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WO2002036769A2 - Acides nucleiques codant des polypeptides chimeriques cd40/cd40l, procedes de production et utilisations de ces acides nucleiques - Google Patents

Acides nucleiques codant des polypeptides chimeriques cd40/cd40l, procedes de production et utilisations de ces acides nucleiques Download PDF

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Publication number
WO2002036769A2
WO2002036769A2 PCT/EP2001/012495 EP0112495W WO0236769A2 WO 2002036769 A2 WO2002036769 A2 WO 2002036769A2 EP 0112495 W EP0112495 W EP 0112495W WO 0236769 A2 WO0236769 A2 WO 0236769A2
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Prior art keywords
cd40l
nucleic acid
tumor
cells
polypeptide
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PCT/EP2001/012495
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WO2002036769A3 (fr
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Frank Dicker
Dirk Moritz
Gerd Maass
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F. Hoffmann-La Roche Ag
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Priority to AU2002221780A priority Critical patent/AU2002221780A1/en
Publication of WO2002036769A2 publication Critical patent/WO2002036769A2/fr
Publication of WO2002036769A3 publication Critical patent/WO2002036769A3/fr

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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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention relates to nucleic acids encoding CD40/CD40L chimeric polypeptides, to methods for their production, pharmaceutical compositions containing said nucleic acids, and to uses thereof.
  • CD40 receptor (hereinafter also referred to as CD40), a cell surface receptor from the family of TNF receptors, was first identified and functionally characterized on B lymphocytes, where it is involved in the regulation of growth and differentiation. It may also be found, however, on other cell types, for example on T cells (activation - ⁇ proliferation stimulation, cyto ine secretion), dendritic cells, and monocytes (activation - ⁇ expression of costimulatory molecules, secretion of inflammatory cytokines, e.g., TNF- ⁇ , IL-8, IL-12) as well as on carcinomas (activation leads to inhibition of growth) (van Kooten, C, and Bancherau, J., Int. Arch. Allergy
  • APC Antigen Presenting Cell
  • CD40L belongs to the group of type II membrane proteins, which implies an intracellular N terminus, a transmembrane region and an extracellular C terminus (Laman, J.D., et al, Critical Reviews in Immunology 16 (1996) 59-108).
  • soluble forms of GD40L which means that they only consist of the C terminal extracellular domain and are capable of activating CD40. Consequently, the extracellular domain is sufficient for the formation of trimers, which, in turn, bind to the extracellular domain of the receptor CD40 and thereby trimerize and activate this receptor.
  • trimers which, in turn, bind to the extracellular domain of the receptor CD40 and thereby trimerize and activate this receptor.
  • qualitative differences have been discussed in the literature with respect to the mediation of signals by soluble CD40L and by membrane-bound CD40L (Laman, J.D., et al., Critical Reviews in Immunology 16 (1996) 59-108).
  • WO 98/26061 describes a method of treating a human neoplasia comprising inserting into a human neoplastic cell a cD ⁇ A which encodes a chimeric protein which contains at least a portion of the murine CD40L gene together with portions of the same or other CD40L ligand genes from either mouse, human or other species.
  • a chimeric gene consisting of the murine CD40L gene transmembrane and cytoplasmatic domains having been attached to the extracellular domains of human CD40L gene.
  • the invention provides a chimera consisting of the fleece which is, the non-membrane-bound, extracellular domain of CD40L covalently bound to the CD40. This is achieved by means of a fusion gene which contains the following components, starting from the ⁇ -terminus:
  • the invention therefore comprises a nucleic acid encoding a chimeric polypeptide comprising nucleic acid fragments encoding i) the signal transduction domain of CD40 and downstream thereof; ii) a transmembrane domain of a type II membrane receptor and downstream thereof iii) the binding and trimerization domain of CD40L;
  • Fragments or modifications of these domains having substantially the same activity in respect of binding, trimerization and signal transduction are also useful according to the invention. Such modifications are, e.g., exchanges of amino acids by other amino acids which do not affect the protein conformation.
  • nucleic acids can be used which due to the degeneracy of the genetic code encode a polypeptide encoded by any of the above-mentioned nucleic acids. Particularly preferred are nucleic acids which use the human codon usage.
  • the encoded polypeptide consists of the signaling domain of CD40 and the binding and trimerization domain of CD 40L as well as the transmembrane domain of CD40L. It must be ensured that the binding domain of CD40L is able to bind and trimerize. For this, it maybe useful to add linkers between the three domains sufficient in length. A length of about 1 to 30 amino acids, preferably 1 to 15 amino acids, is therefore preferred, but also linkage of the three domains without linker amino acids is possible.
  • the chimera according to the invention offer the advantages that the covalently bound, extracellular CD40L domain trimerizes spontaneously and, as a result, trimerizes the covalently bound cy oplasmic CD40 receptor and activates the same.
  • CD40 ligand trimer is still present in an unbound form and can therefore interact in trans with other natural CD40 receptors on different cells, trimerize same and, as a result, activate the same as well, that is to say, this embodiment not only acts directly on the cell expressing the chimeric receptor but can also have an activating effect in trans on another cell that expresses natural CD40 receptors.
  • the chimeric CD40/CD40L polypeptide shows a noirtrans" activity by activation of professional APCs in the same way as CD40L ligated to CD40, that is, it translocates the signal via the JNK/AP-1, JAK/STAT and I-KB/NFkB pathways, which leads to upregulation of genes coding for MHC-I and/or MHC-II, for costimulatory molecules such as B-7.1, B-7.2, immunostimulatory cytokines such as IL-12, IL-8, and proinflammatory cytokines such as IL-l ⁇ , IL-l ⁇ and/or IL-6.
  • fusion proteins of CD40L and CD40 according to the invention are preferably free of typical protease cleavage sites, preferably free of a site cleared by metalloproteinases (mmp).
