EP1355661A2 - Vaccin antitumoral - Google Patents

Vaccin antitumoral

Info

Publication number
EP1355661A2
EP1355661A2 EP02704624A EP02704624A EP1355661A2 EP 1355661 A2 EP1355661 A2 EP 1355661A2 EP 02704624 A EP02704624 A EP 02704624A EP 02704624 A EP02704624 A EP 02704624A EP 1355661 A2 EP1355661 A2 EP 1355661A2
Authority
EP
European Patent Office
Prior art keywords
cells
tumor
sequence
patients
vaccine
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP02704624A
Other languages
German (de)
English (en)
Inventor
Burghardt Wittig
Ingo Schmidt-Wolf
Tomislav Dorbic
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Mologen AG
Original Assignee
Mologen Forschungs- Entwicklungs- und Vertriebs GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Mologen Forschungs- Entwicklungs- und Vertriebs GmbH filed Critical Mologen Forschungs- Entwicklungs- und Vertriebs GmbH
Publication of EP1355661A2 publication Critical patent/EP1355661A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001136—Cytokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5152—Tumor cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5156—Animal cells expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55522—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants

Definitions

  • the invention relates to a vaccine for the therapeutic treatment of tumor diseases, and to a method for producing such a vaccine.
  • Tumor diseases are one of the main causes of death in the industrialized world.
  • the basic idea of the immunological approaches is to present the tumor as an aberrant cell to the immune system, and, based on the expectation that it is the natural task of the immune system to recognize and destroy such aberrant cells, to initiate these mechanisms also against the tumor cell population that is the reason for the disease ,
  • immunological cell system is under equilibrium between cell degradation and cell build-up under physiological conditions.
  • the proliferation, differentiation, function and regulation, the cell-cell contact and the The formation of immunological recognition structures is controlled by cytokines.
  • cytokines control the growth and differentiation of leukocytes, the initial phase of the immune response, and they stimulate and suppress effector functions of the immune system.
  • interleukin 1 a lymphocyte-activating factor
  • interleukin 2 a T cell growth factor
  • interleukin 3 a mast cell growth factor
  • interleukin 4 a B cell stimulating factor
  • interleukin 7 and other interleukins as well as the granulocyte / macrophage colony stimulating factor (GM-CSF)
  • GM-CSF granulocyte / macrophage colony stimulating factor
  • TNF Tumor Factor
  • US 5,589,466 discloses immunizing mammals by injecting DNA or RNA, the DNA or RNA molecules being transported in constructs. It is described that the constructs used are free of viral particles.
  • IL-2 expression levels can be determined.
  • the viral vectors used as the expression vector can be regarded as less advantageous. Due to the instability of the attenuated vaccine strain, a conversion back into a virulent strain cannot be ruled out.
  • the viral components themselves can have an immunogenic effect, which leads to a reduction in their effectiveness by the patient's immune system. These risks are widely used as ten leads. These risks are largely opposed to widespread use as a gene therapy vector.
  • the inventors of the present application were able to show that the transfer of expression plasmids for human interleukin 7 (IL-7) in tumor cells leads to an increased sensitivity to effector cells of the immune system, especially with autologous transfer (Finke et al., 1997, Cancer Gene Ther. 4: 260-268).
  • IL-7 human interleukin 7
  • Plasmids are used as expression vectors. This document shows a concept for treating tumor diseases by immunotherapy. Meaningful in vivo or in vitro data or clinical results that prove the effectiveness of the claimed vaccine are not shown. It should be noted that these plasmid-based vectors are not suitable for use in human gene therapy without reservation, since they carry, in addition to the therapeutic sequences, genetic functional units which they require for their replication. In addition, they have antibiotic resistance genes that are essential for their selection. So there is a permanent expression of therapeutically undesirable mammals - or bacterial proteins.
  • the object of the invention is to provide a vaccine as a medicament for the treatment of diseases related to cytokines, such as cancer, which can be used specifically and efficiently and in particular leads to the induction of tumor-specific immune responses.
  • a corresponding method for producing such a vaccine is also to be provided.
