U.S. patent application number 10/412684 was filed with the patent office on 2005-09-22 for combined therapeutical treatment of hyperproliferative diseases.
Invention is credited to Tocque, Bruno.
Application Number | 20050209177 10/412684 |
Document ID | / |
Family ID | 9475169 |
Filed Date | 2005-09-22 |
United States Patent
Application |
20050209177 |
Kind Code |
A9 |
Tocque, Bruno |
September 22, 2005 |
Combined therapeutical treatment of hyperproliferative diseases
Abstract
A medicinal combination of one or more nucleic acids that at
least partially inhibit oncogenic cell signalling pathways, and a
therapeutic anticancer agent, for use simultaneous, separate or
over a period of time to treat hyperproliferative diseases.
Inventors: |
Tocque, Bruno; (Courbevoie,
FR) |
Correspondence
Address: |
WILEY, REIN & FIELDING, LLP
ATTN: PATENT ADMINISTRATION
1776 K. STREET N.W.
WASHINGTON
DC
20006
US
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Prior
Publication: |
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Document Identifier |
Publication Date |
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US 0127437 A1 |
July 1, 2004 |
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Family ID: |
9475169 |
Appl. No.: |
10/412684 |
Filed: |
April 14, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10412684 |
Apr 14, 2003 |
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09816144 |
Mar 26, 2001 |
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09816144 |
Mar 26, 2001 |
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08875222 |
Jul 17, 1997 |
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6262032 |
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08875222 |
Jul 17, 1997 |
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PCT/FR96/00056 |
Jan 12, 1996 |
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Current U.S.
Class: |
514/44R |
Current CPC
Class: |
A61P 9/10 20180101; A61K
33/243 20190101; A61K 38/191 20130101; A61P 35/00 20180101; A61P
43/00 20180101; A61K 31/70 20130101; A61P 35/02 20180101; A61K
38/191 20130101; A61K 31/70 20130101; A61K 33/24 20130101; A61K
31/70 20130101; A61K 31/70 20130101; A61K 31/70 20130101; A61K
31/70 20130101; A61K 31/70 20130101; A61K 31/70 20130101; A61K
31/475 20130101; A61K 31/70 20130101; A61K 31/47 20130101; A61K
31/70 20130101; A61K 31/335 20130101; A61K 31/70 20130101; A61K
2300/00 20130101; A61K 33/24 20130101; A61K 2300/00 20130101; A61K
38/191 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
514/044 |
International
Class: |
A61K 048/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 17, 1995 |
FR |
FR95/00436 |
Claims
1. Medicinal combination of one or more nucleic acids that at least
partially inhibit the oncogenic cell signalling pathways and an
anticancer therapeutic agent, for use simultaneously, separately or
spread over time for the treatment of hyperproliferative
pathologies.
2. Combination according to claim 1, characterized in that the
nucleic acid is a deoxyribonucleic acid (DNA) or ribonucleic acid
(RNA) coding for a product that at least partially inhibits the
oncogenic cell signalling pathways.
3. Combination according to claim 2, characterized in that the
nucleic acid is a DNA coding for an antisense RNA.
4. Combination according to claim 2, characterized in that the
nucleic acid is a DNA coding for a ligand RNA.
5. Combination according to claim 2, characterized in that the
nucleic acid is a DNA coding for a dominant negative.
6. Combination according to claim 2, characterized in that the
nucleic acid is a DNA coding for an ScFv.
7. Combination according to claim 2, characterized in that the
nucleic acid is a DNA coding for a tumour suppressor protein.
8. Combination according to claim 1, characterized in that the
nucleic acid is an antisense oligonucleotide, where appropriate
chemically modified.
9. Combination according to one of the preceding claims,
characterized in that the nucleic acid is incorporated in a
vector.
10. Combination according to claim 9, characterized in that the
vector is chosen from liposome, nanoparticle, peptide complex,
cationic lipids and lipopolyamines.
11. Combination according to claim 9, characterized in that the
vector is a viral vector derived from retroviruses, adenovirus,
herpesvirus, AAV or vaccinia virus.
12. Combination according to one of the preceding claims,
characterized in that the nucleic acid is administered directly at
the site to be treated.
13. Combination according to claim 1, characterized in that the
anticancer therapeutic agent is a chemotherapeutic agent chosen
from cisplatin, taxoids, etoposide, TNF, adriamycin, camptothecin,
vinca alkaloids and navellein.
14. Combination according to claim 13, characterized in that the
anticancer chemotherapeutic agent is a taxoid.
15. Combination according to claim 14, characterized in that the
anticancer chemotherapeutic agent is chosen from taxol, docetaxel
and paclitaxel.
16. Combination according to one of claims 13 to 15, characterized
in that the anticancer chemotherapeutic agent is administered
parenterally.
17. Combination according to one of the preceding claims,
characterized in that the nucleic acid and the anticancer
chemotherapeutic agent are used simultaneously.
18. Combination according to one of claims 1 to 16, characterized
in that the nucleic acid is administered before the anticancer
chemotherapeutic agent.
19. Medicinal combination of one or more tumour suppressor genes
and a taxoid, for use simultaneously, separately or spread over
time for the treatment of hyperproliferative pathologies.
20. Combination according to claim 19, characterized in that the
suppressor gene codes for the wild-type form of the p53
protein.
21. Combination according to claim 19, characterized in that the
suppressor gene codes for the waf1 protein.
22. Combination between claim 1, characterized in that the
anticancer therapeutic agent is a radiotherapeutic agent.
Description
[0001] The present invention relates to the field of the therapy of
hyperproliferative pathologies. It relates more especially to a new
method of treatment of hyperproliferative pathologies based on the
combined use of two types of therapeutic agents.
[0002] More specifically, the present invention relates to a new
method of treatment of hyperproliferative pathologies based on the
combined use of genes that block oncogenic cell signalling pathways
and chemotherapeutic and or radiotherapeutic agents. The combined
treatments according to the present invention have especially
effective effects for the destruction of hyperproliferating cells,
at relatively low doses. The present invention thus provides an
especially effective new method of treatment of hyperproliferative
pathologies (cancer, restenosis and the like) with limited
side-effects.
