WO2002059291A2 - Materiel biologique pour la preparation de compositions pharmaceutiques destinees au traitement de mammiferes - Google Patents
Materiel biologique pour la preparation de compositions pharmaceutiques destinees au traitement de mammiferes Download PDFInfo
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- WO2002059291A2 WO2002059291A2 PCT/FR2002/000330 FR0200330W WO02059291A2 WO 2002059291 A2 WO2002059291 A2 WO 2002059291A2 FR 0200330 W FR0200330 W FR 0200330W WO 02059291 A2 WO02059291 A2 WO 02059291A2
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Classifications
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/521—Chemokines
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- A—HUMAN NECESSITIES
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- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
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- A—HUMAN NECESSITIES
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- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55522—Cytokines; Lymphokines; Interferons
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- A—HUMAN NECESSITIES
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- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2799/00—Uses of viruses
- C12N2799/02—Uses of viruses as vector
- C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2799/00—Uses of viruses
- C12N2799/02—Uses of viruses as vector
- C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
- C12N2799/022—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from an adenovirus
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the present invention relates to the field of gene therapy applied to specific or non-specific immunotherapy (or gene immunotherapy), more particularly in the context of the treatment of diseases in which the agent responsible is a pathogenic organism, such as in particular an agent bacterial, parasitic or viral, or in the treatment of cancer or prion diseases. More particularly, the invention relates to a biological material comprising at least (i) a nucleic acid coding for all or part of an antibody and (ii) a nucleic acid coding for all or part of a polypeptide involved in the stimulation of immune response and / or influencing cytotoxic effector cells and / or helper T lymphocytes.
- the present invention also relates to a host cell and a composition comprising said biological material as well as their use for medical purposes, more particularly therapeutic or prophylactic.
- gene therapy has been proposed to correct the disorders observed in the context of genetic diseases. These diseases are explained in particular by a dysfunction of the expression of specific genes or by the expression of nonfunctional mutated polypeptides in at least one cell type.
- Gene therapy consists in transferring genetic information capable of correcting the observed defect to the patient to be treated. It may be, for example, the gene coding for the CFTR protein in the case of cystic fibrosis or the gene coding for dystrophin in the case of Duchenne muscular dystrophy.
- genetic information is introduced either in vitro into a specific host cell extracted from the organ, the modified cell then being reintroduced into the organism (ex vivo process), or directly in vivo in or at proximity to the appropriate tissue (eg affected organ).
- Many publications describe the implementation of gene therapy protocols in order to obtain in host cells the expression of a protein of interest by introducing the corresponding genetic information.
- the advantage of this type of approach is not limited to the treatment of purely genetic disorders and can also allow or contribute to the reduction or elimination of tumors, isolated tumor cells, pathogens, such as the bacterial, parasitic or viral agents, or cells infected with such pathogens. Otherwise, such treatment can delay the progression of the conditions indicated, or caused by the agents mentioned above.
- the immune response directed against a specific antigen can be divided into two distinct categories: one involving antibodies (immune response of humoral type), the other cytotoxic effector cells such as for example macrophages, cytotoxic lymphocytes (CTL ) or killer cells ( ⁇ K and ⁇ KT) as well as helper T lymphocytes, in particular LTCD4 (cell type immune response).
- cytotoxic effector cells such as for example macrophages, cytotoxic lymphocytes (CTL ) or killer cells ( ⁇ K and ⁇ KT) as well as helper T lymphocytes, in particular LTCD4 (cell type immune response).
- the two types of response are distinguished in that the antibodies recognize the antigens in their three-dimensional form whereas the T lymphocytes, for example, recognize peptide portions of said antigens, associated with glycoproteins coded by the genes of the major complex d histocompatibility (MHC), in particular the genes of the major histocompatibility complex of type I which are expressed ubiquitously on the surface of cells or the genes of the major histocompatibility complex of type II which are expressed in specific manner on the surface cells involved in the presentation of antigens (APC).
- MHC major complex d histocompatibility
- APC antigens
- the cellular type immune response is characterized in that CD4 + type T cells (“helpeo” T cells), following a well-known activation phenomenon (for a review see Alberola-Ila, 1997, Annu. Rev. Immunol. 15, 125-154), produce cytokines which, in turn, induce the proliferation of APC cells capable of producing said cytokines; cell differentiation of B lymphocytes capable of producing specific antibodies; and stimulation of cytotoxic T lymphocytes (CTL).
- helpeo T cells CD4 + type T cells
- APC cells capable of producing said cytokines
- B lymphocytes capable of producing specific antibodies
- CTL cytotoxic T lymphocytes
- cytotoxic effector cells such as for example CD8 + lymphocytes (CTL) are activated a) after interaction with antigenic peptides fixed on and presented by the glycoproteins carried by ubiquitous cells and encoded by genes belonging to the MHC I system, and b) possibly by the cytokines produced by CD4 +.
- CTLs thus activated are then capable of destroying the cells expressing said antigenic peptide.
- Cytokines are molecules naturally produced following antigenic stimulation or an inflammatory reaction (Gillis and Williams, 1998, Curr. Opin. Immunol., 10, 501-503) whose usefulness in the context of treatment of certain cancers has been shown in particular by Oettger (1991, Curr. Opin. Immunol., 3, 699-705). Leroy et al. (1998, Res. Immunol., 149, 681-684) have described that the production of cytokines at the tumor site, after intra-tumor administration of recombinant viral vectors allows the induction of an immune response associated with inhibition of tumor growth.
- Chemokines are a subclass of the cytokine family. They are distinguished from other cytokines by their chemo-attractive property, in particular during the natural processes of chemotaxis, and in particular of attraction of the cells of the immune system towards the tissues in which the inflammation or the infection are located, they are also distinguished by their anti-angiogenic properties. Chemokines are small proteins (70 to 80 amino acids) whose amino acid sequences have a low rate of homology (varying from 10 to 70% depending on the chemokines considered).
- chemokines have been identified (see for example Zlotnik and Yoshie, 2000, Immunity 12, 121-127 which describes the classification of chemokines and their respective role in the immune mechanism).
- Four families have been defined on the basis of the position of the cysteine residues which they contain.
- the ⁇ families whose N-terminal end comprises 2 cysteines separated by a single amino acid (ie chemokines of the IL-8, NAP-2, GCP-2 type) and 3 whose N-terminal end comprises 2 adjacent cysteines (ie the chemokines of RANTES type, MIP-1, MCP1) are the best characterized (Horuk, R., 1994, Trends Pharmacol. Sci., 15, 159-165; Murphy, 1994, Annu. Rev. Immunol., 12 , 593-633).
- T cells Na ⁇ ve CD4 + require 2 signals for the induction of an effective immune response: a first involving the specific recognition of MHC / peptide by the TCR / CD3 complex and the CD4 co-receptor, and a second called costimulation. Costimulation makes it possible to amplify and prolong the primary activation by the TCR, but also to inhibit the apoptosis of T cells after activation.
- the TCR is made up of two functionally distinct units.
- a heterodimer ( ⁇ or ⁇ ) specific for each lymphocyte is necessary for antigenic recognition.
- the engagement of the TCR with its CD3 ligand initiates a cascade of intracellular signals which results in the adhesion, proliferation, differentiation into mature cells and the increase in the transcriptional activity of cytokine genes, cytokine receptors and proto-oncogenes.
- Another therapeutic strategy has also been proposed combining the advantages of gene therapy and the use of specific antibodies.
- the production by genetic engineering of antibodies, antibody fragments or derivatives of antibodies such as chimeric antibodies, in eukaryotic cells is now a standard technique (EP 0 120 694 or EP 0 125 023).
- international patent application WO 00/24896 describes a method for the treatment or prevention of cancer, based on the expression on the surface of tumor cells of antibodies specific for the TCR / CD3 complex in order to induce stimulation of T cells and the anti-tumor immune response.
- compositions allowing in particular the implementation of effective anti-tumor treatments, easy to set up and allowing a prolonged control of the tumor volume and an increase in the survival rate of the treated patients.
- the depositor has now identified new compositions, the different constituents of which are chosen so as to obtain a synergistic effect from their activities. respective and improved properties of said constituents. More particularly, such compositions allow better recruitment of the various effectors of the anti-tumor immune response (T cells, NK, dendritics, macrophages, etc.) thus making it possible to inhibit or delay cell proliferation and to induce apoptosis (cell death) of target cells.
