U.S. patent application number 11/244067 was filed with the patent office on 2006-06-29 for adenoviral vectors encoding an antibody fused to a cd4 extracellular domain.
This patent application is currently assigned to TRANSGENE S.A.. Invention is credited to Pierre Leroy, Majid Mehtali.
Application Number | 20060140920 11/244067 |
Document ID | / |
Family ID | 9466896 |
Filed Date | 2006-06-29 |
United States Patent
Application |
20060140920 |
Kind Code |
A1 |
Leroy; Pierre ; et
al. |
June 29, 2006 |
Adenoviral vectors encoding an antibody fused to a CD4
extracellular domain
Abstract
Genetically modified cell implant comprising an exogenous
nucleotide sequence coding for all or part of an antibody, method
for the preparation of such an implant and its therapeutic use for
the treatment or prevention of an acquired disease. The invention
also concerns an adenoviral vector for the expression of one or
more proteins capable of forming a multimer, viral particles and
cells containing the adenoviral vector, a pharmaceutical
composition and its therapeutic use.
Inventors: |
Leroy; Pierre;
(Ernolsheim-les-Saverne, FR) ; Mehtali; Majid;
(Plobsheim, FR) |
Correspondence
Address: |
BUCHANAN INGERSOLL PC;(INCLUDING BURNS, DOANE, SWECKER & MATHIS)
POST OFFICE BOX 1404
ALEXANDRIA
VA
22313-1404
US
|
Assignee: |
TRANSGENE S.A.
Strasbourg
FR
|
Family ID: |
9466896 |
Appl. No.: |
11/244067 |
Filed: |
October 6, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09927933 |
Aug 13, 2001 |
6960469 |
|
|
11244067 |
Oct 6, 2005 |
|
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Current U.S.
Class: |
424/93.21 ;
424/133.1 |
Current CPC
Class: |
C12N 2710/10343
20130101; A61K 48/00 20130101; C12N 2840/203 20130101; C07K 2319/00
20130101; C12N 2740/13043 20130101; A61K 38/00 20130101; C07K 16/10
20130101; A61P 31/18 20180101; A61P 43/00 20180101; A61P 35/00
20180101; A61K 9/0024 20130101; C12N 15/86 20130101; C07K 16/3046
20130101; C12N 2840/20 20130101; A61P 31/04 20180101; A61P 37/04
20180101 |
Class at
Publication: |
424/093.21 ;
424/133.1 |
International
Class: |
A61K 48/00 20060101
A61K048/00; A61K 39/395 20060101 A61K039/395 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 13, 1994 |
FR |
94 10911 |
Claims
1. Implant of genetically modified cells comprising an exogenous
nucleotide sequence encoding all or part of an antibody, said
exogenous nucleotide sequence being placed under the control of the
elements necessary for its expression and for the secretion of said
antibody.
2. Implant according to claim 1, wherein said antibody is selected
from the group consisting of: a native antibody, a chimeric
antibody, an antibody fragment, especially a fragment Fab,
F(ab').sub.2, Fc or scFv, and a bispecific antibody.
3. Implant according to claim 1, wherein said antibody is modified
by a toxic or immunopotentiating substance.
4. Implant according to claim 3, wherein said antibody may be
modified by a toxic substance selected from a ribonuclease, and
especially the ribonuclease from Bacillus amyloliquefaciens, ricin,
diphtheria toxin, cholera toxin, herpes simplex virus thymidine
kinase, cytosine deaminase from Escherichia coli or from a yeast of
the genus Saccharomyces, exotoxin from Pseudomonas and human
angiogenin or an analog of said substances.
5. Implant according to claim 1, wherein cells are genetically
modified by transfection of a vector derived from a plasmid, from a
retrovirus or from a herpes virus, from an adenovirus, from an
adenovirus-associated virus comprising said exogenous nucleotide
sequence placed under the control of the elements necessary for its
expression and for the secretion of the said antibody.
6. Implant according to claim 5, wherein said vector is
dicistronic.
7. Implant according to claim 6, wherein said vector is retroviral
and comprises from 5' to 3': (a) a 5' LTR derived from a
retrovirus, (b) an encapsidation region, (c) an exogenous
nucleotide sequence comprising: an internal promoter, a first
sequence encoding the heavy chain of an antibody, a ribosome entry
initiation site, a second sequence encoding the light chain of an
antibody, and (d) a 3' LTR derived from a retrovirus.
8. Implant according to claim 7, wherein said exogenous nucleotide
sequence comprises, in addition, a third sequence encoding a toxic
or immunopotentiating substance fused downstream and operably to
the second sequence.
9. Implant according to claim 1, comprising genetically modified
autologous cells.
10. Implant according to claim 9, comprising genetically modified
fibroblasts.
11. Implant according to claim 1, wherein from 10.sup.6 to
10.sup.12, preferably from 10.sup.7 to 10.sup.11 genetically
modified cells.
12. Method for the preparation of an implant according to claim 1,
wherein the genetically modified cells and an extracellular matrix
are placed in contact.
13. A pharmaceutical composition intended for the treatment or for
the prevention of an acquired disease comprising the implant
according to claim 1.
14. A pharmaceutical composition intended for the treatment or for
the prevention of an infectious disease cancer comprising the
implant according claim 1.
15. Recombinant adenoviral vector comprising an exogenous
nucleotide sequence encoding all or part of an antibody and placed
under the control of the elements necessary for its expression,
wherein said antibody is modified by a toxic or immunopotentiating
substance.
16. Recombinant adenoviral vector according to claim 15, the
wherein said antibody is selected from the group consisting of a
native antibody, a chimeric antibody, an antibody fragment and
especially a fragment F(ab').sub.2, Fc or scFv and a bispecific
antibody.
17. Recombinant adenoviral vector according to claim 15, wherein
said antibody may be modified by a toxic substance selected from a
ribonuclease, and especially the ribonuclease from Bacillus
amyloliquefacienis, ricin, diphtheria toxin, cholera toxin, herpes
simplex virus thymidine kinase, cytosine deaminase from Escberichia
coli or from a yeast of the genus Saccharomyces, exotoxin from
Pseudomonas and human angiogenin or an analog of said
substances.
18. Recombinant adenoviral vector according to claim 15, wherein
said antibody is modified by an immunopotentiating substance.
19. Recombinant adenoviral vector comprising an exogenous
nucleotide sequence encoding all or part of one or more protein(s)
of interest capable of forming a multimer, such as a dimer or a
tetramer, in a host cell; the said exogenous nucleotide sequence
being placed under the control of the elements necessary for its
expression, said vector being derived from an adenovirus of human,
canine, avian, bovine, murine, ovine, porcine or simian origin or a
hybrid comprising adenoviral genome fragments of different
origins.
20. Recombinant adenoviral vector according to claim 15, derived
from an adenovirus of human, canine, avian, bovine, murine, ovine,
porcine or simian origin or from a hybrid comprising adenoviral
genome fragments of different origins.
21. Recombinant adenoviral vector according to claim 15, wherein it
is defective for replication.
22. Recombinant adenoviral vector according to claim 21, wherein it
at least lacks all or part of the E1 region and, optionally, all or
part of the E3 region.
23. Recombinant adenoviral vector according to claim 21, comprising
an exogenous nucleotide sequence encoding the heavy chain of the
2F5 antibody, an IRES element and the light chain of the 2F5
antibody; said exogenous nucleotide sequence being placed under the
control of the elements necessary for its expression.
24. Recombinant adenoviral vector according to claim 21, comprising
an exogenous nucleotide sequence encoding the signal sequence and
the extracellular I and II domains of the CD4 protein operably
fused to the constant .gamma.3 region (hinge region-CH2 and CH3) of
the heavy chain of the 2F5 antibody.
25. Recombinant adenoviral vector according to claim 21, comprising
an exogenous nucleotide sequence encoding the signal sequence and
the extracellular I and II domains of the CD4 protein operably
fused to the constant .gamma.3 region (hinge region-CR2 and CR3) of
the heavy chain of the 2F5 antibody and operably fused to the
mature human angiogenin.
26. Recombinant adenoviral vector according to claim 15, wherein
the elements necessary for the expression comprise a promoter
selected from the group consisting of the adenoviral early promoter
E1A, the late promoter MLP (Major Late Promoter), the murine or
human PGK (Phosphoglycerate kinase) promoter, the SV40 virus early
promoter, the RSV (Rous Sarcoma virus) virus promoter, a promoter
which is specifically active in tumor cells and finally a promoter
which is specifically active in the infected cells.
27. Infectious viral particle comprising a recombinant adenoviral
vector according to claim 15.
28. Eukaryotic host cell comprising a recombinant adenoviral vector
according to claim 15.
29. Pharmaceutical composition comprising a recombinant adenoviral
vector according to claim 15, in association with a
pharmaceutically acceptable carrier.
30. Pharmaceutical composition according to claim 29, comprising
10.sup.4 to 10.sup.14 pfu.
31. Pharmaceutical composition according to claim 29 wherein it is
in injectable form.
32. A method for the preparation of a pharmaceutical composition
intended for the treatment and/or prevention of the human or animal
body by gene therapy comprising using the recombinant adenoviral
vector according to claim 15.
33. The method according to claim 32, wherein said pharmaceutical
composition is intended for the treatment and/or prevention of
acquired diseases and especially cancers and AIDS.
34. The method according to claim 33, wherein said pharmaceutical
composition administrable via the intravenous or intratumor route.
Description
[0001] The present invention relates to a novel type of implant and
its use for the treatment and prevention of cancer or of AIDS. More
particularly, its subject is an implant comprising genetically
modified cells capable of expressing and of secreting specific
antibodies recognizing cancer cells or infected cells so as to
inhibit at least partially their division or propagation as well as
the production of viral particles in the infected cells. The
present invention also relates to an adenoviral vector capable of
directing the expression of a multimeric protein of interest as
well as an antibody or one of its derivatives.
[0002] The possibility of treatments of human diseases by gene
therapy has gone within a few years from the theoretical
considerations stage to that of clinical applications. The first
procedure applied to man was thus initiated in the United States in
September 1990 on a genetically immunodeficient patient because of
a mutation affecting the gene encoding Adenine Deaminase (ADA). The
relative success of this first experimentation encouraged the
development of new gene therapy procedures for various genetic or
acquired diseases. Those currently under experimentation consist,
for the most part, in transferring ex vivo the therapeutic gene
into the patient's cells, for example the stem cells of the
hematopoietic line, and then reinfusing these corrected cells into
the patient. It is therefore a nonreversible, cumbersome technology
which carries the risk of reimplanting transformed cells.
