U.S. patent application number 09/800433 was filed with the patent office on 2002-08-08 for materials and methods for intracellular transport and their uses.
Invention is credited to Elliott, Gillian Daphne, O'Hare, Peter Francis Joseph.
Application Number | 20020106378 09/800433 |
Document ID | / |
Family ID | 26310846 |
Filed Date | 2002-08-08 |
United States Patent
Application |
20020106378 |
Kind Code |
A1 |
O'Hare, Peter Francis Joseph ;
et al. |
August 8, 2002 |
Materials and methods for intracellular transport and their
uses
Abstract
Coupled polypeptides and fusion polypeptides for intracellular
transport and their preparation and use, include (i) an aminoacid
sequence with the transport function of herpesviral VP22 protein
(or a homologue, e.g. from VZV, BHV or MDV) and (ii) another
protein sequence selected from (a) proteins for cell cycle control;
(b) suicide proteins; (c) antigenic sequences or antigenic proteins
from microbial and viral antigens and tumour antigens; (d)
immunomodulating proteins; and (e) therapeutic proteins. The
coupled proteins can be used for intracellular delivery of protein
sequences (ii), to exert the corresponding effector function in the
target cell, and the fusion polypeptides can be expressed from
corresponding polynucleotides, vectors and host cells.
Inventors: |
O'Hare, Peter Francis Joseph;
(Oxted, GB) ; Elliott, Gillian Daphne; (Oxted,
GB) |
Correspondence
Address: |
KLARQUIST SPARKMAN CAMPBELL LEIGH & WHINSTON, LLP
One World Trade Center, Suite 1600
121 S.W. Salmon Street
Portland
OR
97204
US
|
Family ID: |
26310846 |
Appl. No.: |
09/800433 |
Filed: |
March 5, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09800433 |
Mar 5, 2001 |
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09395344 |
Sep 13, 1999 |
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6251398 |
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Current U.S.
Class: |
424/186.1 ;
530/350 |
Current CPC
Class: |
Y10S 530/826 20130101;
C12N 2710/16622 20130101; A61P 37/04 20180101; C07K 2319/00
20130101; C07K 14/005 20130101 |
Class at
Publication: |
424/186.1 ;
530/350 |
International
Class: |
A61K 039/12; C07K
014/03; C07K 001/00; C07K 014/00; C07K 017/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 23, 1997 |
GB |
9701363.5 |
Aug 1, 1997 |
GB |
9716398.4 |
Claims
1. Coupled polypeptides and fusion polypeptides comprising (i) an
aminoacid sequence with the transport function of herpesviral VP22
protein and (ii) another protein sequence selected from (a)
proteins for cell cycle control; (b) suicide proteins (proteins
that are conditionally cytotoxic or lethal upon administration, to
a cell containing them, of a corresponding (pro)drug or activator
compound): (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.
2. A polypeptide according to claim 1 where said other protein
sequence is from a mammalian (e.g. human) cell cycle control
protein.
3. A polypeptide according to claim 2 where said other protein
sequence is from a mammalian (e.g. human) protein for increasing or
inducing cell apoptosis or for conferring on 2 cell the ability to
undergo apoptosis.
4. A polypeptide according to claim 2 where said other protein
sequence is from a mammalian (e.g. human) cell cycle control
protein selected from p53 protein, cyclin dependent kinase
inhibitors, and proteins oil the bcl2 and bax families.
5. A polypeptide according to claim 4, which is a fusion
polypeptides and comprises a sequence from a p53 protein.
6. A fusion polypeptide according to claim 5, comprising
substantially a full length VP22 sequence and substantially a full
length p53 sequence.
7. A polypeptide according to claim 1 where said other protein
sequence is from a suicide protein.
8. A polypeptide according to claim 7 where said suicide protein is
selected from thymidine kinase and nitroreductase.
9. A polypeptide according to claim 7, comprising substantially the
full length VP22 sequence.
