Patent 6,960,469

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, a pharmaceutical composition and its therapeutic use.

Description of the Invention

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.

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.

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.

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™). 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.

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, α₁-antitrypsin, ADA, erythropoietin (EPO) and β-glucuronidase. Up until now, this technology has not been suited to the secretion of more complex therapeutic molecules such as antibodies.

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.

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.

Accordingly, the subject of the present invention is:

(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

(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.

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.

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.

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.

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_L) at the N-terminal position and a constant region (C_L) at the C-terminal position whereas the heavy chain comprises from the N to the C-terminal a variable region (V_H) followed by three constant regions (C_H1, C_H2 and C_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.

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′)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 (see Orignial Patent). As regards more specifically the scFv fragment, it may be obtained from a sequence encoding a V_L region followed by a V_Hregion with optionally a spacer (of 1 to 10 neutral amino acid residues which are not very bulky) between the V_Land V_Hsequences.

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.

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.

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).

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:

	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,
	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,
	an antibody directed against the protein MUG-1, and
	an antibody directed against the E6 or E7 protein of the HPV virus (Human Papillomavirus) especially type 16 or 18.

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.

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.

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(FcγR). 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 FcγR 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.

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 frame-work 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).

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).

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 10⁶or better still 10 to 10⁵and, most preferably, 10²to 10⁴ 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).

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.

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 β-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.

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.

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.

A first vector which is particularly appropriate for the present invention is an adenoviral vector (see below).

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.

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.

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,

	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.

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 γ3 segment of the heavy chain of the antibody 2F5 (sCD4-2F5) and, optionally, a third sequence encoding human angiogenin operably linked to the second.

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.

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.

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.

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.

Advantageously, an implant according to the invention comprises from 10⁶to 10¹², preferably from 10⁷to 10¹¹, and most preferably from 10⁸to 10¹⁰genetically modified cells.

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° 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.

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.

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:

	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,
	of breast cancer using an anti-MUC1 antibody,
	of AIDS using an antibody directed against an envelope glycoprotein epitope conserved in numerous isolates,
	of hepatitis using an antibody directed against an epitope of the hepatitis B or C virus.

Of course, these antibodies may be modified by fusion especially to angiogenin, barnase or TK-HSV-1.

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.

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.

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).

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).

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.

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 β-globin (BGL) gene, the sequences encoding the light chain of 2F5, the IRES site of the EMCV virus and the heavy chain of 2F5 and then the polyadenylation site of the human β-globin gene, or

(b) a promoter, the intron of the human β-globin gene, the sequences encoding the molecule sCD4-2F5 optionally fused at the C-terminus and in the same reading frame to human angiogenin.

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.

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.

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).

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.

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 10⁴and 10¹⁴pfu (plaque forming units), advantageously 10⁵and 10¹³ pfu and, preferably, 10⁶and 10¹¹pfu. The formulation may also include an adjuvant or an excipient which is acceptable from a pharmaceutical point of view.

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.

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.

Claim 1 of 10 Claims

1. A recombinant adenoviral vector derived from a human adenovirus comprising an exogenous nucleotide sequence encoding all or part of an antibody, wherein said all or part of an antibody is capable of recognizing a tumor antigen or an epitope specific for an infectious and pathogenic organism, wherein said all or part of an antibody is modified at the N-terminus by fusion to extracellular domains I and II of CD4, and wherein said exogenous nucleotide sequence is under the control of elements necessary for expression of said modified antibody.

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