WO2016032009A1 - Adjuvant - Google Patents

Adjuvant Download PDF

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WO2016032009A1
WO2016032009A1 PCT/JP2015/075019 JP2015075019W WO2016032009A1 WO 2016032009 A1 WO2016032009 A1 WO 2016032009A1 JP 2015075019 W JP2015075019 W JP 2015075019W WO 2016032009 A1 WO2016032009 A1 WO 2016032009A1
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lam
dectin
man
mice
cells
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PCT/JP2015/075019
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Sho Yamasaki
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Kyushu University, National University Corporation
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Priority to JP2017510682A priority Critical patent/JP2017526673A/ja
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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
    • A61K31/739—Lipopolysaccharides
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • 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
    • A61P31/04—Antibacterial agents
    • A61P31/06—Antibacterial agents for tuberculosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A
    • 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
    • A61K39/02—Bacterial antigens
    • A61K39/04—Mycobacterium, e.g. Mycobacterium tuberculosis

Definitions

  • the present invention relates to an adjuvant comprising a lipoarabinomannan and a method of activating a Dectin-2 -expressing cell by using a lipoarabinomannan.
  • Mycobacteria possess various cell wall components that influence host immune responses, such as trehalose-6,6'-dimycolate (TDM), mycolate, phosphatidyl-w o-inositol mannosides (PIMs), lipomannnan (LM) and lipoarabinomannan (LAM).
  • TDM trehalose-6,6'-dimycolate
  • PIMs phosphatidyl-w o-inositol mannosides
  • LM lipomannnan
  • LAM lipoarabinomannan
  • LAM is a major lipoglycan and important virulence factor of mycobacteria (Mishra et al., 2011), enabling mycobacteria to infect host organisms and survive within host cells.
  • Ethambutol an inhibitor of LAM synthesis, is widely used as an anti-mycobacterial drug (Belanger et al., 1996).
  • LAM consists of four components; a marinosyl-phosphatidyl-myo-inositol (MPI) anchor, a mannose backbone, an arabinan domain and capping moieties.
  • the capping moieties located at the terminal extremity of the arabinan domain differ among mycobacterial species, such as mannose-capped LAM (Man-LAM), phosphoinositol-capped LAM (PI-LAM) and non-capped LAM (Ara-LAM).
  • Man-LAM mannose-capped LAM
  • PI-LAM phosphoinositol-capped LAM
  • Au-LAM non-capped LAM
  • Man-LAM has been intensively studied because it exerts pleiotropic effects on host immunity (Mishra et al., 2011).
  • Pathogenic species including Mycobacterium tuberculosis, possess Man-LAM, which has been shown to suppress host immune system (Briken et al., 2004) and
  • CLRs C-type lectin receptors
  • PRRs pattern recognition receptors
  • DC-SIGN DC-specific intercellular adhesion molecule-3 grabbing nonintegrin
  • SIGNRl also called CD209b
  • SIGNR3 SIGNR3
  • MMR Macrophage mannose receptor
  • CLRs Mincle (gene symbol Clec4e) and MCL (gene symbol Clec4d) are Fc receptor ⁇ chain (FcRy, gene symbol Fcerl g)-coup ⁇ ed activating receptors for mycobacterial glycolipids (Ishikawa et al., 2009; Miyake et al., 2013).
  • FcRy gene symbol Fcerl g
  • Another CLR, Dendritic cell-associated C-type lectin-2 (Dectin-2, gene symbol Clec4n) is located adjacent to Mincle and MCL within the gene cluster on chromosome 6. Dectin-2 is an Fc receptor ⁇ chain (FcRy, gene symbol Fcerl g)-coup ⁇ ed activating receptors for mycobacterial glycolipids (Ishikawa et al., 2009; Miyake et al., 2013).
  • Another CLR, Dendritic cell-associated C-type lectin-2 (Dectin-2, gene symbol Clec
  • Dectin-2 is a direct receptor for Man-LAM.
  • Man-LAM recognition by Dectin-2 induced the production of both pro- and anti-inflammatory cytokines in DCs.
  • the present invention is based on this discovery. As such, the present invention provides the followings:
  • An activating agent of Dectin-2 -expressing cell comprising a lipoarabinomannan.
  • An adjuvant comprising a lipoarabinomannan.
  • a vaccine comprising an antigenic component and the adjuvant according to (3) or (4).
  • a method of increasing the immune response to an antigenic component comprising administering a vaccine comprising the adjuvant according to (3) or (4) and an antigenic component to a subject.
  • a method of activating a Dectin-2 -expressing cell comprising contacting a lipoarabinomannan with said cell.
  • C and D Lumbar (C) and inguinal (D) lymph nodes were collected at 23 days after immunization. Lymph node cells were stimulated with MOG 35-55 peptide for four days. Concentrations of IL-17, IFN- ⁇ and GM-CSF were determined using ELISA.
  • E and F BMDCs obtained from WT, Clec4e ⁇ ' ⁇ , Fcerlg ⁇ ' ⁇ or MyD8S ⁇ ' ⁇ mice were stimulated with plate-coated LAM (0.3 ⁇ 11) or LPS (10 ng/ml) for 48 hours.
  • Dectin-2 recognizes pathogenic mycobacterial species though LAM.
