Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Chemical modification or the reaction product thereof – e.g.,...
Reexamination Certificate
1999-10-06
2002-04-30
Kunz, Gary L. (Department: 1647)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Chemical modification or the reaction product thereof, e.g.,...
C435S235100
Reexamination Certificate
active
06380364
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
This invention relates to a chimeric biotin-binding papillomavirus protein.
BACKGROUND OF THE INVENTION
Papillomavirus are causative agents for several types of epithelial and mucosal diseases. Of particular concern is that certain strains of papillomavirus are associated with genital cancers, cancers of the head and neck, and also rectal cancers (see, e.g., Iwasawa et al.,
J. Urol
., 149, 59-63 (1993); Koutsky et al.,
N. Engl. J. Med
., 327, 1272-78 (1992)). Considerable efforts, therefore, are underway to prevent the spread of this virus by developing a prophylactic vaccine and novel treatments for papillomavirus-induced lesions (see, e.g., Cason et al.,
Vaccine
, 11, 603-11 (1993); Crawford,
Cancer Suev
., 16, 215-29 (1993), Schiller et al., in
Papillomavirus
-
Like Particles: Basic and Applied Studies
(Lacey, C., ed., 101-12 (Leeds Medical Information, Leeds, U.K., 1996)).
Papillomaviruses are nonenveloped double-stranded DNA viruses about 55 nm in diameter with an approximately 8-kb genome in a nucleohistone core (Baker et al.,
Biophys J
. 60, 1445-56 (1991)). The capsids include two viral proteins (L1 and L2) of about 55 kDa and 75 kDa, respectively (Larson et al.,
J. Virol
., 61, 3596-3601 (1987)). L1 is the major capsid protein, and it is arranged in 72 pentameres within the capsid. In fact, L1 has the ability to self-assemble into virus-like particles (VLPs) upon production of the L1 protein in eukaryotic cells (see, e.g., Hagensee et al.,
J. Virol
., 67, 315-22 (1993); Kirnbauer et al.,
J. Virol
., 67, 6929-36 (1993)). The function and position of L2 within the virion are not clear, although the protein is assembled with L1 into VLPs when coexpressed in cells. The ratio of L1 to L2 within VLPs is about 30:1 (Kirnbauer et al.,
J. Virol
., 67, 6929-36 (1993)).
VLPs typically are used for in vitro studies of papillomavirus infection, as opposed to intact papillomavirus (see, e.g., Roden et al.,
J. Virol
., 68, 7260-66 (1994); Volpers et al.,
J. Virol
., 69, 3258-64 (1995)), because of the lack of a suitable in vitro culture system. While it has been suggested that VLPs may prove useful as vectors for targeting drugs, nucleic acids, or other substances to cells subject to papillomavirus infection, they have not proven useful as a vector system. Recently, it has been discovered that chimeric fusion proteins consisting of the amino-terminal portion of the L1 or L2 protein fused to a portion of a non-structural papillomavirus protein are able to assemble into capsomeres and VLPs. VLPs including such chimeric molecules have been demonstrated to elicit an immune response from cells (see, e.g., published international application WO 96/11274; Müller et al.,
Virology
, 234, 93-111 (1997); Greenstone et al.,
Proc. Nat. Acad. Sci
. (
USA
), 95, 1800-05 (1998)). While it is thus possible to use such chimeric capsomeres or VLPs for delivering the non-L1 or L2 portion of such fusion proteins to cells (e.g., as vaccines), the method is of limited utility. For example, such a method does not permit the delivery of nucleic acids or other non-proteinaceous species to cells. Moreover, such a method requires the engineering of a novel chimeric capsid protein for each desired application. In view of the foregoing problems, there exists a need for a vector system able to deliver a wide variety of substances to cells subject to papillomavirus infection.
BRIEF SUMMARY OF THE INVENTION
The present invention provides a chimeric protein including a first domain which includes at least a portion of a papillomavirus L1 or L2 protein and a second domain which includes a biotin-binding polypeptide. The invention also provides papillomaviruses, capsomeres, and VLPs including such chimeric proteins and a method for delivering biotinylated substances to cells using such reagents. These and other advantages of the present invention, as well as additional inventive features, will be apparent from the following detailed description.
