative but surprisingly not in normal cells (Example 3) .

The surprising finding of the tumour cell specific apoptotic effect of overexpressed pl6^INK4 was investigated in more detail. It was observed that the level of the Rb protein (pRb) was considerably reduced over a period of three days when pl6^INK4 was overexpressed (Example 4) . This effect is probably due to autorepression of the Rbl gene by its non- phosphorylated product pRb. Thus, overexpession of pl6^INK4 not only blocks cell division but subsequently reduces the level of pRb which, in turn, leads to an increased ability of p53 -positive tumour cells to undergo apoptosis. The latter can be dramatically increased by simultaneous overexpression of p53. 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. First, 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%) . At the same time, the p53 level should preferably be increased to more than two fold of the original level. For efficient apoptosis in 80-100% of the malignant cells, it is not sufficient to only increase the level of p53 without reducing the level of pRb. 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. Generally, the total amount of pRb will be measured including phosphorylated pRb as well as non-phosphorylated 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 p₁g^iNK4 g_enθ/ 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 .

Thus, 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.

DETAILED DISCLOSURE OF THE INVENTION

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. Thus, 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.

In relation to the present invention, 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. For the first time it is shown that overexpression of pl6^INK4 induces down-modulation of pRb protein synthesis resulting in increased sensitivity of tumour cells to apoptotic stimuli. Thus, 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. Thus, 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. In contrast to this, 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 .

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, however, 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) . Furthermore, 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.

In the present context, the term "growth arrest" means a substantial block of cell division, which is understood as an almost complete block, typically at least 90%.

In preferred embodiments of the present invention, 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.

It has been shown for the first time by the present inventors that 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. By using the method of the present invention, 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.

According to the present invention the ratio of the level of pRb protein to the level of apoptosis-inducing protein (such as pRb/p53) 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. Thus, 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.

In one embodiment of the present invention the malignant cells are p53 positive. By the term "p53 positive" is meant that the normal (wild type) protein is expressed and can be detected by standard techniques such as immunohistochemistry or immunoblotting.

In another embodiment of the present invention, the malignant cells are p53 deficient. By the term "p53 deficient" is meant the lack of expression due to gross rearrangement or deletion of the gene .

In a preferred embodiment of the invention, the level of pRb protein in malignant cells is reduced.

According to the central concept behind the present invention, the ratio pRb/apoptosis-inducing protein (e.g. pRb/p53) is crucial to the induction of apoptosis in malignant cells. Furthermore, 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. Likewise, the presence of p53 is necessary. Thus, according to the invention, it is possible to induce apoptosis of malignant cells which are p53 positive by manipulating the level of pRb and the level of p53. If cells are p53 deficient, it is necessary to make them p53 positive according to the invention.

In another preferred embodiment of the present invention the ratio of level of pRb to level of apoptosis-inducing protein (such as pRb/p53) 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. By the term "genetic material" is meant sequences of nucleic acids.

In especially preferred embodiments of the present invention 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. Also 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.

By the term "modified version" is meant genetic material derived from the original gene by deleting, adding or substituting specific nucleic acids in the genetic sequence.

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.

As shown in Example 4, genetic transfer and subsequent ecto- pic cellular expression of the pl6^INK4 gene in malignant cells will lead to a reduction in the cellular level of pRb. This reduction results in growth arrest of said malignant cells which is, according to the invention, a prerequisite for induction of apoptosis. According to the invention, it is possible to achieve growth arrest followed by apoptosis by any means that mimics the effect of expressing pl6^INK4 in malignant cells. The proteins encoded by the genes for pl5^INK4,

p21^KIP, p27^KIP, p57^KIP are relatives of pl6^INK4 and are capable of mimicking the pl6^INK4 effect in cells to some extent. The genes encoding these proteins can thus, according to the invention, be transferred to malignant cells in order to achieve growth arrest and subsequent indue- tion of apoptosis. The same applies to modified but still functional versions of the pl6^INK4 gene.

In the present context the term "pl6^INK4 relatives" means genes of the same recognized INK4 gene family, such as

In an especially preferred embodiment of the present invention, 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.

By the term "p53 pro-apoptotic downstream gene" is meant a gene which acts downstream of p53 which is on the top of a regulatory cascade (like pRb) . As a transcriptional regulator, 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.