- the present invention offers an advantageous and effective alternative to the approaches of the prior art, in particular for treating or preventing cancer of man or animal.
- the present invention relates firstly to a biological material comprising at least a first and at least a second nucleic acid sequence, said nucleic acid sequences being placed under the control of the elements ensuring their expression in a host cell, according to which:
- Said first nucleic acid sequence codes for all or part of an antibody, characterized in that when this said antibody or this said part of antibody is expressed, it is located on the surface of said host cell and in that said antibody or said part of antibody is capable of binding to a polypeptide present on the surface of a cytotoxic effector cell or of a T helper lymphocyte, said polypeptide being involved in the process of activation of such a cell,
- Said second nucleic acid sequence codes for all or part of a polypeptide involved in the stimulation of the immune response and / or in the attraction to the expression site and in the activation of cytotoxic effector cells and / or of lymphocytes T helper.
- said polypeptide is selected from chemokines and co-stimulation molecules.
- nucleic acid sequence is intended to denote a fragment of DNA and / or RNA, double strand or single strand, linear or circular, natural isolated or synthetic, designating a precise sequence of nucleotides, modified or not. , making it possible to define a fragment or a region of a nucleic acid without limitation of size. According to a preferred embodiment, it is a nucleic acid chosen from the group consisting of a cDNA; genomic DNA; plasmid DNA; a messenger RNA.
- said “first nucleic acid sequence” in particular codes all or part of a native antibody, or for a derivative of such an antibody, provided that said antibody, fragment or derivative of antibody is expressed at the surface of the host cell in which a said first nucleic acid sequence has been introduced and in that said antibody is capable of binding to a polypeptide present on the surface of a cytotoxic effector cell or of a T helper lymphocyte and involved in the process of activation of such a cell.
- fragment of antibodies is intended to denote the fragments F (ab) 2 , Fab ', Fab, Fv, sFv (Blazar et al., 1997, J.
- derivatives of antibodies is intended to denote, for example, a chimeric derivative of such an antibody (see for example the chimeras the anti CD3 Mouse / Man antibodies in Arakawa et al., 1996, J. Biochem., 120, 657- 662 or immunotoxins such as sFv-toxin from Chaudary et al., 1989, Nature, 339, 394-397).
- antibody expressed on the surface of the host cell is meant an antibody whose at least the functional region capable of recognizing and binding to its specific antigen is expressed on the surface of the host cells to allow said recognition and fixation. More particularly, the antibodies used in the context of the present invention consist of fusion polypeptides comprising at least the amino acids defining said functional region and a peptide capable of conferring on the antibody a transmembrane localization in the host cell.
- transmembrane localization refers to an anchoring within the double lipid membrane layer of the host cell or on the external surface of this bilayer. The nucleic acid sequences encoding many transmembrane peptides are described in the literature.
- said transmembrane peptide is isolated or derived from a glycoprotein, a lipoprotein, or a membrane receptor.
- the transmembrane peptide is isolated from a glycoprotein such as the glycoprotein F of the measles virus (European patent EP 0 305 229), CD4 (Weijtens et al., 1998, Gene Therapy, 5, 1195-1203), the gpl60 of the HIV virus (Polydefkis et al, 1990, J. Exp. Med., 171, 875-887), Fc (see below) and more particularly the glycoprotein of the rabies virus (French patent application FR 97 09152).
- a glycoprotein such as the glycoprotein F of the measles virus (European patent EP 0 305 229), CD4 (Weijtens et al., 1998, Gene Therapy, 5, 1195-1203), the gpl60 of the HIV virus (Polydefkis et al, 1990, J. Exp
- the first nucleic acid sequence contains a gene coding for the heavy chain of said antibody fused with the nucleic acid sequence coding for a transmembrane peptide as defined above.
- the first nucleic acid sequence may contain a gene encoding the light chain of said antibody.
- the expression of the heavy and light chains can be controlled by independent regulatory elements but it is also possible to have recourse to common elements (bicistronic cassette) and possibly, to reinitiate the translation of the second cistron (for example the sequences coding for the light chain) by means of an IRES (WO 98/49334).
- the antibody expressed on the surface of host cells is capable of binding to a polypeptide present on the surface of a cytotoxic effector cell or of a T helper lymphocyte, in particular a CD4 T helper lymphocyte, and involved in the process for activating such a cell, and more particularly at a receptor directly involved in such a process.
- this phenomenon of activation of cytotoxic effector cells or of helper T lymphocytes is a determining element of the cell-mediated immune reaction.
- this antibody can also bind to the polypeptides present on cytotoxic effector cells or already activated helper lymphocytes.
- cytotoxic effector cell is meant the macrophages, cytotoxic T lymphocytes (CTL) and killer cells (NK), as well as their derived cells such as for example LAK, (Versteeg, 1992, Immunology Today, 13, 244-247; Brittende et al., 1996, Cancer, 77, 1226-1243; Poplack et al., 1976, Blood, 48, 809-816).
- CTL cytotoxic T lymphocyte
- NK killer cells
- helper T lymphocyte is intended to denote in particular the CD4 cells which, after activation, allow the secretion of activation factors of the effector cells of the immune response (see below).
- polypeptides, and in particular the receptors, expressed on the surface of these cells and which are involved in the activation of such cells consist in particular all or part of the TCR complex, more particularly TCR- ⁇ , TCR- ⁇ or CD3, all or part of the complexes CD8, CD4, CD28, LFA-1, 4-1BB (Melero et al., 1998, Eur. J.
- cytokine receptors Finke et al., 1998, Gene Therapy, 5, 31- 39
- IL-7 IL-4, IL-2, IL-12, IL-15, IL-18, IL-21 or GM-CSF
- NK cells such as for example N ⁇ l4NKT (Kawano et al., 1998, Immunology, 95, 5690-5693), NKAR, Nkp46 (Pessino et al., 1998, J. Exp.
- Nkp44 all or part of the receptors macrophages such as for example the Fc receptor (Deo et al., 1997, Immunology Today, 18, 127-135).
- the cytotoxic effector cells or the T helper lymphocytes so that they express on their surface a polypeptide, naturally not expressed by these cells, and capable of inducing the activation process of such cells, by the introduction into these cells of nucleic acid sequences containing the gene coding for such a polypeptide.
- the present invention is then possible to select a nucleic acid sequence coding for all or part of an antibody capable of being expressed on the surface of the target cells of the patient to be treated, said antibody being capable of binding to such a polypeptide naturally not expressed by these cytotoxic effector cells or helper T lymphocytes. More particularly, the present invention is based on the possibility of cloning the genes coding for all or part of an antibody and of expressing said antibody in cells after transfer of said genes into said cells from conventional expression vectors (see below). -after).
- the literature offers a large number of examples of genes coding for antibodies capable of reacting with such polypeptides or receptors. It is within the ability of those skilled in the art to obtain the nucleic acid sequences encoding such antibodies.
- hybridomas can also be cited as being particularly suitable for isolating the first nucleic acid sequence as defined above:
- - TR310 hybridoma (murine anti-N ⁇ 7 rat (IgG2b); ATCC HB-219; I. L. Weissman; murine myeloma / rat splenocyte fusion); - H57-597 hybridoma (anti-murine TCR ⁇ hamster (IgG); ATCC HB-218; Kubo et al., 1989, J. Immunology, 142, 2736-2742; murine myeloma / hamster splenocyte fusion);
- nucleic acid sequences encoding the heavy and / or light chains of these different antibodies can be obtained by conventional methods in the art, in particular amplification (PCR, RT-PCR, ...), cloning using specific oligonucleotides or techniques using cAD ⁇ libraries (Maniatis et al., 1982, Molecular cloning. A laboratory manual.
- nucleic acid sequence coding for the heavy chain of the antibody is fused with the nucleic acid sequence coding for a transmembrane peptide such as the rabies glycoprotein.
- the "second nucleic acid sequence” codes for all or part of a polypeptide involved in the stimulation of the immune response and / or in the attraction to the expression site and the activation cytotoxic effector cells and / or helper T cells.
- said polypeptide is selected from chemokines and co-stimulation molecules.
- stimulation of the immune response makes it possible to induce or activate an immune response directed specifically against a cell, in particular a tumor or a cell infected with a virus, or to inhibit growth and / or division. of such a cell.
- Attraction to the expression site and activation of cytotoxic effector cells or helper T cells can be defined by the ability to increase the migration of immune cells to the expression site of a chemokine to develop or prolong a response immune.
