Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Method of regulating cell metabolism or physiology
Reexamination Certificate
1999-10-15
2002-08-27
Schwartzman, Robert A. (Department: 1636)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Method of regulating cell metabolism or physiology
C435S325000
Reexamination Certificate
active
06440736
ABSTRACT:
The present invention relates to pharmaceutical compositions, methods, kits, cells and particles containing membranes derived from cells. More specifically, the invention relates to a novel method of altering the properties of cells and of particles with membranes derived from cells.
In 1993 Laukanen et al. have described a method that facilitates the addition of a hydrophobic membrane anchor to single chain variable fragment (scFv) antibody fragments expressed in
E. coli
(Laukanen et al., Protein Engineering 6:449-454,1993). This method relies on the fusion of the signal peptide and 9 N-terminal residues of the bacterial lipoprotein (lpp) to a scFv antibody fragment. It was previously shown by Ghrayeb et al (1984) that only the signal peptide and nine amino acids of the mature lpp are required for the correct processing and transport of an lpp fusion protein to the outer membrane of
E. coli
. Laukanen (Laukanen et al., Biochemistry 33: 11G64-11670,1994) and de Kruif (de Kruif et al., FEBS letters 399:232-236, 1996) demonstrated that the lipid-modified antibody fragment can be expressed in
E. coli
and that the lipid-modified scFv is inserted into he periplasmic leaflet of the outer membrane and not on the outside of tee bacterium. After preparation of membrane extracts from the
E. coli
cells and purification of the lipid-modified scFv, it was shown that these molecules retain their binding specificity and can be functionally reconstituted into artificial vesicles composed of a lipid bilayer.
Although possible, the loading of artificial vesicles requires a lengthy biochemical procedure which is not very efficient and requires large amounts of lipid-modified proteinaceous molecules for effective incorporation of said molecules into the artificial lipid bilayer. Moreover, the process is not very controllable as to the orientation of said molecules in the lipid bilayer. In addition, it was found that the resulting vesicles shed the lipid-modified proteinaceous molecules quite rapidly in vivo. These disadvantages are not easily bypassed by optimising the procedure since the underlying reasons for these phenomena are unknown. The limitations of the procedure severely inhibit the practical applications for artificial lipid bilayers loaded with lipid-modified proteinaceous molecules.
The most common approach to change the properties of cells is the introduction of DNA or RNA into the cells. Subsequent expression of the introduced nucleic acid leads to the presence of novel proteins in intracellular compartments or on the plasma membrane of the cells. DNA is introduced into cells by one of a variety of methods, where upon it is stably integrated in the genomic DNA or remains in the cells as an extra-chromosomal fragment. This strategy is widely used to study the function of the molecule encoded by the introduced DNA or to endow the recipient cell with properties encoded by the introduced DNA.
Cell therapy is a strategy for the treatment of many diseases. The aim of cell therapy is to replace or repair damaged tissue or organs or to enhance the biological function of cells. ?or cell therapy, cells are isolated from a tissue or organ and manipulated ex vivo, for example by adding growth factors to cells in tissue culture with the aim to increase their number (Gage et al, Nature 392 (supplement):18-24, 1998). It is also common practise in cell therapy to introduce DNA into cells and, by using selectable markers such as antibiotic resistance, select cells that stably or transiently express the introduced DNA. The aim of this approach is to endow the recipient cell with novel properties. For example, DNA encoding a growth factor receptor may be introduced ex vivo into bone marrow cells and cells that express the growth factor receptor may be re-infused into a recipient organism. Treatment of the organism with the growth factor then results in the in vivo expansion of manipulated bone marrow cells expressing the growth factor receptor (Jin et al, Proc. Natl. Acad. Sci. USA 95:8092-8097,1998). In another application, T lymphocytes may be retrovirally transduced with DNA encoding a fusion protein consisting of a scFv antibody specific for folate-binding protein joined to the Fc-receptor gamma chain. After selection and expansion of T cells expressing the fusion protein and re-infusion, it was shown in an animal model that the T lymphocytes reacted against tumour cells (Hwu et al., Cancer Res. 55:3369-3373, 1995). The method of introducing DNA into cells with the aim to alter their properties is time-consuming, requires the introduction of foreign genetic material into human cells and is constrained by the inefficiency of DNA transfer into some cell types, especially freshly-isolated cells.
The present invention discloses a novel use of lipid-modified proteinaceous molecules. In the present invention lipid-modified proteinaceous molecules are contacted with cells and/or with particles containing membranes derived from cells. Upon contact the lipid-modified proteinaceous molecules are inserted into the membrane of a cell and/or of a particle with a membrane derived from a cell. This novel use not only enables a completely novel approach to manipulate cells and/or particles containing membranes derived from cells. But in addition, this process is very efficient, requiring relatively low amounts of lipid-modified proteinaceous molecules for effective incorporation by a cell and or a particle derived from a cell. Moreover, the lipid-modified proteinaceous molecules added to said cell and/or said particle are surprisingly stable in vivo. The proteinaceous molecules to be taken up by said cells and/or said particles are produced as lipid-modified molecules that spontaneously attach to membranes without using biochemical coupling procedures. This procedure can be used to endow the recipient cells with molecules that act as signaling molecules or otherwise affect the properties of the cells, for example in their capacity to recognise a target or to home to a particular tissue or organ. The present invention provides a rapid and effective alternative for methods that rely on introduction and expression of nucleic acids into cells, resulting in the expression of plasma membrane-associated proteinaceous molecules. Since the present invention is independent of gene transfer and rapid the invention possesses discrete advantages over the conventional methods based on gene transfer. Although many gene transfer methods have been devised some cell types have remained refractory to efficient gene transfer. The present invention is independent of gene transfer efficiency and thus provides a completely different and versatile method for the insertion of proteinaceous molecules in the membranes of cells and/or of particles with membranes derived from cells. Moreover, the process of adding the proteinaceous molecules is fast, thus obviating the obligatory incubation period for obtaining maximal protein expression following gene transfer.
One of the most prominent applications of the present invention lies in the field of cell therapy as a strategy for the treatment of human diseases.
The present invention provides a novel method to alter the properties of cells and/or of particles containing membranes derived from cells. The present invention does not rely on the transfer of a nucleic acid but instead directly supplies the desired protein to the membrane enabling a rapid alteration of the properties of the cell and/or the particle with a membrane derived from a cell. The present invention offers advant ages over conventional nucleic acid transfer in that it is fast and suitable for a wide variety of cell types including but not limited to primary cells isolated from a human.
SUMMARY OF THE INVENTION
One aspect of the invention is to provide a method for efficiently attaching proteinaceous molecules to the membranes of cells and or of particles containing membranes derived from cells. The method is rapid, leads to the incorporation of high numbers of molecules in the outer membrane, requires minimal han
De Kruif Cornelis Adriaan
Logtenberg Tom
Morrison & Foerster / LLP
Schwartzman Robert A.
U-BiSys B.V.
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