The combined transfer of pl6^INK4 and p53 makes 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. In a similar fashion it is possible to use a modified version of the pl6^INK4 gene in combination with a p53 downstream gene .

In a further preferred embodiment of the present invention, 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) .

In an especially preferred embodiment of the present invention 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.

In the present context, the term "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. Before the disclosure of the present invention, it was not known that ectopic expression of pl6^INK4 in itself is sufficient to obtain an effect. Thus, for the first time, 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.

In an especially preferred embodiment according to the present 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. Preferably 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. Especially preferred is the use according to the invention of the vector Ad-pl6-9 (ECACC accession number V97021335) .

In the present context the term "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 .

In 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. Especially preferred is 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 p53 pro-apoptotic downstream gene.

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. For efficient apoptosis in 80-100% of the malignant cells, it is not sufficient to only increase the level of the apoptosis-inducing protein (e.g. p53) without reducing the level of pRb. However, it may be sufficient to only reduce the level of pRb without altering the level of the apoptosis- inducing protein. Within the scope of the present invention is thus the use wherein pRb reduced and the apoptosis-inducing protein such as p53 maintained at the same level.

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

V97021335 by Humboldt Universitat zu Berlin, Lehrstuhl Mole- kulare Zellbiologie, Max-Delbrϋck-Haus, Robert-Rδssle-Str . 10, D-13122 Berlin, Germany. The deposit was made in accordance with the provisions of the Budapest Treaty.

LEGENDS TO FIGURES

Figure 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.

Figure 2. 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) and 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. 48 hours after infection bromodeoxy- uridine (BrdU) was added to the cell culture medium. Cells were trypsinized 30 minutes later, fixed and analyzed for DNA content by propidium iodine staining and for DNA synthesis by and uptake of BrdU using flow cytometry. There is almost no fraction of proliferating cells in Ad-pl6^INK4 treated HuH7 and LOVO cells whereas this virus has no effect on BT549 and C33A cells. A fraction of cells with a DNA content less than that of Gl cells (shoulder of the Gl peak) is found in Ad- pl6^INK4 treated HuH7 cells (the existence of this shoulder varies between individual experiments in both tumour cell lines) .

Figure 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. Adβgal 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. 25 (HuH7) and 15 (LOVO) for each virus. Whereas only a slight S-phase reduction is found and a normal cell cycle curve is maintained in Ad-p53 treated cells, the Gl and G2 peaks widened in Ad-pl6^INK4 infected cells (typical experiment) . Most of the double infected HuH7 and LOVO cells show a drastically reduced DNA content. The Gl and G2 peaks are absent. No sub-Gl (apoptotic) DNA peak is observed in IMR-90 cells. Figure 4. The figure shows a time-course of pRb expression and phosphorylation after infection of HuH7 cells with Ad- pl6^INK4 virus. Infection was done at an m.o.i. of 50 and extracts were prepared at the time points indicated. Extracts were subjected to SDS-PAA electrophoresis, Western blotting and immunodetection of pRb as described in Methods.

Figure 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 MgCl₂. Cells were incubated at 37°C for three days, washed and extracted in lysis buffer. Protein (50 μg) was separated on a 10% polyacrylamide-SDS gel, transferred to a nitrocellulose membrane by semidry blotting and immunodetection was performed. Primary antibodies were G3245 (Pharmingen) against pRb and Ab-2 (Oncogene Sciences) against p53. Detection of specific immunocomplexes was dome using biotinylated goat anti-mouse antibody and streptavidin-POD conjugate (ECL system, Amersham) . Chemi- luminescence was detected by exposure to X-ray film.

Figure 6. The figure shows tumour size over time as a function of treatment with p53 and pl6^INK4. 3xl0⁶ HuH7 cells were injected into the left inguinal region of nude mice. After 15 days, when visible tumours had grown, adenoviral vectors (6xl0⁹, 150 μl ) were injected into the tumours (day 1) . Injection was repeated 4 days later. Tumour size was measured as described in Methods.