[0003] More recently initiated, the neo-organ technology makes it
possible to overcome the major disadvantages of the conventional
gene therapy procedures. It is based on the reimplantation, in the
patient, of an artificial structure which may be called "implant"
and comprising living cells, which are real "micro-factories" which
make it possible to deliver the therapeutic molecule of interest in
vivo and continuously.
[0004] More precisely, this artificial structure consists of living
cells previously transduced by a viral vector carrying the
therapeutic gene, which are included in a collagen gel coating a
backbone of synthetic fibers of a biocompatible material (PTFE,
polytetrafluoroethylene or Gore-Tex.TM.). This gel also contains an
angiogenic growth factor (bFGF, basic Fibroblast Growth Factor).
After its reimplantation in the animal, the neo-organ is generally
vascularized within a few days by virtue of the angiogenic and
trophic properties of bFGF. It then develops into an autonomous
structure, provided with a connective, sometimes innerated, tissue
and linked to the bloodstream into which the therapeutic molecules
are poured.
[0005] The possibility of using neo-organs for gene therapy has
already been raised in several scientific articles as well as in
international application WO 92/15676. However, the technology
disclosed in the prior art documents deals with only the treatment
of monogenic genetic diseases resulting from the defective and
innate expression of a single gene, and has consequently been used
only for the secretion of monomeric therapeutic molecules such as
factor IX, .alpha..sub.1-antitrypsin, ADA, erythropoietin (EPO) and
.beta.-glucuronidase. Up until now, this technology has not been
suited to the secretion of more complex therapeutic molecules such
as antibodies.
[0006] It has now been found that an implant of fibroblasts,
genetically modified by a retroviral vector for the expression of
the heavy and light chains of an anti-HIV antibody, once
reimplanted in a mouse, is capable of continuously secreting into
the bloodstream a large quantity of functional antibodies
recognizing the infected cells carrying, at their surface, the
antigen against which it is directed. The present invention is
based on the fact that a fibroblast is capable of producing roughly
stoichiometric quantities of heavy and light chains of an antibody
capable of then associating into a tetramer to form a functional
molecule. It offers the possibility of treating, by immunotherapy,
acquired diseases and especially AIDS and cancer, two diseases
whose complexity, seriousness as well as the absence of really
satisfactory treatments, justify the development of novel
technologies, such as that which is the subject of the present
invention.
[0007] The present invention also provides adenoviral vectors
capable of directing the expression of multimeric molecules of
interest as well as of antibodies and derivatives thereof. They can
be used to produce an immunotoxin directed against the HIV virus
and to induce the selective destruction of infected cells.
[0008] Accordingly, the subject of the present invention is:
[0009] (1) an implant of genetically modified cells comprising an
exogenous nucleotide sequence encoding all or part of an antibody,
the said exogenous nucleotide sequence being placed under the
control of the elements necessary for its expression and for the
secretion of the said antibody, and
[0010] (2) a recombinant adenoviral vector comprising an exogenous
nucleotide sequence encoding all or part of one or more protein(s)
of interest capable of forming a multimer in a host cell; the said
exogenous nucleotide sequence being placed under the control of the
elements necessary for its expression in the said host cell.
[0011] For the purposes of the present invention, an implant
designates any set of genetically modified living cells, as defined
below and intended to be implanted in the human or animal body.
Most particularly preferred is the case where the cells are
attached to an extracelluar matrix, the whole forming a
biocompatible and vascularizable structure. The matrix is
preferably composed of collagen. However, other materials may be
used within the framework of the present invention as long as they
are biocompatible. It comprises especially (1) a biocompatible
support such as synthetic fibers PTFE (polytetrafluoroethylene or
Gore-Tex) coated with a collagen film so as to allow cell adhesion
(2), a collagen gel in which the cells inside the implant are
included and (3) an angiogenic agent promoting vascularization in
the host. The term implant is a generic term which includes
especially neo-organs and organoids.
[0012] Moreover, this may also involve encapsulated implants, that
is to say included in a membrane of controlled porosity preventing
especially the passage of cells (cells of the implant and cells of
the host's immune system) but allowing the diffusion of the
therapeutic molecule, nutrients and waste.
[0013] The term "genetically modified cell" refers to a cell having
incorporated exogenous genetic material. The latter may be inserted
into the genome of the cell or be present in episome form either in
the cytoplasm or in the cell nucleus. The technology for
introducing an exogenous genetic material into a cell is
conventional and accessible to persons skilled in the art. In this
regard, numerous vectors have been developed and are widely
described in basic molecular biology manuals accessible to persons
skilled in the art.
[0014] The genetically modified cells in use within the framework
of the present invention comprise especially an exogenous
nucleotide sequence. The latter may be a natural sequence (already
present in the genome of the host cell) or a heterologous sequence,
but it will have been introduced into the host cells by genetic
engineering techniques (and therefore exogenously). Most
particularly preferred is a sequence encoding a product which is
not normally expressed therein or, if it is, at physiologically low
concentrations. In accordance with the aims pursued by the present
invention, the exogenous nucleotide sequence encodes all or part of
an antibody. An antibody is a protein (immunoglobulin) normally
produced by the B lymphocytes and which recognizes a specific
foreign antigen and triggers the immune response. A native antibody
is a tetramer composed of four protein chains: two light (L) chains
and two heavy (H for heavy) chains associated with each other via
disulfide bridges. The light chain consists of a variable region
(V.sub.L) at the N-terminal position and a constant region
(C.sub.L) at the C-terminal position whereas the heavy chain
comprises from the N to the C-terminal a variable region (V.sub.H)
followed by three constant regions (C.sub.H1, C.sub.H2 and
C.sub.H3). The corresponding regions of the light and heavy chains
associate to form distinct domains. The variable domain, formed by
the association of the variable regions of the light and heavy
chains of an immunoglobulin, is responsible for recognizing the
corresponding antigen. The constant domains exert effector
functions involved in the progress of the immune response.
[0015] For the purposes of the present invention, the two heavy and
light chains may be identical (native antibodies). In this context,
an exogenous nucleotide sequence is used which encodes a heavy
chain and a light chain which will associate into a tetramer after
their synthesis. However, a sequence may also be used which encodes
only part of an antibody so as to produce, preferably, a fragment
Fab (ab for antigen binding) or F(ab').sub.2, Fc (c for
crystallizable) or scFv (sc for single chain and v for variable).
Such fragments are described in detail in immunology manuals such
as Immunology (third edition, 1993, Roitt, Brostoff and Male, ed
Gambli, Mosby) and are schematically represented in FIG. 1. As
regards more specifically the scFv fragment, it may be obtained
from a sequence encoding a V.sub.L region followed by a V.sub.H
region with optionally a spacer (of 1 to 10 neutral amino acid
residues which are not very bulky) between the V.sub.L and V.sub.H
sequences.
[0016] It is also possible to generate a chimeric (or hybrid)
antibody derived from the fusion of sequences of diverse origins
(species or types of antibody). In particular, it is possible to
include or exchange constant regions derived from antibodies of
different isotopes so as to confer new properties on the chimeric
antibody, for example an enhancement of the cytotoxic reaction.
This may also be a humanized antibody combining at least part of
the variable regions of a mouse antibody and the constant regions
of a human antibody. It is also possible to fuse one or more
variable and/or constant regions or region parts of any origin, for
example derived from light/or heavy chains in the form of a
single-chain molecule.
[0017] Finally, another approach consists in producing a bispecific
antibody comprising two variable domains, for example a domain
recognizing an antigen carried by an infected or a tumor cell and
the other a structure for activation of the immune response. This
makes it possible to increase the activity of the killer cells in
contact with the tumor or with the infected cell.
[0018] It goes without saying that an antibody in use in the
present invention may have a sequence which is slightly different
from the native sequence of an antibody. In practice, the common
criterion for characterizing an antibody is its function, that is
to say its capacity to bind specifically to the antigen against
which it is directed. Numerous techniques which appear in general
immunology manuals make it possible to demonstrate an antibody
function, for example the ELISA, Western or fluorescence
techniques. The invention extends to an antibody whose sequence has
a degree of homology with the native sequence(s) (in the case of a
chimeric antibody) greater than 70%, advantageously greater than
80%, preferably greater than 90% and, most preferably, greater than
95%. Such an analogue may be obtained by mutation, deletion,
substitution and/or addition of one or more nucleotide(s) of the
corresponding sequence(s).
[0019] In accordance with the aims pursued by the present
invention, it is preferable to use an antibody directed against a
tumor antigen or an epitope specific for an infectious and
pathogenic microrganism, especially a virus and more particularly
the HIV virus and, advantageously, an antigen strongly represented
at the surface of the target cell. This type of antibody is widely
described in the literature. There may be mentioned especially:
[0020] the human monoclonal antibody 2F5 (Buchacher et al., 1992,
Vaccines, 92, 191-195) recognizing a continuous (ELDKWAS) (SEQ ID
NO: 21) and highly conserved epitope of the transmembrane
glycoprotein gp41 of the HIV-1 envelope molecule, [0021] the murine
monoclonal antibody 17-1-A (Sun et al., 1987, Proc. Natl. Acad.
Sci. USA, 84, 214-218) recognizing the GA733 glycoprotein present
at the surface of the human colorectal carcinoma cells, [0022] an
antibody directed against the protein MUC-1, and [0023] an antibody
directed against the E6 or E7 protein of the HPV virus (Human
Papillomavirus) especially type 16 or 18.
[0024] Within the framework of the present invention, the
nucleotide sequences encoding an antibody in use within the
framework of the present invention may be obtained by any
conventional technique in use in the field of genetic engineering,
such as PCR (Polymerase Chain Reaction), cloning and chemical
synthesis. Purely as a guide, the sequences encoding the heavy and
light chains of an antibody may be cloned by PCR using the
degenerate oligonucleotides recognizing the conserved sequences
found at the 5' and 3' ends of most immunoglobulin genes (Persson
et al., 1991, Proc. Natl. Acad. Sci. USA, 88, 2432-2436; Burton et
al., 1991, Proc. Natl. Acad. Sci. USA, 88, 10134-10137). The
antibody function of the expression product is then checked in
relation to a specific antigen as indicated above.
[0025] Another approach, which is moreover preferred, consists in
using an antibody which is modified especially by a toxic substance
or an immunopotentiating protein. This specific embodiment makes it
possible to destroy in vivo, by a local chemotherapy (toxic
substance), the target cell (cancer cell or infected cell) which
carries, at its surface, the specific antigen against which the
antibody part is directed or to enhance the immune reaction with
respect to it (immunopotentiating substance). In the context of the
toxic substance, it may be advantageous to choose antibodies which
may be endocytosed by the target cell. It goes without saying that
the corresponding sequences may be obtained by any conventional
technique in the state of the art.
