WO2023020993A1 - Novel methods - Google Patents

Novel methods Download PDF

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Publication number
WO2023020993A1
WO2023020993A1 PCT/EP2022/072762 EP2022072762W WO2023020993A1 WO 2023020993 A1 WO2023020993 A1 WO 2023020993A1 EP 2022072762 W EP2022072762 W EP 2022072762W WO 2023020993 A1 WO2023020993 A1 WO 2023020993A1
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WO
WIPO (PCT)
Prior art keywords
antigen
subject
sting agonist
administering
adjuvant
Prior art date
Application number
PCT/EP2022/072762
Other languages
French (fr)
Inventor
Arnauld Michel DIDIERLAURENT
Catherine Pascaline Anne Ghislaine Collignon
Cindy GUTZEIT
Margherita COCCIA
Robbert Gerrit Van Der Most
Ronan ROUXEL
Stephane Theophile TEMMERMAN
Original Assignee
Glaxosmithkline Biologicals Sa
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Publication date
Application filed by Glaxosmithkline Biologicals Sa filed Critical Glaxosmithkline Biologicals Sa
Publication of WO2023020993A1 publication Critical patent/WO2023020993A1/en

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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
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/08—Clostridium, e.g. Clostridium tetani
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • 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
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • 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
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • 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
    • C12N2710/10011—Adenoviridae
    • C12N2710/10111—Atadenovirus, e.g. ovine adenovirus D
    • C12N2710/10134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
    • C12N2710/16734—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to methods for improving immune responses with a STING agonist, and to related aspects.
  • Adjuvants are sometimes included in vaccines to improve humoral and cellular immune responses, particularly in the case of poorly immunogenic subunit vaccines. Similar to natural infections by pathogens, adjuvants rely on the activation of the innate immune system to promote long-lasting adaptive immunity. As simultaneous activation of multiple innate immune pathways is a feature of natural infections, adjuvants may combine multiple immunostimulants in order to promote adaptive immune responses to vaccination.
  • STING STimulator of InterferoN Genes
  • PRR pattern-recognition receptor
  • the present invention provides a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
  • a method of administering to a subject a STING agonist and an antigen which is associated with a pathogen wherein the subject was selected for having been previously administered a STING agonist, and optionally wherein the subject’s immune response is of increased level, speed, and/or breadth as compared to that of a subject who had not been previously administered the STING agonist.
  • the present invention also provides a method for preparing the immune system of a subject for (ii) administration of an antigen and a STING agonist, comprising (i) administering to the subject a STING agonist.
  • the present invention provides a STING agonist for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
  • an antigen for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
  • STING agonist for use in a method of reducing the variability of the immune response to an antigen in subjects, wherein the method comprises the following steps:
  • the present invention provides an antigen for use in a method of reducing the variability of the immune response to an antigen in subjects, wherein the method comprises the following steps:
  • STING agonist for use in a method of improving an immune response to an antigen in a subject, wherein the method comprises the following steps:
  • an antigen for use in a method of improving an immune response to an antigen in a subject, wherein the method comprises the following steps: (i) administering to the subject a STING agonist, prior to
  • the present invention also provides a STING agonist for use in a method of reducing the amount of an antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
  • an antigen for use in a method of reducing the amount of an antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
  • a STING agonist for use in a method of preparing the immune system of a subject for (ii) administration of an antigen and a STING agonist, wherein the method comprises (i) administering to the subject a STING agonist.
  • the present invention also provides an antigen for use in a method of preparing the immune system of a subject for administration of an antigen and a STING agonist, wherein the method comprises administering to the subject a STING agonist.
  • the invention provides the use of a STING agonist for the manufacture of a medicament for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
  • an antigen for the manufacture of a medicament for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
  • a STING agonist for the manufacture of a medicament for use in a method of reducing the variability of the immune response to an antigen in subjects, wherein the method comprises the following steps:
  • the present invention provides the use of an antigen for the manufacture of a medicament for use in a method of reducing the variability of the immune response to an antigen in subjects, wherein the method comprises the following steps:
  • a STING agonist in the manufacture of a medicament for use in a method of improving an immune response to an antigen in a subject, wherein the method comprises the following steps:
  • an antigen for the manufacture of a medicament for use in a method of improving an immune response to an antigen in a subject, wherein the method comprises the following steps:
  • the present invention also provides the use of a STING agonist for the manufacture of a medicament for use in a method of reducing the amount of an antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
  • an antigen for the manufacture of a medicament for use in a method of reducing the amount of an antigen required to elicit a desired immune response in a subject comprising the following steps:
  • a STING agonist for the manufacture of a medicament for use in a method of preparing the immune system of a subject for administration of an antigen and a STING agonist, wherein the method comprises administering to the subject a STING agonist.
  • a STING agonist for the manufacture of a medicament for use in a method of preparing the immune system of a subject for administration of an antigen and a STING agonist, wherein the method comprises administering to the subject a STING agonist.
  • the present invention also provides the use of an antigen for the manufacture of a medicament for use in a method of preparing the immune system of a subject for (ii) administration of an antigen and a STING agonist, wherein the method comprises (i) administering to the subject a STING agonist.
  • the present invention provides a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
  • a STING agonist and an antigen which is associated with a pathogen (Antigen A)
  • Antigen B an antigen which is associated with a pathogen
  • Antigen A and Antigen B are different (such as wherein Antigen A and Antigen B are not associated with the same pathogen)
  • the subject’s immune response is of increased level, speed, and/or breadth as compared to that of a subject who had not been previously administered the STING agonist and Antigen B.
  • the present invention also provides a method for preparing the immune system of a subject for (ii) administration of a second antigen and a STING agonist, comprising (i) administering to the subject a STING agonist and a first antigen.
  • the present invention provides a STING agonist for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
  • a first antigen for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
  • a second antigen for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
  • STING agonist for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
  • the present invention also provides a first antigen for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
  • the present invention provides a second antigen for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
  • STING agonist for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
  • first antigen for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
  • a second antigen for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
  • the present invention also provides a STING agonist for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps: (i) administering to the subject a STING agonist and a first antigen, prior to
  • the present invention provides a first antigen for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
  • a second antigen for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
  • a STING agonist for use in a method of preparing the immune system of a subject for (ii) administration of a second antigen and a STING agonist, wherein the method comprises (i) administering to the subject a STING agonist and a first antigen.
  • a first antigen for use in a method of preparing the immune system of a subject for administration of a second antigen and a STING agonist, wherein the method comprises administering to the subject a STING agonist and a first antigen.
  • the present invention also provides a second antigen for use in a method of preparing the immune system of a subject for administration of a second antigen and a STING agonist, wherein the method comprises administering to the subject a STING agonist and a first antigen.
  • the invention provides the use of a STING agonist for the manufacture of a medicament for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
  • a first antigen for the manufacture of a medicament for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
  • a second antigen for the manufacture of a medicament for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps: (i) administering to the subject a STING agonist and a first antigen, prior to
  • a STING agonist for the manufacture of a medicament for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
  • the present invention also provides the use of a first antigen for the manufacture of a medicament for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
  • the present invention provides the use of a second antigen for the manufacture of a medicament for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
  • a STING agonist in the manufacture of a medicament for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
  • a first antigen for the manufacture of a medicament for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
  • a second antigen for the manufacture of a medicament for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
  • the present invention also provides the use of a STING agonist for the manufacture of a medicament for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps: (i) administering to the subject a STING agonist and a first antigen, prior to (ii) administering to the subject the second antigen and a STING agonist.
  • the present invention provides the use of a first antigen for the manufacture of a medicament for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
  • a second antigen for the manufacture of a medicament for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
  • a STING agonist for the manufacture of a medicament for use in a method of preparing the immune system of a subject for administration of a second antigen and a STING agonist, wherein the method comprises administering to the subject a STING agonist and a first antigen.
  • a STING agonist for the manufacture of a medicament for use in a method of preparing the immune system of a subject for administration of a second antigen and a STING agonist, wherein the method comprises administering to the subject a STING agonist and a first antigen.
  • the present invention also provides the use of a second antigen for the manufacture of a medicament for use in a method of preparing the immune system of a subject for (ii) administration of a second antigen and a STING agonist, wherein the method comprises (i) administering to the subject a STING agonist and a first antigen.
  • the present invention provides a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
  • the present invention also provides a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
  • a method of administering to a subject a STING agonist and an antigen which is associated with a pathogen wherein the subject was selected for having been previously administered a STING agonist, and optionally wherein the subject’s immune response is of increased level, speed, and/or breadth as compared to that of a subject who had not been previously administered the STING agonist.
  • a method of reducing the variability of the immune response to an antigen in subjects wherein the method comprises the following steps:
  • a method of reducing the variability of the immune response to an antigen in subjects comprising the following steps:
  • the invention also provides a method of preparing the immune system of a subject for (ii) administration of an antigen, wherein the method comprises (i) administering to the subject a STING agonist.
  • the present invention provides a method for prophylaxis in a subject at risk of infection comprising administering a STING agonist to the subject.
  • the present invention provides a STING agonist for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
  • the present invention also provides a STING agonist for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
  • STING agonist for use in a method of reducing the variability of the immune response to an antigen in subjects, wherein the method comprises the following steps:
  • a STING agonist for use in a method of reducing the variability of the immune response to an antigen in subjects, wherein the method comprises the following steps:
  • STING agonist for use in a method of improving an immune response to an antigen in a subject, wherein the method comprises the following steps:
  • STING agonist for use in a method of improving an immune response to an antigen in a subject, wherein the method comprises the following steps:
  • STING agonist for use in a method of reducing the amount of an antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
  • a STING agonist for use in a method of reducing the amount of an antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
  • the invention also provides a STING agonist for use in a method of preparing the immune system of a subject for (ii) administration of an antigen, wherein the method comprises (i) administering to the subject a STING agonist. Also provided is a STING agonist for use in a method of preparing the immune system of a subject for (ii) administration of an antigen and a second adjuvant, wherein the method comprises (i) administering to the subject a STING agonist.
  • the present invention provides a STING agonist for use in a method for prophylaxis in a subject at risk of infection comprising administering a STING agonist to the subject.
  • the present invention also provides a second adjuvant for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
  • a second adjuvant for use in a method of reducing the variability of the immune response to an antigen in subjects, wherein the method comprises the following steps:
  • a second adjuvant for use in a method of improving an immune response to an antigen in a subject, wherein the method comprises the following steps:
  • a second adjuvant for use in a method of reducing the amount of an antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
  • a second adjuvant for use in a method of preparing the immune system of a subject for (ii) administration of an antigen and a second adjuvant, wherein the method comprises (i) administering to the subject a STING agonist.
  • the present invention provides an antigen for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
  • the present invention also provides an antigen for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
  • an antigen for use in a method of reducing the variability of the immune response to an antigen in subjects, wherein the method comprises the following steps:
  • an antigen for use in a method of reducing the variability of the immune response to an antigen in subjects, wherein the method comprises the following steps:
  • an antigen for use in a method of improving an immune response to an antigen in a subject, wherein the method comprises the following steps:
  • an antigen for use in a method of improving an immune response to an antigen in a subject, wherein the method comprises the following steps:
  • an antigen for use in a method of reducing the amount of an antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
  • an antigen for use in a method of reducing the amount of an antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
  • the invention also provides an antigen for use in a method of preparing the immune system of a subject for administration of an antigen, wherein the method comprises administering to the subject a STING agonist.
  • an antigen for use in a method of preparing the immune system of a subject for administration of an antigen and a second adjuvant, wherein the method comprises administering to the subject a STING agonist.
  • the present invention provides the use of a STING agonist for the manufacture of a medicament for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
  • the present invention also provides the use of a STING agonist for the manufacture of a medicament for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
  • a STING agonist for the manufacture of a medicament for use in a method of reducing the variability of the immune response to an antigen in subjects, wherein the method comprises the following steps:
  • a STING agonist for the manufacture of a medicament for use in a method of reducing the variability of the immune response to an antigen in subjects, wherein the method comprises the following steps:
  • a STING agonist in the manufacture of a medicament for use in a method of improving an immune response to an antigen in a subject, wherein the method comprises the following steps:
  • a STING agonist in the manufacture of a medicament for use in a method of improving an immune response to an antigen in a subject, wherein the method comprises the following steps:
  • a STING agonist for the manufacture of a medicament for use in a method of reducing the amount of an antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
  • a STING agonist for the manufacture of a medicament for use in a method of reducing the amount of an antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
  • the invention also provides the use of a STING agonist for the manufacture of a medicament for use in a method of preparing the immune system of a subject for administration of an antigen, wherein the method comprises administering to the subject a STING agonist.
  • a STING agonist for the manufacture of a medicament for use in a method of preparing the immune system of a subject for administration of an antigen and a second adjuvant, wherein the method comprises administering to the subject a STING agonist.
  • the present invention provides the use of a STING agonist for the manufacture of a medicament for use in a method for prophylaxis in a subject at risk of infection comprising administering a STING agonist to the subject.
  • the present invention also provides the use of a second adjuvant for the manufacture of a medicament for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
  • a second adjuvant for the manufacture of a medicament for use in a method of reducing the variability of the immune response to an antigen in subjects, wherein the method comprises the following steps:
  • a second adjuvant for the manufacture of a medicament for use in a method of reducing the amount of an antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
  • a second adjuvant for the manufacture of a medicament for use in a method of preparing the immune system of a subject for administration of an antigen and the second adjuvant, wherein the method comprises administering to the subject a STING agonist.
  • the invention provides the use of an antigen for the manufacture of a medicament for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
  • the present invention also provides the use of an antigen for the manufacture of a medicament for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
  • an antigen for the manufacture of a medicament for use in a method of reducing the variability of the immune response to an antigen in subjects, wherein the method comprises the following steps:
  • an antigen for the manufacture of a medicament for use in a method of reducing the variability of the immune response to an antigen in subjects, wherein the method comprises the following steps:
  • an antigen for the manufacture of a medicament for use in a method of improving an immune response to an antigen in a subject, wherein the method comprises the following steps:
  • an antigen for the manufacture of a medicament for use in a method of improving an immune response to an antigen in a subject, wherein the method comprises the following steps:
  • an antigen for the manufacture of a medicament for use in a method of reducing the amount of an antigen required to elicit a desired immune response in a subject comprising the following steps:
  • an antigen for the manufacture of a medicament for use in a method of reducing the amount of an antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
  • the invention also provides the use of an antigen for the manufacture of a medicament for use in a method of preparing the immune system of a subject for (ii) administration of an antigen, wherein the method comprises (i) administering to the subject a STING agonist.
  • the present invention provides a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
  • the present invention also provides a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
  • a STING agonist and an antigen which is associated with a pathogen (Antigen A)
  • Antigen B an antigen which is associated with a pathogen
  • Antigen A and Antigen B are different (such as wherein Antigen A and Antigen B are not associated with the same pathogen)
  • the subject’s immune response is of increased level, speed, and/or breadth as compared to that of a subject who had not been previously administered the STING agonist and Antigen B.
  • a method of reducing the variability of the immune response to a second antigen in subjects comprising the following steps:
  • the invention also provides a method of preparing the immune system of a subject for (ii) administration of a second antigen, wherein the method comprises (i) administering to the subject a STING agonist and a first antigen.
  • the present invention provides a method for prophylaxis in a subject at risk of infection comprising administering a STING agonist and a first antigen to the subject.
  • the present invention provides a STING agonist for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
  • the present invention also provides a STING agonist for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
  • STING agonist for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
  • a STING agonist for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
  • STING agonist for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
  • STING agonist for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
  • STING agonist for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
  • a STING agonist for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
  • the invention also provides a STING agonist for use in a method of preparing the immune system of a subject for (ii) administration of a second antigen, wherein the method comprises (i) administering to the subject a STING agonist and a first antigen.
  • a STING agonist for use in a method of preparing the immune system of a subject for (ii) administration of a second antigen and a second adjuvant, wherein the method comprises (i) administering to the subject a STING agonist and a first antigen.
  • the present invention provides a STING agonist or a first antigen for use in a method for prophylaxis in a subject at risk of infection comprising administering a STING agonist and a first antigen to the subject.
  • the present invention provides a first antigen for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
  • the present invention also provides a first antigen for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
  • a first antigen for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
  • a first antigen for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
  • first antigen for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
  • a first antigen for use in a method of improving an immune response to a second antigen in a subject comprising the following steps:
  • first antigen for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
  • a first antigen for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
  • the invention also provides a first antigen for use in a method of preparing the immune system of a subject for administration of a second antigen, comprising administering to the subject a STING agonist and a first antigen. Also provided is a first antigen for use in a method of preparing the immune system of a subject for administration of a second antigen and a second adjuvant, comprising administering to the subject a STING agonist and a first antigen.
  • the present invention also provides a second adjuvant for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
  • a second adjuvant for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
  • a second adjuvant for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
  • a second adjuvant for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
  • a second adjuvant for use in a method of preparing the immune system of a subject for (ii) administration of a second antigen and a second adjuvant, wherein the method comprises (i) administering to the subject a STING agonist and a first antigen.
  • the present invention provides a second antigen for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
  • the present invention also provides a second antigen for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
  • a second antigen for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
  • a second antigen for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
  • a second antigen for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
  • a second antigen for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
  • a second antigen for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
  • the invention also provides a second antigen for use in a method of preparing the immune system of a subject for administration of a second antigen, wherein the method comprises administering to the subject a STING agonist and a first antigen.
  • a second antigen for use in a method of preparing the immune system of a subject for administration of a second antigen and a second adjuvant, wherein the method comprises administering to the subject a STING agonist and a first antigen.
  • the present invention provides the use of a STING agonist for the manufacture of a medicament for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps: (i) administering to the subject a STING agonist and a first antigen, prior to
  • the present invention also provides the use of a STING agonist for the manufacture of a medicament for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
  • a STING agonist for the manufacture of a medicament for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
  • a STING agonist for the manufacture of a medicament for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
  • a STING agonist in the manufacture of a medicament for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
  • a STING agonist in the manufacture of a medicament for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
  • a STING agonist for the manufacture of a medicament for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
  • a STING agonist for the manufacture of a medicament for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps: (i) administering to the subject a STING agonist and a first antigen, prior to (ii) administering to the subject the second antigen and a second adjuvant.
  • the invention also provides the use of a STING agonist for the manufacture of a medicament for use in a method of preparing the immune system of a subject for administration of a second antigen, wherein the method comprises administering to the subject a STING agonist and a first antigen.
  • a STING agonist for the manufacture of a medicament for use in a method of preparing the immune system of a subject for administration of a second antigen and a second adjuvant, wherein the method comprises administering to the subject a STING agonist and a first antigen.
  • the present invention provides the use of a STING agonist or a first antigen for the manufacture of a medicament for use in a method for prophylaxis in a subject at risk of infection comprising administering a STING agonist and a first antigen to the subject.
  • the present invention provides the use of a first antigen for the manufacture of a medicament for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
  • the present invention also provides the use of a first antigen for the manufacture of a medicament for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
  • a first antigen for the manufacture of a medicament for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
  • a first antigen for the manufacture of a medicament for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
  • a first antigen for the manufacture of a medicament for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
  • a first antigen for the manufacture of a medicament for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
  • a first antigen for the manufacture of a medicament for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
  • a first antigen for the manufacture of a medicament for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject comprising the following steps:
  • the invention also provides the use of a first antigen for the manufacture of a medicament for use in a method of preparing the immune system of a subject for administration of a second antigen, wherein the method comprises administering to the subject a STING agonist and a first antigen.
  • the invention also provides the use of a first antigen for the manufacture of a medicament for use in a method of preparing the immune system of a subject for administration of a second antigen and a second adjuvant, wherein the method comprises administering to the subject a STING agonist and a first antigen.
  • the present invention also provides the use of a second adjuvant for the manufacture of a medicament for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
  • a second adjuvant for the manufacture of a medicament for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
  • a second adjuvant for the manufacture of a medicament for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
  • a second adjuvant for the manufacture of a medicament for use in a method of preparing the immune system of a subject for administration of a second antigen and a second adjuvant, wherein the method comprises administering to the subject a STING agonist and a first antigen.
  • the invention provides the use of a second antigen for the manufacture of a medicament for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
  • the present invention also provides the use of a second antigen for the manufacture of a medicament for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
  • a second antigen for the manufacture of a medicament for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
  • a second antigen for the manufacture of a medicament for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
  • a second antigen for the manufacture of a medicament for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
  • a second antigen for the manufacture of a medicament for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
  • a second antigen for the manufacture of a medicament for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
  • the invention also provides the use of a second antigen for the manufacture of a medicament for use in a method of preparing the immune system of a subject for (ii) administration of a second antigen, wherein the method comprises (i) administering to the subject a STING agonist and a first antigen.
  • SEQ ID NO: 1 A polypeptide sequence of varicella-zoster virus (VZV)-gE antigen
  • Toxin B neutralising antibody titre at 15 Post III following administration of VZV- gE, STING agonist and aluminium hydroxide before administration of F2 with an adjuvant comprising a saponin.
  • FIG. 2 Toxin B specific IgG at 15 Post III following administration of VZV-gE, STING agonist and aluminium hydroxide before administration of F2 with an adjuvant comprising a saponin.
  • Fig. 3 Toxin A neutralising antibody titre at 15 Post III following administration of VZV- gE, STING agonist and aluminium hydroxide before administration of F2 with an adjuvant comprising a saponin.
  • Fig. 4 Toxin A specific IgG at 15 Post III following administration of VZV-gE, STING agonist and aluminium hydroxide before administration of F2 with an adjuvant comprising a saponin.
  • Fig. 5 Percentage of F2-specific CD4+ T-cells expressing at least 2 cytokines among IL-2, IFN-gamma and TNF-alpha 15 Post III following administration of VZV-gE, STING agonist and aluminium hydroxide before administration of F2 with an adjuvant comprising a saponin.
  • Fig. 8 Toxin A specific IgG at 15 Post II following administration of VZV-gE, STING agonist and aluminium hydroxide before administration of F2 with an adjuvant comprising a saponin.
  • Toxin A specific IgG at 14 day Pll following administration of STING agonist and aluminium hydroxide or VZV-gE with STING agonist and aluminium hydroxide before administration of F2 with an adjuvant comprising a saponin.
  • Toxin A specific IgG at 14 day Pll following administration of STING agonist or VZV-gE with STING agonist before administration of F2 with an adjuvant comprising a saponin.
  • Fig. 14 Toxin A neutralising antibody titre at 14 day Pll following administration of STING agonist and aluminium hydroxide or VZV-gE with STING agonist and aluminium hydroxide before administration of F2 with an adjuvant comprising a saponin.
  • Fig. 15 Toxin A neutralising antibody titre at 14 day PH following administration of STING agonist or VZV-gE with STING agonist before administration of F2 with an adjuvant comprising a saponin.
  • Toxin B neutralising antibody titre at 14 day PHI following administration of STING agonist and aluminium hydroxide or VZV-gE with STING agonist and aluminium hydroxide before administration of F2 with an adjuvant comprising a saponin.
  • Toxin B neutralising antibody titre at 14 day Pill following administration of STING agonist or VZV-gE with STING agonist before administration of F2 with an adjuvant comprising a saponin.
  • Toxin A neutralising antibody titre at 14 day Pill following administration of STING agonist or VZV-gE with STING agonist before administration of F2 with an adjuvant comprising a saponin.
  • Toxin A specific IgG at 14 day PH following administration of STING agonist and aluminium hydroxide or VZV-gE with STING agonist and aluminium hydroxide before administration of F2 with a squalene emulsion adjuvant.
  • Fig. 25 Toxin A specific IgG at 14 day Pill following administration of STING agonist and aluminium hydroxide or VZV-gE with STING agonist and aluminium hydroxide before administration of F2 with a squalene emulsion adjuvant.
  • Fig. 26 Toxin A neutralising antibody titre at 14 day PH following administration of STING agonist or VZV-gE with STING agonist before administration of F2 with a squalene emulsion adjuvant.
  • Toxin A neutralising antibody titre at 14 day PH following administration of STING agonist and aluminium hydroxide or VZV-gE with STING agonist and aluminium hydroxide before administration of F2 with a squalene emulsion adjuvant.
  • Fig. 30 Toxin B neutralising antibody titre at 14 day Pill following administration of STING agonist and aluminium hydroxide or VZV-gE with STING agonist and aluminium hydroxide before administration of F2 with a squalene emulsion adjuvant.
  • Toxin B neutralising antibody titre at 14 day Pill following administration of STING agonist or VZV-gE with STING agonist before administration of F2 with a squalene emulsion adjuvant.
  • Fig. 32 Toxin A neutralising antibody titre at 14 day Pill following administration of STING agonist and aluminium hydroxide or VZV-gE with STING agonist and aluminium hydroxide before administration of F2 with a squalene emulsion adjuvant.
  • Fig. 33 Toxin A neutralising antibody titre at 14 day PHI following administration of STING agonist or VZV-gE with STING agonist before administration of F2 with a squalene emulsion adjuvant.
  • Fig. 34 Toxin B neutralising antibody titre at 14 day Pill following administration of STING agonist and aluminium hydroxide or VZV-gE with STING agonist and aluminium hydroxide before administration of F2 with STING agonist and aluminium hydroxide.
  • Toxin A neutralising antibody titre at 14 day PHI following administration of STING agonist and aluminium hydroxide or VZV-gE with STING agonist and aluminium hydroxide before administration of F2 with STING agonist and aluminium hydroxide.
  • Toxin A neutralising antibody titre at 14 day Pll following administration of STING agonist and aluminium hydroxide or VZV-gE with STING agonist and aluminium hydroxide before administration of F2 with STING agonist and aluminium hydroxide.
  • Fig. 37 Toxin B specific IgG at 14 day Pill following administration of STING agonist and aluminium hydroxide or VZV-gE with STING agonist and aluminium hydroxide before administration of F2 with STING agonist and aluminium hydroxide.
  • Fig. 38 Toxin A specific IgG at 14 day PH following administration of STING agonist and aluminium hydroxide or VZV-gE with STING agonist and aluminium hydroxide before administration of F2 with STING agonist and aluminium hydroxide.
  • Administration of a STING agonist may be of benefit in respect of subsequent administration of an antigen and a STING agonist or in respect of prophylaxis in a subject at risk of infection.
  • the present invention provides a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
  • the invention also provides a method of preparing the immune system of a subject for (ii) administration of an antigen and a STING agonist, wherein the method comprises (i) administering to the subject a STING agonist.
  • Administration of an antigen and a STING agonist may be of benefit in respect of subsequent administration of an antigen and a STING agonist (‘a second adjuvant’).
  • the present invention provides a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
  • the invention also provides a method of preparing the immune system of a subject for (ii) administration of a second antigen and a STING agonist, wherein the method comprises (i) administering to the subject a STING agonist and a first antigen.
  • Administration of an antigen and a STING agonist may be of benefit in respect of subsequent administration of an antigen or in respect of prophylaxis in a subject at risk of infection.
  • the present invention provides a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
  • the present invention also provides a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
  • a method of reducing the variability of the immune response to an antigen in subjects comprising the following steps:
  • the invention also provides a method of preparing the immune system of a subject for (ii) administration of an antigen, wherein the method comprises (i) administering to the subject a STING agonist.
  • the present invention provides a method for prophylaxis in a subject at risk of infection comprising administering a STING agonist to the subject.
  • Administration of an antigen and a STING agonist may be of benefit in respect of subsequent administration of an antigen or in respect of prophylaxis in a subject at risk of infection.
  • the present invention provides a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
  • the present invention also provides a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
  • a method of reducing the variability of the immune response to a second antigen in subjects comprising the following steps:
  • the invention also provides a method of preparing the immune system of a subject for (ii) administration of a second antigen, wherein the method comprises (i) administering to the subject a STING agonist and a first antigen.
  • the present invention provides a method for prophylaxis in a subject at risk of infection comprising administering a STING agonist and a first antigen to the subject.
  • the invention involves the administration of a STING agonist.
  • the STING agonist may be any appropriate agonist that is capable of binding to and activating STING receptor and STING signalling.
  • the STING agonist binds to STING with an in vitro Kf of less than about 0.750 pM (e.g., less than about 0.500 pM, less than about 0.250 pM or less than about 0.100 pM).
  • the STING agonist activates STING with an in vitro ECso of about 100 pM or less (e.g., about 50 pM or less, about 20 pM or less, or about 10 pM or less) when measured by monitoring phosphorylation of interferon regulatory factor-3 (I RF3).
  • I RF3 interferon regulatory factor-3
  • the STING agonist activates STING with an in vitro ECso of about 100 pM or less (e.g., about 50 pM or less, about 20 pM or less, or about 10 pM or less) as measured by monitoring interferon-p induction.
  • the STING agonist is a nucleic acid, a protein, a peptide, or a small molecule. In one embodiment, the STING agonist is a small molecule selected from a modified or unmodified cyclic dinucleotide. In one embodiment, the cyclic dinucleotide is selected from a compound of Formulae (l)-(lll):
  • R 1 and R 2 are each independently selected from the following groups:
  • R 3 and R 4 are each independently -SH or -OH, R 5 and R 6 are oxygen or sulphur,
  • R 7 and R 8 are each independently halogen, hydrogen, -OH, or OCH3.
  • the STING agonist is the compound:
  • the cyclic dinucleotide is selected from c-di tz GMP, c-di- th GMP, c- G tz GMP, c-GAMP, c- th GMP, c- tz GMP, c-di- th AMP, c-di tz AMP, c-di-AMP, c-di-GMP, c-diXGMP, c-G th XMP, c-GXMP, c-ACMP, c-A th XMP, c-A tz XMP, c-di- th XMP, or c-di tz XMP (as defined in paragraph [0053] of US 2021/0101924).
  • the STING agonist is 2’,3’-cGAMP. In one embodiment, the STING agonist is 3’,3’-cGAMP. In one embodiment, the STING agonist is the compound:
  • the STING agonist is the compound 6-bromo-N-(naphthalen-1- yl)benzo[d][1 ,3]dioxole-5-carboxamide:
  • the STING agonist is a compound of Formula A: wherein
  • X is O or NR 4A ,
  • Y is O, NR 4A , CH2, or absent, n is 0, 1 , 2, or 3,
  • R 1 and R 2 are independently selected from OH, OR 3 , OR 3A , SR 3 , and NR 3 R 4 , R 3 , R 4 , and R 4A are independently selected from hydrogen, C1 -C10 alkyl optionally substituted with 1-6 halogen, C6-C10 aryl or 5-10 membered heteroaryl, or R 3 and R 4 together with the nitrogen atom to which they are attached form a 3 to 7 membered heterocycle or 5 to 10 membered heteroaryl, represents the point of connection of R 3A to the remainder of the molecule, R 5 -R 10 are independently selected from hydrogen, halogen, pseudohalogen, C1-C10 alkyl optionally substituted with 1-6 halogens, C6-C10 aryl, and 5 to 10 membered heteroaryl.
  • the STING agonist is selected from:
  • the STING agonist is a flavonoid.
  • Suitable flavonoids include, but are not limited to, 10-(carboxymethyl)-9(10H)acridone (CMA), 5,6-Dimethylxanthenone-4- acetic acid (DMXAA), methoxyvone, 6,4'-dimethoxyflavone, 4'-methoxyflavone, 3', 6'- dihydroxyflavone, 7,2'-dihydroxyflavone, daidzein, formononetin, retusin 7-methyl ether, xanthone, or any combination thereof.
  • the STING agonist can be 10- (carboxymethyl)-9(10H)acridone (CMA).
  • the STING agonist can be 5,6- Dimethylxanthenone-4-acetic acid (DMXAA). In one aspect, the STING agonist can be methoxyvone. In one aspect, the STING agonist can be 6,4'-dimethoxyflavone. In one aspect, the STING agonist can be 4'-methoxyflavone. In one aspect, the STING agonist can be 3', 6'- dihydroxyflavone. In one aspect, the STING agonist can be 7,2'-dihydroxyflavone. In one aspect, the STING agonist can be daidzein. In one aspect, the STING agonist can be formononetin. In one aspect, the STING agonist can be retusin 7-methyl ether. In one aspect, the STING agonist can be xanthone. In one aspect, the STING agonist can be any combination of the above flavonoids.
  • DMXAA Dimethylxanthenone-4-acetic acid
  • the STING agonist is a compound of Formula B: represents two conjugated double bonds in a five-membered heteroaromatic ring and three conjugated double bonds in a six-membered aromatic or heteroaromatic ring, W 1 is selected from CR 11 and N;
  • X 1 is selected from CR 1 , C(R 1 ) 2 , N, NR 1 , O and S;
  • X 2 is selected from CR 2 , C(R 2 ) 2 , N, NR 2 , O and S;
  • X 3 is selected from CR 3 , C(R 3 ) 2 , N, NR 3 , O and S; where two or three of X 1 , X 2 and X 3 are independently selected from N, NR 1 ,
  • NR 2 , NR 3 , O and S where at least one of X 1 , X 2 and X 3 is selected from N, NR 1 , NR 2 and NR 3 ;
  • Y 1 is selected from N, NR 4 , O, S, CR 4 and C(R 4 ) 2 ;
  • Y 2 is selected from N, NR 5 , O, S, CR 5 and C(R 5 ) 2 ;
  • Y 3 is selected from N, NR 6 , O, S, CR 6 and C(R 6 ) 2 ;
  • Y 4 is selected from C and N;
  • Y 5 is selected from C and N; where at least one and not more than two of Y 1 , Y 2 and Y 3 are independently selected from N, NR 4 , NR 5 and NR 6 ; where when if one of Y 4 or Y 5 is N, the other one of Y 4 or Y 5 is C;
  • Z 1 is selected from C and N;
  • Z 2 is selected from N, NR 8 and CR 8 ;
  • Z 3 is selected from N, NR 9 and CR 9 ;
  • Z 4 is selected from N, NR 10 and CR 10 ;
  • Z 5 is selected from N, NR 7 and CR 7 ; where two or three of Z 1 , Z 2 , Z 3 , Z 4 and Z 5 are independently selected from N, NR 7 , NR 8 , NR 9 , and NR 10 ; each R 1 is independently selected from the group consisting of H, Ci -Cs alkyl, Ci-Cs alkylene-NRR and Ci-Cs alkylene-C(O)OR; each R 2 is independently selected from the group consisting of H, Ci-Cs alkyl, Ci-Cs alkylene-NRR, Ci-Cs alkylene-C(O)OR, Ci-Cs alkylene-OR and Ci-Cs alkylene-O-P(O)(OH) 2 ; each R3 is independently selected from the group consisting of H, Ci-Cs alkyl, Ci-Cs alkylene-NRR, Ci-Cs alkylene-C(O)OR and Ci-Cs alkylene-O-P(O)(OH)
  • the STING agonist is a compound selected from: In one embodiment, the STING agonist is a compound of Formula C: wherein G1 is independently selected from Ring A or ring A is independently selected from optionally substituted heterocyclyl and optionally substituted heteroaryl, ring B is aromatic carbocyclic ring, ring C is optionally substituted five-membered heteroaryl, R 1 is -CON(R 3 )2, R 2 is independently selected from hydrogen, optionally substituted C1-C6 alkyl, and optionally substituted C3-C5 monocyclic cycloalkyl, R 3 is independently selected from hydrogen, and optionally substituted C 1 -C 6 alkyl; m is selected from 0, or 1; n is selected from 0, 1, or 2; o is 1; p is selected from 0, 1, or 2; when 'alkyl's substituted, it is substituted with 1 to 4 substituents independently selected from halogen, alkyl, perhaloalkyl, cycloalkyl, hetero
  • each R 4 is independently selected from hydrogen, alkyl, and cycloalkyl; and each R 4a is independently selected from alkyl, and cycloalkyl.
  • the STING agonist is selected from:
  • the STING agonist is a STING agonist as disclosed in WO 2017/175147 (pages 7 to 92).
  • the STING agonist is a compound according to Formula (l-N):
  • R A1 and R A2 are each independently H, halogen, hydroxy, -O-P(O)(OH)2, -O-P(O)(R I R II )2, -N(R e )(R f ), -CO2R f , -N(R f )COR b , -N(R g )SO2(C1-C4alkyl)-N(R e )(R f ), -N(R g )CO(C1-C4alkyl)-N(R h )(R f ), optionally substituted (C1-C6alkyl), optionally substituted (C1-C6alkyl)oxy-, optionally substituted (C1-C6alkyl)amino-, and optionally
  • the STING compound is selected from: (E)-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1H- benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-7- (3-hydroxypropoxy)-1H-benzo[d]imidazole-5-carboxamide (Example 10 of WO (E)-1-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-2,3- dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-5- carbonyl)imino)-7
  • the STING agonist is 3-(((Z)-6-Carbamoyl-3-((E)-4-((Z)-5- carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1 H- benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-2,3- dihydro-1 H-benzo[d]imidazol-4-yl)oxy)propyl dihydrogen phosphate
  • the STING agonist is (E)-3-((5-carbamoyl-1-(4-(5-carbamoyl-2-(1- ethyl-3-methyl-1 H-pyrazole-5-carboxamido)-7-methoxy-1 H-benzo[d]imidazol-1-yl)but-2-en-1- yl)-2-(1-ethyl-3-methyl-1 H-pyrazole-5-carboxamido)-1 H-benzo[d]imidazol-7-yl)oxy)propyl
  • the STING agonist is by 3-(((E)-6-carbamoyl-3-((E)-4-((E)-5- carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1 H- benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-2,3- dihydro-1 H-benzo[d]imidazol-4-yl)oxy)propyl
  • Compounds may also contain ionisable groups and therefore may be presented as a pharmaceutically acceptable salt.
  • pharmaceutically acceptable salts refer to derivatives of the disclosed compounds wherein the parent compound is modified by making pharmaceutically acceptable acid or base salts thereof.
  • pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic groups such as amines; and alkali or organic salts of acidic groups such as carboxylic acids.
  • the pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.
  • such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, and nitric; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2- acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, and isethionic, and the like.
  • inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, and nitric
  • organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic
  • phrases "pharmaceutically acceptable salt” is employed herein to refer to those salts which are, within the scope of sound medical judgment, suitable for use in a pharmaceutical context, without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • STING agonists with ionisable groups may be in dissociated form.
  • Pharmaceutically acceptable salts of STING agonists may be dissociated in solution.
  • the STING agonist may be in the form of a solvate, such as a hydrate.
  • a typical human dose of STING agonist may be 0.1 to 150 ug, especially 0.5 to 100 ug, such as 1 to 50 ug.
  • More than one STING agonist may be utilised, such as two. Typically only one STING agonist is utilised.
  • a STING agonist may be administered with an aluminium salt, such as AIPO4, AI(OH)3, AIO(OH), or a mixture thereof. More suitably the aluminium salt comprises, consists essentially of, or more suitably consists of, AI(OH)3.
  • the STING agonist may be combined with the aluminium salt in a single formulation and suitably the STING agonist is adsorbed to the aluminium salt.
  • the STING agonist is 3-(((E)-6-carbamoyl-3-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3- methyl-1 H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1 H-benzo[d]imidazol-1-yl)but-2- en-1-yl)-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1 H-benzo[d]imidazol-
  • a STING agonist is administered in step (i) and a second adjuvant is administered in step (ii).
  • the second adjuvant may be an adjuvant comprising a saponin.
  • a suitable saponin for use in the present invention is Quil A and its derivatives.
  • Quil A is a saponin preparation isolated from the South American tree Quillaja saponaria Molina and was first described as having adjuvant activity by Dalsgaard et al. in 1974 (“Saponin adjuvants”, Archiv. fur dieumble Virusforschung, Vol. 44, Springer Verlag, Berlin, p243- 254). Purified fractions of Quil A have been isolated by HPLC which retain adjuvant activity without the toxicity associated with Quil A (see, for example, EP0362278). Fractions of general interest include QS-7, QS-17, QS-18 and QS-21 , for example QS-7 and QS-21 (also known as QA7 and QA21). QS-21 is a saponin fraction of particular interest.
  • the saponin is a derivative of Quillaja saponaria Molina quil A, suitably an immunologically active fraction of Quil A, such as QS-7, QS-17, QS-18 or QS-21 , in particular QS-21.
  • WQ2019/106192 incorporated herein by reference for the purpose of defining saponin fractions which may be of use in the present invention, describes QS-21 fractions of particular interest.
  • the saponin, such as Quil A and in particular QS-21 is at least 90% pure, such as at least 95% pure, especially at least 98% pure, in particular 99% pure w/w.
  • QS-21 contains a plurality of components, with the principal components typically being: ‘QS-21 1988 A component’, which is identified in Kite 2004 as Peak 88 and corresponds to the A-isomer xylose chemotype structures S4 (apiose isomer) and S6
  • the QS-21 1988 A component may consist of QS-21 1988 A V1 (i.e. apiose isomer): - ‘QS-21 1856 A component’, which is identified in Kite 2004 as Peak 86 and corresponds to the A-isomer xylose chemotype structure S2 characterised in Nyberg 2000 and Nyberg 2003.
  • the QS-21 1856 A component may consist of:
  • QS-21 2002 A component which is identified in Kite 2004 as Peak 85 and corresponds to the A-isomer rhamnose chemotype of structures S3 and S5 characterised in Nyberg 2000 and Nyberg 2003.
  • the QS-21 2002 A component may consist of QS-21 2002 A V1 (i.e. apiose isomer): and QS-21 2002 A V2 (i.e. xylose isomer):
  • the saponin desirably comprises at least 40%, such as at least 50%, suitably at least 60%, especially at least 70% and desirably at least 80%, for example at least 90% (as determined by UV absorbance at 214 nm and by relative ion abundance) QS-21 1988 A component, QS-21 1856 A component and/or QS-21 2002 A component.
  • the saponin contains at least 40%, such as at least 50%, in particular at least 60%, especially at least 65%, such as at least 70%, QS-21 1988 A component as determined by UV absorbance at 214 nm and by relative ion abundance.
  • the saponin extracts contain 90% or less, such as 85% or less, or 80% or less, QS-21 1988 A component as determined by UV absorbance at 214 nm and by relative ion abundance.
  • the saponin extracts contain from 40% to 90% QS-21 1988 A component, such as 50% to 85% QS-21 1988 A component, especially 70% to 80% QS-21 1988 A component as determined by UV absorbance at 214 nm and by relative ion abundance.
  • the saponin extracts contain 30% or less, such as 25% or less, QS-21 1856 A as determined by UV absorbance at 214 nm and by relative ion abundance. In certain embodiments the saponin extracts contain at least 5%, such as at least 10% QS-21 1856 A by UV absorbance at 214 nm and by relative ion abundance. In certain embodiments, the saponin extracts contain from 5% to 30% QS-21 1856 A, such as 10% to 25% QS-21 1856 A as determined by UV absorbance at 214 nm and by relative ion abundance.
  • the saponin extracts contain 40% or less, such as 30% or less, in particular 20% or less, especially 10% or less QS-21 2002 A component by UV absorbance at 214 nm and by relative ion abundance. In certain embodiments, the saponin extracts contain at least 0.5%, such as at least 1%, QS-21 2002 A component by UV absorbance at 214 nm and by relative ion abundance. In certain embodiments, the saponin extracts contain from 0.5% to 40% QS-21 2002 A component, such as 1% to 10% QS-21 2002 A component as determined by UV absorbance at 214 nm and by relative ion abundance.
  • UV absorbance at 214 nm and relative ion abundance is meant an estimate for the percentage of a given m/z for co-eluting species.
  • QS-21 1988 A component, QS-21 1856 A component and/or QS-21 2002 A component may be obtained by extraction from Quillaja species or may be prepared synthetically (such as semi-synthetically).
  • TLR agonists may be obtained by extraction from Quillaja species or may be prepared synthetically (such as semi-synthetically).
  • TLR Toll-like receptor
  • TLRs Toll-like receptors
  • PAMPs pathogen associated molecular patterns
  • TLRs that are expressed on the surface of cells include TLR1, TLR2, TLR4, TLR5, and TLR6, while TLR3, TLR7/8, and TLR9 are expressed with the ER compartment.
  • Human dendritic cell (DC) subsets can be identified on the basis of distinct TLR expression patterns.
  • the myeloid or "conventional" subset of DC (mDC) expresses TLRs 1-8 when stimulated, and a cascade of activation markers (e.g. CD80, CD86, MHC class I and II, CCR7), pro-inflammatory cytokines, and chemokines are produced.
  • a cascade of activation markers e.g. CD80, CD86, MHC class I and II, CCR7
  • DCs acquire an enhanced capacity to take up antigens and present them in an appropriate form to T cells.
  • pDC plasmacytoid subset of DC expresses only TLR7 and TLR9 upon activation, with a resulting activation of NK cells as well as T-cells.
  • TLR agonists known in the art include: Pam3Cys, a TLR1/2 agonist; CFA, a TLR2 agonist; MALP2, a TLR2 agonist; Pam2Cys, a TLR2 agonist; FSL-I, a TLR-2 agonist; Hib- OMPC, a TLR-2 agonist; polyinosinic: polycytidylic acid (Poly I :C), a TLR3 agonist; polyadenosine-polyuridylic acid (poly AU), a TLR3 agonist; polyinosinic-polycytidylic acid stabilized with poly-L-lysine and carboxymethylcellulose (Hiltonol), a TLR3 agonist; bacterial flagellin a TLR5 agonist; imiquimod, a TLR7 agonist; resiquimod, a TLR7/8 agonist; loxoribine, a TLR7/8 agonist; and unmethylated Cp
  • Exemplary agonists of TLR7 and/or TLR8 include imiquimod and resiquimod.
  • Other agonists of TLR7 and/or TLR8 include:
  • TLR4 agonist is a lipopolysaccharide, suitably a non-toxic derivative of lipid A, particularly a monophosphoryl lipid A and more particularly 3-de-O- acylated monophosphoryl lipid A (3D-MPL).
  • lipid A particularly a monophosphoryl lipid A and more particularly 3-de-O- acylated monophosphoryl lipid A (3D-MPL).
  • 3D-MPL is sold under the name ‘MPL’ by GlaxoSmithKline Biologicals N.A. and is referred throughout the document as 3D-MPL. See, for example, US Patent Nos. 4,436,727; 4,877,611 ; 4,866,034 and 4,912,094.
  • 3D-MPL can be produced according to the methods described in GB 2 220211 A. Chemically it is a mixture of 3-deacylated monophosphoryl lipid A with 4, 5 or 6 acylated chains.
  • small particle 3D-MPL may be used to prepare the aqueous adjuvant composition.
  • Small particle 3D-MPL has a particle size such that it may be sterile-filtered through a 0.22 urn filter. Such preparations are described in WO94/21292.
  • powdered 3D-MPL is used to prepare aqueous adjuvant compositions of use in the present invention.
  • TLR4 agonists which can be used are aminoalkyl glucosaminide phosphates (AGPs) such as those described in WO98/50399 or US patent No. 6,303,347 (processes for preparation of AGPs are also described). Some AGPs are TLR4 agonists, and some are TLR4 antagonists.
  • AGP of interest is set forth as follows:
  • TLR4 agonists which may be of use in the present invention include Glucopyranosyl Lipid Adjuvant (GLA) such as described in W02008/153541 or WO2009/143457 or the literature articles Coler RN et al. (2011) Development and Characterization of Synthetic Glucopyranosyl Lipid Adjuvant System as a Vaccine Adjuvant. PLoS ONE 6(1): e16333. doi:10.1371/journal.pone.0016333 and Arias MA et al. (2012) Glucopyranosyl Lipid Adjuvant (GLA), a Synthetic TLR4 Agonist, Promotes Potent Systemic and Mucosal Responses to Intranasal Immunization with HIVgp140.
  • GLA Glucopyranosyl Lipid Adjuvant
  • TLR4 agonists of interest include:
  • TLR4 agonist of interest is: deacyl monophosphoryl lipid A.
  • TLR agonist of interest is dLOS (as described in Han, 2014): outer core OS inner core OS Lipid A
  • the TLR4 agonist such as the lipopolysaccharide, such as a monophosphoryl lipid A and in particular 3D-MPL, is at least 90% pure, such as at least 95% pure, especially at least 98% pure, in particular 99% pure w/w.
  • TLR4 agonists may be utilised in combination.
  • Adjuvants comprising a saponin
  • An adjuvant comprising a saponin may additionally comprise further immunostimulants, in particular they may contain a TLR4 agonist. In some embodiments the adjuvant comprising a saponin does not comprise non-saponin immunostimulants.
  • the saponin, such as QS-21 can be used at amounts between 1 and 100 ug per dose, such as per human dose.
  • Saponin, such as QS-21 may be used at a level of about 50 ug. Examples of suitable ranges are 40 to 60 ug, suitably 45 to 55 ug or 49 to 51 ug, such as 50 ug.
  • a dose, such as a human dose comprises saponin, such as QS- 21 , at a level of about 25 ug. Examples of lower ranges include 20 to 30 ug, suitably 22 to 28 ug or 24 to 26 ug, such as 25 ug. Human doses intended for children may be reduced compared to those intended for a human adult (e.g. reduction by 50%).
  • a TLR4 agonist may be a lipopolysaccharide, such as a monophosphoryl lipid A, such as 3D-MPL, and can be used at amounts between 1 and 100 ug per dose, such as per human dose.
  • TLR4 agonist, such as 3D-MPL may be used at a level of about 50 ug. Examples of suitable ranges are 40 to 60 ug, suitably 45 to 55 ug or 49 to 51 ug, such as 50 ug.
  • a dose such as a human dose, comprises TLR4 agonist, such as 3D-MPL, at a level of about 25 ug.
  • Examples of lower ranges include 20 to 30 ug, suitably 22 to 28 ug or 24 to 26 ug, such as 25 ug.
  • Human doses intended for children may be reduced compared to those intended for a human adult (e.g. reduction by 50%).
  • the weight ratio of TLR4 agonist to saponin is suitably between 1 :5 to 5: 1 , suitably 1 :2 to 2:1 , such as about 1:1 , especially 1 :1.
  • suitably QS-21 may also be present at an amount of about 50 ug or about 25 ug per human dose.
  • the saponin is QS-21 and the TLR4 agonist is 3D-MPL.
  • the second adjuvant may be a squalene emulsion adjuvant.
  • squalene emulsion adjuvant refers to a squalene containing oil-in-water emulsion adjuvant.
  • tocopherol-containing squalene emulsion adjuvant refers to a squalene and tocopherol-containing oil-in-water emulsion adjuvant wherein the weight ratio of squalene to tocopherol is 20 or less (i.e. 20 weight units of squalene or less per weight unit of tocopherol or, alternatively phrased, at least 1 weight unit of tocopherol per 20 weight units of squalene).
  • Tocopherol-containing squalene emulsion adjuvants are therefore a subset of squalene emulsion adjuvants and are of particular interest in the present invention.
  • Squalene is readily available from commercial sources or may be obtained by methods known in the art. Squalene shows good biocompatibility and is readily metabolised.
  • Squalene emulsion adjuvants will typically have a submicron droplet size. Droplet sizes below 200 nm are beneficial in that they can facilitate sterilisation by filtration. There is evidence that droplet sizes in the 80 to 200 nm range are of particular interest for potency, manufacturing consistency and stability reasons (Klucker, 2012; Shah, 2014; Shah, 2015; Shah, 2019). Suitably the squalene emulsion adjuvant has an average droplet size of less than 1 um, especially less than 500 nm and in particular less than 200 nm.
  • the squalene emulsion adjuvant has an average droplet size of at least 50 nm, especially at least 80 nm, in particular at least 100 nm, such as at least 120 nm.
  • the squalene emulsion adjuvant may have an average droplet size of 50 to 200 nm, such as 80 to 200 nm, especially 120 to 180 nm, in particular 140 to 180 nm, such as about 160 nm. Uniformity of droplet sizes is desirable.
  • a polydispersity index (PdI) of greater than 0.7 indicates that the sample has a very broad size distribution and a reported value of 0 means that size variation is absent, although values smaller than 0.05 are rarely seen.
  • the squalene emulsion adjuvant has a polydispersity of 0.5 or less, especially 0.3 or less, such as 0.2 or less.
  • the droplet size means the average diameter of oil droplets in an emulsion and can be determined in various ways e.g. using the techniques of dynamic light scattering and/or single-particle optical sensing, using an apparatus such as the AccusizerTM and NicompTM series of instruments available from Particle Sizing Systems (Santa Barbara, USA), the ZetasizerTM instruments from Malvern Instruments (UK), or the Particle Size Distribution Analyzer instruments from Horiba (Kyoto, Japan). See Light Scattering from Polymer Solutions and Nanoparticle Dispersions Schartl, 2007.
  • Dynamic light scattering is the preferred method by which droplet size is determined.
  • the preferred method for defining the average droplet diameter is a Z-average i.e. the intensity-weighted mean hydrodynamic size of the ensemble collection of droplets measured by DLS.
  • the Z-average is derived from cumulants analysis of the measured correlation curve, wherein a single particle size (droplet diameter) is assumed and a single exponential fit is applied to the autocorrelation function.
  • references herein to average droplet size should be taken as an intensity-weighted average, and ideally the Z-average.
  • Polydispersity Index (PdI) values are easily provided by the same instrumentation which measures average diameter.
  • one or more emulsifying agents are generally required.
  • Surfactants can be classified by their ‘HLB’ (Griffin’s hydrophile/lipophile balance), where a HLB in the range 1-10 generally means that the surfactant is more soluble in oil than in water, whereas a HLB in the range 10-20 means that the surfactant is more soluble in water than in oil.
  • HLB values are readily available for many surfactants of interest or can be determined experimentally, e.g. polysorbate 80 has a HLB of 15.0 and TPGS has a HLB of 13 to 13.2. Sorbitan trioleate has a HLB of 1.8.
  • the resulting HLB of the blend is typically calculated by the weighted average e.g. a 70/30 wt% mixture of polysorbate 80 and TPGS has a HLB of (15.0 x 0.70) + (13 x 0.30) i.e. 14.4.
  • a 70/30 wt% mixture of polysorbate 80 and sorbitan trioleate has a HLB of (15.0 x 0.70) + (1.8 x 0.30) i.e. 11.04.
  • Surfactant(s) will typically be metabolisable (biodegradable) and biocompatible, being suitable for use as a pharmaceutical.
  • the surfactant can include ionic (cationic, anionic or zwitterionic) and/or non-ionic surfactants.
  • ionic cationic, anionic or zwitterionic
  • non-ionic surfactants The use of only non-ionic surfactants is often desirable, for example due to their pH independence.
  • the invention can thus use surfactants including, but not limited to: the polyoxyethylene sorbitan ester surfactants (commonly referred to as the Tweens or polysorbates), such as polysorbate 20 and polysorbate 80, especially polysorbate 80; copolymers of ethylene oxide (EG), propylene oxide (PO), and/or butylene oxide (BO), sold under the DOWFAXTM, PluronicTM (e.g., the polyoxyethylene sorbitan ester surfactants (commonly referred to as the Tweens or polysorbates), such as polysorbate 20 and polysorbate 80, especially polysorbate 80; copolymers of ethylene oxide (EG), propylene oxide (PO), and/or butylene oxide (BO), sold under the DOWFAXTM, PluronicTM (e.g.
  • Tweens or polysorbates such as polysorbate 20 and polysorbate 80, especially polysorbate 80
  • SynperonicTM tradenames such as linear EO/PO block copolymers, for example poloxamer 407, poloxamer 401 and poloxamer 188; octoxynols, which can vary in the number of repeating ethoxy (oxy-1, 2-ethanediyl) groups, with octoxynol-9 (Triton X-100, or t-octylphenoxypolyethoxyethanol) being of particular interest;
  • octylphenoxy polyethoxyethanol
  • phospholipids such as phosphatidylcholine (lecithin); polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants), such as polyoxyethylene 4 lauryl ether (Brij 30, Emulgen 104P), polyoxyethylene-9-lauryl ether and polyoxyethylene 12 cetyl/stearyl ether (EumulginTM B1, cetereth-12 or polyoxyethylene cetostearyl ether); sorbitan esters (commonly known as the Spans), such as sorbitan trioleate (Span 85), sorbitan monooleate (Span 80) and sorbitan monolaurate (Span 20); or tocopherol derivative surfactants, such as alpha-tocopherol-polyethylene glycol succinate (TPGS).
  • TPGS alpha-tocopherol-polyethylene glycol succinate
  • the surfactant component has a HLB between 10 and 18, such as between 12 and 17, in particular 13 to 16. This can be typically achieved using a single surfactant or, in some embodiments, using a mixture of surfactants.
  • Surfactants of particular interest include: poloxamer 401, poloxamer 188, polysorbate 80, sorbitan trioleate, sorbitan monooleate and polyoxyethylene 12 cetyl/stearyl ether either alone, in combination with each other or in combination with other surfactants.
  • polysorbate 80, sorbitan trioleate, sorbitan monooleate and polyoxyethylene 12 cetyl/stearyl ether either alone, or in combination with each other.
  • a particular surfactant of interest is polysorbate 80.
  • a particular combination of surfactants of interest is polysorbate 80 and sorbitan trioleate.
  • a further combination of surfactants of interest is sorbitan monooleate and polyoxyethylene cetostearyl ether.
  • the squalene emulsion adjuvant comprises one surfactant, such as polysorbate 80. In some embodiments the squalene emulsion adjuvant comprises two surfactants, such as polysorbate 80 and sorbitan trioleate or sorbitan monooleate and polyoxyethylene cetostearyl ether. In other embodiments the squalene emulsion adjuvant comprises three or more surfactants, such as three surfactants.
  • the amount of squalene in a single dose, such as a human dose, of squalene emulsion adjuvant may be 50 mg or less, especially 40 mg or less, in particular 30 mg or less, such as 20 mg or less (for example 15 mg or less).
  • the amount of squalene in a single dose, such as a human dose, of squalene emulsion adjuvant may be 0.5 mg or more, especially 1 mg or more, in particular 2 mg or more, such as 4 mg or more and desirably 8 mg or more.
  • the amount of squalene in a single dose, such as a human dose, of squalene emulsion adjuvant may be 0.5 to 50 mg, especially 1 to 20 mg, in particular 2 to 15 mg, such as 5 to 15 mg.
