EP1441759A2 - Cellular vaccines comprising adjuvants - Google Patents
Cellular vaccines comprising adjuvantsInfo
- Publication number
- EP1441759A2 EP1441759A2 EP02802659A EP02802659A EP1441759A2 EP 1441759 A2 EP1441759 A2 EP 1441759A2 EP 02802659 A EP02802659 A EP 02802659A EP 02802659 A EP02802659 A EP 02802659A EP 1441759 A2 EP1441759 A2 EP 1441759A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- cells
- tumor
- composition according
- adjuvant
- cpg
- 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- Withdrawn
Links
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Classifications
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- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
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- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
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- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
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Definitions
- the present invention relates to cellular vaccines for use in tumor therapy.
- 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.
- MHC transplantation antigens
- T cells T cells specific for these peptides.
- MHC complexes There are two classes of MHC complexes - class I and class II. MHC-I complexes are expressed on almost all nucleated vertebrate cells, while MHC-II complexes are only found on antigen-presenting cells.
- a T cell When a specific immune response is formed, a T cell recognizes the MHC complex with the presented peptide of an antigen through its T cell receptor and is thereby stimulated to form an immune response. Cytotoxic T cells (CTLs) bind to MHC-I complexes and are then stimulated to proliferation (clonal selection), while T-helper cells bind to MHC-II complexes, which also causes a T cell clone to proliferate.
- CTLs Cytotoxic T cells
- the binding of the T cell receptor to the MHC complex is usually not sufficient for the development of a specific immune response. Rather, additional so-called co-stimulatory molecules are required which increase the signal exchange between the T cell and the MHC-carrying cell.
- the class I MHC complexes are of particular importance for triggering an immune response against tumor cells, since tumor cells in their MHC-I complexes present peptides which (almost) exclusively occur on tumor cells, so-called tumor antigens or peptides derived therefrom. It is known in the prior art that the recognition of peptides which are derived from tumor antigens and which are presented by MHC class I molecules causes the proliferation of cytotoxic T lymphocytes (also called cytotoxic T cells) by certain T cells, which in turn can kill morocells (Janeway C. et al., (1999) in: Immunobiology; Current Biology Publications, 551-554).
- cytotoxic T lymphocytes also called cytotoxic T cells
- T-cell help For an efficient activation of cytotoxic T-lymphocytes (CTLs) and antigen-presenting cells, the right amount of support by T-cells ("T-cell help") is necessary.
- This support can be provided primarily by Thl, but also Th2 cells.
- Thl cells mainly stimulate a CTL response via IL-12 and IFN-gamma, while Th2 cells a B-cell response via IL-4 and Promote IL-10.
- Antigen-presenting cells activate CTLs via so-called cross-sensitization (cross-priming). If this cross-priming does not take place to a sufficient extent, CTLs, which are required for the detection and elimination of tumor cells, are only activated incompletely.
- one (or more) established tumor cell line (s) is generally used to vaccinate the patient (see WO 97/24132).
- the object of the present invention is therefore to provide an improved vaccine in order to efficiently activate the host's immune system in order to combat the growing tumor or to prevent the development of a tumor.
- the object is achieved by a composition for the vaccination of tumors containing at least one tumor cell which expresses at least one cytokine, chemokine and / or a co-stimulating molecule and an effective amount of at least one adjuvant.
- adjuvants were found in the context of the present invention, with the aid of which it is possible to efficiently activate the immune system of the tumor patient and thus to combat the growing tumor or to prevent the development of a tumor.
- the effectiveness of a cellular vaccine - both in an autologous and in an allogeneic situation - can be improved by adding CpG oligonucleotide.
- the time window within which vaccination with a cellular vaccine is effective is increased by the combination of the vaccine with an adjuvant according to the invention. For example, mice in the final stage of a tumor disease still responds to cellular vaccination when the vaccine included an adjuvant.
- the effects of cellular vaccines which expressed transgenes such as cytokines, chemokines and / or co-stimulating molecules were investigated in combination with an adjuvant such as CpG oligonucleotide.
- an adjuvant such as CpG oligonucleotide.
- a co-stimulating molecule, cytokine or chemokine synergistically enhanced the effect of a vaccine with an adjuvant such as CpG.
- B7.2 in particular leads to a surprisingly high effectiveness of the vaccination if an adjuvant such as e.g. CpG was added to the vaccine.
- an adjuvant such as e.g. CpG
- the combination of both a cytokine / chemokine such as GM-CSF and a stimulatory molecule such as B7.2 also led to a surprisingly high effectiveness.
- the present invention thus relates to a composition for the vaccination of tumors comprising at least one tumor cell which expresses at least one cytokine, chemokine and / or co-stimulating molecule, and an effective amount of at least one adjuvant.
- cytokine is a general term for a large group of soluble proteins and peptides that act as humoral regulators in nano- to picomolar concentrations. Under normal or pathological conditions, these modulate the functional activities of individual cells or tissues. They also mediate interactions between cells and regulate processes that take place in the extracellular environment.
- Cyhemokines are a subset of the cytokines. They are smaller proteins or peptides that have a chemotactic effect on cells.
- a “co-stimulating molecule” is a molecule which enhances the signal exchange between the T cell and the MHC-carrying cell.
- an "adjuvant” is a substance which enhances the immunogenic (sensitizing) effect of an antigen.
- an effective amount of adjuvant means an amount which measurably extends the survival time of the treated test object in comparison to a treated test object to which the tumor cell was administered alone or which significantly increases a response in an in vitro immunassay.
- a “vaccination of tumors” in the context of the present invention preferably means that a patient is vaccinated with one of the compositions according to the invention and thereby a tumor of the patient is treated or a tumor is prevented.
- the invention also relates to a composition
- a composition comprising at least one tumor cell which expresses at least one cytokine, chemokine and / or a co-stimulating molecule; and an effective amount of at least one adjuvant.
- the tumor cell is derived from a pre-tumor, from a tumor or from a metastasis.
- tumor means at least one cell or cell mass in the form of a new tissue formation, in particular in the form of spontaneous, differently uninhibited, autonomous and irreversible excess growth of the body's own tissue, which usually has a different degree of loss of specific cell and tissue functions connected is. This cell or cell mass is not effectively inhibited in its growth by itself or by regulating mechanisms of the host organism, e.g. Melanoma, carcinoma.
- pre-tumor means at least one cell or cell mass as defined under the term tumor, in contrast to the tumor, however, this is inhibited by self-regulating mechanisms of the host organism in growth (eg cervical intraepithelial neolepsy of grade 1 (CIN1), CIN2, CIN3).
- CIN1 cervical intraepithelial neolepsy of grade 1 (CIN1), CIN2, CIN3).
- metastasis denotes the spread of tumor cells and the establishment of secondary areas of tumor growth. Malignant cells have the ability to metastasize.
- the tumor cell can be autologous or allogeneic with respect to the vaccinated patient. If the vaccination is carried out in an autologous situation, this means that the tumor cell is re-injected into the same patient from which it originally came, ie the vaccine and the tumor to be treated have the same MHC haplotype.
- the implementation in an allogeneic situation means that the tumor cell used for the vaccination comes from another patient and therefore usually does not have the identical MHC genes as the patient's own cells.
- the tumor cell can be derived from many different types of tumors, for example from melanoma, ovarian cancer, breast cancer, colon cancer, leukemia, lymphoma, kidney cancer, lung cancer, prostate cancer, cervical cancer and / or brain tumor.
- tumor cells such as Leukemia / or lymphoma cells
- cytokines and / or chemokines such as e.g. IL-2 or MCP1 or co-stimulating molecules
- B7.1, B7.2, CD40 or CD70 have to be genetically modified so that they express one or more molecules from the group containing cytokines, chemokines and / or co-stimulating molecules. Methods for the transduction of cells are described in the literature, e.g. in US 6,171,597.
- the cytokine / chemokine is selected from the group consisting of GM-CSF, G-CSF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL -7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19 , IL-20, IL-21, IL-22, IFN-alpha, IFN-beta, IFN-gamma, Flt3 L, Flt3, TNF-alpha, RANTES, MlPl-alpha, MlPl-beta, MIP1- gamma, MlPl- delta, MIP2, MIP2-alpha, MIP2-beta, MIP3-alpha, MIP3-beta, MIP4, MIP5, MCP1, MCPl-beta, MCP2, MCP3,
- the co-stimulating molecule is selected from the group comprising B7.1, B7.2, CD40, LIGHT, Ox40, 4.1.BB, Icos, Icos L, SLAM, ICAM-1, LFA-3, B7.3, CD70, HSA (heat stable antigen), CD84, CD7, B7 RP-1 L, MAdCAM-1, VCAM-1, CS-1, CD82, CD30, CD120a, CD120b and TNFR-RP, whereby B7. 1 and B7.2 are particularly preferred embodiments.
- such expressed cytokines, chemokines and / or co-stimulating molecules are mutated. Such mutations include, but are not limited to, point mutations, deletions, or fusions with other peptides or proteins.
- Adjuvants according to the invention are preferably those which are suitable for shifting the ratio between Th2 and Thl immune responses in favor of the Thl response.
- Her2 neu antibodies are used for the treatment of breast cancer or anti-idiotype antibodies are used for T or B cell lymphomas / leukaemias, which lead to an activation of the Th2 response.
- an adequate CTL response is particularly important for combating tumors and preventing their development in the patient.
- a CTL response is particularly supported by a Thl response, so that the correct ratio between the Thl and Th2 immune response is required to achieve this goal, i.e. to achieve a preferred activation of CTLs.
- the restricted immune response observed in tumor patients is explained as follows: In most cases of tumor patients, the relationship between the Thl and Th2 immune response is shifted towards the Th2 response, especially in patients with tumors in the Final stage (Nieland JD et al. (1998) J Immunother 21, 4, 317-22). There are two mechanisms leading to this problem: First of all, all Th2 cells carry an IL-4 receptor which, when occupied, increases the resistance of the Th2 cells to Fas-induced apoptosis.
- cytokines relevant to a CTL response that specifically stimulate a Thl response such as IFN-gamma and IL-12, and also molecules such as CD40L, are insufficiently expressed.
- adjuvants that can be used to efficiently activate the immune response, which is particularly restricted in tumor patients, in order to fight the tumor or to prevent its development, are toll-like receptor agonists such as CpG oligonucleotide, lipopolysaccharides or Bacillus Calmette-Guerin Cell wall skeleton (CWS) as well as superantigens and agents that inhibit the signaling effect of CTLA-4.
- toll-like receptor agonists such as CpG oligonucleotide, lipopolysaccharides or Bacillus Calmette-Guerin Cell wall skeleton (CWS) as well as superantigens and agents that inhibit the signaling effect of CTLA-4.
- agonist denotes a physiological substance or a medicament which triggers an effect by occupying a membrane receptor.
- toll like receptor denotes receptors which have homology with the Toll receptors known from Drosophila. These receptors are seen as mediators of the Danger signal (Matzinger P., (2002) Ann. NY Acad. Sci. 961: 341-2; Matzinger P., (1994) Annu. Rev. Immunol. 12: 991-1045 ). These react to bacterial or viral signals, e.g. bacterial DNA, CpG motifs, double-stranded RNA, as well as bacterial or viral proteins.
- CpG are synthetic DNA fragments that contain the so-called "CpG motifs" that occur in bacterial DNA.
- Bacterial DNA has the property to have a large number of unmethylated CpG motifs. They have a frequency of 1/16 in bacteria compared to 1 / 50-60 in mammalian DNA where they are suppressed (Chen Y et al. (2001) Int Immunol 13, 1013-20).
- CpG means one or more oligonucleotide (s) containing at least one CpG motif.
- CpG mimic the stimulating effect of bacterial DNA. As a factor of innate immunity, they affect both the non-specific and the specific immune response. It is known from the literature that CpG intervenes in several steps of the immune response. CpG interacts with toll-like receptors on various immune cells such as macrophages, dendritic cells and NK cells. Normal ligands for toll-like receptors are LPS and other PAMPs (pathogen-associated molecular pattern, "pathogen-associated molecular pattern") (Wagner H (2001) Immunity 14, 499-502). As a result of the binding of ligands to toll-like receptors, Thl cytokines such as IFN-gamnia and IL-12 are highly upregulated.
- Inflammatory cytokines including TNF-alpha (tumor necrosis factor-alpha), IL-6 and IFN type I have the same fate.
- NK cells are activated to secrete IFN-gamma and their lytic activity is increased (Chen et al. 2001).
- a polarization of the T helper response from Th2 to Thl is initiated (Krieg AM et al. (1999) Pharmacol Ther 84, 2, 113-20; Kranzer K et al. (2000) Immunology 99, 2, 170 -8th). This leads to the activation of immature dendritic cells by CD40L on Th cells.
- T-helper-1 cells are able to stimulate a response with specific CTLs.
- Synthetic CpG is able to mimic the immunostimulatory effect of bacterial DNA. That is why it seems to be a good adjuvant, surprisingly also in attempts to vaccinate the tumor.
- the adjuvant is therefore an agonist of a toll-like receptor.
- the adjuvant is selected from the group consisting of CpG oligonucleotides, LPS and BCG-CWS.
- oligonucleotide contains at least 8 nucleotides, where C is unmethylated and where X ⁇ and X are 2 nucleotides.
- a “nucleotide” comprises e.g. Adenosine, cytidine, guanosine, thymidine or uridine or modified forms of these.
- the G in the oligonucleotide sequence 5 'X ⁇ CGX 2 3' is also unmethylated.
- the CpG oligonucleotide is an oligonucleotide with a sequence containing at least the following formula:
- nucleotide separates successive CpGs and where Xi is adenine, guanine or thymine, and where X 2 is cytosine, adenine or thymine and where N is any nucleotide and where Ni and N 2 are nucleic acid sequences, each composed of approximately 0 -25 nucleotides.
- Ni and N 2 of the nucleic acid contain no CCGG tetramer (quadramer) or no more than one CCG or CGG trimer.
- nucleic acid where at least one nucleotide separates successive CpGs and where X ⁇ X 2 is selected from the group consisting of GpT, GpA, ApA, GpG and ApT and where X 3 is selected from the group consisting of TpT, CpT, TpC, CpC and ApT and where N is any nucleotide and where Ni and N 2 are nucleic acid sequences, each composed of approximately 0-25 nucleotides. According to a further preferred embodiment, Ni and N of the nucleic acid contain no CCGG tetramer (quadramer) or no more than one CCG or CGG trimer.
- a further embodiment of this invention is that the CpG oligonucleotide has a nucleic acid sequence, wherein Ni and N 2 contain no CCGG tetamer (quadmer) or no more than one CCG or CGG trimer.
- a “CCGG tetramer” means an oligonucleotide consisting of the nucleotide sequence CCGG and a “CCG or CGG trimer” means an oligonucleotide consisting of the nucleotide sequence CCG or CGG.
- the CpG oligonucleotide is an oligonucleotide with the sequence:
- XiX 2 is selected from the group consisting of GpT, GpA, ApA, GpG and ApT and wherein X 3 X 4 is selected from the group consisting of TpT, CpT, TpC, CpC and ApT and where N is any nucleotide and where Ni and N 2 are nucleic acid sequences, each composed of approximately 0-25 nucleotides.
- the CpG oligonucleotides are coupled to the surface of the cell.
- the oligonucleotides can be covalently bound to the surface, for example by cross-linking or, for example, by an interaction between a cell membrane product.
- tein and the CpG oligonucleotide are covalently bound to the surface, for example by cross-linking or, for example, by an interaction between a cell membrane product.
- tein and the CpG oligonucleotide One possibility is to express an IgM immunoglobulin, which is specific for the respective CpG oligonucleotide, and to incubate the tumor cells with the respective CpG oligonucleotides, before the injection into a patient.
- CpG-polylysine complexes could also be coupled to the surface of the tumor cell.
- Bi-specific antibodies can also be used to couple CpG or other adjuvants to a membrane protein of the tumor cell.
- oligonucleotide is used interchangeably and means multiple nucleotides (ie molecules containing a sugar (e.g. ribose or deoxyribose) linked to a phosphate group and an exchangeable organic base which either contains a substituted pyrimidine (e.g. cytosine (C), thymine ( T) or uracil (U)) or a substituted purine (for example adenine (A) or guanine (G)).
- a substituted pyrimidine e.g. cytosine (C), thymine ( T) or uracil (U)
- a substituted purine for example adenine (A) or guanine (G)
- the term refers to both oligoribonucleotides and oligodeoxyribonucleotides.
- nucleic acid molecules can be obtained from existing sources of nucleic acids (eg genomic or cDNA), but are preferably synthetic (eg produced by oligonucleotide synthesis). All or some of the CpG oligonucleotide may be unmethylated, but at least the C of 5 'CG 3' must be unmethylated.
- CpG-containing oligonucleotides are preferably in the range from 8 to 30 bases in length.
- nucleic acids of any size larger than 8 nucleotides are capable of triggering an immune response according to the invention, as long as there are enough immunostimulatory motifs, because larger nucleic acids are broken down into oligonucleotides within cells.
- Preferred synthetic oligonucleotides contain no CCGG tetramer (quadmer) or no more than one CCG or CGG trimer at or near the 5 'and / or 3' ends.
- Stabilized oligonucleotides where the oligonucleotide involves modification of the phosphate backbone are also preferred.
- the modification can be, for example, a phosphorothioate or phosphorodithioate modification.
- the modification of the phosphate backbone preferably takes place at the 5 'end of the nucleic acid, for example on the first two nucleotides of the 5' end of the oligonucleotide.
- the modification of the phosphate backbone can take place at the 3 'end of the nucleic acid, for example on the last 5 nucleotides of the 3' end of the nucleic acid.
- the oligonucleotide can be completely or partially modified.
- the CpG oligonucleotide is preferably large in the range between 8 and 100 and particularly preferably between 8 and 30 nucleotides.
- CpG oligonucleotides can be produced on a large scale in plasmids and broken down into oligonucleotides.
- the CpG oligonucleotide and at least one immunopotentiating cytokine can be administered directly to the treated test object or can be administered together with a nucleic acid delivery complex.
- Nucleic acid / cytokine delivery complex is intended to mean a nucleic acid molecule and / or a cytokine which is associated (for example ionically or covalently bound to or encapsulated within) with a means for targeting (for example a molecule which binds to the target cell in a higher affinity (for example Surfaces of dendritic cells and / or increased cellular uptake by target cells) results).
- nucleic acid / cytokine delivery complexes examples include nucleic acid / cytokines associated with: a sterol (eg cholesterol), a lipid (eg a cationic lipid, virosom or liposome) or a target cell specific binding agent (eg a ligand recognized by a target cell specific receptor) ). It is preferred that the complexes are sufficiently stable in vivo to prevent significant decoupling prior to internalization by the target cell. However, it is preferred that the complex can be cleaved within the cell under suitable conditions, so that the nucleic acid / cytokine is released in functional form.
- a sterol eg cholesterol
- lipid eg a cationic lipid, virosom or liposome
- target cell specific binding agent eg a ligand recognized by a target cell specific receptor
- the CpG oligonucleotide can be an oligonucleotide with palindromic sequences.
- "Palindrome sequence” is intended to mean an inverted repeat (i.e., a sequence like ABCDEE'D'C'B'A ', where A and A' are bases that can form the usual Watson-Crick base pairs). In vivo, such
- the CpG oligonucleotide contains a palindrome sequence.
- a palindrome sequence relates to a palindrome, the CpG being part of the
- Is palindrome and is preferably the center of the palindrome.
- the CpG oligonucleotide is free from a palindrome.
- Oligonucleotide that is free of a palindrome is one in which the CpG
- Dinucleotide is not part of a palindrome.
- Such an oligonucleotide can contain a palindrome in which the CpG is not part of the palindrome.
- the CpG oligonucleotide can be a stabilized nucleic acid molecule.
- a "stabilized nucleic acid molecule” is intended to mean a nucleic acid molecule that is relatively resistant to in vivo degradation (e.g. by an exo- or endo-nuclease). Stabilization can be a function of the length or the secondary structure. Unmethylated CpG oligonucleotides that are several 10 kb to several 100 kb long are relatively resistant to degradation in vivo. With shorter CpG oligonucleotides, the secondary structure can stabilize and increase its effect. If e.g.
- the 3 'end of an oligonucleotide has self-complementarity with a region located higher up, so that it can fold back and form a kind of "stem loop structure", then the oligonucleotide is stabilized and therefore has more activity.
- Preferred stabilized oligonucleotides of the present invention have a modified backbone.
- the modification of the oligonucleotide backbone has been shown to cause increased activity of the CpG oligonucleotide when the it is administered in vivo.
- CpG constructs that have at least two phosphorothioate linkages at the 5 'end of the oligonucleotide and several phosphorothioate linkages at the 3' end, preferably 5 such linkages, brought about maximum activity and protected the oligonucleotide from degradation by intracellular exo- and endonucleases.
- modified oligonucleotides include phosphodiester modified oligonucleotides, combinations of phosphodiester and phosphorothioate oligonucleotides, methylphosphonate, methylphosphorothioate, phosphorodithioate, and combinations thereof. Each of these combinations and their particular effect on immune cells is discussed in greater detail in US 6,207,646 and US 6,239,116 and the entire content of the latter is hereby incorporated by reference into this application. It is believed that these modified oligonucleotides may show greater stimulatory activity due to their increased nuclease resistance, increased cellular uptake, increased protein binding and / or altered intracellular locations.
- phosphorothioate and phosphodiester oligonucleotides containing CpG motifs are found in APCs such as e.g. active dendritic cells. However, based on the concentration needed to induce CpG specific effects, the CpG oligonucleotides with nuclease resistant phosphorothioate backbone are more effective (2 ⁇ g / ml for the phosphorothioates vs. a total of 90 ⁇ g / ml for the phosphodiester).
- oligonucleotides include: non-ionic DNA analogs such as alkyl and aryl phosphates (in which the charged phosphonate oxygen is replaced by an alkyl or aryl group), phosphodiesters and alkyl phosphotriesters, in which the charged oxygen group is alkylated.
- Oligonucleotides containing diol, such as tetraethylene glycol or hexaethylene glycol, at one or both ends are known to be essentially resistant to nuclease degradation.
- Further adjuvants according to the invention which act as toll-like receptor agonists are lipopolysaccharides (LPS) or components thereof, such as the lipid A portion or the poly or oligosaccharide portion.
