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  • 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
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  • C12N2710/00011—Details
  • C12N2710/10011—Adenoviridae
  • C12N2710/10041—Use of virus, viral particle or viral elements as a vector
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  • C12N2840/00—Vectors comprising a special translation-regulating system
  • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
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  • 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
  • C12N2840/206—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES having multiple IRES

Definitions

• the present invention relates to viral vectors which comprise nucleic acid sequences which code for single-chain interleukin-12 (single-chain TIL-12, "single chain IL-12” or scIL-12) and a Kosti ulator protein, and the use of these vectors for gene therapy, in particular for the therapy of tumors.
• HCC hepatocellular carcinoma
• PKI percutaneous ethanol injection
• nucleic acid used for gene therapy contains a gene whose gene product inhibits the growth of the tumor or induces apoptosis in the tumor cells. Most clinical trials are based on transmission of the p53 gene.
• nucleic acid used for gene therapy comprises sequences whose gene products activate the patient's immune system and trigger an immune reaction directed against the tumor cells.
• the immune response itself then leads to the destruction of the tumor.
• Numerous cytokines, Kosti ulator molecules and tumor-specific molecules have been proposed for immunotherapy.
• the nucleic acid used for gene therapy codes for a gene product, for example for an enzyme, which converts a non-toxic active ingredient into an agent cytotoxic for the tumor cell.
• Nucleic acid vectors based on viral sequences are used for this form of gene therapy.
• Vectors with oncolytic, viral sequences have a tumor-specific promoter which controls the replication of the virus, so that a selective growth of the viruses in the tumor lines is made possible.
• nucleotides are Acids administered that include sequences that activate the immune system and target the tumor.
• the immune system also recognizes tumor-specific structures on tumor cells. The activation of the immune system can therefore lead to the destruction of the tumor by the components of the immune system.
• cytokines Numerous molecules are known in the prior art that stimulate the immune system or modulate an immune response, in particular the cytokines. It was discovered early on that cytokines also have anti-tumor activities. For example, IL-12 has been reported to be a stimulator of cellular immunity and has strong anti-tumor activity (Brunda et al., J. Exp. Med. 1993, 178: 1223-1230). However, administration of the recombinant IL-12 protein itself as an anti-tumor agent failed because the cytokine has toxic side effects in therapeutic doses (Lotze et al., Ann.NYAcad.Sci., 1997, 795: 440-454; and Cohen J. Science, 1995, 270: 908).
• IL-12 which is also known as CMLF ("cytotoxic ly phocyte maturation factor") or NKSF ' ("natural killer cell stimulatory factor ") is a heterodimeric cytokine that is naturally formed by peripheral B-lymphocytes after activation.
• the protein consists of two subunits with relative molecular weights of 40 and 35 kDa, which are linked together via disulfide bridges The disulfide bridges are essential for the biological activity. As already indicated by the different names, the protein stimulates the proliferation of activated human lymphoblasts and activates natural killer cells.
• Vectors encoding the various subunits of this protein have been used to treat tumors (Barajas et al., Hepatology, 2001, 33: 52-61; Mazzolini et al., Cancer Gene Therapy, 1999, 6: 514-522 ). Furthermore, these vectors were used in combination with other sequences for immunotherapy, in particular in combination with sequences for a stimulator protein, which were present on the same or a different vector, for the treatment of tumors (Gyorffy et al., J.
• IL-12 has also already been expressed as single-chain IL-12 with good activity, that is to say as a protein in which the various subunits have been linked to form a fusion protein (Lieschke et al., Nature Biotechnology, 1997, 15: 35-40).
• a plasmid which codes for single-chain IL-12 or IL-12 and a costimulator (US2002 / 0018767). After this in vitro treatment, the tumor cells are to be reimplanted.
• the method thus comprises several interventions on the patient and a reimplantation of tumor cells in the patient, which should prevent many patients from appropriate treatment.
• nucleic acids used so far has been able to establish itself for the treatment of mammals, preferably for the treatment of humans.
• the Ruiz et al. described treatment method from a very high dosage of the vector used for the therapy (3xl0 9 -2, 5xl0 13 "plaque forming units", PFU, per dosage)
• PFU plaque forming units
• the dosage of the nucleic acids is j 'edoch for gene therapy is a critical factor, since at too high a dosage, or a release side effects of the vector are to be expected by the leakage of the vector from the tumor.
• the present invention was therefore based on the object of providing vectors which can be used for immunotherapy with improved effectiveness.
• viral vectors which comprise nucleic acid sequences which code for single-chain IL-12 and a costimulator protein, the vector being a viral vector.