  • the amount of squalene in a single dose, such as a human dose, of squalene emulsion adjuvant may be 0.5 to 2 mg, 2 to 4 mg, 4 to 8 mg, 8 to 12 mg, 12 to 16 mg, 16 to 20 mg or 20 to 50 mg.
  • the amount of squalene in a single dose, such as a human dose, of squalene emulsion adjuvant may be 1.2 to 20 mg, in particular 1.2 to 15 mg.
  • the amount of squalene in a single dose, such as a human dose, of squalene emulsion adjuvant may be 1.2 to 2 mg, 2 to 4 mg, 4 to 8 mg or 8 to 12.1 mg.
  • the amount of squalene in a single dose, such as a human dose, of squalene emulsion adjuvant may be 1.21 to 1.52 mg, 2.43 to 3.03 mg, 4.87 to 6.05 mg or 9.75 to 12.1 mg.
  • the weight ratio of squalene to surfactant is 0.73 to 6.6, especially 1 to 5, in particular 1.5 to 4.5.
  • the weight ratio of squalene to surfactant may be 1.5 to 3, especially 1.71 to 2.8, such as 2.2 or 2.4.
  • the weight ratio of squalene to surfactant may be 2.5 to 3.5, especially 3 or 3.1.
  • the weight ratio of squalene to surfactant may be 3 to 4.5, especially 4 or 4.3.
  • the amount of surfactant in a single dose, such as a human dose, of squalene emulsion adjuvant is typically at least 0.4 mg. Generally, the amount of surfactant in a single dose, such as a human dose, of squalene emulsion adjuvant is 18 mg or less. The amount of surfactant in a single dose, such as a human dose, of squalene emulsion adjuvant may be 0.4 to 9.5 mg, in particular 0.4 to 7 mg. The amount of surfactant in a single dose, such as a human dose, of squalene emulsion adjuvant may be 0.4 to 1 mg, 1 to 2 mg, 2 to 4 mg or 4 to 7 mg.
  • the amount of surfactant in a single dose, such as a human dose, of squalene emulsion adjuvant may be 0.54 to 0.71 mg, 1.08 to 1.42 mg, 2.16 to 2.84 mg or 4.32 to 5.68 mg.
  • the squalene emulsion adjuvant may contain one or more tocopherols. Any of the a, P, y, 5, E and/or 5, tocopherols can be used, but a-tocopherol (also referred to herein as alphatocopherol) is typically used. D-alpha-tocopherol and D/L-alpha-tocopherol can both be used. Tocopherols are readily available from commercial sources or may be obtained by methods known in the art. In some embodiments the squalene emulsion adjuvant does not contain tocopherol. In some embodiments the squalene emulsion adjuvant contains tocopherol (i.e. at least one tocopherol, suitably one tocopherol), especially alpha-tocopherol, in particular D/L-alpha-tocopherol.
  • tocopherol i.e. at least one tocopherol, suitably one tocopherol
  • Tocopherols have been used, in relatively small amounts, in squalene emulsion adjuvants as antioxidants. Desirably tocopherols are present a level where the weight ratio of squalene to tocopherol is 20 or less, such as 10 or less. Suitably the weight ratio of squalene to tocopherol is 0.1 or more. Typically the weight ratio of squalene to tocopherol is 0.1 to 10, especially 0.2 to 5, in particular 0.3 to 3, such as 0.4 to 2. Suitably, the weight ratio of squalene to tocopherol is 0.72 to 1.136, especially 0.8 to 1, in particular 0.85 to 0.95, such as 0.9. Alternatively, the weight ratio of squalene to tocopherol is 3.4 to 4.6, especially 3.6 to 4.4, in particular 3.8 to 4.2, such as 4.
  • the amount of tocopherol in a single dose, such as a human dose, of squalene emulsion adjuvant is typically at least 0.5 mg, especially at least 1.3 mg. Generally, the amount of tocopherol in a single dose, such as a human dose, of squalene emulsion adjuvant is 55 mg or less. The amount of tocopherol in a single dose, such as a human dose, of squalene emulsion adjuvant may be 1.3 to 22 mg, in particular 1.3 to 16.6 mg.
  • the amount of tocopherol in a single dose, such as a human dose, of squalene emulsion adjuvant may be 1.3 to 2 mg, 2 to 4 mg, 4 to 8 mg or 8 to 13.6 mg.
  • the amount of tocopherol in a single dose, such as a human dose, of squalene emulsion adjuvant may be 1.33 to 1.69 mg, 2.66 to 3.39 mg, 5.32 to 6.77 mg or 10.65 to 13.53 mg.
  • the squalene emulsion adjuvant may consist essentially of squalene, tocopherol (if present), surfactant and water.
  • squalene emulsion adjuvants may contain additional components as desired or required depending upon the intended final presentation and vaccination strategy, such as buffers and/or tonicity modifying agents, for example modified phosphate buffered saline (disodium phosphate, potassium biphosphate, sodium chloride and potassium chloride).
  • buffers and/or tonicity modifying agents for example modified phosphate buffered saline (disodium phosphate, potassium biphosphate, sodium chloride and potassium chloride).
  • a squalene emulsion of interest in the present invention is known as 'MF59' (WO90/14837; Podda, 2003; Podda, 2001) and is a submicron oil-in-water emulsion of squalene, polysorbate 80 (also known as Tween 80TM), and sorbitan trioleate (also known as Span 85TM). It may also include citrate ions e.g. 10mM sodium citrate buffer.
  • the composition of the emulsion by volume can be about 5% squalene, about 0.5% polysorbate 80 and about 0.5% sorbitan trioleate.
  • Vaccine Design The Subunit and Adjuvant Approach (chapter 10), Vaccine Adjuvants: Preparation Methods and Research Protocols (chapter 12) and New Generation Vaccines (chapter 19).
  • aqueous phase e.g. citrate buffer
  • the emulsion is typically prepared at double-strength (4.3% v/v squalene, 0.5% v/v polysorbate 80 and 0.5% v/v sorbitan trioleate) and is diluted 1:1 (by volume) with an antigen composition to provide a final adjuvanted vaccine composition.
  • An adult human dose of MF59 contains 9.75 mg squalene, 1.17 mg polysorbate 80 and 1.17 mg sorbitan trioleate (O’Hagan, 2013).
  • AF03 includes squalene, sorbitan monooleate, polyoxyethylene cetostearyl ether and mannitol.
  • AF03 is typically prepared by cooling a preheated water-in-oil emulsion until it crosses its emulsion phase inversion temperature, at which point it thermoreversibly converts into an oil-in-water emulsion.
  • the mannitol, cetostearyl ether and a phosphate buffer are mixed in one container to form an aqueous phase, while the sorbitan ester and squalene are mixed in another container to form an oily component.
  • the aqueous phase is added to the oily component and the mixture is then heated to approximately 60°C and cooled to provide the final emulsion.
  • the emulsion is typically initially prepared as a concentrate with a composition of 32.5% squalene, 4.8% sorbitan monooleate, 6.2% polyoxyethylene cetostearyl ether and 6% mannitol and 50.5% phosphate buffered saline.
  • AF03 adjuvant contains 12.4 mg squalene, 1.9 mg sorbitan monooleate, 2.4 mg polyoxyethylene cetostearyl ether and 2.3 mg mannitol per 500 ul human adult dose (HumenzaTM Summary of Product Characteristics).
  • AS03' Another squalene emulsion of interest in the present invention is known as 'AS03' (Gargon, 2012) and is typically prepared by mixing an oil mixture (consisting of squalene and alpha-tocopherol) with an aqueous phase (polysorbate 80 and buffer), followed by microfluidisation (W02006/100109).
  • AS03 is typically prepared at double-strength with the expectation of dilution by an aqueous antigen containing composition prior to administration.
  • An adult human dose of AS03A contains 10.69 mg squalene, 11.86 mg alpha-tocopherol and 4.86 mg polysorbate 80 (Morel, 2011; Fox, 2009).
  • AS03B Certain reduced does of AS03 have also been described (W02008/043774), including AS03B (1/2 dose), AS03c (1/4 dose) and AS03D (1/8 dose) (Carmona Martinez, 2014).
  • squalene based self-emulsifying adjuvant systems have been described.
  • W02015/140138 and WO2016/135154 describe the preparation of oil/surfactant compositions, which when diluted with an aqueous phase spontaneously form oil-in-water emulsions having small droplet particle sizes, such emulsions can be used as immunological adjuvants.
  • An adult human dose of ‘SEA160’ emulsion may include 7.62 mg squalene, 2.01 mg polysorbate 80 and 2.01 mg sorbitan trioleate. (Shah, 2014; Shah, 2015; Shah, 2019)
  • SEAS44 contains 60% v/v squalene, 15% v/v alpha-tocopherol and 25% v/v polysorbate 80.
  • the squalene/tocopherol/polysorbate mixture is intended to be diluted approximately 10-fold with an aqueous medium to form the final emulsion adjuvant. Consequently, an adult human dose of SEAS44 emulsion may include about 13 mg squalene, 3.6 mg alpha-tocopherol and 6.7 mg polysorbate 80.
  • SWE (Younis, 2018) comprising squalene 3.9% w/v, sorbitan trioleate 0.47% w/v, and polysorbate 80 (0.47% w/v) dispersed in 10 mM citrate buffer at pH 6.5. Consequently, an adult human dose of SWE may include about 9.75 mg squalene, 1.175 mg sorbitan trioleate and 1.175 mg polysorbate 80, similar to MF59.
  • SE (Carter, 2016; Sun, 2016) comprising squalene, phosphatidyl choline, poloxamer 188 and an ammonium phosphate buffered aqueous phase also containing glycerol.
  • SE has been described as containing small amounts of tocopherol.
  • An adult human dose of SE may include about 8.6 mg squalene, 2.73 mg phosphatidyl choline and 0.125 mg poloxamer 188, optionally with 0.05 mg tocopherol.
  • CoVaccine (Hilgers, 2006; Hamid, 2011 ; Younis, 2019) comprises squalene, polysorbate 80 and sucrose fatty acid sulfate esters, typically with phosphate buffered saline.
  • An adult human dose of CoVaccine may include about 40 mg squalene, 10 mg polysorbate 80 and 10 mg sucrose fatty acid sulfate esters.
  • the squalene emulsion adjuvant may be derived from MF59. Consequently, the squalene emulsion adjuvant may comprise squalene, polysorbate 80, sorbitan trioleate and water.
  • the squalene emulsion adjuvant may consist essentially of squalene, polysorbate 80, sorbitan trioleate and water.
  • the aqueous phase may contain additional components as desired or required depending upon the intended final presentation and vaccination strategy, such as buffers and/or tonicity modifying agents, in particular citrate ions e.g. 10mM sodium citrate buffer.
  • the weight ratio of squalene to polysorbate 80 is 10 to 6.6, especially 9.1 to 7.5, in particular 8.7 to 7.9, such as 8.3.
  • the weight ratio of squalene to sorbitan trioleate is 10 to 6.6, especially 9.1 to 7.5, in particular 8.7 to 7.9, such as 8.3.
  • a single dose, such as a typical full human dose, of squalene emulsion adjuvant derived from MF59 may comprise 9 to 11 mg of squalene, such as 9.5 to 10 mg, in particular 9.75 mg. Higher or lower doses of squalene emulsion adjuvant derived from MF59 may be used.
  • a single dose is at least 0.1x a typical full human dose, especially at least 0.25x a typical full human dose, in particular at least 0.5x a typical full human dose. Desirably the single dose is less than or equal to a full human dose.
  • the single dose may be 0.1 to 1x a typical full human dose, i.e. comprising 0.9 to 11 mg of squalene.
  • Particular single doses of interest include 0.1x a typical full human dose i.e. comprising 0.9 to 1.1 mg of squalene, 0.125x a typical full human dose i.e. comprising 1.1 to 1.4 mg of squalene, 0.25x a typical full human dose i.e. comprising 2.2 to 2.8 mg of squalene, such as 0.5x a typical full human dose i.e. comprising 4.5 to 5.5 mg of squalene or 1x a typical full human dose i.e. comprising 9 to 11 mg of squalene.
  • Squalene emulsion adjuvant derived from MF59 may include citrate ions e.g. 10mM sodium citrate buffer.
  • the squalene emulsion adjuvant may be derived from AF03. Consequently, the squalene emulsion adjuvant may comprise squalene, sorbitan monooleate, polyoxyethylene cetostearyl ether and water.
  • the squalene emulsion adjuvant may consist essentially of squalene, sorbitan monooleate, polyoxyethylene cetostearyl ether and water.
  • Mannitol has been shown to reduce the phase transition temperature and is therefore desirable for manufacturing reasons, although excessive levels of mannitol may cause heterogeneity in size and larger droplets (Klucker, 2012).
  • the aqueous phase may contain additional components as desired or required depending upon the intended final presentation and vaccination strategy, such as buffers and/or tonicity modifying agents, in particular phosphate buffered saline.
  • the weight ratio of squalene to sorbitan monooleate is 7.8 to 5.2, especially 7.15 to 5.85, in particular 6.8 to 6.2, such as 6.5.
  • the weight ratio of squalene to polyoxyethylene cetostearyl ether is 6.2 to 4.1, especially 5.7 to 4.7, in particular 5.4 to 4.9, such as 5.2.
  • the weight ratio of squalene to mannitol is 6.5 to 4.3, especially 5.9 to 4.9, in particular 5.7 to 5.1 , such as 5.4.
  • a single dose, such as a typical full human dose, of squalene emulsion adjuvant derived from AF03 may comprise 11.2 to 13.6 mg of squalene, such as 12 to 12.8 mg, in particular 12.4 mg. Higher or lower doses of squalene emulsion adjuvant derived from AF03 may be used.
  • a single dose is at least 0.1x a typical full human dose, especially at least 0.25x a typical full human dose, in particular at least 0.5x a typical full human dose. Desirably the single dose is less than or equal to a full human dose.
  • the single dose may be 0.1 to 1x a typical full human dose, i.e. comprising 1.1 to 13.6 mg of squalene.
  • Particular single doses of interest include 0.1x a typical full human dose i.e. comprising 1.1 to 1.35 mg of squalene, 0.125x a typical full human dose i.e. comprising 1.4 to 1.7 mg of squalene, 0.25x a typical full human dose i.e. comprising 2.8 to 3.4 mg of squalene, such as 0.5x a typical full human dose i.e. comprising 5.6 to 6.8 mg of squalene or 1x a typical full human dose i.e. comprising 11.2 to 13.6 mg of squalene.
  • Squalene emulsion adjuvant derived from AF03 may also include in particular phosphate buffered saline.
  • the squalene emulsion adjuvant may be derived from AS03. Consequently, the squalene emulsion adjuvant may comprise squalene, tocopherol, polysorbate 80 and water.
  • the squalene emulsion adjuvant may consist essentially of squalene, tocopherol, polysorbate 80 and water.
  • the aqueous phase may contain additional components as desired or required depending upon the intended final presentation and vaccination strategy, such as buffers and/or tonicity modifying agents.
  • Suitable buffers include Na2HPO4 and KH2PO4.
  • Suitable tonicity modifying agents include NaCI and KCI.
  • Modified phosphate buffered saline may be used, such as comprising Na2HPO4 and KH2PO4, NaCI and KCI.
  • any of the a, p, y, 5, E or 5, tocopherols can be used, but a-tocopherol (also referred to herein as alpha-tocopherol) is typically used.
  • D-alpha-tocopherol and D/L-alpha-tocopherol can both be used.
  • a particularly desirable alpha-tocopherol is D/L-alpha-tocopherol.
  • the weight ratio of squalene to tocopherol is 0.5 to 1 .5, especially 0.6 to 1 .35, in particular 0.7 to 1 .1 , such as 0.85 to 0.95 e.g. 0.9.
  • the tocopherol is alphatocopherol, such as D/L-alpha-tocopherol.
  • the weight ratio of squalene to polysorbate 80 is 1.2 to 3.6, especially 1.46 to 3.3, in particular 1.9 to 2.5 such as 2.1 to 2.3 e.g. 2.2.
  • a single dose, such as a typical full human dose, of squalene emulsion adjuvant derived from AS03 may comprise 9.7 to 12.1 mg of squalene, such as 10.1 to 11.8 mg, in particular 10.7 mg. Higher or lower doses of squalene emulsion adjuvant derived from AS03 may be used.
  • a single dose is at least 0.1x a typical full human dose, especially at least 0.25x a typical full human dose, in particular at least 0.5x a typical full human dose. Desirably the single dose is less than or equal to a full human dose.
  • the single dose may be 0.1 to 1x a typical full human dose, i.e. comprising 0.9 to 12.1 mg of squalene.
  • Particular single doses of interest include 0.1x a typical full human dose i.e. comprising 0.9 to 1.3 mg of squalene (typically with 1 to 1.4 mg tocopherol, such as D/L-alpha tocopherol, and 0.43 to 0.57 mg polysorbate 80), 0.125x a typical full human dose i.e. comprising 1.2 to 1.6 mg of squalene (typically with 1.3 to 1.7 mg tocopherol, such as D/L-alpha tocopherol, and 0.54 to 0.71 mg polysorbate 80), 0.25x a typical full human dose i.e.
  • squalene typically with 2.6 to 3.4 mg tocopherol, such as D/L-alpha tocopherol, and 1 to 1.5 mg polysorbate 80
  • squalene typically with 2.6 to 3.4 mg tocopherol, such as D/L-alpha tocopherol, and 1 to 1.5 mg polysorbate 80
  • 0.5x a typical full human dose i.e. comprising 4.8 to 6.1 mg of squalene (typically with 5.3 to 6.8 mg tocopherol, such as D/L-alpha tocopherol, and 2.1 to 2.9 mg polysorbate 80) or 1x a typical full human dose i.e. comprising 9.7 to 12.1 mg of squalene (typically with 10.6 to 13.6 mg tocopherol, such as D/L-alpha tocopherol, and 4.3 to 5.7 mg polysorbate 80).
  • Squalene emulsion adjuvant derived from AS03 may also include in particular a phosphate buffered saline, such as modified phosphate buffered saline.
  • the squalene emulsion adjuvant may be derived from SE. Consequently, the squalene emulsion adjuvant may comprise squalene, phosphatidyl choline, poloxamer 188 and water, optionally with glycerol.
  • the squalene emulsion adjuvant may consist essentially of squalene, phosphatidyl choline, poloxamer 188 and water, optionally with glycerol.
  • the aqueous phase may contain additional components as desired or required depending upon the intended final presentation and vaccination strategy, such as buffers and/or tonicity modifying agents, in particular ammonium phosphate buffer.
  • Tocopherol, such as alpha-tocopherol may be present as an antioxidant.
  • the weight ratio of squalene to phosphatidyl choline is 2.52 to 3.8, especially 2.85 to 3.5, in particular 3 to 3.3, such as 3.15.
  • the weight ratio of squalene to poloxamer 188 is 55 to 83, especially 62 to 76, in particular 65.5 to 72.5, such as 69.
  • the weight ratio of squalene to tocopherol, if present, is at least 50, especially 137 to 207, in particular 154 to 190, such as 163 to 181, for example 172.
  • a single dose, such as a typical full human dose, of squalene emulsion adjuvant derived from SE may comprise 7.7 to 9.5 mg of squalene, such as 8.1 to 9 mg, in particular 8.6 mg. Higher or lower doses of squalene emulsion adjuvant derived from SE may be used.
  • a single dose is at least 0.1x a typical full human dose, especially at least 0.25x a typical full human dose, in particular at least 0.5x a typical full human dose. Desirably the single dose is less than or equal to a full human dose.
  • the single dose may be 0.1 to 1x a typical full human dose, i.e. comprising 0.77 to 9.5 mg of squalene.
  • Particular single doses of interest include 0.1x a typical full human dose i.e. comprising 0.77 to 0.95 mg of squalene, 0.125x a typical full human dose i.e. comprising 0.96 to 1.2 mg of squalene, 0.25x a typical full human dose i.e. comprising 1.9 to 2.4 mg of squalene, such as 0.5x a typical full human dose i.e. comprising 3.8 to 4.8 mg of squalene or 1x a typical full human dose i.e. comprising 7.7 to 9.5 mg of squalene.
  • Squalene emulsion adjuvant derived from SE may also include in particular ammonium phosphate buffer and glycerol.
  • the squalene emulsion adjuvant may be derived from SEA160. Consequently, the squalene emulsion adjuvant may comprise squalene, polysorbate 80, sorbitan trioleate and water. The squalene emulsion adjuvant may consist essentially of squalene, polysorbate 80, sorbitan trioleate and water.
  • the aqueous phase may contain additional components as desired or required depending upon the intended final presentation and vaccination strategy, such as buffers and/or tonicity modifying agents.
  • the weight ratio of squalene to polysorbate 80 is 4.6 to 3.0, especially 4.2 to 3.4, in particular 4.0 to 3.6, such as 3.8.
  • the weight ratio of squalene to sorbitan trioleate is 4.6 to 3.0, especially 4.2 to 3.4, in particular 4.0 to 3.6, such as 3.8.
  • a single dose, such as a typical full human dose, of squalene emulsion adjuvant derived from SEA160 may comprise 6.8 to 8.4 mg of squalene, such as 7.2 to 8 mg, in particular 7.6 mg. Higher or lower doses of squalene emulsion adjuvant derived from SEA160 may be used.
  • a single dose is at least 0.1x a typical full human dose, especially at least 0.25x a typical full human dose, in particular at least 0.5x a typical full human dose. Desirably the single dose is less than or equal to a full human dose.
  • the single dose may be 0.1 to 1x a typical full human dose, i.e. comprising 0.68 to 8.4 mg of squalene.
  • Particular single doses of interest include 0.1x a typical full human dose i.e. comprising 0.68 to 0.84 mg of squalene, 0.125x a typical full human dose i.e. comprising 0.85 to 1.1 mg of squalene, 0.25x a typical full human dose i.e. comprising 1.7 to 2.1 mg of squalene, such as 0.5x a typical full human dose i.e. comprising 3.4 to 4.2 mg of squalene or 1x a typical full human dose i.e. comprising 6.8 to 8.4 mg of squalene.
  • Squalene emulsion adjuvant derived from SEA160 may also include in particular a phosphate buffered saline, such as modified phosphate buffered saline.
  • the squalene emulsion adjuvant may be derived from SEAS44. Consequently, the squalene emulsion adjuvant may comprise squalene, tocopherol, polysorbate 80 and water.
  • the squalene emulsion adjuvant may consist essentially of squalene, tocopherol, polysorbate 80 and water.
  • the aqueous phase may contain additional components as desired or required depending upon the intended final presentation and vaccination strategy, such as buffers and/or tonicity modifying agents.
  • Suitable buffers include Na2HPO4 and KH2PO4.
  • Suitable tonicity modifying agents include NaCI and KCI.
  • Modified phosphate buffered saline may be used, such as comprising Na2HPO4 and KH2PO4, NaCI and KCI.
  • any of the a, p, y, 5, E or 5, tocopherols can be used, but a-tocopherol is typically used.
  • D-alpha-tocopherol and D/L-alpha-tocopherol can both be used.
  • a particularly desirable alpha-tocopherol is D/L-alpha-tocopherol.
  • the weight ratio of squalene to tocopherol is 2.6 to 4.5, especially 2.8 to 4.3, in particular 3.25 to 4, such as 3.4 to 3.8 e.g. 3.6.
  • the tocopherol is alpha-tocopherol, especially D/L-alpha-tocopherol.
  • the weight ratio of squalene to polysorbate 80 is 1.3 to 2.5, especially 1.56 to 2.3, in particular 1.75 to 2.15 such as 1.85 to 2 e.g. 1.94.
  • a single dose, such as a typical full human dose, of squalene emulsion adjuvant derived from SEAS44 may comprise 11.7 to 14.3 mg of squalene, such as 12.3 to 13.7 mg, in particular 13 mg. Higher or lower doses of squalene emulsion adjuvant derived from SEAS44 may be used.
  • a single dose is at least 0.1x a typical full human dose, especially at least 0.25x a typical full human dose, in particular at least 0.5x a typical full human dose. Desirably the single dose is less than or equal to a full human dose.
  • the single dose may be 0.1 to 1x a typical full human dose, i.e. comprising 1.1 to 14.3 mg of squalene.
  • Particular single doses of interest include 0.1x a typical full human dose i.e. comprising 1.1 to 1.5 mg of squalene, 0.125x a typical full human dose i.e. comprising 1.4 to 1.8 mg of squalene, 0.25x a typical full human dose i.e. comprising 2.9 to 3.6 mg of squalene, such as 0.5x a typical full human dose i.e. comprising 5.8 to 7.2 mg of squalene or 1x a typical full human dose i.e. comprising 11.7 to 14.3 mg of squalene.
  • Squalene emulsion adjuvant derived from SEAS44 may also include in particular a phosphate buffered saline, such as modified phosphate buffered saline.
  • Self-emulsifying adjuvants such as SEA160, SEAS44 and squalene emulsion adjuvant adjuvants derived therefrom, may be provided in dry form.
  • dry self-emulsifying adjuvants may consist essentially of squalene and surfactant(s), such as in the case of SEA160 derived squalene emulsion adjuvants.
  • Such dry self-emulsifying adjuvants may consist essentially of squalene and surfactant(s) or consist essentially of squalene, tocopherol and surfactant(s), such as in the case of SEAS44 derived tocopherol-containing squalene emulsion adjuvants.
  • High pressure homogenization may be applied to yield squalene emulsion adjuvants which demonstrate uniformly small droplet sizes and long-term stability (see EP 0 868 918 B1 and W02006/100109).
  • oil phase composed of squalene and tocopherol may be formulated under a nitrogen atmosphere.
  • Aqueous phase is prepared separately, typically composed of water for injection or phosphate buffered saline, and polysorbate 80.
  • Oil and aqueous phases are combined, such as at a ratio of 1 :9 (volume of oil phase to volume of aqueous phase) before homogenisation and microfluidisation, such as by a single pass through an in-line homogeniser and three passes through a microfluidiser (at around 15000 psi).
  • the resulting emulsion may then be sterile filtered, for example through two trains of two 0.5/0.2 urn filters in series (i.e. 0.5/0.2/0.5/0.2), see WO2011/154444. Operation is desirably undertaken under an inert atmosphere, e.g. nitrogen. Positive pressure may be applied, see WO2011/154443.
  • WO2015/140138 WO2016/135154, Shah, 2014 Shah, 2015, Shah, 2019, W02020/160080 and Lodaya, 2019 describe squalene emulsion adjuvants which are selfemulsifying adjuvant systems (SEAS) and their manufacture.
  • Adjuvant carriers are selfemulsifying adjuvant systems (SEAS) and their manufacture.
  • the STING agonist and/or the second adjuvant may be formulated with one or more carriers.
  • Suitable carriers include ISCOMs, liposomes and emulsions.
  • the saponin and/or the TLR4 agonist if present will typically be formulated with one or more carriers. Suitable carriers include ISCOMs, liposomes and emulsions.
  • the saponin and TLR4 agonist may be formulated with different carriers or may be formulated with the same carrier (whether formulated together or separately). Conveniently, the saponin and TLR4 agonist are formulated together with the same carrier, in particular with liposomes or an emulsion, especially with liposomes.
  • liposome is well known in the art and defines a general category of vesicles which comprise one or more lipid bilayers surrounding an aqueous space. Liposomes thus consist of one or more lipid and/or phospholipid bilayers and can contain other molecules, such as proteins or carbohydrates, in their structure. Because both lipid and aqueous phases are present, liposomes can encapsulate or entrap water-soluble material, lipid-soluble material, and/or amphiphilic compounds.
  • Liposome size may vary from 30 nm to several urn depending on the phospholipid composition and the method used for their preparation.
  • the liposome size will typically be in the range of 50 nm to 200 nm, especially 60 nm to 180 nm, such as 70 to 165 nm.
  • the liposomes should be stable and have a diameter of -100 nm to allow convenient sterilization by filtration.
  • a beneficial feature of the present invention is that the saponin may be presented in a less reactogenic composition where it is quenched with a sterol, such as cholesterol.
  • the liposomes of use in the present invention suitably contain DOPC, such as contain DOPC and sterol, such as cholesterol.
  • the liposomes consist essentially of DOPC and sterol such as cholesterol (with saponin and/or TLR4 agonist).
  • the ratio of saponin: DOPC will typically be in the order of 1 :50 to 1 :10 (w/w), suitably between 1 :25 to 1 :15 (w/w), and preferably 1 :22 to 1 :18 (w/w), such as 1 :20 (w/w).
  • the ratio of DOPC:sterol, such as cholesterol will typically be in the order of 10:1 to 1 :1 (w/w), suitably between 8:1 to 2:1 (w/w), and preferably 6:1 to 2.6:1 (w/w), such as about 4:1 (w/w).
  • Structural integrity of the liposomes may be assessed by methods such as dynamic light scattering (DLS) measuring the size (Z-average diameter, Zav) and polydispersity of the liposomes, or, by electron microscopy for analysis of the structure of the liposomes.
  • DLS dynamic light scattering
  • Zav Z-average diameter
  • polydispersity of the liposomes or, by electron microscopy for analysis of the structure of the liposomes.
  • the average particle size is between 90 and 120 nm, or between 95 and 120 nm.
  • a polydispersity index (Pdl) of greater than 0.7 indicates that the sample has a very broad size distribution and a reported value of 0 means that size variation is absent, although values smaller than 0.05 are rarely seen.
  • the squalene emulsion has a polydispersity of 0.5 or less, especially 0.3 or less, such as 0.2 or less.
  • a particular second adjuvant of interest features liposomes comprising DOPC and cholesterol, with TLR4 agonist and saponin, especially 3D-MPL and QS-21.
  • Another second adjuvant of interest features liposomes comprising DOTAP and DMPC, with TLR4 agonist and saponin, especially dLOS and QS-21.
  • Emulsion carriers will typically be an oil in water emulsion comprising a pharmaceutically acceptable metabolizable oil, such as squalene.
  • Squalene is readily available from commercial sources or may be obtained by methods known in the art. Squalene shows good biocompatibility and is readily metabolised.
  • Squalene emulsions will typically have a submicron droplet size. Droplet sizes below 200 nm are beneficial in that they can facilitate sterilisation by filtration. There is evidence that droplet sizes in the 80 to 200 nm range are of particular interest for potency, manufacturing consistency and stability reasons. (Klucker, 2012; Shah, 2014; Shah, 2015; Shah, 2019).
  • the squalene emulsion may have an average droplet size of 50 to 200 nm, such as 80 to 200 nm, especially 120 to 180 nm, in particular 140 to 180 nm, such as about 160 nm.
  • a polydispersity index (Pdl) of greater than 0.7 indicates that the sample has a very broad size distribution and a reported value of 0 means that size variation is absent, although values smaller than 0.05 are rarely seen.
  • the squalene emulsion has a polydispersity of 0.5 or less, especially 0.3 or less, such as 0.2 or less.
  • the droplet size means the average diameter of oil droplets in an emulsion and can be determined in various ways e.g. using the techniques of dynamic light scattering and/or single-particle optical sensing, using an apparatus such as the AccusizerTM and NicompTM series of instruments available from Particle Sizing Systems (Santa Barbara, USA), the ZetasizerTM instruments from Malvern Instruments (UK), or the Particle Size Distribution Analyzer instruments from Horiba (Kyoto, Japan). See Light Scattering from Polymer Solutions and Nanoparticle Dispersions Schartl, 2007. Dynamic light scattering (DLS) is the preferred method by which droplet size is determined. The preferred method for defining the average droplet diameter is a Z-average i.e.
  • one or more emulsifying agents are generally required.
  • Surfactant(s) will typically be metabolisable (biodegradable) and biocompatible, being suitable for use as a pharmaceutical.
  • the surfactant can include ionic (cationic, anionic or zwitterionic) and/or non-ionic surfactants. The use of only non-ionic surfactants is often desirable, for example due to their pH independence.
  • the invention can thus use surfactants including, but not limited to: the polyoxyethylene sorbitan ester surfactants (commonly referred to as the Tweens or polysorbates), such as polysorbate 20 and polysorbate 80, especially polysorbate 80; copolymers of ethylene oxide (EO), propylene oxide (PO), and/or butylene oxide (BO), sold under the DOWFAXTM, PluronicTM (e.g., the polyoxyethylene sorbitan ester surfactants (commonly referred to as the Tweens or polysorbates), such as polysorbate 20 and polysorbate 80, especially polysorbate 80; copolymers of ethylene oxide (EO), propylene oxide (PO), and/or butylene oxide (BO), sold under the DOWFAXTM, PluronicTM (e.g.
  • Tweens or polysorbates such as polysorbate 20 and polysorbate 80, especially polysorbate 80
  • SynperonicTM tradenames such as linear EO/PO block copolymers, for example poloxamer 407, poloxamer 401 and poloxamer 188; octoxynols, which can vary in the number of repeating ethoxy (oxy-1 , 2-ethanediyl) groups, with octoxynol-9 (Triton X-100, or t-octylphenoxypolyethoxyethanol) being of particular interest;
  • octylphenoxy polyethoxyethanol
  • phospholipids such as phosphatidylcholine (lecithin); polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants), such as polyoxyethylene 4 lauryl ether (Brij 30, Emulgen 104P), polyoxyethylene-9-lauryl ether and polyoxyethylene 12 cetyl/stearyl ether (EumulginTM B1 , cetereth-12 or polyoxyethylene cetostearyl ether); sorbitan esters (commonly known as the Spans), such as sorbitan trioleate (Span 85), sorbitan monooleate (Span 80) and sorbitan monolaurate (Span 20); or tocopherol derivative surfactants, such as alpha-tocopherol-polyethylene glycol succinate (TPGS).
  • TPGS alpha-tocopherol-polyethylene glycol succ
  • Surfactants of particular interest include: poloxamer 401 , poloxamer 188, polysorbate 80, sorbitan trioleate, sorbitan monooleate and polyoxyethylene 12 cetyl/stearyl ether either alone, in combination with each other or in combination with other surfactants.
  • polysorbate 80 sorbitan trioleate, sorbitan monooleate and polyoxyethylene 12 cetyl/stearyl ether either alone, or in combination with each other.
  • a particular surfactant of interest is polysorbate 80.
  • a particular combination of surfactants of interest is polysorbate 80 and sorbitan trioleate.
  • a further combination of surfactants of interest is sorbitan monooleate and polyoxyethylene cetostearyl ether.
  • a STING agonist is administered in step (i) and optionally a STING agonist is administered in step (ii).
  • a second adjuvant is administered in step (ii). In other approaches described herein, a second adjuvant is not administered in step (ii).
  • the second adjuvant may be any adjuvant. Typically, the second adjuvant does not comprise a STING agonist.
  • the second adjuvant comprises one immunostimulant. In some embodiments the second adjuvant comprises two immunostimulants.
  • the second adjuvant comprises, such as consists of, a TLR agonist. In one embodiment the second adjuvant comprises, such as consists of, a TLR1 agonist. In one embodiment the second adjuvant comprises, such as consists of, a TLR1/2 agonist. In one embodiment the second adjuvant comprises, such as consists of, a TLR2 agonist. In one embodiment the second adjuvant comprises, such as consists of, a TLR3 agonist. In one embodiment the second adjuvant comprises, such as consists of, a TLR4 agonist. In one embodiment the second adjuvant comprises, such as consists of, a TLR5 agonist. In one embodiment the second adjuvant comprises, such as consists of, a TLR6 agonist.
  • the second adjuvant comprises, such as consists of, a TLR7 agonist, a TLR8 agonist or a TLR7/TLR8 agonist. In one embodiment the second adjuvant comprises, such as consists of, a TLR9 agonist.
  • the second adjuvant comprises, such as consists of, an adjuvant comprising a saponin.
  • the second adjuvant comprises, such as consists of, a squalene emulsion adjuvant.
  • a STING agonist is administered in step (i) and a STING agonist is administered in step (ii).
  • the STING agonist of step (i) and the STING agonist of step (ii) are desirably essentially the same, such as the same.
  • the STING agonist of step (i) and the STING agonist of step (ii) are essentially the same if they elicit a functionally equivalent immune response.
  • a functionally equivalent immune response may be the level of immune response and/or the nature of the immune response.
  • Immune responses of the same nature may be immune responses which are both innate immune responses (such as both the same profile of innate immune cells, such as the presence of comparable innate immune cell types and the comparable concentration of said innate immune cell types) or both adaptive immune responses (such as both T cell responses (such as both CD8+ T cell responses or both CD4+ T cell responses), or both antibody responses).
  • innate immune responses such as both the same profile of innate immune cells, such as the presence of comparable innate immune cell types and the comparable concentration of said innate immune cell types
  • both adaptive immune responses such as both T cell responses (such as both CD8+ T cell responses or both CD4+ T cell responses), or both antibody responses).
  • Immune responses of the same level may be immune responses which produce essentially the same titre of neutralising antibodies or the same quantity of T cells in the same immunoassay.
  • a STING agonist is administered in step (i) and an antigen is administered in step (ii).
  • the antigen is a polypeptide.
  • the antigen is provided in the form of a polynucleotide, such as a viral vector or mRNA.
  • the antigen is a polysaccharide.
  • the antigen may be derived from (i.e. associated with) a pathogen, especially a human pathogen, (such as a bacterium, virus or parasite) or may be a cancer antigen (such as a tumour antigen and/or a neoantigen).
  • a pathogen especially a human pathogen, (such as a bacterium, virus or parasite) or may be a cancer antigen (such as a tumour antigen and/or a neoantigen).
  • antigen is meant a substance which is capable of eliciting an immune response in a subject.
  • the immune response from the antigen is a protective immune response, e.g. reducing partially or completely the severity of one or more symptoms and/or time over which one or more symptoms are experienced by a subject, reducing the likelihood of developing an established infection after challenge and/or slowing progression of an associated illness (e.g. extending survival).
  • the antigen may be delivered in the same form (i.e. a ‘homologous’ dosage regime), in different forms (i.e. a ‘heterologous’ dosage regime), or any combination of the same form and different forms, over each of the multiple administrations.
  • the antigen in a first administration in step (ii) is a polypeptide and the antigen in a second administration is a polynucleotide encoding the polypeptide then the antigens delivered in each administration are delivered in heterologous forms but are the same antigen for the purposes of the invention.
  • the antigen is derived from a bacterium.
  • the antigen may be derived from a virus.
  • an antigen is derived from a pathogen if it contains a polypeptide sequence which is obtainable from said pathogen.
  • the polypeptide sequence which is obtainable from said pathogen is at least 50, especially at least 100, in particular at least 150, such as at least 200 amino acids long.
  • an antigen comprises at least one B or T cell epitope, suitably an antigen comprises B and T cell epitopes.
  • the elicited immune response may be an antigen specific B cell response which produces neutralizing antibodies.
  • the elicited immune response may be an antigen specific T cell response, which may be a systemic and/or a local response.
  • the antigen specific T cell response may comprise a CD4+ T cell response, such as a response involving CD4+ T cells expressing a plurality of cytokines, e.g. IFNgamma, TNFalpha and/or IL2.
  • the antigen specific T cell response comprises a CD8+ T cell response, such as a response involving CD8+ T cells expressing a plurality of cytokines, e.g., IFNgamma, TNFalpha and/or IL2.
  • cytokines e.g., IFNgamma, TNFalpha and/or IL2.
  • an antigen contains 3000 residues or fewer, especially 2000 residues or fewer, in particular 1500 residues or fewer.
  • An antigen may contain 1000 residues or fewer, 800 residues or fewer, 600 residues or fewer, 400 residues or fewer or 200 residues or fewer.
  • an antigen contains 50 residues or more, especially 100 residues or more, in particular 150 residues or more.
  • an antigen contains 50 to 3000 residues, especially 100 to 1500 residues, in particular 200 to 1000 residues.
  • the antigen is derived from a bacterium.
  • the antigen is derived from a gram-negative bacterium, such as Clostridia.
  • the antigen is derived from C. difficile, such as specifically C. difficile toxin A and/or toxin B or fragments thereof, especially a fusion of fragments of toxin A and toxin B such as an F2 antigen (see, e.g., WO2012163811).
  • the F2 antigen may be a sequence comprising, such as consisting of, a sequence having at least 80%, such as at least 90%, especially at least 95%, in particular at least 98% for example at least 99% or 100% identity to SEQ ID No: 2.
  • the antigen may be an F2 antigen.
  • the antigen is derived from a virus.
  • the antigen may be a respiratory syncytial virus (RSV) antigen.
  • RSV respiratory syncytial virus
  • the antigen is derived from a herpesvirus, such as human alphaherpesvirus 3 (HHV-3), also known as varicella-zoster virus (VZV). More suitably the antigen is the VZV-gE antigen.
  • the VZV-gE antigen may be a sequence comprising, such as consisting of, a sequence having at least 80%, such as at least 90%, especially at least 95%, in particular at least 98% for example at least 99% or 100% identity to SEQ ID No: 1.
  • the antigen is an influenza virus antigen, such as a hemagglutinin or a neuraminidase.
  • the antigen is a coronavirus antigen, such as a spike protein.
  • a STING agonist and a first antigen is administered in step (i) and a STING agonist and a second antigen is administered in step (ii).
  • the antigens are all polypeptides. In another embodiment, the antigens are all provided in the form of polynucleotides, such as a viral vector or mRNA. In a further embodiment, the antigens are all polysaccharides.
  • the first antigen is a polypeptide and the second antigen is provided in the form a polynucleotide, such as a viral vector or mRNA.
  • the second antigen is a polypeptide and the first antigen is provided in the form a polynucleotide, such as a viral vector or mRNA.
  • the antigens are all polypeptides.
  • the antigens may be derived from (i.e. associated with) a pathogen, especially a human pathogen, (such as a bacterium, virus or parasite) or may be a cancer antigen (such as a tumour antigen and/or a neoantigen).
  • a pathogen especially a human pathogen, (such as a bacterium, virus or parasite) or may be a cancer antigen (such as a tumour antigen and/or a neoantigen).
  • antigen is meant a substance which is capable of eliciting an immune response in a subject.
  • the immune response from the first and/or second antigen is a protective immune response, e.g. reducing partially or completely the severity of one or more symptoms and/or time over which one or more symptoms are experienced by a subject, reducing the likelihood of developing an established infection after challenge and/or slowing progression of an associated illness (e.g. extending survival).
  • the immune response to the second antigen in administration under step (ii) is a protective immune response.
  • the first antigen may be delivered in the same form (i.e. a ‘homologous’ dosage regime), in different forms (i.e. a ‘heterologous’ dosage regime), or any combination of the same form and different forms, over each of the multiple administrations.
  • the first antigen in a first administration in step (i) is a polypeptide
  • the first antigen in a second administration in step (i) is a polynucleotide encoding the polypeptide
  • the antigens delivered in each administration are delivered in heterologous forms but nevertheless the same antigen for the purposes of the invention.
  • the second antigen may be delivered in the same form (i.e. a ‘homologous’ dosage regime), in different forms (i.e. a ‘heterologous’ dosage regime), or any combination of the same form and different forms, over each of the multiple administrations.
  • the second antigen in a first administration in step (ii) is a polypeptide and the second antigen in a second administration is a polynucleotide encoding the polypeptide then the antigens delivered in each administration are delivered in heterologous forms but are the same antigen for the purposes of the invention.
  • the first antigen and the second antigen are different, i.e the first antigen has a different polypeptide, different polypeptide encoded by a polynucleotide or different polysaccharide sequence to the second antigen.
  • the first antigen is derived from a different pathogen to the second antigen.
  • a different pathogen is a pathogen which belongs to a different species, especially genus, in particular family, such as order, especially class, in particular phylum, such as kingdom or especially domain.
  • pathogens may be considered different if the first antigen is derived from (such as containing a portion of a polypeptide sequence obtainable from) one of bacteria, viruses or parasites and the second antigen is derived from another of bacteria, viruses or parasites.
  • the first antigen is derived from a virus and the second antigen is derived from a bacterium.
  • the first antigen may be derived from a bacterium and the second antigen may be derived from a virus.
  • an antigen is derived from a pathogen if it contains a polypeptide sequence which is obtainable from said pathogen.
  • the polypeptide sequence which is obtainable from said pathogen is at least 50, especially at least 100, in particular at least 150, such as at least 200 amino acids long.
  • the first antigen and the second antigen may be different antigens if the first antigen does not comprise any epitopes which are cross-reactive with epitopes of the second antigen in a CD4+ T cell, CD8+ T cell and/or antibody response.
  • the first antigen and the second antigen may be different antigens if the first antigen does not comprise any epitopes which are cross-reactive with epitopes of the second antigen in a CD4+ T cell response.
  • the first antigen and the second antigen may be different antigens if the first antigen does not comprise any epitopes which are cross-reactive with epitopes of the second antigen in a CD8+ T cell response.
  • the first antigen and the second antigen may be different antigens if the first antigen does not comprise any epitopes which are cross-reactive with epitopes of the second antigen in an antibody response.
  • the first antigen and the second antigen may be different antigens if the first antigen does not comprise any epitopes which are cross-reactive with epitopes of the second antigen in a CD4+ T cell and CD8+ T cell response.
  • the first antigen and the second antigen may be different antigens if the first antigen does not comprise any epitopes which are cross-reactive with epitopes of the second antigen in a CD4+ T cell and antibody response.
  • the first antigen and the second antigen may be different antigens if the first antigen does not comprise any epitopes which are cross-reactive with epitopes of the second antigen in a CD8+ T cell and antibody response.
  • the first antigen and the second antigen may be different antigens if the first antigen does not comprise any epitopes which are cross-reactive with epitopes of the second antigen in a CD4+ T cell, CD8+ T cell and antibody response.
  • the first antigen and the second antigen are different antigens if the second antigen does not comprise any epitopes which are cross-reactive with epitopes of the first antigen.
  • the CD4+ or CD8+ T cell immunoassay may for example be long- or short-term culture, restimulation, ELISpot, ELISA or bead-based assays for cytokine quantification, cytometry-based phenotyping (including tetramer staining) or cytometry-based intracellular staining.
  • the antibody immunoassay may for example be ELISA, affinity measurements, measurements of antibody functionality (such as virus neutralization assays, virus inhibition assays, ADCC assays, ADCP assays, complement activation or antibody-dependent cytokine secretion).
  • an antigen comprises at least one B or T cell epitope, suitably an antigen comprises B and T cell epitopes.
  • the elicited immune response may be an antigen specific B cell response which produces neutralizing antibodies.
  • the elicited immune response may be an antigen specific T cell response, which may be a systemic and/or a local response.
  • the antigen specific T cell response may comprise a CD4+ T cell response, such as a response involving CD4+ T cells expressing a plurality of cytokines, e.g. IFNgamma, TNFalpha and/or IL2.
  • the antigen specific T cell response comprises a CD8+ T cell response, such as a response involving CD8+ T cells expressing a plurality of cytokines, e.g., IFNgamma, TNFalpha and/or IL2.
  • cytokines e.g., IFNgamma, TNFalpha and/or IL2.
  • an antigen contains 3000 residues or fewer, especially 2000 residues or fewer, in particular 1500 residues or fewer.
  • An antigen may contain 1000 residues or fewer, 800 residues or fewer, 600 residues or fewer, 400 residues or fewer or 200 residues or fewer.
  • an antigen contains 50 residues or more, especially 100 residues or more, in particular 150 residues or more.
  • an antigen contains 50 to 3000 residues, especially 100 to 1500 residues, in particular 200 to 1000 residues.
  • the first antigen is derived from a virus.
  • the first antigen is derived from a herpesvirus, such as human alphaherpesvirus 3 (HHV-3), also known as varicella-zoster virus (VZV).
  • HHV-3 human alphaherpesvirus 3
  • VZV varicella-zoster virus
  • the VZV- gE antigen may be a sequence comprising, such as consisting of, a sequence having at least 80%, such as at least 90%, especially at least 95%, in particular at least 98% for example at least 99% or 100% identity to SEQ ID No: 1 .
  • the first antigen is an influenza virus antigen, such as a hemagglutinin or a neuraminidase.
  • the first antigen is derived from a bacterium.
  • the first antigen is derived from a different organism to the second antigen.
  • the second antigen is derived from a bacterium.
  • the second antigen is derived from a gram-negative bacterium, such as Clostridia. More suitably the second antigen is derived from C. difficile, such as specifically C. difficile toxin A and/or toxin B or fragments thereof, especially a fusion of fragments of toxin A and toxin B such as an F2 antigen (see, e.g., WO2012163811).
  • the F2 antigen may be a sequence comprising, such as consisting of, a sequence having at least 80%, such as at least 90%, especially at least 95%, in particular at least 98% for example at least 99% or 100% identity to SEQ ID No: 2.
  • the first antigen may be a VZV-gE antigen and the second antigen an F2 antigen.
  • the second antigen is derived from a virus.
  • the second antigen may be a respiratory syncytial virus (RSV) antigen.
  • RSV respiratory syncytial virus
  • the second antigen is an influenza virus antigen, such as a hemagglutinin or a neuraminidase.
  • the second antigen is a coronavirus antigen, such as a spike protein.
  • a STING agonist is administered in step (i) and an antigen is administered in step (ii) optionally with a second adjuvant.
  • the antigen is a polypeptide.
  • the antigen is provided in the form of a polynucleotide, such as a viral vector or mRNA.
  • the antigen is a polysaccharide.
  • the antigens may be derived from (i.e. associated with) a pathogen, especially a human pathogen (such as a bacterium, virus or parasite) or may be a cancer antigen (such as a tumour antigen and/or a neoantigen).
  • a pathogen especially a human pathogen (such as a bacterium, virus or parasite) or may be a cancer antigen (such as a tumour antigen and/or a neoantigen).
  • antigen is meant a substance which is capable of eliciting an immune response in a subject.
  • the immune response from antigen is a protective immune response, e.g. reducing partially or completely the severity of one or more symptoms and/or time over which one or more symptoms are experienced by a subject, reducing the likelihood of developing an established infection after challenge and/or slowing progression of an associated illness (e.g. extending survival).
  • the antigen may be delivered in the same form (i.e. a ‘homologous’ dosage regime), in different forms (i.e. a ‘heterologous’ dosage regime), or any combination of the same form and different forms, over each of the multiple administrations.
  • a first administration in step (ii) is a polypeptide
  • the antigen in a second administration is a polynucleotide encoding the polypeptide
  • the antigens delivered in each administration are delivered in heterologous forms but are the same antigen for the purposes of the invention.
  • the antigen is derived from a bacterium.
  • the antigen may be derived from a virus.
  • an antigen is derived from a pathogen if it contains a polypeptide sequence which is obtainable from said pathogen.
  • the polypeptide sequence which is obtainable from said pathogen is at least 50, especially at least 100, in particular at least 150, such as at least 200 amino acids long.
  • an antigen comprises at least one B or T cell epitope, suitably an antigen comprises B and T cell epitopes.
  • the elicited immune response may be an antigen specific B cell response which produces neutralizing antibodies.
  • the elicited immune response may be an antigen specific T cell response, which may be a systemic and/or a local response.
  • the antigen specific T cell response may comprise a CD4+ T cell response, such as a response involving CD4+ T cells expressing a plurality of cytokines, e.g. IFNgamma, TNFalpha and/or IL2.
  • the antigen specific T cell response comprises a CD8+ T cell response, such as a response involving CD8+ T cells expressing a plurality of cytokines, e.g., IFNgamma, TNFalpha and/or IL2.
  • cytokines e.g., IFNgamma, TNFalpha and/or IL2.
  • an antigen contains 3000 residues or fewer, especially 2000 residues or fewer, in particular 1500 residues or fewer.
  • An antigen may contain 1000 residues or fewer, 800 residues or fewer, 600 residues or fewer, 400 residues or fewer or 200 residues or fewer.
  • an antigen contains 50 residues or more, especially 100 residues or more, in particular 150 residues or more.
  • an antigen contains 50 to 3000 residues, especially 100 to 1500 residues, in particular 200 to 1000 residues.
  • the antigen is derived from a bacterium.
  • the antigen is derived from a gram-negative bacterium, such as Clostridia.
  • the antigen is derived from C. difficile, such as specifically C. difficile toxin A and/or toxin B or fragments thereof, especially a fusion of fragments of toxin A and toxin B such as an F2 antigen (see, e.g., WO2012163811).
  • the F2 antigen may be a sequence comprising, such as consisting of, a sequence having at least 80%, such as at least 90%, especially at least 95%, in particular at least 98% for example at least 99% or 100% identity to SEQ ID No: 2.
  • the antigen may be an F2 antigen.
  • the antigen is derived from a virus.
  • the antigen may be a respiratory syncytial virus (RSV) antigen.
  • RSV respiratory syncytial virus
  • the antigen is derived from a herpesvirus, such as human alphaherpesvirus 3 (HHV-3), also known as varicella-zoster virus (VZV).
  • HHV-3 human alphaherpesvirus 3
  • VZV varicella-zoster virus
  • the VZV-gE antigen may be a sequence comprising, such as consisting of, a sequence having at least 80%, such as at least 90%, especially at least 95%, in particular at least 98% for example at least 99% or 100% identity to SEQ ID No: 1.
  • the antigen is an influenza virus antigen, such as a hemagglutinin or a neuraminidase.
  • the antigen is a coronavirus antigen, such as a spike protein.
  • a STING agonist and a first antigen are administered in step (i) and a second antigen and optional second adjuvant are administered in step (ii).
  • the antigens are all polypeptides. In another embodiment, the antigens are all provided in the form of polynucleotides, such as a viral vector or mRNA. In a further embodiment, the antigens are all polysaccharides.
  • the first antigen is a polypeptide and the second antigen is provided in the form a polynucleotide, such as a viral vector or mRNA.
  • the second antigen is a polypeptide and the first antigen is provided in the form a polynucleotide, such as a viral vector or mRNA.
  • the antigens are all polypeptides.
  • the antigens may be derived from (i.e. associated with) a pathogen, especially a human pathogen (such as a bacterium, virus or parasite) or may be a cancer antigen (such as a tumour antigen and/or a neoantigen).
  • a pathogen especially a human pathogen (such as a bacterium, virus or parasite) or may be a cancer antigen (such as a tumour antigen and/or a neoantigen).
  • antigen is meant a substance which is capable of eliciting an immune response in a subject.
  • the immune response from the first and/or second antigen is a protective immune response, e.g. reducing partially or completely the severity of one or more symptoms and/or time over which one or more symptoms are experienced by a subject, reducing the likelihood of developing an established infection after challenge and/or slowing progression of an associated illness (e.g. extending survival).
  • the immune response to the second antigen in administration under step (ii) is a protective immune response.
  • the first antigen may be delivered in the same form (i.e. a ‘homologous’ dosage regime), in different forms (i.e. a ‘heterologous’ dosage regime), or any combination of the same form and different forms, over each of the multiple administrations.
  • the first antigen in a first administration in step (i) is a polypeptide
  • the first antigen in a second administration in step (i) is a polynucleotide encoding the polypeptide
  • the antigens delivered in each administration are delivered in heterologous forms but nevertheless the same antigen for the purposes of the invention.
  • the second antigen may be delivered in the same form (i.e. a ‘homologous’ dosage regime), in different forms (i.e. a ‘heterologous’ dosage regime), or any combination of the same form and different forms, over each of the multiple administrations.
  • the second antigen in a first administration in step (ii) is a polypeptide and the second antigen in a second administration is a polynucleotide encoding the polypeptide then the antigens delivered in each administration are delivered in heterologous forms but are the same antigen for the purposes of the invention.
  • the first antigen and the second antigen are different, i.e the first antigen has a different polypeptide, different polypeptide encoded by a polynucleotide or different polysaccharide sequence to the second antigen.
  • the first antigen is derived from a different pathogen to the second antigen.
  • the first antigen is derived from a different pathogen from the pathogen presenting the risk of infection.
  • a different pathogen is a pathogen which belongs to a different species, especially genus, in particular family, such as order, especially class, in particular phylum, such as kingdom or especially domain.
  • pathogens may be considered different if the first antigen is derived from (such as containing a portion of a polypeptide sequence obtainable from) one of bacteria, viruses or parasites and the second antigen is derived from another of bacteria, viruses or parasites.
  • the first antigen is derived from a virus and the second antigen is derived from a bacterium.
  • the first antigen is derived from a virus and the pathogen presenting the risk of infection is a bacterium.
  • the first antigen may be derived from a bacterium and the second antigen may be derived from a virus.
  • the first antigen is derived from a bacterium and the pathogen presenting the risk of infection is a virus.
  • the first antigen is derived from a virus and the pathogen presenting the risk of infection is a virus.
  • an antigen is derived from a pathogen if it contains a polypeptide sequence which is obtainable from said pathogen.
  • the polypeptide sequence which is obtainable from said pathogen is at least 50, especially at least 100, in particular at least 150, such as at least 200 amino acids long.
  • the first antigen and the second antigen may be different antigens if the first antigen does not comprise any epitopes which are cross-reactive with epitopes of the second antigen in a CD4+ T cell, CD8+ T cell and/or antibody response.
  • the first antigen and the second antigen may be different antigens if the first antigen does not comprise any epitopes which are cross-reactive with epitopes of the second antigen in a CD4+ T cell response.
  • the first antigen and the second antigen may be different antigens if the first antigen does not comprise any epitopes which are cross-reactive with epitopes of the second antigen in a CD8+ T cell response.
  • the first antigen and the second antigen may be different antigens if the first antigen does not comprise any epitopes which are cross-reactive with epitopes of the second antigen in an antibody response.
  • the first antigen and the second antigen may be different antigens if the first antigen does not comprise any epitopes which are cross-reactive with epitopes of the second antigen in a CD4+ T cell and CD8+ T cell response.
  • the first antigen and the second antigen may be different antigens if the first antigen does not comprise any epitopes which are cross-reactive with epitopes of the second antigen in a CD4+ T cell and antibody response.
  • the first antigen and the second antigen may be different antigens if the first antigen does not comprise any epitopes which are cross-reactive with epitopes of the second antigen in a CD8+ T cell and antibody response.
  • the first antigen and the second antigen may be different antigens if the first antigen does not comprise any epitopes which are cross-reactive with epitopes of the second antigen in a CD4+ T cell, CD8+ T cell and antibody response.
  • the first antigen and the second antigen are different antigens if the second antigen does not comprise any epitopes which are cross-reactive with epitopes of the first antigen.