- LPS lipopolysaccharides
- LPS are the main outer membrane components of almost all Gram-negative bacteria and are known to act as strong stimulators of the immune system.
- LPS consist of a poly- or oligosaccharide region, which are anchored by the lipid A in the outer bacterial membrane.
- the specific, cellular recognition of the LPS / Lipid A is mediated by the joint extracellular interaction of the LPS binding protein, the membrane-bound or the soluble form of CD 14 and the Toll-like-receptor 4 * MD2 complex. This leads to the rapid activation of an intracellular signal network that is strongly homologous to the signal cascade of IL-1 and IL-8 (Alexander C and Rietschel ET (2001) J Endotoxin Res 7, 3, 167-202).
- the adjuvant is therefore LPS.
- 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 can trigger the differentiation of immune 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, for example, Okamoto S et al (2001) Infect. Immun. 69, 11, 6633-42).
- Known superantigens are, for example, 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 which inhibits the signaling action of CTLA-4.
- CTLA-4 cytotoxic T-lymphocyte associated antigen 4
- CTLA-4 B7 binds and suppresses T cell-dependent antibodies in vivo immune responses.
- CTLA-4 can e.g. are antibodies or antibody fragments that bind specifically to the extracellular domain of CTLA-4 and inhibit its signaling action.
- the generation or screening of such antibodies or antibody fragments is known to the person skilled in the art (see, for example, WO 0032231).
- Other agents which are suitable for binding CTLA-4 and inhibiting its signaling are small organic molecules, peptide analogs or soluble T cell receptors (see WO 9720574).
- the invention also relates to the use of a composition
- a composition comprising at least one tumor cell which expresses at least one cytokine, chemokine and / or a co-stimulating molecule and an effective amount of at least one adjuvant for the manufacture of a medicament for the treatment or prevention of tumors.
- the above statements apply to the tumor cell, the cytokine, chemokine and / or co-stimulating molecule and to the adjuvant.
- the invention also relates to a method for producing a medicament for the therapy or prevention of tumors, at least one tumor cell containing at least one cytokine, chemokine and / or a co-stimulant Molecule expressed and an effective amount of at least one adjuvant mixed.
- the invention also relates to a method for treating or preventing tumors, in which a patient is administered an effective amount of tumor cells which express at least one cytokine, chemokine and / or a co-stiumlating molecule and an effective amount of at least one adjuvant. This applies to the tumor cell, the cytokine, chemokine and / or co-stimulatory molecule and to the adjuvant.
- the tumor cell which expresses at least one cytokine, chemokine and / or a co-stimulating molecule, is produced by transduction with recombinant adeno-associated virus (AAV).
- AAV vectors were produced as described in WO 00/47757.
- transduction with recombinant adeno-associated virus is understood to mean that the gene (s) for a cytokine, chemokine and / or a co-stimulatory molecule are introduced into the cell by means of one or more recombinant AAVs and as a result of which are expressed.
- AAV adeno-associated virus
- the preparation of suitable recombinant AAV is well known to those skilled in the art (see e.g. WO 00/47757).
- the AAV vectors used in the context of this invention were produced by the processes described in WO 00/47757.
- the adjuvant is added to the cell suspension.
- the cells and adjuvant are mixed together. If necessary, other hooves and additives are added. Examples
- mice Female C3H / He mice, 6-7 weeks old, were obtained from Harlan, Borchen, Germany.
- the K-1735-M2 melanoma cell line was kindly developed by Dr. Souberbielle (King's College, London) and Prof. I. J. Fidler (University of Texas M. D. Anderson Cancer Center, Houston, USA).
- the well-known mouse melanoma cell line B16F10 was also used.
- CpG oligonucleotide nucleotides were provided through a collaboration with Coley Pharmaceuticals Group TM.
- the expression vector pcDNA3neo-HEL was cloned for the generation of stable transfectants of the B16F10 and K-1735 melanoma cells.
- the HEL gene was cut out of the vector pcDNAl-HEL and ligated into the expression vector pcDNA3neo, which is a neomycin resistance gene for selection wearing.
- Transfection of B16F10 and K-1735 cells was performed using Lipofectamine ® in a 15 cm cell culture dish. Positive cells were selected using selection 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. The two clones with the best expression rate were chosen for vaccination experiments.
- RNA preparation was performed using 2-5 x 10 cells, QIAshredder columns (# 79654) and the RNeasy Kit (# 74104) from QIAgen ® .
- DNA eg episomal plasmid DNA
- RNAse-free DNAse # 776785, Röche ®
- RNA was transcribed to cDNA using the Gene Amp RNA PCR Kit Gore (Perkin Elmer ®, # N808-0143).
- PCR for HEL and ß-actin was carried out with the QIAgen Taq master mix kit (# 1007 544) and the following primers:
- the fragments obtained were the HEL fragment (430 bp) and the ⁇ -actin fragment (290 bp).
- Streptavidin-HRP 1 5000 (Sigma ® , # S-5512)
- K1735 and K-1735-HEL cells expressing murine B7.2, GM-CSF or both molecules were produced by transduction with recombinant adeno-associated virus (AAV).
- AAV vectors were produced as described in WO 00/47757.
- B16-HEL cells that cannot be efficiently transduced with recombinant AAV were transfected with Polyfect (QIAgen, # 301107) to transiently express the two molecules B7.2 and / or GM-CSF.
- the expression rates of GM-CSF were comparable to those in K1735-HEL cells, those of B7.2 were slightly worse.
- the vaccine cells were irradiated and stored in liquid nitrogen.
- the cells were thawed, washed three times with PBS and adjusted to a cell number of 3 ⁇ 10 5 cells per dose in PBS.
- GM-CSF secreted GM-CSF was after 48 hours in the supernatant of transduced or transfected cells using the enzyme-linked immunoassay (ELISA) kit OptEIA mouse GM-CSF set from Pharmingen (San Diego, USA) detected. B7.2 expression was detected by flow cytometry using the antibody GL1 (Pharmingen).
- ELISA enzyme-linked immunoassay
- CpG was added to the cell suspension or PBS at a concentration of 10 ⁇ g per dose.
- mice were killed by neck dislocation, the lungs were prepared, weighed and fixed. In the case of C3H mouse lungs, Bouin's reagent was used (refer to: Current protocols in Immunology). The number of metastases was counted on the dissecting microscope.
- mice The spleens of the vaccinated mice were removed when the animals were dissected and stored in medium until further processing. In order to obtain a suspension of single cells, the spleens were disrupted using a "cell strainer" (70 ⁇ l / Nunc ® ). The cells were washed once and then purified from macrophages by passage through nylon wool. The extracted T cells were re-stimulated with irradiated, autologous tumor cells once a week. Rat spleen ConA sup (T stim TM cultures Supplement, Collaborative Biomedical Products, # 354115) was added at a concentration of 1-3% to improve growth.
- T-cell cultures were harvested, washed and as triplets on round-bottom plates with 96 wells with a cell number of 1, 8xl0 5 , 6xl0 4 , 2xl0 4 and 6.7xl0 3 cells per well plated.
- Living target cells were labeled with 51 chromium at 37 ° C for one hour, washed four times and added so that a final effector to target cell ratio of 90: 1, 30: 1, 10: 1 and 3: 1 was obtained.
- unlabeled YAC-1 cells were added in a ratio of 1: 5 to 1:10 to the target cells. After 5 hours of incubation, the supernatant was collected and transferred to LUMA plates. The next day the dried plates were counted in a ⁇ -counter (Packard). Specific lysis was calculated using the following formula:
- mice vaccinated with K-1735 cells co-expressing B7.2 / GM-CSF developed a lower average lung weight than animals vaccinated with PBS (206.4 + 13.6 mg compared to 339.5 ⁇ 75.8 mg and 166.80 ⁇ 10.7 mg compared to 200.75 ⁇ 42.8 mg, respectively).
- the combination of K1735-B7.2-GM-CSF with CpG increased the therapeutic effect. Delaying the start of vaccination reduced the therapeutic effect in all groups, with the exception of K1735-B7.2-GM + CpG. In the latter group, comparable results were seen in all groups, with some variation.
- TV20 the group vaccinated with PBS starting on days 7 and 11 showed strange results that cannot be explained and did not reappear in further experiments.
- mice vaccinated with B16F10-HEL cells co-expressing B7.2 / GM-CSF after tumor induction with wt developed a significantly lower average lung weight than animals vaccinated with control wild-type cells (404 mg compared to 564 mg ( Figure 6A)).
- the therapeutic effect of autologous and allogeneic vaccine cells expressing B7.2 / GM-CSF was comparable. In addition, this indicates that the vaccine exhibits tumor-reducing activity even at a time when the organism already has a growing tumor mass.
- spleen cells were taken in culture and re-stimulated as described in the material and methods.
- a chromium release assay against autologous target cells was carried out as a test system.
- T cells derived from mice vaccinated with K-1735-B7.2-GM-CSF cells with or without CpG could be expanded efficiently.
- PBS or PBS and CpG did not appear to be a sufficient stimulus to induce T cell proliferation sufficient for long-term expansion in vitro. After 2-3 rounds of re-stimulation, only the cell lines shown in Figure 7 grew well.
- K-1735 cells H2-k haplotype
- a tumor model in an autologous therapeutic vaccination scheme in C3H / He mice.
- living tumor cells were injected into the tail vein.
- K-1735 cells transduced with rAAV-muB7.2-GM-CSF or PBS were used as vaccines, without or in combination with CpG.
- Vaccination was started either on days 4, 7 or 11 after the tumor induction (challenge). On day 21 after the tumor induction (challenge), the animals were sacrificed to determine the lung weight and the number of lung metastasis nodes.
- CpG was used in an allogeneic situation using B16F10-HEL (H2-b haplotype) and K-1735-HEL (H2-k haplotype) cells in C3H / He mice.
- B16F10-HEL cells transfected with ⁇ AAV-muB7.2-GM-CSF were used as a completely allogeneic vaccine.
- Chicken egg lysozyme (HEL) is a model antigen from chicken egg white that is known in the literature as a good antigen (Calin-Laurens V et al. (1993) Vaccine 11, 9, 974-8; Cavani A et al .
- CpG enhanced the effect of an autologous as well as an allogeneic vaccine.
- the T cell experiments carried out support the data from animal experiments with autologous vaccines.
- the 5 Cr release test shows that the tumor reduction effects in animals vaccinated with PBS and CpG must be based on innate immunity and cannot be a case of T cell stimulation because no specific lysis was found. Only animals that also received cellular antigen (K-1735-B7.2-GM-CSF) were able to develop a clear T cell response, which can be seen not only in the tumor rejection in vivo, but also is also detectable in a 51 Cr release test, which activity of T cells verified. Since the chromium release data was collected 2-3 weeks after spleen cells were cultured, the responsible cells cannot be NK cells. After 2-3 weeks in culture, NK cells have largely disappeared in normal cases. In addition, unlabelled YAC-1 cells were added in the chromium release experiments to block NK lysis. Therefore, the calculated values for the specific lysis correspond to cytotoxic T cell lysis.
- Figure 1 Therapeutic vaccination (autologous) with B7.2 / GM-CSF and / or CpG (TV20): Mice were sacrificed on day 21 after the challenge and the lung weight was determined on a micro balance. The mean lung weight is shown in mg against the respective test approach. The mean lung weight of a healthy mouse is 140 mg. The numbers in brackets indicate the day of the first vaccination (day 4, 7 or 11).
- FIG. 2 Therapeutic vaccination (autologous) with B7.2 / GM-CSF and / or CpG (TV20): mice were sacrificed on day 21 after the challenge, lungs were fixed in a Bound's solution and the lung metastases were determined using a dissecting microscope , The average number of lung metastases is shown against the respective test approach. The numbers shown in brackets indicate the day of the first vaccination (day 4, 7 or 11).
- Figure 3 Therapeutic vaccination (autologous) with B7.2 / GM-CSF and / or CpG (TV24): Mice were sacrificed on day 21 after the challenge and lungs weighed. The mean lung weight is shown in mg against the respective test approach. The mean lung weight of a healthy mouse is 140 mg. The numbers shown in brackets indicate the day of the first vaccination (day 4, 7 or 11).
- Figure 4 Therapeutic vaccination (autologous) with B7.2 / GM-CSF and / or CpG (TV24): Mice were sacrificed on day 21 after the challenge, lungs were fixed in a Bouin's solution and the lung metastases were determined using a dissection microscope , The average number of lung metastases is shown against the respective test approach. The numbers shown in brackets indicate the day of the first vaccination (day 4, 7 or 11).
- FIG. 5 Therapeutic vaccination of C3H / He mice with transduced melanoma cells; The mean lung weight in mg is shown against the respective test approach. The mean lung weight of a healthy mouse is 140 mg.
- FIG. 5A shows the TV26 experiment and FIG. 5B shows the TV27 experiment.
- FIG. 6A Therapeutic vaccination (allogeneic) of C3H He mice with transduced melanoma cells (TV22): Mice were sacrificed on day 21 after the challenge and the lung weight was determined on a micro balance. The mean lung weight is shown in mg against the respective test approach. The mean lung weight of a healthy mouse is 140 mg.
- FIG 6B Therapeutic vaccination (allogeneic) of C3H / He mice with transduced melanoma cells (TV22): Mice were sacrificed on day 21 after the challenge, lungs were fixed in a Bouin's solution and the lungs were determined using a dissection microscope. The average number of lung metastases is shown against the respective test approach.
- Figure 7 51 Cr release test of spleen cells from TV20: Spleen cells from animals of TV20 were re-stimulated in vitro with irradiated K-1735-HEL cells. On day 5 after the re-stimulation, the cells were incubated with 51 chromium-labeled target cells (K-1735-HEL) for 4 hours with the effector-target cell ratios shown. Supernatants were measured in a ß counter. The% specific lysis is shown against the ratio of effector to target cells (E: T).
Abstract
The invention relates to a composition for vaccination against tumours containing at least one tumour cell, which expresses at least one cytokine, chemokine and/or a co-stimulating molecule and an effective quantity of at least one adjuvant. The invention also relates to the use of a composition of this type for producing a medicament for the treatment or prevention of tumours.
Description
Zelluläre Impfstoffe mit Adj vanzien Cellular vaccines with adjuvants
Die vorliegende Erfindung betrifft zelluläre Impfstoffe zum Einsatz in der Tumortherapie.The present invention relates to cellular vaccines for use in tumor therapy.
Die Aktivierung des körpereigenen Immunsystems zur Behandlung und Präventi- on von Tumoren ist ein vielversprechender Ansatz in der modernen Krebstherapie.Activating the body's immune system to treat and prevent tumors is a promising approach in modern cancer therapy.
Zur Aktivierung des körpereigenen Immunsystems sind im Stand der Technik u.a. autologe und allogene Vakzine bekannt (Pardoll D.M., (1998) Nat. Med. 4 (5 Suppl): 525-31; Wolchock J.D. und Livingston P.O., (2001) Lancet Ocol. 2 (4): 205-11; Schadendorf D. et al., (2000) Immunol. Lert. 15; 74 (1): 67-74)To activate the body's immune system, the prior art includes autologous and allogeneic vaccine known (Pardoll DM, (1998) Nat. Med. 4 (5 Suppl): 525-31; Wolchock JD and Livingston PO, (2001) Lancet Ocol. 2 (4): 205-11; Schadendorf D. et al., (2000) Immunol. Lert. 15; 74 (1): 67-74)
Bei der autologen Vakzine werden Zellen vom patienteneigenen Tumor für die Herstellung der Vakzine verwendet. Dabei werden die Tumorzellen dem Körper entnommen, ggf. genetisch modifiziert und beispielsweise durch Bestrahlung pro- liferationsinkompetent gemacht, bevor sie dem Patienten wieder verabreicht werden. Ziel ist es, dass Immunzellen, insbesondere cytotoxische T-Zellen und Hel- fer-T-Zellen, die verabreichten Zellen erkennen und so eine Immunantwort aufgebaut wird, die sich dann auch gegen den Tumor richten kann.In 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.
Eine Alternative zur autologen Vakzinierung ist die sog. allogene Immunisierung, d.h. die Immunisierung mit Zellen, die nicht vom selben Patienten stammen. Somit unterscheiden sich die Vakzinezellen von den körpereigenen Zellen des Patienten, da sie in der Regel nicht die identischen Transplantationsantigene (MHC- Gene) besitzen.
Der MHC-Komplex auf der Oberfläche von Zellen ist für die Ausbildung der spezifischen Irnmunantwort von besonderer Bedeutung, da im MHC-Komplex Peptide präsentiert werden, die dann von für diese Peptide spezifischen T-Zellen er- kannt werden. Dabei gibt es zwei Klassen von MHC Komplexen - Klasse I und Klasse II. MHC-I Komplexe werden auf nahezu allen kernhaltigen Zellen von Vertebraten exprimiert, während MHC-II Komplexe sich nur auf Antigen- präsentierenden Zellen finden.An alternative to autologous vaccination is so-called allogeneic immunization, ie immunization with cells that do not come from the same patient. Thus, the vaccine cells differ from the patient's own cells, since they usually do not have the identical transplantation antigens (MHC genes). The 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. There are two classes of MHC complexes - class I and class II. MHC-I complexes are expressed on almost all nucleated vertebrate cells, while MHC-II complexes are only found on antigen-presenting cells.
Bei der Ausbildung einer spezifischen Immunantwort erkennt eine T-Zelle durch ihren T-Zellrezeptor den MHC-Komplex mit dem präsentierten Peptid eines Anti- gens und wird dadurch zur Ausbildung einer Irnmunantwort angeregt. Dabei binden cytotoxische T-Zellen (CTLs) an MHC-I Komplexe und werden daraufhin zur Proliferation (klonale Selektion) angeregt, während T-Helfer Zellen an MHC-II Komplexe binden, wodurch ebenfalls eine Proliferation eines T-Zellklons stattfindet.When a specific immune response is formed, a T cell recognizes the MHC complex with the presented peptide of an antigen through its T cell receptor and is thereby stimulated to form an immune response. Cytotoxic T cells (CTLs) bind to MHC-I complexes and are then stimulated to proliferation (clonal selection), while T-helper cells bind to MHC-II complexes, which also causes a T cell clone to proliferate.
Allerdings genügt meistens die Bindung des T-Zellrezeptors an den MHC- Komplex nicht für die Ausbildung einer spezifischen Immunantwort. Vielmehr werden weitere sog. co-stimulatorische Moleküle benötigt, die den Signalaustausch zwischen T-Zelle und MHC-tragender Zelle verstärken.However, the binding of the T cell receptor to the MHC complex is usually not sufficient for the development of a specific immune response. Rather, additional so-called co-stimulatory molecules are required which increase the signal exchange between the T cell and the MHC-carrying cell.
Für die Auslösung einer Immunantwort gegen Tumorzellen sind die MHC- Komplexe der Klasse I von besonderer Bedeutung, da Tumorzellen in ihren MHC-I Komplexen Peptide präsentieren, die (nahezu) ausschließlich auf Tumorzellen vorkommen, sog. Tumorantigene oder von diesen abgeleitete Peptide. Im Stand der Technik ist bekannt, dass die Erkennung von Peptiden, die von Tumorantigenen abgeleitet sind und die von MHC Klasse I Molekülen präsentiert werden, durch bestimmte T-Zellen die Proliferation von cytotoxischen T- Lymphozyten (auch cytotoxische T-Zellen genannt) bewirkt, die wiederum Tu-
morzellen abtöten können (Janeway C. et al., (1999) in: Immunobiology; Current Biology Publications, 551-554).The class I MHC complexes are of particular importance for triggering an immune response against tumor cells, since tumor cells in their MHC-I complexes present peptides which (almost) exclusively occur on tumor cells, so-called tumor antigens or peptides derived therefrom. It is known in the prior art that the recognition of peptides which are derived from tumor antigens and which are presented by MHC class I molecules causes the proliferation of cytotoxic T lymphocytes (also called cytotoxic T cells) by certain T cells, which in turn can kill morocells (Janeway C. et al., (1999) in: Immunobiology; Current Biology Publications, 551-554).
Für eine effiziente Aktivierung cytotoxischer T-Lymphozyten (CTLs) und Anti- gen-präsentierender Zellen ist die richtige Menge an Unterstützung durch T- Zellen ("T-cell help") nötig. Diese Unterstützung kann vor allem durch Thl-, aber auch Th2-Zellen bereitgestellt werden Dabei stimulieren Thl -Zellen hauptsächlich eine CTL-Anwort via IL-12 und IFN-gamma, während Th2-Zellen eine B- Zell- Antwort via IL-4 und IL-10 fördern. Antigen-präsentierende Zellen aktivie- ren CTLs über eine sogenannte Kreuz-Sensibilisierung (Cross-Priming). Findet dieses Cross-Priming nicht in ausreichendem Maße statt, werden CTLs, die für die Erkennung und Eliminierung von Tumorzellen benötigt werden, nur unvollständig aktiviert.For an efficient activation of cytotoxic T-lymphocytes (CTLs) and antigen-presenting cells, the right amount of support by T-cells ("T-cell help") is necessary. This support can be provided primarily by Thl, but also Th2 cells. Thl cells mainly stimulate a CTL response via IL-12 and IFN-gamma, while Th2 cells a B-cell response via IL-4 and Promote IL-10. Antigen-presenting cells activate CTLs via so-called cross-sensitization (cross-priming). If this cross-priming does not take place to a sufficient extent, CTLs, which are required for the detection and elimination of tumor cells, are only activated incompletely.
Bei der allogenen Vakzinierung wird in der Regel eine (oder auch mehrere) etablierte Tumorzelllinie(n) zur Vakzinierung des Patienten verwendet (siehe WO 97/24132).In allogeneic vaccination, one (or more) established tumor cell line (s) is generally used to vaccinate the patient (see WO 97/24132).
Obwohl allein durch die Verabreichung einer allogenen Tumorzelllinie bereits im Patientenkörper eine gewisse Immunreaktion hervorgerufen wird, ist diese Immunreaktion in aller Regel nicht ausreichend, um den patienteneigenen Tumor zu bekämpfen. Daher sind im Stand der Technik verschiedene Versuche unternommen worden, durch genetische Manipulierung der verabreichten Tumorzelllinie eine Verstärkung der Irnmunantwort hervorzurufen. Beispielsweise ist im Stand der Technik bekannt (siehe WO 97/24132), dass eine Verstärkung der Immunantwort dadurch erzielt werden kann, dass eine genetisch modifizierte Tumorzelle verabreicht wird, die GM-CSF exprimiert.Although a certain immune response is already caused in the patient's body simply by administering an allogeneic tumor cell line, this immune response is usually not sufficient to combat the patient's own tumor. Various attempts have therefore been made in the prior art to genetically manipulate the administered tumor cell line to induce an increase in the immune response. For example, it is known in the prior art (see WO 97/24132) that an enhancement of the immune response can be achieved by administering a genetically modified tumor cell that expresses GM-CSF.