• vectors which code for single-chain IL-12 and a costimulator protein are particularly suitable for the treatment of tumors in the context of immunotherapy.
• Immunotherapy not only eliminates the primary tumor but also the metastases.
• the synergistic effect of the proteins encoded by the constructs according to the invention in immunostimulation enables the use of the nucleic acids in lower doses than was suggested in the prior art.
• the lower dosage has fewer side effects, such as a lower risk of an autoimmune disease for the patient, and at the same time improved safety in clinical use.
• the probability of spreading the Virus is less than the known vectors.
• the lower dosage also makes it possible to treat larger tumor masses or multiple tumor foci at the same time without producing side effects.
• the use of the single chain IL-12 gene saves space in the vector for expression of the nucleic acids compared to the use of genes encoding the two subunits of the IL-12.
• This sequence thus enables the use of relatively small vectors, such as the adenoviral vectors, in which two or more foreign genes which enhance immunotherapy are nevertheless present in addition to the gene for single-chain IL-12.
• the vectors according to the invention are intended for the treatment of mammals, in particular for the treatment of humans. Accordingly, the following references to specific gene sequences preferably refer to human sequences. However, the gene sequences can also originate from other species or can be modified within the scope of the specified homology ranges (which are determined for the present application using the BLAST software) using methods known in the art, provided the activity of the proteins (immunostimulating and / or T - cell binding) remains in the range of at least 50%, preferably at least 70% of the corresponding activity of the human gene product.
• the ranges of homology given refer to the range which codes for the biological activity in the native gene. If nucleic acid sequences are used which code for fusion proteins, the homology range thus refers to the part which codes for the biological activity mentioned (IL-12, costimulator). Changes in the gene sequence which lead to an increase in the activity of the proteins are included.
• sequences coding for a Kosti ulator protein is used to designate sequences which, when expressed in human cells, produce proteins which are present as cell surface proteins and are specifically bound by receptors of the T cells.
• costimulator proteins increase the immune response.
• the vector comprises sequences which code for the costimulator protein 4-1BB ligand, in particular sequences with a sequence homology of at least 40%, preferably at least 70%, at least 80% or at least 90%, to that in Fig. 21 shown sequence, wherein the protein encoded by the sequence has the ability to specifically bind T cells.
• Further activity tests for 4-1BBL are known in the prior art (Vinay DS, Kwon BS. Semin. Immunol., 1998, 10: 481-9. Review; Kwon et al., Mol Cells., 2000, 10: 119-26 ).
• a protein is referred to as single chain IL-12 if the protein consists of an amino acid sequence which comprises the two subunits of the natural IL-12 as a fusion protein.
• Nucleic acid sequences encoding a single chain IL-12 will usually have a sequence homology of at least 40%, preferably at least 70%, at least 80% or at least 90% of the sequences shown in FIGS. 19 and 20.
• the sequences shown represent the IL-12 portion of the fusion gene. Sequences which link the subunits are known to the person skilled in the art and are not taken into account when determining the homology.
• the single-chain IL-12 also has an immunostimulating activity which is not significantly worse than the corresponding activity of the natural IL-12 in heterodimeric form.
• One of the effects of human IL-12 in stimulating the immune system is to initiate the release of gamma interferon.
• the immune stimulating activity of the single chain IL-12 is at least 50%, preferably at least 70% of the corresponding activity of the natural IL-12.
• the activity of the proteins can be compared by known in vitro test methods (for example using the in vitro tests to compare the activity of IL-12 in Lieschke et al., Loc. Cit.).
• the immunostimulating activity of the single chain IL-12 is even higher or significantly higher than the corresponding activity of the natural IL-12.
• the present invention further comprises vectors which code for further cytokines, for proteins with cytokine activity and / or for costimulator proteins.
• Proteins with cytokine activity are proteins that have the immunostimulatory activity of a cytokine, but have no structural relationship to the cytokines.
• Corresponding cytokine agonists for example agonistic cytokine receptor antibodies, are known in the prior art.
• the vectors according to the invention can thus have sequences which for one or more further cytokines which activate T and / or B cells, or encode one or more additional costimulator proteins.
• the invention relates to vectors comprising sequences encoding single chain IL-12, 4-1BB ligand and IL-2.
• a protein is referred to as IL-2 if it is encoded by a sequence which has a sequence homology of at least 40%, preferably of at least 70%, at least 80% or at least 90%, to that in FIG. 22 sequence shown.