  • the present invention solves this technical problem by providing a vaccine for the treatment of patients with defined tumor diseases, consisting of tumor cells from a patient, these tumor cells previously ex-vivo with code for interleukin-7 (IL-7) and macrophage-activating factor GM-CSF - Rende expression constructs were transfected. Under transfection, the bringing nucleic acid sequences are understood by means of biological, chemical or physical methods, as a result of which there is a sustained or temporary expression of proteins encoded by these sequences.
  • IL-7 interleukin-7
  • GM-CSF - Rende expression constructs were transfected.
  • immunostimulatory nucleic acid sequences were used as adjuvants.
  • the CpG motifs of the ISS cause an increase in the activity of NK cells and macrophages and a strong stimulation of the cellular TH1 immune response.
  • Covalently closed ISSs with a length of 30 bp are preferably used, as described in WO 01/07055.
  • the constructs according to the invention are referred to below as d-SLIM (double stem-loop immunomodulating oligodeoxyribonucleotides).
  • nucleic acids which code for a macrophage activating factor and an interleukin in a cell in which these nucleic acids are expressed leads to effective and specific vaccine protection being able to be achieved.
  • Plasmids can be used as DNA expression constructs; according to the invention, minimalistic immunologically defined gene expression constructs are preferably used. These are linear, covalently closed expression cassettes which consist only of the CMV promoter, an intron, the corresponding gene sequence and a polyadenylation sequence. These covalently closed minimalistic DNA constructs are referred to below as MIDGE ® vectors (MIDGE ® : MINIMALISTIC jMMUNO-LOGICALLY DEFINED GENE EXPRESSION VECTORS); see. EP 0 941 318 B1.
  • the MIDGE constructs have the advantage that they can be used without structures that are not essential for the therapeutic effect, which ultimately avoids the disadvantages of gene transports of viral origin.
  • the invention thus relates to the combination of at least two expressible nucleic acids, selected from the group of nucleic acids which code for at least one interleukin and at least one macrophage activating factor?
  • the two nucleic acids can be introduced in one or more (preferably two) expression constructs.
  • a number of general terms are understood below as follows:
  • treatment means the prophylactic and / or therapeutic effect of a medicinal substance.
  • macrophage-activating factor and / or interleukin relates both to naturally occurring macrophage-activating factors and / or interleukins and to all modifications, mutants or derivatives of the macrophage-activating factors and / or interleukins, macropage-activating factors produced by recombination techniques and / or interleukins which contain amino acid modifications such as inversions, deletions, insertions, additions, etc., provided that at least some of the essential functions of the wild-type macrophage activating factors and / or interleukins are present.
  • macrophage activating factors and / or interleukins can also include unusual amino acids and / or modifications such as alkylation, oxidation, thiol modification, denaturation and oligomerization and the like.
  • macrophage activating factors and / or interleukins can in particular be proteins, peptides and / or fusion peptides which, in addition to other proteins, peptides or parts thereof, contain macrophage activating factors and / or interleukins in whole or in part.
  • the macrophage activating factors and / or interleukins are shortened forms of the naturally occurring macrophage activating factors and / or interleukins.
  • a tumor cell comprises at least one nucleic acid molecule which codes together for a macrophage activating factor (preferably GM-CSF) and an interleukin (preferably interleukin-7).
  • a macrophage activating factor preferably GM-CSF
  • an interleukin preferably interleukin-7
  • These tumor cells come from patients with a defined tumor disease, such as, for example, preferably renal carcinoma, malignant melanoma and / or colon carcinoma.
  • the DNA expression constructs which lead to expression in the cells are introduced into these cells by means of biological, chemical and / or physical transfection methods.
  • the transfected cells serve as vaccines and are applied to patients with the same clinical picture, to them themselves or the cells were previously taken from other patients with the same clinical picture.
  • the body of the cell extraction can be the body to be treated with the drug; however, the body of the cell extraction cannot be the body to be treated with the drug.
  • the cells to be treated can come from a single body or can be pooled from several bodies with the same clinical picture.
  • the vaccine can comprise at least one immunomodulating oligodeoxyribonucleotide.
  • the immunomodulating oligodeoxyribonucleotide comprises a circular strand of deoxyribonucleic acid with a partially complementary, antiparallel base sequence.
