JP2003501367A - Compositions for the treatment of cytotoxicity, particularly antitumor or antiviral, in mammals - Google Patents

Abstract

  (57) [Summary] The present invention provides a composition for the treatment of cytotoxicity in a mammal, comprising: (i) a nucleic acid sequence encoding all or a portion of a MIP chemokine; and (ii) a polypeptide having at least one cytotoxic activity. Or at least one nucleic acid sequence encoding the nucleic acid sequence, wherein the nucleic acid sequence is under the control of elements necessary for its expression in a mammalian host cell.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION Field of the Invention The present invention comprises a first nucleic acid sequence encoding all or part of the MIP chemokines (MIP chemokine), the polypeptide having at least cytotoxic activity, particularly anti-tumor or anti-viral activity A cytotoxic composition comprising a second nucleic acid sequence encoding all or a portion. The present invention is particularly useful in an environment for treating proliferative diseases or infectious diseases by gene therapy.

[0002] By BACKGROUND Currently, it relates to treatment with a combination of the most promising results obtained in anti-tumor therapy based therapy compounds (chemotherapy) and based on the use of radiation therapy (radiotherapy). This type of treatment results in considerable inconvenience to the patient and, in many cases, metastatic or otherwise metastatic tumor cells may persist in the treated individual and recur, thus not providing complete sedation. It is noticeable.

Recent studies conducted in the field of cancer have proposed adaptation of gene therapy protocols to anti-tumor therapies. In this regard, for example, E6 of the human 16 papillomavirus
And Meneguzzi et al., 1991, Virology, 181, 61-69, on immunization against tumor cells with a vaccinia recombinant vector expressing the E7 gene. Also included is the content of French Patent No. 92/03120 on the use of recombinant adenoviruses expressing cytokines in antitumor gene therapy.

Cytokines are naturally produced after antigenic stimulation or inflammatory responses (Gillis
and Williams, 1998, Curr. Opin. Immunol., 10, 501-503), and especially by Oettger, was shown to be useful in the treatment of certain cancers (Curr. Opin. Immunol., 199).
1, 3, 699-705) molecule. Thus, Leroy et al., (1998, Res. Immunol. 14
9 (7-8): 681-684) have shown that the production of cytokines at tumor sites after intratumoral administration of recombinant vectors induces an immune response associated with inhibition of tumor growth. However, although this antitumor response is promising, it does not lead to complete elimination of tumor cells and therefore cannot be a satisfactory antitumor therapeutic tool.

Regarding chemokines, they constitute a subclass of the cytokine group. They differ from other cytokines in particular by their chemotaxis, and their chemoattractant in the natural process of cellular attraction of the immune system, especially to inflamed or infected tissues, and their anti-angiogenic properties.

Chemokines have low molecular weight (between 8 and 10 kd) and small size (70 to 80 kd).
Amino acid), and the amino acid sequence has low homology (1 according to the chemokine under consideration).
Proteins (ranging from 0-70%), it is now possible to define about 50 different chemokines. However, these chemokines can be subdivided into four major groups with respect to the position of the cysteine residues in which they are included. Α comprising two cysteines at the N-terminus identified by a single amino acid
The groups (IL-8, NAP-2, GCP-2 type chemokines) and the β group (RANTES, MIP1, MCP1 type chemokines) comprising two flanking cysteines at the N-terminus are the most characteristic (Roruk , R., 1994, Trends Pharmacol. Sci.
15, pages 159-165; Murphy, PM, 1994, Annu. Rev. Immunol., 12, pages
593-633).

Furthermore, Dilloo et al. (1996, Nature Medicine, vol. 2, Number 10, 1090-1
(095) group is a specific chemokine, lymphotactin (Lptn) after recombinant administration of ex vivo modified fibroblasts to mice using a retroviral vector.
) And interleukin-2 (IL-2) co-expression was able to stimulate the anti-tumor immune response of treated animals. However, this effect is limited in time, only transient control of tumor burden and not sedation in treated animals.

[0008] Therefore, there is a need for new compositions that can be an easy-to-prepare effective means of anti-tumor therapy, that is, they can provide sustained control of tumor burden and increased survival of treated patients.

[0009]

[Outline of the Invention]

The inventors have now identified a novel cytotoxic composition in which the various components thereof are selected to obtain a synergistic effect with their respective activities and the improved properties of the components. More particularly, such compositions are capable of inhibiting or slowing cell proliferation by inducing specific death of cells, particularly tumor cells, more antigen presentation and / or stimulation of immune cells of the host organism. It is possible. The present invention provides prior art, particularly an advantageous and effective alternative approach for treating human or animal cancer.

The present invention is first of all a composition intended for any purpose for the treatment of cytotoxicity in mammals, eg anti-tumor or anti-viral, or cell death, comprising: (i) a MIP chemokine A nucleic acid sequence coding for all or part thereof, and (ii) at least one nucleic acid sequence coding for all or part of a polypeptide having at least cytotoxic activity, wherein these nucleic acid sequences are mammalian It relates to a composition which is under the control of the elements necessary for its expression in a host cell.

[0011]

[Detailed Description of the Invention]

In the present invention, if the functions and properties of MIP chemokines are preserved,
(I) It is possible to use all of the nucleic acid sequence encoding the MIP chemokine (for macrophage inflammatory protein) or only a part of this polypeptide, or a derived or mutated polypeptide. In the present invention, "mutation" means deletion and / or substitution and / or addition of one or more nucleotides. Similarly, use of a sequence encoding a hybrid chemokine resulting from the fusion of a sequence encoding a MIP-type chemokine with a sequence encoding at least one other form of chemokine (RANTES, MCP1, etc.).

In the present invention, a preferred MIP chemokine is a MIP1 type chemokine,
More specifically, it is selected from the group consisting of MIP1α and MIP1β chemokines, the properties of which are exemplified by Wolpe et al., 1988 J. Exp. Med, 167, 570-581.

MIP1α is described in Obaru et al., 1986, J. Biochem. 99, 885-894, whose nucleic acid and peptide sequences are incorporated herein by reference,
Produced by T lymphocytes and monocytes. It is associated with eosinophils and T during respiratory infections.
Allows chemoattraction of monocytes and neutrophils during rheumatoid arthritis, digestive system inflammation or meningitis of bacterial origin. Furthermore, it inhibits the proliferation of hematopoietic precursors.

MIP1β is described in Brown et al., 1989, J. Immunol. 142, 679-68, the nucleic acid and peptide sequences of which are incorporated by reference.
It is also produced by T lymphocytes and monocytes. It exerts its chemoattractant properties on monocytes and neutrophils in osteoarthritic conditions and bacterial meningitis. MIP1
Like α, it inhibits the proliferation of hematopoietic progenitors.

Natural variants exist in the MIP1α and MIP1β proteins, which are known to the person skilled in the art, eg GOS19, LD7 for MIP1α.
8, PAT464, TY5 (derived from mouse) or SISα (derived from mouse), or MIP1β is named PAT744, Act-2, G-26, II-400 (derived from mouse) or hSISγ (derived from mouse). In the special case of MIP1β, for example, the sequence corresponding to Act-2 (Lipes et al., 1988, PNAS, 85, 9704-9708, which is incorporated herein by reference) is selected.