[0003] In spite of the very substantial progress made in this
field, the methods currently available for the treatment of cancer
still have limited efficacy. Radiotherapy and chemotherapy
admittedly have a very favourable impact on the development of
cancers. However, an acute problem in the treatment of cancer is
the insensitivity of certain primary tumours and/or the appearance
of tumour cells which are resistant, after a first cycle of
effective treatments, both to radio- and to chemotherapy.
[0004] Numerous studies have attempted to elucidate the molecular
mechanisms which may be the source of these events. Generally
speaking, the investigations have been directed towards the manner
in which chemotherapeutic agents entered the cells and the manner
in which they reacted with their cell targets (Chin et al.,
Adv.Cancer Res. 60 (1993) 157-180; Chabner and Meyers in
Cancer/Principles and practices of Oncology, De Vita et al. Eds.,
J.B. Lippencott Co. pp. 349-395, 1989). For example, high levels of
expression of the mdr1 gene can limit the intracellular
concentration of various chemotherapeutic agents and might
contribute to the expression of the multiple drug resistance (Chin
et al., see above).
[0005] A more complete elucidation of the mechanisms of resistance
to chemotherapy and to radiotherapy involves a better knowledge of
the processes of cell death induced by these agents. Since ionizing
radiation and many anticancer agents induce damage in the DNA, the
effect of these therapeutic agents has been attributed to their
genotoxic power. However, the cell damage caused by these agents
does not enable their therapeutic activity to be explained
completely (Chabner and Meyers, see above). In the last few years,
the exploration and understanding of the mechanisms of programmed
death or apoptosis have enabled the mechanisms by which tumour
cells acquire or lose their sensitivity to cytotoxic agents to be
reconsidered. Numerous toxic stimuli induce apoptosis, even at
doses which are insufficient to induce metabolic dysfunctions. The
capacity to induce an apoptotic response in tumour cells might
determine the efficacy of the treatment.
[0006] The applicant has now developed a new method of treatment
which is especially effective for the destruction of
hyperproliferative cells. As mentioned above, the method of
treatment according to the invention is based essentially on the
combined use of two types of therapeutic agents: genes that block
oncogenic cell signalling pathways and chemotherapeutic and/or
radiotherapeutic agents. The present invention is, in effect, the
outcome of the demonstration of an especially large synergistic
effect associated with the combined use of these two types of
agents.
[0007] A first subject of the present invention hence relates to a
medicinal combination of one or more nucleic acids that at least
partially inhibit oncogenic cell signalling pathways and an
anticancer therapeutic agent, for use simultaneously, separately or
spread over time for the treatment of hyperproliferative
pathologies.
[0008] As mentioned above, the invention is based essentially on
the demonstration of a synergistic effect between the product of
certain genes and anticancer therapeutic agents. This combined use
produces more powerful effects at lower doses of agents. This
invention thus affords an especially advantageous means for the
treatment of hyperproliferative pathologies.
[0009] As mentioned later, depending on the gene and the chemo- or
radiotherapeutic agent which are chosen, the two components of the
combined treatment of the present invention may be used
simultaneously, separately or spread over time. In the case of a
simultaneous use, both agents are incubated with the cells or
administered to the patient simultaneously. According to this
embodiment of the present invention, the two agents may be packaged
separately and then mixed at the time of use before being
administered together. More commonly, they are administered
simultaneously but separately. In particular, the administation see
of the two agents can be different. In another embodiment, the two
agents are administered spaced over time.
[0010] The nucleic acid used in the context of the present
invention can be a deoxyribonucleic acid (DNA) or a ribonucleic
acid (RNA). Among DNAs, possible alternatives include a
complementary DNA (cDNA), a genomic DNA (gDNA), a hybrid sequence
or a synthetic or semi-synthetic sequence. A further possibility is
a nucleic acid modified chemically, for example, for the purpose of
increasing its resistance to nucleases, its cell penetration or
cell targeting, its therapeutic efficacy, and the like. These
nucleic acids can be of human, animal, plant, bacterial, viral,
synthetic and the like, origin. They may be obtained by any
technique known to a person skilled in the art, and in particular
by screening of libraries, by chemical synthesis or alternatively
by mixed methods including the chemical or enzymatic modification
of sequences obtained by screening of libraries. As mentioned
later, they can, moreover, be incorporated in vectors such as
plasmic, viral or chemical vectors.
[0011] As mentioned above, the nucleic acid according to the
present invention is a nucleic acid capable of at least partially
inhibiting oncogenic cell signalling pathways. These nucleic acids
are designated hereinafter by the term "oncogene intracellular
neutralization elements" or OINE. The signalling pathways leading
to cell transformation are manifold. Cell proliferation involves a
multitude of factors, such as membrane receptors (G proteins),
oncogenes, enzymes (protein kinases, farnesyl transferases,
phospholipases, and the like), nucleosides (ATP, AMP, GDP, GTP, and
the like), activating factors [guanosine exchange factors (GRF,
GAP, RAF, and the like), transcription factors, and the like],
Disturbances, for example in the structure, activity, conformation,
and the like, of these different factors have been associated with
phenomena of deregulation of cell proliferation. Thus, 90% of
adenocarcinomas of the pancreas possess a Ki-ras oncogene mutated
on the twelfth codon (Almoguera et al., Cell 53 (1988) 549).
Similarly, the presence of a mutated ras gene has been demonstrated
in adenocarcinomas of the colon and thyroid cancers (50%), or in
carcinomas of the lung and myeloid leukaemias (30%, Bos, J. L.
Cancer Res. 49 (1989) 4682). Many other oncogenes have now been
identified (myc, fos, jun, ras, myb, erb, and the like), mutated
forms of which appear to be responsible for a disturbance of cell
proliferation. Similarly, mutated forms of p53 are observed in many
cancers, such as, in particular, colorectal cancer, breast cancer,
lung cancer, stomach cancer, cancer of the oesophagus, B-cell
lymphomas, ovarian cancer, bladder cancer, and the like. The
nucleic acids used in the context of the invention are nucleic
acids capable of interfering with one of these factors involved in
cell proliferation, and of at least partially inhibiting its
activity. The factors towards which the nucleic acids of the
invention are preferentially directed are those which appear
preferentially or specifically during disturbances of cell
proliferation (activated oncogenes, mutant of tumour suppressor,
and the like).
