WO2018211113A1 - Immunostimulating, highly pure oligosaccharides - Google Patents

Immunostimulating, highly pure oligosaccharides Download PDF

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WO2018211113A1
WO2018211113A1 PCT/EP2018/063207 EP2018063207W WO2018211113A1 WO 2018211113 A1 WO2018211113 A1 WO 2018211113A1 EP 2018063207 W EP2018063207 W EP 2018063207W WO 2018211113 A1 WO2018211113 A1 WO 2018211113A1
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chitin
nag
oligosaccharide
immune
subunits
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PCT/EP2018/063207
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German (de)
French (fr)
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Alexander Weber
Katharina Fuchs
Cecile Gouttefangeas
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Eberhard Karls Universitaet Tuebingen Medizinische Fakultaet
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/716—Glucans
    • A61K31/722—Chitin, chitosan
    • 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
    • A61K39/12—Viral antigens
    • 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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • 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
    • A61K2039/55583—Polysaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16111—Cytomegalovirus, e.g. human herpesvirus 5
    • C12N2710/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16211—Lymphocryptovirus, e.g. human herpesvirus 4, Epstein-Barr Virus
    • C12N2710/16234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to a high-purity oligosaccharide for use as an immunostimulant, a pharmaceutical composition comprising the high-purity oligosaccharide according to the invention, and the use of the high-purity oligosaccharide according to the invention in vitro for stimulating immune cells or for activating / binding the TLR2 receptor.
  • Immune stimulation refers to therapeutic measures aimed at activating the immune system.
  • immune stimulants i.
  • Substances that trigger an immune stimulation are used.
  • specific immune stimulation the activity of the immune system is directed to the elimination of a particular disease-causing factor, e.g. on an infectious microorganism, such as a virus or a bacterium, or on a tumor.
  • non-specific immune stimulation attempts to generally stimulate the immune system in order to favorably influence a disease.
  • a potent specific immune activation should be triggered.
  • An unspecific immune stimulation is, for example, from so-called adjuvants, which in drug formulations the specific drug or in vaccine preparations the usually be added to specific antigen to improve its effect or reinforce.
  • the antigen is responsible for the specific immune response and, for the strength of the immune response, essentially the adjuvant.
  • Adjuvants may also act alone, ie without the addition of exogenous specific antigens, by interacting with specific antigens already present in the tumor or the infected tissue.
  • vaccine adjuvants intended for use in humans are intended to activate the immune system in such a manner that the antigen contained in the vaccine is effectively presented to the immune system in the context of a danger signal, thereby providing potent and sustained T cell and / or antibody mediation Vaccine protection produced.
  • Many of the adjuvants currently used constitute so-called "micro-associated molecular pattern" (MAMPs), i. microbial-derived molecules that can be recognized as foreign by the innate immune system through pattern recognition receptors (PRRs).
  • MAMPs micro-associated molecular pattern
  • PRRs pattern recognition receptors
  • classical adjuvants used in already approved vaccines for infectious diseases tend to promote an antibody-mediated immune response but not a Th1-cell or cellular-mediated immune response that would be desirable, especially in cancer therapy.
  • tumor vaccination represents a promising therapeutic approach in the treatment of cancer patients; see. Walter et al. Multipeptide immune response to Cancer Vaccine IMA901 after single-dose cyclophosphamide associates with longer patient survival. Nature medicine 18, 1254-1261 (2012). Cancer vaccines or tumor vaccines are used which contain tumor-specific structures, such as, for example, tumor antigens or tumor-associated antigens, which are intended to induce or assist the immune system in a specific reaction against a specific tumor.
  • tumor antigens or tumor-associated antigens which are intended to induce or assist the immune system in a specific reaction against a specific tumor.
  • T-cell responses are usually relatively weak adjuvants for tumor vaccination available; see. Khong and Overwijk WW.
  • Adjuvants for peptide-based cancer vaccines J Immuno-Cancer 4: 56 (2016). Thus, urgently new immune stimulants and in particular adjuvant molecules are needed.
  • Chitin a homopoly or oligomer of ⁇ (1 -4) -N-acetyl-D-glucosamine (NAG), is the second most abundant polysaccharide in nature after cellulose. It is a component of fungal cell walls, the exoskeleton and digestive tract of insects and shellfish, and the microfilariae of parasitic nematodes. Chitin serves lower life forms as protection against harsh environmental conditions. Chitin does not occur in higher organisms, but not only chitin-degrading enzymes, so-called chitinases, but also many immunological effects on chitin exposure of body cells can be found; see. Mack et al.
  • Chitin differs from chitosan (poly- ⁇ (1 -4) -D-glucosamine) in the presence of an acetyl group on the amino group at position 2 of the ring.
  • the degree of acetylation (DA) in commercially available preparations determines whether the preparation is in the form of "chitin” (with a DA of> 50%) or "chitosan” (with a DA of ⁇ 50%).
  • DA degree of acetylation
  • chitin plays a role in the development of type 2 allergies, in which it contributes to the accumulation of eosinophilic granulocytes and other cells of the innate and adaptive immune system, for example in the respiratory tract ; see. Lee (supra), Reese et al. Chitin induces accumulation in tissue of innate immune cells associated with allergy.
  • chitin is usually in "large”(> 70 ⁇ ), “average” (70-40 ⁇ ), “small” (40-10 ⁇ ) and “very small”( ⁇ 10 ⁇ ) chitin , While sizes over 70 ⁇ are immunologically inert, "small” or very small chitin fragments, especially when inhaled, can activate alveolar macrophages and secretion cytokines such as IL-12, tumor necrosis factor (TNF), and IL-18 to lead; see. Lee (supra), Da Silva et al. Chitin is a size-dependent regulator of macrophage TNF and IL-10 production.
  • chitin as an immune stimulant, in particular as an adjuvant in vaccine compositions, therefore fails because the exact composition of the crude preparations is unknown.
  • the known chitin preparations do not have the purity of the immunostimulating component required for a drug, but are often contaminated with endotoxin or other contaminants.
  • the immune recognition receptor for chitin in humans is unknown, which prevents targeted drug development of chitin as an immune stimulant.
  • the substance should provide sufficient therapeutic safety to be used as an adjuvant in a vaccine can.
  • This object is achieved by a high purity oligosaccharide consisting of> 6 N-acetylglucosamine (NAG) subunits for use as an immune stimulant.
  • NAG N-acetylglucosamine
  • This object is also achieved by providing a pharmaceutical composition, preferably an immunostimulant, comprising a high purity oligosaccharide consisting of> 6 NAG subunits.
  • the problem underlying the invention is hereby completely solved.
  • the inventors were able to establish that in chitin a chemically-structurally well-defined oligosaccharide with> 6 NAG subunits has an immunostimulating effect. Furthermore, the inventors were the first to identify the natural receptor for chitin: Toll-like receptor 2 (TLR2). These findings now allow the development of a chitin-derived adjuvant that can be used in humans as a drug or drug additive or immune stimulant.
  • TLR2 is a recognition receptor of innate immunity (see Kawasaki and Kawai, Toll-like receptor signaling pathways, Front Immunol 5: 461, (2014)), which is expressed on various professional immune cells, but also on tissue cells such as epithelial cells or keratinocytes. Activation of TLR2 by agonists initiates a signaling cascade, e.g. the production of inflammatory cytokines, as well as other immunological phenomena in humans triggers. In general, TLR2 activation leads to an immune stimulation that triggers the activation of the adaptive immune response and thus activates cell- and / or antibody-mediated defense mechanisms. So far, various lipopeptides or lipids have been described as agonists of TLR2, e.g. acylated lipopeptides of various bacteria (e.g., Staphylococcus aureus) or
  • Mycobacteria (eg Mycobacterium tuberculosis), which are recognized by TLR2 together with TLR1 and TLR6 (see Jin et al .: Crystal structure of the TLR1 -TLR2 heterodimerically induced by binding of a tri-acylated lipopeptide.) Cell 130 (6): 1071 - 82 (2007); Kang et al. Recognition of lipopeptide patterns by toll-like receptor 2-toll-like receptor 6 heterodimer. / mmun / fy 31 (6): 873-84, (2009)). The fact that chitin is a specific agonist of TLR2 has not yet been clearly demonstrated, for example, by binding studies.
  • TLR2 is also recognized by endogenous substances associated with certain pathologies, such as oxidized LPL, which is shared by TLR2 with TLR4; see. Cbirvez-Sänchez et al. Activation of TLR2 and TLR4 by minimally modified low density lipoprotein in human macrophages and monocytes triggers the inflammatory response. Hum Immunol. 71 (8): 737-44 (2010). Other TLR2-dependent endogenous, ie endogenous, ligands have been described: biglycan, endoplasmin, HMGB1, HSP60, HSP70, human cardiac myosin, hyaluronan and uric acid crystals; see. Yu et al.
  • TLR2 plays a role in various human diseases, which are influenced by exogenous or endogenous TLR2-mediated signals.
  • the oligosaccharide of the present invention binds and activates the TLR2 receptor in a manner similar to e.g. Lipopeptide agonists.
  • the evoked spectrum after receptor activation of transcribed genes overlaps strongly, but also shows chitin-specific genes.
  • chitin as an immune stimulant in humans is with the previously described chitin fragments of unknown molecular size and structure, usually in the form of crushed exoskeletons of Schieder Stahl ßern, only partially possible or difficult, especially due to the impurities of chitin preparations, especially by endotoxins , inaccurately defined DA and / or heterogeneous particle sizes.
  • oligosaccharide is characterized by its chemically unique structure. This may be purified or synthetic oligosaccharide. The latter is distinguished from naturally isolated oligo- or polysaccharides, which are present as mixtures of structurally not clearly defined structure size, purity and effective molarity.
  • Purified oligosaccharide can be e.g. HF (hydrofluoric acid) cleaved macroscopic chitin, e.g. from crab shells and prepared by HPLC chromatography on an amino column, e.g.
  • the oligosaccharide or the pharmaceutical composition according to the invention has a purity with respect to the oligosaccharide, which at about> 99, preferably about> 99.9, more preferably about> 99.99, further preferably about> 99.999 , more preferably about> 99.9999, and most preferably about 100% by weight.
  • the oligosaccharide according to the invention is consequently high-purity oligosaccharide.
  • NAG N-acetylglucosamine
  • NAG is a monosaccharide and a derivative of D-glucose which has an acetylated amine radical at position 2 of the ring.
  • NAG has the molecular formula C 8 Hi 5 N0 6 , the CAS number 7512-17-6 and has a molar mass of 221, 21 g-mol " and is about 9.3 ⁇ long in the longest dimension.
  • an “immune stimulus” is understood to mean a substance which excites the immune system by inducing its activation and / or increasing the activity of one or more of its components, such as those of T lymphocytes, natural killer cells, etc.
  • composition according to the invention may comprise a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carriers are well known in the art. They include, for example, binders, disintegrants, fillers, lubricants and buffers, salts and other substances suitable for the formulation of medicaments; see. Rowe et al. (2006), Handbook of Pharmaceutical Excipients, 5 th Edition Pharmaceutical Press; or Bauer et al. (1999), Textbook of Pharmaceutical Technology, 6th Edition, Academictinct Verlagsgesellschaft Stuttgart mbH. The content of the present publications is incorporated herein by reference.
  • the immune stimulant is an adjuvant
  • the pharmaceutical composition is a vaccine or a vaccine composition.
  • the immunostimulatory property of the invention is highly pure
  • Oligosaccharide particularly good as an adjuvant, i. as an adjuvant which enhances the action of a reagent or drug.
  • the oligosaccharide provides relief in the search for new adjuvants, but especially not exclusively those for tumor vaccination, where there is an acute need for new vaccine adjuvants.
  • the high-purity oligosaccharide or the pharmaceutical composition is designed for the treatment and / or prevention of a tumor disease or for the prevention of an infectious disease or for the treatment of a chronic infectious disease.
  • Treatment and prevention should be understood as the treatment of a primary diagnosis of cancer or an infectious disease, as well as the prevention of relapse, as well as the prevention of the onset or progression of the disease.
  • the immunostimulatory property of the invention is highly pure
  • Oligosaccharide also for use in other diseases in which a relatively broad activation of the immune system, e.g. chronic viral diseases, is desirable, designed.
