WO2019122050A1 - Méthodes de traitement - Google Patents

Méthodes de traitement Download PDF

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Publication number
WO2019122050A1
WO2019122050A1 PCT/EP2018/086049 EP2018086049W WO2019122050A1 WO 2019122050 A1 WO2019122050 A1 WO 2019122050A1 EP 2018086049 W EP2018086049 W EP 2018086049W WO 2019122050 A1 WO2019122050 A1 WO 2019122050A1
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WIPO (PCT)
Prior art keywords
group
antigen
agonist
receptor
toll
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PCT/EP2018/086049
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English (en)
Inventor
Cornelis Joseph Maria Melief
Gavin Frank PAINTER
Wilhelmus Johannes Theodorus Alexander KREBBER
Ian Francis Hermans
Gijsbertus Gerardus Petrus ZON
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Isa Pharmaceuticals B.V.
Avalia Immunotherapeutics Limited
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Publication of WO2019122050A1 publication Critical patent/WO2019122050A1/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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—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
    • 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
    • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • A61K2039/585—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation wherein the target is cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/80—Vaccine for a specifically defined cancer
    • A61K2039/86—Lung
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/80—Vaccine for a specifically defined cancer
    • A61K2039/876—Skin, melanoma
    • 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
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to novel methods for immunization, in particular methods for immunization comprising administration of an antigen, an iNKT cell agonist and a TLR2 agonist.
  • Immunization involves the administration of a substance (an antigen) to a patient in order to induce an immune response against said antigen.
  • a substance an antigen
  • the purpose of an immunization can be to prevent a disease (prophylactic immunization) or to treat an existing disease (therapeutic immunization).
  • Antigens can be derived from pathogens or can be disease-related, e.g. an antigen found in tumour cells but not in normal cells used for a therapeutic immunization against cancer.
  • Specific immune responses against antigens can often be further stimulated by the co-administration of adjuvants.
  • Adjuvants are known in the art to accelerate, prolong, or enhance the quality of the specific immune response to antigens. Many different types of adjuvants have been described in the art.
  • TLRs toll-like receptors
  • TLRs recognize specific patterns of microbial components, especially those from pathogens, and regulate the activation of both innate and adaptive immunity. Thirteen members of the TLR-family have been identified in man. TLRs are expressed by phagocytic cells such as monocytes, macrophages and dendritic cells.
  • a known lipopeptide adjuvant which interacts with toll- like receptor 2 (TLR2) is the Pam3Cys-lipopeptide. Improved variants of Pam3Cys have been described in Willems et al. (2014) J Med Chem 57 : 6873 and WO2013051936.
  • iNKT invariant natural killer T
  • iNKT cells have the ability to respond rapidly to danger signals and pro- inflammatory cytokines. Once activated, they engage in effector functions, like NK transactivation, T cell activation and differentiation, B cell activation, dendritic cell activation and cross-presentation activity, as well as macrophage activation.
  • Alpha- galactosylceramide a-GalCer
  • a-GalCer is an example of an iNKT cell agonist, which has been shown to function as a potent vaccine adjuvant in the treatment and prophylaxis of cancer and infectious disease.
  • US2011229556 describes lipid-coated polymer particles for immune stimulation, Particles containing a-GalCer and Pam3Cys are described, but particles containing Pam3Cys were not functionally tested.
  • W02010055340 relates to prophylactic and therapeutic treatments involving the use of an iNKT agonist, such as a-GalCer or an analogue thereof, or a TLR agonist, or a combination thereof.
  • an iNKT agonist such as a-GalCer or an analogue thereof
  • TLR agonist a TLR agonist, or a combination thereof.
  • the specifically proposed and tested TLR agonists did not include TLR2 agonists.
  • Osmond et al. (2015) showed that TLR ligands can increase the number of circulating antigen-specific CD8+ T cells that were induced in the presence of NKT cell activation in mice.
  • the effects were dependent on the type of TLR agonist (TLR2, TLR3 or TLR7) and the timing of TLR activation relative to NKT cell activation and antigen administration. It was not evaluated whether the effects induced by TLR2 agonists translated into stronger anti-tumour responses or stronger protective prophylactic immunity.
  • the present inventors have now demonstrated that an immunization strategy which combines an antigen with an iNKT cell agonist and a TLR2 agonist results in remarkable synergistic effects compared to the use of each of these adjuvants alone.
  • immunization with antigen and either of the agonists alone had only modest effects on tumour growth, but solid anti-tumour responses were observed when both agonists were co-administered with the antigen.
  • the invention relates to a method of immunizing a human subject comprising administration of:
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising :
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising :
  • the invention relates to an antigen for use in a method of immunizing a human subject, wherein said antigen is administered in combination with an iNKT cell agonist and a Toll-like receptor 2 agonist.
  • the invention relates to a kit-of-parts comprising :
  • Figure 1 Therapeutic activity in B16.0VA melanoma model using Trp-2-based compounds - Individual growth curves.
  • Figure 1 shows the individual tumour growth curves in tumour challenged mice that have been administered immunogenic Trp-2-based compounds CI041 (Avalia) and AMPLIVANT-Trp2 (AV-Trp2). The data show that strong anti-tumour protection is achieved when CI041 and AMPLIVANT-Trp2 (AV-Trp2) are combined.
  • Groups of C57BL/6 mice ( n 8) were injected into the flank with 5xl0 5 B16.0VA melanoma cells.
  • mice were subcutaneously immunized with 5 nmol total of the indicated compounds (either 5 nmol of one compound or 2.5 nmol of each compound combined). The tumour sizes were monitored over time until the tumours reached ethical limits. Also included was a version of the vaccines that contained a human antigen that would not give an immune response in mice (Avalia-NLV and AV-NLV) (data not shown). There was no anti-tumour response in the mice that received this vaccine combination indicating that the response observed with the melanoma antigen vaccine is an antigen-specific response.
  • Figure 2 Therapeutic activity in a B16.0VA melanoma model using Trp-2-based compounds - Overall survival.
  • Figure 2 shows the overall survival in tumour-challenged mice that have been administered immunogenic Trp-2-based compounds. The data show that strong anti-tumour protection is achieved when the two types of Trp-2-based immunogenic compounds are combined.
  • Groups of C57BL/6 mice ( n 8) were injected into the flank with 5xl0 5 B16.0VA melanoma cells.
  • mice were subcutaneously immunized with 5 nmol total of the indicated compounds, CI041 (Avalia) and AMPLIVANT-Trp2 (AV-Trp2) (either 5 nmol of one compound or 2.5 nmol of each compound combined).
  • the tumour sizes were monitored over time until the tumours reached ethical limits, and the overall survival is presented.
  • Figure 3 Therapeutic activity in a TC-1 HPV16 E6- and E7-expressing tumour model using HPV-based compounds - Overall survival.
  • Figure 3 shows the overall survival in tumour challenged mice that have been administered immunogenic compounds CI078 (Avalia) and AMPLIVANT-HPV (AV-HPV). The data show that, while there was some increase in survival in tumour-bearing mice when they were injected with either compound alone compared to untreated tumour-bearing mice, the combined immunizations had a much greater anti-tumour effect that significantly prolonged survival.
  • Groups of C57BL/6 mice ( n 8) were injected into the flank with lxlO 5 TC-1 cells.
  • mice were subcutaneously immunized with 10 nmol total of the indicated compounds (either 10 nmol of one compound or 5 nmol of each compound combined). The tumour sizes were monitored over time until the tumours reached ethical limits, and the overall survival is presented.
  • FIG. 4 shows percentages of T cells with specificity for an epitope embedded in the HPV-peptide antigen following subcutaneous administration of compounds (single or combined) into mice.
  • Four different iNKT-cell-agonist-coupled peptides CI078, CI079, CI112 and CI113 were used that all had slightly different chemistry to determine if there was a superior combination chemistry.
  • the data show that the alphaGalCer-coupled peptides, with all four chemistry variations, were highly efficient at expanding antigen-specific T cells.
  • FIG. 5 shows the phenotype of T cells with specificity for the HPV- peptide antigen following subcutaneous administration of CI078 (Avalia SPAAC) or AMPLIVANT-HPV SLP (AV-HPV) either on their own or together.
  • CI078 Avalia SPAAC
  • AMPLIVANT-HPV SLP AV-HPV
  • the cells are identified as either terminally differentiated effector T cells using the marker KLRG1, or memory cells using the marker CD62L, or as activated but not yet differentiated into a committed effector or memory phenotype (negative for both KLRG1 and CD62L).
  • FIG. 6 CD8 T cell phenotype in the blood after immunogenic compound administration.
  • Figure 6 shows the phenotype of T cells with specificity for the HPV- peptide antigen following subcutaneous administration of CI079 (Avalia SPAAC FFRK) or AMPLIVANT-HPV SLP (AV-HPV) either on their own or together.
  • CI079 Avalia SPAAC FFRK
  • AMPLIVANT-HPV SLP AV-HPV
  • the cells are identified as either terminally differentiated effector T cells using the marker KLRG1, or memory cells using the marker CD62L, or as activated but not yet differentiated into a committed effector or memory phenotype (negative for both KLRG1 and CD62L).
  • FIG. 7 CD8 T cell phenotype in the blood after immunogenic compound administration.
  • Figure 7 shows the phenotype of T cells with specificity for the HPV- peptide antigen following subcutaneous administration of CI113 (Avalia Oxime) or AMPLIVANT-HPV SLP (AV-HPV) either on their own or together.
  • CI113 Avalia Oxime
  • AMPLIVANT-HPV SLP AV-HPV
  • the cells are identified as either terminally differentiated effector T cells using the marker KLRG1, or memory cells using the marker CD62L, or as activated but not yet differentiated into a committed effector or memory phenotype (negative for both KLRG1 and CD62L).
  • FIG 8. CD8 T cell phenotype in the blood after immunogenic compound administration.
  • Figure 8 shows the phenotype of T cells with specificity for the HPV- peptide antigen following subcutaneous administration of CI112 (Avalia Oxime FFRK) or AMPLIVANT-HPV SLP (AV-HPV) either on their own or together.
  • CI112 Avalia Oxime FFRK
  • AMPLIVANT-HPV SLP AV-HPV
  • the cells are identified as either terminally differentiated effector T cells using the marker KLRG1, or memory cells using the marker CD62L, or as activated but not yet differentiated into a committed effector or memory phenotype (negative for both KLRG1 and CD62L).
  • the term "antigen” refers to a molecule capable of inducing an immune response in a host organism, typically a mammalian host organism, such as a human host.
  • epitopope refers to an antigenic determinant, i.e. a point of interaction on the antigen for specific antibodies or an antigenic determinant that is presented on an MHC molecule and recognised by a T-cell receptor.
  • An antigen may contain more than one antigenic determinant.
  • An antigen may e.g. be a protein, a peptide, a nucleic acid, a polysaccharide or a lipid.
  • antigens used in the present invention are peptides.
  • an antigen when an antigen is a peptide, said peptide may be from 8 to 100 amino acids, or 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 50, 55, 60, 70, 80, 90 amino acids or more.
  • a peptide is a synthetic long peptide (SLP), which is a peptide having a length of 18 to 100 amino acids, more preferably a peptide having a length of 20-45, e.g. 20-35, amino acids.
  • SLP synthetic long peptide
  • a peptide antigen may be derived from a protein which is a suitable target for prophylactic or therapeutic vaccines.
  • a peptide antigen "derived from" a target protein is to be understood herein as to comprise a contiguous amino acid sequence selected from the target protein, which, while preserving its antigenic properties, may be modified by deletion or substitution of one or more amino acids, by extension at the N- and/or C-terminus with additional amino acids or functional groups, which may improve bio-availability, targeting to T-cells, or comprise or release immune modulating substances that provide adjuvant or (co)stimulatory functions.