  • the first antigen does not comprise any epitopes which are cross-reactive with epitopes of the pathogen presenting the risk of infection in a CD4+ T cell, CD8+ T cell and/or antibody response.
  • the first antigen suitably does not comprise any epitopes which are cross-reactive with epitopes of the pathogen presenting the risk of infection in a CD4+ T cell response.
  • the first antigen suitably does not comprise any epitopes which are cross-reactive with epitopes of the pathogen presenting the risk of infection in a CD8+ T cell response.
  • the first antigen suitably does not comprise any epitopes which are cross-reactive with epitopes of the pathogen presenting the risk of infection in an antibody response.
  • the first antigen suitably does not comprise any epitopes which are cross-reactive with epitopes of the pathogen presenting the risk of infection in a CD4+ T cell and CD8+ T cell response.
  • the first antigen suitably does not comprise any epitopes which are cross-reactive with epitopes of the pathogen presenting the risk of infection in a CD4+ T cell and antibody response.
  • the first antigen suitably does not comprise any epitopes which are cross-reactive with epitopes of the pathogen presenting the risk of infection in a CD8+ T cell and antibody response.
  • the first antigen suitably does not comprise any epitopes which are cross-reactive with epitopes of the pathogen presenting the risk of infection in a CD4+ T cell, CD8+ T cell and antibody response.
  • the CD4+ or CD8+ T cell immunoassay may for example be long- or short-term culture, restimulation, ELISpot, ELISA or bead-based assays for cytokine quantification, cytometry-based phenotyping (including tetramer staining) or cytometry-based intracellular staining.
  • the antibody immunoassay may for example be ELISA, affinity measurements, measurements of antibody functionality (such as virus neutralization assays, virus inhibition assays, ADCC assays, ADCP assays, complement activation or antibody-dependent cytokine secretion).
  • an antigen comprises at least one B or T cell epitope, suitably an antigen comprises B and T cell epitopes.
  • the elicited immune response may be an antigen specific B cell response which produces neutralizing antibodies.
  • the elicited immune response may be an antigen specific T cell response, which may be a systemic and/or a local response.
  • the antigen specific T cell response may comprise a CD4+ T cell response, such as a response involving CD4+ T cells expressing a plurality of cytokines, e.g. IFNgamma, TNFalpha and/or IL2.
  • the antigen specific T cell response comprises a CD8+ T cell response, such as a response involving CD8+ T cells expressing a plurality of cytokines, e.g., IFNgamma, TNFalpha and/or IL2.
  • cytokines e.g., IFNgamma, TNFalpha and/or IL2.
  • an antigen contains 3000 residues or fewer, especially 2000 residues or fewer, in particular 1500 residues or fewer.
  • An antigen may contain 1000 residues or fewer, 800 residues or fewer, 600 residues or fewer, 400 residues or fewer or 200 residues or fewer.
  • an antigen contains 50 residues or more, especially 100 residues or more, in particular 150 residues or more.
  • an antigen contains 50 to 3000 residues, especially 100 to 1500 residues, in particular 200 to 1000 residues.
  • the first antigen is derived from a virus.
  • the first antigen is derived from a herpesvirus, such as human alphaherpesvirus 3 (HHV-3), also known as varicella-zoster virus (VZV).
  • HHV-3 human alphaherpesvirus 3
  • VZV varicella-zoster virus
  • the VZV- gE antigen may be a sequence comprising, such as consisting of, a sequence having at least 80%, such as at least 90%, especially at least 95%, in particular at least 98% for example at least 99% or 100% identity to SEQ ID No: 1 .
  • the first antigen is an influenza virus antigen, such as a hemagglutinin or a neuraminidase.
  • the first antigen is derived from a bacterium.
  • the first antigen is derived from a different organism to the second antigen.
  • the second antigen is derived from a bacterium.
  • the second antigen is derived from a gram-negative bacterium, such as Clostridia. More suitably the second antigen is derived from C. difficile, such as specifically C. difficile toxin A and/or toxin B or fragments thereof, especially a fusion of fragments of toxin A and toxin B such as an F2 antigen (see, e.g., WO2012163811).
  • the F2 antigen may be a sequence comprising, such as consisting of, a sequence having at least 80%, such as at least 90%, especially at least 95%, in particular at least 98% for example at least 99% or 100% identity to SEQ ID No: 2.
  • the first antigen may be a VZV-gE antigen and the second antigen an F2 antigen.
  • the second antigen is derived from a virus.
  • the second antigen may be a respiratory syncytial virus (RSV) antigen.
  • the second antigen is an influenza virus antigen, such as a hemagglutinin or a neuraminidase.
  • the second antigen is a coronavirus antigen, such as a spike protein.
  • an antigen is a polynucleotide
  • the antigen may be in the form of mRNA.
  • Messenger RNA can direct the cellular machinery of a subject to produce proteins.
  • mRNA may be circular or branched, but will generally be linear.
  • mRNA used herein are preferably provided in purified or substantially purified form i.e. substantially free from proteins (e.g., enzymes), other nucleic acids (e.g. DNA and nucleoside phosphate monomers), and the like, generally being at least about 50% pure (by weight), and usually at least 90% pure, such as at least 95% or at least 98% pure.
  • mRNA may be prepared in many ways e.g.
  • RNA may be prepared enzymatically using a DNA template.
  • mRNA as used herein includes conventional mRNA or mRNA analogs, such as those containing modified backbones or modified bases (e.g. pseudouridine, or the like). mRNA, may or may not have a 5' cap.
  • the mRNA comprises a sequence which encodes at least one antigen.
  • the nucleic acids of the invention will be in recombinant form, i.e. a form which does not occur in nature.
  • the mRNA may comprise one or more heterologous nucleic acid sequences (e.g. a sequence encoding another antigen and/or a control sequence such as a promoter or an internal ribosome entry site) in addition to the sequence encoding the antigen.
  • sequence or chemical structure of the nucleic acid may be modified compared to a naturally-occurring sequence which encodes the antigen.
  • the sequence of the nucleic acid molecule may be modified, e.g. to increase the efficacy of expression or replication of the nucleic acid, or to provide additional stability or resistance to degradation.
  • mRNA may also be codon optimised.
  • mRNA may be codon optimised for expression in human cells.
  • codon optimised is intended modification with respect to codon usage which may increase translation efficacy and/or half-life of the nucleic acid.
  • a poly A tail (e.g., of about 30 adenosine residues or more) may be attached to the 3' end of the RNA to increase its half-life.
  • the 5' end of the RNA may be capped, for example with a modified ribonucleotide with the structure m7G (5') ppp (5') N (cap 0 structure) or a derivative thereof, which can be incorporated during RNA synthesis or can be enzymatically engineered after RNA transcription (e.g., by using Vaccinia Virus Capping Enzyme (VCE) consisting of mRNA triphosphatase, guanylyl-transferase and guanine-7-methytransferase, which catalyzes the construction of N7- monomethylated cap 0 structures).
  • VCE Vaccinia Virus Capping Enzyme
  • Cap 0 structure plays an important role in maintaining the stability and translational efficacy of the mRNA molecule.
  • the 5' cap of the mRNA molecule may be further modified by a 2'-O-Methyltransferase which results in the generation of a cap 1 structure (m7Gppp [m2'-O] N), which may further increase translation efficacy.
  • mRNA may comprise one or more nucleotide analogs or modified nucleotides.
  • nucleotide analog or “modified nucleotide” refers to a nucleotide that contains one or more chemical modifications (e.g., substitutions) in or on the nitrogenous base of the nucleoside (e.g.
  • a nucleotide analog can contain further chemical modifications in or on the sugar moiety of the nucleoside (e.g. ribose, modified ribose, six-membered sugar analog, or open-chain sugar analog), or the phosphate.
  • the preparation of nucleotides and modified nucleotides and nucleosides are well-known in the art, see the following references: US Patent Numbers 4373071, 4458066, 4500707, 4668777, 4973679, 5047524, 5132418, 5153319, 5262530, 5700642. Many modified nucleosides and modified nucleotides are commercially available.
  • Modified nucleobases which can be incorporated into modified nucleosides and nucleotides and be present in the mRNA molecules include: m5C (5- methylcytidine), m5U (5-methyluridine), m6A (N6-methyladenosine), s2U (2-thiouridine), Um (2'-O-methyluridine), m1A (1 -methyladenosine); m2A (2-methyladenosine); Am (2-1-0- methyladenosine); ms2m6A (2-methylthio-N6-methyladenosine); i6A (N6- isopentenyladenosine); ms2i6A (2-methylthio-N6isopentenyladenosine); io6A (N6-(cis- hydroxyisopentenyl)adenosine); ms2io6A (2-methylthio-N6-(cis-hydroxyisopentenyl)a
  • the mRNA may encode more than one antigen.
  • the mRNA encoding an antigen protein may encode only the antigen or may encode additional proteins.
  • mRNA may be non-replicating or may be replicating, also known as self-amplifying.
  • a self-amplifying mRNA molecule may be an alphavirus-derived mRNA replicon.
  • mRNA amplification can also be achieved by the provision of a non-replicating mRNA encoding an antigen in conjunction with a separate mRNA encoding replication machinery.
  • Self-replicating RNA molecules are well known in the art and can be produced by using replication elements derived from, e.g., alphaviruses, and substituting the structural viral proteins with a nucleotide sequence encoding a protein of interest.
  • a self-replicating RNA molecule is typically a +-strand molecule which can be directly translated after delivery to a cell, and this translation provides a RNA-dependent RNA polymerase which then produces both antisense and sense transcripts from the delivered RNA. Thus the delivered RNA leads to the production of multiple daughter RNAs.
  • RNAs may be translated themselves to provide in situ expression of an encoded antigen, or may be transcribed to provide further transcripts with the same sense as the delivered RNA which are translated to provide in situ expression of the antigen.
  • the overall result of this sequence of transcriptions is a huge amplification in the number of the introduced replicon RNAs and so the encoded antigen becomes a major polypeptide product of the cells.
  • Suitable alphavirus replicons can use a replicase from a Sindbis virus, a Semliki forest virus, an eastern equine encephalitis virus, a Venezuelan equine encephalitis virus, etc.
  • Mutant or wild-type virus sequences can be used e.g. the attenuated TC83 mutant of VEEV has been used in replicons, see the following reference: W02005/113782.
  • the self-replicating RNA molecule described herein encodes (i) a RNA-dependent RNA polymerase which can transcribe RNA from the self-replicating RNA molecule and (ii) an antigen.
  • the polymerase can be an alphavirus replicase e.g. comprising one or more of alphavirus proteins nsPI, nsP2, nsP3 and nsP4.
  • the self-replicating RNA molecules do not encode alphavirus structural proteins.
  • the self-replicating RNA can lead to the production of genomic RNA copies of itself in a cell, but not to the production of RNA-containing virions.
  • the inability to produce these virions means that, unlike a wild-type alphavirus, the self-replicating RNA molecule cannot perpetuate itself in infectious form.
  • alphavirus structural proteins which are necessary for perpetuation in wild-type viruses are absent from self-replicating RNAs of the present disclosure and their place is taken by gene(s) encoding the immunogen of interest, such that the subgenomic transcript encodes the immunogen rather than the structural alphavirus virion proteins.
  • RNA molecule useful with the invention may have two open reading frames.
  • the first (5') open reading frame encodes a replicase; the second (3') open reading frame encodes an antigen.
  • the RNA may have additional (e.g. downstream) open reading frames e.g. to encode further antigens or to encode accessory polypeptides.
  • the self-replicating RNA molecule disclosed herein has a 5' cap (e.g. a 7-methylguanosine). This cap can enhance in vivo translation of the RNA.
  • the 5' sequence of the self-replicating RNA molecule must be selected to ensure compatibility with the encoded replicase.
  • a self-replicating RNA molecule may have a 3' poly-A tail. It may also include a poly-A polymerase recognition sequence (e.g. AALIAAA) near its 3' end.
  • AALIAAA poly-A polymerase recognition sequence
  • Self-replicating RNA molecules can have various lengths, but they are typically 5000- 25000 nucleotides long. Self-replicating RNA molecules will typically be single-stranded. Single-stranded RNAs can generally initiate an adjuvant effect by binding to TLR7, TLR8, RNA helicases and/or PKR. RNA delivered in double-stranded form (dsRNA) can bind to TLR3, and this receptor can also be triggered by dsRNA which is formed either during replication of a single-stranded RNA or within the secondary structure of a single-stranded RNA.
  • dsRNA double-stranded form
  • a self-replicating RNA may comprise two separate RNA molecules, each comprising a nucleotide sequence derived from an alphavirus: one RNA molecule comprises a RNA construct for expressing alphavirus replicase, and one RNA molecule comprises a RNA replicon that can be replicated by the replicase in trans.
  • the RNA construct for expressing alphavirus replicase comprises a 5'-cap. See WO2017/162265.
  • the self-replicating RNA can conveniently be prepared by in vitro transcription (IVT).
  • IVT can use a (cDNA) template created and propagated in plasmid form in bacteria, or created synthetically (for example by gene synthesis and/or polymerase chain-reaction (PCR) engineering methods).
  • a DNA-dependent RNA polymerase such as the bacteriophage T7, T3 or SP6 RNA polymerases
  • Appropriate capping and poly-A addition reactions can be used as required (although the replicon's poly-A is usually encoded within the DNA template).
  • RNA polymerases can have stringent requirements for the transcribed 5' nucleotide(s) and in some embodiments these requirements must be matched with the requirements of the encoded replicase, to ensure that the IVT-transcribed RNA can function efficiently as a substrate for its self-encoded replicase.
  • a self-replicating RNA can include (in addition to any 5' cap structure) one or more nucleotides having a modified nucleobase.
  • a RNA used with the invention ideally includes only phosphodiester linkages between nucleosides, but in some embodiments it can contain phosphoramidate, and/or methylphosphonate linkages.
  • the self-replicating RNA molecule may encode a single heterologous polypeptide antigen (i.e. the antigen) or, optionally, two or more heterologous polypeptide antigens linked together in a way that each of the sequences retains its identity (e.g., linked in series) when expressed as an amino acid sequence.
  • the heterologous polypeptides generated from the self-replicating RNA may then be produced as a fusion polypeptide or engineered in such a manner to result in separate polypeptide or peptide sequences.
  • the self-replicating RNA molecules described herein may be engineered to express multiple nucleotide sequences, from two or more open reading frames, thereby allowing coexpression of proteins, such as one, two or more antigens (e.g. one, two or more coronavirus protein(s), such as SARS-CoV-2 S protein(s)) together with cytokines or other immunomodulators, which can enhance the generation of an immune response.
  • proteins such as one, two or more antigens (e.g. one, two or more coronavirus protein(s), such as SARS-CoV-2 S protein(s)) together with cytokines or other immunomodulators, which can enhance the generation of an immune response.
  • cytokines or other immunomodulators which can enhance the generation of an immune response.
  • Such a selfreplicating RNA molecule might be particularly useful, for example, in the production of various gene products (e.g., proteins) at the same time, for example, as a bivalent or multivalent vaccine.
  • the self-replicating RNA molecules can be screened or analyzed to confirm their therapeutic and prophylactic properties using various in vitro or in vivo testing methods that are known to those of skill in the art.
  • vaccines comprising self-replicating RNA molecule can be tested for their effect on induction of proliferation or effector function of the particular lymphocyte type of interest, e.g., B cells, T cells, T cell lines, and T cell clones.
  • lymphocyte type of interest e.g., B cells, T cells, T cell lines, and T cell clones.
  • spleen cells from immunized mice can be isolated and the capacity of cytotoxic T lymphocytes to lyse autologous target cells that contain a self-replicating RNA molecule that encodes an antigen.
  • T helper cell differentiation can be analyzed by measuring proliferation or production of TH1 (IL-2 and IFN-y) and /or TH2 (IL-4 and IL-5) cytokines by ELISA or directly in CD4+ T cells by cytoplasmic cytokine staining and flow cytometry.
  • TH1 IL-2 and IFN-y
  • TH2 IL-4 and IL-5
  • Self-replicating RNA molecules that encode an antigen can also be tested for ability to induce humoral immune responses, as evidenced, for example, by induction of B cell production of antibodies specific for the antigen of interest.
  • These assays can be conducted using, for example, peripheral B lymphocytes from immunized individuals. Such assay methods are known to those of skill in the art.
  • Other assays that can be used to characterize the self-replicating RNA molecules can involve detecting expression of the encoded antigen by the target cells.
  • FACS can be used to detect antigen expression on the cell surface or intracellularly. Another advantage of FACS selection is that one can sort for different levels of expression; sometimes-lower expression may be desired.
  • Other suitable method for identifying cells which express a particular antigen involve panning using monoclonal antibodies on a plate or capture using magnetic beads coated with monoclonal antibodies.
  • a non-replicating mRNA will typically contain 10000 bases or fewer, especially 8000 bases or fewer, in particular 5000 base or fewer.
  • a replicating mRNA will typically contain 25000 bases or fewer, especially 20000 bases or fewer, in particular 15000 bases or fewer.
  • a single dose of mRNA may be 0.001 to 1000 ug, especially 1 to 500 ug, in particular 10 to 250 ug.
  • a single dose of mRNA may be 0.001 to 75 ug, 1 to 75 ug, 25 to 250 ug, or 250 to 1000 ug.
  • a replicating mRNA dose may be 0.001 to 75 ug, such as 0.1 to 75 ug.
  • a non-replicating mRNA dose may, for example, be 1 to 500 ug, such as 1 to 250 ug.
  • the mRNA is non-replicating mRNA. In a second embodiment the mRNA is replicating mRNA.
  • a range of carrier systems have been described which encapsulate or complex mRNA in order to facilitate mRNA delivery and consequent expression of encoded antigens as compared to mRNA which is not encapsulated or complexed.
  • the present invention may utilise any suitable carrier system.
  • Particular mRNA carrier systems of note are further described below.
  • Lipid nanoparticles are non-virion liposome particles in which mRNA can be encapsulated.
  • LNP delivery systems and methods for their preparation are known in the art.
  • the particles can include some external mRNA (e.g. on the surface of the particles), but desirably at least half of the RNA (and suitably at least 85%, especially at least 95%, such as all of it) is encapsulated.
  • LNP can, for example, be formed of a mixture of (i) a PEG-modified lipid (ii) a non-cationic lipid (iii) a sterol (iv) an ionisable cationic lipid.
  • LNP can for example be formed of a mixture of (i) a PEG-modified lipid (ii) a non-cationic lipid (iii) a sterol (iv) a non-ionisable cationic lipid.
  • the PEG-modified lipid may comprise a PEG molecule with a molecular weight of 10000 Da or less, especially 5000 Da or less, in particular 3000 Da, such 2000 Da or less.
  • the PEG-modified lipid may comprise a PEG molecule with an average molecular weight of 1500 Da to 2500 Da, such as 2000 Da.
  • Examples of PEG-modified lipids include PEG- distearoyl glycerol, PEG-dipalmitoyl glycerol and PEG-dimyristoyl glycerol.
  • the PEG-modified lipid is typically present at around 0.5 to 15 molar %.
  • the non-cationic lipid may be a neutral lipid, such as 1,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2- oleoyl-sn-glycero-3-phosphocholine (POPO), 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and sphingomyelin (SM).
  • DSPC 1,2-distearoyl-sn-glycero-3- phosphocholine
  • DPPC 1,2-dipalmitoyl-sn-glycero-3-phosphocholine
  • POPO 1-palmitoyl-2- oleoyl-sn-glycero-3-phosphocholine
  • DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • SM
  • the sterol may be cholesterol.
  • the sterol is typically present at around 25 to 55 molar %.
  • RNA to lipid can be varied (see for example WO2013/006825).
  • N:P ratio refers to the molar ratio of protonatable nitrogen atoms in the cationic lipids (typically solely in the lipid's headgroup) to phosphates in the RNA.
  • the ratio of nucleotide (N) to phospholipid (P) can be in the range of, e.g., 1 N:1P to 20N:1 P, 1N:1P to 10N:1 P, 2N:1 P to 8N:1 P, 2N:1P to 6N:1 P or 3N:1 P to 5N:1 P.
  • the ratio of nucleotide (N) to phospholipid (P) can be in the range of, e.g., 1 N:1P, 2N:1P, 3N:1P, 4N:1 P, 5N:1 P, 6N:1 P, 7N:1 P, 8N:1P, 9N:1P, or 10N:1P.
  • the ratio of nucleotide (N) to phospholipid (P) is 4N:1 P.
  • WO2017/070620 provides general information on LNP compositions and is incorporated herein by reference.
  • Other useful LNPs are described in the following references: WO2012/006376; WO2012/030901 ; WO2012/031046; WO2012/031043; WO2012/006378; WO2011/076807; WO2013/033563; WO2013/006825; WO2014/136086; WO2015/095340; WO2015/095346; WO2016/037053, which are also incorporated herein by reference.
  • Liposomal particles can, for example, be formed of a mixture of zwitterionic, cationic and anionic lipids which can be saturated or unsaturated, for example; DSPC (zwitterionic, saturated), DlinDMA (cationic, unsaturated), and/or DMG (anionic, saturated).
  • Preferred LNPs for use with the invention include an amphiphilic lipid which can form liposomes, optionally in combination with at least one cationic lipid (such as DOTAP, DSDMA, DODMA, DLinDMA, DLenDMA, etc.).
  • DOTAP DOTAP
  • DSDMA DODMA
  • DLinDMA DLenDMA
  • cholesterol a mixture of DSPC, DlinDMA, PEG-DMG and cholesterol is particularly effective.
  • the LNPs are RV01 liposomes, see the following references: WG2012/006376 and Geall et al. (2012) PNAS USA. Sep 4; 109(36): 14604-9.
  • LNPs are typically 50 to 200 urn in diameter (Z-average).
  • the LNPs have a polydispersity of 0.4 or less, such as 0.3 or less.
  • the carrier is a lipid nanoparticle (LNP).
  • LNP lipid nanoparticle
  • the carrier may be a cationic nanoemulsion (CNE) delivery system.
  • CNE cationic nanoemulsion
  • Such cationic oil- in-water emulsions can be used to deliver the mRNA to the interior of a cell.
  • the emulsion particles comprise a hydrophobic oil core and a cationic lipid, the latter of which can interact with the mRNA, thereby anchoring it to the emulsion particle.
  • the mRNA which encodes the antigen is complexed with a particle of a cationic oil-in-water emulsion.
  • CNE carriers and methods for their preparation are described in WO2012/006380, WO2013/006837 and WO2013/006834 which are incorporated herein by reference.
  • the mRNA may be complexed with a particle of a cationic oil-in-water emulsion.
  • the particles typically comprise an oil core (e.g. a plant oil or squalene) that is in liquid phase at 25°C, a cationic lipid (e.g. phospholipid) and, optionally, a surfactant (e.g. sorbitan trioleate, polysorbate 80); polyethylene glycol can also be included.
  • the CNE comprises squalene and a cationic lipid, such as 1,2-dioleoyloxy-3- (trimethylammonio)propane (DOTAP) (see e.g. Brito, 2014).
  • DOTAP 1,2-dioleoyloxy-3- (trimethylammonio)propane
  • the CNE is an oil-in-water emulsion of DOTAP and squalene stabilised with polysorbate 80 and/or sorbitan trioleate.
  • RNA is complexed with the cationic oil-in-water emulsion carrier.
  • CNE are typically 50 to 200 urn in diameter (Z-average).
  • the CNE have a polydispersity of 0.4 or less, such as 0.3 or less.
  • the carrier is a cationic nanoemulsion (CNE).
  • CNE cationic nanoemulsion
  • a lipidoid-coated iron oxide nanoparticle is capable of delivering mRNA into cells and may be aided after administration to a subject by application of an external magnetic field.
  • a LION is an iron oxide particle with one or more coatings comprising lipids and/or lipidoids wherein mRNA encoding the antigen is incorporated into or associated with the lipid and/or lipidoid coating(s) through electrostatic interactions. The mRNA being embedded within the coating(s) may offer protection from enzymatic degradation.
  • the lipids and/or lipidoids comprised within a LION may for example include those included in Figure S1 of Jiang, 2013, especially lipidoids comprising alkyl tails of 12 to 14 carbons in length and in particular lipidoid C14-200 as disclosed in Jiang, 2013.
  • a LION may typically comprise 200 to 5000, such as 500 to 2000, in particular about 1000 lipid and/or lipidoid molecules.
  • the LIONs are 20 to 200 nm in diameter, especially 50 to 100 nm in diameter.
  • the lipid/lipidoid to mRNA weight ratio may be about 1:1 to 10:1, especially about 5:1.
  • Particularly suitable LIONs, and methods for preparation of LIONs are disclosed in Jiang, 2013.
  • the carrier is a lipidoid-coated iron oxide nanoparticle (LION). Sequence alignments
  • Identity or homology with respect to a sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the reference amino acid sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
  • Sequence identity can be determined by standard methods that are commonly used to compare the similarity in position of the amino acids of two polypeptides. Using a computer program such as BLAST or FASTA, two polypeptides are aligned for optimal matching of their respective amino acids (either along the full length of one or both sequences or along a predetermined portion of one or both sequences). The programs provide a default opening penalty and a default gap penalty, and a scoring matrix such as PAM 250 [a standard scoring matrix; see Dayhoff et al., in Atlas of Protein Sequence and Structure, vol. 5, supp. 3 (1978)] can be used in conjunction with the computer program. For example, the percent identity can then be calculated as: the total number of identical matches multiplied by 100 and then divided by the sum of the length of the longer sequence within the matched span and the number of gaps introduced into the shorter sequences in order to align the two sequences.
  • the present invention may involve a plurality of antigenic components, for example with the objective to elicit a broad immune response e.g. to a pathogen or to elicit responses to multiple pathogens. Consequently, more than one antigen may be present, more than one polynucleotide encoding an antigen may be present, one polynucleotide encoding more than one antigen may be present or a mixture of antigen(s) and polynucleotide(s) encoding antigen(s) may be present. Polysaccharides such as polysaccharide conjugates, may also be present.
  • step (i) a STING agonist and a first antigen are administered in step (i) and a STING agonist and a second antigen are administered in step (ii).
  • step (i) involves only one antigen.
  • step (ii) involves an antigen. In some embodiments step (ii) involves only one antigen.
  • the present invention is generally intended for mammalian subjects, in particular human subjects.
  • the subject may be a wild or domesticated animal.
  • Mammalian subjects include for example cats, dogs, pigs, sheep, horses or cattle.
  • the subject is human.
  • the subject to be treated using the method of the invention may be of any age.
  • the subject is a human infant (up to 12 months of age). In one embodiment the subject is a human child (less than 18 years of age). In one embodiment the subject is an adult human (aged 18-59). In one embodiment the subject is an older human (aged 60 or greater).
  • Doses administered to younger children, such as less than 12 years of age, may be reduced relative to an equivalent adult dose, such as by 50%.
  • the methods of the invention may be intended for prophylaxis of infectious diseases, i.e. for administration to a subject which is not infected with a pathogen.
  • the methods of the invention may be intended for treatment, e.g. for the treatment of infectious diseases, i.e. for administration to a subject which is infected with a pathogen.
  • a STING agonist is administered to a subject at risk of infection.
  • the risk of infection is 2 weeks to 2 years, especially 2 weeks to 1 year, in particular 3 weeks to 9 months, such as 4 weeks to 6 months from completion of administration of STING agonist (i.e. step (i)).
  • the risk of infection is 2 weeks to 2 months from completion of completion of administration of STING agonist. In some embodiments the risk of infection is 1 month to 3 months from completion of administration of STING agonist. In some embodiments the risk of infection is 2 months to 4 months from completion of administration of STING agonist. In some embodiments the risk of infection is 3 months to 6 months from completion of administration of STING agonist. In some embodiments the risk of infection is 4 months to 12 months from completion of administration of STING agonist.
  • the subject at risk of infection may be a diabetic who has experienced an abrasion or injury.
  • the subject at risk of infection may be an individual expected to undergo hospital treatment, suitably the pathogen being Staphylococcus aureus.
  • the pathogen presenting a risk to the subject at risk of infection may be a pathogen causing a pandemic, such as an influenza virus or a coronavirus.
  • a STING agonist and an antigen are administered to a subject at risk of infection.
  • the risk of infection is 2 weeks to 2 years, especially 2 weeks to 1 year, in particular 3 weeks to 9 months, such as 4 weeks to 6 months from completion of administration of STING agonist and the first antigen (i.e. step (i)).

Abstract

The present invention relates inter alia to methods of eliciting an immune response by administration of a STING agonist.

Description

Novel Methods
TECHNICAL FIELD
The present invention relates to methods for improving immune responses with a STING agonist, and to related aspects.
BACKGROUND ART
Adjuvants are sometimes included in vaccines to improve humoral and cellular immune responses, particularly in the case of poorly immunogenic subunit vaccines. Similar to natural infections by pathogens, adjuvants rely on the activation of the innate immune system to promote long-lasting adaptive immunity. As simultaneous activation of multiple innate immune pathways is a feature of natural infections, adjuvants may combine multiple immunostimulants in order to promote adaptive immune responses to vaccination.
STING (STimulator of InterferoN Genes) plays an important role in innate immunity. It is an intracellular receptor, part of the pattern-recognition receptor (PRR) family. Upon activation, it triggers the STING signalling pathway leading (see, e.g., Dubensky et al. 2013).
There remains a need for the provision of further immunisation approaches that may have benefits such as reducing the variability of immune responses, improving immune responses (e.g. magnitude/level of the immune response, speed of the immune response, breadth and/or duration of the immune response) and/or reducing the amount of material, such as antigen, required to elicit a desired immune response.
SUMMARY OF THE INVENTION
The present invention provides a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject a STING agonist and an antigen which is associated with the pathogen.
Provided is also a method of administering to a subject a STING agonist and an antigen which is associated with a pathogen, wherein the subject was selected for having been previously administered a STING agonist, and optionally wherein the subject’s immune response is of increased level, speed, and/or breadth as compared to that of a subject who had not been previously administered the STING agonist.
Also provided is a method of reducing the variability of the immune response to an antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist, prior to
(ii) administering to each of the subjects a STING agonist and the antigen. Further provided is a method of improving an immune response to an antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject a STING agonist and the antigen.
Additionally provided is a method of reducing the amount of an antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the antigen and a STING agonist.
The present invention also provides a method for preparing the immune system of a subject for (ii) administration of an antigen and a STING agonist, comprising (i) administering to the subject a STING agonist.
The present invention provides a STING agonist for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject a STING agonist and an antigen which is associated with the pathogen.
Further provided is an antigen for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject a STING agonist and an antigen which is associated with the pathogen.
Additionally provided is a STING agonist for use in a method of reducing the variability of the immune response to an antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist, prior to
(ii) administering to each of the subjects a STING agonist and the antigen.
The present invention provides an antigen for use in a method of reducing the variability of the immune response to an antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist, prior to
(ii) administering to each of the subjects a STING agonist and the antigen.
Also provided is a STING agonist for use in a method of improving an immune response to an antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject a STING agonist and the antigen.
Additionally provided is an antigen for use in a method of improving an immune response to an antigen in a subject, wherein the method comprises the following steps: (i) administering to the subject a STING agonist, prior to
(ii) administering to the subject a STING agonist and the antigen.
The present invention also provides a STING agonist for use in a method of reducing the amount of an antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the antigen and a STING agonist.
Also provided is an antigen for use in a method of reducing the amount of an antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the antigen and a STING agonist.
Further provided is a STING agonist for use in a method of preparing the immune system of a subject for (ii) administration of an antigen and a STING agonist, wherein the method comprises (i) administering to the subject a STING agonist.
The present invention also provides an antigen for use in a method of preparing the immune system of a subject for administration of an antigen and a STING agonist, wherein the method comprises administering to the subject a STING agonist.
The invention provides the use of a STING agonist for the manufacture of a medicament for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject a STING agonist and an antigen which is associated with the pathogen.
Further provided is the use of an antigen for the manufacture of a medicament for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject a STING agonist and an antigen which is associated with the pathogen.
Additionally provided is the use of a STING agonist for the manufacture of a medicament for use in a method of reducing the variability of the immune response to an antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist, prior to
(ii) administering to each of the subjects a STING agonist and the antigen. The present invention provides the use of an antigen for the manufacture of a medicament for use in a method of reducing the variability of the immune response to an antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist, prior to
(ii) administering to each of the subjects a STING agonist and the antigen.
Also provided is the use of a STING agonist in the manufacture of a medicament for use in a method of improving an immune response to an antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject a STING agonist and the antigen.
Additionally provided is the use of an antigen for the manufacture of a medicament for use in a method of improving an immune response to an antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject a STING agonist and the antigen.
The present invention also provides the use of a STING agonist for the manufacture of a medicament for use in a method of reducing the amount of an antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the antigen and a STING agonist.
Also provided is the use of an antigen for the manufacture of a medicament for use in a method of reducing the amount of an antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the antigen and a STING agonist.
Further provided is the use of a STING agonist for the manufacture of a medicament for use in a method of preparing the immune system of a subject for administration of an antigen and a STING agonist, wherein the method comprises administering to the subject a STING agonist.
Additionally provided is the use of a STING agonist for the manufacture of a medicament for use in a method of preparing the immune system of a subject for administration of an antigen and a STING agonist, wherein the method comprises administering to the subject a STING agonist.
The present invention also provides the use of an antigen for the manufacture of a medicament for use in a method of preparing the immune system of a subject for (ii) administration of an antigen and a STING agonist, wherein the method comprises (i) administering to the subject a STING agonist. The present invention provides a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a STING agonist and a second antigen which is associated with the pathogen.
Provided is also a method of administering to a subject a STING agonist and an antigen which is associated with a pathogen (Antigen A), wherein the subject was selected for having been previously administered a STING agonist and an antigen (Antigen B), optionally wherein Antigen A and Antigen B are different (such as wherein Antigen A and Antigen B are not associated with the same pathogen); and optionally wherein the subject’s immune response is of increased level, speed, and/or breadth as compared to that of a subject who had not been previously administered the STING agonist and Antigen B.
Also provided is a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist and a first antigen, prior to
(ii) administering to each of the subjects a STING agonist and the second antigen.
Further provided is a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a STING agonist and the second antigen.
Additionally provided is a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen and a STING agonist.
The present invention also provides a method for preparing the immune system of a subject for (ii) administration of a second antigen and a STING agonist, comprising (i) administering to the subject a STING agonist and a first antigen.
The present invention provides a STING agonist for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a STING agonist and a second antigen which is associated with the pathogen.
Also provided is a first antigen for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to (ii) administering to the subject a STING agonist and a second antigen which is associated with the pathogen.
Further provided is a second antigen for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a STING agonist and a second antigen which is associated with the pathogen.
Additionally provided is a STING agonist for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist and a first antigen, prior to
(ii) administering to each of the subjects a STING agonist and the second antigen.
The present invention also provides a first antigen for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist and a first antigen, prior to
(ii) administering to each of the subjects a STING agonist and the second antigen.
The present invention provides a second antigen for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist and a first antigen, prior to
(ii) administering to each of the subjects a STING agonist and the second antigen.
Also provided is a STING agonist for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a STING agonist and the second antigen.
Further provided is a first antigen for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a STING agonist and the second antigen.
Additionally provided is a second antigen for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a STING agonist and the second antigen.
The present invention also provides a STING agonist for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps: (i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen and a STING agonist.
The present invention provides a first antigen for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen and a STING agonist.
Also provided is a second antigen for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen and a STING agonist.
Further provided is a STING agonist for use in a method of preparing the immune system of a subject for (ii) administration of a second antigen and a STING agonist, wherein the method comprises (i) administering to the subject a STING agonist and a first antigen.
Additionally provided is a first antigen for use in a method of preparing the immune system of a subject for administration of a second antigen and a STING agonist, wherein the method comprises administering to the subject a STING agonist and a first antigen.
The present invention also provides a second antigen for use in a method of preparing the immune system of a subject for administration of a second antigen and a STING agonist, wherein the method comprises administering to the subject a STING agonist and a first antigen.
The invention provides the use of a STING agonist for the manufacture of a medicament for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a STING agonist and a second antigen which is associated with the pathogen.
Also provided is the use of a first antigen for the manufacture of a medicament for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a STING agonist and a second antigen which is associated with the pathogen.
Further provided is the use of a second antigen for the manufacture of a medicament for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps: (i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a STING agonist and a second antigen which is associated with the pathogen.
Additionally provided is the use of a STING agonist for the manufacture of a medicament for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist and a first antigen, prior to
(ii) administering to each of the subjects a STING agonist and the second antigen.
The present invention also provides the use of a first antigen for the manufacture of a medicament for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist and a first antigen, prior to
(ii) administering to each of the subjects a STING agonist and the second antigen.
The present invention provides the use of a second antigen for the manufacture of a medicament for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist and a first antigen, prior to
(ii) administering to each of the subjects a STING agonist and the second antigen.
Also provided is the use of a STING agonist in the manufacture of a medicament for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a STING agonist and the second antigen.
Further provided is the use of a first antigen for the manufacture of a medicament for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a STING agonist and the second antigen.
Additionally provided is the use of a second antigen for the manufacture of a medicament for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a STING agonist and the second antigen.
The present invention also provides the use of a STING agonist for the manufacture of a medicament for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps: (i) administering to the subject a STING agonist and a first antigen, prior to (ii) administering to the subject the second antigen and a STING agonist.
The present invention provides the use of a first antigen for the manufacture of a medicament for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen and a STING agonist.
Also provided is the use of a second antigen for the manufacture of a medicament for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen and a STING agonist.
Further provided is the use of a STING agonist for the manufacture of a medicament for use in a method of preparing the immune system of a subject for administration of a second antigen and a STING agonist, wherein the method comprises administering to the subject a STING agonist and a first antigen.
Additionally provided is the use of a STING agonist for the manufacture of a medicament for use in a method of preparing the immune system of a subject for administration of a second antigen and a STING agonist, wherein the method comprises administering to the subject a STING agonist and a first antigen.
The present invention also provides the use of a second antigen for the manufacture of a medicament for use in a method of preparing the immune system of a subject for (ii) administration of a second antigen and a STING agonist, wherein the method comprises (i) administering to the subject a STING agonist and a first antigen.
The present invention provides a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject an antigen which is associated with the pathogen.
The present invention also provides a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject a second adjuvant and an antigen which is associated with the pathogen.
Provided is also a method of administering to a subject a STING agonist and an antigen which is associated with a pathogen, wherein the subject was selected for having been previously administered a STING agonist, and optionally wherein the subject’s immune response is of increased level, speed, and/or breadth as compared to that of a subject who had not been previously administered the STING agonist. Also provided is a method of reducing the variability of the immune response to an antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist, prior to
(ii) administering to each of the subjects the antigen.
Provided is a method of reducing the variability of the immune response to an antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist, prior to
(ii) administering to each of the subjects a second adjuvant and the antigen.
Further provided is a method of improving an immune response to an antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the antigen.
There is provided a method of improving an immune response to an antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject a second adjuvant and the antigen.
Additionally provided is a method of reducing the amount of an antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the antigen.
Additionally there is provided a method of reducing the amount of an antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject a second adjuvant and the antigen.
The invention also provides a method of preparing the immune system of a subject for (ii) administration of an antigen, wherein the method comprises (i) administering to the subject a STING agonist.
Also provided is a method of preparing the immune system of a subject for (ii) administration of a second adjuvant and an antigen, wherein the method comprises (i) administering to the subject a STING agonist.
The present invention provides a method for prophylaxis in a subject at risk of infection comprising administering a STING agonist to the subject.
The present invention provides a STING agonist for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject an antigen which is associated with the pathogen. The present invention also provides a STING agonist for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject a second adjuvant and an antigen which is associated with the pathogen.
Also provided is a STING agonist for use in a method of reducing the variability of the immune response to an antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist, prior to
(ii) administering to each of the subjects the antigen.
Provided is a STING agonist for use in a method of reducing the variability of the immune response to an antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist, prior to
(ii) administering to each of the subjects a second adjuvant and the antigen.
Further provided is a STING agonist for use in a method of improving an immune response to an antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the antigen.
There is provided a STING agonist for use in a method of improving an immune response to an antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject a second adjuvant and the antigen.
Additionally provided is a STING agonist for use in a method of reducing the amount of an antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the antigen.
Additionally there is provided a STING agonist for use in a method of reducing the amount of an antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the antigen and a second adjuvant.
The invention also provides a STING agonist for use in a method of preparing the immune system of a subject for (ii) administration of an antigen, wherein the method comprises (i) administering to the subject a STING agonist. Also provided is a STING agonist for use in a method of preparing the immune system of a subject for (ii) administration of an antigen and a second adjuvant, wherein the method comprises (i) administering to the subject a STING agonist.
The present invention provides a STING agonist for use in a method for prophylaxis in a subject at risk of infection comprising administering a STING agonist to the subject.
The present invention also provides a second adjuvant for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the second adjuvant and an antigen which is associated with the pathogen.
Provided is a second adjuvant for use in a method of reducing the variability of the immune response to an antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist, prior to
(ii) administering to each of the subjects the second adjuvant and the antigen.
There is provided a second adjuvant for use in a method of improving an immune response to an antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the second adjuvant and the antigen.
Additionally there is provided a second adjuvant for use in a method of reducing the amount of an antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the antigen and the second adjuvant.
Also provided is a second adjuvant for use in a method of preparing the immune system of a subject for (ii) administration of an antigen and a second adjuvant, wherein the method comprises (i) administering to the subject a STING agonist.
The present invention provides an antigen for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject an antigen which is associated with the pathogen.
The present invention also provides an antigen for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject a second adjuvant and an antigen which is associated with the pathogen. Also provided is an antigen for use in a method of reducing the variability of the immune response to an antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist, prior to
(ii) administering to each of the subjects the antigen.
Provided is an antigen for use in a method of reducing the variability of the immune response to an antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist, prior to
(ii) administering to each of the subjects a second adjuvant and the antigen.
Further provided is an antigen for use in a method of improving an immune response to an antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the antigen.
There is provided an antigen for use in a method of improving an immune response to an antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject a second adjuvant and the antigen.
Additionally provided is an antigen for use in a method of reducing the amount of an antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the antigen.
Additionally there is provided an antigen for use in a method of reducing the amount of an antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the antigen and a second adjuvant.
The invention also provides an antigen for use in a method of preparing the immune system of a subject for administration of an antigen, wherein the method comprises administering to the subject a STING agonist.
Also provided is an antigen for use in a method of preparing the immune system of a subject for administration of an antigen and a second adjuvant, wherein the method comprises administering to the subject a STING agonist.
The present invention provides the use of a STING agonist for the manufacture of a medicament for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject an antigen which is associated with the pathogen. The present invention also provides the use of a STING agonist for the manufacture of a medicament for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject a second adjuvant and an antigen which is associated with the pathogen.
Also provided is the use of a STING agonist for the manufacture of a medicament for use in a method of reducing the variability of the immune response to an antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist, prior to
(ii) administering to each of the subjects the antigen.
Provided is the use of a STING agonist for the manufacture of a medicament for use in a method of reducing the variability of the immune response to an antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist, prior to
(ii) administering to each of the subjects a second adjuvant and the antigen.
Further provided is the use of a STING agonist in the manufacture of a medicament for use in a method of improving an immune response to an antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the antigen.
There is provided the use of a STING agonist in the manufacture of a medicament for use in a method of improving an immune response to an antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject a second adjuvant and the antigen.
Additionally provided is the use of a STING agonist for the manufacture of a medicament for use in a method of reducing the amount of an antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the antigen.
Additionally provided is the use of a STING agonist for the manufacture of a medicament for use in a method of reducing the amount of an antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the antigen and a second adjuvant. The invention also provides the use of a STING agonist for the manufacture of a medicament for use in a method of preparing the immune system of a subject for administration of an antigen, wherein the method comprises administering to the subject a STING agonist.
Also provided is the use of a STING agonist for the manufacture of a medicament for use in a method of preparing the immune system of a subject for administration of an antigen and a second adjuvant, wherein the method comprises administering to the subject a STING agonist.
The present invention provides the use of a STING agonist for the manufacture of a medicament for use in a method for prophylaxis in a subject at risk of infection comprising administering a STING agonist to the subject.
The present invention also provides the use of a second adjuvant for the manufacture of a medicament for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the second adjuvant and an antigen which is associated with the pathogen.
Provided is the use of a second adjuvant for the manufacture of a medicament for use in a method of reducing the variability of the immune response to an antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist, prior to
(ii) administering to each of the subjects the second adjuvant and the antigen.
There is provided the use of a second adjuvant in the manufacture of a medicament for use in a method of improving an immune response to an antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the second adjuvant and the antigen.
Additionally provided is the use of a second adjuvant for the manufacture of a medicament for use in a method of reducing the amount of an antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the antigen and the second adjuvant.
Also provided is the use of a second adjuvant for the manufacture of a medicament for use in a method of preparing the immune system of a subject for administration of an antigen and the second adjuvant, wherein the method comprises administering to the subject a STING agonist. The invention provides the use of an antigen for the manufacture of a medicament for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the antigen which is associated with the pathogen.
The present invention also provides the use of an antigen for the manufacture of a medicament for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject a second adjuvant and the antigen which is associated with the pathogen.
Also provided is the use of an antigen for the manufacture of a medicament for use in a method of reducing the variability of the immune response to an antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist, prior to
(ii) administering to each of the subjects the antigen.
Provided is the use of an antigen for the manufacture of a medicament for use in a method of reducing the variability of the immune response to an antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist, prior to
(ii) administering to each of the subjects a second adjuvant and the antigen.
Further provided is the use of an antigen for the manufacture of a medicament for use in a method of improving an immune response to an antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the antigen.
There is provided the use of an antigen for the manufacture of a medicament for use in a method of improving an immune response to an antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject a second adjuvant and the antigen.
Additionally provided is the use of an antigen for the manufacture of a medicament for use in a method of reducing the amount of an antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the antigen. Additionally there is provided the use of an antigen for the manufacture of a medicament for use in a method of reducing the amount of an antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the antigen and a second adjuvant.
The invention also provides the use of an antigen for the manufacture of a medicament for use in a method of preparing the immune system of a subject for (ii) administration of an antigen, wherein the method comprises (i) administering to the subject a STING agonist.
The present invention provides a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a second antigen which is associated with the pathogen.
The present invention also provides a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a second adjuvant and a second antigen which is associated with the pathogen.
Provided is also a method of administering to a subject a STING agonist and an antigen which is associated with a pathogen (Antigen A), wherein the subject was selected for having been previously administered a STING agonist and an antigen (Antigen B), optionally wherein Antigen A and Antigen B are different (such as wherein Antigen A and Antigen B are not associated with the same pathogen); and optionally wherein the subject’s immune response is of increased level, speed, and/or breadth as compared to that of a subject who had not been previously administered the STING agonist and Antigen B.
Also provided is a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist and a first antigen, prior to
(ii) administering to each of the subjects the second antigen.
Provided is a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist and a first antigen, prior to
(ii) administering to each of the subjects a second adjuvant and the second antigen.
Further provided is a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen. There is provided a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a second adjuvant and the second antigen.
Additionally provided is a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen.
Additionally there is provided a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a second adjuvant and the second antigen.
The invention also provides a method of preparing the immune system of a subject for (ii) administration of a second antigen, wherein the method comprises (i) administering to the subject a STING agonist and a first antigen.
Also provided is a method of preparing the immune system of a subject for (ii) administration of a second adjuvant and a second antigen, wherein the method comprises (i) administering to the subject a STING agonist and a first antigen.
The present invention provides a method for prophylaxis in a subject at risk of infection comprising administering a STING agonist and a first antigen to the subject.
The present invention provides a STING agonist for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a second antigen which is associated with the pathogen.
The present invention also provides a STING agonist for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a second adjuvant and a second antigen which is associated with the pathogen.
Also provided is a STING agonist for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist and a first antigen, prior to
(ii) administering to each of the subjects the second antigen. Provided is a STING agonist for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist and a first antigen, prior to
(ii) administering to each of the subjects a second adjuvant and the second antigen.
Further provided is a STING agonist for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen.
There is provided a STING agonist for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a second adjuvant and the second antigen.
Additionally provided is a STING agonist for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen.
Additionally there is provided a STING agonist for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen and a second adjuvant.
The invention also provides a STING agonist for use in a method of preparing the immune system of a subject for (ii) administration of a second antigen, wherein the method comprises (i) administering to the subject a STING agonist and a first antigen.
Also provided is a STING agonist for use in a method of preparing the immune system of a subject for (ii) administration of a second antigen and a second adjuvant, wherein the method comprises (i) administering to the subject a STING agonist and a first antigen.
The present invention provides a STING agonist or a first antigen for use in a method for prophylaxis in a subject at risk of infection comprising administering a STING agonist and a first antigen to the subject.
The present invention provides a first antigen for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a second antigen which is associated with the pathogen. The present invention also provides a first antigen for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a second adjuvant and a second antigen which is associated with the pathogen.
Also provided is a first antigen for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist and a first antigen, prior to
(ii) administering to each of the subjects the second antigen.
Provided is a first antigen for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist and a first antigen, prior to
(ii) administering to each of the subjects a second adjuvant and the second antigen.
Further provided is a first antigen for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen.
There is provided a first antigen for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a second adjuvant and the second antigen.
Additionally provided is a first antigen for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen.
Additionally there is provided a first antigen for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen and a second adjuvant.
The invention also provides a first antigen for use in a method of preparing the immune system of a subject for administration of a second antigen, comprising administering to the subject a STING agonist and a first antigen. Also provided is a first antigen for use in a method of preparing the immune system of a subject for administration of a second antigen and a second adjuvant, comprising administering to the subject a STING agonist and a first antigen.
The present invention also provides a second adjuvant for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a second adjuvant and a second antigen which is associated with the pathogen.
Provided is a second adjuvant for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist and a first antigen, prior to
(ii) administering to each of the subjects a second adjuvant and the second antigen.
There is provided a second adjuvant for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a second adjuvant and the second antigen.
Additionally there is provided a second adjuvant for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen and a second adjuvant.
Also provided is a second adjuvant for use in a method of preparing the immune system of a subject for (ii) administration of a second antigen and a second adjuvant, wherein the method comprises (i) administering to the subject a STING agonist and a first antigen.
The present invention provides a second antigen for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a second antigen which is associated with the pathogen.
The present invention also provides a second antigen for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a second adjuvant and a second antigen which is associated with the pathogen. Also provided is a second antigen for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist and a first antigen, prior to
(ii) administering to each of the subjects the second antigen.
Provided is a second antigen for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist and a first antigen, prior to
(ii) administering to each of the subjects a second adjuvant and the second antigen.
Further provided is a second antigen for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen.
There is provided a second antigen for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a second adjuvant and the second antigen.
Additionally provided is a second antigen for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen.
Additionally there is provided a second antigen for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen and a second adjuvant.
The invention also provides a second antigen for use in a method of preparing the immune system of a subject for administration of a second antigen, wherein the method comprises administering to the subject a STING agonist and a first antigen.
Also provided is a second antigen for use in a method of preparing the immune system of a subject for administration of a second antigen and a second adjuvant, wherein the method comprises administering to the subject a STING agonist and a first antigen.
The present invention provides the use of a STING agonist for the manufacture of a medicament for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps: (i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a second antigen which is associated with the pathogen.
The present invention also provides the use of a STING agonist for the manufacture of a medicament for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a second adjuvant and a second antigen which is associated with the pathogen.
Also provided is the use of a STING agonist for the manufacture of a medicament for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist and a first antigen, prior to
(ii) administering to each of the subjects the second antigen.
Provided is the use of a STING agonist for the manufacture of a medicament for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist and a first antigen, prior to
(ii) administering to each of the subjects a second adjuvant and the second antigen.
Further provided is the use of a STING agonist in the manufacture of a medicament for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen.
There is provided the use of a STING agonist in the manufacture of a medicament for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a second adjuvant and the second antigen.
Additionally provided is the use of a STING agonist for the manufacture of a medicament for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen.
Additionally provided is the use of a STING agonist for the manufacture of a medicament for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps: (i) administering to the subject a STING agonist and a first antigen, prior to (ii) administering to the subject the second antigen and a second adjuvant.
The invention also provides the use of a STING agonist for the manufacture of a medicament for use in a method of preparing the immune system of a subject for administration of a second antigen, wherein the method comprises administering to the subject a STING agonist and a first antigen.
Also provided is the use of a STING agonist for the manufacture of a medicament for use in a method of preparing the immune system of a subject for administration of a second antigen and a second adjuvant, wherein the method comprises administering to the subject a STING agonist and a first antigen.
The present invention provides the use of a STING agonist or a first antigen for the manufacture of a medicament for use in a method for prophylaxis in a subject at risk of infection comprising administering a STING agonist and a first antigen to the subject.
The present invention provides the use of a first antigen for the manufacture of a medicament for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a second antigen which is associated with the pathogen.
The present invention also provides the use of a first antigen for the manufacture of a medicament for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a second adjuvant and a second antigen which is associated with the pathogen.
Also provided is the use of a first antigen for the manufacture of a medicament for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist and a first antigen, prior to
(ii) administering to each of the subjects the second antigen.
Provided is the use of a first antigen for the manufacture of a medicament for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist and a first antigen, prior to
(ii) administering to each of the subjects the second antigen and a second adjuvant.
Further provided is the use of a first antigen for the manufacture of a medicament for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to (ii) administering to the subject the second antigen.
There is provided the use of a first antigen for the manufacture of a medicament for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a second adjuvant and the second antigen.
Additionally provided is the use of a first antigen for the manufacture of a medicament for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen.
Additionally there is provided the use of a first antigen for the manufacture of a medicament for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen and a second adjuvant.
The invention also provides the use of a first antigen for the manufacture of a medicament for use in a method of preparing the immune system of a subject for administration of a second antigen, wherein the method comprises administering to the subject a STING agonist and a first antigen.
The invention also provides the use of a first antigen for the manufacture of a medicament for use in a method of preparing the immune system of a subject for administration of a second antigen and a second adjuvant, wherein the method comprises administering to the subject a STING agonist and a first antigen.
The present invention also provides the use of a second adjuvant for the manufacture of a medicament for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a second adjuvant and a second antigen which is associated with the pathogen.
Provided is the use of a second adjuvant for the manufacture of a medicament for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist and a first antigen, prior to
(ii) administering to each of the subjects a second adjuvant and the second antigen. There is provided the use of a second adjuvant in the manufacture of a medicament for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a second adjuvant and the second antigen.
Additionally provided is the use of a second adjuvant for the manufacture of a medicament for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen and a second adjuvant.
Also provided is the use of a second adjuvant for the manufacture of a medicament for use in a method of preparing the immune system of a subject for administration of a second antigen and a second adjuvant, wherein the method comprises administering to the subject a STING agonist and a first antigen.
The invention provides the use of a second antigen for the manufacture of a medicament for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a second antigen which is associated with the pathogen.
The present invention also provides the use of a second antigen for the manufacture of a medicament for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a second adjuvant and a second antigen which is associated with the pathogen.
Also provided is the use of a second antigen for the manufacture of a medicament for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist and a first antigen, prior to
(ii) administering to each of the subjects the second antigen.
Provided is the use of a second antigen for the manufacture of a medicament for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist and a first antigen, prior to
(ii) administering to each of the subjects a second adjuvant and the second antigen. Further provided is the use of a second antigen for the manufacture of a medicament for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen.
There is provided the use of a second antigen for the manufacture of a medicament for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a second adjuvant and the second antigen.
Additionally provided is the use of a second antigen for the manufacture of a medicament for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen.
Additionally there is provided the use of a second antigen for the manufacture of a medicament for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen and a second adjuvant.
The invention also provides the use of a second antigen for the manufacture of a medicament for use in a method of preparing the immune system of a subject for (ii) administration of a second antigen, wherein the method comprises (i) administering to the subject a STING agonist and a first antigen.
BRIEF DESCRIPTION OF THE SEQUENCES
SEQ ID NO: 1 - A polypeptide sequence of varicella-zoster virus (VZV)-gE antigen
SEQ ID NO: 2 - A polypeptide sequence of F2 antigen
BRIEF DESCRIPTION OF THE FIGURES
Fig. 1 Toxin B neutralising antibody titre at 15 Post III following administration of VZV- gE, STING agonist and aluminium hydroxide before administration of F2 with an adjuvant comprising a saponin.
Fig. 2 Toxin B specific IgG at 15 Post III following administration of VZV-gE, STING agonist and aluminium hydroxide before administration of F2 with an adjuvant comprising a saponin. Fig. 3 Toxin A neutralising antibody titre at 15 Post III following administration of VZV- gE, STING agonist and aluminium hydroxide before administration of F2 with an adjuvant comprising a saponin.
Fig. 4 Toxin A specific IgG at 15 Post III following administration of VZV-gE, STING agonist and aluminium hydroxide before administration of F2 with an adjuvant comprising a saponin.
Fig. 5 Percentage of F2-specific CD4+ T-cells expressing at least 2 cytokines among IL-2, IFN-gamma and TNF-alpha 15 Post III following administration of VZV-gE, STING agonist and aluminium hydroxide before administration of F2 with an adjuvant comprising a saponin.
Fig. 6 Toxin B specific IgG at 15 Post II following administration of VZV-gE, STING agonist and aluminium hydroxide before administration of F2 with an adjuvant comprising a saponin.
Fig. 7 Toxin B specific IgG at 15 Post I following administration of VZV-gE, STING agonist and aluminium hydroxide before administration of F2 with an adjuvant comprising a saponin.
Fig. 8 Toxin A specific IgG at 15 Post II following administration of VZV-gE, STING agonist and aluminium hydroxide before administration of F2 with an adjuvant comprising a saponin.
Fig. 9 Toxin A specific IgG at 15 Post I following administration of VZV-gE, STING agonist and aluminium hydroxide before administration of F2 with an adjuvant comprising a saponin.
Fig. 10 Toxin A specific IgG at 14 day Pll following administration of STING agonist and aluminium hydroxide or VZV-gE with STING agonist and aluminium hydroxide before administration of F2 with an adjuvant comprising a saponin.
