U.S. patent application number 12/899170 was filed with the patent office on 2011-01-27 for immunovectors which can be used for the intracellular and intranuclear transport.
This patent application is currently assigned to INSTITUT PASTEUR. Invention is credited to Alexandre Avrameas, Stratis AVRAMEAS, Gerard Buttin, Faridabano Nato, Therese Ternynck.
Application Number | 20110020928 12/899170 |
Document ID | / |
Family ID | 9480845 |
Filed Date | 2011-01-27 |
United States Patent
Application |
20110020928 |
Kind Code |
A1 |
AVRAMEAS; Stratis ; et
al. |
January 27, 2011 |
IMMUNOVECTORS WHICH CAN BE USED FOR THE INTRACELLULAR AND
INTRANUCLEAR TRANSPORT
Abstract
A method of transferring a biologically active principle of
interest into a cell by coupling an antibody or a fragment of an
antibody, which recognizes an epitope contained in a nucleic acid,
with the biologically active principle, to form a coupled
biologically active principle; and incubating the coupled
biologically active principle, which is transferred through the
cell membrane and into the cell, with the cell.
Inventors: |
AVRAMEAS; Stratis; (Paris,
FR) ; Buttin; Gerard; (Paris, FR) ; Ternynck;
Therese; (Paris, FR) ; Nato; Faridabano;
(Antony, FR) ; Avrameas; Alexandre; (Vitry Sur
Seine, FR) |
Correspondence
Address: |
OBLON, SPIVAK, MCCLELLAND MAIER & NEUSTADT, L.L.P.
1940 DUKE STREET
ALEXANDRIA
VA
22314
US
|
Assignee: |
INSTITUT PASTEUR
Paris
FR
Universite Pierre et Marie Curie
Paris Cedex
FR
|
Family ID: |
9480845 |
Appl. No.: |
12/899170 |
Filed: |
October 6, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10465654 |
Jun 20, 2003 |
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12899170 |
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08981474 |
Apr 28, 1998 |
6608034 |
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PCT/FR96/01076 |
Jul 10, 1996 |
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10465654 |
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Current U.S.
Class: |
435/346 ;
435/375 |
Current CPC
Class: |
A61K 47/6845 20170801;
A61P 31/18 20180101; A61P 43/00 20180101; A61K 48/00 20130101; C07K
16/18 20130101; A61P 31/00 20180101; A61P 35/00 20180101 |
Class at
Publication: |
435/346 ;
435/375 |
International
Class: |
C12N 5/071 20100101
C12N005/071; C12N 5/16 20060101 C12N005/16 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 10, 1995 |
FR |
95/08316 |
Claims
1. A method of transferring a biologically active principle of
interest into a cell comprising: a. coupling an antibody or a
fragment of an antibody, which recognizes an epitope contained in a
nucleic acid, with the biologically active principle, thereby
forming a coupled biologically active principle; and b. incubating
the coupled biologically active principle with the cell, wherein
said coupled principle is transferred through the cell membrane and
into the cell.
2. The method of claim 1, wherein said biologically active
principle is selected from the group consisting of a hapten having
biological activity, a hormone, and a medicine.
3. The method of claim 2, wherein said biologically active
principle is a medicine allowing the immortalization of selected
type cells including one of macrophages, dendritic cells, B cells,
T cells, and hematopoietic cells.
4. The method of claim 1, wherein said biologically active
principle is an antiviral agent.
5. The method of claim 1, wherein the antibody or fragment of
antibody is a monoclonal IgG, a (Fab').sub.2 fragment, or a (Fab')
fragment.
6. The method of claim 1, wherein the antibody or fragment of
antibody is obtained from individuals presenting auto-immune
syndromes.
7. The method of claim 1, wherein the antibody or fragment of
antibody is obtained from individuals presenting disseminated
erythematous lupus syndromes.
8. A hybridoma deposited at the CNCM on Jun. 30, 1996 under the
registration number I-1605, I-1606 or I-1607.
Description
[0001] 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.
[0002] 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).
[0003] 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).
[0004] 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.
[0005] 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.
[0006] 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.
[0007] 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.
[0008] 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.
[0009] 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).
[0010] 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.
[0011] 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.
[0012] 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.
[0013] Preferably, a chemical mode of coupling, allowing the
formation of covalent or non-covalent bonds, will be used.
[0014] 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.
[0015] 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.
[0016] 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.
[0017] In one embodiment, the lymphoid cells used are auto-immune
mouse splenocytes exhibiting a lupus syndrome.
[0018] A preferred immunovector for the coupling product is chosen
from among monoclonal IgG's, (Fab').sub.2 or (Fab') fragments, or
any polypeptide corresponding to the site(s) of the antibodies
involved in the recognition of the corresponding nucleic acid.
[0019] Preferably, this immunovector is an immunoglobulin, more
particularly an IgG carrying an anti-DNA activity, and obtained
from normal individuals.
[0020] 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 syndromes.
