WO2005000348A2 - Matieres et procedes concernant la modulation de la reponse des lymphocytes t a un antigene soluble - Google Patents

Matieres et procedes concernant la modulation de la reponse des lymphocytes t a un antigene soluble Download PDF

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Publication number
WO2005000348A2
WO2005000348A2 PCT/GB2004/002726 GB2004002726W WO2005000348A2 WO 2005000348 A2 WO2005000348 A2 WO 2005000348A2 GB 2004002726 W GB2004002726 W GB 2004002726W WO 2005000348 A2 WO2005000348 A2 WO 2005000348A2
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antigen
activator
galcer
cells
composition
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PCT/GB2004/002726
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English (en)
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WO2005000348A3 (fr
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Vincenzo Cerundolo
Ian Hermans
Uzi Gileadi
Jonathan Silk
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Isis Innovation Limited
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Publication of WO2005000348A3 publication Critical patent/WO2005000348A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—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
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/542—Mucosal route oral/gastrointestinal
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55583—Polysaccharides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to materials and methods for improving vaccination strategies. Particularly, but not exclusively, the present invention relates to the use of an activator of NKT cells as an adjuvant to enhance T-cell responses to soluble antigen.
  • a significant challenge for the immune system is to be able to distinguish between harmful and benign antigenic stimuli in order to avoid inappropriate adaptive responses .
  • DC reside in peripheral tissues in an immature state defined by significant capacity for antigen uptake and low expression of MHC and costimulatory molecules .
  • APC antigen- presenting cells
  • DC dendritic cells
  • DC reside in peripheral tissues in an immature state defined by significant capacity for antigen uptake and low expression of MHC and costimulatory molecules .
  • DC Upon exposure to inflammatory stimuli, DC undergo a maturation process involving migration to lymphoid sites, down-regulation in rate of antigen uptake and increase in levels of MHC and costimulatory molecules. These changes enable DC to present antigens to naive CD4 + and CD8 + T cells and thereby promote adaptive immune responses (2) .
  • the potency of DC as APC for na ⁇ ve T cells can also be modulated directly by microbial stimuli via pattern recognition receptors, of which toll-like receptors are the prime examples (3), and also by signals such as CD40L (4, 5), RANKL (6) and FasL (7) mediated by T cells themselves.
  • CD40L 4, 5
  • RANKL RANKL
  • FasL FasL (7)
  • CD4 + T cells can activate DC specifically through signals mediated via CD40, inducing further upregulation of surface molecules involved in T cell stimulation and inducing the release of IL-12 (5) .
  • This 'conditioning' of DC is particularly important for the generation of CTL from na ⁇ ve CD8 + T cell precursors (8-10) .
  • T cell help is therefore most likely to be effective if it is achieved through interaction with immature DC early in the evolution of a T cell-mediated response.
  • Exogenous protein antigens can be taken up by APC and processed into peptides for presentation on the cell surface by MHC class II molecules to CD4 + T cells. This has been referred to as the "exogenous" pathway of antigen presentation. In contrast, internal antigens are processed and presented by MHC class I molecules to CD8 + T cells; the so- called “endogenous” pathway of antigen presentation (18, 19) . It is now becoming clear that some APC, including subsets of
  • DC are capable presenting peptides derived from exogenous antigens on MHC class I molecules, resulting in the stimulation of CD8 + T cell responses (20-22) . This would be a desirable outcome for protein-based vaccine development in which CTL responses are sought.
  • NKT cells Another obstacle to the efficacy of help provided by T cells in this manner is the scarcity of na ⁇ ve T cells with reactivity to unique antigens presented by the DC.
  • An alternative source of help is NKT cells, a ⁇ T cells with an invariant TCR and intermediate level NK1 surface expression.
  • CDld-restricted NKT cells expressing the invariant TCR chain encoded by Vc-14Jc-281 gene segments in mice, and VC-24JC-Q in humans, (NKT cells) are found in relative abundance in tissues such as spleen, bone marrow, thymus and liver (12, 13) .
  • NKT cells respond to a common glycolipid presented by CDld molecules that is induced as an innate response to cellular distress.
  • CDld molecules that is induced as an innate response to cellular distress.
  • NKT can express CD40L and secrete significant levels of inflammatory cytokines, it is possible that these cells can be readily induced to activate DC (14) .
  • human NKT cell clones restricted to the group I CD1 molecules CDla, CDlb and CDlc, and ⁇ T cells restricted by CDlc can directly induce maturation of cultured DC in vitro (16, 17) .
  • NKT cells can influence immunological responses to soluble antigen in the presence of ⁇ -galactosyl ceramide ( ⁇ -GalCer) , a CDld-binding glycolipid that has been shown to specifically induce activation of NKT cells (26) .
  • ⁇ -GalCer ⁇ -galactosyl ceramide
  • the inventors show that, while intravenous and oral administration of ovalbumin protein alone can induce activation of CD4 + and CD8 + T cells, these responses are considerably enhanced by concomitant activation of NKT cells leading to modulation of DC function.