Materials and Methods

Construction and Preparation of Recombinant Adenoviruses

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-P0₄ 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 β-galactosidase gene under control of the RSV promoter was kindly provided by Michel Perricaudet (Paris) . For the preparation of large purified virus stocks, 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. After removing cell debris, the virus suspension was subjected to two rounds of 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 MgCl₂ , 10 mM Tris pH 7.4 and 10% glycerol . Titers were determined by plaque assay on 293 cells.

Cell Culture and Virus Infection

HuH7 (human hepatocellular carcinoma), BT549, MCF7 (human breast carcinoma) , IMR-90 (normal human fibroblasts) and 293 cells (human primary embryonal kidney) were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% FCS and 2 mM glutamine at 5% C0₂. C33A (human cervix carcinoma) and LOVO cells (colon carcinoma) were maintained in RPMI contain- ing 10% FCS. 8xl0⁵ cells/10 cm dish plated 1 day before were infected with adenoviruses for 60 minutes in PBS + 1 mM MgCl₂ at an m.o.i. of 50 (HuH7, BT549, C33A) , 30 (LOVO), 100 (IMR- 90) and 300 (MCF7) . Optimum concentrations of virus causing expression in 100% of the cells without visible toxic effect were determined by infection of cells with the β-galactosida- se expressing virus at different doses and subsequent staining with Xgal .

Cell Cycle analysis

For pulse labelling of proliferating cells, 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 Na₂B₄0₇, cells were exposed to a mouse monoclonal Anti-BrdU antibody (Becton Dickinson) followed by a FITC-conjugated goat anti-mouse antibody. For detection of DNA content, propidium iodine was added to PBS-washed cells to a final concentration of 5 μg/ml, and flow cytometric analysis was performed using the EPICS XL-MCL flow cytometer (Coulter) .

EXAMPLE 1

Overexpression of the pl6^INK4 gene by the vector Ad-pl6-9 (ECACC accession number V97021335)

Normal human fibroblasts (IMR-90, ATCC CCL186) were incubated with Ad-pl6-9 (ECACC accession number V97021335) for 1 hour and subsequently incubated at 37°C for two days. Infected as well as untreated control cells were harvested, washed and extracted in lysis buffer. Total protein (50 μg) was run on a 15% SDS-polyacrylamide gel and blotted onto nitrocellulose membranes. The blot was incubated with anti-pl6^INK4 antibody and with secondary antibody afterwards. The result is given in Fig. 1. The blot was scanned to quantify the relative amounts of protein. The experiment resulted in a total in- crease in the amount of pl6^INK4 of 43 fold. 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.

EXAMPLE 2

Overexpression of pl6^JWK4 leads to growth inhibition and apoptosis

An 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

CCL229) (Fig. 3) . Interestingly, a shoulder was observed left from the Gl peak in the DNA profile from HuH7 cells at 2 days after infection. Two days later, this shoulder was even more pronounced in the same experiment but not in several others (not shown) . It was suspected to reflect a small fraction of apoptotic cells. To prove this assumption, a TUNEL assay was carried out which detected a few apoptotic cells (not shown) . Whereas complete growth arrest was observed over a period of ten days, apoptotic cell death was only detectable occasio- nally in the tumour cells tested but not in normal diploid fibroblasts (IMR-90) (not shown) .

EXAMPLE 3

Synergistic effect of pl6^XWK4 and p53 towards apoptosis

Since it appeared that overexpression of pl6^INK4 can not only induce cell cycle arrest but renders Rb-positive carcinoma cells somehow susceptible to apoptotic stimuli, it was interesting to find out if overexpression of the p53 gene would result in efficient induction of apoptosis in pl6^INK4 expressing cells. HuH7 and LOVO tumour cells as well as normal IMR- 90 cells were infected with Ad-pl6^INK4 and Ad-p53 either individually or together at comparable limited multipliciti- es, and the DNA profiles were analyzed by flow cytometry three days later. The results are given in Fig. 3. 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) . Analysis of apoptosis in HuH7 cells by the TUNEL assay at day 3 after viral infection showed that p53 could induce apoptosis in some cells, pl6^INK4 overexpression led to occasional appearance of apoptotic cells, and a combination of both genes induced apoptosis in the majority of cells. Whereas similar results were obtained for LOVO cells, Ad-p53 could induce efficient apoptosis on its own in Rb-deficient BT549 carcinoma cells (not shown) .