  • A NFAT-GFP reporter cells expressing Mincle + FcRy (Mincle) or Dectin-2 + FcRy
  • Reporter cells were stimulated with plate-coated Water extract or C:M extract for 24 hours.
  • C Reporter cells expressing FcRy alone or Dectin-2 + FcRy were stimulated with the indicated amounts of LAM derived from M. tuberculosis strain Aoyama B in plate-coated form for 24 hours.
  • Dectin-2 selectively recognizes mannose-capped LAM.
  • NFAT-GFP reporter cells expressing Mincle + FcRy or Dectin-2 + FcRy were stimulated with the indicated strains of heat-killed NTM.
  • B Reporter cells expressing Dectin-2 + FcRy were stimulated with M. bovis BCG pre-treated with or without a-mannosidase for 24 hours.
  • C and D Reporter cells expressing Dectin-2 WT + FcRy or Dectin-2 QPD + FcRy were stimulated with plate-coated LAM (C) and NTM strains (D) for 24 hours.
  • BMDCs obtained from WT or Clec4n ' ⁇ mice were stimulated with the indicated amounts of plate-coated LAM or TDM for 48 hours (A and C). BMDCs were infected with 1 to 10 x 10 9 of M. bovis BCG for 48 hours (B and D). LPS was used as control. The concentrations of MIP-2, TNF, IL-6 (A and B), IL-10 and IL-2 (C and D) were measured using ELISA.
  • E BMDCs were stimulated for 48 hours with M bovis BCG or clinical isolates of heat-killed M. abscessus derived from ten individual patients. The concentrations of MIP-2, IL-10 and IL-2 were measured using ELISA.
  • D Histology of the lungs from control mice, LAM (50 ⁇ g)- or TDM (50 ⁇ g)-injected mice were was examined by hematoxylin-eosin staining at day 7. Scale bars represent 0.1 mm. Data are representative of three separate experiments.
  • BMDCs obtained from WT, Clec4n ' ⁇ , Clec4e ⁇ ' ⁇ , or FcerJg ⁇ ' ⁇ mice were left untreated or stimulated with plate-coated LAM or LPS for 48 hours. The surface expressions of CD40 and CD80 were analyzed using flow cytometry.
  • B BMDCs were pulsed with OVA323-339 peptides and cocultured with CFSE-labeled CD4 + OT-II T cells for three days in the presence or absence of plate-coated LAM. Cytokine concentrations were determined using ELISA. Cell proliferation was analyzed using flow cytometry for dilution of CFSE within the CD4 + population.
  • FIG. 8 Schematic structure of mannose-capped LAM (related to Figure 1). A typical structure of Man-LAM in M. tuberculosis is shown.
  • Dectin-2 QPD Surface expression level of Dectin-2 QPD is comparable to that of wild-type in reporter cells (related to Figure 3). Wild-type Dectin-2 or Dectin-2 QPD was stained by anti-Dectin-2 raAb (clone: D2.11E4) followed by incubating with phycoerythrin-conjugated donkey anti-rat IgG (H+L), then their surface expressions were measured by flow cytometry.
  • SIGNR1, SIGNR3 and MMR are dispensable for Man-LAM-induced cytokine production in DCs (related to Figure 4).
  • A Generation of SIGNR1 -deficient (CD209b OTIUOTK ) mice. Genomic SIGNR1 structure and targeting construct with diphtheria toxin receptor (DTR) and neomycin resistance insertion. The Cd209b exons are shown as black boxes. TK; thymidine kinase, E; EcoRI site, B; BamHI site. (B) BMDCs obtained from WT and Cd209b ⁇ ' ⁇ mice were stimulated with indicated amounts of plate-coated LAM for 48 hours. The concentrations of TNF, IL-6, IL-2 and IL-10 were measured using ELISA.
  • C RT-PCR analyses of Clec4n, Cd209d and Cd206 in splenic DCs and BMDCs.
  • D BMDCs from WT and Clec4n ' ' mice were transduced with vector alone (-) or SIGNR3 (R3) through lentivirus vector. The expression of SIGNR3 was confirmed by RT-PCR and staining with anti-Flag mAb. Cells were stimulated with Man-LAM for 48 hours and concentrations of TNF and IL-10 were determined by ELISA.
  • BMDCs obtained from WT mice were treated with anti-MMR mAb or rat IgGl as a contorol and then stimulated with indicated amounts of plate-coated LAM for 48 hours.
  • the concentrations of IL-10 and IL-2 were measured using ELISA.
  • F BMDCs obtained from WT, Fcerlg '1' , Clec4e ⁇ ' ⁇ or Clec4d ⁇ ' ⁇ mice were stimulated with indicated amounts of plate-coated LAM for 48 hours.
  • the concentrations of TNF and IL-10 were measured using ELISA.
  • G BMDCs obtained from WT or MyD88 ⁇ ' ⁇ mice were stimulated with indicated amounts of plate-coated LAM for 48 hours.
  • the concentration of IL-10 was measured using ELISA.
  • H WT BMDCs were stimulated with plate-coated anti-Dectin-2 mAb or anti-Mincle mAb for 48 hours. The concentration of IL-10 was measured using ELISA.
  • B and D to H All data are presented as the means ⁇ SD of triplicate and are representative of three separate experiments.
  • Man-LAM promotes T cell responses in PBMCs from tuberculosis patients (related to Figure 6).