  • the cleavage sites described for CD40L exist in a region of the CD40L-molecule which is preferably deleted in the fusion constructs. Therefore the CD40/CD40L chimeric molecules are less sensitive to proteolytic cleavage and thereby less sensitive to downmodulation of their signaling capacity by proteolytic cleavage.
  • the nucleic acid contains a further gene fragment encoding a cytokine such as IL-2, IL-12, lymphotactin (Dilloo, D., et al., Nature Medicine 2 (1996) 1090-1095, and Emtage, P.C., et al.,
  • a cytokine such as IL-2, IL-12, lymphotactin (Dilloo, D., et al., Nature Medicine 2 (1996) 1090-1095, and Emtage, P.C., et al.
  • the invention further comprises a recombinant vector for the expression of said nucleic acid, wherein the expression of said nucleic acid is under the control of a mammalian promoter, preferably of a CMN promoter or a cytokine-inducible (inflammatory regulated) promoter, more preferably, under the control of an acute phase protein gene promoter, and particularly preferably, under the control of the human acute phase serum amyloid A gene promoter SAAl or SAA2 (hereafter referred to as contextSSAA promoter").
  • a mammalian promoter preferably of a CMN promoter or a cytokine-inducible (inflammatory regulated) promoter
  • an acute phase protein gene promoter preferably, under the control of an acute phase protein gene promoter
  • SAAl or SAA2 hereafter referred to as staSAA promoter
  • the invention further comprises, in a preferred embodiment, a combination of the vector according to the invention with one or more vectors, which expresses, one or more additional genes selected from the group consisting of the genes encoding a cytokine such as IL-2, IL-12 and Interferon-alpha.
  • a cytokine such as IL-2, IL-12 and Interferon-alpha.
  • genes can be under the control of the same promoter on the same vector, under the control of two identical or different promoters on the same vector, or on different expression vectors.
  • the invention further comprises compositions, preferably pharmaceutical compositions, containing at least one expression vector according to the invention as an essential component.
  • compositions comprise nucleic acids/expression vectors according to the invention together with a pharmaceutically acceptable excipient and/or preservative.
  • compositions are produced by the use of the nucleic acids/expression vectors according to the invention as the essential constituents of such compositions.
  • the compositions are useful for activating antigen presenting cells and T cells.
  • the composition contains at least two genes on one or more vectors.
  • Preferred examples of genes encoded by such vectors are listed below in Tables 1 and 2.
  • Genes from Vector 1 and Vector 2 can also be co-expressed on one vector.
  • the following combinations are also preferred:
  • Genes fromVectors 1 to 3 can also be combined on one or two vectors.
  • IL-12 as gene or as protein
  • the invention further comprises methods for the production of such expression vectors and of compositions, preferably pharmaceutical compositions, containing such vectors.
  • the pharmaceutical compositions are used for ex vivo and in vivo treatment, preferably for in vivo treatment of tumor cells of a patient (gene therapy treatment).
  • vectors containing the chimeric CD40/CD40L gene under the control of the SAA promoter are preferred and improved therapeutic agents for the treatment of tumor diseases.
  • the expression vectors or host cells according to the invention are combined, for the treatment of tumor disease, with the proteins of Interleukin-2 (IL-2), Interleukin-12 (IL-12) and/or Interferon- alpha (preferably interferon- ⁇ 2A) and/or with 5-fluorouracil, preferably for application in vivo preferably for the treatment of solid tumors.
  • IL-2 Interleukin-2
  • IL-12 Interleukin-12
  • Interferon- alpha preferably interferon- ⁇ 2A
  • 5-fluorouracil preferably for application in vivo preferably for the treatment of solid tumors.
  • the invention further comprises a mammalian host cell transfected with an expression vector according to the invention and a process for the production of a chimeric CD40/CD40L polypeptide according to the invention by culturing a host cell of the invention under conditions promoting the expression of the CD40/CD40L chimeric gene and presenting said polypeptide on the surface of the host cell.
  • the transfected host cell can also be used as a pharmaceutical agent.
  • the invention further comprises a process for the production of a modified human tumor cell containing an expression vector encoding a chimeric CD40/CD40L polypeptide under conditions in which said CD40/CD40L polypeptide is produced in the tumor cell and presented on the surface of said cell.
  • a further object of the invention is a chimeric polypeptide encoded by a nucleic acid according to the invention, wherein the CD40L part consists of amino acids 2- 261 of human CD40L or 2-260 of murine CD40L and the CD40 part consists of amino acids 217-277 of human CD40 or 217-289 of murine CD40.
  • the invention has to be understood in that way that these amino acid ranges of the preferred embodiments of the invention may vary within a few amino acids as long as the function of the chimeras according to the invention is not considerably altered.
  • the CD40 part can be prolongated at its N-terms with a few (i.e., one to three) aminoacids.
  • a further object of the invention is a method for the production of a composition, preferably a pharmaceutical composition for activating antigen presenting cells and
  • T cells comprising a nucleic acid according to the invention, characterized by the use of a nucleic acid according to the invention as an essential constituent of said composition.
  • a promoter also designated as an expression control region
  • a promoter is understood as a nucleic acid region which causes the expression of DNA and hence transcription into mRNA and which usually has a length of 0.5-5 kb.
  • Such expression control regions usually contain enhancer regions and promoter regions to which transcription factors or repressors can bind.
  • Expression control regions can be regulated via binding of activating or repressing factors.
  • a regulatory region according to the invention is understood as a region which influences expression due to induction by cytokines. Based on this, the expression is stimulated.
  • a minimal promoter combined with enhancing elements is used.
  • Minimal promoters and methods for their construction are described, for example, in Luckow, B., and Sch ⁇ tz, G., Nucleic Acids Res. 10 (1987) 5490 and Spear, B.T., et al., DNA Cell Biol. 14 (1995) 635-642.
  • a minimal promoter useful in the expression vectors according to the invention contains at least a TATA-box, one ore more cytokine-responsive elements (CRE) and one or more binding sites for transactivators such as NFkB, CEBP/NF-IL6 and also others such as YY1, SAF and API. Such binding sites usually consist of 4 to 12 nucleotides in length.