EXAMPLE 4

Effect over time on pRb of pl6^INK4 overexpression

Surprisingly, overexpression of pl6^INK4 over a period of three days did not only result in the disappearance of phosphorylated forms of pRb but also led to a considerable decrease in the amount of total Rb protein which was most dramatic in the hepatocellular carcinoma line HuH7. A time- course experiment demonstrated that the pRb level gradually decreased over a period of five days (Fig. 4) .

Immunob1otting

Cells were washed twice with PBS, scraped off from the plate and lysed in cell lysis buffer. Protein concentration was determined by a Bradford assay. 50 μg of protein were elec- trophoretically separated on either 8% (for pRB) , 10% (p53) or 15% (pl6^INK4 and p21) polyacrylamide-SDS gels. Proteins were transferred to nitrocellulose by semidry blotting and immunodetection was performed using the enhanced chemilumine- scence system ECL. Mouse monoclonal antibodies against pl6^INK4, Rb (G3245, Pharmingen) , p53 (Ab-2, Oncogene Sciences) and p21 (DCS60) were used as primary antibodies, and biotinylated goat anti-mouse antibody and a streptavidine-POD conjugate (Amersham) were used for secondary detection.

pRb down-modulation and apoptosis

The results demonstrate that overexpression of the tumour suppressor gene product pl6^INK4 does not only block the cell cycle in Gl but can also induce repression of pRb synthesis afterwards. This, in turn, seems to render tumour cells susceptible to apoptotic stimuli. Such stimuli probably occur also under conditions of prolonged growth arrest in culture. Since pl6^INK4 induces cell cycle arrest also in normal cells but does not lead to subsequent apoptosis [Lukas et al., (1995) and this example] , the data suggest that the apoptotic response to sustained growth arrest is specific for tumour cells. Thus, the apoptotic control machinery probably recognizes other changes which have occurred in the development of a tumour cell. It is known that the presence of a functional p53 is not absolutely required for induction of apoptosis

[White et al. (1996), Bates et al . (1996)], a notion to some extent supported by the experiments with HuH7 cells harbouring mutant p53. However, a low degree of apoptosis was more consistently observed in Ad-pl6^INK4-transduced p53 -positive tumour cells (LOVO, data not shown) . Nevertheless, apoptosis was dramatically facilitated by overexpression of wild-type p53 in cells in which the Rb pathway was blocked by over- expressed pl6^INK4. Although the current knowledge of apop- tosis-controlling pathways is insufficient to fully explain this observed synergism, one possible scenario is suggested by the gradual reduction of pRb level (Fig. 2) . This unex- pected effect of sustained Gl block by pl6^INK4 is probably caused by autorepression of RB-1 gene expression [Gill et al . (1994)] in response to accumulation of nonphosphorylated pRb and/or its proteolytic degradation [An and Dou (1996)] . When the pRb level is sufficiently reduced, the protective role of Rb towards apoptosis can no longer be maintained. This, in turn, might be due to release of the transcription factor E2F [Weinberg (1995), Nevins (1992)] which was shown to induce apoptosis under certain circumstances [Qin et al . (1994), Shan and Lee (1994), Wu and Levine (1994)]. 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 . (1995), Roth et al . (1996)]. Both Rb-positive tumour cell lines tested in this example (HuH7, LOVO) were neither strongly arrested in Gl by p53 overexpression nor did they respond by apoptosis. It could be concluded that, at least in these Rb-positive tumour cell lines, Gl arrest via block of the Rb pathway and/or subsequent down-regulation of pRb level is a prerequisite for apoptosis which is accelerated by over- expression of p53. Thus, it is assumed that a low pRb/p53 ratio is required for apoptosis and Gl arrest alone is not sufficient. Expression of 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) . Thus, 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 . The molecular basis for this 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. In normal cells, down-modulation of pRb after Gl arrest is presumably not unusual and may serve as a differentiation-promoting event.