  • E PBMCs from a tuberculosis patient who responded most potently to antigen peptide (CFP-10) were incubated in the presence or absence of synthetic peptide from CFP-10, plate-coated LAM or anti-human Dectin-2 antibody for four days.
  • C to F The concentration of each cytokine was measured using ELISA. The data are presented as the means ⁇ SD. *, p ⁇ 0.05. **, p ⁇ 0.01.
  • an adjuvant is defined as a substance that non-specifically enhances the immune response to an antigen. Depending on the nature of the adjuvant it can promote either a cell-mediated immune response, a humoral immune response or a mixture of the two. Since the enhancement of the immune response is non-specific, it is well understood in the art that the adjuvant of the present invention can be used with different antigens to promote responses against different targets e.g. with an antigen from Mycobacterium tuberculosis to promote immunity against Mycobacterium tuberculosis or with an antigen derived from a tumor, to promote immunity against tumors of that specific kind. The adjuvant is added to the antigenic component and used as a vaccine.
  • an antigenic component or substance is a molecule, which reacts with specific receptors on dendritic cells, macrophages and granulocytes.
  • a molecule that can stimulate the development of specific dendritic, T, B or NKT cells leading to the formation of a memory population of immune cells that will promote a faster "memory" response if the antigen is encountered a second time by immune cells. Since memory populations are rarely clonal, in practice this means that an antigen is any molecule or collection of molecules, which can stimulate an increase in immune responses when it is re-encountered by immune cells from an individual who has previously been exposed to it.
  • examples of Dectin-2 -expressing cell are, but not limited to, dendritic cells, T cells, B cells or NKT cells.
  • the antigenic component can be a polypeptide or a part of the polypeptide, which elicits an immune response in an animal or a human being, and/or in a biological sample determined by any of the biological assays.
  • the antigenic component may be any pure chemical species such as a protein or a fragment thereof or artificial mixtures prepared of such species. But it can also be any naturally occurring mixture of chemical species such as e.g. a cell homogenate or fractions thereof, a culture filtrate from microorganisms or cell tissues from multicellular organisms, e.g. higher animals.
  • the antigenic substance may be derived from a culture of metabolising Mycobacterium tuberculosis, Mycobacterium bovis and other environmental mycobacteria such as e.g. Mycobacterium avium and Mycobacterium intracellulars
  • immune response is illustratively any alteration of a subject's immune system in response to challenge from a vaccine, infectious or otherwise foreign organism, tissue, cell, antigen, antibody, nucleotide strand, or other immune stimulating substance recognized in the art.
  • Non-limiting examples of immune responses include in vitro activation of dendritic cells, in vitro secretion of IL-2, IL-4, IFN- ⁇ , IL-17 or GM-CSF in CD4 + or CD8 + T-cells; protection from challenge after M.
  • tuberculosis or other infectious organism alteration in nitrite levels; Thl and Th2 cytokine responses in various immune compartments; alteration in allotype and isotype antibody levels; in vitro recognition of antigen; B-cell responses; inhibition of growth of M. tuberculosis in infected macrophages; survival; or other response known in the art.
  • a vaccine is defined as a suspension of dead, attenuated, or otherwise modified microorganisms (bacteria, viruses, or rickettsiae) or parts thereof for inoculation to produce immunity to a disease.
  • the vaccine can be administered either prophylactic to prevent disease or as a therapeutic vaccine to combat already existing diseases such as cancer or latent infectious diseases but also in connection with allergy and autoimmune diseases.
  • the vaccine can be emulsified in the adjuvant of the present invention for potentiating the immune response.
  • the vaccine (vaccine composition) of the present invention can be formulated as freeze-dried or liquid preparations according to any means suitable in the art.
  • liquid form preparations include solutions, suspensions, syrups, slurries, and emulsions.
  • Suitable liquid carriers include any suitable organic or inorganic solvent, for example, water, alcohol, saline solution, buffered saline solution, physiological saline solution, dextrose solution, water propylene glycol solutions, and the like, preferably in sterile form.
  • the vaccine can be formulated in either neutral or salt forms.
  • Pharmaceutically acceptable salts include the acid addition salts and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, and the like.
  • inorganic acids such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, mandelic, and the like.
  • Salts formed from free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol,
  • the vaccine is preferably formulated for inoculation or injection into the subject.
  • the vaccine of the invention can be formulated in aqueous solutions such as water or alcohol, or in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or
  • physiological saline buffer The solution can contain formulatory agents such as suspending, preserving, stabilizing and/or dispersing agents.
  • Injection formulations can also be prepared as solid form preparations which are intended to be converted, shortly before use, to liquid form preparations suitable for injection, for example, by constitution with a suitable vehicle, such as sterile water, saline solution, or alcohol, before use.
  • the vaccine of the present invention can also be formulated in sustained release vehicles or depot preparations. Such long acting formulations can be administered by inoculation or implantation (for example subcutaneously or intramuscularly) or by injection.
  • the vaccine can be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • suitable polymeric or hydrophobic materials for example, as an emulsion in an acceptable oil
  • ion exchange resins for example, as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • Liposomes and emulsions are well-known examples of delivery vehicles suitable for use as carriers.
  • the vaccine is administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic.
  • the dosage of the vaccine will depend on the route of administration and will vary according to the age of the person to be vaccinated and, to a lesser degree, the size of the person to be vaccinated, the capacity of the individual's immune system to mount an immune response, and the degree of protection desired .