[0021] In a specific embodiment of the invention, the immunovector
coupled to the active principle is a bi-specific antibody
recognizing on the one hand the DNA, and on the other hand a
protein such as Tat, Rev of the HIV retrovirus, as well as surface
markers such as CD3, CD4, CD8, CD19 and CD34.
[0022] Preferably, the biologically active principle coupled to the
immunovector is a molecule selected from especially nucleic acids,
proteins especially enzymes, for example peroxidase, haptens
especially biotin or fluorescein, enzyme activators or inhibitors
and medicaments.
[0023] In a preferred embodiment, the coupled nucleic acid is a
polynucleotide, the immunovector being an IgG, with the coupling
being carried out via p-benzoquinone at the rate of one molecule of
immunovector per 4 molecules of polynucleotide.
[0024] A biologically active principle preferably used consists of
a plasmid intended to integrate into the nucleus of the target
cells for the expression of a protein encoded by a gene contained
in the said plasmid. When such a plasmid is coupled to an
immunovector of the antibody type, having affinity for DNA, this
immunovector is, preferably, previously coupled to an agent capable
of inducing a compacting effect on the DNA, this agent being
preferably polylysine.
[0025] Indeed, polylysine, which by virtue of its cationic
properties is capable of compacting DNA, promotes the transfection
of cells. The coupling of polylysine to the immunovector, which is
carried out with the aid of a coupling agent, more particularly a
carbodiimide such as EDC
(1-(3-dimethylaminopropyl)-1'-ethylcarbodiimide), allows the DNA to
react therewith. This has the effect of inducing the liberation of
the active site of the antibody which may sometimes be masked when
the antibody is coupled to an active principle of the size of a
plasmid.
[0026] Other agents capable of having, by virtue of their cationic
properties, a compacting effect on DNA (2-6) may also be coupled to
the immunovector to fulfil such a function.
[0027] More specifically, a biologically active principle
preferably used consists of a gene intended to integrate in the
genome of target cells, especially by homologous recombination,
more particularly a "nude" DNA containing a nucleic acid sequence
coding for a polypeptide originating from bacterial or eucaryotic
cells, fungal cells or viruses, this polypeptide having vaccinating
properties.
[0028] Advantageously, this active principle allows the
immortalization of selected types of cells, particularly
macrophages, dendritic cells, B and T cells, and hematopoietic
cells, especially of human origin.
[0029] Still more preferably, this biologically active principle is
an antisense oligonucleotide allowing the inhibition of protein or
nucleotide synthesis, for example in cells infectable by an HIV
retrovirus, or in tumour cells.
[0030] Thus, the inventors have, on the one hand, tried to obtain,
from the spleen of autoimmune mice (NZB.times.NZW)F1, having a
lupus syndrome, IgG monoclonal antibodies which have been selected
for their capacity to react with DNA but also for their capacity to
penetrate as far as the nucleus of the cells. In parallel,
polyclonal antibodies reacting with the DNA and capable of
penetrating into the nuclei of the cells have been isolated by
affinity chromatography. This chromatography was applied either to
a "pool" of sera of normal patients or to a normal individual
serum, preferably to sera obtained from patients suffering from
infections, particularly to a serum of normal patients suffering
from DLE, or to a mouse (NZB.times.NZW)F1 serum.
[0031] In a method of selection of immunovectors according to the
present invention, the test of cellular penetration of the
immunovectors comprises a first incubation of eucaryotic cell lines
in a medium comprising the said immunovectors preferably in
increasing concentration, then the fixing, and, if necessary, the
permeabilization, or vice versa, of these cells, followed by an
incubation of the said cell lines with anti-immunovector antibodies
preferably labelled with fluorescein or with peroxidase, and the
localization of the antibodies thus labelled close to the nuclei of
the said cells or better still inside the nuclei. The cell lines
are especially chosen from among fibroblasts, thymocytes or
splenocytes.
[0032] Without the following reaction conditions having a limiting
character, it may be mentioned that the first incubation is often
carried out at 37.degree. C., for about 2 to 8 hours with
immunovector concentrations of about 1 to 70 .mu.g/ml, on cell
lines inoculated at a concentration ranging from 5.times.10.sup.3
to 5.times.10.sup.6 cells per millilitre.
[0033] In one of the embodiments of the method, the cell line is a
fibroblast line in exponential growth inoculated at a concentration
of 2.times.10.sup.4 cells per millilitre, or thymocytes or
splenocytes of BALB/c mice, which are suspended at the rate of
about 10.sup.6 cells per millitre.
[0034] Moreover, the invention relates to a method of preparation
of immunovector-molecule(s) coupling product, the immunovectors
being chosen from among antibodies, more particularly the IgG's
obtained according to the method of selection, (Fab').sub.2 or
(Fab') fragments, or any polypeptide corresponding to the site of
the antibodies or fragments of antibodies involved in the transport
of the molecules.
[0035] In the method of preparation of a coupling product according
to the invention, it is ensured that for each immunovector is
coupled at least one molecule of biologically active product, the
said molecule being preferably covalently linked to the
immunovector.