  • the immunomodulating oligodeoxyribonucleotide is dumbbell-shaped.
  • the immunostimulatory nucleic acid sequences are only a few bases long and do not act via the expression of proteins encoded on them.
  • Most known immunomodifying short oligodeoxyribonucleotide sequences contain an unmethylated cytosine guanosine motif.
  • double-stranded molecules with the relevant immunostimulatory sequence in the double-strand region exert a noteworthy stimulatory effect.
  • the short deoxyribonucleic acid molecules consist in particular of a ring-shaped, closed sequence of nucleoside residues.
  • Such ring-shaped closed deoxyribonucleic acid molecules can advantageously be obtained from open-chain deoxyribonucleic acid molecules which have a partially self-complementary sequence and which can form an intermediate stable hybrid with one another or with a second molecule.
  • the molecule can also advantageously be obtained by intramolecular ligation of a molecule which has at least two self-complementary regions which are separated by only one gap in the phosphate backbone. The molecules obtained in this way have no free 5 'or 3' ends and are therefore not accessible to exonucleases.
  • the immunomodulating oligodeoxyribonucleotide can preferably comprise a sequence with the base sequence N 1 N 2 CGN 3 N 4 , where N 1 N 2 is an element selected from the group comprising GT, GG, GA, AT or AA, N 3 N 4 is an element out- is selected from the group comprising CT or TT, and C is deoxycytosine, G deoxyguanosine, A deoxyadenosine and T deoxythymidine.
  • the sequence with the base sequence N 1 N 2 CGN 3 N 4 is preferably positioned in the single-stranded region of the oligodeoxyribonucleotide.
  • the oligodeoxyribonucleotide advantageously comprises 20 to 200 nucleotides.
  • An advantage of the invention is that the selected and used nucleic acid sequences (d-SLIM) additionally have an enormous immunostimulatory effect.
  • These immunostimulatory nucleic acids have their effect according to the invention, for example in the vaccines, in particular via a secondary stimulation of the release of co-stimulatory factors.
  • the transfection into a cell preferably takes place.
  • the cells are expediently an allogeneic cell, a heterotopic cell, a syngeneic cell, a xenogeneic cell, an autologous cell, a virus-infected cell, a degenerate cell, a transformed cell, an antibody-loaded cell and / or an undifferentiated cell.
  • the cells are preferably mammalian cells, especially human cells.
  • the nucleic acid molecule is a DNA, in particular a cDNA or a genomic DNA.
  • the nucleic acid molecule can also be advantageous for the nucleic acid molecule to be an RNA.
  • transfection for example transfection by means of ballistic transfer, polycation transfection, calcium phosphate precipitation, microinjection, protoplast fusion, liposome fusion, viral transfection systems, lipofection and / or Electroporation in vivo and / or in vitro.
  • the transfection takes place by means of ballistic transfer.
  • Biological transfection methods such as receptor-mediated gene transfer are further advantageous methods.
  • a DNA sequence such as a sequence of DNA sequences.
  • Expression construct that codes for at least one macrophage activatable factor and at least one interleukin, covalently linked to an oligopeptide, which is preferably the nuclear localization signal (NLS) from Simian Virus SV-40.
  • NLS nuclear localization signal
  • the age range was 46 to 69 years. Eight patients were male, two female. One patient suffered from colon cancer, four from malignant melanoma and five patients from renal cell carcinoma. Renal cell carcinomas have very poor healing prospects. The location of the metastases is also shown.
  • Table 2 shows the transfection efficiency. After in vitro transfection with expression constructs coding for IL-7 and GM-CSF, all cells secrete the cytokines IL-7 and GM-CSF.
  • Table 3 shows the cytokine profiles of the sera of the
  • IL-7 interferon-gamma
  • TGF-ß Transforming Growth Factor-ß
  • CD3 and CD 56 positive PBL The cytotoxicity of these cells increased significantly as a result of the treatment.
  • Table 5 shows the increase in the cytotoxic activity of the
  • ten patients with metastatic solid tumors were taken from tumor material, these were transfected outside the body with expression vectors coding for IL-7 and GM-CSF and these cells were fed back to the patient after cultivation ex vivo and after the addition of immunostimulatory sequences.
  • the patients received four subcutaneous injections on the 0th, 14th, 28th and 56th day.
  • Renal cell carcinoma diseases belong to the group of those with very poor healing prospects among neoplasias. The patients were followed for 84 days. Table 1 Characteristics of the patients