  • the inventors were able to establish that the highly pure oligosaccharide with> 6 NAG subunits in particular activates those components of the immune system which are also required for effective control of tumor cells or an infection.
  • the invention not only the clinical picture of a cancer but also its precursors, carcinogenic cells, tumor cells, metastases, etc. are understood to mean a tumor disease.
  • the high-purity oligosaccharide is in
  • the combination of the high purity oligosaccharide of the invention with> 6 NAG subunits with one or more tumor antigens or microbial antigens in a composition provides the conditions for a particularly effective tumor vaccination or vaccination against infection.
  • the high-purity oligosaccharide according to the invention provides a general stimulation of the immune system, in particular of CD8 + T cells, whereas the tumor antigen directs the immune system in a targeted manner to the particular tumor entity to be addressed.
  • the antigens can be added, for example, in the form of proteins or peptides or already be present in the tissue.
  • the combination of the high purity oligosaccharide of the invention with one or more other or known adjuvants is included.
  • Tumor antigen is understood to mean a structure which is produced by cancer cells and is able to trigger an immune response in the affected organism.
  • Tumor antigens include, for example, tumor-specific antigens (TSA), also called neoantigens, such as BCR-ABL and ABL-BCR, modified p53, ras, etc., as well as tumor-associated antigens (TAA), such as tyrosinase, mucin-1, etc., in question.
  • TSA tumor-specific antigens
  • TAA tumor-associated antigens
  • the high-purity oligosaccharide consists of> 7, preferably> 8, more preferably> 9, more preferably> 10, more preferably> 1 1, further preferably> 12, more preferably> 13, with further preference> 14, more preferably > 15, and most preferably 10-15 NAG subunits.
  • This measure has the advantage that the high-purity oligosaccharide according to the invention is provided in a size which, according to the findings of the inventors, ensures a particularly high immunostimulating activity. It is understood, however, that also 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 , 85, 85, 90 and 95 NAG subunits may be advantageous and a correspondingly long high-purity oligosaccharide is also included according to the invention.
  • various oligosaccharides of the invention may be present in parallel, e.g. Oligosaccharides with 6, oligosaccharides with 7, with 8, 9, 10, 1 1, 12, etc. ... NAG subunits, each with certain relative proportions.
  • Another object of the invention relates to the use of a high purity
  • Oligosaccharide consisting of> 6 NAG subunits in vitro for the stimulation of immune cells, preferably selected from the group consisting of: macrophages, neutrophilic granulocytes, and B cells.
  • the immune cells are brought into contact with the high-purity oligosaccharide according to the invention in vitro under suitable conditions known to the person skilled in the art.
  • the high-purity oligosaccharide according to the invention or the pharmaceutical composition according to the invention apply correspondingly to the use according to the invention.
  • Another object of the invention relates to the use of a high purity
  • Oligosaccharide consisting of> 6 NAG subunits in vitro for the activation and / or binding of the TLR2 receptor.
  • the TLR2 receptor is brought into contact with the high-purity oligosaccharide according to the invention in vitro under suitable conditions known to the person skilled in the art.
  • the high-purity oligosaccharide according to the invention or the pharmaceutical composition according to the invention apply correspondingly to the use according to the invention.
  • Another object of the invention relates to a method for the therapeutic and / or prophylactic treatment of a living being, including a farm animal and humans, for stimulating the immune system, which comprises the administration of the high-purity oligosaccharide and / or the pharmaceutical composition according to the invention in the subject.
  • the method also includes vaccinating the subject, including tumor vaccination.
  • the high-purity oligosaccharide or the pharmaceutical composition according to the invention apply correspondingly to the process according to the invention.
  • FIG. 1 Current hypothesis on the size dependence of crude chitin
  • Fig. 2 Chemical structures of inventive high purity and reference oligonucleotides.
  • FIG. 3 Size-dependent immune activation of defined oligosaccharides derived from chitin fragments by isolated human and murine immune cells and in human whole blood.
  • TLR2 is the receptor for chitin-derived oligosaccharides in mouse
  • Fig. 5 In vitro adjuvant effect of high purity chitin-derived oligosaccharides with> 6 NAG subunits.
  • FIG. 1A The supposed dependence of the triggered immune response on the size of the chitin fragments is shown in FIG. 1A.
  • the chitin fragment size is determinative of the triggered immune effects. While sizes of over 70 ⁇ should be immunologically inert, it is believed that small and very small chitin fragments, especially when inhaled, trigger inflammatory reactions.
  • FIG. 1B particles of 10 ⁇ m are already as large as a whole human macrophage. Only oligosaccharides of 5-20 subunits are comparable in size to the size of a TLR recognition domain (shown to scale in Figure 1C).
  • Figure 2 illustrates the chemical structures of some of the high purity oligonucleotides of this invention, C6, 07, and C10-15 ( Figure 2B), and some reference oligonucleotides, 04, C5 ( Figure 2A).
  • oligosaccharides with 3 NAG subunits (03) to 7 NAG subunits (C7). The purity of the preparations was verified by mass spectrometry.
  • oligosaccharides having 10-15 NAG subunits (010-15) were prepared by acetylation from a mixture of chitosan polymers of chain lengths 10 to 15. The degree of acetylation was determined by mass spectrometry to about 90%.
  • the oligosaccharides were assayed for endotoxin-free by Limulus amebocyte assay ( ⁇ 0.25 EU / ml in stock solutions of> 1 mM, i.e., 100-fold higher than in experiments).
  • a human macrophage cell line, THP-1 was stimulated with chitin and LPS alone and in the presence of polymyxin B for 18h.
  • primary macrophages on eg C10-15 also react in the presence of polymyxin with IL-6 release while LPS activation is blocked.
  • Figure 3B TLR4-deficient mouse macrophages
  • Macrophages of volunteer donors were stimulated with different sized NAG oligosaccharides and controls for 18 hours and cytokine release of IL-6 (Fig. 3C, D) and TNF (not shown) were determined.
  • B-cells which are important for antibody production in the context of vaccination, could also be dose-dependently activated for cytokine production by C10-15.
  • Microarray transcription analyzes of whole blood samples of healthy donors, a particularly physiological system for the determination of immune responses previously stimulated with chitin 10-15 or known TLR4 (LPS) or TLR2 (Pam2, Pam3) ligands, (Fig. 3G), that chitin induced a variety of genes and elicited specific effects compared to other TLR2 ligands (as evidenced by a principal component analysis), but also overlapped (numbers in the Venn diagram) with genes induced by other TLR2 ligands, indicating broad immunostimulatory activity occupied with a special gene transcription profile.
  • cytokines such as CCL3, IL12B, IL6, and IL8, which are responsible for activating a robust immune response necessary for an effective adjuvant effect, were robustly induced by chitin 10-15.
  • the induction was higher than the equimolar used other TLR2 ligands Pam2 and Pam3.
  • the activity of defined chitin fragments was also tested in the animal model by in vivo application to the lungs (trachea, Fig. 3H) of mice and could also be confirmed here by inflammatory cell infiltration (neutrophils, PMN). Highly pure oligosaccharides of ⁇ 6NAG show no immunostimulating effect.
  • TLR2 is the receptor for NAG oligosaccharides derived from molecular chitin fragments
  • TLR2 was reconstituted in HEK-293 cells by transfecting 10 ng TLR2 plasmid ( Figure 4D top) or empty vector (EV, bottom) and using oligosaccharide C10-15 and TLR2 ligand controls (Pam 2 CSK 4 , Pam 3 CSK 4 ).
  • the activity of the transcription factor NF- ⁇ was determined by means of dual luciferase assays. It was shown that the expression of TLR2 was sufficient to make the cells chitin-responsive.
  • oligosaccharide C10-15 also interacted with recombinantly produced murine mTLR2 ectodomain protein in a flow cytometric assay (Figure 4E).
  • Alexa647-coupled chitin was stained more strongly with mTLR2-Fc-PE than with control IgG1 -PE. Single measurements and a quantification of four measurements are shown. This was also confirmed by the latest measurements with the Microscale Thermophoresis (MST) method: MST titration of mTLR2-Fc protein with Alexa647-labeled chitin 10-15 showed binding with nanomolar affinity; Fig. 4F. Similar binding data were obtained for binding to human TLR2.
  • MST Microscale Thermophoresis
  • TLR2 is the receptor for molecular chitin or NAG oligosaccharides derived therefrom in the human and murine systems, so that future immunological signal transduction and therapeutic approaches can now address this well-defined receptor pathway ,
  • PBMC peripheral blood mononuclear cells
  • certain peptide antigens either from cytomegalovirus (CMV), influenza A (Flu) or Epstein-Barr virus (EBV BMLF1 and EBV-LMP2)
  • IL-2 T-cell growth factor
  • TLR2 stimulants standard
  • TLR2 ligands Pam 3 CSK 4 or active ingredient 1, 10 ⁇ g ml
  • oligosaccharides C10-15 according to the invention (10 and 50 ⁇ g ml).
  • the cells were harvested, stained with relevant antibodies and tetramers, and analyzed by flow cytometry.
  • FIG. 5A Panel A shows the percentage of antigen-specific T cells for the 4 specificities tested under the different conditions (duplicate approaches, respectively).
  • Panel B shows representative dot plots for EBV-BMLF1-specific CD8 + T cells following the standard (top) or chitin 10 ⁇ g ml (bottom) stimulations.
  • the oligosaccharides of the present invention resulted in a strong proliferation of antigen-specific CD8 + in 2/3 of the tested subjects compared to a comparative drug 1 (another human adjuvant candidate) and Pam 3 CSK 4 T cells as shown by tetramer staining; see.
  • Fig. 5A-B These results of the inventors show that the oligosaccharides according to the invention are promising immune stimulants and adjuvant candidates.

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Abstract

The present invention relates to a highly pure oligosaccharide for use as an immunostimulant, to a pharmaceutical composition comprising the highly pure oligosaccharide according to the invention, to the use in vitro of the highly pure oligosaccharide according to the invention for stimulating immune cells or for activating/binding the TLR2 receptor.

Description

Immunstimulierende hochreine Oligosaccharide  Immunostimulant high purity oligosaccharides
Die vorliegende Erfindung betrifft ein hochreines Oligosaccharid zur Verwendung als Immunstimulanz, eine pharmazeutische Zusammensetzung, die das erfindungsgemäße hochreine Oligosaccharid aufweist, sowie die Verwendung des erfindungsgemäßen hochreinen Oligosaccharids in vitro zur Stimulation von Immunzellen bzw. zur Aktivierung/Bindung des TLR2-Rezeptors. The present invention relates to a high-purity oligosaccharide for use as an immunostimulant, a pharmaceutical composition comprising the high-purity oligosaccharide according to the invention, and the use of the high-purity oligosaccharide according to the invention in vitro for stimulating immune cells or for activating / binding the TLR2 receptor.
Eine Immunstimulation bezeichnet therapeutische Maßnahmen, die darauf gerichtet sind, das Immunsystem zu aktivieren. Dabei kommen sogenannte Immunstimulantien, d.h. Substanzen, die eine Immunstimulation auslösen, zum Einsatz. Bei der spezifischen Immunstimulation wird die Aktivität des Immunsystems auf die Beseitigung eines bestimmten krankheitsauslösenden Faktors ausgerichtet, z.B. auf einen infektiösen Mikroorganismus, wie einen Virus oder ein Bakterium, oder auf einen Tumor. Die unspezifische Immunstimulation versucht hingegen, das Immunsystem allgemein zu stimulieren, um eine Erkrankung günstig zu beeinflussen. In Kombination mit spezifischer Immunstimulation soll beispielsweise eine potente spezifische Immunaktivierung ausgelöst werden. Immune stimulation refers to therapeutic measures aimed at activating the immune system. There are so-called immune stimulants, i. Substances that trigger an immune stimulation are used. In specific immune stimulation, the activity of the immune system is directed to the elimination of a particular disease-causing factor, e.g. on an infectious microorganism, such as a virus or a bacterium, or on a tumor. By contrast, non-specific immune stimulation attempts to generally stimulate the immune system in order to favorably influence a disease. In combination with specific immune stimulation, for example, a potent specific immune activation should be triggered.