10. A polypeptide according to claim 1, which is a fusion
polypeptide, and comprises a cleavage-inducing linker sequence
located between the VP22 sequence and said other protein
sequence.
11. A polypeptide according to claim 1, comprising a sub-sequence
of HSV VP22 starting from about aa 159 and extending to about aa
301, and having (relative to the full VP22 sequence) at least one
deletion of at least part of the VP22 sequence extending for
example from the N-terminal to the sequence of about aa 1-158.
12. A polypeptide according to claim 1, which comprises a sequence
corresponding to aminoacids 60-301 or 159-301 of the full HSV VP22
sequence.
Description
FIELD OF THE INVENTION
[0001] 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
[0002] Relevant to the present application is the inventors' own
earlier international patent application WO 97/05255 (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.
[0003] 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 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 teen 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.
[0004] 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.
[0005] For example, among cell cycle control proteins, protein p53
is known as a tumour suppressor. p53 is a 53kDa nuclear
phosphoproprotein (FIG. 1c). Wild-type and mutant p.sup.53 proteins
have been expressed by means of recombinant vaccinia viruses,
(Ronen et al., Nucleic Acids Research, 20:3435-3441, 1592). 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., 1999) 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 at., 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.
[0006] Also known from U.S. Pat. No. 5,434,710 (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 or cell cycle control.
[0007] It remains desirable to provide particular further
cell-delivery constructs for useful proteins.
SUMMARY AND DESCRIPTION OF THE INVENTION
[0008] 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.)
[0009] 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.
[0010] 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.
[0011] 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 par, 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 WO97/05255, or
of those mentioned or described in WO 92/05263, WO 94/21807, 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.
[0012] 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.
[0013] A fusion polypeptide as desired 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.
[0014] 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.
[0015] 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 VP22 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.
[0016] Among sub-sequences of herpesviral VF22 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 97/05275. 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 1-5 (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 1-153.
(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.
[0017] 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 EHV (e.g. all or homologous parts
of the sequence from aa 1-258). The sequences of the corresponding
proteins from HSV2, VZV, BHV 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. Z86059 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 L10233 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 95/12573, page 9.
[0018] 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.
[0019] 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)
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. 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 VF22 are also provided.
[0020] 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,
RTASR, RSRAR, RTRAR, ATATR, and wherein the third or fourth residue
A can be duplicated, e.g. 25 in RSAASR. Corresponding fusion
polypeptides with proteins of the kinds mentioned herein are also
provided.
[0021] 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.
[0022] (`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)
[0023] 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.
[0024] It is sometimes preferred that the VP22 sequence is fused at
its N-termninus to the sequence of the chosen other protein of one
of the kinds mentioned herein. C-terminal fusions can sometimes be
correspondingly less preferred.
[0025] 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 polynucleotide 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 dec 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.)
[0026] Coupling with cell cycle control proteins:
[0027] 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 call 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.
[0028] VP22 can also usefully be coupled with cyclin-dependent
kinnase inhibitors, e.g. p15, 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.
[0029] VP22 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
apoptic domain coupled to VF,22, such as e.g. apoptosis protein
bak, or its known identified apoptosis 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 neuorodegeneration.
[0030] 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.
[0031] 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.
[0032] 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.
[0033] 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.
[0034] 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 97/05255 or Elliott and O'Hare (1997).
[0035] 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.
[0036] 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.
[0037] 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 SAOCS-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 lines where p53 is present
but contains specific and well characterized point mutations
leading to non-functionality.
[0038] 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 cell-cycle
control proteins will be apparent to the skilled reader.
[0039] 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.
[0040] 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.
[0041] 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 S A: 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 murine 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 or 75-335 of human wild-type p53.
[0042] 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 96/15989 and U.S. Pat. No. 5,573,925 (Wistar
Institute of Anatomy & Biology: Halazonetis T D) 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 2 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.
[0043] 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 suppressor 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).
[0044] 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 California:
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. leukaemia cells, soft tissue
sarcoma cells or breast, bladder or prostate carcinoma cells.
[0045] 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 95/12560 (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.