- the term “chemokine” can be defined as a cytokine with a chemoattractor effect (ability to attract immune cells to the expression site of said chemokine).
- the chemokine in use in the context of the present invention has one or more complementary functions, in particular the activation of immune cells making it possible to stimulate the immune response and / or an anti-angiogenic function making it possible to inhibit vascularization within of a tumor.
- the chemoattraction activity of a given polypeptide, in particular derived from the chemokine MIP, on cells involved in immune reactions can be evaluated by a test.
- co-stimulation molecules refers to a molecule making it possible to amplify an immune response and / or to prolong the state of activation of an immune cell or involved in an immune response , in particular by stabilizing the TCR complex after recognition of the peptide linked to the MHC.
- the co-stimulation activity can be evaluated by conventional techniques such as those described in the example part of the present application (for example by measuring the proliferation of naive splenocytes after incorporation of thymidine H as described in point 5 of the example 2).
- nucleic acid sequence coding for the entirety of said polypeptide in question or only part of this polypeptide, or a derived or mutated polypeptide, insofar as the function and properties in terms of stimulation of the immune response, of attraction to the site of expression and / or activation of cytotoxic effector cells or of helper T lymphocytes are conserved.
- mutation refers to the deletion and / or substitution and / or addition of one or more nucleotides or any combination of this type of mutation.
- sequence coding for a hybrid polypeptide originating from the fusion of sequences of various origins for example coding for two different chemokines).
- a preferred chemokine is a chemokine of the MIP-1 type, and more particularly selected from the group consisting of the chemokines MIP-la and MlP-l ⁇ , the properties of which have been demonstrated by Wolpe et al. (1988, J. Exp. Med. 167, 570-581).
- MIP 1 ⁇ is produced by T lymphocytes and monocytes. It allows chemoattraction of eosinophils and T lymphocytes during respiratory tract infections; monocytes and neutrophils in rheumatoid arthritis, inflammation of the digestive system or meningitis of bacterial origin. In addition, it inhibits the proliferation of hematopoietic precursors.
- MlPl ⁇ The nucleotide and amino acid sequences of MlPl ⁇ are described in Obaru et al. (1986, J. Biochem. 99, 885-894), the content of which is incorporated by reference in the present application.
- MlP-l ⁇ whose nucleotide and amino acid sequences are described in Brown et al. (1989, J. Immunol. 142, 679-88, the content of which is incorporated by reference in the present application), is also produced by T lymphocytes and monocytes. It exerts its chemoattractive properties on monocytes and neutrophils in cases of bone arthritis and bacterial meningitis. Like MIP-la, it inhibits the proliferation of hematopoietic precursors.
- MIP- la and MlP-l ⁇ There are natural variants of said proteins MIP- la and MlP-l ⁇ which are known to those skilled in the art and which bear, for example, the names GOS19, LD78, pAT464, TY5 (for mice) or SIS ⁇ (for mice) for MIP-la and pAT744, Act-2, G-26, H- 400 (mouse) or hSIS ⁇ (mouse) for MlP-l ⁇ .
- the sequence corresponding to Act-2 will preferably be chosen (Lipes et al., 1988, Proc. Natl. Acad. Sci. USA 85, 9704-9708, the content of which is incorporated herein by reference).
- co-stimulation molecules which can be used in the context of the present invention, it is preferred to use those acting on the co-stimulation of T cells and, in particular B7-1 (GenBank XM002948) and B7-H1 (GenBank AF 177937) .
- the costimulation of T cells decreases their apoptosis generated by the first activation signal (AICD; Activation-Induced Cell Death), by inducing the expression of anti-apoptotic factors such as Bcl-2, c-FLIP, Bcl-xL and by reducing the activity of the Fas apoptotic complex and procaspase-8. It also makes it possible to promote the differentiation of activated T cells into memory cells.
- the present invention also relates to any other molecule allowing said co-stimulation of T cells.
- the second nucleic acid sequence codes for a polypeptide secreted from the host cell.
- the means for obtaining the secretion of a polypeptide from the host cell are known to those skilled in the art.
- Many functional signal peptides in eukaryotic cells, and in particular mammals, are disclosed in the state of the art.
- the biological material according to the present invention also comprises at least a third nucleic acid sequence coding for all or part of a polypeptide having cytotoxic activity.
- polypeptide having cytotoxic activity means any polypeptide capable of inhibiting the growth and / or division of a host cell, in particular a tumor cell (the cytotoxic activity is then called anti-tumor activity) or infected (cytotoxic activity is then called antiviral, parasitic or bacterial activity depending on the infecting pathogen). According to a preferred case, the cytotoxic activity results in the death of said cell. According to a particular case, it would also be possible to use the biological material according to the present invention in pathological cases associated with cell proliferation, such as for example restenosis phenomena.
- the cytotoxic activity of a given polypeptide in particular with regard to tumor cells, can be evaluated in vitro by measuring cell survival either by viability tests in the short term (such as for example the tryptan blue test or MTT), either by clonogenic survival tests (colony formation) (Brown and Wouters, 1999, Cancer Research 59, 1391-1399) or in vivo by measuring tumor growth (size and / or volume) in an appropriate animal model (Ovejera and Houchens, 1981, Semin. Oncol. 8, 386-393).
- said polypeptide having cytotoxic activity is chosen from cytokines, proteins encoded by suicide genes and anti-angiogenic protein factors.
- said polypeptide is a cytokine
- it is preferably a cytokine chosen from interferons ⁇ , ⁇ and ⁇ , interleukins, and in particular IL-2, IL-4, IL-6, IL-7, ⁇ IL-IO, IL-12, 1TL-15, 1TL-18 or riL-21, tumor necrosis factors (TNF) and colony stimulating factors (GM-CSF, C-CSF, M -CSF, .
- said cytokine is selected from interleukin-2 (IL-2) and interleukin-12 (IL-12).
- Interleukin-2 is in particular responsible for the proliferation of activated T lymphocytes, the multiplication and activation of cells of the immune system (for the nucleic acid sequence see in particular FR 85 09480).
- Interleukin-12 has been identified and isolated in macrophages as a stimulating factor for cytotoxic and NK T cells, which induces the differentiation of CD4 + T lymphocytes by participating in the homeostasis of the Thl and Th2 populations.
- Two cell types (NK and NKT cells) are involved in the anti-tumor activity mediated by 1TL-12, depending on the dose used or its injection scheme.
- the polypeptide product of expression of a suicide gene exhibits at least one enzymatic activity selected from thymidine kinase activity, purine nucleoside phosphorylase activity, guanine or uracil activity or orotate phosphoribosyl transferase and the cytosine deaminase activity.
- polypeptides are not toxic as such but have catalytic enzymatic properties capable of transforming an inactive substance (predrogue), for example a nucleoside or a nucleoside analog, into a substance which is highly toxic to the cell, for example a modified nucleoside which may be incorporated into the DNA or RNA chains in elongation, with the consequence, in particular, of the inhibition of cell division or of cellular dysfunctions leading to the death of the cell containing such polypeptides.
- the genes coding for such polypeptides are called "suicide genes". Of Many suicide / predrole gene couples are currently available.
- TK HSN-1 herpes simplex virus type 1
- GCN Pacyclovir or ganciclovir
- FCY1 genes of Saccharomyces cerevisiae (S. cerevisiae) and codA of E.
- CDase is used in combination with the enzyme uracilephosphoribosyl transferase (UPRTase) which has the property of converting the 5-FU produced by the action of CDase into highly toxic 5-FUMP.
- UPRTase uracilephosphoribosyl transferase
- the upp and FUR1 genes coding for UPRTase respectively from E. coli and S. cerevisiae have been cloned and sequenced (Andersen et al., 1992, ⁇ ur. J.
- the biological material according to the invention comprises a first nucleic acid sequence coding for the antibody KT3 expressed in transmembrane form and capable of fixing the TCR complex and a second nucleic acid sequence coding for the chemokine MlP-l ⁇ .
- Another preferred biological material comprises a first nucleic acid sequence coding for the antibody KT3 expressed in transmembrane form and capable of fixing the TCR complex and a second nucleic acid sequence coding for the chemokine BRAK.
- the biological material also includes at least a third nucleic acid sequence coding for 1TL-2 or 1TL-12.