Eine unspezifische Immunstimulation geht bspw. von sogenannten Adjuvantien aus, die in Arzneimittelzubereitungen dem spezifischen Wirkstoff bzw. in Impfstoffzubereitungen dem i.d.R. spezifischen Antigen zugesetzt werden, um dessen Wirkung zu verbessern bzw. zu verstärken. Im letzteren Fall ist für die spezifische Immunantwort das Antigen und für die Stärke der Immunantwort im Wesentlichen das Adjuvans verantwortlich. Adjuvantien können auch allein, d.h. ohne Zugabe von exogenen spezifischen Antigenen wirken, indem sie mit bereits im Tumor oder dem infizierten Gewebe vorhandenen spezifischen Antigenen zusammen wirken. An unspecific immune stimulation is, for example, from so-called adjuvants, which in drug formulations the specific drug or in vaccine preparations the usually be added to specific antigen to improve its effect or reinforce. In the latter case, the antigen is responsible for the specific immune response and, for the strength of the immune response, essentially the adjuvant. Adjuvants may also act alone, ie without the addition of exogenous specific antigens, by interacting with specific antigens already present in the tumor or the infected tissue.
Für die Anwendung im Menschen vorgesehene Impfstoff-Adjuvantien sollen vorzugsweise das Immunsystem in einer solchen Weise aktivieren, dass das im Impfstoff enthaltene Antigen im Kontext eines Gefahrensignals effektiv dem Immunsystem präsentiert wird und so einen potenten und anhaltenden T-Zell- und/oder Antikörper-vermittelten Impfschutz erzeugt. Viele der momentan eingesetzten Adjuvantien stellen sogenannte "microbe- associated molecular pattern" (MAMPs) dar, d.h. von Mikroben-stammende Moleküle, die vom angeborenen Immunsystem durch sogenannte "pattern recognition receptors" (PRRs) als fremd erkannt werden können. Allgemein gibt es für die Anwendung im Menschen derzeit nur eine geringe Zahl von Adjuvantien. Manche dieser Adjuvantien sind zudem relativ schwach und führen zu einer suboptimalen Impfreaktion. In der Regel fördern klassische Adjuvantien, die in bereits zugelassenen Vakzinen für Infektionserkrankungen verwendet werden, eher eine Antikörper-vermittelte Immunantwort, nicht jedoch eine Th1 -Zellen- bzw. zellulär-vermittelte Immunantwort, die besonders in der Krebstherapie wünschenswert wäre. Preferably, vaccine adjuvants intended for use in humans are intended to activate the immune system in such a manner that the antigen contained in the vaccine is effectively presented to the immune system in the context of a danger signal, thereby providing potent and sustained T cell and / or antibody mediation Vaccine protection produced. Many of the adjuvants currently used constitute so-called "micro-associated molecular pattern" (MAMPs), i. microbial-derived molecules that can be recognized as foreign by the innate immune system through pattern recognition receptors (PRRs). In general, there are currently only a small number of adjuvants for use in humans. Some of these adjuvants are also relatively weak and lead to a suboptimal vaccination reaction. Typically, classical adjuvants used in already approved vaccines for infectious diseases tend to promote an antibody-mediated immune response but not a Th1-cell or cellular-mediated immune response that would be desirable, especially in cancer therapy.
Die sog. Tumorvakzinierung stellt einen vielversprechenden therapeutischen Ansatz in der Behandlung von Krebspatienten dar; vgl. Walter et al. Multipeptide immune response to Cancer Vaccine IMA901 after single-dose cyclophosphamide associates with longer patient survival. Nature medicine 18, 1254-1261 (2012). Dabei kommen Krebsimpfstoffe bzw. Tumorvakzine zum Einsatz, die tumorspezifische Strukturen, wie bspw. Tumorantigene oder Tumor-assoziierte Antigene, enthalten, welche das Immunsystem zu einer spezifischen Reaktion gegen einen bestimmten Tumor veranlassen oder es dabei unterstützen sollen. Allerdings stehen derzeit nur einige wenige und für robuste T-Zell- Antworten meist relativ schwache Adjuvantien für die Tumorvakzinierung zur Verfügung; vgl. Khong und Overwijk WW. Adjuvants for peptide-based Cancer vaccines. J Immuno- ther Cancer 4: 56 (2016). Somit werden dringend neue Immunstimulanzien und insbesondere Adjuvantien-Moleküle benötigt. The so-called tumor vaccination represents a promising therapeutic approach in the treatment of cancer patients; see. Walter et al. Multipeptide immune response to Cancer Vaccine IMA901 after single-dose cyclophosphamide associates with longer patient survival. Nature medicine 18, 1254-1261 (2012). Cancer vaccines or tumor vaccines are used which contain tumor-specific structures, such as, for example, tumor antigens or tumor-associated antigens, which are intended to induce or assist the immune system in a specific reaction against a specific tumor. However, currently only a few and for robust T-cell responses are usually relatively weak adjuvants for tumor vaccination available; see. Khong and Overwijk WW. Adjuvants for peptide-based cancer vaccines. J Immuno-Cancer 4: 56 (2016). Thus, urgently new immune stimulants and in particular adjuvant molecules are needed.
Chitin, ein Homopoly- oder oligomer aus ß(1 -4)-N-Acetyl D-Glucosamin (NAG), ist nach Zellulose das am zweithäufigsten vorkommende Polysaccharid in der Natur. Es ist ein Bestandteil von Pilzzellwänden, dem Exoskelett und Digestionstrakt von Insekten und Schalentieren und den Mikrofilarien parasitischer Nematoden. Chitin dient niederen Lebensformen als Schutz vor rauen Umweltbedingungen. In höheren Organismen kommt Chitin nicht vor, aber es lassen sich nicht nur Chitin abbauende Enzyme, sogenannte Chitinasen, sondern auch viele immunologische Effekte auf eine Chitin-Exposition von Körperzellen hin finden; vgl. Mack et al. The role of chitin, chitinases, and chitinase-like proteins in pediatric lung diseases. Mol Cell Pediatr 2(1 ): 3 (2015); Lee. Chitin, chitinases and chitinase-like proteins in allergic inflammation and tissue remodeling. Yonsei medical journal 50(1 ): 22-30 (2009). Chitin, a homopoly or oligomer of β (1 -4) -N-acetyl-D-glucosamine (NAG), is the second most abundant polysaccharide in nature after cellulose. It is a component of fungal cell walls, the exoskeleton and digestive tract of insects and shellfish, and the microfilariae of parasitic nematodes. Chitin serves lower life forms as protection against harsh environmental conditions. Chitin does not occur in higher organisms, but not only chitin-degrading enzymes, so-called chitinases, but also many immunological effects on chitin exposure of body cells can be found; see. Mack et al. The role of chitin, chitinases, and chitinase-like proteins in pediatric lung diseases. Mol Cell Pediatr 2 (1): 3 (2015); Lee. Chitin, chitinases and chitinase-like proteins in allergic inflammation and tissue remodeling. Yonsei medical journal 50 (1): 22-30 (2009).
Chitin unterscheidet sich von Chitosan (poly-ß(1 -4)-D-Glucosamin) im Vorhandensein einer Acetylgruppe an der Aminogruppe an Position 2 des Ringes. Der Grad der Acetylie- rung (engl, "degree of acetylation", DA) bei kommerziell verfügbaren Präparationen entscheidet darüber, ob die Präparation als "Chitin" (bei einem DA von >50%) oder "Chitosan" (bei einem DA von <50%) bezeichnet wird. Somit enthalten i.d.R. kommerzielle Präparationen von Chitin zumindest Spuren von Chitosan und umgekehrt. Chitin differs from chitosan (poly-β (1 -4) -D-glucosamine) in the presence of an acetyl group on the amino group at position 2 of the ring. The degree of acetylation (DA) in commercially available preparations determines whether the preparation is in the form of "chitin" (with a DA of> 50%) or "chitosan" (with a DA of < 50%). Thus, i.d.R. commercial preparations of chitin at least traces of chitosan and vice versa.
Es konnte gezeigt werden, dass aus Krabben oder Pilzwänden hergestellte Roh-Chitin- Präparationen ungenau bestimmter Größe, Reinheit und/oder DA z.B. peritoneale Makrophagen und natürliche Killerzellen der Maus aktivieren und Zytokine wie Interleukin (IL)- 1 ß, Colony-Stimulating-Factor (CSF) und Interferon γ gebildet wurden. In zahlreichen weiteren in vivo Studien in der Maus konnte festgestellt werden, dass Chitin eine Rolle bei der Entstehung von Typ 2 Allergien zu spielen scheint, in denen es zu einer Akkumulation von eosinophilen Granulozyten sowie weiteren Zellen des angeborenen und adaptiven Immunsystems z.B. in den Atemwegen beiträgt; vgl. Lee (a.a.O.), Reese et al. Chitin induces accumulation in tissue of innate immune cells associated with allergy. Nature 447(7140): 92-6 (2007). Im Stand der Technik erfolgt die Größeneinteilung von Chitin üblicherweise in "großes" (>70 μπι), "mittleres" (70-40 μπι), "kleines" (40-10 μηι) und "sehr kleines" (< 10 μπι) Chitin. Während Größen von über 70 μηι immunologisch inert sind, können "kleine" oder sehr kleine Chitin-Fragmente, insbesondere wenn sie eingeatmet werden, alveoläre Makrophagen aktivieren und zur Sekretion von Zytokinen wie IL-12, Tumor Nekrose Faktor (TNF) und IL-18 führen; vgl. Lee (a.a.O.), Da Silva et al. Chitin is a size-dependent regulator of macrophage TNF and IL-10 production. J Immunol 182(6): 3573-82 (2009). In Studien am Menschen oder der Maus wurden bisher nur makroskopische, durch physikalischen Aufbruch generierte Roh-Chitin Präparationen untersucht, wobei die kleinste untersuchte "definierte" Größe meist bei ca. 1 μηι lag; vgl. Wagener et al. Fungal chitin dampens inflammation through IL-10 induction mediated by NOD2 and TLR9 activation. PLoS pathogens 10(4): e1004050 (2014). Die kleinste bislang genau beschrieben Partikelgröße beträgt 0.2 μιτι, vgl. Alvarez. The effect of chitin size, shape, source and purifica- tion method on immune recognition. Molecules 19(4): 4433-51 (2014). Da die N- Acetylglucosamin-Untereinheit des Chitins eine längste Dimension von <10 Ä (1 Ä = 0,1 nm) aufweist, entspricht diese Partikelgröße vermutlich ca. 200 NAG-Untereinheiten. It could be shown that raw chitin preparations prepared from crab or mushroom walls inaccurately activate specific size, purity and / or DA eg peritoneal macrophages and natural killer cells of the mouse and cytokines such as interleukin (IL) - 1β, colony-stimulating factor (CSF) and interferon γ were formed. Numerous other in vivo studies in mice have shown that chitin plays a role in the development of type 2 allergies, in which it contributes to the accumulation of eosinophilic granulocytes and other cells of the innate and adaptive immune system, for example in the respiratory tract ; see. Lee (supra), Reese et al. Chitin induces accumulation in tissue of innate immune cells associated with allergy. Nature 447 (7140): 92-6 (2007). In the prior art, the size division of chitin is usually in "large"(> 70 μπι), "average" (70-40 μπι), "small" (40-10 μηι) and "very small"(<10 μπι) chitin , While sizes over 70 μηι are immunologically inert, "small" or very small chitin fragments, especially when inhaled, can activate alveolar macrophages and secretion cytokines such as IL-12, tumor necrosis factor (TNF), and IL-18 to lead; see. Lee (supra), Da Silva et al. Chitin is a size-dependent regulator of macrophage TNF and IL-10 production. J Immunol 182 (6): 3573-82 (2009). In human or mouse studies, only macroscopic crude chitin preparations generated by physical disruption have so far been investigated, with the smallest investigated "defined" size usually being about 1 μm; see. Wagener et al. Fungal chitin inflammation through IL-10 induction mediated by NOD2 and TLR9 activation. PLoS pathogens 10 (4): e1004050 (2014). The smallest particle size exactly described so far is 0.2 μιτι, cf. Alvarez. The effect of chitin size, shape, source and purifica- tion method on immune recognition. Molecules 19 (4): 4433-51 (2014). Since the N-acetylglucosamine subunit of chitin has a longest dimension of <10 Å (1 λ = 0.1 nm), this particle size probably corresponds to about 200 NAG subunits.