EXAMPLE 5

Co-expression of pl6^INK4 and p53 reduces the ratio pRb/p53 and leads to apoptosis

HuH7 cells (8xl0⁵ cells/10 cm dish) were seeded the day before infection. Adenoviral infection with Ad-pl6^INK4 (15 m.o.i.) and Ad-p53 (15 m.o.i.) was carried out for 1 hour in phosphate-buffered saline (PBS) in the presence of 1 mM MgCl₂. 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 μg 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

HuH7 control HuH7 + Ad-pl6^INK4/Ad-p53 Changed levels O.D. (-background) O.D. (-background)

pRb 821,896 150,797 - 5,5 fold

(18% of normal)

p53 78,134 306,874 + 4 fold

(400 % of normal)

Thus, 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.

EXAMPLE 6

Inhibition of tumour development by pl6^JOT4 and p53

It was investigated whether adenoviral transduction of carcinoma cells in vi tro would prevent tumour growth in nude mice. To this end, 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. In a second experiment, half the amount of Ad-pl6^INK4 and Ad-p53 was used and Ad-βgal was added to a total m.o.i. of 100. Tumour size was measured after 6 weeks. The results are summarized in table 2. 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.

Nude mouse experiments

HuH7 cells were seeded on culture flasks and grown to 80% confluence. Adenoviruses as indicated in table 2 below were applied at an m.o.i. of 100. After 1.5 hours of infection in PBS, medium was applied. Another 7 hours later, cells were harvested and injected subcutaneously into the left inguinal region of nude mice at 3xl0⁶ cells per animal (female CD1- nu/nu, Charles River (Sulzfeld, Germany), in experiment #1, female NMRI-I-nu/nu, bred at MDC, Berlin in experiment #2) . After 6 weeks tumour volumes were measured according to the formula V=axb²/2 [Carlsson et al . (1983)].

Table 2: Inhibition of tumour growth by transduction with pl6^INK4 and p53 genes

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. In experiment #1, 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. In experiment #2, Ad- pl6^INK4 or Ad-p53 were each administered at an m.o.i. of 50 if given alone and Adβgal was used to adjust the total viral m.o.i. to 100.

EXAMPLE 7

Inhibition of tumour growth by pl6^INK4 and p53

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.

Nude mouse experiments

For in vivo gene transfer experiments, 3xl0⁶ 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 μl/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. Transfer of the two genes to hepatocel- lular carcinoma cells (HuH7) and colon carcinoma cells (LOVO) completely prevented development of subcutaneous tumours in nude mice. Under these conditions even pl6^INK4 on its own was sufficient to cause inhibition of tumour growth (table 1) .

Injection of adenoviral vectors into tumours in vivo resulted in different outcomes. Some growth retardation was observed with pl6^INK4, little effect was seen with p53 , but strong inhibition of tumour growth was seen with the combination of the two genes (Fig. 6) . Even multiple injections of the tumours did not result in 100% infection as checked by Adβgal control infection. Slow increase in tumour size after 18 days probably reflects outgrowth of non-infected cells.

Both efficient vectors and biological principles for cancer gene therapy are being established, but better ways of distributing the vector within the rigid tumour tissue need to be developed. In this regard, the application of electric pulses with surface electrodes may be helpful (Rols, M. P. et al . , 1998). The requirement of efficient distribution is also shared with classical chemotherapy and with suicide gene therapy where accessibility of the inner tumour mass is the major problem in tumours which would otherwise respond to the treatment. However, the strategy for gene therapy presented has the advantage of being selective for tumour cells and, moreover, might be applicable to the majority of human malignancies . The data presented here also point to the importance of detailed knowledge of the status of both the apoptosis- inducing pathway such as p53 and members of the Rb pathway of a particular tumour in selecting the appropriate treatment. Both diagnostics and future therapy should benefit from the current understanding of basic mechanisms of cell cycle control [Weinberg (1995), Strauss et al . (1995), Sherr and Roberts (1995)] and apoptosis [White (1996)]. The integrated knowledge of both mechanisms will aid in further improvements of strategies aiming at selective elimination of tumour cells.

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INDICATIONS RELATING TO A DEPOSITED MICROORGANISM

(PCT Rule 13«s)

A. Tbe indications made below relate to the microorganism referred to in the description on page 21 . line s 17 ~ 24

B. mENTDTICATION OF DEPOSIT Further deposits are identified on an additional sheet [ [

Name of depositary institution

European Collection of Animal Cell Cultures (ECACC) , CAMR

Address of depositary institution (including postal code and country)

Porton Down

Salisbury, Wiltshire SP4 OJG

Great Britain

Date of deposit Accession Number

13 February 1997 V97021335

C ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet Q

As regards the respective Patent Offices of the respective designated states, 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.