[0036] The following examples illustrate conditions in which
haptens such as fluorescein and biotin, small molecules such as
hormones, proteins preferably enzymes, enzyme inhibitors or
activators and medicines, for example antivirals such as acyclovir
or AZT, may be actively transported through the cytoplasm of the
treated cells and transferred into the nucleus of the said cells.
In particular, fluorescein has been coupled to the free amino
groups of the immunovectors via an active isothiocyanate group and
the biotin via an active succinimide ester. The coupling of the
haptens and the like to the immunovector may be carried out by
means of other homo- or heterobifunctional bridging groups or
reagents known in the literature, such as the imido esters and
N-hydroxysuccinimidyl esters which are capable of reacting with the
amino groups, for example derivatives of alkyl, haloaryl,
haloacetyl and pyridyl disulphide groups reacting preferentially,
maleimides, or by means of sulphydryl groups, carbodiimides, as
well as molecules having photoactivable groups such as azidobenzoyl
hydrazide (13).
[0037] In addition, the inventors have prepared products of
coupling where the immunovector is a bi-specific antibody against a
target antigen, constructed either by the chemical route or by the
genetic engineering route, and have in parallel immunized
individuals, for example mice, with the said target antigens, and
selected immunovectors which are bi-specific antibodies preferably
reacting with the said target antigens, such that the action of the
immunovectors is directed specifically.
[0038] Techniques relating to the synthesis of bi-specific
antibodies have especially been described by Porstmann et al. in
1984 (16), a study entitled "Development of a bispecific monoclonal
antibody for use in molecular hybridisation" having moreover been
published in 1984 by Auriol et al. (17).
[0039] Advantageously, the bi-specific antibodies used in this
method recognize, inter alia, the proteins Tat, Rev of the HIV
retrovirus, as well as surface markers CD3, CD4, CD8, CD19 and
CD34.
[0040] Moreover, the present invention relates to a method for
transferring an active principle in the nuclei of selected
eucaryotic cells, characterized by coupling this active principle
with an immunovector having both the capacity of allowing
internalization of this active principle in these eucaryotic cells
and an affinity for the DNA of these cells to such a point that the
said immunovector is rendered capable of transporting this
biologically active principle immediately close to or into the
nuclei of these cells.
[0041] This biologically active principle may be covalently or
non-covalently coupled to the immunovector.
[0042] Preferably, this method of transfer is characterized in that
the immunovector entering into the composition of this product is
selected from those which are selectable by a cellular penetration
test comprising a first incubation of the immunovector of interest
in the presence of cells 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 the detection immediately close to the
nucleus or even in the nucleus of the antigen-antibody type
immunologic reaction between the immunovector and the
anti-immunovector antibody.
[0043] In a preferred embodiment of the method of transfer
according to the invention, the immunovector used is formed of an
antibody having an affinity for a nucleic acid, or of a fragment of
this antibody retaining this affinity.
[0044] This immunovector used in this method is preferably selected
from among antibodies, preferably monoclonal IgG's, (Fab').sub.2 or
(Fab') fragments, or any polypeptide corresponding to the site(s)
of the antibodies involved in the recognition of the corresponding
nucleic acid.
[0045] Advantageously, once the product of immunovector/active
principle coupling is prepared, it may be used for the intranuclear
transfer of other molecules. Thus, the fluorescein/immunovector
conjugate tested may be associated with an anti-fluorescein
antibody coupled with a third molecule, and thus transfer the said
third molecule into the nuclei of the cells. Similarly, the
biotin/immunovector conjugate may allow the binding of an
anti-biotin antibody or of avidin-streptavidin coupled with a third
molecule to be transferred into the nuclei.
[0046] In the present invention, an enzyme such as horseradish
peroxidase was transferred into the nucleus, but other proteins
having varied biological activities may also be used. Peroxidase
coupled to the immunovector via glutaraldehyde has also been used.
However, other methods known in the literature, such as those
described in the case of haptens, may also be used.
[0047] As in the case of the hapten/immunovector conjugates, there
may be used, in association with the protein/immunovector
conjugates, an anti-protein antibody coupled with a third molecule
for the intranuclear transfer of the said molecule.
[0048] In the invention described herein, although a polynucleotide
has been transferred into the nucleus, a wide variety of nucleic
acids having appropriate biological activities may also be actively
transferred at the intranuclear level.
[0049] Thus, a method of transfer of active principles according to
the invention allows in particular the transfer of genes intended
to integrate into the genome of the target cells, especially by
homologous recombination, more particularly the transfer of "nude"
DNA, it being possible for the latter especially to be used as "DNA
vaccine".
[0050] One of the homologous recombination techniques possible is
that described by Mouellic et al. in 1990 (18).
[0051] Recent studies carried out by Whalen R. G. et al. have made
it possible to show the existence of an immune response following a
DNA transfer. These studies, which have been the subject of patent
application WO 95/11307, were more particularly applied to the
expression of monoclonal molecules of the IL2 cytokine type
(19).
