Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
2000-03-10
2004-01-20
Low, Christopher S. F. (Department: 1653)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C424S183100, C424S178100, C530S300000, C530S350000, C530S370000, C530S391700, C514S002600
Reexamination Certificate
active
06680296
ABSTRACT:
FIELD OF THE INVENTION
The invention discloses a new type-1 ribosome-inactivating protein (RIP), referred to as bouganin, isolated from the leaves of Bougainvillea species, especially
B. spectabilis
Willd. Bouganin differs from other type-1 RIP by its unique amino acid composition. Bouganin has a molecular weight of about 26,200 daltons. Bouganin is useful as an immunomodulator, anti-viral agent or anti-tumour agent. Compositions comprising bouganin and a cell binding ligand are particularly useful to kill cells of a target population.
BACKGROUND OF THE INVENTION
Ribosome-inactivating proteins
It has been known for a long time that extracts from many plant tissues possess anti-viral activity, which in several cases is due to proteins identified as inhibitors of protein synthesis, called ribosome-inactivating proteins (RIP, reviewed by Barbieri et al.,
Biochim. Biophys. Acta
1154:237 (1993)). The pokeweed anti-viral protein (PAP) was the first anti-viral protein to be identified as a RIP (reviewed by Irvin, in
Antiviral Proteins in Higher Plants
65 (1994)). Subsequently, all other RIP tested possess anti-viral activity not only against plant viruses, but also against animal viruses, including HIV (reviewed by Battelli and Stirpe, in
Antiviral Proteins in Higher Plants
(1994)).
All RIP, either single-chain (type-1) or two-chain (type-2), enzymatically release adenine from a single nucleotide in a precise position (A
4324
in the case of rat liver 28S rRNA, A
2660
of
E. coli
rRNA) in a universally conserved GAGA tetraloop of the major rRNA (Endo and Tsurugi,
J. Biol. Chem
. 262:8128 (1987); reviewed by Barbieri et al.,
Biochim. Biophys. Acta
1154:237 (1993)). Depurinated ribosomes become unable to elongate the nascent peptide chain.
The anti-viral activity of these proteins was commonly attributed to the inactivation of ribosomes, with inhibition of protein synthesis of the host cell and consequent arrest of viral replication. However a degradation of supercoiled DNA in the presence of RIP was reported (Li et al.,
Nucleic Acid Res
. 22:6309 (1991); Ling et al.,
FEBS Lett
. 345:143 (1994); Roncuzzi and Gasperi-Campani,
FEBS Let
. 392:16 (1996)). Moreover, at least some RIP release more than one adenine residue from ribosomes (Barbieri et al.,
Biochem. J
. 286:1 (1992)) and act on RNA species other than ribosomal, including viral RNAs, on poly(A), and on DNA (Barbieri et al.,
Nature
372.624 (1994),
Nucleic Acid Res
. 25:518 (1997); Stirpe et al.,
FEBS Lett
. 382:309 (1996)). Thus many, if not all, RIP have polynucleotide:adenosine glycosidase activity, which may have a role in the anti-viral activity besides the inactivation of the host cell ribosomes.
Immunotoxins
Immunotoxins are chimeric molecules in which cell-binding ligands are coupled to toxins or their subunits. The ligand portion of the immunotoxin is usually an antibody that binds to selected target cells. The toxin portion of the immunotoxin can be derived form various sources. Most commonly, toxins are derived from plants or bacteria, but toxins of human origin or synthetic toxins (drugs) have been used as well. Toxins used for immunotoxins derived from plants or bacteria all inhibit protein synthesis of eukaryotic cells. The most widely used plant toxin, ricin, consist of two disulfide-linked polypeptides A and B (Olsnes et al., in
Molecular Action of Toxins and Viruses
51 (1982)). Another group of plant-derived toxins used in immunotoxins are the type-1 RIP. These molecules are single-chain proteins found in plants and have similar enzymatic properties as the A-chain of ricin (reviewed in Stirpe and Barbieri
FEBS Lett
. 195:1 (1986)).
