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Description  |
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FIELD OF THE INVENTION
The present invention concerns improvements, modifications and developments in relation to transport proteins, intracellular transport and their applications. In particular embodiments, the invention relates to fusion proteins comprising
transport proteins comprising sequences from herpesviral VP22 or from homologues or fragments thereof together with sequences from other proteins; and to methods for their preparation and use. In particular embodiments, the invention relates to fusion
proteins for cell cycle control, and to materials and methods for their preparation and their use. In particular examples the invention relates to fusion proteins having both mammalian p53 functionality and herpesviral VP22 functionality. Other aspects
of the invention will be apparent from the description and claims.
BACKGROUND OF THE INVENTION, AND PRIOR ART
Relevant to the present application is the inventors' own earlier international patent application WO 97/05265 (O'Hare and Elliott) (published after the priority date claimed for this application), which relates to VP22 protein and its properties
and uses. Similarly the inventors' paper (Elliott and O'Hare (1997), in Cell, vol 88 pp 223-233 (1997), relates to intercellular trafficking and protein delivery by a herpesvirus structural protein. Both these documents are hereby incorporated in their
entirety by reference and made an integral part of this disclosure,
The inventors have shown that the HSV-1 virion protein VP22 possesses an unusual intercellular trafficking mechanism, an effect particularly described in specification WO 97/05265. VP22 is a 38 kDa protein which in primary-expressing transfected
mammalian cells is located predominantly in the cytoplasm where it associates with cellular microtubules (see accompanying drawing, FIG. 1b). However a remarkable property of VP22 is its ability to spread throughout a monolayer of non-expressing cells.
VP22 is transported from the cytoplasm of an expressing cell into neighbouring cells where it accumulates in the nucleus (FIG. 1b). The mechanism of this transport is still incompletely understood, but has been shown to be via a golgi-independent
pathway and may utilise the actin cytoskeleton. HIV-1 Tat (Ensoli et al., 1993, Fawell et. al., 1994) and a small number of other non-viral proteins (Jackson et al., 1992) have been attributed with intercellular trafficking properties, but none appears
to demonstrate this phenomenon as strikingly as VP22. A further important property of VP22 is that when applied exogenously to the medium of an untransfected cell monolayer, it can be taken up by those untransfected cells where it accumulates in the
cell nucleus.
The prior art generally includes a variety of antigens, immunomodulating proteins, proteins that are conditionally cytotoxic or lethal upon administration (to a cell containing them) of a corresponding drug or activator compound, proteins for
cell cycle control, and other therapeutic and diagnostic proteins, especially in the forms of protein and polynucleotide sequences enabling genetic manipulation by standard techniques. References to some examples of these materials are given below.
For example, among cell cycle control proteins, protein p53 is known as a tumour suppressor. p53 is a 53 kDa nuclear phosphoproprotein (FIG. 1c). Wild-type and mutant p53 proteins have been expressed by means of recombinant vaccinia viruses,
(Ronen et al., Nucleic Acids Research, 20:3435-3441, 1992). p53 functions to regulate cell cycle progression and under conditions of DNA damage through a complex signal transduction mechanism can induce cell cycle arrest or apoptosis (Levine 1997).
Failure to synthesize p53, or more commonly synthesis of a mutated form of the protein can result in uncontrolled cell proliferation and tumour formation. It has been shown by several groups that exogenous addition of functional wild type p53 can
promote cell cycle arrest and/or apoptosis resulting in tumour regression with examples including cervical carcinomas (Hamada et al., 1996) and breast cancer xenografts (Nielsen et al., 1997). A number of p53 delivery systems have been utilised in vivo
and in vitro such as intravenous injection of a p53:liposome complex (Kumar et al., 1997), direct transfection (Zheng et al., 1996) and adenoviral mediated transfer (Hamada et al., 1996, Sandig et al., 1997) but delivery of functional protein into a
sufficiently high percentage of surviving cells remains a difficulty.