[0046] 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).
[0047] 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.
[0048] Coupling with `suicide protein`:
[0049] 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.
[0050] 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 Anzelare et al), and WO 93/01231 (US DHHS; R
M Blaese et al), and include, besides thymidine kinase (suicide
gene) and ganciclovir/acyclovir (prodrug), nitroreductase (suicide
gene) and CE1954 (prodrug), and cytosine deaminase (suicide gene)
and 5-fluorcytosine (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, August 1997, pp 359-366.
[0051] 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.
[0052] Coupling with antigens:
[0053] 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 (eg. of greater than 12 aminoacid residues in length)
selected for example from any of the antigenic materials or other
proteins and peptides mentioned below.
[0054] 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.
[0055] Coupling with antigens VP22 can usefully be coupled with
examples of microbial and viral antigens and of tumour antigens
such as those mentioned below.
[0056] 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, pol, env and
nef, chlamydia antigens (such as the chlamydia Major Outer Membrane
Protein MOMP) and Chlamydia heat shock proteins.
[0057] VP22 can also usefully be coupled with antigens from
mycobacteria such as antigen from Mycobacterium tuberculosis.
[0058] 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, interleukin-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 Gruggen et al., Current Opinion in
Immunology, 4(5) (1992) 608-612. Particular examples of such
antigens which are envisaged for use in the contest of the present
application are E6 and E7 antigens of human papillomavirus
(especially for example of types 5, 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 Res 58 (1992), pp
177-210 and/or MZ2-E and other antigens as identified in P. van der
Bruggen et al, Science 254 (1991) 1643-1647; melanoma proteins,
e.g. human tyrosinase; and mucins, such as those identified in P.
O. Livingston, in Current Opinion in Immunology 4 (:5) (1992) pp
624-629; e.g. MUC1 as identified in J Burchell et al. Int J Cancer
44 (1959) pp 591-696.
[0059] VP22 can also be usefully coupled with viral proteins such
as glycoprotein antigens, e.g. from herpesviruses, such as gH or gD
or gB of herpes simplex virus; or gp50 of pseudorabies virus, as an
example of an antigen of a veterinary pathogen, in this case a
veterinary virus.
[0060] VP22 thus can be usefully coupled with antigens known from
the prior art of malignant tumour treatment, including studies that
have highlighted the potential for therapeutic vaccination against
tumours using autologous material derived from a patient's own
tumour. The theory behind this approach is that tumour cells may
express one or more proteins or other biological macromolecules
that are distinct from normal healthy cells, and which might
therefore be used to target an immune response to recognise and
destroy the tumour cells.
[0061] These tumour targets may be present ubiquitously in tumours
of a certain type. A good example of this in cervical cancer, where
the great majority of tumours express the human papillomavirus E6
and E7 proteins. In this case the tumour target is not a self
protein, and hence its potential as a unique tumour-specific marker
for cancer immunotherapy is clear.
[0062] There is increasing evidence that certain self proteins can
also be used as tumour target antigens. This is based on the
observation that they are expressed consistently in tumour cells,
but not in normal healthy cells. Examples of these include the MAGE
family of proteins. It is expected that more self proteins useful
as tumour targets remain to be identified.
[0063] Tumour associated antigens and their role in the
immunobiology of certain cancers are discussed for example by P van
der Bruggen et al, in Current Opinion in Immunology, 4(5) (1992)
606-612. Other such antigens, of the MAGE series, are identified in
T. Boon, Adv Cancer Res 511 (1992) pp 177-210, and MZ2-E and other
related tumour antigens are identified in P. van der Bruggen et al,
Science 254 (1991) 1643-1647: tumour-associated mucins are
mentioned in P O Livingston, in Current Opinion in Immunology 4 (5)
(1992). pp 624-629: e.g. MUC1 as mentioned in J Burchell et al, Int
J Cancer 44 (1989) pp 691-696.