[0012] Nucleic acids used in the context of the invention can be of
different types. Preferential possibilities are:
[0013] antisense nucleic acids,
[0014] oligoribonucleotides capable of binding oncogenic target
proteins directly in order to neutralize them (ligand RNA),
[0015] nucleic acids coding for proteins having dominant negative
character, capable of oligomerizing and thereby producing an
inactive complex,
[0016] nucleic acids coding for intracellular antibodies (for
example single-chain variable fragments originating from an
antibody) directed against an oncogenic protein (ScFv).
[0017] tumour suppressor genes.
[0018] According to a first preferred embodiment of the present
invention, the nucleic acid is a DNA or an RNA coding for a
polypeptide or protein that at least partially inhibits oncogenic
cell signalling pathways. More especially, the polypeptide or
protein are chosen from dominant negatives, ScFvs and tumour
suppressors.
[0019] Still more preferably, the dominant negative is a
constituent of the N-terminal region of the GAP protein, of the
Gbr3-3 protein or of the mutants of Ets proteins. As regards ScFv,
this is preferably an ScFv directed against a mutated ras protein
or against the GAP factor. The tumour suppressor protein is
advantageously p53, Rb, waf1, p21, DCC or MTS.
[0020] According to another preferred embodiment of the present
invention, the nucleic acid is a DNA coding for an RNA that at
least partially inhibits oncogenic cell signalling pathways. More
especially, the RNA is an RNA complementary to a target nucleic
acid and capable of blocking its transcription and/or its
translation (antisense RNA); a ribozyme or a ligand RNA. A
preferred example is an anti-Kiras antisense RNA.
[0021] Still according to a preferred embodiment of the present
invention, the nucleic acid is an antisense oligonucleotide, where
appropriate chemically modified. Possible oligonucleotides are ones
whose phosphodiester skeleton has been chemically modified, such
as, for example, the oligonucleotide phosphonates,
phosphotriesters, phosphoramidates and phosphorothioates which are
described, for example, in Patent Application WO94/08003. Other
possibilities are alpha oligonucleotides or oligonucleotides
conjugated to agents such as acrylating compounds.
[0022] In an especially preferred embodiment of the present
invention, the nucleic acid is incorporated in a vector. The vector
used can be of chemical origin (liposome, nanoparticle, peptide
complex, cationic lipids, and the like), viral origin (retrovirus,
adenovirus, herpesvirus, AAV, vaccinia virus, and the like) or
plasmid origin. The nucleic acid used in the present invention may
be formulated with a view to topical, oral, parenteral, intranasal,
intravenous, intramuscular, subcutaneous, intraocular, transdermal,
and the like, administration. Preferably, the nucleic acid is used
in an injectable form. It may hence be mixed with any vehicle which
is pharmaceutically acceptable for an injectable formulation, in
particular for direct injection at the site to be treated. Possible
formulations are, in particular, sterile isotonic solutions, or
dry, in particular lyophilized, compositions which, on adding
physiological saline or sterilized water as appropriate, enable
injectable solutions to be made up. Direct injection of the nucleic
acid into the patient's tumour is advantageous, since it enables
the therapeutic effect to be concentrated in the affected tissues.
The doses of nucleic acid used can be adapted in accordance with
various parameters, and in particular in accordance with the gene,
the vector, the method of administration used, the pathology in
question or the desired treatment period.
[0023] The anticancer therapeutic agent used for carrying out the
present invention can be any agent currently used by a person
skilled in the art in chemotherapy or radiotherapy. It can, in
particular, be cisplatin, taxoid, etoposide, TNF, adriamycin,
camptothecin, a mitotic spindle poison (vinca alkaloids),
navellein, and the like), X-rays, UV, and the like. Especially
advantageous results have been obtained using a taxoid as
chemotherapeutic agent. The anticancer chemotherapeutic agent is
administered by the traditional routes. Generally, it is
administered parenterally.
[0024] As mentioned above, the two agents may be used
simultaneously, separately or spread over time. In an especially
preferred embodiment of the invention, the nucleic acid is
administered first and then, when the nucleic acid can be expressed
by the cells or some cells, the anticancer therapeutic agent is
administered.
[0025] An especially preferred embodiment of the present invention
relates to a medicinal combination of one or more tumour suppressor
genes and a taxoid, for use simultaneously, separately or spread
over time for the treatment of hyperproliferative pathologies.
Still more preferably, the suppressor gene codes for the wild-type
form of the p53 protein or for the waf1 (p21) protein.
[0026] The present invention thus provides a method which is
especially effective for the destruction of hyperproliferative
cells. It may be used in vitro or ex vivo, by incubating the cells
simultaneously or spread over time in the presence of the nucleic
acid or acids and the chemotherapeutic agents. In this connection,
the subject of the invention is also a method of destruction of
hyperproliferative cells, comprising the bringing of the said cells
or of a portion of them into contact with a nucleic acid and a
chemotherapeutic agent as are defined above.
[0027] The present invention is advantageously used in vivo for the
destruction of hyperproliferating (i.e. abnormally proliferating)
cells. It is thus applicable to the destruction of tumour cells or
of smooth muscle cells of the vascular wall (restenosis). It is
most especially suitable for the treatment of cancers in which an
activated oncogene is involved. As an example, there may be
mentioned adenocarcinoma of the colon, thyroid cancer, carcinoma of
the lung, myeloid leukaemias, colorectal cancer, breast cancer,
lung cancer, stomach cancer, cancer of the oesophagus, B-cell
lymphomas, ovarian cancer, bladder cancer, gliobastomas, and the
like.
[0028] Use of Antisense Nucleic Acids
[0029] Regulation of the expression of target genes by means of
antisense nucleic acids constitutes a therapeutic approach
undergoing increasing development. This approach is based on the
capacity of nucleic acids to hybridize specifically with
complementary regions of another nucleic acid, and thereby to
inhibit specifically the expression of particular genes. This
inhibition can take place either at translational level or at
transcriptional level.
[0030] Antisense nucleic acids are nucleic acid sequences capable
of hybridizing selectively with target cell messenger RNAs to
inhibit their translation to protein. These nucleic acids form
locally, with the target mRNA, RNA/mRNA or even DNA/mRNA type
double-stranded regions by classical Watson-Crick type interaction.
Possible examples are small synthetic oligonucleotides
complementary to cellular mRNAs and which are introduced into the
target cells. Such oligonucleotides have, for example, been
described in Patent No. EP 92 574. Other possible sequences are DNA
sequences whose expression in the target cell generates RNAs
complementary to cellular mRNAs. Such sequences have, for example,
been described in Patent No. EP 140 308.