Häufig ist bei den im Stand der Technik beschriebenen Untersuchungen unklar, welche Reinheit das eingesetzte Chitin aufweist bzw. wie frei es von anderen MAMPs ist, ob es makroskopische Größenunterschiede gibt, welche Kettenlängen oder Molekulargewichte bzw. welcher Grad der Acetylierung vorliegen. Die im Stand der Technik in Bezug auf Chitin beschriebenen Eigenschaften sind folglich widersprüchlich, z.B. diskutiert in Bueter et al. Innate sensing of chitin and chitosan. PLoS Pathog 9(1 ): e1003080 (2013). It is often unclear in the investigations described in the prior art which purity the chitin used has or how free it is from other MAMPs, whether there are macroscopic size differences, which chain lengths or molecular weights or what degree of acetylation are present. The properties described in the prior art with respect to chitin are therefore contradictory, e.g. discussed in Bueter et al. Innate sensing of chitin and chitosan. PLoS Pathog 9 (1): e1003080 (2013).
Ein therapeutischer Einsatz des Chitins als Immunstimulanz, insbesondere als Adjuvans in Impfstoffzusammensetzungen, scheitert deshalb daran, dass die genaue Zusammensetzung der Rohpräparationen unbekannt ist. Au ßerdem weisen die bekannten Chitinpräparationen nicht die für ein Arzneimittel erforderliche Reinheit der immunstimulierenden Komponente auf sondern sind häufig mit Endotoxin oder anderen Kontaminanten verunreinigt. Schließlich ist der Immun-Erkennungsrezeptor für Chitin im Menschen unbekannt, was eine gezielte Wirkstoffentwicklung des Chitins als Immunstimulanz verhindert. Vor diesem Hintergrund ist es eine Aufgabe der vorliegenden Erfindung, eine neue immunstimulierende Substanz bereitzustellen, die strukturell genau charakterisiert ist und chemisch einfach synthetisiert werden kann. Insbesondere soll die Substanz ausreichende therapeutische Sicherheit bieten, um als Adjuvans in einem Impfstoff eingesetzt werden zu können. Therapeutic use of chitin as an immune stimulant, in particular as an adjuvant in vaccine compositions, therefore fails because the exact composition of the crude preparations is unknown. In addition, the known chitin preparations do not have the purity of the immunostimulating component required for a drug, but are often contaminated with endotoxin or other contaminants. Finally, the immune recognition receptor for chitin in humans is unknown, which prevents targeted drug development of chitin as an immune stimulant. Against this background, it is an object of the present invention to provide a novel immunostimulating substance which is structurally well characterized and can be readily synthesized chemically. In particular, the substance should provide sufficient therapeutic safety to be used as an adjuvant in a vaccine can.
Diese Aufgabe wird durch ein hochreines Oligosaccharid bestehend aus > 6 N- Acetylglucosamin(NAG)-Untereinheiten zur Verwendung als Immunstimulanz gelöst. Diese Aufgabe wird au ßerdem durch die Bereitstellung einer pharmazeutischen Zusammensetzung, vorzugsweise eines Immunstimulanz, aufweisend ein hochreines Oligosaccharid bestehend aus > 6 NAG-Untereinheiten, gelöst. This object is achieved by a high purity oligosaccharide consisting of> 6 N-acetylglucosamine (NAG) subunits for use as an immune stimulant. This object is also achieved by providing a pharmaceutical composition, preferably an immunostimulant, comprising a high purity oligosaccharide consisting of> 6 NAG subunits.
Die der Erfindung zugrundeliegende Aufgabe wird hiermit vollkommen gelöst. Die Erfinder konnten feststellen, dass im Chitin ein chemisch-strukturell genau definiertes Oligosaccharid mit > 6 NAG-Untereinheiten immunstimulierend wirkt. Ferner konnten die Erfinder erstmals den natürlichen Rezeptor für Chitin identifizieren: Toll-like-Rezeptor 2 (TLR2). Diese Erkenntnisse erlauben nun die Entwicklung eines vom Chitin abgeleiteten Adjuvans, das im Menschen als Arzneimittel bzw. Arzneimittelzusatz oder Immunstimulanz einsetzbar ist. The problem underlying the invention is hereby completely solved. The inventors were able to establish that in chitin a chemically-structurally well-defined oligosaccharide with> 6 NAG subunits has an immunostimulating effect. Furthermore, the inventors were the first to identify the natural receptor for chitin: Toll-like receptor 2 (TLR2). These findings now allow the development of a chitin-derived adjuvant that can be used in humans as a drug or drug additive or immune stimulant.
TLR2 ist ein Erkennungsrezeptor der angeborenen Immunität (vgl. Kawasaki und Kawai. Toll-like receptor signaling pathways. Front lmmunol 5: 461 , (2014)), der auf verschiedenen professionellen Immunzellen, aber auch Gewebszellen wie Epithelzellen oder Kera- tinozyten exprimiert wird. Die Aktivierung von TLR2 durch Agonisten setzt eine Signalkaskade in Gang, die z.B. die Produktion inflammatorischer Zytokine, sowie weiterer immunologischer Phänomene im Menschen auslöst. Allgemein führt die TLR2-Aktivierung zu einer Immunstimulation, die die Aktivierung der adaptiven Immunantwort auslöst und somit zell- und/oder antikörpervermittelte Abwehrmechanismen einschaltet. Bislang sind verschiedene Lipopeptide oder Lipide als Agonisten von TLR2 beschrieben worden, z.B. acylierte Lipopeptide verschiedener Bakterien (z.B. Staphylococcus aureus) oder TLR2 is a recognition receptor of innate immunity (see Kawasaki and Kawai, Toll-like receptor signaling pathways, Front Immunol 5: 461, (2014)), which is expressed on various professional immune cells, but also on tissue cells such as epithelial cells or keratinocytes. Activation of TLR2 by agonists initiates a signaling cascade, e.g. the production of inflammatory cytokines, as well as other immunological phenomena in humans triggers. In general, TLR2 activation leads to an immune stimulation that triggers the activation of the adaptive immune response and thus activates cell- and / or antibody-mediated defense mechanisms. So far, various lipopeptides or lipids have been described as agonists of TLR2, e.g. acylated lipopeptides of various bacteria (e.g., Staphylococcus aureus) or
Mycobakterien (z.B. Mycobakterium tuberculosis), die von TLR2 gemeinsam mit TLR1 und TLR6 erkannt werden (vgl. Jin et al. Crystal structure of the TLR1 -TLR2 heterodimer induced by binding of a tri-acylated lipopeptide. Cell 130(6): 1071 -82 (2007); Kang et al. Recognition of lipopeptide patterns by Toll-like receptor 2-Toll-like receptor 6 heterodimer. /mmun/fy 31 (6): 873-84, (2009)). Dass Chitin ein spezifischer Agonist von TLR2 ist, war bislang nicht eindeutig, z.B. durch Bindungsstudien, belegt. Neben exogenen Substanzen wird TLR2 auch durch körpereigene, mit bestimmten Pathologien in Verbindung gebrachten Substanzen, wie oxidiertem LPL, welches von TLR2 gemeinsam mit TLR4, erkannt; vgl. Chävez-Sänchez et al. Activation of TLR2 and TLR4 by minimally modified low- density lipoprotein in human macrophages and monocytes triggers the inflammatory response. Hum Immunol. 71 (8):737-44 (2010). Als weitere TLR2-abhängige endogene, d.h. körpereigene, Liganden wurden beschrieben: biglycan, endoplasmin, HMGB1 , HSP60, HSP70, human cardiac myosin, hyaluronan und Harnsäurekristalle; vgl. Yu et al. Endogenous toll-like receptor ligands and their biological significance. J Cell Mol Med 14(1 1 ): 2592-603 (2010). Wie die Phänotypen von TLR2-defizienten Mäusen nahelegen, spielt TLR2 eine Rolle in verschiedenen Erkrankungen des Menschen, die durch exogene oder endogene TLR2-vermittelte Signale beeinflusst werden. Mycobacteria (eg Mycobacterium tuberculosis), which are recognized by TLR2 together with TLR1 and TLR6 (see Jin et al .: Crystal structure of the TLR1 -TLR2 heterodimerically induced by binding of a tri-acylated lipopeptide.) Cell 130 (6): 1071 - 82 (2007); Kang et al. Recognition of lipopeptide patterns by toll-like receptor 2-toll-like receptor 6 heterodimer. / mmun / fy 31 (6): 873-84, (2009)). The fact that chitin is a specific agonist of TLR2 has not yet been clearly demonstrated, for example, by binding studies. In addition to exogenous substances, TLR2 is also recognized by endogenous substances associated with certain pathologies, such as oxidized LPL, which is shared by TLR2 with TLR4; see. Chävez-Sänchez et al. Activation of TLR2 and TLR4 by minimally modified low density lipoprotein in human macrophages and monocytes triggers the inflammatory response. Hum Immunol. 71 (8): 737-44 (2010). Other TLR2-dependent endogenous, ie endogenous, ligands have been described: biglycan, endoplasmin, HMGB1, HSP60, HSP70, human cardiac myosin, hyaluronan and uric acid crystals; see. Yu et al. Endogenous toll-like receptor ligands and their biological significance. J Cell Mol Med 14 (11): 2592-603 (2010). As the phenotypes of TLR2-deficient mice suggest, TLR2 plays a role in various human diseases, which are influenced by exogenous or endogenous TLR2-mediated signals.
Gemäß den Erkenntnissen der Erfinder bindet und aktiviert das erfindungsgemäße Oligosaccharid den TLR2-Rezeptor in ähnlicher Weise wie z.B. Lipopeptid-Agonisten. Das hervorgerufene Spektrum nach Rezeptoraktivierung transkribierter Gene überlappt stark, zeigt jedoch auch Chitin-spezifische Gene. According to the inventors of the present invention, the oligosaccharide of the present invention binds and activates the TLR2 receptor in a manner similar to e.g. Lipopeptide agonists. The evoked spectrum after receptor activation of transcribed genes overlaps strongly, but also shows chitin-specific genes.
Dabei war besonders überraschend, dass das hochreine Oligosaccharid eine It was particularly surprising that the high-purity oligosaccharide a
Mindestgröße von 6 NAG-Untereinheiten benötigt, um eine immunstimulierende Wirkung auszuüben. Kleinere hochreine Oligosaccharide mit < 6 NAG-Untereinheiten weisen keine immunstimulierende Wirkung. Dies war aufgrund der im Stand der Technik getroffenen Annahmen, wonach gerade besonders kleine Chitinfragmente immunstimulierend wirken, nicht zu erwarten gewesen. Minimum size of 6 NAG subunits needed to exert an immunostimulating effect. Smaller high-purity oligosaccharides with <6 NAG subunits have no immunostimulating effect. This was unexpected due to the assumptions made in the prior art that particularly small chitin fragments are immunostimulating.
Auch konnten die Erfinder feststellen, dass kleinere Oligosaccharide mit < 6 NAG antagonistische Wirkungen aufweisen, folglich überraschenderweise gegensätzliche Aktivität ausüben. Eine Verwendung von Chitin als Immunstimulanz im Menschen ist mit den bisher beschriebenen Chitin-Fragmenten unbekannter molekularer Größe und Struktur, meist in Form von zerkleinerten Exoskeletten von Gliederfü ßern, nur bedingt möglich oder erschwert, insbesondere auch aufgrund der Verunreinigungen der Chitinpräparationen, vor allem durch Endotoxine, ungenau definierten DA und/oder heterogenen Partikelgrö ßen. Diese Nachteile weisen das erfindungsgemäße Oligosaccharid bzw. die erfindungsgemäße pharmazeutische Zusammensetzung nicht auf, was für zusätzliche therapeutische Sicherheit sorgt. Also, the inventors have found that smaller <6 NAG oligosaccharides have antagonistic effects, thus surprisingly exerting opposing activity. A use of chitin as an immune stimulant in humans is with the previously described chitin fragments of unknown molecular size and structure, usually in the form of crushed exoskeletons of Schiederfü ßern, only partially possible or difficult, especially due to the impurities of chitin preparations, especially by endotoxins , inaccurately defined DA and / or heterogeneous particle sizes. These disadvantages are not exhibited by the oligosaccharide according to the invention or the pharmaceutical composition according to the invention, which provides additional therapeutic safety.