Also known from U.S. Pat. No. 5,484,710 (La Jolla: J. C. Reed et al) are regulatory elements linked to genes involved in cell death, as regulated by p53 tumour suppressor protein, and further proteins and their analogues for cell cycle control.
It remains desirable to provide particular further cell-delivery constructs for useful proteins.
SUMMARY AND DESCRIPTION OF THE INVENTION
According to an aspect of the present invention, there are provided coupled proteins comprising transport protein sequences comprising sequences from herpesviral VP22 or from homologues or fragments thereof, together with sequences from other
proteins selected from: (a) proteins for cell cycle control; (b) proteins that are conditionally cytotoxic or lethal upon administration (to a cell containing them) of a corresponding drug, pro-drug or activator compound (otherwise described herein as
suicide proteins); (c) antigenic sequences or antigenic proteins (e.g. of greater than 12 aminoacid residues in length) from microbial and viral antigens and tumour antigens; (d) immunomodulating proteins; and (e) therapeutic proteins. Examples of these
kinds of proteins mentioned below. Thus, coupling or fusion to an aminoacid sequence with the transport function of VP22 protein can provide a useful cell delivery construct for proteins of the kinds mentioned. (Where the context admits, `coupling
products` and similar expressions include reference to fusion proteins.)
Preferably the coupled proteins are fusion proteins, which can conveniently be expressed in known suitable host cells. Corresponding polynucleotide sequences can be prepared and manipulated using elements of per-se known and standard recombinant
DNA technique and readily available adaptations thereof. However, chemically-coupled products can for certain applications be used if desired, and can be prepared from the individual protein components according to any of a variety of per-se known
chemical coupling techniques.
VP22 or a functional sub-sequence thereof, optionally with an additional polypeptide tail for coupling, can be linked to other proteins or nucleic acid by chemical coupling in any known suitable standard manner.
Also provided by the invention are polynucleotides encoding the fusion proteins as described herein, including sequences corresponding to VP22 and another protein of one of the kinds mentioned above, and expression cassettes, plasmids, vectors
and recombinant cells comprising the polynucleotides. These can be formed and used in ways analogous to or readily adaptable from standard recombinant DNA technique. Thus, corresponding polynucleotides can encode a fusion polypeptide that comprises a
sequence with the transport function of herpesviral VP22 protein and a sequence with one of the functions specified herein. The polynucleotide can be comprised in an open reading frame operably linked to a suitable promoter sequence, and can according
to examples of the invention form part of an expression vector, e.g. comprising the polynucleotide carried in a plasmid. The expression vector can be for example a recombinant virus vector or a non-viral transfection vector. The vectors can for example
be analogues or examples of those vectors mentioned or described in WO97/05265, or of those mentioned or described in WO 92/05263, WO 94121807, or WO 96/26267. For nucleotide sequence that are capable of being transcribed and translated to produce a
functional polypeptide, degeneracy of the genetic code results in a number of nucleotide sequences that encode the same polypeptide. The invention includes all such sequences.
Thus products described herein can be used according to the invention as transportable proteins capable of being taken up by a target population of cells, e.g. so that an effector function corresponding to the polypeptide sequence coupled to the
VP22, from among the kinds mentioned above, can take place within the target cells that have taken up the product. Thus, for example, the target cells may present desired tumour antigen epitopes in a case where the polypeptide sequence is from a chosen
tumour antigen, or become subject to cell cycle control effects where the the polypeptide sequence is from a cell cycle control protein, or become in some degree susceptible to cell killing or injury after additional treatment with a prodrug where the
polypeptide sequence is from a corresponding `suicide protein`. In use, many of the products described herein can be expressed as fusion proteins in a first part of the target population of cells, exported therefrom, and taken up by a second part of the
target population of cells not directly producing the protein. Also within the invention are mammalian and microbial host cells comprising such vectors or other polynucleotides encoding the fusion proteins, and their production and use.