“Polypeptide having at least cytotoxic activity” means tumor cells (the cytotoxic activity at this time is called antitumor activity) or cells infected with a virus (the cytotoxic activity at this time is referred to as antiviral activity). Any peptide substance capable of inducing or activating an immune response specifically directed against (referred to) otherwise inhibiting the growth and / or division of such cells, in particular such tumors or infected cells. Also means. According to one preferred case, the cytotoxic activity induces the cell death. According to one particular case, it would also be possible to use the compositions of the invention in pathological cases associated with cell proliferation, such as the phenomenon of restenosis.

Chemoattractant activity in cells associated with the immune response of certain polypeptides, particularly those derived from MIP chemokines (eg, eosinophils, T lymphocytes, monocytes or neutrophils) is chemotactic. Can be evaluated using (Maghazachi, 1993, N
ature Immunity, 12, 57). Similarly, this type of chemokine inhibits the proliferation of hematopoietic progenitors, so it is possible to assess such properties in vitro according to Graham et al., 1992, Growth Factors, 7, 151.

The cytotoxic activity of a given polypeptide, in particular the anti-tumor activity, is measured using either a short-term activity assay (such as the triptan blue or MTT assay) or a clonogenic survival assay (colony formation) to enhance cell survival. Tumor growth (size and / or amount) in vivo by measuring in vitro (Brown and Wouters, 1999, Cancer Research, 59, 1391-1399) or in animal models.
(Ovejera and Houchens, 1981, Semin. Oncol., 8, 386-393)
It is possible to evaluate it.

According to a first variant, the invention is characterized in that the polypeptide having cytotoxic activity is selected from cytokines, proteins encoded by genes called “suicide genes” and anti-angiogenic protein factors. And to a composition.

More specifically, when the polypeptide in (ii) is a cytokine, interferons α, β and γ, interleukins, especially IL-2, IL-4,
Preferably, it is a cytokine selected from IL-6, IL-10, IL-12, tumor necrosis factor (TNF) and colony stimulating factor (GM-CSF, C-CSF, M-CSF etc.).

In a preferred embodiment, the cytokine is interleukin-2 (IL-2)
And interferon gamma (IFN-γ). Interleukin-
2 (IL-2) is responsible for the proliferation of specifically activated T lymphocytes, and the proliferation and activation of cells of the immune system (for nucleic acid sequences, especially French patent FR8).
5/09480). IFN-γ activates phagocytes and enhances the expression of type I and type II surface antigens of the major histocompatibility complex (for nucleic acid sequences see in particular French patent 85/09225). The nucleic acid sequences are incorporated herein by reference.

According to another aspect, the composition of the invention comprises in (ii) all or part of interleukin-2 (IL-2) and all or part of interferon gamma (IFN-γ). Comprising at least two nucleic acid sequences encoding

According to a second variant, the invention also provides that the polypeptide in (ii) is selected from thymidine kinase activity, purine nucleoside phosphorylase activity, guanine or uracil or orotate phosphoribosyl transferase activity and cytosine deaminase activity. It relates to such a composition characterized in that it has at least one enzymatic activity.

Some studies have shown that non-toxic substances (prodrugs) such as nucleosides or nucleoside analogues that are not toxic on their own are highly toxic to cells, eg DNA or RNA upon elongation. Enables the identification of polypeptides that are incorporated into chains and, as a result, have catalytic enzymatic properties that can convert cells containing such polypeptides into modified nucleosides that can lead to death, particularly with inhibition of cell division or cell dysfunction. It's coming. The gene encoding such a polypeptide is
It's called the suicide gene. Many suicide gene / prodrug combinations are readily available. This combination can be elaborated further: type-1 herpes simplex virus thymidine kinase (HSV-1 TK) and acyclovir or ganciclovir (GCV) (Caruso et al., 1993, Proc. Natl.
Acad. Sci. USA 90, 7024-7028; Culvet et al., 1992, Science 256, 1550-1.
552; Ram et al., 1997, Nat. Med. 3, 1354-1361);-rat cytochrome p450 and cyclophosphamide (Wei et al., 1994, H.
uman Gene Therapy 5, 969-978);-Escherichia coli (E. coli) purine nucleoside phosphorylase and 6-methylpurine deoxyribonucleoside (Sorscher et al., 1994, Gene
Therapy 1, 233-238);-E. Coli guanine phosphoribosyl transferase and 6-thioxanthine (Mzoz and Moolten, 1993, Human Gene Therapy 4, 589-595) and-cytosine deaminase (CDase) and 5-fluorocytosine (5 -F
C).

More specifically, CDase is an enzyme involved in the metabolic pathway of primidine and converting exocytosine into uracil by hydrolytic deamination. CDase activity has been exemplified in prokaryotes and lower eukaryotes (Jund and Lacroute, 1970.
, J. Bacteriol. 102, 607-615; Beck et al., 1972, J. Bacteriol. 110, 219-
228; De Haan et al., 1972, Antonie van Leeuwenhoek 38, 257-263; Hoeprich
et al., 1974, J. Inf. Dis. 130, 112-118; Esders and Lynn, 1985, J. Biol.
Chem. 260, 3915-3922), but these do not exist in mammals (Koechlin e
t al., 1966, Biochem Pharmacol. 15, 435-446; Polak et al., 1976, Chemoth.
erapy 22, 137-153). Saccharomyces cerevisiae ( S. cerevisiae ) FCY1 encoding each of the CDases of these two organisms
And the E. coli codA gene are known and their sequences are publicly available (
EP402 108; Erbs et al., 1997, Curr. Genet. 31, 1-6; WO93 /
01281).

CDase also becomes a cytosine analogue, 5-fluorocytosine (5-FC), a highly cytotoxic compound, especially when it is converted to 5-fluoro-UMP (5-FUMP) 5. -Deamination to fluorouracil (5-FU). Cells lacking CDase activity, either due to inactivating mutations in the gene encoding the enzyme or due to a natural deficiency of this enzyme (eg, mammalian cells), are 5-FC resistant.
(Jund and Lacroute, 1970, J. Bacteriol. 102, 607-615; Kilstrup et al., 1
989, J. Bacteriol. 1989, 171, 2124-2127). On the other hand, it has been shown that it is possible to transmit susceptibility to 5-FC to mammalian cells having altered sequences encoding CDase activity (Huber et al., 1993, Cancer Res. 53, 4619-4626). ; Mu
llen et al., 1992, Proc. Natl. Acad. Sci. USA 89, 33-37; WO93 / 01
281). Furthermore, in this case neighboring non-transformed cells are also susceptible to 5-FC (Huber et al., 1994, Proc. Natl. Acad. Sci. USA 91, 8302-8306).
This phenomenon is called the bystander effect and is due to the excretion of 5-FU, which excites neighboring cells by monodiffusion through the cell membrane, by cells expressing CDase activity. This passive diffusion property of 5-FU is an advantageous property for the reference system tk / GCV and the bystander effect for this system requires contact with cells expressing tk (Mesnil et al., 1996). , Proc. Natl. Acad. Sci. USA 93, 183
1-1835). This effect is therefore due to CD for gene therapy, especially anti-cancer gene therapy.
There are additional advantages of using ase.