  • the vaccine according to the invention made from modified tumor cells produced by the method according to the invention shows surprising effects. It is advantageous that the vaccine according to the invention induces a tumor-specific immune response.
  • the serum level of IL-7 and interferon gamma of the patients after treatment was measurably increased (see Table 3).
  • the cytotoxicity of this cell population increased significantly during the treatment from 21.5% to 25.6% (calculated from mean values on the 84th day) in all patients after completion of the treatment.
  • the increase in PBLs that expressed CD 56 was comparable from 16.3% to 27.5% (calculated from mean values on the 84th day).
  • the significance was p 0.03 (see Table 4).
  • a partial response refers to the decline in detectable tumor areas by more than 50% in the past four weeks and the suppression of new tumor areas.
  • a stable disease course SD is to be understood as the steady state. All patients tolerated the treatment well and there were no undesirable side effects.
  • kidney cancer four of the successfully treated patients came from the group with the initial diagnosis of renal cell carcinoma.
  • the success of the treatment means that there was a minimal response in one patient, a stable disease course (SD) in two patients and even a complete response (CR) in one patient.
  • SD stable disease course
  • CR complete response
  • the patient with the complete reaction showed the following initial picture: metastases up to 3 cm in size in the lungs, a 4x4x3 cm size metastasis infiltrated the neighboring rib, metastases in ribs IV and VII and a large kidney tumor. After its removal, the kidney tumor was checked for confirmation of the diagnosis of renal cell carcinoma. Part of the tumor was used for vaccine production. After four vaccinations with transfected cells, a decrease in metastases in the lungs and bones was observed. At the following initial picture: metastases up to 3 cm in size in the lungs, a 4x4x3 cm size metastasis infiltrated the neighboring rib, metastases in ribs IV and VII and a large kidney tumor. After its removal, the kidney tumor was checked for confirmation of the diagnosis of renal cell carcinoma. Part of the tumor was used for vaccine production. After four vaccinations with transfected cells, a decrease in metastases in the lungs and bones was observed. At the following initial picture: metastases up to 3 cm in size in the
  • Treatment In addition to the medical history, the following parameters were determined before the start of treatment: physical examination, hematology (hemoglobin, hematocrit, leukocytes and platelet count), chemical blood values and urine analysis. Blood was taken for immune status determination. DHT (Delayed type hypersensitivity) skin tests were carried out with the Multitest Merieux Test (Leimen, Germany). X-rays of the upper body and computed tomography of the upper body and abdomen were taken. The patients received four subcutaneous injections with at least 1x10 6 cells of their previously ex-vivo treated tumor cells. Comprehensive immunological examinations, haematological examinations and rough clinical examinations (physical examination, ultrasound examination and examination of the abdomen, if necessary) were repeated on the 14th, 28th and 56th day. A complete clinical and immunological examination, comparable to the selection examination carried out at the beginning, took place on the 84th day. Preparation of the tumor cell suspension
  • the tumor cell suspension was treated as described for primary cell cultures (Finke, et al., 1997, Cancer Gene Ther. 4: 260-268). Tumor samples taken sterile from the patients were immediately transferred to ice-cooled suspension medium in 50 ml Falcon centrifuge tubes with screw caps. The transport of the cooled samples to the laboratory, in which the ex-vivo treatment of the patient cells took place, took between 1-3 hours.
  • Suspension medium for renal carcinoma cells undiluted L-15 medium (BioWhittaker), 10% FCS (BioWhittaker), 1x MEM vitamins (Biochrom), 1x insulin transferrin-Selenium-X (Gibco), 1mM L-glutamine (BioWhittaker), 1g / l NaHCO3 (Gibco), 1g / l glucose for DMEM (Gibco), 50 ⁇ g / ⁇ l gentamicin (BioWhittaker).
  • Suspension medium for malignant melanoma cells undiluted RPMI medium 1640 (with ultraglutamine, BioWhittaker), 10% FCS (BioWhittaker), 50 ⁇ g / ⁇ l gentamicin (BioWhittaker)
  • MIDGE Two covalently closed minimalistic DNA expression vectors, called "MIDGE" were used.
  • One expression vector was used for the expression of human interleukin-7 (IL-7), the other for the expression of the human granulocyte macrophage colony stimulating factor (GM-CSF).
  • IL-7 human interleukin-7
  • GM-CSF human granulocyte macrophage colony stimulating factor
  • the MIDGE vectors were synthesized according to the SOP regulation and in the class B corresponding laboratory with subsequent quality control.