Unter einem Oligosaccharid" wird erfindungsgemäß ein aus mehreren durch Under an oligosaccharide "according to the invention one of several
glycosidische Bindungen miteinander verbunden Monosacchariden bestehendes Kohlenhydrat verstanden. Das erfindungsgemäße " hochreine" Oligosaccharid ist durch seine chemisch eindeutige Struktur gekennzeichnet. Dabei kann es sich um aufgereinigtes oder synthetisches Oligosaccharid handeln. Letzteres grenzt sich ab von aus der Natur isolierten Oligo- oder Polysacchariden, die als Gemische mit strukturell nicht eindeutig definierter Struktur Grö ße, Reinheit und effektiver Molarität vorliegen. Aufgereinigtes Oligosaccharid lässt sich z.B. mittels HF (Hydrofluor-Säure) gespaltenem makroskopischem Chitin, z.B. aus Krabbenschalen stammend, herstellen und mittels HPLC-Chromatographie auf einer Amino-Säule, z.B. aus den Materialien Pronto-Sil oder Varian AX-5, und einem Wasser-Acetonitril-Gemisch aus mobiler Phase hochrein auch im Gro ßmaßstab darstellen. Dadurch weist das erfindungsgemäße Oligosaccharid bzw. die erfindungsgemäße pharmazeutische Zusammensetzung eine Reinheit in Bezug auf das Oligosaccharid auf, die bei ca. > 99, vorzugsweise ca. > 99,9, weiter vorzugsweise ca. > 99,99, weiter vorzugsweise ca. > 99,999, weiter vorzugsweise ca. > 99,9999, und höchst vorzugsweise ca. 1 00 Gew.-% liegt. Das erfindungsgemäße Oligosaccharid ist folglich hochreines Oligosaccharid. glycosidic bonds interconnected monosaccharides understood carbohydrate. The "high-purity" oligosaccharide according to the invention is characterized by its chemically unique structure. This may be purified or synthetic oligosaccharide. The latter is distinguished from naturally isolated oligo- or polysaccharides, which are present as mixtures of structurally not clearly defined structure size, purity and effective molarity. Purified oligosaccharide can be e.g. HF (hydrofluoric acid) cleaved macroscopic chitin, e.g. from crab shells and prepared by HPLC chromatography on an amino column, e.g. from the materials Pronto-Sil or Varian AX-5, and a water-acetonitrile mixture of mobile phase highly pure even on a large scale. As a result, the oligosaccharide or the pharmaceutical composition according to the invention has a purity with respect to the oligosaccharide, which at about> 99, preferably about> 99.9, more preferably about> 99.99, further preferably about> 99.999 , more preferably about> 99.9999, and most preferably about 100% by weight. The oligosaccharide according to the invention is consequently high-purity oligosaccharide.
"N-Acetylglucosamin (NAG)" ist erfindungsgemäß ein Monosaccharid und ein Derivat der D-Glucose, das an der Position 2 des Ringes einen acetylierten Aminrest aufweist. NAG hat die Summenformel C8Hi5N06, die CAS-Nummer 7512-17-6 und weist eine molare Masse von 221 ,21 g-mol" auf und ist in der längsten Dimension etwa 9.3 Ä lang. Erfindungsgemäß wird unter einem "Immunstimulanz" eine Substanz verstanden, die das Immunsystem anregt, indem sie dessen Aktivierung induziert und/oder die Aktivität einer oder mehrerer ihrer Komponenten erhöht, wie bspw. die der T- Lymphozyten, Natürlichen Killer-Zellen etc. According to the invention, "N-acetylglucosamine (NAG)" is a monosaccharide and a derivative of D-glucose which has an acetylated amine radical at position 2 of the ring. NAG has the molecular formula C 8 Hi 5 N0 6 , the CAS number 7512-17-6 and has a molar mass of 221, 21 g-mol " and is about 9.3 Ä long in the longest dimension. According to the invention, an "immune stimulus" is understood to mean a substance which excites the immune system by inducing its activation and / or increasing the activity of one or more of its components, such as those of T lymphocytes, natural killer cells, etc.
Es versteht sich, dass die erfindungsgemäße pharmazeutische Zusammensetzung einen pharmazeutisch akzeptablen Träger aufweisen kann. Pharmazeutisch akzeptable Träger sind im Stand der Technik hinreichend bekannt. Sie umfassen bspw. Bindemittel, Sprengmittel, Füllmittel, Gleitmittel sowie Puffer, Salze und sonstige zur Formulierung von Arzneimitteln geeignete Substanzen; vgl. Rowe et al. (2006), Handbook of Pharmaceuti- cal Excipients, 5th Edition Pharmaceutical Press; oder Bauer et al. (1999), Lehrbuch der pharmazeutischen Technologie, 6. Auflage, Wissenschaftliche Verlagsgesellschaft Stuttgart mbH. Der Inhalt der vorliegenden Publikationen ist durch Inbezugnahme Bestandteil der vorliegenden Anmeldung. It is understood that the pharmaceutical composition according to the invention may comprise a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art. They include, for example, binders, disintegrants, fillers, lubricants and buffers, salts and other substances suitable for the formulation of medicaments; see. Rowe et al. (2006), Handbook of Pharmaceutical Excipients, 5 th Edition Pharmaceutical Press; or Bauer et al. (1999), Textbook of Pharmaceutical Technology, 6th Edition, Wissenschaftliche Verlagsgesellschaft Stuttgart mbH. The content of the present publications is incorporated herein by reference.
Nach einer bevorzugten Ausgestaltung handelt es sich bei dem Immunstimulanz um ein Adjuvans, bei der pharmazeutischen Zusammensetzung um einen Impfstoff bzw. eine Impfstoffzusammensetzung. In a preferred embodiment, the immune stimulant is an adjuvant, and the pharmaceutical composition is a vaccine or a vaccine composition.
Durch die immunstimulierende Eigenschaft ist das erfindungsgemäße hochreine The immunostimulatory property of the invention is highly pure
Oligosaccharid besonders gut als Adjuvans, d.h. als Hilfsstoff, der die Wirkung eines Reagenz oder eines Arzneistoffes verstärkt, geeignet. Das Oligosaccharid schafft Abhilfe bei der Suche nach neuen Adjuvantien, insbesondere jedoch nicht ausschließlich solcher für die Tumorvakzinierung, wo akuter Bedarf an neuen Impfstoffadjuvantien besteht. Oligosaccharide particularly good as an adjuvant, i. as an adjuvant which enhances the action of a reagent or drug. The oligosaccharide provides relief in the search for new adjuvants, but especially not exclusively those for tumor vaccination, where there is an acute need for new vaccine adjuvants.
In einer weiteren erfindungsgemäßen Ausgestaltung ist das hochreine Oligosaccharid bzw. die pharmazeutische Zusammensetzung zur Behandlung und/oder Prävention einer Tumorerkrankung oder zur Prävention einer Infektionserkrankung oder zur Behandlung einer chronischen Infektionserkrankung ausgestaltet. Unter Behandlung und Prävention ist sowohl die Behandlung einer primären Diagnose von Krebs oder einer Infektionserkrankung zu verstehen, als auch die Prävention eines Rezidivs, als auch die Prävention einer Entstehung oder Progression der Erkrankung zu verstehen. Durch die immunstimulierende Eigenschaft ist das erfindungsgemäße hochreine In a further embodiment according to the invention, the high-purity oligosaccharide or the pharmaceutical composition is designed for the treatment and / or prevention of a tumor disease or for the prevention of an infectious disease or for the treatment of a chronic infectious disease. Treatment and prevention should be understood as the treatment of a primary diagnosis of cancer or an infectious disease, as well as the prevention of relapse, as well as the prevention of the onset or progression of the disease. The immunostimulatory property of the invention is highly pure
Oligosaccharid auch für die Anwendung in weiteren Erkrankungen, in denen eine relativ breite Aktivierung des Immunsystems, wie z.B. chronischen Viruserkrankungen, wünschenswert ist, ausgestaltet. Oligosaccharide also for use in other diseases in which a relatively broad activation of the immune system, e.g. chronic viral diseases, is desirable, designed.
Die Erfinder konnten feststellen, dass das hochreine Oligosaccharid mit > 6 NAG- Untereinheiten insbesondere solche Komponenten des Immunsystems aktiviert, die auch für eine effektive Bekämpfung von Tumorzellen oder einer Infektion erforderlich sind. Dabei werden erfindungsgemäß unter einer Tumorerkrankung nicht nur das klinische Bild einer Krebserkrankung sondern auch ihre Vorstufen, kanzerogene Zellen, Tumorzellen, Metastasen etc. verstanden. The inventors were able to establish that the highly pure oligosaccharide with> 6 NAG subunits in particular activates those components of the immune system which are also required for effective control of tumor cells or an infection. According to the invention, not only the clinical picture of a cancer but also its precursors, carcinogenic cells, tumor cells, metastases, etc. are understood to mean a tumor disease.
In einer Ausführungsform der Erfindung liegt das hochreine Oligosaccharid in In one embodiment of the invention, the high-purity oligosaccharide is in
Kombination mit zumindest einem oder mehreren Tumor- und/oder mikrobiellen Antigenen vor. Combination with at least one or more tumor and / or microbial antigens.
Die Kombination des erfindungsgemäßen hochreinen Oligosaccharids mit > 6 NAG- Untereinheiten mit einem oder mehreren Tumorantigenen oder mikrobiellen Antigenen in einer Zusammensetzung schafft die Voraussetzungen für eine besonders wirksame Tumorvakzinierung oder Vakzinierung gegen eine Infektion. Das erfindungsgemäße hochreine Oligosaccharid sorgt für eine allgemeine Stimulierung des Immunsystems, insbesondere von CD8+-T-Zellen, wohingegen das Tumorantigen das Immunsystem zielgerichtet auf die jeweils zu adressierende Tumorentität ausrichtet. Die Antigene können z.B. in Form von Proteinen oder Peptiden beigefügt werden können oder bereits im Gewebe vorhanden sein. Die Kombination des erfindungsgemäßen hochreinen Oligosaccharids mit einem oder mehreren anderen bzw. bekannten Adjuvantien ist eingeschlossen. The combination of the high purity oligosaccharide of the invention with> 6 NAG subunits with one or more tumor antigens or microbial antigens in a composition provides the conditions for a particularly effective tumor vaccination or vaccination against infection. The high-purity oligosaccharide according to the invention provides a general stimulation of the immune system, in particular of CD8 + T cells, whereas the tumor antigen directs the immune system in a targeted manner to the particular tumor entity to be addressed. The antigens can be added, for example, in the form of proteins or peptides or already be present in the tissue. The combination of the high purity oligosaccharide of the invention with one or more other or known adjuvants is included.
Dabei wird erfindungsgemäß unter einem "Tumorantigen" eine Struktur verstanden, die von Krebszellen produziert wird und in der Lage ist, im betroffenen Organismus eine Immunantwort auszulösen. Als Tumorantigene kommen bspw. tumorspezifische Antigene (TSA), auch Neoantigene genannt, wie z.B. BCR-ABL und ABL-BCR, modifiziertes p53, ras, etc., sowie tumorassoziierte Antigene (TAA), wie z.B. Tyrosinase, Mucin-1 etc., in Frage. According to the invention, a "tumor antigen" is understood to mean a structure which is produced by cancer cells and is able to trigger an immune response in the affected organism. Tumor antigens include, for example, tumor-specific antigens (TSA), also called neoantigens, such as BCR-ABL and ABL-BCR, modified p53, ras, etc., as well as tumor-associated antigens (TAA), such as tyrosinase, mucin-1, etc., in question.
Nach einer Weiterbildung der Erfindung besteht das hochreine Oligosaccharid aus > 7, bevorzugt > 8, weiter bevorzugt > 9, weiter bevorzugt > 10, weiter bevorzugt > 1 1 , weiter bevorzugt > 12, weiter bevorzugt > 13, weiter bevorzugt > 14, weiter bevorzugt > 15, und höchst bevorzugt 10-15 NAG-Untereinheiten. According to a development of the invention, the high-purity oligosaccharide consists of> 7, preferably> 8, more preferably> 9, more preferably> 10, more preferably> 1 1, further preferably> 12, more preferably> 13, with further preference> 14, more preferably > 15, and most preferably 10-15 NAG subunits.