[0064] Coupling with immunomodulating proteins:
[0065] Embodiments of the invention of use in immune modulation
include for example the following. VP22 can usefully be coupled
with examples of cytokines or of other immunomodulatory compounds
as mentioned below. Thus, VP22 can also be usefully coupled with
immuno modulating proteins, e.g. those which enhance the immune
response including the cytokine, interleukin 1, interleukin 2 and
granulocyte-macrophage colony stimulating factor (GM-CSF). Such
products can ,for example be used in ways analogous to those
mentioned in for example WO 96/26257 or WO 97/14808, to alter, e.g.
to increase, an immune response specific to a target cell type,
e.g. a tumour cell type, which has been exposed to the product
either in-vitro or in-vivo.
[0066] As used herein, the expression "immunomodulatory protein"
and related terms includes a protein or proteins which either
enhance or suppress a host immune response to a mutant virus or
protein encoded thereby, or to an antigen such as an immunogen from
a pathogen or source exogenous to the virus, or a tumour-associated
antigen. The immunomodulating proteins are not normally those
proteins presently used as immunogens (antigens) in themselves. An
immunomodulatory protein can be a natural member of a human or non
human animal immune system, e.g. of a mammalian immune system, with
a functional binding capacity for another natural constituent of
such an immune system. Alternatively an immunomodulatory protein
can be a protein encoded by a pathogen, which has a functional
binding capacity for a natural constituent of such an immune
system.
[0067] Alternatively an immunomodulatory protein can be an
artificial protein, for example a fragment of a natural
immunomodulatory protein or a mutein of such a protein or fragment,
or a fusion protein incorporating any of these. Many
immunomodulatory proteins, and genetic materials encoding them, and
their nucleotide and aminoacid sequences, are known to the
literature of this subject, and available in genetic sequence
databases such as the EMBL database, and several are commercially
available in the form of engineered genetic material for cloning
and other manipulation.
[0068] Immunomodulating proteins coupled with VP22 as described
herein can usefully for example be of sequences native to the
species which is to receive treatment with these coupling products
or with DNA e.g. in the form of recombinant viruses, e.g. an
immunomodulating protein of human type for treatment of a human
subject.
[0069] Examples of useful known immunomodulating proteins in this
connection include cytokines, chemokines, complement components,
immune system accessory and adhesion molecules and their receptors
of human or non-human animal specificity. Useful examples include
GM-CSF, IL-2, IL-12, lymphotactin, CD40, and CD40L. Further useful
examples include interleukins for example interleukins 1 to 15,
interferons alpha, beta or gamma, tumour necrosis factor,
granulocyte-macrophage colony stimulating factor (GM-CSF),
macrophage colony stimulating factor (M-CSF granulocyte colony
stimulating factor (G-CSF), chemokines such as neutrophil
activating protein (NAP), macrophage chemoattractant and activating
factor (MCAF), RANTES, macrophage inflammatory peptides MIP-1 and
MIP-1b, complement components and their receptors, or an accessory
molecule such as B7.1, B7.2, ICAM-1, 2 or 3 and cytokine
receptors.
[0070] OX40 and OX40-ligand (OX40L) (gp34) (see e.g. WO 95/12573,
WO 95/21251 and WO 21915) are further useful examples of
immunomodulatory proteins. Immunomodulatory proteins can for
various purposes be of human or non-human animal specificity and
can be represented for present purposes, as the case may be and as
may be convenient, by extracellular domains and other fragments
with the binding activity of the naturally occurring proteins, and
muteins thereof, and their fusion proteins with other polypeptide
sequences, e.g. with immunoglobulin heavy chain constant domains.
Where nucleotide sequences encoding more than one immunomodulating
protein are inserted, they can for example comprise more than one
cytokine or a combination of cytokine(s) and accessory/adhesion
molecule(s).