  • TLR Toll Like Receptors
  • TLR4 Stimulation of NKT cells leads to the maturation of DC upon the interaction of CD40L (expressed by activated NKT cells) and CD40 (expressed by DC) .
  • the inventors have found that stimulation of TLR4 has a synergistic effect with the CD40 dependent stimulation pathway and results in a much stronger immune response.
  • TLR4 can be stimulated by LPS and a derivative of LPS "MPL", which is non-toxic and can be injected into patients.
  • ⁇ -GalCer Injection of ⁇ -GalCer has been used to enhance anti-tumour immune responses, chiefly by activating NKT cells which kill tumour cells in an NK-like way (47) or by activating NK cells themselves (51) . It has also been reported that the anti- tumour effect of c--GalCer may in part be due to stimulation of CD8+ T cells by IFN ⁇ produced by activated NKT cells (50) . O03/009812 suggests the use of glycosylceramides, including ⁇ -GalCer, as activators of NKT cells in immunisation protocols designed to elicit a T-cell response.
  • isolated and purified DC cells loaded with antigenic peptide may be used for immunisation.
  • peptide-expressing DC cells have been treated with c-- GalCer before reinjection (15) .
  • the method of the present invention has several advantages . Firstly, whole antigen is used for vaccination, so both CD4+,
  • CD8+ T-cell and antibody responses are obtained, resulting in an enhanced T-cell response overall.
  • the strategy is much more simple then existing strategies as it does not require that DC cells be isolated, cultured or reinjected.
  • it can be used with any individual, independent of MHC haplotype.
  • the invention provides materials and methods for inducing an immune response to soluble polypeptide antigen using an NKT cell activator.
  • compositions for inducing an immune response in an individual to an antigen comprising said antigen in combination with an activator of NKT cells.
  • the invention also provides the use of an activator of NKT cells in the preparation of a medicament for inducing an immune response to soluble polypeptide antigen in an individual .
  • the invention also provides for a kit having first and second containers, wherein the first container comprises a composition comprising an activator of NKT cells, and the second container comprises a composition comprising a soluble polypeptide antigen.
  • the activator of NKT cells may be a glycolipid presented by CDld molecules or any molecule which activates NKT cells . Activation of NKT cells may be detected by, for example, increased expression of CD40L or secretion of cytokine such as IFN ⁇ or IL-4.
  • the activator of NKT cells may be a natural ligand of NKT cells.
  • the activator of NKT cells may be a- GalCer, or an analogue of ⁇ -GalCer such as OCH (described in ref . 30) or alpha-glucosyl ceramide, or it may be a TCR specific antibody.
  • OCH described in ref . 30
  • alpha-glucosyl ceramide alpha-glucosyl ceramide
  • the immune response is preferably a T cell response. It may be a CD4+ T cell response or CD8+ T cell response.
  • the immune response is both a CD4+ and a CD8+ T cell response .
  • the immune response may also be an antibody response .
  • the antigen may be any antigen of choice, but is preferably a soluble protein.
  • the composition may further comprise a TLR activator, that is to say a substance which activates a Toll-like receptor. It is believed that use of TLR activators in conjunction with activators of NKT cells provide a synergistic adjuvant effect.
  • the TLR activator is an activator of TLR3, TLR4, TLR5, TLR7 or TLR9.
  • a suitable TLR activator is MPL (monophosphoryl lipid A), which binds TLR4.
  • Other TLR activators which may be used are LTA (lipoteichoic acid) and PGN (peptidoglycan) , which bind TLR2 and TLR6 ; Poly I:C
  • TLRs in accordance with the invention is not restricted to use in compositions comprising NKT cell activators .
  • NKT cell activator and the TLR activator may be administered in separate compositions.
  • the invention provides the use of an activator of NKT cells in the preparation of a medicament for inducing an immune response to soluble polypeptide antigen in an individual, wherein the medicament is for administration in conjunction with a TLR activator.
  • TLR activator in the preparation of a medicament for inducing an immune response to soluble polypeptide antigen in an individual, wherein the medicament is for administration in conjunction with an activator of NKT cells.
  • the activator of NKT cells is a glycosylceramide
  • the TLR activator is not CpG or MPL.
  • glycosylceramide is used herein to refer to compounds of the formula I as disclosed in WO03/009812.
  • R_ . , R 2 and R 5 represent H or a specific monosaccharide
  • R 3 and R s represent H or OH, respectively
  • R 4 represents H, OH or a specific monosaccharide
  • X denotes an integer from 1 to 23
  • R 7 represents any one of the following groups (a) - (g) : (a) - (CH 2 .
  • the activator of NKT cells may be ⁇ -GalCer, OCH, a- glucosylceramide, or any of the other NKT activators referred to in this specification.
  • ⁇ GalCer, c--glucosylceramide and OCH have the following formulae: ⁇ -GalCer
  • the TLR activator may be an activator of TLR3 , TLR4 , TLR5 , TLR7 or TLR9, including any of the specific activators referred to above.
