EP1441758A2 - Vaccin allogenique comportant une cellule tumorale qui exprime un polypeptide co-stimulant - Google Patents

Vaccin allogenique comportant une cellule tumorale qui exprime un polypeptide co-stimulant

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Publication number
EP1441758A2
EP1441758A2 EP02787620A EP02787620A EP1441758A2 EP 1441758 A2 EP1441758 A2 EP 1441758A2 EP 02787620 A EP02787620 A EP 02787620A EP 02787620 A EP02787620 A EP 02787620A EP 1441758 A2 EP1441758 A2 EP 1441758A2
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EP
European Patent Office
Prior art keywords
cells
tumor
use according
tumor cell
cell
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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.)
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EP02787620A
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German (de)
English (en)
Inventor
John Nieland
Claudia Breidenstein
Ute Sartorius
Ulrich Moebius
Christoph Bogedain
Adelheid Dinkel
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Medigene AG
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Medigene AG
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • 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
    • 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
    • A61K2039/55516—Proteins; Peptides
    • 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
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • 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
    • A61K2039/80—Vaccine for a specifically defined cancer

Definitions

  • the present invention relates to the use of genetically modified tumor cells for the production of vaccines.
  • the autologous vaccine cells from the patient's own tumor are used to produce the vaccine.
  • the tumor cells are removed from the body, genetically modified if necessary, and made proliferation incompetent, for example by radiation, before they are re-administered to the patient.
  • the aim is that immune cells, in particular cytotoxic T cells and helper T cells, recognize the administered cells and thus build up an immune response that can then also be directed against the tumor.
  • autologous cell vaccines also have a number of strong disadvantages. Often, especially with smaller neoplasms, it is very difficult or almost impossible to cultivate the tumor cells. In addition, a vaccine must be made individually for each patient. It is therefore very difficult to standardize the production of autologous vaccines, which can be a considerable disadvantage for the approval of such a vaccine. Further The production of an autologous vaccine means a long waiting time for the patient, since after the tumor material has been removed, the cells must first be processed and manipulated before they can be administered to the patient again. In the meantime there is a risk that (further) metastases have formed in the patient's body.
  • allogeneic immunization i.e. immunization with non-patient cells.
  • the vaccine cells differ from the patient's own cells, since they usually do not have the identical transplantation antigens (MHC genes).
  • MHC complex on the surface of cells is of particular importance for the development of the specific immune response, since peptides are presented in the MHC complex, which are then recognized by T cells specific for these peptides.
  • MHC complexes There are two classes of MHC complexes - Class I and Class II.
  • a T cell When a specific immune response is formed, a T cell recognizes the MHC complex through its T cell receptor and is thereby stimulated to form an immune response.
  • the binding of the T cell receptor to the MHC complex is usually not sufficient for the development of a specific immune response. Rather, further so-called costimulatory molecules are required which increase the signal exchange between the T cell and the MHC-carrying cell.
  • the MHC complexes of class I are of particular importance for triggering an immune response against tumor cells, since tumor cells in their MHC I complexes present peptides that (almost) exclusively occur on tumor cells, so-called tumor antigens or peptides derived from them. It is known in the prior art that the detection of peptides by tumor antigen are derived from MHC class I molecules and cause certain T cells to proliferate cytotoxic T cells, which in turn can kill tumor cells. (Janeway C. et al. (1999) in: Immunobiology; Current Biology Publications, pages 551-554)
  • HLA A There are three genes in humans that code for three different MHC class I molecules, HLA A, HLA B and HLA C. Each of these genes is highly polymorphic, i.e. for each of the genes there are a number of different alleles in the population that lead to different MHC molecules. In the Caucasian population, for example, there are 95 different HLA A, 207 HLA B and 50 HLA C alleles according to current knowledge. Some of the alleles are very common, such as the allele HLA A2, which occurs in approximately 50% of the population, while others are very rare.
  • a person's HLA type can be determined by two different methods. On the one hand, antibodies are available which specifically recognize certain MHC proteins which are encoded by HLA alleles and which are thus used for a specific staining of cells in a subject. On the other hand, there are specific oligonucleotide primers for the various alleles, which are used in PCR reactions to determine the HLA type of a test person. (Welsh K and Bunce M (1999) Rev Immunogenet 1 (2): 157-76; Parham P (1992) Eur J Immunogenet 19 (5): 347-59)
  • T cell recognizes only one type of MHC complex, usually the body's own MHC complex. This is due to the fact that in the context of the positive selection of T cells in the thymus, the production site of the T cells, only those T cells that recognize the body's own MHC complexes survive. However, there are also alloreactive T cells in the body that recognize foreign MHC complexes, for example on organs transplanted on cells.
  • one (or more) established tumor cell line is usually used to vaccinate the patient (see WO 97/24132).
  • the object is achieved by using a tumor cell to produce a vaccine for the treatment or prevention of a tumor in a patient, characterized in that the tumor cell expresses a stimulatory polypeptide and that the tumor cell and the patient do not match their MHC molecules exhibit.