D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications are not for all designated Stales)

E. SEPARATE FURNISHING ©^IN ICATIONS (leave blank if not applicable)

The indications lbted below will be submitted to the International Bureau later (specify the general nature of ^'theindeationscg^ 'Accession Number of Deposit")

For International Bureau use only

I I This sheet was received by tbe International Bureau on:

Authorized officer

Claims

1. Use of a vector containing 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 phosphorylation of the pRb protein.

2. Use according to claim 1, wherein the vector is a non- viral vector.

3. Use according to claim 1, wherein the vector is a viral vector.

4. Use according to claim 1, wherein the viral vector is chosen from the group consisting of an adenoviral vector, a retroviral vector, a herpes viral vector and a hybrid vectors .

5. Use according to claim 1, wherein the vector is ECACC accession number V97021335.

6. Use according to claim 1, wherein the genetic material is one or more genes chosen from the group consisting of _P16^INK4,

p21^KIP, p27^KIP, p57^KIP, p53, bax, bak and bcl-X.

7. Use according to any of the preceding claims, wherein the malignant cells are positive for an apoptosis-inducing protein such as p53.

8. Use according to claim 7, wherein the genetic material is a modified version of the pl6^INK4 gene.

9. Use according to claim 7, wherein the genetic material is a modified version of the pl6^INK4 gene which only encodes the cdk-binding domain of the pl6^INK4 protein.

10. Use according to any of the preceding claims, wherein apoptosis-inducing protein-deficient malignant cells are made positive for an apoptosis-inducing protein by transfer of one or more genes chosen from the group consisting of p53, bax, bak and bcl-X.

11. Use according to claim 10, wherein the genetic material is a pl6^INK4 gene in combination with a gene coding for an apoptosis-inducing protein.

12. Use according to claim 10, wherein the genetic material is a modified version of the pl6^INK4 gene which only encodes the cdk-binding domain of the pl6^INK4 protein in combination with a gene coding for an apoptosis-inducing protein.

13. Use according to any of the preceding claims, wherein high levels of overexpression of the transferred genetic material is achieved.

14. Use according to claim 13, wherein more than 2 fold levels of overexpression of the chosen gene or genes is achieved.

15. Use according to claim 13, wherein more than 4 fold levels of overexpression of the chosen gene or genes is achieved.

16. Use according to claim 13, wherein more than 6 fold levels of overexpression of the chosen gene or genes is achieved.

17. Use according to claim 13, wherein more than 8 fold levels of overexpression of the chosen gene or genes is achieved.

18. Use according to claim 13, wherein more than 10 fold levels of overexpression of the chosen gene or genes is achieved.

19. Use according to any of the preceding claims, wherein the ratio (pRb/apoptosis-inducing protein) is reduced to below

0,4.

20. Use according to any of the preceding claims, wherein the ratio (pRb/apoptosis-inducing protein) is reduced by reducing the level of pRb at least 5 fold and increasing the level of the apoptosis-inducing protein at least 2 fold.

21. Use according to claim 20, wherein the ratio (pRb/apopto- sis-inducing protein) is reduced by reducing the level of pRb at least 10 fold and increasing the level of the apoptosis- inducing protein at least 2 fold.

22. Use according to claim 20, wherein the ratio (pRb/apopto- sis-inducing protein) is reduced by reducing the level of pRb at least 20 fold and increasing the level of the apoptosis- inducing protein at least 2 fold.

23. Use according to any of the preceding claims, wherein the malignant disease is a solid tumour.

24. Use according to claim 23, wherein the malignant disease is chosen 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.

25. A vector comprising the nucleic acid sequence encoding the pl6 protein or a functional equivalent thereof, and further optionally comprising a nucleic acid sequence encoding an apoptosis-inducing protein, in combination with a genetic element inherent to the vector, said inherent element effecting significant overexpression of said gene sequences.

26. The vector Ad-pl6-9 (ECACC accession number V97021335)