A fusion polypeptide as described herein can be transported to a target population of cells, by introducing a polynucleotide or other vector encoding the fusion polypeptide into a first part of the target population of cells, e.g. by transfection
or microinjection; expressing the encoding polynucleotide to produce the fusion polypeptide, thereby to cause it to be exported from said first part of said target population, and to cause it to be taken up by a second part of the target population of
cells not directly producing the fusion polypeptide.
Coupling products (including chemically coupled products) can also be transported into a target population of cells by directly exposing the cells to a preparation of the coupling products, thereby to cause the target cells to take them up.
In this specification, `VP22` denotes: protein VP22 of HSV, e.g. of HSV1, and transport-active fragments and homologues thereof, including transport-active homologues from other herpesviruses including varicella zoster virus VZV, equine
herpesvirus EHV and bovine herpesvirus BHV; modified and mutant proteins and fusion polypeptides and coupling products having homology therewith and a transport function corresponding to a transport function of VF22 of HSV1; and in context also relates
to nucleic acid sequences encoding any of the above whether in the form of naked DNA or RNA or of a vector, or of larger nucleic acid sequences including such sequences as sub-sequences.
Among sub-sequences of herpesviral VP22 protein with transport activity we have found that for example transport activity is present in polypeptides corresponding to aminoacids 60-301 and 159-301 of the full HSV1 VP22 sequence (1-301). For the
sequence, see e.g. FIG. 4 in WO 97/05265. A polypeptide consisting of aa 175-301 of the VP22 sequence has markedly less transport activity, and is less preferred in connection with the present invention. Accordingly, the present invention relates in
one aspect to coupled and fusion proteins comprising a sub-sequence of VP22 containing a sequence starting preferably from about aa 159 (or earlier, towards the N-terminal, in the native VP22 sequence), to about aa301, and having (relative to the full
VP22 sequence) at least one deletion of at least part of the VP22 sequence which can extend for example from the N-terminal to the cited starting point, e.g. a deletion of all or part of the sequence of about aa 1-158. (Less preferably, such a deletion
can extend further in the C-terminal direction, e.g. to about aa 175.) For example, partial sequences in the range from about aa 60-301 to about aa 159-301 are provided.
VP22 sequences as contemplated herein extend to homologous proteins and fragments based on sequences of VP22 protein homologues from other herpesviruses, e.g. the invention provides corresponding derivatives and uses of the known VP22-homologue
sequences from VZV (e.g. all or homologous parts of the sequence from aa 1-302), from MDV (e.g. all or homologous parts of the sequence from aa 1-249) and from BHV (e.g. all or homologous parts of the sequence from aa 1-258). The sequences of the
corresponding proteins from HSV2, VZV, SHV and MDV are available in public protein/nucleic acid sequence databases. Thus, for example, within the EMBL/Genbank database, a VP22 sequence from HSV2 is available as gene item UL49 under accession no. Z86099
containing the complete genome of HSV2 strain HG52; the complete genome of VZV including the homologous gene/protein is available under accession numbers X04370, M14891. M16612; the corresponding protein sequence from BHV is available as `bovine
herpesvirus 1 virion tegument protein` under accession number U21137; and the corresponding sequence from MDV is available as gene item UL49 under accession number L10283 for `gallid herpesvirus type 1 homologous sequence genes`. In these proteins,
especially those from HSV2 and VZV, corresponding deletions can be made, e.g. of sequences homologous to aa 1-159 of VP22 from HSV1. These cited sequences are hereby incorporated herein by reference. Homologies between them are readily accessible by
the use of standard algorithms and software, for example those mentioned in WO 95112673, page 9.
Furthermore, chimeric VP22 proteins and protein sequences are also useful within the context of the present invention, e.g. a protein sequence from VP22 of HSV1 for part of which a homologous sequence from the corresponding VP22 homologue of
another herpesvirus has been substituted. For example, into the sequence of polypeptide 159-301 from VP22 of HSV1, C-terminal sequences can be substituted from VP22 of HSV2 or from the VP22 homologue of BHV.