  • the term “allogeneic” in the context of the present invention means that two individuals (or one individual and the cell used for vaccination) are different with respect to their antigens. This usually, but not necessarily, means that they their HLA antigens are different, expressly including the fact that the two individuals partially match in their HLA genes, or complete or no match of the HLA genes is included, in the former case there is at least one other antigen between the Individuals (or the cell and the patient) differ.
  • no match between their MHC / HLA molecules means that two individuals (or one individual and the cell used for vaccination) have no alleles in common with regard to their MHC-I complexes.
  • the costimulatory polypeptide is selected from the group consisting of B7.1, B7.2, CD40, Light, Ox40, 4.1.BB, Icos L, SLAM, ICAM 1, LFA-3, B7.3, CD70, HSA, CD84, CD7, B7 RP-1 L, MAdCAM-1, VCAM-1, CS-1, CD82, CD30, CD120a, CD120b and TNFR-RP, CD40L.
  • the costimulatory polypeptide is selected from the group consisting of B7.1 and B7.2. According to a very particularly preferred embodiment, the costimulatory polypeptide is B7.2. According to a preferred embodiment of the use according to the invention, the patient has at least one tumor or is to be protected against a tumor which is of the same type as that from which the tumor cell is derived. Methods for determining the type of tumor are known from pathological textbooks.
  • tumor antigens which are presented in the MHC complexes of the tumor cell used for vaccination.
  • the immune response can also be based on the recognition of other molecules, e.g. tissue-specific differentiation antigens or glycoproteins / peptides.
  • the tumor cell used according to the invention is of a different type than the tumor which is to be treated in the patient or is to be prevented from occurring.
  • the immune cells recognize the proteins / peptides on the tumor cell surface, which are also present on the surface of the tumors. These can be bound to MHC molecules or not bound.
  • the tumor cell is derived from a primary tumor or a metastasis.
  • the tumor cell preferably originates from a tumor which is selected from the group consisting of melanoma, breast cancer, colon cancer, ovarian cancer, lymphoma, leukemia, prostate cancer, lung cancer, bronchial cancer or pancreatic cancer.
  • the tumor cell used according to the invention expresses at least one tumor antigen which is characteristic of the respective tumor, for example a cellular or viral tumor antigen.
  • This tumor antigen is preferably recognized by the immune system, which leads to activation of the immune system and then to treatment or prevention of the patient.
  • the tumor antigen is selected from the group consisting of MART, Her2neu, tyrosinase, tyrosinase-related proteins (TRP), MARTl / MelanA, Ny-ESO-1, CEAl, CEA2, CEA3, ⁇ -feto protein, MAGE X2, BAGE, GAGE1, GAGE2, GAGE3, GAGE4, GAGE5, GAGE6, GAGE7, GAGE7a, GAGE8, MAGE A4, MAGE A5, MAGE A8, MAGE A9, MAGE A10, MAGE AI 1, MAGE A12, MAGE1, MAGE2, MAGE3, MAGE3b, MAGE4a, MAGE4b, MAGE5, MAGE5a, MAGE5b, MAGE6, MAGE7, MAGE8, MAGE9, PAGE1, PAGE4, CAMEL, PRAME, LAGE1, g lOO, Her2neu, ras, p53, E6, E7 as well as SV40 large and small T-ant
  • the tumor cell is derived from a melanoma and the tumor antigen is selected from the group consisting of tyrosinase, MARTl / MelanA, Ny-ESO-1, MAGE3 and gplOO.
  • the tumor cell expresses at least one cytokine and / or one chemokine, preferably selected from the group consisting of GM-CSF, G-CSF, IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, ILl l, IL12, IL13, IL14, IL15, IL16, IL17, IL18, IL19, IL20, IL21, IL22, IFN ⁇ , IFNß, IFN ⁇ , Flt3 L, Flt3, TNF, RANTES, MlPl ⁇ , MlPlß, MlPl ⁇ , MlPl ⁇ , MIP2, MIP2, MIP2ß, MIP3 ⁇ , MIP3ß, MIP4, MIP5, MCP1, MCPlß, MCP2, MCP3, MCP4, MCP5, MCP6, 6cykine, Dcckl and DCDF.
  • cytokine and chemokine preferably
  • the tumor cell used according to the invention can also express a fusion protein from the abovementioned polypeptides. It is furthermore included according to the invention that the tumor cell expresses functional variants of the above-mentioned polypeptides, functional variants being characterized in that they have essentially the same biological activity as the mentioned polypeptides. In the prior art, tests are known for the respective polypeptides, how these can be detected or their respective activity can be measured.
  • the tumor cell used according to the invention expresses B7.2 and GM-CSF.
  • B7.2 and GM-CSF expresses B7.2 and GM-CSF.
  • melanoma cells that have been genetically modified to express the two polypeptides B7.2 and GMCSF are more effective than tumor cells that only express GMCSF even in the context of allogeneic vaccination without agreement of the MHC molecules.
  • mice were injected intravenously with vital, unchanged tumor cells to provoke the formation of lung metastases.
  • the animals were then vaccinated twice with irradiated tumor cells which either expressed unchanged or expressed GMCSF, or B7.2 and GMCSF.