  • the invention further provides the use of ⁇ -GalCer in the preparation of a medicament for inducing an immune response to The invention further provides the use of ⁇ -GalCer in the preparation of a medicament for inducing an immune response to soluble polypeptide antigen in an individual, wherein the medicament is for administration in conjunction with CpG or MPL.
  • the invention further provides the use of ⁇ -glucosylceramide in the preparation of a medicament for inducing an immune response to soluble polypeptide antigen in an individual, wherein the medicament is for administration in conjunction with CpG or MPL.
  • the invention provides a composition comprising an activator of NKT cells and a TLR activator, with the proviso that, when the activator of NKT cells is a glycosylceramide, the TLR activator is not CpG or MPL.
  • composition comprising ⁇ -GalCer and MPL or CpG, and, further, a composition comprising ⁇ -glucosylceramide and MPL or CpG.
  • compositions of the invention may further comprise a purified soluble polypeptide antigen.
  • composition comprising OCH and a purified soluble polypeptide antigen.
  • kits containing combinations of some or all of NKT cell activators, TLR activators and soluble antigens are provided.
  • the invention provides a kit having first and second containers, wherein the first container comprises a composition comprising an activator of NKT cells, and the second container comprises a composition comprising a TLR activator, with the proviso that, when the activator of NKT cells is a glycosylceramide, the TLR activator is not CpG or MPL.
  • kits having first and second containers, wherein the first container comprises a composition comprising ⁇ -GalCer, and the second container comprises a composition comprising CpG or MPL.
  • kits having first and second containers, wherein the first container comprises a composition comprising ⁇ -glucosylceramide, and the second container comprises a composition comprising CpG or MPL.
  • the kit may additionally have a third container, which container comprises a composition comprising a purified polypeptide antigen.
  • kits having first and second containers, wherein the first container comprises a composition comprising
  • the second container comprises a composition comprising a purified polypeptide antigen.
  • any or all of said compositions may further comprise a pharmaceutically acceptable carrier or diluent .
  • the invention provides for a kit having first, second and third containers, wherein the first container comprises a composition comprising an activator of NKT cells, the second container comprises a composition comprising a soluble polypeptide antigen, and the third container comprises a composition comprising a TLR activator.
  • compositions of the invention are preferably pharmaceutical compositions and, if necessary, further comprise a pharmaceutically acceptable carrier or diluent.
  • the pharmaceutical composition may comprise a liposome which helps to target the peptide antigen to particular cells or tissue, e.g. lymphoid tissue, and increases the half life of the antigen. Liposomes include emulsions, foams, micelles etc.
  • the pharmaceutical composition may also comprise at least one component which assists in the induction of any immune response.
  • the one or more components may include lipids, e.g. palmitic acid residues attached to the peptide antigen, or E.coli lipoproteins such as tripalmitoyl-S-glycerylcystein lyseryl-serine (P 3 CSS) ; and adjuvants, e.g. Freund's adjuvant.
  • the pharmaceutical composition according to the invention may be used as a vaccine .
  • the vaccine may comprise one or more protein, polypeptide or peptide antigens in combination with the NKT cell activator.
  • the one or more peptides may be linked to a carrier such as thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino acids such as poly L-lysine, poly L-glutamic acid, influenza, hepatitis B virus care protein etc .
  • the peptide may be present in the vaccine as a homopolymer comprising multiple copies of the same peptide, or as a heteropolymer of various peptides comprising antigens or epitopes of interest.
  • the vaccines may further comprise physiologically tolerable/acceptable diluent such as water or saline, e.g. phosphate buffered saline.
  • physiologically tolerable/acceptable diluent such as water or saline, e.g. phosphate buffered saline.
  • the vaccine preferably includes an adjuvant such as incomplete Freund's adjuvant, aluminium phosphate, aluminium hydroxide, or alum.
  • a method of immunising an individual against an antigen comprising the step of administering to said individual said antigen and a NKT cell activator, optionally in combination with a TLR activator as already described.
  • the activator of NKT cells and the antigen may be administered simultaneously, preferably as a composition according the invention.
  • the activator of NKT cells may be administered before the polypeptide antigen is administered, preferably less than about 2 hours before.
  • the activator of NKT cells may be administered after the polypeptide antigen is administered, preferably less than about 8 hours after.
  • the vaccine composition may be administered via injection, aerosol, oral, transdermal, transmucosal, intrapleural, intrathecal, or other suitable routes, e.g. intravenously, subcutaneously, intradermally, or intramuscularly.
  • an activator of a TLR receptor may be used in combination with the activator of NKT cells.
  • a suitable TLR activator is MPL, which binds TLR4.
  • Other TLR activators which may be used are LTA and PGN, which bind TLR2 and TLR6; Poly I:C, which binds TLR3 ; flagellin, which binds TLR5; imiquimod, which binds TLR7 and CpG, which binds TLR9; or any other component which binds and activates a TLR.
  • the soluble antigen is a tumour antigen.
  • the antigen may be a polypeptide unique to a virus or bacterial cell, a parasite cell, or cellular products .