• the sequences coding for IL-2 used in the context of the present invention also essentially have the immunostimulating activity of natural IL-2, ie within the scope of the present invention, sequences are used which code for IL-2 and which have an immunostimulatory activity in vitro which corresponds to at least about 70% of the activity of natural IL-2. Corresponding in vitro test methods for determining the IL-2 activity are known in the prior art. It is preferred that in Gillis et al. (J Immunol., 1978, 120 (6): 2027-32) uses the methods described for the activity determination.
• the vectors according to the invention comprise, in addition to the sequences which code for single-chain IL-12, 4-1BB ligand and IL-2, furthermore sequences which are for the costimulator protein B7-1 and / or B7-2 encode.
• a costimulator protein is referred to as B7-1 (or B7-2) if it is encoded by a gene which has a sequence homology of at least 40%, preferably at least 70%, at least 80% or at least 90% to the sequence shown in Fig. 23 A (or B).
• the vectors further comprise sequences which enable expression of the coding sequences.
• the vectors according to the invention can thus comprise a promoter and one or more internal ribosome entry points (IRES).
• the promoters can have tumor specificity, that is to say they can only be expressed in the tumor, or they cannot be active in all cells.
• non-specific promoters are preferred, since corresponding promoters are generally better expressed and these vectors can be used to treat various tumors.
• the cistrons When using tetracistronic vectors, it may be advantageous to divide the cistrons over several expression cassettes (see examples). In this case there is preferably one promoter per expression cassette. By dividing into two expression cassettes, which are preferably at a maximum distance from one another in the vector, the promoters are spatially separated and the mutual inhibition is thus reduced.
• sequences according to the invention have the particular advantage that the proteins are expressed particularly well in human cells. According to this embodiment, the advantageous effect of the sequences according to the invention for immunotherapy is therefore also due to the high expression of the coding sequences.
• the vectors preferably consist of DNA or RNA.
• a vector is referred to as a "viral vector” if it is a nucleic acid sequence which comprises sequences of viral origin which allow the packaging of the nucleic acid in virus envelopes.
• the vectors can be used as adenoviral vectors, adeno-associated vectors, lentiviral vectors, HSV vectors, retroviral vectors, bacu loviral vectors or Semliki Forrest virus vectors are present.
• the adenoviral vectors can be adenoviral vectors of the first (deletions in regions El and E3 of the AdEasy cloning system; for example available from QBiogene GmbH, Heidelberg) or second generation (deletions i El, E2, E3, E4, etc .) or helper-dependent adenoviral vectors.
• Corresponding vectors are comprehensively known in the prior art (Nicklin SA, Baker AH. Curr Gene Ther., 2002, 2: 273-93; Mah et al., Clin Pharmacokinet., 2002, 41: 901-11).
• the invention relates to an adenoviral vector which comprises sequences which code for single-chain IL-12, 4-1BB ligand and IL-2.
• Adenoviral vectors have the particular advantage that there are corresponding vectors which are approved for use in human gene therapy. The vectors are therefore safe for certain applications (e.g. tumor treatment with local administration).
• Adenoviral vectors are among the vector systems that are most commonly used in the clinic and for which most data on the safety of use are available.
• adenoviral vectors are also made available for the first time, express the 4 genes, which in the examples were divided into two expression cassettes.
• the present invention further relates to virus particles which comprise the vectors according to the invention.
• virus particles As virus particles or. Virions are called nucleic acids that are surrounded by the envelope proteins of a virus.
• the present invention relates to medicaments which comprise the vectors or virus particles according to the invention.
• the vectors or virus particles according to the invention can simply be mixed with a carrier or administered together with other auxiliaries.
• the vectors or virus particles according to the invention can be introduced, for example, into liposomes or liposomes with replication-competent adenoviruses (RCAs; see Yoon et al., Curr Cancer Drug Targets, 2001, 1: 85-107), as polyethylene glycol-coated adenoviruses, as antibody-bridged adenoviruses (that is, as viruses that are bound to an antibody which has specificity for the virus and a cell marker, preferably a tumor cell marker), mixed with RCAs, as a cassette in an RCA or as for reproduction in Tumor conditional RCA is present (conditional RCA: RCA with the El function under the regulation of a tumor-specific promoter; van der Poel et al., J Urol., 2002, 168
• the exact dosage of the virus particles depends on the disease to be treated, the type of application and the structure of the vector used and can be determined in individual cases by a person skilled in the art using standard methods.
• the nucleic acids according to the invention enable the tumor to be destroyed or significantly reduced even with a particularly low dosage.
• the medicament preferably has a concentration per dosage unit of not more than 1 ⁇ 10 , preferably not more than 10 ⁇ 10 , no more than 10 ⁇ 9 or no more than 10 ⁇ 7 . However, the dosage can also be well below that mentioned ranges are and do not even exceed lxl0 ⁇ .