It has been found that deletion of the 34-aminoacid C-terminal sequence from VP22 of HSV1 abolishes transport-activity, thus this sequence region contains essential elements for transport activity. According to a further aspect of the invention,
there are provided coupled and fusion polypeptides comprising. the 34-aminoacid C-terminal sequence from VP22, or a variant thereof, together with a sequence from another protein selected from: (a) proteins for cell cycle control; (b) proteins that are
conditionally cytotoxic or lethal upon administration (to a cell containing them) of a corresponding drug or activator compound; (c) entigenic sequences or antigenic proteins (e.g. of greater than 12 aminoacid residues in length) from microbial and viral
antigens and tumour antigens; (d) immunomodulating proteins; and (e) therapeutic proteins. These are provided for example for use by administration in the form of protein to cells that will take them up. Coupled products of modified terminal fragments
having at least one mutation insertion or deletion relative to the C-terminal 34 aminoacid sequence of HSV1 VP22 are also provided.
It has also been found that sequences necessary for transport activity contain one or a plurality of aminoacid sequence motifs or their homologues from the C-terminal sequence of VP22 of HSV1 or other herpesviruses, which can be selected from
RSASR SEQ ID NO:1, RTASR SEQ ID NO:3, RSRAR SEQ ID NO:5, RTRAR SEQ ID NO:7, ATATR SEQ ID NO:9, and wherein the third or fourth residue A can be duplicated, e.g. as in RSAASR SEQ ID NO:2 and SEQ ID NOS:4, 6, 8, 10. Corresponding fusion polypeptides with
proteins of the kinds mentioned herein are also provided.
In addition to their uses as indicated elsewhere herein, the coupled and fusion polypeptides can also be used to raise antibodies which can be used in diagnostic and monitoring specific binding assays in per-se known manner, e.g for monitoring
the intracellular localization of the coupled or fusion proteins themselves or their components.
(`VP22` herein is not intended to include natural unmodified VP22 protein or corresponding gene in its natural and unmodified association with herpes virus in its various natural lifecycle stages, e.g. in association with herpesvirus which has
not been subjected to genomic alteration. However, `VP22` does for example refer to the corresponding protein or gene of a virus which has for example been altered in respect of its UL49/VP22 gene or function, or which has had inserted into its genome
an additional and/or hybrid VP22 gene.)
The coupling products or fusion proteins based on VP22 can have a range of molecular sizes. The products can in practice be for example up to about 70 kDa or more, e.g. 90 kDa or 100 kDa or more in respect of the size of the protein to be
coupled or fused to VP22. The embodiments of the invention include examples where the fusion peptide is e.g. at least about 13 residues long, or more than about 12 aminoacid residues long, e.g. other than a 12-residue antigenic epitope peptide. The
proteins to be fused can sometimes also be more than about 27 or 32 kDa. e.g. they can be other than 27 kDa in size. For example, one of the proteins that can be thus coupled, p53. itself has a size of about 53 kDa. The coupled polypeptide or fusion
protein, including the VP22 component can have a size up to about 120 kDa, e.g. up to about 80 kDa or 100 kDa.
It is sometimes preferred that the VP22 sequence is fused at its N-terrninus to the sequence of the chosen other protein of one of the kinds mentioned herein. C-terminal fusions can sometimes be correspondingly less preferred.
In the polypeptides of the invention, mutations of the constituent aminoacid sequences (including those of the immunomodulatory and other proteins mentioned herein) can be incorporated in the fusion polypeptides and other coupled proteins.