[0071] Immune response evoked by the use of such VP22 coupling
products or by vectors encoding them can include immune responses
of a variety of types, e.g. a response against a virally-encoded
protein, and/or a response against a host antigen, being a response
stimulated by a viral vector or by the expression of a heterologous
gene encoded thereby, e.g. the coupling product with VP22. Among
the uses of the mutant virus vectors as described herein is e.g. to
protect a subject of a susceptible species against infection by a
corresponding wild-type virus when the subject is treated
therewith, e.g. infected therewith, e.g. by direct
immunisation.
[0072] An immunomodulatory protein to be coupled with VP22 can be
itself a hybrid or fusion protein which comprises a polypeptide
region having homology to and functionality of an immunomodulatory
protein, linked to a polypeptide region having another homology and
optionally another functionality. For example, the immunomodulatory
protein can be comprise, or correspond in functionality to the gp34
protein identified as a binding partner to human Ox-40 (see W
Godfrey et al, J Exp Med 180(2) 1994 pp 757-702, and references
cited therein, including S Miura et al, Mol Cell Biol 11(3) 1991,
pp 113-132). The version of this protein functionality that can be
encoded in the mutant viral genome can correspond to the natural
gp34 sequence itself, or to a fragment therefore to a hybrid
expression product e.g. based on the (C terminal) extra cellular
(binding) domain of gp34 fused to another protein, e.g. to the
constant region of an immunoglobulin heavy chain such as human
IgG1, e g. with the extracellular domain of gp34 (a type 2 membrane
protein) fused at its N-terminal to the C-terminal of the
immunoglobulin constant domain.
[0073] Others of the immunomodulatory proteins can also be carried
and expressed in such derivative and hybrid forms, including
mutated forms as mentioned herein.
[0074] In certain examples the immunomodulating protein can
comprise a cytokine, preferably granulocyte macrophage colony
stimulating factor (GM-CSF), e.g. murine or preferably human
GM-CSF.
[0075] Murine and human GM-CSFs are both known: the murine GM-CSF
gene encodes a polypeptide of 141 amino acids, the mature secreted
glycoprotein having a molecular weight of between 14 k-30 k daltons
depending on the degree of glycosylation. GM-CSF generically is a
member of the haematopoietic growth factor family and was first
defined and identified by its ability to stimulate in vitro colony
formation in haematopoietic progenitors. GM-CSF is a patent
activator of neutrophils, eosinophils and macrophage-monocyte
function, enhancing migration, phagocytosis, major
histocompatibility complex (MHC) expression, and initiating a
cascade of bioactive molecules which further stimulate the immune
system. GM- CSF is currently being clinically evaluated for
treatment of neutropenia following chemotherapy and as an adjuvant
in cancer therapy.
[0076] The heterologous nucleotide sequence employed can comprise a
heterologous gene, gene fragment or combination of genes provided
it encodes an immunomodulation protein as defined above.
[0077] According to examples of the invention, combinations of two
or more immunomodulatory proteins can be used for the purposes
described herein, in particular examples, given for illustration
only and not limitation, combinations involving IL2, GMCSF,
lymphotactin and/or CD40L can be used with each other or with
others of the immunomodulatory proteins cited above Each of the
other binary combinations of the immunomodulatory proteins
mentioned above are also given by, and within the scope of, this
disclosure.
[0078] Other coupling products:
[0079] In certain embodiments the invention can be useful in gene
therapy applications: thus, for example, VP22 can also be usefully
coupled with examples of genes used or proposed to be used in-gene
therapy including: the gene for human adenosine deaminase (ADA), as
mentioned in for example WO 92/10504 (K W Culver et al: US
Secretary for Commerce & Celico Inc), WO 89/12109 & EP 0
420 911 (I H Pastan et al): the cystic fibrosis gene and variants
described in WO 91/02796 (L-C Tsui et al: HSC Research &
University of Michigan), in WO 92/05273 (F S Collins & J M
Wilson: University of Michigan) and in WO 94/12649 (R J Gregory et
al: Genzyme Corp).
[0080] VP22 can also usefully be coupled with known transcriptional
regulatory proteins such as NF-AT, which becomes activated by
translocation to the nucleus and induces transcription of
interleukin e.g. of IL1. The coupling with VP22 can be used here to
avoid retention of the coupled product in the cytoplasm.