  • Suitable dosage ranges are of the order of several hundred micrograms active ingredient per vaccination with a preferred range from about 0.1 ⁇ g/kg/day to 1000 ⁇ g/kg/day, such as in the range from about 1 ⁇ g/kg/day to 300 ⁇ g/kg/day, and especially in the range from about 10 ⁇ g/kg day to 50 ⁇ g/kg/day.
  • Suitable regimens for initial administration and booster shots are also variable but are typified by an initial administration followed by subsequent inoculations or other administrations.
  • Dectin-2 is a direct receptor for Man-LAM.
  • Man-LAM recognition by Dectin-2 induced the production of both pro- and anti-inflammatory cytokines in DCs.
  • Man-LAM potently promoted T cell-mediated acquired immunity as an adjuvant without causing detrimental inflammation.
  • Dectin-2-deficient mice that Dectin-2 plays a critical role in host responses against mycobacterial infection.
  • Man-LAM Mannose-capped lipoarabinomannan
  • BMDCs bone marrow-derived dendritic cells
  • LAM promotes experimental autoimmune encephalomyelitis (EAE) through FcRy.
  • EAE was not induced by incomplete Freunds' adjuvant (IF A) alone, a single injection of LAM elicited EAE with 100% incidence ( Figures 1A and IB).
  • the EAE symptoms were completely abrogated in Fcerlg ⁇ ' ⁇ mice ( Figures 1 A and IB), suggesting that FcRy-coupled receptor(s) may contribute to LAM-induced EAE.
  • lymphocytes from Fcerlg " mice exhibited impaired ex vivo recall responses to MOG peptides as judged by the production of IL-17 interferon- ⁇ (IFN-y) and granulocyte macrophage-colony stimulating factor (GM-CSF) ( Figures 1C and ID).
  • IFN-y interferon- ⁇
  • GM-CSF granulocyte macrophage-colony stimulating factor
  • soluble LAM did not induce cytokine production
  • plate-coated LAM could stimulate BMDCs to secrete a large amount of the pro-inflammatory cytokines, macrophage
  • Dectin-2 recognizes mycobacterial LAM.
  • Mincle, MCL and Dectin-2 are FcRy-coupled activating receptors within the same gene cluster, and two of these receptors, Mincle and MCL, recognize mycobacteria (Ishikawa et al., 2009; Miyake et al., 2013). We therefore assumed that Dectin-2 may also be evolved as a receptor for mycobacteria. Indeed, Dectin-2 was demonstrated to recognize the virulent strain M. tuberculosis H37Rv and the vaccine strain M. bovis Bacille de Calmette et Guerin (BCG) to activate the reporter cells, in a similar manner to Mincle (Figure 2A). However, the ligand for Dectin-2 was distinct from Mincle ligand trehalose-6,6'-dimycolate (TDM) ( Figure 2A).
  • TDM Mincle ligand trehalose-6,6'-dimycolate
  • LAM constitutes the most abundant hydrophilic lipoglycan (Leopold and Fischer, 1993) ( Figure 8).
  • LAM derived from M. tuberculosis activated reporter cells expressing Dectin-2 ( Figure 2C).
  • Dectin-2 directly recognized the LAM, as soluble Dectin-2 protein (Dectin-2 -Ig) bound to purified LAM in a dose-dependent manner ( Figure 9).
  • Dectin-2 recognizes Mycobacteria through mannose-capped LAM.
  • LAM The structure of LAM differs depending on the mycobacterial species, particularly with the respect to the capping moieties (Briken et al., 2004).
  • M. smegmatis possesses PI-LAM.
  • NTM non-tuberculosis mycobacteria
  • Dectin-2 recognized slow growing strains, such as M. intracellular and M. gordonae, which possess Man-LAM. In contrast, Dectin-2 did not recognize M. abscesssus and M.
  • Man-LAM induces cytokines production by DCs in a Dectin-2-dependent manner.
  • Man-LAM similar to TDM, induced the expression of inflammatory cytokines, such as MIP-2, TNF and IL-6, in a dose-dependent manner ( Figure 4A).
  • the LAM-induced cytokine production was abolished in Clec4rf' ⁇ BMDCs, whereas the TDM-mediated cytokine production was not altered ( Figure 4A).
  • Man-LAM also slightly enhanced IL-12 p40 in a Dectin-2 dependent manner. The production of TNF and IL-6 upon M.
  • Man-LAM immune-suppressive action of Man-LAM (Geijtenbeek et al., 2003; Wieland et al., 2007).
  • Man-LAM potently induced the production of anti-inflammatory cytokine, IL-10, in BMDCs ( Figure 4C, left).
  • Other pathogen-associated molecular patterns (PAMPs) TDM ( Figure 4C, right) and LPS did not induce the secretion of IL-10 and IL-2, indicating that Man-LAM has a unique profile of the cytokine production.
  • Man-LAM-induced release of these cytokines was completely suppressed in Clec4n ⁇ BMDCs ( Figure 4C). Although TNF production during M.
  • Macrophage mannose receptor could also bind to Man-LAM (Nigou et al., 2001). Since MMR expression was detected in BMDCs ( Figure 1 1C), we assessed its role in Man-LAM-induced cytokine release using anti-MMR blocking monoclonal antibody (mAb). However, the mAb treatment did not influence the production of IL-10 and IL-2 in BMDCs ( Figure HE).