Fig. 11 Toxin A specific IgG at 14 day Pll following administration of STING agonist or VZV-gE with STING agonist before administration of F2 with an adjuvant comprising a saponin.
Fig. 12 Toxin A specific IgG at 14 day Pill following administration of STING agonist and aluminium hydroxide or VZV-gE with STING agonist and aluminium hydroxide before administration of F2 with an adjuvant comprising a saponin.
Fig. 13 Toxin A specific IgG at 14 day Pill following administration of STING agonist or VZV-gE with STING agonist before administration of F2 with an adjuvant comprising a saponin.
Fig. 14 Toxin A neutralising antibody titre at 14 day Pll following administration of STING agonist and aluminium hydroxide or VZV-gE with STING agonist and aluminium hydroxide before administration of F2 with an adjuvant comprising a saponin.
Fig. 15 Toxin A neutralising antibody titre at 14 day PH following administration of STING agonist or VZV-gE with STING agonist before administration of F2 with an adjuvant comprising a saponin.
Fig. 16 Toxin B specific IgG at 14 day Pill following administration of STING agonist and aluminium hydroxide or VZV-gE with STING agonist and aluminium hydroxide before administration of F2 with an adjuvant comprising a saponin.
Fig. 17 Toxin B specific IgG at 14 day Pill following administration of STING agonist or VZV-gE with STING agonist before administration of F2 with an adjuvant comprising a saponin.
Fig. 18 Toxin B neutralising antibody titre at 14 day PHI following administration of STING agonist and aluminium hydroxide or VZV-gE with STING agonist and aluminium hydroxide before administration of F2 with an adjuvant comprising a saponin.
Fig. 19 Toxin B neutralising antibody titre at 14 day Pill following administration of STING agonist or VZV-gE with STING agonist before administration of F2 with an adjuvant comprising a saponin.
Fig. 20 Toxin A neutralising antibody titre at 14 day Pill following administration of STING agonist and aluminium hydroxide or VZV-gE with STING agonist and aluminium hydroxide before administration of F2 with an adjuvant comprising a saponin.
Fig. 21 Toxin A neutralising antibody titre at 14 day Pill following administration of STING agonist or VZV-gE with STING agonist before administration of F2 with an adjuvant comprising a saponin.
Fig. 22 Toxin A specific IgG at 14 day PH following administration of STING agonist or VZV-gE with STING agonist before administration of F2 with a squalene emulsion adjuvant.
Fig. 23 Toxin A specific IgG at 14 day PH following administration of STING agonist and aluminium hydroxide or VZV-gE with STING agonist and aluminium hydroxide before administration of F2 with a squalene emulsion adjuvant.
Fig. 24 Toxin A specific IgG at 14 day Pill following administration of STING agonist or VZV-gE with STING agonist before administration of F2 with a squalene emulsion adjuvant.
Fig. 25 Toxin A specific IgG at 14 day Pill following administration of STING agonist and aluminium hydroxide or VZV-gE with STING agonist and aluminium hydroxide before administration of F2 with a squalene emulsion adjuvant. Fig. 26 Toxin A neutralising antibody titre at 14 day PH following administration of STING agonist or VZV-gE with STING agonist before administration of F2 with a squalene emulsion adjuvant.
Fig. 27 Toxin A neutralising antibody titre at 14 day PH following administration of STING agonist and aluminium hydroxide or VZV-gE with STING agonist and aluminium hydroxide before administration of F2 with a squalene emulsion adjuvant.
Fig. 28 Toxin B specific IgG at 14 day Pill following administration of STING agonist or VZV-gE with STING agonist before administration of F2 with a squalene emulsion adjuvant.
Fig. 29 Toxin B specific IgG at 14 day Pill following administration of STING agonist and aluminium hydroxide or VZV-gE with STING agonist and aluminium hydroxide before administration of F2 with a squalene emulsion adjuvant.
Fig. 30 Toxin B neutralising antibody titre at 14 day Pill following administration of STING agonist and aluminium hydroxide or VZV-gE with STING agonist and aluminium hydroxide before administration of F2 with a squalene emulsion adjuvant.
Fig. 31 Toxin B neutralising antibody titre at 14 day Pill following administration of STING agonist or VZV-gE with STING agonist before administration of F2 with a squalene emulsion adjuvant.
Fig. 32 Toxin A neutralising antibody titre at 14 day Pill following administration of STING agonist and aluminium hydroxide or VZV-gE with STING agonist and aluminium hydroxide before administration of F2 with a squalene emulsion adjuvant.
Fig. 33 Toxin A neutralising antibody titre at 14 day PHI following administration of STING agonist or VZV-gE with STING agonist before administration of F2 with a squalene emulsion adjuvant.
Fig. 34 Toxin B neutralising antibody titre at 14 day Pill following administration of STING agonist and aluminium hydroxide or VZV-gE with STING agonist and aluminium hydroxide before administration of F2 with STING agonist and aluminium hydroxide.
Fig. 35 Toxin A neutralising antibody titre at 14 day PHI following administration of STING agonist and aluminium hydroxide or VZV-gE with STING agonist and aluminium hydroxide before administration of F2 with STING agonist and aluminium hydroxide.
Fig. 36 Toxin A neutralising antibody titre at 14 day Pll following administration of STING agonist and aluminium hydroxide or VZV-gE with STING agonist and aluminium hydroxide before administration of F2 with STING agonist and aluminium hydroxide.
Fig. 37 Toxin B specific IgG at 14 day Pill following administration of STING agonist and aluminium hydroxide or VZV-gE with STING agonist and aluminium hydroxide before administration of F2 with STING agonist and aluminium hydroxide.
Fig. 38 Toxin A specific IgG at 14 day PH following administration of STING agonist and aluminium hydroxide or VZV-gE with STING agonist and aluminium hydroxide before administration of F2 with STING agonist and aluminium hydroxide.
Fig. 39 Toxin A specific IgG at 14 day Pill following administration of STING agonist and aluminium hydroxide or VZV-gE with STING agonist and aluminium hydroxide before administration of F2 with STING agonist and aluminium hydroxide.
DETAILED DESCRIPTION OF THE INVENTION
Administration of a STING agonist (a ‘first adjuvant’) may be of benefit in respect of subsequent administration of an antigen and a STING agonist or in respect of prophylaxis in a subject at risk of infection.
Consequently, the present invention provides a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject a STING agonist and an antigen which is associated with the pathogen.
Also provided is a method of reducing the variability of the immune response to an antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist, prior to
(ii) administering to each of the subjects a STING agonist and the antigen.
Further provided is a method of improving an immune response to an antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject a STING agonist and the antigen.
Additionally provided is a method of reducing the amount of an antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the antigen and a STING agonist. The invention also provides a method of preparing the immune system of a subject for (ii) administration of an antigen and a STING agonist, wherein the method comprises (i) administering to the subject a STING agonist.
Administration of an antigen and a STING agonist (a ‘first adjuvant’) may be of benefit in respect of subsequent administration of an antigen and a STING agonist (‘a second adjuvant’).
Consequently, the present invention provides a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a STING agonist and a second antigen which is associated with the pathogen.
Also provided is a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist and a first antigen, prior to
(ii) administering to each of the subjects a STING agonist and the second antigen.
Further provided is a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a STING agonist and the second antigen.
Additionally provided is a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen and a STING agonist.
The invention also provides a method of preparing the immune system of a subject for (ii) administration of a second antigen and a STING agonist, wherein the method comprises (i) administering to the subject a STING agonist and a first antigen.
Administration of an antigen and a STING agonist (a ‘first adjuvant’) may be of benefit in respect of subsequent administration of an antigen or in respect of prophylaxis in a subject at risk of infection.
The present invention provides a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject an antigen which is associated with the pathogen.
The present invention also provides a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to (ii) administering to the subject a second adjuvant and an antigen which is associated with the pathogen.
Also provided is a method of reducing the variability of the immune response to an antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist, prior to
(ii) administering to each of the subjects the antigen.
Provided is a method of reducing the variability of the immune response to an antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist, prior to
(ii) administering to each of the subjects a second adjuvant and the antigen.
Further provided is a method of improving an immune response to an antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the antigen.
There is provided a method of improving an immune response to an antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject a second adjuvant and the antigen.
Additionally provided is a method of reducing the amount of an antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the antigen.
Additionally there is provided a method of reducing the amount of an antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject a second adjuvant and the antigen.
The invention also provides a method of preparing the immune system of a subject for (ii) administration of an antigen, wherein the method comprises (i) administering to the subject a STING agonist.
Also provided is a method of preparing the immune system of a subject for (ii) administration of a second adjuvant and an antigen, wherein the method comprises (i) administering to the subject a STING agonist.
The present invention provides a method for prophylaxis in a subject at risk of infection comprising administering a STING agonist to the subject. Administration of an antigen and a STING agonist (a ‘first adjuvant’) may be of benefit in respect of subsequent administration of an antigen or in respect of prophylaxis in a subject at risk of infection.
The present invention provides a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a second antigen which is associated with the pathogen.
The present invention also provides a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a second adjuvant and a second antigen which is associated with the pathogen.
Also provided is a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist and a first antigen, prior to
(ii) administering to each of the subjects the second antigen.
Provided is a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist and a first antigen, prior to
(ii) administering to each of the subjects a second adjuvant and the second antigen.
Further provided is a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen.
There is provided a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a second adjuvant and the second antigen.
Additionally provided is a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen.
Additionally there is provided a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to (ii) administering to the subject a second adjuvant and the second antigen.
The invention also provides a method of preparing the immune system of a subject for (ii) administration of a second antigen, wherein the method comprises (i) administering to the subject a STING agonist and a first antigen.
Also provided is a method of preparing the immune system of a subject for (ii) administration of a second adjuvant and a second antigen, wherein the method comprises (i) administering to the subject a STING agonist and a first antigen.
The present invention provides a method for prophylaxis in a subject at risk of infection comprising administering a STING agonist and a first antigen to the subject.
Figure imgf000036_0001
The invention involves the administration of a STING agonist.
The STING agonist may be any appropriate agonist that is capable of binding to and activating STING receptor and STING signalling.
In one embodiment, the STING agonist binds to STING with an in vitro Kf of less than about 0.750 pM (e.g., less than about 0.500 pM, less than about 0.250 pM or less than about 0.100 pM).
In one embodiment, the STING agonist activates STING with an in vitro ECso of about 100 pM or less (e.g., about 50 pM or less, about 20 pM or less, or about 10 pM or less) when measured by monitoring phosphorylation of interferon regulatory factor-3 (I RF3).
In one embodiment, the STING agonist activates STING with an in vitro ECso of about 100 pM or less (e.g., about 50 pM or less, about 20 pM or less, or about 10 pM or less) as measured by monitoring interferon-p induction.
In one embodiment, the STING agonist is a nucleic acid, a protein, a peptide, or a small molecule. In one embodiment, the STING agonist is a small molecule selected from a modified or unmodified cyclic dinucleotide. In one embodiment, the cyclic dinucleotide is selected from a compound of Formulae (l)-(lll):
Figure imgf000037_0001
wherein
5 R1 and R2 are each independently selected from the following groups:
Figure imgf000037_0002
Figure imgf000038_0001
R3 and R4 are each independently -SH or -OH, R5 and R6 are oxygen or sulphur,
R7 and R8 are each independently halogen, hydrogen, -OH, or OCH3.
In one embodiment, the STING agonist is the compound:
Figure imgf000038_0002
In one embodiment, the cyclic dinucleotide is selected from c-ditzGMP, c-di-thGMP, c- GtzGMP, c-GAMP, c-thGMP, c-tzGMP, c-di-thAMP, c-ditzAMP, c-di-AMP, c-di-GMP, c-diXGMP, c-GthXMP, c-GXMP, c-ACMP, c-AthXMP, c-AtzXMP, c-di-thXMP, or c-ditzXMP (as defined in paragraph [0053] of US 2021/0101924).
In one embodiment, the STING agonist is 2’,3’-cGAMP. In one embodiment, the STING agonist is 3’,3’-cGAMP. In one embodiment, the STING agonist is the compound:
Figure imgf000039_0001
In one embodiment, the STING agonist is the compound 6-bromo-N-(naphthalen-1- yl)benzo[d][1 ,3]dioxole-5-carboxamide:
Figure imgf000039_0002
In one embodiment, the STING agonist is a compound of Formula A:
Figure imgf000039_0003
wherein
X is O or NR4A,
Y is O, NR4A, CH2, or absent, n is 0, 1 , 2, or 3,
R1 and R2 are independently selected from OH, OR3, OR3A, SR3, and NR3R4, R3, R4, and R4A are independently selected from hydrogen, C1 -C10 alkyl optionally substituted with 1-6 halogen, C6-C10 aryl or 5-10 membered heteroaryl, or R3 and R4 together with the nitrogen atom to which they are attached form a 3 to 7 membered heterocycle or 5 to 10 membered heteroaryl,
Figure imgf000039_0004
represents the point of connection of R3A to the remainder of the molecule, R5-R10 are independently selected from hydrogen, halogen, pseudohalogen, C1-C10 alkyl optionally substituted with 1-6 halogens, C6-C10 aryl, and 5 to 10 membered heteroaryl.
In one embodiment, the STING agonist is selected from:
Figure imgf000040_0001
In one embodiment, the STING agonist is a flavonoid. Suitable flavonoids include, but are not limited to, 10-(carboxymethyl)-9(10H)acridone (CMA), 5,6-Dimethylxanthenone-4- acetic acid (DMXAA), methoxyvone, 6,4'-dimethoxyflavone, 4'-methoxyflavone, 3', 6'- dihydroxyflavone, 7,2'-dihydroxyflavone, daidzein, formononetin, retusin 7-methyl ether, xanthone, or any combination thereof. In one aspect, the STING agonist can be 10- (carboxymethyl)-9(10H)acridone (CMA). In one aspect, the STING agonist can be 5,6- Dimethylxanthenone-4-acetic acid (DMXAA). In one aspect, the STING agonist can be methoxyvone. In one aspect, the STING agonist can be 6,4'-dimethoxyflavone. In one aspect, the STING agonist can be 4'-methoxyflavone. In one aspect, the STING agonist can be 3', 6'- dihydroxyflavone. In one aspect, the STING agonist can be 7,2'-dihydroxyflavone. In one aspect, the STING agonist can be daidzein. In one aspect, the STING agonist can be formononetin. In one aspect, the STING agonist can be retusin 7-methyl ether. In one aspect, the STING agonist can be xanthone. In one aspect, the STING agonist can be any combination of the above flavonoids.
In one embodiment, the STING agonist is a compound of Formula B:
Figure imgf000040_0002
Figure imgf000041_0001
represents two conjugated double bonds in a five-membered heteroaromatic ring and three conjugated double bonds in a six-membered aromatic or heteroaromatic ring, W1 is selected from CR11 and N;
X1 is selected from CR1, C(R1)2, N, NR1, O and S;
X2 is selected from CR2, C(R2)2, N, NR2, O and S;
X3 is selected from CR3, C(R3)2, N, NR3, O and S; where two or three of X1, X2 and X3 are independently selected from N, NR1,
NR2, NR3, O and S; and where at least one of X1, X2 and X3 is selected from N, NR1, NR2 and NR3;
Y1 is selected from N, NR4, O, S, CR4 and C(R4)2;
Y2 is selected from N, NR5, O, S, CR5 and C(R5)2;
Y3 is selected from N, NR6, O, S, CR6 and C(R6)2;
Y4 is selected from C and N;
Y5 is selected from C and N; where at least one and not more than two of Y1, Y2 and Y3 are independently selected from N, NR4, NR5 and NR6; where when if one of Y4 or Y5 is N, the other one of Y4 or Y5 is C;
Z1 is selected from C and N;
Z2 is selected from N, NR8 and CR8;
Z3 is selected from N, NR9 and CR9;
Z4 is selected from N, NR10 and CR10;
Z5 is selected from N, NR7 and CR7; where two or three of Z1, Z2, Z3, Z4 and Z5 are independently selected from N, NR7, NR8, NR9, and NR10; each R1 is independently selected from the group consisting of H, Ci -Cs alkyl, Ci-Cs alkylene-NRR and Ci-Cs alkylene-C(O)OR; each R2 is independently selected from the group consisting of H, Ci-Cs alkyl, Ci-Cs alkylene-NRR, Ci-Cs alkylene-C(O)OR, Ci-Cs alkylene-OR and Ci-Cs alkylene-O-P(O)(OH)2; each R3 is independently selected from the group consisting of H, Ci-Cs alkyl, Ci-Cs alkylene-NRR, Ci-Cs alkylene-C(O)OR and Ci-Cs alkylene-O-P(O)(OH)2; each R4 is independently selected from the group consisting of H, -OR, -NRR, Ci-Cs alkyl optionally substituted with one or two -OR, Ci-Cs alkylene-NRR, - C(O)OR, C1-C8 alkylene-C(O)OR, 3-10 membered heterocycle, C1-C8 alkylene- 3-10 membered heterocycle optionally substituted with one 3-10 membered heterocycle, (C3 -C10)-cycloalkyl, and C1 -C8 alkylene-(C3-C10)-cycloalkyl; each R5 is independently selected from the group consisting of H, OR, C1-C8 alkyl, -NRR, C1-C8 alkylene-NRR, -C(O)OR, C1-C8 alkylene-C(O)OR, 3-10 membered heterocycle, C1-C8 alkylene-3-10 membered heterocycle optionally substituted with one 3-10 membered heterocycle, and C1-C8 alkylene-OR; each R6 is H; R7 is selected from the group consisting of H, halo, hydroxy or NH2; R8 is selected from the group consisting of H, C1-C8 alkyl optionally substituted with one or two -NRR or -OR, C1-C8 alkylene-C(O)OR and C1-C8 alkylene- SO2R; R9 is H; R10 is selected from the group consisting of H, C1-C8 alkyl optionally substituted with one or two -OR, and halo; R11 is selected from the group consisting of H, C1-C8 alkyl, -OR and halo; R12 is -C(O)N(R)2 or -C(O)NHR; R13 is H; each R is independently selected from the group consisting of H or C1-C8 alkyl, or C1-C8 haloalkyl, or two R join to form, together with the atom or atoms to which they are bound, a -C3-C10 cycloalkyl or 3-10 membered heterocycle, where said 3-10 membered heterocycle contains one, two or three atoms selected from N, O and S; and where, when two R join to form, together with the atom or atoms to which they are bound, a –(C3-C10) cycloalkyl or 3-10 membered heterocycle, said -C3-C10 cycloalkyl or 3-10 membered heterocycle is optionally substituted with one or more substituents each independently selected from C1-C8 alkyl, hydroxy, C1-C8 alkoxy, -(C3-C10) cycloalkyl, 3-10 membered heterocycle, halo and cyano. In one embodiment, the STING agonist is a compound selected from:
Figure imgf000043_0001
Figure imgf000044_0001
Figure imgf000045_0001
Figure imgf000046_0001
Figure imgf000047_0001
Figure imgf000048_0001
Figure imgf000049_0001
In one embodiment, the STING agonist is a compound of Formula C:
Figure imgf000050_0001
wherein G1 is independently selected from Ring A or
Figure imgf000050_0002
ring A is independently selected from optionally substituted heterocyclyl and optionally substituted heteroaryl, ring B is aromatic carbocyclic ring, ring C is optionally substituted five-membered heteroaryl, R1 is -CON(R3)2, R2 is independently selected from hydrogen, optionally substituted C1-C6 alkyl, and optionally substituted C3-C5 monocyclic cycloalkyl, R3 is independently selected from hydrogen, and optionally substituted C1-C6 alkyl; m is selected from 0, or 1; n is selected from 0, 1, or 2; o is 1; p is selected from 0, 1, or 2; when 'alkyl's substituted, it is substituted with 1 to 4 substituents independently selected from halogen, alkyl, perhaloalkyl, cycloalkyl, heterocyclyl, -N(R4)2, and -OR4; when ‘carbocycle’ or 'cycloalkyl' is substituted, it is substituted with 1 to 4 substituents independently selected from halogen, alkyl, perhaloalkyl, -N(R4)2, and -OR4; when 'heterocycle' or 'heterocyclyl' is substituted, it is substituted with 1 to 4 substituents independently selected from oxo (=0), halogen, cyano, alkyl, perhaloalkyl, -OR4, -C(=O)OH, -OP(O)(OR4)2, -P(O)(OR4)2, -P(O)(OR4)R4a, - SO2R4a, -SO2NH2, -C(=O)N(H)R4, -C(=O)N(alkyl)R4, -N(H)C(=O)R4a, -N(H)R4, and -N(alkyl)R4; when the 'heteroaryl' group is substituted, it is substituted with I to 4 substituents selected from halogen, cyano, alkyl, perhaloalkyl, -O-alkyl, -O-perhaloalkyl, - N(alkyl)alkyl, -N(H)R4, -SO2-alkyl, -N(alkyl)C(=O)alkyl, -N(H)C(=O)alkyl, -
C(=O)N(alkyl)alkyl, -C(=O)N(H)alkyl, -C(=O)NH2, -SO2N(alkyl)alkyl, - SO2N(H)alkyl, -SO2NH2, -C(=O)OH, -OP(O)(OR4)2, -P(O)(OR4)2, and - P(O)(OR4)R4a; each R4 is independently selected from hydrogen, alkyl, and cycloalkyl; and each R4a is independently selected from alkyl, and cycloalkyl.
In one embodiment, the STING agonist is selected from:
Figure imgf000051_0001
Figure imgf000052_0001
Figure imgf000053_0001
Figure imgf000054_0001
8,
SB-11285, SRCB-0074, TAK-676, and TTI-10001.
In one embodiment, the STING agonist is a STING agonist as disclosed in WO 2017/175147 (pages 7 to 92). In particular, the STING agonist is a compound according to Formula (l-N):
Figure imgf000055_0001
wherein: q is 0 or 1; r is 0 or 1; s is 0 or 1; wherein q + r + s = 1 or 2; when q is 0, RA1 and RA2 are each independently H, halogen, hydroxy, -O-P(O)(OH)2, -O-P(O)(RIRII)2, -N(Re)(Rf), -CO2Rf, -N(Rf)CORb, -N(Rg)SO2(C1-C4alkyl)-N(Re)(Rf), -N(Rg)CO(C1-C4alkyl)-N(Rh)(Rf), optionally substituted (C1-C6alkyl), optionally substituted (C1-C6alkyl)oxy-, optionally substituted (C1-C6alkyl)amino-, and optionally substituted (C1-C6alkyl)(C1-C4alkyl)amino-, wherein the (C1-C6alkyl) of said optionally substituted (C1-C6alkyl), optionally substituted (C1-C6alkyl)oxy-, optionally substituted (C1-C6alkyl)amino- and optionally substituted (C1-C6alkyl)(C1-C4alkyl)amino- is optionally substituted by 1-4 substituents each independently selected from hydroxy, –O-P(O)(OH)2, -O-P(O)(RIRII)2, C1-C4alkoxy-, -N(Re)(Rf), -CO2(Rf), -CON(Re)(Rf), optionally substituted phenyl, optionally substituted 5-6 membered heterocycloalkyl and optionally substituted 5-6 membered heteroaryl group, wherein said optionally substituted phenyl, 5-6 membered heterocycloalkyl or 5-6 membered heteroaryl is optionally substituted by 1-4 substituents each independently selected from halogen, hydroxy, -O-P(O)(OH)2, -O- P(O)(RIRII)2, amino, (C1-C6alkyl)amino-, (C1-C6alkyl)(C1-C6alkyl)amino-, -(C1-C6alkyl)- NH2, halo(C1-C6alkyl), hydroxy-(C1-C4alkyl)-, -(C1-C4alkyl)-O-P(O)(OH)2, - (C1-C4alkyl)-O-P(O)(RIRII)2, halo(C1-C4alkoxy)-, C1-C4alkoxy-, hydroxy-(C2-C4alkoxy)-, -(C2-C4alkoxy)-O-P(O)(OH)2, -(C2-C4alkoxy)-O-P(O)(RIRII)2, -C1-C4alkyl-(C1-C4alkoxy) or C1-C4alkoxy-(C1-C4alkoxy)-; when r is 0, RB1 and RB2 are each independently H, optionally substituted C1-C6alkyl, halo(C1-C6alkyl), optionally substituted C2-C6alkenyl, optionally substituted C2-C6alkynyl, optionally substituted C3-C6cycloalkyl, optionally substituted 4-6 membered heterocycloalkyl, optionally substituted phenyl, optionally substituted 5-6 membered heteroaryl, or optionally substituted 9-10 membered heteroaryl, wherein said optionally substituted C1-C6alkyl, optionally substituted C2-C6alkenyl, optionally substituted C2-C6alkynyl, optionally substituted C3-C6cycloalkyl, optionally substituted 4-6 membered heterocycloalkyl, optionally substituted phenyl, optionally substituted 5-6 membered heteroaryl, or optionally substituted 9-10 membered heteroaryl is optionally substituted by 1-4 substituents each independently selected from halogen, nitro, -Rc, -OH, -O-P(O)(OH)2, -O- P(O)(RIRII)2, -ORc, -NH2, -NRcRc, -NRcRd, -OCORc, -CO2H, -CO2Rc, -SORc, -SO2Rc, -CONH2, -CONRcRd, -SO2NH2, -SO2NRcRd, -OCONH2, -OCONRcRd, -NRdCORc, -NRdSORc, -NRdCO2Rc, and -NRdSO2Rc; when s is 0, RC1 is H, halogen, or C1-C4alkyl and RC2 is optionally substituted C1-C4alkyl, wherein said optionally substituted C1-C4alkyl group is optionally substituted by a substituent selected from -ORc, -NRcRd, -CO2Rc, -CONRcRd, -SO2NRcRd, and -OCONRcRd; when q is 1, RA1 and RA2 are each independently -CH2-, -NRe-, or -O-, and A, taken together with RA1 and RA2, forms a linking group, wherein A is -halo(C1-C12alkyl)-, optionally substituted -C1-C12alkyl-, optionally substituted -C2-C12alkenyl-, optionally substituted -C2-C12alkynyl-, optionally substituted -C1-C6alkyl-O-C1-C6alkyl-, optionally substituted -C1-C6alkyl-NRa-C1-C6alkyl-, optionally substituted -C1-C6alkyl-(C3-C6cycloalkyl)-C1-C6alkyl-, optionally substituted -C1-C6alkyl-phenyl-C1-C6alkyl-, optionally substituted -C1-C6alkyl-(4-6 membered heterocycloalkyl)-C1-C6alkyl-, or optionally substituted -C1-C6alkyl-(5-6 membered heteroaryl)-C1-C6alkyl-, wherein the alkyl moiety of said optionally substituted -C1-C12alkyl-, optionally substituted -C2-C12alkenyl-, optionally substituted -C2-C12alkynyl-, optionally substituted -C1-C6alkyl-O-C1-C6alkyl-, optionally substituted -C1-C6alkyl-NRa-C1-C6alkyl-, optionally substituted -C1-C6alkyl-(C3-C6cycloalkyl)-C1-C6alkyl-, optionally substituted -C1-C6alkyl-phenyl-C1-C6alkyl-, optionally substituted -C1-C6alkyl-(4-6 membered heterocycloalkyl)-C1-C6alkyl-, or optionally substituted -C1-C6alkyl-(5-6 membered heteroaryl)-C1-C6alkyl- is optionally substituted by 1-4 substituents each independently selected from halogen, halo(C1-C4alkyl), -OH, -O-P(O)(OH)2, -O-P(O)(RIRII)2, -ORc, -NH2, -NRcRd, -OCORc, -CO2H, -CO2Rc, -SORc, -SO2Rc, -CONH2, -CONRcRd, -SO2NH2, -SO2NRcRd, -OCONH2, -OCONRcRd, -NRdCORc, -NRdSORc, -NRdCO2Rc, and -NRdSO2Rc, and the C3-C6cycloalkyl, phenyl, 4-6 membered heterocycloalkyl, or 5-6 membered heteroaryl moiety of said optionally substituted -C1-C6alkyl-(C3-C6cycloalkyl)-C1-C6alkyl-, optionally substituted -C1-C6alkyl-phenyl-C1-C6alkyl-, optionally substituted -C1-C6alkyl-(4-6 membered heterocycloalkyl)-C1-C6alkyl-, or optionally substituted -C1-C6alkyl-(5-6 membered heteroaryl)-C1-C6alkyl- is optionally substituted by 1-4 substituents each independently selected from halogen, hydroxy, -O-P(O)(OH)2, -O-P(O)(RIRII)2, amino, (C1-C4alkyl)amino-, (C1-C4alkyl)(C1-C4alkyl)amino-, C1-C4alkyl, halo(C1-C4alkyl), halo(C1-C4alkoxy)-, C1-C4alkoxy-, hydroxy-(C1-C4alkoxy)-, -(C1-C4alkoxyl)-O-P(O)(OH)2, -(C1-C4alkoxyl)-O-P(O)(RIRII)2 and C1-C4alkoxy-(C1-C4alkoxy)-; when r is 1, RB1 and RB2 are each independently -CH -, and B, taken together wit B1 2 h R and RB2, forms a linking group, wherein B is a bond or B is -halo(C1-C10alkyl)-, optionally substituted -C1-C10alkyl-, optionally substituted -C2-C10alkenyl-, optionally substituted -C2-C10alkynyl-, optionally substituted -C1-C6alkyl-O-C1-C6alkyl-, optionally substituted -C1-C6alkyl-NRa-C1-C6alkyl-, optionally substituted C3-C6cycloalkyl, optionally substituted phenyl, optionally substituted 4-6 membered heterocycloalkyl, optionally substituted 5-6 membered heteroaryl, optionally substituted -C1-C4alkyl-(C3-C6cycloalkyl)-C1-C4alkyl-, optionally substituted -C1-C4alkyl-phenyl-C1-C4alkyl-, optionally substituted -C1-C4alkyl-(4-6 membered heterocycloalkyl)-C1-C4alkyl-, or optionally substituted -C1-C4alkyl-(5-6 membered heteroaryl)-C1-C4alkyl-, wherein the alkyl moiety of said optionally substituted -C1-C10alkyl-, optionally substituted -C2-C10alkenyl-, optionally substituted -C2-C10alkynyl-, optionally substituted -C1-C6alkyl-O-C1-C6alkyl-, optionally substituted -C1-C6alkyl-NRa-C1-C6alkyl-, optionally substituted -C1-C4alkyl-(C3-C6cycloalkyl)-C1-C4alkyl-, optionally substituted -C1-C4alkyl-phenyl-C1-C4alkyl-, optionally substituted -C1-C4alkyl-(4-6 membered heterocycloalkyl)-C1-C4alkyl-, or optionally substituted -C1-C4alkyl-(5-6 membered heteroaryl-C1-C4alkyl)- is optionally substituted by 1 or 2 substituents each independently selected from halogen, halo(C1-C4alkyl), -OH, -O-P(O)(OH)2, -O-P(O)(RIRII)2, -ORc, -NH2, -NRcRd, -OCORc, -CO2H, -CO2Rc, -SORc, -SO2Rc, -CONH2, -CONRcRd, -SO2NH2, -SO2NRcRd, -OCONH2, -OCONRcRd, -NRdCORc, -NRdSORc, -NRdCO2Rc, and -NRdSO2Rc, and the C3-C6cycloalkyl, phenyl, 4-6 membered heterocycloalkyl, or 5-6 membered heteroaryl moiety of said optionally substituted C3-C6cycloalkyl, optionally substituted phenyl, optionally substituted 4-6 membered heterocycloalkyl, optionally substituted 5-6 membered heteroaryl, optionally substituted -C1-C4alkyl-(C3-C6cycloalkyl)-C1-C4alkyl-, optionally substituted -C1-C4alkyl-phenyl-C1-C4alkyl-, optionally substituted -C1-C4alkyl-(4-6 membered heterocycloalkyl)-C1-C4alkyl-, or optionally substituted -C1-C4alkyl-(5-6 membered heteroaryl)-C1-C4alkyl- is optionally substituted by 1-4 substituents each independently selected from halogen, hydroxy, -O-P(O)(OH)2, -O- P(O)(RIRII)2, amino, (C1-C4alkyl)amino-, (C1-C4alkyl)(C1-C4alkyl)amino-, C1-C4alkyl, halo(C1-C4alkyl), halo(C1-C4alkoxy)-, C1-C4alkoxy-, hydroxy-(C2-C4alkoxy)-, -(C2-C4alkoxy) O-P(O)(OH)2, -(C2-C4alkoxy)-O-P(O)(RIRII)2, and C1-C4alkoxy-(C1-C4alkoxy)-; when s is 1, RC1 and RC2 are each independently -CH -, and C, taken toget C1 2 her with R and RC2, forms a linking group, wherein C is -halo(C1-C12alkyl)-, optionally substituted -C1-C12alkyl-, optionally substituted -C2-C12alkenyl-, optionally substituted -C2-C12alkynyl-, optionally substituted -C1-C6alkyl-O-C1-C6alkyl-, optionally substituted -C1-C6alkyl-NRa-C1-C6alkyl-, optionally substituted -C1-C6alkyl-(C3-C6cycloalkyl)-C1-C6alkyl-, optionally substituted -C1-C6alkyl-phenyl-C1-C6alkyl-, optionally substituted -C1-C6alkyl-(4-6 membered heterocycloalkyl)-C1-C6alkyl-, or optionally substituted -C1-C6alkyl-(5-6 membered heteroaryl)-C1-C6alkyl-, wherein the alkyl moiety of said optionally substituted -C1-C12alkyl-, optionally substituted -C2-C12alkenyl-, optionally substituted -C2-C12alkynyl-, optionally substituted -C1-C6alkyl-O-C1-C6alkyl-, optionally substituted -C1-C6alkyl-NRa-C1-C6alkyl-, optionally substituted -C1-C6alkyl-(C3-C6cycloalkyl)-C1-C6alkyl-, optionally substituted -C1-C6alkyl-phenyl-C1-C6alkyl-, optionally substituted -C1-C6alkyl-(4-6 membered heterocycloalkyl)-C1-C6alkyl-, or optionally substituted -C1-C6alkyl-(5-6 membered heteroaryl)-C1-C6alkyl- is optionally substituted by 1 or 2 substituents each independently selected from halogen, halo(C1-C4alkyl), -OH, -O-P(O)(OH)2, -O-P(O)(RIRII)2, -ORc, -NH2, -NRcRd, -OCORc, -CO2H, -CO2Rc, -SORc, -SO2Rc, -CONH2, -CONRcRd, -SO2NH2, -SO2NRcRd, -OCONH2, -OCONRcRd, -NRdCORc, -NRdSORc, -NRdCO2Rc, and -NRdSO2Rc, and the C3-C6cycloalkyl, phenyl, 4-6 membered heterocycloalkyl, or 5-6 membered heteroaryl moiety of said optionally substituted -C1-C6alkyl-(C3-C6cycloalkyl)-C1-C6alkyl-, optionally substituted -C1-C6alkyl-phenyl-C1-C6alkyl-, optionally substituted -C1-C6alkyl-(4-6 membered heterocycloalkyl)-C1-C6alkyl-, or optionally substituted -C1-C6alkyl-(5-6 membered heteroaryl)-C1-C6alkyl- is optionally substituted by 1-4 substituents each independently selected from halogen, hydroxy, -O-P(O)(OH)2, -O-P(O)(RIRII)2, amino, (C1-C4alkyl)amino-, (C1-C4alkyl)(C1-C4alkyl)amino-, C1-C4alkyl, halo(C1-C4alkyl), halo(C1-C4alkoxy)-, C1-C4alkoxy-, hydroxy-(C2-C4alkoxy)-, -(C2-C4alkoxy)-O-P(O)(OH)2, -(C2-C4alkoxy)-O-P(O)(RIRII)2,and C1-C4alkoxy-(C1-C4alkoxy)-; R3 and R5 are each independently -CON(Rd)(Rf), or one of R3 and R5 is -CON(Rd)(Rf), and the other of R3 and R5 is H, COOH or -CO2(Rc); R4 and R6 are each independently selected from H, halogen, halo(C1-C6alkyl), halo(C1-C6alkoxy)-, hydroxy, -O-P(O)(OH)2, -O-P(O)(RIRII)2, -NH2, -NRcRc, -NRcRd, -CORc, -CO2Rc, -N(Rd)CORc, -N(Rd)SO2Rc, -N(Rg)SO2(C1-C2alkyl)-N(Rh)(Rf), -N(Rg)CO(C1-C2alkyl)-N(Rh)(Rf), optionally substituted (C1-C6alkyl), optionally substituted (C1-C6alkyl)oxy-, optionally substituted (C1-C6alkyl)amino-, and optionally substituted (C1-C6alkyl)(C1-C4alkyl)amino-, wherein the (C1-C6alkyl) of said optionally substituted (C1-C6alkyl), optionally substituted (C1-C6alkyl)oxy-, optionally substituted (C1-C6alkyl)amino- and optionally substituted (C1-C6alkyl)(C1-C4alkyl)amino- is optionally substituted by 1-4 substituents each independently selected from -OH, -O-P(O)(OH)2, -O-P(O)(RIRII)2,-ORc, -NH2, -NRcRc, -NRcRd, -CO2H, -CO2Rc, -OCORc, -CO2H, -CO2Rc, -SORc, -SO2Rc, -CONH2, -CONRcRd, -SO2NH2, -SO2NRcRd, -OCONH2, -OCONRcRd, -NRdCORc, -NRdSORc, -NRdCO2Rc, -NRdSO2Rc, optionally substituted phenyl, optionally substituted 5-6 membered heterocycloalkyl and optionally substituted 5-6 membered heteroaryl group, wherein said optionally substituted phenyl, 5-6 membered heterocycloalkyl or 5-6 membered heteroaryl is optionally substituted by 1-4 substituents each independently selected from halogen, hydroxy, -O-P(O)(OH)2, -O- P(O)(RIRII)2, amino, (C1-C4alkyl)amino-, (C1-C4alkyl)(C1-C4alkyl)amino-, C1-C4alkyl, halo(C1-C4alkyl), hydroxy-(C1-C4alkyl)-, -(C1-C4alkyl)-O-P(O)(OH)2, -(C1-C4alkyl)-O-P(O)(RIRII)2, halo(C1-C4alkoxy)-, C1-C4alkoxy-, hydroxy-(C2-C4alkoxy)-, -(C2-C4alkoxy)-O-P(O)(OH)2, -(C2-C4alkoxy)-O-P(O)(RIRII)2, C1-C4alkoxy-(C1-C4alkoxy)-, -CORd, -CON(Rd)(Rf), and -CO2Rd; R14 is optionally substituted C1-C4alkyl, wherein said optionally substituted C1-C4alkyl is optionally substituted by a substituent selected from -ORc, -NRcRd, -CO2Rc, -CONRcRd, -SO2NRcRd, and -OCONRcRd; R16 is H, halogen, or C1-C4alkyl; R15 and R17 are each independently H, cyclopropyl, or C1-C4alkyl; Ra is H, -Rc, -CORc, -CO2H, -CO2Rc, -SORc, -SO2Rc, -CONH2, -CONRcRd, -SO2NH2, or -SO2NRcRd; each Rb is independently C1-C4alkyl, halo(C1-C4alkyl), -(C1-C4alkyl)-OH, -(C1-C4alkyl)-O-P(O)(OH)2, -(C1-C4alkyl)-O-P(O)(RIRII)2, -(C1-C4alkyl)-O-(C1-C4alkyl), -(C1-C4alkyl)-N(Re)(Rf), -(C1-C4alkyl)-O-CO(C1-C4alkyl), or -(C1-C4alkyl)-CO-O- (C1-C4alkyl); each Rc is independently C1-C4alkyl, halo(C1-C4alkyl), -(C1-C4alkyl)-OH, -(C1-C4alkyl)-O-P(O)(OH)2, -(C1-C4alkyl)-O-P(O)(RIRII)2,-(C1-C4alkyl)-O-(C1-C4alkyl), -(C1-C4alkyl)-N(Re)(Rf), -(C1-C4alkyl)-O-CO(C1-C4alkyl), -(C1-C4alkyl)-CO-O- (C1-C4alkyl), optionally substituted C3-C6cycloalkyl, optionally substituted phenyl, optionally substituted 4-6 membered heterocycloalkyl, optionally substituted 5-6 membered heteroaryl, optionally substituted 9-10 membered heteroaryl, optionally substituted -C1-C4alkyl-C3-C6cycloalkyl, optionally substituted -C1-C4alkyl-phenyl, optionally substituted -C1-C4alkyl-4-6 membered heterocycloalkyl, optionally substituted -C1-C4alkyl-5-6 membered heteroaryl, or optionally substituted -C1-C4alkyl-9-10 membered heteroaryl, wherein the C3-C6cycloalkyl, phenyl, 4-6 membered heterocycloalkyl, 5-6 membered heteroaryl or 9-10 membered heteroaryl moiety of said optionally substituted C3-C6cycloalkyl, optionally substituted phenyl, optionally substituted 4-6 membered heterocycloalkyl, optionally substituted 5-6 membered heteroaryl, optionally substituted 9-10 membered heteroaryl optionally substituted -C1-C4alkyl-C3-C6cycloalkyl, optionally substituted -C1-C4alkyl-phenyl, optionally substituted -C1-C4alkyl-4-6 membered heterocycloalkyl, optionally substituted -C1-C4alkyl-5-6 membered heteroaryl, or optionally substituted -C1-C4alkyl-9-10 membered heteroaryl is optionally substituted by 1-4 substituents each independently selected from halogen, hydroxy, -O-P(O)(OH)2, -O-P(O)(RIRII)2, amino, -(C1-C4alkyl)NH2, (C1-C4alkyl)amino-, (C1-C4alkyl)(C1-C4alkyl)amino-, C1-C4alkyl, halo(C1-C4alkyl), halo(C1-C4alkoxy)-, C1-C4alkoxy-, hydroxy-(C2-C4alkoxy)-, -(C2-C4alkoxy)-O-P(O)(OH)2, -(C2-C4alkoxy)-O-P(O)(RIRII)2, C1-C4alkoxy-(C1-C4alkoxy)-, -CORd, -CON(Rd)(Rf), and -CO2Rd; each Rd is independently H or C1-C4alkyl; each Re is independently H, (C1-C4alkyl), -CO(C1-C4alkyl), -OCO(C1-C4alkyl), -CO2(C1-C4alkyl), -(C1-C4alkyl)NH2, -(C1-C4alkyl) C1-C4alkoxy, -CO-(optionally substituted 5-6 membered heterocycloalkyl), -CO(C1-C4alkyl)-(optionally substituted 5-6 membered heterocycloalkyl), -CO(optionally substituted 5-6 membered heteroaryl), -CO(C1-C4alkyl)-(optionally substituted 5-6 membered heteroaryl), wherein the optionally substituted 5-6 membered heterocycloalkyl or optionally substituted 5-6 membered heteroaryl is optionally substituted 1-4 substituents each independently selected from halogen, hydroxy, -O-P(O)(OH)2, -O-P(O)(RIRII)2, amino, (C1-C4alkyl)amino-, (C1-C4alkyl)(C1-C4alkyl)amino-, C1-C4alkyl, halo(C1-C4alkyl), halo(C1-C4alkoxy)-, C1-C4alkoxy-, hydroxy-(C2-C4alkoxy)-, -(C2-C4alkoxy) O-P(O)(OH)2, -(C2-C4alkoxy)-O-P(O)(RIRII)2, C1-C4alkoxy-(C1-C4alkoxy)- , -CORd, -CON(Rd)(Rf), and -CO2Rd; each Rf is independently H or (C1-C4alkyl); Rg and Rh are each independently H or (C1-C4alkyl) or Rg and Rh, taken together with the atom or atoms through which they are connected, form a 5-6 membered ring; and each occurrence of RI and RII are independently (C1-C6alkyl)oxy-; or a tautomer thereof. In one embodiment, the STING compound is selected from: (E)-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1H- benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-7- (3-hydroxypropoxy)-1H-benzo[d]imidazole-5-carboxamide (Example 10 of WO
Figure imgf000061_0001
(E)-1-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-2,3- dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-5- carbonyl)imino)-7-(3-hydroxypropoxy)-2,3-dihydro-1H-benzo[d]imidazole-5- carboxamide (Example 10 of WO 2017/175147))
Figure imgf000062_0001
(Z)-1-((E)-4-((Z)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-2,3- dihydro-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-7-(3-hydroxypropoxy)-2,3-dihydro-1 H-benzo[d]imidazole-5- carboxamide (Example 10 of WO 2017/175147)
Figure imgf000062_0002
(E)-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1 H-pyrazole-5-carboxamido)-7-(3- hydroxypropoxy)-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1 H- pyrazole-5-carboxamido)-7-methoxy-1 H-benzo[d]imidazole-5-carboxamide (Example 11 of WO 2017/175147)
Figure imgf000062_0003
(E)-1-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-7-(3- hydroxypropoxy)-2,3-dihydro-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3- methyl-1 H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1 H-benzo[d]imidazole-5- carboxamide (Example 11 of WO 2017/175147)
Figure imgf000063_0001
(Z)-1-((E)-4-((Z)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-7-(3- hydroxypropoxy)-2,3-dihydro-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3- methyl-1 H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1 H-benzo[d]imidazole-5- carboxamide (Example 11 of WO 2017/175147)
Figure imgf000063_0002
(E)-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1 H-pyrazole-5-carboxamido)-1 H- benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1 H-pyrazole-5-carboxamido)-7-
(3-morpholinopropoxy)-1 H-benzo[d]imidazole-5-carboxamide (Example 13 of WO
Figure imgf000063_0003
(E)-1-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-2,3- dihydro-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-7-(3-morpholinopropoxy)-2,3-dihydro-1 H-benzo[d]imidazole-5- carboxamide (Example 13 of WO 2017/175147)
Figure imgf000064_0001
(Z)-1-((E)-4-((Z)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-2,3- dihydro-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-7-(3-morpholinopropoxy)-2,3-dihydro-1 H-benzo[d]imidazole-5- carboxamide (Example 13 of WO 2017/175147)
Figure imgf000064_0002
(E)-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1 H-pyrazole-5-carboxamido)-7-(3- morpholinopropoxy)-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1 H- pyrazole-5-carboxamido)-7-methoxy-1 H-benzo[d]imidazole-5-carboxamide (Example
Figure imgf000064_0003
(E)-1-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-7-(3- morpholinopropoxy)-2,3-dihydro-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3- methyl-1 H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1 H-benzo[d]imidazole-5- carboxamide (Example 14 of WO 2017/175147)
Figure imgf000065_0001
(Z)-1-((E)-4-((Z)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-7-(3- morpholinopropoxy)-2,3-dihydro-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3- methyl-1 H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1 H-benzo[d]imidazole-5- carboxamide (Example 14 of WO 2017/175147)
Figure imgf000065_0002
3-(((Z)-6-carbamoyl-3-((E)-4-((Z)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-7-methoxy-2,3-dihydro-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1- ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1 H-benzo[d]imidazol-4- yl)oxy)propyldihydrogen phosphate (Example 19 of WO 2017/175147)
Figure imgf000065_0003
(E)-3-((5-carbamoyl-1 -(4-(5-carbamoyl-2-(1 -ethyl-3-methyl-1 H-pyrazole-5- carboxamido)-7-methoxy-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-
1 H-pyrazole-5-carboxamido)-1 H-benzo[d]imidazol-7-yl)oxy)propyl dihydrogen phosphate (Example 19 of WO 2017/175147)
Figure imgf000066_0001
3-(((E)-6-carbamoyl-3-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-7-methoxy-2,3-dihydro-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1- ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1 H-benzo[d]imidazol-4- yl)oxy) propyl dihydrogen phosphate (Example 19 of WO 2017/175147)
Figure imgf000066_0002
(E)-4-((5-carbamoyl-1 -(4-(5-carbamoyl-2-(1 -ethyl-3-methyl-1 H-pyrazole-5- carboxamido)-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1 H-pyrazole- 5-carboxamido)-1 H-benzo[d]imidazol-7-yl)oxy)butanoic acid (Example 52 of WO 2017/175147)
Figure imgf000067_0001
(E)-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1 H-pyrazole-5-carboxamido)-7-methoxy-1 H- benzo[d]imidazol-1-yl)but-2-en-1-yl)-7-(3-(dimethylamino)propoxy)-2-(1-ethyl-3-methyl- 1 H-pyrazole-5-carboxamido)-1 H-benzo[d]imidazole-5-carboxamide (Example 39 of WO
Figure imgf000067_0002
(E)-1-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-7-(3-
(4-(2-hydroxyethyl)piperazin-1-yl)propoxy)-2,3-dihydro-1 H-benzo[d]imidazol-1-yl)but-2- en-1-yl)-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1 H- benzo[d]imidazole-5-carboxamide (Example 43 of WO 2017/175147)
Figure imgf000067_0003
In one embodiment, the STING agonist is 3-(((Z)-6-Carbamoyl-3-((E)-4-((Z)-5- carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1 H- benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-2,3- dihydro-1 H-benzo[d]imidazol-4-yl)oxy)propyl dihydrogen phosphate
Figure imgf000068_0001
In one embodiment, the STING agonist is (E)-3-((5-carbamoyl-1-(4-(5-carbamoyl-2-(1- ethyl-3-methyl-1 H-pyrazole-5-carboxamido)-7-methoxy-1 H-benzo[d]imidazol-1-yl)but-2-en-1- yl)-2-(1-ethyl-3-methyl-1 H-pyrazole-5-carboxamido)-1 H-benzo[d]imidazol-7-yl)oxy)propyl
Figure imgf000068_0002
In one embodiment, the STING agonist is by 3-(((E)-6-carbamoyl-3-((E)-4-((E)-5- carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1 H- benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-2,3- dihydro-1 H-benzo[d]imidazol-4-yl)oxy)propyl
Figure imgf000068_0003
Those skilled in the art will appreciate that STING agonists may contain one or more asymmetrically substituted atom(s), furthermore, geometric isomers of double bonds such as olefins and C=N double bonds may also be present in certain STING agonists, and all chiral, diastereomeric, racemic forms and all geometric isomeric forms of a structure are intended, unless the specific stereochemistry or isomeric form is specifically indicated. Additionally, certain compounds may exist as tautomers, and all such tautomers of a structure are intended unless the context requires otherwise.
Compounds may also contain ionisable groups and therefore may be presented as a pharmaceutically acceptable salt.
As used herein, "pharmaceutically acceptable salts" refer to derivatives of the disclosed compounds wherein the parent compound is modified by making pharmaceutically acceptable acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic groups such as amines; and alkali or organic salts of acidic groups such as carboxylic acids. The pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, and nitric; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2- acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, and isethionic, and the like.
Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, 1985, p.1418, the disclosure of which is hereby incorporated by reference.
The phrase "pharmaceutically acceptable salt" is employed herein to refer to those salts which are, within the scope of sound medical judgment, suitable for use in a pharmaceutical context, without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
If provided in solution, STING agonists with ionisable groups may be in dissociated form. Pharmaceutically acceptable salts of STING agonists may be dissociated in solution.
If provided in solid form, the STING agonist may be in the form of a solvate, such as a hydrate.
A typical human dose of STING agonist may be 0.1 to 150 ug, especially 0.5 to 100 ug, such as 1 to 50 ug.
More than one STING agonist may be utilised, such as two. Typically only one STING agonist is utilised. A STING agonist may be administered with an aluminium salt, such as AIPO4, AI(OH)3, AIO(OH), or a mixture thereof. More suitably the aluminium salt comprises, consists essentially of, or more suitably consists of, AI(OH)3. In one embodiment, the STING agonist may be combined with the aluminium salt in a single formulation and suitably the STING agonist is adsorbed to the aluminium salt. In a particular embodiment of a combined formulation the STING agonist is 3-(((E)-6-carbamoyl-3-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3- methyl-1 H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1 H-benzo[d]imidazol-1-yl)but-2- en-1-yl)-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1 H-benzo[d]imidazol-
4-yl)oxy)propyl
Figure imgf000070_0001
pharmaceutically acceptable salt thereof, and most suitably the aluminum salt is AI(OH)3.
Saponins
In certain embodiments a STING agonist is administered in step (i) and a second adjuvant is administered in step (ii). In certain embodiments the second adjuvant may be an adjuvant comprising a saponin.
A suitable saponin for use in the present invention is Quil A and its derivatives. Quil A is a saponin preparation isolated from the South American tree Quillaja saponaria Molina and was first described as having adjuvant activity by Dalsgaard et al. in 1974 (“Saponin adjuvants”, Archiv. fur die gesamte Virusforschung, Vol. 44, Springer Verlag, Berlin, p243- 254). Purified fractions of Quil A have been isolated by HPLC which retain adjuvant activity without the toxicity associated with Quil A (see, for example, EP0362278). Fractions of general interest include QS-7, QS-17, QS-18 and QS-21 , for example QS-7 and QS-21 (also known as QA7 and QA21). QS-21 is a saponin fraction of particular interest.
In certain embodiments of the present invention, the saponin is a derivative of Quillaja saponaria Molina quil A, suitably an immunologically active fraction of Quil A, such as QS-7, QS-17, QS-18 or QS-21 , in particular QS-21. WQ2019/106192, incorporated herein by reference for the purpose of defining saponin fractions which may be of use in the present invention, describes QS-21 fractions of particular interest. Typically the saponin, such as Quil A and in particular QS-21, is at least 90% pure, such as at least 95% pure, especially at least 98% pure, in particular 99% pure w/w.
QS-21 contains a plurality of components, with the principal components typically being: ‘QS-21 1988 A component’, which is identified in Kite 2004 as Peak 88 and corresponds to the A-isomer xylose chemotype structures S4 (apiose isomer) and S6
(xylose isomer) characterised in Nyberg 2000 and Nyberg 2003. The QS-21 1988 A component may consist of QS-21 1988 A V1 (i.e. apiose isomer):
Figure imgf000071_0001
- ‘QS-21 1856 A component’, which is identified in Kite 2004 as Peak 86 and corresponds to the A-isomer xylose chemotype structure S2 characterised in Nyberg 2000 and Nyberg 2003. The QS-21 1856 A component may consist of:
Figure imgf000072_0001
- ‘QS-21 2002 A component’, which is identified in Kite 2004 as Peak 85 and corresponds to the A-isomer rhamnose chemotype of structures S3 and S5 characterised in Nyberg 2000 and Nyberg 2003. The QS-21 2002 A component may consist of QS-21 2002 A V1 (i.e. apiose isomer):
Figure imgf000072_0002
and QS-21 2002 A V2 (i.e. xylose isomer):
Figure imgf000073_0001
Consequently, the saponin desirably comprises at least 40%, such as at least 50%, suitably at least 60%, especially at least 70% and desirably at least 80%, for example at least 90% (as determined by UV absorbance at 214 nm and by relative ion abundance) QS-21 1988 A component, QS-21 1856 A component and/or QS-21 2002 A component.
In certain embodiments, the saponin contains at least 40%, such as at least 50%, in particular at least 60%, especially at least 65%, such as at least 70%, QS-21 1988 A component as determined by UV absorbance at 214 nm and by relative ion abundance. In certain embodiments the saponin extracts contain 90% or less, such as 85% or less, or 80% or less, QS-21 1988 A component as determined by UV absorbance at 214 nm and by relative ion abundance. In certain embodiments, the saponin extracts contain from 40% to 90% QS-21 1988 A component, such as 50% to 85% QS-21 1988 A component, especially 70% to 80% QS-21 1988 A component as determined by UV absorbance at 214 nm and by relative ion abundance.
In certain embodiments, the saponin extracts contain 30% or less, such as 25% or less, QS-21 1856 A as determined by UV absorbance at 214 nm and by relative ion abundance. In certain embodiments the saponin extracts contain at least 5%, such as at least 10% QS-21 1856 A by UV absorbance at 214 nm and by relative ion abundance. In certain embodiments, the saponin extracts contain from 5% to 30% QS-21 1856 A, such as 10% to 25% QS-21 1856 A as determined by UV absorbance at 214 nm and by relative ion abundance.
In certain embodiments, the saponin extracts contain 40% or less, such as 30% or less, in particular 20% or less, especially 10% or less QS-21 2002 A component by UV absorbance at 214 nm and by relative ion abundance. In certain embodiments, the saponin extracts contain at least 0.5%, such as at least 1%, QS-21 2002 A component by UV absorbance at 214 nm and by relative ion abundance. In certain embodiments, the saponin extracts contain from 0.5% to 40% QS-21 2002 A component, such as 1% to 10% QS-21 2002 A component as determined by UV absorbance at 214 nm and by relative ion abundance.
By the term ‘UV absorbance at 214 nm and relative ion abundance’ is meant an estimate for the percentage of a given m/z for co-eluting species. (Percentage area for given UV peak) x (relative ion abundance for m/z of interest in given peak)/(sum of all relative ion abundance for given peak) = percentage of m/z of interest in the given UV peak, assumes relative ion abundance included for all coeluting species.
QS-21 1988 A component, QS-21 1856 A component and/or QS-21 2002 A component may be obtained by extraction from Quillaja species or may be prepared synthetically (such as semi-synthetically). TLR agonists
The term "Toll-like receptor" (or "TLR") refers to a member of the Toll-like receptor family of proteins which sense microbial products and/or initiate adaptive immune responses. Toll-like receptors (TLRs) are a family of pattern recognition receptors that were initially identified as sensors of the innate immune system that recognize microbial pathogens. TLRs recognize distinct structures in microbes, often referred to as "PAMPs" (pathogen associated molecular patterns). Ligand binding to TLRs invokes a cascade of intra-cellular signaling pathways that induce the production of factors involved in inflammation and immunity. In humans, ten TLRs have been identified. TLRs that are expressed on the surface of cells include TLR1, TLR2, TLR4, TLR5, and TLR6, while TLR3, TLR7/8, and TLR9 are expressed with the ER compartment. Human dendritic cell (DC) subsets can be identified on the basis of distinct TLR expression patterns. By way of example, the myeloid or "conventional" subset of DC (mDC) expresses TLRs 1-8 when stimulated, and a cascade of activation markers (e.g. CD80, CD86, MHC class I and II, CCR7), pro-inflammatory cytokines, and chemokines are produced. A result of this stimulation and resulting expression is antigen-specific CD4+ and CD8+ T cell priming. These DCs acquire an enhanced capacity to take up antigens and present them in an appropriate form to T cells. In contrast, the plasmacytoid subset of DC (pDC) expresses only TLR7 and TLR9 upon activation, with a resulting activation of NK cells as well as T-cells.