[0052] In addition, this method of transfer makes it possible to
introduce nucleotide sequences involved in the immortalization of
different cell types, particularly macrophages, dendritic cells, B
and T cells, and haemotopoietic cells especially of human origin.
As nucleotide sequences, there may be mentioned the oncogenic
sequences or viral sequences associated with cell transformation
phenomena.
[0053] It also allows the transfer of antisense oligonucleotides
allowing the inhibition of protein or nucleotide synthesis, for
example in cells infectible by a retrovirus such as HIV, or tumour
cells.
[0054] In the present invention, the polynucleotide was coupled to
the immunovector via p-benzoquinone. However, other methods known
in the literature may also be used.
[0055] Moreover, the present invention also relates to the
eucaryotic cells containing active molecules preferably at the
nuclear level, characterized in that the said molecules cannot be
naturally incorporated into the nuclei of the said cells or have a
weak expression level in the said cells. These molecules are
presented in these cells immediately close to their nuclei or into
their nuclei, and are coupled to an immunovector characterized by
its affinity for the DNA of these cells, in the form of a coupling
product according to the invention.
[0056] Among the cells to which the present invention relates,
there are especially the cells which can be infected by a virus or
tumour cells.
[0057] Also entering within the framework of the present invention
are the hybridomas producing the antibodies according to the
present invention, as deposited at the CNCM on 30 Jun. 1995 under
the numbers 1-1605, 1-1606 and 1-1607.
[0058] In addition, the invention relates to a pharmaceutical
composition, characterized in that it contains, in combination with
a physiologically acceptable vehicle, a coupling product according
to the invention in which the biologically active principle is a
medicine or vaccinal active principle and the immunovector is
compatible with the host organism to which the medicine is
directed.
[0059] Also entering into the framework of the present invention is
the use of the coupling product of the invention for the
expression, in receiving cells, of a nucleotide sequence which is
heterologous to the DNA of the host.
EXAMPLES
1. Preparation of Immunovectors
A) Polyclonal Immunovectors
[0060] The human or murine IgG's are first isolated by passage of a
mixture of sera obtained from individuals suffering from
disseminated erythematous lupus--or from lupus mice
(NZB.times.NZW)F1--on protein A immobilized on Sepharose (14).
[0061] The isolated IgG's are passed over a column of DNA
immobilized on cellulose. The specific anti-DNA antibodies present
in these IgG's are attached to this DNA-cellulose immunoabsorbent
and eluted with a 20 .mu.M sodium carbonate-bicarbonate buffer, pH
10, containing 5% dimethyl sulphoxide (15). 1 to 2 mg of antibodies
are thus isolated from 10 mg of IgG. The eluted antibodies are
dialysed, concentrated and stored at +4.degree. C. until they are
used.
B) Murine Monoclonal Immunovectors
[0062] Splenocytes obtained from lupus mice (NZB.times.NZW)F1 are
fused with the X63 myeloma according to the method of Kohler and
Milstein. The hybridomas produced are tested by ELISA for the
secretion of IgG and for their anti-DNA activity. The anti-DNA IgG
secreting hybridomas are subcloned at least twice and the clones
which remain doubly positive (IgG+anti-DNA) are bulk cultured or
alternatively ascites are prepared from these clones in mice. In a
typical experiment, starting with the spleen of a mouse
(NZB.times.NZW)F1, approximately 300 positive wells secreting IgG's
were obtained of which 60 were capable of reacting with DNA. After
cloning, 20 clones secreted IgG recognizing DNA. Of these 20
clones, approximately half secreted antibodies capable of
penetrating into the nucleus of the cells whereas the others were
not capable of this (see C: test of intranuclear penetration of the
immunovectors). The monoclonal IgG's are isolated from culture
supernatants or ascitic fluids by precipitation with 45% ammonium
sulphate followed, after dialysis, by passage on protein A
immobilized on Sepharose. After neutralization of the eluted IgG's,
the preparations are dialysed, concentrated and stored at
-20.degree. C. until they are used.
[0063] The mouse antibodies mentioned above will be subsequently
humanized using one of known techniques, for example that described
by Riechmann et al. (20).
C) Selection of the Immunovectors
[0064] 1) Test of Intranuclear Penetration of the Immunovectors
[0065] Two fibroblast lines--PtK2 obtained from kangaroo rat kidney
and GMA-32 obtained from hamster kidney--were mainly used. The
slides carrying the fibroblasts in the exponential growth phase,
inoculated at 2.times.10.sup.4 cells/ml, 24 hours beforehand and
cultured in RPMI 1640 or MEM medium (containing 10% foetal calf
serum, 2 mM L-glutamine and 1% sodium pyruvate) are incubated at
37.degree. C. in renewed culture medium, containing selected
quantities of immunovector (1 to 70 .mu.g/ml). After 2 to 4 hours
of incubation, the cells are washed with PBS and fixed with either
ethanol for 10 minutes at -20.degree. C., or with 0.2%
glutaraldehyde and 2% formaldehyde in PBS for 20 minutes. After
three washes with PBS, the cells are permeabilized for 20 minutes
in PBS containing 0.2% bovine serum albumin and 0.5% saponin.
