WO1999050411A2 - Tumour-specific expression control region and the use thereof - Google Patents

Tumour-specific expression control region and the use thereof Download PDF

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Publication number
WO1999050411A2
WO1999050411A2 PCT/EP1999/002031 EP9902031W WO9950411A2 WO 1999050411 A2 WO1999050411 A2 WO 1999050411A2 EP 9902031 W EP9902031 W EP 9902031W WO 9950411 A2 WO9950411 A2 WO 9950411A2
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tumour
nucleic acid
seq
fragment
cells
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WO1999050411A3 (en
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Reinhard BÜTTNER
Anja-Katrin Bosserhoff
Stefan Seeber
Georg Tiefenthaler
Rüdiger Rüger
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Roche Diagnostics Gmbh
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    • 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
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/42—Vector systems having a special element relevant for transcription being an intron or intervening sequence for splicing and/or stability of RNA
    • 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
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
    • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES

Definitions

  • Tumour-specific expression control region and the use thereof
  • the invention concerns a tumour-specific expression control region, vectors containing this region and its use especially for in vivo expression.
  • tumouricidal foreign genes in tumours is a promising approach for the therapeutic treatment of tumour diseases.
  • IGF-I regulates CD-RAP gene expression via an AP-2 binding site (bp -475 to -458).
  • Bosserhoff et al. Proc. Am. Association for Cancer Research Annual Meeting 39 (1980) p. 250, XP002087909, Abstract 1711, and Lederer et al., J. Dermatol. Sci. 16 (1998) Suppl. 1 S48, describe that a region of about 30 bp lying within the region -210 to -1 for the expression control region of the MIA gene is responsible for the tumor-specific expression pattern. However, the authors do not describe the region itself.
  • Bosserhoff et al. Proc. Am. Association for Cancer Research Annual Meeting 37 (1996) p. 512, Abstract 3565, describe that a 300 bp partial region of the MIA expression control region is melanoma-specifically active. However, the authors do not mention that the MIA promotor has a tumor specificity.
  • the object of the invention is to improve the MIA promoter so that it is able in vivo to express genes specifically in tumour cells.
  • tumour-cell-specific expression vector containing a gene which codes for a transcription or translation product that is therapeutically active in tumour cells wherein this gene is under the control of an expression control region of the SEQ ID NO:l or a fragment thereof which comprises at least bp -224 to -214 and/or -197 to -207 from SEQ ID NO:l.
  • the segment is bp -224 to -197 of said sequence.
  • bp -224 from SEQ ID NO:l means the base or the base pair (bp) 157 from SEQ ID NO:l in the single or double strand.
  • An expression control region 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 to 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.
  • An expression control region according to the invention contains at least the nucleic acid fragment (oligonucleotide) bp - 224 to -197 from SEQ ID NO:l or active regulatory parts thereof.
  • oligonucleotide contains two highly conserved binding sites, region X (bp -197 to -207) and region Y (bp -224 to -214, TCF-Box).
  • These regulatory nucleic acid fragments (regulatory regions) are also suitable as an expression control region according to the invention in combination with other promoters such as for example the TK promoter, the minimal early SV40 or the CMV immediate early promoter in expression vectors.
  • a regulatory region is understood as a region which influences expression in a negative or positive manner. If the expression control sequence according to the invention contains a negative regulatory region, the tumour-specific expression is achieved by abolishing such an inhibition in tumour cells and vice versa. The tumour-specific expression can similarly be inhibited by elements that bind to a positive regulatory region (antisense).
  • the distance between the regulatory nucleic acid fragments region X and region Y is not very large.
  • the distance is preferably between 0 and 20 bps.
  • (homologous) MIA regions from mammals such as for example the mouse or rat are suitable as an expression control region.
  • the human MIA sequence is described in the EMBL data base under the number X84707, the murine sequence under the number 485612.
  • a therapeutically active translation product is understood as a polypeptide (protein) which immediately causes a regression or ablation of tumour cells or results in this via stimulation of the immune system.
  • Suitable genes code for example for tumour suppressor proteins, for proteins which induce apoptosis (e.g. p53), pro-drug activators (suicide genes, such as TK or cytosine deaminase
  • a therapeutically active transcription product is preferably understood as an antisense sequence (e.g. ribozyme or antisense RNA) which is directed against an oncogene, a gene inhibiting apoptosis or another tumour gene such as MIA (WO 95/03328).
  • an antisense sequence e.g. ribozyme or antisense RNA
  • Such a transcription product is therapeutically active because it causes a regression or ablation of tumour cells. This can for example be achieved by inhibition of the expression of a tumour gene or of an oncogene.
  • Such therapeutically active transcription or translation products differ from so-called indicator genes like the CAT or luciferase gene which are derived from prokaryotes or insects and are not expressed in a therapeutically active manner in mammals and only serve as an expression test.
  • indicator genes like the CAT or luciferase gene which are derived from prokaryotes or insects and are not expressed in a therapeutically active manner in mammals and only serve as an expression test.
  • the expression vectors and regulatory regions according to the invention are tumour-specific or tumour-cell-specific and are in particular specific for metastatic cells since the MIA promoter is particularly active in those tumour cells which have become detached from the primary tumour and have thus become mobile.
  • the therapeutically active transcription product which is preferably an antisense nucleic acid binds in vitro under stringent conditions to a nucleic acid of the sequence SEQ ID NO:l.
  • stringent standard conditions and methods for hybridization are known to a person skilled in the art and described for example by Sambrook J., et al. in Expression of clones genes in E. coli" in Molecular Cloning: A laboratory manual (1989), Cold Spring Harbor Laboratory Press, New York, USA and Hames, B.D., and Higgins, S.J., in Nucleic Acid Hybridisation - A Practical
  • Preferred stringent conditions are present when hybridizing in the presence of 1 mol/1 NaCl, 1 % SDS and 10 % dextran sulfate and subsequently washing the filter twice for 5 minutes at room temperature in 2 x SSC and carrying out one wash step for 30 minutes.