  • Peptides that comprises the antigen or epitopes of interest can be prepared synthetically, or by recombinant DNA technology or from natural sources such as tumours or viruses .
  • the peptide will preferably be substantially free of other naturally occurring host cell proteins and fragments thereof, in some embodiments the peptides can be synthetically conjugated to native fragments or particles.
  • C ELISpot assays were used to assess induction of IFN ⁇ by CD8 + cells responding to the MHC class I-binding peptide OVA 257 . 2S4 , or CD4 + T cells responding to the class II-binding peptides OvA 2S5 -29o and OVA 323 . 339 , 11 days after ovalbumin administration.
  • splenocytes isolated from animals of the different treatment groups were stimulated with each of the peptides in vitro for 48 h.
  • FIG. 3 Enhancement of T cell responses to soluble antigen with injection of ⁇ -GalCer requires the involvement of NKT cells.
  • DC-induced T cell responses can be enhanced by concomitant, ⁇ -GalCer-mediated, activation of NKT cells capable of substituting for CD4 + T cell help.
  • One group (first panel) received DC that were loaded with LCMV GP 34 _ 4 ⁇ and ⁇ -GalCer separately, and then combined before injection.
  • NKT "/_ mice were used as recipients (right panel) , and in others the DC were matured with LPS prior to injection (lower panel) .
  • OVA 257 . 264 -specific T cell responses were assessed in animals that received ⁇ -GalCer- or vehicle-treated DC loaded with the MHC class I-binding peptide OVA 257 _ 2S4 alone, or together with the class II-binding peptide OVA 323 _ 339 . Animals injected with DC without peptides served as negative controls.
  • C OVA 2S7-26 4-specific T cell responses were assessed in animals that received ⁇ -GalCer- or vehicle-treated DC exposed to whole ovalbumin for 20 h.
  • FIG. 7 Activation of NKT cells enhances T cell responses to soluble protein via a CD40-mediated mechanism independent of IFN ⁇ production.
  • OVA 257 OVA 257 .
  • 264 -specific T cell responses were similarly assessed in IFN ⁇ R _ " animals and 129S6/SvEv controls (C) , or in C57BL/6 animals injected with ovalbumin and the c--GalCer analogue OCH (D) . Serum cytokine levels were determined for the latter experiment (E) .
  • FIG. 8 Oral administration of soluble antigen and alpha- GalCer induces functional CTL.
  • (a) Whole OVA was administered by gavage (30 mg/mouse) together with either alpha-GalCer (8 ⁇ g/mouse) or vehicle. Induction of OVA 2s7 - 2s4 -CD8 + T cell responses was assessed in the blood 7 days later, (b)
  • Cytolytic activity of the induced OVA 257 _ 2S4 -specific response was assessed in vivo 11 days after OVA administration against various CFSE-labeled syngeneic splenocytes loaded with titrated doses of OVA 257 - 2s4 -peptide as indicated, and a control splenocyte population without peptide labelled with CMTMR. Representative FACS profiles for each treatment group are shown. Analysis of antigen specific lysis was calculated at 16 h after target cell administration, with percent specific lysis calculated as the mean proportion of antigen loaded cells depleted relative to control populations without antigen.
  • FIG. 9 Co-stimulation of TLR4 on DC further enhances T-cell response to soluble protein
  • Liposomes containing alphaGalcer + ovalblumin + MPL an LPS derivative which binds TLR4 were injected i.v. into mice and 7 days later frequency of OVA specific CTL was measured by ex- vivo tetramer staining. Liposomes containing either ovalbumin and MPL or ovalbumin and alphaGalceramide were used as control. FACS profiles of na ⁇ ve animals and those receiving ovalbumin +/- c--GalCer and/or MPL are shown.
  • mice immunized with liposomes containing OVA alone showed: 0.1% of tetramer ⁇ cells; OVA + alphaGalcer: 2% of tetramer+ cells; OVA+alphaGalcer+MPL : 13% of tetramer ⁇ cells.
  • iNKT cell ligands Injection of iNKT cell ligands leads to maturation of splenic DCs, increasing their immunostimulatory capacity.
  • Animals were treated i.v. with either 100 ng of iNKT cell ligand alpha-GalCer, or 25 ⁇ g of TLR4 ligand monophosphoryl lipid A (MPL), or 2.5 ⁇ g.poly I:C, or 2.5 ⁇ g. flagellin, or 50 ⁇ g.CpG.
  • MPL TLR4 ligand monophosphoryl lipid A
  • surface expression of CD86 was assessed on splenic CDllc + cells 24 h later.
  • OCH + ovalblumin + MPL were injected i.v. into mice and 7 days later frequency of OVA specific CTL was measured by ex-vivo tetramer staining.
  • FIG. 14 Injection of OCH and MPL leads to maturation of splenic DCs, increasing their immunostimulatory capacity.
  • Animals were treated i.v. with either 1 ⁇ g of OCH, or 25 ⁇ g of MPL, or OCH plus MPL.