- each nucleic acid sequence can be directed by elements (identical or different, homologous or heterologous with respect to the nucleic acid sequence in question, constitutive or inducible) which are specific to it, but it is possible to use common elements to direct the expression of two, or even three, nucleic acid sequences used in the part of the present invention. In this case, we can use merged sequences or IRES to reinitiate the translation of cistrons.
- elements ensuring expression is intended to denote the elements necessary to ensure the expression of the nucleic acid sequence after its transfer into a target cell.
- These include promoter sequences and / or regulatory sequences effective in said cell.
- the promoter used can be a viral or cellular, ubiquitous or tissue-specific promoter or even a synthetic promoter. Of course, it can be modified with respect to the native promoter sequence in order to include or delete one or more restriction sites or else to delete negative sequences reducing the levels of transcription, etc.
- viral promoters in particular RSN (Rous Sarcoma Virus), SV40 (Simian Virus), CMV (Cytomegalovirus), early adenoviral (Ela, E3, ...) and late (MLP) viruses.
- RSN Raster Sarcoma Virus
- SV40 Simian Virus
- CMV Cytomegalovirus
- Ela, E3, ...) and late (MLP) viruses for Major Late Promoter
- retroviral LTRs like that of the MLV virus for Murine Leukemia Virus
- TK-HSV-1 promoter the TK-HSV-1 promoter.
- the promoters that can be used can be chosen from promoters 7.5K, H5R, TK, p28, pl i, K1L of vaccinia virus as well as early-late hybrid promoters and synthetic promoters (Chakrabarti et al., 1997, Biotechniques 23, 1094-1097; Hammond et al., 1997, J. Virological Methods 66, 135-138; Kumar and Boyle, 1990, Virology 179, 151-158). It is also possible to choose a promoter sequence specific for a given cell type, or activatable under defined conditions. The literature provides a great deal of information relating to such promoter sequences.
- promoters of the MT genes (metallothionein; Me Ivor et al., 1987, Mol. Cell Biol. 7, 838-848), and those coding for ⁇ -1 antitrypsin, CFTR, the pulmonary surfactant , immunoglobulins, muscle creatine kinase, ⁇ -actin, SR ⁇ , protein SM22 (Moessler et al, 1996, Development, 122, 2415-2425) and desmin (WO 96/26284). It is also possible to use a promoter which can be activated in dividing cells, for example governing the transcription of genes overexpressed in tumor cells.
- the elements ensuring expression can also include so-called enhancer sequences, LCR (for Locus Control Region) making it possible to improve or stabilize the expression of the nucleic acid sequence in the host cell or to confer expression tissue-specific.
- the elements ensuring the expression of said nucleic acid sequences may also include sequences required for intracellular transport, for replication and / or for integration, for transcription or translation.
- the literature provides a great deal of information relating to such regulatory elements.
- the nucleic acid sequences used in the context of the present invention may include “neutral” sequences or introns which do not interfere with transcription and are spliced before the translation step. Such sequences and their uses are described in the literature (WO 94/29471).
- nucleic acids which can be used according to the present invention can also be nucleic acids modified so that it is not possible for them to integrate into the genome of the target cell or nucleic acids stabilized using agents, such as for example spermine, which as such have no effect on the efficiency of the transfection.
- Said nucleic acid sequences can be controlled by identical or different elements, can be located in relation to one another in a contiguous, distant, in the same direction or in opposite directions, provided that their expression in the cell host is not affected.
- the biological material according to the invention is in the form of naked DNA or RNA, that is to say free of any compound facilitating its introduction into cells (transfer nucleic acid sequence).
- said first and second (and optionally third) nucleic acid sequences of the biological material according to the invention can be included in at least one vector allowing their transfer into the host cell.
- they can be included in the same vector or, according to a preferred embodiment, in independent vectors allowing their transfer into the host cell.
- such a vector could be a plasmid.
- plasmids which can be used in the context of the present invention is vast. They may be cloning and / or expression vectors. In general, they are known to those skilled in the art and many of them are commercially available. However, it is also possible to construct or modify them by genetic manipulation techniques. Examples of plasmids derived from pBR322 (Gibco BRL), pUC (Gibco BRL), pBluescript (Stratagene), pREP4, pCEP4 (Invitrogene) or even p Poly (Lathe et al., 1987, Gene, 57) , 193-201).
- a plasmid used in the context of the present invention contains an origin of replication ensuring the initiation of replication in a producer cell and / or a host cell (for example, the ColEl origin will be retained for a plasmid intended to be produced in E. coli and the oriP / EBNAl system if it is desired to be self-replicating in a mammalian host cell, Lupton and Levine, 1985, Mol. Cell. Biol, 5, 2533-2542; Yates and al., 1985, Nature 313, 812-815). It can also comprise a selection gene making it possible to select or identify the transfected cells (complementation of an auxotrophy mutation, gene coding for resistance to an antibiotic, etc.).
- cer sequence which promotes the monomeric maintenance of a plasmid (Summers and Sherrat, 1984, Cell 36, 1097-1103, sequences of integration into the cell genome).
- the vectors used in the context of the present invention are viral vectors, for example selected from adenoviral, retroviral, poxviral vectors, in particular derived from the vaccinia virus (Goebel et al., 1990, Virol., 179, 247-266 and 517-563; Johnson et al., 1993, Virol., 196, 381-401), from the Modifed Virus Ankara (MVA) (Antoine et al., 1998, Virol., 244, 365-396 ) or canarypox, or vectors derived from herpes viruses, alphaviruses, foamyviruses or viruses associated with adenoviruses.
- adenoviral, retroviral, poxviral vectors in particular derived from the vaccinia virus (Goebel et al., 1990, Virol., 179, 247-266 and 517-563; Johnson et al., 1993, Virol., 196, 381-401), from
- a non-replicative and non-integrative vector will be used.
- the nature of the vector is of little importance since the transfer only takes place to allow the transfer of the sequences (first, second, even third ) of acids nucleic acids inside host cells.
- transfer models are widely used in the literature.
- the basic technology for inserting a nucleic acid sequence and its regulatory elements into a poxviral genome is described in numerous documents accessible to those skilled in the art (Piccini et al., 1987, Methods of Enzymology, 153, 545-563; US 4,769,330; US 4,772,848; US 4,603,112; US 5,100,587 and US 5,179,993).
- This technique is based on homologous recombination between the common sequences present in the viral genome and a transfer plasmid carrying the nucleic acid sequence to be transferred.
- the insertion site in the poxviral genome preferably consists of a nonessential locus.
- nonessential loci reside in the non-coding intergenic regions or within a gene whose deletion or non-functionality has little or no effect on viral growth, replication or infection. It is also possible to consider insertion into an essential locus provided that the affected function is complemented in trans during the production of the viral particles, by means for example of a complementation line or of a helper virus carrying the sequences. coding for deficient function.
- a preferred insertion site is located within the thymidine kinase (TK) gene (Hruby et al., 1983, Proc. Natl. Acad. Sci. USA, 80, 3411-3415; Weir et al, 1983, J.
- TK thymidine kinase
- Virol, 46, 530-537 Being an MVA vector, the insertion of the nucleic acid sequence will take place within one of deletions I to VII, and preferably within deletion II or III (Meyer et al, 1991, J. Gen. Virol, 72, 1031-1038; Sutter et al, 1994, Vaccine, 12, 1032-1040).
- the conditions for constructing a recombinant vaccinia virus are known to those skilled in the art (see for example EP 83,286 and EP 206,920 for the vaccinia virus, and Mayr et al, 1975, Infection, 3, 6 -14 and Sutter and Moss, 1992, Proc. Natl. Acad. Sci. USA, 89, 10847-10851 for MVA).
- Retroviruses have the property of infecting and integrating mainly in dividing cells and in this respect are particularly suitable for cancer application.
- a recombinant retrovirus according to the invention generally comprises the LTR sequences, an encapsidation region and at least one of the nucleic acid sequences in use in the context of the invention, placed under the control of the retroviral LTR or of a promoter internal such as those described below. It can be derived from a retrovirus of an origin any (murine, primate, feline, human, etc.) and in particular MoMuLV (Moloney murine leukemia virus; Gilboa et al, 1988, Adv. Exp. Med.
- the retroviral vector according to the invention may include modifications in particular at the level of the LTRs (replacement of the promoter region with a eukaryotic promoter) or of the packaging region (replacement with a heterologous packaging region, for example of the VL30 type). (see French applications 94 08300 and 97 05203 and US 5,747,323).