  • This further wash step can be carried out at 65°C at 0.5 x SSC, 0.1 % SDS, preferably at 0.2 x SSC and 0.1 % SDS and especially preferably at 0.1 x SSC, 0.1 % SDS.
  • a further subject matter of the invention is a process for the production of a tumour-specific expression vector according to the invention wherein a gene is inserted into a suitable vector in such a way that it is expressed under the control of the described expression control and/or regulatory region according to the invention.
  • Such vectors are expediently vectors which are suitable for gene therapy.
  • Such vectors can be either naked or formulated plasmid DNA (e.g. formulated with transfer reagents such as liposomes) or viral nucleic acids or can be artificial chromosomes.
  • tumour-cell-specific expression vectors produced according to the invention can be advantageously used ex vivo or in vivo for gene therapy to regress or ablate tumours and tumour cells such as, e.g., melanoma, colon carcinoma and/or mamma carcinoma cells and metastasising cells derived therefrom.
  • tumour cells such as, e.g., melanoma, colon carcinoma and/or mamma carcinoma cells and metastasising cells derived therefrom.
  • the regression or ablation of tumour cells can take place immediately (for example by expression of p53) or indirectly (by expression of immunostimulating agents such as cytokines
  • costimulatory molecules e.g. B7-1, B7-2, CD40.
  • a further subject matter of the invention is a process for the production of a pharmaceutical agent for the regression or ablation of primary tumours, residual tumours, metastases and minimal residual disease in vivo or ex vivo of tumour or leukaemia cells which contains an expression vector according to the invention as an essential component, and still another subject matter of the invention is the said pharmaceutical agent itself.
  • a further subject matter of the invention is a nucleic acid fragment with a length of
  • nucleic acid fragment 10 to 28 bases from the region bp -224 to -197 from SEQ ID NO:l or a complementary nucleic acid hybridizing under stringent conditions with said fragment.
  • Such a nucleic acid fragment is suitable as a tumour cell-specific regulatory element in an expression control sequence.
  • Such a nucleic acid fragment is also suitable for the detection and identification of substances binding to this nucleic acid fragment. Such detection methods are described for example in the US patent No. 5,578,444, US patent No. 5,716,760 and the Canadian patent No. 2,112,130.
  • nucleic acid fragment is covalently bound to the DNA and a binding partner of the nucleic acid fragment influences the DNA protein binding.
  • a further subject matter of the invention is based on the binding of elements that can bind in a complementary manner to the nucleic acid fragment (bp -197 to - 224) (preferably under physiological conditions e.g. in tumour cells) in order to inhibit the expression of the MIA promoter.
  • elements are for example antisense RNA, ribozyme, PNA or chimeraplasts and can be used in a modified form in the same sense as described above and/or used directly (without requiring an expression vector as a vehicle).
  • a further subject matter of the invention is a first nucleic acid fragment which specifically binds under physiological conditions to a second nucleic acid fragment of sequence SEQ ID NO:l or a fragment thereof which comprises at least the bases from bp -224 to -197 from SEQ ID NO:l and forms a triple helix with the double-stranded second nucleic acid fragment in the region of the bases bp -224 to -197 from SEQ ID NO:l or contains one or several point mutations in the region of the bases bp -224 to -197 from SEQ ID NO:l.
  • Such a first nucleic acid fragment is preferably 10 to 40 nucleotides long and can be used to inhibit MIA expression in tumour cells.
  • the nucleic acid fragment (preferably factor X) represents a conserved expression control element.
  • the mutation of a few bases preferably 1 to 3 bases
  • a chimeraplast is understood as a chimeric DNA-RNA hybrid molecule (expediently 60 to 90 bp) which is able to bind sequence- specifically to DNA and induce the point mutations described above.
  • chimeraplasts and the production thereof are described in the US patent No. 5,565,350. Their in vivo application is described by Kren, B.T., et al., Nature Medicine 4 (1998) 285-290.
  • An expression vector according to the invention can also be used advantageously for an ex vivo purging of leukaemia cells or tumour cells in autologous bone marrow transplantations. Since the expression vector is active specifically in tumour cells, this would enable otherwise non-detectable tumour cells to be labelled in autologous transplantation preparations. It is expedient to carry this out by using a suitable tumour cell marker as described for example in WO 95/06723. It is particularly advantageous to use the LNGFR gene as a marker gene for tumour cells from autologous bone marrow transplantation preparations. Additionally an expression vector according to the invention is used which in this case does not code for a therapeutically active product but rather for a selectable indicator gene. The preferred LNGFR gene expresses the LNGF receptor which labels the cell surface and is reliably detected by means of antibodies. This enables the undesired tumour cells to be removed from autologous bone marrow preparations.
  • Analogously suicide genes, toxin genes and apoptosis-inducing genes can also be introduced and used to kill the tumour or leukaemia cells for purging.
  • FIG. 1 Restriction map of the plasmid pCMVhl2-bgh-cat.
  • pMIA380hl2-bgh-cat is prepared as follows: The CMV promoter is removed from pCMVhl2-bgh-cat by a Pstl/Xhol double digestion. The shortened MIA promoter fragment (0 to -380 bp) is amplified from pBL-MIA1386 (which contains the entire MIA promoter region described in Bosserhoff, A.K., et al, J. Biol. Chem.