  • iNKT cell agonist when used herein refers to any natural or non-natural compound which has the ability to stimulate NKT cells. Typically the activation is assayed by the production of cytokines. In particular, NKT cells are activated when they produced interferon gamma, IL-4, IL-10, IL-13 or IL-22. More particularly, a compound is considered as a NKT cell agonist when the compound is able to induce production of IL-22 by NKT cells.
  • An iNKT cell agonist is typically a ligand of CDld.
  • CDld when used herein, refers to the human CDld protein (UniProtKB-P15813).
  • TLR2 Toll-like receptor 2 (UniProtKB-060603) is a member of the Toll-like receptor family.
  • TLR2 is required for recognition of diverse microbial molecules from broad groups of species such as Gram-positive and Gram-negative bacteria, as well as mycoplasma and yeast. Specifically, TLR2 is involved in the recognition of cell-wall components, lipoteichoic acid and lipoprotein, from gram-positive bacteria; lipoarabinomannan, from mycobacteria; and zymosan, from yeast. TLR2 is involved in the specific recognition of a wide range of ligands, either as a homodimer or as a heterodimer with TLR1 or TLR6.
  • TLR2 agonist refers to a compound that binds to TLR2 and activates it to produce a biological response.
  • the term includes compounds that bind to TLR2 homodimers as well as compounds that bind to TLR1/2 or TLR2/6 heterodimers.
  • Treatment refers to methods and compositions to prevent, cure, or ameliorate a medical disease, disorder, or condition, and/or reduce at least a symptom of such disease or disorder.
  • this includes methods and compositions to prevent or delay onset of a medical disease, disorder, or condition; to cure, correct, reduce, slow, or ameliorate the physical or developmental effects of a medical disease, disorder, or condition; and/or to prevent, end, reduce, or ameliorate the pain or suffering caused by the medical disease, disorder, or condition.
  • amino acid includes both natural and non-natural amino acids.
  • hydrophobic amino acid when used herein is an amino acid selected from the group consisting of: Ala, Phe, Leu, lie, Val, Tyr and Trp.
  • a “linker” in the context of the invention is understood to mean a low molecular weight moiety with at least two attachment points for moieties.
  • a divalent linker has two such attachment points and a multivalent linker has at least three such attachment points. Via one of these attachment points, the linker is attached to an adjuvant moiety, and via one of the other attachment point to the antigen.
  • These attachment points originate from functional groups in the precursor of the linker and allow at least one antigen to be attached to the adjuvant compound.
  • a linker preferably has a molecular weight of at most 800 Da. This is all conventional chemistry.
  • Suitable chemical linkages in which the antigen is attached to the linker and/or in which the linker is attached to the adjuvant moiety are organic molecules containing an aliphatic chain and optionally including single or repetitive thioether, amide, amine, oxime, disulfide, thiazolidine, thiourea, ester, thioester, ether, carbamate, thiocarbamate, carbonate, thiocarbonate, hydrazone, sulfate, sulfamidate, sulfone, sulfonamide, phosphate, phosphorothioate, glyoxylic-oxime, or a bond obtained via Diels-Alder cycloaddition, Staudinger ligation, native ligation or Huisgen 1,3-dipolar cycloaddition.
  • linker may include natural and non- natural amino acid residues, alicyclic compounds such as cyclohexane and cyclopentane derivatives and (hetero) aromatic rings, such as substituted phenyl and substituted triazole.
  • the antigen may be coupled directly to the remainder of the compound according to the invention.
  • suitable bonds between the Km peptide part and the antigen are thioether, disulphide, amide or ester bonds.
  • the linker merely serves as a spacer to increase the distance between the antigen and the TLR2- or iNKT cell agonist moiety. In other embodiments, the linker is cleavable.
  • self-immolative linker means any chemical group that, by covalent attachment, bridges a second and a third chemical group, wherein the covalent bond between the self-immolative linker and the second chemical group is metabolically cleavable in vivo and wherein, upon cleavage of this covalent bond in vivo, the self- immolative linker is detached from the second chemical group through spontaneous chemical bond rearrangements.
  • At least one, preferably both, of the second and third chemical groups is a biologically active, e.g. pharmaceutically active, agent or prodrug thereof.
  • each of the second and third chemical groups is independently an immune stimulant (e.g.
  • TLR2 agonist or iNKT-cell agonist or an antigen (e.g. peptide, protein or carbohydrate).
  • an antigen e.g. peptide, protein or carbohydrate.
  • self-immolative linkers are described in Philip L. Carl, Prasun K. Chakravarty, John A. Katzenellenbogen, Journal of Medicinal Chemistry, 1981 , Vol. 24, No. 5, pg 479; and Simplicio et al., Molecules, 2008, vol. 13, pg 519.
  • the covalent bond between the self-immolative linker and the second chemical group may be cleaved by, for example, an esterase, a peptidase, a phosphatase, a phospholipase or a hydrolase, or by way of a redox or pH-dependent process.
  • alkyl means any saturated hydrocarbon radical having up to 30 carbon atoms and includes any C1-C25, C1-C20, C1-C15, C1-C10, or C1-C6 alkyl group, and, unless specified otherwise, is intended to include cyclic (including fused bicyclic) alkyl groups (sometimes referred to herein as "cycloalkyl”), straight-chain and branched-chain alkyl groups, and straight or branched chain alkyl groups substituted with cyclic alkyl groups.
  • alkyl groups include: methyl group (Me), ethyl group, n-propyl group, iso-propyl group, cyclopropyl group, n-butyl group, iso-butyl group, sec-butyl group, f- butyl group, n-pentyl group, 1,1-dimethylpropyl group, 1,2-dimethylpropyl group, 2,2- dimethy!propyl group, 1-ethylpropyl group, 2-ethyIpropyl group, n-hexyl group, cyclohexyl group, cyclooctyl group, and l-methyl-2-ethylpropyl group.
  • Me methyl group
  • ethyl group ethyl group
  • n-propyl group iso-propyl group
  • cyclopropyl group iso-butyl group
  • sec-butyl group sec-butyl group
  • alkylene means a diradical corresponding to an alkyl group.
  • alkylene groups include methylene group, cyclohexylene group, ethylene group.
  • An alkylene group can incorporate one or more cyclic alkylene group(s) in the alkylene chain, for example, "alkylene” can include a cyclohexylene group attached to a methylene group.
  • Any alkylene group may be optionally substituted with one or more substituents selected from the group consisting of hydroxyl, halogen, e.g. fluorine, alkyl, e.g. methyl, and aryl.
  • Any alkylene may optionally include one or more arylene moieties within the alkylene chain, for example, a phenylene group may be included within an alkylene chain.
  • lower alkyl means any saturated hydrocarbon radical having from 1 to 6 carbon atoms and is intended to include both straight- and branched-chain alkyl groups.
  • Any alkyl group may optionally be substituted with one or more substituents selected from the group consisting of SC H (or a salt thereof), hydroxy and halogen, e.g. fluorine.
  • alkenyl means any hydrocarbon radical having at least one double bond, and having up to 30 carbon atoms, and includes any C2-C25, C2-C20, C2-C15, C2-C10 or C2-C6 alkenyl group, and is intended to include both straight- and branched-chain alkenyl groups.
  • alkenyl groups include: ethenyl group, n-propenyl group, iso-propenyl group, n- butenyl group, iso-butenyl group, sec-butenyl group, f-butenyl group, n-pentenyl group, 1,1- dimethylpropenyl group, 1,2-dimethylpropenyl group, 2,2-dimethylpropenyl group, 1- ethylpropenyl group, 2-ethylpropenyl group, n-hexenyl group and l-methyl-2 - ethylpropenyl group.
  • lower alkenyl means any hydrocarbon radical having at least one double bond, and having from 2 to 6 carbon atoms, and is intended to include both straight- and branched- chain alkenyl groups. Any alkenyl group may optionally be substituted with one or more substituents selected from the group consisting of alkoxy, hydroxy and halogen, e.g. fluorine.
  • aryl means an aromatic radical having 4 to 18 carbon atoms and includes heteroaromatic radicals. Examples include monocyclic groups, as well as fused groups such as bicyclic groups and tricyclic groups. Examples include a phenyl group (Ph), indenyl group, 1-naphthyl group, 2-naphthyl group, azulenyl group, heptalenyl group, biphenyl group, indacenyl group, acenaphthyl group, fluorenyl group, phenalenyl group, phenanthrenyl group, anthracenyl group, cyclopentacyclooctenyl group, and benzocyclooctenyl group, pyridyl group (Py), pyrrolyl group, pyridazinyl group, pyrimidinyl group, pyrazinyl group, triazolyl group (including a l-/-/-l,2,3
  • arylene means a diradical corresponding to an aryl group. Examples include phenylene group.
  • aralkyl means an aryl group which is attached to an alkylene moiety, where aryl and alkylene are as defined above. Examples include benzyl group.
  • Any aryl or aralkyl group may optionally be substituted with one or more substituents selected from the group consisting of alkyl, halogen, cyano, dialkylamino, amide (both N-linked and C-linked : -NHC(0)R and -C(O)NHR), nitro, alkoxy, acyloxy and thioalkyl.
  • alkoxy means an OR group, where R is alkyl as defined above.
  • lower alkoxy means an OR group, where R is “lower alkyl” as defined above.
  • fatty-acyl group is a group formed by loss of OH from the carboxy group of a fatty acid.
  • acyloxy means an OR" group, where R" is acyl as defined above.
  • glycosyl means a radical derived from a cyclic monosaccharide, disaccharide or oligosaccharide by removal of the hemiacetal hydroxy group. Examples include a-D-glucopyranosyl, a-D-galactopyranosyl, b-D-galactopyranosyl, and a-D-2- deoxy-2-acetamidogalactopyranosyl.
  • amide includes both N-linked (-NHC(O)R) and C-linked (-C(O)NHR) amides.
  • pharmaceutically acceptable salt is intended to apply to non-toxic salts derived from inorganic or organic acids, including, for example, the following acid salts: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycerophosphate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2- hydroxyethanesulfonate, lactate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, oxalate
  • particle refers to a particle comprising a polymer core and a lipid bilayer coat.
  • the particles are synthetic and not naturally occurring.
  • the particles may be nanoparticles or microparticles. These terms are used to denote the size of the particles, typically characterized by particle diameter.
  • any reference to the disclosed compounds includes all possible formulations, configurations, and conformations, for example, in free form (e.g. as a free acid or base), in the form of salts or hydrates, in the form of isomers (e.g. cis/trans isomers), stereoisomers such as enantiomers, diastereomers and epimers, in the form of mixtures of enantiomers or diastereomers, in the form of racemates or racemic mixtures, or in the form of individual enantiomers or diastereomers. Specific forms of the compounds are described in detail herein.
  • the invention relates to a method of immunizing a human subject comprising administration of:
  • antigen refers to a molecule capable of inducing an immune response in a host organism, typically a mammalian host organism, such as a human host.
  • An antigen may be presented in different molecular entities and e.g. be a protein, a peptide, a nucleic acid, a polysaccharide or a lipid.
  • antigens used in the present invention are peptides.
  • a peptide antigen may be derived from a protein.
  • Proteins that are specifically expressed by infected, pre-cancerous and/or cancerous cells are suitable targets for therapeutic vaccines.
  • Such target proteins may be viral or non-viral proteins.
  • viral proteins that are targets for prophylactic and therapeutic vaccines are proteins derived from Epstein Bar virus induced lymphoma's (EBV), Human T lymphotrophic virus I, Hepatitis B virus (HBV), Human papilloma virus (HPV), Kaposi sarcoma herpes virus (KSHV), Hepatitis C virus (HCV), and Merkel cell carcinoma virus.