Insgesamt sind im Stand der Technik eine Vielzahl von allogenen und autologen Vakzinen bekannt, die genetisch modifizierte Tumorzellen umfassen (PardollOverall, a large number of allogeneic and autologous vaccines are known in the prior art, which comprise genetically modified tumor cells (Pardoll
D.M., (1998) Nat. Med. 4 (5 Suppl): 525-31; Wolchock J.D. und Livingston P.O.,
(2001) Lancet Ocol. 2 (4): 205-11; Schadendorf D. et al., (2000) Immunol. Lett. 15; 74 (1): 67-74).DM, (1998) Nat. Med. 4 (5 Suppl): 525-31; Wolchock JD and Livingston PO, (2001) Lancet Ocol. 2 (4): 205-11; Schadendorf D. et al., (2000) Immunol. Lett. 15; 74 (1): 67-74).
Trotz dieser Vielzahl potentieller Vakzine ist im Stand der Technik keine Vakzine bekannt, die beim Einsatz im Patienten eine befriedigende Wirkung erzielt. Ein gemeinsamer Nachteil aller im Stand der Technik bekannten Vakzine ist, dass die im Patienten ausgelöste Immunantwort in aller Regel zu schwach ist, um den patienteneigenen Tumor wirksam zu bekämpfen.Despite this large number of potential vaccines, no vaccine is known in the prior art which achieves a satisfactory effect when used in patients. A common disadvantage of all vaccines known in the prior art is that the immune response triggered in the patient is usually too weak to effectively fight the patient's own tumor.
Aufgabe der vorliegenden Erfindung ist es daher, eine verbesserte Vakzine be- reutzustellen, um das Immunsystem des Wirts effizient zu aktivieren, um den wachsenden Tumor zu bekämpfen oder der Entstehung eines Tumors vorzubeugen.The object of the present invention is therefore to provide an improved vaccine in order to efficiently activate the host's immune system in order to combat the growing tumor or to prevent the development of a tumor.
Erfindungsgemäß wird die Aufgabe gelöst durch eine Zusammensetzung für die Impfung von Tumoren enthaltend zumindest eine Tumorzelle, die zumindest ein Cytokin, Chemokin und/oder ein co-stimulierendes Molekül exprimiert und eine wirksame Menge von zumindest einem Adjuvans.According to the invention, the object is achieved by a composition for the vaccination of tumors containing at least one tumor cell which expresses at least one cytokine, chemokine and / or a co-stimulating molecule and an effective amount of at least one adjuvant.
Überraschenderweise wurden im Rahmen der vorliegenden Erfindung Adjuvanzi- en aufgefunden, mit Hilfe derer es gelingt, das Immunsystem des Tumorpatienten effizient zu aktivieren und damit den wachsenden Tumor zu bekämpfen oder der Entstehung eines Tumors vorzubeugen.Surprisingly, adjuvants were found in the context of the present invention, with the aid of which it is possible to efficiently activate the immune system of the tumor patient and thus to combat the growing tumor or to prevent the development of a tumor.
Insbesondere konnte gezeigt werden, dass die Wirksamkeit eines zellulären Impfstoffes - sowohl in einer autologen als auch in einer allogenen Situation - durch die Zugabe von CpG Oligonukleotid verbessert werden kann. Des weiteren wird das Zeitfenster, innerhalb dessen eine Impfung mit einem zellulären Impfstoff wirksam ist, durch die Kombination des Impfstoffs mit einem erfindungsgemäßen Adjuvans vergrößert. Beispielsweise zeigten Mäuse im Endstadium einer Tumo-
rerkrankung noch immer eine Antwort auf die zelluläre Impfung, wenn der Impfstoff ein Adjuvans umfasste.In particular, it was shown that the effectiveness of a cellular vaccine - both in an autologous and in an allogeneic situation - can be improved by adding CpG oligonucleotide. Furthermore, the time window within which vaccination with a cellular vaccine is effective is increased by the combination of the vaccine with an adjuvant according to the invention. For example, mice in the final stage of a tumor disease still responds to cellular vaccination when the vaccine included an adjuvant.
Im Rahmen der vorliegenden Erfindung wurden die Auswirkungen von zellulären Impfstoffen untersucht, die Transgene wie Cytokine, Chemokine und/oder co- stimulierende Moleküle exprimierten, in Kombination mit einem Adjuvans wie CpG Oligonukleotid. Überraschenderweise konnte gezeigt werden, dass die Gegenwart eines co-stimulierenden Moleküls, Cytokins oder Chemokins die Wirkung eines Impfstoffes mit einem Adjuvans wie CpG in synergetischer Weise verstärkte. Dies gilt z.B. für die Expression von einem co-stimulatorischen Molekül wie B7.2 oder einem Cytokin/Chemokin wie z.B. GM-CSF. Insbesondere die Expression von B7.2 führt zu einer überraschend großen Wirksamkeit der Impfung, wenn ein Adjuvans wie z.B. CpG dem Impfstoff zugegeben war. Auch die Kombination von sowohl einem Cytokin/Chemokin wie GM-CSF und einem co- stimulatorischen Molekül wie B7.2 führte zu einer überraschend großen Wirksamkeit.In the context of the present invention, the effects of cellular vaccines which expressed transgenes such as cytokines, chemokines and / or co-stimulating molecules were investigated in combination with an adjuvant such as CpG oligonucleotide. Surprisingly, it was shown that the presence of a co-stimulating molecule, cytokine or chemokine synergistically enhanced the effect of a vaccine with an adjuvant such as CpG. This applies e.g. for the expression of a co-stimulatory molecule such as B7.2 or a cytokine / chemokine such as e.g. GM-CSF. The expression of B7.2 in particular leads to a surprisingly high effectiveness of the vaccination if an adjuvant such as e.g. CpG was added to the vaccine. The combination of both a cytokine / chemokine such as GM-CSF and a stimulatory molecule such as B7.2 also led to a surprisingly high effectiveness.
Gegenstand der vorliegenden Erfindung ist somit eine Zusammensetzung für die Impfung von Tumoren enthaltend zumindest eine Tumorzelle, die zumindest ein Cytokin, Chemokin und/oder co-stimulierendes Molekül exprimiert, und eine wirksame Menge von zumindest einem Adjuvans.The present invention thus relates to a composition for the vaccination of tumors comprising at least one tumor cell which expresses at least one cytokine, chemokine and / or co-stimulating molecule, and an effective amount of at least one adjuvant.
Im Rahmen der vorliegenden Erfindung sind die vorliegenden Definitionen von allgemeiner Bedeutung:In the context of the present invention, the present definitions are of general importance:
Der Begriff "Cytokin" ist eine allgemeine Bezeichnung für eine große Gruppe löslicher Proteine und Peptide, die als humorale Regulatoren in nano- bis pico- molaren Konzentrationen fungieren. Diese modulieren unter normalen oder pathologischen Zuständen die funktioneilen Aktivitäten von einzelnen Zellen oder Geweben. Ferner vermitteln sie direkt Interaktionen zwischen Zellen und regulieren Prozesse, die in der extrazellulären Umgebung ablaufen.
"Chemokine" sind eine Untergruppe der Cytokine. Es sind kleinere Proteine bzw. Peptide, die u.a. chemotaktisch auf Zellen wirken.The term "cytokine" is a general term for a large group of soluble proteins and peptides that act as humoral regulators in nano- to picomolar concentrations. Under normal or pathological conditions, these modulate the functional activities of individual cells or tissues. They also mediate interactions between cells and regulate processes that take place in the extracellular environment. "Chemokines" are a subset of the cytokines. They are smaller proteins or peptides that have a chemotactic effect on cells.
Ein "co-stimulierendes Molekül" ist im Rahmen der vorliegenden Erfindung ein Molekül, das den Signalaustausch zwischen T-Zelle und MHC-tragender Zelle verstärkt.In the context of the present invention, a “co-stimulating molecule” is a molecule which enhances the signal exchange between the T cell and the MHC-carrying cell.
Ein "Adjuvans" ist im Rahmen der vorliegenden Erfindung eine Substanz, die die immunogene (sensibilisierende) Wirkung eines Antigens verstärkt.In the context of the present invention, an "adjuvant" is a substance which enhances the immunogenic (sensitizing) effect of an antigen.
"Eine wirksame Menge" an Adjuvans bedeutet erfindungsgemäß eine Menge, die messbar die Überlebensdauer des behandelten Versuchsobjekts verlängert im Vergleich zu einem behandelten Versuchsobjekt, dem die Tumorzelle allein ver- abreicht wurde oder die signifikant eine Antwort in einem in vitro Immunassay erhöht.According to the invention, “an effective amount” of adjuvant means an amount which measurably extends the survival time of the treated test object in comparison to a treated test object to which the tumor cell was administered alone or which significantly increases a response in an in vitro immunassay.
Eine "Impfung von Tumoren" bedeutet im Rahmen der vorliegenden Erfindung vorzugsweise, dass ein Patient mit einer der erfindungsgemäßen Zusammenset- zungen geimpft wird und dadurch ein Tumor des Patienten behandelt oder einem Tumor vorgebeugt wird.A “vaccination of tumors” in the context of the present invention preferably means that a patient is vaccinated with one of the compositions according to the invention and thereby a tumor of the patient is treated or a tumor is prevented.
Gegenstand der Erfindung ist auch eine Zusammensetzung enthaltend zumindest eine Tumorzelle, die zumindest ein Cytokin, Chemokin und/oder ein co- stimulierendes Molekül exprimiert; und eine wirksame Menge von zumindest einem Adjuvans.The invention also relates to a composition comprising at least one tumor cell which expresses at least one cytokine, chemokine and / or a co-stimulating molecule; and an effective amount of at least one adjuvant.
Die folgenden bevorzugten Ausführungsformen gelten für beide erfindungsgemäße Zusammensetzungen.
Gemäß einer bevorzugten Ausführungsform der Erfindung stammt die Tumorzelle von einem Prä-Tumor, von einem Tumor oder von einer Metastase ab.The following preferred embodiments apply to both compositions according to the invention. According to a preferred embodiment of the invention, the tumor cell is derived from a pre-tumor, from a tumor or from a metastasis.
Der Begriff "Tumor" bedeutet mindestens eine Zelle bzw. Zellmasse in Form ei- ner geweblichen Neubildung insbesondere in Form eines spontanen verschieden- gradig enthemmten, autonomen und irreversiblen Überschusswachstums von körpereigenem Gewebe, das in der Regel mit unterschiedlich ausgeprägtem Verlust spezifischer Zeil- und Gewebefunktionen verbunden ist. Diese Zelle bzw. Zellmasse ist nicht durch sich selbst oder regulierende Mechanismen des Wirtsorganimus in seinem Wachstum wirksam gehemmt, z.B. Melanom, Karzinom.The term "tumor" means at least one cell or cell mass in the form of a new tissue formation, in particular in the form of spontaneous, differently uninhibited, autonomous and irreversible excess growth of the body's own tissue, which usually has a different degree of loss of specific cell and tissue functions connected is. This cell or cell mass is not effectively inhibited in its growth by itself or by regulating mechanisms of the host organism, e.g. Melanoma, carcinoma.
Der Begriff "Prä-Tumor" bedeutet mindestens eine Zelle oder Zellmasse wie definiert unter dem Begriff Tumor, im Gegensatz zum Tumor wird diese allerdings durch sich selbst oder regulierende Mechanismen des Wirtsorganismus im Wachstum gehemmt (z.B. Zervikale Intraepitheliale Neolepsie des Grades 1 (CIN1), CIN2, CIN3).The term "pre-tumor" means at least one cell or cell mass as defined under the term tumor, in contrast to the tumor, however, this is inhibited by self-regulating mechanisms of the host organism in growth (eg cervical intraepithelial neolepsy of grade 1 (CIN1), CIN2, CIN3).
Der Begriff "Metastase" bezeichnet die Ausbreitung von Tumorzellen und die Einrichtung von sekundären Bereichen des Tumorwachstums. Maligne Zellen haben die Fähigkeit zur Metastasierung.The term "metastasis" denotes the spread of tumor cells and the establishment of secondary areas of tumor growth. Malignant cells have the ability to metastasize.
Gemäß einer weiteren bevorzugten Ausführungsform kann die Tumorzelle autolog oder allogen bezüglich des geimpften Patienten sein. Wird die Impfung in einer autologen Situation durchgeführt, bedeutet dies, dass die Tumorzelle in den- selben Patienten, aus dem sie ursprünglich stammte, wieder injiziert wird, der Impfstoff und der zu behandelnde Tumor also den selben MHC-Haplotyp aufweisen. Die Durchführung in einer allogenen Situation bedeutet, dass die Tumorzelle die für die Vakzinierung eingesetzt wird, von einem anderen Patienten stammt und somit in der Regel nicht die identischen MHC-Gene wie die körpereigenen Zellen des Patienten besitzt.
Gemäß einer weiteren bevorzugten Ausführungsform kann die Tumorzelle von vielen verschiedenen Arten von Tumoren abstammen, z.B. von einem Melanom, Eierstockkrebs, Brustkrebs, Kolonkarzinom, Leukämie, Lymphom, Nierenkarzinom, Lungenkarzinom, Prostatakrebs, Gebärmutterhalskrebs und/oder Gehirntu- mor.According to a further preferred embodiment, the tumor cell can be autologous or allogeneic with respect to the vaccinated patient. If the vaccination is carried out in an autologous situation, this means that the tumor cell is re-injected into the same patient from which it originally came, ie the vaccine and the tumor to be treated have the same MHC haplotype. The implementation in an allogeneic situation means that the tumor cell used for the vaccination comes from another patient and therefore usually does not have the identical MHC genes as the patient's own cells. According to a further preferred embodiment, the tumor cell can be derived from many different types of tumors, for example from melanoma, ovarian cancer, breast cancer, colon cancer, leukemia, lymphoma, kidney cancer, lung cancer, prostate cancer, cervical cancer and / or brain tumor.
Während bestimmte Tumorzellen wie z.B. Leukämie-/ oder Lymphom-Zellen, selbst bestimmte Cytokine und/oder Chemokine wie z.B. IL-2 oder MCP1 oder co-stimulierende Moleküle wie z.B. B7.1, B7.2, CD40 oder CD70 exprimieren, müssen — in einer bevorzugten Ausführungsform - andere Tumorzellen genetisch modifiziert werden, so dass sie ein oder mehrere Moleküle aus der Gruppe enthaltend Cytokine, Chemokine und/oder co-stimulierende Moleküle exprimieren. Verfahren für die Transduktion von Zellen sind in der Literatur beschrieben wie z.B. in US 6,171,597.While certain tumor cells such as Leukemia / or lymphoma cells, even certain cytokines and / or chemokines such as e.g. IL-2 or MCP1 or co-stimulating molecules such as e.g. In a preferred embodiment, B7.1, B7.2, CD40 or CD70 have to be genetically modified so that they express one or more molecules from the group containing cytokines, chemokines and / or co-stimulating molecules. Methods for the transduction of cells are described in the literature, e.g. in US 6,171,597.
Gemäß einer bevorzugten Ausführungsform dieser Erfindung ist das Cytokin/Chemokin ausgewählt aus der Gruppe bestehend aus GM-CSF, G-CSF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IFN-alpha, IFN-beta, IFN- gamma, Flt3 L, Flt3, TNF-alpha, RANTES, MlPl-alpha, MlPl-beta, MIP1- gamma, MlPl-delta, MIP2, MIP2-alpha, MIP2-beta, MIP3-alpha, MIP3-beta, MIP4, MIP5, MCP1, MCPl-beta, MCP2, MCP3, MCP4, MCP5, MCP6, 6cykine, Dcckl und DCDF, wobei GM-CSF, RANTES und/oder MlPl-alpha besonders bevorzugte Ausführungsformen sind.According to a preferred embodiment of this invention, the cytokine / chemokine is selected from the group consisting of GM-CSF, G-CSF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL -7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19 , IL-20, IL-21, IL-22, IFN-alpha, IFN-beta, IFN-gamma, Flt3 L, Flt3, TNF-alpha, RANTES, MlPl-alpha, MlPl-beta, MIP1- gamma, MlPl- delta, MIP2, MIP2-alpha, MIP2-beta, MIP3-alpha, MIP3-beta, MIP4, MIP5, MCP1, MCPl-beta, MCP2, MCP3, MCP4, MCP5, MCP6, 6cykine, Dcckl and DCDF, being GM-CSF , RANTES and / or MlPl-alpha are particularly preferred embodiments.
Gemäß einer weiteren Ausfuhrungsform dieser Erfindung ist das co-stimulierende Molekül ausgewählt aus der Gruppe enthaltend B7.1, B7.2, CD40, LIGHT, Ox40, 4.1.BB, Icos, Icos L, SLAM, ICAM-1, LFA-3, B7.3, CD70, HSA (heat stable antigen), CD84, CD7, B7 RP-1 L, MAdCAM-1, VCAM-1, CS-1, CD82, CD30, CD120a, CD120b und TNFR-RP, wobei B7.1 und B7.2 besonders bevorzugte Ausführungsformen sind.
Gemäß einer weiteren Ausführungsform dieser Erfindung sind solche exprimier- ten Cytokine, Chemokine und/oder co-stimulierenden Moleküle mutiert. Solche Mutationen schließen ein, sind aber nicht beschränkt auf, Punktmutationen, Dele- tionen oder Fusionen mit anderen Peptiden oder Proteinen.According to a further embodiment of this invention, the co-stimulating molecule is selected from the group comprising B7.1, B7.2, CD40, LIGHT, Ox40, 4.1.BB, Icos, Icos L, SLAM, ICAM-1, LFA-3, B7.3, CD70, HSA (heat stable antigen), CD84, CD7, B7 RP-1 L, MAdCAM-1, VCAM-1, CS-1, CD82, CD30, CD120a, CD120b and TNFR-RP, whereby B7. 1 and B7.2 are particularly preferred embodiments. According to a further embodiment of this invention, such expressed cytokines, chemokines and / or co-stimulating molecules are mutated. Such mutations include, but are not limited to, point mutations, deletions, or fusions with other peptides or proteins.
Erfindungsgemäße Adjuvanzien sind bevorzugt solche, die geeignet sind, das Verhältnis zwischen Th2- und Thl -Immunantwort zugunsten der Thl -Antwort zu verschieben.Adjuvants according to the invention are preferably those which are suitable for shifting the ratio between Th2 and Thl immune responses in favor of the Thl response.
Diese stehen im Gegensatz zu anderen Adjuvanzien, deren Ziel eine Th2- Aktivierung ist. Beispielsweise werden für die Behandlung von Brustkrebs Her2 neu-Antikörper oder bei T- oder B-Zell-Lymphomen/Leukämien anti-idiotyp- Antikörper eingesetzt, die zu einer Aktivierung der Th2-Antwort fuhren.These are in contrast to other adjuvants that aim to activate Th2. For example, Her2 neu antibodies are used for the treatment of breast cancer or anti-idiotype antibodies are used for T or B cell lymphomas / leukaemias, which lead to an activation of the Th2 response.
Wie oben erwähnt, ist für die Bekämpfung von Tumoren und zur Verhinderung von deren Entstehung im Patienten vor allem eine ausreichende CTL-Antwort von Bedeutung. Eine CTL-Antwort wird wiederum besonders von einer Thl -Antwort unterstützt, so dass das richtige Verhältnis zwischen der Thl- und Th2- Immunantwort erforderlich dafür ist, dieses Ziel, d.h. eine bevorzugte Aktivierung von CTLs, zu erreichen.As mentioned above, an adequate CTL response is particularly important for combating tumors and preventing their development in the patient. A CTL response, in turn, is particularly supported by a Thl response, so that the correct ratio between the Thl and Th2 immune response is required to achieve this goal, i.e. to achieve a preferred activation of CTLs.
Ohne auf die folgende Theorie beschränkt zu sein, erklärt man sich die bei Tumorpatienten beobachtete eingeschränkte Immunantwort folgendermaßen: In den meisten Fällen von Tumorpatienten ist das Verhältnis zwischen Thl- und Th2- Immunanwort zur Th2-Antwort hin verschoben ist, insbesondere bei Patienten mit Tumoren im Endstadium (Nieland JD et al. (1998) J Immunother 21, 4, 317-22). Es gibt zwei Mechanismen, die zu diesem Problem führen: Als erstes tragen alle Th2-Zellen einen IL-4 Rezeptor der, wenn er besetzt ist, die Resistenz der Th2- Zellen gegen Fas-induzierte Apoptose verstärkt.
In Tumorpatienten ist zwar in der Regel keine direkte Erhöhung der IL4 und ILIO-Spiegel messbar, allerdings im Vergleich zu Thl-Cytokinen wie IFN, IL2 und TNFα, deren Spiegel in Tumorpatienten häufig absinkt, verschiebt sich somit das Verhältnis zugunsten von IL10 und IL4, so dass die Immunantwort zur Th2- Antwort hin verschoben ist.Without being limited to the following theory, the restricted immune response observed in tumor patients is explained as follows: In most cases of tumor patients, the relationship between the Thl and Th2 immune response is shifted towards the Th2 response, especially in patients with tumors in the Final stage (Nieland JD et al. (1998) J Immunother 21, 4, 317-22). There are two mechanisms leading to this problem: First of all, all Th2 cells carry an IL-4 receptor which, when occupied, increases the resistance of the Th2 cells to Fas-induced apoptosis. Although there is usually no direct increase in IL4 and ILIO levels in tumor patients, however, the ratio shifts in favor of IL10 and IL4 compared to Thl cytokines such as IFN, IL2 and TNFα, whose levels often drop in tumor patients. so that the immune response is shifted towards the Th2 response.