  • CREs are described, for example, in Dendorfer, U., Artif. Organs 20 (1996) 437-444; Birt, D.F., et al., J. Nutr. 129 (1999) 25 Suppl, 5715- 5745; Kang, D.C., et al., Int. J. Oncol. 13 (1998) 1117-1126; Weber-Nordt, R.M., et al., Leuk. Lymphoma 28 (1998) 459-467; Sen, C.K., FASEB J. 10 (1996) 709-720.
  • Useful promoters according to the invention are exogenous viral promoters such as
  • Simian virus 40 Rous sarcoma virus and cytomegalovirus (CMV).
  • CMV cytomegalovirus
  • cytokine-inducible promoters useful in the invention are highly sensitive to cytokine induction during local or systemic inflammation due to the action of pro -inflammatory proteins like IL-l ⁇ , IL-6 and TNF ⁇ . Also due to the fact that expression of these cytokines has been found in several tumors (e.g., squamous cell carcinomas) but not in normal uninflamed tissue, a specific expression of such promoters in these types of tumors can be found.
  • tumors e.g., squamous cell carcinomas
  • Several tumors have been described to express high amounts of pro-inflammatory cytokines, e.g.
  • A-SAAs acute phase serum amyloid As [A-SAAs] and C-reactive protein (CRP)
  • A-SAAs acute phase serum amyloid As [A-SAAs]
  • C-reactive protein C-reactive protein
  • Promoters of genes encoding the major APRs are extremely responsive to such signals causing them to be massively induced during the acute phase response (Varley, A.W., et al., Proc. NatL Acad. Sci. USA 92 (1995) 5346- 5356; U.S. Patent No. 5,744,304; U.S. Patent No. 5,851,822; Kushner, I., Ann. NY Acad. Sci. USA 389 ( 1982) 39-48; Kushner, I., and Mackiewicz, A., Disease Markers
  • the human acute-phase serum amyloid A promoter (SAA2 -promoter) have been cloned by Ublar, CM., et al., J. Immunol. Methods 203 (1997) 123-130.
  • the SAA2- promoter is described to be active in inflamed tissue and can be highly activated (by factor 70) in vitro by monocyte conditioned medium as well as by IL-l ⁇ IL-6 and TNF- ⁇ IL-6.
  • IL-l ⁇ mediated stimulation can be blocked by IL-1 receptor antagonist (Uhlar, CM., et al., J. Immunol. Methods 203 (1997) 123-130 and Steel, D.M., et al., Biochem. Journal 291 (1993) 701-707).
  • a translation control element within the SAA2 5 C -UTR that plays a crucial role in modulating A-SAA production.
  • This element is a cell- and/or tissue-specific translational enhancer. Its efficiency could be mediated by an intracellular factor that is activated or synthesized de novo after cytokine treatment.
  • the sequence of this enhancer element is shown in SEQ ID NO:4 of WO 98/40506.
  • the enhancer is used in conjunction with the A-SAA promo tor (i.e., downstream of the promotor and upstream of the gene encoding the product of interest).
  • the SAA2 promoter which is preferred according to the invention is a promoter which is very silent in healthy noninflammated tissues but which is specifically active in inflammated but also in tumor tissue.
  • a murine squamous carcinoma cell line like SCC-VII is a model system to show the functionality and tumor specificity of the SAA2 promoter in tumors expressing preinflammatory cytokines as it is already described for human squamous carcinomas.
  • "SAA promoter" according to the invention therefore means a promoter which has the function of an A-SAA promoter and which is therefore inducible by a cytokine in substantially the same manner as the SAA promoter described in WO 98/40506 and which is essentially identical to the sequence of the SAAl and/or SAA2 promoter.
  • promoters which are coded by DNA sequences which hybridize with SEQ ID NO:l shown in WO 98/40506 under stringent conditions and have the ability to act as an expression control sequence inducible by cytokines.
  • hybridize under stringent conditions means that two nucleic acid fragments are capable of hybridization to one another under standard hybridization conditions described in Sambrook et al., "Expression of cloned genes in E.coli" in
  • stringent conditions refers to hybridization in 6.0 x SSC at about 45°C followed by a wash of 2.0 x SSC at 50°C
  • salt concentration in the wash step can be selected, for example, from about 2.0 x SSC at 50°C for low stringency to about 0.2 x SSC at 50°C for high stringency.
  • temperature in the wash step can be increased from low stringency conditions at room temperature, about 22°C, to high stringency conditions, about 65°C
  • under the control means that the promoter is located in a position relative to that of the DNA encoding the desired polypeptide that allows the promoter to efficiently direct transcription of the structural gene or the genes for the desired polypeptide(s).
  • the CD40L domains of the fusion genes trimerize and, therefore, trimerize the intramolecularly bound CD40 receptor which causes activation of receptor- mediated signal transduction in the same manner as a transactivating CD40 ligand trimer in a natural system.
  • CD40 according to the invention is understood as a polypeptide with the activity and biochemical characteristics of CD40 receptor.
  • Such polypeptides are, for example, human or murine CD40 or modifications thereof such as mutations, deletions or substitutions.
  • Preferred modified CD40 polypeptides are at least 80% homologous to human CD40 preferably within the intracellular domain. Homology according to the invention can be determined with the aid of the computer programs Gap or BestFit (University of Wisconsin; Needleman, S.B., and Wunsch, CD., J. Mol. Biol. 48 (1970) 443-453; Smith and Waterman, Adv. Appl. Math. 2 (1981) 482-489).
  • the extracellular domain is responsible for the ligand binding and for the thereby mediated trimerization of the active complex. It consists of two subdomains (Hgand binding subdomain and stalk subdomain). According to the invention, the extracellular domain is to be understood as the full length domain, or at least as part thereof, which causes ligand (CD40L) binding.