[0031] More recently, a new type of nucleic acid capable of
regulating the expression of target genes has been demonstrated.
These nucleic acids do not hybridize with cellular mRNAs, but
directly with the double-stranded genomic DNA. This new approach is
based on the demonstration that some nucleic acids are capable of
interacting specifically in the major groove of the DNA double
helix to form triple helices locally, leading to an inhibition of
the transcription of target genes. These nucleic acids selectively
recognize the DNA double helix at oligopurine.oligopyrimidine
sequences, that is to say at regions possessing an oligopurine
sequence on one strand and an oligopyrimidine sequence on the
complementary strand, and form a triple helix locally thereat. The
bases of the third strand (the oligonucleotide) form hydrogen bonds
(Hoogsteen or reverse Hoogsteen bonds) with the purines of the
Watson-Crick base pairs. Such nucleic acids have, in particular,
been described by Helene in Anti-Cancer drug design 6 (1991)
569.
[0032] The antisense nucleic acids according to the present
invention can be DNA sequences coding for antisense RNAs or for
ribozymes. The antisense RNAs thereby produced can interact with a
target mRNA or genomic DNA and form double or triple helices
therewith. Other possible sequences are antisense (oligonucleotide)
sequences, where appropriate chemically modified, capable of
interacting directly with the target gene or RNA.
[0033] Preferably, the antisense sequences according to the
invention are directed against activated oncogenes or specific
regions of activated oncogenes, especially the ras oncogene.
[0034] Use of Ligand RNAs
[0035] Ligand RNAs are small oligoribonucleotides which are very
specific and have very high affinity for a given target, in
particular protein target. The preparation and identification of
such ligand RNAs has been described, in particular, in Application
WO91/19813. According to a particular embodiment of the present
invention, it is possible to combine a small RNA specific for the
Ki-ras protein, expressed in the cells by means of a suitable viral
or non-viral vector, with the chemotherapeutic or radiotherapeutic
agents described.
[0036] Dominant Negatives
[0037] A dominant negative is a polypeptide antagonist of an
oncogenic signalling pathway. This antagonism takes place when the
polypeptide becomes positioned in contact with a key element of the
oncogenic signalling and enters into competition with the
polypeptide naturally used in the cell for this signalling. The
polypeptide antagonist used is very frequently a mimic of the
natural polypeptide but which lacks domains that enable the
oncogenic signal to be propagated through it.
[0038] Among dominant negatives preferred for carrying out the
present invention, there may be mentioned the nucleic acids coding
for the NH2-terminal domain of the GAP protein, for the Grb3-3
protein or for mutated forms of the ETS proteins.
[0039] It has been demonstrated in Patent Application WO94/03597
that the overexpression of the NH2-terminal domain of the GAP-Ras
protein could specifically block the tumorigenicity of cells
transformed following the expression of a mutated ras gene. Example
1 of the present application now shows that an overexpression of
the GAP(170-702) domain induces an apoptosis of human cells,
so-called non-small cell carcinoma of the lung (H460). Example 2
shows, furthermore, that the apoptotic effect induced by the GAP
(170-702) construction is very greatly increased by the addition of
products such as cisplatin, camptothecin or taxotere to the culture
medium of human tumour cells, at concentrations of these products
which are without effect on cell viability.
[0040] Example 1 of the present application describes, moreover,
the activity of the grb3-3 gene in H460 cells. The sequence and the
presumed function of Grb3-3 have been described in Science 1994.
Example 2 also shows that the apoptotic effect induced by the
transfer of the Grb3-3 gene is very greatly increased by the
addition of products such as cisplatin, camptothecin or taxotere to
the culture medium of human tumour cells, at concentrations of
these products which are without effect on cell viability.
[0041] These examples demonstrate clearly that different
chemotherapeutic agents can be effectively combined with strategies
for induction of apoptosis by means of gene transfer.
[0042] ScFvs
[0043] ScFvs are intracellularly active molecules having binding
property comparable to that of an antibody. They are, more
especially, molecules consisting of a peptide corresponding to the
binding site of the light chain variable region of an antibody,
linked via a peptide linker to a peptide corresponding to the
binding site of the heavy chain variable region of an antibody. It
has been shown by the applicant that such ScFvs could be produced
in vivo by gene transfer (see Application WO94/29446).
[0044] More especially, this application shows that it is possible
to neutralize oncogenic proteins by expressing ScFvs in different
cell compartments. According to an embodiment of the present
invention, a nucleic acid permitting the intracellular production
of an ScFv which neutralizes the transforming power of the ras
proteins is used in combination with a chemotherapeutic agent. Such
a combination produces substantial synergistic effects (see Example
2).
[0045] Tumour Suppressors
[0046] Among the tumour suppressor genes which can be used in the
context of the present invention, the p53, p21, Rb, rap1A, DDC, WAF
and MTS genes may be mentioned more especially. More especially,
the p53, Rb or Waf genes are used.
[0047] The p53 gene codes for a nuclear protein of 53 kDa. The form
of this gene mutated by deletion and/or mutation is involved in the
development of most human cancers (Baker et al., Science 244 (1989)
217). Its mutated forms are also capable of cooperating with the
ras oncogenes to transform mouse fibroblasts. The wild-type gene
coding for native p53 inhibits, on the other hand, the formation of
foci of transformation in rodent fibroblasts transfected with
various combinations of oncogenes. Recent data emphasize the fact
that the p53 protein could itself be a transcription factor and
could stimulate the expression of other tumour suppressor genes.
Moreover, an effect of p53 on the proliferation of vascular smooth
muscle cells has been demonstrated recently (Epstein et al.,
Science 151 (1994)).
[0048] The Rb gene determines the synthesis of a nuclear
phosphoprotein of approximately 927 amino acids (Friend et al.,
Nature 323 (1986) 643) whose function is to repress cell division
by making the cells enter a quiescent phase. Inactivated forms of
the Rb gene have been implicated in various tumours, and in
particular in retinoblastomas or in mesenchymal cancers such as
osteosarcoma. Reintroduction of this gene into the tumour cells in
which it was inactivated produces a return to the normal state and
a loss of the tumorigenicity (Huang et al., Science 242 (1988)
1563). Recently, it has been demonstrated that the normal Rb
protein, but not its mutated forms, represses the expression of the
c-fos proto-oncogene, a gene essential for cell proliferation.