  • Non-limiting examples of viral target proteins are proteins from EBV, e.g. LMP1 or late membrane protein 1 (e.g. UniProtKB P03230) and LMP2 or late membrane protein 2 (e.g. UniProtKB P13285); target proteins from Human T lymphotrophic virus I, e.g. Tax protein (e.g. UniProtKB P14079; P0C213; P03409); target proteins from HBV e.g. genotypes A, B, C or D, e.g. Core protein (HBcAg) (e.g. UniProtKB Q21360), Polymerase (P protein) (e.g. UniProtKB 011885), protein HBsAg (e.g.
  • UniProtKB Q773S4 X-protein
  • X-protein e.g. UniProtKB Q8V1H6
  • Large envelope protein e.g. UniProtKB P03138
  • capsid protein e.g. UniProtKB P03147
  • target proteins from HCV e.g. genome polyprotein (e.g. UniProtKB P26663; Q99IB8; A3EZI9) and HCV protein (e.g. UniProtKB Q99398)
  • protein antigens from HPV e.g. genotypes 6 and 11 which induce genital warts and oncogenic genotypes 16, 18, 31, 33, 45, 52 and 58 e.g. L2 protein (e.g.
  • E2 protein e.g. UniProtKB P03120, P06790
  • E6 oncoprotein e.g. UniProtKB P03126; P06463
  • E7 oncoprotein e.g. UniProtKB P03129; P06788
  • target proteins from KSHV e.g. protein ORF36 (e.g. UniProtKB F5HGH5), Core gene UL42 family protein (e.g. UniProtKB Q77ZG5), Virion egress protein UL31 homolog (e.g. UniProtKB F5H982), Triplex capsid protein VP19C homolog (e.g. UniProtKB F5H8Y5), Viral macrophage inflammatory protein 2 (e.g. UniProtKB Q98157), mRNA export factor ICP27 homolog (e.g.
  • UniProtKB Q2HR73 Bcl-2 (e.g. UniProtKB Q76RI8), Large tegument protein deneddylase (e.g. UniProtKB Q2HR64), V-cyclin (e.g. UniProtKB 040946), VIRF-1 (e.g. UniProtKB F5HF68) and E3 ubiquitin-protein ligase MIR1 (e.g. UniProtKB P90495) and target proteins from Merkel cell carcinoma virus, e.g. large T protein (e.g. UniProtKB E2IPT4; K4P159), e.g. small T protein (e.g. UniProtKB B6DVX0; B6DVX6).
  • large T protein e.g. UniProtKB E2IPT4; K4P159
  • small T protein e.g. UniProtKB B6DVX0; B6DVX6
  • the target protein is an HBV protein, in particular X- protein (e.g. UniProtKB Q8V1H6), Polymerase (P protein) (e.g. UniProtKB 011885) or Core protein (HBcAg) (e.g. UniProtKB Q21360) or splice variants thereof.
  • HBV-derived antigens have been described in W02015187009 (ISA Pharmaceuticals) (herein incorporated by reference).
  • Non-viral proteins that are suitable targets for prophylactic and therapeutic vaccines may be tumour specific proteins and/or tumour associated proteins, often termed tumour specific antigens and tumour associate antigens.
  • Tumour specific antigens are antigens that are exclusively expressed by tumour cells and not by any other cell and are often mutated proteins, such as Kras G12D and mutant p53, or neo-antigens developed in due course by DNA mutations and malfunctioning DNA repair mechanisms.
  • Tumour associated antigens are endogenous antigens present in both tumour and normal cells but are dysregulated in their expression or cellular localization, such as the HER-2/neu receptor.
  • Non limiting examples of such non-viral proteins that may be targets for therapeutic vaccines are Her-2/neu (or ErbB-2, Human Epidermal growth factor Receptor 2 (e.g. UniProtKB P04626); WT-1 or Wilms tumour protein (e.g. UniProtKB P19544); NY-ESO-1 or cancer/testis antigen 1 (e.g. UniProtKB P78358); MAGE-A3 or melanoma-associated antigen-A3 (e.g. UniProtKB P43357); BAGE or B melanoma antigen (e.g. UniProtKB Q13072); CEA or carcinoembryonic antigen (e.g.
  • UniProtKB Q13984 AFP or a-fetoprotein (e.g. UniProtKB P02771); XAGE-1B or X antigen family member 1 (e.g. UniProtKB Q9HD64); survivin or BIRC5, Baculoviral IAP repeat-containing protein 5 (e.g. UniProtKB 015392); p53 (e.g. UniProtKB P04637); h-TERT or Telomerase reverse transcriptase (e.g. UniProtKB 014746); mesothelin (e.g. UniProtKB H3BR90); PRAME or Melanoma antigen preferentially expressed in tumours (e.g.
  • Neo-antigen is to be understood herein as a tumour antigen that arises from a tumour-specific mutation(s) which alters the amino acid sequence of genome-encoded proteins. Neo-antigens can be identified by whole-genome sequencing elucidating all, or nearly all, mutated neo-antigens that are uniquely present in a cancer (or neoplasia or tumour) of an individual patient.
  • mutated neo-antigens may be analysed to identify a specific, optimized subset of mutated neo-epitopes for use as an antigen source for the development of a personalized cancer vaccine for treatment of the patient's cancer. Methods to identify such neo-antigens are described in WO2014/168874, which is incorporated herein by reference.
  • iNKT cell agonists are described in WO2014/168874, which is incorporated herein by reference.
  • the method of the invention comprises administration of an iNKT cell agonist.
  • said iNKT cell agonist is a CDld ligand.
  • the iNKT cell agonist used in the method of the invention may or may not be covalently coupled (conjugated) to an antigen.
  • the iNKT cell agonist is covalently coupled to the antigen.
  • said iNKT cell agonist may be directly coupled to said antigen or it may be coupled to said antigen via a linker, such as a self-immolative linker.
  • said linker is a proteolytically-cleavable linker.
  • Suitable iNKT cell agonists for use in the method of the invention have e.g. been described in WO2014017928 and W020140088432 (both herein incorporated by reference).
  • the method of the invention comprises administration of a molecule of the formula (I):
  • A is a self-immolative linker group
  • D is selected from the group consisting of:
  • R 15 is a side-chain of one of the following amino acids: L-lysine, L-citrulline, L-arginine, L- glutamine or L-threonine;
  • R 16 is a side chain of a hydrophobic amino acid
  • R 19 is an alkylene group
  • R 32 is an alkylene group or an O-alkylene group wherein the O is attached to the carbonyl group of D2;
  • E is selected from the group consisting of:
  • R 20 is H or lower alkyl
  • R 21 is an alkylene group
  • g is 0 when R 20 is H or g is 1 when R 20 is lower alkyl;
  • E is E18 only when D is Dl, D2 or D3 and provided that E is El, E2, E3, E4, E5, E6, E7, E8, E9, E10, El l , E12, E13, E15, E20, E21, E93, E94 or E96 only when D is Dl, D2, D3 or D4; and provided that E is E91, E92 or E95 only when D is D5 and provided that E is E97 only when D is D2;
  • G is absent or G is an amino-acid sequence of up to 6 amino acids, attached through its N-terminus to group E and through its C-terminus to group J;
  • J is said antigen
  • R 1 is H or glycosyl, provided that if R 1 is glycosyl then R 2 and R 3 are both OH and R 4 is CH2OH;
  • R 2 is selected from the group consisting of H, OH, F and OR 10 ; provided that if R 2 is H, F or OR 10 , then R 1 is H, R 3 is OH and R 4 is CH2OH;
  • R 3 is selected from the group consisting of H, OH, F and OR 10 ; provided that if R 3 is H, F or OR 10 , then R 1 is H, R 2 is OH and R 4 is CH2OH;
  • R 4 is CHs, CH2OH, CH2OCOR 11 , CH2OR 10 , CH2OR 11 , CH2OSO3H, CH2SH, CH2SR 11 , CH2SOR 11 , CH2SO2R 11 , CH2PO3H2, CH 2 0P(0)(0H)2, CH 2 0P(0)(0H)(0R 11 ), CH 2 0P(0)(0R 11 )2, CO2H, CH2NHCOR 11 , CH2NHCO2R 11 , CH2NHCONH2, CH2NHCONH R 11 , CH 2 NHCON(R 11 ) 2 , CH 2 N(R 11 ) 2 , or CH2NHSO2R 11 ; provided that if R 4 is other than CH2OH, then R 1 is H and R 2 and R 3 are OH;
  • R 6 is OR 12 , OH or H
  • R 7 is OR 12 , OH or H; provided that at least one of R 6 and R 7 is OR 12 ; wherein, when R 6 is
  • R 7 is H
  • R 8 is C1-C15 alkyl and X is 0, denotes an optional double bond linking the carbon adjacent to R 7 with the carbon adjacent to R 8 ;
  • R 8 is H or C1-C15 alkyl having a straight or branched carbon chain, wherein the carbon chain optionally incorporates one or more double bonds, one or more triple bonds, one or more oxygen atoms and/or a terminal or non-terminal optionally substituted aryl group;
  • R 10 is glycosyl
  • R 11 is lower alkyl, lower alkenyl or aralkyl
  • R 12 is C6-C30 acyl having a straight or branched carbon chain optionally substituted with one or more hydroxy groups at positions 2 and/or 3 of the acyl group and/or an optionally substituted chain terminating aryl group and which optionally incorporates one or more double bonds, one or more triple bonds, and/or one or more optionally substituted arylene groups and wherein the carbon chain is optionally substituted with one or more deuterium atoms; wherein the optional substituents on the aryl and arylene groups may be selected from halogen, cyano, dialkylamino, C1-C6 amide, nitro, C1-C6 alkoxy, C1-C6 acyloxy and Ci- C 6 thioalkyl;
  • X is 0, CH2 or S; n is 1 when X is 0 or S; or n is 0 or 1 when X is CH2; wherein where X is CH2 then the following must all be true: the stereochemistry of the 6- membered sugar ring in formula (I) is alpha-D-galacto; R 1 is H; R 2 and R 3 are both OH; R 4 is CH2OH, CH2OR 10 or CH2OR 11 ; and :
  • R 6 is OH and R 7 is OR 12 and the stereochemistry at carbon atoms 2, 3 and 4 is (2S, 3S, 4R), (2S, 3S, 4S), (2R, 3S, 4S), (2R, 3S, 4R) or (2S, 3R, 4S) or R 6 is OR 12 and R 7 is H, and R 8 is C13H27 and the stereochemistry at carbon atoms 2 and 3 is (2S, 3S); wherein where X is S then the following must all be true: the stereochemistry of the 6- membered sugar ring in formula (I) is alpha-D-galacto; R 1 is H; R 2 and R 3 are both OH; R 4 is CH2OH, CH2OR 10 , CH2OR 1 1 or CO2H; and : either R 6 is OH and R 7 is OR 12 and the stereochemistry at carbon atoms 2, 3 and 4 is (2S, 3S, 4R); or R 6 is OR 12 and R 7 is H and the stereochemistry at the carbon atoms 2 and 3 is (2
  • A is selected from the group consisting of:
  • each Q 1 is independently selected from the group consisting of: H, alkyl, alkoxy, halogen, nitro, aryl; or, together with the ring to which it is attached, forms a fused bicyclic aryl group;
  • p is an integer from 1 to 4.
  • Aik 1 is C 1 -C 4 straight chain alkyl
  • R 28 is H or lower alkyl
  • A is A1 only when D is D1 and provided that A is A2 only when D is D2, D3 or D5 and provided that A is A3 only when D is D1 , D3 or D4 and provided that A is A4 only when D is D2, D3 or D5 and provided that A is A5 only when D is D1 , D3 or D4.
  • A is A2, Q 1 is H and p is 4.
  • D is D2 wherein preferably, R 15 is a side-chain of L-citrulline, R 16 is a side-chain of valine and R 32 is a pentylene or hexylene group.
  • E is E4
  • R 20 is H or methyl and R 21 is methylene.
  • E is E97 and wherein R 21 is butylene group.
  • G is
  • R 1 is H
  • R 2 is OH
  • R 3 is OH
  • R 4 is CH2OH
  • X is oxygen and n is 1.