- an adenoviral vector can be non-defective but preferably replicative in a conditional manner (CRAd; Heise and Kirn, 2000, J. Clin. Invest, 105, 847851; Alemany et al, 2000, Nature Biotechnology, 18, 723-727; Hernandez- Alcoceba et al, 2000, Human Gene Ther, 11, 2009-2024) or defective for replication, that is to say devoid of all or part of at least one region essential for replication selected from the El regions, E2, and E4. A deletion from the El region is preferred.
- the adenoviral vector can also be devoid of all or part of the non-essential E3 region. According to another alternative, it is possible to use a minimal adenoviral vector retaining the sequences essential for the packaging, namely the ITRs (Inverted Terminal Repeat) 5 'and 3' and the packaging region. Furthermore, the origin of the adenoviral vector according to the invention can be varied both from the point of view of the species and of the serotype.
- adenovirus of human or animal origin canine, avian, bovine, murine, ovine, porcine, simian, ...) or a hybrid comprising fragments of the adenoviral genome from at least two different origins.
- Mention may more particularly be made of the adeno virus CAV-1 or CAV-2 of canine origin, DAV of avian origin or else Bad of type 3 of bovine origin (Zakharchuk et al, Arch. Virol, 1993, 128: 171- 176; Spibey and Cavanagh, J. Gen. Virol, 1989, 70: 165-172; Jouvenne et al.
- an adenoviral vector of human origin preferably deriving from a serotype C adenovirus, in particular of type 2 or 5, is preferred.
- An adenoviral vector according to the present invention can be generated in vitro in Escherichia coli (E. coli) by homologous ligation or recombination (see for example international application WO 96/17070) or alternatively by recombination in a complementation line (see for example Graham and Prevect, 1991, in Methods in Molecular Biology, vol. 7, p 109-128; Ed .: EJ Murey, The Human Press Inc.).
- the first nucleic acid sequence is carried by a poxviral vector derived from the Modifed Virus Ankara (MVA) strain of the vaccinia virus and the second nucleic acid sequence is carried by an adenoviral vector.
- the biological material according to the invention comprises a third nucleic acid sequence, this is preferably carried by an adenoviral vector.
- the term “biological material” includes the viral vector (recombinant genome) and the infectious viral particles comprising said viral vector.
- a viral particle can be generated from a viral vector according to any conventional technique in the art. Its propagation is carried out in particular in a complementation cell adapted to the deficiencies of said vector.
- an adenoviral vector use will be made, for example, of a complementation line as described in application WO 94/28152, to line 293 established from human embryonic kidney cells, which effectively complements the El function (Graham et al, 1977, J. Gen. Virol.
- helper viruses can also be used to at least partially complement defective functions.
- cell complementation means a cell capable of providing in trans the early and / or late factors necessary for the packaging of the viral genome in a viral capsid to generate a viral particle containing the recombinant vector.
- Said cell may not by itself complement all the defective functions of the vector and in this case may be transfected / transduced by a vector / helper virus providing the complementary functions.
- the viral particles containing poxviral vectors are prepared by infection of permissive cells (for example primary fibroblasts of chicken embryos) according to the techniques of the art widely detailed in the documents cited in the field of poxviruses.
- the invention also relates to a process for preparing a viral particle, according to which:
- a biological material according to the invention is introduced into a cell, in particular a complementation cell capable of complementing said vector in trans, so as to obtain a said transfected cell, (ii) culturing said transfected cell under appropriate conditions to allow the production of said viral particle, and
- the viral particle can be recovered from the culture supernatant but also from the cells.
- One of the commonly used methods consists in lysing the cells (chemical lysis, freezing / thawing, osmotic shocks, mechanical shocks, sonication, etc.), to collect the virions in the lysis supernatant. These can be amplified and purified according to the techniques of the art (chromatographic process, ultracentrifugation in particular through a cesium chloride gradient, ).
- the vector used according to the invention can be a non-viral vector such as for example a vector consisting of at least one said nucleic acid sequence or a plasmid vector as defined above, complexed or conjugated to at least one molecule or carrier substance selected from the group consisting of a cationic amphiphile, in particular a cationic lipid, a cationic or neutral polymer, a polar protic compound in particular chosen from propylene glycol, polyethylene glycol, glycerol, ethanol, 1-methyl L -2-pyrrolidone or their derivatives, and an aprotic polar compound chosen in particular from dimethylsulfoxide (DMSO), diethylsulfoxide, di-n-propylsulfoxide, dimethylsulfone, sulfolane, dimethylformamide, dimethylacetamide, tetramethylurea, acetonitrile or their derivatives.
- DMSO dimethylsulfoxide
- DMSO dimethylsul
- cationic lipids have a great affinity for nucleic acids and have the capacity to interact with the cell membrane (Felgner et al, 1989, Nature, 337, 387-388).
- the cationic lipids which are particularly suitable for the implementation of the present invention include in particular DOTMA (Felgner et al, 1987, Proc. Natl. Acad. Sci. USA, 84, 7413-7417), DOGS or Transfectam TM ( Behr et al, 1989, Proc. Natl. Acad. Sci.
- the polymers which are suitable for implementing the invention are preferably cationic, such as polyamidoamine (Haensler and Szoka, 1993, Bioconjugate Chem, 4, 372-379), dendritic polymers (WO 95/24221), polyethylene imine or polypropylene imine (WO 96/02655), polylysine (US 5,595,897 or FR 2,719,316), chitosan (US 5,744,166) or DEAE dextran (Lopata et al, 1984, Nucleic Acid Res, 12, 5707- 5717).
- polyamidoamine Hyensler and Szoka, 1993, Bioconjugate Chem, 4, 372-379
- dendritic polymers WO 95/24221
- polyethylene imine or polypropylene imine WO 96/02655
- polylysine US 5,595,897 or FR 2,719,316
- chitosan US 5,744,166
- the vectors used in the context of the present invention may also comprise targeting elements which can make it possible to direct the transfer of said nucleic acid sequences to certain cell types or certain particular tissues (tumor cells, cells of the pulmonary epithelium, hematopoietic cell, muscle cell, nerve cell, ). They can also make it possible to direct the transfer of an active substance to certain preferred intracellular compartments such as the nucleus and the mitochondria. They may also be elements which facilitate penetration into the interior of the cell or the lysis of endosomes. Such targeting elements are widely described in the literature.
- lectins may for example be all or part of lectins, peptides, in particular the peptide JTS-1 (see patent application WO 94/40958), oligonucleotides, lipids, hormones, vitamins, antigens, antibodies, ligands specific for membrane receptors, ligands capable of reacting with an anti-ligand, fusogenic peptides, nuclear localization peptides, or a combination of such compounds.
- they may be galactosyl residues making it possible to target the asialoglycoprotein receptor on the surface of liver cells, ligands which can interact with receptors such as growth factor receptors, cytokine receptors, lectins, adhesion proteins, it can also be an antibody fragment such as the Fab fragment, a fusogenic peptide INF-7 derived from the HA-2 subunit of the hemagglutinin of the influenza virus (Plank and al, 1994, J. Biol. Chem, 269, 12918-12924), of a nuclear localization signal derived from the T antigen of the SV40 virus or of the EBNA-1 protein of the Epstein Barr virus.
- tumor cells cells infected with a viral, parasitic or even bacterial pathogen.
- the expression on the surface of these cells of all or part of an antibody capable of binding to a polypeptide present on the surface of a cytotoxic effector cell or of a T helper lymphocyte and involved in the method of activating such a cell makes it possible to direct the cytotoxic immune response towards a given target, and more particularly to direct this response at the level of a tumor or an infectious focus.
- the invention also relates to a host cell comprising a biological material according to the invention. It is preferably a tumor mammalian cell or a mammalian cell infected with a viral pathogen or a mammalian cell infected with a bacterial pathogen.
- the host cell according to the invention does not naturally express said first nucleic acid sequence (coding for an antibody).
- a said cell is present in a form allowing its administration in the organism of a mammal, human or animal, as well as possibly its prior culture and said cell being genetically modified in vitro by introduction:
- At least one said second nucleic acid sequence encoding all or part of a polypeptide allowing the activation of the immune response and / or the chemoattraction of a cytotoxic effector cell and / or of a T helper lymphocyte.
- said polypeptide is selected from chemokines and co-stimulation molecules, and
- nucleic acid sequence encoding all or part of a polypeptide having cytotoxic activity.