  • pMIA380hl2-bgh-cat Based on the construct pMIA380hl2-bgh-cat the construct pMIA380hl2-bgh- HSV-TK is prepared as follows: The cat gene is removed from pMIA380hl2-bgh- cat by a Notl digestion. The HSV-TK gene is amplified from pLTl (Fig. 2) by means of PCR and primers against the 5' and the 3' end of the gene, which carry overhanging ends with the Notl site and is cloned into the Notl cleavage site.
  • pMIA380hl2-bgh-cat Based on the construct pMIA380hl2-bgh-cat the construct pMIA380hl2-bgh-hIL- 2 is prepared as follows: The cat gene is removed from pMIA380hl2-bgh-cat by a Notl digestion. The human IL-2 gene is amplified from pCMVhIL2IREShB7-l (Fig. 3) by means of PCR and primers against the 5' and the 3' end of the cDNA, which carry overhanging ends with the Notl sites and is cloned into the Notl cleavage site.
  • pMIA380hl2-bgh-GM- CSF is prepared as follows: The cat gene is removed from pMIA380hl2-bgh-cat by a Notl digestion. GM-CSF is described in EP-B 0 202 300 and EP-B 0 188 479 (see also (Dranoff, G., et al, Proc. Natl. Acad. Sci. USA 90 ( 1993) 3539-3543). The GM- CSF gene is amplified by means of PCR and primers against the 5' and the 3' end of the cDNA, which carry overhanging ends with the Notl sites and is cloned into the Notl cleavage site.
  • the murine B16 melanoma cell line (ATCC# CRL 6322) is cultured in DMEM + 10 % FCS and L-glutamine.
  • DOSPER Roche Diagnostics GmbH, Mannheim, DE
  • pCMVhl2-bgh-cat or pMIA380hl2-bgh-cat is used as the transfection reagent according to the manufacturer's instructions; serum free transfection medium.
  • the B16 cells are transfected; the cat activity is measured after 2 to 3 days.
  • the test for cat activity is carried out with a cat ELISA (Roche Diagnostics GmbH, Mannheim, DE) according to the manufacturer's instructions and the results obtained for the shortened MIA promoter fragment (example 1.1) are compared with those of the CMV promoter.
  • the B16 cells are transfected; the IL-2 activity is measured after 2 to 3 days.
  • the test for IL-2 activity is carried out with an IL-2 ELISA (Roche Diagnostics GmbH,
  • the transfection into the B16 cells is carried out with an Asp700I linearized plasmid.
  • the plasmid pCDNA3 (Invitrogen) which contains a NeoR expression cassette is co-transfected in a 10-fold molar deficit. It is selected for 2 - 3 weeks with 50 ⁇ g/ml G418. Subsequently HSV-TK gene-positive B16 melanoma cell clones are isolated by limited dilution and PCR screening of the individual clones obtained.
  • HSV-TK activity is carried out with Ganciclovir (Cymeven®, Syntex/10 ⁇ g/ml; Beck, C, et al., Human Gene Therapy 6 (1995) 1525-1530); HSV- TK expressing positive cells die.
  • Ganciclovir Cymeven®, Syntex/10 ⁇ g/ml; Beck, C, et al., Human Gene Therapy 6 (1995) 1525-1530
  • HSV- TK expressing positive cells die.
  • the results obtained for the shortened MIA promoter fragment (example 1.1) are compared with those for the CMV promoter.
  • a stable B16 melanoma cell line is set up for pMIA380hl2-bgh-GM-CSF analogously to the HSV TK gene; positive clones are characterized by PCR and a GM-CSF ELISA (endogenous).
  • HSV-TK 1 x 10" B16/HSV-TK cells are washed in PBS and injected subcutaneously in a volume of 200 ⁇ l into the abdominal wall of C57B16 mice (Fidler, I.J., Cancer Research 35 (1975) 218-224). The stably transfected tumour has started to grow after 4 - 6 days.
  • Variant 1 x 10 ⁇ B16/HSV-TK cells are washed in PBS and injected intravenously into the C57B16 mice. After 10 - 14 days the lung metastases have started to grow.
  • the B16 melanomas expressing HSV-TK but not the non-expressing B16 melanomas are killed by GCV doses (2 x daily; for 5 days; 150 mg/kg GCV in 200 ⁇ l 0.9 % NaCl solution (Beck, C, et al., Human Gene Therapy 6 (1995) 1525-1530) or only 0.9 % NaCl solution as a control).
  • the mice are sacrificed, dissected and examined histologically; the GCV-treated animals are compared with the non- treated animals.
  • GM-CSF(3) The syngenic C57B16 mice are vaccinated by subcutaneous injection of 5 x 10-> live pMIA380hl2-bgh-GM-CSF transfected B16 cells into the abdomen. After 7 - 14 days the animals are challenged by subcutaneously injecting 5 x 10 ⁇ live, non- transduced B16 melanoma cells into the back. The mice are sacrificed, dissected and examined when tumours of 2 - 3 cm in size occur or after at most 100 days. The mice which have received an injection of B16 cells stably transfected with the
  • GM-CSF gene under MIA promoter control are compared with mice that have received untransfected B16 cells.
  • CSF as a formulation with DOTAP (Roche Diagnostics GmbH, DE) into established B16 melanoma tumours or into normal muscle tissue of syngenic C57B16 mice
  • pMIA380hl2-bgh-HSV-TK is formulated with DOTAP and injected into the pre-formed B16 melanoma tumours or into healthy muscle tissue.
  • Ganciclovir is administered after 2 - 3 days (2 x daily; for 5 days; 150 mg/kg GCV in
  • the tumour cells transfected by the HSV-TK gene and therefore expressing HSV-TK are killed by the prodrug activation but not the non-expressing cells (normal body cells or normal body cells which have been transduced by pMIA380hl2-bgh-HSV-TK in which the MIA promoter is, however, not activated i.e. HSV-TK is also not expressed).