Included here are proteins having mutated sequences such that they remain homologous, e.g. in sequence, function, and antigenic character or other function, with a protein having the corresponding parent sequence. Such mutations can preferably for
example be mutations involving conservative aminoacid changes. e.g. changes between aminoacids of broadly similar molecular properties. For example, interchanges within the aliphatic group alanine, valine, leucine and isoleucine can be considered as
conservative. Sometimes substitution of glycine for one of these can also be considered conservative. Interchanges within the aliphatic group aspartate and glutamate can also be considered as conservative. Interchanges within the amide group
asparagine and glutamine can also be considered as conservative. Interchanges within the hydroxy group serine and threonine can also be considered as conservative. Interchanges within the aromatic group phenylaalanine, tyrosine and tryptophan can also
be considered as conservative. Interchanges within the basic group lysine, arginine and histidine can also be considered conservative. Interchanges within the sulphur-containing group methionine and cysteine can also be considered conservative.
Sometimes substitution within the group methionine and leucine can also be considered conservative. Preferred conservative substitution groups are aspartate-glutamate; asparagine-glutamine; valine-leucine-isoleucine; alanine-valine; phenylalanine-
tyrosine; and lysine-arginine. In other respects, mutated sequences can comprise insertion and/or deletions. The mutated protein sequences can additionally or alternatively be encoded by polynucleotides that hybridize under stringent conditions with
the appropriate strand of the naturally-occurring polynucteotide encoding the parent protein, and can be tested for positive results in known functional tests relevant to the parent protein. (`Stringent conditions` are sequence dependent and will be
different in different circumstances. Generally, stringent conditions can be selected to be about 5 deg C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined
ionic strength and Ph) at which 50% of the target sequence hybridizes to a perfectly matched probe. Typically, stringent conditions will be those in which the salt concentration is at least about 0.02 molar at pH 7 and the temperature is at least about
60 deg C. As other factors may affect the stringency of hybridization, including, among others, base composition and size of the complementary strands, the presence of organic solvents and the extent of base mismatching, the combination of parameters is
more important than the absolute measure of any one.)
Coupling with cell cycle control proteins:
In one useful class of embodiments of the invention, VP22 can be coupled with per-se known cell cycle control proteins. Thus, in an example of the invention concerned with cell cycle control, as particularly described in an example below, VP22
can be coupled with p53 protein. A purpose and use here can be to block cell cycle progression, especially in malignant cells.
VP22 can also usefully be coupled with cyclin-dependent kinase inhibitors, e.g. p16, p21 or p27. Normal cell cycle progression requires these proteins; absence of these can derepress the cell cycle, and corresponding coupling products can be
used for treatment of cancer cells.
VFP22 coupling products can also usefully be used in the modulation of apoptosis, e.g. to induce cell death, of the apoptosis type, by the introduction into a cell of a protein apoptotic domain coupled to VP22, such as e.g. apoptosis protein bax,
or its known identified apoptotis inducing peptide; or known related protein bad or bak. Here too the coupling product can be applied in the form either of protein or DNA encoding it. VP22 coupling products can be used in the form of VP22 with known
proteins of the bcl2 family, such as bcl2 itself, bcl-xL, or bclw, to mask or inhibit apoptosis where this is desired, e.g. in treatment of neurodegeneration.
Other VP22 coupling products can be used to promote apoptosis, comprising VP22 linked with known ICE-like proteases. VP22 linkage products with inhibitors of ICE-like proteases, eg pseudosubstrates, can be used to mask or overcome the
apoptosis-stimulating effects the proteases themselves.
Thus, according to an embodiment of the invention there is provided a fusion polypeptide comprising an aminoacid sequence with the transport function of herpesviral VP22 protein and a sequence with the cell cycle control functionality of p53
protein. The fusion polypeptide can include for example substantially the full length p53 sequence or substantially the full length VP22 sequence, or both.
Fusion with VP22 can thus be used for delivery of an agent for cell cycle control such as p53. (Where the description given herein refers to p53 and related peptides, it will be understood that, where the context admits, alternative cell cycle
control agents, such as for example those p53 analogues and other cell cycle control proteins mentioned and referred to herein, are also contemplated, as are, more generally, alternative fusion or coupling partners for VP22, of any of the other types
mentioned herein.) Once expressed in a subpopulation of expressing cells, such a fusion protein can be transported by the VP22 transport mechanism from the expressing cell into a significant proportion of surrounding cells, and the foreign attached
polypeptide can then exert its functionality.