[0066] The cellular preparations are then washed with PBS and
incubated for 45 minutes at 24.degree. C. with anti-mouse
immunoglobulin (or anti-human immunoglobulin) rabbit or sheep
antibodies labelled with fluorescein or with peroxidase (20
.mu.g/ml). After washing, the cellular preparations incubated with
the fluorescent antibody are examined under a fluorescence
microscope. The cellular preparations incubated with the
peroxidase-labelled antibody are first incubated in the
cytochemical substrate of peroxidase (diaminobenzidine
(DAB)+H.sub.2O.sub.2) and, after washing, the preparation is
examined under an optical microscope (14). The number of positive
cells is counted.
[0067] As described above, mouse thymocytes were also used to test
the penetration of the immunovectors into the nucleus. Suspensions
of thymocytes were prepared from BALB/c mouse thymus. The
thymocytes, at a concentration of 1.times.10.sup.6 cells/ml, are
incubated at 37.degree. C. for 3 hours in a culture medium
containing increasing quantities of immunovector (1 to 70 .mu./ml).
After washing and fixing, the lymphocytes are treated like the
preparations of fibroblasts above for the intranuclear detection of
the antibodies.
[0068] 2) Test of Attachment of the Anti-DNA Antibodies to the
Lymphoid Cells
[0069] To demonstrate a reaction with the cell membranes, 10.sup.6
mouse thymocytes or splenocytes were incubated at cold temperature
for 45 minutes with 0.1 ml of different monoclonal antibodies
diluted in a solution of bovine albumin at 0.1% containing 0.2%
sodium azide. After washing, the cells are incubated with
fluorescent anti-mouse IgG antibodies for minutes at cold
temperature. After washing, the cells are examined by FACS and the
number of positive cells determined in each population.
[0070] Of the 20 monoclonal antibodies examined, approximately half
secrete antibodies which are capable of penetrating into the
nucleus of the cells whereas the other half do not have this
capacity. A correlation was able to be established between the
monoclonal antibodies penetrating with a high efficiency into the
nucleus of the cells (number of labelled cells, limiting dilution
to obtain a labelling) and their ability to label the thymocytes
and the splenocytes.
Ii Preparation of Immunovectors Carrying Haptens, Proteins or
Nucleic Acids
[0071] A) Preparation of F(ab').sub.2 and Fab' Fragments of
Immunovectors
[0072] The F(ab').sub.2 fragments of the immunovectors are prepared
according to described methods involving proteolysis with pepsin
followed by reduction by cysteine to obtain the Fab' fragment (14).
Thus in 5 ml of 0.1 M citrate-citric acid buffer, pH 3.5,
containing 5 mg of immunovector, 150 .mu.g of pepsin are added and
the mixture is incubated for 2 hours at 37.degree. C. The medium is
adjusted to pH 8 and the preparation filtered on a protein
A-Sepharose column in order to remove the undigested IgG's. After
dialysis against PBS, this F(ab').sub.2 preparation is stored at
-20.degree. C. until it is used. To obtain the Fab' fragments,
cysteine is added to a final concentration of 0.02 M to the
F(ab').sub.2 preparation. After 10 minutes of incubation at
37.degree. C., 0.04 M iodoacetamide is added and the mixture is
incubated for 30 minutes. This Fab' preparation is dialysed against
PBS and stored at -20.degree. C. until it is used.
B) Immunovectors/Haptens
[0073] Coupling to Biotin
[0074] 2 .mu.l of a 0.1 M solution of d-biotin-N-hydroxysuccinimide
ester in dimethylformamide (1 mg of the active ester in 30 .mu.l of
dimethylformamide) are added to 0.5 ml of 0.1 M phosphate buffer,
pH 7, containing 1 mg of antibody. The solution is left for 1 hour
at laboratory temperature and dialysed against PBS at +4.degree. C.
overnight.
[0075] Coupling to Fluorescein
[0076] 20 .mu.l of a solution of fluorescein isothiocyanate in
dimethyl sulphoxide (10 mg/ml) are added to 1 ml of a 0.1 M
solution of sodium carbonate containing 1 mg of antibody. The
solution is left for 3 hours at laboratory temperature and dialysed
against PBS at +4.degree. C.
C) Immunovectors/Proteins
[0077] Coupling with Peroxidase
[0078] Ten milligrammes of peroxidase are dissolved in 0.2 ml of 1%
glutaraldehyde in 0.1 M phosphate buffer pH 6.8. After incubation
at laboratory temperature for 18 hours, the solution is filtered on
a Sephadex G25 column (0.9.times.60 cm) equilibrated with 0.15 M
NaCl to remove the excess glutaraldehyde. To this activated
peroxidase solution, there is added 1 ml of a 0.15 M NaCl solution
containing 5 mg of antibody and 0.2 ml of 1 M carbonate-bicarbonate
buffer pH 9.5. The solution is stored at +4.degree. C. for 24 hours
and then supplemented with lysine to the final concentration of 0.1
M, and then dialysed against PBS at 4.degree. C.