However, the sensitivity to 5-FC varies widely depending on the cell line.
For example, PAN transduced with a retrovirus expressing the Escherichia coli codA gene
Poor sensitivity is found in the C-1 (pancreatic cancer) and SK-BR-3 (breast adenocarcinoma) human tumor lines (Harris et al., 1994, Gene Therapy 1, 170-175). This undesired phenomenon is caused by the enzymatic action of CDase when 5-FU is a cytotoxic 5-FUMP.
It is explained by the fact that there is no endogenous conversion to or there is insufficient endogenous conversion. This step is normally performed by orotate phosphoribosyl transferase in mammalian cells (Peters et al., 1991, Cancer 68, 1903-1909) and is a CDase-based gene as it is not present in some tumors. The therapy is ineffective.

In prokaryotes and lower eukaryotes, uracil is converted to UMP by the action of uracil phosphoribosyl transferase (and consequently has UPRTase activity). This enzyme also converts 5-FU to 5-FUMP. U
In the absence of PRTase activity, it starts with the deamination of 5-FC by CDase, and 5-FU is not converted to cytotoxic 5-FUMP. The fur1 mutant of S. cerevisiae yeast is resistant to high concentrations of 5-FU (10 mM) and 5-FC (10 mM) (Jund and Lacroute, 1970, J. Bacteriol.
102, 607-615). E. coli and S. The upp and FUR1 genes encoding the UPRTase of S. cerevisiae have been cloned and sequenced
(Andersen et al., 1992, Eur. J. Biochem. 204, 51-56; Kern et al., 1990 G
ene 88, 149-157).

For the purposes of the present invention, a polypeptide having UPRTase activity means uracil, or one of its derivatives, into a monophosphate analog, in particular 5-FU.
-Refers to a polypeptide that can be converted to FUMP. “Mutation” should be understood to mean the addition, deletion and / or substitution of one or more residues at any position of the polypeptide.

The native UPRTase according to the invention may be of any origin, especially of prokaryotic, fungal or yeast origin. In the context of the present invention, by way of example, E. coli (Andersen et al., 1992, Eur. J. Biochem. 204, 51-56), Lactococcus
Lactococcus lactis (Martinussen and Hammer, 1994, J. Bacteriol.
176, 6457-6463), Mycobacterium bovis (Kimn et al., 1997, Bio
chem Mol. Biol. Int 41, 1117-1124) and Bacillus subtilis (Martin
The nucleic acid sequence encoding the UPRTase of ussen et al., 1995, J. Bacteriol. 177, 271-274) can be used. However, most particularly preferably yeast UPRTase, especially S. The use of that encoded by the S. cerevisiae FUR1 gene, the sequence of which is disclosed in Kern et al., (1990, Gene 88, 149-157), is hereby incorporated by reference. Gene sequences and corresponding UPRTases as indicators can be found in the literature and expert databanks (SWISSPROT
, EMBL, Genbank, Medline, etc.).

Furthermore, in application PCT / FR99 / 00904, 1 in 5 ′ of the coding region
FUR1 lacking 05 nucleotides and synthesized UPRTase lacking the first 35 residues at the N-terminal position, starting with methionine at position 36 in the native protein
Genes are described. The expression product of the mutant gene called FUR1Δ105 is able to complement the S. cerevisiae fur1 mutant. Furthermore, the truncated mutants have higher UPRTase activity than that of the native enzyme. Thus, according to a particularly advantageous embodiment, the encoded polypeptide of the invention is a deletion mutant of the native UPRTase. The deletion is preferably present in the N-terminal region of the original UPRTase. It can be all (related to all residues in the N-terminal region) or partial (
Related to one or more residues in the primary structure, which may or may not be contiguous. Generally, a polypeptide consists of an N-terminal, central, C-terminal portion,
Each represents approximately one-third of the molecule. For example, S. Since the S. cerevisiae UPRTase has 251 amino acids, its N-terminal part consists of the first 83 residues, starting with the so-called initiation methionine located in natural position 1. As far as E. coli UPRTase is concerned, its N-terminal part extends from position 1 to position 69.

Furthermore, patent applications WO 96/16183 and PCT / FR99 / 00904
Describes the use of a fusion protein encoding an enzyme with two domains having CDase and UPRTase activity, carried out by an expression plasmid, the hybrid gene codA :: upp or FCY1 :: FU.
It has been shown that transfection of R1 or FCY1 :: FUR1Δ105 sensitizes transfected B16 cells to 5-FC. These two
The protein and nucleic acid sequences described in one application are incorporated into the description of this application. According to this aspect, the polypeptide is in-frame with at least one second
Is a polypeptide fused with the polypeptide of. The fusion can be at any site in the first polypeptide, but is preferably N- or C-terminal, particularly preferably N-terminal. Fusion of CDase and UPRTase activities allows improved sensitivity of target cells to 5-FC and 5-FU.

Those skilled in the art can use published data to clone the CDase or UPRTase sequence and make mutations as desired, according to techniques in the art or in application PCT.
/ FR99 / 009004 to examine the enzymatic activity of mutant forms in non-cell or cell lines by following the protocol set out in US Pat. It is possible to fuse all or part.

Thus, in a particular case, the composition of the invention comprises a nucleic acid sequence (ii) of CodA, u
characterized in that it is selected from the nucleic acid sequences of the pp, FUR1, FCY1 and FUR1Δ105 genes or by a combination of all or part of said sequences. The present invention more particularly relates to a composition characterized in that the polypeptide in (ii) has at least CDase activity and UPRTase activity.

“Nucleic acid sequence combination” refers to both different sequences that encode at least two different polypeptides and fused sequences that encode a fusion polypeptide, such that the production of such polypeptides is the same. Under the control of regulatory elements (polycistronic cassettes), or independent elements that may be the same or different, homologous or heterologous with respect to the vector containing them, and may be constitutive or inducible It is understood that what can be done in.

According to a particular embodiment, the composition of the invention is such that the first polypeptide having UPRTase or CDase activity has at least one second polypeptide which in frame respectively has CDase or UPRTase activity. It comprises at least one nucleic acid sequence (ii) encoding a fusion polypeptide fused to a peptide. More specifically, such a polypeptide is characterized in that the fusion with the second polypeptide is carried out at the N-terminus of the first polypeptide.

According to one preferred case, the composition is such that the nucleic acid sequence coding for the fusion polypeptide is -UPRTase or a first nucleic acid sequence coding for a first polypeptide having CDase activity, -CDase or A second nucleic acid sequence encoding a second polypeptide having an UPRTase activity, respectively.