- said host cell comes either from the mammal to be treated, or from a mammal other than that to be treated. In the latter case, it should be noted that said host cell will have undergone a treatment making it compatible with the mammal to be treated. According to a preferred case, by “mammal” is intended to denote a human mammal.
- Such biological material when administered to a patient, and more particularly administered intratumorally, is capable of inducing or stimulating in the latter a cell-mediated immune response which can lead to the production of cytokines and to cytotoxic effect of effector cells which result not only in the elimination of the administered cells but also in the elimination of neighboring cells presenting the antigens, in particular tumor, capable of being recognized by said activated cytotoxic effector cells.
- the invention also relates to a method for preparing a cell according to the invention, characterized in that said first and second nucleic acid sequences as described above are introduced by any suitable means into a mammalian cell. , and, optionally, said third nucleic acid sequence, said nucleic acid sequences being placed under the control of the elements ensuring their expression in said host cell, then in that one selects from these cells those genetically modified by said nucleic acid sequences.
- the invention further relates to the use of a biological material or of a cell according to the invention, for the preparation of a pharmaceutical composition intended for the treatment or prevention of cancers or viral infections. More specifically, the invention relates to the co-use on the one hand, of a nucleic acid sequence coding for all or part of an antibody expressed on the surface of said target cell and capable of binding to a polypeptide present at the surface of a cytotoxic effector cell or a helper T lymphocyte and involved in the activation process of such a cell and, on the other hand, of a second nucleic acid sequence coding for a polypeptide of chemokine type or co-stimulation molecule, involved in the stimulation of the immune response or in the attraction to the expression site and the activation of cytotoxic effector cells or T helper lymphocytes, for the preparation of pharmaceutical compositions intended for treating a mammal by gene transfer.
- the invention also relates to the use of said first and second nucleic acid sequences and of a third nucleic acid sequence encoding a polypeptide having cytotoxic activity.
- a preferred use relates to (i) a vector or a viral particle MVA comprising a nucleic acid sequence coding for the antibody KT3 expressed in transmembrane manner, (ii) a vector or an adenoviral particle comprising a nucleic acid sequence coding for the Act-2 variant of the chemokine MlP-l ⁇ and (iii) a vector or an adenoviral particle comprising a nucleic acid sequence coding for IL-2 or lTL-12.
- compositions comprising a biological material or a cell as previously described, advantageously combined with a pharmaceutically acceptable vehicle for administration to the man or animal.
- a pharmaceutically acceptable vehicle for administration to the man or animal.
- This pharmaceutically acceptable vehicle is preferably isotonic, hypotonic or has low hypertonicity and has a relatively low ionic strength, such as for example a sucrose solution.
- said composition may contain solvents, aqueous or partially aqueous vehicles such as sterile water, free of pyrogenic agent and dispersion media for example.
- the pH of these pharmaceutical compositions is suitably adjusted and buffered according to conventional techniques.
- the medicament can be administered directly in vivo (for example in an accessible tumor or at its periphery, intravenously, in the vascular system by means of an appropriate probe).
- vivo for example in an accessible tumor or at its periphery, intravenously, in the vascular system by means of an appropriate probe.
- host cells from the mammal to be treated (bone marrow stem cells, peripheral blood lymphocytes, etc.), in transfecting or infecting them in vitro according to the techniques of the art and in re-administer to said mammal.
- the biological material according to the invention can be administered in vivo in particular in injectable form, in particular by intratumoral route.
- intratracheal intranasal, epidermal, intravenous, intraarterial, intracardiac, intramuscular, intrapleural, intraperitoneal, intracerebral by syringe or any other equivalent means.
- systems suitable for the treatment of the airways or mucous membranes such as inhalation, instillation, or aerosolization, topically, by oral administration or any other perfectly known means of those skilled in the art and applicable to the present invention.
- the administration can take place in single or repeated dose, one or more times after a certain interval of interval.
- compositions based on viral particles can be formulated in the form of doses of between 10 4 and 10 14 iu (infectious units) or pfu (particles forming plaques), preferably 10 6 to 10 12 iu or pfu and , most preferably, 10 7 to 10 11 iu or pfu.
- doses comprising from 0.01 to 100 mg of DNA, preferably 0.05 to 10 mg and, most preferably, from 0.5 to 5 mg can be considered.
- a preferred use is to treat or prevent cancers, tumors and diseases resulting from unwanted cell proliferation.
- cancers of the breast, the uterus in particular those induced by papillomaviruses
- the prostate in particular those induced by papillomaviruses
- the lung the bladder
- the liver in particular those induced by papillomaviruses
- the lung the bladder
- the liver in particular those induced by papillomaviruses
- the stomach the liver, the colon, the pancreas, the stomach
- esophagus larynx
- central nervous system and blood lymphomas, leukemia
- cardiovascular diseases for example to inhibit or delay the proliferation of smooth muscle cells of the vascular wall (restenosis).
- infectious diseases application to AIDS, hepatitis, cancer induced by viruses (retrovirus, papillomavirus, etc.) can be considered.
- a composition according to the invention is more particularly intended for preventive or curative treatment of diseases by
- the pharmaceutical composition of the invention can be used in combination with at least one compound naturally responsible for the co-stimulation of cytotoxic effector cells or of lymphocytes
- T helper According to this embodiment, it is possible to envisage administering said composition with a polypeptide of the cytokine or chemokine type, for example 1TL-2 or 1TL-12.
- the invention also extends to a method for the treatment and prevention of diseases by gene transfer (gene therapy), characterized in that a host organism or cell, in particular a mammal, in need of administration is administered.
- a biological material or a host cell according to the invention When the treatment method implements a biological material or a host cell comprising a third nucleic acid sequence coding for a cytotoxic gene of the suicide gene type, the treatment method also comprises an additional step according to which acceptable amounts of d are administered. '' a pharmaceutical point of view of the predrogue acting in concert with the selected suicide gene.
- a cytosine analog such as 5-FC is preferably used.
- the predrogue is administered according to standard practices and this in a prior manner, concomitant or even after that of the therapeutic agent according to the invention.
- the oral route is preferred.
- Either a single dose of the pre-drug or repeated doses may be administered long enough to allow the production of the toxic metabolite within the host organism or cell.
- the therapeutic use or the method of treatment can be associated with a second treatment of the patient by surgery (in particular by removing the tumor partially or totally), by radiotherapy or chemotherapy.
- the treatment according to the invention is applied beforehand, concomitantly or following said second treatment.
- this treatment will be applied following said second treatment.
- the therapeutic use or the treatment method according to the invention can be combined with additional treatment of the patient with molecules intended to reduce the inflammatory response induced at the tumor site (for example a vasoactive compound such as serotonin) or a molecule that inhibits reactive forms of oxygen (for example histamine).
- molecules intended to reduce the inflammatory response induced at the tumor site for example a vasoactive compound such as serotonin
- a molecule that inhibits reactive forms of oxygen for example histamine.
- the treatment according to the invention is applied beforehand, concomitantly or following said additional treatment of the patient.
- FIG. 1 is a schematic representation of the combined strategies using the vector MVA-KT3 and the vectors Ad-cytokines and / or Ad-chemokines in the RenCa model.
- the injection protocol is 2x10 iu of adenovirus injected at OJ, J2 and J4 and 2x10 7 pfu of MVA virions injected at D2, J3 and J4.
- the statistical values are calculated between the 2 curves linked by a brace (Statistica 5.1 software, Mat & Met). A p value ⁇ 0.05 is considered to be statistical.
- FIG. 2 is a schematic representation of the combined strategies using the MVA-KT3 vector and the Ad-cytokines and / or Ad-chemokines vectors in the RenCa model.
- the injection protocol is 1x10 iu of adenoviruses injected on D0, D1 and D2 and lxl O 7 pfu of MVA virions injected on D2, D3 and D4.
- the statistical values are calculated between the 2 curves linked by a brace (Statistica 5.1 software, Mat & Met). A p value ⁇ 0.05 is considered to be statistical.
- the constructions described below are carried out according to the general techniques of genetic engineering and molecular cloning, detailed in Maniatis et al, (1989, Laboratory Manual, Cold Spring Harbor, Laboratory Press, Cold Spring Harbor, NY) or according to the recommendations of the manufacturer when using a commercial kit.
- the homologous recombination steps are preferably carried out in the E. coli BJ 5183 strain. (Hanahan, 1983, J. Mol. Biol, 166, 557-580).