  • the mice are sacrificed, dissected and examined; the GCV-treated animals are compared histologically with non-treated animals.
  • the treated tissue is also compared by means of PCR and RT-PCR for the presence or expression of the transgenic HSV-TK gene.
  • GM-CSF 1 x 10" B16 cells are washed in PBS and injected subcutaneously in a volume of 200 ⁇ l into the abdominal wall of syngenic C57B16 mice. The tumour has started to grow after 4 - 6 days.
  • pMIA380hl2-bgh-GM-CSF is formulated with DOTAP and injected into the pre-formed B16 melanoma tumours or into muscle tissue.
  • the mice are sacrificed, dissected and histologically examined for the immunostimulatory effect of the GM-CSF gene (tumour size, macrophage infiltration) and for the presence of the plasmid DNA by means of PCR or for GM- CSF expression by means of RT-PCR.
  • the mice which have received pMIA380hl2- bgh-GM-CSF/ DOTAP into the pre-formed B16 melanoma tumours are compared with mice which have received only the empty vector plasmid or an injection into muscle tissue.
  • Mutations are introduced by site-directed mutagenesis (site directed mutagenesis kit, Clontech) in region X of the expression control sequence according to Table 1 in a reporter plasmid which contains the luciferase gene under the control of the MIA promoter fragment according to SEQ ID NO:l and expression in malignant melanoma cells (MM, B16) and non-melanoma cells (nonMM, HeLa) is examined.
  • site-directed mutagenesis site directed mutagenesis kit, Clontech
  • the reporter plasmid is prepared by inserting the MIA promoter fragment as well as the luciferase indicator gene via Hindlll/Bglll into the vector pGL3 basic (Promega GmbH, Mannheim, DE). Table 1

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Abstract

An expression vector containing a gene which codes for a transcription or translation product which is therapeutically active, wherein this gene is under the control of an expression control region having the sequence SEQ ID NO:1 or a fragment thereof which comprises at least the bases bp -224 to -214 and/or -197 to -207 from SEQ ID NO:1 is tumour-cell-specific and suitable for in vivo and in vitro ablation or regression of tumour cells.

Description

Tumour-specific expression control region and the use thereof
The invention concerns a tumour-specific expression control region, vectors containing this region and its use especially for in vivo expression.
The specific expression of tumoricidal foreign genes in tumours is a promising approach for the therapeutic treatment of tumour diseases.
The structure and the promoter analysis of the gene which codes for the human melanoma-inhibiting protein MIA (also referred to as CD-RAP) is known from Bosserhoff, A.K., et al, J. Biol. Chem. 271 (1996) 490-495. MIA is expressed in melanoma cell lines and has growth-inhibiting effects on melanoma cells in vitro (Bogdahn et al., Cancer Res. 49 (1989) 5358-5363; International Application No. WO 95/03328). Furthermore it is known from Bosserhoff and WO 95/03328 that a region of about 500 base pairs of the 5' untranslated region of the MIA gene causes the expression of MIA in malignant melanoma cells.
From Kondo, S., et al., 44th Annual Meeting, Orthopaedic Research Society, March 16-19, 1998, New Orleans, Louisiana, p. 178-30 it is known that IGF-I regulates CD-RAP gene expression via an AP-2 binding site (bp -475 to -458).
From Xie, W.F., 44tn Annual Meeting, Orthopaedic Research Society, March 16-19, 1998, New Orleans, Louisiana, p. 207-35 it is known that a 2.2 kb CD-RAP promoter fragment causes expression of lacZ in transgenic mice. A blue colouration shows that all regulatory elements of CD-RAP are present in order to determine the natural CD-RAP expression.
Bosserhoff et al., Proc. Am. Association for Cancer Research Annual Meeting 39 (1980) p. 250, XP002087909, Abstract 1711, and Lederer et al., J. Dermatol. Sci. 16 (1998) Suppl. 1 S48, describe that a region of about 30 bp lying within the region -210 to -1 for the expression control region of the MIA gene is responsible for the tumor-specific expression pattern. However, the authors do not describe the region itself. Bosserhoff et al., Proc. Am. Association for Cancer Research Annual Meeting 37 (1996) p. 512, Abstract 3565, describe that a 300 bp partial region of the MIA expression control region is melanoma-specifically active. However, the authors do not mention that the MIA promotor has a tumor specificity.
The object of the invention is to improve the MIA promoter so that it is able in vivo to express genes specifically in tumour cells.
The object is achieved by a tumour-cell-specific expression vector containing a gene which codes for a transcription or translation product that is therapeutically active in tumour cells wherein this gene is under the control of an expression control region of the SEQ ID NO:l or a fragment thereof which comprises at least bp -224 to -214 and/or -197 to -207 from SEQ ID NO:l. Preferably, the segment is bp -224 to -197 of said sequence.
Details of the numbering of the bases (e.g. bp -224) correspond to the numbering system for expression control sequences (upstream numbering) familiar to a person skilled in the art. The following classification applies:
SEQ ID NO:l upstream numbering system
1 -380
157 -224
184 -197
380 -1
Hence according to the invention "bp -224 from SEQ ID NO:l" means the base or the base pair (bp) 157 from SEQ ID NO:l in the single or double strand.
Surprisingly, it has turned out that such a vector enables a tumour-specific expression of therapeutically active genes in tumour cells, which enable exclusive expression of therapeutically effective translation- or transcription products in tumour cells.
An expression control region 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 to 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.