  • Man-LAM induces minimal inflammation in vivo.
  • BALF bronchoalveolar lavage fluid
  • Figures 5A and 5B LPS induced a significant increase in TNF production and cell infiltration in WT mice.
  • Man-LAM did not induce marked inflammatory responses. Consistent with this observation, FcRy deficiency had no apparent effect compared with Man-LAM-treated WT mice.
  • Man-LAM stimulation enhances APC functions to promote IL-17 production in vitro.
  • BMDCs were pulsed with the ovalbumin (OVA) antigen peptides and co-cultured with T cells obtained from OVA-specific OT-II TCR transgenic mice in the presence or absence of Man-LAM.
  • OVA ovalbumin
  • T cells do not express Dectin-2 (Ariizumi et al., 2000)
  • this system enables us to evaluate the role of Man-LAM in APC functions toward T cell priming and activation.
  • the antigen-specific secretion of IL-17 from CD4 + OT-II T cells was significantly augmented when the cells were co-cultured with Man-LAM-treated APCs ( Figure 6B).
  • Man-LAM promotes antigen-specific human T cell responses through human Dectin-2.
  • Man-LAM influences human T cell responses as observed in murine T cells. Importantly, Man-LAM activated reporter cells expressing hDectin-2, and this activity was blocked in the presence of anti-hDectin-2 mAb ( Figure 12B).
  • PBMCs Peripheral blood mononuclear cells
  • WRFQEAANKQKQEL(SEQ ID NO: l) CIO peptide
  • CFP-10 10 kDa culture filtrate antigen
  • T cells from tuberculosis patients presumably through the activation of myeloid cells in PBMCs.
  • Dectin-2 is a direct and functional receptor for mycobacterial Man-LAM. Mincle, MCL and Dectin-2 are located in the same gene cluster, and we found that all these CLRs recognize mycobacteria (Ishikawa et al., 2009;
  • Dectin-2 preferentially binds to high mannose structures, similar to DC-SIGN, SIGNR1, SIGNR3 and MMR (McGreal et al., 2006), all of which possess the mannose-binding EPN sequence within their carbohydrate recognition domains (CRDs) (Drickamer, 1992).
  • Man-LAM possesses polysaccharide chains that terminates in a cc-l,2-mannose cap (Mishra et al., 201 1). It is highly likely that the a-l,2-linked mannose residues of Man-LAM are the direct determinant recognized by Dectin-2.
  • Man-LAM is densely distributed in the envelopes of mycobacteria with the specific configuration, thereby their polar mannose-caps are exposed on the bacterial surface with oligomeric valency.
  • the multivalent a-l,2-mannose residue is not inconsistent with the characteristics of pathogen associatd molecular patterns (PAMPs) recognized by Dectin-2 (Ishikawa et al., 2013; Saijo et al., 2010).
  • PAMPs pathogen associatd molecular patterns
  • TLR-bound lipoprotein may provide a scaffold for soluble Man-LAM through hydrophilic interaction in aqueous media, which leads to ligand multimerization sufficient for the engagement of Dectin-2.
  • multimerized Man-LAM in oil emulsion exhibited potent adjuvant activity in vivo.
  • Dectin-2 may discriminate multivalent PAMPs presented on "real" pathogens, presumably to prevent false recognition of their targets.
  • PIMs phosphatidyl-wyo-inositol mannosides
  • Dectin-2 may not be able to access "short" PIMs within "tall” cell wall components, such as long-chain mycolic acids, glycolipids, lipoglycans and polysaccharides (Mishra et al., 2011 ; Torrelles et al., 2006).
  • SIGNR1, SIGNR3 and MMR have been reported as murine receptor for Man-LAM (Koppel et al., 2004; Schlesinger et al, 1994; Tanne et al., 2009).
  • Peritoneal macrophages from Cd209b ⁇ ' ⁇ mice produce less, but detectable, IL-10 in response to Man-LAM (Wieland et al., 2007).
  • gene ablation of SIGNR1 and anti-MMR blocking mAb did not influence the LAM-induced cytokine production in BMDCs.
  • SIGNR3 is not expressed on BMDCs and forced expression of SIGNR3 did not rescue cytokine production in Clec4rf' ⁇ BMDCs.
  • Man-LAM-induced in vivo responses were also completely abolished in Clec4n ' ⁇ mice, it is possible that SIGNR3 plays a role in Man-LAM responses in particular cells, such as dermal DCs, that express SIGNR3 (Nagaoka et al., 2010).
  • these Man-LAM-binding molecules may promote the binding of myeloid cells to Man-LAM-bearing bacteria (Tanne et al., 2009), thereby leading to the efficient phagocytosis of mycobacteria.
  • IL-10 production during infection correlates with susceptibility to M. tuberculosis.
  • Large amounts of IL-10 can be detected in the serum of active tuberculosis patients, particularly in response to hyper virulent strains of M. tuberculosis
  • IL-10 may limit excessive damage to the host tissue (Redford et al., 201 1).
  • CARD9 also mediates signaling through Mincle, the effect of Mincle deficiency on the pathologies during mycobacterial infection was modest compared with those of CARD9 ⁇ ⁇ mice (Behler et al., 2012; Heitmann et al., 2013; Lee et al., 2012).