D) Immunovectors/Nucleic Acids
[0079] Polynucleotides
[0080] The polynucleotide used was composed of 15 nucleotides and
carried a fluorescein in 5' and a free NH.sub.2 group in 3'. It was
prepared according to conventional methods of nucleic synthesis.
This nucleotide was coupled to the immunovector via p-benzoquinone
(14). 0.1 ml of ethanol containing 3 mg of p-benzoquinone is added
to 0.4 ml of 0.1 M phosphate buffer pH 6 containing 1 mg of
immunovector (whole molecule, F(ab').sub.2 or Fab'). After an
incubation of one hour at laboratory temperature, the preparation
is filtered on a Sephadex G-25 column. The fraction containing the
activated immunovector is supplemented with the polynucleotide in a
ratio of one molecule of immunovector to 4 molecules of
polynucleotide, and the solution is adjusted to pH 9.2 with
carbonate-bicarbonate buffer. After 18 hours incubation at
laboratory temperature, the reaction is stopped by addition of
lysine to the final concentration of 0.1 M, followed by dialysis
against PBS. This preparation is stored at +4.degree. C. until it
is used.
[0081] Plasmids
[0082] Two plasmids were tested, a first carrying the vimentin
promoter upstream of the gene encoding the SV40 T, t antigens
(pHuVim 830 T,t) (21) and a second carrying the luciferase gene
(22) under the control of a cytomegalovirus promoter (pCMV-Luc)
(5).
[0083] These plasmids are maintained in the E. coli strain and are
prepared after a bacterial culture by the standard lysis method in
the presence of detergent and in alkaline medium. The plasmids are
then purified by chromatography on a resin column (Qiagen Plasmid
Kits).
[0084] The immunovectors J-20.8 and F-14.6 are used for this work.
These antibodies are prepared by the method of preparation of
monoclonal immunovectors previously described in paragraph I.B/.
The antibodies are coupled to poly-L-lysine with the aid of a
coupling agent, especially a carbodiimide, such as EDC
(1-(3-dimethylaminopropyl)-1'-ethylcarbodiimide). In some cases,
polyclonal IgG's are added to the anti-DNA antibody in a 10:1 ratio
so as to increase the concentration of IgG in the medium and
thereby to promote the coupling with the polylysine. [0085] 2 mg of
monoclonal antibody (J-20.8 or F-14.6) in 1 ml of PBS or of
concentrated polyclonal IgG's at 20 mg/ml are dialysed overnight
against a 10 mM MES buffer, pH 5. Two mg of poly-L-lysine
(MW=18,000) are dissolved in 1 ml of this same buffer and then
supplemented with 0.2 mg of EDC (in 50 .mu.l of MES buffer) for 30
seconds. The poly-L-lysine solution is then added to the
antibody/EDC mixture and the incubation is continued for 2 hours.
[0086] the preparation is then filtered on a protein A-Sepharose
column in order to separate the excess poly-L-lysine from the
antibodies conjugated to the polylysine which are eluted at pH 3
under the usual conditions, neutralized and dialysed against
PBS.
III. Examples of Transfer of Substances into the Nuclei oF Cells by
Immunovectors Associated with these Substances
A) Transfer In Vitro of Fluorescein
[0087] Fibroblasts of the GM A-32 line in culture on glass
coverslips are incubated at. 37.degree. C. for 2 to 4 hours in RPMI
culture medium containing increasing quantities of monoclonal
immunovectors (J-20.8 antibody or Fab'2 fragments) labelled with
fluorescein. At the end of this time, the cells are washed and
fixed as described in IC1. After inclusion in Mowiol medium, they
are examined under a fluorescence microscope. Practically all the
nuclei of the fibroblasts show a fluorescent labelling. On the
other hand, the nuclei of fibroblasts incubated with a control
monoclonal antibody Ig 2a without anti-DNA activity, which does not
penetrate to the nuclei, do not exhibit any fluorescence (FIGS. 1
and 2).
B) Transfer In Vivo of Fluorescein into Mouse Peripheral
Lymphocytes
[0088] One mg of immunovector (monoclonal antibody C-2.1 or F-4.1
or control antibody (monoclonal antibody G-14) labelled with
fluorescein is injected into two mice in an amount of 0.2 ml
intraveneously and 0.3 ml intraperitoneally. After 5 hours, the
mice are bled and sacrificed and the circulating blood lymphocytes
are analysed by FACS. It is noted that 60% of the peripheral blood
lymphocytes obtained from the mouse injected with the immunovector
are fluorescent whereas none of the lymphocytes from the control
animal are (FIG. 3). Microscopic examination shows a fluorescence
at the level of the nuclei in the majority of the cells.