Such a hybrid nucleic acid sequence encoding said fusion polypeptide is also an IRE
It also contains S-type sequences.

The invention is particularly concerned when its first nucleic acid sequence is upp, FUR1 and FUR1Δ10.
5 and the second nucleic acid sequence is selected from CodA and FCY1;
Vice versa also relates to such compositions. Most preferably, such hybrid nucleic acid sequences are selected from the sequences described in patent applications WO 96/16183 and PCT / FR99 / 009004.

According to a third variant, the composition of the invention comprises a polypeptide having cytotoxic activity (
ii) is an anti-angiogenic protein factor. Angiogenesis is the process responsible for the formation of new capillaries from an already existing vascular network. This complex process is finely regulated in healthy tissue by the balance of action of numerous angiogenic and anti-angiogenic factors. However, in certain pathologies, especially in the formation of tumours, this process is disrupted: Angiogenic factors exceed anti-angiogenic factors, which leads to numerous neovascularization of the tumor, which results in its rapid spread and / or metastasis. Will occur. Therefore, in the present invention, the anti-angiogenic factor is considered to be a cytotoxic agent, especially an antitumor agent. Among the various anti-angiogenic factors known at present, angiostatin, endostatin, platelet factor PF4, thrombospondin-1, PRP (proliferin-related protein), VEGI (vascular endothelial growth inhibitor), metalloprotease and urokinase Is mentioned.

The nucleic acid sequence (i) or (ii) can be easily obtained by conventional cloning, PCR, or chemical synthesis. They may be natural genes or mutations, ie genes derived from the latter by deletion, substitution and / or addition of one or more nucleotides. Moreover, the sequences are widely described in the literature and can be searched for by a person skilled in the art.

The invention also relates to said composition, characterized in that the nucleic acid sequences (i) and (ii) are inserted into a recombinant vector of plasmid or viral origin, and control of the elements necessary for its expression in the host cell. It relates to such a recombinant vector having such a nucleotide sequence located therebelow. Nucleic acid sequences (i) and (ii) may be present in more than one copy of the same vector.

More specifically, the composition of the invention may comprise nucleic acid sequences (i) and (ii) inserted in the same recombinant vector or different recombinant vectors.

According to the invention, “recombinant vector” refers to a vector of plasmid or viral origin, optionally related to the transfection efficiency and / or stability of the vector and / or the immune system of the host organism. It may be such a vector linked to one or more substances which improve the protection of the vector in vivo. These materials are available in the literature available to the person skilled in the art (for example Felgner et al., 1987, Proc.
t. Pharmacol. Soc. 32, 115-121; Hodgson and Solaiman, 1996, Nature Biote
chnology 14, 339-342; Remy et al., 1994, Bioconjugate Chemistry 5, 647-6
54)). By way of non-limiting example, they may be polymers, lipids, especially cationic lipids, liposomes, nuclear or viral proteins or neutral lipids. These substances can be used alone or in combination. Examples of such compounds are in particular patent applications WO 98/08489, WO 9
8/17693, WO98 / 34910, WO98 / 37916, WO98 / 5
3853, EP 890362 or WO 99/05183. A possible combination is a recombinant plasmid vector combined with a cationic lipid (DOGS, DC-CHOL, spermine-col, spermidine-col, etc.) and a neutral lipid (DOPE).

The plasmids that can be used in the present invention are widely selected. They may be cloning and / or expression vectors. In general they are known to the person skilled in the art, many of which are commercially available, but it is also possible to construct or modify them using genetic engineering techniques. For example, pBR
322 (Gibco BRL), pUC (Gibco BRL), pBluescript (Stratagene)
, PREF4, pCEF4 (Invitrogen) or pPoly (Lathe et al., 1987,
Gene 57, 193-201). Preferably, the plasmid used in the present invention contains an origin of replication that ensures the initiation of replication in the production cell and / or host cell (for example, the ColE1 origin is selected for the plasmid to be produced in E. coli, If plasmid self-renewal in mammalian host cells is desired, the oriP / EBNA1 system is selected Lupt
on and Levine, 1985, Mol. Cell. Biol. 5, 2533-2542; Yates et al., Nature
313, 812-815). It may also comprise a selection gene allowing the selection or identification of transfected cells (complementation of auxotrophic mutations, genes encoding antibiotic resistance, etc.). Of course, it has additional elements that enhance its persistence and / or stability in a given cell (cer sequences that promote the persistence of plasmid monomers (Summer and Sherrat, 1984, Cell 36, 1097-1.
103, sequences for integration into the cell genome))).

Examples of the viral vector include poxvirus (vaccinia virus, especially MVA, canarypox, etc.), adenovirus, retrovirus, herpesvirus, alphavirus, foam virus, or adeno-associated virus-derived vector. It is preferred to use non-replicating and non-integrating vectors. In this regard, adenovirus vectors are most particularly suitable for use in the present invention. However, it is noted herein that the nature of the vector is relatively unimportant for use in the present invention.

Retroviruses have the property of mainly infecting and integrating dividing cells, which makes them particularly suitable for cancer applications. Recombinant retroviruses of the invention generally comprise an LTR sequence, an inclusion region, and a nucleotide sequence of the invention under the control of an internal promoter such as the retroviral LTR or those described below. It is derived from a retrovirus of any origin (murine, primate, cat, human, etc.), in particular MoMuLV (Molony murine leukemia virus), MSV (murine sarcoma virus) or friend murine retrovirus (Fb29). Can be derived from This is the g needed for the construction of the viral particle.
The ag, pol and / or env viral polypeptides are grown in inclusion lines which can provide in trans. Such lines are described in the literature (PA317, Psi
CRIP GP + Am-12 etc.). The retroviral vector of the invention comprises in particular a mutation in the LTR (replacement of the promoter region with a eukaryotic promoter) or the inclusion region (replacement with a heterologous inclusion region, eg VL30) (
See French patent applications 94/06300 and 97/05203).