[0026] The term "toxic substance" refers to a molecule having a
degradation activity drastically inhibiting cell growth or inducing
cell death. This may be a molecule which is toxic by itself or
indirectly, for example a protein catalyzing the synthesis of a
toxic substance. These molecules may be derived from plants,
animals or microorganisms. Of course, the toxic function may be
fulfilled by a native toxic substance (as found in nature) or an
analog thereof, which may be conventionally obtained by mutation,
deletion, substitution and/or addition of one or more nucleotide(s)
of the native sequence. Among the preferred toxic substances, there
may be mentioned a ribonuclease, ricin, diphtheria toxin, cholera
toxin, herpes simplex virus type 1 thymidine kinase (TK-HSV-1),
cytosine deaminase from Escherichia coli or from a yeast of the
genus Saccharomyces and the exotoxin from Pseudomonas. To
illustrate an immunopotentiating protein (whose function is to
improve the host organism's immune reaction toward the target
cell), there may be mentioned the CD4 protein, the high-affinity
receptor for the HIV-1 virus or an Fc receptor for IgG(Fc.gamma.R).
Its coupling to an antibody directed against an HIV virus antigen
or a tumor antigen will make it possible, consequently, to generate
a hybrid molecule having a ligand recognizing a killer cell and a
ligand recognizing the target cell so as to promote its elimination
more efficiently. In this context, a hybrid molecule may be used
which is obtained from the fusion between an anti-HIV antibody and
Fc.gamma.R or between the extracellular domain of the CD4 molecule
and an anti-CD3 antibody. However, these examples are not limiting
and such immunopotentiating proteins are known to persons skilled
in the art.
[0027] Advantageously, the toxic function is provided by a
ribonuclease which may be of prokaryotic or eukaryotic origin.
Among those which may be used within the framework of the present
invention, there may be mentioned colicin E6, cloacin from
Escherichia coli, nuclease from Staphylococcus, birnase from
Bacillus intermedius and nuclease from Bacillus amyloliquefaciens,
also designated by the name barnase, whose sequence is disclosed in
Hartley (1988, J. Mol. Biol., 202, 913-915). However, the use of
human angiogenin is most particularly preferred (Saxena et al.,
1991, J. Biol. Chem., 266, 21208-21214; Saxena et al., 1992, J.
Biol. Chem., 267, 21982-21986).
[0028] According to another variant, the toxic function may be
exerted by TK-HSV-1. It exhibits a greater affinity, compared with
the mammalian TK enzyme, for certain nucleoside analogs such as
acyclovir and ganciclovir and it converts them to nucleotide
precursors which are toxic for the cell. Consequently, their
incorporation into the DNA of replicating cells makes it possible
to kill specifically dividing cells, such as cancer cells, by a
toxic effect and/or by a proximity effect ("bystander" effect).
[0029] According to another embodiment of the invention, an
attenuated analog may be used which also exhibits a toxic function
but to a lesser degree compared with the native toxic substance.
Any mutant having an attenuated degradation activity may be used
within the framework of the invention. In this context, an
attenuated mutant of a ribonuclease may be used which exhibits an
activity attenuated by a factor of 10 to 10.sup.6 or better still
10 to 10.sup.5 and, most preferably, 10.sup.2 to 10.sup.4 compared
to the native ribonuclease from which it is derived. This variant
is based on the high toxicity of the ribonucleases to cellular
RNAs, which makes the molecular construction stages difficult. By
way of examples, there may be mentioned the attenuated mutants of
barnase K27A (Mossakowska et al., 1989, Biochemistry, 28,
3843-3850) and K27A, L89F (Natsoulis and Boeke, 1991, Nature, 352,
1632-1635). The nuclease activity may be evaluated in accordance
with the method described by Shapiro et al. (1987, Proc. Natl.
Acad. Sci. USA, 84, 8783-8787). Of course, it is possible to
measure it by other techniques too, as indicated in Example 2.
[0030] A particularly preferred construction consists in including
the nucleotide sequence encoding the said toxic or
immunopotentiating substance in 5' or in 3' of the nucleotide
sequence encoding all or part of an antibody. There is especially
preferred the case where it is introduced downstream of the
sequence encoding the heavy chain of an antibody, the said chain
being deleted of the stop codon for translation and the fusion
taking place in the correct reading frame. The fusion of two
sequences operably constitutes a conventional molecular biology
technique accessible to persons skilled in the art. Moreover, it is
possible to include, at the level of the fusion, a binding sequence
capable of being cleaved inside the target cell in order to release
the toxin. In this context, the term "exogenous nucleotide
sequence" refers to a sequence encoding all or part of an antibody
optionally fused to the said substance.
[0031] Of course, the said exogenous nucleotide sequence is placed
under the control of elements which are necessary for its
expression. "Elements which are necessary" is understood to mean
all the elements which are necessary for its transcription into
messenger RNA (mRNA) and for the translation of the latter into
protein. Among the elements which are necessary for the
transcription, the promoter is of particular importance. In
general, a promoter will be used which is functional in a
eukaryotic, and especially human, cell. This may be a constitutive
promoter or a regulatable promoter and it may be isolated from any
gene of eukaryotic or viral origin. Moreover, a promoter in use in
the present invention may be modified so as to contain regulatory
sequences, such as "enhancer" type activating sequences.
Alternatively, a promoter derived from immunoglobulin genes may be
used when it is desired to target a lymphocytic host cell.
Nevertheless, it will be preferable to use a constitutive promoter
allowing expression in a large number of cell types and especially
a promoter of a housekeeping gene such as the promoter of the
TK-HSV-1 gene, the adenoviral promoter E1A, MLP (for Major Late
promoter), the murine or human PGK (phosphoglycerate kinase)
promoter, the promoter of the rat .beta.-actin (ACT) gene, the HPRT
(Hypoxantyl Phosphoribosyl Transferase) promoter, the HMG
(Hydroxymethyl-Glutaryl coenzyme-A) promoter, the RSV (Rous Sarcoma
Virus) promoter, the SV40 virus (Simian Virus) early promoter or
the DHFR (Dihydrofolate Reductase) promoter. As a guide, when the
nucleotide sequence is incorporated into a retroviral vector, the
5' LTR may be used as promoter. However, it is most particularly
preferable to use an internal nonretroviral promoter, such as those
specified earlier.
[0032] The exogenous nucleotide sequence may, in addition, contain
other elements contributing to its expression both at the level of
transcription and translation, especially an intron sequence
bordered by appropriate splicing signals, a nuclear localization
sequence, a sequence for initiation of translation, the elements
for termination of transcription (polyadenylation signal), and/or a
sequence encoding a secretory signal. The said sequence may be
homologous, that is to say derived from the gene encoding the
antibody in question, or heterologous, that is to say derived from
any gene encoding a precursor of a secreted expression product. The
choice of such elements is wide and accessible to persons skilled
in the art.
[0033] For the purposes of the present invention, the exogenous
nucleotide sequence provided with the elements necessary for its
expression is introduced into a host cell to give a genetically
modified cell. All the procedures which make it possible to
introduce a nucleic acid into a cell may be used, such as for
example precipitation with calcium phosphate, DEAE dextran
technique, direct injection of nucleic acid into the host cell, the
bombardment of gold microparticles covered with nucleic acid or the
use of liposomes or of cationic lipids. However, within the
framework of the present invention, the exogenous nucleotide
sequence is preferably inserted into an expression vector. In
particular, it may be of the plasmid type or derived from an animal
virus and especially a retrovirus, an adenovirus, an
adenovirus-associated virus or a herpes virus. However, the use of
an integrative vector is preferred. The choice of such a vector is
wide and the techniques for cloning into the vector selected are
accessible to persons skilled in the art. Likewise, the process to
be used to generate infectious viral particles is known.
[0034] A first vector which is particularly appropriate for the
present invention is an adenoviral vector (see below).
[0035] According to another, also advantageous, alternative, a
retroviral vector is used. The numerous vectors described in the
literature may be used within the framework of the present
invention and especially those derived from the Moloney murine
leukemia virus (MoMuLV) or from the Friend's virus (FrMuLV). In
general, a retroviral vector in use in the present invention is
deleted of all or part of the viral genes gag, pol and/or env and
comprises a 5' LTR, an encapsidation region and a 3' LTR. The
exogenous nucleotide sequence is inserted preferably downstream of
the encapsidation region. The propagation of such a vector requires
the use of complementation lines described in the prior art, such
as the lines CRE, GP+E-86, PG13, Psi Env-am-12, pA317 and
psi-CRIP.
[0036] According to a preferred embodiment and as regards producing
an antibody which is other than a single chain (comprising for
example two heavy and light protein chains), the use of a
dicistronic vector allowing the synthesis of two translational
products from a single mRNA is preferred. The initiation of
translation of the second translational product is preferably
provided by an IRES site (for Internal Ribosome Entry Site, that is
to say an internal site for entry of the ribosomes). A number of
IRES sites have so far been identified and there may be mentioned
that of the poliomyelitis virus (Pelletier et al., 1988, Mol. Cell.
Biol., 8, 1103-1112), of EMCV (Encephalomyocarditis Virus) (Jang et
al., J. Virol., 1988, 62, 2636-2643) or those described in
international application WO 93/03143. But other IRES sites may
also be used. This type of construction may be appropriate for any
vector in use within the framework of the invention.
[0037] One of the preferred vectors within the framework of the
present invention is a retroviral vector which comprises from 5' to
3':
(a) a 5' LTR derived from a retrovirus,
(b) an encapsidation region,
(c) an exogenous nucleotide sequence comprising:
[0038] an internal promoter
[0039] a first sequence encoding the heavy chain of an
antibody,
[0040] a ribosome entry initiation site,
[0041] a second sequence encoding the light chain of an antibody,
and
(d) a 3' LTR derived from a retrovirus.
[0042] Another preferred retroviral vector comprises an exogenous
nucleotide sequence provided with the murine PGK promoter followed
by a first sequence encoding the extracellular I and II domains of
the CD4 molecule and a second sequence fused in phase with the
first and encoding the .gamma.3 segment of the heavy chain of the
antibody 2F5 (sCD4-2F5) and, optionally, a third sequence encoding
human angiogenin operably linked to the second.