C) Transfer of Biotin
[0089] Fibroblasts of the PtK2 line (10.sup.5/ml) cultured for 24
hours beforehand are incubated in a complete RPMI culture medium
with human anti-DNA polyclonal IgG's labelled with biotin in
increasing quantities (5-100 .mu.g). After 3 hours, the cells are
washed, fixed and permeabilized as described in IC. The cells are
then incubated with RPMI containing 1 .mu.g/ml of
peroxidase-labelled streptavidin. After one hour, the cells are
washed three times with PBS and the peroxidase associated with the
cells is revealed using the DAB+H.sub.2O.sub.2 medium. The
preparations are included in Mowiol and examined under an optical
microscope. A large number of nuclei of the fibroblasts incubated
with anti-DNA IgG are positive whereas the cells incubated with
IgG's obtained from normal individuals and labelled with biotin are
negative.
D) Transfer of Peroxidase
[0090] Under the conditions defined in paragraph IIIC, the PtK2
fibroblasts are incubated with increasing quantities of Fab'
fragments of an immunovector (antibody J-20.8) labelled with
peroxidase. After 3 hours, the cells are washed three times with
PBS and fixed for 20 minutes with 0.2% glutaraldehyde and 2%
formaldehyde in PBS. After washing, the peroxidase activity is
revealed by the coloured DAB+H.sub.2O.sub.2 test and the
preparations are examined under an optical microscope. A large
proportion of nuclei of the fibroblasts incubated with the Fab'
fragments of the J-20.8 antibody are positive for peroxidase,
whereas those incubated with the control antibody 48.9 are
negative.
E) Transfer of Polynucleotide Labelled with Fluorescein
[0091] 3.times.10.sup.6 splenocytes, prepared from BALB/c mouse
spleen, are incubated in 1 ml of RPMI containing 40 .mu.g/ml of
immunovector (J-20.8) or of its Fab' fragment covalently coupled to
the polynucleotide. After three hours of incubation at 37.degree.
C., the cells are washed with PBS, fixed in 4% paraformaldehyde and
examined under a microscope. Eight to 10% of the cells show an
intranuclear fluorescence (FIG. 4).
F) Transfer of Plasmid
[0092] The transfection efficiency was evaluated by demonstrating
the synthesis of the proteins encoded by these genes, either with
the aid of anti-T antigen antibodies coupled to peroxidase, or by a
luminometric assay of the activity of luciferase on its substrate,
luciferin.
[0093] The cells used are fibroblasts of the GMA-32 line and Hep 2
carcinoma cells. They are cultured in a complete medium (RPMI 1640
medium containing 10% foetal calf serum, 2 mM L-glutamine, 1%
sodium pyruvate and antibiotics), at 37.degree. C. of 5%
CO.sub.2.
[0094] Plasmid pHuVim 830 T, t: The Hep2 cells are inoculated the
day before in an amount of 2.times.10.sup.4 cells in 0.5 ml of
complete medium per well of a 24-well plate. For the transfection,
the medium is removed and replaced with 0.3 ml of complete medium
containing 20 .mu.g of antibody-poly-L-lysine and 2 .mu.g of
plasmid, or 20 .mu.g of native antibodies and 2 .mu.g of plasmid or
2 .mu.g of plasmid alone. After 6 hours, the medium is changed and
the culture is continued by changing the medium every two days and
by subdividing the cells into two if necessary. The transfection
efficiency is tested at variable times.
[0095] Plasmid pCMV-Luc: The GMA-32 cells are inoculated the day
before in an amount of 7 to 10.times.10.sup.4 cells/0.5 ml of
complete medium per well of a 24-well plate of complete culture
medium. For the transfection, the medium is removed and replaced
with 0.5 ml of complete medium containing 8 .mu.g of
J-20.8/polylysine or of F-14.6/polylysine, or 20 .mu.g of J-20.8
polyclonal IgG and 2 .mu.g of plasmid or 2 .mu.g of plasmid alone.
After 6 hours, the medium is changed. The transfection efficiency
is tested 24 hours after the start of transfection.
Control of Transfection
[0096] Plasmid pHuVim 830 T, t: The synthesis of the T antigen in
the nucleus of the transfected cells is demonstrated by an
immunocytochemical method. The cells are washed 3 times with PBS
and then fixed for minutes in methanol at -20.degree. C. They are
then incubated with the anti-T antigen antibody coupled to
peroxidase for 1 hour. After washing, the peroxidase is revealed
with the DAB+H.sub.2O.sub.2 mixture. In the well incubated with the
J-20.8 polylysine and plasmid complex, isolated cells and a few
clusters of cells have a nucleus which is intensely coloured brown
after 48 hours and after 2 weeks. The control with the native
antibody or the plasmid alone is negative.
[0097] Plasmid pCMV-Luc: The transfection efficiency is
demonstrated by the luciferase synthesis detectable in the lysates
of the transfected cells. This enzyme catalyses the oxidation of
luciferin which results in a product detectable in a luminometre.
