Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Amino acid sequence disclosed in whole or in part; or...
Reexamination Certificate
1999-09-13
2001-06-26
Mosher, Mary E. (Department: 1648)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Amino acid sequence disclosed in whole or in part; or...
C424S192100, C424S204100, C424S208100, C424S248100, C424S263100, C530S350000, C530S826000, C536S023400, C435S235100, C435S325000, C435S317100, C435S252300
Reexamination Certificate
active
06251398
ABSTRACT:
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 38kDa protein which in primary-expressing transfected mammalian cells is located predominantly in the cytoplasm where it associates with cellular microtubules (see accompanying drawing,
FIG. 1
b
). However a remarkable property of VP22 is its ability to spread throughout a monolayer of non-expressing cells. VP22 is transported from the cytosplasm of an expressing cell into neighbouring cells where it accumulates in the nucleus (
FIG. 1
b
). The mechanism of this transport is still incompletely understood, but has been shown to be via a golgi-independent pathway and may utilise the acting 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 exogenuously 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 53kDa nuclear phosphoproprotein (
FIG. 1
c
). 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 adapatable 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 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 inclu
Elliott Gillian Daphne
O'Hare Peter Francis Joseph
Klarquist Sparkman Campbell & Leigh & Whinston, LLP
Marie Curie Cancer Care
Mosher Mary E.
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