[0049] The WAF and MTS genes and their antitumour properties have
been described in the literature (Cell 75 (1993) 817; Science 264
(1994) 436).
[0050] Example 3 demonstrates an effective type of combination
between a taxol derivative and the p53 gene. Taxol induces
apoptosis in various tumour cell lines in culture (Proceedings of
the American Association for cancer Research Vol. 35, march 1994,
Bhalla et al., p306, Seiter et al., p314, Saunders et al., p317).
p53 triggers an apoptosis in various cell types. We have now been
able to show that the combination of a taxol derivative and p53
induces an apoptosis of human tumour cells. Notably, particular
clones of H460 cells which were resistant to the effect of p53 were
cultured in the presence of increasing doses of taxotere. Example 3
demonstrates clearly that the cells die following the treatment
with taxotere at concentrations which are completely ineffective on
cells that do not express wild-type p53.
[0051] Waf 1 (wild-type p53 activated fragment Cell, 75,817, 1993),
or alternatively p21 (Nature, 366,701,1993), is induced by the
overexpression of wild-type p53. Waf 1 appears in cells which have
stopped in the G1 phase or in apoptosis following an overexpression
of wild-type p53, but not in cells which have stopped in G1 or in
apoptosis in a p53-independent manner (Cancer Res, 54, 1169, 1994).
Waf 1 decreases the growth of tumour cells as effectively as
wild-type p53. The combined use of a Waf 1 gene and taxol
derivatives also induces a synergistic effect on the destruction of
hyperproliferative cells.
[0052] Anticancer Therapeutic Agent
[0053] The anticancer therapeutic agents which can be used in the
combined therapy according to the present invention can be chosen
from all chemotherapeutic or radiotherapeutic agents known to a
person skilled in the art. Possible agents are, in particular,
cisplatin, taxoid, etoposide, TNF, adriamycin, camptothecin, a
mitotic spindle poison, and the like. These various agents may be
obtained from a commercial source.
[0054] Among these agents, the taxoids constitute a preferred
embodiment. In this connection, the taxoids which can be used more
especially in the context of the present invention are those which
are represented by the general formula: 1
[0055] in which:
[0056] the symbols R.sub.1 and R.sub.2 each represent a hydrogen
atom, or alternatively one of the radicals R.sub.1 or R.sub.2
represents a hydrogen atom and the other represents a hydroxyl,
acyloxy or acylcarbonyloxy radical, or alternatively R.sub.2
represents a hydrogen atom and R.sub.1 forms a bond with the carbon
atom of the methyl radical at the a-position, so as to form a
cyclopropane ring,
[0057] one of the symbols R.sub.3 or R.sub.4 represents a hydrogen
atom, and the other represents a hydroxyl radical, or alternatively
R.sub.3 and R.sub.4 together form a carbonyl radical,
[0058] the symbols R.sub.5 and R.sub.6 each represent a hydrogen
atom, or alternatively one of the symbols R.sub.5 or R.sub.6
represents a hydrogen atom and the other represents a hydroxyl,
acyloxy, acylcarbonyloxy or alkoxymethylcarbonyloxy radical, or
alternatively R.sub.5 and R.sub.6 together form a carbonyl
radical,
[0059] the symbols R.sub.8 and R.sub.9 each represent a hydrogen
atom, or alternatively R.sub.1 and R.sub.8 together form a
bond,
[0060] the symbol R.sub.7 represents an alkoxy, alkenyloxy or
cycloalkyloxy radical or a phenyl radical, and
[0061] Ar represents a phenyl radical optionally substituted with
one or more identical or different atoms or radicals chosen from
halogen atoms and alkyl, alkoxy, dialkylamino, acylamino,
alkoxycarbonylamino or trifluoromethyl radicals, or a 5-membered
aromatic heterocyclic radical containing one or more identical or
different hetero atoms chosen from nitrogen, oxygen and sulphur
atoms,
[0062] on the understanding that the alkyl radicals and the alkyl
portions of the other radicals contain 1 to 8 carbon atoms in an
unbranched or branched chain and that the alkenyl radicals contain
2 to 8 carbon atoms.
[0063] More especially advantageous are the taxoids for which,
R.sub.2 representing a hydrogen atom, R.sub.1 represents a hydrogen
atom or a hydroxyl radical or alternatively R.sub.1 forms a single
bond with the carbon atom of the methyl radical at the
.alpha.-position, R.sub.3 and R.sub.4 together form a carbonyl
radical, R.sub.5 represents a hydrogen atom and R.sub.6 represents
a hydrogen atom or a hydroxyl, acetyloxy or methoxyacetyloxy
radical or alternatively R.sub.5 and R.sub.6 together form a
carbonyl radical, and R.sub.7 represents a t-butoxy radical or a
phenyl radical.
[0064] The following products may be mentioned more especially:
[0065]
4-acetoxy-2.alpha.-benzoyloxy-5.beta.,20-epoxy-1.beta.,7.beta.,10.b-
eta.-trihydroxy-9-oxo-l1-taxen-13.alpha.-yl
(2R,3S)-3'-t-butoxycarbonylami- no-3'-phenyl-2'-hydroxypropionate
(docetaxel or Taxotre.RTM.)