  • the coding gene sequence for IL-7 and GM-CSF was amplified from human white blood cell mRNA using reverse transcriptase-PCR (RT-PCR).
  • RT-PCR reverse transcriptase-PCR
  • the amplificates were inserted into the plasmid pMOK, a pUC19 derivative, between the SstI and Kpnl interfaces.
  • PMOK has an intron and a polyadenylation sequence from the SV40 virus (MOLOGEN, Berlin, Germany).
  • the plasmid pMOK was completely digested with the restriction enzyme Eco31 I overnight at 37 ° C. Restriction digestion generated two DNA fragments. One consisted of the kanamycin resistance gene and other sequences necessary for the propagation of the plasmid in bacteria. The other fragment consisted of the sequences that were to become part of the MIDGE DNA: enhanced CMV promoter, chimeric intron, the corresponding gene sequence and the polyadenylation sequence from SV-40.
  • the 5 'phosphorylated hairpin-shaped oligodeoxynucleotides (TIB-MolBiol, Berlin) became 5' -PH-GGG AGT CCA GTT TTC TGG AC-3 'and 5' PH-AGG GGT CCA GTT TTC TGG AC-3 using the enzyme T4-DNA ligase 'Ligated in the presence of the restriction enzyme Eco31 I overnight at 37 D C to the DNA fragment forming the MIDGE DNA.
  • the reaction was stopped by heating to 70 ° C.
  • the resulting mixture of nucleic acids was treated with the enzyme T7 DNA polymerase.
  • the MIDGE DNA was purified by anion exchange chromatography and precipitated with isopropanol (cf. EP 0 941 318 B1).
  • Double-stranded immunolatory oligodeoxynucleotides (d-SLIM)
  • Double-stranded immunomodulators d-SLIMs of the ISS30 type were produced after (SOP) and final quality control in the class B laboratory.
  • ODN Single-stranded, hairpin-shaped, 5'-phosphorylated oligodeoxyribonucleotides
  • a modified device for cell acceleration the Biolistic PDS-1000 / He (Bio-Rad, Hercules, CA, USA), was used for the ex-vivo gene transfer into the cell nucleus of the tumor cells and the control cells.
  • the modification relates to a device for pressure distribution (cf. EP 0732 395 B1). More than 1.5 Fix10 7 cells can be treated per shot.
  • the ballistic method is combined with the magnetic separation of the transfected cells. Although the magnetic separation was not carried out, the magnetic particles were nevertheless also introduced into the cells, since they increase the binding capacity of the gold particles for DNA. So far, this method of ballistic DNA transfer could only be used with adherently growing cells.
  • 1-2x10 7 cells In order to ensure the DNA transfer into the tumor cells, they were plated on a 44 mm 2 polycarbonate membrane (Transwell TM, pore size 3.0 ⁇ m, from Costar). 1-2x10 7 cells, depending on their size, can be loaded onto such a membrane, compared to 1-2x10 6 for adherent cell cultures in culture vessels with comparable surface contents. Cell recovery and viability are significantly improved, and the treatment time is greatly reduced. Standard conditions, depending on the morphological and functional differences of the tumor line types, were developed.
  • the tumor cell suspensions were adjusted to a concentration of 5x10 6 cells / ml in the cell suspension media.
  • a Transwell membrane was washed with 15 ml medium from both sides for 5 min. equilibrated. The medium layer above the membrane was then removed and 2-4 ml of the tumor cell suspension was applied uniformly to the polycarbonate membrane. The cells colonized the membrane, while the suspension medium was able to drain through the membrane pores into deeper membrane layers. Excess medium in the deeper layers was removed through an opening until no liquid residues were visible over the monolayer layer.
  • the medium below the membrane is a very important technical support during the transfection process and keeps the cells moist and viable.
  • the membranes coated with the particles were attached at a distance of 15 mm from the stop screen.
  • the monolayer layer of the tumor cells on the Transwell membrane was placed approximately 90 mm below the stop screen.
  • the ballistic transfer accelerated the gold particles towards the tumor cells with a pressure of 1550 psi.
  • the tumor cells were covered with 10 ml of prewarmed cell suspension medium and transferred into 15 ml centrifuge tubes by careful pipetting. After sedimentation of the cells at 300xg and 4 ° C for 5 min, they were dissolved in medium. Aliquots were again retained for sterility tests, cell recovery and viability tests, for FACS analyzes, and for determining cytokine expression. The transfection efficiency was determined as described (Foa et al., 1994, Nat. Immun. 13: 65-75)).
  • the transfected tumor cell suspension was dried again, taken up in 1.5 ml freezing medium (80% FCS, 10% DMSO, 10% suspension medium) and stored in portions of 5x10 6 to 2x10 7 cells in sealed 2 ml freezer tubes. The tubes were stored at -80 ° C at 1 ° C / min in liquid nitrogen.