An expression control region according to the invention contains at least the nucleic acid fragment (oligonucleotide) bp - 224 to -197 from SEQ ID NO:l or active regulatory parts thereof. Such an oligonucleotide contains two highly conserved binding sites, region X (bp -197 to -207) and region Y (bp -224 to -214, TCF-Box). These regulatory nucleic acid fragments (regulatory regions) are also suitable as an expression control region according to the invention in combination with other promoters such as for example the TK promoter, the minimal early SV40 or the CMV immediate early promoter in expression vectors.
A regulatory region is understood as a region which influences expression in a negative or positive manner. If the expression control sequence according to the invention contains a negative regulatory region, the tumour-specific expression is achieved by abolishing such an inhibition in tumour cells and vice versa. The tumour-specific expression can similarly be inhibited by elements that bind to a positive regulatory region (antisense).
For a tumour-specific expression it is important that the distance between the regulatory nucleic acid fragments region X and region Y is not very large. The distance is preferably between 0 and 20 bps.
The human MIA region described in SEQ ID NO:l as well as corresponding
(homologous) MIA regions from mammals such as for example the mouse or rat are suitable as an expression control region. The human MIA sequence is described in the EMBL data base under the number X84707, the murine sequence under the number 485612.
A therapeutically active translation product is understood as a polypeptide (protein) which immediately causes a regression or ablation of tumour cells or results in this via stimulation of the immune system. Suitable genes code for example for tumour suppressor proteins, for proteins which induce apoptosis (e.g. p53), pro-drug activators (suicide genes, such as TK or cytosine deaminase
(CDA)), immunostimulators (e.g. cytokines), co-stimulators (e.g. B7-1 or B7-2), CD40 and/or CD40 ligand, or toxic proteins such as choleratoxin. A therapeutically active transcription product is preferably understood as an antisense sequence (e.g. ribozyme or antisense RNA) which is directed against an oncogene, a gene inhibiting apoptosis or another tumour gene such as MIA (WO 95/03328). Such a transcription product is therapeutically active because it causes a regression or ablation of tumour cells. This can for example be achieved by inhibition of the expression of a tumour gene or of an oncogene.
Hence such therapeutically active transcription or translation products differ from so-called indicator genes like the CAT or luciferase gene which are derived from prokaryotes or insects and are not expressed in a therapeutically active manner in mammals and only serve as an expression test.
Surprisingly, the expression vectors and regulatory regions according to the invention are tumour-specific or tumour-cell-specific and are in particular specific for metastatic cells since the MIA promoter is particularly active in those tumour cells which have become detached from the primary tumour and have thus become mobile.
The therapeutically active transcription product which is preferably an antisense nucleic acid binds in vitro under stringent conditions to a nucleic acid of the sequence SEQ ID NO:l. Such stringent standard conditions and methods for hybridization are known to a person skilled in the art and described for example by Sambrook J., et al. in Expression of clones genes in E. coli" in Molecular Cloning: A laboratory manual (1989), Cold Spring Harbor Laboratory Press, New York, USA and Hames, B.D., and Higgins, S.J., in Nucleic Acid Hybridisation - A Practical
Approach, Hames and Higgins publishers (1985), IRL Press. The standard protocols described in this manual are usually used for this. Particular reference is made to Sambrook, Section IX.
Preferred stringent conditions are present when hybridizing in the presence of 1 mol/1 NaCl, 1 % SDS and 10 % dextran sulfate and subsequently washing the filter twice for 5 minutes at room temperature in 2 x SSC and carrying out one wash step for 30 minutes. This further wash step can be carried out at 65°C at 0.5 x SSC, 0.1 % SDS, preferably at 0.2 x SSC and 0.1 % SDS and especially preferably at 0.1 x SSC, 0.1 % SDS. A further subject matter of the invention is a process for the production of a tumour-specific expression vector according to the invention wherein a gene is inserted into a suitable vector in such a way that it is expressed under the control of the described expression control and/or regulatory region according to the invention. Such vectors are expediently vectors which are suitable for gene therapy.
Such vectors can be either naked or formulated plasmid DNA (e.g. formulated with transfer reagents such as liposomes) or viral nucleic acids or can be artificial chromosomes.
The tumour-cell-specific expression vectors produced according to the invention can be advantageously used ex vivo or in vivo for gene therapy to regress or ablate tumours and tumour cells such as, e.g., melanoma, colon carcinoma and/or mamma carcinoma cells and metastasising cells derived therefrom. The regression or ablation of tumour cells can take place immediately (for example by expression of p53) or indirectly (by expression of immunostimulating agents such as cytokines
(e.g. IL-2, GM-CSF or IL-12) or costimulatory molecules (e.g. B7-1, B7-2, CD40).
A further subject matter of the invention is a process for the production of a pharmaceutical agent for the regression or ablation of primary tumours, residual tumours, metastases and minimal residual disease in vivo or ex vivo of tumour or leukaemia cells which contains an expression vector according to the invention as an essential component, and still another subject matter of the invention is the said pharmaceutical agent itself.
A further subject matter of the invention is a nucleic acid fragment with a length of
10 to 28 bases from the region bp -224 to -197 from SEQ ID NO:l or a complementary nucleic acid hybridizing under stringent conditions with said fragment. Especially preferred are fragments consisting of bp -207 to -197 or -224 to 214. Such a nucleic acid fragment is suitable as a tumour cell-specific regulatory element in an expression control sequence. Such a nucleic acid fragment is also suitable for the detection and identification of substances binding to this nucleic acid fragment. Such detection methods are described for example in the US patent No. 5,578,444, US patent No. 5,716,760 and the Canadian patent No. 2,112,130. In these methods the nucleic acid fragment is covalently bound to the DNA and a binding partner of the nucleic acid fragment influences the DNA protein binding. A further subject matter of the invention is based on the binding of elements that can bind in a complementary manner to the nucleic acid fragment (bp -197 to - 224) (preferably under physiological conditions e.g. in tumour cells) in order to inhibit the expression of the MIA promoter. Such elements are for example antisense RNA, ribozyme, PNA or chimeraplasts and can be used in a modified form in the same sense as described above and/or used directly (without requiring an expression vector as a vehicle).