  • GMCSF GMCSF
  • B7.2 and GMCSF irradiated tumor cells which either expressed unchanged or expressed GMCSF, or B7.2 and GMCSF.
  • antigen-specific T cells takes place according to the general state of knowledge via two signaling pathways: On the one hand the antigen fragment loaded on the body's own MHC is presented to the T cell receptor, on the other hand there is a receptor-ligand binding between the B7.2 on the Antigen-presenting cell and CD28 are held on the T cell. Both signals are required to activate the T cell (two-signal model, see e.g. Bretscher P (1992) Immunol Today 1992 Feb; 13 (2): 74-6).
  • NK cells natural killer cells
  • CD28 i.e. the receptor for B7.2
  • NK cells are also activated by B7.2 interaction. However, it is generally believed that this interaction only increases the cytokine pro- production leads (see Nandi D supra, Amakata Y supra, Marin-Fontecha A supra and Yeh KY supra), but not to an increased lytic activity. This increase in the lytic activity of NK cells was surprisingly demonstrated in the context of the present invention. In the case of an allogeneic vaccination without agreement of the MHC molecules, this can lead to increased lysis of the vaccine cells and thus to the release of antigens, which can subsequently be more effectively taken up by the patient's antigen-presenting cells and presented to T cells, as well for the already observed release of various cytokines that stimulate T cells and antigen-presenting cells.
  • the tumor cell used according to the invention contains one or more vectors which cause the expression of one or more of the polypeptides defined above.
  • the vector can comprise control sequences which bring about the expression of the polypeptide starting from the endogenous gene of the polypeptide.
  • control sequences which bring about the expression of the polypeptide starting from the endogenous gene of the polypeptide.
  • the vector comprises nucleic acid sequences which code for the above-mentioned polypeptides.
  • the respective nucleic acid sequences are known in the prior art and can be found, for example, in the literature references listed above. The state of the art basically knows how to construct a vector so that a polypeptide can be expressed. (Sambrook J et al. (1989) Molecular Cloning: A Laboratory Manual, 2 ⁇ d ed., Cold Spring Harbor: Cold Spring Harbor Laboratory).
  • the vector is of non-viral origin, for example common expression plasmids, or viral origin, preferably derived from AAV, HSV, retrovirus, lentivirus, adenovirus, SV40.
  • the vector is episomal or integrated into the genome of the cell.
  • the vector derives from AAV.
  • AAV vectors and vectors derived therefrom are known in the prior art (see, for example, WO 00/47757, WO 02/20748).
  • the AAV vector contained in the tumor cell used according to the invention is present as a concatemer in the AAV-S1 acceptor site.
  • expression of the polypeptide is carried out by a constitutive, for example the CMN promoter (Vincent et al (1990) Vaccine 90, 353, the SV40 promoter (Samulski et al (1989) J Virol 63, 3822) or the LTR Promoter of retroviruses (Lipkowski et al (1988) Mol Cell Biol 8, 3988), an inducible, for example the tet promoter, and / or a tissue-specific, for example the elongation factor promoter, the Ig promoter or the IL2 / ⁇ FAT Promoter controlled.
  • a constitutive for example the CMN promoter (Vincent et al (1990) Vaccine 90, 353, the SV40 promoter (Samulski et al (1989) J Virol 63, 3822) or the LTR Promoter of retroviruses (Lipkowski et al (1988) Mol Cell Biol 8, 3988), an inducible, for example the
  • the promoters can control the expression by either controlling the expression of the polypeptide starting from the endogenous genes or by coding the expression of the polypeptides starting from the nucleic acid sequences included in the vector.
  • a variety Known number of promoters that can be used according to the invention are known. (Sambrook J supra).
  • the tumor cell used according to the invention is incapable of proliferation, for example by radiation or chemical inactivation.
  • proliferation incompetent means that the tumor cell is no longer able to proliferate.
  • tumor cells become incompetent for proliferation by garnma radiation with 25-100 Gy (see, for example, WO 97/32988).
  • chemical inactivation for example, the addition of 40 ⁇ g / ml mitomycin can be used.
  • an adjuvant is defined as a compound which can intensify the triggering of an immune reaction.
  • the drug also contains an adjuvant.
  • the medicament thus contains an adjuvant, preferably those adjuvants which act as toll-like receptor agonists. These are e.g. CpG oligonucleotides. These are oligonucleotides that contain at least one CpG motif (see e.g. Wagner H (2001) Immunity 14, 499-502).
  • the adjuvant is derived from Bacillus Calmette-Guerin cell wall structure (BCG-CWS).
  • BCG-CWS is known to be a ligand of toll-like receptors 2 and 4 and to differentiate Im- can trigger mun cells (Matsumoto M et al (2001) Int Immunopharmacol 1, 8, 1559-69).
  • the adjuvant is a superantigen.
  • Superantigens are antigens that bind directly to T cell receptors and MHC molecules and cause direct activation of the T cells.
  • Superantigens are known to have an adjuvant effect (see e.g. Okamoto S et al (2001) Infect. Immun. 69, 11, 6633-42).