  • said iNKT cell agonist is not covalently coupled to an antigen.
  • Suitable compounds for this embodiment have e.g. been described in W020140088432.
  • the iNKT cell agonist used in the method of the invention is a compound of the formula (II) :
  • Z is selected from the group consisting of:
  • R 23 is aryl, aralkyl or optionally substituted alkyl
  • R 24 is lower alkyl
  • R 25 is p-C 6 H 4 L wherein L is H, methoxy, COOH, C(0)NHCH 2 C00H or CH2CH 2 NMe2;
  • R 26 is aralkyl
  • R 27 is H or lower alkyl
  • R 28 is alkylene
  • R 31 is (CH 2 CH 2 0)k
  • k is an integer from 2 to 100;
  • W is an optionally substituted cyclooctynyl ring; or W is a fused bicyclic or tricyclic ring system comprising an optionally substituted cyclooctynyl ring fused to one or more aryl groups or one or more cycloalkyl groups; wherein the cyclooctynyl ring optionally contains a N atom within the ring, which N atom is optionally substituted with an acyl group; and wherein the cyclooctynyl ring is optionally substituted with one or more substituents selected from the group consisting of halogen, hydroxyl, alkoxy and aralkyl wherein the aryl part of this group is optionally substituted with a carboxylic acid; and wherein * or one of the optional substituents comprises a point of attachment of Z23 to group D; provided that Z is Z1 , Z2, Z3, Z4, Z7, Z8, Z9, Z10, Zll , Z13,
  • said iNKT cell agonist is not covalently coupled to an antigen.
  • suitable compounds for this embodiment have e.g. been described in W020140017928.
  • the method of the invention comprises administration of a molecule of the formula (III) :
  • R 1 is H or glycosyl, provided that if R 1 is glycosyl then R 2 and R 3 are both OH and R 4 is CH2OH;
  • R 2 is selected from the group consisting of H, OH, F and OR 10 , provided that if R 2 is H, F or OR 10 , then R 1 is H, R 3 is OH and R 4 is CH2OH;
  • R 3 is selected from the group consisting of H, OH, F and OR 10 ; provided that if R 3 is H, F or OR 10 , then R 1 is H, R 2 is OH and R 4 is CH2OH; R4 is CHs, CH2OH, CH2OCOR 11 , CH2OR 10 , CH2OR 11 , CH2OSO3H, CH2SH, CH2SR 11 , CH2SOR 11 , CH2SO2R 11 , CH2PO3H2, CH 2 0P(0)(0H)2, CH 2 0P(0)(0H)(0R 11 ), CH 2 0P(0)(0R 11 )2, CO2H, CH2NHCOR 11 , CH2NHCO2R 11 , CH2N HCONH2, CH2NHCONHR 11 , CH 2 NHCON(R 11 ) 2 , CH 2 N(R 11 ) 2 , CH2NHSO2R 11 ; provided that if R 4 is other than CH2OH, then R 1 is H and R 2 and R
  • R 5 is H; or R 5 is a radical of formula (i) :
  • each E 1 is independently selected from the group consisting of H, alkyl, alkoxy, halogen, nitroaryl; or, together with the ring to which it is attached, forms a fused bicyclic aryl group; p is an integer from 1 to 4; t is an integer from 1 to 2;
  • Aik 1 is C1-C4 straight chain alkyl; wherein when Y is a radical of formula (a) or (b) then Z is:
  • u is 1 or 2; each A 1 , the same or different, is independently selected from the group consisting of: alkyl which may be optionally substituted with one or more substituents selected from the group consisting of (OCH2CH2)mOMe, N HC(0)0R 14 , alkoxyimino, oxo, halogen, alkoxy, NHCOCH2(OCH 2 CH2)mOMe, alkenyl which may be optionally substituted with one or more substituents selected from the group consisting of (OCH2CH2)mOMe, alkoxyimino, oxo, halogen and alkoxy; aryl which may be optionally substituted with one or more substituents selected from the group consisting of (OCH2CH2)mOMe, alkyl, alkoxy, dialkylamino, nitro, halogen; or aralkyl which may be optionally substituted with one or more substituents selected from the group consisting of (OCH2CH2)mOMe, alk
  • E 2 and A 2 are each independently selected from H and A 1 ;
  • a 5 is H or benzyloxycarbonyl ;
  • R 6 is OR 12 , OH or H;
  • R 7 is OR 12 , OH or H; provided that at least one of R 6 and R 7 is OR 12 ; wherein when R 6 is
  • R 7 is H
  • R 8 is C1-C15 alkyl and X is 0, then - denotes an optional double bond linking the carbon adjacent to R 7 with the carbon adjacent to R 8 ;
  • R 8 is H or C1-C15 alkyl having a straight or branched carbon chain, wherein the carbon chain optionally incorporates one or more double bonds, one or more triple bonds, one or more oxygen atoms and/or a terminal or non-terminal optionally substituted aryl group;
  • R 10 is glycosyl
  • R 11 is lower alkyl, lower alkenyl or aralkyl
  • R 12 is C6-C30 acyl having a straight or branched carbon chain optionally substituted with one or more hydroxy groups at positions 2 and/or 3 of the acyl group and/or an optionally substituted chain terminating aryl group and which optionally incorporates one or more double bonds, one or more triple bonds, and/or one or more optionally substituted arylene groups and wherein the carbon chain is optionally substituted with one or more deuterium atoms; wherein the optional substituents on the aryl and arylene groups may be selected from halogen, cyano, dialkylamino, C1-C6 amide, nitro, C1-C6 alkoxy, C1-C6 acyloxy and Ci- C 6 thioalkyl;
  • R 14 is an optionally substituted alkyl, aryl or aralkyl group
  • X is 0, CH2 or S; n is 1 when X is 0 or S; or n is 0 or 1 when X is CH2;
  • R 6 is OH and R 7 is OR 12 and the stereochemistry at carbon atoms 2, 3 and 4 is (2S, 3S, 4R), (2S, 3S, 4S), (2R, 3S, 4S), (2R, 3S, 4R) or (2S, 3R, 4S); or
  • R 6 is OR 12 and R 7 is H, and R 8 is C13H27 and the stereochemistry at carbon atoms 2 and 3 is (2S, 3S); wherein where X is S then the following must all be true :
  • the stereochemistry of the 6- membered sugar ring in formula (III) is a-D-galacto;
  • R 1 is H;
  • R 2 and R 3 are both OH;
  • R 4 is CH2OH, CH2OR 10 or CH2OR 1 1 or CO2H; and :
  • R 6 is OH and R 7 is OR 12 and the stereochemistry at carbon atoms 2, 3 and 4 is (2S, 3S, 4R) or
  • R 6 is OR 12 and R 7 is H and the stereochemistry at the carbon atoms 2 and 3 is (2S, 3S); or a pharmaceutically acceptable salt thereof.
  • the stereochemistry of the 6- membered sugar ring of formula (I) is a-D-galacto.
  • n in formula (I) is 1
  • the stereochemistry of the 6-membered sugar ring of formula (I) is a-D-galacto
  • R 6 is OH
  • R 7 is OR 12
  • the stereochemistry at carbon atoms 2 3 and 4 is (2S, 3S, 4R).
  • n in formula (I) is 0, the stereochemistry of the 6-membered sugar ring of formula (I) is a -D-galacto, R 6 is OH, R 7 is OR 12 and the stereochemistry at carbon atoms 2, 3 and 4 is (2S, 3S, 4R).
  • X is O
  • R 6 is OR 12
  • R 7 is H
  • R 8 is
  • C1-C15 alkyl and - is a double bond linking the carbon adjacent to R 7 with the carbon adjacent to R 8 , and the stereochemistry at carbon atoms 2, 3 is (2S, 3S).
  • R 1 is H.
  • R 2 is OH.
  • R 3 is OH.
  • R 4 is CH2OH.
  • R 5 is a radical of formula (i).
  • R 6 is OH.
  • R 7 is OR 12 .
  • R 8 is C1-C15 alkyl having a straight or branched carbon chain, wherein the carbon chain optionally incorporates one or more double bonds, one or more triple bonds, one or more oxygen atoms and/or a terminal or non-terminal optionally substituted aryl group.
  • the iNKT cell agonist is a compound described in W02014200363 or WO 2015187040 (herein both incorporated by reference).
  • the iNKT cell agonist is the following compound, covalently linked to an antigen, described in W02015187040 (FFRK is SEQ ID NO: l) :
  • the iNKT cell agonist is the following compound, described in W02014200363 :
  • the iNKT cell agonist is selected from the group consisting of a- GalCer (Morita et al. 1995 J. Med Chem 38:2176), 7DW8-5 (Li et al. 2010 PNAS 107: 13010; Padte et al. 2013 PLoS One 8:e78407), C-34 (Huang et al. 2013 PNAS 110:2517; Hung et al. 2014 Antiviral Res 107: 110), threitolceramide (ThrCer) (Silk et al. 2008 J Immunol 180:6452), b-ManCer (O'Konek et al. 2011 J Clin Invest 121 :683, ABX196 (Tefit et al. 2014 Vaccine 32:6138) and C20:2 (Yu et al. 2005 PNAS 102: 3383).
  • a- GalCer Morita et al. 1995 J. Med Chem 38:2176
  • the iNKT cell agonist is selected from the group consisting of: plakoside A and analogs thereof (Tyznik et al. 2011 Chem Biol 18: 1620), ceramide chain aryl analogs and phytosphingosine C6Ph, containing a 6-phenylhexanoyl group (East et al. 2014 Med Res Rev 34:45), NU-alpha-GalCer (Aspeslagh et al. 2011 EMBO J 30:2294), RCAI-56 (Tashiro et al. 2007 Tetrahedron Lett 48:3343), GCK152 (Li et al. 2009 J Immunol 183:4415) and a-GalCer analogs with shorter acyl chain lengths as described by Fujio et al. 2006 JACS 128:9022. Toll-like receptor 2 agonists
  • the method of the invention comprises administration of an toll-like receptor 2 agonist.
  • said Toll-like receptor 2 agonist is a lipopeptide.
  • said Toll-like receptor 2 agonist is a di- or triacylated lipopeptide, preferably wherein one, two or all of the acyl groups is a fatty-acyl group having 8 or more carbon atoms, e.g. from 12 to 20 carbon atoms.
  • said Toll-like receptor 2 agonist comprises a N- tetradecylcarbamyl chain.
  • said Toll-like receptor 2 agonist further comprises one or two palmitoyl chains.
  • the Toll-like receptor 2 used in the method of the invention may or may not be covalently coupled (conjugated) to an antigen.
  • the Toll-like receptor 2 agonist is covalently coupled to the antigen.
  • said Toll-like receptor 2 agonist may be directly coupled to said antigen or it may be coupled to said antigen via a linker, such as a self-immolative linker.
  • said linker is a proteolytically-cleavable linker.
  • Suitable Toll-like receptor 2 agonists for use in the method of the invention have e.g. been described in WO2013051936 (herein incorporated by reference).
  • the method of the invention comprises administration of a molecule of the formula (IV) :
  • R 33 and R 34 are each independently a branched or straight group having up to 17 atoms selected from C (carbon), N (nitrogen), 0 (oxygen) and S (sulphur), q is from 0 to and including 18, Y 1 is S or Se (Selene), X 1 is S or 0 and R 35 is -OH or a group comprising said antigen;
  • X 1 is 0.
  • Y 1 is S.
  • R 33 and R 34 are preferably each independently branched or straight aliphatic groups having up to 17 atoms (i.e. each 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 atoms) in length containing one or more selected from carbon, nitrogen, oxygen and sulphur.
  • up to 17 atoms in length means in the context of the invention that the backbone of the branched or straight group comprises up to 17 atoms; thus the hydrogen atoms of an aliphatic group are not included in calculating the number of up to 17 atoms.