  • Man-LAM could induce IL-23 production. This activity is lost in the lipophilic extract of the strain (Jonsson et al., 2012), supporting the idea that hydrophilic Man-LAM is involved in the promotion of Thl7 cell differentiation.
  • Man-LAM enhanced IFN- ⁇ production induced by mycobacterial antigen. The undetectable concentration of IL-17 secretion is consistent with previous observation (Yamashita et al., 2013), although the underlying mechanism is currently unknown.
  • T cells in tuberculosis patients may have already skewed to a Thl cell phenotype upon repetitive antigen exposure during infection.
  • Dectin-2 ligand Man-LAM uniquely induces the production of IL-10 and IL-2, despite the fact that both CLRs share the same signaling subunit FcRy.
  • IL-2 production from DCs may contribute to the adjuvanticity by promoting T cell priming (Granucci et al., 2001).
  • Man-LAM In addition to the functions of Man-LAM described above, Man-LAM is known to have pleiotropic functions during mycobacterial infection. Mycobacteria limit
  • Man-LAM is one of the candidate involved in this process (Fratti et al, 2003; Vergne et al., 2004), while more detailed studies are needed to determine whether Dectin-2 -mediated signaling affect phagosome-lysosome fusion.
  • a recent study has demonstrated that Man-LAM treatment inhibits T cell migration from the draining lymph nodes (Richmond et al., 2012). This effect seems to occur independently of Dectin-2, as its expression was not detected in any subsets of T cells (Ariizumi et al., 2000).
  • Lipids extract M. bovis BCG was fractionated by distilled water with repeated washing five times. After centrifugation, the soluble fraction was collected. The insoluble fraction was further delipidated with C:M (2: 1, vol/vol). Each fraction was resuspended in a volume of isopropanol at equivalent amount of 0.1 mg as the original M. bovis BCG weight.
  • Mycobacterial lipid extracts LAM in aqueous solution (1 mg/ml), TDM dissolved in C:M at 1 mg/ml and Candida albicans cell wall mannan (5 mg/ml) were diluted in isopropanol and added to 96-well plates at 20 ⁇ /well, followed by evaporation of the solvents as previously described (Ishikawa et al., 2009).
  • Reporter cells were stimulated for 24 hours and the activation of NFAT-GFP was monitored using flow cytometry.
  • BMDCs were stimulated for two days, then the culture supernatants were collected. The concentrations of each cytokine were determined by ELISA. Activation was determined using surface staining of the costimulatory molecules CD40 and CD80 by flow cytometry.
  • BMDCs were generated from WT and Clec4n ' ⁇ mice as described above. BMDCs were left untreated or stimulated with indicated amount of plate-coated LAM in the presence of OVA323-339 peptides (ABGENT).
  • CD4 + T cells from OT-II Tg mice were purified with anti-CD4-conjugated magnetic beads (MACS, Miltenyi), and then labeled with CFSE (DOJINDO) and cocultured with OVA-pulsed DCs in 96-well plates. On day 3, the supernatants were harvested and determined the concentration of IFN- ⁇ , IL-17 and IL-10 using ELISA. CFSE-labeled T cells were analyzed for dilution of CFSE within the CD4 + T cell population using flow cytometry.
  • EAE Experimental autoimmune encephalomyelitis
  • mice received three daily intraperitoneal administrations of 500 ng of pertussis toxin (List Biological Laboratories) starting on day 1. The disease severity was scored as previously described (Miyake et al., 2013).
  • cells were collected from the axillary, inguinal and lumbar (paraaortic) lymph nodes on day 23. Lymphocytes (5 x 10 s cells/well) were stimulated with MOG 35 _55 peptides (0, 3, 10 and 30 ⁇ g/ml) for four days.
  • concentrations of IL-17, IFN- ⁇ and GM-SCF in culture supernatants were determined by ELISA.
  • BMDCs were infected with 1 to 10 x 10 9 CFU of M. bovis BCG. After 48 hours, the culture supernatants were collected and cytokine concentration was determined by ELISA.
  • WT mice and Clec4n ' ⁇ mice were anesthetized with isoflurane, and each mouse was subsequently infected intranasally with 2.5 x 10 6 CFU M. avium complex (MAC) per mouse.
  • MAC avium complex
  • Lipoarabinomannan mannose caps do not affect mycobacterial virulence or the induction of protective immunity in experimental animal models of infection and have minimal impact on in vitro inflammatory responses.
  • embAB genes of Mycobacterium avium encode an arabinosyl transferase involved in cell wall arabinan biosynthesis that is the target for the antimycobacterial drug ethambutol. Proc Natl Acad Sci U S A 93, 1 1919-11924.
  • lipoarabinomannan and related lipoglycans from biogenesis to modulation of the immune response. Mol Microbiol 53, 391 -403. Chan, E.D., Morris, K.R., Belisle, J.T., Hill, P., Remigio, L.K., Brennan, P.J., and Riches, D.W. (2001). Induction of inducible nitric oxide synthase-NO* by lipoarabinomannan of
  • Mycobacterium tuberculosis is mediated by MEK1-ERK, MKK7-JNK, and NF- ⁇ signaling pathways.
  • Infect Immun 69, 2001 -2010. Dorhoi, A., Desel, C, Yeremeev, V., Pradl, L., Brinkmann, V., Mollenkopf, H.J., Hanke, K., Gross, O., Ruland, J., and Kaufmann, S.H. (2010).