[0081] The invention also includes coupled and fusion proteins in
which a linker sequence is provided that enables the fusion protein
to be split intracellularly to enable separation of the antigenic
part, such as that mentioned above, from the transport protein
part. A cleavage-inducing sequence can comprise for example the
aminoacid sub-sequence RVCSNPPCETHETGTTNTATATSN or other cleavage
sequences indicated for example in A C Wilson et al, in Genes and
Development 9 (1995) 2445-2453.
[0082] Also provided by the present invention are processes for
treating cells with coupling products as described herein, so as to
produce immunogenic, immunomodulatory, cytotoxic/lethal or
therapeutic effects.
[0083] Examples of materials and processes as described herein are
useful in the modulation of cellular activity, e g. with the aim
and effect of producing or altering immune responses, for example
for the prophylaxis or therapy of disease, e.g. the production of
immune responses against pathogens or tumours.
[0084] Other uses for certain of the materials and processes hereof
include the regulation of gene expression in calls. e.g. for
purposes of corrective gene therapy and/or for reducing or
controlling tumour cell growth and activity. Cell treatments
according to the invention can be in-vitro, ex-vivo or in-vivo.
[0085] Among the derivatives of VP22 that can be used according to
aspects of the invention as transport active substances and for
coupling with materials to be transported, for the purposes set
forth elsewhere herein, are peptides comprising a transport-active
functional sequence from the C terminal section of VP22.
[0086] Non-limitative examples of treatment methods using the
materials described herein comprise treatment of antigen-presenting
cells or cell preparations containing them with a fusion of VP22
and an antigen, e.g. one of those antigens mentioned above, (or
with a vector, e.g. a viral vector, encoding such a fusion), so as
to procure processing of the antigen and presentation by the MHCl
route so as to procure a CTL response to the antigen. The methods
so provided include priming and expansion of T cells and adoptive
immunotherapy using the materials so obtained, in a manner
otherwise analogous to known priming, expansion and adoptive
immunotherapy methods.
[0087] A number of vector systems such as retroviral or adenoviral
infection or the injection of protein-liposome complexes, as well
as herpesviral vector systems, can be readily adapted to form
examples of this invention. For example, naked DNA for a
VP22-protein fusion with a protein so one of the kinds mentioned
herein can be injected into a tissue to be treated, according to
the nature and purpose of the protein to be delivered. Recombinant
viruses can be used as mentioned, encoding and able to express VP22
fusion proteins. 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.
[0088] Vectors and fusion proteins of examples of the invention can
be useful in gene therapy, and to treat or protect against abnormal
cell proliferation, esp. cancer but also psoriasis, atherosclerosis
and arterial restenosis, and to induce apoptosis of e.g.
proliferating lymphocytes, i.e. to induce tolerance, e.g. to
prevent transplant rejection or for treatment of autoimmune
diseases such as systemic lupus erythematosus or rheumatoid
arthritis.
[0089] In addition to medical therapeutic applications, the effect
shown herein can also be exploited by assays, provided by the
invention, which rely on substrate-enzyme interactions or the
interaction of proteins expressed in different cellular
populations.
[0090] An embodiment of the invention is further described, without
intent to limit the invention, with reference to the accompanying
drawings and to the materials and methods described below.
[0091] In the accompanying drawings,
[0092] FIG. 1 illustrates that:
[0093] Mock-transfected cos-1 cells were labelled by indirect
immunofluorescence with antibodies for VP22 (FIG. 1a), p53 (FIG.
1c) and the CMV epitope (FIG. 1d) to establish the levels of
background label Cells transfected with pc49epB (FIG. 1b) and
labelled for P22 demonstrate a typical VP22 cytoplasmic pattern
with clear spread to the nuclei of adjacent cells. Cells
transfected with the VP22-p53 fusion protein construct p4955ep+10
were labelled for VP22 and p53 (FIGS. 1e and 1f) or VP22 and
epitope (FIGS. 1g and h): the fusion protein can be detected in the
nuclei of cells adjacent to the primary expressing cell.