  • Known superantigens are e.g. Staphylococcus aureus Enterotoxins A, B, C, D and E (SEA, SEB, SEC, SED, SEE), Staphylococcal aureus toxic shock syndrome toxin 1 (TSST-1), Staphylococcal exfoliating toxin or Streptococcal pyrogenic exotoxins.
  • the adjuvant is an agent that inhibits the signaling effect of CTLA-4.
  • the medicament contains suitable additives and / or binders.
  • the additive or binder preferably comprises 0.3 to approx. 4 M, preferably 0.4 to approx. 3 M, in particular approx. 0.5 to 2 M, very particularly preferably approx. 1 to approx. 2 M of a salt a pH of approx. 7.3-7.45, in particular 7.4.
  • the salt is preferably an alkali or an alkaline earth metal salt, in particular a halogenite or a phosphate, in particular an alkali metal halite, very particularly preferably NaCl or KCl.
  • the pH is adjusted by means of a buffer, for example by means of a phosphate buffer, a Tris buffer, a HEPES buffer or a MOPS buffer.
  • the tumor cells are administered in amounts of preferably at least 1 ⁇ 10 5 , preferably 1 ⁇ 0 6 , in particular 1 ⁇ 0 7 cell per dose. These amounts apply to both prophylactic and therapeutic vaccination.
  • the cells are preferably administered at least twice, particularly preferably at least three times at intervals of at least 2 weeks, preferably 4 weeks, in particular 8 weeks.
  • Administration is usually subcutaneous, intracutaneous or intranodal.
  • the tumor cell is derived from an individual of the same species as the patient.
  • the patient is a mammal, preferably a human.
  • the vaccine produced in the context of the use according to the invention activates the lytic activity of NK cells.
  • the invention further relates to the use of a tumor cell expressing a costimulatory polypeptide for the production of a vaccine for increasing the lytic activity of NK cells in the treatment or prevention of a tumor in a patient allogeneic to the tumor cell.
  • the term "allogeneic" in the context of the present invention means that two individuals (or one individual and those for Cell used) are different with respect to their antigens. This usually, but not necessarily, means that they are different in their HLA antigens. It is expressly included that the two individuals partially agree in their HLA genes. Full or no match of the HLA genes is also included. In the former case, at least one other antigen is then different between the individuals (or the cell and the patient).
  • the same embodiments apply to the costimulatory polypeptide, the tumor cell, the tumor, the vaccine and the patient as described above in the context of the other use according to the invention. According to the invention, it is also included that the patient and the tumor cell partially agree in their HLA antigens.
  • the invention further relates to a method for treatment or prevention in a patient, in which the patient is administered a therapeutically effective amount of a tumor cell expressing a costimulatory polypeptide, the tumor cell and the patient having no match in their MHC complexes.
  • the invention further relates to a method for treatment or prevention in a patient, in which the patient is administered a therapeutically effective amount of a tumor cell expressing a costimulatory polypeptide, which activates the lytic activity of NK cells, whereby the tumor cell is used Patient is allogeneic.
  • the invention opens up completely new perspectives for the treatment of tumor patients.
  • IFN- ⁇ test describes the graphical evaluation of an intracellular IFN- ⁇ test.
  • the IFN ⁇ release measured by immunostaining was determined from HLA-A2 positive donor T cells which had been stimulated with Mel29 tumor cells with HLA agreement (top) or with Mel 62 tumor cells without HLA agreement (bottom). In both cases, B7.2 transduced and untransduced tumor cells were compared.
  • NK cells effector cells
  • target cells melanoma cells
  • FIG. 5 describes the graphical evaluation of an experiment in which untransduced melanoma cells or transduced by means of rAAV with B7.2 or with B7.2 and GM-CSF were incubated with PBLs for 5 days and their proliferation on the basis of the incorporation of H- Tdr was measured.
  • FIG. 6 describes the graphic evaluation of a mouse vaccination experiment with pre-implanted tumors, in which autologous and allogeneic vaccination strategies are compared with one another.
  • Cell lines used for vaccination and transgenes expressed by them are plotted on the X axis; the relative tumor load was evaluated in%.
  • Mel 29 Melanoma cell line derived from melanoma patient, HLA-A2.01 positive, untransduced or B7.2 / GMCSF transduced;
  • Mel 62 Melanoma cell line derived from melanoma patient, HLA-A2.01 negative, untransduced or B7.2 / GMCSF transduced;
  • TAP-deficient lymphoblastoid cells TAP-deficient lymphoblastoid cells; these were loaded with a complex of HLA-A2.01 restricted melanoma peptides;
  • PBLs Peripheral Blood Lymphocytes
  • Melanoma cells are sown at a density of 3.8x10 4 cells in 1 ml medium (DMEM with 10% FCS, 2 mM L-glutamine, 1 x antibiotic-antimycotic, 1 x MEM vitamins; Gibco-BRL) in three wells of a 24-well Plate. The next day, the cells are irradiated with 100 Gy and infected with 20 ⁇ L rAAVB7.2 / GM-CSF virus (corresponds to MOI 84). After incubation for 48 h at 37 ° C., 5% CO 2 , the culture supernatant is aspirated from the transduced melanoma cells and 2.5x10 6 PBMC from an HLA-A2 positive donor are added.