- the technique used consists of filling the protruding 5 ′ ends with the large fragment of DNA polymerase I from E. coli (Klenow).
- the adenoviral genome fragments used in the various constructions described below are indicated precisely according to their position in the nucleotide sequence of the Ad5 genome as disclosed in the Genbank database under the reference M73260.
- the cells are transfected or transduced and cultured according to standard techniques well known to those skilled in the art. I. Tumor models
- P815 mastocytoma H-2d, described in Dunn et al, 1957, J. Natl. Cancer Inst, 18, 587-590
- B16FO melanoma H-2b, described in Wu et al, 1996, Cancer Res, 56, 21-26
- RENCA renal cell carcinoma H-2d, described in Murphy et al, 1973, J. Natl. Cancer Inst, 50, 1013-1025.
- mice In order to establish tumors in mice, the tumor cells P815, B16F10 or RENCA are trypsinized, washed 3 times in PBS and resuspended at 3 x 10 cells / ml. 100 ⁇ l of this cell suspension are then injected into the right flank of immunocompetent B6D2 mice [(C57BL / 6 x DBA / 2) F1] aged 6-7 weeks. After the appearance of a tumor with a palpable volume of between 5 and 25 mm 3 , each mouse receives three intratumoral injections (100 ⁇ l) of a defined quantity of virus in 10 mM Tris-HCl pH 7.5, 1 mM MgCl 2 . Each condition is evaluated in groups of 10 mice. The tumor volume and the survival of the mice are determined twice a week. After a primary rejection, the mice are reinjected on the opposite flank, with the same dose of tumor cells (3E + 5 cells / mouse).
- composition of the invention administered is controlled by measuring the size of the tumors as well as by measuring the survival time of the mice treated with, if necessary, a control of the immunological status of the animal by ELISPOT, test CTL, ....
- the animals can then be subjected to a lateral challenge during which a lethal dose of tumor cells is administered to the pre-treated animal.
- the cloning of the sequences coding for the entire heavy and light chains of the antibodies KT3 and H57 is described in international application WO 00/24896.
- the chains thus isolated are subcloned by recombination in a recombinant MVA virus containing the nucleic acid sequence coding for the transmembrane region of the rabies virus (Modified Vaccinia Ankara; Antoine et al, 1998, Virology, 244, 365-396 and French patent application FR 97 09152), in order to obtain the MVATG14240 virus expressing the rat antibody KT3 (rat anti-CD3 epsilon) and MVATG14237 expressing the hamster antibody H57-597 (anti TCR alpha / beta of hamster).
- the expression cassettes are introduced into deletion II of MVA as described in application WO 00/24896. Briefly, the light chain of the antibodies is placed under the control of the early late promoter p7.5 (Goebel et al, 1990, Virol, 179, 247-266). The sequence encoding the heavy chain is placed under the control of the early late promoter pH5R (Goebel et al, 1990, Virol, 179, 247-266). The C-terminal end of the heavy chain is fused with the transmembrane and intracytoplasmic domain of the rabbit glycoprotein in order to allow the anchoring of the antibody in the plasma membrane.
- KT3 and H57 antibodies in infected cells are verified by Western blot and flow cytometry. The results observed show that there is indeed expression of immunoglobulin of the IgG type of rat and hamster on the surface of the infected cells (see example 2 of WO 00/24896).
- the functionality of MVA viruses expressing anti TCR / CD3 antibodies is tested by proliferation tests on murine spenocytes.
- the expression of the antibody KT3 or H57 on the surface of the murine cells makes it possible to induce a strong proliferation of naive T cells (see example 2 of WO 00/24896).
- the human chemokine genes used were reconstituted by assembly of oligonucleotides according to their sequence published in the database of the "National Center for Biotechnology Information”. This cloning strategy consists of the assembly, in two stages, of 8 oligonucleotides of approximately 80 bases each. The set covers the entire nucleotide sequence of the cDNA of the Act-2 variant MlP-l ⁇ genes (Accession: J04130, described in application WO 00/74629, hereinafter called MIP-1 ⁇ ), IP-10 (Accession N °: X02530), and DC-CK1 (Accession N °: AB000221).
- IL-18 does not have a natural signal sequence and is produced as a mature protein only after cleavage by caspase-1.
- the sequence encoding the mature protein (nucleotides 108-582) has therefore been cloned in phase with the signal peptide BM40 previously described (Yamaguchi et al, 1999, EMBO J, 18, 4414-1423).
- the chemokine BRAK was cloned on the basis of the sequence described by Frederick et al. (2000, Am. J. Pathol, 156, 1937-50) and Sleeman et al. (2000, Int. Immunol, 12, 677-689). These genes were cloned into an adenoviral transfer vector.
- the transfer vectors expressing the genes of human 1TL-2, of the two murine 1TL-12 subunits (p40 and p35) separated by a ribosome entry site, the mGM-CSF (leader sequence human in phase with the murine sequence) and murine B7-1 were constructed by inserting into the transfer vector the cloned sequence on the basis of the published sequence.
- the transfer vector consists of an expression cassette containing the promoter / enhancer sequences of human cytomegalo virus (CMV), a chimeric ⁇ -globin / human IgG unintron and a polyadenylation sequence of the SV40 virus.
- CMV human cytomegalo virus
- the flanking sequences of the E1 region (5 ′ nucleotide fragment (nt) 1 to 458 and 3 ′ fragment nt 3328 to 5788) of the human adenovirus type 5 were placed on either side of this cassette. These sequences make it possible to generate an "infectious" plasmid by homologous recombination, with the complete adenoviral genome ⁇ E1 / ⁇ E3 containing the candidate gene, in E.
- Coli (Chartier et al, 1996, J. Virol, 70, 4805-4810)
- the adenoviruses recombinants are then produced by transfection of a Pacl digestion of the "infectious" plasmid in a complementation line (Graham et al, 1977, J. Gen. Virol, 36, 59-74)
- a recombinant adenovirus not containing a transgene in the expression cassette (Ad-empty) will be used control adenovirus in all experiments.
- the propagation of the virions, their purification and the titration were carried out according to standard techniques (Lusky et al, 1998, J. Virol, 72, 2022-32).
- the purified viruses are stored at ⁇ 80 ° C. in 1M sucrose, 10 mM Tris-HCl pH8.5, 1 mM MgCl 2 , 150 mM NaCl, 0.005% Tween 80. 2. Cell lines and primary cells.
- the murine tumor lines P815, B16F0 and B 16F 10 were obtained from the ATCC (American Type Culture Collection; Rockville, MD, USA).
- the P815 cells (ATCC number: TIB-64) originate from a mouse mastocytoma DBA / 2 (H2-K d ).
- the highly metastatic B16-F10 (ATCC, CRL-6475) come from a mouse melanoma C57BL / 6J (H2-K). RenCa cells are derived from a murine renal cell carcinoma (BalB / cCr (H2-K d )).
- the tumor line A549 (ATCC, CCL-185) comes from a human pulmonary carcinoma.
- Human monocytes are obtained by leukapheresis and elutriation. The cells were cultured in DMEM medium (Gibco-BRL) supplemented with 10% fetal calf serum (SVF) at 37 ° C, 5% CO 2 . Human dermal microvascular cells (HDMEC) were cultured as recommended by the supplier (PromoCell, Heidelberg, Germany).
- the in vitro expression of the huIP-10 proteins, huMIP-l ⁇ and huIL-18 was analyzed on culture supernatants of infected HDMEC cells for 48 h at an MOI of 50. Indeed, the supernatants of HDMEC are much less rich in contaminating proteins than those of other cell lines.
- the RENCA cells are infected at MOI 50 with Ad.huB7H-1 and then treated for 1 hour with 50 ⁇ g / ml of mitomycin C (SIGMA-Aldrich, Saint-Quentin
- naive splenocytes are prepared from a spleen of DBA / 2 mice (Paul et al, 1999, Cancer Immunol. Immunother, 48, 22-28). After washing, the tumor cells and the splenocytes are co-cultured.
- the positive and negative controls consist of splenocytes activated with 10 ⁇ g / ml of ConA or unstimulated respectively (R&D Systems, Minneapolis, USA). After 96 hours at 37 ° C and 5% CO2,
- thymidine [ 3 H] / well 1 ⁇ Ci of tritiated thymidine [ 3 H] / well is added.