  • the immunostimulatory capacity of CDllc + cells isolated from the spleens of these group of animals was assessed by loading with influenza NP 3 ⁇ 6 - 374 peptide ex vivo, and then by adding splenocytes transgenic for the NP 366 _ 374 specific T cell receptor.
  • A) proliferation of CFSE labelled transgenic splenocytes is shown
  • B) secretion of gamma interferon by transgenic splenocytes is shown.
  • the NKT activators of the invention may be formulated in pharmaceutical compositions, and administered to patients in a variety of forms .
  • compositions for oral administration may be in tablet, capsule, powder or liquid form.
  • a tablet may include a solid carrier such as gelatin or an adjuvant or an inert diluent.
  • Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. Such compositions and preparations generally contain at least 0. lwt% of the compound.
  • Parenteral administration includes administration by the following routes: intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraocular, transepithelial, intraperitoneal and topical (including dermal, ocular, rectal, nasal, inhalation and aerosol), and rectal systemic routes.
  • intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • suitable solutions using, for example, solutions of the compounds or a derivative thereof, e.g. in physiological saline, a dispersion prepared with glycerol, liquid polyethylene glycol or oils.
  • compositions can comprise one or more of a pharmaceutically acceptable excipient, carrier, buffer, stabiliser, isotonicizing agent, preservative or anti-oxidant or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient .
  • a pharmaceutically acceptable excipient e.g. orally or parenterally.
  • Liquid pharmaceutical compositions are typically formulated to have a pH between about 3.0 and 9.0, more preferably between about 4.5 and 8.5 and still more preferably between about 5.0 and 8.0.
  • the pH of a composition can be maintained by the use of a buffer such as acetate, citrate, phosphate, succinate, Tris or histidine, typically employed in the range from about 1 mM to 50 mM.
  • the pH of compositions can otherwise be adjusted by using physiologically acceptable acids or bases.
  • Preservatives are generally included in pharmaceutical compositions to retard microbial growth, extending the shelf life of the compositions and allowing multiple use packaging.
  • preservatives include phenol, meta-cresol, benzyl alcohol, para-hydroxybenzoic acid and its esters, methyl paraben, propyl paraben, benzalconium chloride and benzethonium chloride.
  • Preservatives are typically employed in the range of about 0.1 to 1.0 % (w/v) .
  • mice C57BL/6, 129S6/Sv ⁇ v and CD40L “7" mice were from breeding pairs originally obtained from Jackson Laboratories, Bar Harbor, Maine. Also used were mice lacking the J ⁇ 281 TCR gene segment (27) which were devoid of Vc-14 + NKT cells while having other lymphoid cell lineages intact (referred to in the text as NKT " _ mice) , CDld " mice (28) and IFNDR "/ ⁇ mice (29) . All animals were maintained at the Biomedical Services Unit of John Radcliffe Hospital by brother x sister mating; in vivo experimental protocols were performed according to institutional guidelines .
  • CM complete medium
  • RPMI Sigma-Aldrich, Dorset, England
  • 2 mM glutamine 1 % penicillin-streptomycin
  • 5 x 10 " ⁇ M 2 -mercapto-ethanol all Invitrogen Ltd, Paisley, UK
  • 10 % fetal bovine serum 10 % fetal bovine serum
  • Chicken ovalbumin grade VII was from Sigma-Aldrich.
  • Peptides derived from chicken ovalbumin, including OVA 257 _ 2S4 (binding to H-2K b ) , OVA 323-339 and OVA 26S-28o (both binding and I-A b ) , and peptide LCMV 34 _ 41 (H-2K b ) derived from lymphocytic choriomeningitis virus glycoprotein were prepared in-house.
  • the glycolipids ⁇ -galactosylceramide (c-GalCer) and /3-galactosylceramide (5-GalCer) were from Kirin Brewery Co. Ltd, Japan and Fluka Chemie AG, Switzerland respectively.
  • the glycolipids were solubilized in 0.5 % Tween/PBS, hereafter referred to as 'vehicle' .
  • Ovalbumin protein was diluted in PBS and admixed with PBS- diluted glycolipid solutions ( ⁇ -GalCer, 3-GalCer or OCH) , or PBS-diluted vehicle, immediately prior to injection into the lateral vein. Each animal received 400 ⁇ g protein and either 1 ⁇ g of glycolipid, or equivalent final volume of vehicle.
  • IL-4 and 20 ng/ml GM-CSF for 7 d.
  • Cultures typically contained 70-100 % DC as determined by fluorescent staining with the anti-CDllc (BD Pharmingen, San Diego, CA) .
  • DC were loaded with peptide by incubation in CM containing 10 ⁇ M synthetic peptide for 2 h, or loaded with whole protein by incubation in CM containing 100 ⁇ g/ml protein for 24 h.
  • DC were loaded with a- GalCer by incubation in CM containing 100 ng/ml for 40 h. Cells were washed to remove excess Ag prior to i.v. injection (6 x 10 s DC per animal) .