We also used adenovirus vectors defective in replication, ie lacking all or part of at least one region essential for replication, selected from the E1, E2, E4 and [defective] regions. May be. Deletion of the E1 region is preferred. However, to the extent that defective essential functions are complemented by trans-complementary strains and / or accessory viruses, the production of the viral particles of interest is guaranteed, in particular E2, E4 and / or L1. -Can be combined with other mutations / deletions that act on all or part of the L5 region. In this regard, state-of-the-art second generation vectors can be used (eg International Application WO94).
/ 26152 and WO 97/04119). As an example, E1 region and E
Most advantageous is the deletion of most of the 4 transcription units. In addition, the adenovirus vector may lack all or part of the non-essential E3 region in order to enhance the cloning ability. According to another alternative, the sequences essential for encapsulation, namely the 5'and 3'IT
A minimal adenovirus vector with R (inverted terminal repeats) and inclusion region can be used. Furthermore, the origin of the adenoviral vectors of the present invention may vary both in terms of species and serotype. It can be human or animal (dog,
It can be derived from an adenovirus genome of avian, bovine, murine, ovine, porcine, simian, etc.) origin, or a hybrid comprising at least two fragments of an adenovirus genome of different origin. More specifically, CAV-1 or CAV-2 adenovirus of dog origin, DAV adenovirus of avian origin or type 3 Bad adenovirus of bovine origin can be mentioned (Zakharchuk et al., Arch. Virol. 1).
993, 128: 171-176; Spibey and Cavanagh, J. Gen. Virol., 1989, 70: 165-17.
2; Jouvenne et al., Gene, 1967. 60: 21-28; Mittal et al., J. Gen. Virol.
, 1995, 76: 93-102). However, adenovirus vectors of human origin are preferred, and those derived from serotype C adenovirus, especially type 2 or 5 adenovirus. The adenoviral vector of the present invention can be obtained in E. coli in vitro by ligation or homologous recombination (see, eg, International Application WO96 / 17070) or by recombination in a complementary strain. . Various adenovirus vectors and techniques for preparing them are well known (eg, Graham and Prevect, 1991, in Methods in Molecular Biology, vol 7, p1.
09-128; Ed: EJ Murey, The Human Press Inc.).

Elements required for expression are transcription of the nucleotide sequence into RNA and mRNA
All of the elements that allow translation of the polypeptide into a polypeptide, in particular, consist of promoter and / or regulatory sequences that are effective in the cell and optionally sequences required for excretion or expression of the polypeptide on the surface of the target cell. These elements may be adjustable and constitutive. Of course, the promoter is suitable for the chosen vector and host cell. For example, PGR (phosphoglycerate kinase), MT (metallothionein; McIvor et al., 1987, Mol. Cell. Biol
.7, 838-848), a eukaryotic promoter of α-1 antitrypsin and CFTR gene, a gene encoding muscle creatine kinase, actin, a lung surfactant,
Immunoglobulin, β-actin (Tabin et al., 1982, Mol. Cell. Biol. 2, 426
-436) and SRα (Takebe et al., 1988, Mol. Cell. Biol. 8, 466-472), SV40 virus (simian virus) early promoter, RSV (Rous sarcoma virus) LTR, MPSV promoter, HSV- 1 TK promoter, CMV virus (cytomegalovirus) early promoter, vaccinia virus promoter p7.5k, pH5R, Pk1L, p28 and p11
, And E1A and MLP adenovirus promoters, or a combination of said promoters. It may also be a promoter that stimulates expression in cancerous or tumor cells. MUC-1 gene overexpressed especially in breast cancer and prostate cancer (Chen et al., 1995, J. Clin. Invest. 96, 2775-2782)
, CEA (carcinoembryonic antigen) gene overexpressed in colon cancer (Schrewe et al.,
1990, Mol. Cell. Biol. 10, 2738-2748), tyrosinase gene overexpressed in melanoma (Vile et al., 1993, Cancer Res. 53, 3860-3864), ERB overexpressed in breast cancer and pancreatic cancer. -2 gene (Harris et al., 1994, Gene Thera
py 1, 170-175) and the promoter of the α-fetoprotein gene (Kanai et al., 1997, Cancer Res. 57, 461-465) overexpressed in liver cancer. Most particularly preferred is the cytomegalovirus (CMV) early promoter or RSV. It is also possible to use promoter regions which are tissue-specific, especially when the tumor to be treated is derived from a particular cell type, or which can be activated under defined conditions. The literature provides a great deal of information regarding such promoter sequences.

The necessary elements may also include additional elements that enhance the expression of the nucleotide sequences of the invention, or their persistence in host cells. In particular, intron sequence (WO94 / 29471), secretory signal sequence, nuclear localization sequence, I
RES-type internal translation re-initiation site and poly A transcription termination sequence.

According to a preferred embodiment, the present invention more particularly relates to recombinant vectors, in particular viral vectors, more particularly replication-defective adenoviral vectors, wherein (i) all or part of a MIP chemokine. A coding nucleic acid sequence, and (ii) at least one nucleic acid sequence coding for all or part of a polypeptide having at least cytotoxic activity, the nucleic acid sequence being necessary for its expression in a host cell. Which is under the control of and is defined above.

An object of the invention is also a virus particle, in particular an adenovirus particle, which comprises the recombinant viral vector of the invention. Such viral particles can be obtained from viral vectors by conventional methods in the art. The propagation is carried out in cells which are especially complementary to the defects of the vector. As for the adenovirus vector, for example, the complementary line described in application WO94 / 28152, the 293 line established from human embryonic kidney cells that effectively complements E1 function (Graham et al., 1977, J. Gen.
Virol. 36, 59-72), A549-E1 strain (Imler et al., 1996, Gene Therapy.
3, 75-84) or strains that confer double complementarity (Yeh et al., 1996, J. Virol. 70, 5
59-565; Krougliak and Graham, 1995, Human Gene Therapy 6, 1575-1586; Wan
g et al., 1995 Gene Therapy 2, 775-783; International Application WO 97/04119). Auxiliary viruses can also be used to supplement defective functions, at least in part. "Complementary cells" refer to cells that can provide in trans the early and / or late factors required for inclusion of the viral genome in the viral capsid to obtain viral particles containing the recombinant vector. The cells do not by themselves complement all the defective functions of the vector, but in this case they are transfected / transduced with an accessory virus / vector which provides a complementary function.

The present invention also provides a method for preparing a virus particle, which comprises (i) introducing the recombinant vector of the present invention into a cell, particularly a complementing cell capable of complementing with trans, to obtain a transfected cell. And (ii) culturing the transfected cells under suitable conditions to produce viral particles, and (iii) recovering the viral particles from the cell culture.

Of course, viral particles can be recovered from the culture supernatant and also from cells. One commonly used method is to lyse cells by successive cycles of freeze / thaw and recover virions in the lysed supernatant. These virions can be amplified and purified by techniques in the art (chromatography, in particular ultracentrifugation with a cesium chloride gradient).

The invention also relates to a eukaryotic host cell comprising the DNA fragment present in the composition of the invention. The host cell is conveniently a mammalian cell, preferably a human cell. It is preferably 293, LCA4 or PERC6 cells. The cells are particularly useful for producing high titers of viral particles, but replication competent particles are not obtained. The invention also relates to host cells which comprise the nucleotide sequences of the invention or recombinant vectors or are infected with the viral particles of the invention. In the present invention, host cells consist of cells either transfected with a recombinant vector or capable of being infected by viral particles, as described above. Mammalian, especially human cells are most particularly preferred. It may comprise the vector in a genomically integrated or non-genomically integrated (episomal) form. It can be of any origin, especially hematopoiesis (differentiated totipotent stem cells, white blood cells, lymphocytes, monocytes or macrophages), muscles (satellite cells, myocytes, myoblasts, smooth muscle cells, etc.), heart,
It may be a primary or tumor cell of lung, trachea, liver, epithelial or fibroblast origin.