[0043] It goes without saying that the order of the first, second
and third sequences may be reversed. Moreover, as indicated above,
the exogenous nucleotide sequence may comprise a sequence encoding
a toxic or immunopotentiating substance. The latter will be
preferably inserted downstream of the first sequence encoding the
heavy chain of an antibody. However, the present invention is not
limited to this specific embodiment.
[0044] Moreover, a vector in use within the framework of the
invention may also contain other elements, for example, a gene
encoding a selectable marker which makes it possible to select or
identify the host cells transfected. There may be mentioned the neo
gene which confers resistance to the antibiotic G418, the dhfr
gene, the CAT (chloramphenicol Acetyl Transferase) gene, the
puromycin acetyl transferase (pac or PURO) gene or the gpt
(xanthine guanine phosphoribosyl transferase) gene.
[0045] A genetically modified cell is preferably chosen so as to be
tolerated by the immune system of the host organism in which it is
envisaged to graft an implant according to the invention. In this
context, a nontumor and transfectable cell is most particularly
preferred. They may be autologous cells removed or derived from
this host organism, but also cells which are capable of being
tolerated following an appropriate chemical or genetic treatment
(it is for example possible to envisage repressing the expression
of the surface antigens normally recognized by the host organism's
immune system). It is also possible to use a syngenic cell or an
allogenic cell of the same haplotype as the host organism as
regards the major histocompatibility complex class II antigens.
[0046] Preferably, a genetically modified cell results from the
introduction of the exogenous nucleotide sequence into autologous
fibroblasts and, in particular, fibroblasts removed from the skin
of a host organism. However, other cell types may be used, such as
endothelial cells, myoblasts, lymphocytes and hepatocytes. Although
not a preferred embodiment, it is also possible to use tumor cells
(optionally attenuated by radiotherapy) removed from a host
organism having tumors, in order to modify their gene pool and make
them capable of inhibiting or slowing down tumor progression.
[0047] Advantageously, an implant according to the invention
comprises from 10.sup.6 to 10.sup.12, preferably from 10.sup.7 to
10.sup.11, and most preferably from 10.sup.8 to 10.sup.10
genetically modified cells.
[0048] The present invention also relates to a method for the
preparation of an implant according to the invention in which the
genetically modified cells and an extracellular matrix are placed
in contact. Various techniques may be used to generate an implant
according to the invention. The procedure is preferably carried out
in the following manner: the genetically modified cells are brought
into contact with a liquid collagen solution, preferably of type I,
with a biocompatible support consisting, for example, of synthetic
Gore-Tex fibers coated with collagen and with at least one
angiogenic growth factor, for example bFGF or VEGF (Vascular
Endothelial Growth Factor). The whole is placed at 37.degree. C. so
that the collagen solution forms a gel with a dense meshwork which
includes the cells and then cultured for 4 to 5 days in vitro so as
to allow the genetically modified cells to colonize the implant. It
is desirable to carry out the last stage of culture in a medium
containing at least one angiogenic factor or a combination of two
or more. In general, the techniques which make it possible to
generate an implant and the culture conditions are known to persons
skilled in the art.
[0049] An implant according to the invention is intended to be
transplanted in a host, animal or, preferably, human organism so as
to produce a therapeutic (curative and/or preventive) effect
therein. Transplanted in a laboratory animal, it will make it
possible, in particular, to evaluate therapeutic procedures
applicable to man. The site of reimplantation is preferably the
peritoneal or subcutaneous, intrarachidian or intraabdominal
cavity.
[0050] The invention also extends to the therapeutic use of an
implant according to the invention for the preparation of a
pharmaceutical composition intended more particularly for the
treatment and/or prevention of an acquired disease such as cancer
or an infectious disease caused by a pathogenic microorganism
(virus, parasite or bacterium). It relates especially to the
treatment: [0051] of cancer of the uterus induced by a
papillomavirus against which an implant will be used comprising
autologous fibroblasts into which a sequence encoding an anti-HPV
(in particular of type 16 or 18) E6 or E7 antibody has been
introduced, [0052] of breast cancer using an anti-MUC1 antibody,
[0053] of AIDS using an antibody directed against an envelope
glycoprotein epitope conserved in numerous isolates, [0054] of
hepatitis using an antibody directed against an epitope of the
hepatitis B or C virus.
[0055] Of course, these antibodies may be modified by fusion
especially to angiogenin, barnase or TK-HSV-1.
[0056] The invention also relates to a method for the treatment or
prevention of acquired diseases according to which an implant
according to the invention is generated in vitro and it is
transplanted into a patient requiring such a treatment. The sites
of reimplantation may be varied as mentioned above. Once the
desired therapeutic effect is obtained, the implant simply has to
be surgically removed from the patient.
[0057] Naturally, the modalities of the therapeutic procedure have
to be developed by the clinician according to the patient and the
disease to be treated. This procedure may be subject to numerous
variants such as the number of implants according to the invention
to be transplanted, the site of implantation and the type of
antibody secreted as well as the level of expression. Purely as a
guide, a level of expression in the patient's serum of at least 50
ng/ml of functional antibody, advantageously of at least 100 ng/ml,
preferably of at least 200 ng/ml and, most preferably, of at least
500 ng/ml, is preferred. A functional antibody is an antibody
capable of recognizing the antigen against which it is directed.
The functionality may be determined for example by ELISA or FACS.
On the other hand, when an antibody fused to TK-HSV-1 is used, it
is desirable to include in the therapeutic procedure the
administration of acyclovir or of ganciclovir so that its toxic
effect may be exerted.
[0058] Moreover, the present invention also relates to a
recombinant adenoviral vector comprising an exogenous nucleotide
sequence encoding all or part of one or more protein(s) capable of
forming a multimer in a host cell and, preferably, a dimer or a
tetramer. For the purposes of the present invention, a recombinant
adenoviral vector according to the invention may be used alone to
combat an infection induced by a pathogenic organism or the
establishment/propagation of a tumor in an organism or a host cell.
According to a completely preferred embodiment, a recombinant
adenoviral vector according to the invention comprises an exogenous
nucleotide sequence as defined above (intended to express an
antibody or one of its derivatives such as a fragment, a modified,
chimeric antibody and the like).
[0059] A recombinant adenoviral vector according to the invention
is preferably derived from a human adenovirus serotype C and, more
particularly, type 2, 5 or 7. However, there may also be used other
adenoviruses, especially of animal (canine, bovine, murine, avian,
ovine, porcine or simian) origin or a hybrid between a variety of
species. There may be mentioned more particularly the canine
adenovirus CAV-1 or CAV-2, the avian adenovirus DAV or the bovine
adenovirus Bad type 3 (Zakharchuk et al., 1993, Arch. Virol., 128,
171-176; Spibey and Cavanagh, 1989, J. Gen. Virol., 70, 165-172;
Jouvenne et al., 1987, Gene, 60, 21-28; Mittal et al., 1995, J.
Gen. Virol., 76, 93-102). The general technology relating to
adenoviruses is disclosed in Graham and Prevec (1991, Methods in
Mol. Biol., Vol. 7, Gene Transfer and Expression Protocols, Ed:
Murray, The Human Press Inc., p109-118).
[0060] An advantageous embodiment of the present invention consists
in using a vector which is defective for one or more viral
function(s) which is (are) essential for replication, because of
the deletion or non-functionality of one or more viral genes
encoding the said function. Such a vector, which is incapable of
autonomous replication, will be propagated in a complementation
cell capable of providing en trans the early and/or late proteins
which it cannot itself produce and which are necessary for the
constitution of an infectious viral particle. The latter term
designates a viral particle having the capacity to infect a host
cell and to cause the viral genome to penetrate therein. By way of
illustration, to propagate an adenoviral vector which is defective
for the E1 function, there will be used a complementation cell such
as the line 293 capable of providing en trans all the proteins
encoded by the E1 region (Graham et al., 1977, J. Gen. Virol. 36,
59-72). Of course, a vector according to the invention may comprise
additional deletions, especially in the nonessential E3 region so
as to increase the cloning capacities, but also in the essential
E2, E4, L1-L5 regions (see international application WO 94/28152).
The defective functions may be complemented with the aid of a cell
line or a helper virus.
[0061] A preferred adenoviral vector according to the invention is
deleted of most of the E1 and E3 regions and carries, in place of
the E1 region, an expression cassette comprising:
(a) a promoter, the intron of the human .beta.-globin (BGL) gene,
the sequences encoding the light chain of 2F5, the IMES site of the
EMCV virus and the heavy chain of 2F5 and then the polyadenylation
site of the human .beta.-globin gene, or
(b) a promoter, the intron of the human .beta.-globin gene, the
sequences encoding the molecule sCD4-2F5 optionally fused at the
C-terminus and in the same reading frame to human angiogenin.
[0062] Among the promoters which may be envisaged within the
framework of the present invention, there may be mentioned the
adenoviral early promoter E1A, the late promoter MLP (Major Late
Promoter), the murine or human PGK (Phosphoglycerate Kinase)
promoter, the SV40 virus early promoter, the RSV (Rous Sarcoma
Virus) virus promoter, a promoter which is specifically active in
tumor cells and finally a promoter which is specifically active in
the infected cells.
[0063] The invention also relates to an infectious adenoviral
particle as well as to a eukaryotic host cell comprising a
recombinant adenoviral vector according to the invention. The said
host cell is advantageously a mammalian cell and, preferably, a
human cell and may comprise the said vector in a form integrated in
the genome or nonintegrated (episome). This may be a primary or
tumor cell of hematopoietic origin (totipotent stem cell,
leukocyte, lymphocyte, monocyte or macrophage and the like), or of
muscle, hepatic, epithelial or fibroblast origin.
[0064] An infectious viral particle according to the invention may
be prepared according to any conventional technique in the state of
the art (Graham and Prevect, 1991, supra), for example, by
cotransfection of a vector and of an adenoviral fragment into an
appropriate cell or by means of a helper virus providing en trans
the non-functional viral functions. It is also possible to envisage
generating the viral vector in vitro in Escherichia coli (E. coli)
by ligation or homologous recombination (see for example French
Application 94 14470).
[0065] The subject of the invention is also a pharmaceutical
composition comprising, as therapeutic or prophylactic agent, an
adenoviral vector, an infectious viral particle or a eukaryotic
host cell according to the invention in combination with a
pharmaceutically acceptable carrier. The composition according to
the invention is in particular intended for the preventive or
curative treatment of acquired diseases such as cancers, viral
diseases such as AIDS, hepatitis B or C or recurrent viral
infections caused by the herpes virus.