  • mice were infected with 10 s plaque forming units of the recombinant virus W-OVA (31) , expressing full length chicken ovalbumin, by intravenous injection in PBS.
  • Tetrameric H-2 K b /OVA 2S7 -. 2S4 peptide, or tetrameric H-2 K b /LCMV 34 _ 41 peptide complexes were prepared as outlined in Altman et al . (32) , and used to stain fresh PBL isolated from the lateral tail vein. Approximately 5 x 10 5 PBL were suspended in 20 ⁇ l CM and incubated with 0.5 ⁇ g of tetramer complexes at 37 °C for 20 min. The cells were then incubated with anti-CD8 ⁇ (BD Pharmingen) for 10 min at 4 °C, washed twice with PBS, and resuspended in PBS for FACS analysis . Cells were analysed with FACScan hardware and CellQuest software (BD Biosciences , Mountain View, CA) .
  • Cytotoxicity was assessed on fluorescence-labeled syngeneic spleen cell populations loaded with specific antigen administered by i.v. injection.
  • Cytotoxicity was assessed by FACS analysis on blood taken from the lateral tail vein. The mean percent survival of peptide-loaded targets cells was calculated relative to antigen-negative controls . Cytotoxic activity was then expressed as percent specific lysis, calculated by the equation 100 - mean percent survival of peptide-loaded targets.
  • Phenotypic analysis of splenic DC DC were isolated from the spleens of animals that had been injected with ⁇ -GalCer or vehicle by using anti-CDllc magnetic beads (Miltenyi Biotech, Gladbach, Germany) according to the manufacturer's instructions. The isolated cells were assessed for expression of CD8D, CD8 ⁇ and CD86 with specific monoclonal antibodies (BD Pharmingen) .
  • Serum cytokine levels were determined by ELISA using commercial ELISA kits (R&D Systems, Minneapolis, USA) following the manufacturers instructions.
  • NKT cells have the potential to influence the quality of adaptive immune responses
  • Immune responses were therefore monitored in C57BL/6 mice injected with chicken ovalbumin protein in the presence or absence of the NKT cell ligand ⁇ -GalCer.
  • This glycolipid has been shown to rapidly induce the release of a NKT-cell derived cytokines, leading to a 'cytokine storm' characterized by high levels of IFN ⁇ and IL-4 in the serum (26, 34).
  • co-administration of 1 ⁇ g of ⁇ -GalCer with 400 ⁇ g ovalbumin induced a substantial increase of IFN ⁇ in the serum over a 48 h period.
  • NKT cell mediated signals could lead to cross presentation of peptides derived from the administered ovalbumin protein to peptide- specific CD8 + T cells, thereby perhaps providing a potent, specific, CTL response.
  • immune responses were initially screened in the blood using MHC class I/peptide tetramers capable of highlighting CD8 + T cells reactive to the H-2 K b -binding peptide OVA 2S7 _ 2S4 .
  • the functional state of the induced ovalbumin-reactive CD8 + T cells was investigated by assessing cytotoxic capacity, cytokine induction and ability to respond to further antigenic stimulation. Cytotoxic capacity was assessed against 0VA 257 _ 264 peptide-loaded syngeneic splenocyte targets that had been labeled with fluorescent dye and injected into the lateral vein 13 days after initial priming with ovalbumin + ⁇ -GalCer. Specific lysis of the antigen-loaded population was monitored relative to a differentially labeled control splenocyte population that was not loaded with peptide. This assay, presented in Figure IB, indicated that the ovalbumin-specific CD8 + T cells induced were indeed capable of mediating specific cell lysis.
  • peptide- specifc ELISPOT assays were used to assess IFN ⁇ production from the induced OVA 2S7 - 2 64 CD8- T cell population.
  • ELISPOT assays were also used to determine whether responses to the MHC class II binding peptides OVA 323 _ 339 and OVA 2 65- 2 s o were similarly affected by ⁇ -GalCer treatment .
  • splenocytes were taken from animals 11 days after antigen administration and stimulated with the specific peptides in vitro for 48 h (Fig. IC) . IFN ⁇ responses to the class I peptide, OVA 257 .
  • FIG. 2 OVA 257 _ 264 -specific populations were greatly expanded one week after restimulation in vivo. The responses were proportional to the initial responses following protein administration, so that in both assays the groups initially treated with ovalbumin and ⁇ -GalCer produced the most significant restimulatory responses. OVA 257 _ 264 -specific CD8 + T cells induced in the absence of ⁇ -GalCer could also be restimulated, but the smaller populations induced likely reflect the initially smaller populations induced in the absence of ⁇ -GalCer.
  • ⁇ -GalCer is a CDld- binding ligand of NKT cells
  • the increased immune responses to i.v. injection of ovalbumin protein seen in the presence of ⁇ -GalCer was not directly related to stimulation of activity from CDld/c-GalCer-reactive NKT cells.
  • increased T cell responses may result from interaction between ovalbumin protein and the glycolipid leading to efficient absorption of antigen by resident APC, perhaps via CDld molecules.
