WO2018211113A1 - Oligosaccharides de grande pureté immunstimulants - Google Patents

Oligosaccharides de grande pureté immunstimulants 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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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

La présente invention concerne un oligosaccharide de grande pureté destiné à être utilisé comme immunostimulant, une composition pharmaceutique qui contient l'oligosaccharide de grande pureté selon l'invention, l'utilisation de l'oligosaccharide de grande pureté selon l'invention in vitro aux fins de stimulation de cellules immunitaires ou encore à l'activation/la liaison du récepteur TLR2.
PCT/EP2018/063207 2017-05-19 2018-05-18 Oligosaccharides de grande pureté immunstimulants WO2018211113A1 (fr)

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DE102017110933.1A DE102017110933A1 (de) 2017-05-19 2017-05-19 Immunstimulierende hochreine Oligosaccharide
DE102017110933.1 2017-05-19

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