  • the ligand binding subdomain consists of about 100 amino acids (Pos. 20-120), and the stalk subdomain consists of about 73 amino acids (Pos. 121-193) according to Bajorath, J., and Aruffo, A., Proteins 27 (1997) 59-70.
  • the stalk subdomain is a domain which is necessary for steric reasons. This subdomain ensures an optimal distance between the binding domain and the cell surface which is necessary for trimerization.
  • the cytoplasmic domain mediates the signal transduction of the complex that is activated by trimerization. Signal transduction is mediated by the socalled TRAF proteins (TNF-R Associated Factor), whose binding sites are located in the cytoplasmic domain of CD40, and ultimately leads to the activation of various transcription factors, NF- ⁇ B being the most prominent of these transcription factors.
  • TRAF proteins TRAF-R Associated Factor
  • CD40L (CD 154) according to the invention is understood as a polypeptide with the activity and biochemical characteristics of CD40L.
  • Such polypeptides are, for example, human or murine CD40L or modifications thereof such as mutations, deletions or substitutions.
  • Preferred modified CD40L polypeptides are at least 80% homologous to human CD40L preferably 'within the extracellular domain.
  • the extracellular domain of CD40L consists of two subdomains: the stalk domain (Pos. 47-119) and the trimerization domain (Pos. 120-261).
  • the mature glycoprotein has a molecular weight of 35 kDa.
  • the mature murine glycoprotein has a molecular weight of 33 kDa.
  • CD40L The three-dimensional structure of CD40L has been elucidated:
  • the structure of the CD40 / CD40L complex has been derived from the known homologous structure of TNF-R / TNF- ⁇ and from a crystal-structure determination of the soluble extracellular domain of the CD40L trimer (Karpusas, M., et al., Structure 3
  • a transmembrane of a type II receptor according to the invention is a domain of a polypeptide which secures the correct membrane insertion and orientation of the chimeric construct according to the invention.
  • Such domains are preferably transmembrane domains of TNF and TNF-related ligands (ligands of the TNF- family) like TNF- ⁇ , FASLigand, TRAIL, CD70, CD40L, Lymphotoxin (LT), whereby the tiansmenbrane domain of the CD40L is preferred.
  • TNF and TNF-related ligands ligands of the TNF- family
  • Such receptors of the TNF -family are described for example by Orlinick, J.R., et al., Cell Signal. 10
  • nucleic acid segments according to the invention may be introduced ex vivo or in vivo into a mammalian cell or a mammalian host by any of several means, including vector transfection.
  • Viral vectors may be used to infect human and animal cells with the recombinant DNA; certain adenoviral vectors have proved particularly useful. Adenoviral vectors are preferred.
  • DNA segments need not be introduced into cells by a viral vector: Direct transfection may be performed by electroporation, gene gun techniques, or DNA-liposome complexes, for example.
  • DNA/liposome complexes have been used to introduce DNA encoding prostaglandin synthase into rabbits, with subsequent production of prostaglandin E2 and prostacyclin (Conary, J.T., et al., J. Clin. Invest. 93 (1994) 1834-1840).
  • An appropriate vector includes the gene encoding the selected protein or proteins such that the nucleic acid or acids according to the invention is or are under the transcriptional control of a promoter which is active in mammalian, preferably in human cells such as the CMV promoter, the SV40 promoter or the SAA promoter.
  • the vector is introduced into a host cell by any of a number of procedures known to those skilled in the art, such as direct introduction of DNA by gene gun techniques, liposomal transfection or direct local injection. Direct infusion is preferred in the case of bladder carcinomas and infusion with an endoscopic probe is preferred in the case of colon carcinomas.
  • the chimeric nucleic acid and the genes encoding IL-2, IL-12 and/or Interferon-alpha are being co-expressed on the same or on different vectors. This can be effected by means of cotransfection or cotransduction of two viral or plasmid vectors which carry both genes or of a single vector on which both genes are present in coded form.
  • both genes can be expressed by separate promoters (the promoters may be identical or different from one another), they may be present coupled via an IRES sequence (internal ribosomal entry site) in the expression, or coupled via a splice-donor sequence before the first gene and a splice-acceptor sequence after the first gene, that is, before the second gene.
  • IRES sequence internal ribosomal entry site
  • Genes encoding chimeric nucleic acids according to the invention represent prominentimmunological master genes", that is to say, after ligand binding through CD40L, CD40 induces a great number of co-stimulatory factors, inflammation-inducing or maintaining factors, and thus, a strong immune reaction (Stout, R.D., and Suttles, J., Immunology Today 17 (1996) 487-492).
  • CD40 also induces the NFkB transduction pathway, at the end of which there are a great number of inflammation mediators such as, for instance, IL-12, ILl- ⁇ , IL-6 or TNF- ⁇ .
  • the preferred embodiment of the invention therefore consists of a chimeric nucleic acid under the expression control of the cytokine-inducible promoter.
  • cytokine- inducible promoter the binding sites for pro-inflammatory transactiyators, such as NFkB or NF-IL6 (e.g., the region from -190 to -78 of the SAA2 promoter or only the individual binding sites), preferably in multimeric form.
  • pro-inflammatory transactiyators such as NFkB or NF-IL6
  • a gene therapy agent according to the invention is understood to mean a pharmaceutical composition which contains one or more expression vectors according to the invention as essential components, in an amount needed by the tumor patient to ensure an effective treatment.
  • a composition preferably contains at least one vector together with a non-viral delivery system, as an adenoviral vector or as a retroviral vector.
  • the delivery system or the viral vector per se or the expressed CD40/CD40L gene will cause a local or systemic inflammatory response in the tumor and/or in the tissue surrounding the tumor.
  • cytokine mobilization caused by the administration of the delivery/targeting vehicle would lead to the promoter-driven production of the therapeutic agent at up to at least a hundred times over its uninduced basal level.