  • DMEM 1 ml medium
  • FCS 1 mM L-glutamine
  • 1 x antibiotic-antimycotic 1 x MEM vitamins
  • Gibco-BRL Gibco-BRL
  • the PBMC are restimulated with peptide-loaded PBMC.
  • 1.5 ⁇ 10 7 PBMC from the same donor are mixed with 10 ⁇ g / ⁇ l MART mut peptide with the sequence ELAGIGILTV and incubated for 4 h at 37 ° C., 5% CO 2 .
  • the cells are then diluted with T-cell medium with 10% human serum and 0.4 U / mL IL-2 (Boehringer Ingelheim) to the final concentration of the peptides of 0.5 ⁇ g / ml and cells of 3-4x10 5 / ml , From the primary stimulation approach, 1 ml of culture supernatant is aspirated and 3 ⁇ 4 ⁇ 10 5 peptide-loaded PBMC in 1 ml are added.
  • T2 cells T2 cells (TAP-deficient B-cell lymphoma) are loaded with peptide as described above, then irradiated with 100 Gy and 1 ⁇ 10 5 cells are added to the stimulation approach. The restimulation takes place without IL-2.
  • An intracellular IFN- ⁇ staining is performed the day after the third restimulation.
  • the cells are cultured further and the assay is repeated a week later.
  • T2 cells In order to stimulate the production of IFN- ⁇ in the T cells, they are incubated with peptide-loaded antigen-presenting cells. T2 cells are adjusted to a concentration of lxl 0 6 cells / ml, and 10 ug / ml Mart mu t- peptide or HIV peptide. 50 ⁇ l of this are sown per well in a 96-well round-bottom plate and incubated overnight at 37 ° C. and 5% CO 2 .
  • PBMC restimulated PBMC
  • assay medium RPMI 1640, Gibco BRL, Cat # 21875-034; 1 mM Na pyruvate; 2 mM L-glutamine; 1 x MEM non-essential amino acids; 50 ⁇ g / ml gentamicin ; freshly mixed with 10% human serum; 0.8 U / ml IL-2) resuspended and adjusted to lx 10 7 / ml. 50 ⁇ l of this cell suspension are added to the T2 cells loaded with peptide. To improve the interaction of the cells, the plate is centrifuged for 1 min at 1200 rpm, 4 ° C. After incubation for 1 h at 37 ° C.
  • the cells are stained.
  • 20 ⁇ l of the following antibodies, diluted 1/50 in 1 ⁇ perm / wash solution, are added: anti-IFN- ⁇ -FITC (Caltag, Cat # MHCIFG01), anti-CD8-PE (Becton Dickinson, Cat # 30325X) and anti-CD4-Cychrome (Becton Dickinson, Cat # 30158X), the plate vortexed briefly and incubated for 30 min on ice.
  • the cells are washed 2x with 100 ⁇ l perm / wash solution each, the cell pellets resuspended in 150 ⁇ l PBS / 0.5% BSA and transferred to Micronic tubes. The samples are then measured in the FACS.
  • IFN- ⁇ staining was found in T cells which had originally been stimulated with Mel62-B7.2 / GM-CSF, while stimulation with untransduced Mel62 had no effect. IFN- ⁇ production was peptide-specific because T2 cells loaded with HIV peptide were ineffective. The result was confirmed a week later.
  • the antigenic signal comes from tumor antigens that are endogenously expressed by the melanoma cell and are presented by an MHC molecule that is common to both the stimulator cell and the T cell (HLA-A2 agreement) .
  • HLA-A2 agreement the activation of a T cell is dependent on the presentation of the melanoma antigens by induced antigen-presenting cells (APCs), such as, for example, dendritic cells (“cross-priming”).
  • APCs induced antigen-presenting cells
  • Test components B16F10-HEL-wt cells (H-2b) transfected with B7.2 and / or GM-CSF pAAV plasmid K-1735-HEL (H-2k), transduced with rAAV-B7.2 / GM-CSF
  • the expression vector pcDNA3neo-HEL was cloned for the production of stable transfectants of the melanoma cell lines B16F10 (Prof. Judith J. Fidler, MD Anderson Cancer Center, Texas, USA) and K-1735 (Dr. Souberbielle, King's College, London).
  • the HEL gene was cut out of the vector pcDNA1-HEL (Shastri, University of CA, Berkeley, CA, USA) and ligated into the expression vector pcDNA3neo (Invitrogen, Carlsbad CA, USA), which contains a neomycin resistance gene, which makes the selection more positive Serves clones.
  • the transfection of B16F10 and K-1735 cells was carried out using Lipofectamine® (# 11668, Invitrogen, Carlsbad CA, USA) on 15 cm culture dishes. Positive cells were selected using medium containing G418 (800 ⁇ g / ml). After 2-3 weeks, individual clones were picked and expanded. The clones were tested for expression of the transgene using RT-PCR and Western blot examined. The two clones with the best expression rate were selected for vaccination experiments.