  • the adaptor molecule CARD9 is essential for tuberculosis control. J Exp Med 207, 777-792.
  • Mycobacterium tuberculosis lipoarabinomannan enhances LPS-induced TNF-a production and inhibits NO secretion by engaging scavenger receptors.
  • Microb Pathog 50 350-359.
  • Dectin-2 is a C-type lectin with specificity for high mannose. Glycobiology 16, 422-430. Mishra, A.K., Driessen, N.N., Appelmelk, B.J., and Besra, G.S. (2011). Lipoarabinomannan and related glycoconjugates: structure, biogenesis and role in Mycobacterium tuberculosis
  • C-type Lectin MCL Is an FcRy-Coupled Receptor that Mediates the Adjuvanticity of Mycobacterial Cord Factor. Immunity.
  • Mannosylated lipoarabinomannans inhibit IL-12 production by human dendritic cells: evidence for a negative signal delivered through the mannose receptor. J Immunol 166, 7477-7485.
  • Mannose-Capped Lipoarabinomannan from Mycobacterium tuberculosis Preferentially Inhibits Sphingosine-1 -Phosphate-Induced Migration of Thl Cells. J Immunol 189, 5886-5895.
  • Dectin-2 is a Syk-coupled pattern recognition receptor crucial for Thl 7 responses to fungal infection. J Exp Med 206, 2037-2051.
  • Dectin-2 is a pattern recognition receptor for fungi that couples with the Fc receptor ⁇ chain to induce innate immune responses. J Biol Chem 281, 38854-38866.
  • DC-SIGN is the major Mycobacterium tuberculosis receptor on human dendritic cells. J Exp Med 797, 121-127.
  • C-type lectin Mincle is an activating receptor for pathogenic fungus, Malassezia. Proc Natl Acad Sci U S A 106, 1897-1902.
  • C-type lectin receptors Dectin-3 and Dectin-2 form a heterodimeric pattern-recognition receptor for host defense against fungal infection. Immunity 39, 324-334.
  • mice were obtained from Japan Clea (Tokyo, Japan) or Kyudo (Fukuoka,
  • Clec4n ⁇ ' ⁇ mice were backcrossed for at least nine generations to C57BL/6J (Saijo et al., 2010).
  • Fcerlg ⁇ ' ⁇ mice (Park et al., 1998), MyD88 ⁇ j - mice (Kawai et al., 1999) and OVA-specific TCR OT-II transgenic mice (Barnden et al., 1998) were used on a C57BL/6 background.
  • Cd209b OT TR mice were established with Rl embryonic stem cells (the Fl offspring of 129Xl/SvJ and 129Sl/Sv mice) and used as C57BL/6-129 mixed genetic background (Figure 11 A). All mice were maintained in a filtered-air laminar-flow enclosure and given standard laboratory food and water ad libitum. All animal protocols were approved by the committee of Ethics on Animal Experiment, Faculty of Medical Sciences, Kyushu University, Chiba
  • M. smegmatis strain mc2155 was cultured in Middlebrook 7H9 broth as previously described (Morita et al., 2005).
  • M. bovis Bacille de Calmette et Guerin (BCG) was purchased from Japan BCG Laboratory.
  • Ten NTM strains including M. avium, M. intracellulare, M. abscessus and M. gordonae were provided by the Division of Respiratory Medicine, Respiratory and Stress Care Center, agoshima University Hospital. These strains were isolated from patients diagnosed with NTM lung disease according to the American Thoracic Society's criteria (Griffith et al., 2007).
  • Virulent strain M. tuberculosis H37Rv and NTM strains were heat-killed before use.
  • LAM from M. tuberculosis strain Aoyama B was purchased from Nacalai tesque.
  • TDM TDM, LPS (L4516) and a-mannosidase were purchased from Sigma- Aldrich.
  • the concentrations of TNF, IL-6, IL-10, IL-2, IFN- ⁇ , GM-CSF (BD Biosciences, Franklin Lakes, NJ), MIP-2 and IL-17 (R&D Systems, Abingdon, UK) were determined by ELISA.
  • the concentrations of human IFN- ⁇ from PBMCs were analyzed using Human IFN- ⁇ High Sensitivity ELISA Kit (Abeam, US).
  • Mycobacterial antigen M. avium sensitin PPD was purchased from Statens Serum Instit (Copenhagen, Denmark). Recombinant MAC Ag85A was kindly provided from M. Sugita (Kyoto Univ.) (Matsunaga et al., 2008).
  • PBMCs (2 x 10 5 /well) were incubated in 96-well plates with or without synthetic peptide from ESAT-6 or CFP-10, LAM or anti-human Dectin-2 Ab for four days.
  • the culture supernatants were collected for cytokine titrations.
  • the concentration of IFN- ⁇ was determined using ELISA.
  • Antibodies Anti-Mannose Receptor mAb (15-2) for neutralization was purchased from Abeam (Da Silva et al., 2009) and anti-human Dectin-2 mAb (545943) for neutralization was from R&D Systems (Gringhuis et al., 2011). Anti-mouse Dectin-2 mAb (D2.1 1E4) was purchased from Abeam. Anti-Flag mAb (1E6) was purchased from Wako. Phycoerythrin-conjugated donkey anti-rat IgG (H+L) (712-116-153) and donkey anti-mouse IgG (H+L) (715-116-151) were obtained from Jackson ImmunoResearch. Neutralizing anti-IL-2 mAb (JES6-1 A 12) and isotype-matched control mAb Rat IgG2a ⁇ (eBR2a) was purchased from eBioscience.