DETAILED DESCRIPTION OF THE INVENTION
The first domain of the chimeric protein includes at least a portion of a papillomavirus L1 or L2 protein. The sequence of the L1 and L2 proteins of many papillomaviruses is known, and the chimeric protein can include all or a portion of the L1 or L2 protein from any papillomavirus strain. The second domain includes a biotin-binding polypeptide, and it can be derived from any protein known to bind biotin, many of which are known in the art (see, e.g., Green et al.,
Meth. Enzymol
., 184, 51-67 (1990); Howard et al.,
Gene
, 35, 321-31 (1985); Li et al.,
J. Biol. Chem
., 267, 855-63 (1992); Saggio et al.,
Biochem. J
., 293, 613-16 (1993); Hiller et al.,
Biochem. J
., 278, 573-85 (1991)). Examples of proteins from which the biotin-binding domain of the inventive chimeric protein, thus, include avidin, streptavidin, biotin operon repressor and biotin holoenzyme synthase, biotin carboxylase, biotin-binding phage, and the like.
Each domain of the chimeric protein contributes its respective function to the chimeric protein of the present invention. Thus, an L1 domain is able to interact as a native L1 protein (i.e., with either native L1 proteins from the same strain as the chimeric protein is derived or with other chimeric proteins) to form papillomaviruses, capsomeres, or VLPs. The L1 protein can be further modified (e.g., by deleting the residue corresponding to Cys 428 of the wild-type HPV 16 protein) to render it more able to form capsomeres than VLPs. Such modification is preferable in some applications to ensure that the biotin-binding domain is exposed, rather than hidden in the internal region of a VLP. Depending on how the protein is produced, however, an L1 domain can represent wild-type L1 sequence to permit the resulting chimeric protein to assemble into whole papillomaviruses or VLPs. Moreover, an L1 domain includes sequences able to bind receptors present on target cell surfaces. To provide these functions, where the chimeric protein includes an L1 domain, typically it includes at least the amino-terminal portion of the L1 protein. Similarly, where the chimeric protein includes an L2 domain, the L2 domain permits the chimeric protein to be incorporated into VLPs (see, e.g., WO 96/11274; Greenstone et al., supra). Similarly, the domain derived from a biotin-binding protein function as a ligand for biotin, thereby conferring biotin-binding ability to the chimeric protein of the present invention. Importantly, the biotin-binding domain should not be so large so as to inhibit the capsomere-, virus-, or VLP-forming ability of the L1- or L2-derived domain. Thus, where the chimeric protein includes a L1 domain, preferably, the biotin-binding domain is smaller than about 50 amino acids (e.g., smaller than about 30 amino acids), such as about 20 amino acids or smaller. Where the chimeric protein includes an L2 domain, the biotin-binding domain can be (but need not be) somewhat larger, for example, smaller than about 150 amino acids (e.g., smaller than about 100 amino acids), such as about 75 amino acids or smaller.
Chimeric proteins of the present invention can be synthesized using standard direct peptide synthesizing techniques (see, e.g., Bodanszky,
Principles of Peptide Synthesis
(Springer-Verlag, Heidelberg: 1984)), such as via solid-phase synthesis (see, e.g., Merrifield,
J. Am. Chem. Soc
., 85, 2149-54 (1963); Barany et al.,
Int. J. Peptide Protein Res
., 30, 705-739 (1987); and U.S. Pat. No. 5,424,398). Alternatively, such modified proteins can be chemically crosslinked, and a variety of cross-linking agents are known in the art and widely available (e.g., succinimidyl or maleimidyl cross-linkers). Methods for conjugating peptides and polyamines are also well-known in the art (see, e.g., Staros,
Biochem
., 21, 3990 (1982)). Alternatively, a DNA fragment encoding the chimeric protein can be subcloned into an appropriate vector using well known molecular genetic techniques. The fragment then is transcribed and the peptide subsequently translated in vitro within a host cell. Any
Kast W. Martin
Mueller Martin
Nieland John D.
Velders Markwin P.
Kunz Gary L.
Landsman Robert S.
Leydig Voit & Mayer Ltd
Loyola University of Chicago
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