[0066] A pharmaceutical composition according to the invention may
be produced in a conventional manner. In particular, a
therapeutically effective quantity of a therapeutic or prophylactic
agent is combined with a carrier such as a diluent. A composition
according to the invention may be administered locally or
systemically or by aerosol. Especially preferred is the
intramuscular, intratumor and intrapulmonary administration and,
most particularly, intravenous injection. The administration may
take place in a single dose or in a dose which is repeated once or
several times after a certain interval of time. The appropriate
route of administration and dosage vary according to various
parameters, for example, the individual or the disease to be
treated or the gene(s) of interest to be transferred. In
particular, the viral particles according to the invention may be
formulated in the form of doses of between 10.sup.4 and 10.sup.14
pfu (plaque forming units), advantageously 10.sup.5 and 10.sup.13
pfu and, preferably, 10.sup.6 and 10.sup.11 pfu. The formulation
may also include an adjuvant or an excipient which is acceptable
from a pharmaceutical point of view.
[0067] Finally, the present invention relates to the therapeutic or
prophylactic use of an adenoviral vector, an infectious viral
particle or a eukaryotic host cell according to the invention for
the preparation of a medicament intended for the treatment of the
human or animal body and, preferably, by gene therapy. According to
a first possibility, the medicament may be administered directly in
vivo (for example by intravenous injection, into an accessible
tumor, into the lungs by aerosol and the like). The ex vivo
approach may also be adopted which consists in removing cells from
the patient (bone marrow stem cells, peripheral blood lymphocytes,
muscle cells and the like), in infecting them in vitro according to
prior art techniques and in readministering them to the
patient.
[0068] The invention also relates to a method for the treatment or
prevention of acquired diseases according to which a
therapeutically effective quantity of a recombinant adenoviral
vector, an infectious adenoviral particle or a host cell according
to the invention is administered to a patient requiring such a
treatment.
[0069] The invention is illustrated, without however being limited,
by the following examples and with reference to the following
figures:
[0070] FIG. 1 is a schematic representation of the structure of an
antibody and of the F(ab) and Fc fragments.
[0071] FIG. 2 is a schematic representation of the vector pTG4370
allowing the expression of the antibody 2F5.
[0072] FIG. 3 is a schematic representation of the vector pTG6356
allowing the expression of the antibody 17-1-A coupled to the
native barnase.
[0073] FIG. 4 is a schematic representation of the vector pTG6357
allowing the expression of the antibody 17-1-A coupled to the
attenuated barnase K27A.
[0074] FIG. 5 is a schematic representation of the vector pTG6355
allowing the expression of the antibody 17-1-A.
[0075] FIG. 6 is a schematic representation of the structure of the
CD4 membrane protein.
[0076] FIG. 7 is a representation of the scheme for the
construction of sequences encoding the hybrid molecule sCD4-2F5
using primers OTG7094, OTG7095, OTG7096 and OTG7097 (SEQ ID NOS:
13, 14, 16 and 15).
[0077] FIG. 8 is a schematic representation of the retroviral
vector pTG8338 allowing the expression of the hybrid molecule
sCD4-2F5.
[0078] FIG. 9 is a schematic representation of the vector pTG83 73
comprising the sequences encoding the fusion molecule
sCD4-2F5-Angiogenin.
[0079] FIG. 10 is a schematic representation of the adenoviral
vector pTG8357 allowing the expression of the hybrid molecule
sCD4-2F5.
[0080] FIG. 11 is a schematic representation of the adenoviral
vector pTG8376 allowing the expression of the fusion molecule
sCD4-2F5-Angiogenin.
EXAMPLES
[0081] The constructions described below are carried out according
to the general genetic engineering and molecular cloning techniques
detailed in Maniatis et al. (1989, Laboratory Manual, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y.) or according to
the recommendations of the manufacturer when a commercial kit is
used. As regards the repair of the restriction sites, the filling
of the protruding 5' ends may be performed with the aid of the
Klenow fragment of DNA polymerase of Escherichia coli (E. Coli) and
the destruction of the protruding 3' ends in the presence of the T4
phage DNA polymerase or by treatment with S1 nuclease followed by
repair with Klenow. The PCR techniques are known to persons skilled
in the art and are abundantly described in PCR Protocols, a guide
to methods and applications (Ed: Inis, Gelfand, Sninsky and White,
Academic Press, Inc.).
[0082] The cloning steps using bacterial plasmids are preferably
carried out by passage on the E. coli XL1-Blue strain (Stratagene),
and those relating to the vectors derived from the M13 phage in E.
coli NM522. The mutageneses are performed using synthetic
oligodeoxynucleotides with the aid of a kit of commercial origin
(for example Amersham, RPN1523) and according to the
recommendations of the manufacturer.
Example 1
Preparation of an Implant Secreting the Antibody 2F5 and Intended
for an Anti-AIDS Immunotherapy
[0083] A. Construction of a Dicistronic Retroviral Vector for the
Expression and Secretion of Anti-HIV Antibody 2F5.
[0084] The vector which forms the basis of the constructions is
pLXSP which is derived from pLXSN (Miller and Rosman, 1989,
BioTechniques, 7, 980-988). The latter is a retroviral vector which
comprises the 5' LTR of MoMuSV (Moloney Murine Sarcoma Virus), a
retroviral encapsidation region, multiple restriction sites, the
neo gene for resistance to neomycin under the control of the SV40
promoter and the MoMuLV 3' LTR. The vector pLXSP is obtained after,
on the one hand, the replacement of the NheI-KpnI fragment of the
3' LTR of pLXSN by an analogous fragment obtained from the MPSV
(Myelo Proliferative Sarcoma Virus) 3' LTR isolated from the vector
pMPSV.H-2K.IL-2R (Takeda et al., 1988, Growth Factors, 1, 59-66)
and, on the other hand, introduction of the puromycin resistance
gene as replacement for the neo gene. The puromycin gene is
obtained from pBabe Puro described in Morgenstern and Land (1990,
Nucleic Acids Res., 18, 3587-3596).
[0085] The vector pLXSP is digested with EcoRI and HpaI and an
EcoRI-PstI fragment (after repairing the PstI site) isolated from
pKJ-1 (Adra et al., 1987, Gene, 60, 65-74) is introduced therein.
This fragment carries the promoter of the mouse PGK gene. After
ligation, the vector pTG2663 is obtained. This is subjected to a
digestion with the enzymes ClaI and BamHI in order to eliminate the
cassette for expression of the puromycin gene. After treatment with
Klenow and ligation, the vector pTG2673 is obtained in which the
BamHI site is reconstituted.
[0086] The vector pTG2676 is obtained by cloning the HindIII-EcoRI
fragment comprising the cDNA encoding the light chain (LC) of the
monoclonal antibody 2F5 into the plasmid Bluescript SK+
(Stratagene). It may be cloned by PCR with a cDNA library derived
from the mRNA of the hybridoma 2F5 (Buchacher et al., 1994, AIDS
Research and Human Retroviruses, 10, 359-369; Katinger, 1992,
Seventh Cent Gardes Conference, 299-303) using appropriate primers
complementary to the sequences surrounding the codon for initiation
of translation and the stop codon, such as the primers OTG5168 and
OTG5169 (SEQ ID NO: 1 and 2). There is isolated from pTG2676 an
XhoI-BamHI fragment carrying the cDNA LC 2F5 which is inserted into
the vector pTG2673 previously digested with the same enzymes in
order to generate pTG4336.
[0087] The latter is linearized with the NcoI enzyme and subjected
to ligation with, on the one hand, the NcoI-EcoRI fragment obtained
from pTG2677 and carrying the cDNA encoding the heavy chain (HC) of
the antibody 2F5 and, on the other hand, the EcoRI-NcoI fragment
purified from pTG4369 and carrying the EMCV IRES site. The vector
pTG2677 is a pBluescript SK+ in which the cDNA HC 2F5 carrying the
same ends has been introduced between the HindIII and EcoRI sites.
It was obtained by PCR on the preceding library with the aid of the
primers OTG5170 and OTG5171 (SEQ ID NO: 3 and 4). As for the vector
pTG4369, it is obtained from the cloning, also into pBluescript
SK+, of the XbaI-ClaI fragment corresponding to the EMCV IRES (Jang
et al., 1988, supra). The triple ligation generates the vector
pTG4370 (FIG. 2). The cassette for expression of the puromycin gene
may be optionally reintroduced into the plasmid part of pTG4370 so
as to facilitate the selection stages. pTG6368 is obtained.
[0088] Infectious viral particles are generated in the following
manner:
[0089] The ecotropic complementation line GP+E-86 (Markowitz et
al., 1988, J. Virol., 62, 1120-1124) and the target cells NIH3T3
(mouse fibroblast cells), available from ATCC, are cultured at
37.degree. C. in the presence of 5% CO.sub.2 in DMEM medium
(Dulbecco's Modified Eagle's Medium) containing 10% foetal calf
serum (FCS) (GibcoBRL), 1 mM Glutamine, 1% nonessential amino acids
and 40 .mu.g/l of gentamicin (complete DMEM medium). The day before
the transfection, the GP+E-86 cells are cultured in an amount of
5.times.10.sup.5 cells per 10 cm dish. The next day, 20 .mu.g of
linearized plasmid pTG4370 and 1 .mu.g of selection vector (for
example the vector pLXSP carrying the puromycin resistance gene)
are transfected according to the traditional calcium phosphate
method. The following day (D+1), the cells are washed according to
the prior art methods and placed in a new medium for 48 hours,
before being cultured from D+3 in selective medium (5 .mu.g/ml of
puromycin).
[0090] At the end of about 2 weeks of selection, cell clones
resistant to the antibiotic are visible on the dish. They are
isolated according to prior art techniques and the cells
resuspended in selective medium in 96-well culture plates. The
clones which are the best producers of antibodies are selected
using the ELISA method described below and the supernatants are
titrated on NIH3T3 cells. To do this, on the day before the
infection, they are inoculated at 10.sup.5 cells per well. The
viral infections are performed according to the conventional
procedure described in the literature. The titration method is the
so-called limiting point method. The ELISA method makes it possible
to titrate the quantity of functional 2F5 antibodies recognizing
the target epitope (ELDKWAS) (SEQ ID NO: 21). The latter is
chemically synthesized.
[0091] Briefly, a peptide solution (2 mg/ml) is diluted 2000 fold
in a buffer (15 mM Na.sub.2CO.sub.3, 35 mM NaHCO.sub.3, pH 9.6) and
100 .mu.l are deposited at the bottom of each well of a microtiter
plate and incubated at 4.degree. C. for 16 h. After extensive
washes in a PBS buffer, 0.05% Tween-20, the wells are saturated
with 50 .mu.l of 1% BSA dissolved in a PBS buffer 1 h at 37.degree.