Weiterhin führt das veränderte Redox-Potential in Tumorpatienten zu einer höheren Anzahl von Makrophagen, die die Anzahl von Thl -Zellen reduzieren und die Anzahl von Th2-Zellen erhöhen. Darum werden für eine CTL-Antwort relevante Cytokine, die spezifisch eine Thl -Antwort stimulieren, wie IFN-gamma und IL- 12 sowie auch Moleküle wie CD40L unzureichend exprimiert.Furthermore, the changed redox potential in tumor patients leads to a higher number of macrophages, which reduce the number of Thl cells and increase the number of Th2 cells. For this reason, cytokines relevant to a CTL response that specifically stimulate a Thl response, such as IFN-gamma and IL-12, and also molecules such as CD40L, are insufficiently expressed.
Beispiele für Adjuvanzien, mit denen es gelingt, die besonders bei Tumorpatienten eingeschränkte Immunantwort effizient zu aktivieren, um den Tumor zu be- kämpfen oder dessen Entstehung zu verhindern, sind Toll-like-receptor-Agonisten wie CpG Oligonukleotid, Lipopolysaccharide oder Bacillus Calmette-Guerin Zellwandgerüst (cell wall skeleton, CWS) sowie Superantigene und Agenzien, die die Signalwirkung von CTLA-4 inhibieren.Examples of adjuvants that can be used to efficiently activate the immune response, which is particularly restricted in tumor patients, in order to fight the tumor or to prevent its development, are toll-like receptor agonists such as CpG oligonucleotide, lipopolysaccharides or Bacillus Calmette-Guerin Cell wall skeleton (CWS) as well as superantigens and agents that inhibit the signaling effect of CTLA-4.
Der Begriff "Agonist" bezeichnet eine physiologische Substanz bzw. ein Arzneimittel, das durch Besetzung eines Membranrezeptors eine Wirkung auslöst.The term "agonist" denotes a physiological substance or a medicament which triggers an effect by occupying a membrane receptor.
Der Begriff "Toll like receptor" bezeichnet Rezeptoren, die eine Homologie zu den aus Drosophila bekannten Toll-Rezeptoren aufweisen. Diese Rezeptoren wer- den als Vermittler des Danger Signals gesehen (Matzinger P., (2002) Ann. N. Y. Acad. Sei. 961: 341-2; Matzinger P., (1994) Annu. Rev. Immunol. 12: 991-1045). Diese reagieren auf bakterielle oder virale Signale, wie z.B. bakterielle DNA, CpG-Motive, doppelsträngige RNA, sowie bakterielle oder virale Proteine.The term "toll like receptor" denotes receptors which have homology with the Toll receptors known from Drosophila. These receptors are seen as mediators of the Danger signal (Matzinger P., (2002) Ann. NY Acad. Sci. 961: 341-2; Matzinger P., (1994) Annu. Rev. Immunol. 12: 991-1045 ). These react to bacterial or viral signals, e.g. bacterial DNA, CpG motifs, double-stranded RNA, as well as bacterial or viral proteins.
CpG sind synthetische DNA-Fragmente, die die sogenannten "CpG-Motive" enthalten, die in bakterieller DNA vorkommen. Bakterielle DNA hat die Eigenschaft,
eine hohe Anzahl an unmethylierten CpG-Motiven zu haben. Sie haben eine Häufigkeit von 1/16 in Bakterien im Vergleich zu 1/50-60 in Säuger-DNA, wo sie unterdrückt sind (Chen Y et al. (2001) Int Immunol 13, 1013-20).CpG are synthetic DNA fragments that contain the so-called "CpG motifs" that occur in bacterial DNA. Bacterial DNA has the property to have a large number of unmethylated CpG motifs. They have a frequency of 1/16 in bacteria compared to 1 / 50-60 in mammalian DNA where they are suppressed (Chen Y et al. (2001) Int Immunol 13, 1013-20).
"CpG" bedeutet im Rahmen der vorliegenden Erfindung ein oder mehrere Oligo- nukleotid(e) enthaltend mindestens ein CpG Motiv.In the context of the present invention, “CpG” means one or more oligonucleotide (s) containing at least one CpG motif.
CpG ahmen den stimulierenden Effekt bakterieller DNA nach. Als ein Faktor der angeborenen Immunität beeinflussen sie sowohl die unspezifische als auch die spezifische Immunantwort. Es ist aus der Literatur bekannt, dass CpG bei mehreren Schritten der Immunantwort eingreift. CpG wechselwirkt mit Toll-like- receptors auf verschiedenen Immunzellen wie Makrophagen, dendritische Zellen und NK Zellen. Normale Liganden für Toll-like-receptors sind LPS und andere PAMPs (pathogen-assoziiertes molekulares Muster, "pathogen associated mo- lecular pattern") (Wagner H (2001) Immunity 14, 499-502). Als Folge der Bindung von Liganden an Toll-like-receptors sind Thl -Cytokine wie IFN-gamnia und IL-12 stark hochreguliert. Inflammationsfördernde (proinflammatory) Cytokine einschließlich TNF-alpha (tumor necrosis factor-alpha), IL-6 und IFN-Typ-I haben dasselbe Schicksal. Zusätzlich werden NK-Zellen aktiviert, um IFN-gamma zu sekretieren und ihre lytische Aktivität ist verstärkt (Chen et al. 2001). Eine Polarisierung der T-Helfer-Antwort von Th2 zu Thl hin wird eingeleitet (Krieg AM et al. (1999) Pharmacol Ther 84, 2, 113- 20; Kranzer K et al. (2000) Immu- nology 99, 2, 170-8). Das führt zur Aktivierung von unreifen dendritischen Zellen durch CD40L auf Th-Zellen. T-Helfer-1 -Zellen sind in der Lage eine Antwort mit spezifischen CTLs zu stimulieren. Wie oben diskutiert scheint dies, ohne auf diese Theorie beschränkt zu sein, besonders im Falle einer Impfung gegen Krebs nützlich zu sein, denn dort sind die Antworten der T-Helferzellen oft in Richtung der Th2-vermittelten Immunantwort verschoben, insbesondere bei Patienten mit Tumoren im Endstadium. Des weiteren werden auch Antigen-präsentierende Zellen durch CpG aktiviert, was in einer besseren Sensibilisierung (priming) von CTLs
resultiert (Hacker H et al. (2000) J Exp Med 192, 4, 595-600), Kranzer et al. su- pra).CpG mimic the stimulating effect of bacterial DNA. As a factor of innate immunity, they affect both the non-specific and the specific immune response. It is known from the literature that CpG intervenes in several steps of the immune response. CpG interacts with toll-like receptors on various immune cells such as macrophages, dendritic cells and NK cells. Normal ligands for toll-like receptors are LPS and other PAMPs (pathogen-associated molecular pattern, "pathogen-associated molecular pattern") (Wagner H (2001) Immunity 14, 499-502). As a result of the binding of ligands to toll-like receptors, Thl cytokines such as IFN-gamnia and IL-12 are highly upregulated. Inflammatory cytokines including TNF-alpha (tumor necrosis factor-alpha), IL-6 and IFN type I have the same fate. In addition, NK cells are activated to secrete IFN-gamma and their lytic activity is increased (Chen et al. 2001). A polarization of the T helper response from Th2 to Thl is initiated (Krieg AM et al. (1999) Pharmacol Ther 84, 2, 113-20; Kranzer K et al. (2000) Immunology 99, 2, 170 -8th). This leads to the activation of immature dendritic cells by CD40L on Th cells. T-helper-1 cells are able to stimulate a response with specific CTLs. As discussed above, without being limited to this theory, this appears to be particularly useful in the case of a vaccination against cancer, since the responses of the T helper cells are often shifted towards the Th2-mediated immune response, particularly in patients with tumors in the final stage. Furthermore, antigen-presenting cells are activated by CpG, which results in better sensitization (priming) of CTLs results (Hacker H et al. (2000) J Exp Med 192, 4, 595-600), Kranzer et al. supra).
Synthetisches CpG ist in der Lage, den immunstimulierenden Effekt von bakteri- eller DNA nachzuahmen. Darum scheint es ein gutes Adjuvans zu sein, überraschenderweise auch in Versuchen zur Tumorimpfung.Synthetic CpG is able to mimic the immunostimulatory effect of bacterial DNA. That is why it seems to be a good adjuvant, surprisingly also in attempts to vaccinate the tumor.
Gemäß einer weiteren bevorzugten Ausführungsform der Erfindung ist das Adjuvans daher ein Agonist eines Toll-like-receptors.According to a further preferred embodiment of the invention, the adjuvant is therefore an agonist of a toll-like receptor.
Gemäß einer weiteren bevorzugten Ausführungsform ist das Adjuvans ausgewählt aus der Gruppe bestehend aus CpG Oligonukleotiden, LPS und BCG-CWS.According to a further preferred embodiment, the adjuvant is selected from the group consisting of CpG oligonucleotides, LPS and BCG-CWS.
Es gibt eine Serie von verschiedenen möglichen CpG-Motiven, die die verschie- denen Immunzellen in unterschiedlichem Maße stimulieren. Wir verwenden ein spezielles Motiv, das uns von Coley Pharmaceuticals im Rahmen unserer Zusammenarbeit zur Verfügung gestellt wurde, und derartig gestaltet ist, Thl und NK-Zellen optimal zu stimulieren (erhalten von Coley Pharmaceuticals, #M426).There are a number of different possible CpG motifs that stimulate the different immune cells to different degrees. We use a special motif provided by Coley Pharmaceuticals as part of our collaboration that is designed to optimally stimulate Thl and NK cells (obtained from Coley Pharmaceuticals, # M426).
Aus dem US-Patent Nr. 6,218,371 ist bekannt, dass man einen synergetischen Effekt beobachten kann, wenn man immun-stimulierende CpG Oligonukleotide und immun- verstärkende Cytokine wie z.B. GM-CSF kombiniert. Die Kombination mit zellulären Impfstoffen ist hingegen nicht beschrieben.From US Patent No. 6,218,371 it is known that a synergetic effect can be observed when immunostimulatory CpG oligonucleotides and immune-enhancing cytokines such as e.g. GM-CSF combined. However, the combination with cellular vaccines has not been described.
Eine weitere Ausführungsform dieser Erfindung besteht darin, dass das CpG Oli- gonukleotid ein Oligonukleotid mit einer Sequenz ist, enthaltend zumindest die folgende Formel:Another embodiment of this invention is that the CpG oligonucleotide is an oligonucleotide with a sequence containing at least the following formula:
5' X,CGX2 3'
wobei das Oligonukleotid zumindest 8 Nukleotide enthält, wobei C unmethyliert ist und wobei X} und X2 Nukleotide sind.5 'X, CGX 2 3' wherein the oligonucleotide contains at least 8 nucleotides, where C is unmethylated and where X} and X are 2 nucleotides.
Im Rahmen der vorliegenden Erfindung umfasst ein "Nukleotid" z.B. Adenosin, Cytidin, Guanosin, Thymidin oder Uridin oder modifizierte Formen dieser.In the context of the present invention, a "nucleotide" comprises e.g. Adenosine, cytidine, guanosine, thymidine or uridine or modified forms of these.
Gemäß einer weiteren Ausführungsform der Erfindung ist zusätzlich das G in der Oligonukleotidsequenz 5' XιCGX2 3' unmethyliert.According to a further embodiment of the invention, the G in the oligonucleotide sequence 5 'XιCGX 2 3' is also unmethylated.
Gemäß einer weiteren Ausführungsform der Erfindung ist das CpG Oligonukleotid ein Oligonukleotid mit einer Sequenz, enthaltend zumindest die folgende Formel:According to a further embodiment of the invention, the CpG oligonucleotide is an oligonucleotide with a sequence containing at least the following formula:
5' NιXιCGX2N2 3'5 'NιXιCGX 2 N 2 3'
wobei zumindest ein Nukleotid aufeinanderfolgende CpGs trennt und wobei Xi Adenin, Guanin oder Thymin ist, und wobei X2 Cytosin, Adenin oder Thymin ist und wobei N ein beliebiges Nukleotid ist und wobei Ni und N2 Nukleinsäurese- quenzen sind, zusammengesetzt aus je ungefähr 0-25 Nukleotiden. Gemäß einer besonders bevorzugten Ausführungsform enthalten Ni und N2 der Nukleinsäure kein CCGG Tetramer (Quadramer) oder nicht mehr als ein CCG oder CGG Tri- mer.wherein at least one nucleotide separates successive CpGs and where Xi is adenine, guanine or thymine, and where X 2 is cytosine, adenine or thymine and where N is any nucleotide and where Ni and N 2 are nucleic acid sequences, each composed of approximately 0 -25 nucleotides. According to a particularly preferred embodiment, Ni and N 2 of the nucleic acid contain no CCGG tetramer (quadramer) or no more than one CCG or CGG trimer.
Eine weitere Ausführungsform dieser Erfindung besteht darin, dass das CpG Oli- gonukleotid ein isoliertes Oligonukleotid mit einer Sequenz ist, enthaltend zumindest die folgende Formel:Another embodiment of this invention is that the CpG oligonucleotide is an isolated oligonucleotide with a sequence containing at least the following formula:
wobei zumindest ein Nukleotid aufeinanderfolgende CpGs trennt und wobei XιX2 ausgewählt ist aus der Gruppe bestehend aus GpT, GpA, ApA, GpG und ApT und
wobei X3 ausgewählt ist aus der Gruppe bestehend aus TpT, CpT, TpC, CpC und ApT und wobei N jedes beliebige Nukleotid ist und wobei Ni und N2 Nu- kleinsäuresequenzen sind, zusammengesetzt aus je ungefähr 0-25 Nukleotiden. Gemäß einer weiteren bevorzugten Ausführungsform enthalten Ni und N der Nukleinsäure kein CCGG Tetramer (Quadramer) oder nicht mehr als ein CCG oder CGG Trimer.where at least one nucleotide separates successive CpGs and where XιX 2 is selected from the group consisting of GpT, GpA, ApA, GpG and ApT and where X 3 is selected from the group consisting of TpT, CpT, TpC, CpC and ApT and where N is any nucleotide and where Ni and N 2 are nucleic acid sequences, each composed of approximately 0-25 nucleotides. According to a further preferred embodiment, Ni and N of the nucleic acid contain no CCGG tetramer (quadramer) or no more than one CCG or CGG trimer.
Eine weitere Ausfuhrungsform dieser Erfindung besteht darin, dass das CpG Oligonukleotid eine Nukleinsäuresequenz hat, wobei Ni und N2 kein CCGG Te- tramer (quadmer) oder nicht mehr als ein CCG oder CGG Trimer enthalten.A further embodiment of this invention is that the CpG oligonucleotide has a nucleic acid sequence, wherein Ni and N 2 contain no CCGG tetamer (quadmer) or no more than one CCG or CGG trimer.
Ein "CCGG Tetramer" bedeutet im Rahmen der vorliegenden Erfindung ein Oligonukleotid bestehend aus der Nukleotidfolge CCGG und ein "CCG bzw. CGG Trimer" bedeutet ein Oligonukleotid bestehend aus der Nukleotidfolge CCG bzw. CGG.In the context of the present invention, a “CCGG tetramer” means an oligonucleotide consisting of the nucleotide sequence CCGG and a “CCG or CGG trimer” means an oligonucleotide consisting of the nucleotide sequence CCG or CGG.
Gemäß einer weiteren Ausführungsform dieser Erfindung ist das CpG Oligonukleotid ein Oligonukleotid mit der Sequenz:According to a further embodiment of this invention, the CpG oligonucleotide is an oligonucleotide with the sequence:
5* TCN1TX1X2CGX3X4 3'5 * TCN 1 TX 1 X 2 CGX 3 X 4 3 '
wobei zumindest ein Nukleotid aufeinanderfolgende CpGs trennt und wobei XiX2 ausgewählt ist aus der Gruppe bestehend aus GpT, GpA, ApA, GpG und ApT und wobei X3X4 ausgewählt ist aus der Gruppe bestehend aus TpT, CpT, TpC, CpC und ApT und wobei N jedes beliebige Nukleotid ist und wobei Ni und N2 Nu- kleinsäuresequenzen sind, zusammengesetzt aus je ungefähr 0-25 Nukleotiden.wherein at least one nucleotide separates successive CpGs and wherein XiX 2 is selected from the group consisting of GpT, GpA, ApA, GpG and ApT and wherein X 3 X 4 is selected from the group consisting of TpT, CpT, TpC, CpC and ApT and where N is any nucleotide and where Ni and N 2 are nucleic acid sequences, each composed of approximately 0-25 nucleotides.
Gemäß einer weiteren Ausführungsform dieser Erfindung sind die CpG Oligonu- kleotide an die Oberfläche der Zelle gekoppelt. Die Oligonukleotide können ko- valent an die Oberfläche gebunden sein, z.B. durch Quervernetzungen (cross- linking) oder z.B. durch eine Wechselwirkung zwischen einem Zellmembranpro-
tein und dem CpG Oligonukleotid. Eine Möglichkeit besteht darin, ein IgM Im- munglobulin, das spezifisch für das jeweilige CpG Oligonukleotid ist, zu exprimieren und die Tumorzellen mit den jeweiligen CpG Oligonukleotiden zu inkubieren, und zwar vor der Injektion in einen Patienten. Man könnte auch CpG- Polylysin-Komplexe an die Oberfläche der Tumorzelle koppeln. Bi-spezifische Antikörper können auch verwendet werden, um CpG oder andere Adjuvanzien an ein Membranprotein der Tumorzelle zu koppeln.According to a further embodiment of this invention, the CpG oligonucleotides are coupled to the surface of the cell. The oligonucleotides can be covalently bound to the surface, for example by cross-linking or, for example, by an interaction between a cell membrane product. tein and the CpG oligonucleotide. One possibility is to express an IgM immunoglobulin, which is specific for the respective CpG oligonucleotide, and to incubate the tumor cells with the respective CpG oligonucleotides, before the injection into a patient. CpG-polylysine complexes could also be coupled to the surface of the tumor cell. Bi-specific antibodies can also be used to couple CpG or other adjuvants to a membrane protein of the tumor cell.
Der Begriff "Oligonukleotid" wird austauschbar verwendet und bedeutet multiple Nukleotide (d.h. Moleküle enthaltend einen Zucker (z.B. Ribose oder Desoxyri- bose) verbunden mit einer Phosphatgruppe und einer austauschbaren organischen Base, die entweder ein substituiertes Pyrimidin (z.B. Cytosin (C), Thymin (T) oder Uracil (U)) oder ein substituiertes Purin (z.B. Adenin (A) oder Guanin (G)) ist). Wie hierin verwendet bezieht sich der Begriff sowohl auf Oligoribonukleoti- de als auch auf Oligodesoxyribonukleotide. Der Begriff soll auch Polynukleoside (d.h. ein Polynukleotid minus dem Phosphat) umfassen und jedes andere Polymer enthaltend organische Basen. Nukleinsäuremoleküle können aus bestehenden Quellen für Nukleinsäuren (z.B. genomisch oder cDNA) erhalten werden, sind aber vorzugsweise synthetisch (z.B. produziert durch Oligonukleotidsynthese). Das gesamte CpG Oligonukleotid kann vollständig oder teilweise unmethyliert sein, aber zumindest das C des 5' CG 3' muss unmethyliert sein. Bevorzugt enthält das erfindungsgemäße CpG Oligonukleotid XιX2 ausgewählt aus der Gruppe bestehend aus GpT, GpG, GpA und ApA und X3X4 ausgewählt aus der Gruppe bestehend aus TpT, CpT und GpT. Um die Aufnahme in Zellen zu erleichtern sind CpG enthaltende Oligonukleotide vorzugsweise im Bereich von 8 bis 30 Basen Länge. Allerdings sind Nukleinsäuren jeder beliebigen Größe größer als 8 Nukleotide (sogar viele kb lang) in der Lage eine erfindungsgemäße Immunantwort auszulösen, solange genügend viele immunstimulierende Motive vorhanden sind, denn größere Nukleinsäuren werden innerhalb von Zellen zu Oligonukleotiden abgebaut. Bevorzugte synthetische Oligonukleotide enthalten kein CCGG Tetramer (Quadmer) oder nicht mehr als ein CCG oder CGG Trimer am oder nahe
den 5' und/oder 3' Enden. Stabilisierte Oligonukleotide, wo das Oligonukleotid eine Modifikation des Phosphatrückgrates beinhaltet, wie detaillierter unten diskutiert, sind auch bevorzugt. Die Modifikation kann, z.B., eine Phosphorthioat- oder Phosphordithioat-Modifikation sein. Bevorzugt geschieht die Modifikation des Phosphatrückgrates am 5' Ende der Nukleinsäure, z.B. an den ersten zwei Nukleotiden des 5' Endes des Oligonukleotides. Des weiteren kann die Modifikation des Phosphatrückgrates am 3' Ende der Nukleinsäure erfolgen, z.B. an den letzten 5 Nukleotiden des 3' Endes der Nukleinsäure. Alternativ dazu kann das Oligonukleotid komplett oder teilweise modifiziert sein.The term "oligonucleotide" is used interchangeably and means multiple nucleotides (ie molecules containing a sugar (e.g. ribose or deoxyribose) linked to a phosphate group and an exchangeable organic base which either contains a substituted pyrimidine (e.g. cytosine (C), thymine ( T) or uracil (U)) or a substituted purine (for example adenine (A) or guanine (G)). As used herein, the term refers to both oligoribonucleotides and oligodeoxyribonucleotides. The term is also meant to include polynucleosides (ie, a polynucleotide minus the phosphate) and any other polymer containing organic bases. Nucleic acid molecules can be obtained from existing sources of nucleic acids (eg genomic or cDNA), but are preferably synthetic (eg produced by oligonucleotide synthesis). All or some of the CpG oligonucleotide may be unmethylated, but at least the C of 5 'CG 3' must be unmethylated. Preferably, the CpG oligonucleotide XιX 2 selected from the group consisting of GpT, GpG, GpA and ApA and X 3 X 4 selected from the group consisting of TpT, CpT and GpT. In order to facilitate the uptake in cells, CpG-containing oligonucleotides are preferably in the range from 8 to 30 bases in length. However, nucleic acids of any size larger than 8 nucleotides (even many kb long) are capable of triggering an immune response according to the invention, as long as there are enough immunostimulatory motifs, because larger nucleic acids are broken down into oligonucleotides within cells. Preferred synthetic oligonucleotides contain no CCGG tetramer (quadmer) or no more than one CCG or CGG trimer at or near the 5 'and / or 3' ends. Stabilized oligonucleotides where the oligonucleotide involves modification of the phosphate backbone, as discussed in more detail below, are also preferred. The modification can be, for example, a phosphorothioate or phosphorodithioate modification. The modification of the phosphate backbone preferably takes place at the 5 'end of the nucleic acid, for example on the first two nucleotides of the 5' end of the oligonucleotide. Furthermore, the modification of the phosphate backbone can take place at the 3 'end of the nucleic acid, for example on the last 5 nucleotides of the 3' end of the nucleic acid. Alternatively, the oligonucleotide can be completely or partially modified.