  • R 33 and R 34 are each independently a straight alkyl group having 10 to 17 carbon atoms, preferably 15 carbon atoms.
  • q is from 11 to and including 18, e.g. 11 to and including 15, such as 11, 12, 13 or 14, e.g. 12 or 14.
  • R 35 comprises a peptide of two or more amino acids. Such peptides have been described to promote agonistic activity (Bessler et al. (1985) J Immunol 135 : 1900).
  • group R 35 is represented by
  • R 36 is a (Lys)r peptide part, wherein r is 0, 1, 2, 3, 4 or 5, and wherein R 36 is optionally covalently coupled to an antigen and in which R 37 is hydrogen or a group comprising one to six atoms chosen from carbon, nitrogen and oxygen.
  • X F, Cl, Br
  • R 37 is not hydrogen and the asymmetric carbon to which R 37 is attached has the L configuration.
  • R 35 comprises said antigen.
  • the antigen may be coupled to the peptide, in particular to the (Lys)r, such as the (Lys)4 part, either directly or via a linker molecule. Examples of suitable chemical linkages in which the antigen is attached to the linker and/or in which the linker is attached to the remainder of the R 35 group are described in WO2013051936.
  • R 35 is a group comprising said antigen and a peptide of two or more amino acids covalently coupled to said antigen.
  • the R 35 group comprises a Ser(Lys) r peptide wherein r is 1, 2, 3, 4 or 5.
  • said peptide is Ser(Lys) 4 covalently coupled to said antigen at the C- terminal lysine (Reitermann et al. (1989) Biol Chem Hoppe-Seyler 370:343).
  • said Toll-like receptor 2 agonist is selected from the group consisting of: Parr Cys, Parri 3 CysSer(Lys)4 (also termed Pam3CSK4), Parri2CysSer(Lys)4 (also termed Pam2CSK4), PamiCysSer(l_ys)4 (also termed PamlCSK4), MALP-2 (e.g. from Mycoplasma sutans), OspA (e.g. from Borrelia burgdorferi), lipoteichoic acid, a porin (e.g.
  • LcrV protein e.g. the LcrV protein of Yersinia
  • lipomannan e.g. lipomannan of Mycobacterium tuberculosis
  • protozoan GPI anchor e.g. of Trypanosoma cruzi or Plasmodium falciparum
  • zymosan e.g. of Trypanosoma cruzi or Plasmodium falciparum
  • zymosan e.g. of Trypanosoma cruzi or Plasmodium falciparum
  • zymosan e.g. of Trypanosoma cruzi or Plasmodium falciparum
  • zymosan e.g. of Trypanosoma cruzi or Plasmodium falciparum
  • zymosan e.g. of Trypanosoma cruzi or Plasmodium falciparum
  • zymosan e.g. of Trypanosoma cruzi or Plasmodium falciparum
  • zymosan
  • both the iNKT cell agonist and the Toll-like receptor 2 agonist are covalently linked to the antigen.
  • the method comprises administration of:
  • the method may comprise administration of two molecules, (a) a conjugate of the iNKT cell agonist and the antigen, and (b) a conjugate of the Toll-like receptor 2 agonist and the antigen
  • both agonists are covalently bound to the same antigen molecule, i.e. each antigen molecule is covalently coupled to said iNKT cell agonist as well as to said Toll-like receptor 2 agonist.
  • Such molecules could be synthesized using, as a starting point, a compound of formula (IV) covalently coupled to a peptide antigen and attaching the iNKT cell agonist towards the C-terminus of the antigen.
  • Such a structure could e.g. be of the type illustrated below for an HPV antigen (SEQ ID NO:2) :
  • the antigen (i), the iNKT cell agonist (ii) and the Toll-like receptor 2 agonist (iii) are not administered together as part of a single particle. In another embodiment, neither of (i), (ii) or (iii) is administered as part of a particle.
  • said iNKT cell agonist is covalently coupled to said antigen, said antigen is a peptide and said iNKT cell agonist is administered at a dose of 0.1 to 200 micrograms per immunization.
  • said toll-like receptor 2 agonist is covalently coupled to said antigen, said antigen is a peptide and said toll-like receptor 2 agonist is administered at a dose of 0.3 to 200 micrograms per immunization, such as 0.5 to 100 micrograms, e.g. 1 to 50, such as 2 to 20 micrograms per immunization.
  • the expected therapeutic dose lies in the 50-4800 (pg/m 2 ) range (Giaccone, Punt et al. 2002). Note, 0.23 nmol in a mouse is a human equivalent dose of 30 pg/m 2 for a-GalCer. Higher doses are likely for other administration routes.
  • the compounds specified in (ii) and (iii) are administered in a molar ratio from 10: 1 to 1 : 10, such as a molar ratio between 5: 1 and 1 : 5, e.g. a molar ratio between 2: 1 and 1 :2, such as an equimolar ratio.
  • the compounds specified in (a) and (b) are administered in a molar ratio of between 10: 1 and 1 : 10, such as a molar ratio between 5 : 1 and 1 : 5, e.g. a molar ratio between 2: 1 and 1 :2, such as equimolar ratio.
  • administration of all ((i), (ii) and (iii)) or ((a) and (b)) of the components is simultaneous or sequential within a time interval or 24 hrs or less, such as 2 hrs or less, e.g. 1 hr or less, such as 30 min or less.
  • all components are administered in the same limb, preferably at the same site, i.e. wherein the administration sites, e.g. the injection sites, are within a distance of 1 cm, such as within 0.5 cm, e.g. within 0.25 cm.
  • two or all components are mixed prior to administration.
  • Preferably all components are provided together in one formulation.
  • Administration of the antigen (i), the iNKT cell agonist (ii) and the Toll-like receptor 2 agonist (iii) to the subject may be done by any suitable route of administration.
  • (i), (ii) and (iii) or (a) (a conjugate of the iNKT cell agonist and the antigen) and (b) (a conjugate of the Toll-like receptor 2 agonist and the antigen) are administered parenterally, such as intradermally, intramuscularly or subcutaneously.
  • (ii) is administered intravenously and (iii) is administered subcutaneously or (a) is administered intravenously and (b) is administered subcutaneously.
  • the method of the invention may comprise administration of further compounds, e.g. further antigens or further adjuvants.
  • said iNKT cell agonist is coupled to a first antigen and said Toll-like receptor 2 agonist is coupled to a second antigen, wherein said first and second antigens are not identical.
  • the said first and second antigens are not identical, but they are derived from the same target protein, i.e. different overlapping or non-overlapping fragments of the same target protein.
  • the method of immunization of the invention may be a therapeutic immunization or a prophylactic immunization.
  • a therapeutic immunization aims at combatting an existing disease or disorder in the subject, e.g. a chronic infection or a benign or malignant neoplasia.
  • a prophylactic immunization aims at inducing a response that protects against a disease, e.g. a disease caused by a pathogen or is preventive by e.g. preventing progression of a benign neoplasia to a malignant form, e.g. preventing progression of cervical intraepithelial neoplasia (CIN) to cervical cancer.
  • CIN cervical intraepithelial neoplasia
  • the immunization is a therapeutic or prophylactic immunization against cancer.
  • the antigen is a cancer antigen, e.g. a neo-antigen or an antigen derived from a target that is preferentially expressed in tumour tissue/cells as compared to healthy tissue/cells.
  • said antigen(s) is/are from a pathogen, such as a virus, bacterium, fungus, parasite and the immunization is a prophylactic or therapeutic immunization against a disease or infection associated with said pathogen.
  • a pathogen such as a virus, bacterium, fungus, parasite
  • the immunization is a prophylactic or therapeutic immunization against a disease or infection associated with said pathogen.
  • the immunization induces an immune response wherein the relative proportion of the CD62L- KLRG1+ subpopulation of CD8 T-cells is increased as compared to an immune response obtained by an otherwise identical immunization lacking component (ii) or (iii).
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising :
  • the invention relates to pharmaceutical composition
  • pharmaceutical composition comprising
  • the antigens in (a) and (b) may be the same or different (a) and (b) may be separate molecules or in some embodiments, the iNKT cell agonist and the Toll-like receptor 2 agonist may be covalently linked to the same antigen molecule.
  • the invention relates to a n antigen for use in a method of immunizing a human subject, wherein said antigen is administered in combination with an iNKT cell agonist and a Toll-like receptor 2 agonist.
  • the invention relates to the use of an antigen in the manufacture of a medicament for the immunization of a human subject, wherein said antigen is administered in combination with an iNKT cell agonist and a Toll-like receptor 2 agonist.
  • the invention relates to a kit-of-parts comprising :
  • Said kit may further comprise instructions to the user to co-administer the components in a method of immunization.
  • Embodiment 1 A method of immunizing a human subject comprising administration of:
  • Embodiment 2 The method according to embodiment 1, wherein (i), (ii) and (iii) are not administered together as part of a single particle.
  • Embodiment 3. The method according to embodiment 1 or 2, wherein neither of (i), (ii) or (iii) is administered as part of a particle.
  • Embodiment 4 The method according to any one of the preceding embodiments, wherein said iNKT cell agonist is a CDld ligand.
  • Embodiment 5 The method according to any one of the preceding embodiments, wherein said iNKT cell agonist is covalently coupled to said antigen.
  • Embodiment 6 The method according to embodiment 5, wherein said iNKT cell agonist is covalently coupled to said antigen via a linker, such as a self-immolative linker.
  • a linker such as a self-immolative linker.
  • Embodiment 7 The method according to embodiment 5 or 6, wherein iNKT cell agonist is covalently coupled to said antigen via a proteolytically-cleavable linker.