  • NKT stimulation is dependent on timing of administration of c-GalCer relative to protein
  • the kinetics of NKT activation, cytokine release and subsequent activation induced cell death following ⁇ -GalCer injection have been well documented (26, 35, 37) . Screens were performed to establish at which time-point (s) within these kinetics that the most significant enhancing effect of a-
  • GalCer on immune responses to soluble antigen was observed.
  • hallmark time-points for injection of - GalCer relative to ovalbumin were chosen on the basis of the known serum cytokine profiles in response to ⁇ -GalCer.
  • ovalbumin was injected 2 h after c-GalCer injection when IL-4 levels were known to be peaking in the serum, and 24 h after c--GalCer injection, when IFN ⁇ levels were peaking.
  • ⁇ -GalCer administration had no enhancing effect on 0VA 257-2S4 CD8 + T cells responses measured by tetramer in the blood on day 7 post-ovalbumin injection (Fig. 4A) .
  • NKT cell stimunlation is dependent upon simultaneous presentation of ⁇ -GalCer and processed protein antigen by dendritic cells . That NKT induced enhancement of T cell responses required close timing of ⁇ -GalCer and protein administration could reflect the need for simultaneous uptake of these molecules, and perhaps a requirement for co-localization of their antigenic components on the same APC.
  • immune responses to injection of cultured bone marrow-derived DC loaded with glycolipid and peptide antigens in vitro were examined.
  • FIG. 6A Representative FACS profiles are shown in Fig. 6A. While injection of vehicle had no effect upon levels of CD80 and CD86 on DC relative to na ⁇ ve controls, injection of c--GalCer induced an upregulation of expression of these co-stimulatory molecules that was equal to LPS, or in the case of CD80, even greater than LPS. These data suggest, therefore, that NKT cell activation provides a potent DC maturation stimulus .
  • the enhancing effect of NKT cell stimulation is CD40L dependent, but does not require INF ⁇
  • CD40L is upregulated on CD4 + T cells in response to T cell activation, and signals mediated by via CD40 on DC lead to DC activation.
  • NKT cells have also been reported to express CD40L upon activation (40) and it has therefore been suggested that NKT cells may also induce DC maturation and activation through interaction with CD40 (14, 16) .
  • ovalbumin protein was injected into CD40L "/_ recipients together with a- GalCer or vehicle.
  • OVA 2S7 _ 264 -specific CD8 + T cell responses were monitored in the blood seven days later with tetramers .
  • the c-- GalCer-mediated enhancement of responses to ovalbumin administration was not observed in CD40L _/" recipients (Fig. 7A) .
  • Analysis of cytokine levels in serum in these recipients indicated that whereas ⁇ -GalCer induced both IFN ⁇ and IL-4 release in wild-type animals, only IL-4 was induced in CD40L _ " recipients (Fig. 7B) .
  • NKT cells were activated in CD40L _ ⁇ animals, they were unable to produce of IFN ⁇ and enhance T cell responses in the absence of subsequent CD40/CD40L mediated signals.
  • ovalbumin protein was injected into IFN ⁇ R _ " recipients together with ⁇ -GalCer or vehicle (Fig 7C) .
  • Lack of a receptor for IFN ⁇ made no difference to the induction of OVA 2S7-264 -specific CD8 + T cell responses measured in the blood, and importantly, did not affect the enhancement of this response by ⁇ -GalCer.
  • Murine iNKT cells are found with relative abundance in bone marrow, thymus, spleen and liver, while in low numbers in the blood and peripheral lymph nodes. It is likely, therefore, that this distribution of cells will determine the efficacy of administration of iNKT cell agonists by different routes .
  • vaccine administration via mucosal surfaces is generally seen as a preferred option in terms of likely patient uptake and ease of administration we examined the possibility of inducing immune responses by the oral route.
  • Proteolytic activity in the digestive tract meant that the dose of OVA used was necessarily high, with 30 mg administered per animal.
  • the enhanced immune response observed after stimulating NKT cells can be significantly further enhanced by stimulating simultaneously the TLR4, which is expressed on DC.
  • Stimulation of NKT cells via either alphagal Cer or TCR stimulation leads to the maturation of DC upon the interaction of CD40L (expressed by activated NKT cells) and CD40 (expressed by DC) .
  • Stimulation of TLR4 (expressed by DC) has a synergistic effect with the CD40 dependent stimulation pathway and results in a much stronger immune response (Fig. 9) .
  • TLR4 binds LPS and a derivative of LPS "MPL", which is not toxic and can be injected into patients.
  • the experiment shown in Fig. 9 was carried out by incorporating alphaGalcer + ovalblumin + MPL into liposomes .
  • the liposomes were injected i.v. into mice and 7 days later frequency of OVA specific CTL was measured by ex-vivo tetramer staining.
  • Liposomes containing either ovalbumin and MPL or ovalbumin and alphaGalceramide were used a control.
  • the results of these experiments show a very significant enhancement of the immune response in mice immunized with liposomes containing alphaGalcer + ovalblumin + MPL (i.e. OVA: 0.1% of tetramer ⁇ - cells; OVA + alphaGalcer: 2% of tetramer+ cells; OVA+alphaGalcer+MPL: 13% of tetramer ⁇ cells.
