EP1441759A2 - Vaccins cellulaires comprenant des adjuvants - Google Patents

Vaccins cellulaires comprenant des adjuvants

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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
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EP
European Patent Office
Prior art keywords
cells
tumor
composition according
adjuvant
cpg
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EP02802659A
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German (de)
English (en)
Inventor
Claudia Breidenstein
John Nieland
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Medigene AG
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Medigene AG
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Publication of EP1441759A2 publication Critical patent/EP1441759A2/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5152—Tumor cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5156—Animal cells expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55516—Proteins; Peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55522—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/80—Vaccine for a specifically defined cancer

Definitions

  • the present invention relates to 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).

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Abstract

La présente invention concerne une composition permettant la vaccination contre des tumeurs contenant au moins une cellule tumorale qui exprime au moins une cytokine, une chimiokine et/ou une molécule co-stimulante, et une quantité efficace d'au moins un adjuvant, ainsi que l'utilisation d'une composition de ce type pour préparer un produit pharmaceutique permettant le traitement ou la prévention des tumeurs.
EP02802659A 2001-11-09 2002-11-08 Vaccins cellulaires comprenant des adjuvants Withdrawn EP1441759A2 (fr)

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PT1699480E (pt) * 2003-12-30 2011-08-30 Mologen Ag AGENTE TERAPjUTICO ANTI-TUMORAL ALOGÉNICO
EP2296705A1 (fr) * 2008-06-24 2011-03-23 Hadasit Medical Research Services And Development Ltd. Anticorps spécifiques de ccl20 pour la thérapie du cancer
WO2010051502A2 (fr) * 2008-10-31 2010-05-06 Biogen Idec Ma Inc. Molécules ciblant light et leurs utilisations
US8709417B2 (en) 2009-09-30 2014-04-29 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
CN107530428B (zh) 2015-03-23 2022-05-13 震动疗法股份有限公司 Icos的抗体
AU2016342269A1 (en) 2015-10-22 2018-03-29 Jounce Therapeutics, Inc. Gene signatures for determining icos expression
SG11201808058PA (en) * 2016-03-18 2018-10-30 Nantcell Inc Multimodal vector for dendritic cell infection
US9567399B1 (en) 2016-06-20 2017-02-14 Kymab Limited Antibodies and immunocytokines
CN116640214A (zh) 2016-08-09 2023-08-25 科马布有限公司 分离抗体及其应用
EP3534947A1 (fr) 2016-11-03 2019-09-11 Kymab Limited Anticorps, combinaisons comprenant des anticorps, biomarqueurs, utilisations et procédés
GB201709808D0 (en) 2017-06-20 2017-08-02 Kymab Ltd Antibodies
EP3728314A1 (fr) 2017-12-19 2020-10-28 Kymab Limited Anticorps bispécifique pour icos et pd-l1
CN114807229B (zh) * 2022-05-27 2024-09-24 中国科学院长春应用化学研究所 细胞膜、纳米疫苗及其制备方法和应用

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