  • Embodiment 8 The method according to any one of the preceding embodiments, comprising administration of a molecule of the formula (I) :
  • A is a self-immolative linker group
  • D is selected from the group consisting of: wherein * denotes a point of attachment of group D to group A;
  • R 15 is a side-chain of one of the following amino acids: L-lysine, L-citrulline, L-arginine, L- glutamine or L-threonine;
  • R 16 is a side chain of a hydrophobic amino acid
  • R 19 is an alkylene group
  • R 32 is an alkylene group or an O-alkylene group wherein the O is attached to the carbonyl group of D2;
  • E is selected from the group consisting of:
  • R 20 is H or lower alkyl
  • R 21 is an alkylene group
  • g is 0 when R 20 is H or g is 1 when R 20 is lower alkyl;
  • E is E18 only when D is Dl, D2 or D3 and provided that E is El, E2, E3, E4, E5, E6, E7, E8, E9, E10, Ell , E12, E13, E15, E20, E21, E93, E94 or E96 only when D is Dl, D2, D3 or D4; and provided that E is E91, E92 or E95 only when D is D5 and provided that E is E97 only when D is D2;
  • G is absent or G is an amino-acid sequence of up to 6 amino acids, attached through its N-terminus to group E and through its C-terminus to group J;
  • J is said antigen
  • R 1 is H or glycosyl, provided that if R 1 is glycosyl then R 2 and R 3 are both OH and R 4 is CH2OH;
  • R 2 is selected from the group consisting of H, OH, F and OR 10 ; provided that if R 2 is H, F or OR 10 , then R 1 is H, R 3 is OH and R 4 is CH 2 OH;
  • R 3 is selected from the group consisting of H, OH, F and OR 10 ; provided that if R 3 is H, F or OR 10 , then R 1 is H, R 2 is OH and R 4 is CH 2 OH;
  • R 4 is CHs, CH2OH, CH2OCOR 11 , CH2OR 10 , CH2OR 11 , CH2OSO3H, CH2SH, CH2SR 11 , CH2SOR 11 , CH2SO2R 11 , CH2PO3H2, CH 2 0P(0)(0H)2, CH 2 0P(0)(0H)(0R 11 ), CH 2 0P(0)(0R 11 )2, CO2H, CH2NHCOR 11 , CH2NHCO2R 11 , CH2NHCONH2, CH2NHCONH R 11 , CH 2 NHCON(R 11 ) 2 , CH2N(R n )2, or CH2NHSO2R 11 ; provided that if R 4 is other than CH2OH, then R 1 is H and R 2 and R 3 are OH;
  • R 6 is OR 12 , OH or H
  • R 7 is OR 12 , OH or H; provided that at least one of R 6 and R 7 is OR 12 ; wherein, when R 6 is
  • R 7 is H
  • R 8 is C1-C15 alkyl and X is 0, denotes an optional double bond linking the carbon adjacent to R 7 with the carbon adjacent to R 8 ;
  • R 8 is H or C 1 -C 15 alkyl having a straight or branched carbon chain, wherein the carbon chain optionally incorporates one or more double bonds, one or more triple bonds, one or more oxygen atoms and/or a terminal or non-terminal optionally substituted aryl group;
  • R 10 is glycosyl
  • R 1 1 is lower alkyl, lower alkenyl or aralkyl
  • R 12 is C6-C30 acyl having a straight or branched carbon chain optionally substituted with one or more hydroxy groups at positions 2 and/or 3 of the acyl group and/or an optionally substituted chain terminating aryl group and which optionally incorporates one or more double bonds, one or more triple bonds, and/or one or more optionally substituted arylene groups and wherein the carbon chain is optionally substituted with one or more deuterium atoms; wherein the optional substituents on the aryl and arylene groups may be selected from halogen, cyano, dialkylamino, C1-C6 amide, nitro, C1-C6 alkoxy, C1-C6 acyloxy and Ci- C6 thioalkyl;
  • X is 0, CH2 or S; n is 1 when X is 0 or S; or n is 0 or 1 when X is CH2; wherein where X is CH2 then the following must all be true: the stereochemistry of the 6- membered sugar ring in formula (I) is alpha-D-galacto; R 1 is H; R 2 and R 3 are both OH; R 4 is CH2OH, CH2OR 10 or CH2OR 11 ; and :
  • R 6 is OH and R 7 is OR 12 and the stereochemistry at carbon atoms 2, 3 and 4 is (2S, 3S, 4R), (2S, 3S, 4S), (2R, 3S, 4S), (2R, 3S, 4R) or (2S, 3R, 4S) or R 6 is OR 12 and R 7 is H, and R 8 is C13H27 and the stereochemistry at carbon atoms 2 and 3 is (2S, 3S); wherein where X is S then the following must all be true: the stereochemistry of the 6- membered sugar ring in formula (I) is alpha-D-galacto; R 1 is H; R 2 and R 3 are both OH; R 4 is CH2OH, CH2OR 10 , CH2OR 1 1 or CO2H; and :
  • R 6 is OH and R 7 is OR 12 and the stereochemistry at carbon atoms 2, 3 and 4 is (2S, 3S, 4R); or R 6 is OR 12 and R 7 is H and the stereochemistry at the carbon atoms 2 and 3 is (2S, 3S); or a pharmaceutically acceptable salt thereof.
  • Embodiment 9 The method according to embodiment 8, wherein A is selected from the group consisting of:
  • each Q 1 is independently selected from the group consisting of: H, alkyl, alkoxy, halogen, nitro, aryl; or, together with the ring to which it is attached, forms a fused bicyclic aryl group;
  • p is an integer from 1 to 4.
  • Aik 1 is C 1 -C 4 straight chain alkyl
  • R 28 is H or lower alkyl
  • A is A1 only when D is D1 and provided that A is A2 only when D is D2, D3 or D5 and provided that A is A3 only when D is D1 , D3 or D4 and provided that A is A4 only when D is D2, D3 or D5 and provided that A is A5 only when D is D1 , D3 or D4.
  • Embodiment 10 The method according to embodiment 9, wherein A is A2, Q 1 is H and p is 4.
  • Embodiment 11 The method according to any one of embodiments 8 to 10, wherein D is D2.
  • Embodiment 12 The method according to embodiment 11, wherein R 15 is a side-chain of L-citrulline, R 16 is a side-chain of valine and R 32 is a pentylene or hexylene group.
  • Embodiment 13 The method according to any one of embodiments 8 to 12, wherein E is E4,
  • R 20 is H or methyl and R 21 is methylene.
  • Embodiment 14 The method according to any one of embodiments 8 to 12, wherein E is E97 and wherein R 21 is butylene group.
  • Embodiment 15 The method according to any one of embodiments 8 to 14, wherein G is
  • Embodiment 16 The method according to any one of embodiments 8 to 15, wherein R 1 is H, R 2 is OH, R 3 is OH, R 4 is CH2OH, X is oxygen and n is 1.
  • Embodiment 17 The method according to any one of embodiments 8 to 16, wherein R 6 is OH, R 7 is OH and R 8 is C13H27.
  • Embodiment 18 The method according to any one embodiments 1 to 7, wherein said iNKT cell agonist is selected from the group consisting of a-GalCer, 7DW8-5, C-34, threitolrcar (ThrCer), b-ManCer, ABX196, C20:2, plakoside A, phytosphingosine C6Ph, NU-alpha- GalCer, RCAI-56 and GCK152.
  • a-GalCer 7DW8-5, C-34, threitolrcar (ThrCer), b-ManCer, ABX196, C20:2, plakoside A, phytosphingosine C6Ph, NU-alpha- GalCer, RCAI-56 and GCK152.
  • Embodiment 19 The method according to any one of the preceding embodiments, wherein said Toll-like receptor 2 agonist is a lipopeptide.
  • Embodiment 20 The method according to any one of the preceding embodiments, wherein said Toll-like receptor 2 agonist is a di- or triacylated lipopeptide.
  • Embodiment 21 The method according to embodiment 20, wherein one, two or all of the acyl groups is a fatty-acyl group having 8 or more carbon atoms, e.g. from 12 to 20 carbon atoms.
  • Embodiment 22 The method according to any one of the preceding embodiments, wherein said Toll-like receptor 2 agonist comprises a N-tetradecylcarbamyl chain.
  • Embodiment 23 The method according to embodiment 22, wherein said Toll-like receptor 2 agonist further comprises one or two palmitoyl chains.
  • Embodiment 24 The method according to any one of the preceding embodiments, wherein said Toll-like receptor 2 agonist is covalently coupled to said antigen.
  • Embodiment 25 The method according to embodiment 24, wherein said Toll-like receptor 2 agonist and said antigen are coupled via a linker, such as a self-immolative linker.
  • a linker such as a self-immolative linker.
  • Embodiment 26 The method according to any one of the preceding embodiments, wherein said Toll-like receptor 2 agonist is a compound of the formula (IV) :
  • R 33 and R 34 are each independently a branched or straight group having up to 17 atoms selected from C, N, 0 and S, q is from 0 to and including 18, Y 1 is S or Se, X 1 is S or 0 and R 35 is a group comprising said antigen;
  • Embodiment 27 The method according to embodiment 26, wherein X 1 is 0.
  • Embodiment 28 The method according to embodiment 26 or 27, wherein Y 1 is S.
  • Embodiment 29 The method according to any one of embodiments 26 to 28, wherein R 33 and R 34 are each independently a straight alkyl group having 10 to 17 carbon atoms.
  • Embodiment 30 The method according to any one of embodiments 26 to 29, wherein R 33 and R 34 are straight alkyl groups having 15 carbon atoms.
  • Embodiment 31 The method according to any one of embodiments 26 to 30, wherein q is from 11 to and including 18, e.g. 11 to and including 15, such as 12 or 14.
  • Embodiment 32 The method according to any one of embodiments 26 to 31, wherein R 35 is an organic group comprising a peptide of two or more amino acids, preferably R 35 is Ser(Lys) r , wherein r is 1, 2, 3, 4 or 5.
  • Embodiment 33 The method according to embodiment 32, wherein R 35 is Ser(Lys) 4 covalently coupled to said antigen at the C-terminal lysine.
  • Embodiment 34 The method according to any one of embodiments 1 to 18, wherein said Toll-like receptor 2 agonist is selected from the group consisting of: Parri 3 Cys, Parri 3 CysSer(Lys)4, Par Cys Ser(l_ys) 4 , PamiCys Ser(l_ys) 4 , MALP-2, OspA, lipoteichoic acid, a porin, MALP-404, LcrV protein, lipomannan, a protozoan GPI anchor, zymosan, lysophosphatidylserine, lipophosphoglycan, hsp60, glycoprotein gH/gL or gB of herpes simplex virus, hemagglutinin protein of measles virus.
  • Embodiment 35 The method according to any one of the preceding embodiments, wherein the method comprises administration of:
  • Embodiment 36 The method according to any one of the preceding embodiments, wherein each antigen molecule is covalently coupled to said iNKT cell agonist as well as to said Toll- like receptor 2 agonist.
  • Embodiment 37 The method according to any one of the preceding embodiments, wherein said antigen is a peptide.
  • Embodiment 38 The method according to embodiment 37, wherein said peptide is from 8 to 100 amino acids in length, e.g. from 20 to 45 amino acids in length.
  • Embodiment 39 The method according to any one of the preceding embodiments, wherein said iNKT cell agonist is covalently coupled to said antigen, wherein said antigen is a peptide and wherein said iNKT cell agonist is administered at a dose of 0.1 to 200 micrograms per immunization.
  • Embodiment 40 The method according to any one of the preceding embodiments, wherein said toll-like receptor 2 agonist is covalently coupled to said antigen, wherein said antigen is a peptide and wherein said toll-like receptor 2 agonist is administered at a dose of 0.3 to 200 micrograms per immunization.
  • Embodiment 41 The method according to any one of the preceding embodiments, wherein the compounds specified in (ii) and (iii) are administered in a molar ratio from 10: 1 to 1 : 10, such as a molar ratio between 5: 1 and 1 : 5, e.g. a molar ratio between 2: 1 and 1 :2, such as an equimolar ratio.
  • Embodiment 42 The method according to any one of embodiments 35, 37 or 38, wherein the compounds specified in (a) and (b) are administered in a molar ratio of between 10: 1 and 1 : 10, such as a molar ratio between 5 : 1 and 1 : 5, e.g. a molar ratio between 2: 1 and 1 :2, such as equimolar ratio.
  • Embodiment 43 The method according to any one of the preceding embodiments wherein the administration of two or all of the components is simultaneous or sequential within a time interval of 24 hrs or less, such as 2 hrs or less, e.g. 1 hr or less, such as 30 min or less.
  • Embodiment 44 The method according to any one of the preceding embodiments, wherein all components are administered in the same limb, preferably at the same site.
  • Embodiment 45 The method according to any one of the preceding embodiments, wherein all components are mixed prior to administration, preferably wherein all components are provided together in one formulation.
  • Embodiment 46 The method according to any one of the preceding embodiments, wherein (i), (ii) and (iii) or (a) and (b) are administered parenterally, such as intradermally, intramuscularly or subcutaneously.
  • Embodiment 47 The method according to any one of embodiments 1 to 45, wherein (ii) is administered intravenously and (iii) is administered subcutaneously or wherein (a) is administered intravenously and (b) is administered subcutaneously.
  • Embodiment 48 The method according to any one of the preceding embodiments, wherein the immunization induces an immune response wherein the relative proportion of the CD62L- KLRG1+ subpopulation of CD8 T-cells is increased as compared to an immune response obtained by an otherwise identical immunization lacking component (ii) or (iii).
  • Embodiment 49 The method according to any one of the preceding embodiments, wherein the method comprises administration of one or more further antigens.
  • Embodiment 50 The method according to embodiment 49, wherein said iNKT cell agonist is coupled to a first antigen and said Toll-like receptor 2 agonist is coupled to a second antigen, wherein said first and second antigens are not identical.
  • Embodiment 51 The method according to embodiment 50, wherein the first and second antigens are derived from the same target protein.
  • Embodiment 52 The method according to any one of the preceding embodiments, wherein the method is for therapy.