Vorzugsweise ist das CpG Oligonukleotid im Bereich zwischen 8 und 100 und besonders bevorzugt zwischen 8 und 30 Nukleotiden groß. Alternativ dazu können CpG Oligonukleotide in großem Maßstab in Plasmiden produziert werden und zu Oligonukleotiden abgebaut werden.The CpG oligonucleotide is preferably large in the range between 8 and 100 and particularly preferably between 8 and 30 nucleotides. Alternatively, CpG oligonucleotides can be produced on a large scale in plasmids and broken down into oligonucleotides.
Das CpG Oligonukleotid und mindestens ein immunverstärkendes (immunopo- tentiating) Cytokin können dem behandelten Versuchsobjekt direkt verabreicht werden oder können zusammen mit einem Nukleinsäurenabgabekomplex (nucleic acid delivery complex) verabreicht werden. "Nukleinsäure/Cytokin- Abgabekomplex" soll ein Nukleinsäuremolekül und/oder ein Cytokin bedeuten, das assoziiert ist (z.B. ionisch oder kovalent gebunden an oder eingekapselt innerhalb) mit einem Mittel zur Zielsteuerung (z.B. ein Molekül das in einer höheraffinen Bindung an die Zielzelle (z.B. Oberflächen von dendritischen Zellen und/oder erhöhte zelluläre Aufnahme durch Zielzellen) resultiert). Beispiele für Nuklein- säure/Cytokin- Abgabekomplexe beinhalten Nukleinsäure/Cytokine assoziiert mit: einem Sterol (z.B. Cholesterol), einem Lipid (z.B. ein kationisches Lipid, Viro- som oder Liposom) oder einem zielzellspezifischen Bindemittel (z.B. ein Ligand erkannt von einem zielzellspezifischen Rezeptor). Es ist bevorzugt, dass die Komplexe in vivo genügend stabil sind, so dass signifikantes Entkoppeln vor der Internalisierung durch die Zielzelle verhindert wird. Allerdings ist es bevorzugt,
dass der Komplex unter geeigneten Bedingungen innerhalb der Zelle spaltbar ist, so dass das Nukleinsäure/Cytokin in funktionaler Form freigesetzt wird.The CpG oligonucleotide and at least one immunopotentiating cytokine can be administered directly to the treated test object or can be administered together with a nucleic acid delivery complex. "Nucleic acid / cytokine delivery complex" is intended to mean a nucleic acid molecule and / or a cytokine which is associated (for example ionically or covalently bound to or encapsulated within) with a means for targeting (for example a molecule which binds to the target cell in a higher affinity (for example Surfaces of dendritic cells and / or increased cellular uptake by target cells) results). Examples of nucleic acid / cytokine delivery complexes include nucleic acid / cytokines associated with: a sterol (eg cholesterol), a lipid (eg a cationic lipid, virosom or liposome) or a target cell specific binding agent (eg a ligand recognized by a target cell specific receptor) ). It is preferred that the complexes are sufficiently stable in vivo to prevent significant decoupling prior to internalization by the target cell. However, it is preferred that the complex can be cleaved within the cell under suitable conditions, so that the nucleic acid / cytokine is released in functional form.
Das CpG Oligonukleotid kann ein Oligonukleotid mit palindromen Sequenzen sein. "Palindrome Sequenz" soll eine invertierte Wiederholung (inverted repeat) bedeuten (d.h. eine Sequenz wie ABCDEE'D'C'B'A', wobei A und A' Basen sind, die die üblichen Watson-Crick-Basenpaare bilden können). In vivo können solcheThe CpG oligonucleotide can be an oligonucleotide with palindromic sequences. "Palindrome sequence" is intended to mean an inverted repeat (i.e., a sequence like ABCDEE'D'C'B'A ', where A and A' are bases that can form the usual Watson-Crick base pairs). In vivo, such
Sequenzen doppelsträngige Strukturen bilden. In einer Ausführungsform enthält das CpG Oligonukleotid eine palindrome Sequenz. Eine palindrome Sequenz be- zieht sich in diesem Zusammenhang auf ein Palindrom, wobei das CpG Teil desSequences form double-stranded structures. In one embodiment, the CpG oligonucleotide contains a palindrome sequence. In this context, a palindrome sequence relates to a palindrome, the CpG being part of the
Palindroms ist, und bevorzugt das Zentrum des Palindroms ist. In einer anderenIs palindrome, and is preferably the center of the palindrome. In another
Ausführungsform ist das CpG Oligonukleotid frei von einem Palindrom. Ein CpGIn one embodiment, the CpG oligonucleotide is free from a palindrome. A CpG
Oligonukleotid, das frei von einem Palindrom ist, ist eines, bei dem das CpGOligonucleotide that is free of a palindrome is one in which the CpG
Dinukleotid nicht Teil eines Palindroms ist. Ein derartiges Oligonukleotid kann ein Palindrom enthalten, bei dem das CpG nicht Teil des Palindroms ist.Dinucleotide is not part of a palindrome. Such an oligonucleotide can contain a palindrome in which the CpG is not part of the palindrome.
Das CpG Oligonukleotid kann ein stabilisiertes Nukleinsäuremolekül sein. Ein "stabilisiertes Nukleinsäuremolekül" soll ein Nukleinsäuremolekül bezeichnen, das relativ resistent gegen in vivo Abbau ist (z.B. durch eine Exo- oder Endo- Nuklease). Stabilisierung kann eine Funktion der Länge oder der Sekundärstruktur sein. Unmethylierte CpG Oligonukleotide, die mehrere 10 kb bis mehrere 100 kb lang sind, sind relativ resistent gegen einen Abbau in vivo. Bei kürzeren CpG Oligonukleotide kann die Sekundärstruktur stabilisieren und deren Effekt erhöhen. Falls z.B. das 3' Ende eines Oligonukleotids Selbstkomplementarität zu einer weiter oben gelegenen Region aufweist, so dass es sich zurückfalten und eine Art "stem loop structure" ausbilden kann, dann wird das Oligonukleotid stabilisiert und weist daher mehr Aktivität auf.The CpG oligonucleotide can be a stabilized nucleic acid molecule. A "stabilized nucleic acid molecule" is intended to mean a nucleic acid molecule that is relatively resistant to in vivo degradation (e.g. by an exo- or endo-nuclease). Stabilization can be a function of the length or the secondary structure. Unmethylated CpG oligonucleotides that are several 10 kb to several 100 kb long are relatively resistant to degradation in vivo. With shorter CpG oligonucleotides, the secondary structure can stabilize and increase its effect. If e.g. the 3 'end of an oligonucleotide has self-complementarity with a region located higher up, so that it can fold back and form a kind of "stem loop structure", then the oligonucleotide is stabilized and therefore has more activity.
Bevorzugte stabilisierte Oligonukleotide der vorliegenden Erfindung haben ein modifiziertes Rückgrat. Es wurde gezeigt, dass die Modifikation des Oligonu- kleotid-Rückgrates erhöhte Aktivität des CpG Oligonukleotids bewirkt, wenn die-
ses in vivo verabreicht wird. CpG Konstrukte, die zumindest zwei Phosphorthioat- Verknüpfungen am 5 '-Ende des Oligonukleotids aufweisen und mehrere Phosphorthioat- Verknüpfungen am 3'-Ende, bevorzugt 5 solcher Verknüpfungen, bewirkten maximale Aktivität und schützten das Oligonukleotid vor Abbau durch intrazelluläre Exo- und Endonukleasen. Andere modifizierte Oligonukleotide schließen Phosphodiester modifizierte Oligonukleotide, Kombinationen von Phosphodiester und Phosphorthioat Oligonukleotide, Methylphosphonat, Methyl- phosphorthioat, Phosphordithioat und Kombinationen aus diesen mit ein. Jede dieser Kombinationen und ihr besonderer Effekt auf Immunzellen ist detaillierter in US 6,207,646 und US 6,239,116 diskutiert und der gesamte Inhalt der letzteren wird hiermit durch diese Bezugnahme in diese Anmeldung eingeschlossen. Es wird angenommen, dass diese modifizierten Oligonukleotide wegen ihrer erhöhten Nukleaseresistenz, erhöhter zellulärer Aufnahme, erhöhter Proteinbindung und/oder veränderter intrazellulärer Lokalisationen eine größere stimulierende Aktivität zeigen können.Preferred stabilized oligonucleotides of the present invention have a modified backbone. The modification of the oligonucleotide backbone has been shown to cause increased activity of the CpG oligonucleotide when the it is administered in vivo. CpG constructs that have at least two phosphorothioate linkages at the 5 'end of the oligonucleotide and several phosphorothioate linkages at the 3' end, preferably 5 such linkages, brought about maximum activity and protected the oligonucleotide from degradation by intracellular exo- and endonucleases. Other modified oligonucleotides include phosphodiester modified oligonucleotides, combinations of phosphodiester and phosphorothioate oligonucleotides, methylphosphonate, methylphosphorothioate, phosphorodithioate, and combinations thereof. Each of these combinations and their particular effect on immune cells is discussed in greater detail in US 6,207,646 and US 6,239,116 and the entire content of the latter is hereby incorporated by reference into this application. It is believed that these modified oligonucleotides may show greater stimulatory activity due to their increased nuclease resistance, increased cellular uptake, increased protein binding and / or altered intracellular locations.
Sowohl Phosphorthioat als auch Phosphodiester Oligonukleotide, die CpG-Motive enthalten, sind in APCs wie z.B. dendritischen Zellen aktiv. Allerdings sind, basierend auf der Konzentration, die benötigt ist um CpG spezifische Effekte zu induzieren, die CpG Oligonukleotide mit nukleaseresistentem Phosphorthioat- Rückgrat wirksamer (2 μg/ml für die Phosphorthioate vs. eine Gesamtmenge von 90 μg/ml für Phosphodiester).Both phosphorothioate and phosphodiester oligonucleotides containing CpG motifs are found in APCs such as e.g. active dendritic cells. However, based on the concentration needed to induce CpG specific effects, the CpG oligonucleotides with nuclease resistant phosphorothioate backbone are more effective (2 μg / ml for the phosphorothioates vs. a total of 90 μg / ml for the phosphodiester).
Andere stabilisierte Oligonukleotide schließen ein: nicht-ionische DNA-Analoga, wie z.B. Alkyl- und Aryl-Phosphate (bei denen der geladene Phosphonatsauer- stoff ersetzt ist durch eine Alkyl- oder Aryl-Gruppe), Phosphodiester und Alkyl- Phosphotriester, bei denen die geladene Sauerstoffgruppe alkyliert ist. Oligonukleotide, die Diol enthalten, wie z.B. Tetraethylenglycol oder Hexaethylenglycol, an je einem oder an beiden Enden sind bekannt als im Wesentlichen resistent ge- gen Nukleaseabbau.
Weitere erfindungsgemäße Adjuvanzien, die als Toll-like-receptor-Agonisten wirken, sind Lipopolysaccharide (LPS) bzw. Komponenten davon, wie z.B. der Lipid A-Anteil oder der Poly- oder Oligosaccharidanteil. LPS sind die hauptsächlichen Außenmembrankomponenten von nahezu allen Gram-negativen Bakterien und sind bekannt dafür, als starke Stimulatoren des Immunsystems zu wirken. LPS bestehen aus einer Poly- oder Oligosaccharidregion, die durch das Lipid A in der äußeren Bakterienmembran verankert sind. Die spezifische, zelluläre Erkennung des LPS/Lipid A wird durch die gemeinsame extrazelluläre Interaktion des LPS binding protein, der membrangebundenen oder der löslichen Form von CD 14 und dem Toll-like-receptor 4*MD2-Komplex vermittelt. Dies führt zu einer raschen Aktivierung eines intrazellulären Signalnetzwerks, das stark homolog zu der Signalkaskade von IL-1 und IL-8 ist (Alexander C and Rietschel ET (2001) J Endotoxin Res 7, 3, 167-202).Other stabilized oligonucleotides include: non-ionic DNA analogs such as alkyl and aryl phosphates (in which the charged phosphonate oxygen is replaced by an alkyl or aryl group), phosphodiesters and alkyl phosphotriesters, in which the charged oxygen group is alkylated. Oligonucleotides containing diol, such as tetraethylene glycol or hexaethylene glycol, at one or both ends are known to be essentially resistant to nuclease degradation. Further adjuvants according to the invention which act as toll-like receptor agonists are lipopolysaccharides (LPS) or components thereof, such as the lipid A portion or the poly or oligosaccharide portion. LPS are the main outer membrane components of almost all Gram-negative bacteria and are known to act as strong stimulators of the immune system. LPS consist of a poly- or oligosaccharide region, which are anchored by the lipid A in the outer bacterial membrane. The specific, cellular recognition of the LPS / Lipid A is mediated by the joint extracellular interaction of the LPS binding protein, the membrane-bound or the soluble form of CD 14 and the Toll-like-receptor 4 * MD2 complex. This leads to the rapid activation of an intracellular signal network that is strongly homologous to the signal cascade of IL-1 and IL-8 (Alexander C and Rietschel ET (2001) J Endotoxin Res 7, 3, 167-202).
Gemäß einer weiteren Ausführungsform der Erfindung ist das Adjuvans daher LPS.According to a further embodiment of the invention, the adjuvant is therefore LPS.
Gemäß einer weiteren Ausführungsform ist das Adjuvans von Bacillus Calmette- Guerin Zellwandgerüst (BCG-CWS) abgeleitet. Von BCG-CWS ist bekannt, dass es ein Ligand der Toll-like-receptors 2 und 4 ist und die Differenzierung von Immunzellen auslösen kann (Matsumoto M et al (2001) Int Immunopharmacol 1, 8, 1559-69).According to a further embodiment, 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 can trigger the differentiation of immune cells (Matsumoto M et al (2001) Int Immunopharmacol 1, 8, 1559-69).
Gemäß einer weiteren Ausführungsform der Erfindung ist das Adjuvans ein Supe- rantigen. Superantigene sind Antigene, die direkt an T-Zellrezeptoren und MHC- Moleküle binden und eine direkte Aktivierung der T-Zellen bewirken. Von Superantigenen ist bekannt, dass diese auch eine adjuvante Wirkung haben können (siehe z.B. Okamoto S et al (2001) Infect. Immun. 69,11, 6633-42). Bekannte Superantigene sind z.B. Staphylococcus aureus Enterotoxine A, B, C, D und E (SEA, SEB, SEC, SED, SEE), Staphylococcal aureus toxic shock syndrome toxin 1
(TSST-1), Staphylococcal exfoliating toxin oder Streptococcal pyrogenic exoto- xins.According to a further embodiment of the invention, 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, for example, Okamoto S et al (2001) Infect. Immun. 69, 11, 6633-42). Known superantigens are, for example, 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.
Gemäß einer weiteren erfindungsgemäßen Ausführungsform ist das Adjuvans ein Agens, das die Signalwirkung von CTLA-4 inhibiert.According to a further embodiment according to the invention, the adjuvant is an agent which inhibits the signaling action of CTLA-4.
CTLA-4 (cytotoxic T-lymphocyte associated antigen 4) ist ein Rezeptor, der nach Aktivierung die Immunantwort bremst, da er als Antagonist zu CD28 fungiert. Er wird in niedriger Kopienzahl durch aktivierte T-Zellen exprimiert, bindet aber mit einer ca. 20-fach höheren Affinität an B7 als dessen eigentlicher Rezeptor CD28. Es ist bekannt, dass eine lösliche Form der extrazellulären Domäne von CTLA-4 B7 bindet und in vivo T-Zell-abhängige Antikörper Immunantworten unterdrückt.CTLA-4 (cytotoxic T-lymphocyte associated antigen 4) is a receptor that, after activation, slows down the immune response because it acts as an antagonist to CD28. It is expressed in a low copy number by activated T cells, but binds to B7 with an approximately 20-fold higher affinity than its actual receptor CD28. It is known that a soluble form of the extracellular domain of CTLA-4 B7 binds and suppresses T cell-dependent antibodies in vivo immune responses.
Dabei kann es sich z.B. um Antikörper oder Antikörperfragmente handeln, die spezifisch an die extrazelluläre Domäne von CTLA-4 binden und dessen Signalwirkung inhibieren. Die Generierung bzw. das Screening derartiger Antikörper bzw. Antikörperfragmente ist dem Fachmann bekannt (siehe z.B. WO 0032231). Weitere Agenzien, die geeignet sind, CTLA-4 zu binden und dessen Signaling zu inhibieren, sind kleine organische Moleküle, Peptidanaloga oder lösliche T- Zellrezeptoren (siehe WO 9720574).It can e.g. are antibodies or antibody fragments that bind specifically to the extracellular domain of CTLA-4 and inhibit its signaling action. The generation or screening of such antibodies or antibody fragments is known to the person skilled in the art (see, for example, WO 0032231). Other agents which are suitable for binding CTLA-4 and inhibiting its signaling are small organic molecules, peptide analogs or soluble T cell receptors (see WO 9720574).
Gegenstand der Erfindung ist auch die Verwendung einer Zusammensetzung enthaltend zumindest eine Tumorzelle, die zumindest ein Cytokin, Chemokin und/oder ein co-stimulierendes Molekül exprimiert und eine wirksame Menge von zumindest einem Adjuvans zur Herstellung eines Arzneimittels zur Behandlung oder Vorbeugung von Tumoren. Für die Tumorzelle, das Cytokin, Chemokin und/oder co-stimulierende Molekül und für das Adjuvans gilt das oben gesagte.The invention also relates to the use of a composition comprising at least one tumor cell which expresses at least one cytokine, chemokine and / or a co-stimulating molecule and an effective amount of at least one adjuvant for the manufacture of a medicament for the treatment or prevention of tumors. The above statements apply to the tumor cell, the cytokine, chemokine and / or co-stimulating molecule and to the adjuvant.
Gegenstand der Erfindung ist auch ein Verfahren zur Herstellung eines Arznei- mittels zur Therapie oder Vorbeugung von Tumoren, wobei zumindest eine Tumorzelle, die zumindest ein Cytokin, Chemokin und/oder ein co-stimulierendes
Molekül exprimiert und eine wirksame Menge von zumindest einem Adjuvans gemischt werden.The invention also relates to a method for producing a medicament for the therapy or prevention of tumors, at least one tumor cell containing at least one cytokine, chemokine and / or a co-stimulant Molecule expressed and an effective amount of at least one adjuvant mixed.
Gegenstand der Erfindung ist auch ein Verfahren zum Behandeln oder Vorbeugen von Tumoren, bei dem man einen Patienten eine wirksame Menge von Tumorzellen, die zumindest ein Cytokin, Chemokin und/ ein co-stiumlierendes Molekül exprimieren und eine wirksame Menge von zumindest einem Adjuvans verabreicht. Für die Tumorzelle, das Cytokin, Chemokin und/oder co-stimulatorische Molekül und für das Adjuvans gilt das oben gesagt.The invention also relates to a method for treating or preventing tumors, in which a patient is administered an effective amount of tumor cells which express at least one cytokine, chemokine and / or a co-stiumlating molecule and an effective amount of at least one adjuvant. This applies to the tumor cell, the cytokine, chemokine and / or co-stimulatory molecule and to the adjuvant.
Gemäß einer bevorzugten Ausführungsform wird die Tumorzelle, die zumindest ein Cytokin, Chemokin und/oder ein co-stimulierendes Molekül exprimiert, durch Transduktion mit rekombinantem Adeno-assoziiertem Virus (AAV) hergestellt. AAV- Vektoren wurden wie in WO 00/47757 beschrieben hergestellt.According to a preferred embodiment, the tumor cell, which expresses at least one cytokine, chemokine and / or a co-stimulating molecule, is produced by transduction with recombinant adeno-associated virus (AAV). AAV vectors were produced as described in WO 00/47757.
Unter dem Begriff "Transduktion mit rekombinantem Adenoassoziiertem Virus (AAV)" wird verstanden, dass das/die Gen(e) für ein Cytokin, Chemokin und/oder ein co-stimulatorisches Molekül mittels eines oder mehrerer rekombinanter AAV in die Zelle eingebracht und als Folge dessen exprimiert werden. Die Herstellung geeigneter rekombinanter AAV ist dem Fachmann wohlbekannt (siehe z.B. WO 00/47757). Die im Rahmen dieser Erfindung verwendeten AAV-Vektoren wurden nach den Verfahren beschrieben in WO 00/47757 hergestellt.The term “transduction with recombinant adeno-associated virus (AAV)” is understood to mean that the gene (s) for a cytokine, chemokine and / or a co-stimulatory molecule are introduced into the cell by means of one or more recombinant AAVs and as a result of which are expressed. The preparation of suitable recombinant AAV is well known to those skilled in the art (see e.g. WO 00/47757). The AAV vectors used in the context of this invention were produced by the processes described in WO 00/47757.
Gemäß einer weiteren bevorzugten Ausführungsform wird das Adjuvans der Zell- Suspension zugegeben. Dabei werden Zellen und Adjuvans miteinander vermischt. Gegebenenfalls werden weitere Hufs- und Zusatzstoffe hinzugegeben.
BeispieleAccording to a further preferred embodiment, the adjuvant is added to the cell suspension. The cells and adjuvant are mixed together. If necessary, other hooves and additives are added. Examples
Beispiel 1example 1
1. Material1. Material
1. Zelllinien und Tiere:1. Cell lines and animals:
Weibliche C3H/He Mäuse, 6-7 Wochen alt, wurden von Harlan, Borchen, Deutschland bezogen. Die Melanomzelllinie K-1735-M2 wurde freundlicherweise von Dr. Souberbielle (King's College, London) und Prof. I. J. Fidler (University of Texas M. D. Anderson Cancer Center, Houston, USA) zur Verfügung gestellt. Desweiteren wurde die bekannte Maus-Melanomzelllinie B16F10 verwendet.Female C3H / He mice, 6-7 weeks old, were obtained from Harlan, Borchen, Germany. The K-1735-M2 melanoma cell line was kindly developed by Dr. Souberbielle (King's College, London) and Prof. I. J. Fidler (University of Texas M. D. Anderson Cancer Center, Houston, USA). The well-known mouse melanoma cell line B16F10 was also used.