Also provided by this aspect of the invention are corresponding polynucleotides, encoding a fusion polypeptide that comprises a sequence with the transport function of herpesviral VP22 protein and a sequence with the human/mammalian cell
cycle-regulating function of p53 protein. The polynucleotide can be comprised in an open reading frame operably linked to a suitable promoter sequence.
The polynucleotide can according to examples of the invention form part of an expression vector, e.g. comprising the polynucleotide carried in a plasmid. The expression vector can be for example a virus vector or a non-viral transfection vector. The vectors can for example be analogues or examples of those described and referred to in WO 97/05265 or Elliott and O'Hare (1997).
Also provided by the invention are methods of inhibiting cell division, which comprise exposing cells that have insufficient active/free p53 to arrest their cell cycle, to contact with a fusion polypeptide as described herein.
Among the methods of the invention is a method of inhibiting tumour cell division, which comprises exposing a tumour cell present in a tumour cell mass, the tumour cell comprising insufficient active/free p53 to arrest its cell cycle, to contact
with a vector as described herein, thereby causing the cell to express a fusion polypeptide as described herein and to expose other cells of the tumour cell mass to contact with the fusion polypeptide.
We have shown (see description below) that VP22-p53 can be transported to many untransfected cells in a monolayer. The fusion protein can be functional in cell cycle arrest and/or induction of apoptosis, for example both in primary expressing
cells and in cells which have received VP22 via cell-to-cell spread. For example, the fusion protein can be applied to a p53 negative osteosarcoma cell line SAOS-2 (Diller et al., 1990). Functional p53 expressed in these cells causes cell cycle arrest
at the G.sub.1 -S boundary and ultimately cell death, this can be assayed using confocal microscopy and antibodies against specific cell cycle markers. Function of the p53 fusion protein can also be used and assessed in other tumorigenic cell fines
where p53 is present but contains specific and well characterized point mutations leading to non-functionality.
A number of vector systems such as retroviral or adenoviral infection or the injection of protein-liposome complexes can be readily adapted to form examples of this invention for the administration of cell-cycle control proteins to cells and
tissues of human and non-human animal subjects to be treated. For example, in relation to work on p53 protein alone, these have clearly demonstrated that addition of wild type p53 protein can curtail cancerous cell growth in vivo. A number of
therapeutic applications of non-invasive delivery of VP22 coupling products with celleycle control proteins will be apparent to the skilled reader.
For example, naked DNA for a VP22-protein fusion with a tumour effector protein such as p53 can be injected into a tumour, e.g. a solid tumour, e.g. a solid tumor selected by molecular diagnostics for lack of functional p53.
Recombinant viruses can be used as mentioned, encoding and able to express VP22-p53 and equivalently-functioning fusion proteins. For example an adenovirus can express VP22-p53 and can be made dependent on a tumour-specific promoter to drive an
essential viral gene such as E1a. More generally, a recombinant virus vector carrying such a fusion can be defective, non-replicating or replication-restricted so that replication is dependent on conditions prevailing in the target tissue or cell but
not in normal or non-target cells.
In certain examples of the invention, the protein having p53 functionality can for example comprise variants or mutants of p53, for example those variants as described in specification WO 97/04092 (Rhone Poulenc Rorer SA: Bracco L., Conseiller
E.) ("New p53 variants e.g. with oligomerisation domain replaced by leucine zipper--useful for treating hyper-proliferative disorders, especially cancer and restenosis"), which describes inter alia the following variant proteins: (a) variants of protein
p53 having at least part of the oligomerisation domain deleted and replaced by a leucine zipper domain; (b) variants of p53 preferentially active in transformed cells, where all or part of at least one functional domain has been deleted and replaced by a
heterologous domain preferentially active in such cells; (c) variants of p53 with a deletion in the C-terminal part, from residue 366, followed by a 19 amino acid sequence (encoded by a 76 bp fragment reproduced in the specification) representing the
last part of the alternatively spliced part of nurine p53; and (d) chimeric protein containing a transactivating domain, a DNA-binding domain, a nuclear localisation domain and an oligomerisation domain, in which DNA-binding domain and the nuclear
localisation domain comprise amino acids 75-325 6r 75-336 of human wild-type p53.