Diese Maßnahme hat den Vorteil, dass das erfindungsgemäße hochreine Oligosaccharid in einer solchen Größe bereitgestellt wird, die nach den Erkenntnissen der Erfinder eine besonders hohe immunstimulierende Aktivität gewährleistet. Es versteht sich jedoch, dass auch 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 85, 90 und 95 NAG-Untereinheiten vorteilhaft sein können und ein entsprechend langes hochreines Oligosaccharid erfindungsgemäß ebenfalls umfasst ist. This measure has the advantage that the high-purity oligosaccharide according to the invention is provided in a size which, according to the findings of the inventors, ensures a particularly high immunostimulating activity. It is understood, however, that also 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 , 85, 85, 90 and 95 NAG subunits may be advantageous and a correspondingly long high-purity oligosaccharide is also included according to the invention.
In der erfindungsgemäßen pharmazeutischen Zusammensetzung können gemäß einem Ausführungsbeispiel verschiedene erfindungsgemäße Oligosaccharide parallel vorliegen, z.B. Oligosaccharide mit 6, Oligosaccharide mit 7, mit 8, 9, 10, 1 1 , 12, usw. ... NAG- Untereinheiten, jeweils mit bestimmten relativen Anteilen. In the pharmaceutical composition according to the invention, according to one embodiment, various oligosaccharides of the invention may be present in parallel, e.g. Oligosaccharides with 6, oligosaccharides with 7, with 8, 9, 10, 1 1, 12, etc. ... NAG subunits, each with certain relative proportions.
Ein weiterer Gegenstand der Erfindung betrifft die Verwendung eines hochreinen Another object of the invention relates to the use of a high purity
Oligosaccharids bestehend aus > 6 NAG-Untereinheiten in vitro zur Stimulation von Immunzellen, vorzugsweise ausgewählt aus der Gruppe bestehend aus: Makrophagen, neutrophile Granulozyten, und B-Zellen. Oligosaccharide consisting of> 6 NAG subunits in vitro for the stimulation of immune cells, preferably selected from the group consisting of: macrophages, neutrophilic granulocytes, and B cells.
Dabei werden die Immunzellen in vitro unter geeigneten, dem Fachmann bekannten Bedingungen mit dem erfindungsgemäßen hochreine Oligosaccharid in Kontakt gebracht. In this case, the immune cells are brought into contact with the high-purity oligosaccharide according to the invention in vitro under suitable conditions known to the person skilled in the art.
Die Eigenschaften, Vorteile, Weiterbildungen und Ausgestaltungen des The features, advantages, developments and refinements of
erfindungsgemäßen hochreinen Oligosaccharids bzw. der erfindungsgemäßen pharmazeutischen Zusammensetzung gelten für die erfindungsgemäße Verwendung entsprechend. Ein weiterer Gegenstand der Erfindung betrifft die Verwendung eines hochreinen The high-purity oligosaccharide according to the invention or the pharmaceutical composition according to the invention apply correspondingly to the use according to the invention. Another object of the invention relates to the use of a high purity
Oligosaccharids bestehend aus > 6 NAG-Untereinheiten in vitro zur Aktivierung und/oder Bindung des TLR2-Rezeptors. Oligosaccharide consisting of> 6 NAG subunits in vitro for the activation and / or binding of the TLR2 receptor.
Dabei wird der TLR2-Rezeptor in vitro unter geeigneten, dem Fachmann bekannten Bedingungen mit dem erfindungsgemäßen hochreinen Oligosaccharid in Kontakt gebracht. In this case, the TLR2 receptor is brought into contact with the high-purity oligosaccharide according to the invention in vitro under suitable conditions known to the person skilled in the art.
Die Eigenschaften, Vorteile, Weiterbildungen und Ausgestaltungen des The features, advantages, developments and refinements of
erfindungsgemäßen hochreinen Oligosaccharids bzw. der erfindungsgemäßen pharmazeutischen Zusammensetzung gelten für die erfindungsgemäße Verwendung entsprechend. The high-purity oligosaccharide according to the invention or the pharmaceutical composition according to the invention apply correspondingly to the use according to the invention.
Ein weiterer Gegenstand der Erfindung betrifft ein Verfahren zur therapeutischen und/oder prophylaktischen Behandlung eines Lebewesens, einschließlich eines Nutztiers und Menschen, zur Stimulierung des Immunsystems, das die Verabreichung des erfindungsgemäßen hochreinen Oligosaccharids und/oder der erfindungsgemäßen pharmazeutischen Zusammensetzung in das Lebewesen umfasst. Das Verfahren umfasst auch die Vakzinierung des Lebewesens, einschließlich die Tumorvakzinierung. Another object of the invention relates to a method for the therapeutic and / or prophylactic treatment of a living being, including a farm animal and humans, for stimulating the immune system, which comprises the administration of the high-purity oligosaccharide and / or the pharmaceutical composition according to the invention in the subject. The method also includes vaccinating the subject, including tumor vaccination.
Die Eigenschaften, Vorteile, Weiterbildungen und Ausgestaltungen des The features, advantages, developments and refinements of
erfindungsgemäßen hochreinen Oligosaccharids bzw. der erfindungsgemäßen pharmazeutischen Zusammensetzung gelten für das erfindungsgemäße Verfahren entsprechend. According to the invention, the high-purity oligosaccharide or the pharmaceutical composition according to the invention apply correspondingly to the process according to the invention.
Es versteht sich, dass die vorstehend genannten und die nachstehend noch zu It is understood that the above and the still to follow
erläuternden Merkmale nicht nur in der jeweils angegebenen Kombination, sondern auch in anderen Kombinationen oder in Alleinstellung verwendbar sind, ohne den Rahmen der vorliegenden Erfindung zu verlassen. explanatory features are usable not only in the combination given, but also in other combinations or alone, without departing from the scope of the present invention.
Die vorliegende Erfindung wird nun anhand von Ausführungsbeispielen näher erläutert, aus denen sich weitere Merkmale, Eigenschaften und Vorteile der Erfindung ergeben. Die Ausführungsbeispiele sind dabei nicht einschränkend. Es versteht sich au ßerdem, dass einzelne Merkmale, die in den Ausführungsbeispielen offenbart sind, nicht nur im Kontext der jeweiligen spezifischen Ausführungsform sondern in einer Allgemeingültigkeit offenbart sind und für sich genommenen einen eigenen Beitrag zur Erfindung liefern. Der Fachmann kann deshalb diese Merkmale frei mit anderen Merkmalen der Erfindung kombinieren. The present invention will now be explained in more detail by means of exemplary embodiments from which further features, properties and advantages of the invention result. The embodiments are not limiting. It is understood, by the way, that individual features disclosed in the embodiments are disclosed not only in the context of the specific embodiment but in generality and in themselves provide a separate contribution to the invention. The person skilled in the art can therefore freely combine these features with other features of the invention.
Dabei wird Bezug genommen auf die beigefügten Abbildungen, in denen Folgendes dargestellt ist: Reference is made to the accompanying drawings, in which:
Fig. 1 : Aktuelle Hypothese zur Größenabhängigkeit der durch Roh-Chitin-FIG. 1: Current hypothesis on the size dependence of crude chitin
Präparationen verschiedener Partikelgröße hervorgerufenen Immunreaktionen. Preparations of different particle size induced immune reactions.
Fig. 2: Chemischen Strukturen erfindungsgemäßer hochreiner und Referenz- Oligonukleotiden. Fig. 2: Chemical structures of inventive high purity and reference oligonucleotides.
Fig. 3: Größenabhängige Immunaktivierung von definierten von Chitin-Fragmenten abgeleiteten Oligosacchariden durch isolierte humane und murine Immunzellen und in humanem Vollblut. FIG. 3: Size-dependent immune activation of defined oligosaccharides derived from chitin fragments by isolated human and murine immune cells and in human whole blood. FIG.
Fig. 4: TLR2 ist der Rezeptor für von Chitin abgeleitete Oligosaccharide in Maus und Fig. 4: TLR2 is the receptor for chitin-derived oligosaccharides in mouse and
Mensch und bindet direkt von Chitin abgeleitete Oligosaccharide.  Human and binds directly from chitin-derived oligosaccharides.
Fig. 5: In vitro Adjuvans-Wirkung von hochreinem vom Chitin abgeleitete Oligosacchariden mit > 6 NAG-Untereinheiten. Fig. 5: In vitro adjuvant effect of high purity chitin-derived oligosaccharides with> 6 NAG subunits.
Ausführunqsbeispiele EXEMPLARY EMBODIMENTS
1 . Chitinfraqmente und Immunreaktion 1 . Chitin proteins and immune reaction
Die vermeintliche Abhängigkeit der ausgelösten Immunantwort von der Größe der Chitinfragmente ist in der Fig. 1 A dargestellt. So wird im Stand der Technik die Auffas- sung vertreten, dass die Chitinfragmentgröße bestimmend für die ausgelösten Immuneffekte sei. Während Größen von über 70 μηι immunologisch inert sein sollen, so wird angenommen, dass kleine und sehr kleine Chitin-Fragmente, insbesondere wenn sie eingeatmet werden, Entzündungsreaktionen auslösen. Wie Fig. 1 B zeigt, sind jedoch Partikel von 10 μηι bereits so groß wie ein ganzer humaner Makrophage. Nur Oligosaccharide von 5-20 Untereinheiten sind größenmäßig mit der Größe einer TLR Erkennungsdomäne zu vergleichen (maßstabsgetreu dargestellt in Fig. 1 C) The supposed dependence of the triggered immune response on the size of the chitin fragments is shown in FIG. 1A. Thus, in the prior art, the that the chitin fragment size is determinative of the triggered immune effects. While sizes of over 70 μηι should be immunologically inert, it is believed that small and very small chitin fragments, especially when inhaled, trigger inflammatory reactions. As shown in FIG. 1B, however, particles of 10 μm are already as large as a whole human macrophage. Only oligosaccharides of 5-20 subunits are comparable in size to the size of a TLR recognition domain (shown to scale in Figure 1C).
2. Hochreine Oligosaccharide mit > 6 NAG-Untereinheiten aktivieren verschiedene primäre Immunzellen des Menschen und der Maus 2. Highly pure oligosaccharides with> 6 NAG subunits activate various human and mouse primary immune cells
In der Fig. 2 sind die chemischen Strukturen einiger erfindungsgemäßer hochreiner Oligonukleotide, C6, 07, und C10-15 (Fig. 2B), und einiger Referenz-Oligonukleotide, 04, C5 (Fig. 2A), dargestellt. Figure 2 illustrates the chemical structures of some of the high purity oligonucleotides of this invention, C6, 07, and C10-15 (Figure 2B), and some reference oligonucleotides, 04, C5 (Figure 2A).
Die Erfinder haben kommerziell verfügbare Oligosaccharide mit 3 NAG-Untereinheiten (03) bis 7 NAG-Untereinheiten (C7) akquiriert. Die Reinheit der Präparationen wurde massenspektrometrisch verifiziert. Daneben wurden Oligosaccharide mit 10-15 NAG- Untereinheiten (010-15) mittels Acetylierung aus einer Mischung von Chitosan-Polymeren der Kettenlängen 10 bis 15 hergestellt. Der Acetylierungsgrad wurde massenspektrometrisch auf ca. 90% bestimmt. Die Oligosaccharide wurden auf Endotoxin-Freiheit mittels Limulus-Amöbozyten-Assay (<0,25 EU/ml in Stammlösungen von >1 mM, d.h. 100-fach höher als in Experimenten eingesetzt). The inventors have acquired commercially available oligosaccharides with 3 NAG subunits (03) to 7 NAG subunits (C7). The purity of the preparations was verified by mass spectrometry. In addition, oligosaccharides having 10-15 NAG subunits (010-15) were prepared by acetylation from a mixture of chitosan polymers of chain lengths 10 to 15. The degree of acetylation was determined by mass spectrometry to about 90%. The oligosaccharides were assayed for endotoxin-free by Limulus amebocyte assay (<0.25 EU / ml in stock solutions of> 1 mM, i.e., 100-fold higher than in experiments).