  • RNA preparation was prepared from 2 - 5 x 10 6 cells performed with QIAshredder column (# 79654, Qiagen ®, Hilden) and the RNeasy Kit (# 74104, Qiagen ®, Hilden).
  • DNA eg episomal plasmid DNA
  • RNAse-free DNAse # 776785, Röche ® , Basel
  • the amplified HEL fragment had a length of 430 bp
  • the amplified fragment of murine ⁇ -actin had a length of 290 bp.
  • Antibodies biotinylated anti HEL used in a dilution of 1: 200 (RDI, # RDI-Lyszym-BT, Flanders, NJ). Streptavidin-HRP: used in a dilution of 1: 5,000 (Sigma ® , # S-5512, Deisenhofen)
  • K1735-HEL cells expressing murine B7.2, GM-CSF or both molecules were generated by transduction with recombinant adeno-associated virus (AAV).
  • AAV adeno-associated virus
  • the plasmids pAAV-muGMCSF and pAAV-muB7.2 were cloned: The cDNA of GM-CSF and B7.2 were cloned into the vector pCI (Promega, Madison, WI, USA), which contains one CMV promoter and one SV40 3'- untranslated region.
  • plasmid pAAV-GM-CSF two expression cassettes - which contain GM-CSF with the CMV promoter and the SV40-pA site - were ligated in tandem into the plasmid pAAV base vector (see WO 00/47757, Example 4 ).
  • an additional 400 bp from pUC19 (bp 1516-1910) were also integrated into the vector.
  • the expression cassette - with B7.2, the CMV promoter and the S V40-pA site - was ligated into the plasmid pAAV base vector.
  • An additional 700 bp from pUC19 (bp 1201-1910) were required to generate the optimal size of the vector.
  • Heia T cells were transfected simultaneously with 2 plasmids by means of calcium phosphate coprecipitation: with the vector plasmid pAAV-muGM-CSF or pAAV-muB7.2 and the AAV helper plasmid pUC "rep / cap” (RBS) D37, which contains the AAV genes for 'rep' and 'cap' of AAV2 wears (see WO 00/47757).
  • K-1735-HEL cells were irradiated (10OGy) and infected with the AAV.
  • GM-CSF expression 200-300 ng / 10 6 cells in 48 h
  • approximately 700 ⁇ l of the virus rAAV-muGMCSF for 5 ⁇ 10 6 cells were normally used.
  • B7.2 expression 70% - 96%)
  • approx. 4 ml of the virus rAAV-muB7.2 was necessary for 5 x 10 6 cells.
  • the cells were harvested, frozen (FCS, 10% DMSO) and stored in liquid nitrogen.
  • mice To prepare cells for application to mice, these were thawed in a 37 ° C. water bath, washed three times in PBS and adjusted to the correct number of cells (3 ⁇ 10 5 cells per dose in PBS).
  • B16F10 cells cannot be efficiently transduced with rAAN. Therefore, the transfection with the Liposome Polyfect (QIAgen ® 'Hilden) was carried out in order to generate vaccine cells for allogeneic vaccination experiments.
  • the melanoma cell line B16F10-HEL was transfected individually or in combination with the vectors pAAV-muGMCSF and pAAV-muB7.2. The cells were seeded in cell culture flasks (1.66 x 10 per T75) and transfected the following day according to the manufacturer's instructions.
  • the expression of the transgenes was measured for GM-CSF using ELISA and for B7.2 using flow cytometry.
  • mice To prepare cells for injection into mice, these were thawed in a 37 ° C. water bath, washed three times in PBS and adjusted to the correct cell number (3 ⁇ 10 5 cells per dose in PBS).
  • GM-CSF Secreted GM-CSF was determined 48 hours after sowing in the supernatant of transduced or transfected cells.
  • mice were sacrificed by cervical dislocation on day 21 after challenge. The lungs were removed immediately afterwards, weighed on an analytical balance and then fixed in Bouin's Solution (85% picric acid, 10% formaldehyde, 5% glacial acetic acid). The number of metastasis nodes was determined by counting under a microscope.
  • Bouin's Solution 85% picric acid, 10% formaldehyde, 5% glacial acetic acid. The number of metastasis nodes was determined by counting under a microscope.
  • Fig. 2 shows that the group B16 B7.2 / GMCSF (allogeneic) does not differ statistically from the group Kl 735 B7.2 / GM-CSF (autologous).
  • the coexpression of B7.2 and GM-CSF also shows a synergistic effect.
  • FIG. 6 The joint evaluation of three similar but independent animal experiments is shown in FIG. 6. This shows the clear superiority of the combination of B7.2 and GMCSF over the single molecules. This synergistic effect is particularly significant.
  • the synergistic effect of the molecules occurs both in autologous vaccination, where B7.2 can have a direct effect on T cell activation, and in allogeneic vaccination without agreement of the MHC molecules, where only indirect effects via NK cells and allo- reactive T cells are conceivable. This underscores the interesting discovery that B7.2 has an immunostimulatory effect even with allogeneic vaccination, despite the contrary opinion to date.