The cross-linker used to join the ligand (antibody) and the toxin must remain stable when extracellular, but labile when intracellular, so that the toxin fragment can enter the cytosol. The choice of cross-linker depends on whether intact toxins, A-chains or type-1 RIP are used. A-chains and type-1 RIP are generally coupled to the ligand using Linkers that introduce a disulfide bond between the ligand and the A-chain (Myers et al.,
J. Immunol. Meth
. 136:221 (1991)). Intact toxins are usually linked to ligands using non-reducible linkages (such as thioether) to prevent release of the active free toxin in vivo. Recombinant immunotoxins have been prepared by splicing the genes encoding the toxin to the gene encoding the ligand (for instance a recombinant antibody fragment) and expressing the entire immunotoxin as a fusion protein (Pastan et al.,
Ann. Rev. Biochem
. 61:331 (1992)). Recombinant immunotoxins are highly stable in vivo because they contain non-reducible peptide bonds.
Various types of immunotoxins directed against different cellular targets have been evaluated in vivo, both in animal models and in phase I or II clinical trials. The results of a number of these studies are reviewed in Ghetie and Vitetta
Curr. Opin. Inmmunol
. 6:707 (1994) and Thrush et al.,
Ann. Rev. Immunol
. 14:49 (1996).
SUMMARY OF THE INVENTION
Ribosome inactivating proteins (RIP) comprise a class of proteins with potent inhibitory activity of eukaryotic protein synthesis. RIP can be classified in two groups. Type-1 RIP consist of a single peptide chain having ribosome inactivating activity, whereas type-2 RIP consist of an A chain with ribosome inactivating activity and a B chain having cell binding activity. Here we describe the isolation of a novel type-1 RIP, referred to as bouganin, with a low non-specific toxicity, making it very suitable for the incorporation as the toxin part in various immnunotoxin molecules. The invention pertains to this novel protein and biologically active peptide parts and equivalents thereof, to immunotoxins based On this protein, to the production of such proteins and immunotoxins, and to their use in the medical and plant-protection fields. The invention is defined in the appending claims.
DETAILED DESCRIPTION OF THE INVENTION
The invention described herein draws on previously published work. By way of example, such work consists of scientific papers, patents and pending patent applications. All of these publications and applications, cited previously or below, are hereby incorporated by reference.
The protein according to the invention corresponds to the bouganin protein as described below in more detail, as well as to biologically active fragments and equivalents thereof. The term “biologically active” means being capable of inhibiting protein synthesis in vitro or in vivo. Such fragments generally comprise one or more active sites of the protein or the encoding polynucleotide and generally comprise a sequence at least 8 amino acids, preferably at least 10, at least 15 or even at least 30 amino acids, of the protein, or the corresponding number of nucleotides of the polynucleotide.
The term “ligand” refers to any molecule capable of binding with or otherwise recognizing a receptor on a target cell. The ligand may be a protein or a non-protein molecule. Examples of such ligands include, but are not limited to, antibodies, growth factors, cytokines, hormones and the like, that specifically bind desired target cells.
As used herein, the term “immunotoxin” refers to chimeric molecules in which a cell binding ligand is coupled to the novel type-1 RIP bouganin or fragments thereof
As used herein, the term “antibody” refers to polyclonal antibodies, monoclonal antibodies, humanized antibodies, single-chain antibodies, and fragments thereof such as. Fab, F(ab′)2, Fv, and other fragments which retain the antigen binding function of the parent antibody.
As used herein, the term “monoclonal antibody” refers to an antibody composition having a homogeneous antibody population. The term is not limited regarding the species or source of the antibody, nor is it intended to be limited by the manner in which it is made. The term encompasses whole immunoglobulins as well as fragments such as Fab, F(ab′)2, Fv, and others which retain the antigen binding function of the antibody. Monoclonal antibodies of any mammalian species can be used in this invent
Bolognesi Andrea
Stirpe Fiorenzo
Handal Anthony H.
Kirkpatrick & Lockhart LLP
Low Christopher S. F.
Schnizer Holly
Tanox Pharma B.V.
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