[0066]
4,10.beta.-diacetoxy-2.alpha.-benzoyloxy-5.beta.,20-epoxy-1.beta.,7-
.beta.-dihydroxy-9-oxo-11-taxen-13.alpha.-yl
(2R,3S)-3'-benzoylamino-3'-ph- enyl-2'-hydroxypropionate
(paclitaxel)
[0067]
4-acetoxy-2.alpha.-benzoyloxy-5.beta.,20-epoxy-1.beta.,10.beta.-dih-
ydroxy-7.beta.,10.beta.-methylene-19-nor-9-oxo-11-taxen-13.alpha.-yl
(2R,3S)-3'-t-butoxycarbonylamino-3'-phenyl-2'-hydroxypropionate
[0068]
4,10.beta.-diacetoxy-2.alpha.-benzoyloxy-5.beta.,20-epoxy-1.beta.-h-
ydroxy-7.beta.,10.beta.-methylene-19-nor-9-oxo-11-taxen-13.alpha.-yl
(2R,3S)-3'-t-butoxycarbonylamino-3'-phenyl-2'-hydroxypropionate,
[0069]
4-acetoxy-2.alpha.-benzoyloxy-5.beta.,20-epoxy-1.beta.,7.beta.,10.b-
eta.-trihydroxy-9-oxo-11-taxen-13.alpha.-yl
(2R,3S)-3'-t-butoxycarbonylami-
no-3'-(2-fluorophenyl)-2'-hydroxypropionate
[0070]
4-acetoxy-2.alpha.-benzoyloxy-5.beta.,20-epoxy-1.beta.,7.beta.,10.b-
eta.-trihydroxy-9-oxo-11-taxen-13.alpha.-yl
(2R,3S)-3'-t-butoxycarbonylami-
no-3'-(4-chlorophenyl)-2'-hydroxypropionate
[0071]
4-acetoxy-2.alpha.-benzoyloxy-5.beta.,20-epoxy-1.beta.,7.beta.,10.b-
eta.-trihydroxy-9-oxo-11-taxen-13.alpha.-yl
(2R,3S)-3'-t-butoxycarbonylami-
no-3'-(4-methoxyphenyl)-2'-hydroxypropionate
[0072]
4-acetoxy-2.alpha.-benzoyloxy-5.beta.,20-epoxy-1.beta.,7.beta.,10.b-
eta.-trihydroxy-9-oxo-11-taxen-13.alpha.-yl
(2R,3S)-3'-t-butoxycarbonylami-
no-3'-(4-fluorophenyl)-2'-hydroxypropionate
[0073]
4-acetoxy-2.alpha.-benzoyloxy-5.beta.,20-epoxy-1.beta.,7.beta.,10.b-
eta.-trihydroxy-9-oxo-11-taxen-13.alpha.-yl
(2R,3S)-3'-adamantyloxycarbony-
lamino-3'-phenyl-2'-hydroxypropionate
[0074]
4-acetoxy-2.alpha.-benzoyloxy-5.beta.,20-epoxy-1.beta.,7.beta.,10.b-
eta.-trihydroxy-9-oxo-11-taxen-13.alpha.-yl
(2R,3S)-3'-tert-pentyloxycarbo-
nylamino-3'-phenyl-2'-hydroxypropionate
[0075]
4-acetoxy-2.alpha.-benzoyloxy-5.beta.,20-epoxy-1.beta.,7.beta.,10.b-
eta.-trihydroxy-9-oxo-11-taxen-13.alpha.-yl
(2R,3S)-3'-(1-methylcyclohexyl-
)oxycarbonylamino-3'-phenyl-2'-hydroxypropionate
[0076]
4-acetoxy-2.alpha.-benzoyloxy-5.beta.,20-epoxy-1.beta.,7.beta.,10.b-
eta.-trihydroxy-9-oxo-11-taxen-13.alpha.-yl
(2R,3S)-3'-(1-methylcyclopropy-
l)oxycarbonylamino-3'-phenyl-2'-hydroxypropionate
[0077]
4-acetoxy-2.alpha.-benzoyloxy-5.beta.,20-epoxy-1.beta.,7.beta.,10.b-
eta.-trihydroxy-9-oxo-11-taxen-13.alpha.-yl
(2R,3S)-3'-(1-methylcyclopenty-
l)oxycarbonylamino-3'-phenyl-2'-hydroxypropionate
[0078]
4-acetoxy-2.alpha.-benzoyloxy-5.beta.,20-epoxy-1.beta.,7.beta.,10.b-
eta.-trihydroxy-9-oxo-11-taxen-13.alpha.-yl
(2R,3S)-3'-(1,1-dimethyl-2-pro-
pyn)yloxycarbonylamino-3'-phenyl-2'-hydroxypropionate
[0079]
4-acetoxy-2.alpha.-benzoyloxy-5.beta.,20-epoxy-1.beta.,7.beta.,9.be-
ta.,10.beta.-tetrahydroxy-11-taxen-13.alpha.-yl
(2R,3S)-3'-t-butoxycarbony-
lamino-3'-phenyl-2'-hydroxypropionate
[0080]
4-acetoxy-2.alpha.-benzoyloxy-5.beta.,20-epoxy-1.beta.,7.beta.-dihy-
droxy-9-oxo-11-taxen-13.alpha.-yl
(2R,3S)-3'-t-butoxycarbonylamino-3'-phen-
yl-2'-hydroxypropionate
[0081]
4-acetoxy-2.alpha.-benzoyloxy-5.beta.,20-epoxy-1.beta.,7.beta.,10.b-
eta.-trihydroxy-9-oxo-11-taxen-13.alpha.-yl
(2R,3S)-3'-t-butoxycarbonylami-
no-3'-(2-thienyl)-2'-hydroxypropionate
[0082]
4-acetoxy-2.alpha.-benzoyloxy-5.beta.,20-epoxy-1.beta.,7.beta.,10.b-
eta.-trihydroxy-9-oxo-11-taxen-13.alpha.-yl
(2R,3S)-3'-t-butoxycarbonylami-
no-3'-(2-furyl)-2'-hydroxypropionate
[0083]
4-acetoxy-2.alpha.-benzoyloxy-5.beta.,20-epoxy-1.beta.,7.beta.,10.b-
eta.-trihydroxy-9-oxo-11-taxen-13.alpha.-yl
(2R,3S)-3'-t-butoxycarbonylami-
no-3'-(3-thienyl)-2'-hydroxypropionate
[0084]
4-acetoxy-2.alpha.-benzoyloxy-5.beta.,20-epoxy-1.beta.,10.beta.-dih-
ydroxy-9-oxo-11-taxen-13.alpha.-yl
(2R,3S)-3'-t-butoxycarbonylamino-3'-phe-
nyl-2'-hydroxypropionate
[0085]
4-acetoxy-2.alpha.-benzoyloxy-5.beta.,20-epoxy-1.beta.,7.beta.-dihy-
droxy-9,10-dioxo-11-taxen-13.alpha.-yl
(2R,3S)-3'-t-butoxycarbonylamino-3'-
-phenyl-2'-hydroxypropionate
[0086]
4-acetoxy-2.alpha.-benzoyloxy-5.beta.,20-epoxy-1.beta.-hydroxy-9-ox-
o-11-taxen-13.alpha.-yl
(2R,3S)-3'-t-butoxycarbonylamino-3'-phenyl-2'-hydr-
oxypropionate
[0087] These different compounds may be obtained according to the
methods described in Applications WO94/13654 and WO92/09589, for
example, which are incorporated in the present application by
reference.