  • results of the three experiments were weighted relative to one another by setting the mean lung weight of the groups in animals without any manipulation (blank value) to be 0% and that of the group which had been vaccinated with wild-type cells to be 100%.
  • the individual test groups were then weighted as a percentage of their lung weight. An average was then formed for all three experiments.
  • the experimental analysis of a postulated function of a melanoma vaccine in a human experimental setup harbors several restrictions with regard to the methods that can be used and the parameters that can be investigated.
  • the measurement of an effect of cellular-produced GM-CSF is limited to the induction of the differentiation of monocytes to (pre-) dendritic cells.
  • the chemotactic effect is a parameter that can only be analyzed in vivo.
  • B7.2 CD86
  • Cells / material Melanoma cell line Mel 29 derived from a patient
  • PBLs Peripheral blood lymphocytes
  • Test components Mel 29 cells: untransduced or transduced with B7.2. Aim of the experiment: Analysis of the effect of B7.2 on the NK cell-mediated
  • Lysis of melanoma cells Design PBLs were freshly isolated, purified using a Ficoll gradient and incubated overnight at different E / T ratios with 2x10 3 51 Cr-labeled cells that were either untransduced or transduced with B7.2. The release of 51 Cr from lysed cells was measured.
  • Cells in a T80 bottle are detached and centrifuged at 175xg for 5 min.
  • the cell pellet is resuspended in 2-5 ml assay medium (RPMI1640, Gibco with 5% FCS, 2 mM L-glutamine, 1 mM Na pyruvate, 1 x non-essential arninoacids, 50 ⁇ g / ml gentamicin) and the cells are counted ,
  • 2-5 ml assay medium RPMI1640, Gibco with 5% FCS, 2 mM L-glutamine, 1 mM Na pyruvate, 1 x non-essential arninoacids, 50 ⁇ g / ml gentamicin
  • 100-200 ⁇ l of the cell suspension are transferred to a 1.5 ml Eppendorf tube (cell number: up to 1E + 6). After adding 20-50 ⁇ l 51 Cr, the cells are incubated for 45 min to 60 min at 37 ° C., 5% CO2. The cells are washed twice and diluted to 2000 cells per 100 ⁇ l medium.
  • PBMNC are adjusted to a cell number of 6.7E + 6 / ml in assay medium, of which 150 ⁇ l are pipetted into the first row of a 96-well round-bottom MTP. A total of 6 titer levels are produced in 1: 3 dilution steps.
  • 100 ⁇ l of target cell suspension are pipetted into 100 ⁇ l effector titration per well.
  • 100 ⁇ l target cell suspension mixed with 100 ⁇ l assay medium.
  • the cells are incubated for about 16 h at 37 ° C., 5% CO 2 .
  • 100 ⁇ l of target cell suspension are mixed with 100 ⁇ l of 2% Triton X-100 to determine the maximum release.
  • the NK cell-mediated lysis of melanoma cells was significantly increased by the expression of B7.2 by the Mel29 cells. Similar results were obtained with a second melanoma cell line, Mel 62 (data not shown). In total, three out of five donors showed a comparable increase in their NK cell activity comparable to that from FIG. 4. These results thus show the increase in NK cell-mediated lysis of tumor cells as a result of B7.2 expression on corresponding tumor cells. Consequences of such an increase in NK cell activity are (1) the release of cytokines, (2) the release of tumor antigen and (3) the activation of DC cells through the interaction of CD40 and CD40L. As already stated, it is known that these consequences support the efficient activation of T cells against the tumor antigens derived from the melanoma cells.
  • Test summary Cells / material Melanoma cell lines Mel derived from a patient
  • Test components Melanoma cells derived from a patient, transduced with:
  • the samples are precipitated on glass fiber filter mats using a semi-automatic sample harvesting device.
  • the filters are dried in a drying cabinet at 60 ° C for at least 1 h (or overnight).
  • the dried filters are sealed in foil and wetted with ⁇ -scintillator liquid.
  • the ⁇ -radiation of the samples in the filter is then measured in a radioactivity meter for ⁇ -radiation (cpm).
  • melanoma cells with increasing amounts of B7.2 expression were used to induce T cell proliferation. It was found that the maximum of the measured T cell activation was close to a B7.2 expression rate of 30% positive cells (data not shown). It should also be noted that the transduction of melanoma cells with the control proteins GFP (green fluorescence protein) or lacZ (due to infection with rAAV) had only an insignificant influence on T cell proliferation, and significantly less than the influence by B7. 2 Expression. This confirms that the observed increase in T cell proliferation was dependent on the expression of B7.2.
  • GFP green fluorescence protein
  • lacZ due to infection with rAAV
  • Cells / material Melanoma cell lines derived from a patient Mel 29 (HLA A2.01 positive), Mel 62 (HLA A2.01 negative) T2 cells: TAP-deficient lymphoblastoid cells PBLs from a healthy, HLA A2.01 positive donor Test components: Mel 62, Mel 29 cells:
  • Aim of the experiment induction of an immune response against peptide epitopes from known tumor antigens in a test arrangement with and without match of the MHC haplotypes (“match” or “mismatched”) between melanoma cell lines and PBLs.