CpG Oligondinukleotide wurden durch eine Kollaboration mit der Coley Pharmaceuticals Group ™ zur Verfügung gestellt.CpG oligonucleotide nucleotides were provided through a collaboration with Coley Pharmaceuticals Group ™.
2. Methoden2. Methods
1. Erzeugung von HEL-exprimierenden Tumorzellen:1. Generation of HEL-Expressing Tumor Cells:
Für ein allogenes Impfschema: zwei stabile HEL-exprimierende Zelllinien, die für das allogene Impfexperiment erzeugt wurden, wurden verwendet: B16-HEL-61 (H2-b) und K-1735-HEL-48 (H2-k).For an allogeneic vaccination scheme: two stable HEL-expressing cell lines generated for the allogeneic vaccination experiment were used: B16-HEL-61 (H2-b) and K-1735-HEL-48 (H2-k).
Für die Erzeugung von stabilen Transfektanten der B16F10 und K-1735 Me- lanomzellen, wurde der Expressionsvektor pcDNA3neo-HEL kloniert. Dazu wur- de das HEL Gen aus dem Vektor pcDNAl-HEL ausgeschnitten und in den Expressionsvektor pcDNA3neo ligiert, der ein Neomycinresistenzgen zur Selektion
trägt. Die Transfektion von B16F10 und K-1735 Zellen wurde unter Verwendung von Lipofectamine® in einer 15 cm Zellkulturschale durchgeführt. Positive Zellen wurden unter Verwendung von G418-enthaltendem Selektionsmedium (800 μg/ml) selektiert. Nach 2-3 Wochen wurden einzelne Klone gepickt und expan- diert. Die Klone wurden auf die Expression des Transgens mit RT-PCR und Western Blot getestet. Die zwei Klone mit der besten Expressionsrate wurden für Impfexperimente gewählt.The expression vector pcDNA3neo-HEL was cloned for the generation of stable transfectants of the B16F10 and K-1735 melanoma cells. For this purpose, the HEL gene was cut out of the vector pcDNAl-HEL and ligated into the expression vector pcDNA3neo, which is a neomycin resistance gene for selection wearing. Transfection of B16F10 and K-1735 cells was performed using Lipofectamine ® in a 15 cm cell culture dish. Positive cells were selected using selection medium containing G418 (800 μg / 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. The two clones with the best expression rate were chosen for vaccination experiments.
RT-PCR: RNA-Präparation wurde unter Verwendung von 2-5 x 10 Zellen, QIAshredder Säulen (#79654) und dem RNeasy Kit (#74104) von QIAgen® durchgeführt. DNA (z.B. episomale Plasmid DNA) wurde mit RNAse-freier DNAse entfernt (#776785, Röche®). RNA wurde zu cDNA transkribiert unter Verwendung des Gene Amp RNA PCR Gore Kit (Perkin Eimer®, #N808-0143). PCR für HEL und ß-Actin wurde mit dem QIAgen Taq-Mastermix Kit (# 1007 544) und den folgenden Primern durchgeführt:RT-PCR: RNA preparation was performed using 2-5 x 10 cells, QIAshredder columns (# 79654) and the RNeasy Kit (# 74104) from QIAgen ® . DNA (eg episomal plasmid DNA) was removed with RNAse-free DNAse (# 776785, Röche ® ). RNA was transcribed to cDNA using the Gene Amp RNA PCR Kit Gore (Perkin Elmer ®, # N808-0143). PCR for HEL and ß-actin was carried out with the QIAgen Taq master mix kit (# 1007 544) and the following primers:
HEL-up (5'-AGG TCT TTG CTA ATC TTG GTG C-3 ')HEL-up (5'-AGG TCT TTG CTA ATC TTG GTG C-3 ')
HEL-down (5'-GGC AGC CTC TGA TCC ACG-3 ') mu ß-Actin-up (5'-GAT CCT GAC CGA GCG TGG CTA C-3* mu ß-Actin-down (5'-CAA CGT CAC ACT TCA TGA TGG AAT TG-3')HEL-down (5'-GGC AGC CTC TGA TCC ACG-3 ') must-actin-up (5'-GAT CCT GAC CGA GCG TGG CTA C-3 * must-actin-down (5'-CAA CGT CAC ACT TCA TGA TGG AAT TG-3 ')
Die erhaltenen Fragmente waren das HEL-Fragment (430 bp) und das ß-Actin- Fragment (290 bp).The fragments obtained were the HEL fragment (430 bp) and the β-actin fragment (290 bp).
Western Blot:Western blot:
Zellen wurden mit Zelllysepuffer lysiert. Lysate wurden mit DTT enthaltendem Ladepuffer auf 12% Pofyacrylamidgelen laufen gelassen. Hühnereierlysozym (Sigma®, #L4631) wurde als Standard verwendet. Der Transfer auf Nitrozellulosemembranen wurde mit einem Semi-dry Transfersystem durchgeführt. Blockie- rung und Markierung mit Antikörpern wurde in 5% Trockenmagermilchlösung (in Tris gepufferter Salzlösung, 0,01% Tween (TBST)) durchgeführt.
Antikörper: biotinylierter anti-HEL 1 :200 (RDI, #RDI-Lyszym-BT)Cells were lysed with cell lysis buffer. Lysates were run with loading buffer containing DTT on 12% pofyacrylamide gels. Hühnereierlysozym (Sigma ®, # L4631) was used as standard. The transfer to nitrocellulose membranes was carried out with a semi-dry transfer system. Blocking and labeling with antibodies was carried out in 5% dry skimmed milk solution (in Tris buffered saline, 0.01% Tween (TBST)). Antibody: biotinylated anti-HEL 1: 200 (RDI, # RDI-Lyszym-BT)
Streptavidin-HRP 1 :5000 (Sigma®, #S-5512)Streptavidin-HRP 1: 5000 (Sigma ® , # S-5512)
Super Signal (Pierce®, #34080) wurde als Substrat verwendet. Röntgenfilme wur- den 30 Sekunden lang bis zu eine Stunde lang exponiert.Super Signal (Pierce ®, # 34080) was used as substrate. X-ray films were exposed for 30 seconds to an hour.
2. Erzeugung von B7.2 und GM-CSF exprimierenden Kl 735 (-HEL) und B16F10-HEL Impfzellen:2. Generation of B7.2 and GM-CSF expressing Kl 735 (-HEL) and B16F10-HEL vaccine cells:
K1735 und K-1735-HEL Zellen, die das murine B7.2, GM-CSF oder beide Moleküle exprimieren, wurden durch Transduktion mit rekombinantem Adeno- assoziiertem Virus (AAV) hergestellt. AAV-Vektoren wurden hergestellt wie beschrieben in WO 00/47757.K1735 and K-1735-HEL cells expressing murine B7.2, GM-CSF or both molecules were produced by transduction with recombinant adeno-associated virus (AAV). AAV vectors were produced as described in WO 00/47757.
B16-HEL Zellen, die nicht effizient mit rekombinantem AAV transduziert werden können, wurden mit Polyfect (QIAgen, #301107) transfiziert, um transient die zwei Moleküle B7.2 und/oder GM-CSF zu exprimieren. Die Expressionsraten von GM-CSF waren vergleichbar mit denen in K1735-HEL Zellen, die von B7.2 waren etwas schlechter.B16-HEL cells that cannot be efficiently transduced with recombinant AAV were transfected with Polyfect (QIAgen, # 301107) to transiently express the two molecules B7.2 and / or GM-CSF. The expression rates of GM-CSF were comparable to those in K1735-HEL cells, those of B7.2 were slightly worse.
Die Impfzellen wurden bestrahlt und in flüssigem Stickstoff gelagert.The vaccine cells were irradiated and stored in liquid nitrogen.
Um Zellen für eine Applikation in der Maus vorzubereiten, wurden die Zellen aufgetaut, drei Mal mit PBS gewaschen und auf eine Zellzahl von 3xl05 Zellen pro Dosis in PBS eingestellt.In order to prepare cells for application in the mouse, the cells were thawed, washed three times with PBS and adjusted to a cell number of 3 × 10 5 cells per dose in PBS.
3. Detektion der Expression von GM-CSF und B7.2:3. Detection of the Expression of GM-CSF and B7.2:
Sekretiertes GM-CSF wurde nach 48 Stunden im Überstand von transduzierten oder transfizierten Zellen unter Verwendung des Enzym-gekoppelten Immunas- says (ELISA) Kits OptEIA Maus GM-CSF Set von Pharmingen (San Diego,
USA) delektiert. B7.2 Expression wurde durch Flusscytometrie unter Verwendung des Antikörpers GL1 (Pharmingen) nachgewiesen.Secreted GM-CSF was after 48 hours in the supernatant of transduced or transfected cells using the enzyme-linked immunoassay (ELISA) kit OptEIA mouse GM-CSF set from Pharmingen (San Diego, USA) detected. B7.2 expression was detected by flow cytometry using the antibody GL1 (Pharmingen).
4. Verwendung von CpG Oligonukleotiden als Adjuvans:4. Use of CpG oligonucleotides as adjuvant:
Für Gruppen, die mit Adjuvans geimpft wurden, wurde CpG der Zellsuspension oder PBS in einer Konzentration von 10 μg pro Dosis zugegeben.For groups vaccinated with adjuvant, CpG was added to the cell suspension or PBS at a concentration of 10 μg per dose.
5. Analyse der Lungenmetastasen:5. Analysis of lung metastases:
Mäuse wurden durch Genickdislokation getötet, die Lungen wurden präpariert, gewogen und fixiert. Im Falle der Lungen von C3H Mäusen wurde Bouin's Reagenz verwendet (Bezug auf: Current protocols in Immunology). Die Anzahl der Metastasen wurde am Seziermikroskop gezählt.Mice were killed by neck dislocation, the lungs were prepared, weighed and fixed. In the case of C3H mouse lungs, Bouin's reagent was used (refer to: Current protocols in Immunology). The number of metastases was counted on the dissecting microscope.
6. Präparation von Milzzellen T-Zellen6. Preparation of spleen cells T cells
Die Milzen der geimpften Mäuse wurden beim Sezieren der Tiere herausgenommen und in Medium bis zur weiteren Verarbeitung gelagert. Um eine Suspension von Einzelzellen zu erhalten wurden die Milzen unter Verwendung eines "Cell strainer" (70 μl/Nunc®) aufgeschlossen. Die Zellen wurden einmal gewaschen und dann von Makrophagen durch Passage durch Nylonwolle aufgereinigt. Die extrahierten T-Zellen wurden einmal pro Woche mit bestrahlten, autologen Tumorzellen re-stimuliert. ConA sup aus Rattenmilz (T stim™ cultures Supplement, Colla- borative Biomedical Products, #354115) wurde in einer Konzentration von 1-3 % zugesetzt, um das Wachstum zu verbessern.The spleens of the vaccinated mice were removed when the animals were dissected and stored in medium until further processing. In order to obtain a suspension of single cells, the spleens were disrupted using a "cell strainer" (70 μl / Nunc ® ). The cells were washed once and then purified from macrophages by passage through nylon wool. The extracted T cells were re-stimulated with irradiated, autologous tumor cells once a week. Rat spleen ConA sup (T stim ™ cultures Supplement, Collaborative Biomedical Products, # 354115) was added at a concentration of 1-3% to improve growth.
7. 51Cr-Freissetzungstest:7. 51 Cr release test:
5 Tage nach der Re-Stimulation wurden T-Zellkulturen geemtet, gewaschen und als Triplets auf Rundbodenplatten mit 96 Vertiefungen bei einer Zellzahl von
l,8xl05, 6xl04, 2xl04 und 6,7xl03 Zellen pro Vertiefung ausplattiert. Lebende Zielzellen wurden eine Stunde lang bei 37°C mit 51Chrom markiert, vier Mal gewaschen und zugesetzt, so dass ein Endverhältnis von Effektor zu Zielzelle von 90:1, 30:1 , 10:1 und 3:1 erhalten wurde. Um NK-Lyse zu blockieren wurden unmarkierte YAC-1 Zellen in einem Verhältnis von 1:5 bis 1:10 zu den Zielzellen zugegeben. Nach 5-stündiger Inkubation wurde der Überstand gesammelt und auf LUMA-Platten überführt. Am nächsten Tag wurden die getrockneten Platten in einem ß-Zähler (Packard) gezählt. Spezifische Lyse wurde mit der folgenden Formel berechnet:5 days after the re-stimulation, T-cell cultures were harvested, washed and as triplets on round-bottom plates with 96 wells with a cell number of 1, 8xl0 5 , 6xl0 4 , 2xl0 4 and 6.7xl0 3 cells per well plated. Living target cells were labeled with 51 chromium at 37 ° C for one hour, washed four times and added so that a final effector to target cell ratio of 90: 1, 30: 1, 10: 1 and 3: 1 was obtained. In order to block NK lysis, unlabeled YAC-1 cells were added in a ratio of 1: 5 to 1:10 to the target cells. After 5 hours of incubation, the supernatant was collected and transferred to LUMA plates. The next day the dried plates were counted in a β-counter (Packard). Specific lysis was calculated using the following formula:
% spezifische Lyse = (Testlyse - Spontane Lyse) / (Maximale Lyse - Spontane Lyse) * 100% specific lysis = (test lysis - spontaneous lysis) / (maximum lysis - spontaneous lysis) * 100
Beispiel 2Example 2
Therapeutische, autologe Impfung gegen K1735 Melanom mit und ohne CpG:Therapeutic, autologous vaccination against K1735 melanoma with and without CpG:
Mäuse, die mit K-1735 Zellen, die B7.2/GM-CSF coexprimierten, geimpft wurden, entwickelten ein geringeres durchschnittliches Lungengewicht als Tiere, die mit PBS geimpft wurden (206,4 + 13,6 mg im Vergleich zu 339,5 ± 75,8 mg und 166,80 ± 10,7 mg im Vergleich zu 200,75 ± 42,8 mg, respektiv). Die Kombination von K1735-B7.2-GM-CSF mit CpG erhöhte den therapeutischen Effekt. In allen Gruppen reduzierte eine Verzögerung des Impfbeginns den therapeutischen Effekt, mit Ausnahme von K1735-B7.2-GM+CpG. In letzterer Gruppe wurden in allen Gruppen vergleichbare Ergebnisse gesehen, mit gewisser Schwankung. In TV20 zeigte die Gruppe, die mit PBS beginnend an Tag 7 und 11 geimpft wurde, seltsame Ergebnisse, die nicht erklärt werden können und in weiteren Experimenten nicht wieder auftauchten. Mice vaccinated with K-1735 cells co-expressing B7.2 / GM-CSF developed a lower average lung weight than animals vaccinated with PBS (206.4 + 13.6 mg compared to 339.5 ± 75.8 mg and 166.80 ± 10.7 mg compared to 200.75 ± 42.8 mg, respectively). The combination of K1735-B7.2-GM-CSF with CpG increased the therapeutic effect. Delaying the start of vaccination reduced the therapeutic effect in all groups, with the exception of K1735-B7.2-GM + CpG. In the latter group, comparable results were seen in all groups, with some variation. In TV20, the group vaccinated with PBS starting on days 7 and 11 showed strange results that cannot be explained and did not reappear in further experiments.
Bislang wurden zwei autologe, therapeutische Experimente in C3H/He Mäusen durchgeführt. Die Ergebnisse sind in der folgenden Tabelle zusammengefasst. Experiment TV24 zeigte eine geringere Tumorlast und darum keine klaren Unterschiede zwischen den Gruppen:
So far, two autologous, therapeutic experiments have been carried out in C3H / He mice. The results are summarized in the following table. Experiment TV24 showed a lower tumor burden and therefore no clear differences between the groups:
aten: Durchschnittswert und Standardfehler
In zwei separaten Experimenten (TV26 und TV27) wurde eine Tumorinduktion in C3H/He Mäuse am Tag 0 mit 6xl04 K-1735 Zellen durch intravenöse Injektion ausgelöst. Die Impfung wurde an den Tagen 4 und 11 in 3 Gruppen mit 3xl05 K- 1735 Zellen durchgeführt. Bei der Gruppe 1 (WT) waren es K-1735 Zellen, bei der Gruppe 2 waren es K-1735 Zellen, die B7.2 exprimieren, und bei der Gruppe 3 waren es K-1735 Zellen, die B7.2 exprimieren, zusammen mit CpG. Am Tag 21 wurden die Tiere getötet und das durchschnittliche Lungengewicht und die Anzahl der Metastasen bestimmt. aten: average value and standard error In two separate experiments (TV26 and TV27), tumor induction in C3H / He mice was triggered on day 0 with 6 × 10 4 K-1735 cells by intravenous injection. The vaccination was carried out on days 4 and 11 in 3 groups with 3 × 10 5 K- 1735 cells. Group 1 (WT) was K-1735 cells, Group 2 was K-1735 cells expressing B7.2, and Group 3 was K-1735 cells expressing B7.2 together with CpG. The animals were sacrificed on day 21 and the average lung weight and number of metastases were determined.
In beiden Experimenten zeigte die Kombination von B7.2 und CpG den besten therapeutischen Effekt (TV26, siehe Figur 5A), wobei Experiment TV27 einen klaren synergetischen Effekt von B7.2 und CpG zeigt (Figur 5B). Allerdings waren im Experiment TV26 (Figur 5A) die Unterschiede zwischen den Gruppen mit B7.2 und B7.2/CpG nicht signifikant, was durch die recht geringe Anzahl an Me- tastasen erklärt werden kann, die in allen Gruppen dieses speziellen Experiments auftrat. Darum zeigt die Kombination von CpG mit B7.2 einen synergetischen Effekt in diesem Maus-Modell.In both experiments, the combination of B7.2 and CpG showed the best therapeutic effect (TV26, see FIG. 5A), experiment TV27 showing a clear synergetic effect of B7.2 and CpG (FIG. 5B). However, in the TV26 experiment (FIG. 5A) the differences between the groups with B7.2 and B7.2 / CpG were not significant, which can be explained by the rather small number of metastases which occurred in all groups of this special experiment. Therefore the combination of CpG with B7.2 shows a synergetic effect in this mouse model.
Im Vergleich zu ähnlichen Experimenten mit Tumorzellen, die andere Cytokine und/oder co-stimulierende Moleküle exprimieren, schien die Kombination von B7.2 und CpG einen der stärksten bislang beobachteten synergetischen Effekt zu erzielen.
Compared to similar experiments with tumor cells that express other cytokines and / or co-stimulating molecules, the combination of B7.2 and CpG appeared to achieve one of the strongest synergistic effects observed to date.
2. Therapeutische, allogene Impfung gegen K1735-hel Melanom mit und ohne CpG Oligonukleotid:2. Therapeutic, allogeneic vaccination against K1735-hel melanoma with and without CpG oligonucleotide:
Mäuse, die nach der Tumorinduktion mit wt Tumorzellen mit B16F10-HEL Zellen, die B7.2/GM-CSF coexprimieren, geimpft wurden, entwickelten ein signifikant geringeres durchschnittliches Lungengewicht als Tiere, die mit Kontroll- Wildtypzellen geimpft wurden (404 mg im Vergleich zu 564 mg (Figur 6A)). Der therapeutische Effekt von B7.2/GM-CSF exprimierenden autologen und allogenen Impfzellen war vergleichbar. Darüber hinaus weist dies daraufhin, dass der Impfstoff Tumor-reduzierende Aktivität zeigt, selbst zu einem Zeitpunkt, zu dem der Organismus bereits eine wachsende Tumormasse hat.Mice vaccinated with B16F10-HEL cells co-expressing B7.2 / GM-CSF after tumor induction with wt developed a significantly lower average lung weight than animals vaccinated with control wild-type cells (404 mg compared to 564 mg (Figure 6A)). The therapeutic effect of autologous and allogeneic vaccine cells expressing B7.2 / GM-CSF was comparable. In addition, this indicates that the vaccine exhibits tumor-reducing activity even at a time when the organism already has a growing tumor mass.
Ein ähnlicher Effekt, allerdings weniger ausgeprägt, wurde beim Vergleich der Metastasenknoten in der Lunge von behandelten Mäusen und Kontrolltieren beobachtet (Figur 6B).
A similar effect, albeit less pronounced, was observed when comparing the metastatic nodules in the lungs of treated mice and control animals (FIG. 6B).
Daten: Durchschnittswerte und StandardfehlerData: averages and standard errors
3. T-Zellexperimente:3. T cell experiments:
Um das lytische Potential der T-Zellen aus geimpften Mäusen zu testen, wurden Milzzellen in Kultur genommen und wie in Material und Methoden beschrieben re-stimuliert. Als Testsystem wurde ein Chrom-Freisetzungsassay gegen autologe Zielzellen durchgeführt.In order to test the lytic potential of the T cells from vaccinated mice, spleen cells were taken in culture and re-stimulated as described in the material and methods. A chromium release assay against autologous target cells was carried out as a test system.
Aus dem Experiment TV20 wurden die Milzzellen von 2 Tieren der folgenden Gruppen in Kultur genommen:
From the experiment TV20, the spleen cells of 2 animals from the following groups were taken in culture:
Es stellte sich heraus, dass nur T-Zellen, die aus Mäusen stammten, die mit K- 1735-B7.2-GM-CSF Zellen mit oder ohne CpG geimpft worden waren, effizient expandiert werden konnten. PBS oder PBS und CpG schienen kein ausreichender Stimulus zu sein um eine T-Zell-Proliferation zu induzieren, die für langfristige Expansion in vitro ausreicht. Nach 2-3 Runden Re-Stimulation wuchsen nur die Zelllinien, die in Figur 7 gezeigt sind, gut.It was found that only T cells derived from mice vaccinated with K-1735-B7.2-GM-CSF cells with or without CpG could be expanded efficiently. PBS or PBS and CpG did not appear to be a sufficient stimulus to induce T cell proliferation sufficient for long-term expansion in vitro. After 2-3 rounds of re-stimulation, only the cell lines shown in Figure 7 grew well.