- the quantity of thymidine incorporated is measured after 8 hours by precipitating the cellular DNA on glossy filter paper (PHD harvester, Cambridge Technologie, Plainfield, USA).
- the radioactivity emitted is measured using a ⁇ counter (Beckman Instruments Inc., Palo Alto, USA).
- mice In order to establish tumors in mice, the P815, B16F10 or RENCA tumor cells are trypsinized, washed 3 times in PBS and resuspended at 3 ⁇ 10 6 cells / ml. 100 ⁇ l of this cell suspension are then injected into the right flank of immunocompetent B6D2 mice [(C57BL / 6 x DBA 2) F1] aged 6-7 weeks. After the appearance of a tumor with a palpable volume of between 5 and 25 mm, each mouse receives three intratumoral injections (100 ⁇ l) of a defined quantity of virus in 10 mM Tris-HCl pH 7.5, 1 mM MgCl 2 . Each condition is evaluated in groups of 10 mice.
- mice The tumor volume and the survival of the mice are determined twice a week. For ethical reasons, animals are sacrificed when the tumor volume becomes greater than or equal to 3000 mm 3 . After a primary rejection, the mice are re-injected on the opposite flank, with the tumor cells (3 ⁇ 10 5 cells / mouse). Statistical studies on the figures obtained make it possible to draw a Kaplan-Meier type survival curve. Statistical significance is calculated using Fisher's exact test (Statistica 5.1-Statsoft Inc. software, Tulsa, USA). Results.
- RENCA cells were infected with Ad-vacuum or Ad-B7-Hl for 48 h, then treated with mitomycin C in order to stop their proliferation. The infected cells were then brought into contact with naive splenocytes (of the same haplotype as the RenCa cells), in order to assess whether the membrane expression of B7-H1 on the surface of tumor cells enabled the proliferation of lymphocytes. .
- naive splenocytes proliferate (stimulation factor: 12x) in the presence of RenCa / Ad-B7-Hl cells but no RenCa / Ad-empty cells, indicating that the vector Ad-B7-Hl is functional in vitro .
- the established protocol consists in injecting a dose of adenoviral vector (from 1.10 to 4.10 iu depending on the tumor models) on D0, D1 and D2 and a dose of MNA vector (1.10 7 or 2.10 7 pfu depending on the tumor models) on D2 , J3 and J4.
- This protocol considerably reduces the anti-tumor activity induced by the co-injection of control viruses in the three tumor models.
- the simple change consisting in injecting adenovirus first makes it possible to reduce from 20 to 0 the percentage of mice having rejected their tumor (comparison of FIGS. 1 and 2).
- the results obtained in these combined antitumor strategies are always compared with those obtained in the groups treated with the viruses alone for the double combinations and with those obtained in the groups treated with two of the three viruses for the combinations triplets.
- cytokines IL-2, IL-12 and IL-18
- IL-2, IL-12 and IL-18 have been described as molecules allowing the activation of unrestricted effector cells by MHC of the NK or LAK type.
- the antitumor efficacy of these molecules could be added to that of the vectors MVA-KT3 and MVA-H57.
- the co-injection of Ad-huIL-2 and MVA-H57 increases the survival of the animals by 30 to 45 days compared to the groups treated with MNA-H57 or Ad-huIL-2 alone and improves statistically animal survival (15 to 35-40% rejection depending on experience).
- the results obtained show that, in different models of murine tumors, the co-injection of an adenoviral vector coding for 1TL-12 increases the anti-tumor effect induced by the in vivo membrane expression of the vector MVA-KT3. This synergy can be explained by several non-exclusive mechanisms.
- the apparent anti-tumor efficacy of 1TL-12 can be mediated by macrophages, NK cells (CD3 " ) and / or NKT cells (NK1.1 + , CD3 + , TCR ⁇ + , CD4 + , CD8 " , CD28 + , V ⁇ l4).
- CMHI and CD 1 d on the surface of these different models of tumors could also influence the importance of NK and NKT cells in the process of tumor rejection mediated by 1TL-12
- the anti-angiogenic activity of 1TL-12 mediated by the induction of the chemokine IP-10 could reduce tumor growth by inhibiting its birth This would allow the different immune effectors induced by 1TL-12 and KT3 on a tumor whose growth is slowed down.
- the anti-tumor effect of triple combinations was evaluated by adding an adenoviral vector expressing chemokines to these interesting double combinations.
- the adenoviral vectors were administered on D0, D1 and D2 (1 to 2 x 10 8 iu vector / injection) and the vector MVA-KT3 to D2, D3 and D4 (1 to 2 x 10 7 / injection).
- the injection of the Ad-huMIPl ⁇ + Ad-huIL-2 + MVA-KT3 viruses slightly increases the survival (by 15 to 25%) of the animals in the P815 model.
- the survival rate of the animals was also increased after administration of the triple combination MVA-KT3 / Ad-huBRAK / Ad-huIL-2 reaching 75%, while the survival rates observed after administration of Ad -huBRAK and Ad-huIL-2, Ad-huIL-2 alone and MVA-KT3 alone are 63%, 50% and 20% respectively.
- adenoviral vectors coding for a chemokine (MlP-1 ⁇ or BRAK) and for a cytokine (IL-12 or IL-2) in combination with the vector MNA- coding for the antibody KT3 expressed in transmembrane form heals 75 to 100% of animals in the RenCa model ( Figure 1 and above).
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WO1995009241A2 (fr) * | 1993-09-29 | 1995-04-06 | Transgene S.A. | Therapie genique anticancereuse par modulation de la reponse immunitaire et/ou inflammatoire |
WO2000024896A2 (fr) * | 1998-10-22 | 2000-05-04 | Transgene S.A. | Materiel biologique pour la preparation de compositions pharmaceutiques destinees au traitement de mammiferes |
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WO2000024896A2 (fr) * | 1998-10-22 | 2000-05-04 | Transgene S.A. | Materiel biologique pour la preparation de compositions pharmaceutiques destinees au traitement de mammiferes |
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DE INES C. ET AL.: "Apoptosis of a human melanoma cell line specifically induced by a membrane-bound single-chain antibodies" THE JOURNAL OF IMMUNOLOGY, vol. 163, 1999, pages 3948-3956, XP002204615 * |
KIM J J ET AL: "CHEMOKINE GENE ADJUVANTS CAN MODULATE IMMUNE RESPONSES INDUCED BY DNA VACCINES" JOURNAL OF INTERFERON AND CYTOKINE RESEARCH, MARY ANN LIEBERT, NEW YORK, NY, US, no. 20, 2000, pages 487-498, XP002908473 ISSN: 1079-9907 * |
PAUL S ET AL: "TUMOR GENE THERAPY BY MVA-MEDIATED EXPRESSION OF T-CELL-STIMULATING ANTIBODIES" CANCER GENE THERAPY, NORWALK, CT, US, vol. 5, no. 9, mai 2002 (2002-05), pages 470-477, XP001080563 ISSN: 0929-1903 * |
TAUB D D ET AL: "BETA CHEMOKINES COSTIMULATE LYMPHOCYTE CYTOLYSIS, PROLIFERATION, AND LYMPHOKINE PRODUCTION" JOURNAL OF LEUKOCYTE BIOLOGY, FEDERATION OF AMERICAN SOCIETIES FOR EXPERIMENTAL, US, vol. 59, no. 1, janvier 1996 (1996-01), pages 81-89, XP001088166 ISSN: 0741-5400 * |
TAUB D. ET AL: "Chemokines and T lymphocyte activation: I. beta chemokines costimulate human T lymphocyte activation in vitro." JOURNAL OF IMMUNOLOGY, vol. 156, no. 6, 1996, pages 2095-2103, XP001084176 * |
WARD S.G ET AL.: "Chemokines and T lymphocytes: more than an attraction" IMMUNITY, vol. 9, 1998, pages 1-11, XP002221891 * |
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WO2007014977A1 (fr) * | 2005-07-30 | 2007-02-08 | Consejo Superior De Investigaciones Científicas | Vecteurs recombines bases sur le virus ankara modifie (mva) utilises en tant que vaccin contre la leishmaniasis |
ES2313807A1 (es) * | 2005-07-30 | 2009-03-01 | Consejo Superior De Investigaciones Cientificas | Vectores recombinantes basados en el virus vaccinia modificado de ankara (mva) como vacunas contra la leishmaniasis. |
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CA2435949A1 (fr) | 2002-08-01 |
JP2005501512A (ja) | 2005-01-20 |
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