C. After washing, 100 .mu.l of the sample to be tested or of a
standard solution are added. The plate is incubated for 2 h at room
temperature and washed thoroughly with the PBS buffer-Tween 20. 100
.mu.l of a peroxidase-conjugated anti-human IgG goat antibody are
then added (concentration 0.8 mg/ml; Jackson Immuno Research
Laboratories Inc, PA) diluted 1000 fold in PBS buffer, 1% BSA.
After 2 hours at room temperature and extensive washes, the
peroxidase enzymatic activity is revealed by addition of 100 .mu.l
of the following preparation (0.066 M Na.sub.2HPO.sub.4, 0.035 M
C.sub.6H.sub.8O.sub.7 pH5, 0.04% ortho-phenylenediamine and 0.014%
hydrogen peroxide). The reaction is stopped by 150 .mu.l of 1 M
H.sub.2SO.sub.4. The absorbance is measured at 490 nm.
[0092] The standard solution is obtained either from a commercial
source (Virus Testing Systems, Houston) or from a hybridoma
supernatant concentrated on Centricon. The antibody solution (1
.mu.g/ml) is 2-fold serially diluted in fetal calf serum. The
absorbance is measured for each of the dilutions and the
calibration curve is plotted (ng of antibody as a function of the
absorbance). The most productive clones are thus determined.
[0093] B. Preparation of the Implant
1/Removal and Culture of Primary Fibroblasts.
[0094] Skin biopsies are performed on young 2- to 3-day old BALB/C
female mice. However, other mouse lines may also be suitable. After
a rough mechanical dissociation, the sample is placed in 30 ml of
complete DMEM medium in the presence of 5000 units of dispase
(Collaborative Medical Products) and of 1% collagenase (Sigma).
After 2 hours at 37.degree. C., the mixture is diluted and the
cells are harvested by centrifugation, carefully washed before
being resuspended in RPMI 1640 medium (Gibco BRL). After about one
week of culture, the primary fibroblasts are infected with the
culture supernatants of the producing clones selected as in the
preceding stage (A) according to the conventional procedure.
Fibroblast cells NIH3T3 may also be reimplanted by way of a model.
They are cultured as indicated above and infected conventionally.
The presence of the 2F5 antibody in the supernatants of NIH3T3
cells infected with one of the producing clones was monitored by
the ELISA test and was evaluated at 500 ng/ml/24 h, that is to say
a productivity of more than 1 .mu.g/10.sup.6 cells/24 h.
2/Preparation of a Neo-Organ
[0095] The PTFE fibers previously autoclaved (Gore Inc, AZ) are
first of all placed in contact with a rat tail collagen solution
(solution at 0.5 mg/ml in 0.1 N acetic acid) for 2 hours under
vacuum. They are then spread at the bottom of a well (12-well
plate), UV-sterilized, rehydrated with PBS buffer overnight before
being treated for 2 hours at room temperature with angiogenic
factors (10 ml of PBS containing 2 .mu.g of bFGF and 1 .mu.g of
VEGF per 100 mg of fibers approximately).
[0096] In parallel, the infected fibroblasts (primary fibroblasts
or NIH3T3) are briefly trypsinized. 1.5.times.10.sup.7 cells are
resuspended in 0.2 ml of medium and then 2 ml of the following
mixture are added per well: 200 .mu.l of 10.times.RPMI, 24 .mu.l of
7.5% sodium bicarbonate, 5 .mu.l of 1 M Hepes, 20 .mu.l of
Gentamicin, 20 .mu.l of Glutamine, 2 .mu.l of bFGF (10 ng/.mu.l), 2
.mu.l of EGF (Epidermal Growth Factor) (10 ng/.mu.l), 1.5 ml of
collagen (2 mg/ml), 12 .mu.l of NaOH (10 N) and 15 .mu.l of
H.sub.2O. After 30 to 60 minutes of incubation at 37.degree. C., a
polymerization of the mixture is observed. The latter is cultured
at 37.degree. C. for about 4 days in the presence, for the last
night, of angiogenic factors (bFGF and VEGF).
[0097] C. Reimplantation of the Implant
[0098] One or two implants are introduced into the peritoneal
cavity either of BALB/c female mice or of nude Swiss mice. One
month after their implantation, it is checked that they have
anchored to the adipose tissue of the abdominal cavity and are
vascularized. Moreover, blood samples are collected regularly
during the month following the implantation and the assay of the
2F5 antibody by ELISA (according to the technique described above)
reveals values of the order of 20 ng/ml of serum in syngenic BALB/c
mice and exceeding 100 ng/ml of serum in the nude mice. The
antibody level is maintained over a period of more than 6 to 7
weeks after the implantation.
[0099] The efficacy of the 2F5 antibody to inhibit the HIV viral
infection is evaluated on SCID (Severe Combined Immuno Deficiency)
mice. They are immunodeficient mice possessing no T cells or mature
B cells which, moreover, may be humanized by introduction of human
cells or tissues. This treatment makes them injectable by HIV
(Namikawa et al., 1988, Science 242, 1684-1686)
[0100] One to two neo-organs secreting the 2F5 antibody are
implanted in the abdominal cavity of humanized SCID mice by
intraperitoneal injection of human lymphocyte cells CEM A3
(40.times.10.sup.6 cells). 3 to 5 weeks after the transplant, the
mice are challenged with the HIV virus (1000 TCID.sub.50 of HIV1
Bru isolate intravenously). The cells are recovered from the animal
3 days post-infection and cultured. The cell supernatant is
collected at regular time intervals and the reverse transcriptase
activity determined. It is observed that the human cells are
protected against infection by HIV and that the protection is
maintained for the entire duration of the experiment (50 days).
Indeed, the reverse transcriptase activity is below the detection
threshold in mice which have received a transplant of the implant
secreting the 2F5 antibody (behavior similar to the noninfected
control). These data reflect an inhibition of the replication of
HIV in the animals producing 2F5 in their bloodstream.
Example 2
Preparation of an Implant for an Anticancer Immunotherapy
[0101] A. Construction of the Dicistronic Retroviral Vector for the
Expression and Secretion of the 17-1-A Antibody.
[0102] In the first place, the plasmid pBluescript SK+ is digested
with NotI and then subjected to a treatment with the large Klenow
fragment of DNA polymerase before being self-religated. The vector
pTG6336 is generated in which the NotI site has been destroyed. In
parallel, the cDNA encoding the light chain of the 17-1-A antibody
is isolated by PCR from a cDNA library constructed from mRNA
isolated from 17-1-A hybridoma cells (Sun et al., 1987, Proc. Natl.
Acad. Sci. USA, 84, 214-218; Herlyn et al., 1979, Proc. Natl. Acad.
Sci. USA 76, 1438-1442). As a guide, this antibody is directed
against an epitope of the transmembrane glycoprotein GA733-2 (Szala
et al., 1990, Proc. Natl. Acad. Sci. USA, 87, 3542-3546) present at
the surface of human colorectal carcinoma cells. The PCR uses the
primers OTG6114 and OTG6115 (SEQ ID NO: 5 and 6) designed so as to
introduce restriction sites facilitating subsequent cloning steps,
the EcoRI and NcoI sites in 5' and the BglII and XbaI sites in 3'
respectively. After checking on agarose gel, the PCR fragment thus
generated is digested with EcoRI and XbaI and then cloned into
pTG6336 between the same sites. pTG6339 is generated.
[0103] The latter is digested with EcoRI and NcoI and ligated to
the EcoRI-NcoI fragment of pTG4369 carrying the IRES site, to give
pTG6343. There is introduced into the latter the cDNA encoding the
heavy 17-1-A antibody chain lacking the stop codon in the place of
which there is inserted a small spacer encoding the residues
Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 22). The cDNA HC 17-1-A is obtained
by PCR from the preceding cDNA library and using the
oligonucleotides OTG6192 and OTG6194 (SEQ ID NO: 7 and 8). The
insertion of the PCR fragment digested with SalI-EcoRI makes it
possible to generate pTG6346.
[0104] The latter is linearized with NotI and then ligated to the
NotI fragment carrying the sequence encoding barnase to give
pTG6347. The gene encoding barnase is obtained by PCR from a
preparation of Bacillus amyloliquefaciens genomic DNA and the
primers OTG5147 and OTG5148 (SEQ ID NO: 9 and 10). The
oligonucleotides were designed so as to introduce an NotI
restriction site in 5' of the codon corresponding to the first
amino acid of the mature barnase and in 3' of the stop codon. It is
checked that the sequence of the PCR fragment thus generated is in
conformity with that published in Hartley (1988, J. Mol. Biol.,
202, 913-915).
[0105] In parallel, the NotI fragment is inserted into an Ml 3-type
vector, for example the vector M13TG130 (Kieny et al., 1983, Gene,
26, 91-99) in which an NotI site has been previously introduced
inside the cloning site by site-directed mutagenesis. The
modification of the restriction sites by site-directed mutagenesis
is a technique known to persons skilled in the art. The vector thus
obtained is subjected to a site-directed mutagenesis with the aid
of the oligonucleotide OTG5299 (SEQ ID NO: 11) so as to modify the
lysine residue (Lys or K) at position 27 of the native barnase by
an alanine residue (Ala or A). Next, the modified NotI fragment is
isolated and introduced, as above, into the vector pTG6346 to give
pTG6348.
[0106] The SalI-BglII fragment isolated from the vector pTG6347 or
pTG6348 is transferred into the vector pTG2673 previously digested
with XhoI and BamHI. The vectors pTG6356 and pTG6357 are obtained
respectively (FIGS. 3 and 4).
[0107] Moreover, a dicistronic vector is constructed which contains
the sequences encoding HC 17-1-A, the EMCV IRES followed by LC
17-1-A. The HC fragment provided with a stop codon is obtained by
PCR from the vector pTG6346 with the oligonucleotides OTG6192 (SEQ
ID NO: 7) and OTG6193 (SEQ ID NO: 12). This PCR fragment, which is
equipped with SalI and EcorI sites at its 5' and 3' ends
respectively, is inserted into the vector pTG6343 digested with the
same enzymes to give pTG6345. The SalI-BglII fragment of the latter
is cloned between the XhoI and BamHI sites of pTG2673. The vector
pTG6355 is obtained (FIG. 5).