[0088] It is especially advantageous, for the purposes of the
present invention, to use taxol, docetaxel or paclitaxel.
[0089] Vectors for Administration of the Nucleic Acid
[0090] The nucleic acid may be injected as it is at the site to be
treated, or incubated directly with the cells to be destroyed or
treated. It has, in effect, been reported that naked nucleic acids
could enter cells without a special vector. Nevertheless, it is
preferable in the context of the present invention to use an
administration vector, enabling (i) the efficacy of cell
penetration, (ii) targeting and (iii) extra- and intracellular
stability to be improved.
[0091] Different types of vectors can be used. The vectors can be
viral or non-viral.
[0092] Viral Vectors
[0093] The use of viral vectors is based on the natural properties
of transfection of viruses. It is thus possible to use
adenoviruses, herpesviruses, retroviruses and, more recently,
adeno-associated viruses. These vectors prove especially
efficacious from the standpoint of transfection.
[0094] As regards adenoviruses more especially, different
serotypes, the structure and properties of which vary somewhat,
have been characterized. Among these serotypes, it is preferable to
use, in the context of the present invention, human adenoviruses
type 2 or 5 (Ad 2 or Ad 5) or adenoviruses of animal origin (see
Application WO94/26914). Among adenoviruses of animal origin which
can be used in the context of the present invention, adenoviruses
of canine, bovine, murine (for example: Mav1, Beard et al.,
Virology 75 (1990) 81), ovine, porcine, avian or alternatively
simian (for example: SAV) origin may be mentioned. Preferably, the
adenovirus of animal origin is a canine adenovirus, and more
preferably a CAV2 adenovirus [strain Manhattan or A26/61 (ATCC
VR-800), for example] . It is preferable to use adenoviruses of
human or canine or mixed origin in the context of the
invention.
[0095] Preferably, the defective adenoviruses of the invention
comprise the ITRs, a sequence permitting encapsidation and the
nucleic acid of interest. Still more preferably, in the genome of
the adenoviruses of the invention, the E1 region at least is
non-functional. The viral gene in question may be rendered
non-functional by any technique known to a person skilled in the
art, and in particular by total elimination, substitution, partial
deletion or addition of one or more bases in the.gene or genes in
question. Such modifications may be obtained in vitro (on the
isolated DNA) or in situ, for example by means of genetic
engineering techniques, or alternatively by treatment by means of
mutagenic agents. Other regions may also be modified (E2, E3, E4,
L1-L5, and the like).
[0096] The defective recombinant adenoviruses according to the
invention may be prepared by any technique known to a person
skilled in the art (Levrero et al., Gene 101 (1991) 195, EP 185
573; Graham, EMBO J. 3 (1984) 2917). In particular, they may be
prepared by homologous recombination between an adenovirus and a
plasmid carrying, inter alia, the DNA sequence of interest.
Homologous recombination takes place after cotransfection of the
said adenovirus and said plasmid into a suitable cell line. The
cell line used should preferably (i) be transformable by the said
elements, and (ii) contain the sequences capable of complementing
the portion of the genome of the defective adenovirus, preferably
in integrated form in order to avoid risks of recombination. As an
example of a line, there may be mentioned the human embryonic
kidney line 293 (Graham et al., J. Gen. Virol. 36 (1977) 59) which
contains, in particular, integrated in its genome, the left-hand
portion of the genome of an Ad5 adenovirus (12%). Strategies of
construction of vectors derived from adenoviruses have also been
described in Applications Nos. WO 94/26914 and FR 93 08596.
[0097] Thereafter, the adenoviruses which have multiplied are
recovered and purified according to standard techniques of
molecular biology, as illustrated in the examples.
[0098] Adeno-associated viruses (AAV) are, for their part,
relatively small-sized DNA viruses which integrate stably and in a
site-specific manner in the genome of the cells they infect. They
are capable of infecting a broad range of cells without inducing an
effect on cell growth, morphology or differentiation. Moreover,
they do not appear to be implicated in pathologies in man. The AAV
genome has been cloned, sequenced and characterized. It comprises
approximately 4700 bases, and contains at each end an inverted
repeat region (ITR) of approximately 145 bases, serving as origin
of replication for the virus. The remainder of the genome is
divided into 2 essential regions carrying the encapsidation
functions: the left-hand portion of the genome, which contains the
rep gene involved in the viral replication and expression of the
viral genes; and the right-hand portion of the genome, which
contains the cap gene coding for the capsid proteins of the
virus.
[0099] The use of vectors derived from AAVs for the transfer of
genes in vitro and in vivo has been described in the literature
(see, in particular, WO 91/18088; WO 93/09239; U.S. Pat. No.
4,797,368, U.S. Pat. No. 5,139,941, EP 488 528). These applications
describe different constructions derived from AAVs, in which the
rep and/or cap genes are deleted and replaced by a gene of
interest, and their use for transferring the said gene of interest
in vitro (to cells in culture) or in vivo (directly into a body).
The defective recombinant AAVs according to the invention may be
prepared by cotransfection, into a cell line infected with a human
helper virus (for example an adenovirus), of a plasmid containing
the nucleic acid sequence of interest flanked by two inverted
repeat regions (ITR) of AAV, and a plasmid carrying the
encapsidation genes (rep and cap genes) of AAV. The recombinant
AAVs produced are then purified by standard techniques.
[0100] Regarding herpesviruses and retroviruses, the construction
of recombinant vectors has been amply described in the literature:
see, in particular, Breakfield et al., New Biologist 3 (1991) 203;
EP 453242, EP178220, Bernstein et al. Genet. Eng. 7 (1985) 235;
McCormick, BioTechnology 3 (1985) 689, and the like. In particular,
retroviruses are integrative viruses which selectively infect
dividing cells. They hence constitute vectors of interest for
cancer applications. The retrovirus genome essentially comprises
two LTRs, an encapsidation sequence and three coding regions (gag,
pol and env). In the recombinant vectors derived from retroviruses,
the gag, pol and env genes are generally deleted wholly or
partially, and replaced by a heterologous nucleic acid sequence of
interest. These vectors may be produced from different types of
retrovirus such as, in particular, MoMuLV (Moloney murine leukaemia
virus; also designated MOMLV), MSV (Moloney murine sarcoma virus),
HaSV (Harvey sarcoma virus), SNV (spleen necrosis virus), RSV (Rous
sarcoma virus) or alternatively Friend virus.
