Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
1998-04-28
2003-08-19
Crouch, Deborah (Department: 1632)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C536S023100, C530S388100, C530S388210, C530S391100
Reexamination Certificate
active
06608034
ABSTRACT:
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates to the active transfer of haptens, proteins, nucleic acids and other molecules into the nucleus of eucaryotic cells. This invention is of major importance since it can be applied to various fields, especially those of gene therapy and vaccines.
Gene therapy continues to be dependent on a considerable number of parameters among which are the development of vectors capable of transporting through the cytoplasm of these cells of the host organism active principles endowed with predetermined specific properties into the nuclei of cells of the organism in the absence of genetic alterations associated with the use of these vectors, and the non-degradation of the biological activity of the active principles transferred. It is known that so far all these conditions are far from being fulfilled (1).
Indeed, the current methods commonly used to transfer DNA into cells are the following: general, non-selective methods which use the property of DNA to coprecipitate with calcium phosphate or DEAE-dextran, or alternatively the direct introduction of DNA into cells under the effect of an electric field (electroporation). These methods are very toxic for the cells, leading to a high mortality and a high variability according to the cells used. Other methods use targeting of the entry of the gene into cells by receptors present on their membrane. The DNA may then penetrate into the cell via either a ligand specific for these receptors: asialorosomucoid (2), insulin (3) or transferrin (4), or antibodies specific for membrane constituents (5). The DNA/ligand complex penetrates into the cell by a process of endocytosis. The transfection is therefore limited by a substantial destruction of the complex in the lysosomal vesicles, and different methods have been proposed to overcome these disadvantages, especially the blocking of the lysosomal compartment by chloroquine or the simultaneous addition of adenoviruses which escape the endosomal compartment by destroying the membrane of the endocytosis vesicles (6).
The aim of the present invention is to provide a new type of vectors which are both more efficient and safer than the viral vectors whose use has been envisaged until now.
The invention therefore relates to a product of coupling between a biologically active principle and one of these new vectors, hereinafter called “immunovectors”, the said product of coupling being characterized both by the capacity of the immunovector to allow the internalization in eucaryotic cells of biologically active principles linked covalently or non-covalently to these immunovectors, and by their affinity for the DNA of these cells to such a point that the said immunovector is rendered capable of transferring the biologically active principle immediately close to the nuclei of these cells or into the nuclei of these cells.
These immunovectors preferably consist of antibodies or fragments of antibodies capable of recognizing DNA sequences inside these cells, and to which biologically active principles may be covalently or non-covalently linked, these antibodies or fragments of antibodies being, in addition, capable of transporting in vitro and in vivo these biologically active principles through the membranes and the cytoplasm of these cells, and transferring them close to or even into the nucleus of these cells.
It is understood that in the present description the term “biologically active principle” relates to any molecule, macromolecule or group of molecules having biological activity of the type in question.
The invention also relates to a method of transferring especially haptens, proteins and/or nucleic acids into the nucleus of cells, particularly eucaryotic cells, this method being based on the use of the properties of the said immunovectors.
The existence of antibodies capable of penetrating inside the nuclei of human lymphocytes when these cells are incubated in vitro in a culture medium containing a serum obtained from patients suffering from disseminated erythematous lupus (DLE) was reported for the first time by Alarcon-Segovia et al. in 1978 (7). Subsequently, the same team demonstrated that these antibodies are of the IgG isotype and are capable of reacting with ribonucleic acids, free or complexed with proteins (8). Recently, this type of antibody was detected in MRL lpr/lpr lupus mice, but also in NZB mice having a haemolytic auto-immune disease syndrome and even in normal BALB/c mice. Some monoclonal antibodies, prepared from the spleen of these mice, have proved capable of penetrating in vitro into the nucleus of cells maintained in culture (10-13). As in humans, it was noted that these monoclonal antibodies were capable of recognizing nucleic acids. Furthermore, it was shown that these antibodies are also capable, when they are injected into mice, of penetrating into several types of cells, ending up in their nuclei (11).
The invention results from the discovery that this type of antibody or fragments of these antibodies could also be used as vectors, hereinafter “immunovectors” capable of transporting biologically active principles, such as haptens, proteins, and nucleic acids through the membranes and the cytoplasm of the corresponding cells, and ensuring their transfer into the nucleus of the said cells.
These antibodies may be obtained in polyclonal form from a serum, in particular from an animal previously immunized against nucleic acid fragments having the corresponding epitope, or in monoclonal form from hybridomas secreting such antibodies.
Any type of bonding, chemical or otherwise, may be used to ensure the coupling of an immunovector of antibody or antibody fragment type having an affinity for the nucleic acids to the biologically active principle, for example a hapten or a nucleic acid, for the purpose of transporting it through the membranes and the cytoplasm of the cells, and to ensure the transfer of these active principles into the nucleus.
Preferably, a chemical mode of coupling, allowing the formation of covalent or non-covalent bonds, will be used.
Preferred coupling products are those in which the immunovectors are selectable by a cellular penetration test comprising a first incubation of the immunovector of interest in the presence of cells in culture in the nucleus of which the active principle capable of being associated with the immunovector has to be transported, followed, after fixing and permeabilization of these cells, by another incubation with labelled anti-immunovector antibodies, and finally by a detection immediately close to the nucleus or even inside the nucleus of the antigen-antibody type immunologic reaction between the immunovector and the anti-immunovector antibody.
Among the preferred immunovectors of the present invention, there may be mentioned the antibodies having an affinity for a nucleic acid, or a fragment thereof, the latter retaining this affinity.
Antibodies also having the capacity to bind to the cells, in particular lymphoid cells, can also be used. The latter category of immunovectors may also be selected by a test, which may then also comprise the incubation of the immunovectors of interest with lymphoid cells, washing the said lymphoid cells, incubating them with labelled anti-immunovector antibodies, and determining the number of positive cells in each population.
In one embodiment, the lymphoid cells used are auto-immune mouse splenocytes exhibiting a lupus syndrome.
A preferred immunovector for the coupling product is chosen from among monoclonal IgG's, (Fab′)2 or (Fab′) fragments, or any polypeptide corresponding to the site(s) of the antibodies involved in the recognition of the corresponding nucleic acid.
Preferably, this immunovector is an immunoglobulin, more particularly an IgG carrying an anti-DNA activity, and obtained from normal individuals.
In addition, this immunovector may be an IgG carrying an anti-DNA activity and obtained from individuals presenting auto-immune syndromes, more particularly disseminated erythematous lupus syndr
Avrameas Alexandre
Avrameas Stratis
Buttin Gerard
Nato Faridabano
Ternynck Therese
Crouch Deborah
Institut Pasteur
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
Woitach Joseph
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