In further examples of the invention, vectors and fusion proteins can encode or comprise variant p53 polypeptides comprising chimaeric p53 sequences including heterologous tetramerisation domains, which can be adapted from those described in
specifications WO 96/16989 and U.S. Pat. No. 5,573.925 (Wistar Institute of Anatomy & Biology: Halazonetis TD) and used in corresponding ways. In such examples of the invention, the p53 sequences can comprise chimaeric p53 protein having a native p53
sequence and a heterologous tetramerisation domain that forms homotetramers such that the resulting chimaeric protein cannot hetero-oligomerise with wild-type or tumour derived mutant p53 and does not interfere with the native p53 tumour suppressing
functionality.
Fusion proteins and vectors according to further examples of the present invention can be used for treatment of hyperproliferative disease, especially cancer and autoimmune disease, e.g. restenosis, and particularly for treatment of cells having
a p53 mutation and which also express protein MDM2 at high level, including for example HPV-related cancer cells. They may also be used to kill hyperproliferating cells in vitro. Such variants can involve active and stable tumour suppressors and
apoptosis-inducing agents and are proposed to be active where the wild type protein is not, i.e. not inactivated by dominant negative or oncogenic mutants, nor by other cellular proteins (because the leucine zipper domain prevents formation of inactive
mixed oligomers).
Fusion proteins and vectors can also be used, according to further examples of the present invention, in medicaments for suppressing neoplastic phenotype of cancer cells lacking wild-type p53 protein, in ways e.g. corresponding to the use of
wild-type p35 gene as described in specification EP 0 710 722 (Univ Califomia: Chen P., Lee W.), which describes genes and retroviral vectors for the purposes inter alia of suppressing neoplastic phenotype in cancer cells such as osteosarcoma cells, lung
carcinoma cells, colon carcinoma cells, lymphoma cells, leukemia cells, soft tissue sarcoma cells or breast, bladder or prostate carcinoma cells.
Fusion proteins and vectors can also be used according to further examples of the present invention. e.g. in ways corresponding to those described in specification WO 95/12660 (Univ Texas System: Roth J. A. et al), which describes recombinant
adenovirus which carries an adenovirus vector construct comprising an expression region encoding p53. and which is capable of expressing the p53 in for example human malignant cells, and which can be used inter alia for regional delivery of tumour
suppressor gene p53, to diseased cells, either to restore p53 function to p53 deficient cells, or to suppress tumour growth in cells having abnormal p53. and thus to treat human malignancies such as breast and lung cancer. Such adenovirus may also be
used for in vitro analyses and mutagenesis studies of various p53 genes.
Fusion proteins and vectors can also be used, according to further examples of the present invention as inhibitors of hepatitis B virus (HBV) replication, in ways corresponding to those described in U.S. Pat. No. 5,635,473 and WO 96/11017
(Mogam Biotechnology Research Institute: H. S. Lee et al).
Screening assays for identifying agents that effectively increase the level of cell death, and which can act as p53 analogues and can induce apoptosis in cells, are described for example in U.S. Pat. No. 5,484,710 (La Jolla: J. C. Reed et al),
particularly in example IV thereof. Also contemplated as alternative embodiments of the invention are fusion proteins and related materials incorporating VP22 functionality and Bax protein functionality. In relation to Bax protein, reference is made to
U.S. Pat. No. 5,484,710 and references cited therein, incorporated herein by reference.