  • Embodiment 53 The method according to any one of embodiments 1 to 51, wherein the method is for prophylaxis.
  • Embodiment 54 The method according to embodiment 52 or 53, wherein the immunization is an immunization against cancer.
  • Embodiment 55 The method according to embodiment 54, wherein the antigen is a neo- antigen or an antigen derived from a target that is overexpressed in tumour tissue/cells.
  • Embodiment 56 The method according to any one of the preceding embodiments, wherein said antigen(s) is/are from a pathogen and the immunization is a prophylactic or therapeutic immunization against a disease or infection associated with said pathogen.
  • Embodiment 57 A pharmaceutical composition comprising :
  • Embodiment 58 A pharmaceutical composition comprising
  • Embodiment 59 The pharmaceutical composition according to embodiment 57 or 58, wherein said iNKT cell agonist is a CDld ligand.
  • Embodiment 60 The pharmaceutical composition according to any one of embodiments 57 to 59, wherein said iNKT cell agonist is covalently coupled to said antigen.
  • Embodiment 61 The pharmaceutical composition according to any one of embodiments 57 to 60, wherein said iNKT cell agonist is covalently coupled to said antigen via a linker, such as a self-immolative linker.
  • Embodiment 62 The pharmaceutical composition according to any one of embodiments 57 to 61, wherein iNKT cell agonist is covalently coupled to said antigen via a proteolytically- cleavable linker.
  • Embodiment 63 The pharmaceutical composition according to any one of embodiments 57 to 62, comprising administration of a molecule of the formula (I):
  • A is a self-immolative linker group
  • D is selected from the group consisting of:
  • R 15 is a side-chain of one of the following amino acids: L-lysine, L-citrulline, L-arginine, L- glutamine or L-threonine;
  • R 16 is a side chain of a hydrophobic amino acid
  • R 19 is an alkylene group
  • R 32 is an alkylene group or an O-alkylene group wherein the 0 is attached to the carbonyl group of D2;
  • E is selected from the group consisting of:
  • R 20 is H or lower alkyl
  • R 21 is an alkylene group
  • g is 0 when R 20 is H or g is 1 when R 20 is lower alkyl;
  • E is E18 only when D is Dl, D2 or D3 and provided that E is El, E2, E3, E4, E5, E6, E7, E8, E9, E10, El l , E12, E13, E15, E20, E21, E93, E94 or E96 only when D is Dl, D2, D3 or D4; and provided that E is E91, E92 or E95 only when D is D5 and provided that E is E97 only when D is D2;
  • G is absent or G is an amino-acid sequence of up to 6 amino acids, attached through its N-terminus to group E and through its C-terminus to group J;
  • J is said antigen
  • R 1 is H or glycosyl, provided that if R 1 is glycosyl then R 2 and R 3 are both OH and R 4 is CH2OH;
  • R 2 is selected from the group consisting of H, OH, F and OR 10 ; provided that if R 2 is H, F or OR 10 , then R 1 is H, R 3 is OH and R 4 is CH2OH;
  • R 3 is selected from the group consisting of H, OH, F and OR 10 ; provided that if R 3 is H, F or OR 10 , then R 1 is H, R 2 is OH and R 4 is CH2OH;
  • R 4 is CHs, CH2OH, CH2OCOR 11 , CH2OR 10 , CH2OR 11 , CH2OSO3H, CH2SH, CH2SR 11 , CH2SOR 11 , CH2SO2R 11 , CH2PO3H2, CH 2 0P(0)(0H)2, CH 2 0P(0)(0H)(0R 11 ), CH 2 0P(0)(0R 11 )2, CO2H, CH2NHCOR 11 , CH2NHCO2R 11 , CH2NHCONH2, CH2NHCONH R 11 , CH 2 NHCON(R 11 ) 2 , CH 2 N(R 11 ) 2 , or CH2NHSO2R 11 ; provided that if R 4 is other than CH2OH, then R 1 is H and R 2 and R 3 are OH;
  • R 6 is OR 12 , OH or H
  • R 7 is OR 12 , OH or H; provided that at least one of R 6 and R 7 is OR 12 ; wherein, when R 6 is
  • R 7 is H
  • R 8 is C1-C15 alkyl and X is 0, denotes an optional double bond linking the carbon adjacent to R 7 with the carbon adjacent to R 8 ;
  • R 8 is H or C1-C15 alkyl having a straight or branched carbon chain, wherein the carbon chain optionally incorporates one or more double bonds, one or more triple bonds, one or more oxygen atoms and/or a terminal or non-terminal optionally substituted aryl group;
  • R 10 is glycosyl
  • R 11 is lower alkyl, lower alkenyl or aralkyl
  • R 12 is C6-C30 acyl having a straight or branched carbon chain optionally substituted with one or more hydroxy groups at positions 2 and/or 3 of the acyl group and/or an optionally substituted chain terminating aryl group and which optionally incorporates one or more double bonds, one or more triple bonds, and/or one or more optionally substituted arylene groups and wherein the carbon chain is optionally substituted with one or more deuterium atoms; wherein the optional substituents on the aryl and arylene groups may be selected from halogen, cyano, dialkylamino, C1-C6 amide, nitro, C1-C6 alkoxy, C1-C6 acyloxy and Ci- C 6 thioalkyl;
  • X is 0, CH2 or S; n is 1 when X is 0 or S; or n is 0 or 1 when X is CH2; wherein where X is CH2 then the following must all be true: the stereochemistry of the 6- membered sugar ring in formula (I) is alpha-D-galacto; R 1 is H; R 2 and R 3 are both OH; R 4 is CH2OH, CH2OR 10 or CH2OR 11 ; and :
  • R 6 is OH and R 7 is OR 12 and the stereochemistry at carbon atoms 2, 3 and 4 is (2S, 3S, 4R), (2S, 3S, 4S), (2R, 3S, 4S), (2R, 3S, 4R) or (2S, 3R, 4S) or R 6 is OR 12 and R 7 is H, and R 8 is C13H27 and the stereochemistry at carbon atoms 2 and 3 is (2S, 3S); wherein where X is S then the following must all be true: the stereochemistry of the 6- membered sugar ring in formula (I) is alpha-D-galacto; R 1 is H; R 2 and R 3 are both OH; R 4 is CH2OH, CH2OR 10 , CH2OR 1 1 or CO2H; and :
  • R 6 is OH and R 7 is OR 12 and the stereochemistry at carbon atoms 2, 3 and 4 is (2S, 3S, 4R); or R 6 is OR 12 and R 7 is H and the stereochemistry at the carbon atoms 2 and 3 is (2S, 3S); or a pharmaceutically acceptable salt thereof.
  • Embodiment 64 The pharmaceutical composition according to embodiment 63, wherein A is selected from the group consisting of:
  • each Q 1 is independently selected from the group consisting of: H, alkyl, alkoxy, halogen, nitro, aryl; or, together with the ring to which it is attached, forms a fused bicyclic aryl group;
  • p is an integer from 1 to 4.
  • Aik 1 is C 1 -C 4 straight chain alkyl
  • R 28 is H or lower alkyl
  • Embodiment 65 The pharmaceutical composition according to embodiment 64, wherein A is A2, Q 1 is H and p is 4.
  • Embodiment 66 The pharmaceutical composition according to any one of embodiments 63 to 65, wherein D is D2.
  • Embodiment 67 The pharmaceutical composition according to embodiment 66, wherein R 15 is a side-chain of L-citrulline, R 16 is a side-chain of valine and R 32 is a pentylene or hexylene group.
  • Embodiment 68 The pharmaceutical composition according to any one of embodiments 63 to 67, wherein E is E4, R 20 is H or methyl and R 21 is methylene.
  • Embodiment 69 The pharmaceutical composition according to any one of embodiments 63 to 67, wherein E is E97 and wherein R 21 is butylene group.
  • Embodiment 70 The pharmaceutical composition according to any one of embodiments 63 to 69, wherein G is
  • Embodiment 71 The pharmaceutical composition according to any one of embodiments 63 to 70, wherein R 1 is H, R 2 is OH, R 3 is OH, R 4 is CH2OH, X is oxygen and n is 1.
  • Embodiment 72 The pharmaceutical composition according to any one of embodiments 63 to 71, wherein R 6 is OH, R 7 is OH and R 8 is C13H 27.
  • Embodiment 73 The pharmaceutical composition according to any one of embodiments 57 to 63, wherein said iNKT cell agonist is selected from the group consisting of a-GalCer, 7DW8-5, C-34, threitolceramide (ThrCer), b-ManCer, ABX196, C20:2, plakoside A, phytosphingosine C6Ph, NU-alpha-GalCer, RCAI-56 and GCK152.
  • Embodiment 74 The pharmaceutical composition according to any one of embodiments 57 to 73, wherein said Toll-like receptor 2 agonist is a lipopeptide.
  • Embodiment 75 The pharmaceutical composition according to any one of embodiments 57 to 73, wherein said Toll-like receptor 2 agonist is a di- or triacylated lipopeptide.
  • Embodiment 76 The pharmaceutical composition according to embodiment 75, wherein one, two or all of the acyl groups is a fatty-acyl group having 8 or more carbon atoms, e.g. from 12 to 20 carbon atoms.
  • Embodiment 77 The pharmaceutical composition according to any one of embodiments 57 to 76, wherein said Toll-like receptor 2 agonist comprises a N-tetradecylcarbamyl chain.
  • Embodiment 78 The pharmaceuticals composition according to embodiment 77, wherein said Toll-like receptor 2 agonist further comprises one or two palmitoyl chains.
  • Embodiment 79 The pharmaceutical composition according to any one of embodiments 57 to 78, wherein said Toll-like receptor 2 agonist is covalently coupled to said antigen.
  • Embodiment 80 The pharmaceutical composition according to embodiment 79, wherein said Toll-like receptor 2 agonist and said antigen are coupled via a linker, such as a self- immolative linker.
  • a linker such as a self- immolative linker.
  • Embodiment 81 The pharmaceutical composition according to any one of embodiments 57 to 80, wherein said Toll-like receptor 2 agonist is a compound of the formula (IV) :
  • R 33 and R 34 are each independently a branched or straight group having up to 17 atoms selected from C, N, 0 and S, q is from 0 to and including 18, Y 1 is S or Se, X 1 is S or 0 and R 35 is a group comprising said antigen; or a pharmaceutically acceptable salt thereof.
  • Embodiment 82 The pharmaceutical composition according to embodiment 81, wherein X 1 is 0.
  • Embodiment 83 The pharmaceutical composition according to embodiment 81 or 82, wherein Y 1 is S.
  • Embodiment 84 The pharmaceutical composition according to any one of embodiments 81 to 83, wherein R 33 and R 34 are each independently a straight alkyl group having 10 to 17 carbon atoms.
  • Embodiment 85 The pharmaceutical composition according to any one of embodiments 81 to 84, wherein R 33 and R 34 are straight alkyl groups having 15 carbon atoms.
  • Embodiment 86 The pharmaceutical composition according to any one of embodiments 81 to 85, wherein q is from 11 to and including 18, e.g. 11 to and including 15, such as 12 or 14.
  • Embodiment 87 The pharmaceutical composition according to any one of embodiments 81 to 86, wherein R 35 is an organic group comprising a peptide of two or more amino acids, preferably R 35 is Ser(Lys) r , wherein r is 1, 2, 3, 4 or 5.
  • Embodiment 88 The pharmaceutical composition according to embodiment 87, wherein R 35 is Ser(l_ys)4 covalently coupled to said antigen at the C-terminal lysine.
  • Embodiment 89 The pharmaceutical composition according to any one of embodiments 57 to 80, wherein said Toll-like receptor 2 agonist is selected from the group consisting of: Parri 3 Cys, Parri 3 CysSer(Lys)4, Par Cys Ser(l_ys)4, PamiCys Ser(l_ys)4, MALP-2, OspA, lipoteichoic acid, a porin, MALP-404, LcrV protein, lipomannan, a protozoan GPI anchor, zymosan, lysophosphatidylserine, lipophosphoglycan, hsp60, glycoprotein gH/gL or gB of herpes simplex virus, hemagglutinin protein of measles virus.