In einem Chrom-Freisetzungstest zeigten die Zellen aus mit K-1735-B7.2-GM- CSF (+/- CpG) geimpften Mäusen eine Lyse der Zielzellen. In diesem Fall zeigte die Kombination eines Impfstoffes mit CpG die besten Effekte (Tier Nummer 79). Spezifische Lyse von bis zu 70 % konnte beobachtet werden. Ein Beispiel für einen derartigen Chrom- Assay ist in Figur 7 gezeigt.In a chromium release test, the cells from mice vaccinated with K-1735-B7.2-GM-CSF (+/- CpG) showed lysis of the target cells. In this case the combination of a vaccine with CpG showed the best effects (animal number 79). Specific lysis of up to 70% could be observed. An example of such a chromium assay is shown in FIG. 7.
4. Auswertung4. Evaluation
In den Experimenten, die wir durchgeführt haben, versuchten wir den Effekt von CpG als Adjuvans mit zellulären Melanom-Impfstoffen zu kombinieren, die B7.2 und GM-CSF trugen. Dies sollte in einer direkter Aktivierung von naiven CD8 T- Zellen und NK-Zellen durch B7.2 auf der Tumorzelle resultieren und außerdem zur Rekrutierung und Reifung von dendritischen Zellen durch GM-CSF führen. Letztere greifen Antigene auf, prozessieren und präsentieren sie im richtigen Zusammenhang, um CD8 T-Zellen via MHC-I und CD4 T-Zellen via MHC-II zu aktivieren. Außerdem aktiviert das CpG Motiv, das wir benutzten, APCs, NK- Zellen und CD4 Thl Zellen, die für sich schon Th2 Zellen blockieren und außer-
dem DC aktivieren, indem sie CD40L exprimieren. Das Zusammenwirken all dieser Faktoren sollte zu einer wirksamen Immunaktivierung führen, die in der Lage ist einen existierenden Tumor zu bekämpfen.In the experiments we performed, we tried to combine the effect of CpG as an adjuvant with cellular melanoma vaccines that carried B7.2 and GM-CSF. This should result in direct activation of naive CD8 T cells and NK cells by B7.2 on the tumor cell and also lead to recruitment and maturation of dendritic cells by GM-CSF. The latter take up antigens, process and present them in the right context to activate CD8 T cells via MHC-I and CD4 T cells via MHC-II. In addition, the CpG motif that we used activates APCs, NK cells and CD4 Thl cells, which in themselves block Th2 cells and also activate the DC by expressing CD40L. The interaction of all these factors should lead to an effective immune activation that is able to fight an existing tumor.
Dieses Potential wurde in einer autologen Situation getestet (derselbe MHC- Haplotyp zwischen Impfstoff und Tumor). Des weiteren wurde wurde CpG auch in einer allogenen Situation verwendet, bei der keine MHC-Übereinstimmung vorlag. Für dieses Experiment wurden Tumorzelllinien etabliert, die HEL (Hüh- nerei-Lysozym) als gemeinsames Modelltumorantigen trugen.This potential was tested in an autologous situation (the same MHC haplotype between vaccine and tumor). Furthermore, CpG was also used in an allogeneic situation in which there was no MHC agreement. For this experiment, tumor cell lines were established that carried HEL (chicken egg lysozyme) as a common model tumor antigen.
In dieser Reihe von Experimenten wurde das Potential der als Adjuvans wirkenden CpG Oligonukleotide, eine aufkommende Immunantwort gegen den Tumor, ausgelöst durch den Impfstoff, zu unterstützen in Impfstudien in einem Maus- Melanom-Modell untersucht.In this series of experiments, the potential of the adjuvant CpG oligonucleotides to support an emerging immune response against the tumor triggered by the vaccine was investigated in vaccination studies in a mouse melanoma model.
Als Tumormodell verwendeten wir K- 1735 -Zellen (H2-k Haplotyp) in einem autologen therapeutischen Impfschema in C3H/He Mäusen. Um Lungenmetastasen zu erhalten, wurden lebende Tumorzellen in die Schwanzvene injiziert. Als Impfstoffe wurden K- 1735 -Zellen, die mit rAAV-muB7.2-GM-CSF transduziert waren oder PBS verwendet, ohne oder in Kombination mit CpG. Die Impfung wurde entweder an Tag 4, 7 oder 11 nach der Tumorinduktion (challenge) begonnen. Am Tag 21 nach der Tumorinduktion (challenge) wurden die Tiere getötet um das Lungengewicht und die Anzahl an Lungenmetastaseknoten zu bestimmen.We used K-1735 cells (H2-k haplotype) as a tumor model in an autologous therapeutic vaccination scheme in C3H / He mice. To obtain lung metastases, living tumor cells were injected into the tail vein. K-1735 cells transduced with rAAV-muB7.2-GM-CSF or PBS were used as vaccines, without or in combination with CpG. Vaccination was started either on days 4, 7 or 11 after the tumor induction (challenge). On day 21 after the tumor induction (challenge), the animals were sacrificed to determine the lung weight and the number of lung metastasis nodes.
Alternativ dazu wurde in einem Fall CpG in einer allogenen Situation unter Verwendung von B16F10-HEL (H2-b Haplotyp) und K-1735-HEL (H2-k Haplotyp) Zellen in C3H/He Mäusen verwendet. Hier wurden, als völlig allogener Impfstoff, mit ρAAV-muB7.2-GM-CSF transfizierte B16F10-HEL Zellen verwendet. Hüh- nerei-Lysozym (HEL) ist ein Modellantigen aus Hühnereiweiß, das in der Litera- tur als gutes Antigen bekannt ist (Calin-Laurens V et al. (1993) Vaccine 11, 9, 974-8; , Cavani A et al. (1995) J Immunol 154, 3, 1232-8; Forquet F et al. (1990)
Eur J Immunol 20, 2325-32; Schneider SC et al. (2000) J Immunol 165, 1, 20-3; Thatcher TH et al. (2000) Immunology 99, 2, 235-42). Es wurde als Modell- Tumorantigen in der allogenen Situation verwendet. CpG wurde zusammen mit B16-HEL- Wildtypzellen appliziert oder in Kombination mit B7.2-GM-CSF trans- fizierten Zellen. Die transduzierten/transfizierten Impfzellen wurden vor der sub- cutanen Applikation in die Tiere bestrahlt, um Tumorwachstum zu verhindern.Alternatively, in one case, CpG was used in an allogeneic situation using B16F10-HEL (H2-b haplotype) and K-1735-HEL (H2-k haplotype) cells in C3H / He mice. Here, as a completely allogeneic vaccine, B16F10-HEL cells transfected with ρAAV-muB7.2-GM-CSF were used. Chicken egg lysozyme (HEL) is a model antigen from chicken egg white that is known in the literature as a good antigen (Calin-Laurens V et al. (1993) Vaccine 11, 9, 974-8; Cavani A et al . (1995) J Immunol 154, 3, 1232-8; Forquet F et al. (1990) Eur J Immunol 20, 2325-32; Schneider SC et al. (2000) J Immunol 165, 1, 20-3; Thatcher TH et al. (2000) Immunology 99, 2, 235-42). It has been used as a model tumor antigen in the allogeneic situation. CpG was applied together with B16-HEL wild-type cells or in combination with B7.2-GM-CSF transfected cells. The transduced / transfected vaccine cells were irradiated before the subcutaneous application in the animals in order to prevent tumor growth.
CpG verstärkte den Effekt eines autologen, wie auch eines allogenen Impfstoffes.CpG enhanced the effect of an autologous as well as an allogeneic vaccine.
PBS alleine sowie PBS und CpG hatten keinen oder nur einen geringen Effekt auf die Metastasenbildung. Eine Verzögerung der Impfung auf den Tag 7 oder 11 minimierte den "Impfeffekt". Eine Impfung mit bestrahlten Tumorzellen, die B7.2/GM-CSF exprimierten, reduzierte zwar die Anzahl an Lungenmetastasen allerdings nicht so stark wie in Kombination mit CpG.PBS alone as well as PBS and CpG had little or no effect on metastasis. Delaying vaccination to day 7 or 11 minimized the "vaccination effect". Vaccination with irradiated tumor cells that expressed B7.2 / GM-CSF did not, however, reduce the number of lung metastases as much as in combination with CpG.
Femer wurde im Gegensatz zur Vakzinierung ohne CpG bei Verwendung von CpG nach der Applikation der lebenden Tumorzellen (Tumorinduktion) eine Zeitverzögerung in Bezug auf den Beginn der Impfung toleriert. So war das Lungengewicht und die Anzahl an Metastasen bei einem Impfbeginn an Tag 4 so hoch wie bei Beginn an Tag 11. Dies erweitert folglich das zeitliche Fenster einer erfolgreichen Therapie. Dieser Effekt war daran gebunden, dass ebenso Tumorzellen, die ein Transgen trugen, appliziert wurden, so dass der Vakzinierungser- folg antigen-abhängig war.In contrast to vaccination without CpG, when using CpG after application of the living tumor cells (tumor induction), a time delay with regard to the start of vaccination was tolerated. The lung weight and the number of metastases at the start of vaccination on day 4 were as high as at the start on day 11. This consequently extends the time window for successful therapy. This effect was linked to the fact that tumor cells carrying a transgene were also applied, so that the success of the vaccination was dependent on the antigen.
Im Falle einer allogenen Situation erhöhte CpG ebenfalls den Effekt eines zellulären Impfstoffes. In diesem Fall zeigte eine Kombination mit allogenen Wildtypzellen alleine fast keinen Effekt, wohingegen eine Kombination mit B7.2-GM- CSF exprimierenden Zellen den Impfeffekt im Vergleich zu Zellen ohne CpG drastisch erhöhte.
Die zwei Maus-Melanom-Modelle B16F10 und Kl 735 wurden als relevantes Tumormodell verwendet, um den Effekt eines allogenen mit einem autologen Impfstoff bei malignem Melanom zu vergleichen. Dennoch muss der experimentelle Charakter von tierischen Tumormodellen im Generellen und die Heteroge- neität zwischen verschiedenen Tumoren bedacht werden (Maus vs. Mensch, zwischen einzelnen Patienten). Solche Modelle können eher qualitative Informationen vergleichender Natur geben als absolute, quantitative Informationen über die therapeutische Wirksamkeit in einer humanen Situation, wenn dies berücksichtigt wird.In the case of an allogeneic situation, CpG also increased the effect of a cellular vaccine. In this case, a combination with allogeneic wild-type cells alone showed almost no effect, whereas a combination with cells expressing B7.2-GM-CSF drastically increased the vaccination effect compared to cells without CpG. The two mouse melanoma models B16F10 and Kl 735 were used as a relevant tumor model to compare the effect of an allogeneic with an autologous vaccine in malignant melanoma. Nevertheless, the experimental character of animal tumor models in general and the heterogeneity between different tumors must be considered (mouse vs. human, between individual patients). Such models can provide qualitative information of a comparative nature rather than absolute, quantitative information about the therapeutic effectiveness in a human situation if this is taken into account.
Der Zweck dieser Experimente war es, den stimulierenden Effekt von CpG als Adjuvans bei zellulärer Tumorimpfung gegen Melanom zu beweisen. Gemäß der oben diskutierten Theorie, auf die die Erfindung nicht beschränkt sein soll, sollte die Wirksamkeit des Impfstoffes erhöht sein, wenn der polarisierende Effekt von CpG zugunsten einer Thl -Antwort und auch die Aktivierung von Antigen- präsentierenden Zellen effizient ist.The purpose of these experiments was to demonstrate the stimulatory effect of CpG as an adjuvant in cellular tumor vaccination against melanoma. According to the theory discussed above, to which the invention should not be limited, the effectiveness of the vaccine should be increased if the polarizing effect of CpG in favor of a Thl response and also the activation of antigen-presenting cells is efficient.
Mit CpG in einer autologen Situation konnte der Beginn der Impfung in Richtung Ende des Experiments verschoben werden. Im Experiment dargestellt in Fig. 1 zeigen die drei Gmppen K-1735-B7.2-GM-CSF + CpG fast das gleiche Lungengewicht, während in den drei Gruppen K-1735-B7.2-GM-CSF das mittlere Lungengewicht ansteigt, je nachdem ob die Impfung an Tag 4, 7 oder 11 nach Tumorinduktion durchgeführt wurde. Das zeigt, dass mit CpG der Beginn der Impfung nicht so kritisch ist als ohne. Selbst bei einem Beginn an Tag 11 - an dem Tiere ihre zweite Impfung drei Tage vor dem Sezieren bekommen - war die Tumorlast nicht höher als bei einem Beginn an Tag 4.With CpG in an autologous situation, the start of vaccination could be shifted towards the end of the experiment. In the experiment shown in FIG. 1, the three groups K-1735-B7.2-GM-CSF + CpG show almost the same lung weight, while in the three groups K-1735-B7.2-GM-CSF the mean lung weight increases, depending on whether the vaccination was carried out on day 4, 7 or 11 after tumor induction. This shows that the start of vaccination is not as critical with CpG as without. Even at a start on day 11 - when animals received their second vaccination three days before dissection - the tumor burden was not higher than when they started on day 4.
Dieser Effekt scheint besonders bei der CpG-Impfung kombiniert mit Antigenen und Transgenen zu gelten. Dies kann im Experiment dargestellt in Fig. 6 gesehen werden, in dem autologe Tumorzellen zusammen mit CpG einen geringeren Effekt haben, wobei derselbe Impfstoff, der aber B7.2 und GM-CSF exprimiert, dra-
stisch die Tumorlast der Tiere reduziert. CpG alleine zeigte einen gewissen Effekt (siehe Beispiel, Punkt 3). Aber in diesem Fall wie auch im Fall einer Impfung nur mit B7.2-GM-CSF-exprimierenden Zellen verminderte sich der therapeutische Effekt bei einer Zeitverzögerung der ersten Impfung nach der Tumorinduktion (Challenge). Dieses Szenario kann in den beiden Experimenten gesehen werden, die in den Figuren 1 und 2 sowie 3 und 4 dargestellt sind. Der Zeitpunkt der ersten Impfung scheint in diesen Fällen kritisch zu sein, wie dies von van Elsas et al. (1999, J Exp Emd 190, 355-66) gezeigt wurde. Gemäß einer Erklärungsmöglichkeit, auf die die Erfindung nicht beschränkt sein soll, könnte dies auf eine Herun- terregulierung der CD3 ζ Kette im T-Zellrezeptor der T-Helferzellen aufgrund ihrer Aktivierung oder auf eine Fas L induzierte Herunterregulierung bevorzugt von T-Helfer-1 -Antworten zurückzuführen sein.This effect appears to apply particularly in the case of CpG vaccination combined with antigens and transgenes. This can be seen in the experiment shown in FIG. 6, in which autologous tumor cells together with CpG have a lower effect, the same vaccine, but which expresses B7.2 and GM-CSF, drastically reduces the tumor burden of the animals. CpG alone showed some effect (see example, point 3). But in this case as well as in the case of vaccination only with cells expressing B7.2-GM-CSF, the therapeutic effect was reduced if the first vaccination was delayed after tumor induction (challenge). This scenario can be seen in the two experiments shown in FIGS. 1 and 2 and 3 and 4. The timing of the first vaccination appears to be critical in these cases, as described by van Elsas et al. (1999, J Exp Emd 190, 355-66). According to one possible explanation, to which the invention is not to be restricted, this could be based on a downregulation of the CD3 ζ chain in the T cell receptor of the T helper cells due to their activation or on a Fas L induced downregulation, preferably of T helper 1 - Responses.
Bei einer therapeutischen Impfung konnten besonders gute Effekte durch eine Impfung beginnend am Tag 4 nach der Tumorinduktion (Challenge) ausgelöst werden. Ein späterer Start war weniger vorteilhaft. Die ausgeführten Experimente zeigen, dass eine Kombination eines Impfstoffes, der B7.2 und GM-CSF zusammen mit CpG exprimiert, diese negative Korrelation beenden kann. Somit ist bei den erfindungsgemäßen Zusammensetzungen der Zeitpunkt der Impfung nicht kritisch, was einen wesentlichen Vorteil der vorliegen Erfindung darstellt.With a therapeutic vaccination, particularly good effects could be triggered by vaccination starting on day 4 after the tumor induction (challenge). A later start was less advantageous. The experiments carried out show that a combination of a vaccine that expresses B7.2 and GM-CSF together with CpG can end this negative correlation. Thus, the timing of the vaccination is not critical in the compositions according to the invention, which represents a significant advantage of the present invention.
Die durchgeführten T-Zell-Experimente unterstützen die Daten aus den Tierexperimenten mit autologen Vakzinen.The T cell experiments carried out support the data from animal experiments with autologous vaccines.
Der 5 Cr-Freisetzungstest zeigt, dass die Tumorreduktionseffekte in Tieren, die mit PBS und CpG geimpft worden waren, auf einer angeborenen Immunität beruhen müssen und kein Fall von T-Zell-Stimulierung sein können, denn es wurde keine spezifische Lyse gefunden. Nur Tiere, die auch zelluläres Antigen bekamen (K-1735-B7.2-GM-CSF), waren in der Lage, eine deutliche T-Zell-Antwort zu entwickeln, was nicht nur an der Tumorabweisung in vivo gesehen werden kann, sondern auch in einem 51 Cr-Freisetzungstest detektierbar ist, was die lytische Ak-
tivität von T-Zellen verifiziert. Da die Daten zur Chrom-Freisetzung 2-3 Wochen nachdem Milzzellen in Kultur genommen worden waren, aufgenommen wurden, können die verantwortlichen Zellen nicht NK-Zellen sein. Nach 2-3 Wochen in Kultur sind NK-Zellen in normalen Fällen zum großen Teil verschwunden. Au- ßerdem wurden unmarkierte YAC-1 -Zellen bei den Chrom- Freisetzungsexperimenten zugesetzt um NK-Lyse zu blockieren. Darum korrespondieren die berechneten Werte für die spezifische Lyse mit cytotoxischer T- Zell-Lyse.The 5 Cr release test shows that the tumor reduction effects in animals vaccinated with PBS and CpG must be based on innate immunity and cannot be a case of T cell stimulation because no specific lysis was found. Only animals that also received cellular antigen (K-1735-B7.2-GM-CSF) were able to develop a clear T cell response, which can be seen not only in the tumor rejection in vivo, but also is also detectable in a 51 Cr release test, which activity of T cells verified. Since the chromium release data was collected 2-3 weeks after spleen cells were cultured, the responsible cells cannot be NK cells. After 2-3 weeks in culture, NK cells have largely disappeared in normal cases. In addition, unlabelled YAC-1 cells were added in the chromium release experiments to block NK lysis. Therefore, the calculated values for the specific lysis correspond to cytotoxic T cell lysis.
Insgesamt unterstützen alle Experimente die Rolle von CpG als Adjuvans für autologe und allogene Impfung, um die Stimulation von spezifischen cytotoxischen T-Zellen zu erhöhen und die Abweisung des Tumors zu induzieren.Overall, all experiments support the role of CpG as an adjuvant for autologous and allogeneic vaccination to increase the stimulation of specific cytotoxic T cells and to induce tumor rejection.
Beschreibung der Figuren:Description of the figures:
Figur 1: Therapeutische Impfung (autolog) mit B7.2/GM-CSF und/oder CpG (TV20): Mäuse wurden am Tag 21 nach der Herausforderung getötet und das Lungengewicht wurde auf einer Mikro-Waage bestimmt. Dargestellt ist das mittlere Lungengewicht in mg gegen den jeweiligen Versuchsansatz. Dabei beträgt das mittlere Lungengewicht einer gesunden Maus 140 mg. Die in Klammern gezeigten Zahlen benennen den Tag der ersten Impfung (Tag 4, 7 oder 11).Figure 1: Therapeutic vaccination (autologous) with B7.2 / GM-CSF and / or CpG (TV20): Mice were sacrificed on day 21 after the challenge and the lung weight was determined on a micro balance. The mean lung weight is shown in mg against the respective test approach. The mean lung weight of a healthy mouse is 140 mg. The numbers in brackets indicate the day of the first vaccination (day 4, 7 or 11).
Figur 2: Therapeutische Impfung (autolog) mit B7.2/GM-CSF und/oder CpG (TV20): Mäuse wurden am Tag 21 nach der Herausforderung getötet, Lungen wurden in einer Bound's Lösung fixiert und die Lungenmetastasen wurden unter Verwendung eines Seziermikroskops bestimmt. Dargestellt ist die mittlere Anzahl an Lungenmetastasen gegen den jeweiligen Versuchsansatz. Die in Klammem gezeigten Zahlen benennen den Tag der ersten Impfung (Tag 4, 7 oder 11).Figure 2: Therapeutic vaccination (autologous) with B7.2 / GM-CSF and / or CpG (TV20): mice were sacrificed on day 21 after the challenge, lungs were fixed in a Bound's solution and the lung metastases were determined using a dissecting microscope , The average number of lung metastases is shown against the respective test approach. The numbers shown in brackets indicate the day of the first vaccination (day 4, 7 or 11).
Figur 3: Therapeutische Impfung (autolog) mit B7.2/GM-CSF und/oder CpG (TV24): Mäuse wurden am Tag 21 nach der Herausforderung getötet und Lungen
gewogen. Dargestellt ist das mittlere Lungengewicht in mg gegen den jeweiligen Versuchsansatz. Dabei beträgt das mittlere Lungengewicht einer gesunden Maus 140 mg. Die in Klammem gezeigten Zahlen benennen den Tag der ersten Impfung (Tag 4, 7 oder 11).Figure 3: Therapeutic vaccination (autologous) with B7.2 / GM-CSF and / or CpG (TV24): Mice were sacrificed on day 21 after the challenge and lungs weighed. The mean lung weight is shown in mg against the respective test approach. The mean lung weight of a healthy mouse is 140 mg. The numbers shown in brackets indicate the day of the first vaccination (day 4, 7 or 11).
Figur 4: Therapeutische Impfung (autolog) mit B7.2/GM-CSF und/oder CpG (TV24): Mäuse wurden am Tag 21 nach der Herausforderung getötet, Lungen wurden in einer Bouin's Lösung fixiert und die Lungenmetastasen wurden unter Verwendung eines Seziermikroskops bestimmt. Dargestellt ist die mittlere Anzahl an Lungenmetastasen gegen den jeweiligen Versuchsansatz. Die in Klammem gezeigten Zahlen benennen den Tag der ersten Impfung (Tag 4, 7 oder 11).Figure 4: Therapeutic vaccination (autologous) with B7.2 / GM-CSF and / or CpG (TV24): Mice were sacrificed on day 21 after the challenge, lungs were fixed in a Bouin's solution and the lung metastases were determined using a dissection microscope , The average number of lung metastases is shown against the respective test approach. The numbers shown in brackets indicate the day of the first vaccination (day 4, 7 or 11).