• the dosage information relates to the number of infectious virus particles.
• the drug is formulated so that the vectors can be delivered to the tumor well.
• the medicament is preferably in the form of a solution for intratumoral injection.
• the production of corresponding solutions is known in the prior art.
• the medicament can be formulated as a carrier material which releases the vector over a certain period of time after implantation in the tumor.
• Corresponding carrier materials such as cellulose sulfate or the like, are known in the prior art.
• the present invention relates to the use of the vectors or virus particles for the treatment of tumors, in particular for the treatment of solid tumors such as HCC, colon cancer, breast cancer, etc. in humans.
• the present invention relates to the use of the vectors or virus particles for the treatment of infectious diseases or prion diseases.
• Immunostimulating therapy also in the form of gene therapy, has already been proposed for the treatment of corresponding infectious diseases (cf. van der Meide et al ., Vaccine., 2002, 20: 2296-302).
• the immunostimulatory effect of the vectors and virus particles according to the invention thus also have therapeutic potential for the treatment of infectious diseases, such as, for example, for the treatment of infections by the human immunodeficiency virus (HIV), by hepatitis viruses type A, B, C (HAV, HBV) , HCV), by cytomegaloviruses (CMV) and by human papillomaviruses HPV, which can lead to cervical cancer, among other things.
• HIV human immunodeficiency virus
• HCV hepatitis viruses type A, B, C (HAV, HBV) , HCV)
• CMV cytomegaloviruses
• HPV human papillomaviruses
• the vector is present in a concentration of not more than 1x10, preferably not more than lxlO 10 , no more than lxlO 9 or no more than lxlO 7 .
• the dosage can also be significantly below the ranges mentioned and may even not exceed lxlO 5 .
• the dosage information here again relates to the number of infectious virus particles.
• the tricistronic expression cassette thus constructed was cloned into the pShuttle-CMV plasmid of the AdEasy system (QBiogene GmbH, Heidelberg) without the promoter and the 3'-untranslated sequences.
• the result is the plasmid pShuttle [CMV] scIL12 [IRES] 4-1BBL [IRES] IL2 (FIG. 2).
• the cassette [CMV] scIL12 [IRES] 4-1BBL [IRES] IL2 from the latter plasmid was completely checked for correctness by DNA sequencing.
• this construct was then recombined to the plasmid pAd-3 (see FIG.), which contains the entire recombinant DNA for Ad-3.
• adenoviruses were transfected from the plasmid precursors after release by means of Pacl digestion into HEK293 cells and the resulting viral plaques (ie as virus particles) were isolated and multiplied. Overview of the expression cassettes: Fig. 1.
• Ad-1 and Ad-2 served as controls here since no IL-2 expression can be expected (not shown).
• the vector Ad-3 (Fig. 5 a) was tested in our rat model for hepatocellular carcinoma (HCC).
• HCC hepatocellular carcinoma
• McA-RH7777 hepatocellular carcinoma of the rat
• Buffalo Rat is used to subcapsul ren rule hepatic transplantation of tumors.
• the tumor growth after implantation of 1 million cells is carried out by means of magnetic resonance imaging (MRI) in collaboration with Prof. Dr. Gerrit Krupski-Berdien from the radiological clinic of the UKE monitored before and after injection of the virus particles.
• 5 b shows the course of the tumor volumes between the 3rd and 12th day after virus injection.
• the very clear effect of the vector in this short period of time is dose-dependent and, in the highest dose, the tumor volumes are reduced to 27% of the size when the virus is injected.
• FIG. 6 with selected MRT images of individual animals in order to better illustrate the tumor size in relation to the size of the animal.
• the dose for long-term treatment of larger groups of animals was set at 5 x 10 7 infectious particles per tumor.
• the effect of the individual vectors (Ad-1 to Ad-3) was determined at this low dose.
• tumors were implanted, 6 animals in the A groups were then treated with the vector after 14 days and the parameters of the antitumor immune response were analyzed after a further 2 weeks.
• the B groups (long-term group) consist of 10 animals each.
• two tumors were set, only one of which was treated with the virus to determine the distal effects of immune stimulation.
• the B groups were used to analyze the long-term kinetics of tumor reduction and survival.
• IL-12 was detected in the serum of the rats.
• Interferon gamma was also determined, which is released by immune cells after IL-12 stimulation and is responsible for a large part of the antitumor effects. Interferon gamma was also clearly detectable.
• T-cells that are specifically directed against the tumor were xicity test demonstrated.