  • a tube was removed from the liquid nitrogen and thawed in a water bath with gentle shaking at 37 ° C. Then it was briefly disinfected from the outside with 70% ethanol. The thawed cell suspension was slowly and dropwise transferred into 50 ml PBS (without Ca 2+ and Mg 2+ ) at 4 ° C, and centrifuged at 300xg, at 4 ° C for 5 min. The pellet was carefully taken up in 50 ml of PBS, dried again, dissolved in 1 ml of PBS and transferred to 2 ml vessels (Biopure TM, Eppendorf).
  • the contents were drawn onto a 1 ml syringe, sealed with a screw cap, irradiated 3 times with gamma radiation for 667 sec, which corresponded to a total dose of 105 gray. Then the syringe was transported to the clinic in a sterile, double-walled and shockproof container at 4 ° C.
  • Peripheral blood lymphocytes were treated with various monoclonal antibodies to human surface antigens.
  • the antibodies were AK against human CD3, CD4, CD8, CD16, CD11a, CD14, CD19, CD25, CD28, CD45, CD54, CD56 and HLA-DR (Immunotech Hamburg, Germany).
  • AKs from the same isotype were used as controls.
  • the labeled cells were washed and then analyzed with the FACScan (Becton Dickinson, San Jose, CA). Irrelevant AKs were used to determine the background for this study, it was less than 2%. 10 4 cells of each sample were analyzed.
  • CytoTox 96 a non-radioactive, cytotoxic test (Promega, Madison, Wisconsin) was used to determine and compare the cytotoxic activity of the peripheral blood lymphocytes. These examinations were carried out on days 0, 14, 28, 56 and 84.
  • the test is a calorimetric alternative to the 51 chromium release test.
  • the quantitative measure is lactate dehydrogenase (LDH), which is released by cells like 51 Cr in cell lysis.
  • the free LDH is determined in the supernatant after a 30 minute incubation using a coupled enzymatic test.
  • the amount of dye released is proportional to the amount of lysed cells. The absorption was measured at 490 nm.
  • 5000 target cells were applied in triplicate in V-shaped 96-well tissue culture plates and incubated for four hours with different ratios of effector and target cells. After the incubation, 50 ⁇ l aliquots were transferred to a new 96 well plate. 50 ⁇ l of the substrate mix was added to each chamber and subsequently incubated in the dark for 30 min at RT. 50 ⁇ l stop solution were added before the determination. Autologous tumor cells were targeted used. Each experiment was carried out in triplicate and an average was formed. The lytic unit (LU) was determined by titration curves. An LU is defined as the number of effector cells that are required to achieve a 10% lysis of 10 4 target cells.
  • the proliferation test "Easy for you” (Biomedica, Vienna, Austria) was used to determine the proliferation of the peripheral blood lymphocytes (PBL), which were taken from the patients before, during and after the treatment.
  • PBL peripheral blood lymphocytes
  • PBL peripheral blood lymphocytes
  • ELISA reader a chromophoric substrate solution with the aid of a spectrophotometer (ELISA reader).
  • the incubation with the substrate solution was carried out for four hours, depending on the metabolic performance of the cells. Converted derivative, the absorption maximum is at 490 nm.
  • the experiments were carried out in triplicate and averaged.
  • Cytokine levels in the patient's sera from day 0, 14, 28, 56 and 84 were determined by an enzyme test (ELISA).
  • the IL-7 ELISA kit purchased from Laboserv (Staufenberg, Germany); the R&D Systems IFN-gamma, TNF-alpha and TGF-beta1 ELISA kit (Quantikine, Minneapolis, Minnesota); and the GM-CSF-ELISA from Promocell (Heidelberg, Germany). Soluble IL-7, IFN-gamma, TNF-alpha, TGF-beta1 and GM-CSF in concentrations of 9, 3, 4.4, 7, 1.5 pg / ml to 1000, 500, 500, 1000, 500 pg / ml could be used be determined.
  • the microtiter plates were coated with a monoclonal AK, which bound the cytokines after incubation in the serum. After several washing steps to
  • DTH delayed type hypersensitivity
  • PBMC Peripheral blood monocytes
  • a HATF multititer plate (Millipore, Eschborn, Germany) was dissolved with 500ng anti-lFN-gamma antibody (AK) (Biozol, Eching, Germany) in coating solution (3.8g / l NaHCO 3, 1.9g / l Na 2 CO 3 ), coated at 4 ° C overnight. After washing three times, the plate was blocked with 200 ⁇ l blocking solution (5% BSA in phosphate buffer) at 37 ° C. for 30 min. After washing, 10 5 peripheral blood lymphocytes were applied per well and incubated overnight (at 37 ° C, 5%
  • AK anti-lFN-gamma antibody
  • the plate was washed and incubated with 500ng of a second biotin-labeled IFN-gamma-AK (Biozol) overnight at 4 ° C.
  • Avidin and BCIP / NBT solution (Sigma, Deisenhofen, Germany) were used for the detection.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • Chemical & Material Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Mycology (AREA)
  • Oncology (AREA)
  • Epidemiology (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