The present invention also provides a composition for use in anti-tumor or anti-viral therapy in mammals, or for any purpose for cell death, comprising: (i) all or part of a MIP polypeptide, (Ii) A composition comprising at least all or part of a polypeptide having cytotoxic activity, wherein the polypeptide is as defined above.

Another object of the invention is a formulation intended as a means of cytotoxicity in mammals, in particular antitumor or antiviral therapy, comprising a composition (based on said nucleic acid or polypeptide), an adenovirus according to the invention. A formulation, characterized in that it comprises a viral vector or viral particle and a pharmaceutically acceptable carrier. Such carriers are preferably isotonic, hypotonic or slightly hypertonic and have a relatively low ionic strength, for example sucrose solutions. In addition, such carriers may include any solvent or aqueous or partially aqueous lipid such as a non-pyrogenic lipid. Also adjust the pH of the formulation and treat with a buffer
Meet the requirements for in vivo use. The formulation may also include pharmaceutically acceptable diluents, adjuvants or excipients, as well as solubilizing, stabilizing and preservative agents. For injectable administration, aqueous, non-aqueous or isotonic preparations are preferred. It may be in single or multiple doses in liquid form or in dry form (powder, lyophilisate etc.) which can be reconstituted extemporaneously with a suitable diluent.

According to a particular aspect of the invention, the formulation also comprises a pharmaceutically acceptable amount of a prodrug which is at least convertible by the polypeptide having cytotoxic activity into a cytotoxic molecule.

Such a prodrug consists especially of acyclovir or ganciclovir (GCV), cyclophosphamide, 6-methylpurine deoxyribonucleoside, 6-thioxanthine, cytosine or one of its derivatives, or uracil or one of its derivatives. Selected from the group. Most preferably, the prodrug is 5-
Fluorocytosine (5-FC) or 5-fluorouracil (5-FU).

Furthermore, it should be noted that especially in the formulations containing the composition according to the second variant, the formulation may also comprise one or more substances which enhance the cytotoxic effect of 5-FU. . In particular, thymidylate which reduces the pool of dTMP required for replication in the presence of 5-FU metabolites (5-FdUMP), agents that inhibit enzymes of the pyrimidine's new biosynthetic pathway (eg, those described below). Leucovorin increases the inhibition of synthase (Waxman et al., 1982, Eur. J. Cancer Clin. Oncol. 18
, 685-692) and finally inhibits dihydrofolate reductase and promotes PR
Methotrexate (Cadman et a, which results in an increase in 5-FU in cellular RNA by increasing the pool of uptake of PP (phosphoribosyl pyrophosphate)
l., 1979, Science 250, 1135-1137).

The formulations of the present invention are more particularly directed to the prevention or therapeutic treatment of diseases by gene therapy, more particularly to proliferative diseases (cancer, tumors, restenosis etc.) and especially to infected cells (B Or hepatitis C virus, HIV, herpes virus,
(Induced by retroviruses, etc.) is directed to infectious diseases of viral origin that need to limit their growth.

The formulations of the present invention may be conveniently prepared for topical, parenteral or digestive administration. Many routes of administration are possible. For example, intragastric, subcutaneous, intracardiac, intramuscular,
Intravenous, intraperitoneal, intratumoral, intranasal, intrapulmonary or intratracheal routes are included. Latter three
In one aspect, administration by aerosol or infusion is advantageous. The administration may be performed once or may be repeated one or more times with optionally delayed time. The preferred route of administration and viral load will vary as a function of various parameters such as the individual, the disease to be treated, or the gene of interest to be transferred. Preparations based on the viral particles of the invention are between 10 ^{4 and} 10 ¹⁴ pfu (plaque forming units),
Advantageously 10 ⁵ and 10 ¹³ pfu, preferably 10 ⁶ and 10 ¹² pfu
Can be formulated into a dosage form of For the recombinant vector of the present invention, the DNA is 0.01 to 100 mg, preferably 0.05 to 10 mg, and most preferably 0.0.
A dosage comprising 5-5 mg is envisaged. Compositions based on polypeptides preferably comprise 0.05 to 10 g of polypeptide, most preferably 0.5 to 5 g. Of course, the doctor can adjust the dose.

The invention also relates to the manufacture of a medicament intended for the treatment of the human or animal body by gene therapy, in particular cells aimed at inhibiting growth or leading to the elimination of tumors or the death of infected cells. It relates to the use of the composition, recombinant vector or viral particle of the invention for the treatment or prevention of injuries, in particular antitumor or antiviral medicaments. According to the first possibility, the drug may be administered directly in vivo (for example to the tumor or its surroundings accessible by intravenous infusion, to the lungs with an aerosol, to the vasculature with a suitable probe, etc.). It also consists in removing the patient's cells (bone marrow stem cells, peripheral blood lymphocytes, muscle cells, etc.) and transfecting them by methods in the art, or infecting them in vitro and re-administering them to the patient. It is also possible to adopt the ex vivo approach. A preferred use is in treating or preventing cancers, tumors and diseases that result in unwanted cell proliferation. Possible uses include breast cancer, uterine cancer (especially those induced by papillomavirus), prostate cancer, lung cancer, bladder cancer,
It includes liver cancer, colon cancer, pancreatic cancer, gastric cancer and cancer of the esophagus, throat, central nervous system and blood (lymphoma, leukemia, etc.). In cardiovascular disease, it is useful, for example, to inhibit or delay the proliferation (restenosis) of smooth muscle cells of the blood vessel wall. Finally, for infectious diseases one can consider its use in AIDS.

It is further envisaged that, where appropriate, simultaneous or sequential administration of the various components recited in the pharmaceutical formulations or compositions of the present invention by various routes and without departing from the present invention.

The invention also provides a method of treating a disease by gene therapy, which comprises administering the nucleic acid sequences, recombinant vectors, viral particles or host cells of the invention to an organism or host cell in need of such treatment. Spread to the way you do. Recombinant vector encoding a second polypeptide having CDase activity when using a nucleic acid sequence, a recombinant vector or a viral particle that causes expression of a polypeptide of the invention having UPRTase activity in a therapeutic method Alternatively, it may be advantageous to administer a viral particle, or a second nucleic acid sequence carried by the vector or an independent viral particle. Administration of the UPRTase and CDase sequences in the latter case may be simultaneous or sequential and the order of administration is not critical.

According to an advantageous embodiment, the therapeutic use or method also comprises according to the steps a prodrug, preferably a cytosine analogue, in particular a pharmaceutically acceptable amount of 5-FC, is administered to a host cell or organism. Comprising further steps. As an example, 50 to 500
A dose of mg / kg / day may be used, preferably 200 mg / kg / day. In the present invention, the prodrug is administered prior to administration of the therapeutic agent of the present invention, at the same time as, or after administration by standard practice. The oral route is preferred. The prodrug can be administered in a single dose or in repeated doses for a long enough period to produce toxic metabolites in the host cell or organism.

According to an advantageous aspect of the invention, the therapeutic use or method is combined with a second treatment of the patient by surgery (in particular by partial or total excision of the tumor), radiation therapy or chemotherapy. In this particular case, the treatment of the invention is used before, at the same time as, or after the second treatment. This treatment is preferably used after the second treatment.