Eine humane Makrophagen-Zelllinie, THP-1 , wurde mit Chitin und LPS alleine und in Anwesenheit von Polymyxin B für 18h stimuliert. Wie in der Fig. 3A dargestellt, reagieren primäre Makrophagen auf z.B. C10-15 auch in der Gegenwart von Polymyxin mit IL-6- Freisetzung, während die LPS-Aktivierung blockiert ist. Diese und Experimente in TLR4- defizienten Mausmakrophagen (Fig. 3B) lassen eine LPS-Kontamination als Ursache der immunstimulatorischen Wirkung des Chitins ausschließen. Anschließend wurden von Chitin-abgeleitete NAG-Oligosaccharide verschiedener Größe an primären humanen (Fig. 3C) und murinen (Fig. 3D) Makrophagen weiter untersucht. Makrophagen freiwilliger Spender (n=8) wurden mit unterschiedlich großen NAG- Oligosacchariden und Kontrollen für 18 Stunden stimuliert und die Zytokinfreisetzung von IL-6 (Fig. 3C, D) und TNF (nicht gezeigt) bestimmt. Wie zu sehen ist, führte eine Mindestlänge von 6 NAG (C6) bzw. 7 NAG (C7), idealerweise 10 bis 15 NAG, zu einer starken inflammatorischen Zytokin an twort, z.B. der Bildung von TNF oder Interleukin (IL-) 6. A human macrophage cell line, THP-1, was stimulated with chitin and LPS alone and in the presence of polymyxin B for 18h. As shown in Figure 3A, primary macrophages on eg C10-15 also react in the presence of polymyxin with IL-6 release while LPS activation is blocked. These and experiments in TLR4-deficient mouse macrophages (Figure 3B) exclude LPS contamination as the cause of the immunostimulatory effect of chitin. Subsequently, different sized chitin-derived NAG oligosaccharides were further tested on primary human (Figure 3C) and murine (Figure 3D) macrophages. Macrophages of volunteer donors (n = 8) were stimulated with different sized NAG oligosaccharides and controls for 18 hours and cytokine release of IL-6 (Fig. 3C, D) and TNF (not shown) were determined. As can be seen, a minimum length of 6 NAG (C6) or 7 NAG (C7), ideally 10 to 15 NAG, led to a strong inflammatory cytokine at twort, eg the formation of TNF or interleukin (IL-) 6.
Neben primären humanen und murinen Makrophagen (Fig. 3C und D) reagierten auch primäre Neutrophile mit CD62L Shedding; vgl. Fig. 3E. B-Lymphozyten wurden mit Chitin und unterschiedlichen Kontrollen stimuliert und IL-6 im Zellüberstand mittels ELISA gemessen. Demnach reagieren auch primäre B Zellen mit IL-6 Sekretion. Neutrophile Granulozyten wurden auch mit den Oligosacchariden und Kontrollen für 1 Stunde inkubiert und der Grad der CD62L-Abspaltung, ein Zeichen ihrer Aktivierung, wurde durch- flusszytometrisch gemessen. Auch hier führten C10-15 Fragmente zu einer potenten Aktivierung der Neutrophilen (Fig. 3E). Auch B-Zellen, die für die Antikörper-Produktion im Rahmen einer Vakzinierung wichtig sind, konnten zur Zytokinproduktion mittels C10-15 dosisabhängig aktiviert werden. Microarray Transkriptionsanalysen von Vollblut-Proben gesunder Spender, einem besonders physiologischen System zur Bestimmung von Immunantworten, die zuvor mit Chitin 10-15 oder bekannten TLR4 (LPS) oder TLR2 (Pam2, Pam3) Liganden stimuliert worden waren, zeigte (Fig. 3G), dass Chitin eine Vielzahl von Genen induzierte und spezifische Effekte im Vergleich zu anderen TLR2 Liganden hervorrief (belegt durch eine Principal Component Analyse), jedoch auch mit den durch andere TLR2 Liganden induzierten Genen überlappte (Zahlen im Venn- Diagram), was eine breite immunstimulatorische Aktivität mit besonderem Gentranskriptionsprofil belegt. Es wurden zudem mehrere für die Aktivierung einer robusten für eine effektive Adjuvantswirkung notwendige Immunantwort verantwortliche Zytokine wie CCL3, IL12B, IL6 und IL8 robust durch Chitin 10-15 induziert. Die Induktion lag dabei höher als die bei equimolar eingesetzten anderen TLR2-Liganden Pam2 und Pam3. Die Aktivität definierter Chitin-Fragmente wurde auch im Tiermodell durch in vivo Applikation in die Lunge (Trachea; Fig. 3H) von Mäusen überprüft und konnte auch hier durch inflammatorische Zellinfiltration (Neutrophile, PMN) bestätigt werden. Hochreine Oligosaccharide von < 6NAG zeigen keine immunstimulierende Wirkung. Somit aktivieren hochreine Oligosaccharide mit einer Kettenlänge von > 6 NAG bzw. > 7 NAG, insbesondere 10-15 NAG, stark mehrere verschiedene Arten von Immunzellen des Menschen und in der Maus und rufen ein für eine robuste Immunantwort wünschenswertes Muster an pro-inflammatorischen Zytokinen hervor. In addition to primary human and murine macrophages (Figures 3C and D), primary neutrophils also reacted with CD62L shedding; see. Fig. 3E. B lymphocytes were stimulated with chitin and various controls and IL-6 was measured in the cell supernatant by ELISA. Accordingly, primary B cells also react with IL-6 secretion. Neutrophil granulocytes were also incubated with the oligosaccharides and controls for 1 hour, and the degree of CD62L cleavage, a sign of their activation, was measured by flow cytometry. Again, C10-15 fragments led to potent neutrophil activation (Figure 3E). B-cells, which are important for antibody production in the context of vaccination, could also be dose-dependently activated for cytokine production by C10-15. Microarray transcription analyzes of whole blood samples of healthy donors, a particularly physiological system for the determination of immune responses previously stimulated with chitin 10-15 or known TLR4 (LPS) or TLR2 (Pam2, Pam3) ligands, (Fig. 3G), that chitin induced a variety of genes and elicited specific effects compared to other TLR2 ligands (as evidenced by a principal component analysis), but also overlapped (numbers in the Venn diagram) with genes induced by other TLR2 ligands, indicating broad immunostimulatory activity occupied with a special gene transcription profile. Several cytokines, such as CCL3, IL12B, IL6, and IL8, which are responsible for activating a robust immune response necessary for an effective adjuvant effect, were robustly induced by chitin 10-15. The induction was higher than the equimolar used other TLR2 ligands Pam2 and Pam3. The activity of defined chitin fragments was also tested in the animal model by in vivo application to the lungs (trachea, Fig. 3H) of mice and could also be confirmed here by inflammatory cell infiltration (neutrophils, PMN). Highly pure oligosaccharides of <6NAG show no immunostimulating effect. Thus, highly pure oligosaccharides with a chain length of> 6 NAG or> 7 NAG, especially 10-15 NAG, strongly activate several different types of human and mouse immune cells and elicit a pattern of pro-inflammatory cytokines desirable for a robust immune response ,
3. TLR2 ist der Rezeptor für von molekularen Chitin-Fragmenten abgeleitete NAG- Oligosaccharide 3. TLR2 is the receptor for NAG oligosaccharides derived from molecular chitin fragments
Als nächstes sollte geklärt werden, über welche "pattern recognition receptors" [PRR(s)] die bereits vielfach für Undefinierte Chitin-Fragmente beschriebenen sowie die hier beobachteten Effekte der erfindungsgemäßen und Referenz-Oligosacchariden vermittelt werden. Da diese Frage bislang nicht geklärt ist, untersuchten die Erfinder die Wirkung der Oligosaccharide zunächst in Zellen mit genetischer Deletion oder Rekonstitution. Zunächst wurden Makrophagen von C57BL/6 WT, Myd88 Gen-Knockout (KO; Fig. 4A) und Tlr2 KO (Fig. 4B) Mäusen mit Chitin und Kontrollen für 18 Stunden stimuliert und die TNF-Freisetzung wurde mit der ELISA-Methode gemessen (n=3 pro Gruppe). Es zeigte sich eine starke Reduktion der durch Chitin 10-15 und den bekannten TLR2-Liganden Pam3 verursachten TNF-Sekretion. Nicht-TLR2-abhängige Stimuli wurden durch Myd88 und Tlr2 KO nicht beeinflusst. Im humanen System konnte die IL-6-Freisetzung in THP-1 - Zellen ohne (WT) und mit genetischer TLR2-Deletion (CRISPR#1 und #2) mittels Cas9- CRISPR untersucht werden (Fig. 4C, Bereitstellung der Zelllinien mit Genehmigung durch Prof. Dr. Veit Hornung, BioSys München, Ludwigs-Maximilians-Universität München, beschrieben unter Schmid-Burgk et al. (2014), OutKnocker: a web tool for rapid and simple genotyping of designer nuclease edited cell lines, Genome Res. Oct. 24(10): 1719- 1723). Auch hier zeigte sich eine selektive und starke Reduktion der durch Chitin 10-15 und den bekannten TLR2-Liganden Pam2 und Pam3 verursachten IL-6 Sekretion. Next, it should be clarified about which "pattern recognition receptors" [PRR (s)] already described many times for undefined chitin fragments and the effects observed here of the inventive and reference oligosaccharides are mediated. Since this question has not yet been clarified, the inventors first investigated the effect of the oligosaccharides in cells with genetic deletion or reconstitution. First, macrophages from C57BL / 6 WT, Myd88 gene knockout (KO; Fig. 4A) and Tlr2 KO (Fig. 4B) were stimulated with chitin and controls for 18 hours, and TNF release was measured by the ELISA method ( n = 3 per group). There was a marked reduction in TNF secretion caused by chitin 10-15 and the known TLR2 ligand Pam3. Non-TLR2 dependent stimuli were unaffected by Myd88 and Tlr2 KO. In the human system, IL-6 release into THP-1 cells without (WT) and with TLR2 genetic deletion (CRISPR # 1 and # 2) could be assayed by Cas9CRISPR (Figure 4C, providing cell lines with permission by Prof. Dr. Veit Hornung, BioSys Munich, Ludwigs-Maximilians-University Munich, described under Schmid-Burgk et al. (2014), OutKnocker: a web tool for rapid and simple genotyping of designer nuclease-edited cell lines, Genome Res. Oct. 24 (10): 1719-1723). Here, too, a selective and strong reduction of IL-6 secretion caused by chitin 10-15 and the known TLR2 ligands Pam2 and Pam3 was shown.
Schließlich wurde TLR2 in HEK-293 Zellen durch Transfektion von 10 ng TLR2 Plasmid (Fig. 4D oben) oder leerem Vektor (EV, unten) rekonstitutiert und mit Oligosaccharid C10- 15 und TLR2-Liganden-Kontrollen (Pam2CSK4, Pam3CSK4) stimuliert. Die Aktivität des Transkriptionsfaktors NF-κΒ wurde mittels Dual Luciferase Assays bestimmt. Es zeigte sich, dass die Expression von TLR2 ausreichte, um die Zellen Chitin-responsiv zu machen. Zudem beobachteten die Erfinder, dass eine Expression der bisher diskutierten Kandidaten Dectin-1 , Nod2 und TLR9 in HEK293T-Zellen überraschenderweise nicht zu Sensitivität gegenüber Chitin führt (nicht gezeigt), während die Expression von TLR2 zu einer Dosis-abhängigen NF-κΒ Aktivierung führte. Finally, TLR2 was reconstituted in HEK-293 cells by transfecting 10 ng TLR2 plasmid (Figure 4D top) or empty vector (EV, bottom) and using oligosaccharide C10-15 and TLR2 ligand controls (Pam 2 CSK 4 , Pam 3 CSK 4 ). The activity of the transcription factor NF-κΒ was determined by means of dual luciferase assays. It was shown that the expression of TLR2 was sufficient to make the cells chitin-responsive. In addition, the inventors observed that surprisingly, expression of the previously discussed candidate Dectin-1, Nod2 and TLR9 in HEK293T cells not to Sensitivity to chitin results (not shown) while expression of TLR2 resulted in dose-dependent NF-κΒ activation.