[0094] FIG. 2 is a plasmid map to illustrate p4953ep+10, encoding a
fusion protein comprising sequences VP22, p53 and an epitope
tag.
[0095] FIG. 3 illustrates that
[0096] Protein extracts from cos-1 cells transfected with a range
of plasmid constructs were analysed by western blot The panel shown
leftmost has been probed with an antibody against VP22 and
demonstrates that pUL49epB and pc49epB plasmids encoding VP22 alone
generate a protein of 38kDa, the VP22-p53 fusion protein expressed
from p4953ep+10 produces a protein of approx. 50 kDa with very
little degradation.
[0097] The panel shown rightmost has been probed with an antibody
against p53 and demonstrates that cells transfected with plasmids
encoding either p53 alone (pcB6+p53) or the p4953ep+10 fusion
protein construct produce p53 protein at 53 kDa. The p4953ep+10
construct also synthesises the VP22-p53 fusion protein at 90 kDa,
the p53 in this sample may be a degradation product or more likely
endogenously induced p53.
MATERIALS AND METHODS
[0098] Cell culture and transfection
[0099] Cos-1 cells were grown in Dulbecco's modified MEM
supplemented with 10% new born calf serum at 37.degree. C. with 5%
CO.sub.2.
[0100] Transfections were performed using the BES/CaCl.sub.2 method
(Elliott and O'Hare, 1997) with 200 ng test plasmid with 1800 ng
pUB19. Transfections were allowed to proceed for 48 h at which
point the monolayers were harvested for immunofluorescence or
western blot analysis.
[0101] Immunofluorescence and antibodies
[0102] Cell monolayers on coverslips were fixed with 100% methanol
for 15 mins at room temperature and labelled as described in
Elliott and O'Hare (1997). All antibodies were diluted in PBS+10%
serum. VP22 was detected using a rabbit polyclonal antibody AGV30
(1:500), p53 was detected using a mouse monoclonal antibody DO-1
(Santa-Cruz Ltd), the CMV epitope was detected using a mouse
monoclonal antibody CMV LNA (Capricorn Ltd). images were obtained
using a Bio-Rad MRC500 confocal microscope.
[0103] Plasmid Constructs
[0104] The VP22-p53 fusion protein construct was generated by
cloning a full length p53 PCR fragment C-terminal to VP22 into a
unique Bam site, keeping both VP22 and the CMV epitope in
frame.
[0105] Western blot analysis
[0106] Western blots were prober with anti-VP22 (1:10,000),
anti-p53.(1:1000).
[0107] We constructed an epitope-tagged full length in-frame
VP22-p53 fusion protein construct (FIG. 2). This vector generates a
fusion protein of approx 90 kDa when expressed in Cos-1 cells, with
very little protein degradation as judged by western blot analysis
(FIG. 3). When tested for delivery by intercellular trafficking,
the fusion protein appears to function exactly as VP22 alone. It is
located in the cytoplasm of primary transfected cells as shown by
immunofluorescence using methanol-fixed Cos-1 cell monolayers
labelled with anti-VP22 (FIGS. 1e and 1g), p53 (FIG. 1f) or
-epitope (FIG. 1h) antibodies and is able to move very efficiently
into the nuclei of neighbouring cells. The relative efficiency of
transport has not been empirically determined but appears only
slightly less than VP22 alone.
[0108] In further experiments, p53-negative osteosarcoma cells were
transfected (using the calcium phosphate technique) with naked DNA
expressibly encoding either (a) wild-type VP22, (b) wild-type p53)
or (c) the VP22-p53 fusion protein described above. The transfected
cells (b) and (c) showed ability to undergo apoptosis, unlike the
control cells (a), indicating that the VP22-p53 fusion protein
retains the functionality of p53.
[0109] In variants of the example given here, VP22 deletion
constructs with decreased fusion protein size can be made if
desired. e.g. to improve rate or extent of transport, and without
loss of protein function.