[0101] To construct recombinant retroviruses containing a sequence
of interest, a plasmid containing, in particular, the LTRs, the
encapsidation sequence and the said sequence of interest is
generally constructed, and then used to transfect a so-called
encapsidation cell line capable of providing in trans the
retroviral functions which are deficient in the plasmid. Generally,
the encapsidation lines are hence capable of expressing the gag,
pol and env genes. Such encapsidation lines have been described in
the prior art, and in particular the line PA317 (U.S. Pat. No.
4,861,719), the line PsiCRIP (WO90/02806) and the line GP+envAm-12
(WO89/07150). Moreover, the recombinant retroviruses can contain
modifications in the LTRs to eliminate transcriptional activity, as
well as extended encapsidation sequences containing a portion of
the gag gene (Bender et al., J. Virol. 61 (1987) 1639). The
recombinant retroviruses produced are then purified by standard
techniques.
[0102] To implement the present invention, it is most especially
advantageous to use a defective recombinant adenovirus or
retrovirus. These vectors possess, in effect, especially
advantageous properties for the transfer of genes into tumour
cells.
[0103] Non-viral Vectors
[0104] The vector according to the invention can also be a
non-viral agent capable of promoting the transfer of nucleic acids
to eukaryotic cells and their expression therein. Chemical or
biochemical vectors represent an advantageous alternative to
natural viruses, especially for reasons of convenience and safety
and also on account of the absence of theoretical limit regarding
the size of the DNA to be transfected.
[0105] These synthetic vectors have two main functions, to compact
the nucleic acid which is to be transfected and to promote its
binding to the cell as well as its passage through the plasma
membrane and, where appropriate, both nuclear membranes. To
compensate for the polyanionic nature of nucleic acids, non-viral
vectors all possess polycationic charges.
[0106] Among the synthetic vectors developed, cationic polymers of
the polylysine, (LKLK)n, (LKKL)n, polyethylenimine and DEAE-dextran
type, or alternatively cationic lipids or lipofectants are the most
advantageous. They possess the property of condensing DNA and of
promoting its association with the cell membrane. Among the latter
compounds, there may be mentioned lipopolyamines (lipofectamine,
transfectam, and the like) and various cationic or neutral lipids
(DOTMA, DOGS, DOPE, and the like). More recently, the concept of
receptor-mediated, targeted transfection has been developed, which
turns to good account the principle of condensing DNA by means of
the cationic polymer while directing the binding of the complex to
the membrane as a result of a chemical coupling between the
cationic polymer and the ligand for a membrane receptor present at
the surface of the cell type which it is desired to graft.
Targeting of the transferrin or insulin receptor or of the
asialoglycoprotein receptor of hepatocytes has thus been
described.
[0107] Administration Protocol
[0108] A preferred administration protocol according to the
invention comprises first the nucleic acid and then the therapeutic
agent. In a preferential use, administration of the transgene is
repeated in order to obtain a maximum expression in a maximum
number of dividing cells (for example 5 days in succession), and
the chemotherapeutic treatment is then administered.
Advantageously, the nucleic acid or acids are administered in
contact with the lesion, either by direct intratumoral injection
into multiple sites of the lesion, or in contact with the
atheromatous lesion by means of a cushion suited to this type of
operation. The chemotherapeutic agent is administered according to
the clinical protocols in force.
EXAMPLES
Example 1
[0109] H460 cells, cultured in RPMI 1640 medium containing 10% of
foetal calf serum, are transfected with cDNAs coding for the
GAP[170-702] domain or for the Grb3-3 protein in combination with a
gene conferring resistance to geneticin (Neo) on the positively
transfected cells. These cDNAs, placed in plasmids and whose
expression is under the control of viral promoters
(pSV.sub.2-GAP[170-702], pSV.sub.2-Grb3-3 and pSV.sub.2-Neo), are
introduced into the H460 cells using lipofectAMINE as transfecting
agent. The H460/Neo.sup.R cells (resistant to the presence of 400
.mu.g/ml of geneticin in the culture medium) are selected and
quantified 15-20 days after transfection. The results of an
experiment which is representative of quantification of the number
of Neo.sup.R colonies under the different transfection conditions
are summarized in FIG. 1 (pSV.sub.2-Oli: control plasmid not
possessing any cDNA of interest and thus permitting monitoring of
the efficacy of the selection by geneticin).
Example 2
[0110] H460 cells transfected as described in Example 1 are
subjected during selection by geneticin to a treatment for several
days at different concentrations of taxotere, of cisplatin or of
camptothecin. The H460/Neo.sup.R cells and which are resistant to
the chemotherapeutic agents are quantified as described in Example
1. The sensitivity to taxotere (A), to cisplatin (B) or to
camptothecin (C) of the H460 cells transfected with pSV.sub.2-Neo
(.circle-solid.), pSV.sub.2-Neo+pSV.sub.2-- GAP[170-702]
(.tangle-solidup.) or pSV.sub.2-Neo+pSV.sub.2-Grb3-3
(.tangle-soliddn.) is depicted relative to cells transfected
identically but in the absence of treatment with the
chemotherapeutic agents. The results of an experiment which is
representative of quantification of the number of colony after the
different treatments mentioned are summarized in FIG. 2.
Example 3
[0111] H460 cells are transfected with cDNA coding for the
wild-type p53 protein (p53.sup.WT) placed in the plasmid pcDNA3
under the control of the CMV promoter. Plasmid pcDNA3 also contains
the Neo gene placed under the control of the SV40 promoter. Cells
transfected with pcDNA3 or pcDNA3-p53.sup.WT are selected and
isolated as described in Example 1. In the cells transfected with
pcDNA3-p53.sup.WT and which are resistant to geneticin, the
presence of p53 is verified by western blotting using specific
antibodies. FIG. 3 summarizes the results of a representative
experiment in which are depicted, on the one hand the number of
colonies obtained after pcDNA3 or pcDNA3-p53.sup.WT transfection
(A), and on the other hand the sensitivity of the clones isolated
to a treatment with taxotere (B).
* * * * *