Drug – bio-affecting and body treating compositions – Immunoglobulin – antiserum – antibody – or antibody fragment,... – Structurally-modified antibody – immunoglobulin – or fragment...
Reexamination Certificate
2000-02-04
2003-10-21
Park, Hankyel T. (Department: 1648)
Drug, bio-affecting and body treating compositions
Immunoglobulin, antiserum, antibody, or antibody fragment,...
Structurally-modified antibody, immunoglobulin, or fragment...
C424S130100, C424S133100, C424S134100, C424S178100, C530S300000, C530S350000, C530S387100
Reexamination Certificate
active
06635248
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to active transfer of haptens, proteins, peptides, nucleic acids and other molecules into cells. More particularly, the present invention relates to novel polypeptides which can effectively penetrate into cells, in particular eukaryotic cells, and transport thereto a substance of interest which is capable of constituting novel antiviral compositions. This invention is of major importance as it has application in a variety of fields, in particular that of gene therapy and vaccines.
2. Description of the Background
Gene therapy remains dependent on a considerable number of parameters, among them the development of vectors which are capable of transferring active principles endowed with predetermined specific properties to the cytoplasm of cells of the host organism under consideration in the absence of genetic alterations associated with the use of such vectors, and with no degradation of the biological activity of the transferred active principles. Current knowledge is that in spite of the effort achieved in developing vectors of viral or non viral origins, not all of these conditions have been satisfactorily fulfilled.
Further, the possibility of transporting substances efficiently into cells is also important for all biotechnological applications. Thus transferring substances into cells in vitro or ex vivo can be used either to produce proteins or peptides, or to regulate gene expression, or to analyse the properties of a given substance in that cell. In vivo, the transfer of a substance to a cell can also act to create models for studying diseases in animals or for studying the effect of a given compound on an organism.
The present invention thus aims to provide a novel type of vector which is both effective and is more innocuous than viral vectors in current use.
International patent application WO 97/02840 describes the use of antibodies or their F(ab′)2 and Fab′ fragments which can penetrate into the interior of living cells, as immunovectors for intracytoplasmic and intranuclear transfer of biologically active substances. While such vectors are highly effective, their use can produce problems in some applications. The use of antibodies or F(ab′)2 antibody fragments involves the production of high titers of these molecules with qualities which are compatible with therapeutic use. Further, the use of molecules with the size and complexity of antibodies can constitute a further disadvantage, in particular as regards use. U.S. Pat. No. 5,635,383 illustrates a further type of complex vector based on polylysine for transferring nucleic acids into cells.
The present application relates to novel polypeptides with advantageous properties both for transferring of substances into cells and as antiviral agents. The primary structure of these polypeptides is much simpler than antibodies and they are of reduced size. Further, preparation is easy and their potential applications are highly varied.
More particularly, the present invention stems from the discovery by the inventors that it is possible to identify, from whole antibodies, limited regions carrying a cellular penetration activity. The invention also stems from the discovery that it is possible to isolate, from whole antibodies, in particular from a single chain of these antibodies, peptides or polypeptides endowed with cell penetration activity. The present invention constitutes the first demonstration that a fragment of a single chain of an antibody can effectively penetrate into cells. The present invention also constitutes the first demonstration that such a fragment is also capable, advantageously, of transporting a substance of interest into said cell, and can preferably have an antiviral activity.
SUMMARY OF THE INVENTION
The present invention thus provides novel molecules which are particularly adapted to transfer biologically active substances into eukaryotic cells, particularly mammalian cells.
In a first aspect, the invention provides a polypeptide characterized in that:
it is constituted by a unique or repeated peptide motif; and
it comprises an amino acid sequence endowing it with the capacity to penetrate into cells and, if necessary, to transport thereto a substance of interest.
In this regard, the invention concerns a polypeptide characterized in that:
it is constituted by a unique or repeated peptide motif; and
it comprises an amino acid sequence constitute by one or more different antibody fragment(s); and
it is capable of penetrating into cells.
In one implementation of the invention, the polypeptides thus comprise one or more fragment(s) of an antibody which may or may not be different. In their simplest form, antibodies (molecules from the immunoglobulin superfamily) are constituted by four chains which are associated together (for example IgG) two heavy chains H, and two light chains L (FIG.
1
). These four chains are associated together post-synthesis to form a molecule with a molecular weight of about 150,000 kD. The antigenic specificity of antibodies is provided by variable domains involving a number of regions of a heavy chain and a number of regions of a light chain (FIG.
1
).
Polypeptides can also be constituted by sequences originating from other immunoglobulin representatives such as IgM.
Each heavy chain of an antibody is composed of about 450 amino acids, and comprises different domains termed the constant domain (C), variable domain (V) and joining domains (D and J). Particular motifs are found in the variable domains, termed CDR (Complementarity Determining Region) which can readily be localised by sequence alignment (C. Janeway and P. Travers, 1996, Immunobiology, Academic Press, “The Structure of a Typical Antibody Molecule”). For an analysis of the sequences of the variable regions, reference should also be made to the article by T. T. Wu and E. Kabat (J. Exp. Med., 1970, Vol. 132, p. 211-250). CDR motifs themselves comprise hypervariable regions.
The present application stems from the demonstration that it is possible to obtain regions which are limited in size and of simple structure with particularly advantageous properties from the antibody structure. Thus, starting from a molecule which is complex (four associated chains) and large (150000 kD), the Applicant has succeeded in constructing polypeptides with a single chain, with the capability of penetrating into cells and of transporting thereto substances of interest. The properties of the polypeptides of the invention are all the more remarkable since their sequences corresponding to those of one or more fragments of only one of the chains of an antibody and thus in order to be active, there is no need for constant regions originating from a heavy chain and a light chain. Polypeptides of the invention obtained by chemical synthesis have the same properties.
The term “polypeptide” as used in the present invention defines a molecule comprising a concatenation of amino acids, with a size in the range 3 to 100 amino acids, for example less than 60 amino acids. Still more preferably, it is a molecule comprising a concatenation of 3 to 60 amino acids, advantageously 3 to 30. Particularly preferred polypeptides advantageously comprise more than about 10 amino acids. The polypeptide of the invention can also comprise certain structural modifications, of a chemical or enzymatic nature for example. Thus the polypeptide of the invention can comprise certain functional groups which, by chemical or enzymatic reaction, can couple with another substance. The polypeptides of the invention can also be chemically modified in order to render them more resistant to proteases or less visible to the immune system. The polypeptides of the invention can be obtained by any method which is known to the skilled person, in particular by chemical synthesis, for example using peptide synthesisers, or by fragmentation or deletion from larger polypeptides, natural or otherwise. They can also be prepared using recombinant DNA techniques, by expression of a corresp
Avrameas Alexandre
Avrameas Stratis
Blondel Bruno
Buttin Gerard
Couderc Therese
Brown Stacy S.
Institut Pasteur
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
Park Hankyel T.
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