[0108] Viral particles are generated by transfection of the GP+E-86
cells with the vectors pTG6355, pTG6356 and pTG6357 (cotransfection
with pLXSP) according to the technique described in Example 1, the
only difference being that the transfected clones are tested for
the production of 17-1-A antibodies as indicated below. 100 .mu.l
of anti-murine immunoglobulin antibody (Southern Biotechnology)
previously diluted 100 fold in a buffer (80 mM Na.sub.2CO.sub.3,
200 mM NaHCO.sub.3, pH9.6) are distributed in the wells of a
microtiter plate (Nunc) and incubated overnight at 4.degree. C. The
nonadsorbed antibodies are removed by extensive washes with
1.times.PBS buffer, 10 mM EDTA, 0.05% Tween 20. The wells are
saturated by addition of 200 .mu.l of a 1.times.PBS solution, 1%
BSA (Bovine Serum Albumin) 1 h at 37.degree. C. After this stage,
the plate is again washed, and then the control series (diluted in
1.times.PBS, 1% BSA) or the culture supernatants to be tested are
deposited and incubated for 2 h at room temperature, with
agitation. After several washes, the plates are incubated with 100
.mu.l of an anti-Ig.sub.2a goat antibody (isotype of 17-1-A),
coupled to biotin (Southern Biotechnology) diluted 5000 fold in
1.times.PBS buffer, 1% BSA. After 1 h of incubation at room
temperature with agitation, the excess is removed by 8 washes and
then 100 .mu.l of a peroxidase-streptavidin solution (Amersham),
diluted 1000 fold, are distributed. After incubation for 45 min
followed by extensive washes, the enzymatic activity is revealed by
addition of 100 .mu.l of substrate solution (for one plate: 12.5 ml
of 25 mM citrate buffer, 50 mM Na.sub.2HPO.sub.4, pH 5; 1 pastille
of 5 mg of OPD (ortho-phenoldiamine, Sigma); 5 .mu.l of 35%
H.sub.2O.sub.2). The reaction is stopped by addition of 25 .mu.l of
3M H.sub.2SO.sub.4 per well. The absorbance is then read at 490
nm.
[0109] The quantity of antibody present in the culture supernatants
is established as a function of a calibration series prepared as
follows: the 17-1-A hybridoma cells are injected into "nude" mice.
After formation of ascites, the ascitic fluid is collected and the
17-1-A antibody is purified therefrom by passage on a protein A
sepharose column. This is a conventional technique accessible to
persons skilled in the art. A solution of purified 17-1-A antibody
is prepared at a concentration of 1 .mu.g/ml and then 2-fold
serially diluted in a PBS buffer. The absorbance is measured for
each of the dilutions and the calibration curve is established (ng
of antibody as a function of the absorbance). The most productive
clones secrete, according to the transfected vector, from 200 to
900 ng of 17-1-A antibody/10.sup.6 cells/24 h.
[0110] B. Preparation of the Implant
[0111] The NIH3T3 cells are infected with the most productive
clones (derived from the transfection of the GP+E-86 cells with the
vectors pTG6355, pTG6356 and pTG6357) and the culture supernatants
tested by ELISA for the secretion of 17-1-A antibody. An antibody
level varying from 100 to 1000 ng/10.sup.6 cells/24 hours is
detected according to the constructs.
[0112] Moreover, it is checked that the antibody produced
recognizes the GA733 antigen expressed by the SW948 cells. For
that, the flow cytometry technique is used. The SW948 cells (ATCC
CCL237) are cultured in complete DMEM medium. They are detached by
the action of trypsin, counted and then 5.times.10.sup.5 cells are
distributed in wells of a 96-well plate. This plate is centrifuged
for 1 min at 1000 rpm, without brakes, to pellet the cells. The
supernatant is removed and then the plate is vortexed and the cells
resuspended in 100 .mu.l of FACS buffer (cationic 1.times.PBS, 1%
BSA, 0.1% human .gamma. globulins, 5 mM EDTA). The plate is again
centrifuged and the supernatant removed. The cells are then
resuspended in the culture supernatant to be tested or in a
dilution of the control antibody in FACS buffer and incubated for 1
h at 4.degree. C. 4 washes are then carried out under the
conditions described above and then the cells are resuspended in
100 .mu.l of a fluorescein-coupled anti-mouse immunoglobulin goat
F(ab').sub.2 fragment (DTAF) (Jackson Immuno Research Laboratories)
diluted 100 fold in FACS buffer. Another incubation of 1 h at
4.degree. C. allows the antibody to bind. The excess is then
removed by 4 washes and the cells are finally resuspended in 300
.mu.l of cationic 1.times.PBS before being analyzed with a flow
cytometer FACScan (Becton Dickinson). It is observed that all the
NIH3T3 supernatants tested (resulting from the infection with 3
types of viral particles) secrete an antibody capable of binding to
the target GA733 protein.
[0113] In the case of the constructs where the antibody is fused to
barnase or to the attenuated version thereof (pTG6356 and pTG6357),
it is advantageous to be able to check that the fused antibody has
a nuclease activity. For that, the degradation of a tRNA is
monitored. 100 .mu.l of supernatants to be tested or of an RNAseA
solution at a known concentration (as reaction control) are added
to 200 .mu.l of 0.5 M Tris-HCl, pH 7.5, 5 mM EDTA, 0.5 mg/mg of BSA
and 1 mg/ml final of tRNA and incubated at 37.degree. C. for 30
min. The tubes are then placed on ice and 700 .mu.l of 6%
perchloric acid are added to precipitate the tRNA for 10 min on
ice. A 10 min centrifugation at a maximum speed at 4.degree. C.
makes it possible to pellet the tRNA, leaving in suspension the
free nucleotides released by the action of the enzyme. The
absorbance of these nucleotides is then read at 260 nm.
[0114] The implant may be constituted according to the method
indicated in Example 1 by incorporation of primary murine
fibroblasts or of NIH3T3 cells, transduced with the vectors
pTG6355, pTG6356 or pTG6357.
Example 3
Preparation of an Implant Secreting an Immunoadhesin and Intended
for an Anti-AIDS Immunotherapy
[0115] The aim is to produce an immunoadhesin resulting from the
fusion of an "adhesive" molecule binding the HIV virus glycoprotein
and of an immunoglobulin stabilizing the structure and conferring a
nonspecific immunity. The adhesive part is derived from the CD4
membrane protein (structure represented in FIG. 6) from which the
N-terminal part is retained (signal sequence and I and II domains
of the extracellular region). Several studies have shown that they
are capable, by themselves, of binding gp120, of blocking the
interaction and the penetration of HIV into CD4.sup.+ target cells
(Traunecker et al., 1988, Nature 331, 84-86; Deen et al., 1988,
Nature 331, 82-84; Hussey et al., 1988, Nature 331, 78-81; Fisher
et al., 1988, Nature 331, 76-78). The immunoglobulin part consists
of the constant .gamma.3 region (hinge region --CH2-CH3) of the 2F5
antibody. The hybrid molecule is subsequently designated
sCD4-2F5.
[0116] The construction is carried out in the following manner (see
FIG. 7):
[0117] The sequences encoding the sCD4 region (signal sequence--I
and II domains) are conventionally isolated by PCR. As template,
there is used the cDNA CD4 described in the literature (obtained
from mRNA of CD4.sup.+ cells) or a prior art plasmid in which the
cDNA is cloned (Jay Maddon et al., 1985, Cell 42, 93-104), which is
hybridized with the primers OTG7094 and OTG7095 (SEQ ID NO: 13 and
14). The first makes it possible to introduce an XhoI site and
Kozak type consensus sequences upstream of the CD4 initiator ATG
and the second carries nucleotides corresponding, on the one hand,
to the C-terminal end of the II CD4 domain and, on the other hand,
to the N-terminal end of the hinge region of the 2F5 HC. The
reaction occurs over 25 cycles (1 min at 94.degree. C., 2 min at
50.degree. C. and 3 min at 72.degree. C.).
[0118] The sequences encoding the constant .gamma.3 segment of the
2F5 HC are also amplified by PCR. The plasmid pTG2677 and the
primers OTG7097 and OTG7096 (SEQ ID NO: 15 and 16) are used. The
first is complementary to OTG7095 and the second covers the XmaI
site situated within the CH3 region.
[0119] The PCR products thus generated overlap over 30 bp. They are
rehybridized and subjected to a second amplification reaction which
occurs in 2 stages, in the first instance, a linear amplification
to extend the rehybridized product (10 cycles: 1 min at 94.degree.
C., 2 min at 37.degree. C. and 3 min at 72.degree. C.) followed by
an exponential amplification in the presence of the primers OTG7094
and OTG7096 (SEQ ID NO: 13 and 16) (20 cycles: 1 min at 94.degree.
C., 2 min at 50.degree. C. and 3 min at 72.degree. C.).
[0120] The final product is inserted between the XhoI and XmaI
sites of pTG2677 to give pTG8332 in order to reconstitute the
complete sCD4-2F5 molecule. The latter is excised by XhoI-BamHI
digestion and cloned downstream of the murine PGK promoter into the
vector pTG6368. pTG8338 is obtained (FIG. 8).
[0121] The NIH3T3 cells are transfected with 10 .mu.g of
BglII-linearized pTG8338. Two days later, the cells are cultured in
an increasing puromycin concentration (5 to 75 .mu.g/ml). The
expression of the sCD4-2F5 protein is verified by
immunofluorescence using an antibody which recognizes either the
CD4 part (Leu3A; Becton Dickinson) or the 2F5 part (mouse
monoclonal antibody to the hinge region of a human IgG3; Interchim)
and a conjugate consisting of a fluorescein-coupled anti-mouse Ig
donkey antibody (Jackson Laboratories). It is noted that about 30%
of the cellular pool expresses a detectable level of immunoadhesin.
For this reason, producing clones are isolated by clonal
dilution.
[0122] The production of the viral particles is carried out in
transfected GP+E-86 cells according to the conventional procedure
and selected in the presence of puromycin. The target NIH3T3 cells
are then infected with the cell supernatant and the
immunofluorescence analyses confirm the expression of the transgene
in 100% of the cells.
[0123] The quantification is performed by ELISA. In the first
place, 500 ng of HIV-1 virus gp160 envelope glycoprotein are
deposited so as to bind the CD4 part of the CD4-2F5 molecule. It is
produced by the recombinant route as indicated in international
application WO 92/19742. The supernatant to be assayed is then
added and, finally, an antibody directed against the 2F5 part
(peroxidase-conjugated anti-human Ig goat antibody; Interchim). The
calibration solution consists of the recombinant immunoadhesin
purified from the culture supernatants on a fast-flow sepharose-G
protein column (Pharmacia). A productivity greater than 10
.mu.g/ml/24 h/10.sup.6 cells is measured on the supernatants of the
infected NIH 3T3 cells. This test also indicates that the protein
is capable of binding gp160 and, consequently, would be capable of
binding the HIV virus in order to exert its therapeutic
function.