  • tumour cell line EG7.0VA a derivative of the murine thymoma EL4 transfected with chicken ovalbumin cDNA (33), grows progressively when injected s.c. into C57BL/6 mice .
  • recipient were treated with ovalbumin together with vehicle 11 days prior to tumour challenge, no notable anti-tumour response was initiated, and tumour engraftment and progression was observed. This was despite a weak, but 'measurable OVA 257 _ 2S4 -specific CD8 + T cell response in the blood at day 7 post ovalbumin injection (data not shown) .
  • NKT cells responding to glycolipids presented by monomorphic CDld molecules can significantly regulate T cell responses to soluble antigen in vivo, and that this modulation is through direct interaction with DC. NKT cells therefore represent an abundant source of the T cell derived factors required for DC activation, and that these cells can potentially be rapidly mobilized in the generation of T cell mediated responses.
  • NKT cells can provide an early source of CD40L signals that are known to be required for Thl-biased responses, and CTL induction.
  • NKT cells Activation of NKT cells by administration of ⁇ -GalCer together with intravenous delivery of soluble ovalbumin protein results in considerable enhancement of ovalbumin-specific T cell responses, including CTL responses reliant upon cross presentation.
  • appropriately timed administration of protein relative to ⁇ -GalCer allowed processed protein to be presented on DC with enhanced stimulatory function.
  • CD40L signals generally presumed to come from CD4 + T helper cells (8-10) .
  • Schulz et al . (41) have reported that effective CD40 triggering of DC, as measured by IL-12 induction, requires initial microbial priming of the DC.
  • intravenous injection of soluble ovalbumin alone induced cross-priming it is most likely that low levels of endotoxin in the commercial source of ovalbumin used was providing such a microbial signal. This signal could then combine with conventional CD4 + T cell-mediated help to induce priming of CD8 + responses.
  • the fact that all cross priming was abrogated in CD40L "/" animals highlights the importance of integrating microbial and CD40-mediated signals in this system. Importantly, however, cross-priming in CD40 proficient animals was always significantly enhanced when a-
  • GalCer was administered together with ovalbumin, most likely reflecting a greater supply of CD40L signals from stimulated NKT cells.
  • a dramatic increase in the quantity of CTL (5-10 times in number) was observed when NKT were concomitantly activated with c-GalCer.
  • CTL induced in the presence of ⁇ -GalCer provided resistance to challenge with ovalbumin + tumours.
  • activated NKT cells can provide a potent CD40L-mediated boost to an otherwise weakly cross-presented response, providing functionally superior CTL.
  • NKT cell and DC will ultimately determine the quality of an adaptive response.
  • Initial engagement between NKT and APC bearing glycolipid ligand leads to release of a burst of IL-4 by NKT cells, which can be achieved in the absence of CD40 signalling ( (42) and Fig. 7) .
  • This initial engagement may then lead to increased expression of CD40L on the NKT cell surface and subsequent interaction with DC expressing CD40.
  • DC will be induced to release IL-12, which, in turn, stimulates NKT cells to release IFN ⁇ .
  • IFN ⁇ produced by NKT cells can induce upregulation of IL-12 receptors . on NKT cells in an autocrine manner (14) .
  • Vc-24/V / 311 cells restricted by CDld can stimulate human Vc-24/V / 311 cells restricted by CDld.
  • Activated Vc-24/V ll NKT cells can exert cytotoxic activity against a number of human tumours in vitro, regardless of histological origin (46) .
  • Animal studies have also highlighted the utility of this glycolipid in the induction of anti-tumour responses. However, these latter studies have suggested that direct NKT cell-mediated cytoxixicty does not play a significant role in responses to tumours in vivo (47) .
  • NKT cell-mediated activation of NK cells and rapid induction of IFN ⁇ , that exerts the anti-tumour activity (48) , with the anti-angiogenic property of IFN ⁇ playing a key role (49) .
  • anti-tumour CTL activity may also be invoked through the NKT activation with ⁇ -GalCer (50, 51) . This activity may be dependent upon NKT-mediated activation of DC that have acquired unique tumour antigens.

Abstract

L'invention concerne des matières et des procédés pour améliorer des stratégies de vaccination. En particulier, l'invention concerne, mais n'est pas limitée à, l'utilisation d'un activateur de cellules NKT, en combinaison avec un activateur des récepteurs de type Toll, ce qui permet d'améliorer les réponses des lymphocytes T à des antigènes solubles. L'invention concerne également l'utilisation d'activateurs cellulaires NKT, par exemple OCH, pour améliorer les réponses des lymphocytes T à des antigènes solubles. Les matières et procédés de l'invention conviennent en particulier pour améliorer la présentation de l'antigène soluble par l'intermédiaire des molécules MHC I des cellules dentritiques et ainsi stimuler les réponses des lymphocytes T cytotoxiques contre l'antigène.
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