• the immune cell response was currently also characterized on tissue preparations from the treated animals. In treated tumor tissue, CD8 + cells, CD4 + cells, macrophages and natural killer cells were detected in an increased amount compared to tissue treated with control vector.
• an expression cassette for the B7-1 is inserted into the position of the adenoviral E3 region.
• This region is functionally inactive, since large portions of this region have been deleted in the vectors used here.
• the expression cassette has its own promoter of human phosphoglycerate kinase or a promoter of similar 'strength.
• Fig. 2 shows the time course of expression over 3 days in the rat hepatoma cells MCA-RH7777. Procedure: MCA-RH7777 cells were infected with Ad-1, Ad-2 or Ad-3 in MOIs of 10. The supernatants were collected on days 0, 1, 2 and 3 after infection. scIL-12 concentrations were determined by ELISA with an anti-mouse IL-12p70 antibody (Pharmingen).
• Fig. 3 Detection of 4-1BBL in the McA-RH7777 cell cultures. Flow cytometric determination of 4-1BBL expression. Ad-2 and Ad-3 express 4-1BBL, Ad-1 does not express.
• McA-RH7777 cells were infected with the matched virus concentrations at MOI 10 with Ad-1, Ad-2 or Ad-3. The cells were harvested 24 h after infection and incubated with a rat anti-mouse 4- 1BBL monoclonal antibody (TKS-1, Pharmingen) and stained for detection with R-PE-conjugated goat anti-rat Ig polyclonal antibody (Pharmingen).
• TKS-1 rat anti-mouse 4- 1BBL monoclonal antibody
• R-PE-conjugated goat anti-rat Ig polyclonal antibody Pharmingen
• Fig. 4 Expression of IL-2 in the MCA-RH7777 cell cultures over 3 days.
• Ad-3 expresses molar 466 times more IL-12 than IL-2 (calculated for day 3).
• McA-RH7777 cells were infected with Ad-3 at MOI 10. Supernatants were collected on day 0, 1, 2, 3. IL-2 concentrations were determined by ELISA using an anti-mouse IL-2 antibody (Pharmingen).
• Fig. 5 dose escalation study Change in tumor size within 9 days after treatment with Ad3.
• the tumor volumes were measured by means of MRI in an interval of 9 days.
• the reference value of 100% relates to the size of the tumor on day 3 after the virus injection (1st MRI), the final size shown here was measured on day 12 after administration of the virus (2nd MRI).
• Fig. 6 MRI images of the dose escalation study.
• Fig. 7 Schematic representation of the sequence of animal experiments using Ad-1, Ad-2 and Ad-3.
• 11 shows the course of the tumor sizes, calculated from the MRI data.
• Fig. 12 Long-term survival rate of the test animals up to 100 days after virus injection.
• Fig. 13 Map of the vector pTrident3.
• FIG. 14 Map of the vector pShuttle [CMV] IL12 [IRES] 4- 1BBL [IRE-S] IL-2.
• FIG. 15 Map of the vector pShuttle [CMV] IL12 [IRES] 4-1BBL.
• FIG. 16 Map of the vector pShuttle [CMV] IL12.
• Fig. 17 Map of the vector pAd-3.
• FIG. 18 sequence of the tricistronic expression cassette which contains the murine cDNAs corresponds to insert Ad-3 from FIG. 1.
• Figure 19 Coding sequence of human IL-12 40 kDa.
• Figure 20 Coding sequence of human IL-12 35 kDa.
• Fig. 21 Coding sequence of the human 4-1BBL.
• FIG. 29 shows the effects on tumor growth after, treatment with Ad-1 and Ad-3 in comparison to a control vector (Ad-GFP).
• n 3
• the figure shows the change in tumor volumes at different vector doses of Ad-1 and Ad-3.
• 1 x 10 s MH-7777A tumor cells were applied in the right lobe of the liver and two weeks later the Vek- injected into the left tumor.
• MRI scans were done one day before and 13 days after vector application.
• 5 x 10 iu Ad-1 the mean tumor volume increases significantly, whereas with 1 x 10 7 iu Ad-1 it increases only minimally. This only minimal increase can be determined for Ad-3 at a dose of 5 x 10 ⁇ iu, whereas a decrease is already evident at 1 x 10 7 iu.
• Ad-3 much more effective than Ad-1.
• the values for 1 x 10 7 iu Ad-3 were determined on day 3 and day 12 after vector injection.
• the controls (Ad-GFP) increase massively within the observation period of 2 weeks. Extrahepatic metastases were not taken into account in the animals in which they occurred.

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