  • said Toll-like receptor 2 agonist is selected from the group consisting of: Parri 3 Cys, Parri 3 CysSer(Lys)4, Par Cys Ser(l_ys)4, PamiCys
  • Embodiment 90 The pharmaceutical composition according to any one of embodiments 57 to 89, wherein :
  • Embodiment 91 The pharmaceutical composition according to any one of embodiments 57 to 90, wherein each antigen molecule is covalently coupled to said iNKT cell agonist as well as to said Toll-like receptor 2 agonist.
  • Embodiment 92 The pharmaceutical composition according to any one of embodiments 57 to 91, wherein said antigen is a peptide.
  • Embodiment 93 The pharmaceutical composition according to embodiment 92, wherein said peptide is from 8 to 100 amino acids in length, e.g. from 20 to 45 amino acids in length.
  • Embodiment 94 The pharmaceutical composition according to any one of embodiments 57 to 93, wherein the pharmaceutical composition comprises one or more further antigens.
  • Embodiment 95 The pharmaceutical composition according to embodiment 94, wherein said iNKT cell agonist is coupled to a first antigen and said Toll-like receptor 2 agonist is coupled to a second antigen, wherein said first and second antigens are not identical.
  • Embodiment 96 An antigen for use in a method of immunizing a human subject, wherein said antigen is administered in combination with an iNKT cell agonist and a Toll-like receptor 2 agonist.
  • Embodiment 97 The antigen for use according to embodiment 96, wherein the use comprises one or more of the further features as described in embodiments 2 to 56.
  • Embodiment 98 Use of an antigen in the manufacture of a medicament for the immunization of a human subject, wherein said antigen is administered in combination with an iNKT cell agonist and a Toll-like receptor 2 agonist.
  • Embodiment 99 The use of embodiment 98, wherein the use comprises one or more of the further features as described in embodiments 2 to 56.
  • Embodiment 100 A kit-of-parts comprising :
  • Embodiment 101 The kit-of-parts of embodiment 100, wherein the kit-of-parts or its use comprises one or more of the further features as described in embodiments 2 to 56.
  • NKT agonist-SLP conjugate compounds Avalia, NKT cell dependent
  • AMPLIVANT-SLP conjugate compounds ISA Pharmaceuticals, TLR-2 dependent
  • TLR-2 dependent The combination of NKT agonist-SLP conjugate compounds and AMPLIVANT-SLP conjugate compounds gives a more powerful response compared to either component on its own as assessed by increased endogenous T cell priming and anti-tumour activity in two mouse models of cancer.
  • the combination also leads to changes in the T cell phenotype (i.e. increased proportion of CD62L-KLRG1+ cells) that have been reported to predict superior anti- tumour activity.
  • Mouse melanoma cells (B16) transfected with chicken ovalbumin (OVA) were injected s.c. into C57BL/6 mice.
  • the tumour cells also express self-antigens such as tyrosinase-related protein 2 (Trp-2).
  • Trp-2 tyrosinase-related protein 2
  • the immunogenic compounds administered in this case contained a fusion peptide of the self-antigen Trp-2. It is generally more difficult to generate immune responses and anti-tumour activity against a self-antigen compared to transfected foreign antigens.
  • the two compounds were mixed and co-administered via the subcutaneous (s.c.) route at a 2.5 nmol dose (each).
  • a mouse lung epithelial tumour cell line transfected with human papilloma virus (HPV) was injected s.c. into C57BL/6 mice. After the tumours had established and were palpable, the compounds incorporating an HPV SLP were administered (day 8). The two compounds were mixed immediately prior to administration via the subcutaneous (s.c.) route, at a 5 nmol dose for each compound in the combination group and 10 nmol of each compound in the monotherapy group.
  • NKT-agonist-HPV-SLP conjugate constructs containing the FFRK cleavage sequence or not and manufactured using Strain Promoted Alkyne Azide Cycloaddition (SPAAC) or oxime ligation methodologies were synthesised and analysed for their ability to induce an HPV T CD8 cell response.
  • Naive C57BL/6 mice were immunized with NKT-agonist-HPV-SLP conjugates CI078, CI079, CI112, CI113, AMPLIVANT-HPV-SLP conjugates (AV-HPV), or the combinations.
  • the HPV CD8 T cell response in blood was analysed on day 9 for the presence of antigen-specific T cells, using a fluorescent MHCI pentamer.
  • the NKT-agonist-HPV-SLP conjugates alone or in combination with AMPLIVANT- HPV-SLP conjugate induced the greatest proportion of antigen-specific T cells.
  • NKT-agonist-HPV-SLP conjugates regardless of chemistry methodology, induced strong HPV-specific CD8 T cell responses - either alone or in combination with the AMPLIVANT-HPV-SLP conjugate (Figure 4).
  • Figure 4 Experiment overview: When the CD8 T cell population was gated on cell surface markers CD62L and KLRG1, all the NKT-agonist-HPV-SLP conjugates induced a population of CD62L- KLRG1+ cells that was further enhanced in the combination groups ( Figures 5-8). This population has been reported to correlate to increased anti-tumour activity (Van Duikeren et al. 2012 J Immunol 189:3397).
  • NKT-agonist-HPV-SLP conjugates induce strong T cell responses irrespective of the chemistry and their combination with an AMPLIVANT-HPV-SLP conjugate shifts the sub-population further towards a CD62L- KLRG1+ phenotype.
  • CN209 was prepared as described in Example 11 of WO2014088432.
  • Peptide 5- azidopentanoyl-GQAEPDRAHYNIVTFCCKCDS was synthesized using standard SPPS Fmoc/tBu chemistry using appropriate amino acid side-chain protection groups. Synthesis commenced on HMPB ChemMatrix resin pre-loaded with the first (C-terminal) amino acid. Amino acid coupling was achieved by successive addition of the appropriate Fmoc-amino acid (0.2M, 5 eq.), coupling reagent (PyBOP, 0.2M, 5 eq.) and base (/V-methylmorpholine (NMM), 0.8M, 10 eq.). A solution containing 20% piperidine in NMP ( v/v ) was used for Fmoc deprotection.
  • TCEP.HCI 100 mg/ml_, 20 mI_
  • TCEP.HCI 100 mg/ml_, 20 mI_
  • semi-preparative HPLC Phenomenex Luna C18(2), 5 pm, 250 x 21.2 mm, 40 °C, 20 mL/min
  • Mobile phase A 100:0.05 water/TFA
  • Mobile phase B 100:0.0.05 MeOH/TFA
  • HRMS-ESI m/z calcd for C183H294N38O50S3 [M + H + Na] 2+ 1972.0385, found 1972.0354.
  • TCEP.HCI 100 mg/mL, 40 pL
  • TCEP.HCI 100 mg/mL, 40 pL
  • semi-preparative HPLC Phenomenex Luna C18(2), 5 pm, 250 x 21.2 mm, 40 °C, 20 mL/min
  • Mobile phase A 100:0.05 water/TFA
  • Mobile phase B 100:0.0.05 MeOH/TFA
  • HRMS-ESI m/z calcd for C213H337N46O54S3 [M+3H, disulphide] 3+ 1499.8067, found 1499.8099.
  • Compound 1 was prepared as described in Example 10 of WO2014088432.
  • MaGC-PAB-CV-Nonanoyl Ketone (compound 2 described above in section 3.1) (2.0 mg, 1.4 pmol) in THF/MeOH (2: 1, 400 mI_) and anilinium acetate buffer (300 mM, pH 4.8, 200 mI_) was added to peptide 2-(aminooxy)acetyl- FFRKGQAEPDRAHYNIVTFCCKCDS (6.6 mg, 2.2 pmol) under an Ar atmosphere and the reaction mixture was stirred at 50 °C for 6 h.
  • TCEP.HCI 100 mg/ml_, 50 mI_
  • TCEP.HCI 100 mg/ml_, 50 mI_
  • MS-ESI m/z calcd for C178H290N36O50S3 [M+2H] 2+ : 1915.0282, found 1915.0293.
  • the Amplivant moiety consists of five standard amino acids (SKKKK), followed by coupling of Fmoc-(Pam)2Cys-OH which is coupled using 2.5 equivalents of PyBOP and amino acid, and 5 equivalents of NMM. After coupling of Fmoc-(Pam)2Cys-OH, the N- terminal Fmoc-group was removed with 20% piperidine in NMP and a solution of tetradecylisocyanate (150 pl_, 9.1 eq) in 1 ml_ DCM is added to the peptide resin in 2 ml_ NMP and allowed to react for 10 hours.
  • SKKKK standard amino acids
  • the crude peptide conjugate was removed from the resin by treatment with 10 ml_ TFA/TIPS/ET/H2O for 3 hours followed by precipitation in 2x 40 ml_ Et20/Heptane (1 : 1 v/v). The resulting pellet was dissolved in 25%ACN/H20 and lyophilized.
  • Amplivant Trp2 43 mg, 17% overall synthesis yield. Purity: 98.3% by UPLC. ESI-MS calcd C215H 339N43O43S3 : 4310.52, [ M +3H ] 3 + calcd : 1437.84, found 1437.98.
  • mice Breeding pairs of the inbred strain C57BL/6 (CD45.2 + ) were obtained from Jackson Laboratories, Bar Harbor, ME or Envigo, Horst, The Netherlands. All mice were maintained in the Biomedical Research Unit of the Malaghan Institute of Medical Research, Wellington, New Zealand or at the Animal Facility of the Leiden University Medical Center, Leiden, The Netherlands. Experiments were approved by a national Animal Ethics Committee and performed according to established national guidelines. Administration of compounds. Each compound was supplied as an unformulated product, solubilised in DMSO and then water. The stock was diluted in phosphate-buffered saline (PBS) for injection (0-10.0 nmol/mouse) by subcutaneous injection into the tail base.
  • PBS phosphate-buffered saline
  • mice received a subcutaneous injection into the flank of 1 x 10 5 TC-1 lung epithelial cells (which express the HPV16 E6 and E7 proteins that cause cervical cancer) or 5x10 s B16.0VA melanoma cells (which express a cDNA encoding the chicken ovalbumin (OVA) sequence).
  • the different groups were treated 5-9 days later, when tumours were fully engrafted, by subcutaneous injection of the indicated immunogenic compounds or PBS. Mice were monitored for tumour growth every 2-3 days, and were euthanised at the ethical tumour size of 200 mm 2 .

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Abstract

L'invention concerne une méthode d'immunisation d'un sujet humain comprenant l'administration combinée d'un antigène, d'un agoniste des cellules iNKT et d'un agoniste du récepteur 2 de type Toll. L'invention porte en outre sur des compositions et des trousses utilisées dans la méthode de l'invention.
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WO2020231274A1 (fr) * 2019-05-10 2020-11-19 Victoria Link Ltd Vaccin glycopeptidique
CN116606352A (zh) * 2023-07-14 2023-08-18 诺赛联合(北京)生物医学科技有限公司 一种抗菌修复功能的组合物凝胶在妇科疾病中的应用
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020231274A1 (fr) * 2019-05-10 2020-11-19 Victoria Link Ltd Vaccin glycopeptidique
WO2024068636A1 (fr) 2022-09-27 2024-04-04 Isa Pharmaceuticals B.V. Peptides immunogènes à adjuvant pour administration intradermique
CN116606352A (zh) * 2023-07-14 2023-08-18 诺赛联合(北京)生物医学科技有限公司 一种抗菌修复功能的组合物凝胶在妇科疾病中的应用
CN116606352B (zh) * 2023-07-14 2023-09-29 诺赛联合(北京)生物医学科技有限公司 一种抗菌修复功能的组合物凝胶在妇科疾病中的应用

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