The purpose of the following examples is to exemplify the various objects of this invention, and thus not to limit their scope.

[0069]

【Example】

The constructs below are from Maniatis et al., (1989, Laboratory Manual, Cold Spring H
(Arbor, Laboratory Press, Cold Spring Harbor, NY). Manufactured according to standard methods of genetic engineering and molecular cloning detailed in manufacturer's instructions or when using commercially available kits, follow manufacturer's recommended standards. The homologous recombination step is preferably performed in E. coli strain BJ5163 (Hanahan, 1983, J. Mol. Biol. 166, 557-580). The technique used for restriction site repair consists in filling the prodrug 5'end with a large fragment of E. coli DNA polymerase I (Klenow). Fragments of the adenovirus genome used in various constructs described further below are referred to in reference M73260.
Are precisely indicated by their position in the nucleotide sequence of the Ad5 genome disclosed as Genbank databank.

For cell biology, cells are transfected or transduced and cultured according to standard techniques well known to those of skill in the art.

Tumor Models: Three tumor models: p815 (H-2d mastocytoma, Dunn et al, 1957, J. Na.
tl. Cancer Inst., 18, 587-590), B16F0 (H-2b melanoma, Wu et.
al, 1996, Cancer Res., 56, 21-26), and RENCA (H-2d kidney cancer, Murphy et al, 1973, J. Natl. Cancer Inst., 50 (4), 1023-1025. ) Was selected to evaluate the activity of the composition of the present invention. Cells (3 for each tumor model)
Is subcutaneously transplanted to the right flank of 6-8 week old B6D2 mice at D-7 / D-11.

Administration of the cytotoxic composition of the present invention: When the tumor volume is in the range of 4-10 mm ³ , 100 μl volume of adenovirus vector (5 × 10 ⁸ infectious units) is directly injected into the tumor. Repeat this injection under the same conditions at D1 and D2.

Efficacy of the administered compositions of the present invention is controlled by measuring tumor size and survival time of treated mice, optionally controlling the immunological status of the animals by ELISPOT, CTL assays and the like. To do. The animal may then be given contralateral administration, during which time a lethal dose of tumor cells is administered to the pretreated animal.

Example 1 Construction of pTG13010 (Human MIP1α) Human MIP1α cDNA (Genbank Accession No .: X03754; sequence incorporated herein by reference) Obaru, K. et al., 1986, J. . Bio
It was constructed with synthetic oligonucleotides according to the sequence described in chem. 99 (3), 885-894. This cDNA was introduced into a vector derived from pBluescript to obtain the vector pTG13006.

The NotI-Asp718 fragment of pTG13006 containing the MIP1α gene is isolated and introduced into vector pTG8347 cleaved with these same enzymes to obtain transfer vector pTG13008. As an index, pTG8347 is Ad5 sequence 1-4.
58, RSV promoter, splicing sequence of intron 2 of rabbit β-globulin 1, polyadenylation sequence of rabbit β-globulin 1 and Ad5 sequence 3
P-polyII vector inserting 328-5788 (Lathe et al., 1987, Gene 57,
193-201). Such constructs are in the area of the person skilled in the art, in particular French patent application
Based on 7/06757. The adenovirus vector pTG13010 is recombined in E. coli strain BJ5183 with the PacI-BstEII fragment of pTG13008 and the vector pTG6624 linearized with ClaI (described in French patent application 97/06757). As an index, pTG6624 is E1 (nt
459-3327) and E3 (nt 28592-30470) region deleted Ad5 genome, corresponding to p-polyII plasmid with MIP expression cassette inserted in place of E1.

The final construct pTG13010 contains the Ad5 genome lacking the majority of the E1 (nt459-3328) and E3 (nt28249-30758) regions, and E.
Instead of 1, it contains an RSV promoter and an expression cassette for the MIP1α gene which is under the control of the splicing sequence of intron 2 of rabbit β-globulin 1. Adenovirus particles are E1 functional complement strains according to techniques in the art,
For example, it is caused by transfection with 293 strain (ATCC CRL1573) (Graham and Prevec, 1991, Methods in Molecular Biology Vol 7, Ge).
ne Transfer and Expression Protocols; Ed EJ Murray, The Human Press I
nc., Clinton, NJ).

Construction of pTG13023 (Human MIP1β / Mutant Act2) Human MIP1β cDNA (Genbank accession number: J04130; sequence incorporated herein by reference) in Lipes, MA et al. 1988, Proc.
. Natl. Acad. Sci. USA 85 (24), 9704-9708 with synthetic oligonucleotides according to the sequence described. This cDNA was introduced into a vector derived from M13TG130 (Kieny et al. 1983, Gene, 26, 91-99) to obtain vector M13TG13013.

The NotI-Asp718 fragment of M13TG13013 containing the MIP1β gene is isolated and introduced into vector pTG8347 cleaved with these same enzymes to obtain transfer vector pTG13015. Such constructs are in the field of the person skilled in the art, in particular based on French patent application 97/06757. Adenovirus vector pTG13
023 is a recombinant of Pac of pTG13015 in E. coli strain BJ5183.
It is reconstructed with the I-BstEII fragment and the vector pTG6624 linearized with ClaI (described in French patent application 97/06757).

The final construct pTG13023 contains the Ad5 genome deleted in the majority of the E1 (nt459-3328) and E3 (nt28249-30758) regions, and E.
Instead of 1, it contains an RSV promoter and a cassette for expression of the MIP1 β gene which is under the control of the splicing sequence of intron 2 of rabbit β-globulin 1. Adenovirus particles are E1 functional complement strains according to techniques in the art,
For example, it is caused by transfection with 293 strain (ATCC CRL1573) (Graham and Prevec, 1991, Methods in Molecular Biology Vol 7, Ge).
ne Transfer and Expression Protocols; Ed EJ Murray, The Human Press I
nc., Clinton, MJ).

Example 2 In Vivo Testing To evaluate the ability of the compositions of the present invention to inhibit tumor growth in vivo, 3
X10 ⁵ B16F0, RENCA or p815 cells (D = -10 / -7)
Inject into B6D2 immunocompetent mice at. As soon as the tumor became palpable (D = 0), various compositions (see legend to Figures 1-6) were injected into the tumor three times (D).
0, D1, D2) infused at a dose of 5 × 10 ⁸ infectious units.

The results obtained show that MIP1α or MIP1β associated with IL2 or IFNγ
Increased survival rate (FIGS. 3, 5 and 7) associated with reduced tumor burden (FIGS. 2, 4 and 6) in mice treated with a composition comprising an adenovirus that expresses adenovirus.
Shows. These results clearly confirm the advantage of the composition of the present invention in the means of anti-tumor therapy.

The mice thus treated are then given a contralateral administration consisting of a lethal dose of tumor cells (3 × 10 ⁵ cells) at D80 / D100 under the conditions described above.
Thus, the above results have also been shown to be associated with the immune conditions of the mice such that there are no tumors that can progress after this challenge.