  • Gene therapy of somatic cells can be accomplished by using, e.g., retroviral vectors, other viral vectors or by non-viral gene transfer (cf. Friedman, T., Science 244 (1989) 1275-1281; Morgan 1993, RAC Data Management Report, June 1993).
  • Vector systems suitable for gene therapy are, for instance, retroviruses (Mulligan, R.C., (1991) in Nobel Symposium 8: Etiology of human disease at the DNA level (Lindsten, J., and Pattersun, eds.) 143-189, Raven Press), adeno-associated virus (McLaughlin, S.K., et al., J. Virol. 92 (1988) 1963-1973), Vaccinia virus (Moss, B., and Flexner, C, Ann. Rev. Immunol. 5 (1987) 305-324), bovine papilloma virus
  • viruses from the group of the Herpes viruses such as Epstein-Barr virus (Margolskee, R.F., et al., Mol. Cell. Biol. 8 (1988) 2837-2847) or Herpes simplex virus.
  • Epstein-Barr virus Margolskee, R.F., et al., Mol. Cell. Biol. 8 (1988) 2837-2847
  • Herpes simplex virus Herpes simplex virus.
  • adenoviruses are preferred.
  • adenoviral vectors could also be applied as formulations of cationic lipids (e.g.,
  • DOSPER DOSPER with ADV as described by Fasbender, A., et al., J. Biol. Chem. 272 (1997) 6479-6489 and Dodds, E., et al., J. Neurochem. 72 (1999) 2105-2112.
  • nucleic acid preferably DNA
  • auxiliary agent such as, e.g., transfer reagents (liposomes, dendromers, polylysine transferrin conjugates
  • tumor-specific T cells which are cytolytically active for a very long period of time.
  • the combination of CD40 and CD40L expression preferably in combination with the expression of IL-2, IL-12 and/or Interferon-alpha or in combination with a therapeutically active amount of IL-2, IL-12 and/or Interferon-alpha polypeptides or 5-fluorouracil further lead to a synergistic enhancement of the level and duration of the activation phase of said tumor-specific T cells.
  • compositions may be administered parenterally, using, for example, injectable solutions, preferably for intratumoral injection and preferably into head and neck cancer (a squamous cell carcinoma).
  • injectable solutions preferably for intratumoral injection and preferably into head and neck cancer (a squamous cell carcinoma).
  • the vectors according to the invention are admixed with pharmaceutical inert, inorganic or organic excipients, buffers and/or preservatives.
  • excipients are, for example, water, alcohols, polyols, glycerol, preferably having a neutral pH value (pH 6-8).
  • Pharmaceutically acceptable buffers are, for example, phosphate, lactate, phosphate buffered saline, Tris.
  • the pharmaceutical compositions may also contain preserving agents, toxicity agents, stabilizing agents, wetting agents, clarification agents, viscosity agents, salts for the variation of osmotic pressure, buffers or antioxidants. They may also contain other therapeutically valuable agents.
  • Suitable preservatives for use in such preparations include benzalkonium chloride, benzethonium chloride, chlorobutanol, thimerosal, and the like.
  • Suitable buffers include boric acid, sodium and potassium bicarbonate, sodium and potassium borates, sodium and potassium carbonate, sodium acetate, sodium biphosphate and the like, in amounts sufficient to maintain the pH between about pH 6 and pH 8, preferably between about pH 7 and pH 7.5.
  • Suitable tonicity agents are dextran 40, dextran 70, dextrose, glycerine, potassium chloride, propylene glycol, sodium chloride, and the like.
  • Suitable antioxidant and stabilizers include sodium bisulfite, sodium metabisulfite, sodium thiosulfate, thiourea and the like.
  • Suitable wetting and clarifying agents include polysorbate 80, polysorbate 20, poloxamer 282, and tyloxapol.
  • Suitable viscosity increasing agents include dextran 40, gelatin, glycerin, hydroxyethyl cellulose, hydroymethylpropyl cellulose, lanolin, methylcellulose, petrolatum, polyethylene glycol, polyvinyl alcohol, polyvinyl polyvinylpyrrolidone, carboxymethyl cellulose and the like.
  • CD40 on antigen-presenting cells APC
  • CD40L antigen-presenting cells
  • APC antigen-presenting cells
  • B7 surface expression and IL-12 synthesis two proteins which cooperate in the induction of an effective anti-tumor response. It was found that tumor cells from basal and squamous cell carcinoma exhibit a down-regulation or a dramatic loss of CD40 which may account for a tumor escape mechanism, by which activated T cells expressing CD 154 are not able to kill the tumor cells any longer.
  • the potential of the substances according to the invention in inducing an anti-tumor response can be shown by a stable transfection of a non- immunogenic CD40- tumor cell (e.g., a squamous cell carcinoma cell line like SCCVII) with such constructs.
  • a non- immunogenic CD40- tumor cell e.g., a squamous cell carcinoma cell line like SCCVII
  • Wild-type SCCVII, the neo control and CD40 + cells were indistinguishable in their proliferation rate and morphology in vitro.
  • Syngeneic C3H/HeN mice injected s.c. with CD40 + SCCVII had a significant reduction in tumor growth compared to the CD40- wild-type SCCVII or the neo control.
  • CD40-transfected cells injected into nude mice exerted the same tumor growth kinetic as the two controls indicating that immunocompetent T cells are necessary for anti-tumor activity.
  • Histological analysis of tumor explants from CD40 + tumors revealed predominant infiltration of CD4 + T cells and expression of the CD25 T cell activation marker. These changes were not detectable in control group tumors.
  • Chimeras according to the invention induce apoptosis and inhibition of the proliferation rate in tumor cells
  • the vectors according to the invention can be used in the control or treatment of tumor diseases, preferably in treatment of vascularized tumors.
  • the dosage is a biologically effective amount of the vector and can vary within wide limits and is, of course, fitted to the individual requirements in each particular case.
  • the preferable dose for viral vectors is 10 8 -10 n pFU/injection and 50-1000 ⁇ g DNA/injection (see also Kikuchi, T., and
  • the preferable volume per injection is between 1 and 10 ml.