TLR agonists known in the art include: Pam3Cys, a TLR1/2 agonist; CFA, a TLR2 agonist; MALP2, a TLR2 agonist; Pam2Cys, a TLR2 agonist; FSL-I, a TLR-2 agonist; Hib- OMPC, a TLR-2 agonist; polyinosinic: polycytidylic acid (Poly I :C), a TLR3 agonist; polyadenosine-polyuridylic acid (poly AU), a TLR3 agonist; polyinosinic-polycytidylic acid stabilized with poly-L-lysine and carboxymethylcellulose (Hiltonol), a TLR3 agonist; bacterial flagellin a TLR5 agonist; imiquimod, a TLR7 agonist; resiquimod, a TLR7/8 agonist; loxoribine, a TLR7/8 agonist; and unmethylated CpG dinucleotide (CpG-ODN), a TLR9 agonist. The skilled person will appreciate that a plurality of TLR agonists may be utilised in combination.
TLR7/8 agonist
Exemplary agonists of TLR7 and/or TLR8 include imiquimod and resiquimod. Other agonists of TLR7 and/or TLR8 include:
Figure imgf000075_0001
Figure imgf000076_0001
Figure imgf000077_0001
TLR4 agonists
A suitable example of a TLR4 agonist is a lipopolysaccharide, suitably a non-toxic derivative of lipid A, particularly a monophosphoryl lipid A and more particularly 3-de-O- acylated monophosphoryl lipid A (3D-MPL).
3D-MPL is sold under the name ‘MPL’ by GlaxoSmithKline Biologicals N.A. and is referred throughout the document as 3D-MPL. See, for example, US Patent Nos. 4,436,727; 4,877,611 ; 4,866,034 and 4,912,094. 3D-MPL can be produced according to the methods described in GB 2 220211 A. Chemically it is a mixture of 3-deacylated monophosphoryl lipid A with 4, 5 or 6 acylated chains. In the context of the present invention small particle 3D-MPL may be used to prepare the aqueous adjuvant composition. Small particle 3D-MPL has a particle size such that it may be sterile-filtered through a 0.22 urn filter. Such preparations are described in WO94/21292. Suitably, powdered 3D-MPL is used to prepare aqueous adjuvant compositions of use in the present invention.
Other TLR4 agonists which can be used are aminoalkyl glucosaminide phosphates (AGPs) such as those described in WO98/50399 or US patent No. 6,303,347 (processes for preparation of AGPs are also described). Some AGPs are TLR4 agonists, and some are TLR4 antagonists. A particular AGP of interest is set forth as follows:
Figure imgf000078_0001
Other TLR4 agonists which may be of use in the present invention include Glucopyranosyl Lipid Adjuvant (GLA) such as described in W02008/153541 or WO2009/143457 or the literature articles Coler RN et al. (2011) Development and Characterization of Synthetic Glucopyranosyl Lipid Adjuvant System as a Vaccine Adjuvant. PLoS ONE 6(1): e16333. doi:10.1371/journal.pone.0016333 and Arias MA et al. (2012) Glucopyranosyl Lipid Adjuvant (GLA), a Synthetic TLR4 Agonist, Promotes Potent Systemic and Mucosal Responses to Intranasal Immunization with HIVgp140. PLoS ONE 7(7): e41144. doi:10.1371/journal. pone.0041144. WO2008/153541 or W02009/143457 are incorporated herein by reference for the purpose of defining TLR4 agonists which may be of use in the present invention.
TLR4 agonists of interest include:
Figure imgf000079_0001
-deacyl monophosphoryl hexa-acyl lipid A.
Another TLR4 agonist of interest is:
Figure imgf000079_0002
deacyl monophosphoryl lipid A.
A TLR agonist of interest is dLOS (as described in Han, 2014):
Figure imgf000079_0003
outer core OS inner core OS Lipid A Typically the TLR4 agonist, such as the lipopolysaccharide, such as a monophosphoryl lipid A and in particular 3D-MPL, is at least 90% pure, such as at least 95% pure, especially at least 98% pure, in particular 99% pure w/w.
The skilled person will appreciate that a plurality of TLR4 agonists may be utilised in combination.
Adjuvants comprising a saponin
An adjuvant comprising a saponin may additionally comprise further immunostimulants, in particular they may contain a TLR4 agonist. In some embodiments the adjuvant comprising a saponin does not comprise non-saponin immunostimulants.
The saponin, such as QS-21, can be used at amounts between 1 and 100 ug per dose, such as per human dose. Saponin, such as QS-21 , may be used at a level of about 50 ug. Examples of suitable ranges are 40 to 60 ug, suitably 45 to 55 ug or 49 to 51 ug, such as 50 ug. In a further embodiment, a dose, such as a human dose, comprises saponin, such as QS- 21 , at a level of about 25 ug. Examples of lower ranges include 20 to 30 ug, suitably 22 to 28 ug or 24 to 26 ug, such as 25 ug. Human doses intended for children may be reduced compared to those intended for a human adult (e.g. reduction by 50%).
A TLR4 agonist, if present, may be a lipopolysaccharide, such as a monophosphoryl lipid A, such as 3D-MPL, and can be used at amounts between 1 and 100 ug per dose, such as per human dose. TLR4 agonist, such as 3D-MPL, may be used at a level of about 50 ug. Examples of suitable ranges are 40 to 60 ug, suitably 45 to 55 ug or 49 to 51 ug, such as 50 ug. In a further embodiment, a dose, such as a human dose, comprises TLR4 agonist, such as 3D-MPL, at a level of about 25 ug. Examples of lower ranges include 20 to 30 ug, suitably 22 to 28 ug or 24 to 26 ug, such as 25 ug. Human doses intended for children may be reduced compared to those intended for a human adult (e.g. reduction by 50%).
The weight ratio of TLR4 agonist to saponin is suitably between 1 :5 to 5: 1 , suitably 1 :2 to 2:1 , such as about 1:1 , especially 1 :1. For example, where 3D-MPL is present at an amount of about 50 ug or about 25 ug, then suitably QS-21 may also be present at an amount of about 50 ug or about 25 ug per human dose.
Suitably the saponin is QS-21 and the TLR4 agonist is 3D-MPL.
Squalene emulsion adjuvants
In certain embodiments, the second adjuvant may be a squalene emulsion adjuvant.
The term ‘squalene emulsion adjuvant’ as used herein refers to a squalene containing oil-in-water emulsion adjuvant. The term ‘tocopherol-containing squalene emulsion adjuvant’ as used herein refers to a squalene and tocopherol-containing oil-in-water emulsion adjuvant wherein the weight ratio of squalene to tocopherol is 20 or less (i.e. 20 weight units of squalene or less per weight unit of tocopherol or, alternatively phrased, at least 1 weight unit of tocopherol per 20 weight units of squalene). Tocopherol-containing squalene emulsion adjuvants are therefore a subset of squalene emulsion adjuvants and are of particular interest in the present invention. Squalene, is a branched, unsaturated terpenoid ([(CH3)2C[=CHCH2CH2C(CH3)]2=CHCH2-]2; C30H50; 2,6,10,15,19,23-hexamethyl- 2,6,10,14,18,22-tetracosahexaene; CAS Registry Number 7683-64-9). Squalene is readily available from commercial sources or may be obtained by methods known in the art. Squalene shows good biocompatibility and is readily metabolised. Squalene emulsion adjuvants will typically have a submicron droplet size. Droplet sizes below 200 nm are beneficial in that they can facilitate sterilisation by filtration. There is evidence that droplet sizes in the 80 to 200 nm range are of particular interest for potency, manufacturing consistency and stability reasons (Klucker, 2012; Shah, 2014; Shah, 2015; Shah, 2019). Suitably the squalene emulsion adjuvant has an average droplet size of less than 1 um, especially less than 500 nm and in particular less than 200 nm. Suitably the squalene emulsion adjuvant has an average droplet size of at least 50 nm, especially at least 80 nm, in particular at least 100 nm, such as at least 120 nm. The squalene emulsion adjuvant may have an average droplet size of 50 to 200 nm, such as 80 to 200 nm, especially 120 to 180 nm, in particular 140 to 180 nm, such as about 160 nm. Uniformity of droplet sizes is desirable. A polydispersity index (PdI) of greater than 0.7 indicates that the sample has a very broad size distribution and a reported value of 0 means that size variation is absent, although values smaller than 0.05 are rarely seen. Suitably the squalene emulsion adjuvant has a polydispersity of 0.5 or less, especially 0.3 or less, such as 0.2 or less. The droplet size, as used herein, means the average diameter of oil droplets in an emulsion and can be determined in various ways e.g. using the techniques of dynamic light scattering and/or single-particle optical sensing, using an apparatus such as the Accusizer™ and Nicomp™ series of instruments available from Particle Sizing Systems (Santa Barbara, USA), the Zetasizer™ instruments from Malvern Instruments (UK), or the Particle Size Distribution Analyzer instruments from Horiba (Kyoto, Japan). See Light Scattering from Polymer Solutions and Nanoparticle Dispersions Schartl, 2007. Dynamic light scattering (DLS) is the preferred method by which droplet size is determined. The preferred method for defining the average droplet diameter is a Z-average i.e. the intensity-weighted mean hydrodynamic size of the ensemble collection of droplets measured by DLS. The Z-average is derived from cumulants analysis of the measured correlation curve, wherein a single particle size (droplet diameter) is assumed and a single exponential fit is applied to the autocorrelation function. Thus, references herein to average droplet size should be taken as an intensity-weighted average, and ideally the Z-average. Polydispersity Index (PdI) values are easily provided by the same instrumentation which measures average diameter. In order to maintain a stable submicron emulsion, one or more emulsifying agents (i.e. surfactants) are generally required. Surfactants can be classified by their ‘HLB’ (Griffin’s hydrophile/lipophile balance), where a HLB in the range 1-10 generally means that the surfactant is more soluble in oil than in water, whereas a HLB in the range 10-20 means that the surfactant is more soluble in water than in oil. HLB values are readily available for many surfactants of interest or can be determined experimentally, e.g. polysorbate 80 has a HLB of 15.0 and TPGS has a HLB of 13 to 13.2. Sorbitan trioleate has a HLB of 1.8. When two or more surfactants are blended, the resulting HLB of the blend is typically calculated by the weighted average e.g. a 70/30 wt% mixture of polysorbate 80 and TPGS has a HLB of (15.0 x 0.70) + (13 x 0.30) i.e. 14.4. A 70/30 wt% mixture of polysorbate 80 and sorbitan trioleate has a HLB of (15.0 x 0.70) + (1.8 x 0.30) i.e. 11.04.
Surfactant(s) will typically be metabolisable (biodegradable) and biocompatible, being suitable for use as a pharmaceutical. The surfactant can include ionic (cationic, anionic or zwitterionic) and/or non-ionic surfactants. The use of only non-ionic surfactants is often desirable, for example due to their pH independence. The invention can thus use surfactants including, but not limited to: the polyoxyethylene sorbitan ester surfactants (commonly referred to as the Tweens or polysorbates), such as polysorbate 20 and polysorbate 80, especially polysorbate 80; copolymers of ethylene oxide (EG), propylene oxide (PO), and/or butylene oxide (BO), sold under the DOWFAX™, Pluronic™ (e.g. F68, F127 or L121 grades) or Synperonic™ tradenames, such as linear EO/PO block copolymers, for example poloxamer 407, poloxamer 401 and poloxamer 188; octoxynols, which can vary in the number of repeating ethoxy (oxy-1, 2-ethanediyl) groups, with octoxynol-9 (Triton X-100, or t-octylphenoxypolyethoxyethanol) being of particular interest;
(octylphenoxy)polyethoxyethanol (IGEPAL CA-630/NP-40); phospholipids such as phosphatidylcholine (lecithin); polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants), such as polyoxyethylene 4 lauryl ether (Brij 30, Emulgen 104P), polyoxyethylene-9-lauryl ether and polyoxyethylene 12 cetyl/stearyl ether (Eumulgin™ B1, cetereth-12 or polyoxyethylene cetostearyl ether); sorbitan esters (commonly known as the Spans), such as sorbitan trioleate (Span 85), sorbitan monooleate (Span 80) and sorbitan monolaurate (Span 20); or tocopherol derivative surfactants, such as alpha-tocopherol-polyethylene glycol succinate (TPGS). Many examples of pharmaceutically acceptable surfactants are known in the art e.g. see Handbook of Pharmaceutical Excipients 6th edition, 2009. Methods for selecting an optimising the choice of surfactant used in a squalene emulsion adjuvant are illustrated in Klucker, 2012.
In general, the surfactant component has a HLB between 10 and 18, such as between 12 and 17, in particular 13 to 16. This can be typically achieved using a single surfactant or, in some embodiments, using a mixture of surfactants. Surfactants of particular interest include: poloxamer 401, poloxamer 188, polysorbate 80, sorbitan trioleate, sorbitan monooleate and polyoxyethylene 12 cetyl/stearyl ether either alone, in combination with each other or in combination with other surfactants. Especially of interest are polysorbate 80, sorbitan trioleate, sorbitan monooleate and polyoxyethylene 12 cetyl/stearyl ether either alone, or in combination with each other. A particular surfactant of interest is polysorbate 80. A particular combination of surfactants of interest is polysorbate 80 and sorbitan trioleate. A further combination of surfactants of interest is sorbitan monooleate and polyoxyethylene cetostearyl ether.
In certain embodiments the squalene emulsion adjuvant comprises one surfactant, such as polysorbate 80. In some embodiments the squalene emulsion adjuvant comprises two surfactants, such as polysorbate 80 and sorbitan trioleate or sorbitan monooleate and polyoxyethylene cetostearyl ether. In other embodiments the squalene emulsion adjuvant comprises three or more surfactants, such as three surfactants.
The amount of squalene in a single dose, such as a human dose, of squalene emulsion adjuvant may be 50 mg or less, especially 40 mg or less, in particular 30 mg or less, such as 20 mg or less (for example 15 mg or less). The amount of squalene in a single dose, such as a human dose, of squalene emulsion adjuvant may be 0.5 mg or more, especially 1 mg or more, in particular 2 mg or more, such as 4 mg or more and desirably 8 mg or more. The amount of squalene in a single dose, such as a human dose, of squalene emulsion adjuvant may be 0.5 to 50 mg, especially 1 to 20 mg, in particular 2 to 15 mg, such as 5 to 15 mg. The amount of squalene in a single dose, such as a human dose, of squalene emulsion adjuvant may be 0.5 to 2 mg, 2 to 4 mg, 4 to 8 mg, 8 to 12 mg, 12 to 16 mg, 16 to 20 mg or 20 to 50 mg.
The amount of squalene in a single dose, such as a human dose, of squalene emulsion adjuvant may be 1.2 to 20 mg, in particular 1.2 to 15 mg. The amount of squalene in a single dose, such as a human dose, of squalene emulsion adjuvant may be 1.2 to 2 mg, 2 to 4 mg, 4 to 8 mg or 8 to 12.1 mg. For example, the amount of squalene in a single dose, such as a human dose, of squalene emulsion adjuvant may be 1.21 to 1.52 mg, 2.43 to 3.03 mg, 4.87 to 6.05 mg or 9.75 to 12.1 mg.
Typically the weight ratio of squalene to surfactant is 0.73 to 6.6, especially 1 to 5, in particular 1.5 to 4.5. The weight ratio of squalene to surfactant may be 1.5 to 3, especially 1.71 to 2.8, such as 2.2 or 2.4. The weight ratio of squalene to surfactant may be 2.5 to 3.5, especially 3 or 3.1. The weight ratio of squalene to surfactant may be 3 to 4.5, especially 4 or 4.3.
The amount of surfactant in a single dose, such as a human dose, of squalene emulsion adjuvant is typically at least 0.4 mg. Generally, the amount of surfactant in a single dose, such as a human dose, of squalene emulsion adjuvant is 18 mg or less. The amount of surfactant in a single dose, such as a human dose, of squalene emulsion adjuvant may be 0.4 to 9.5 mg, in particular 0.4 to 7 mg. The amount of surfactant in a single dose, such as a human dose, of squalene emulsion adjuvant may be 0.4 to 1 mg, 1 to 2 mg, 2 to 4 mg or 4 to 7 mg. For example, the amount of surfactant in a single dose, such as a human dose, of squalene emulsion adjuvant may be 0.54 to 0.71 mg, 1.08 to 1.42 mg, 2.16 to 2.84 mg or 4.32 to 5.68 mg.
The squalene emulsion adjuvant may contain one or more tocopherols. Any of the a, P, y, 5, E and/or 5, tocopherols can be used, but a-tocopherol (also referred to herein as alphatocopherol) is typically used. D-alpha-tocopherol and D/L-alpha-tocopherol can both be used. Tocopherols are readily available from commercial sources or may be obtained by methods known in the art. In some embodiments the squalene emulsion adjuvant does not contain tocopherol. In some embodiments the squalene emulsion adjuvant contains tocopherol (i.e. at least one tocopherol, suitably one tocopherol), especially alpha-tocopherol, in particular D/L-alpha-tocopherol.
Tocopherols have been used, in relatively small amounts, in squalene emulsion adjuvants as antioxidants. Desirably tocopherols are present a level where the weight ratio of squalene to tocopherol is 20 or less, such as 10 or less. Suitably the weight ratio of squalene to tocopherol is 0.1 or more. Typically the weight ratio of squalene to tocopherol is 0.1 to 10, especially 0.2 to 5, in particular 0.3 to 3, such as 0.4 to 2. Suitably, the weight ratio of squalene to tocopherol is 0.72 to 1.136, especially 0.8 to 1, in particular 0.85 to 0.95, such as 0.9. Alternatively, the weight ratio of squalene to tocopherol is 3.4 to 4.6, especially 3.6 to 4.4, in particular 3.8 to 4.2, such as 4.
The amount of tocopherol in a single dose, such as a human dose, of squalene emulsion adjuvant is typically at least 0.5 mg, especially at least 1.3 mg. Generally, the amount of tocopherol in a single dose, such as a human dose, of squalene emulsion adjuvant is 55 mg or less. The amount of tocopherol in a single dose, such as a human dose, of squalene emulsion adjuvant may be 1.3 to 22 mg, in particular 1.3 to 16.6 mg. The amount of tocopherol in a single dose, such as a human dose, of squalene emulsion adjuvant may be 1.3 to 2 mg, 2 to 4 mg, 4 to 8 mg or 8 to 13.6 mg. For example, the amount of tocopherol in a single dose, such as a human dose, of squalene emulsion adjuvant may be 1.33 to 1.69 mg, 2.66 to 3.39 mg, 5.32 to 6.77 mg or 10.65 to 13.53 mg. In certain embodiments the squalene emulsion adjuvant may consist essentially of squalene, tocopherol (if present), surfactant and water. In addition to squalene, tocopherol, surfactant and water, squalene emulsion adjuvants may contain additional components as desired or required depending upon the intended final presentation and vaccination strategy, such as buffers and/or tonicity modifying agents, for example modified phosphate buffered saline (disodium phosphate, potassium biphosphate, sodium chloride and potassium chloride).
A squalene emulsion of interest in the present invention is known as 'MF59' (WO90/14837; Podda, 2003; Podda, 2001) and is a submicron oil-in-water emulsion of squalene, polysorbate 80 (also known as Tween 80™), and sorbitan trioleate (also known as Span 85™). It may also include citrate ions e.g. 10mM sodium citrate buffer. The composition of the emulsion by volume can be about 5% squalene, about 0.5% polysorbate 80 and about 0.5% sorbitan trioleate. The adjuvant and its production are described in more detail in Vaccine Design: The Subunit and Adjuvant Approach (chapter 10), Vaccine Adjuvants: Preparation Methods and Research Protocols (chapter 12) and New Generation Vaccines (chapter 19). As described in O’Hagan, 2007, MF59 is typically manufactured on a commercial scale by dispersing sorbitan trioleate in the squalene, dispersing polysorbate 80 in an aqueous phase (e.g. citrate buffer), then mixing these two phases to form a coarse emulsion which is then microfluidised. The emulsion is typically prepared at double-strength (4.3% v/v squalene, 0.5% v/v polysorbate 80 and 0.5% v/v sorbitan trioleate) and is diluted 1:1 (by volume) with an antigen composition to provide a final adjuvanted vaccine composition. An adult human dose of MF59 contains 9.75 mg squalene, 1.17 mg polysorbate 80 and 1.17 mg sorbitan trioleate (O’Hagan, 2013). An adult human dose of MF59C.1, as used in the seasonal influenza vaccine Fluad™, contains 9.75 mg squalene, 1.175 mg polysorbate 80 and 1.175 mg sorbitan trioleate, 0.66 mg sodium citrate and 0.04 mg citric acid (O’Hagan, 2013) in 0.5 ml of water for injection (Fluad™ Summary of Product Characteristics).
A further squalene emulsion of interest in the present invention is known as ‘AF03’ (US2007/0014805; Klucker, 2012). AF03 includes squalene, sorbitan monooleate, polyoxyethylene cetostearyl ether and mannitol. AF03 is typically prepared by cooling a preheated water-in-oil emulsion until it crosses its emulsion phase inversion temperature, at which point it thermoreversibly converts into an oil-in-water emulsion. The mannitol, cetostearyl ether and a phosphate buffer are mixed in one container to form an aqueous phase, while the sorbitan ester and squalene are mixed in another container to form an oily component. The aqueous phase is added to the oily component and the mixture is then heated to approximately 60°C and cooled to provide the final emulsion. The emulsion is typically initially prepared as a concentrate with a composition of 32.5% squalene, 4.8% sorbitan monooleate, 6.2% polyoxyethylene cetostearyl ether and 6% mannitol and 50.5% phosphate buffered saline. AF03 adjuvant contains 12.4 mg squalene, 1.9 mg sorbitan monooleate, 2.4 mg polyoxyethylene cetostearyl ether and 2.3 mg mannitol per 500 ul human adult dose (Humenza™ Summary of Product Characteristics).
Another squalene emulsion of interest in the present invention is known as 'AS03' (Gargon, 2012) and is typically prepared by mixing an oil mixture (consisting of squalene and alpha-tocopherol) with an aqueous phase (polysorbate 80 and buffer), followed by microfluidisation (W02006/100109). AS03 is typically prepared at double-strength with the expectation of dilution by an aqueous antigen containing composition prior to administration. An adult human dose of AS03A contains 10.69 mg squalene, 11.86 mg alpha-tocopherol and 4.86 mg polysorbate 80 (Morel, 2011; Fox, 2009). Certain reduced does of AS03 have also been described (W02008/043774), including AS03B (1/2 dose), AS03c (1/4 dose) and AS03D (1/8 dose) (Carmona Martinez, 2014).
As discussed above, high pressure homogenization (HPH or microfluidisation) and a phase inversion temperature method (PIT) may be applied to yield squalene emulsion adjuvants which demonstrate uniformly small droplet sizes and long-term stability. More recently, squalene based self-emulsifying adjuvant systems (SEAS) have been described. W02015/140138 and WO2016/135154 describe the preparation of oil/surfactant compositions, which when diluted with an aqueous phase spontaneously form oil-in-water emulsions having small droplet particle sizes, such emulsions can be used as immunological adjuvants. An adult human dose of ‘SEA160’ emulsion may include 7.62 mg squalene, 2.01 mg polysorbate 80 and 2.01 mg sorbitan trioleate. (Shah, 2014; Shah, 2015; Shah, 2019)
W02020/160080 and Lodaya, 2019 describe further squalene based self-emulsifying adjuvant systems (SEAS), specifically systems comprising a tocopherol in addition to squalene. ‘SEAS44’ contains 60% v/v squalene, 15% v/v alpha-tocopherol and 25% v/v polysorbate 80. The squalene/tocopherol/polysorbate mixture is intended to be diluted approximately 10-fold with an aqueous medium to form the final emulsion adjuvant. Consequently, an adult human dose of SEAS44 emulsion may include about 13 mg squalene, 3.6 mg alpha-tocopherol and 6.7 mg polysorbate 80.
Other squalene emulsion adjuvants have been described including:
SWE (Younis, 2018) comprising squalene 3.9% w/v, sorbitan trioleate 0.47% w/v, and polysorbate 80 (0.47% w/v) dispersed in 10 mM citrate buffer at pH 6.5. Consequently, an adult human dose of SWE may include about 9.75 mg squalene, 1.175 mg sorbitan trioleate and 1.175 mg polysorbate 80, similar to MF59.
SE (Carter, 2016; Sun, 2016) comprising squalene, phosphatidyl choline, poloxamer 188 and an ammonium phosphate buffered aqueous phase also containing glycerol. Sometimes SE has been described as containing small amounts of tocopherol. An adult human dose of SE may include about 8.6 mg squalene, 2.73 mg phosphatidyl choline and 0.125 mg poloxamer 188, optionally with 0.05 mg tocopherol.
CoVaccine (Hilgers, 2006; Hamid, 2011 ; Younis, 2019) comprises squalene, polysorbate 80 and sucrose fatty acid sulfate esters, typically with phosphate buffered saline. An adult human dose of CoVaccine may include about 40 mg squalene, 10 mg polysorbate 80 and 10 mg sucrose fatty acid sulfate esters.
The squalene emulsion adjuvant may be derived from MF59. Consequently, the squalene emulsion adjuvant may comprise squalene, polysorbate 80, sorbitan trioleate and water. The squalene emulsion adjuvant may consist essentially of squalene, polysorbate 80, sorbitan trioleate and water. Optionally the aqueous phase may contain additional components as desired or required depending upon the intended final presentation and vaccination strategy, such as buffers and/or tonicity modifying agents, in particular citrate ions e.g. 10mM sodium citrate buffer.
Typically, the weight ratio of squalene to polysorbate 80 is 10 to 6.6, especially 9.1 to 7.5, in particular 8.7 to 7.9, such as 8.3.
Typically, the weight ratio of squalene to sorbitan trioleate is 10 to 6.6, especially 9.1 to 7.5, in particular 8.7 to 7.9, such as 8.3.
A single dose, such as a typical full human dose, of squalene emulsion adjuvant derived from MF59 may comprise 9 to 11 mg of squalene, such as 9.5 to 10 mg, in particular 9.75 mg. Higher or lower doses of squalene emulsion adjuvant derived from MF59 may be used. Suitably a single dose is at least 0.1x a typical full human dose, especially at least 0.25x a typical full human dose, in particular at least 0.5x a typical full human dose. Desirably the single dose is less than or equal to a full human dose. For example, the single dose may be 0.1 to 1x a typical full human dose, i.e. comprising 0.9 to 11 mg of squalene.
Particular single doses of interest include 0.1x a typical full human dose i.e. comprising 0.9 to 1.1 mg of squalene, 0.125x a typical full human dose i.e. comprising 1.1 to 1.4 mg of squalene, 0.25x a typical full human dose i.e. comprising 2.2 to 2.8 mg of squalene, such as 0.5x a typical full human dose i.e. comprising 4.5 to 5.5 mg of squalene or 1x a typical full human dose i.e. comprising 9 to 11 mg of squalene.
Squalene emulsion adjuvant derived from MF59 may include citrate ions e.g. 10mM sodium citrate buffer.
The squalene emulsion adjuvant may be derived from AF03. Consequently, the squalene emulsion adjuvant may comprise squalene, sorbitan monooleate, polyoxyethylene cetostearyl ether and water. The squalene emulsion adjuvant may consist essentially of squalene, sorbitan monooleate, polyoxyethylene cetostearyl ether and water. Mannitol has been shown to reduce the phase transition temperature and is therefore desirable for manufacturing reasons, although excessive levels of mannitol may cause heterogeneity in size and larger droplets (Klucker, 2012). Optionally the aqueous phase may contain additional components as desired or required depending upon the intended final presentation and vaccination strategy, such as buffers and/or tonicity modifying agents, in particular phosphate buffered saline.
Typically, the weight ratio of squalene to sorbitan monooleate is 7.8 to 5.2, especially 7.15 to 5.85, in particular 6.8 to 6.2, such as 6.5.
Typically, the weight ratio of squalene to polyoxyethylene cetostearyl ether is 6.2 to 4.1, especially 5.7 to 4.7, in particular 5.4 to 4.9, such as 5.2.
Typically, the weight ratio of squalene to mannitol is 6.5 to 4.3, especially 5.9 to 4.9, in particular 5.7 to 5.1 , such as 5.4.
A single dose, such as a typical full human dose, of squalene emulsion adjuvant derived from AF03 may comprise 11.2 to 13.6 mg of squalene, such as 12 to 12.8 mg, in particular 12.4 mg. Higher or lower doses of squalene emulsion adjuvant derived from AF03 may be used. Suitably a single dose is at least 0.1x a typical full human dose, especially at least 0.25x a typical full human dose, in particular at least 0.5x a typical full human dose. Desirably the single dose is less than or equal to a full human dose. For example, the single dose may be 0.1 to 1x a typical full human dose, i.e. comprising 1.1 to 13.6 mg of squalene.
Particular single doses of interest include 0.1x a typical full human dose i.e. comprising 1.1 to 1.35 mg of squalene, 0.125x a typical full human dose i.e. comprising 1.4 to 1.7 mg of squalene, 0.25x a typical full human dose i.e. comprising 2.8 to 3.4 mg of squalene, such as 0.5x a typical full human dose i.e. comprising 5.6 to 6.8 mg of squalene or 1x a typical full human dose i.e. comprising 11.2 to 13.6 mg of squalene.
Squalene emulsion adjuvant derived from AF03 may also include in particular phosphate buffered saline.
The squalene emulsion adjuvant may be derived from AS03. Consequently, the squalene emulsion adjuvant may comprise squalene, tocopherol, polysorbate 80 and water. The squalene emulsion adjuvant may consist essentially of squalene, tocopherol, polysorbate 80 and water. Optionally the aqueous phase may contain additional components as desired or required depending upon the intended final presentation and vaccination strategy, such as buffers and/or tonicity modifying agents. Suitable buffers include Na2HPO4 and KH2PO4. Suitable tonicity modifying agents include NaCI and KCI. Modified phosphate buffered saline may be used, such as comprising Na2HPO4 and KH2PO4, NaCI and KCI.
Any of the a, p, y, 5, E or 5, tocopherols can be used, but a-tocopherol (also referred to herein as alpha-tocopherol) is typically used. D-alpha-tocopherol and D/L-alpha-tocopherol can both be used. A particularly desirable alpha-tocopherol is D/L-alpha-tocopherol. Typically, the weight ratio of squalene to tocopherol is 0.5 to 1 .5, especially 0.6 to 1 .35, in particular 0.7 to 1 .1 , such as 0.85 to 0.95 e.g. 0.9. Suitably the tocopherol is alphatocopherol, such as D/L-alpha-tocopherol.
Typically, the weight ratio of squalene to polysorbate 80 is 1.2 to 3.6, especially 1.46 to 3.3, in particular 1.9 to 2.5 such as 2.1 to 2.3 e.g. 2.2.
A single dose, such as a typical full human dose, of squalene emulsion adjuvant derived from AS03 may comprise 9.7 to 12.1 mg of squalene, such as 10.1 to 11.8 mg, in particular 10.7 mg. Higher or lower doses of squalene emulsion adjuvant derived from AS03 may be used. Suitably a single dose is at least 0.1x a typical full human dose, especially at least 0.25x a typical full human dose, in particular at least 0.5x a typical full human dose. Desirably the single dose is less than or equal to a full human dose. For example, the single dose may be 0.1 to 1x a typical full human dose, i.e. comprising 0.9 to 12.1 mg of squalene.
Particular single doses of interest include 0.1x a typical full human dose i.e. comprising 0.9 to 1.3 mg of squalene (typically with 1 to 1.4 mg tocopherol, such as D/L-alpha tocopherol, and 0.43 to 0.57 mg polysorbate 80), 0.125x a typical full human dose i.e. comprising 1.2 to 1.6 mg of squalene (typically with 1.3 to 1.7 mg tocopherol, such as D/L-alpha tocopherol, and 0.54 to 0.71 mg polysorbate 80), 0.25x a typical full human dose i.e. comprising 2.4 to 3 mg of squalene (typically with 2.6 to 3.4 mg tocopherol, such as D/L-alpha tocopherol, and 1 to 1.5 mg polysorbate 80), such as 0.5x a typical full human dose i.e. comprising 4.8 to 6.1 mg of squalene (typically with 5.3 to 6.8 mg tocopherol, such as D/L-alpha tocopherol, and 2.1 to 2.9 mg polysorbate 80) or 1x a typical full human dose i.e. comprising 9.7 to 12.1 mg of squalene (typically with 10.6 to 13.6 mg tocopherol, such as D/L-alpha tocopherol, and 4.3 to 5.7 mg polysorbate 80).
Squalene emulsion adjuvant derived from AS03 may also include in particular a phosphate buffered saline, such as modified phosphate buffered saline.
The squalene emulsion adjuvant may be derived from SE. Consequently, the squalene emulsion adjuvant may comprise squalene, phosphatidyl choline, poloxamer 188 and water, optionally with glycerol. The squalene emulsion adjuvant may consist essentially of squalene, phosphatidyl choline, poloxamer 188 and water, optionally with glycerol. Optionally the aqueous phase may contain additional components as desired or required depending upon the intended final presentation and vaccination strategy, such as buffers and/or tonicity modifying agents, in particular ammonium phosphate buffer. Tocopherol, such as alpha-tocopherol may be present as an antioxidant.
Typically, the weight ratio of squalene to phosphatidyl choline is 2.52 to 3.8, especially 2.85 to 3.5, in particular 3 to 3.3, such as 3.15.
Typically, the weight ratio of squalene to poloxamer 188 is 55 to 83, especially 62 to 76, in particular 65.5 to 72.5, such as 69. Typically, the weight ratio of squalene to tocopherol, if present, is at least 50, especially 137 to 207, in particular 154 to 190, such as 163 to 181, for example 172.
A single dose, such as a typical full human dose, of squalene emulsion adjuvant derived from SE may comprise 7.7 to 9.5 mg of squalene, such as 8.1 to 9 mg, in particular 8.6 mg. Higher or lower doses of squalene emulsion adjuvant derived from SE may be used. Suitably a single dose is at least 0.1x a typical full human dose, especially at least 0.25x a typical full human dose, in particular at least 0.5x a typical full human dose. Desirably the single dose is less than or equal to a full human dose. For example, the single dose may be 0.1 to 1x a typical full human dose, i.e. comprising 0.77 to 9.5 mg of squalene.
Particular single doses of interest include 0.1x a typical full human dose i.e. comprising 0.77 to 0.95 mg of squalene, 0.125x a typical full human dose i.e. comprising 0.96 to 1.2 mg of squalene, 0.25x a typical full human dose i.e. comprising 1.9 to 2.4 mg of squalene, such as 0.5x a typical full human dose i.e. comprising 3.8 to 4.8 mg of squalene or 1x a typical full human dose i.e. comprising 7.7 to 9.5 mg of squalene.
Squalene emulsion adjuvant derived from SE may also include in particular ammonium phosphate buffer and glycerol.
The squalene emulsion adjuvant may be derived from SEA160. Consequently, the squalene emulsion adjuvant may comprise squalene, polysorbate 80, sorbitan trioleate and water. The squalene emulsion adjuvant may consist essentially of squalene, polysorbate 80, sorbitan trioleate and water. Optionally the aqueous phase may contain additional components as desired or required depending upon the intended final presentation and vaccination strategy, such as buffers and/or tonicity modifying agents.
Typically, the weight ratio of squalene to polysorbate 80 is 4.6 to 3.0, especially 4.2 to 3.4, in particular 4.0 to 3.6, such as 3.8.
Typically, the weight ratio of squalene to sorbitan trioleate is 4.6 to 3.0, especially 4.2 to 3.4, in particular 4.0 to 3.6, such as 3.8.
A single dose, such as a typical full human dose, of squalene emulsion adjuvant derived from SEA160 may comprise 6.8 to 8.4 mg of squalene, such as 7.2 to 8 mg, in particular 7.6 mg. Higher or lower doses of squalene emulsion adjuvant derived from SEA160 may be used. Suitably a single dose is at least 0.1x a typical full human dose, especially at least 0.25x a typical full human dose, in particular at least 0.5x a typical full human dose. Desirably the single dose is less than or equal to a full human dose. For example, the single dose may be 0.1 to 1x a typical full human dose, i.e. comprising 0.68 to 8.4 mg of squalene.
Particular single doses of interest include 0.1x a typical full human dose i.e. comprising 0.68 to 0.84 mg of squalene, 0.125x a typical full human dose i.e. comprising 0.85 to 1.1 mg of squalene, 0.25x a typical full human dose i.e. comprising 1.7 to 2.1 mg of squalene, such as 0.5x a typical full human dose i.e. comprising 3.4 to 4.2 mg of squalene or 1x a typical full human dose i.e. comprising 6.8 to 8.4 mg of squalene.
Squalene emulsion adjuvant derived from SEA160 may also include in particular a phosphate buffered saline, such as modified phosphate buffered saline.
The squalene emulsion adjuvant may be derived from SEAS44. Consequently, the squalene emulsion adjuvant may comprise squalene, tocopherol, polysorbate 80 and water. The squalene emulsion adjuvant may consist essentially of squalene, tocopherol, polysorbate 80 and water. Optionally the aqueous phase may contain additional components as desired or required depending upon the intended final presentation and vaccination strategy, such as buffers and/or tonicity modifying agents. Suitable buffers include Na2HPO4 and KH2PO4. Suitable tonicity modifying agents include NaCI and KCI. Modified phosphate buffered saline may be used, such as comprising Na2HPO4 and KH2PO4, NaCI and KCI.
Any of the a, p, y, 5, E or 5, tocopherols can be used, but a-tocopherol is typically used. D-alpha-tocopherol and D/L-alpha-tocopherol can both be used. A particularly desirable alpha-tocopherol is D/L-alpha-tocopherol.
Typically, the weight ratio of squalene to tocopherol is 2.6 to 4.5, especially 2.8 to 4.3, in particular 3.25 to 4, such as 3.4 to 3.8 e.g. 3.6. Suitably the tocopherol is alpha-tocopherol, especially D/L-alpha-tocopherol.
Typically, the weight ratio of squalene to polysorbate 80 is 1.3 to 2.5, especially 1.56 to 2.3, in particular 1.75 to 2.15 such as 1.85 to 2 e.g. 1.94.
A single dose, such as a typical full human dose, of squalene emulsion adjuvant derived from SEAS44 may comprise 11.7 to 14.3 mg of squalene, such as 12.3 to 13.7 mg, in particular 13 mg. Higher or lower doses of squalene emulsion adjuvant derived from SEAS44 may be used. Suitably a single dose is at least 0.1x a typical full human dose, especially at least 0.25x a typical full human dose, in particular at least 0.5x a typical full human dose. Desirably the single dose is less than or equal to a full human dose. For example, the single dose may be 0.1 to 1x a typical full human dose, i.e. comprising 1.1 to 14.3 mg of squalene.
Particular single doses of interest include 0.1x a typical full human dose i.e. comprising 1.1 to 1.5 mg of squalene, 0.125x a typical full human dose i.e. comprising 1.4 to 1.8 mg of squalene, 0.25x a typical full human dose i.e. comprising 2.9 to 3.6 mg of squalene, such as 0.5x a typical full human dose i.e. comprising 5.8 to 7.2 mg of squalene or 1x a typical full human dose i.e. comprising 11.7 to 14.3 mg of squalene.
Squalene emulsion adjuvant derived from SEAS44 may also include in particular a phosphate buffered saline, such as modified phosphate buffered saline.
Self-emulsifying adjuvants, such as SEA160, SEAS44 and squalene emulsion adjuvant adjuvants derived therefrom, may be provided in dry form. For example, such dry self- emulsifying adjuvants may consist essentially of squalene and surfactant(s), such as in the case of SEA160 derived squalene emulsion adjuvants. Such dry self-emulsifying adjuvants may consist essentially of squalene and surfactant(s) or consist essentially of squalene, tocopherol and surfactant(s), such as in the case of SEAS44 derived tocopherol-containing squalene emulsion adjuvants.
High pressure homogenization (HPH or microfluidisation) may be applied to yield squalene emulsion adjuvants which demonstrate uniformly small droplet sizes and long-term stability (see EP 0 868 918 B1 and W02006/100109). Briefly, oil phase composed of squalene and tocopherol may be formulated under a nitrogen atmosphere. Aqueous phase is prepared separately, typically composed of water for injection or phosphate buffered saline, and polysorbate 80. Oil and aqueous phases are combined, such as at a ratio of 1 :9 (volume of oil phase to volume of aqueous phase) before homogenisation and microfluidisation, such as by a single pass through an in-line homogeniser and three passes through a microfluidiser (at around 15000 psi). The resulting emulsion may then be sterile filtered, for example through two trains of two 0.5/0.2 urn filters in series (i.e. 0.5/0.2/0.5/0.2), see WO2011/154444. Operation is desirably undertaken under an inert atmosphere, e.g. nitrogen. Positive pressure may be applied, see WO2011/154443.
WO2015/140138, WO2016/135154, Shah, 2014 Shah, 2015, Shah, 2019, W02020/160080 and Lodaya, 2019 describe squalene emulsion adjuvants which are selfemulsifying adjuvant systems (SEAS) and their manufacture. Adjuvant carriers
The STING agonist and/or the second adjuvant (if present) may be formulated with one or more carriers. Suitable carriers include ISCOMs, liposomes and emulsions.
The saponin and/or the TLR4 agonist if present will typically be formulated with one or more carriers. Suitable carriers include ISCOMs, liposomes and emulsions. The saponin and TLR4 agonist may be formulated with different carriers or may be formulated with the same carrier (whether formulated together or separately). Conveniently, the saponin and TLR4 agonist are formulated together with the same carrier, in particular with liposomes or an emulsion, especially with liposomes.
The term ‘liposome’ is well known in the art and defines a general category of vesicles which comprise one or more lipid bilayers surrounding an aqueous space. Liposomes thus consist of one or more lipid and/or phospholipid bilayers and can contain other molecules, such as proteins or carbohydrates, in their structure. Because both lipid and aqueous phases are present, liposomes can encapsulate or entrap water-soluble material, lipid-soluble material, and/or amphiphilic compounds.
Liposome size may vary from 30 nm to several urn depending on the phospholipid composition and the method used for their preparation. In the present invention, the liposome size will typically be in the range of 50 nm to 200 nm, especially 60 nm to 180 nm, such as 70 to 165 nm. Optimally, the liposomes should be stable and have a diameter of -100 nm to allow convenient sterilization by filtration.
A beneficial feature of the present invention is that the saponin may be presented in a less reactogenic composition where it is quenched with a sterol, such as cholesterol.
The liposomes of use in the present invention suitably contain DOPC, such as contain DOPC and sterol, such as cholesterol. In some embodiments the liposomes consist essentially of DOPC and sterol such as cholesterol (with saponin and/or TLR4 agonist).
The ratio of saponin: DOPC will typically be in the order of 1 :50 to 1 :10 (w/w), suitably between 1 :25 to 1 :15 (w/w), and preferably 1 :22 to 1 :18 (w/w), such as 1 :20 (w/w).
The ratio of DOPC:sterol, such as cholesterol, will typically be in the order of 10:1 to 1 :1 (w/w), suitably between 8:1 to 2:1 (w/w), and preferably 6:1 to 2.6:1 (w/w), such as about 4:1 (w/w).
Structural integrity of the liposomes may be assessed by methods such as dynamic light scattering (DLS) measuring the size (Z-average diameter, Zav) and polydispersity of the liposomes, or, by electron microscopy for analysis of the structure of the liposomes. In one embodiment the average particle size is between 90 and 120 nm, or between 95 and 120 nm.
Uniformity of particle sizes is desirable. A polydispersity index (Pdl) of greater than 0.7 indicates that the sample has a very broad size distribution and a reported value of 0 means that size variation is absent, although values smaller than 0.05 are rarely seen. Suitably the squalene emulsion has a polydispersity of 0.5 or less, especially 0.3 or less, such as 0.2 or less.
A particular second adjuvant of interest features liposomes comprising DOPC and cholesterol, with TLR4 agonist and saponin, especially 3D-MPL and QS-21.
Another second adjuvant of interest features liposomes comprising DOTAP and DMPC, with TLR4 agonist and saponin, especially dLOS and QS-21.
Emulsion carriers will typically be an oil in water emulsion comprising a pharmaceutically acceptable metabolizable oil, such as squalene. Squalene, is a branched, unsaturated terpenoid ([(CH3)2C[=CHCH2CH2C(CH3)]2=CHCH2-]2; C30H50; 2,6,10,15,19,23- hexamethyl-2,6,10,14,18,22-tetracosahexaene; CAS Registry Number 7683-64-9). Squalene is readily available from commercial sources or may be obtained by methods known in the art. Squalene shows good biocompatibility and is readily metabolised.
Squalene emulsions will typically have a submicron droplet size. Droplet sizes below 200 nm are beneficial in that they can facilitate sterilisation by filtration. There is evidence that droplet sizes in the 80 to 200 nm range are of particular interest for potency, manufacturing consistency and stability reasons. (Klucker, 2012; Shah, 2014; Shah, 2015; Shah, 2019). The squalene emulsion may have an average droplet size of 50 to 200 nm, such as 80 to 200 nm, especially 120 to 180 nm, in particular 140 to 180 nm, such as about 160 nm.
Uniformity of droplet sizes is desirable. A polydispersity index (Pdl) of greater than 0.7 indicates that the sample has a very broad size distribution and a reported value of 0 means that size variation is absent, although values smaller than 0.05 are rarely seen. Suitably the squalene emulsion has a polydispersity of 0.5 or less, especially 0.3 or less, such as 0.2 or less.
The droplet size, as used herein, means the average diameter of oil droplets in an emulsion and can be determined in various ways e.g. using the techniques of dynamic light scattering and/or single-particle optical sensing, using an apparatus such as the Accusizer™ and Nicomp™ series of instruments available from Particle Sizing Systems (Santa Barbara, USA), the Zetasizer™ instruments from Malvern Instruments (UK), or the Particle Size Distribution Analyzer instruments from Horiba (Kyoto, Japan). See Light Scattering from Polymer Solutions and Nanoparticle Dispersions Schartl, 2007. Dynamic light scattering (DLS) is the preferred method by which droplet size is determined. The preferred method for defining the average droplet diameter is a Z-average i.e. the intensity-weighted mean hydrodynamic size of the ensemble collection of droplets measured by DLS. The Z-average is derived from cumulants analysis of the measured correlation curve, wherein a single particle size (droplet diameter) is assumed and a single exponential fit is applied to the autocorrelation function. Thus, references herein to average droplet size should be taken as an intensity-weighted average, and ideally the Z-average. Pdl values are easily provided by the same instrumentation which measures average diameter.
In order to maintain a stable submicron emulsion, one or more emulsifying agents (i.e. surfactants) are generally required. Surfactant(s) will typically be metabolisable (biodegradable) and biocompatible, being suitable for use as a pharmaceutical. The surfactant can include ionic (cationic, anionic or zwitterionic) and/or non-ionic surfactants. The use of only non-ionic surfactants is often desirable, for example due to their pH independence. The invention can thus use surfactants including, but not limited to: the polyoxyethylene sorbitan ester surfactants (commonly referred to as the Tweens or polysorbates), such as polysorbate 20 and polysorbate 80, especially polysorbate 80; copolymers of ethylene oxide (EO), propylene oxide (PO), and/or butylene oxide (BO), sold under the DOWFAX™, Pluronic™ (e.g. F68, F127 or L121 grades) or Synperonic™ tradenames, such as linear EO/PO block copolymers, for example poloxamer 407, poloxamer 401 and poloxamer 188; octoxynols, which can vary in the number of repeating ethoxy (oxy-1 , 2-ethanediyl) groups, with octoxynol-9 (Triton X-100, or t-octylphenoxypolyethoxyethanol) being of particular interest;
(octylphenoxy)polyethoxyethanol (IGEPAL CA-630/NP-40); phospholipids such as phosphatidylcholine (lecithin); polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants), such as polyoxyethylene 4 lauryl ether (Brij 30, Emulgen 104P), polyoxyethylene-9-lauryl ether and polyoxyethylene 12 cetyl/stearyl ether (Eumulgin™ B1 , cetereth-12 or polyoxyethylene cetostearyl ether); sorbitan esters (commonly known as the Spans), such as sorbitan trioleate (Span 85), sorbitan monooleate (Span 80) and sorbitan monolaurate (Span 20); or tocopherol derivative surfactants, such as alpha-tocopherol-polyethylene glycol succinate (TPGS).
Surfactants of particular interest include: poloxamer 401 , poloxamer 188, polysorbate 80, sorbitan trioleate, sorbitan monooleate and polyoxyethylene 12 cetyl/stearyl ether either alone, in combination with each other or in combination with other surfactants. Especially of interest are polysorbate 80, sorbitan trioleate, sorbitan monooleate and polyoxyethylene 12 cetyl/stearyl ether either alone, or in combination with each other. A particular surfactant of interest is polysorbate 80. A particular combination of surfactants of interest is polysorbate 80 and sorbitan trioleate. A further combination of surfactants of interest is sorbitan monooleate and polyoxyethylene cetostearyl ether.
Second adjuvants
In certain embodiments a STING agonist is administered in step (i) and optionally a STING agonist is administered in step (ii). In some of the approaches described herein, a second adjuvant is administered in step (ii). In other approaches described herein, a second adjuvant is not administered in step (ii).
The second adjuvant may be any adjuvant. Typically, the second adjuvant does not comprise a STING agonist.
In some embodiments the second adjuvant comprises one immunostimulant. In some embodiments the second adjuvant comprises two immunostimulants.
In certain embodiments the second adjuvant comprises, such as consists of, a TLR agonist. In one embodiment the second adjuvant comprises, such as consists of, a TLR1 agonist. In one embodiment the second adjuvant comprises, such as consists of, a TLR1/2 agonist. In one embodiment the second adjuvant comprises, such as consists of, a TLR2 agonist. In one embodiment the second adjuvant comprises, such as consists of, a TLR3 agonist. In one embodiment the second adjuvant comprises, such as consists of, a TLR4 agonist. In one embodiment the second adjuvant comprises, such as consists of, a TLR5 agonist. In one embodiment the second adjuvant comprises, such as consists of, a TLR6 agonist. In one embodiment the second adjuvant comprises, such as consists of, a TLR7 agonist, a TLR8 agonist or a TLR7/TLR8 agonist. In one embodiment the second adjuvant comprises, such as consists of, a TLR9 agonist.
In certain embodiments the second adjuvant comprises, such as consists of, an adjuvant comprising a saponin.
In certain embodiments the second adjuvant comprises, such as consists of, a squalene emulsion adjuvant.
In certain embodiments a STING agonist is administered in step (i) and a STING agonist is administered in step (ii). In one such embodiment, the STING agonist of step (i) and the STING agonist of step (ii) are desirably essentially the same, such as the same.
In one embodiment, the STING agonist of step (i) and the STING agonist of step (ii) are essentially the same if they elicit a functionally equivalent immune response. A functionally equivalent immune response may be the level of immune response and/or the nature of the immune response.
Immune responses of the same nature may be immune responses which are both innate immune responses (such as both the same profile of innate immune cells, such as the presence of comparable innate immune cell types and the comparable concentration of said innate immune cell types) or both adaptive immune responses (such as both T cell responses (such as both CD8+ T cell responses or both CD4+ T cell responses), or both antibody responses).
Immune responses of the same level may be immune responses which produce essentially the same titre of neutralising antibodies or the same quantity of T cells in the same immunoassay.
Antigen
In certain embodiments a STING agonist is administered in step (i) and an antigen is administered in step (ii).
In one embodiment, the antigen is a polypeptide. In another embodiment, the antigen is provided in the form of a polynucleotide, such as a viral vector or mRNA. In a further embodiment, the antigen is a polysaccharide.
The antigen may be derived from (i.e. associated with) a pathogen, especially a human pathogen, (such as a bacterium, virus or parasite) or may be a cancer antigen (such as a tumour antigen and/or a neoantigen).
By the term antigen is meant a substance which is capable of eliciting an immune response in a subject. Suitably the immune response from the antigen is a protective immune response, e.g. reducing partially or completely the severity of one or more symptoms and/or time over which one or more symptoms are experienced by a subject, reducing the likelihood of developing an established infection after challenge and/or slowing progression of an associated illness (e.g. extending survival).
If multiple administrations of the STING agonist and antigen are performed in step (ii), then the antigen may be delivered in the same form (i.e. a ‘homologous’ dosage regime), in different forms (i.e. a ‘heterologous’ dosage regime), or any combination of the same form and different forms, over each of the multiple administrations. For example, if the antigen in a first administration in step (ii) is a polypeptide and the antigen in a second administration is a polynucleotide encoding the polypeptide then the antigens delivered in each administration are delivered in heterologous forms but are the same antigen for the purposes of the invention. In one embodiment the antigen is derived from a bacterium. Alternatively, the antigen may be derived from a virus. Suitably an antigen is derived from a pathogen if it contains a polypeptide sequence which is obtainable from said pathogen. Desirably, the polypeptide sequence which is obtainable from said pathogen is at least 50, especially at least 100, in particular at least 150, such as at least 200 amino acids long.
Suitably an antigen comprises at least one B or T cell epitope, suitably an antigen comprises B and T cell epitopes. The elicited immune response may be an antigen specific B cell response which produces neutralizing antibodies. The elicited immune response may be an antigen specific T cell response, which may be a systemic and/or a local response. The antigen specific T cell response may comprise a CD4+ T cell response, such as a response involving CD4+ T cells expressing a plurality of cytokines, e.g. IFNgamma, TNFalpha and/or IL2. Alternatively, or additionally, the antigen specific T cell response comprises a CD8+ T cell response, such as a response involving CD8+ T cells expressing a plurality of cytokines, e.g., IFNgamma, TNFalpha and/or IL2.
Suitably an antigen contains 3000 residues or fewer, especially 2000 residues or fewer, in particular 1500 residues or fewer. An antigen may contain 1000 residues or fewer, 800 residues or fewer, 600 residues or fewer, 400 residues or fewer or 200 residues or fewer. Suitably an antigen contains 50 residues or more, especially 100 residues or more, in particular 150 residues or more. Suitably an antigen contains 50 to 3000 residues, especially 100 to 1500 residues, in particular 200 to 1000 residues.
In one embodiment the antigen is derived from a bacterium. Suitably the antigen is derived from a gram-negative bacterium, such as Clostridia. More suitably the antigen is derived from C. difficile, such as specifically C. difficile toxin A and/or toxin B or fragments thereof, especially a fusion of fragments of toxin A and toxin B such as an F2 antigen (see, e.g., WO2012163811). The F2 antigen may be a sequence comprising, such as consisting of, a sequence having at least 80%, such as at least 90%, especially at least 95%, in particular at least 98% for example at least 99% or 100% identity to SEQ ID No: 2. The antigen may be an F2 antigen.
In one embodiment the antigen is derived from a virus. The antigen may be a respiratory syncytial virus (RSV) antigen.
Suitably the antigen is derived from a herpesvirus, such as human alphaherpesvirus 3 (HHV-3), also known as varicella-zoster virus (VZV). More suitably the antigen is the VZV-gE antigen. The VZV-gE antigen may be a sequence comprising, such as consisting of, a sequence having at least 80%, such as at least 90%, especially at least 95%, in particular at least 98% for example at least 99% or 100% identity to SEQ ID No: 1.
In one embodiment the antigen is an influenza virus antigen, such as a hemagglutinin or a neuraminidase.
In one embodiment the antigen is a coronavirus antigen, such as a spike protein.
In certain embodiments a STING agonist and a first antigen is administered in step (i) and a STING agonist and a second antigen is administered in step (ii).
In one embodiment, the antigens are all polypeptides. In another embodiment, the antigens are all provided in the form of polynucleotides, such as a viral vector or mRNA. In a further embodiment, the antigens are all polysaccharides.
In some embodiments the first antigen is a polypeptide and the second antigen is provided in the form a polynucleotide, such as a viral vector or mRNA. In other embodiments the second antigen is a polypeptide and the first antigen is provided in the form a polynucleotide, such as a viral vector or mRNA. Most suitably the antigens are all polypeptides.
The antigens may be derived from (i.e. associated with) a pathogen, especially a human pathogen, (such as a bacterium, virus or parasite) or may be a cancer antigen (such as a tumour antigen and/or a neoantigen).
By the term antigen is meant a substance which is capable of eliciting an immune response in a subject. Suitably the immune response from the first and/or second antigen is a protective immune response, e.g. reducing partially or completely the severity of one or more symptoms and/or time over which one or more symptoms are experienced by a subject, reducing the likelihood of developing an established infection after challenge and/or slowing progression of an associated illness (e.g. extending survival). Most suitably the immune response to the second antigen in administration under step (ii) is a protective immune response.
If multiple administrations of the STING agonist and first antigen are performed in step (i), then the first antigen may be delivered in the same form (i.e. a ‘homologous’ dosage regime), in different forms (i.e. a ‘heterologous’ dosage regime), or any combination of the same form and different forms, over each of the multiple administrations. For example, if the first antigen in a first administration in step (i) is a polypeptide and the first antigen in a second administration in step (i) is a polynucleotide encoding the polypeptide then the antigens delivered in each administration are delivered in heterologous forms but nevertheless the same antigen for the purposes of the invention.
If multiple administrations of the STING agonist and second antigen are performed in step (ii), then the second antigen may be delivered in the same form (i.e. a ‘homologous’ dosage regime), in different forms (i.e. a ‘heterologous’ dosage regime), or any combination of the same form and different forms, over each of the multiple administrations. For example, if the second antigen in a first administration in step (ii) is a polypeptide and the second antigen in a second administration is a polynucleotide encoding the polypeptide then the antigens delivered in each administration are delivered in heterologous forms but are the same antigen for the purposes of the invention.