Figur 5: Therapeutische Impfung von C3H/He Mäusen mit transduzierten Melanomzellen; Dargestellt ist das mittlere Lungengewicht in mg gegen den jeweili- gen Versuchsansatz. Dabei beträgt das mittlere Lungengewicht einer gesunden Maus 140 mg. Fig. 5 A zeigt das Experiment TV26 und Fig. 5 B zeigt das Experiment TV27.Figure 5: Therapeutic vaccination of C3H / He mice with transduced melanoma cells; The mean lung weight in mg is shown against the respective test approach. The mean lung weight of a healthy mouse is 140 mg. FIG. 5A shows the TV26 experiment and FIG. 5B shows the TV27 experiment.
Figur 6A: Therapeutische Impfung (allogen) von C3H He Mäusen mit trans- duzierten Melanomzellen (TV22): Mäuse wurden am Tag 21 nach der Herausforderung getötet und das Lungengewicht auf einer Mikro-Waage bestimmt. Dargestellt ist das mittlere Lungengewicht in mg gegen den jeweiligen Versuchsansatz. Dabei beträgt das mittlere Lungengewicht einer gesunden Maus 140 mg.Figure 6A: Therapeutic vaccination (allogeneic) of C3H He mice with transduced melanoma cells (TV22): Mice were sacrificed on day 21 after the challenge and the lung weight was determined on a micro balance. The mean lung weight is shown in mg against the respective test approach. The mean lung weight of a healthy mouse is 140 mg.
Figur 6B: Therapeutische Impfung (allogen) von C3H/He Mäusen mit transduzierten Melanomzellen (TV22): Mäuse wurden am Tag 21 nach der Herausforderung getötet, Lungen wurden in einer Bouin's Lösung fixiert und die Lungen unter Verwendung eines Sezemiermikroskops bestimmt. Dargestellt ist die mittlere Anzahl an Lungenmetastasen gegen den jeweiligen Versuchansatz.
Figur 7: 51Cr-Freisetzungstest von Milzzellen aus TV20: Milzzellen aus Tieren von TV20 wurden in vitro re-stimuliert mit bestrahlten K-1735-HEL Zellen. Am Tag 5 nach der Re-Stimulation wurden die Zellen inkubiert mit 51 -Chrommarkierten Zielzellen (K-1735-HEL), 4 Stunden lang mit den gezeigten Effektor- Zielzell- Verhältnissen. Überstände wurden in einem ß-Zähler gemessen. Dargestellt sind die % spezifische Lyse gegen das Verhältnis von Effektor- zu Zielzellen (E:T).
Figure 6B: Therapeutic vaccination (allogeneic) of C3H / He mice with transduced melanoma cells (TV22): Mice were sacrificed on day 21 after the challenge, lungs were fixed in a Bouin's solution and the lungs were determined using a dissection microscope. The average number of lung metastases is shown against the respective test approach. Figure 7: 51 Cr release test of spleen cells from TV20: Spleen cells from animals of TV20 were re-stimulated in vitro with irradiated K-1735-HEL cells. On day 5 after the re-stimulation, the cells were incubated with 51 chromium-labeled target cells (K-1735-HEL) for 4 hours with the effector-target cell ratios shown. Supernatants were measured in a ß counter. The% specific lysis is shown against the ratio of effector to target cells (E: T).
Claims
1. Zusammensetzung für die Impfung von Tumoren enthaltend zumindest eine Tumorzelle, die zumindest ein Cytokin, Chemokin und/oder ein co-stimulie- rendes Molekül exprimiert; und eine wirksame Menge von zumindest einem Adjuvans.1. Composition for the vaccination of tumors containing at least one tumor cell which expresses at least one cytokine, chemokine and / or a co-stimulating molecule; and an effective amount of at least one adjuvant.
2. Zusammensetzung enthaltend zumindest eine Tumorzelle, die zumindest ein Cytokin, Chemokin und/oder ein co-stimulierendes Molekül exprimiert; und eine wirksame Menge von zumindest einem Adjuvans.2. Composition comprising at least one tumor cell which expresses at least one cytokine, chemokine and / or a co-stimulating molecule; and an effective amount of at least one adjuvant.
3. Zusammensetzung nach Anspmch 1 oder 2, wobei die Tumorzelle von einem Prä-Tumor, einem Tumor oder einer Metastase abstammt.3. Composition according to Claim 1 or 2, the tumor cell being derived from a pre-tumor, a tumor or a metastasis.
4. Zusammensetzung nach einem der Ansprüche 1 bis 3, wobei die Tumorzelle autolog oder allogen bezüglich des geimpften Patienten ist.4. The composition according to any one of claims 1 to 3, wherein the tumor cell is autologous or allogeneic with respect to the vaccinated patient.
5. Zusammensetzung nach einem der Ansprüche 1 bis 4, wobei die Tumorzelle von einem Melanom, Eierstockkrebs, Brustkrebs, Kolonkarzinom, Leukämie,5. The composition according to any one of claims 1 to 4, wherein the tumor cell from melanoma, ovarian cancer, breast cancer, colon cancer, leukemia,
Lymphom, Nierenkarzinom, Lungenkarzinom, Prostatakrebs, Gebärmutterhalskrebs und/oder Gehirntumor abstammt.Lymphoma, kidney carcinoma, lung carcinoma, prostate cancer, cervical cancer and / or brain tumor.
6. Zusammensetzung nach einem der Ansprüche 1 bis 5, wobei die Tumorzelle genetisch modifiziert ist, so dass sie ein oder mehrere Moleküle aus der Grup- pe enthaltend Cytokine, Chemokine und/oder co-stimulierende Moleküle exprimiert.6. Composition according to one of claims 1 to 5, wherein the tumor cell is genetically modified so that it expresses one or more molecules from the group containing cytokines, chemokines and / or co-stimulating molecules.
7. Zusammensetzung nach einem der Ansprüche 1 bis 6, wobei das Cytokin/Chemokin ausgewählt ist aus der Gruppe enthaltend GM-CSF, G-CSF, IL- 1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IFN-alpha, IFN- beta, IFN-gamma, Flt3 L, Flt3, TNF-alpha, RANTES, MlPl-alpha, MIP1- beta, MIP1 -gamma, MlPl-delta, MIP2, MIP2-alpha, MIP2-beta, MIP3-alpha, MIP3-beta, MIP4, MIP5, MCP1, MCPl-beta, MCP2, MCP3, MCP4, MCP5, MCP6, 6cykine, Dcckl und DCDF.7. Composition according to one of claims 1 to 6, wherein the cytokine / chemokine is selected from the group comprising GM-CSF, G-CSF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IFN-alpha, IFN-beta, IFN-gamma, Flt3 L , Flt3, TNF-alpha, RANTES, MlPl-alpha, MIP1- beta, MIP1 -gamma, MlPl-delta, MIP2, MIP2-alpha, MIP2-beta, MIP3-alpha, MIP3-beta, MIP4, MIP5, MCP1, MCPl -beta, MCP2, MCP3, MCP4, MCP5, MCP6, 6cykine, Dcckl and DCDF.
8. Zusammensetzung nach einem der Ansprüche 1 bis 7, wobei das co- stimulierende Molekül ausgewählt ist aus der Gruppe enthaltend B7.1, B7.2, CD40, LIGHT, Ox40, 4.1.BB, Icos, 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 und TNFR-RP.8. Composition according to one of claims 1 to 7, wherein the co-stimulating molecule is selected from the group comprising B7.1, B7.2, CD40, LIGHT, Ox40, 4.1.BB, Icos, 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.
9. Zusammensetzung nach einem der Ansprüche 1 bis 8, wobei zumindest ein Cytokin, Chemokin und/oder co-stimulierendes Molekül ein mutiertes Molekül ist, einschließlich Punktmutationen, Deletionen oder Fusionen mit anderen Peptiden oder Proteinen.9. The composition according to any one of claims 1 to 8, wherein at least one cytokine, chemokine and / or co-stimulating molecule is a mutated molecule, including point mutations, deletions or fusions with other peptides or proteins.
10. Zusammensetzung nach einem der Ansprüche 1 bis 9, wobei das Adjuvans ein Agonist eines Toll-like-receptors ist.10. The composition according to any one of claims 1 to 9, wherein the adjuvant is an agonist of a toll-like receptor.
11. Zusammensetzung nach einem der Ansprüche 1 bis 10, wobei das Adjuvans ausgewählt ist aus der Gruppe bestehend aus CpG Oligonukleotiden, LPS und BCG-CWS.11. The composition according to any one of claims 1 to 10, wherein the adjuvant is selected from the group consisting of CpG oligonucleotides, LPS and BCG-CWS.
12. Zusammensetzung nach einem der Ansprüche 1 bis 11, wobei das CpG Oligonukleotid eine Sequenz hat, enthaltend zumindest die folgende Formel:12. The composition according to any one of claims 1 to 11, wherein the CpG oligonucleotide has a sequence containing at least the following formula:
5' XtCGXa 3'5 'XtCGXa 3'
wobei das Oligonukleotid zumindest 8 Nukleotide enthält, wobei C unmethyliert ist und wobei Xi und X2 Nukleotide sind. wherein the oligonucleotide contains at least 8 nucleotides, where C is unmethylated and where Xi and X are 2 nucleotides.
13. Zusammensetzung nach Anspmch 12, wobei zusätzlich das G unmethyliert ist.13. Composition according to Claim 12, the G additionally being unmethylated.
14. Zusammensetzung nach einem der Ansprüche 1 bis 11, wobei das CpG Oligonukleotid eine Sequenz hat, enthaltend zumindest die folgende Formel:14. The composition according to any one of claims 1 to 11, wherein the CpG oligonucleotide has a sequence containing at least the following formula:
5' NιX!CGX2N2 3'5 'NιX ! CGX 2 N 2 3 '
wobei zumindest ein Nukleotid aufeinanderfolgende CpGs trennt und wobei Xi Adenin, Guanin oder Thymin ist, und wobei X2 Cytosin, Adenin, oder Thymin ist und wobei N ein beliebiges Nukleotid ist und wobei Ni und N2 Nukleinsäuresequenzen sind, zusammengesetzt aus je ungefähr 0-25 Nukleo- tiden.where at least one nucleotide separates successive CpGs and where Xi is adenine, guanine or thymine, and where X 2 is cytosine, adenine, or thymine and where N is any nucleotide and where Ni and N 2 are nucleic acid sequences, each composed of about 0- 25 nucleotides.
15. Zusammensetzung nach einem der Ansprüche 1 bis 11, wobei das CpG Oligonukleotid eine Sequenz hat, enthaltend zumindest die folgende Formel:15. The composition according to any one of claims 1 to 11, wherein the CpG oligonucleotide has a sequence containing at least the following formula:
5' N1X1X2CGX3X4N2 3*5 'N 1 X 1 X 2 CGX 3 X 4 N 2 3 *
wobei zumindest ein Nukleotid aufeinanderfolgende CpGs trennt und wobei X1X2 ausgewählt ist aus der Gruppe bestehend aus GpT, GpA, ApA, GpG undwhere at least one nucleotide separates successive CpGs and where X 1 X 2 is selected from the group consisting of GpT, GpA, ApA, GpG and
ApT und wobei X3X4 ausgewählt ist aus der Gmppe bestehend aus TpT, CpT, TpC, CpC und ApT und wobei N jedes beliebige Nukleotid ist und wobei Ni und N2 Nukleinsäuresequenzen sind, zusammengesetzt aus je ungefähr 0-25 Nukleotiden.ApT and where X 3 X 4 is selected from the group consisting of TpT, CpT, TpC, CpC and ApT and where N is any nucleotide and where Ni and N 2 are nucleic acid sequences, each composed of about 0-25 nucleotides.
16. Zusammensetzung nach Anspruch 14 oder 15, wobei Ni und N2 der Nukleinsäure kein CCGG Tetramer (Quadramer) oder nicht mehr als ein CCG oder CGG Trimer enthalten.16. The composition of claim 14 or 15, wherein Ni and N 2 of the nucleic acid contain no CCGG tetramer (quadramer) or no more than one CCG or CGG trimer.
17. Zusammensetzung nach einem der Ansprüche 1 bis 11, wobei das CpG Oligonukleotid die Sequenz hat: 5' TCN1TX1X2CGX3X4 3'17. The composition according to any one of claims 1 to 11, wherein the CpG oligonucleotide has the sequence: 5 'TCN 1 TX 1 X 2 CGX 3 X 4 3'
wobei zumindest ein Nukleotid aufeinanderfolgende CpGs trennt und wobei XiX2 ausgewählt ist aus der Gruppe bestehend aus GpT, GpA, ApA, GpG und ApT und wobei X3X ausgewählt ist aus der Gruppe bestehend aus TpT, CpT, TpC, CpC und ApT und wobei N jedes beliebige Nukleotid ist und wobei Ni und N2 Nukleinsäuresequenzen sind, zusammengesetzt aus je ungefähr 0-25 Nukleotiden.wherein at least one nucleotide separates successive CpGs and where XiX 2 is selected from the group consisting of GpT, GpA, ApA, GpG and ApT and where X 3 X is selected from the group consisting of TpT, CpT, TpC, CpC and ApT and where N is any nucleotide and where Ni and N are 2 nucleic acid sequences, each composed of approximately 0-25 nucleotides.
18. Zusammensetzung nach einem der Ansprüche 12 bis 17, wobei das CpG Oligonukleotid an die Oberfläche der Zelle gekoppelt ist.18. The composition according to any one of claims 12 to 17, wherein the CpG oligonucleotide is coupled to the surface of the cell.
19. Zusammensetzung nach einem der Ansprüche 1 bis 9, wobei das Adjuvans ein Superantigen ist.19. The composition according to any one of claims 1 to 9, wherein the adjuvant is a superantigen.
20. Zusammensetzung nach einem der Ansprüche 1 bis 9, wobei das Adjuvans ein Agens ist, das die Signalwirkung von CTLA-4 inhibiert.20. The composition according to any one of claims 1 to 9, wherein the adjuvant is an agent that inhibits the signaling effect of CTLA-4.
21. Verwendung einer Zusammensetzung enthaltend zumindest eine Tumorzelle, die zumindest ein Cytokin, Chemokin und/oder ein co-stimulierendes Molekül exprimiert und eine wirksame Menge von zumindest einem Adjuvans zur Herstellung eines Arzneimittels zur Behandlung oder Vorbeugung von Tumoren.21. Use of a composition containing at least one tumor cell which expresses at least one cytokine, chemokine and / or a co-stimulating molecule and an effective amount of at least one adjuvant for the manufacture of a medicament for the treatment or prevention of tumors.
22. Verwendung nach Ansprach 21, wobei die Tumorzelle die in einem der Ansprüche 3 bis 6 aufgeführten Eigenschaften aufweist.22. Use according spoke 21, wherein the tumor cell has the properties listed in one of claims 3 to 6.
23. Verwendung nach Ansprach 21 oder 22, wobei das Cytokin/Chemokin die in Ansprach 7 oder 9 aufgeführten Eigenschaften aufweist.23. Use according to spoke 21 or 22, wherein the cytokine / chemokine has the properties listed in spoke 7 or 9.
24. Verwendung nach einem der Ansprüche 21 bis 23, wobei das co-stimulierende Molekül die in Ansprach 8 oder 9 aufgeführten Eigenschaften aufweist. 24. Use according to any one of claims 21 to 23, wherein the co-stimulating molecule has the properties listed in spoke 8 or 9.
25. Verwendung nach einem der Ansprüche 21 bis 24, wobei das Adjuvans die in einem der Ansprüche 10 bis 20 aufgeführten Eigenschaften aufweist.25. Use according to one of claims 21 to 24, wherein the adjuvant has the properties listed in one of claims 10 to 20.
26. Verfahren zur Herstellung eines Arzneimittels zur Therapie oder Vorbeugung von Tumoren, wobei zumindest eine Tumorzelle, die zumindest ein Cytokin, Chemokin und/oder ein co-stimulierendes Molekül exprimiert und eine wirksame Menge von zumindest einem Adjuvans gemischt werden.26. A method for producing a medicament for the therapy or prevention of tumors, wherein at least one tumor cell, which expresses at least one cytokine, chemokine and / or a co-stimulating molecule, and an effective amount of at least one adjuvant are mixed.
27. Verfahren nach Ansprach 26, wobei die Tumorzelle, die zumindest ein Cytokin, Chemokin und/oder ein co-stimulierendes Molekül exprimiert, durch Transduktion mit rekombinantem Adeno-assoziiertem Virus (AAV) herge- stellt wird.27. The method according to approached 26, wherein the tumor cell, which expresses at least one cytokine, chemokine and / or a co-stimulating molecule, is produced by transduction with recombinant adeno-associated virus (AAV).
28. Verfahren nach einem der Ansprüche 26 oder 27, wobei das Adjuvans der Zellsuspension zugegeben wird. 28. The method according to any one of claims 26 or 27, wherein the adjuvant is added to the cell suspension.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
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US33249701P | 2001-11-09 | 2001-11-09 | |
US332497P | 2001-11-09 | ||
PCT/EP2002/012527 WO2003039592A2 (en) | 2001-11-09 | 2002-11-08 | Cellular vaccines comprising adjuvants |
Publications (1)
Publication Number | Publication Date |
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EP1441759A2 true EP1441759A2 (en) | 2004-08-04 |
Family
ID=23298485
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
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EP02802659A Withdrawn EP1441759A2 (en) | 2001-11-09 | 2002-11-08 | Cellular vaccines comprising adjuvants |
EP02787620A Withdrawn EP1441758A2 (en) | 2001-11-09 | 2002-11-08 | Allogenic vaccine that contains a costimulatory polypeptide-expressing tumor cell |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
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EP02787620A Withdrawn EP1441758A2 (en) | 2001-11-09 | 2002-11-08 | Allogenic vaccine that contains a costimulatory polypeptide-expressing tumor cell |
Country Status (4)
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US (1) | US20050085433A1 (en) |
EP (2) | EP1441759A2 (en) |
CA (2) | CA2466698A1 (en) |
WO (2) | WO2003039591A2 (en) |
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DK1699480T3 (en) * | 2003-12-30 | 2011-10-10 | Mologen Ag | Allogeneic therapeutic tumor agent |
EP2296705A1 (en) * | 2008-06-24 | 2011-03-23 | Hadasit Medical Research Services And Development Ltd. | Ccl20-specific antibodies for cancer therapy |
AU2009308707A1 (en) * | 2008-10-31 | 2010-05-06 | Biogen Idec Ma Inc. | LIGHT targeting molecules and uses thereof |
EP3375791A1 (en) | 2009-09-30 | 2018-09-19 | Memorial Sloan Kettering Cancer Center | Combination immunotherapy for the treatment of cancer |
GB201403775D0 (en) | 2014-03-04 | 2014-04-16 | Kymab Ltd | Antibodies, uses & methods |
PL3273992T3 (en) | 2015-03-23 | 2020-11-16 | Jounce Therapeutics, Inc. | Antibodies to icos |
TW201723190A (en) | 2015-10-22 | 2017-07-01 | 永斯醫療股份有限公司 | Gene signatures for determining ICOS expression |
CN109312364A (en) * | 2016-03-18 | 2019-02-05 | 河谷细胞有限公司 | For infecting the multi-mode carrier of Dendritic Cells |
US9567399B1 (en) | 2016-06-20 | 2017-02-14 | Kymab Limited | Antibodies and immunocytokines |
TWI760352B (en) | 2016-08-09 | 2022-04-11 | 英商克馬伯有限公司 | Anti-icos antibodies |
WO2018083248A1 (en) | 2016-11-03 | 2018-05-11 | Kymab Limited | Antibodies, combinations comprising antibodies, biomarkers, uses & methods |
GB201709808D0 (en) | 2017-06-20 | 2017-08-02 | Kymab Ltd | Antibodies |
EP3728314A1 (en) | 2017-12-19 | 2020-10-28 | Kymab Limited | Bispecific antibody for icos and pd-l1 |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
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US6239116B1 (en) * | 1994-07-15 | 2001-05-29 | University Of Iowa Research Foundation | Immunostimulatory nucleic acid molecules |
US6207646B1 (en) * | 1994-07-15 | 2001-03-27 | University Of Iowa Research Foundation | Immunostimulatory nucleic acid molecules |
EP0869803B1 (en) * | 1995-12-28 | 2003-04-16 | The Johns Hopkins University School Of Medicine | Allogeneic paracrine cytokine tumor vaccines |
DE19608751B4 (en) * | 1996-03-06 | 2006-05-18 | Medigene Ag | Use of an adeno-associated virus vector to increase the immunogenicity of cells |
US6051428A (en) * | 1997-03-21 | 2000-04-18 | Sloan-Kettering Institute For Cancer Research | Rapid production of autologous tumor vaccines |
US6218371B1 (en) * | 1998-04-03 | 2001-04-17 | University Of Iowa Research Foundation | Methods and products for stimulating the immune system using immunotherapeutic oligonucleotides and cytokines |
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2002
- 2002-11-08 CA CA002466698A patent/CA2466698A1/en not_active Abandoned
- 2002-11-08 WO PCT/EP2002/012526 patent/WO2003039591A2/en not_active Application Discontinuation
- 2002-11-08 WO PCT/EP2002/012527 patent/WO2003039592A2/en not_active Application Discontinuation
- 2002-11-08 EP EP02802659A patent/EP1441759A2/en not_active Withdrawn
- 2002-11-08 CA CA002466530A patent/CA2466530A1/en not_active Abandoned
- 2002-11-08 EP EP02787620A patent/EP1441758A2/en not_active Withdrawn
- 2002-11-08 US US10/494,716 patent/US20050085433A1/en not_active Abandoned
Non-Patent Citations (1)
Title |
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See references of WO03039592A2 * |
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US20050085433A1 (en) | 2005-04-21 |
EP1441758A2 (en) | 2004-08-04 |
WO2003039591A2 (en) | 2003-05-15 |
CA2466530A1 (en) | 2003-05-15 |
CA2466698A1 (en) | 2003-05-15 |
WO2003039591A3 (en) | 2004-03-11 |
WO2003039592A2 (en) | 2003-05-15 |
WO2003039592A3 (en) | 2003-10-23 |
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