  • Viral vectors are preferably formulated in a pharmaceutical composition containing phosphate buffered saline, pH 7.4 or other buffers of pH 6 to 8 (Caruso, M., et al.,
  • Non-viral vectors are preferably formulated in a pharmaceutical composition with liposomes also at a pH value of 6 to 8 (cf. Yanagihara, I., et al., Mol. Cell Biol. Hum. Dis. Ser. 5 (1995) 64- 82; Thierry, A.R., et al., Gene Ther. 4 (1997) 226-237; Gao, X., and Huang, L., Gene Ther. 2 (1995) 710-722; Abdallah, B., et al., Biol. Cell 85 (1995) 1-7; Treco, D.A., and Selden, R.F., Mol. Med. Today 1 (1995) 314-321).
  • the patient is preferably treated in such a way that 1-2 injections per week are administered directly into the tumor over a period of 3-10 weeks, which induces a cytotoxic immune response against the tumor cells. After that period, the extent of change of the tumor is examined and, if necessary, as well as if possible, the tumor is removed.
  • a therapy regimen of this kind is of particular importance with respect to improving the results of therapy, because after post-operative treatment, a sufficient number of tumor cells will still be present.
  • the presurgical immunization results, however, in a cytotoxic immune response also after the removal of the tumor and therefore cytotoxic T cells are formed which are capable of destroying metastasizing cells and cells of minimal residual disease.
  • the individual injections into a plurality of sites in the tumor and/or in the vicinity of the tumor, which allows the pharmaceutical agent according to the invention to reach not only the actual tumor cells but also other non-tumoral cells of the tumor tissue, for example fibroblasts, macrophages, T cells or dendritic cells.
  • the inflammatory reaction on the tumor should be intensified locally. This can be accomplished by means of, for example, local thermal treatment (microwaves), pressure, or by injection of the pharmaceutical agent into a plurality of sites in and in the vicinity of the tumor.
  • microwaves local thermal treatment
  • pressure or by injection of the pharmaceutical agent into a plurality of sites in and in the vicinity of the tumor.
  • the vectors according to the invention may be injected as formulations with transfer reagents directly into tumors, post-operatively into tumor caves, or systemically.
  • the vector-containing pharmaceutical agent according to the invention preferably containing no expression vector for
  • IL-2, IL-12 and/or Interferon-alpha is administered as an adjuvant and in combination with a polypeptide having the activity of IL-2, IL-12 and/or Interferon-alpha.
  • the vector-containing agent is administered as described above, whereas the polypeptide is preferably administered systemically. In this connection, it is preferred to administer the polypeptide before and after
  • Table 3 shows the preferred administration scheme for the adjuvant administration ofpolypeptides.
  • 5-fluorouracil is administered with 12 mg/kg body weight per day for the first four days of the tumor therapy and with 6 mg/kg body weight on days 6, 8, 10 and 12.
  • endotoxin-free DNA it is important to use endotoxin-free DNA and to avoid any inflammatory reactions of the tissue. This would induce preinflammatoy cytokine expression which on the other hand would lead to a stimulation of the inducible promoter in other tissues but squamous cell carcinomas.
  • endotoxin-free DNA can be produced according to WO 97/29113.
  • tumor specificity can be achieved by either targeting of the transfection reagents to tumors or tumor-specific gene expression or combinations of both. Targeting of the transfection reagents is not yet feasible with sufficient specificity and efficiency but tumor specific promoters are available.
  • this invention will have great impact on the treatment of tumors, metastases and minimal residual disease by vectors which code for immune stimulatory genes.
  • the use of the promoters according to the invention allows high-level, long-lasting, potentially inducible/repressable and tumor specific expression. Therefore in cancer treatment regimens using vector DNA injection it could optimize the results due to longer lasting and higher amounts of the immune stimulatory proteins in the tumor, the possibility of systemic treatment which would reach not only the primary tumors but also metastases, and a wider therapeutic window (higher doses and multiple dosing) due to reduced toxic side effects.
  • Fig. 1 shows a schematic view of the chimeric CD40/CD40L molecule.
  • C57BL/6 mice Six to eight week old female C57BL/6 mice are delivered from Charles River. All animals were IVC's with daily cycles of 12 h light/12 h darkness according to international guidelines. C57BL/6 mice are syngeneic to B16F10 mouse melanoma cells and used for implantation experiments with this cell line.
  • mice Female syngeneic C3H/HeN (H-2K k ) mice were purchased from Charles River, Germany. The mice were between 8-12 weeks old. Female Balb/c nu/nu mice (20 weeks old) were obtained from Bomholtgard Breeding, Denmark.
  • B16F10 mouse melanoma cells were obtained from ATCC (CRL 6475) and cultured in DMEM (Biochrom Ltd., UK) supplemented with 10% fetal bovine serum (PAA Laboratories, Austria), 4 mM L-glutamine (Gibco - Life Tech. / Europe), 1 mM sodium pyruvate and 4,5 g/1 glucose.
  • the culture conditions are 37°C in a water saturated atmosphere containing 5% CO 2 . Culture passage is performed with trypsin/EDTA lx (Roche Diagnostics GmbH, DE) splitting twice a week.
  • HEK293 cells were obtained from ATCC (CRL-1573) and cultured in DMEM (Biochrom Ltd., UK) supplemented with 10% fetal bovine serum (PAA Laboratories, Austria), 2 mM L-glutamine (Gibco - Life Tech. / Europe).
  • a murine squamous carcinoma cell line e.g., SCCVII/SF; O'Malley, B., et al., J. Cancer Res. 56 (1996) 1737-1741 and Suit, H.D., et al., Radiation Research 104
  • a murine colon carcinoma cell line (e.g., CT26; Xiang, R., et al., Cancer Res. 58 (1998) 3918-3925), both being CD40 " and CD40L " , were cultured in DMEM high glutamax ® medium (GIBCO Life Technologies) supplemented with 10% heat-inactivated FCS (GIBCO Life Technologies) penicillin and streptomycin (Roche Diagnostics GmbH, DE).