The first antigen and the second antigen are different, i.e the first antigen has a different polypeptide, different polypeptide encoded by a polynucleotide or different polysaccharide sequence to the second antigen.
Suitably, the first antigen is derived from a different pathogen to the second antigen. Suitably a different pathogen is a pathogen which belongs to a different species, especially genus, in particular family, such as order, especially class, in particular phylum, such as kingdom or especially domain. Alternatively, pathogens may be considered different if the first antigen is derived from (such as containing a portion of a polypeptide sequence obtainable from) one of bacteria, viruses or parasites and the second antigen is derived from another of bacteria, viruses or parasites. In one embodiment the first antigen is derived from a virus and the second antigen is derived from a bacterium. Alternatively, the first antigen may be derived from a bacterium and the second antigen may be derived from a virus. Suitably an antigen is derived from a pathogen if it contains a polypeptide sequence which is obtainable from said pathogen. Desirably, the polypeptide sequence which is obtainable from said pathogen is at least 50, especially at least 100, in particular at least 150, such as at least 200 amino acids long.
The first antigen and the second antigen may be different antigens if the first antigen does not comprise any epitopes which are cross-reactive with epitopes of the second antigen in a CD4+ T cell, CD8+ T cell and/or antibody response. The first antigen and the second antigen may be different antigens if the first antigen does not comprise any epitopes which are cross-reactive with epitopes of the second antigen in a CD4+ T cell response. The first antigen and the second antigen may be different antigens if the first antigen does not comprise any epitopes which are cross-reactive with epitopes of the second antigen in a CD8+ T cell response. The first antigen and the second antigen may be different antigens if the first antigen does not comprise any epitopes which are cross-reactive with epitopes of the second antigen in an antibody response. The first antigen and the second antigen may be different antigens if the first antigen does not comprise any epitopes which are cross-reactive with epitopes of the second antigen in a CD4+ T cell and CD8+ T cell response. The first antigen and the second antigen may be different antigens if the first antigen does not comprise any epitopes which are cross-reactive with epitopes of the second antigen in a CD4+ T cell and antibody response. The first antigen and the second antigen may be different antigens if the first antigen does not comprise any epitopes which are cross-reactive with epitopes of the second antigen in a CD8+ T cell and antibody response. The first antigen and the second antigen may be different antigens if the first antigen does not comprise any epitopes which are cross-reactive with epitopes of the second antigen in a CD4+ T cell, CD8+ T cell and antibody response.
In an alternative embodiment, the first antigen and the second antigen are different antigens if the second antigen does not comprise any epitopes which are cross-reactive with epitopes of the first antigen.
The CD4+ or CD8+ T cell immunoassay may for example be long- or short-term culture, restimulation, ELISpot, ELISA or bead-based assays for cytokine quantification, cytometry-based phenotyping (including tetramer staining) or cytometry-based intracellular staining. The antibody immunoassay may for example be ELISA, affinity measurements, measurements of antibody functionality (such as virus neutralization assays, virus inhibition assays, ADCC assays, ADCP assays, complement activation or antibody-dependent cytokine secretion).
Suitably an antigen comprises at least one B or T cell epitope, suitably an antigen comprises B and T cell epitopes. The elicited immune response may be an antigen specific B cell response which produces neutralizing antibodies. The elicited immune response may be an antigen specific T cell response, which may be a systemic and/or a local response. The antigen specific T cell response may comprise a CD4+ T cell response, such as a response involving CD4+ T cells expressing a plurality of cytokines, e.g. IFNgamma, TNFalpha and/or IL2. Alternatively, or additionally, the antigen specific T cell response comprises a CD8+ T cell response, such as a response involving CD8+ T cells expressing a plurality of cytokines, e.g., IFNgamma, TNFalpha and/or IL2.
Suitably an antigen contains 3000 residues or fewer, especially 2000 residues or fewer, in particular 1500 residues or fewer. An antigen may contain 1000 residues or fewer, 800 residues or fewer, 600 residues or fewer, 400 residues or fewer or 200 residues or fewer. Suitably an antigen contains 50 residues or more, especially 100 residues or more, in particular 150 residues or more. Suitably an antigen contains 50 to 3000 residues, especially 100 to 1500 residues, in particular 200 to 1000 residues.
In one embodiment the first antigen is derived from a virus. Suitably the first antigen is derived from a herpesvirus, such as human alphaherpesvirus 3 (HHV-3), also known as varicella-zoster virus (VZV). More suitably the first antigen is the VZV-gE antigen. The VZV- gE antigen may be a sequence comprising, such as consisting of, a sequence having at least 80%, such as at least 90%, especially at least 95%, in particular at least 98% for example at least 99% or 100% identity to SEQ ID No: 1 . In one embodiment the first antigen is an influenza virus antigen, such as a hemagglutinin or a neuraminidase.
In one embodiment the first antigen is derived from a bacterium.
Suitably, the first antigen is derived from a different organism to the second antigen.
In one embodiment the second antigen is derived from a bacterium. Suitably the second antigen is derived from a gram-negative bacterium, such as Clostridia. More suitably the second antigen is derived from C. difficile, such as specifically C. difficile toxin A and/or toxin B or fragments thereof, especially a fusion of fragments of toxin A and toxin B such as an F2 antigen (see, e.g., WO2012163811). The F2 antigen may be a sequence comprising, such as consisting of, a sequence having at least 80%, such as at least 90%, especially at least 95%, in particular at least 98% for example at least 99% or 100% identity to SEQ ID No: 2.
The first antigen may be a VZV-gE antigen and the second antigen an F2 antigen.
In one embodiment the second antigen is derived from a virus. The second antigen may be a respiratory syncytial virus (RSV) antigen.
In one embodiment the second antigen is an influenza virus antigen, such as a hemagglutinin or a neuraminidase.
In one embodiment the second antigen is a coronavirus antigen, such as a spike protein.
In certain embodiments a STING agonist is administered in step (i) and an antigen is administered in step (ii) optionally with a second adjuvant.
In one embodiment, the antigen is a polypeptide. In another embodiment, the antigen is provided in the form of a polynucleotide, such as a viral vector or mRNA. In a further embodiment, the antigen is a polysaccharide.
The antigens may be derived from (i.e. associated with) a pathogen, especially a human pathogen (such as a bacterium, virus or parasite) or may be a cancer antigen (such as a tumour antigen and/or a neoantigen).
By the term antigen is meant a substance which is capable of eliciting an immune response in a subject. Suitably the immune response from antigen is a protective immune response, e.g. reducing partially or completely the severity of one or more symptoms and/or time over which one or more symptoms are experienced by a subject, reducing the likelihood of developing an established infection after challenge and/or slowing progression of an associated illness (e.g. extending survival).
If multiple administrations of the antigen (with or without second adjuvant as appropriate) are performed in step (ii), then the antigen may be delivered in the same form (i.e. a ‘homologous’ dosage regime), in different forms (i.e. a ‘heterologous’ dosage regime), or any combination of the same form and different forms, over each of the multiple administrations. For example, if the antigen in a first administration in step (ii) is a polypeptide and the antigen in a second administration is a polynucleotide encoding the polypeptide then the antigens delivered in each administration are delivered in heterologous forms but are the same antigen for the purposes of the invention.
In one embodiment the antigen is derived from a bacterium. Alternatively, the antigen may be derived from a virus. Suitably an antigen is derived from a pathogen if it contains a polypeptide sequence which is obtainable from said pathogen. Desirably, the polypeptide sequence which is obtainable from said pathogen is at least 50, especially at least 100, in particular at least 150, such as at least 200 amino acids long.
Suitably an antigen comprises at least one B or T cell epitope, suitably an antigen comprises B and T cell epitopes. The elicited immune response may be an antigen specific B cell response which produces neutralizing antibodies. The elicited immune response may be an antigen specific T cell response, which may be a systemic and/or a local response. The antigen specific T cell response may comprise a CD4+ T cell response, such as a response involving CD4+ T cells expressing a plurality of cytokines, e.g. IFNgamma, TNFalpha and/or IL2. Alternatively, or additionally, the antigen specific T cell response comprises a CD8+ T cell response, such as a response involving CD8+ T cells expressing a plurality of cytokines, e.g., IFNgamma, TNFalpha and/or IL2.
Suitably an antigen contains 3000 residues or fewer, especially 2000 residues or fewer, in particular 1500 residues or fewer. An antigen may contain 1000 residues or fewer, 800 residues or fewer, 600 residues or fewer, 400 residues or fewer or 200 residues or fewer. Suitably an antigen contains 50 residues or more, especially 100 residues or more, in particular 150 residues or more. Suitably an antigen contains 50 to 3000 residues, especially 100 to 1500 residues, in particular 200 to 1000 residues.
In one embodiment the antigen is derived from a bacterium. Suitably the antigen is derived from a gram-negative bacterium, such as Clostridia. More suitably the antigen is derived from C. difficile, such as specifically C. difficile toxin A and/or toxin B or fragments thereof, especially a fusion of fragments of toxin A and toxin B such as an F2 antigen (see, e.g., WO2012163811). The F2 antigen may be a sequence comprising, such as consisting of, a sequence having at least 80%, such as at least 90%, especially at least 95%, in particular at least 98% for example at least 99% or 100% identity to SEQ ID No: 2.
The antigen may be an F2 antigen.
In one embodiment the antigen is derived from a virus. The antigen may be a respiratory syncytial virus (RSV) antigen. Suitably the antigen is derived from a herpesvirus, such as human alphaherpesvirus 3 (HHV-3), also known as varicella-zoster virus (VZV). More suitably the antigen is the VZV-gE antigen. The VZV-gE antigen may be a sequence comprising, such as consisting of, a sequence having at least 80%, such as at least 90%, especially at least 95%, in particular at least 98% for example at least 99% or 100% identity to SEQ ID No: 1.
In one embodiment the antigen is an influenza virus antigen, such as a hemagglutinin or a neuraminidase.
In one embodiment the antigen is a coronavirus antigen, such as a spike protein.
In certain embodiments a STING agonist and a first antigen are administered in step (i) and a second antigen and optional second adjuvant are administered in step (ii).
In one embodiment, the antigens are all polypeptides. In another embodiment, the antigens are all provided in the form of polynucleotides, such as a viral vector or mRNA. In a further embodiment, the antigens are all polysaccharides.
In some embodiments the first antigen is a polypeptide and the second antigen is provided in the form a polynucleotide, such as a viral vector or mRNA. In other embodiments the second antigen is a polypeptide and the first antigen is provided in the form a polynucleotide, such as a viral vector or mRNA. Most suitably the antigens are all polypeptides.
The antigens may be derived from (i.e. associated with) a pathogen, especially a human pathogen (such as a bacterium, virus or parasite) or may be a cancer antigen (such as a tumour antigen and/or a neoantigen).
By the term antigen is meant a substance which is capable of eliciting an immune response in a subject. Suitably the immune response from the first and/or second antigen is a protective immune response, e.g. reducing partially or completely the severity of one or more symptoms and/or time over which one or more symptoms are experienced by a subject, reducing the likelihood of developing an established infection after challenge and/or slowing progression of an associated illness (e.g. extending survival). Most suitably the immune response to the second antigen in administration under step (ii) is a protective immune response.
If multiple administrations of the STING agonist and first antigen are performed in step (i), then the first antigen may be delivered in the same form (i.e. a ‘homologous’ dosage regime), in different forms (i.e. a ‘heterologous’ dosage regime), or any combination of the same form and different forms, over each of the multiple administrations. For example, if the first antigen in a first administration in step (i) is a polypeptide and the first antigen in a second administration in step (i) is a polynucleotide encoding the polypeptide then the antigens delivered in each administration are delivered in heterologous forms but nevertheless the same antigen for the purposes of the invention. If multiple administrations of the second antigen (with or without second adjuvant as appropriate) are performed in step (ii), then the second antigen may be delivered in the same form (i.e. a ‘homologous’ dosage regime), in different forms (i.e. a ‘heterologous’ dosage regime), or any combination of the same form and different forms, over each of the multiple administrations. For example, if the second antigen in a first administration in step (ii) is a polypeptide and the second antigen in a second administration is a polynucleotide encoding the polypeptide then the antigens delivered in each administration are delivered in heterologous forms but are the same antigen for the purposes of the invention.
The first antigen and the second antigen are different, i.e the first antigen has a different polypeptide, different polypeptide encoded by a polynucleotide or different polysaccharide sequence to the second antigen.
Suitably, the first antigen is derived from a different pathogen to the second antigen. In methods for prophylaxis in a subject at risk of infection, suitably the first antigen is derived from a different pathogen from the pathogen presenting the risk of infection.
Suitably a different pathogen is a pathogen which belongs to a different species, especially genus, in particular family, such as order, especially class, in particular phylum, such as kingdom or especially domain. Alternatively, pathogens may be considered different if the first antigen is derived from (such as containing a portion of a polypeptide sequence obtainable from) one of bacteria, viruses or parasites and the second antigen is derived from another of bacteria, viruses or parasites. In one embodiment the first antigen is derived from a virus and the second antigen is derived from a bacterium. In one embodiment the first antigen is derived from a virus and the pathogen presenting the risk of infection is a bacterium. Alternatively, the first antigen may be derived from a bacterium and the second antigen may be derived from a virus. In one embodiment the first antigen is derived from a bacterium and the pathogen presenting the risk of infection is a virus. In one embodiment the first antigen is derived from a virus and the pathogen presenting the risk of infection is a virus. Suitably an antigen is derived from a pathogen if it contains a polypeptide sequence which is obtainable from said pathogen. Desirably, the polypeptide sequence which is obtainable from said pathogen is at least 50, especially at least 100, in particular at least 150, such as at least 200 amino acids long.
The first antigen and the second antigen may be different antigens if the first antigen does not comprise any epitopes which are cross-reactive with epitopes of the second antigen in a CD4+ T cell, CD8+ T cell and/or antibody response. The first antigen and the second antigen may be different antigens if the first antigen does not comprise any epitopes which are cross-reactive with epitopes of the second antigen in a CD4+ T cell response. The first antigen and the second antigen may be different antigens if the first antigen does not comprise any epitopes which are cross-reactive with epitopes of the second antigen in a CD8+ T cell response. The first antigen and the second antigen may be different antigens if the first antigen does not comprise any epitopes which are cross-reactive with epitopes of the second antigen in an antibody response. The first antigen and the second antigen may be different antigens if the first antigen does not comprise any epitopes which are cross-reactive with epitopes of the second antigen in a CD4+ T cell and CD8+ T cell response. The first antigen and the second antigen may be different antigens if the first antigen does not comprise any epitopes which are cross-reactive with epitopes of the second antigen in a CD4+ T cell and antibody response. The first antigen and the second antigen may be different antigens if the first antigen does not comprise any epitopes which are cross-reactive with epitopes of the second antigen in a CD8+ T cell and antibody response. The first antigen and the second antigen may be different antigens if the first antigen does not comprise any epitopes which are cross-reactive with epitopes of the second antigen in a CD4+ T cell, CD8+ T cell and antibody response.
In an alternative embodiment, the first antigen and the second antigen are different antigens if the second antigen does not comprise any epitopes which are cross-reactive with epitopes of the first antigen.
In methods for prophylaxis in a subject at risk of infection, suitably the first antigen does not comprise any epitopes which are cross-reactive with epitopes of the pathogen presenting the risk of infection in a CD4+ T cell, CD8+ T cell and/or antibody response. The first antigen suitably does not comprise any epitopes which are cross-reactive with epitopes of the pathogen presenting the risk of infection in a CD4+ T cell response. The first antigen suitably does not comprise any epitopes which are cross-reactive with epitopes of the pathogen presenting the risk of infection in a CD8+ T cell response. The first antigen suitably does not comprise any epitopes which are cross-reactive with epitopes of the pathogen presenting the risk of infection in an antibody response. The first antigen suitably does not comprise any epitopes which are cross-reactive with epitopes of the pathogen presenting the risk of infection in a CD4+ T cell and CD8+ T cell response. The first antigen suitably does not comprise any epitopes which are cross-reactive with epitopes of the pathogen presenting the risk of infection in a CD4+ T cell and antibody response. The first antigen suitably does not comprise any epitopes which are cross-reactive with epitopes of the pathogen presenting the risk of infection in a CD8+ T cell and antibody response. The first antigen suitably does not comprise any epitopes which are cross-reactive with epitopes of the pathogen presenting the risk of infection in a CD4+ T cell, CD8+ T cell and antibody response.
The CD4+ or CD8+ T cell immunoassay may for example be long- or short-term culture, restimulation, ELISpot, ELISA or bead-based assays for cytokine quantification, cytometry-based phenotyping (including tetramer staining) or cytometry-based intracellular staining. The antibody immunoassay may for example be ELISA, affinity measurements, measurements of antibody functionality (such as virus neutralization assays, virus inhibition assays, ADCC assays, ADCP assays, complement activation or antibody-dependent cytokine secretion).
Suitably an antigen comprises at least one B or T cell epitope, suitably an antigen comprises B and T cell epitopes. The elicited immune response may be an antigen specific B cell response which produces neutralizing antibodies. The elicited immune response may be an antigen specific T cell response, which may be a systemic and/or a local response. The antigen specific T cell response may comprise a CD4+ T cell response, such as a response involving CD4+ T cells expressing a plurality of cytokines, e.g. IFNgamma, TNFalpha and/or IL2. Alternatively, or additionally, the antigen specific T cell response comprises a CD8+ T cell response, such as a response involving CD8+ T cells expressing a plurality of cytokines, e.g., IFNgamma, TNFalpha and/or IL2.
Suitably an antigen contains 3000 residues or fewer, especially 2000 residues or fewer, in particular 1500 residues or fewer. An antigen may contain 1000 residues or fewer, 800 residues or fewer, 600 residues or fewer, 400 residues or fewer or 200 residues or fewer. Suitably an antigen contains 50 residues or more, especially 100 residues or more, in particular 150 residues or more. Suitably an antigen contains 50 to 3000 residues, especially 100 to 1500 residues, in particular 200 to 1000 residues.
In one embodiment the first antigen is derived from a virus. Suitably the first antigen is derived from a herpesvirus, such as human alphaherpesvirus 3 (HHV-3), also known as varicella-zoster virus (VZV). More suitably the first antigen is the VZV-gE antigen. The VZV- gE antigen may be a sequence comprising, such as consisting of, a sequence having at least 80%, such as at least 90%, especially at least 95%, in particular at least 98% for example at least 99% or 100% identity to SEQ ID No: 1 . In one embodiment the first antigen is an influenza virus antigen, such as a hemagglutinin or a neuraminidase.
In one embodiment the first antigen is derived from a bacterium.
Suitably, the first antigen is derived from a different organism to the second antigen.
In one embodiment the second antigen is derived from a bacterium. Suitably the second antigen is derived from a gram-negative bacterium, such as Clostridia. More suitably the second antigen is derived from C. difficile, such as specifically C. difficile toxin A and/or toxin B or fragments thereof, especially a fusion of fragments of toxin A and toxin B such as an F2 antigen (see, e.g., WO2012163811). The F2 antigen may be a sequence comprising, such as consisting of, a sequence having at least 80%, such as at least 90%, especially at least 95%, in particular at least 98% for example at least 99% or 100% identity to SEQ ID No: 2.
The first antigen may be a VZV-gE antigen and the second antigen an F2 antigen.
In one embodiment the second antigen is derived from a virus. The second antigen may be a respiratory syncytial virus (RSV) antigen. In one embodiment the second antigen is an influenza virus antigen, such as a hemagglutinin or a neuraminidase.
In one embodiment the second antigen is a coronavirus antigen, such as a spike protein. mRNA
If an antigen is a polynucleotide, the antigen may be in the form of mRNA. Messenger RNA (mRNA) can direct the cellular machinery of a subject to produce proteins. mRNA may be circular or branched, but will generally be linear. mRNA used herein are preferably provided in purified or substantially purified form i.e. substantially free from proteins (e.g., enzymes), other nucleic acids (e.g. DNA and nucleoside phosphate monomers), and the like, generally being at least about 50% pure (by weight), and usually at least 90% pure, such as at least 95% or at least 98% pure. mRNA may be prepared in many ways e.g. by chemical synthesis in whole or in part, by digesting longer nucleic acids using nucleases (e.g. restriction enzymes), by joining shorter nucleic acids or nucleotides (e.g. using ligases or polymerases), from genomic or cDNA libraries, etc. In particular, mRNA may be prepared enzymatically using a DNA template.
The term mRNA as used herein includes conventional mRNA or mRNA analogs, such as those containing modified backbones or modified bases (e.g. pseudouridine, or the like). mRNA, may or may not have a 5' cap.
The mRNA comprises a sequence which encodes at least one antigen.
Typically, the nucleic acids of the invention will be in recombinant form, i.e. a form which does not occur in nature. For example, the mRNA may comprise one or more heterologous nucleic acid sequences (e.g. a sequence encoding another antigen and/or a control sequence such as a promoter or an internal ribosome entry site) in addition to the sequence encoding the antigen.
Alternatively, or in addition, the sequence or chemical structure of the nucleic acid may be modified compared to a naturally-occurring sequence which encodes the antigen. The sequence of the nucleic acid molecule may be modified, e.g. to increase the efficacy of expression or replication of the nucleic acid, or to provide additional stability or resistance to degradation. mRNA may also be codon optimised. In some embodiments, mRNA may be codon optimised for expression in human cells. By “codon optimised” is intended modification with respect to codon usage which may increase translation efficacy and/or half-life of the nucleic acid.
A poly A tail (e.g., of about 30 adenosine residues or more) may be attached to the 3' end of the RNA to increase its half-life. The 5' end of the RNA may be capped, for example with a modified ribonucleotide with the structure m7G (5') ppp (5') N (cap 0 structure) or a derivative thereof, which can be incorporated during RNA synthesis or can be enzymatically engineered after RNA transcription (e.g., by using Vaccinia Virus Capping Enzyme (VCE) consisting of mRNA triphosphatase, guanylyl-transferase and guanine-7-methytransferase, which catalyzes the construction of N7- monomethylated cap 0 structures). Cap 0 structure plays an important role in maintaining the stability and translational efficacy of the mRNA molecule. The 5' cap of the mRNA molecule may be further modified by a 2'-O-Methyltransferase which results in the generation of a cap 1 structure (m7Gppp [m2'-O] N), which may further increase translation efficacy. mRNA may comprise one or more nucleotide analogs or modified nucleotides. As used herein, "nucleotide analog" or "modified nucleotide" refers to a nucleotide that contains one or more chemical modifications (e.g., substitutions) in or on the nitrogenous base of the nucleoside (e.g. cytosine (C), thymine (T) or uracil (II)), adenine (A) or guanine (G)). A nucleotide analog can contain further chemical modifications in or on the sugar moiety of the nucleoside (e.g. ribose, modified ribose, six-membered sugar analog, or open-chain sugar analog), or the phosphate. The preparation of nucleotides and modified nucleotides and nucleosides are well-known in the art, see the following references: US Patent Numbers 4373071, 4458066, 4500707, 4668777, 4973679, 5047524, 5132418, 5153319, 5262530, 5700642. Many modified nucleosides and modified nucleotides are commercially available.
Modified nucleobases (chemical modifications) which can be incorporated into modified nucleosides and nucleotides and be present in the mRNA molecules include: m5C (5- methylcytidine), m5U (5-methyluridine), m6A (N6-methyladenosine), s2U (2-thiouridine), Um (2'-O-methyluridine), m1A (1 -methyladenosine); m2A (2-methyladenosine); Am (2-1-0- methyladenosine); ms2m6A (2-methylthio-N6-methyladenosine); i6A (N6- isopentenyladenosine); ms2i6A (2-methylthio-N6isopentenyladenosine); io6A (N6-(cis- hydroxyisopentenyl)adenosine); ms2io6A (2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine); g6A (N6-glycinylcarbamoyladenosine); t6A (N6-threonyl carbamoyladenosine); ms2t6A (2-methylthio-N6-threonyl carbamoyladenosine); m6t6A (N6-methyl-N6- threonylcarbamoyladenosine); hn6A(N6-hydroxynorvalylcarbamoyl adenosine); ms2hn6A (2- methylthio-N6-hydroxynorvalyl carbamoyladenosine); Ar(p) (2'-O-ribosyladenosine (phosphate)); I (inosine); mil (1-methylinosine); m'lm (1 ,2'-O-dimethylinosine); m3C (3- methylcytidine); Cm (2’-O-methylcytidine); s2C (2-thiocytidine); ac4C (N4-acetylcytidine); f5C (5-fonnylcytidine); m5Cm (5,2-O-dimethylcytidine); ac4Cm (N4-acetyl-2-O-methylcytidine); k2C (lysidine); m1G (1 -methylguanosine); m2G (N2-methylguanosine); m7G (7-methylguanosine); Gm (2'-O-methylguanosine); m22G (N2,N2-dimethylguanosine); m2Gm (N2,2'-O- dimethylguanosine); m22Gm (N2,N2,2'-O-trimethylguanosine); Gr(p) (2'-O-ribosylguanosine (phosphate)); yW (wybutosine); o2yW (peroxywybutosine); OHyW (hydroxywybutosine); OHyW* (undermodified hydroxywybutosine); imG (wyosine); mimG (methylguanosine); Q (queuosine); oQ (epoxyqueuosine); galQ (galtactosyl-queuosine); manQ (mannosyl- queuosine); preQo (7-cyano-7-deazaguanosine); preQi (7-aminomethyl-7-deazaguanosine); G* (archaeosine); D (dihydrouridine); m5Um (5,2'-O-dimethyluridine); s4U (4-thiouridine); m5s2U (5-methyl-2-thiouridine); s2Um (2-thio-2'-O-methyluridine); acp3U (3-(3-amino-3- carboxypropyl)uridine); ho5U (5-hydroxyuridine); mo5U (5-methoxyuridine); cmo5U (uridine 5- oxyacetic acid); mcmo5U (uridine 5-oxyacetic acid methyl ester); chm5U (5- (carboxyhydroxymethyl)uridine)); mchm5U (5-(carboxyhydroxymethyl)uridine methyl ester); mcm5U (5-methoxycarbonyl methyluridine); mcm5Um (S-methoxycarbonylmethyl-2-O- methyluridine); mcm5s2U (5-methoxycarbonylmethyl-2-thiouridine); nm5s2U (5-aminomethyl- 2-thiouridine); mnm5U (5-methylaminomethyluridine); mnm5s2U (5-methylaminomethyl-2- thiouridine); mnm5se2U (5-methylaminomethyl-2-selenouridine); ncm5U (5-carbamoylmethyl uridine); ncm5Um (5-carbamoylmethyl-2'-O-methyluridine); cmnm5U (5- carboxymethylaminomethyluridine); cnmm5Um (5-carboxymethy 1 aminomethyl-2-L-O-methyl uridine); cmnm5s2U (5-carboxymethylaminomethyl-2-thiouridine); m62A (N6,N6- dimethyladenosine); Tm (2'-O-methylinosine); m4C (N4-methylcytidine); m4Cm (N4,2-O- dimethylcytidine); hm5C (5-hydroxymethylcytidine); m3U (3-methyluridine); cm5U (5- carboxymethyluridine); m6Am (N6,2’-O-dimethyladenosine); rn62Am (N6,N6,0-2- trimethyladenosine); m2'7G (N2,7-dimethylguanosine); m2'2'7G (N2,N2,7-trimethylguanosine); m3Um (3,2’-O-dimethyluridine); m5D (5-methyldihydrouridine); f5Cm (5-formyl-2'-O- methylcytidine); m1Gm (1 ,2'-O-dimethylguanosine); m'Am (1 ,2-O-dimethyl adenosine) irinomethyluridine); tm5s2U (S-taurinomethyl-2-thiouridine)); iniG-14 (4-demethyl guanosine); imG2 (isoguanosine); ac6A (N6-acetyladenosine), hypoxanthine, inosine, 8-oxo-adenine, 7- substituted derivatives thereof, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5- aminouracil, 5-(Ci-C6)-alkyluracil, 5-methyluracil, 5-(C2-C6)-alkenyluracil, 5-(C2-Ce)- alkynyluracil, 5-(hydroxymethyl)uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil, 5- hydroxycytosine, 5-(Ci-C6)-alkylcytosine, 5-methylcytosine, 5-(C2-C6)-alkenylcytosine, 5-(C2- C6)-alkynylcytosine, 5-chlorocytosine, 5-fluorocytosine, 5-bromocytosine, N2-dimethylguanine, 7-deazaguanine, 8-azaguanine, 7-deaza-7-substituted guanine, 7-deaza-7-(C2- C6)alkynylguanine, 7-deaza-8-substituted guanine, 8-hydroxyguanine, 6-thioguanine, 8- oxoguanine, 2-aminopurine, 2-amino-6-chloropurine, 2,4-diaminopurine, 2,6-diaminopurine, 8- azapurine, substituted 7-deazapurine, 7-deaza-7-substituted purine, 7-deaza-8-substituted purine, hydrogen (abasic residue), m5C, m5U, m6A, s2U, W, or 2'-O-methyl-U. Many of these modified nucleobases and their corresponding ribonucleosides are available from commercial suppliers.
The mRNA may encode more than one antigen. For example, the mRNA encoding an antigen protein may encode only the antigen or may encode additional proteins. mRNA may be non-replicating or may be replicating, also known as self-amplifying. A self-amplifying mRNA molecule may be an alphavirus-derived mRNA replicon. mRNA amplification can also be achieved by the provision of a non-replicating mRNA encoding an antigen in conjunction with a separate mRNA encoding replication machinery.
Self-replicating RNA molecules are well known in the art and can be produced by using replication elements derived from, e.g., alphaviruses, and substituting the structural viral proteins with a nucleotide sequence encoding a protein of interest. A self-replicating RNA molecule is typically a +-strand molecule which can be directly translated after delivery to a cell, and this translation provides a RNA-dependent RNA polymerase which then produces both antisense and sense transcripts from the delivered RNA. Thus the delivered RNA leads to the production of multiple daughter RNAs. These daughter RNAs, as well as collinear subgenomic transcripts, may be translated themselves to provide in situ expression of an encoded antigen, or may be transcribed to provide further transcripts with the same sense as the delivered RNA which are translated to provide in situ expression of the antigen. The overall result of this sequence of transcriptions is a huge amplification in the number of the introduced replicon RNAs and so the encoded antigen becomes a major polypeptide product of the cells.
Suitable alphavirus replicons can use a replicase from a Sindbis virus, a Semliki forest virus, an eastern equine encephalitis virus, a Venezuelan equine encephalitis virus, etc. Mutant or wild-type virus sequences can be used e.g. the attenuated TC83 mutant of VEEV has been used in replicons, see the following reference: W02005/113782.
In certain embodiments, the self-replicating RNA molecule described herein encodes (i) a RNA-dependent RNA polymerase which can transcribe RNA from the self-replicating RNA molecule and (ii) an antigen. The polymerase can be an alphavirus replicase e.g. comprising one or more of alphavirus proteins nsPI, nsP2, nsP3 and nsP4.
Whereas natural alphavirus genomes encode structural virion proteins in addition to the non-structural replicase polyprotein, in certain embodiments, the self-replicating RNA molecules do not encode alphavirus structural proteins. Thus, the self-replicating RNA can lead to the production of genomic RNA copies of itself in a cell, but not to the production of RNA-containing virions. The inability to produce these virions means that, unlike a wild-type alphavirus, the self-replicating RNA molecule cannot perpetuate itself in infectious form. The alphavirus structural proteins which are necessary for perpetuation in wild-type viruses are absent from self-replicating RNAs of the present disclosure and their place is taken by gene(s) encoding the immunogen of interest, such that the subgenomic transcript encodes the immunogen rather than the structural alphavirus virion proteins.
Thus a self-replicating RNA molecule useful with the invention may have two open reading frames. The first (5') open reading frame encodes a replicase; the second (3') open reading frame encodes an antigen. In some embodiments the RNA may have additional (e.g. downstream) open reading frames e.g. to encode further antigens or to encode accessory polypeptides.
In certain embodiments, the self-replicating RNA molecule disclosed herein has a 5' cap (e.g. a 7-methylguanosine). This cap can enhance in vivo translation of the RNA. In some embodiments the 5' sequence of the self-replicating RNA molecule must be selected to ensure compatibility with the encoded replicase.
A self-replicating RNA molecule may have a 3' poly-A tail. It may also include a poly-A polymerase recognition sequence (e.g. AALIAAA) near its 3' end.
Self-replicating RNA molecules can have various lengths, but they are typically 5000- 25000 nucleotides long. Self-replicating RNA molecules will typically be single-stranded. Single-stranded RNAs can generally initiate an adjuvant effect by binding to TLR7, TLR8, RNA helicases and/or PKR. RNA delivered in double-stranded form (dsRNA) can bind to TLR3, and this receptor can also be triggered by dsRNA which is formed either during replication of a single-stranded RNA or within the secondary structure of a single-stranded RNA.
In another embodiment, a self-replicating RNA may comprise two separate RNA molecules, each comprising a nucleotide sequence derived from an alphavirus: one RNA molecule comprises a RNA construct for expressing alphavirus replicase, and one RNA molecule comprises a RNA replicon that can be replicated by the replicase in trans. In some embodiments, the RNA construct for expressing alphavirus replicase comprises a 5'-cap. See WO2017/162265.
The self-replicating RNA can conveniently be prepared by in vitro transcription (IVT). IVT can use a (cDNA) template created and propagated in plasmid form in bacteria, or created synthetically (for example by gene synthesis and/or polymerase chain-reaction (PCR) engineering methods). For instance, a DNA-dependent RNA polymerase (such as the bacteriophage T7, T3 or SP6 RNA polymerases) can be used to transcribe the self-replicating RNA from a DNA template. Appropriate capping and poly-A addition reactions can be used as required (although the replicon's poly-A is usually encoded within the DNA template). These RNA polymerases can have stringent requirements for the transcribed 5' nucleotide(s) and in some embodiments these requirements must be matched with the requirements of the encoded replicase, to ensure that the IVT-transcribed RNA can function efficiently as a substrate for its self-encoded replicase.
A self-replicating RNA can include (in addition to any 5' cap structure) one or more nucleotides having a modified nucleobase. A RNA used with the invention ideally includes only phosphodiester linkages between nucleosides, but in some embodiments it can contain phosphoramidate, and/or methylphosphonate linkages.
The self-replicating RNA molecule may encode a single heterologous polypeptide antigen (i.e. the antigen) or, optionally, two or more heterologous polypeptide antigens linked together in a way that each of the sequences retains its identity (e.g., linked in series) when expressed as an amino acid sequence. The heterologous polypeptides generated from the self-replicating RNA may then be produced as a fusion polypeptide or engineered in such a manner to result in separate polypeptide or peptide sequences.
The self-replicating RNA molecules described herein may be engineered to express multiple nucleotide sequences, from two or more open reading frames, thereby allowing coexpression of proteins, such as one, two or more antigens (e.g. one, two or more coronavirus protein(s), such as SARS-CoV-2 S protein(s)) together with cytokines or other immunomodulators, which can enhance the generation of an immune response. Such a selfreplicating RNA molecule might be particularly useful, for example, in the production of various gene products (e.g., proteins) at the same time, for example, as a bivalent or multivalent vaccine.
If desired, the self-replicating RNA molecules can be screened or analyzed to confirm their therapeutic and prophylactic properties using various in vitro or in vivo testing methods that are known to those of skill in the art. For example, vaccines comprising self-replicating RNA molecule can be tested for their effect on induction of proliferation or effector function of the particular lymphocyte type of interest, e.g., B cells, T cells, T cell lines, and T cell clones. For example, spleen cells from immunized mice can be isolated and the capacity of cytotoxic T lymphocytes to lyse autologous target cells that contain a self-replicating RNA molecule that encodes an antigen. In addition, T helper cell differentiation can be analyzed by measuring proliferation or production of TH1 (IL-2 and IFN-y) and /or TH2 (IL-4 and IL-5) cytokines by ELISA or directly in CD4+ T cells by cytoplasmic cytokine staining and flow cytometry.
Self-replicating RNA molecules that encode an antigen can also be tested for ability to induce humoral immune responses, as evidenced, for example, by induction of B cell production of antibodies specific for the antigen of interest. These assays can be conducted using, for example, peripheral B lymphocytes from immunized individuals. Such assay methods are known to those of skill in the art. Other assays that can be used to characterize the self-replicating RNA molecules can involve detecting expression of the encoded antigen by the target cells. For example, FACS can be used to detect antigen expression on the cell surface or intracellularly. Another advantage of FACS selection is that one can sort for different levels of expression; sometimes-lower expression may be desired. Other suitable method for identifying cells which express a particular antigen involve panning using monoclonal antibodies on a plate or capture using magnetic beads coated with monoclonal antibodies.
A non-replicating mRNA will typically contain 10000 bases or fewer, especially 8000 bases or fewer, in particular 5000 base or fewer. A replicating mRNA will typically contain 25000 bases or fewer, especially 20000 bases or fewer, in particular 15000 bases or fewer. A single dose of mRNA may be 0.001 to 1000 ug, especially 1 to 500 ug, in particular 10 to 250 ug. A single dose of mRNA may be 0.001 to 75 ug, 1 to 75 ug, 25 to 250 ug, or 250 to 1000 ug. Specifically, a replicating mRNA dose may be 0.001 to 75 ug, such as 0.1 to 75 ug. A non-replicating mRNA dose may, for example, be 1 to 500 ug, such as 1 to 250 ug.
In one embodiment the mRNA is non-replicating mRNA. In a second embodiment the mRNA is replicating mRNA. mRNA Carriers
A range of carrier systems have been described which encapsulate or complex mRNA in order to facilitate mRNA delivery and consequent expression of encoded antigens as compared to mRNA which is not encapsulated or complexed. The present invention may utilise any suitable carrier system. Particular mRNA carrier systems of note are further described below.
LNP
Lipid nanoparticles (LNPs) are non-virion liposome particles in which mRNA can be encapsulated. LNP delivery systems and methods for their preparation are known in the art. The particles can include some external mRNA (e.g. on the surface of the particles), but desirably at least half of the RNA (and suitably at least 85%, especially at least 95%, such as all of it) is encapsulated. LNP can, for example, be formed of a mixture of (i) a PEG-modified lipid (ii) a non-cationic lipid (iii) a sterol (iv) an ionisable cationic lipid. Alternatively, LNP can for example be formed of a mixture of (i) a PEG-modified lipid (ii) a non-cationic lipid (iii) a sterol (iv) a non-ionisable cationic lipid.
The PEG-modified lipid may comprise a PEG molecule with a molecular weight of 10000 Da or less, especially 5000 Da or less, in particular 3000 Da, such 2000 Da or less. The PEG-modified lipid may comprise a PEG molecule with an average molecular weight of 1500 Da to 2500 Da, such as 2000 Da. Examples of PEG-modified lipids include PEG- distearoyl glycerol, PEG-dipalmitoyl glycerol and PEG-dimyristoyl glycerol. The PEG-modified lipid is typically present at around 0.5 to 15 molar %.
The non-cationic lipid may be a neutral lipid, such as 1,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2- oleoyl-sn-glycero-3-phosphocholine (POPO), 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and sphingomyelin (SM). The non-cationic lipid is typically present at around 5 to 25 molar %.
The sterol may be cholesterol. The sterol is typically present at around 25 to 55 molar %.
A range of suitable ionizable cationic lipids are known in the art, which are typically present at around 20 to 60 molar %. The ratio of RNA to lipid can be varied (see for example WO2013/006825). "N:P ratio" refers to the molar ratio of protonatable nitrogen atoms in the cationic lipids (typically solely in the lipid's headgroup) to phosphates in the RNA. The ratio of nucleotide (N) to phospholipid (P) can be in the range of, e.g., 1 N:1P to 20N:1 P, 1N:1P to 10N:1 P, 2N:1 P to 8N:1 P, 2N:1P to 6N:1 P or 3N:1 P to 5N:1 P. The ratio of nucleotide (N) to phospholipid (P) can be in the range of, e.g., 1 N:1P, 2N:1P, 3N:1P, 4N:1 P, 5N:1 P, 6N:1 P, 7N:1 P, 8N:1P, 9N:1P, or 10N:1P. Alternatively or additionally, the ratio of nucleotide (N) to phospholipid (P) is 4N:1 P.
WO2017/070620 provides general information on LNP compositions and is incorporated herein by reference. Other useful LNPs are described in the following references: WO2012/006376; WO2012/030901 ; WO2012/031046; WO2012/031043; WO2012/006378; WO2011/076807; WO2013/033563; WO2013/006825; WO2014/136086; WO2015/095340; WO2015/095346; WO2016/037053, which are also incorporated herein by reference.
LNP delivery systems, and methods for their preparation are described in the following reference: Geall et al. (2012) PNAS USA. Sep 4; 109(36): 14604-9 (LNP delivery system); Liposomal particles can, for example, be formed of a mixture of zwitterionic, cationic and anionic lipids which can be saturated or unsaturated, for example; DSPC (zwitterionic, saturated), DlinDMA (cationic, unsaturated), and/or DMG (anionic, saturated). Preferred LNPs for use with the invention include an amphiphilic lipid which can form liposomes, optionally in combination with at least one cationic lipid (such as DOTAP, DSDMA, DODMA, DLinDMA, DLenDMA, etc.). A mixture of DSPC, DlinDMA, PEG-DMG and cholesterol is particularly effective.
In some embodiments, the LNPs are RV01 liposomes, see the following references: WG2012/006376 and Geall et al. (2012) PNAS USA. Sep 4; 109(36): 14604-9.
LNPs are typically 50 to 200 urn in diameter (Z-average). Suitably the LNPs have a polydispersity of 0.4 or less, such as 0.3 or less.
In one embodiment the carrier is a lipid nanoparticle (LNP). CNE
The carrier may be a cationic nanoemulsion (CNE) delivery system. Such cationic oil- in-water emulsions can be used to deliver the mRNA to the interior of a cell. The emulsion particles comprise a hydrophobic oil core and a cationic lipid, the latter of which can interact with the mRNA, thereby anchoring it to the emulsion particle. In a CNE delivery system, the mRNA which encodes the antigen is complexed with a particle of a cationic oil-in-water emulsion. CNE carriers and methods for their preparation are described in WO2012/006380, WO2013/006837 and WO2013/006834 which are incorporated herein by reference.
Thus, the mRNA may be complexed with a particle of a cationic oil-in-water emulsion. The particles typically comprise an oil core (e.g. a plant oil or squalene) that is in liquid phase at 25°C, a cationic lipid (e.g. phospholipid) and, optionally, a surfactant (e.g. sorbitan trioleate, polysorbate 80); polyethylene glycol can also be included. Alternatively or additionally, the CNE comprises squalene and a cationic lipid, such as 1,2-dioleoyloxy-3- (trimethylammonio)propane (DOTAP) (see e.g. Brito, 2014). In an embodiment, the CNE is an oil-in-water emulsion of DOTAP and squalene stabilised with polysorbate 80 and/or sorbitan trioleate.
Desirably at least half of the RNA (and suitably at least 85%, such as all of it) is complexed with the cationic oil-in-water emulsion carrier.
CNE are typically 50 to 200 urn in diameter (Z-average). Suitably the CNE have a polydispersity of 0.4 or less, such as 0.3 or less.
In one embodiment the carrier is a cationic nanoemulsion (CNE). LION
A lipidoid-coated iron oxide nanoparticle (LION) is capable of delivering mRNA into cells and may be aided after administration to a subject by application of an external magnetic field. A LION is an iron oxide particle with one or more coatings comprising lipids and/or lipidoids wherein mRNA encoding the antigen is incorporated into or associated with the lipid and/or lipidoid coating(s) through electrostatic interactions. The mRNA being embedded within the coating(s) may offer protection from enzymatic degradation. The lipids and/or lipidoids comprised within a LION may for example include those included in Figure S1 of Jiang, 2013, especially lipidoids comprising alkyl tails of 12 to 14 carbons in length and in particular lipidoid C14-200 as disclosed in Jiang, 2013. A LION may typically comprise 200 to 5000, such as 500 to 2000, in particular about 1000 lipid and/or lipidoid molecules. Typically the LIONs are 20 to 200 nm in diameter, especially 50 to 100 nm in diameter. The lipid/lipidoid to mRNA weight ratio may be about 1:1 to 10:1, especially about 5:1. Particularly suitable LIONs, and methods for preparation of LIONs are disclosed in Jiang, 2013.
In one embodiment the carrier is a lipidoid-coated iron oxide nanoparticle (LION). Sequence alignments
Identity or homology with respect to a sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the reference amino acid sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
Sequence identity can be determined by standard methods that are commonly used to compare the similarity in position of the amino acids of two polypeptides. Using a computer program such as BLAST or FASTA, two polypeptides are aligned for optimal matching of their respective amino acids (either along the full length of one or both sequences or along a predetermined portion of one or both sequences). The programs provide a default opening penalty and a default gap penalty, and a scoring matrix such as PAM 250 [a standard scoring matrix; see Dayhoff et al., in Atlas of Protein Sequence and Structure, vol. 5, supp. 3 (1978)] can be used in conjunction with the computer program. For example, the percent identity can then be calculated as: the total number of identical matches multiplied by 100 and then divided by the sum of the length of the longer sequence within the matched span and the number of gaps introduced into the shorter sequences in order to align the two sequences.
Additional antigens
The present invention may involve a plurality of antigenic components, for example with the objective to elicit a broad immune response e.g. to a pathogen or to elicit responses to multiple pathogens. Consequently, more than one antigen may be present, more than one polynucleotide encoding an antigen may be present, one polynucleotide encoding more than one antigen may be present or a mixture of antigen(s) and polynucleotide(s) encoding antigen(s) may be present. Polysaccharides such as polysaccharide conjugates, may also be present.
In certain embodiments a STING agonist and a first antigen are administered in step (i) and a STING agonist and a second antigen are administered in step (ii). In more particular embodiments, step (i) involves only one antigen.
In certain embodiments step (ii) involves an antigen. In some embodiments step (ii) involves only one antigen.
Subjects
The present invention is generally intended for mammalian subjects, in particular human subjects. The subject may be a wild or domesticated animal. Mammalian subjects include for example cats, dogs, pigs, sheep, horses or cattle. In one embodiment the invention, the subject is human.
The subject to be treated using the method of the invention may be of any age.
In one embodiment the subject is a human infant (up to 12 months of age). In one embodiment the subject is a human child (less than 18 years of age). In one embodiment the subject is an adult human (aged 18-59). In one embodiment the subject is an older human (aged 60 or greater).
Doses administered to younger children, such as less than 12 years of age, may be reduced relative to an equivalent adult dose, such as by 50%.
The methods of the invention may be intended for prophylaxis of infectious diseases, i.e. for administration to a subject which is not infected with a pathogen. In other embodiments the methods of the invention may be intended for treatment, e.g. for the treatment of infectious diseases, i.e. for administration to a subject which is infected with a pathogen.
In certain embodiments a STING agonist is administered to a subject at risk of infection.
By the term subject at risk of infection as used herein is meant a subject believed to be at risk or which may later be at risk of exposure to a pathogen. Suitably the risk of infection is 2 weeks to 2 years, especially 2 weeks to 1 year, in particular 3 weeks to 9 months, such as 4 weeks to 6 months from completion of administration of STING agonist (i.e. step (i)).
In some embodiments the risk of infection is 2 weeks to 2 months from completion of completion of administration of STING agonist. In some embodiments the risk of infection is 1 month to 3 months from completion of administration of STING agonist. In some embodiments the risk of infection is 2 months to 4 months from completion of administration of STING agonist. In some embodiments the risk of infection is 3 months to 6 months from completion of administration of STING agonist. In some embodiments the risk of infection is 4 months to 12 months from completion of administration of STING agonist.
The subject at risk of infection may be a diabetic who has experienced an abrasion or injury.
The subject at risk of infection may be an individual expected to undergo hospital treatment, suitably the pathogen being Staphylococcus aureus.
The pathogen presenting a risk to the subject at risk of infection may be a pathogen causing a pandemic, such as an influenza virus or a coronavirus.
In certain embodiments a STING agonist and an antigen are administered to a subject at risk of infection.
By the term subject at risk of infection as used herein is meant a subject believed to be at risk or which may later be at risk of exposure to a pathogen. Suitably the risk of infection is 2 weeks to 2 years, especially 2 weeks to 1 year, in particular 3 weeks to 9 months, such as 4 weeks to 6 months from completion of administration of STING agonist and the first antigen (i.e. step (i)).
In some embodiments the risk of infection is 2 weeks to 2 months from completion of completion of administration of STING agonist and the first antigen. In some embodiments the risk of infection is 1 month to 3 months from completion of administration of STING agonist and the first antigen. In some embodiments the risk of infection is 2 months to 4 months from completion of administration of STING agonist and the first antigen. In some embodiments the risk of infection is 3 months to 6 months from completion of administration of STING agonist and the first antigen. In some embodiments the risk of infection is 4 months to 12 months from completion of administration of STING agonist and the first antigen.
The subject at risk of infection may be a diabetic who has experienced an abrasion or injury.
The subject at risk of infection may be an individual expected to undergo hospital treatment, suitably the pathogen being Staphylococcus aureus.
The pathogen presenting a risk to the subject at risk of infection may be a pathogen causing a pandemic, such as an influenza virus or a coronavirus. In certain embodiments a STING agonist is administered to a subject at risk of infection. In other embodiments a STING agonist and an antigen are administered to a subject at risk of infection.
In certain embodiments a STING agonist is administered to a subject at risk of infection. In other embodiments a STING agonist and an antigen are administered to a subject at risk of infection.
The subject at risk of infection may be a diabetic who has experienced an abrasion or injury.
The subject at risk of infection may be an individual expected to undergo hospital treatment, suitably the pathogen being Staphylococcus aureus.
The pathogen presenting a risk to the subject at risk of infection may be a pathogen causing a pandemic, such as an influenza virus or a coronavirus.
The subject at risk of infection may be an immunocompromised subject, i.e. a subject having impaired immunity relative to a healthy individual. The subject may be immunocompromised due to e.g. a medical disorder or as a result of treatment for a medical disorder.
Administrations
In certain embodiments a STING agonist is administered in step (i) and a STING agonist and an antigen are administered in step (ii).
Step (i)
The STING agonist may be administered intradermally, intramuscularly, intraperitoneally or subcutaneously. Suitably the STING agonist is administered intramuscularly.
An administration according to step (i) will comprise delivery of the STING agonist. In one embodiment, only one administration is performed. Multiple administrations may alternatively be performed. If so, each administration will comprise a delivery of a STING agonist. Suitably 1 to 5, especially 1 to 4 or in particular 1 to 3 administrations are performed. Suitably, at least 2 administrations are performed. Most suitably, 2 administrations are performed.
If only one administration is performed, then step (i) is completed at the end of the one administration. If multiple administrations are performed, then step (i) is completed at the end of the last administration. The start of step (ii) is when either the STING agonist or the antigen of step (ii) have been administered.
The STING agonist may be provided in liquid or dry form. The preferred form will depend on the precise nature of the STING agonist and any associated carrier, e.g. if capable of reconstitution from dry form, and any other components present. The STING agonist is typically provided in liquid form. Compositions comprising the STING agonist intended for combination with other compositions prior to administration need not themselves have a physiologically acceptable pH or a physiologically acceptable tonicity; a formulation intended for administration should have a physiologically acceptable pH and should have a physiologically acceptable osmolality.
The pH of a liquid preparation is adjusted in view of the components of the composition and necessary suitability for administration to the human subject. The pH of a formulation is generally at least 4, especially at least 5, in particular at least 5.5 such as at least 6. The pH of a formulation is generally 9 or less, especially 8.5 or less, in particular 8 or less, such as 7.5 or less. The pH of a formulation may be 4 to 9, especially 5 to 8.5, in particular 5.5 to 8, such as 6.5 to 7.4 (e.g. 6.5 to 7.1).
For parenteral administration, solutions should have a physiologically acceptable osmolality to avoid excessive cell distortion or lysis. A physiologically acceptable osmolality will generally mean that solutions will have an osmolality which is approximately isotonic or mildly hypertonic. Suitably the formulations for administration will have an osmolality of 250 to 750 mOsm/kg, especially 250 to 550 mOsm/kg, in particular 270 to 500 mOsm/kg, such as 270 to 400 mOsm/kg. Osmolality may be measured according to techniques known in the art, such as by the use of a commercially available osmometer, for example the Advanced® Model 2020 available from Advanced Instruments Inc. (USA).
Liquids used for reconstitution will be substantially aqueous, such as water for injection, phosphate buffered saline and the like. As mentioned above, the requirement for buffer and/or tonicity modifying agents will depend on the on both the contents of the container being reconstituted and the subsequent use of the reconstituted contents. Buffers may be selected from acetate, citrate, histidine, maleate, phosphate, succinate, tartrate and TRIS. The buffer may be a phosphate buffer such as Na/Na2PC>4, Na/K2PO4 or K/K2PO4.
It will be appreciated that some of the components used may form salts under appropriate conditions, therefore such components may be present as a salt, in particular a pharmaceutically acceptable salt.
Suitably, the formulations used in the present invention have a dose volume per administration of between 0.05 ml and 1 ml, such as between 0.1 and 0.6 ml, in particular a dose volume of 0.45 to 0.55 ml, such as 0.5 ml. The volumes of the compositions used may depend on the subject, delivery route and location, with smaller doses being given by the intradermal route or if the STING agonist are delivered separately to the same location. A typical human dose for administration through routes such as intramuscular, is in the region of 200 ul to 750 ml, such as 400 to 600 ul, in particular about 500 ul, such as 500 ul.
If two liquids are intended to be combined, the volume of each liquid may be the same or different. Volumes for combination will typically be in the range of 10:1 to 1:10, such as 2:1 to 1 :2. Suitably the volume of each liquid will be substantially the same, such as the same. The STING agonist may be prepared as a concentrate with the expectation of dilution by a liquid containing composition prior to administration. For example, the STING agonist may be prepared at double-strength with the expectation of dilution by an equal volume of other composition prior to administration.
The STING agonist may be provided in the form of various physical containers such as vials or pre-filled syringes.
It is common where liquids are to be transferred between containers, such as from a vial to a syringe, to provide ‘an overage’ which ensures that the full volume required can be conveniently transferred. The level of overage required will depend on the circumstances but excessive overage should be avoided to reduce wastage and insufficient overage may cause practical difficulties. Overages may be of the order of 20 to 100 ul per dose, such as 30 ul or 50 ul. For example, a typical 5 dose container of doubly concentrated STING agonist (500 ul per dose) may contain around 2.6 to 3 ml of STING agonist.
Stabilisers may be present. Stabilisers may be of particular relevance where multidose containers are provided as doses of the final formulation(s) may be administered to subjects over a period of time.
The STING agonist in liquid form may be provided in the form of pre-filled syringe. Formulations are preferably sterile.
Step (ii)
For a given administration of STING agonist and the antigen, the STING agonist and the antigen may be delivered in co-formulation or in separate formulations. If delivered in separate formulations, the STING agonist and the antigen may be delivered simultaneously or at different times. If delivered in separate formulations, the STING agonist and the antigen may be delivered to the same location or to different locations.
When administered as separate formulations, the STING agonist and antigen are administered to locations with sufficient spatial proximity such that the adjuvant effect of the STING agonist is adequately maintained. For example, spatial proximity is sufficient to maintain at least 50%, especially at least 75% and in particular at least 90% of the adjuvant effect seen with administration to (essentially) the same location. The adjuvant effect seen with administration to the same location is defined as the level of increase observed as a result of administration of STING agonist and antigen to the same location compared with administration of antigen alone. The STING agonist and antigen are desirably administered to a location draining to the same lymph node, such as to the same limb, in particular to the same muscle.
In certain embodiments, the STING agonist and antigen are administered to the same location. The spatial separation of administration locations may be at least 5 mm, such as at least 1 cm. The spatial separation of administration locations may be less than 10 cm, such as less than 5 cm apart.
The STING agonist and antigen may be administered intradermally, intramuscularly, intraperitoneally or subcutaneously. The STING agonist and antigen may be administered via different routes. Suitably the STING agonist and antigen are administered via the same route, in particular intramuscularly. More suitably, the STING agonist and antigen are administered intramuscularly to the same muscle.
When administered as separate formulations, the STING agonist and antigen are administered with sufficient temporal proximity such that the adjuvant effect is adequately maintained. For example, temporal proximity is sufficient to maintain at least 50%, especially at least 75% and in particular at least 90% of the adjuvant effect seen with administration at the same time. The adjuvant effect seen with administration at the same time is defined as the level of increase observed as a result of administration of STING agonist and antigen at (essentially) the same time compared with administration of antigen without STING agonist.
When administered as separate formulations, the STING agonist and antigen may be administered within 84 hours, such as within 60 hours, especially within 36 hours, in particular within 24 hours, for example within 12 hours of each other. Suitably the STING agonist and antigen are administered within 6 hours, especially within 2 hours, in particular within 1 hour, such as within 30 minutes and especially within 15 minutes (e.g. within 5 minutes) of each other.
When administered as separate formulations, if the STING agonist and antigen are administered with a delay, the STING agonist may be administered first and the antigen administered second. Alternatively, the antigen is administered first and the STING agonist administered second. Desirably, the STING agonist and antigen are administered without intentional delay (accounting for the practicalities of multiple administrations).
An administration according to step (ii) will comprise delivery of the STING agonist and antigen separately or together (e.g. in co-formulation). In one embodiment, only one administration is performed. Multiple administrations may alternatively be performed. If so, each administration will comprise a delivery of a STING agonist and a delivery of the antigen. Suitably 1 to 5, especially 1 to 4 or in particular 1 to 3 administrations are performed. Suitably, at least 2 administrations are performed. Most suitably, 2 administrations are performed.
The start of an administration is when either the STING agonist or the antigen has been administered. The completion of an administration is when both the STING agonist and the antigen have been administered. The start of a further administration is when either the STING agonist or the antigen has been administered again. If multiple administrations are performed, each administration may be delivered after a suitable period of time after completion of the previous administration. Suitably, the period between administrations to a human subject is 2 weeks to 12 months, especially 2 weeks to 6 months, in particular 2 weeks to 3 months, such as 2 weeks to 2 months, for example 3 to 5 weeks.