[0124] The infected NIH3T3 cells are amplified by culturing in F175
flasks. 4 organoids each containing about 10.sup.7 cells are
generated by applying the technology detailed in Example 1 and
transplanted in the peritoneal cavity of 4 nude BALB/c female mice.
The secretion of immunoadhesin into the serum is monitored up to 5
weeks post implantation (assay by ELISA). The results indicate a
concentration of the order of 100 to 200 .mu.g/ml and a continuous
secretion during the duration of the experiment.
Example 4
Preparation of an Implant Secreting a Protein Resulting from the
Fusion of the Immunoadhesin sCD4-2F5 and of the Human
Angiogenin
[0125] Angiogenin is a 14.1 kDa plasma protein belonging to the
family of ribonucleolytic enzymes. However, its lytic action is
more limited than that of the reference ribonuclease A (RNase A)
and shows a marked preference for certain RNAs (especially the 18S
and 28S ribosomal RNAs and the transfer RNAs). The gene and the
cDNA were cloned about ten years ago (Kurachi et al., 1985,
Biochemistry 74, 5494-5499).
[0126] The fusion of the sequences encoding angiogenin downstream
of sCD4-2F5 should allow the synthesis of a protein capable of
targeting and destroying the cells infected with HIV. They were
obtained by PCR from the plasmid pHAG1 (Kurachi et al., 1985,
supra) with the aid of the primers OTG10089 and OTG100090 (SEQ ID
NO: 17 and 18) comprising at their 5' ends the EcoRI and BamHI
restriction sites situated in 5' of the first codon of the mature
protein and in 3' of the stop codon, respectively.
[0127] For reasons of stearic hindrance, it is chosen to introduce
a spacer between the two entities. The PCR reaction is carried out
with the aid of the template pTG2677 and the oligonucleotides
OTG10087 and OTG10088 (SEQ ID NO: 19 and 20) in order to amplify
the part of the 2F5 gene stretching from the XmaI site (inside CH3)
to the stop codon. The primer OTG10088 is designed to eliminate the
stop codon and introduce in 3' a BamHI site as well as the spacer
encoding the residues Gly-Gly-Gly-Gly-Ser (SEQ ID NO 22).
[0128] The two PCR fragments obtained, bordered by a BamHI site are
ligated together. The XmaI-EcoRI fragment is isolated and inserted,
first into pBluescript in order to verify the sequence and, finally
into the vector pTG8332 in order to reconstitute the complete
fusion sequence "sCD4-2F5-Angiogenin". pTG8373 is obtained (FIG.
9). The complete unit may be excised by XhoI-BglII digestion and
cloned into the retroviral vector pTG6368 linearized with XhoI and
BamHI. The viral particles may be constituted as above and the
organoids generated from infected target cells NIH3T3 or primary
fibroblasts.
Example 5
Preparation of an Adenoviral Vector Expressing the Immunoadhesin
sCD4-2F5
[0129] The adenoviral genome fragments used in the constructions
described below are indicated precisely according to their position
in the nucleotide sequence of the type 5 adenovirus (Ad5) genome as
disclosed in the Genebank data bank under the reference M73260.
[0130] The intron and the polyadenylation signal (pA) of the
.beta.-globin human gene are obtained from the vector pBCMG/Neo
(Karasuyama, 1988, Eur J. Immuno. 18, 97-104; Karasuyama, 1989, J.
Exp. Med. 169, 13-25) and introduced into the plasmid pREP4
(InVitogen.TM.) downstream of the RSV virus 3' LTR. The cassette
"RSV-intron-pA .beta.-globin promoter" is isolated from the
preceding vector in the form of a SalI-BamHI fragment and inserted
into the vector pTG9350. The latter is obtained from the cloning of
the Ad5 genomic sequences stretching from nucleotides 1 to 458 and
3328 to 5788 in p polyII (Lathe et al., 1987, Gene 57,
193-201).
[0131] A polylinker provided with multiple cloning sites (EcoRI,
XhoI, NotI, XbaI, SpeI, BamHI, EcoRV, HindIII, ClaI, KpnI and
BglII) is introduced into the vector pTG8346 obtained in the
preceding stage (between the intron and pA), to create pTG8347.
[0132] The sequences encoding the hybrid protein sCD4-2F5 are
isolated from the vector pTG8338 (Example 3) by XhoI-BamHI
digestion and introduced into the vector pTG8347 cleaved with XhoI
and BglII1, to generate pTG8349 which allows their expression under
the control of the RSV promoter.
[0133] The in vitro homologous recombination technique (described
in French Application 94 14470) is used to reconstitute the
complete genome of the recombinant vector. There is used to this
end the vector pTG4656 which comprises the Ad5 genome deleted of
the E1 and E3 regions (Ad5 1 to 458-Ad2 MLP promoter-LacZ gene-pA
SV40-Ad5 3329 to 28529 and 30470 to 35935). Any other E1.sup.-
E3.sup.- adenoviral vector may also be suitable, such as those
described in international application WO 94/28152. The BJ5183
cells (Hanahan, 1983, J. Mol. Biol. 166, 557-580) are cotransformed
by pTG4656 linearized with the enzyme ClaI (10 to 20 ng) and the
PacI-BstXI fragment purified from pTG8349 (about 10-fold molar
excess). The recombination at the level of the homologous
adenoviral sequences causes the replacement of the LacZ cassette of
pTG4656 by that of sCD4-2F5 (RSV promoter-.beta.-globin
intron-sCD4-2F5 gene-pA .beta.-globin) carried the fragment derived
from pTG8349. The vector pTG8357 is generated (FIG. 10).
[0134] The recombinant viruses are obtained by transfection of
pTG8357 into the cells 293 (ATCC CRL1573). 5 plaques are selected
which are amplified by culturing in F25 flasks in the presence of
fresh 293 cells. After 5 days, the infected cells are harvested and
subjected to a HIRTH analysis (Gluzman and Van Doren, 1983, J.
Virol. 45, 91-103), in order to verify the presence of the
transgene. Briefly, the HIRTH analysis consists of an extraction of
the adenoviral genome, a precipitation of the viral DNA, a
digestion with an appropriate restriction enzyme, a transfer onto a
membrane and a hybridization with a radioactive probe capable of
hybridizing with the sCD4-2F5 sequences. A positive signal is
observed for the 5 adenoviruses analyzed.
[0135] A substantial adenoviral stock is constituted by successive
amplification in the 293 cells, purification on two cesium chloride
gradients and dialyses. This stock may be used in the context of
clinical anti-AIDS tests.
Example 6
Preparation of an Adenoviral Vector Expressing the Cytotoxic
Immunoadhesin sCD4-2F5-Angiogenin
[0136] The sequences encoding the fusion protein
sCD4-2F5-angiogenin are excised from the vector pTG8373 (Example 4)
by XhoI-BglII digestion and cloned at the level of the same sites
in the vector pTG8347 (Example 5). The vector pTG8376 is obtained
(FIG. 11) which may be subjected to homologous recombination with
an adenoviral vector, for example pTG4656, to produce the defective
recombinant adenoviruses expressing the cytotoxic immunoadhesin
directed against the cells infected with the HIV virus.
Sequence CWU 1
1
22 1 25 DNA Artificial Sequence synthetic oligonucleotide OTG5168 1
ggaagcttcc atggacatga gggtc 25 2 25 DNA Artificial Sequence
synthetic oligonucleotide OTG5169 2 aagaattcct aacactctcc cctgt 25
3 25 DNA Artificial Sequence synthetic oligonucleotide OTG5170 3
aaaagcttcc atggagttgg gtctg 25 4 25 DNA Artificial Sequence
synthetic oligonucleotide OTG5171 4 gggaattctc atttagccgg agaca 25
5 27 DNA Artificial Sequence synthetic oligonucleotide OTG6114 5
gggaattcca ccatgggcat caagatg 27 6 30 DNA Artificial Sequence
synthetic oligonucleotide OTG6115 6 ggtctagatc taacactcat
tcctgttgaa 30 7 27 DNA Artificial Sequence synthetic
oligonucleotide OTG6192 7 ctgtcgacca ccatggatgg agcagag 27 8 43 DNA
Artificial Sequence synthetic oligonucleotide OTG6194 8 acgaattcgc
ggccgcgctc cctccgccac ctttacccgg agt 43 9 26 DNA Artificial
Sequence synthetic oligonucleotide OTG5147 9 ctgtggcggc cgccgcacag
gttatc 26 10 28 DNA Artificial Sequence synthetic oligonucleotide
OTG5148 10 caggcggccg cttttttcgt tatctgat 28 11 21 DNA Artificial
Sequence synthetic oligonucleotide OTG5299 11 tacattacag cctcagaagc
a 21 12 23 DNA Artificial Sequence synthetic oligonucleotide
OTG6193 12 acgaattctc atttacccgg agt 23 13 35 DNA Artificial
Sequence human CD4 cDNA 13 ccgctcgagc caccatgaac cggggagtcc ctttt
35 14 30 DNA Artificial Sequence human CD4 cDNA 14 acaagatttg
ggctcctgga aagctagcac 30 15 30 DNA Artificial Sequence cDNA of
heavy chain of antibody 2F5 15 gtgctagctt tccaggagcc caaatcttgt 30
16 36 DNA Artificial Sequence cDNA of heavy chain of antibody 2F5
16 tgggcccggg atgggggcag ggtgtacacc tgtggt 36 17 27 DNA Artificial
Sequence human angiogenin cDNA 17 gggggatccc aggataactc caggtac 27
18 27 DNA Artificial Sequence human angiogenin cDNA 18 ggggaattct
tacggacgac ggaaaat 27 19 30 DNA Artificial Sequence cDNA of heavy
chain of antibody 2F5 19 tgcccccatc ccgggaggag atgaccaaga 30 20 36
DNA Artificial Sequence cDNA of heavy chain of antibody 2F5 20
gggggatccc ccgccacctt tagccggaga caggga 36 21 7 PRT Artificial
Sequence HIV gp41 2F5 epitope 21 Glu Leu Asp Lys Trp Ala Ser 1 5 22
5 PRT Artificial Sequence Linker 22 Gly Gly Gly Gly Ser 1 5
* * * * *