Consequently a further subject matter of the invention is a first nucleic acid fragment which specifically binds under physiological conditions to a second nucleic acid fragment of sequence SEQ ID NO:l or a fragment thereof which comprises at least the bases from bp -224 to -197 from SEQ ID NO:l and forms a triple helix with the double-stranded second nucleic acid fragment in the region of the bases bp -224 to -197 from SEQ ID NO:l or contains one or several point mutations in the region of the bases bp -224 to -197 from SEQ ID NO:l. Such a first nucleic acid fragment is preferably 10 to 40 nucleotides long and can be used to inhibit MIA expression in tumour cells.
The nucleic acid fragment (preferably factor X) represents a conserved expression control element. The mutation of a few bases (preferably 1 to 3 bases) inhibits or reduces the expression activation of the promoter element located downstream. Therefore for a therapeutic application it is preferable to introduce antisense RNA, ribozyme, PNA or chimeraplasts into tumour cells which cause such an inhibition or mutation in the MIA promoter and consequently inhibit MIA expression in such cells and as a result reduce or abolish the metastasising potential of these tumour cells (preferably melanoma, mamma carcinoma, colon carcinoma).
According to the invention a chimeraplast is understood as a chimeric DNA-RNA hybrid molecule (expediently 60 to 90 bp) which is able to bind sequence- specifically to DNA and induce the point mutations described above. Such chimeraplasts and the production thereof are described in the US patent No. 5,565,350. Their in vivo application is described by Kren, B.T., et al., Nature Medicine 4 (1998) 285-290.
An expression vector according to the invention can also be used advantageously for an ex vivo purging of leukaemia cells or tumour cells in autologous bone marrow transplantations. Since the expression vector is active specifically in tumour cells, this would enable otherwise non-detectable tumour cells to be labelled in autologous transplantation preparations. It is expedient to carry this out by using a suitable tumour cell marker as described for example in WO 95/06723. It is particularly advantageous to use the LNGFR gene as a marker gene for tumour cells from autologous bone marrow transplantation preparations. Additionally an expression vector according to the invention is used which in this case does not code for a therapeutically active product but rather for a selectable indicator gene. The preferred LNGFR gene expresses the LNGF receptor which labels the cell surface and is reliably detected by means of antibodies. This enables the undesired tumour cells to be removed from autologous bone marrow preparations.
Analogously suicide genes, toxin genes and apoptosis-inducing genes can also be introduced and used to kill the tumour or leukaemia cells for purging.
The invention whose protective scope results from the claims is further elucidated by the following examples, publications, the sequence protocol and the figures. The described procedures are understood as examples which still describe the subject matter of the invention even after modifications.
Description of the Figures
Fig. 1 Restriction map of the plasmid pCMVhl2-bgh-cat.
Fig. 2 Restriction map of the plasmid pLTl.
Fig. 3 Restriction map of the plasmid pCMVhil2ireshb7-l.
Example 1
Construction of the expression vectors
1.1 Cat reporter gene as control:
Based on the reporter gene plasmid pCMVhl2-bgh-cat (Fig. 1) the construct pMIA380hl2-bgh-cat is prepared as follows: The CMV promoter is removed from pCMVhl2-bgh-cat by a Pstl/Xhol double digestion. The shortened MIA promoter fragment (0 to -380 bp) is amplified from pBL-MIA1386 (which contains the entire MIA promoter region described in Bosserhoff, A.K., et al, J. Biol. Chem. 271 (1996) 490-495) by means of PCR using appropriate primers which carry overhanging ends with the Pstl/Xhol sites and cloned into the Pstl/Xhol cleavage site. 1.2 Prodrug-activatable suicide gene HSV-TK:
Based on the construct pMIA380hl2-bgh-cat the construct pMIA380hl2-bgh- HSV-TK is prepared as follows: The cat gene is removed from pMIA380hl2-bgh- cat by a Notl digestion. The HSV-TK gene is amplified from pLTl (Fig. 2) by means of PCR and primers against the 5' and the 3' end of the gene, which carry overhanging ends with the Notl site and is cloned into the Notl cleavage site.
1.3. Immunostimulatory IL-2 gene:
Based on the construct pMIA380hl2-bgh-cat the construct pMIA380hl2-bgh-hIL- 2 is prepared as follows: The cat gene is removed from pMIA380hl2-bgh-cat by a Notl digestion. The human IL-2 gene is amplified from pCMVhIL2IREShB7-l (Fig. 3) by means of PCR and primers against the 5' and the 3' end of the cDNA, which carry overhanging ends with the Notl sites and is cloned into the Notl cleavage site.
1.4 Immunostimulatory GM-CSF gene:
Based on the construct pMIA380hl2-bgh-cat the construct pMIA380hl2-bgh-GM- CSF is prepared as follows: The cat gene is removed from pMIA380hl2-bgh-cat by a Notl digestion. GM-CSF is described in EP-B 0 202 300 and EP-B 0 188 479 (see also (Dranoff, G., et al, Proc. Natl. Acad. Sci. USA 90 ( 1993) 3539-3543). The GM- CSF gene is amplified by means of PCR and primers against the 5' and the 3' end of the cDNA, which carry overhanging ends with the Notl sites and is cloned into the Notl cleavage site.