L'invention concerne un vaccin pour traiter des patients souffrant de maladies tumorales définies, ainsi que la production de celui-ci. Ce vaccin comprend des cellules tumorales qui ont été transfectées ex vivo avec des constructions d'expression codant pour certaines cytokines. Des oligodésoxyribonucléotides immunostimulants sont utilisés comme adjuvants.
EP02704624A 2001-01-31 2002-01-30 Vaccin antitumoral Withdrawn EP1355661A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10105421 2001-01-31
DE10105421 2001-01-31
PCT/DE2002/000380 WO2002060476A2 (fr) 2001-01-31 2002-01-30 Vaccin antitumoral

Publications (1)

Publication Number Publication Date
EP1355661A2 true EP1355661A2 (fr) 2003-10-29

Family

ID=7673082

Family Applications (1)

Application Number Title Priority Date Filing Date
EP02704624A Withdrawn EP1355661A2 (fr) 2001-01-31 2002-01-30 Vaccin antitumoral

Country Status (3)

Country Link
EP (1) EP1355661A2 (fr)
DE (1) DE10290185D2 (fr)
WO (1) WO2002060476A2 (fr)

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103173460A (zh) * 2003-12-30 2013-06-26 莫洛根股份公司 同种异基因肿瘤治疗剂
EP1716234B1 (fr) * 2004-02-20 2013-10-02 Mologen AG Molecule d'acide nucleique substituee et non codante pour la stimulation immunitaire therapeutique et prophylactique chez l'homme et les animaux superieurs
WO2006015560A1 (fr) * 2004-08-09 2006-02-16 Mologen Ag Agent immunomodulateur associe a des mesures de chimiotherapie
EA017860B1 (ru) * 2006-05-11 2013-03-29 Мологен Аг Мультимер для иммуностимуляции
LU92821B1 (en) 2015-09-09 2017-03-20 Mologen Ag Combination comprising immunostimulatory oligonucleotides
GB2542425A (en) 2015-09-21 2017-03-22 Mologen Ag Means for the treatment of HIV