  • mice Intratracheal injection.
  • WT and Fcerlg '1 " mice were anesthetized, then 100 ⁇ g LAM and 10 ⁇ g LPS in 100 ⁇ of aqueous solution were administered to mice intratracheally.
  • 100 ⁇ of sterile saline was administered to mice as a control.
  • Eight hours after administrations bronchoalveolar lavage fluid (BALF) was collected by flushing the lungs two times with 800 ⁇ of PBS.
  • the concentrations of TNF in the BALF was measured by ELISA.
  • Man-LAM was prepared as water-in-oil-in-water emulsion (Takimoto et al., 2006) and TDM was prepared as oil-in-water emulsion as previously described (Ishikawa et al., 2009).
  • 100 ⁇ of emulsion containing 50 ⁇ g of LAM or TDM was injected intravenously into 6 to 10-week-old mice.
  • Emulsion without LAM nor TDM was injected as a vehicle control.
  • Lung weight index (LWI) was calculated as previously described (Ishikawa et al., 2009) and lungs were fixed in 10% formaldehyde for hematoxylin-eosin staining.
  • SIGNR3 gene was introduced into expression vector (CSII-CMV-MCS-IRES-Bsd).
  • HEK293T cells were transfected with SIGNR3 expression vector together with packaging vector (pCMV-VAV-G-RSV-Rev and pCAG-HIVgp). Culture supernatant was collected at three days after transfection. Virus was concentrated by
  • BMDCs were incubated with lentivirus with 8 ⁇ g/ml polybrene (Sigma-Aldrich). After 24 hours, the medium was replaced with fresh culture medium. Lentivirus-infected cells were selected by 10 g/ml of blastcidin S (InvivoGen).
  • Human monocytes Preperation of human monocytes and monocyte-derived DCs.
  • human CD14 + monocytes were purified from PBMCs using anti-human CD 14 MicroBeads (Miltenyi Biotech, Germany).
  • Dendritic cells were obtained from CD14 + monocytes after culture in RPMI1640 supplemented with 10% FBS, non-essential amino acid, anti-biotics, 10 ng/ml human GM-CSF and 10 ng/ml human IL-4 for seven days.
  • Human monocytes (2.5 x 10 5 /well) were incubated in 96-well plates with LAM in the presence or absence of anti -human Dectin-2 mAb for four days.
  • Human dendritic cells (2 x 10 5 /well) were incubated in 96-well plates with BCG in the presence or absence of anti-human Dectin-2 mAb for 48 hours. The culture supernatants were collected for cytokine titrations. The concentration of TNF, IL-10 and IL- ⁇ ⁇ was determined using ELISA.

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Abstract

La présente invention concerne un agent activateur de cellule exprimant la dectine-2, comprenant une lipoarabinomannane, et un adjuvant comprenant un lipoarabinomannane.
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CN109843327A (zh) * 2016-07-07 2019-06-04 小利兰·斯坦福大学托管委员会 抗体佐剂缀合物
CN111032087A (zh) * 2017-06-28 2020-04-17 小利兰·斯坦福大学托管委员会 用于dectin-2刺激和癌症免疫治疗的方法和组合物
WO2022108897A1 (fr) * 2020-11-20 2022-05-27 Vanderbilt University Utilisation de la microvirine dans l'identification de lipoarabinomannane coiffé de mannose de mycobacterium tuberculosis
US11400164B2 (en) 2019-03-15 2022-08-02 Bolt Biotherapeutics, Inc. Immunoconjugates targeting HER2

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JP2008143830A (ja) * 2006-12-08 2008-06-26 Institute Of Physical & Chemical Research Ｔｈ１細胞分化促進剤

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109843327A (zh) * 2016-07-07 2019-06-04 小利兰·斯坦福大学托管委员会 抗体佐剂缀合物
US11110178B2 (en) 2016-07-07 2021-09-07 The Board Of Trustees Of The Leland Standford Junior University Antibody adjuvant conjugates
US11547761B1 (en) 2016-07-07 2023-01-10 The Board Of Trustees Of The Leland Stanford Junior University Antibody adjuvant conjugates
CN111032087A (zh) * 2017-06-28 2020-04-17 小利兰·斯坦福大学托管委员会 用于dectin-2刺激和癌症免疫治疗的方法和组合物
JP2020526482A (ja) * 2017-06-28 2020-08-31 ザ ボード オブ トラスティーズ オブ ザ レランド スタンフォード ジュニア ユニバーシティー デクチン−２刺激及び癌免疫療法のための方法及び組成物
EP3645043A4 (fr) * 2017-06-28 2021-04-07 The Board of Trustees of the Leland Stanford Junior University Procédés et compositions pour la stimulation de la dectine 2 et l'immunothérapie contre le cancer
US11400164B2 (en) 2019-03-15 2022-08-02 Bolt Biotherapeutics, Inc. Immunoconjugates targeting HER2
WO2022108897A1 (fr) * 2020-11-20 2022-05-27 Vanderbilt University Utilisation de la microvirine dans l'identification de lipoarabinomannane coiffé de mannose de mycobacterium tuberculosis

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