Example 2 Transfection of the aforementioned vectors into a murine B16 melanoma cell line
The murine B16 melanoma cell line (ATCC# CRL 6322) is cultured in DMEM + 10 % FCS and L-glutamine. DOSPER (Roche Diagnostics GmbH, Mannheim, DE) is used as the transfection reagent according to the manufacturer's instructions; serum free transfection medium. pCMVhl2-bgh-cat or pMIA380hl2-bgh-cat:
The B16 cells are transfected; the cat activity is measured after 2 to 3 days. The test for cat activity is carried out with a cat ELISA (Roche Diagnostics GmbH, Mannheim, DE) according to the manufacturer's instructions and the results obtained for the shortened MIA promoter fragment (example 1.1) are compared with those of the CMV promoter.
pCMVhl2-bgh-IL-2 or pMIA380hl2-bgh-IL-2:
The B16 cells are transfected; the IL-2 activity is measured after 2 to 3 days. The test for IL-2 activity is carried out with an IL-2 ELISA (Roche Diagnostics GmbH,
Mannheim, DE) according to the manufacturer's instructions and the results obtained for the shortened MIA promoter fragment (example 1.1) are compared with those of the CMV promoter.
pCMVhl2-bgh-HSV-TK or pMIA380hl2-bgh-HSV-TK:
The transfection into the B16 cells is carried out with an Asp700I linearized plasmid. The plasmid pCDNA3 (Invitrogen) which contains a NeoR expression cassette is co-transfected in a 10-fold molar deficit. It is selected for 2 - 3 weeks with 50 μg/ml G418. Subsequently HSV-TK gene-positive B16 melanoma cell clones are isolated by limited dilution and PCR screening of the individual clones obtained.
The in vitro test for HSV-TK activity is carried out with Ganciclovir (Cymeven®, Syntex/10 μg/ml; Beck, C, et al., Human Gene Therapy 6 (1995) 1525-1530); HSV- TK expressing positive cells die. The results obtained for the shortened MIA promoter fragment (example 1.1) are compared with those for the CMV promoter.
A stable B16 melanoma cell line is set up for pMIA380hl2-bgh-GM-CSF analogously to the HSV TK gene; positive clones are characterized by PCR and a GM-CSF ELISA (endogenous).
Example 3
Injection of the stable pMIA380hl2-bgh-HSV-TK and pMIA380hl2-bgh-GM-CSF transfected B16 melanoma line in syngenic C57B16 mice
HSV-TK: 1 x 10" B16/HSV-TK cells are washed in PBS and injected subcutaneously in a volume of 200 μl into the abdominal wall of C57B16 mice (Fidler, I.J., Cancer Research 35 (1975) 218-224). The stably transfected tumour has started to grow after 4 - 6 days.
Variant: 1 x 10^ B16/HSV-TK cells are washed in PBS and injected intravenously into the C57B16 mice. After 10 - 14 days the lung metastases have started to grow.
The B16 melanomas expressing HSV-TK but not the non-expressing B16 melanomas are killed by GCV doses (2 x daily; for 5 days; 150 mg/kg GCV in 200 μl 0.9 % NaCl solution (Beck, C, et al., Human Gene Therapy 6 (1995) 1525-1530) or only 0.9 % NaCl solution as a control). The mice are sacrificed, dissected and examined histologically; the GCV-treated animals are compared with the non- treated animals.
GM-CSF(3): The syngenic C57B16 mice are vaccinated by subcutaneous injection of 5 x 10-> live pMIA380hl2-bgh-GM-CSF transfected B16 cells into the abdomen. After 7 - 14 days the animals are challenged by subcutaneously injecting 5 x 10^ live, non- transduced B16 melanoma cells into the back. The mice are sacrificed, dissected and examined when tumours of 2 - 3 cm in size occur or after at most 100 days. The mice which have received an injection of B16 cells stably transfected with the
GM-CSF gene under MIA promoter control are compared with mice that have received untransfected B16 cells.
Example 4 Injection of the plasmids pMIA380hl2-bgh-HSV-TK and pMIA380hl2-bgh-GM-
CSF as a formulation with DOTAP (Roche Diagnostics GmbH, DE) into established B16 melanoma tumours or into normal muscle tissue of syngenic C57B16 mice
HSV-TK:
1 x lθ6 B16 cells are washed in PBS and injected subcutaneously in a volume of 200 μl into the abdominal wall of syngenic C57B16 mice. The tumour has started to grow after 4 - 6 days. pMIA380hl2-bgh-HSV-TK is formulated with DOTAP and injected into the pre-formed B16 melanoma tumours or into healthy muscle tissue. Ganciclovir is administered after 2 - 3 days (2 x daily; for 5 days; 150 mg/kg GCV in
200 μl 0.9 % NaCl solution or only 0.9 % NaCl solution as a control); the tumour cells transfected by the HSV-TK gene and therefore expressing HSV-TK are killed by the prodrug activation but not the non-expressing cells (normal body cells or normal body cells which have been transduced by pMIA380hl2-bgh-HSV-TK in which the MIA promoter is, however, not activated i.e. HSV-TK is also not expressed). The mice are sacrificed, dissected and examined; the GCV-treated animals are compared histologically with non-treated animals. The treated tissue is also compared by means of PCR and RT-PCR for the presence or expression of the transgenic HSV-TK gene.
GM-CSF: 1 x 10" B16 cells are washed in PBS and injected subcutaneously in a volume of 200 μl into the abdominal wall of syngenic C57B16 mice. The tumour has started to grow after 4 - 6 days. pMIA380hl2-bgh-GM-CSF is formulated with DOTAP and injected into the pre-formed B16 melanoma tumours or into muscle tissue. After 7 - 14 days the mice are sacrificed, dissected and histologically examined for the immunostimulatory effect of the GM-CSF gene (tumour size, macrophage infiltration) and for the presence of the plasmid DNA by means of PCR or for GM- CSF expression by means of RT-PCR. The mice which have received pMIA380hl2- bgh-GM-CSF/ DOTAP into the pre-formed B16 melanoma tumours are compared with mice which have received only the empty vector plasmid or an injection into muscle tissue.