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994004196A1 (fr) * 1992-08-14 1994-03-03 Imperial Cancer Research Technology Limited Therapie de tumeurs
US6207646B1 (en) * 1994-07-15 2001-03-27 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US6051428A (en) * 1997-03-21 2000-04-18 Sloan-Kettering Institute For Cancer Research Rapid production of autologous tumor vaccines
AU5458500A (en) * 1999-06-02 2000-12-18 Cell Genesys, Inc. Regulation of systemic immune responses utilizing cytokines and antigens
DE19935756A1 (de) * 1999-07-27 2001-02-08 Mologen Forschungs Entwicklung Kovalent geschlossenes Nukleinsäuremolekül zur Immunstimulation

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO02060476A2 *

Also Published As

Publication number Publication date
WO2002060476A2 (fr) 2002-08-08
DE10290185D2 (de) 2004-04-15
WO2002060476A3 (fr) 2003-01-23

Similar Documents

Publication Publication Date Title
EP1615662B1 (fr) Transfection de globules sanguins avec un arnm pour l'immunostimulation et la therapie genique
DE69434708T2 (de) Antitumor-Gentherapie bei Immuno-und/oder Entzündungsmodulation
US7635468B2 (en) Allogeneic tumor therapeutic agent, a vaccine using allogeneic tumor cells for the therapeutic treatment of tumor diseases, and a method for the making of such a vaccine, and transfected human tumor cells for use as a vaccine
DE69916581T2 (de) Eine universelle immunmodulatorische cytokin-expressierende zelllinie in wartestelling. entsprechende zusammensetzungen und methoden ihrer herstellung und verwendungen
DE69836643T2 (de) Zusammensetzungen für die zufuhr von genen an antigen-präsentierende zellen der haut
DE60038503T2 (de) AKTIVIERUNG DES IMMUNSYSTEMS DURCH EINEN ANTIGEN UND EINEN NF-kappaB AUSLÖSER
EP1311658B1 (fr) Procede de fabrication de cellules dendritiques matures, cryoconservees, chargees d'antigenes ou non, pretes a l'emploi
DE69433921T2 (de) Rekombinante zellen der monozyten-makrophagen zelllinie zur gen-therapie
DE69735643T2 (de) IL12 zur Gentherapie der Tumoren
EP2220214B1 (fr) Procédé d'augmentation de la réactivité immunitaire
EP2658566B1 (fr) Sirna contre la cbl-b combine avec des cytokines et des interferones dans le traitement du cancer
DE69731756T2 (de) Melanomzellinien, welche immundominante melanomantigene teilen und verfahren zu deren anwendung
DE60209915T2 (de) Prozess zur herstellung reifer dendritischer zellen und eines impfstoffes
WO2006015560A1 (fr) Agent immunomodulateur associe a des mesures de chimiotherapie
DE69627527T2 (de) Allogene parakrine cytokine tumor impfstoffe
DE60117167T2 (de) Verfahren zur herstellung von submikropartikel-suspensionen pharmazeutischer substanzen
EP1355661A2 (fr) Vaccin antitumoral
DE10139428A1 (de) Herstellung und Verwendung von humanen CD124 und CD116 positiven Tumorzelllinien zur Herstellung von allogenen oder semi-allogenen Immuntherapeutika
EP2633034B1 (fr) CELLULES DENDRITIQUES À VOIE DE SIGNALISATION NFkB MANIPULÉE
AT412145B (de) Verfahren zur herstellung eines zellulären immuntherapeutikums auf basis von il-12-freisetzenden dendritischen zellen
DE60130206T2 (de) Zusammensetzungen und verfahren zur herstellung von antigen-präsentierenden zellen
WO2003045428A2 (fr) Utilisation d'une cellule techniquement modifiee en tant que vaccin pour traiter une tumeur
DE60223232T2 (de) Mucinpeptid mit immunstärkenden eigenschaften
DE69731950T2 (de) Tumorvakzinierung mittels Verwendung autologer oder HLA-verwandter Antigen präsentierender Zellen (APC), die mit einem Tumorantigen sowie einem eine Immunantwort hervorrufenden Fremdantigen transduziert sind
DE102018108996B4 (de) Verfahren zur Herstellung autologer T-Zellen

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20030804

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE TR

AX Request for extension of the european patent

Extension state: AL LT LV MK RO SI

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: MOLOGEN AG

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: MOLOGEN AG

17Q First examination report despatched

Effective date: 20040825

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20050105