  • This cell line can also be used instead of
  • the CD40/CD40L fusion protein (CD40/CD40L fusion protein:
  • the fusion of the different parts of the cDNA constructs was performed by standard PCR techniques. DNA sequence was verified by sequencing the resulting PCR products.
  • the chimeric cDNA was cloned into the pcDNA3 (Invitrogen Corp., San Diego, US) mammalian expression vector. Transfection of HEK293 cells was carried out using transfection reagents FUGENETM (Roche Diagnostics GmbH, DE)
  • Cells were characterized for the expression of relevant ceE surface markers by direct immunofluorescence using either PE and/or FITC conjugated monoclonal antibodies against CD40, MHC-I (H-2K d ⁇ BALB/C), CD40L, ICAM-I (CD54), B7.1 (CD80), B7.2 (CD86), Fas receptor (Fas, APO-1/CD95), Fas ligand (FasL, CD95L) or tags (e.g. HA-tag, Flag-tag) fused to the N-termini of the CD40-CD40L fusion proteins (-> CD40-CD40L tag ).
  • the corresponding isotype controls were used for determination of unspecific binding.
  • Antibody binding was carried out for 20 min. at room temperature, followed by washing and analyzing using a FACScan ® (Becton Dickinson, DE).
  • Tumor cells were inoculated in a concentration of 5 x 10 5 /200 ⁇ l subcutaneously (s.c.) in the right hind flank of syngenic or athymic animals. Tumor growth was quantitated five days post-inoculation three times a week using a calipper. Tumor volumes were calculated by the approximately ellipsoid formula: 4/3 x ⁇ /
  • Tumor growth was quantitated five days post-inoculation three times a week using a caliper. Tumor volumes were calculated by the approximately ellipsoid formula: 4/3 x ⁇ / 8 x (length x width x depth) .
  • the intracellular domain of human CD40 was amplified by PCR using pcDNA3-hCD40 (pcDNA3 with cloned hCD40 cDNA) as template.
  • pcDNA3-hCD40 pcDNA3 with cloned hCD40 cDNA
  • the upstream oligonucleotide introduced a Kpnl-restriction site and an ATG-start codon upstream of the coding sequence of the human CD40 portion.
  • the downstream oligonucleotide introduced a EcoRI -restriction site downstream of the human CD40 portion.
  • the murine CD40L (aa Pos. 2-260) was amplified by PCR using pcDNA3-mCD40L (pcDNA3 with cloned mCD40L cDNA) as template.
  • pcDNA3-mCD40L pcDNA3 with cloned mCD40L cDNA
  • the upstream oligonucleotide introduced an EcoRI-restriction site upstream of the murine CD40L portion.
  • the downstream oligonucleotide introduced a Xhol- restriction site downstream of the murine CD40L portion.
  • the human CD40 PCR-product was cut with the restriction enzymes Kpnl and EcoRI, whereas the murine CD40L PCR-product was cut with the restriction enzymes EcoRI and Xhol. In a three fragment ligation, these fragments were inserted into a pcDNA3 plasmid expression vector cut with the restriction enzymes Kpnl and Xhol.
  • the resulting vector was pcDNA3-hCD40/mCD40L.
  • AdV-CD40-CD40L AdV-CD40-CD40L
  • AdV Adenovirus vectors
  • AdV Adenovirus vectors
  • the AdV transfer vector and the AdV genome are linearised and cotransfected into 293A cells (ATCC CRL 1573.1). There they recombine to form AdV vectors which can be packed into viral particles but cannot replicate due to deletions in their El region. This deletions result in the cloning capacity of 7 kb.
  • the AdV vectors can only be propagated and therefore be produced in 293A cells which are stable transfected by sheared AdV DNA and therefore provide the El gene products in trans.
  • the adenovirus vector AdV-CD40-CD40L was constructed by insertion of the whole expression cassette from pcDNA3-CD40-CD40L into the adenovirus transfer vector pQBI-AdCMV5 (QUANTUM Biotechnologies Inc.) from which the CMV5 promoter / enhancer and the globin-poly-A site have been removed. Due to the lack of suitable restriction sites the whole expresssion cassette from pcDNA3-CD40- CD40L (CMV promoter, CD40, CD40-L, poly-A site) was amplified by PCR and also the pQBI-AdCMV5 vector without the CMV5 promoter/enhancer and the globin polyA was amplified by PCR. The 2 amplified DNA fragments were restricted with Pmel (the recognition sites were provided by the overhangs of each PCR-primer) and ligated by T4-DNA ligase.
  • Pmel the recognition sites were provided by the overhangs of each PCR-primer
  • the adenovirus transfer vector AdV-CD40-CD40L was linearised by Clal digestion at the linearization site and cotransfected with linear QBI -viral DNA into QBI-293A cells according to manufacturer's protocol (QUANTUM Biotechnologies Inc.).
  • the transfer vector and the cotransfected adenoviral DNA recombine in their overlapping regions to form the adenovirus vector AdV-CD40-CD40L.
  • AdV-CD40-CD40L has deletions in the El region of human adenovirus type 5; the CD40-CD40L expression cassette is cloned instead of the deleted El region. Due to the El deletion the adenovirus vector is not able to replicate alone. Only in 293A cells which contain the sheared genomic DNA from human adenovirus type 5, the El A and E1B genes which are essential for virus replication are provided in trans and therefore the virus could be produced in that cell line. The virus vector was purified via several cycles of plaque purification and isolated in large scale from the infected 293A cells by methods described in the QUANTUM Adeno-Quest Kit protocol or reviewed by Graham, F.L., and Prevec, L., Mol. Biotechnol. 3 (1995) 207-220.
  • AdV-IL2 The adenovirus vector AdV-IL2 was constructed analogous AdV-CD40-CD40L by using the expression cassette of pcDNA3-H2 instead of pcDNA3-CD40-CD40L.