Example 5
Influence of mutations in the region X on MIA expression
Mutations are introduced by site-directed mutagenesis (site directed mutagenesis kit, Clontech) in region X of the expression control sequence according to Table 1 in a reporter plasmid which contains the luciferase gene under the control of the MIA promoter fragment according to SEQ ID NO:l and expression in malignant melanoma cells (MM, B16) and non-melanoma cells (nonMM, HeLa) is examined. The result is shown in Table 1.
The reporter plasmid is prepared by inserting the MIA promoter fragment as well as the luciferase indicator gene via Hindlll/Bglll into the vector pGL3 basic (Promega GmbH, Mannheim, DE). Table 1
Influence of mutations in region X on the MIA expression
Figure imgf000014_0001
List of References
Beck, C, et al., Human Gene Therapy 6 (1995) 1525-1530
Bogdahn et al., Cancer Res. 49 (1989) 5358-5363
Bosserhoff et al., Proc. Am. Association for Cancer Research Annual Meeting 39 ( 1980) p. 250, XP002087909, Abstract 1711
Bosserhoff et al., Proc. Am. Association for Cancer Research Annual Meeting 37 (1996) p. 512, Abstract 3565
Bosserhoff, A.K., et al., J.Biol.Chem. 271 (1996) 490-495
CA Patent No. 2,112,130 Dranoff, G., et al., Proc.Natl.Acad.Sci. USA 90 ( 1993) 3539-3543
EP-B 0 188 479
EP-B 0 202 300
Fidler, I.J., Cancer Research 35 ( 1975) 218-224
Hames, B.D., and Higgins, S.J., in Nucleic Acid Hybridisation - A Practical Approach, publisher Hames and Higgins (1985), IRL Press
Kondo, S., et al., 44tn Annual Meeting, Orthopaedic Research Society, March 16-19, 1998, New Orleans, Louisiana, p. 178-30
Kren, B.T., et al., Nature Medicine 4 ( 1998) 285-290
Lederer et al, J. Dermatol. Sci. 16 (1998) Suppl. 1 S48 Sambrook, J., et al., in "Expression of cloned genes in E. coli" in Molecular Cloning: A laboratory manual (1989), Cold Spring Harbor Laboratory Press, New York, USA
US Patent No. 5,565,350
US Patent No. 5,578,444 US Patent No. 5,716,760 Xie, W.F., 44tn Annual Meeting, Orthopaedic Research Society, March 16-19, 1998, New Orleans, Louisiana, pp. 207-35

Claims

Patent Claims
1. Tumour cell-specific expression vector containing a gene which codes for a transcription or translation product that is therapeutically active in tumour cells, wherein this gene is under the control of an expression control region with the sequence SEQ ID NO:l or a fragment thereof which comprises at least the bases bp -224 to -214 and/or -197 to -207 from SEQ ID NO:l.
2. Expression vector as claimed in claim 1, wherein the therapeutically active transcription or translation product causes tumour regression, tumour ablation or immunostimulation in tumour cells.
3. Expression vector as claimed in claim 1, wherein the therapeutically active transcription product is an antisense nucleic acid or a ribozyme which binds in vitro under stringent conditions to a nucleic acid of sequence SEQ ID NO:l.
4. Expression vector as claimed in claim 1 or 2, wherein the therapeutically active translation product is a prodrug-activating, an apoptosis-inducing, tumour-suppressing, immunostimulating, co-stimulatory or toxic polypeptide.
5. Process for the production of a tumour-specific expression vector as claimed in claims 1 to 4, wherein the therapeutically active gene is inserted in such a way that it is expressed under the control of the expression control region.
6. Use of an expression vector as claimed in claims 1 to 4 for the regression or ablation of primary tumours, residual tumours, metastases and minimal residual disease in vivo or ex vivo of tumour and leukaemia cells.
7. Process for the production of a pharmaceutical agent for the regression or ablation of primary tumours, residual tumours, metastases and minimal residual disease in vivo or ex vivo of tumour and leukaemia cells, wherein an expression vector as claimed in claims 1 to 4 is used as an essential component of the agent.
8. Pharmaceutical agent for the regression or ablation of primary tumours, residual tumours, metastases and minimal residual disease in vivo or ex vivo of tumour and leukaemia cells, wherein it contains an expression vector as claimed in claims 1 to 4 as an essential component.
9. Nucleic acid fragment with a length of 10 to 30 bases, wherein the fragment hybridizes under stringent conditions with the base sequence -224 to -197 from SEQ ID NO:l or with a nucleic acid which is complementary thereto.
10. Use of a nucleic acid fragment of sequence SEQ ID NO:l or a fragment thereof which comprises at least the bases bp -224 to -197 from SEQ ID NO:l in a method for the detection of a DNA-protein binding, wherein the nucleic acid fragment is covalently bound to the DNA and a binding partner of the nucleic acid fragment influences the DNA-protein binding.
11. Nucleic acid fragment which specifically binds under physiological conditions to a second nucleic acid fragment of sequence SEQ ID NO:l or a fragment thereof which comprises at least the bases bp -224 to -197 from
SEQ ID NO:l and forms a triple helix with the double-stranded second nucleic acid fragment in the region of the bases bp -224 to -197 from SEQ ID NO:l or contains one or several point mutations in the region of the bases bp -224 to -197 from SEQ ID NO:l.
12. Use of a first nucleic acid fragment as claimed in claim 11 for the inhibition of the MIA expression in tumour cells.
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