  • Melanoma cells transduced with rAAV-B7.2 induced the activation of T cell lines (Mel29 and Mel62) with higher efficiency than non-transduced cells, which recognized specifically known peptide epitopes from known melanoma antigens (see FIG. 6).
  • control vectors showed that this effect was dependent on B7.2 expression.
  • the antigens tested here included Marti / MelanA, gplOO and tyrosinase (together in the peptide pool).
  • the specificity for these melanoma-derived antigens was demonstrated by comparison to a ControUpeptide derived from HIV-gpl20.
  • Increasing amounts of specific T cells were observed when either HLA-A2 matched allogeneic stimulation (with Mel29, left diagram of FIG. 6) or HLA mismatched stimulation (Mel62, right diagram of FIG. 6) of the T cellsteurkelt.
  • the antigen-acting signal is derived from endogenously expressed tumor antigens of a melanoma cell (stimulator cell), which are presented on the cell surface by MHC molecules.
  • the T cells also originate from an HLA-A2 positive donor and thus the T cell receptor of the T cells recognizes the antigenic peptide directly by presenting the antigen with an appropriate MHC molecule that is common to the stimulation cell and the donor of the T cells.

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Abstract

L'invention concerne l'utilisation d'une cellule tumorale pour préparer un vaccin destiné au traitement thérapeutique ou prophylactique de tumeurs chez des patients, ladite cellule tumorale exprimant un polypeptide co-stimulant et les molécules MHC de cette cellule tumorale et du patient ne correspondant pas. La présente invention porte également sur l'utilisation d'une cellule tumorale exprimant un polypeptide co-stimulant pour préparer un vaccin destiné à augmenter l'activité lytique de cellules NK lors du traitement thérapeutique ou prophylactique d'une tumeur chez un patient allogénique relativement à ladite cellule tumorale.
EP02787620A 2001-11-09 2002-11-08 Vaccin allogenique comportant une cellule tumorale qui exprime un polypeptide co-stimulant Withdrawn EP1441758A2 (fr)

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BRPI0418157A (pt) * 2003-12-30 2007-04-17 Mologen Ag terapêutica de tumores alogênicos
EP2296705A1 (fr) * 2008-06-24 2011-03-23 Hadasit Medical Research Services And Development Ltd. Anticorps spécifiques de ccl20 pour la thérapie du cancer
WO2010051502A2 (fr) * 2008-10-31 2010-05-06 Biogen Idec Ma Inc. Molécules ciblant light et leurs utilisations
WO2011041613A2 (fr) 2009-09-30 2011-04-07 Memorial Sloan-Kettering Cancer Center Immunothérapie combinée pour le traitement du cancer
GB201403775D0 (en) 2014-03-04 2014-04-16 Kymab Ltd Antibodies, uses & methods
SG11201707769VA (en) 2015-03-23 2017-10-30 Jounce Therapeutics Inc Antibodies to icos
CA2998208A1 (fr) 2015-10-22 2017-04-27 Jounce Therapeutics, Inc. Signatures geniques pour determiner l'expression d'icos
JP2019508044A (ja) * 2016-03-18 2019-03-28 ナントセル，インコーポレイテッド 樹状細胞感染のための多モードベクター
US9567399B1 (en) 2016-06-20 2017-02-14 Kymab Limited Antibodies and immunocytokines
CN116640214A (zh) 2016-08-09 2023-08-25 科马布有限公司 分离抗体及其应用
EP3534947A1 (fr) 2016-11-03 2019-09-11 Kymab Limited Anticorps, combinaisons comprenant des anticorps, biomarqueurs, utilisations et procédés
GB201709808D0 (en) 2017-06-20 2017-08-02 Kymab Ltd Antibodies
WO2019122882A1 (fr) 2017-12-19 2019-06-27 Kymab Limited Anticorps bispécifique pour icos et pd-l1

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US6207646B1 (en) * 1994-07-15 2001-03-27 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US6239116B1 (en) * 1994-07-15 2001-05-29 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
WO1997024132A1 (fr) * 1995-12-28 1997-07-10 The Johns Hopkins University School Of Medicine Vaccins a base de tumeurs de cytokine paracrine allogenique
DE19608751B4 (de) * 1996-03-06 2006-05-18 Medigene Ag Verwendung eines Adeno-assoziierten Virus-Vektors zur Steigerung der Immunogenität von Zellen
US6051428A (en) * 1997-03-21 2000-04-18 Sloan-Kettering Institute For Cancer Research Rapid production of autologous tumor vaccines
ATE356630T1 (de) * 1998-04-03 2007-04-15 Univ Iowa Res Found Verfahren und produkte zur stimulierung des immunsystems mittels immunotherapeutischer oligonukleotide und zytokine

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Title
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WO2003039592A3 (fr) 2003-10-23
CA2466530A1 (fr) 2003-05-15
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CA2466698A1 (fr) 2003-05-15

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