  • AdV-IL-12 The adenovirus vector AdV-IL-12 was constructed analogous AdV-CD40-CD40L by using the expression cassette of pcDNA3-IL-12 instead of pcDNA3-CD40- CD40L.
  • Vectors as well as adenoviral vectors coexpressing two genes were constructed by using one of the expression plasmids above, inserting an IRES sequence from EMCV (derived from pIRESlneo; Clontech) directly downstream of the first gene to be expressed and inserting the second gene to be expressed between the IRES sequence and the poly-(A) sequence.
  • IRES-sequence Internal Ribosome Entry Site
  • the pcDNA3-hCD40/mCD40L-chimera was transiently transfected into HEK293 cells. Two days after transfection, the expression of the fusion protein and the cell surface localization was verified by FACS analysis using an anti-murine CD40L antibody.
  • NF-kB e.g., CAT-gene (CAT-ELISA, Roche Diagnostic GmbH, DE) -» pNF-kB-
  • CAT reporter plasmid to test the activation of the transcription factor NF-kB, one of the principal outcomes of CD40 signaling.
  • the pCDNA3- hCD40L/mCD40- plasmid was cotransfected with the reporter plasmid into HEK293 cells and tested for CAT-activity after 48 h hours.
  • an up to 6-fold enhancement of NF-kB-activation could be acchieved by hCD40L/mCD40.
  • the reporter plasmid pNF-kB-CAT was cotransfected with pcDNA3-hCD40 (expression of CD40 gene alone) into HEK293 cells and the pcDNA3-hCD40/mCD40L or the pcDNA3- mCD40L plasmids were seperately transfected into HEK293 cells.
  • the CD40 cell populations were coincubated with the hCD40/mCD40L or mCD40L cell populations for additional 24 h and than tested for CAT-expression. An identical increase in NF-kB-activation of the CD40 cell population was obtained in both cases compared to the basal CD40-activity.
  • SCCVII, CT26 and B16-F10 cells were infected with adenoviruses expressing the CD40-CD40L chimera or LNGFR (or lacZ) as a control.
  • the expression of relevant cell surface markers (CD40-CD40L, MHCI, ICAM-1, 7) in response to viral infection and fusion protein expression was characterized. Further, growth kinetics and apoptotic behavior of the infected cells was examined.
  • FuGENETM6 was added to 100 ⁇ l serum-free DMEM medium, incubated for 5 min at room temperature and then mixed with 2 ⁇ g DNA. This mixture was incubated for 15 min at room temperature and added dropwise to the cells with a volume of 2 ml cell culture medium (DMEM, 10% FCS, 2mM glutamine, 4.5 g/1 glucose).
  • DMEM 10% FCS, 2mM glutamine, 4.5 g/1 glucose
  • mice were anesthetized with ether. Tumor growth was quantitated five days post-inoculation three times a week using a caliper. Body weight was monitored three times a week. General disturbance was checked daily. Tumor volumes were calculated by the approximately ellipsoid formula: 4/3 x ⁇ / 8 x (length x width x depth) .
  • Murine B16F10 cells were injected at a dose of 5 x 10 5 cells / 200 ⁇ l per mouse. 14 to 16 days after tumor cell inoculation Ad-CD40-CD40L (versions A and B) or AdCMV-LacZ as a control were injected intratumorally and the tumor size was measured every third day for a period of about 21 days. Dose response curves with different concentrations of viral vector (1 x 10 7 to 1 x 10 9 / 50 ⁇ l) and comparison of single vs. multiple injections were performed. After termination of the experiment, histopathology is performed with explanted tumors and checked for activated T ceUs (CD4, CD25).
  • Example 6 was performed according to Example 5 however in the case of tumor regression, treated mice are challenged with tumor cells i.p. into the contralateral side. Growth of secondary tumors is measured to monitor memory T-cell response.
  • Ad-CD40-CD40L- or AdCMV-LacZ-transduced SCCVII tumor cells were irradiated (5000 rad) and implanted in syngeneic mice with a preexisting B16F10 -tumor (established according to Example 4) at a distant site. Tumor growth of the primary tumor was monitored to show the antigen- specificity of the anti- tumor effect.
  • Ad-CD40-CD40L- or AdCMV-LacZ- transduced different (e.g. CT-26) tumor cells were implanted, which should give no anti- B16F10 reaction.
  • tumor-specific CTL cytotoxic T cell
  • a nonviral vector according to the invention For the production of a pharmaceutical composition 100 ⁇ g of a nonviral vector according to the invention are dissolved in 1 ml of phosphate buffered saline (PBS: 8 g sodium chloride, 1.44 g di-sodium-hydrogen-phosphate, 0.24 g potassium-di- hydrogen-phosphate per liter H 2 O at a pH of 7.4).
  • PBS phosphate buffered saline
  • l ⁇ '° pFU adenovirus vector according to the invention in a volume of 1 ml PBS was added to 1 ml of liposomal transfection reagent (Roche Diagnostics GmbH, DE; DOSPER: l,3-di-oleoyloxy-2- (6-Carboxy-spermyl)-propylamide) in PBS, incubated for 15 min. at room temperature and stored at 4°C

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Abstract

L'invention concerne un acide nucléique codant un polypeptide chimérique CD40/CD40L comprenant des fragments d'acide nucléique codant i) le domaine de signalisation de CD40 et en aval de celui-ci, ii) un domaine transmembranaire d'un récepteur de type II et en aval de celui-ci, iii) le domaine de liaison et de trimérisation de CD40L, cet acide nucléique pouvant être utilisé comme agent de thérapie génique pour le traitement local de tumeurs solides.
PCT/EP2001/012495 2000-10-31 2001-10-29 Acides nucleiques codant des polypeptides chimeriques cd40/cd40l, procedes de production et utilisations de ces acides nucleiques WO2002036769A2 (fr)

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