After the culture, the cells are washed in PBS and then lysed in 25
mM tris-phosphate buffer, pH 7.8, containing 8 mM MgCl.sub.2, 1 mM
DTT, 1% triton X100, 1% BSA and 15% glycerol. The lysate is assayed
in a luminometre by automated addition of a solution of luciferin
(0.25 mM) and of ATP (1 mM). An aliquot of the same lysate is
assayed for its protein concentration using a Coomassie (Bio-Rad
Protein Assay) reagent. The results are expressed in units (RLU)
per mg of proteins. As shown in the table, a gene transfer takes
place in the presence of the antibody preparations
J-20.8/polylysine and F-14.6/polylysine whereas the IgG/polylysine
preparations have no effect.
[0098] The transfection efficiency for the same antibody/plasmid
ratio is 10 times higher with the J-20.8 preparation than with
F-14.6. Furthermore, the addition of concentrated polyclonal IgG's
during the coupling to polylysine appears to increase the
transfection efficiency since 2 .mu.g of J-20.8 (complex 20:0.5) of
the J-20.8-IgG/polylysine preparation give results of the same
order of magnitude as 8 .mu.g (complex 8:0.5) of the
J-20.8/polylysine preparation.
[0099] The entire results obtained are summarized in the following
table:
TABLE-US-00001 Antibody/plasmid Assay Immunovector ratio
.mu.g/.mu.g RLU/mg .times. 10.sup.4 J-20.8-IgG/ 20:0.5 5.8
polylysine 20:0.5 63.00 J-20.8/ 8:2 2.00 polylysine 8:1 15.00 8:1
38.00 8:0.5 13.00 F-14.6/polylysine 8:0.5 1.3 IgG/polylysine 20:0.5
<0.1 20:0.5 <0.1 Plasmid 2 <0.1 alone (.mu.g) 1 <0.1
0.5 <0.1
[0100] In the preceding text, the preferred immunovectors consisted
essentially of the anti-DNA antibodies or of fragments of these
antibodies, with the proviso that these fragments retain the site
of recognition for the whole DNA. Naturally, it goes without saying
that the immunovectors which can be used within the framework of
the invention may be prepared in any other way, as long as they
would also allow the transport of the biologically active principle
which would be associated therewith through the membranes of these
cells and their cytoplasm and its transfer close to the nucleus of
the cells, or even inside this nucleus.
[0101] By way of examples of such immunovectors, there may be
mentioned conjugates between a nuclear protein, for example a
histone, a protein hnRNP, a polymerase or a factor associated with
this polymerase, and the active product, this nuclear protein being
itself (unless it is capable, on its own, of bringing about the
internalization and the transfer of a biologically active principle
into the nucleus of the cells, conjugated to the anti-cell membrane
receptor antibody or to any other molecule allowing the
internalization into the cell of the conjugate thus produced. As
long as this conjugate is capable of diffusing as far as the
nucleus of the cells and that, moreover, it may in turn transport
and transfer, as was defined above, a biologically active principle
which would be coupled to this conjugate, it constitutes an
immunovector entering within the framework of the present
invention.
[0102] The selection techniques which have been described above for
the choice of efficient immunovectors for the transfer of an active
principle to the nucleus of the cells are equally applicable to the
selection of the abovementioned conjugates.
[0103] Persons skilled in the art will understand that an
additional criterion for choice may, at least for some of the
conjugates used, lie in the absence of an undesirable action of the
nuclear protein which it contains with the cell function. It is in
fact to be noted that only the part of the intranuclear protein
carrying its site of recognition of the corresponding DNA is
essential for the embodiment in accordance with the invention.
LEGEND TO THE FIGURES
[0104] FIG. 1: Transfer of fluorescein "in vitro"
[0105] Labelling of the nuclei of the GMA 32 fibroblasts with an
immunovector labelled with fluorescein (antibody J-20.8) in A and B
(.times.100 magnification).
[0106] C: Absence of labelling with the fluorescent control
antibody (.times.100).
[0107] D: Another field observed at low magnification
(.times.40).
[0108] FIG. 2: Same preparation as that of FIG. 1A analysed under a
confocal microscope. The GMA 32 fibroblasts are labelled
essentially in the nucleus. A total fluorescence may be noted in
FIG. 2A. FIG. 2B corresponds to the analysis of the fluorescence
intensity.
[0109] FIG. 3: Transfer of fluorescein "in vivo" FACS analysis of
mouse peripheral blood cells injected hours beforehand in FIG. 3A,
the fluorescent control antibody and in FIG. 3B with a fluorescent
immunovector the antibody (J-20.8). Histogrammes representing on
the x-axis the fluoresence intensity (in arbitrary units) and on
the y-axis the number of cells.
[0110] FIG. 4: Analysis of the peripheral blood cells (of FIG. 3)
by confocal microscopy. A total fluorescence can be noted in FIG.
4A. FIG. 4B corresponds to analysis of the fluorescence
intensity.
[0111] FIG. 5: Transfer of a nucleotide "in vitro" Confocal
microscopy examination of a mouse splenocyte into which has
penetrated the Fab' fragment of an immunovector (antibody J-20.8)
coupled to a fluorescent nucleotide. A total fluorescence is
observed in FIG. 5A. FIG. 5B corresponds to analysis of the
fluorescence intensity.
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