If only one administration is performed, then step (ii) is completed at the end of the one administration. If multiple administrations are performed, then step (ii) is completed at the end of the last administration.
Before administration, the antigen may be provided in liquid or dry (e.g. lyophilised) form. The preferred form will depend on factors such as the precise nature of the antigen, e.g. if the antigen is amenable to drying, or other components which may be present, the antigen is typically provided in liquid form.
The STING agonist may be provided in liquid or dry form. The preferred form will depend on the precise nature of the STING agonist and any associated carrier, e.g. if capable of reconstitution from dry form, and any other components present. The STING agonist is typically provided in liquid form.
Compositions comprising the STING agonist or the antigen intended for combination with other compositions prior to administration need not themselves have a physiologically acceptable pH or a physiologically acceptable tonicity; a formulation intended for administration should have a physiologically acceptable pH and should have a physiologically acceptable osmolality.
The pH of a liquid preparation is adjusted in view of the components of the composition and necessary suitability for administration to the human subject. The pH of a formulation is generally at least 4, especially at least 5, in particular at least 5.5 such as at least 6. The pH of a formulation is generally 9 or less, especially 8.5 or less, in particular 8 or less, such as 7.5 or less. The pH of a formulation may be 4 to 9, especially 5 to 8.5, in particular 5.5 to 8, such as 6.5 to 7.4 (e.g. 6.5 to 7.1).
For parenteral administration, solutions should have a physiologically acceptable osmolality to avoid excessive cell distortion or lysis. A physiologically acceptable osmolality will generally mean that solutions will have an osmolality which is approximately isotonic or mildly hypertonic. Suitably the formulations for administration will have an osmolality of 250 to 750 mOsm/kg, especially 250 to 550 mOsm/kg, in particular 270 to 500 mOsm/kg, such as 270 to 400 mOsm/kg. Osmolality may be measured according to techniques known in the art, such as by the use of a commercially available osmometer, for example the Advanced® Model 2020 available from Advanced Instruments Inc. (USA).
Liquids used for reconstitution will be substantially aqueous, such as water for injection, phosphate buffered saline and the like. As mentioned above, the requirement for buffer and/or tonicity modifying agents will depend on the on both the contents of the container being reconstituted and the subsequent use of the reconstituted contents. Buffers may be selected from acetate, citrate, histidine, maleate, phosphate, succinate, tartrate and TRIS. The buffer may be a phosphate buffer such as Na/Na2PC>4, Na/K2PO4 or K/K2PO4.
It will be appreciated that some of the components used may form salts under appropriate conditions, therefore such components may be present as a salt, in particular a pharmaceutically acceptable salt.
Suitably, the formulations used in the present invention have a dose volume of between 0.05 ml and 1 ml, such as between 0.1 and 0.6 ml, in particular a dose volume of 0.45 to 0.55 ml, such as 0.5 ml. The volumes of the compositions used may depend on the subject, delivery route and location, with smaller doses being given by the intradermal route or if the STING agonist and antigen are delivered separately to the same location. A typical human dose for administration through routes such as intramuscular, is in the region of 200 ul to 750 ml, such as 400 to 600 ul, in particular about 500 ul, such as 500 ul.
If two liquids are intended to be combined, for example for co-formulation if the STING agonist is in liquid form and the antigen is in liquid form, the volume of each liquid may be the same or different. Volumes for combination will typically be in the range of 10:1 to 1:10, such as 2:1 to 1:2. Suitably the volume of each liquid will be substantially the same, such as the same. For example a 250 ul volume of STING agonist composition in liquid form may be combined with a 250 ul volume antigen composition in liquid form to provide a co-formulation dose with a 500 ul volume, each of the STING agonist and antigen being diluted 2-fold during the combination.
The STING agonist may be prepared as a concentrate with the expectation of dilution by a liquid antigen containing composition prior to administration. For example, the STING agonist may be prepared at double-strength with the expectation of dilution by an equal volume of antigen containing composition prior to administration.
The STING agonist and antigen, whether intended for co-formulation or separate formulation, may be provided in the form of various physical containers such as vials or prefilled syringes.
It is common where liquids are to be transferred between containers, such as from a vial to a syringe, to provide ‘an overage’ which ensures that the full volume required can be conveniently transferred. The level of overage required will depend on the circumstances but excessive overage should be avoided to reduce wastage and insufficient overage may cause practical difficulties. Overages may be of the order of 20 to 100 ul per dose, such as 30 ul or 50 ul. For example, a typical 10 dose container of doubly concentrated adjuvant (250 ul per dose) may contain around 2.85 to 3.25 ml of adjuvant.
Stabilisers may be present. Stabilisers may be of particular relevance where multidose containers are provided as doses of the final formulation(s) may be administered to subjects over a period of time. The STING agonist and antigen in liquid form may be provided in the form of a multichamber syringe. The use of multi-chamber syringes provides a convenient method for the separate sequential administration of the STING agonist and antigen. Multi-chamber syringes may be configured to provide concurrent but separate delivery of the STING agonist and antigen, or they may be configured to provide sequential delivery (in either order).
In other configurations of multichambered syringes, the antigen may be provided in dry form (e.g., freeze-dried) in one chamber and reconstituted by the STING agonist contained in the other chamber before administration.
Examples of multi-chamber syringes may be found in disclosures such as WO2016/172396, although a range of other configurations are possible.
Formulations are preferably sterile.
Conveniently, the STING agonist and the antigen are administered as a co-formulation.
Steps (i) and (ii)
Step (ii) is initiated after an interval from completion of step (i). The interval is sufficient to allow the immune system of the subject to have mounted an appropriate immune response to the administration of step (i). The interval is desirably short enough such that the immune response to the administration of step (i) does not excessively wane before the administration of step (ii). Suitably, step (ii) is initiated at an interval of 2 weeks to 2 years, especially 2 weeks to 1 year, in particular 3 weeks to 9 months, such as 4 weeks to 6 months from completion of step (i).
In some embodiments step (ii) is initiated at an interval of 2 weeks to 2 months from completion of step (i). In some embodiments step (ii) is initiated at an interval of 1 month to 3 months from completion of step (i). In some embodiments step (ii) is initiated at an interval of 2 months to 4 months from completion of step (i). In some embodiments step (ii) is initiated at an interval of 3 months to 6 months from completion of step (i). In some embodiments step (ii) is initiated at an interval of 4 months to 12 months from completion of step (i).
The combined duration of step (i), step (ii) and the interval between the steps is suitably 1 month to 2 years, especially 6 weeks months to 12 months, in particular 2 months to 10 months, such as 10 weeks to 9 months.
Alternatively, the combined duration of step (i), step (ii) and the interval between the steps is suitably at least 2 months, especially at least 3 months, in particular at least 4 months, such as at least 5 months, especially at least 6 months, in particular at least 7 months.
Alternatively, the combined duration of step (i), step (ii) and the interval between the steps is suitably 5 years or less, especially 3 years or less, in particular 2 years or less, such as 18 months or less, especially 12 months or less, in particular 10 months or less. Immune responses The methods and uses of the invention elicit an immune response in the subject against an antigen. The elicited immune response may be an antigen specific B cell response which produces neutralizing antibodies. The elicited immune response may be an antigen specific T cell response, which may be a systemic and/or a local response. The antigen specific T cell response may comprise a CD4+ T cell response, such as a response involving CD4+ T cells expressing a plurality of cytokines, e.g. IFNgamma, TNFalpha and/or IL2. Alternatively, or additionally, the antigen specific T cell response comprises a CD8+ T cell response, such as a response involving CD8+ T cells expressing a plurality of cytokines, e.g., IFNgamma, TNFalpha and/or IL2.
An immune response may be characterised by various parameters.
In one embodiment, the immune response is characterised by the level of response, suitably as indicated by the serum titer of IgG antibodies discussed above which bind and/or are specific to the antigen. In particular, the antibodies are serum neutralising antibodies against the antigen. Suitably the level of the immune response is increased by at least 25%, especially at least 50%, in particular at least 100% (twofold), such as 400% (fivefold), for example 900% (tenfold). The increase in the level of the immune response is relative to the level of immune response achieved wherein step (ii) alone is carried out, without carrying out preceding step (i) of the invention.
In a further embodiment, the immune response is characterised by the breadth of response, suitably as indicated by the antibodies discussed above binding to (a) an increased range of variants of the antigen (such as variants of the antigen from an increased number of strains of the pathogen) or (b) elicit a response in a wider variety of human HLA types).
In a further embodiment, the immune response is characterised by the speed of response, i.e. the time taken to achieve a certain threshold of immune response, such as a threshold level associated with a protective immune response.
In a further embodiment, the immune response is characterised by the duration of immune response, suitably as indicated by the period of time over which the subject maintains a serum titer of antibodies as detailed discussed above binding and/or specific to the antigen. Suitably the duration of the immune response is increased by at least 1 year, especially at least 3 years. Alternatively, or in addition, the immune response is maintained for at least 1 year or especially 3 years.
In a further embodiment, there is provided a method of reducing the variability of the immune response to an antigen in subjects. Suitably, the standard deviation of the level, breadth, speed and/or duration of response between subjects is decreased. Alternatively, the proportion of subjects meeting a specified threshold of immune response to the antigen is increased. The specified threshold may be that which indicates protective efficacy depending on the pathogen to which an immune response is being elicited. In one embodiment there is provided a method of improving an immune response to an antigen in a subject. The immune response may be improved as indicated by the level, breadth or duration of the immune response as set out above.
There is also provided a method of prophylaxis of infection by a pathogen in a subject. ‘Prophylaxis’ as used herein pertains to an action taken in advance of a subject being infected. A subject may be at risk of infection by a pathogen. Suitably, the prophylaxis reduces the likelihood of established infection by the pathogen.
More suitably, prophylaxis prevents infection e.g. reducing partially or completely the severity of one or more symptoms and/or time over which one or more symptoms are experienced by a subject, reducing the likelihood of developing an established infection after challenge and/or slowing progression of an associated illness (e.g. extending survival).
The term ‘preparing the immune system’ includes modification of the innate cell repertoire, including at a genetic level, thereby influencing the immune response resulting from step (ii).
Methods of reducing the amount of an antigen required to elicit a desired immune response are suitably reducing the amount of antigen required to elicit a desired immune response relative to the amount required to elicit the desired immune response without performing step (i).
The present invention also provides kits of parts, comprising the components utilised in the methods described herein, such as a kit of parts comprising (i) a STING agonist; (ii) a first antigen; and (iii) a second antigen. Each of (i), (ii) and (iii) may be provided in separate containers, or (i) and (ii) may optionally be provided together.
In certain embodiments a STING agonist and a first antigen are administered in step (i) and a STING agonist and a second antigen are administered in step (ii).
Step (i)
For a given administration of STING agonist and the first antigen, the STING agonist and the first antigen may be delivered in co-formulation or in separate formulations. If delivered in separate formulations, the STING agonist and the first antigen may be delivered simultaneously or at different times. If delivered in separate formulations, the STING agonist and the first antigen may be delivered to the same location or to different locations.
When administered as separate formulations, the STING agonist and first antigen are administered to locations with sufficient spatial proximity such that the adjuvant effect of the STING agonist is adequately maintained. For example, spatial proximity is sufficient to maintain at least 50%, especially at least 75% and in particular at least 90% of the adjuvant effect seen with administration at (essentially) the same location. The adjuvant effect seen with administration to the same location is defined as the level of increase observed as a result of administration of STING agonist and first antigen to the same location compared with administration of antigen alone. The STING agonist and antigen are desirably administered to a location draining to the same lymph node, such as to the same limb, in particular to the same muscle.
In certain embodiments, the STING agonist and first antigen are administered to the same location. The spatial separation of administration locations may be at least 5 mm, such as at least 1 cm. The spatial separation of administration locations may be less than 10 cm, such as less than 5 cm apart.
The STING agonist and first antigen may be administered intradermally, intramuscularly, intraperitoneally or subcutaneously. The STING agonist and first antigen may be administered via different routes. Suitably the STING agonist and antigen are administered via the same route, in particular intramuscularly. More suitably, the STING agonist and first antigen are administered intramuscularly to the same muscle.
When administered as separate formulations, the STING agonist and first antigen are administered with sufficient temporal proximity such that the adjuvant effect is adequately maintained. For example, temporal proximity is sufficient to maintain at least 50%, especially at least 75% and in particular at least 90% of the adjuvant effect seen with administration at the same time. The adjuvant effect seen with administration at the same time is defined as the level of increase observed as a result of administration of STING agonist and first antigen at (essentially) the same time compared with administration of first antigen without STING agonist.
When administered as separate formulations, the STING agonist and first antigen may be administered within 84 hours, such as within 60 hours, especially within 36 hours, in particular within 24 hours, for example within 12 hours of each other. Suitably the STING agonist and antigen are administered within 6 hours, especially within 2 hours, in particular within 1 hour, such as within 30 minutes and especially within 15 minutes (e.g. within 5 minutes) of each other. When administered as separate formulations, if the STING agonist and first antigen are administered with a delay, the STING agonist may be administered first and the antigen administered second. Alternatively, the antigen is administered first and the STING agonist administered second. Desirably, the STING agonist and antigen are administered without intentional delay (accounting for the practicalities of multiple administrations).
An administration according to step (i) will comprise delivery of the STING agonist and first antigen separately or together (e.g. in co-formulation). In one embodiment, only one administration is performed. Multiple administrations may alternatively be performed. If so, each administration will comprise a delivery of a STING agonist and a delivery of the first antigen. Suitably 1 to 5, especially 1 to 4 or in particular 1 to 3 administrations are performed. Suitably, at least 2 administrations are performed. Most suitably, 2 administrations are performed.
The start of an administration is when either the STING agonist or the first antigen has been administered. The completion of an administration is when both the STING agonist and the first antigen have been administered. The start of a further administration is when either the STING agonist or the first antigen has been administered again. If multiple administrations are performed, each administration may be delivered after a suitable period of time after completion of the previous administration. Suitably, the period between administrations to a human subject is 2 weeks to 12 months, especially 2 weeks to 6 months, in particular 2 weeks to 3 months, such as 2 weeks to 2 months, for example 3 to 5 weeks.
If only one administration is performed, then step (i) is completed at the end of the one administration. If multiple administrations are performed, then step (i) is completed at the end of the last administration. The start of step (ii) is when either the STING agonist or the second antigen of step (ii) have been administered.
Before administration, the first antigen may be provided in liquid or dry (e.g. lyophilised) form. The preferred form will depend on factors such as the precise nature of the antigen, e.g. if the antigen is amenable to drying, or other components which may be present. The first antigen is typically provided in liquid form.
The STING agonist may be provided in liquid or dry form. The preferred form will depend on the precise nature of the STING agonist and any associated carrier, e.g. if capable of reconstitution from dry form, and any other components present. The STING agonist is typically provided in liquid form.
Compositions comprising the STING agonist or the first antigen intended for combination with other compositions prior to administration need not themselves have a physiologically acceptable pH or a physiologically acceptable tonicity; a formulation intended for administration should have a physiologically acceptable pH and should have a physiologically acceptable osmolality.
The pH of a liquid preparation is adjusted in view of the components of the composition and necessary suitability for administration to the human subject. The pH of a formulation is generally at least 4, especially at least 5, in particular at least 5.5 such as at least 6. The pH of a formulation is generally 9 or less, especially 8.5 or less, in particular 8 or less, such as 7.5 or less. The pH of a formulation may be 4 to 9, especially 5 to 8.5, in particular 5.5 to 8, such as 6.5 to 7.4 (e.g. 6.5 to 7.1).
For parenteral administration, solutions should have a physiologically acceptable osmolality to avoid excessive cell distortion or lysis. A physiologically acceptable osmolality will generally mean that solutions will have an osmolality which is approximately isotonic or mildly hypertonic. Suitably the formulations for administration will have an osmolality of 250 to 750 mOsm/kg, especially 250 to 550 mOsm/kg, in particular 270 to 500 mOsm/kg, such as 270 to 400 mOsm/kg. Osmolality may be measured according to techniques known in the art, such as by the use of a commercially available osmometer, for example the Advanced® Model 2020 available from Advanced Instruments Inc. (USA).
Liquids used for reconstitution will be substantially aqueous, such as water for injection, phosphate buffered saline and the like. As mentioned above, the requirement for buffer and/or tonicity modifying agents will depend on the on both the contents of the container being reconstituted and the subsequent use of the reconstituted contents. Buffers may be selected from acetate, citrate, histidine, maleate, phosphate, succinate, tartrate and TRIS. The buffer may be a phosphate buffer such as Na/Na2PO4, Na/K2PO4 or K/K2PO4.
It will be appreciated that some of the components used may form salts under appropriate conditions, therefore such components may be present as a salt, in particular a pharmaceutically acceptable salt.
Suitably, the formulations used in the present invention have a dose volume per administration of between 0.05 ml and 1 ml, such as between 0.1 and 0.6 ml, in particular a dose volume of 0.45 to 0.55 ml, such as 0.5 ml. The volumes of the compositions used may depend on the subject, delivery route and location, with smaller doses being given by the intradermal route or if the STING agonist and antigen are delivered separately to the same location. A typical human dose for administration through routes such as intramuscular, is in the region of 200 ul to 750 ml, such as 400 to 600 ul, in particular about 500 ul, such as 500 ul.
If two liquids are intended to be combined, for example for co-formulation if the STING agonist is in liquid form and the antigen is in liquid form, the volume of each liquid may be the same or different. Volumes for combination will typically be in the range of 10:1 to 1:10, such as 2:1 to 1:2. Suitably the volume of each liquid will be substantially the same, such as the same. For example a 250 ul volume of STING agonist in liquid form may be combined with a 250 ul volume antigen composition in liquid form to provide a co-formulation dose with a 500 ul volume, each of the STING agonist and antigen being diluted 2-fold during the combination.
The STING agonist may be prepared as a concentrate with the expectation of dilution by a liquid antigen containing composition prior to administration. For example, the STING agonist may be prepared at double-strength with the expectation of dilution by an equal volume of antigen containing composition prior to administration.
The first antigen may be prepared as a concentrate with the expectation of dilution by a liquid STING agonist containing composition prior to administration. For example, the first antigen may be prepared at double-strength with the expectation of dilution by an equal volume of STING agonist containing composition prior to administration. The STING agonist and first antigen, whether intended for co-formulation or separate formulation, may be provided in the form of various physical containers such as vials or prefilled syringes.
It is common where liquids are to be transferred between containers, such as from a vial to a syringe, to provide ‘an overage’ which ensures that the full volume required can be conveniently transferred. The level of overage required will depend on the circumstances but excessive overage should be avoided to reduce wastage and insufficient overage may cause practical difficulties. Overages may be of the order of 20 to 100 ul per dose, such as 30 ul or 50 ul. For example, a typical 10 dose container of doubly concentrated STING agonist (250 ul per dose) may contain around 2.85 to 3.25 ml of STING agonist.
Stabilisers may be present. Stabilisers may be of particular relevance where multidose containers are provided as doses of the final formulation(s) may be administered to subjects over a period of time.
The STING agonist and first antigen in liquid form may be provided in the form of a multichamber syringe. The use of multi-chamber syringes provides a convenient method for the separate sequential administration of the STING agonist and antigen. Multi-chamber syringes may be configured to provide concurrent but separate delivery of the STING agonist and first antigen, or they may be configured to provide sequential delivery (in either order).
In other configurations of multichambered syringes, the antigen may be provided in dry form (e.g., freeze-dried) in one chamber and reconstituted by the STING agonist contained in the other chamber before administration.
Examples of multi-chamber syringes may be found in disclosures such as WO2016/172396, although a range of other configurations are possible.
Formulations are preferably sterile.
Conveniently, the STING agonist and the first antigen are administered as a coformulation.
Step (ii)
For a given administration of STING agonist and the second antigen, the STING agonist and the second antigen may be delivered in co-formulation or in separate formulations. If delivered in separate formulations, the STING agonist and the second antigen may be delivered simultaneously or at different times. If delivered in separate formulations, the STING agonist and the second antigen may be delivered to the same location or to different locations.
When administered as separate formulations, the STING agonist and second antigen are administered to locations with sufficient spatial proximity such that the adjuvant effect of the STING agonist is adequately maintained. For example, spatial proximity is sufficient to maintain at least 50%, especially at least 75% and in particular at least 90% of the adjuvant effect seen with administration to (essentially) the same location. The adjuvant effect seen with administration to the same location is defined as the level of increase observed as a result of administration of STING agonist and second antigen to the same location compared with administration of antigen alone. The STING agonist and antigen are desirably administered to a location draining to the same lymph node, such as to the same limb, in particular to the same muscle.
In certain embodiments, the STING agonist and second antigen are administered to the same location. The spatial separation of administration locations may be at least 5 mm, such as at least 1 cm. The spatial separation of administration locations may be less than 10 cm, such as less than 5 cm apart.
The STING agonist and second antigen may be administered intradermally, intramuscularly, intraperitoneally or subcutaneously. The STING agonist and second antigen may be administered via different routes. Suitably the STING agonist and antigen are administered via the same route, in particular intramuscularly. More suitably, the STING agonist and second antigen are administered intramuscularly to the same muscle.
When administered as separate formulations, the STING agonist and second antigen are administered with sufficient temporal proximity such that the adjuvant effect is adequately maintained. For example, temporal proximity is sufficient to maintain at least 50%, especially at least 75% and in particular at least 90% of the adjuvant effect seen with administration at the same time. The adjuvant effect seen with administration at the same time is defined as the level of increase observed as a result of administration of STING agonist and second antigen at (essentially) the same time compared with administration of second antigen without STING agonist.
When administered as separate formulations, the STING agonist and second antigen may be administered within 84 hours, such as within 60 hours, especially within 36 hours, in particular within 24 hours, for example within 12 hours of each other. Suitably the STING agonist and antigen are administered within 6 hours, especially within 2 hours, in particular within 1 hour, such as within 30 minutes and especially within 15 minutes (e.g. within 5 minutes) of each other.
When administered as separate formulations, if the STING agonist and second antigen are administered with a delay, the STING agonist may be administered first and the antigen administered second. Alternatively, the antigen is administered first and the STING agonist administered second. Desirably, the STING agonist and antigen are administered without intentional delay (accounting for the practicalities of multiple administrations).
An administration according to step (ii) will comprise delivery of the STING agonist and second antigen separately or together (e.g. in co-formulation). In one embodiment, only one administration is performed. Multiple administrations may alternatively be performed. If so, each administration will comprise a delivery of a STING agonist and a delivery of the second antigen. Suitably 1 to 5, especially 1 to 4 or in particular 1 to 3 administrations are performed. Suitably, at least 2 administrations are performed. Most suitably, 2 administrations are performed.
The start of an administration is when either the STING agonist or the second antigen has been administered. The completion of an administration is when both the STING agonist and the second antigen have been administered. The start of a further administration is when either the STING agonist or the second antigen has been administered again. If multiple administrations are performed, each administration may be delivered after a suitable period of time after completion of the previous administration. Suitably, the period between administrations to a human subject is 2 weeks to 12 months, especially 2 weeks to 6 months, in particular 2 weeks to 3 months, such as 2 weeks to 2 months, for example 3 to 5 weeks.
If only one administration is performed, then step (ii) is completed at the end of the one administration. If multiple administrations are performed, then step (ii) is completed at the end of the last administration.
Before administration, the second antigen may be provided in liquid or dry (e.g. lyophilised) form. The preferred form will depend on factors such as the precise nature of the antigen, e.g. if the antigen is amenable to drying, or other components which may be present. The second antigen is typically provided in liquid form.
The STING agonist may be provided in liquid or dry form. The preferred form will depend on the precise nature of the STING agonist and any associated carrier, e.g. if capable of reconstitution from dry form, and any other components present. The STING agonist is typically provided in liquid form.
Compositions comprising the STING agonist or the second antigen intended for combination with other compositions prior to administration need not themselves have a physiologically acceptable pH or a physiologically acceptable tonicity; a formulation intended for administration should have a physiologically acceptable pH and should have a physiologically acceptable osmolality.
The pH of a liquid preparation is adjusted in view of the components of the composition and necessary suitability for administration to the human subject. The pH of a formulation is generally at least 4, especially at least 5, in particular at least 5.5 such as at least 6. The pH of a formulation is generally 9 or less, especially 8.5 or less, in particular 8 or less, such as 7.5 or less. The pH of a formulation may be 4 to 9, especially 5 to 8.5, in particular 5.5 to 8, such as 6.5 to 7.4 (e.g. 6.5 to 7.1).
For parenteral administration, solutions should have a physiologically acceptable osmolality to avoid excessive cell distortion or lysis. A physiologically acceptable osmolality will generally mean that solutions will have an osmolality which is approximately isotonic or mildly hypertonic. Suitably the formulations for administration will have an osmolality of 250 to 750 mOsm/kg, especially 250 to 550 mOsm/kg, in particular 270 to 500 mOsm/kg, such as 270 to 400 mOsm/kg. Osmolality may be measured according to techniques known in the art, such as by the use of a commercially available osmometer, for example the Advanced® Model 2020 available from Advanced Instruments Inc. (USA). Liquids used for reconstitution will be substantially aqueous, such as water for injection, phosphate buffered saline and the like. As mentioned above, the requirement for buffer and/or tonicity modifying agents will depend on the on both the contents of the container being reconstituted and the subsequent use of the reconstituted contents. Buffers may be selected from acetate, citrate, histidine, maleate, phosphate, succinate, tartrate and TRIS. The buffer may be a phosphate buffer such as Na/Na2PO4, Na/K2PO4 or K/K2PO4. It will be appreciated that some of the components used may form salts under appropriate conditions, therefore such components may be present as a salt, in particular a pharmaceutically acceptable salt. Suitably, the formulations used in the present invention have a dose volume of between 0.05 ml and 1 ml, such as between 0.1 and 0.6 ml, in particular a dose volume of 0.45 to 0.55 ml, such as 0.5 ml. The volumes of the compositions used may depend on the subject, delivery route and location, with smaller doses being given by the intradermal route or if the STING agonist and antigen are delivered separately to the same location. A typical human dose for administration through routes such as intramuscular, is in the region of 200 ul to 750 ml, such as 400 to 600 ul, in particular about 500 ul, such as 500 ul. If two liquids are intended to be combined, for example for co-formulation if the STING agonist is in liquid form and the antigen is in liquid form, the volume of each liquid may be the same or different. Volumes for combination will typically be in the range of 10:1 to 1:10, such as 2:1 to 1:2. Suitably the volume of each liquid will be substantially the same, such as the same. For example a 250 ul volume of STING agonist composition in liquid form may be combined with a 250 ul volume antigen composition in liquid form to provide a co-formulation dose with a 500 ul volume, each of the STING agonist and antigen being diluted 2-fold during the combination. The STING agonist may be prepared as a concentrate with the expectation of dilution by a liquid antigen containing composition prior to administration. For example, the STING agonist may be prepared at double-strength with the expectation of dilution by an equal volume of antigen containing composition prior to administration. The second antigen may be prepared as a concentrate with the expectation of dilution by a liquid STING agonist containing composition prior to administration. For example, the second antigen may be prepared at double-strength with the expectation of dilution by an equal volume of STING agonist containing composition prior to administration. The STING agonist and second antigen, whether intended for co-formulation or separate formulation, may be provided in the form of various physical containers such as vials or pre-filled syringes.
It is common where liquids are to be transferred between containers, such as from a vial to a syringe, to provide ‘an overage’ which ensures that the full volume required can be conveniently transferred. The level of overage required will depend on the circumstances but excessive overage should be avoided to reduce wastage and insufficient overage may cause practical difficulties. Overages may be of the order of 20 to 100 ul per dose, such as 30 ul or 50 ul. For example, a typical 10 dose container of doubly concentrated adjuvant (250 ul per dose) may contain around 2.85 to 3.25 ml of adjuvant.
Stabilisers may be present. Stabilisers may be of particular relevance where multidose containers are provided as doses of the final formulation(s) may be administered to subjects over a period of time.
The STING agonist and second antigen in liquid form may be provided in the form of a multichamber syringe. The use of multi-chamber syringes provides a convenient method for the separate sequential administration of the STING agonist and antigen. Multi-chamber syringes may be configured to provide concurrent but separate delivery of the STING agonist and antigen, or they may be configured to provide sequential delivery (in either order).
In other configurations of multichambered syringes, the antigen may be provided in dry form (e.g., freeze-dried) in one chamber and reconstituted by the STING agonist contained in the other chamber before administration.
Examples of multi-chamber syringes may be found in disclosures such as WO2016/172396, although a range of other configurations are possible.
Formulations are preferably sterile.
Conveniently, the STING agonist and the second antigen are administered as a coformulation.
Steps (i) and (ii)
Step (ii) is initiated after an interval from completion of step (i). The interval is sufficient to allow the immune system of the subject to have mounted an appropriate immune response to the administration of step (i). The interval is desirably short enough such that the immune response to the administration of step (i) does not excessively wane before the administration of step (ii). Suitably, step (ii) is initiated at an interval of 2 weeks to 2 years, especially 2 weeks to 1 year, in particular 3 weeks to 9 months, such as 4 weeks to 6 months from completion of step (i).
In some embodiments step (ii) is initiated at an interval of 2 weeks to 2 months from completion of step (i). In some embodiments step (ii) is initiated at an interval of 1 month to 3 months from completion of step (i). In some embodiments step (ii) is initiated at an interval of 2 months to 4 months from completion of step (i). In some embodiments step (ii) is initiated at an interval of 3 months to 6 months from completion of step (i). In some embodiments step (ii) is initiated at an interval of 4 months to 12 months from completion of step (i).
The combined duration of step (i), step (ii) and the interval between the steps is suitably 1 month to 2 years, especially 6 weeks months to 12 months, in particular 2 months to 10 months, such as 10 weeks to 9 months.
Alternatively, the combined duration of step (i), step (ii) and the interval between the steps is suitably at least 2 months, especially at least 3 months, in particular at least 4 months, such as at least 5 months, especially at least 6 months, in particular at least 7 months.
Alternatively, the combined duration of step (i), step (ii) and the interval between the steps is suitably 5 years or less, especially 3 years or less, in particular 2 years or less, such as 18 months or less, especially 12 months or less, in particular 10 months or less. Immune responses
The methods and uses of the invention elicit an immune response in the subject against a second antigen. The elicited immune response may be an antigen specific B cell response which produces neutralizing antibodies. The elicited immune response may be an antigen specific T cell response, which may be a systemic and/or a local response. The antigen specific T cell response may comprise a CD4+ T cell response, such as a response involving CD4+ T cells expressing a plurality of cytokines, e.g. IFNgamma, TNFalpha and/or IL2. Alternatively, or additionally, the antigen specific T cell response comprises a CD8+ T cell response, such as a response involving CD8+ T cells expressing a plurality of cytokines, e.g., IFNgamma, TNFalpha and/or IL2.
An immune response may be characterised by various parameters.
In one embodiment, the immune response is characterised by the level of response, suitably as indicated by the serum titer of IgG antibodies discussed above which bind and/or are specific to the second antigen. In particular, the antibodies are serum neutralising antibodies against the second antigen. Suitably the level of the immune response is increased by at least 25%, especially at least 50%, in particular at least 100% (twofold), such as 400% (fivefold), for example 900% (tenfold). The increase in the level of the immune response is relative to the level of immune response achieved wherein step (ii) alone is carried out, without carrying out preceding step (i) of the invention.
In a further embodiment, the immune response is characterised by the breadth of response, suitably as indicated by the antibodies discussed above binding to (a) an increased range of variants of the second antigen (such as variants of the second antigen from an increased number of strains of the pathogen) or (b) elicit a response in a wider variety of human HLA types). In a further embodiment, the immune response is characterised by the speed of response, i.e. the time taken to achieve a certain threshold of immune response, such as a threshold level associated with a protective immune response.
In a further embodiment, the immune response is characterised by the duration of immune response, suitably as indicated by the period of time over which the subject maintains a serum titer of antibodies as detailed discussed above binding and/or specific to the second antigen. Suitably the duration of the immune response is increased by at least 1 year, especially at least 3 years. Alternatively, or in addition, the immune response is maintained for at least 1 year or especially 3 years.
In a further embodiment, there is provided a method of reducing the variability of the immune response to a second antigen in subjects. Suitably, the standard deviation of the level, breadth, speed and/or duration of response between subjects is decreased. Alternatively, the proportion of subjects meeting a specified threshold of immune response to the second antigen is increased. The specified threshold may be that which indicates protective efficacy depending on the pathogen to which an immune response is being elicited.
In one embodiment there is provided a method of improving an immune response to a second antigen in a subject. The immune response may be improved as indicated by the level, breadth or duration of the immune response as set out above.
There is also provided a method of prophylaxis of infection by a pathogen in a subject. ‘Prophylaxis’ as used herein pertains to an action taken in advance of a subject being infected. A subject may be at risk of infection by a pathogen. Suitably, the prophylaxis reduces the likelihood of established infection by the pathogen.
More suitably, prophylaxis prevents infection e.g. reducing partially or completely the severity of one or more symptoms and/or time over which one or more symptoms are experienced by a subject, reducing the likelihood of developing an established infection after challenge and/or slowing progression of an associated illness (e.g. extending survival).
The term ‘preparing the immune system’ includes modification of the innate cell repertoire, including at a genetic level, thereby influencing the immune response resulting from step (ii).
Methods of reducing the amount of an antigen required to elicit a desired immune response are suitably reducing the amount of antigen required to elicit a desired immune response relative to the amount required to elicit the desired immune response without performing step (i).
The present invention also provides kits of parts, comprising the components utilised in the methods described herein, such as a kit of parts comprising (i) a STING agonist; (ii) a first antigen; and (iii) a second antigen. Each of (i), (ii) and (iii) may be provided in separate containers, or (i) and (ii) may optionally be provided together. In certain embodiments a STING agonist is administered in step (i) and an antigen and optionally a second adjuvant are administered in step (ii).
Step (i)
The STING agonist may be administered intradermally, intramuscularly, intraperitoneally or subcutaneously. Suitably the STING agonist is administered intramuscularly.
An administration according to step (i) will comprise delivery of the STING agonist. In one embodiment, only one administration is performed. Multiple administrations may alternatively be performed. If so, each administration will comprise a delivery of a STING agonist. Suitably 1 to 5, especially 1 to 4 or in particular 1 to 3 administrations are performed. Suitably, at least 2 administrations are performed. Most suitably, 2 administrations are performed.
Suitably, the period between administrations to a human subject is 2 weeks to 12 months, especially 2 weeks to 6 months, in particular 2 weeks to 3 months, such as 2 weeks to 2 months, for example 3 to 5 weeks.
If only one administration is performed, then step (i) is completed at the end of the one administration. If multiple administrations are performed, then step (i) is completed at the end of the last administration. The start of step (ii) is when either the second adjuvant or the antigen of step (ii) have been administered.
The STING agonist may be provided in liquid or dry form. The preferred form will depend on the precise nature of the STING agonist and any associated carrier, e.g. if capable of reconstitution from dry form, and any other components present. The STING agonist is typically provided in liquid form.
Compositions comprising the STING agonist intended for combination with other compositions prior to administration need not themselves have a physiologically acceptable pH or a physiologically acceptable tonicity; a formulation intended for administration should have a physiologically acceptable pH and should have a physiologically acceptable osmolality.
The pH of a liquid preparation is adjusted in view of the components of the composition and necessary suitability for administration to the human subject. The pH of a formulation is generally at least 4, especially at least 5, in particular at least 5.5 such as at least 6. The pH of a formulation is generally 9 or less, especially 8.5 or less, in particular 8 or less, such as 7.5 or less. The pH of a formulation may be 4 to 9, especially 5 to 8.5, in particular 5.5 to 8, such as 6.5 to 7.4 (e.g. 6.5 to 7.1).
For parenteral administration, solutions should have a physiologically acceptable osmolality to avoid excessive cell distortion or lysis. A physiologically acceptable osmolality will generally mean that solutions will have an osmolality which is approximately isotonic or mildly hypertonic. Suitably the formulations for administration will have an osmolality of 250 to 750 mOsm/kg, especially 250 to 550 mOsm/kg, in particular 270 to 500 mOsm/kg, such as 270 to 400 mOsm/kg. Osmolality may be measured according to techniques known in the art, such as by the use of a commercially available osmometer, for example the Advanced® Model 2020 available from Advanced Instruments Inc. (USA).
Liquids used for reconstitution will be substantially aqueous, such as water for injection, phosphate buffered saline and the like. As mentioned above, the requirement for buffer and/or tonicity modifying agents will depend on the on both the contents of the container being reconstituted and the subsequent use of the reconstituted contents. Buffers may be selected from acetate, citrate, histidine, maleate, phosphate, succinate, tartrate and TRIS. The buffer may be a phosphate buffer such as Na/Na2PO4, Na/K2PO4 or K/K2PO4.
It will be appreciated that some of the components used may form salts under appropriate conditions, therefore such components may be present as a salt, in particular a pharmaceutically acceptable salt.
Suitably, the formulations used in the present invention have a dose volume per administration of between 0.05 ml and 1 ml, such as between 0.1 and 0.6 ml, in particular a dose volume of 0.45 to 0.55 ml, such as 0.5 ml. The volumes of the compositions used may depend on the subject, delivery route and location, with smaller doses being given by the intradermal route. A typical human dose for administration through routes such as intramuscular, is in the region of 200 ul to 750 ml, such as 400 to 600 ul, in particular about 500 ul, such as 500 ul.
If two liquids are intended to be combined, the volume of each liquid may be the same or different. Volumes for combination will typically be in the range of 10:1 to 1:10, such as 2:1 to 1 :2. Suitably the volume of each liquid will be substantially the same, such as the same.
The STING agonist may be prepared as a concentrate with the expectation of dilution by a liquid containing composition prior to administration. For example, the STING agonist may be prepared at double-strength with the expectation of dilution by an equal volume of other composition prior to administration.
The STING agonist may be provided in the form of various physical containers such as vials or pre-filled syringes.
It is common where liquids are to be transferred between containers, such as from a vial to a syringe, to provide ‘an overage’ which ensures that the full volume required can be conveniently transferred. The level of overage required will depend on the circumstances but excessive overage should be avoided to reduce wastage and insufficient overage may cause practical difficulties. Overages may be of the order of 20 to 100 ul per dose, such as 30 ul or 50 ul. For example, a typical 10 dose container of doubly concentrated adjuvant (250 ul per dose) may contain around 2.85 to 3.25 ml of adjuvant. Stabilisers may be present. Stabilisers may be of particular relevance where multidose containers are provided as doses of the final formulation(s) may be administered to subjects over a period of time.
The STING agonist in liquid form may be provided in the form of a pre-filled syringe Formulations are preferably sterile.
Step (ii)
For a given administration of second adjuvant and the antigen, the second adjuvant and the antigen may be delivered in co-formulation or in separate formulations. If delivered in separate formulations, the second adjuvant and the antigen may be delivered simultaneously or at different times. If delivered in separate formulations, the second adjuvant and the antigen may be delivered to the same location or to different locations.
When administered as separate formulations, the second adjuvant and antigen are administered to locations with sufficient spatial proximity such that the adjuvant effect of the second adjuvant is adequately maintained. For example, spatial proximity is sufficient to maintain at least 50%, especially at least 75% and in particular at least 90% of the adjuvant effect seen with administration at to (essentially) the same location. The adjuvant effect seen with administration to the same location is defined as the level of increase observed as a result of administration of second adjuvant and antigen to the same location compared with administration of antigen alone. The second adjuvant and antigen are desirably administered to a location draining to the same lymph node, such as to the same limb, in particular to the same muscle.
In certain embodiments, the second adjuvant and antigen are administered to the same location. The spatial separation of administration locations may be at least 5 mm, such as at least 1 cm. The spatial separation of administration locations may be less than 10 cm, such as less than 5 cm apart.
The second adjuvant and antigen may be administered intradermally, intramuscularly, intraperitoneally or subcutaneously. The second adjuvant and antigen may be administered via different routes. Suitably the second adjuvant and antigen are administered via the same route, in particular intramuscularly. More suitably, the second adjuvant and antigen are administered intramuscularly to the same muscle.
When administered as separate formulations, the second adjuvant and antigen are administered with sufficient temporal proximity such that the adjuvant effect is adequately maintained. For example, temporal proximity is sufficient to maintain at least 50%, especially at least 75% and in particular at least 90% of the adjuvant effect seen with administration at the same time. The adjuvant effect seen with administration at the same time is defined as the level of increase observed as a result of administration of second adjuvant and antigen at (essentially) the same time compared with administration of antigen without second adjuvant. When administered as separate formulations, the second adjuvant and antigen may be administered within 84 hours, such as within 60 hours, especially within 36 hours, in particular within 24 hours, for example within 12 hours of each other. Suitably the second adjuvant and antigen are administered within 6 hours, especially within 2 hours, in particular within 1 hour, such as within 30 minutes and especially within 15 minutes (e.g. within 5 minutes) of each other.
When administered as separate formulations, if the second adjuvant and antigen are administered with a delay, the second adjuvant may be administered first and the antigen administered second. Alternatively, the antigen is administered first and the second adjuvant administered second. Desirably, the second adjuvant and antigen are administered without intentional delay (accounting for the practicalities of multiple administrations).
As noted above WO2018114892 describes how saponin and TLR4 agonist components of an adjuvant may be administered separately while maintaining a synergistic adjuvant effect. Consequently, the skilled person will appreciate that an adjuvant comprising multiple immunostimulants may itself be administered separately or together (e.g. in coformulation) in an analogous manner to the adjuvant and antigen.
An administration according to step (ii) will comprise delivery of the antigen. In one embodiment, only one administration is performed. Multiple administrations may alternatively be performed. If so, each administration will comprise a delivery of the antigen. Suitably 1 to 5, especially 1 to 4 or in particular 1 to 3 administrations are performed. Suitably, at least 2 administrations are performed. Most suitably, 2 administrations are performed.
An administration according to step (ii) may comprise delivery of the second adjuvant and antigen, which may be separately or together (e.g. in co-formulation). In one embodiment, only one administration of the second adjuvant and antigen is performed. Multiple administrations may alternatively be performed. If so, each administration will comprise a delivery of a second adjuvant and a delivery of the antigen. Suitably 1 to 5, especially 1 to 4 or in particular 1 to 3 administrations are performed. Suitably, at least 2 administrations are performed. Most suitably, 2 administrations are performed.
The start of an administration of the second adjuvant and antigen is when either the second adjuvant or the antigen has been administered. The completion of an administration is when both the second adjuvant and the antigen have been administered. The start of a further administration of the second adjuvant and antigen is when either the second adjuvant or the antigen has been administered again. If multiple administrations are performed, each administration may be delivered after a suitable period of time after completion of the previous administration. Suitably, the period between administrations to a human subject is 2 weeks to 12 months, especially 2 weeks to 6 months, in particular 2 weeks to 3 months, such as 2 weeks to 2 months, for example 3 to 5 weeks. If only one administration is performed, then step (ii) is completed at the end of the one administration. If multiple administrations are performed, then step (ii) is completed at the end of the last administration.
Before administration, the antigen may be provided in liquid or dry (e.g. lyophilised) form. The preferred form will depend on factors such as the precise nature of the antigen, e.g. if the antigen is amenable to drying, or other components which may be present. The antigen is typically provided in liquid form.
The second adjuvant may be provided in liquid or dry form. The preferred form will depend on the precise nature of second adjuvant and any associated carrier, e.g. if capable of reconstitution from dry form, and any other components present. The second adjuvant is typically provided in liquid form.
Compositions comprising the second adjuvant or the antigen intended for combination with other compositions prior to administration need not themselves have a physiologically acceptable pH or a physiologically acceptable tonicity; a formulation intended for administration should have a physiologically acceptable pH and should have a physiologically acceptable osmolality.
The pH of a liquid preparation is adjusted in view of the components of the composition and necessary suitability for administration to the human subject. The pH of a formulation is generally at least 4, especially at least 5, in particular at least 5.5 such as at least 6. The pH of a formulation is generally 9 or less, especially 8.5 or less, in particular 8 or less, such as 7.5 or less. The pH of a formulation may be 4 to 9, especially 5 to 8.5, in particular 5.5 to 8, such as 6.5 to 7.4 (e.g. 6.5 to 7.1).
For parenteral administration, solutions should have a physiologically acceptable osmolality to avoid excessive cell distortion or lysis. A physiologically acceptable osmolality will generally mean that solutions will have an osmolality which is approximately isotonic or mildly hypertonic. Suitably the formulations for administration will have an osmolality of 250 to 750 mOsm/kg, especially 250 to 550 mOsm/kg, in particular 270 to 500 mOsm/kg, such as 270 to 400 mOsm/kg. Osmolality may be measured according to techniques known in the art, such as by the use of a commercially available osmometer, for example the Advanced® Model 2020 available from Advanced Instruments Inc. (USA).
Liquids used for reconstitution will be substantially aqueous, such as water for injection, phosphate buffered saline and the like. As mentioned above, the requirement for buffer and/or tonicity modifying agents will depend on the on both the contents of the container being reconstituted and the subsequent use of the reconstituted contents. Buffers may be selected from acetate, citrate, histidine, maleate, phosphate, succinate, tartrate and TRIS. The buffer may be a phosphate buffer such as Na/Na2PO4, Na/K2PO4 or K/K2PO4. It will be appreciated that some of the components used may form salts under appropriate conditions, therefore such components may be present as a salt, in particular a pharmaceutically acceptable salt.
Suitably, the formulations used in the present invention have a dose volume of between 0.05 ml and 1 ml, such as between 0.1 and 0.6 ml, in particular a dose volume of 0.45 to 0.55 ml, such as 0.5 ml. The volumes of the compositions used may depend on the subject, delivery route and location, with smaller doses being given by the intradermal route or if the second adjuvant and antigen are delivered separately to the same location. A typical human dose for administration through routes such as intramuscular, is in the region of 200 ul to 750 ml, such as 400 to 600 ul, in particular about 500 ul, such as 500 ul.
If two liquids are intended to be combined, for example for co-formulation if the second adjuvant is in liquid form and the antigen is in liquid form, the volume of each liquid may be the same or different. Volumes for combination will typically be in the range of 10:1 to 1:10, such as 2: 1 to 1 :2. Suitably the volume of each liquid will be substantially the same, such as the same. For example a 250 ul volume of second adjuvant composition in liquid form may be combined with a 250 ul volume antigen composition in liquid form to provide a co-formulation dose with a 500 ul volume, each of the second adjuvant and antigen being diluted 2-fold during the combination.
The second adjuvant may be prepared as a concentrate with the expectation of dilution by a liquid antigen containing composition prior to administration. For example, the second adjuvant may be prepared at double-strength with the expectation of dilution by an equal volume of antigen containing composition prior to administration.
The antigen may be prepared as a concentrate with the expectation of dilution by a liquid second adjuvant containing composition prior to administration. For example, the antigen may be prepared at double-strength with the expectation of dilution by an equal volume of second adjuvant containing composition prior to administration.
The second adjuvant and antigen, whether intended for co-formulation or separate formulation, may be provided in the form of various physical containers such as vials or prefilled syringes.
It is common where liquids are to be transferred between containers, such as from a vial to a syringe, to provide ‘an overage’ which ensures that the full volume required can be conveniently transferred. The level of overage required will depend on the circumstances but excessive overage should be avoided to reduce wastage and insufficient overage may cause practical difficulties. Overages may be of the order of 20 to 100 ul per dose, such as 30 ul or 50 ul. For example, a typical 10 dose container of doubly concentrated adjuvant (250 ul per dose) may contain around 2.85 to 3.25 ml of adjuvant. Stabilisers may be present. Stabilisers may be of particular relevance where multidose containers are provided as doses of the final formulation(s) may be administered to subjects over a period of time.
The second adjuvant and antigen in liquid form may be provided in the form of a multichamber syringe. The use of multi-chamber syringes provides a convenient method for the separate sequential administration of the second adjuvant and antigen. Multi-chamber syringes may be configured to provide concurrent but separate delivery of the second adjuvant and antigen, or they may be configured to provide sequential delivery (in either order).
In other configurations of multichambered syringes, the antigen may be provided in dry form (e.g., freeze-dried) in one chamber and reconstituted by the second adjuvant contained in the other chamber before administration.
Examples of multi-chamber syringes may be found in disclosures such as WO2016/172396, although a range of other configurations are possible.
Formulations are preferably sterile.
Conveniently, if a second adjuvant is administered, the second adjuvant and the antigen are administered as a co-formulation.
Steps (i) and (ii)
Step (ii) is initiated after an interval from completion of step (i). The interval is sufficient to allow the immune system of the subject to have mounted an appropriate immune response to the administration of step (i). The interval is desirably short enough such that the immune response to the administration of step (i) does not excessively wane before the administration of step (ii). Suitably, step (ii) is initiated at an interval of 2 weeks to 2 years, especially 2 weeks to 1 year, in particular 3 weeks to 9 months, such as 4 weeks to 6 months from completion of step (i).
In some embodiments step (ii) is initiated at an interval of 2 weeks to 2 months from completion of step (i). In some embodiments step (ii) is initiated at an interval of 1 month to 3 months from completion of step (i). In some embodiments step (ii) is initiated at an interval of 2 months to 4 months from completion of step (i). In some embodiments step (ii) is initiated at an interval of 3 months to 6 months from completion of step (i). In some embodiments step (ii) is initiated at an interval of 4 months to 12 months from completion of step (i).
The combined duration of step (i), step (ii) and the interval between the steps is suitably 1 month to 2 years, especially 6 weeks months to 12 months, in particular 2 months to 10 months, such as 10 weeks to 9 months.
Alternatively, the combined duration of step (i), step (ii) and the interval between the steps is suitably at least 2 months, especially at least 3 months, in particular at least 4 months, such as at least 5 months, especially at least 6 months, in particular at least 7 months. Alternatively, the combined duration of step (i), step (ii) and the interval between the steps is suitably 5 years or less, especially 3 years or less, in particular 2 years or less, such as 18 months or less, especially 12 months or less, in particular 10 months or less. Immune responses
The methods and uses of the invention elicit an immune response in the subject against an antigen. The elicited immune response may be an antigen specific B cell response which produces neutralizing antibodies. The elicited immune response may be an antigen specific T cell response, which may be a systemic and/or a local response. The antigen specific T cell response may comprise a CD4+ T cell response, such as a response involving CD4+ T cells expressing a plurality of cytokines, e.g. IFNgamma, TNFalpha and/or IL2. Alternatively, or additionally, the antigen specific T cell response comprises a CD8+ T cell response, such as a response involving CD8+ T cells expressing a plurality of cytokines, e.g., IFNgamma, TNFalpha and/or IL2.
An immune response may be characterised by various parameters.
In one embodiment, the immune response is characterised by the level of response, suitably as indicated by the serum titer of IgG antibodies discussed above which bind and/or are specific to the antigen. In particular, the antibodies are serum neutralising antibodies against the antigen. Suitably the level of the immune response is increased by at least 25%, especially at least 50%, in particular at least 100% (twofold), such as 400% (fivefold), for example 900% (tenfold). The increase in the level of the immune response is relative to the level of immune response achieved wherein step (ii) alone is carried out, without carrying out preceding step (i) of the invention.
In a further embodiment, the immune response is characterised by the breadth of response, suitably as indicated by the antibodies discussed above binding to (a) an increased range of variants of the antigen (such as variants of the antigen from an increased number of strains of the pathogen) or (b) elicit a response in a wider variety of human HLA types).
In a further embodiment, the immune response is characterised by the speed of response, i.e. the time taken to achieve a certain threshold of immune response, such as a threshold level associated with a protective immune response.
In a further embodiment, the immune response is characterised by the duration of immune response, suitably as indicated by the period of time over which the subject maintains a serum titer of antibodies as detailed discussed above binding and/or specific to the antigen. Suitably the duration of the immune response is increased by at least 1 year, especially at least 3 years. Alternatively, or in addition, the immune response is maintained for at least 1 year or especially 3 years.
In a further embodiment, there is provided a method of reducing the variability of the immune response to an antigen in subjects. Suitably, the standard deviation of the level, breadth, speed and/or duration of response between subjects is decreased. Alternatively, the proportion of subjects meeting a specified threshold of immune response to the antigen is increased. The specified threshold may be that which indicates protective efficacy depending on the pathogen to which an immune response is being elicited.
In one embodiment there is provided a method of improving an immune response to an antigen in a subject. The immune response may be improved as indicated by the level, breadth or duration of the immune response as set out above.
There is also provided a method of prophylaxis of infection by a pathogen in a subject. ‘Prophylaxis’ as used herein pertains to an action taken in advance of a subject being infected. A subject may be at risk of infection by a pathogen. Suitably, the prophylaxis reduces the likelihood of established infection by the pathogen.
More suitably, prophylaxis prevents infection e.g. reducing partially or completely the severity of one or more symptoms and/or time over which one or more symptoms are experienced by a subject, reducing the likelihood of developing an established infection after challenge and/or slowing progression of an associated illness (e.g. extending survival).
The term ‘preparing the immune system’ includes modification of the innate cell repertoire, including at a genetic level, thereby influencing the immune response resulting from step (ii).
Methods of reducing the amount of an antigen required to elicit a desired immune response are suitably reducing the amount of antigen required to elicit a desired immune response relative to the amount required to elicit the desired immune response without performing step (i).
The present invention also provides kits of parts, comprising the components utilised in the methods described herein, such as a kit of parts comprising (i) a STING agonist; (ii) a second adjuvant; and (iii) an antigen. Each of (i), (ii) and (iii) may be provided in separate containers, or (ii) and (ii) may optionally be provided together.
In certain embodiments a STING agonist and a first antigen are administered in step (i) and a second antigen and optionally a second adjuvant are administered in step (ii).
Step (i)
For a given administration of STING agonist and the first antigen, the STING agonist and the first antigen may be delivered in co-formulation or in separate formulations. If delivered in separate formulations, the STING agonist and the first antigen may be delivered simultaneously or at different times. If delivered in separate formulations, the STING agonist and the first antigen may be delivered to the same location or to different locations.
When administered as separate formulations, the STING agonist and first antigen are administered to locations with sufficient spatial proximity such that the adjuvant effect of the STING agonist is adequately maintained. For example, spatial proximity is sufficient to maintain at least 50%, especially at least 75% and in particular at least 90% of the adjuvant effect seen with administration at (essentially) the same location. The adjuvant effect seen with administration to the same location is defined as the level of increase observed as a result of administration of STING agonist and first antigen to the same location compared with administration of antigen alone. The STING agonist and antigen are desirably administered to a location draining to the same lymph node, such as to the same limb, in particular to the same muscle.
In certain embodiments, the STING agonist and first antigen are administered to the same location. The spatial separation of administration locations may be at least 5 mm, such as at least 1 cm. The spatial separation of administration locations may be less than 10 cm, such as less than 5 cm apart.
The STING agonist and first antigen may be administered intradermally, intramuscularly, intraperitoneally or subcutaneously. The STING agonist and first antigen may be administered via different routes. Suitably the STING agonist and antigen are administered via the same route, in particular intramuscularly. More suitably, the STING agonist and first antigen are administered intramuscularly to the same muscle.
When administered as separate formulations, the STING agonist and first antigen are administered with sufficient temporal proximity such that the adjuvant effect is adequately maintained. For example, temporal proximity is sufficient to maintain at least 50%, especially at least 75% and in particular at least 90% of the adjuvant effect seen with administration at the same time. The adjuvant effect seen with administration at the same time is defined as the level of increase observed as a result of administration of STING agonist and first antigen at (essentially) the same time compared with administration of first antigen without STING agonist.
When administered as separate formulations, the STING agonist and first antigen may be administered within 84 hours, such as within 60 hours, especially within 36 hours, in particular within 24 hours, for example within 12 hours of each other. Suitably the STING agonist and antigen are administered within 6 hours, especially within 2 hours, in particular within 1 hour, such as within 30 minutes and especially within 15 minutes (e.g. within 5 minutes) of each other. When administered as separate formulations, if the STING agonist and first antigen are administered with a delay, the STING agonist may be administered first and the antigen administered second. Alternatively, the antigen is administered first and the STING agonist administered second. Desirably, the STING agonist and antigen are administered without intentional delay (accounting for the practicalities of multiple administrations).
An administration according to step (i) will comprise delivery of the STING agonist and first antigen separately or together (e.g. in co-formulation). In one embodiment, only one administration is performed. Multiple administrations may alternatively be performed. If so, each administration will comprise a delivery of a STING agonist and a delivery of the first antigen. Suitably 1 to 5, especially 1 to 4 or in particular 1 to 3 administrations are performed. Suitably, at least 2 administrations are performed. Most suitably, 2 administrations are performed.
The start of an administration is when either the STING agonist or the first antigen has been administered. The completion of an administration is when both the STING agonist and the first antigen have been administered. The start of a further administration is when either the STING agonist or the first antigen has been administered again. If multiple administrations are performed, each administration may be delivered after a suitable period of time after completion of the previous administration. Suitably, the period between administrations to a human subject is 2 weeks to 12 months, especially 2 weeks to 6 months, in particular 2 weeks to 3 months, such as 2 weeks to 2 months, for example 3 to 5 weeks.
If only one administration is performed, then step (i) is completed at the end of the one administration. If multiple administrations are performed, then step (i) is completed at the end of the last administration. The start of step (ii) is when either the second adjuvant or the second antigen of step (ii) have been administered.
Before administration, the first antigen may be provided in liquid or dry (e.g. lyophilised) form. The preferred form will depend on factors such as the precise nature of the antigen, e.g. if the antigen is amenable to drying, or other components which may be present. The first antigen is typically provided in liquid form.
The STING agonist may be provided in liquid or dry form. The preferred form will depend on the precise nature of the STING agonist and any associated carrier, e.g. if capable of reconstitution from dry form, and any other components present. The STING agonist is typically provided in liquid form.
Compositions comprising the STING agonist or the first antigen intended for combination with other compositions prior to administration need not themselves have a physiologically acceptable pH or a physiologically acceptable tonicity; a formulation intended for administration should have a physiologically acceptable pH and should have a physiologically acceptable osmolality.