Example 3 In Vivo Testing To evaluate the ability of the compositions of the present invention to inhibit tumor growth in vivo, 4
X10 ⁵ RENCA cells are injected (B = 0) into B6D2 immunocompetent mice. As soon as the tumor became palpable (D = 6), 2 × 10 ⁸ infectious units (iu) of adenovirus expressing the human MIP1β gene (huMIP1β)
And adenovirus 2 × 10 ⁸ iu expressing the murine IL12 gene (muIL12), or adenovirus 2 × 10 ⁸ iu expressing the murine IL12 gene and an adenovirus containing no transgene (empty adenovirus (empty Ad)) Various compositions comprising either either in combination with 2 × 10 ⁸ iu or empty adenovirus 4 × 10 ⁸ iu are injected intratumorally three times (D6, D7, D8). In all of these compositions, adenovirus is dissolved in a solution containing 100 mM Tris and 10 mM MgCl ₂ . Furthermore, mice treated with a composition comprising adenovirus expressing the MIP1β gene alone under the same conditions have the same or more volume as found in mice treated with a composition comprising empty adenovirus. It was confirmed to present a large tumor.

The results obtained in this example (FIG. 7) show that MIP1β-expressing adenovirus and I
Mice treated with a composition of the invention comprising a combination with an adenovirus expressing L12 show a reduction in tumor burden, most particularly compared to the results seen during treatment of mice with murine IL2 alone. . These results clearly confirm the advantage of the composition of the present invention in the means of anti-tumor therapy.

[Detailed Description of Drawings]

[Figure 1]   8 shows changes in tumor burden in B6D2 mice transplanted with B16FO tumor cells.

FIG. 2 shows the survival rate of these same mice. A group of 15 mice was given the following gene: human MIP
Treatment is with a composition comprising an adenovirus expressing 1α, human IL2, human MIP1α + human IL2, no Ad (adenovirus).

[Figure 3]   8 shows changes in tumor burden of B6D2 mice transplanted with RENCA tumor cells.

FIG. 4 shows the survival rate of these same mice. A group of 15 mice was given the following gene: human MIP
Treatment is with a composition comprising an adenovirus expressing 1β, human IL2, human MIP1β + human IL2, no Ad (adenovirus).

[Figure 5]   7 shows changes in tumor burden of B6D2 mice transplanted with p815 tumor cells.

FIG. 6 shows the survival rate of these same mice. A group of 15 mice was given the following genes: Tris buffer, human MIP1α + human IL2, human MIP1α + murine IFNγ, human I.
Treatment with a composition comprising an adenovirus expressing L2 + murine IFNγ.

FIG. 7 shows changes in tumor burden of B6D2 mice transplanted with RENCA tumor cells. These mice are combined with an adenovirus expressing the human MIP1β (huMIP1β) gene and an adenovirus expressing the murine IL12 (muIL12) gene, or an adenovirus expressing the murine IL12 gene or an adenovirus containing no transgene ( Empty adenovirus (empty Ad
)) Is used for the treatment.

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Claims (22)

[Claims] 1. A composition for the treatment of cytotoxicity in a mammal comprising: (i) a nucleic acid sequence encoding all or part of a MIP chemokine; and (ii) a polypeptide having at least cytotoxic activity. Or at least one nucleic acid sequence encoding the same, wherein these nucleic acid sequences are under the control of the elements necessary for their expression in a mammalian host cell. 2. The composition according to claim 1, wherein the MIP chemokine is selected from the group consisting of MIP1 chemokines, more particularly MIP1α and MIP1β chemokines. 3. The polypeptide having cytotoxic activity is selected from cytokines, proteins encoded by suicide genes and anti-angiogenic protein factors.
Or the composition according to 2. 4. The composition according to claim 3, wherein the polypeptide having cytotoxic activity is a cytokine selected from interferons α, β and γ, interleukins, tumor necrosis factor and colony stimulating factor. 5. The composition according to claim 4, wherein the polypeptide having cytotoxic activity is interleukin-2 (IL-2). 6. The composition according to claim 4, wherein the polypeptide having cytotoxic activity is interferon γ (IFN-γ). 7. (ii) comprising at least two nucleic acid sequences encoding all or part of interleukin-2 (IL-2) and all or part of interferon γ (IFN-γ). The composition according to any one of claims 1 to 6, which comprises: 8. The composition of claim 4, wherein the polypeptide has at least one cytotoxic activity selected from thymidine kinase activity, purine nucleoside phosphorylase activity, guanine phosphoribosyl transferase activity and cytosine deaminase activity. 9. The composition of claim 8, wherein the polypeptide has at least CDase activity and UPRTase activity. 10. The polypeptide having cytotoxic activity is angiostatin, endostatin,
The composition according to claim 4, which is an anti-angiogenic protein factor selected from platelet factor PF4, thrombospondin-1, PRP, VEGI, metalloprotease and urokinase. 11. The composition according to claim 1, wherein the nucleic acid sequences (i) and (ii) are inserted into a recombinant vector of plasmid or viral origin. 12. The nucleic acid sequences (i) and (ii) are inserted into the same recombinant vector.
The composition according to. 13. The nucleic acid sequence (i) and (ii) are inserted into different recombinant vectors.
The composition according to 1. 14. (i) a nucleic acid sequence encoding all or part of a MIP chemokine, and (ii) at least one nucleic acid sequence encoding all or part of a polypeptide having at least cytotoxic activity. A vector comprising a nucleic acid sequence which is under the control of the elements necessary for its expression in a host cell. 15.   The vector according to claim 14, which is a viral vector. 16.   A viral particle comprising the vector of claim 15. 17. A method for preparing the virus particle according to claim 16, comprising: (i) introducing the virus vector according to claim 15 into a cell capable of producing the vector to obtain a transfected cell. , (Ii) culturing the transfected cells under suitable conditions to produce viral particles, and (iii) recovering the viral particles from the cell culture. 18. A composition for treating cytotoxicity in a mammal, which comprises (i) all or part of a MIP polypeptide and (ii) all or part of a polypeptide having at least cytotoxic activity. A composition comprising: and these polypeptides (i) and (ii) are of claims 1-10. 19. A formulation for the treatment of cytotoxicity in a mammal, the composition according to any one of claims 1 to 13, the vector according to claim 14 or 15, and the vector according to claim 16. A formulation comprising viral particles, or the composition of claim 18 and a pharmaceutically acceptable carrier. 20. The formulation according to claim 19, comprising a pharmaceutically acceptable amount of a prodrug that can be converted into a cytotoxic molecule by at least a polypeptide having cytotoxic activity. 21. The formulation of claim 20, wherein the prodrug is selected from 5-fluorouracil (5-FU) and 5-fluorocytosine (5-FC). 22. The composition according to claim 1-13, the vector according to claim 14 or 15, the viral particle according to claim 16, or the composition according to claim 18, for producing a cytotoxic drug. Use of the described composition.