Die Zudem stellten die Erfinder fest, dass das Oligosaccharid C10-15 auch mit rekombinant hergestelltem murine mTLR2-Ektodomänen-Protein in einem durchflusszyto- metrischen Assay interagierte (Fig. 4E). Alexa647-gekoppeltes Chitin konnte mit mTLR2- Fc-PE stärker als mit Kontroll-lgG1 -PE angefärbt werden. Einzelne Messungen sowie eine Quantifizierung von vier Messungen sind gezeigt. Dies konnte auch mittels neuester Messungen mit der Methode Microscale Thermophoresis (MST) bestätigt werden: MST- Titration von mTLR2-Fc Protein mit Alexa647-markiertem Chitin 10-15 zeigten eine Bindung mit nanomolarer Affinität; Fig. 4F. Ähnliche Bindungsdaten wurden für Bindung an humanes TLR2 erhalten. In addition, the inventors found that the oligosaccharide C10-15 also interacted with recombinantly produced murine mTLR2 ectodomain protein in a flow cytometric assay (Figure 4E). Alexa647-coupled chitin was stained more strongly with mTLR2-Fc-PE than with control IgG1 -PE. Single measurements and a quantification of four measurements are shown. This was also confirmed by the latest measurements with the Microscale Thermophoresis (MST) method: MST titration of mTLR2-Fc protein with Alexa647-labeled chitin 10-15 showed binding with nanomolar affinity; Fig. 4F. Similar binding data were obtained for binding to human TLR2.
Zusammenfassend belegen die Ergebnisse der Erfinder, dass TLR2 der Rezeptor für molekulares Chitin bzw. hiervon abgeleitete NAG-Oligosaccharide im menschlichen und murinen System ist, so dass zukünftige Analysen der immunologischen Signaltransduk- tion sowie therapeutische Ansätze nun an diesem gut definierten Rezeptor-Signalweg ansetzen können. In summary, the inventors' findings demonstrate that TLR2 is the receptor for molecular chitin or NAG oligosaccharides derived therefrom in the human and murine systems, so that future immunological signal transduction and therapeutic approaches can now address this well-defined receptor pathway ,
4. Hochreine Oligosaccharide bestehend aus > 6 NAG-Untereinheiten weisen 4. High purity oligosaccharides consisting of> 6 NAG subunits
vielversprechende Adiuvans-Wirkunq auf  promising Adiuvans-Wirkunq on
Es ist seit langem bekannt, dass die im letzten Jahrhundert identifizierten Adjuvantien i.d.R. alle aktivierenden Liganden der PRR darstellen. Dennoch sind für die Anwendung am Menschen bislang nur wenige Adjuvantien zugelassen, so dass in der Impfstoffforschung, gerade auch für die Tumorvakzinierung, akuter Bedarf neuer Impf-Adjuvantien existiert. Auf Grund des hier beschriebenen potenten immunologischen Potenzials von hochreinen Oligosacchariden, die sich von Chitin-Fragmenten ableiten, stellten sich die Erfinder die Frage, ob diese als Adjuvans für die Herausbildung einer effizienten T- zytotoxischen oder T-Helferzell-Antwort und damit Antikörperproduktion und Impfschutz geeignet sein können. Hierfür stimulierten die Erfinder "peripheral blood mononuclear cells" (PBMC) eines repräsentativen gesunden Spenders für 12 Tage in vitro mit bestimm- ten Peptid-Antigenen (entweder von Cytomegalo- (CMV), Influenza A (Flu)- oder Epstein- Barr-Virus (EBV BMLF1 und EBV-LMP2)), IL-2 als T-Zell Wachstumsfaktor und mit oder ohne erfindungsgemäßen Oligosacchariden und anderen PRR-Agonisten. Diese Stimulation wurde entweder ohne TLR2 Stimulanzien durchgeführt ("Standard"), zusammen mit TLR2 Liganden (P3C=Pam3CSK4 oder Wirkstoff 1 , 10 μg ml), oder mit erfindungsgemäßen Oligosacchariden C10-15 (10 und 50 μg ml). Anschließend wurden die Zellen geerntet, mit relevanten Antikörpern und Tetrameren gefärbt, und mittels Durchflu ßzytometrie analysiert. It has long been known that the adjuvants identified in the last century are generally all activating ligands of the PRR. However, only a few adjuvants have been approved for use in humans, so that there is an acute need for new vaccine adjuvants in vaccine research, especially for tumor vaccination. Because of the potent immunological potential of high-purity oligosaccharides derived from chitin fragments described herein, the inventors wondered whether this would serve as an adjunct to the development of an efficient T-cytotoxic or T-helper cell response and thus antibody production and vaccine protection may be suitable. For this purpose, the inventors stimulated peripheral blood mononuclear cells (PBMC) of a representative healthy donor for 12 days in vitro with certain peptide antigens (either from cytomegalovirus (CMV), influenza A (Flu) or Epstein-Barr virus (EBV BMLF1 and EBV-LMP2)), IL-2 as T-cell growth factor and with or without oligosaccharides according to the invention other PRR agonists. This stimulation was carried out either without TLR2 stimulants ("standard"), together with TLR2 ligands (P3C = Pam 3 CSK 4 or active ingredient 1, 10 μg ml) or with oligosaccharides C10-15 according to the invention (10 and 50 μg ml). Subsequently, the cells were harvested, stained with relevant antibodies and tetramers, and analyzed by flow cytometry.
Das Ergebnis ist in der Fig. 5A dargestellt. In der Teilabbildung A ist der prozentuale Anteil der Antigen-spezifischen T-Zellen für die 4 getesteten Spezifitäten bei den verschiedenen Bedingungen dargestellt (jeweils Duplikat-Ansätze). Teilabbildung B zeigt repräsentative Dot-plots für EBV-BMLF1 spezifische CD8+ T-Zellen nach der Standard- (oben) oder Chitin- 10 μg ml (unten) Stimulationen. Wie Fig. 5A zeigt, führten die erfindungsgemäßen Oligosaccharide in 2/3 der getesteten Probanden im Vergleich zu einem Vergleichswirkstoff 1 (einem anderen Adjuvans-Kandidaten für die Anwendung im Menschen) und Pam3CSK4 zu einer starken Proliferation von Antigen-spezifischen CD8+-T- Zellen, wie durch Tetramerfärbung gezeigt; vgl. Fig. 5A-B. Diese Ergebnisse der Erfinder zeigen, dass die erfindungsgemäßen Oligosaccharide vielversprechende Immunstimula- zien und Adjuvans-Kandidaten sind. The result is shown in FIG. 5A. Panel A shows the percentage of antigen-specific T cells for the 4 specificities tested under the different conditions (duplicate approaches, respectively). Panel B shows representative dot plots for EBV-BMLF1-specific CD8 + T cells following the standard (top) or chitin 10 μg ml (bottom) stimulations. As shown in Fig. 5A, the oligosaccharides of the present invention resulted in a strong proliferation of antigen-specific CD8 + in 2/3 of the tested subjects compared to a comparative drug 1 (another human adjuvant candidate) and Pam 3 CSK 4 T cells as shown by tetramer staining; see. Fig. 5A-B. These results of the inventors show that the oligosaccharides according to the invention are promising immune stimulants and adjuvant candidates.

Claims

Patentansprüche claims
1 . Hochreines Oligosaccharid bestehend aus > 6 N-Acetylglucosamin(NAG)- Untereinheiten zur Verwendung als Immunstimulanz. 1 . High purity oligosaccharide consisting of> 6 N-acetylglucosamine (NAG) subunits for use as an immune stimulant.
2. Hochreines Oligosaccharid nach Anspruch 1 , dadurch gekennzeichnet, dass das Immunstimulanz ein Adjuvans ist. 2. Highly pure oligosaccharide according to claim 1, characterized in that the immune stimulant is an adjuvant.
3. Hochreines Oligosaccharid nach Anspruch 1 oder 2, zur Behandlung und/oder Prävention einer Tumor- oder Infektionserkrankung. 3. Highly pure oligosaccharide according to claim 1 or 2, for the treatment and / or prevention of a tumor or infectious disease.
4. Hochreines Oligosaccharid nach einem der vorherigen Ansprüche in Kombination mit einem Tumor- oder mikrobiellen Antigen. 4. A high purity oligosaccharide according to any one of the preceding claims in combination with a tumor or microbial antigen.
5. Hochreines Oligosaccharid nach einem der vorherigen Ansprüche, dadurch 5. Highly pure oligosaccharide according to one of the preceding claims, characterized
gekennzeichnet, dass es aus > 7, bevorzugt > 8, weiter bevorzugt > 9, weiter bevorzugt > 10, weiter bevorzugt > 1 1 , weiter bevorzugt > 12, weiter bevorzugt > 13, weiter bevorzugt > 14, weiter bevorzugt > 15, und höchst bevorzugt 10-15 NAG- Untereinheiten besteht.  in that it is selected from> 7, preferably> 8, more preferably> 9, more preferably> 10, more preferably> 1 1, more preferably> 12, more preferably> 13, more preferably> 14, with further preference> 15, and most preferably 10-15 NAG subunits.
6. Pharmazeutische Zusammensetzung aufweisend ein hochreines Oligosaccharid bestehend aus > 6 NAG-Untereinheiten. 6. A pharmaceutical composition comprising a high purity oligosaccharide consisting of> 6 NAG subunits.
7. Pharmazeutische Zusammensetzung nach Anspruch 6, die ein Immunstimulanz ist. The pharmaceutical composition of claim 6, which is an immune stimulant.
8. Pharmazeutische Zusammensetzung nach Anspruch 6 oder 7, die ein Adjuvans ist. A pharmaceutical composition according to claim 6 or 7, which is an adjuvant.
9. Pharmazeutische Zusammensetzung nach einem der vorherigen Ansprüche, die ein Impfstoff ist. A pharmaceutical composition according to any preceding claim, which is a vaccine.
10. Pharmazeutische Zusammensetzung nach einem der vorherigen Ansprüche zur Behandlung und/oder Prävention einer Tumorerkrankung oder zur Prävention einer Infektionserkrankung oder zur Behandlung einer chronischen Infektionserkrankung. 10. Pharmaceutical composition according to one of the preceding claims for the treatment and / or prevention of a tumor disease or for the prevention of an infectious disease or for the treatment of a chronic infectious disease.
1 1 . Pharmazeutische Zusammensetzung nach einem der vorherigen Ansprüche, die ferner zumindest ein oder mehrere Tumor- und/oder mikrobielles Antigene aufweist. 1 1. A pharmaceutical composition according to any one of the preceding claims further comprising at least one or more tumor and / or microbial antigens.
12. Pharmazeutische Zusammensetzung nach einem der vorherigen Ansprüche, dadurch gekennzeichnet, dass das hochreine Oligosaccharid aus > 7, bevorzugt > 8, weiter bevorzugt > 9, weiter bevorzugt > 10, weiter bevorzugt > 1 1 , weiter bevorzugt > 12, weiter bevorzugt > 13, weiter bevorzugt > 14, weiter bevorzugt > 15, und höchst bevorzugt 10-15 NAG-Untereinheiten besteht. 12. Pharmaceutical composition according to one of the preceding claims, characterized in that the highly pure oligosaccharide of> 7, preferably> 8, more preferably> 9, more preferably> 10, more preferably> 1 1, more preferably> 12, further preferably> 13 , more preferably> 14, more preferably> 15, and most preferably 10-15 NAG subunits.
13. Verwendung eines hochreinen Oligosaccharids bestehend aus > 6 NAG- Untereinheiten in vitro zur Stimulation von Immunzellen, vorzugsweise ausgewählt aus der Gruppe bestehend aus: Makrophagen, neutrophile Granulozyten, und B- Zellen. 13. Use of a high purity oligosaccharide consisting of> 6 NAG subunits in vitro for the stimulation of immune cells, preferably selected from the group consisting of: macrophages, neutrophilic granulocytes, and B cells.
14. Verwendung eines hochreinen Oligosaccharids bestehend aus > 6 NAG- Untereinheiten in vitro zur Aktivierung und/oder Bindung des TLR2-Rezeptors. 14. Use of a high purity oligosaccharide consisting of> 6 NAG subunits in vitro for activation and / or binding of the TLR2 receptor.
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