Coupling with `suicide protein`:
In a further class of embodiments of the invention, VP22 or a functional sub-sequence thereof can be usefully coupled or fused with for example a `suicide protein` such as for example the known thymidine kinase, nitroreductase, or other enzyme or
functional fragment thereof known as applicable for a similar purpose. The coupling product can penetrate into cells which are to be treated with (in the case of thymidine kinase) ganciclovir or another drug (prodrug) of the same family, so that the
prodrug is converted in the cells containing the `suicide gene` product to an active form to kill the cells.
Suitable examples of useful known suicide genes and corresponding pro-drugs are given and referred to for example in WO 94/13824 (Univ Curie Paris, M. Caruso et al), in WO 95/05835 (Baylor College: S. Chen et al), and in WO 93/08288 (Cancer
Research Campaign Technology: G. Anzelark et al), and WO 93/01281 (US DHHS: R. M. Blaese et al), and include, besides thymidine kinase (suicide gene) and ganciclovir/acyclovir (prodrug), nitroreductase (suicide gene) and CB1954 (prodrug), and cytosine
deaminase (suicide gene) and 5-fluorocytosine (prodrug). These and other suicide proteins and corresponding (pro)drugs are also reviewed and their uses mentioned in `Genetic Prodrug Activation Therapy`, A. Rigg and K. Sikora, Molecular Medicine Today,
Aug 1997, pp 359-366.
Where the VP22-TK fusion is presented in the form of DNA in any of the ways described in WO 97/05265 or Elliott and O'Hare (1997). a target cell can be transfected with the gene encoding this fusion, and the expressed fusion can then be
translocated out of the cell in which it was expressed and into surrounding cells--producing a killing effect on such cells when treated with ganciclovir etc, an effect which is different from, and can be additional to known bystander effects.
Alternatively, as with other embodiments, such a VP22-TK fusion can be applied directly as protein.
Coupling with antigens:
In further embodiments, the invention concerns for example transport proteins related to VP22 or its active fragments fused in fusion polypeptides or otherwise coupled with antigenic sequences or proteins (e.g. of greater than 12 aminoacid
residues in length) selected for example from any of the antigenic materials or other proteins and peptides mentioned below.
In addition to the fusion polypeptides and coupling products, the invention provides coupling hybrids comprising VP22 coupled to a DNA that can for example comprise suitable known regulatory elements so that it can be transcribed and translated,
and containing an open reading frame encoding any of the proteins mentioned below.
Coupling with antigens- VP22 can usefully be coupled with examples of microbial and viral antigens and of tumour antigens such as those mentioned below.
Treatment with coupling products of VP22 involving antigens of pathogens as provided hereby can evoke useful immune response against corresponding pathogens. Examples of such antigens are papilloma virus proteins L1 and L2. HIV proteins, gag,
pot, env and nef, chlamydia antigens (such as the chiamydia Major Outer Membrane Protein MOMP) and Chlamydia heat shock proteins.
VP22 can also usefully be coupled with antigens from mycobacteria such as antigen from Mycobacteriurn tuberculosis.
Alternatively the antigen can be a tumour associated antigen, whereby the anti- tumour activity of the CTLs associated with tumour cell depletion is enhanced. It has been found that specific cytokines such as tumour necrosis factor-.alpha.,
interferon gamma, interleukin-2, interieukin-4 and interleukin-7 are particularly useful in this regard. Tumour associated antigens and their role in the immunobiology of certain cancers is discussed for example by P. van der Bruggen et al., Current
Opinion in Immunology, 4(5) (1992) 608-612. Particular examples of such antigens which are envisaged for use in the context of the present application are E6 and E7 antigens of human papillomavirus (especially for example of types 6, 11, 16, 18, etc);
Epstein-Barr virus-derived proteins, e.g. those identified in references 24 and 25 in P. van der Bruggen et al., cited above: antigens of the MAGE series as identified in T. Boon. Adv Cancer | | |