[0110] In further variants, the order of the components of the
fusion can be varied, e.g. the p53 and VP22 sequences can readily
be included in the order opposite to the order involved in the
plasmid shown in FIG. 2, with satisfactory results.
[0111] The present disclosure extends to modifications and
variations of the description given herein inclusive of the
attached claims that will be apparent to the reader skilled in the
art. The disclosure hereof, incorporating WO 97/05265 and of
Elliott and O'Hare (1997) which are made an integral par hereof, is
intended to extend in particular to classes and subclasses of the
products and generally to combinations and to subcombinations of
the features mentioned, described and referred to in the
disclosure. Documents cited herein, including the references below,
are hereby incorporated in their entirety by reference for all
purposes.
[0112] Additional References
[0113] Diller, L., Kassel, J. Nelson, C. E., Gryka, M. A., Litwak,
G., Gebhardt, M. Bressac, B., Ozturk, M., Baker, S. J., Vogelstein,
B. and S. H. Friend, (1990) p53 functions as a cell cycle control
protein in osteosarcomas. Mol. Cell. Bio. 10:5772-5781.
[0114] Elliott G. and P. O'Hare (1997) Intercellular trafficking
and protein delivery by a herpesvirus structural protein. Cell
82:223-233.
[0115] Ensoli, B.. Buonaguro, L., Barillari, G., Fiorelli, V.,
Gendelman, R., Morgan, R. A., Wingfield, P. and R. C. Gallo, (1993)
Release , uptake and effects of extracellular human
immunodeficiency virus Tat protein on cell groves and viral
transactivation. J. Virol. 67:277-287.
[0116] Fawell, S., Seery, J., Daikh, Y., Moore, C., Chen, L. L.,
Pepinsky, B. and J. Barsoum. (1994) Tat-mediated delivery of
heterologous proteins into cells. Proc. Natl. Acad. Sci.
91:664-668,
[0117] Hamada, K., Alemany, R., Zhang, W-W, Hittelman, W. N.,
Lotan, R., Roth, J. A. and M. F. Mitchell. (1996)
Adenovirus-mediated transfer of a wild-type p53 gene and induction
of apoptosis in cervical cancer. Cancer Research 56:3047-3054.
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Sequence CWU 1
1
11 1 5 PRT Artificial Sequence Description of Artificial Sequence
peptide 1 Arg Ser Ala Ser Arg 1 5 2 6 PRT Artificial Sequence
Description of Artificial Sequence peptide 2 Arg Ser Ala Ala Ser
Arg 1 5 3 5 PRT Artificial Sequence Description of Artificial
Sequence peptide 3 Arg Thr Ala Ser Arg 1 5 4 6 PRT Artificial
Sequence Description of Artificial Sequence peptide 4 Arg Thr Ala
Ala Ser Arg 1 5 5 5 PRT Artificial Sequence Description of
Artificial Sequence peptide 5 Arg Ser Arg Ala Arg 1 5 6 6 PRT
Artificial Sequence Description of Artificial Sequence peptide 6
Arg Ser Arg Ala Ala Arg 1 5 7 5 PRT Artificial Sequence Description
of Artificial Sequence peptide 7 Arg Thr Arg Ala Arg 1 5 8 6 PRT
Artificial Sequence Description of Artificial Sequence peptide 8
Arg Thr Arg Ala Ala Arg 1 5 9 5 PRT Artificial Sequence Description
of Artificial Sequence peptide 9 Ala Thr Ala Thr Arg 1 5 10 6 PRT
Artificial Sequence Description of Artificial Sequence peptide 10
Ala Thr Ala Ala Thr Arg 1 5 11 24 PRT Artificial Sequence
Description of Artificial Sequence linker sequence 11 Arg Val Cys
Ser Asn Pro Pro Cys Glu Thr His Glu Thr Gly Thr Thr 1 5 10 15 Asn
Thr Ala Thr Ala Thr Ser Asn 20
* * * * *