The pH of a liquid preparation is adjusted in view of the components of the composition and necessary suitability for administration to the human subject. The pH of a formulation is generally at least 4, especially at least 5, in particular at least 5.5 such as at least 6. The pH of a formulation is generally 9 or less, especially 8.5 or less, in particular 8 or less, such as 7.5 or less. The pH of a formulation may be 4 to 9, especially 5 to 8.5, in particular 5.5 to 8, such as 6.5 to 7.4 (e.g. 6.5 to 7.1). For parenteral administration, solutions should have a physiologically acceptable osmolality to avoid excessive cell distortion or lysis. A physiologically acceptable osmolality will generally mean that solutions will have an osmolality which is approximately isotonic or mildly hypertonic. Suitably the formulations for administration will have an osmolality of 250 to 750 mOsm/kg, especially 250 to 550 mOsm/kg, in particular 270 to 500 mOsm/kg, such as 270 to 400 mOsm/kg. Osmolality may be measured according to techniques known in the art, such as by the use of a commercially available osmometer, for example the Advanced® Model 2020 available from Advanced Instruments Inc. (USA).
Liquids used for reconstitution will be substantially aqueous, such as water for injection, phosphate buffered saline and the like. As mentioned above, the requirement for buffer and/or tonicity modifying agents will depend on the on both the contents of the container being reconstituted and the subsequent use of the reconstituted contents. Buffers may be selected from acetate, citrate, histidine, maleate, phosphate, succinate, tartrate and TRIS. The buffer may be a phosphate buffer such as Na/Na2PO4, Na/K2PO4 or K/K2PO4.
It will be appreciated that some of the components used may form salts under appropriate conditions, therefore such components may be present as a salt, in particular a pharmaceutically acceptable salt.
Suitably, the formulations used in the present invention have a dose volume per administration of between 0.05 ml and 1 ml, such as between 0.1 and 0.6 ml, in particular a dose volume of 0.45 to 0.55 ml, such as 0.5 ml. The volumes of the compositions used may depend on the subject, delivery route and location, with smaller doses being given by the intradermal route or if the STING agonist and antigen are delivered separately to the same location. A typical human dose for administration through routes such as intramuscular, is in the region of 200 ul to 750 ml, such as 400 to 600 ul, in particular about 500 ul, such as 500 ul.
If two liquids are intended to be combined, for example for co-formulation if the STING agonist is in liquid form and the antigen is in liquid form, the volume of each liquid may be the same or different. Volumes for combination will typically be in the range of 10:1 to 1:10, such as 2: 1 to 1 :2. Suitably the volume of each liquid will be substantially the same, such as the same. For example a 250 ul volume of STING agonist in liquid form may be combined with a 250 ul volume antigen composition in liquid form to provide a co-formulation dose with a 500 ul volume, each of the STING agonist and antigen being diluted 2-fold during the combination.
The STING agonist may be prepared as a concentrate with the expectation of dilution by a liquid antigen containing composition prior to administration. For example, the STING agonist may be prepared at double-strength with the expectation of dilution by an equal volume of antigen containing composition prior to administration.
The first antigen may be prepared as a concentrate with the expectation of dilution by a liquid STING agonist containing composition prior to administration. For example, the first antigen may be prepared at double-strength with the expectation of dilution by an equal volume of STING agonist containing composition prior to administration.
The STING agonist and first antigen, whether intended for co-formulation or separate formulation, may be provided in the form of various physical containers such as vials or prefilled syringes.
It is common where liquids are to be transferred between containers, such as from a vial to a syringe, to provide ‘an overage’ which ensures that the full volume required can be conveniently transferred. The level of overage required will depend on the circumstances but excessive overage should be avoided to reduce wastage and insufficient overage may cause practical difficulties. Overages may be of the order of 20 to 100 ul per dose, such as 30 ul or 50 ul. For example, a typical 10 dose container of doubly concentrated adjuvant (250 ul per dose) may contain around 2.85 to 3.25 ml of adjuvant.
Stabilisers may be present. Stabilisers may be of particular relevance where multidose containers are provided as doses of the final formulation(s) may be administered to subjects over a period of time.
The STING agonist and first antigen in liquid form may be provided in the form of a multichamber syringe. The use of multi-chamber syringes provides a convenient method for the separate sequential administration of the STING agonist and antigen. Multi-chamber syringes may be configured to provide concurrent but separate delivery of the STING agonist and first antigen, or they may be configured to provide sequential delivery (in either order).
In other configurations of multichambered syringes, the antigen may be provided in dry form (e.g., freeze-dried) in one chamber and reconstituted by the STING agonist contained in the other chamber before administration.
Examples of multi-chamber syringes may be found in disclosures such as WO2016/172396, although a range of other configurations are possible.
Formulations are preferably sterile.
Conveniently, the STING agonist and the first antigen are administered as a coformulation.
Step (ii)
For a given administration of second adjuvant and the second antigen, the second adjuvant and the second antigen may be delivered in co-formulation or in separate formulations. If delivered in separate formulations, the second adjuvant and the second antigen may be delivered simultaneously or at different times. If delivered in separate formulations, the second adjuvant and the second antigen may be delivered to the same location or to different locations.
When administered as separate formulations, the second adjuvant and second antigen are administered to locations with sufficient spatial proximity such that the adjuvant effect of the second adjuvant is adequately maintained. For example, spatial proximity is sufficient to maintain at least 50%, especially at least 75% and in particular at least 90% of the adjuvant effect seen with administration at to (essentially) the same location. The adjuvant effect seen with administration to the same location is defined as the level of increase observed as a result of administration of second adjuvant and second antigen to the same location compared with administration of antigen alone. The second adjuvant and antigen are desirably administered to a location draining to the same lymph node, such as to the same limb, in particular to the same muscle.
In certain embodiments, the second adjuvant and second antigen are administered to the same location. The spatial separation of administration locations may be at least 5 mm, such as at least 1 cm. The spatial separation of administration locations may be less than 10 cm, such as less than 5 cm apart.
The second adjuvant and second antigen may be administered intradermally, intramuscularly, intraperitoneally or subcutaneously. The second adjuvant and second antigen may be administered via different routes. Suitably the second adjuvant and antigen are administered via the same route, in particular intramuscularly. More suitably, the second adjuvant and second antigen are administered intramuscularly to the same muscle.
When administered as separate formulations, the second adjuvant and second antigen are administered with sufficient temporal proximity such that the adjuvant effect is adequately maintained. For example, temporal proximity is sufficient to maintain at least 50%, especially at least 75% and in particular at least 90% of the adjuvant effect seen with administration at the same time. The adjuvant effect seen with administration at the same time is defined as the level of increase observed as a result of administration of second adjuvant and second antigen at (essentially) the same time compared with administration of second antigen without second adjuvant.
When administered as separate formulations, the second adjuvant and second antigen may be administered within 84 hours, such as within 60 hours, especially within 36 hours, in particular within 24 hours, for example within 12 hours of each other. Suitably the second adjuvant and antigen are administered within 6 hours, especially within 2 hours, in particular within 1 hour, such as within 30 minutes and especially within 15 minutes (e.g. within 5 minutes) of each other.
When administered as separate formulations, if the second adjuvant and second antigen are administered with a delay, the second adjuvant may be administered first and the antigen administered second. Alternatively, the antigen is administered first and the second adjuvant administered second. Desirably, the second adjuvant and antigen are administered without intentional delay (accounting for the practicalities of multiple administrations). As noted above WO2018114892 describes how saponin and TLR4 agonist components of an adjuvant may be administered separately while maintaining a synergistic adjuvant effect. Consequently, the skilled person will appreciate that an adjuvant comprising multiple immunostimulants may itself be administered separately or together (e.g. in coformulation) in an analogous manner to the adjuvant and antigen.
An administration according to step (ii) will comprise delivery of the second antigen. In one embodiment, only one administration is performed. Multiple administrations may alternatively be performed. If so, each administration will comprise a delivery of the second antigen. Suitably 1 to 5, especially 1 to 4 or in particular 1 to 3 administrations are performed. Suitably, at least 2 administrations are performed. Most suitably, 2 administrations are performed.
An administration according to step (ii) may comprise delivery of the second adjuvant and second antigen, which may be separately or together (e.g. in co-formulation). In one embodiment, only one administration of the second adjuvant and second antigen is performed. Multiple administrations may alternatively be performed. If so, each administration will comprise a delivery of a second adjuvant and a delivery of the second antigen. Suitably 1 to 5, especially 1 to 4 or in particular 1 to 3 administrations are performed. Suitably, at least 2 administrations are performed. Most suitably, 2 administrations are performed.
The start of an administration of the second adjuvant and second antigen is when either the second adjuvant or the second antigen has been administered. The completion of an administration is when both the second adjuvant and the second antigen have been administered. The start of a further administration of the second adjuvant and second antigen is when either the second adjuvant or the second antigen has been administered again. If multiple administrations are performed, each administration may be delivered after a suitable period of time after completion of the previous administration. Suitably, the period between administrations to a human subject is 2 weeks to 12 months, especially 2 weeks to 6 months, in particular 2 weeks to 3 months, such as 2 weeks to 2 months, for example 3 to 5 weeks.
If only one administration is performed, then step (ii) is completed at the end of the one administration. If multiple administrations are performed, then step (ii) is completed at the end of the last administration.
Before administration, the second antigen may be provided in liquid or dry (e.g. lyophilised) form. The preferred form will depend on factors such as the precise nature of the antigen, e.g. if the antigen is amenable to drying, or other components which may be present. The second antigen is typically provided in liquid form.
The second adjuvant may be provided in liquid or dry form. The preferred form will depend on the precise nature of second adjuvant and any associated carrier, e.g. if capable of reconstitution from dry form, and any other components present. The second adjuvant is typically provided in liquid form. Compositions comprising the second adjuvant or the second antigen intended for combination with other compositions prior to administration need not themselves have a physiologically acceptable pH or a physiologically acceptable tonicity; a formulation intended for administration should have a physiologically acceptable pH and should have a physiologically acceptable osmolality. The pH of a liquid preparation is adjusted in view of the components of the composition and necessary suitability for administration to the human subject. The pH of a formulation is generally at least 4, especially at least 5, in particular at least 5.5 such as at least 6. The pH of a formulation is generally 9 or less, especially 8.5 or less, in particular 8 or less, such as 7.5 or less. The pH of a formulation may be 4 to 9, especially 5 to 8.5, in particular 5.5 to 8, such as 6.5 to 7.4 (e.g.6.5 to 7.1). For parenteral administration, solutions should have a physiologically acceptable osmolality to avoid excessive cell distortion or lysis. A physiologically acceptable osmolality will generally mean that solutions will have an osmolality which is approximately isotonic or mildly hypertonic. Suitably the formulations for administration will have an osmolality of 250 to 750 mOsm/kg, especially 250 to 550 mOsm/kg, in particular 270 to 500 mOsm/kg, such as 270 to 400 mOsm/kg. Osmolality may be measured according to techniques known in the art, such as by the use of a commercially available osmometer, for example the Advanced® Model 2020 available from Advanced Instruments Inc. (USA). Liquids used for reconstitution will be substantially aqueous, such as water for injection, phosphate buffered saline and the like. As mentioned above, the requirement for buffer and/or tonicity modifying agents will depend on the on both the contents of the container being reconstituted and the subsequent use of the reconstituted contents. Buffers may be selected from acetate, citrate, histidine, maleate, phosphate, succinate, tartrate and TRIS. The buffer may be a phosphate buffer such as Na/Na2PO4, Na/K2PO4 or K/K2PO4. It will be appreciated that some of the components used may form salts under appropriate conditions, therefore such components may be present as a salt, in particular a pharmaceutically acceptable salt. Suitably, the formulations used in the present invention have a dose volume of between 0.05 ml and 1 ml, such as between 0.1 and 0.6 ml, in particular a dose volume of 0.45 to 0.55 ml, such as 0.5 ml. The volumes of the compositions used may depend on the subject, delivery route and location, with smaller doses being given by the intradermal route or if the second adjuvant and antigen are delivered separately to the same location. A typical human dose for administration through routes such as intramuscular, is in the region of 200 ul to 750 ml, such as 400 to 600 ul, in particular about 500 ul, such as 500 ul. If two liquids are intended to be combined, for example for co-formulation if the second adjuvant is in liquid form and the antigen is in liquid form, the volume of each liquid may be the same or different. Volumes for combination will typically be in the range of 10:1 to 1 :10, such as 2:1 to 1:2. Suitably the volume of each liquid will be substantially the same, such as the same. For example a 250 ul volume of second adjuvant composition in liquid form may be combined with a 250 ul volume antigen composition in liquid form to provide a co-formulation dose with a 500 ul volume, each of the second adjuvant and antigen being diluted 2-fold during the combination.
The second adjuvant may be prepared as a concentrate with the expectation of dilution by a liquid antigen containing composition prior to administration. For example, the second adjuvant may be prepared at double-strength with the expectation of dilution by an equal volume of antigen containing composition prior to administration.
The second antigen may be prepared as a concentrate with the expectation of dilution by a liquid second adjuvant containing composition prior to administration. For example, the second antigen may be prepared at double-strength with the expectation of dilution by an equal volume of second adjuvant containing composition prior to administration.
The second adjuvant and second antigen, whether intended for co-formulation or separate formulation, may be provided in the form of various physical containers such as vials or pre-filled syringes.
It is common where liquids are to be transferred between containers, such as from a vial to a syringe, to provide ‘an overage’ which ensures that the full volume required can be conveniently transferred. The level of overage required will depend on the circumstances but excessive overage should be avoided to reduce wastage and insufficient overage may cause practical difficulties. Overages may be of the order of 20 to 100 ul per dose, such as 30 ul or 50 ul. For example, a typical 10 dose container of doubly concentrated adjuvant (250 ul per dose) may contain around 2.85 to 3.25 ml of adjuvant.
Stabilisers may be present. Stabilisers may be of particular relevance where multidose containers are provided as doses of the final formulation(s) may be administered to subjects over a period of time.
The second adjuvant and second antigen in liquid form may be provided in the form of a multichamber syringe. The use of multi-chamber syringes provides a convenient method for the separate sequential administration of the second adjuvant and antigen. Multi-chamber syringes may be configured to provide concurrent but separate delivery of the second adjuvant and antigen, or they may be configured to provide sequential delivery (in either order).
In other configurations of multichambered syringes, the antigen may be provided in dry form (e.g., freeze-dried) in one chamber and reconstituted by the second adjuvant contained in the other chamber before administration. Examples of multi-chamber syringes may be found in disclosures such as WO2016/172396, although a range of other configurations are possible.
Formulations are preferably sterile.
Conveniently, if a second adjuvant is administered, the second adjuvant and the second antigen are administered as a co-formulation.
Steps (i) and (ii)
Step (ii) is initiated after an interval from completion of step (i). The interval is sufficient to allow the immune system of the subject to have mounted an appropriate immune response to the administration of step (i). The interval is desirably short enough such that the immune response to the administration of step (i) does not excessively wane before the administration of step (ii). Suitably, step (ii) is initiated at an interval of 2 weeks to 2 years, especially 2 weeks to 1 year, in particular 3 weeks to 9 months, such as 4 weeks to 6 months from completion of step (i).
In some embodiments step (ii) is initiated at an interval of 2 weeks to 2 months from completion of step (i). In some embodiments step (ii) is initiated at an interval of 1 month to 3 months from completion of step (i). In some embodiments step (ii) is initiated at an interval of 2 months to 4 months from completion of step (i). In some embodiments step (ii) is initiated at an interval of 3 months to 6 months from completion of step (i). In some embodiments step (ii) is initiated at an interval of 4 months to 12 months from completion of step (i).
The combined duration of step (i), step (ii) and the interval between the steps is suitably 1 month to 2 years, especially 6 weeks months to 12 months, in particular 2 months to 10 months, such as 10 weeks to 9 months.
Alternatively, the combined duration of step (i), step (ii) and the interval between the steps is suitably at least 2 months, especially at least 3 months, in particular at least 4 months, such as at least 5 months, especially at least 6 months, in particular at least 7 months.
Alternatively, the combined duration of step (i), step (ii) and the interval between the steps is suitably 5 years or less, especially 3 years or less, in particular 2 years or less, such as 18 months or less, especially 12 months or less, in particular 10 months or less. Immune responses
The methods and uses of the invention elicit an immune response in the subject against a second antigen. The elicited immune response may be an antigen specific B cell response which produces neutralizing antibodies. The elicited immune response may be an antigen specific T cell response, which may be a systemic and/or a local response. The antigen specific T cell response may comprise a CD4+ T cell response, such as a response involving CD4+ T cells expressing a plurality of cytokines, e.g. IFNgamma, TNFalpha and/or IL2. Alternatively, or additionally, the antigen specific T cell response comprises a CD8+ T cell response, such as a response involving CD8+ T cells expressing a plurality of cytokines, e.g., IFNgamma, TNFalpha and/or IL2.
An immune response may be characterised by various parameters.
In one embodiment, the immune response is characterised by the level of response, suitably as indicated by the serum titer of IgG antibodies discussed above which bind and/or are specific to the second antigen. In particular, the antibodies are serum neutralising antibodies against the second antigen. Suitably the level of the immune response is increased by at least 25%, especially at least 50%, in particular at least 100% (twofold), such as 400% (fivefold), for example 900% (tenfold). The increase in the level of the immune response is relative to the level of immune response achieved wherein step (ii) alone is carried out, without carrying out preceding step (i) of the invention.
In a further embodiment, the immune response is characterised by the breadth of response, suitably as indicated by the antibodies discussed above binding to (a) an increased range of variants of the second antigen (such as variants of the second antigen from an increased number of strains of the pathogen) or (b) elicit a response in a wider variety of human HLA types).
In a further embodiment, the immune response is characterised by the speed of response, i.e. the time taken to achieve a certain threshold of immune response, such as a threshold level associated with a protective immune response.
In a further embodiment, the immune response is characterised by the duration of immune response, suitably as indicated by the period of time over which the subject maintains a serum titer of antibodies as detailed discussed above binding and/or specific to the second antigen. Suitably the duration of the immune response is increased by at least 1 year, especially at least 3 years. Alternatively, or in addition, the immune response is maintained for at least 1 year or especially 3 years.
In a further embodiment, there is provided a method of reducing the variability of the immune response to a second antigen in subjects. Suitably, the standard deviation of the level, breadth, speed and/or duration of response between subjects is decreased. Alternatively, the proportion of subjects meeting a specified threshold of immune response to the second antigen is increased. The specified threshold may be that which indicates protective efficacy depending on the pathogen to which an immune response is being elicited.
In one embodiment there is provided a method of improving an immune response to a second antigen in a subject. The immune response may be improved as indicated by the level, breadth or duration of the immune response as set out above.
There is also provided a method of prophylaxis of infection by a pathogen in a subject. ‘Prophylaxis’ as used herein pertains to an action taken in advance of a subject being infected. A subject may be at risk of infection by a pathogen. Suitably, the prophylaxis reduces the likelihood of established infection by the pathogen.
More suitably, prophylaxis prevents infection e.g. reducing partially or completely the severity of one or more symptoms and/or time over which one or more symptoms are experienced by a subject, reducing the likelihood of developing an established infection after challenge and/or slowing progression of an associated illness (e.g. extending survival).
The term ‘preparing the immune system’ includes modification of the innate cell repertoire, including at a genetic level, thereby influencing the immune response resulting from step (ii).
Methods of reducing the amount of an antigen required to elicit a desired immune response are suitably reducing the amount of antigen required to elicit a desired immune response relative to the amount required to elicit the desired immune response without performing step (i).
The present invention also provides kits of parts, comprising the components utilised in the methods described herein, such as a kit of parts comprising (i) a STING agonist; (ii) a first antigen; and (iii) a second antigen. Each of (i), (ii) and (iii) may be provided in separate containers, or (i) and (ii) may optionally be provided together. Also provided is a kit further comprising (iv) a second adjuvant as described herein. Likewise, each of (i), (ii), (iii) and (iv) may be provided in separate containers, or (i) and (ii) may optionally be provided together and/or (iii) and (iv) may optionally be provided together.
“Pharmaceutically acceptable salts” are referred to herein. As used herein, "pharmaceutically acceptable salts" refer to derivatives of the disclosed compounds wherein the parent compound is modified by making pharmaceutically acceptable acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic groups such as amines; and alkali or organic salts of acidic groups such as carboxylic acids. The pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, and nitric; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, and isethionic, and the like.
Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, 1985, p.1418, the disclosure of which is hereby incorporated by reference. The phrase "pharmaceutically acceptable salt" is employed herein to refer to those salts which are, within the scope of sound medical judgment, suitable for use in a pharmaceutical context, without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
Throughout the specification, including the claims, where the context permits, the term “comprising” and variants thereof such as “comprises” are to be interpreted as including the stated element (e.g., integer) or elements (e.g., integers) without necessarily excluding any other elements (e.g., integers). Thus a composition “comprising” X may consist exclusively of X or may include something additional e.g. X + Y.
The word “substantially” does not exclude “completely” e.g. a composition which is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition of the invention.
The term “about” in or “approximately” in relation to a numerical value x is optional and means, for example, x+10% of the given figure, such as x+5% of the given figure, in particular the given figure.
As used herein, the singular forms “a,” “an” and “the” include plural references unless the content clearly dictates otherwise.
As used herein, ng refers to nanograms, ug or pg refers to micrograms, mg refers to milligrams, mL or ml refers to milliliter, and mM refers to millimolar. Similar terms, such as urn, are to be construed accordingly.
Unless specifically stated, a process comprising a step of mixing two or more components does not require any specific order of mixing. Thus components can be mixed in any order. Where there are three components then two components can be combined with each other, and then the combination may be combined with the third component, etc.
The invention is further illustrated by reference to the following clauses:
These clauses relate to embodiments wherein a STING agonist is administered in step (i) and a STING agonist and an antigen are administered in step (ii).
Clause 1 . A method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject a STING agonist and an antigen which is associated with the pathogen.
Clause 2. A method of reducing the variability of the immune response to an antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist, prior to
(ii) administering to each of the subjects a STING agonist and the antigen. Clause 3. A method of improving an immune response to an antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject a STING agonist and the antigen.
Clause 4. A method of reducing the amount of an antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the antigen and a STING agonist.
Clause 5. A method for preparing the immune system of a subject for (ii) administration of an antigen and a STING agonist, comprising (i) administering to the subject a STING agonist.
Clause 6. A STING agonist for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject a STING agonist and an antigen which is associated with the pathogen.
Clause 7. An antigen for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject a STING agonist and an antigen which is associated with the pathogen.
Clause 8. A STING agonist for use in a method of reducing the variability of the immune response to an antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist, prior to
(ii) administering to each of the subjects a STING agonist and the antigen.
Clause 9. An antigen for use in a method of reducing the variability of the immune response to an antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist, prior to
(ii) administering to each of the subjects a STING agonist and the antigen.
Clause 10. A STING agonist for use in a method of improving an immune response to an antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject a STING agonist and the antigen.
Clause 11. An antigen for use in a method of improving an immune response to an antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to (ii) administering to the subject a STING agonist and the antigen.
Clause 12. A STING agonist for use in a method of reducing the amount of an antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the antigen and a STING agonist.
Clause 13. An antigen for use in a method of reducing the amount of an antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the antigen and a STING agonist.
Clause 14. A STING agonist for use in a method of preparing the immune system of a subject for (ii) administration of an antigen and a STING agonist, wherein the method comprises (i) administering to the subject a STING agonist.
Clause 15. An antigen for use in a method of preparing the immune system of a subject for administration of an antigen and a STING agonist, wherein the method comprises administering to the subject a STING agonist.
Clause 16. Use of a STING agonist for the manufacture of a medicament for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject a STING agonist and an antigen which is associated with the pathogen.
Clause 17. Use of an antigen for the manufacture of a medicament for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject a STING agonist and an antigen which is associated with the pathogen.
Clause 18. Use of a STING agonist for the manufacture of a medicament for use in a method of reducing the variability of the immune response to an antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist, prior to
(ii) administering to each of the subjects a STING agonist and the antigen.
Clause 19. Use of an antigen for the manufacture of a medicament for use in a method of reducing the variability of the immune response to an antigen in subjects, wherein the method comprises the following steps: (i) administering to each of the subjects a STING agonist, prior to
(ii) administering to each of the subjects a STING agonist and the antigen.
Clause 20. Use of a STING agonist in the manufacture of a medicament for use in a method of improving an immune response to an antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject a STING agonist and the antigen.
Clause 21. Use of an antigen for the manufacture of a medicament for use in a method of improving an immune response to an antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject a STING agonist and the antigen.
Clause 22. Use of a STING agonist for the manufacture of a medicament for use in a method of reducing the amount of an antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the antigen and a STING agonist.
Clause 23. Use of an antigen for the manufacture of a medicament for use in a method of reducing the amount of an antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the antigen and a STING agonist.
Clause 24. Use of a STING agonist for the manufacture of a medicament for use in a method of preparing the immune system of a subject for administration of an antigen and a STING agonist, wherein the method comprises administering to the subject a STING agonist.
Clause 25. Use of a STING agonist for the manufacture of a medicament for use in a method of preparing the immune system of a subject for administration of an antigen and a STING agonist, wherein the method comprises administering to the subject a STING agonist.
Clause 26. Use of an antigen for the manufacture of a medicament for use in a method of preparing the immune system of a subject for (ii) administration of an antigen and a STING agonist, wherein the method comprises (i) administering to the subject a STING agonist.
Clause 27. The method, STING agonist, antigen or use of any one of clauses 1 to 26, wherein the STING agonist binds to STING with an in vitro Kf of less than 0.750 pM, such as less than 0.500 pM. Clause 28. The method, STING agonist, antigen or use of clause 27, wherein the STING agonist binds to STING with an in vitro Kf of less than 0.250 pM, such as less than 0.100 pM.
Clause 29. The method, STING agonist, antigen or use of any one of clauses 1 to 28, wherein the STING agonist activates STING with an in vitro EC50 of 100 pM or less, such as 50 pM or less, when measured by monitoring phosphorylation of interferon regulatory factor-3 (IRF3).
Clause 30. The method, STING agonist, antigen or use of clause 29, wherein the STING agonist activates STING with an in vitro EC50 of 20 pM or less, such as 10 pM or less, when measured by monitoring phosphorylation of interferon regulatory factor-3 (IRF3).
Clause 31. The method, STING agonist, antigen or use of any one of clauses 1 to 30, wherein the STING agonist activates STING with an in vitro EC50 of 100 pM or less, such as 50 pM or less, as measured by monitoring interferon-p induction.
Clause 32. The method, STING agonist, antigen or use of clause 31, wherein the STING agonist activates STING with an in vitro EC50 of 20 pM or less, such as 10 pM or less, as measured by monitoring interferon-p induction.
Clause 33. The method, STING agonist, antigen or use of any one of clauses 1 to 32, wherein the STING agonist is a nucleic acid, a protein, a peptide, or a small molecule.
Clause 34. The method, STING agonist, antigen or use of clause 33, wherein the STING agonist is a small molecule, such as a modified or unmodified cyclic dinucleotide.
Clause 35. The method, STING agonist, antigen or use of clause 33, wherein the STING agonist is selected from a compound of Formulae (l)-(lll):
Figure imgf000161_0001
Figure imgf000162_0001
wherein R1 and R2 are each independently selected from the following groups:
Figure imgf000162_0002
R3 and R4 are each independently -SH or -OH,
R5 and R6 are oxygen or sulphur,
R7 and R8 are each independently halogen, hydrogen, -OH, or OCH3; or a pharmaceutically acceptable salt thereof and/or a tautomer thereof. Clause 36. The method, STING agonist, antigen or use of clause 33, wherein the STING agonist is the compound:
Figure imgf000163_0001
or a pharmaceutically acceptable salt thereof and/or a tautomer thereof.
Clause 37. The method, STING agonist, antigen or use of clause 33, wherein the STING agonist is selected from c-di^GMP. c-di-thGMP, c-GtzGMP, c-GAMP, c-thGMP, c-tzGMP, c-di-thAMP, c-ditzAMP, c-di-AMP, c-di-GMP, c-diXGMP, c-GthXMP, c-GXMP, c-ACMP, c- AthXMP, c-AtzXMP, c-di-thXMP, or c-ditzXMP.
Clause 38. The method, STING agonist, antigen or use of clause 33, wherein the STING agonist is 2’,3’-cGAMP, or a pharmaceutically acceptable salt thereof.
Clause 39. The method, STING agonist, antigen or use of clause 33, wherein the STING agonist is 3’,3’-cGAMP, or a pharmaceutically acceptable salt thereof.
Clause 40. The method, STING agonist, antigen or use of clause 33, wherein the STING agonist is the compound:
Figure imgf000163_0002
, or a pharmaceutically acceptable salt thereof.
Clause 41 . The method, STING agonist, antigen or use of clause 33, wherein the STING agonist is 6-bromo-N-(naphthalen-1-yl)benzo[d][1 ,3]dioxole-5-carboxamide:
Figure imgf000163_0003
Clause 42. The method, STING agonist, antigen or use of any one of clause 33, wherein the STING agonist is a compound of Formula A:
Figure imgf000164_0001
wherein
X is O or NR4A,
Y is O, NR4A, CH2, or absent, n is 0, 1 , 2, or 3,
R1 and R2 are independently selected from OH, OR3, OR3A, SR3, and NR3R4,
R3, R4, and R4A are independently selected from hydrogen, C1 -C10 alkyl optionally substituted with 1-6 halogen, C6-C10 aryl or 5-10 membered heteroaryl, or R3 and R4 together with the nitrogen atom to which they are attached form a 3 to 7 membered heterocycle or 5 to 10 membered heteroaryl,
Figure imgf000164_0002
represents the point of connection of R3A to the remainder of the molecule,
R5-R10 are independently selected from hydrogen, halogen, pseudohalogen, C1-C10 alkyl optionally substituted with 1-6 halogens, C6-C10 aryl, and 5 to 10 membered heteroaryl; or a pharmaceutically acceptable salt thereof and/or a tautomer thereof.
Clause 43. The method, STING agonist, antigen or use of clause 33, wherein the STING agonist is selected from:
Figure imgf000164_0003
pharmaceutically acceptable salt thereof. Clause 44. The method, STING agonist, antigen or use of clause 33, wherein the STING agonist is a flavonoid.
Clause 45. The method, STING agonist, antigen or use of clause 44, wherein the STING agonist is 10-(carboxymethyl)-9(10H)acridone (CMA), 5,6-Dimethylxanthenone-4- acetic acid (DMXAA), methoxyvone, 6,4'-dimethoxyflavone, 4'-methoxyflavone, 3', 6'- dihydroxyflavone, 7,2'-dihydroxyflavone, daidzein, formononetin, retusin 7-methyl ether, xanthone, or any combination thereof.
Clause 46. The method, STING agonist, antigen or use of clause 33, wherein the STING agonist is a compound of Formula B:
Figure imgf000165_0001
wherein
Figure imgf000165_0002
represents two conjugated double bonds in a five-membered heteroaromatic ring and three conjugated double bonds in a six-membered aromatic or heteroaromatic ring,
W1 is selected from CR11 and N;
X1 is selected from CR1, C(R1)2, N, NR1, O and S;
X2 is selected from CR2, C(R2)2, N, NR2, O and S;
X3 is selected from CR3, C(R3)2, N, NR3, O and S; where two or three of X1, X2 and X3 are independently selected from N, NR1, NR2, NR3, O and S; and where at least one of X1, X2 and X3 is selected from N, NR1, NR2 and NR3;
Y1 is selected from N, NR4, O, S, CR4 and C(R4)2;
Y2 is selected from N, NR5, O, S, CR5 and C(R5)2;
Y3 is selected from N, NR6, O, S, CR6 and C(R6)2;
Y4 is selected from C and N;
Y5 is selected from C and N; where at least one and not more than two of Y1, Y2 and Y3 are independently selected from N, NR4, NR5 and NR6; where when if one of Y4 or Y5 is N, the other one of Y4 or Y5 is C;
Z1 is selected from C and N;
Z2 is selected from N, NR8 and CR8; Z3 is selected from N, NR9 and CR9; Z4 is selected from N, NR10 and CR10; Z5 is selected from N, NR7 and CR7; where two or three of Z1, Z2, Z3, Z4 and Z5 are independently selected from N, NR7, NR8, NR9, and NR10; each R1 is independently selected from the group consisting of H, C1-C8 alkyl, C1-C8 alkylene-NRR and C1-C8 alkylene-C(O)OR; each R2 is independently selected from the group consisting of H, C1-C8 alkyl, C1-C8 alkylene-NRR, C1-C8 alkylene-C(O)OR, C1-C8 alkylene-OR and C1-C8 alkylene-O- P(O)(OH)2; each R3 is independently selected from the group consisting of H, C1-C8 alkyl, C1-C8 alkylene-NRR, C1-C8 alkylene-C(O)OR and C1-C8 alkylene-O-P(O)(OH)2; each R4 is independently selected from the group consisting of H, -OR, -NRR, C1-C8 alkyl optionally substituted with one or two -OR, C1-C8 alkylene-NRR, -C(O)OR, C1-C8 alkylene-C(O)OR, 3-10 membered heterocycle, C1-C8 alkylene-3-10 membered heterocycle optionally substituted with one 3-10 membered heterocycle, (C3 -C10)- cycloalkyl, and C1-C8 alkylene-(C3-C10)-cycloalkyl; each R5 is independently selected from the group consisting of H, OR, C1 -C8 alkyl, - NRR, C1-C8 alkylene-NRR, -C(O)OR, C1-C8 alkylene-C(O)OR, 3-10 membered heterocycle, C1-C8 alkylene-3-10 membered heterocycle optionally substituted with one 3-10 membered heterocycle, and C1-C8 alkylene-OR; each R6 is H; R7 is selected from the group consisting of H, halo, hydroxy or NH2; R8 is selected from the group consisting of H, C1-C8 alkyl optionally substituted with one or two -NRR or -OR, C1-C8 alkylene-C(O)OR and C1-C8 alkylene-SO2R; R9 is H; R10 is selected from the group consisting of H, C1-C8 alkyl optionally substituted with one or two -OR, and halo; R11 is selected from the group consisting of H, C1-C8 alkyl, -OR and halo; R12 is -C(O)N(R)2 or -C(O)NHR; R13 is H; each R is independently selected from the group consisting of H or C1-C8 alkyl, or C1-C8 haloalkyl, or two R join to form, together with the atom or atoms to which they are bound, a -C3-C10 cycloalkyl or 3-10 membered heterocycle, where said 3-10 membered heterocycle contains one, two or three atoms selected from N, O and S; and where, when two R join to form, together with the atom or atoms to which they are bound, a –(C3-C10) cycloalkyl or 3-10 membered heterocycle, said -C3-C10 cycloalkyl or 3-10 membered heterocycle is optionally substituted with one or more substituents each independently selected from C1-C8 alkyl, hydroxy, C1-C8 alkoxy, -(C3-C10) cycloalkyl, 3-10 membered heterocycle, halo and cyano; or a pharmaceutically acceptable salt thereof and/or a tautomer thereof. Clause 47. The method, STING agonist, antigen or use of clause 33, wherein the STING agonist is a compound selected from:
Figure imgf000167_0001
Figure imgf000168_0001
Figure imgf000169_0001
Figure imgf000170_0001
Figure imgf000171_0001
Figure imgf000172_0001
Figure imgf000173_0001
or a pharmaceutically acceptable salt thereof and/or a tautomer thereof.
Clause 48. The method, STING agonist, antigen or use of clause 33, wherein the STING agonist is a compound of Formula C:
Figure imgf000174_0001
wherein — CH-(CH2)n-ring A
Gi is independently selected from Ring A or * ring A is independently selected from optionally substituted heterocyclyl and optionally substituted heteroaryl, ring B is aromatic carbocyclic ring, ring C is optionally substituted five-membered heteroaryl,
R1 is -CON(R3)2,
R2 is independently selected from hydrogen, optionally substituted Ci-Ce alkyl, and optionally substituted C3-C5 monocyclic cycloalkyl,
R3 is independently selected from hydrogen, and optionally substituted Ci-Ce alkyl; m is selected from 0, or 1 ; n is selected from 0, 1 , or 2; o is 1 ; p is selected from 0, 1 , or 2; when 'alkyl's substituted, it is substituted with 1 to 4 substituents independently selected from halogen, alkyl, perhaloalkyl, cycloalkyl, heterocyclyl, -N(R4)2, and -OR4; when ‘carbocycle’ or 'cycloalkyl' is substituted, it is substituted with 1 to 4 substituents independently selected from halogen, alkyl, perhaloalkyl, -N(R4)2, and -OR4; when 'heterocycle' or 'heterocyclyl' is substituted, it is substituted with 1 to 4 substituents independently selected from oxo (=0), halogen, cyano, alkyl, perhaloalkyl, -OR4, -C(=O)OH, -OP(O)(OR4)2, -P(O)(OR4)2, -P(O)(OR4)R4a, -SO2R4a, -SO2NH2, - C(=O)N(H)R4, -C(=O)N(alkyl)R4, -N(H)C(=O)R4a, -N(H)R4, and -N(alkyl)R4; when the 'heteroaryl' group is substituted, it is substituted with 1 to 4 substituents selected from halogen, cyano, alkyl, perhaloalkyl, -O-alkyl, -O-perhaloalkyl, - N(alkyl)alkyl, -N(H)R4, -SO2-alkyl, -N(alkyl)C(=O)alkyl, -N(H)C(=O)alkyl, - C(=O)N(alkyl)alkyl, -C(=O)N(H)alkyl, -C(=O)NH2, -SO2N(alkyl)alkyl, -SO2N(H)alkyl, - SO2NH2, -C(=O)OH, -OP(O)(OR4)2, -P(O)(OR4)2, and -P(O)(OR4)R4a; each R4 is independently selected from hydrogen, alkyl, and cycloalkyl; and each R4a is independently selected from alkyl, and cycloalkyl; or a pharmaceutically acceptable salt thereof and/or a tautomer thereof.
Clause 49. The method, STING agonist, antigen or use of clause 33, wherein the STING agonist is selected from:
Figure imgf000175_0001
Figure imgf000176_0001
Figure imgf000177_0001
Figure imgf000178_0001
or a pharmaceutically acceptable salt thereof and/or a tautomer thereof.
Clause 50. The method, STING agonist, antigen or use of clause 33, wherein the STING agonist is selected from IMSA101 , ADU-S100 (MIW815), BMS-986301, CRD5500, CMA (IO-carboxymethyl-9-acridanone), diABZI STING agonist-1 (e.g., CAS No.: 2138299-34- 8), DMXAA (ASA404/vadimezan), E7766 (Cas no. 2242635-02-3)
Figure imgf000178_0002
, MK-1454, MK-2118, SB-11285,
SRCB-0074, TAK-676, and TTI-10001 or a pharmaceutically acceptable salt thereof. Clause 51. The method, STING agonist, antigen or use of clause 33, wherein the
STING agonist is a compound according to Formula (l-N):
Figure imgf000179_0001
wherein: q is 0 or 1; r is 0 or 1; s is 0 or 1; wherein q + r + s = 1 or 2; when q is 0, RA1 and RA2 are each independently H, halogen, hydroxy, –O-P(O)(OH)2, -O-(O)(RIRII)2, -N(Re)(Rf), -CO2Rf, -N(Rf)CORb, -N(Rg)SO2(C1-C4alkyl)-N(Re)(Rf), -N(Rg)CO(C1-C4alkyl)-N(Rh)(Rf), optionally substituted (C1-C6alkyl), optionally substituted (C1-C6alkyl)oxy-, optionally substituted (C1-C6alkyl)amino-, and optionally substituted (C1-C6alkyl)(C1-C4alkyl)amino-, wherein the (C1-C6alkyl) of said optionally substituted (C1-C6alkyl), optionally substituted (C1-C6alkyl)oxy-, optionally substituted (C1-C6alkyl)amino- and optionally substituted (C1-C6alkyl)(C1-C4alkyl)amino- is optionally substituted by 1-4 substituents each independently selected from hydroxy, -O-P(O)(OH)2, -O-P(O)(RIRII)2, C1-C4alkoxy-, -N(Re)(Rf), -CO2(Rf), -CON(Re)(Rf), optionally substituted phenyl, optionally substituted 5-6 membered heterocycloalkyl and optionally substituted 5-6 membered heteroaryl group, wherein said optionally substituted phenyl, 5-6 membered heterocycloalkyl or 5-6 membered heteroaryl is optionally substituted by 1-4 substituents each independently selected from halogen, hydroxy, -O-P(O)(OH)2, -O- P(O)(RIRII)2, amino, (C1-C6alkyl)amino-, (C1-C6alkyl)(C1-C6alkyl)amino-, -(C1-C6alkyl)- NH2, halo(C1-C6alkyl), hydroxy-(C1-C4alkyl)-, -(C1-C4alkyl)-O-P(O)(OH)2, - (C1-C4alkyl)-O-P(O)(RIRII)2, halo(C1-C4alkoxy)-, C1-C4alkoxy-, hydroxy-(C2-C4alkoxy)-, -(C2-C4alkoxy)-O-P(O)(OH)2, -(C2-C4alkoxy)-O-P(O)(RIRII)2, -C1-C4alkyl-(C1-C4alkoxy) or C1-C4alkoxy-(C1-C4alkoxy)-; when r is 0, RB1 and RB2 are each independently H, optionally substituted C1-C6alkyl, halo(C1-C6alkyl), optionally substituted C2-C6alkenyl, optionally substituted C2-C6alkynyl, optionally substituted C3-C6cycloalkyl, optionally substituted 4-6 membered heterocycloalkyl, optionally substituted phenyl, optionally substituted 5-6 membered heteroaryl, or optionally substituted 9-10 membered heteroaryl, wherein said optionally substituted C1-C6alkyl, optionally substituted C2-C6alkenyl, optionally substituted C2-C6alkynyl, optionally substituted C3-C6cycloalkyl, optionally substituted 4-6 membered heterocycloalkyl, optionally substituted phenyl, optionally substituted 5-6 membered heteroaryl, or optionally substituted 9-10 membered heteroaryl is optionally substituted by 1-4 substituents each independently selected from halogen, nitro, -Rc, -OH, -O-P(O)(OH)2, -O- P(O)(RIRII)2, -ORc, -NH2, -NRcRc, -NRcRd, -OCORc, -CO2H, -CO2Rc, -SORc, -SO2Rc, -CONH2, -CONRcRd, -SO2NH2, -SO2NRcRd, -OCONH2, -OCONRcRd, -NRdCORc, -NRdSORc, -NRdCO2Rc, and -NRdSO2Rc; when s is 0, RC1 is H, halogen, or C1-C4alkyl and RC2 is optionally substituted C1-C4alkyl, wherein said optionally substituted C1-C4alkyl group is optionally substituted by a substituent selected from -ORc, -NRcRd, -CO2Rc, -CONRcRd, -SO2NRcRd, and -OCONRcRd; when q is 1, RA1 and RA2 are each independently -CH2-, -NRe-, or -O-, and A, taken together with RA1 and RA2, forms a linking group, wherein A is -halo(C1-C12alkyl)-, optionally substituted -C1-C12alkyl-, optionally substituted -C2-C12alkenyl-, optionally substituted -C2-C12alkynyl-, optionally substituted -C1-C6alkyl-O-C1-C6alkyl-, optionally substituted -C1-C6alkyl-NRa-C1-C6alkyl-, optionally substituted -C1-C6alkyl-(C3-C6cycloalkyl)-C1-C6alkyl-, optionally substituted -C1-C6alkyl-phenyl-C1-C6alkyl-, optionally substituted -C1-C6alkyl-(4-6 membered heterocycloalkyl)-C1-C6alkyl-, or optionally substituted -C1-C6alkyl-(5-6 membered heteroaryl)-C1-C6alkyl-, wherein the alkyl moiety of said optionally substituted -C1-C12alkyl-, optionally substituted -C2-C12alkenyl-, optionally substituted -C2-C12alkynyl-, optionally substituted -C1-C6alkyl-O-C1-C6alkyl-, optionally substituted -C1-C6alkyl-NRa-C1-C6alkyl-, optionally substituted -C1-C6alkyl-(C3-C6cycloalkyl)-C1-C6alkyl-, optionally substituted -C1-C6alkyl-phenyl-C1-C6alkyl-, optionally substituted -C1-C6alkyl-(4-6 membered heterocycloalkyl)-C1-C6alkyl-, or optionally substituted -C1-C6alkyl-(5-6 membered heteroaryl)-C1-C6alkyl- is optionally substituted by 1-4 substituents each independently selected from halogen, halo(C1-C4alkyl), -OH, -O-P(O)(OH)2, -O-P(O)(RIRII)2, -ORc, -NH2, -NRcRd, -OCORc, -CO2H, -CO2Rc, -SORc, -SO2Rc, -CONH2, -CONRcRd, -SO2NH2, -SO2NRcRd, -OCONH2, -OCONRcRd, -NRdCORc, -NRdSORc, -NRdCO2Rc, and -NRdSO2Rc, and the C3-C6cycloalkyl, phenyl, 4-6 membered heterocycloalkyl, or 5-6 membered heteroaryl moiety of said optionally substituted -C1-C6alkyl-(C3-C6cycloalkyl)-C1-C6alkyl-, optionally substituted -C1-C6alkyl-phenyl-C1-C6alkyl-, optionally substituted -C1-C6alkyl-(4-6 membered heterocycloalkyl)-C1-C6alkyl-, or optionally substituted -C1-C6alkyl-(5-6 membered heteroaryl)-C1-C6alkyl- is optionally substituted by 1-4 substituents each independently selected from halogen, hydroxy, -O-P(O)(OH)2, -O-P(O)(RIRII)2, amino, (C1-C4alkyl)amino-, (C1-C4alkyl)(C1-C4alkyl)amino-, C1-C4alkyl, halo(C1-C4alkyl), halo(C1-C4alkoxy)-, C1-C4alkoxy-, hydroxy-(C1-C4alkoxy)-, -(C1-C4alkoxyl)-O-P(O)(OH)2, -(C1-C4alkoxyl)-O-P(O)(RIRII)2 and C1-C4alkoxy-(C1-C4alkoxy)-; when r is 1, RB1 and RB2 are each independently -CH -, and B, taken together wit B1 2 h R and RB2, forms a linking group, wherein B is a bond or B is -halo(C1-C10alkyl)-, optionally substituted -C1-C10alkyl-, optionally substituted -C2-C10alkenyl-, optionally substituted -C2-C10alkynyl-, optionally substituted -C1-C6alkyl-O-C1-C6alkyl-, optionally substituted -C1-C6alkyl-NRa-C1-C6alkyl-, optionally substituted C3-C6cycloalkyl, optionally substituted phenyl, optionally substituted 4-6 membered heterocycloalkyl, optionally substituted 5-6 membered heteroaryl, optionally substituted -C1-C4alkyl-(C3-C6cycloalkyl)-C1-C4alkyl-, optionally substituted -C1-C4alkyl-phenyl-C1-C4alkyl-, optionally substituted -C1-C4alkyl-(4-6 membered heterocycloalkyl)-C1-C4alkyl-, or optionally substituted -C1-C4alkyl-(5-6 membered heteroaryl)-C1-C4alkyl-, wherein the alkyl moiety of said optionally substituted -C1-C10alkyl-, optionally substituted -C2-C10alkenyl-, optionally substituted -C2-C10alkynyl-, optionally substituted -C1-C6alkyl-O-C1-C6alkyl-, optionally substituted -C1-C6alkyl-NRa-C1-C6alkyl-, optionally substituted -C1-C4alkyl-(C3-C6cycloalkyl)-C1-C4alkyl-, optionally substituted -C1-C4alkyl-phenyl-C1-C4alkyl-, optionally substituted -C1-C4alkyl-(4-6 membered heterocycloalkyl)-C1-C4alkyl-, or optionally substituted -C1-C4alkyl-(5-6 membered heteroaryl-C1-C4alkyl)- is optionally substituted by 1 or 2 substituents each independently selected from halogen, halo(C1-C4alkyl), -OH, -O-P(O)(OH)2, -O-P(O)(RIRII)2, -ORc, -NH2, -NRcRd, -OCORc, -CO2H, -CO2Rc, -SORc, -SO2Rc, -CONH2, -CONRcRd, -SO2NH2, -SO2NRcRd, -OCONH2, -OCONRcRd, -NRdCORc, -NRdSORc, -NRdCO2Rc, and -NRdSO2Rc, and the C3-C6cycloalkyl, phenyl, 4-6 membered heterocycloalkyl, or 5-6 membered heteroaryl moiety of said optionally substituted C3-C6cycloalkyl, optionally substituted phenyl, optionally substituted 4-6 membered heterocycloalkyl, optionally substituted 5-6 membered heteroaryl, optionally substituted -C1-C4alkyl-(C3-C6cycloalkyl)-C1-C4alkyl-, optionally substituted -C1-C4alkyl-phenyl-C1-C4alkyl-, optionally substituted -C1-C4alkyl-(4-6 membered heterocycloalkyl)-C1-C4alkyl-, or optionally substituted -C1-C4alkyl-(5-6 membered heteroaryl)-C1-C4alkyl- is optionally substituted by 1-4 substituents each independently selected from halogen, hydroxy, -O-P(O)(OH)2, -O- P(O)(RIRII)2, amino, (C1-C4alkyl)amino-, (C1-C4alkyl)(C1-C4alkyl)amino-, C1-C4alkyl, halo(C1-C4alkyl), halo(C1-C4alkoxy)-, C1-C4alkoxy-, hydroxy-(C2-C4alkoxy)-, -(C2-C4alkoxy) O-P(O)(OH)2, -(C2-C4alkoxy)-O-P(O)(RIRII)2, and C1-C4alkoxy-(C1-C4alkoxy)-; when s is 1, RC1 and RC2 are each independently -CH -, and C, taken toget C1 2 her with R and RC2, forms a linking group, wherein C is -halo(C1-C12alkyl)-, optionally substituted -C1-C12alkyl-, optionally substituted -C2-C12alkenyl-, optionally substituted -C2-C12alkynyl-, optionally substituted -C1-C6alkyl-O-C1-C6alkyl-, optionally substituted -C1-C6alkyl-NRa-C1-C6alkyl-, optionally substituted -C1-C6alkyl-(C3-C6cycloalkyl)-C1-C6alkyl-, optionally substituted -C1-C6alkyl-phenyl-C1-C6alkyl-, optionally substituted -C1-C6alkyl-(4-6 membered heterocycloalkyl)-C1-C6alkyl-, or optionally substituted -C1-C6alkyl-(5-6 membered heteroaryl)-C1-C6alkyl-, wherein the alkyl moiety of said optionally substituted -C1-C12alkyl-, optionally substituted -C2-C12alkenyl-, optionally substituted -C2-C12alkynyl-, optionally substituted -C1-C6alkyl-O-C1-C6alkyl-, optionally substituted -C1-C6alkyl-NRa-C1-C6alkyl-, optionally substituted -C1-C6alkyl-(C3-C6cycloalkyl)-C1-C6alkyl-, optionally substituted -C1-C6alkyl-phenyl-C1-C6alkyl-, optionally substituted -C1-C6alkyl-(4-6 membered heterocycloalkyl)-C1-C6alkyl-, or optionally substituted -C1-C6alkyl-(5-6 membered heteroaryl)-C1-C6alkyl- is optionally substituted by 1 or 2 substituents each independently selected from halogen, halo(C1-C4alkyl), -OH, -O-P(O)(OH)2, -O-P(O)(RIRII)2, -ORc, -NH2, -NRcRd, -OCORc, -CO2H, -CO2Rc, -SORc, -SO2Rc, -CONH2, -CONRcRd, -SO2NH2, -SO2NRcRd, -OCONH2, -OCONRcRd, -NRdCORc, -NRdSORc, -NRdCO2Rc, and -NRdSO2Rc, and the C3-C6cycloalkyl, phenyl, 4-6 membered heterocycloalkyl, or 5-6 membered heteroaryl moiety of said optionally substituted -C1-C6alkyl-(C3-C6cycloalkyl)-C1-C6alkyl-, optionally substituted -C1-C6alkyl-phenyl-C1-C6alkyl-, optionally substituted -C1-C6alkyl-(4-6 membered heterocycloalkyl)-C1-C6alkyl-, or optionally substituted -C1-C6alkyl-(5-6 membered heteroaryl)-C1-C6alkyl- is optionally substituted by 1-4 substituents each independently selected from halogen, hydroxy, -O-P(O)(OH)2, -O-P(O)(RIRII)2, amino, (C1-C4alkyl)amino-, (C1-C4alkyl)(C1-C4alkyl)amino-, C1-C4alkyl, halo(C1-C4alkyl), halo(C1-C4alkoxy)-, C1-C4alkoxy-, hydroxy-(C2-C4alkoxy)-, -(C2-C4alkoxy)-O-P(O)(OH)2, -(C2-C4alkoxy)-O-P(O)(RIRII)2,and C1-C4alkoxy-(C1-C4alkoxy)-; R3 and R5 are each independently -CON(Rd)(Rf), or one of R3 and R5 is -CON(Rd)(Rf), and the other of R3 and R5 is H, COOH or -CO2(Rc); R4 and R6 are each independently selected from H, halogen, halo(C1-C6alkyl), halo(C1-C6alkoxy)-, hydroxy, –O-P(O)(OH)2, –O-P(O)(RIRII)2, -NH2, -NRcRc, -NRcRd, -CORc, -CO2Rc, -N(Rd)CORc, -N(Rd)SO2Rc, -N(Rg)SO2(C1-C2alkyl)-N(Rh)(Rf), -N(Rg)CO(C1-C2alkyl)-N(Rh)(Rf), optionally substituted (C1-C6alkyl), optionally substituted (C1-C6alkyl)oxy-, optionally substituted (C1-C6alkyl)amino-, and optionally substituted (C1-C6alkyl)(C1-C4alkyl)amino-, wherein the (C1-C6alkyl) of said optionally substituted (C1-C6alkyl), optionally substituted (C1-C6alkyl)oxy-, optionally substituted (C1-C6alkyl)amino- and optionally substituted (C1-C6alkyl)(C1-C4alkyl)amino- is optionally substituted by 1-4 substituents each independently selected from -OH, -O-P(O)(OH)2, -O-P(O)(RIRII)2,-ORc, -NH2, -NRcRc, -NRcRd, -CO2H, -CO2Rc, -OCORc, -CO2H, -CO2Rc, -SORc, -SO2Rc, -CONH2, -CONRcRd, -SO2NH2, -SO2NRcRd, -OCONH2, -OCONRcRd, -NRdCORc, -NRdSORc, -NRdCO2Rc, -NRdSO2Rc, optionally substituted phenyl, optionally substituted 5-6 membered heterocycloalkyl and optionally substituted 5-6 membered heteroaryl group, wherein said optionally substituted phenyl, 5-6 membered heterocycloalkyl or 5-6 membered heteroaryl is optionally substituted by 1-4 substituents each independently selected from halogen, hydroxy, -O-P(O)(OH)2, -O- P(O)(RIRII)2, amino, (C1-C4alkyl)amino-, (C1-C4alkyl)(C1-C4alkyl)amino-, C1-C4alkyl, halo(C1-C4alkyl), hydroxy-(C1-C4alkyl)-, -(C1-C4alkyl)-O-P(O)(OH)2, -(C1-C4alkyl)-O-P(O)(RIRII)2, halo(C1-C4alkoxy)-, C1-C4alkoxy-, hydroxy-(C2-C4alkoxy)-, -(C2-C4alkoxy)-O-P(O)(OH)2, -(C2-C4alkoxy)-O-P(O)(RIRII)2, C1-C4alkoxy-(C1-C4alkoxy)-, -CORd, -CON(Rd)(Rf), and -CO2Rd; R14 is optionally substituted C1-C4alkyl, wherein said optionally substituted C1-C4alkyl is optionally substituted by a substituent selected from -ORc, -NRcRd, -CO2Rc, -CONRcRd, -SO2NRcRd, and -OCONRcRd; R16 is H, halogen, or C1-C4alkyl; R15 and R17 are each independently H, cyclopropyl, or C1-C4alkyl; Ra is H, -Rc, -CORc, -CO2H, -CO2Rc, -SORc, -SO2Rc, -CONH2, -CONRcRd, -SO2NH2, or -SO2NRcRd; each Rb is independently C1-C4alkyl, halo(C1-C4alkyl), -(C1-C4alkyl)-OH, -(C1-C4alkyl)-O-P(O)(OH)2, -(C1-C4alkyl)-O-P(O)(RIRII)2, -(C1-C4alkyl)-O-(C1-C4alkyl), -(C1-C4alkyl)-N(Re)(Rf), -(C1-C4alkyl)-O-CO(C1-C4alkyl), or -(C1-C4alkyl)-CO-O- (C1-C4alkyl); each Rc is independently C1-C4alkyl, halo(C1-C4alkyl), -(C1-C4alkyl)-OH, -(C1-C4alkyl)-O-P(O)(OH)2, -(C1-C4alkyl)-O-P(O)(RIRII)2,-(C1-C4alkyl)-O-(C1-C4alkyl), -(C1-C4alkyl)-N(Re)(Rf), -(C1-C4alkyl)-O-CO(C1-C4alkyl), -(C1-C4alkyl)-CO-O- (C1-C4alkyl), optionally substituted C3-C6cycloalkyl, optionally substituted phenyl, optionally substituted 4-6 membered heterocycloalkyl, optionally substituted 5-6 membered heteroaryl, optionally substituted 9-10 membered heteroaryl, optionally substituted -C1-C4alkyl-C3-C6cycloalkyl, optionally substituted -C1-C4alkyl-phenyl, optionally substituted -C1-C4alkyl-4-6 membered heterocycloalkyl, optionally substituted -C1-C4alkyl-5-6 membered heteroaryl, or optionally substituted -C1-C4alkyl-9-10 membered heteroaryl, wherein the C3-C6cycloalkyl, phenyl, 4-6 membered heterocycloalkyl, 5-6 membered heteroaryl or 9-10 membered heteroaryl moiety of said optionally substituted C3-C6cycloalkyl, optionally substituted phenyl, optionally substituted 4-6 membered heterocycloalkyl, optionally substituted 5-6 membered heteroaryl, optionally substituted 9-10 membered heteroaryl optionally substituted -C1-C4alkyl-C3-C6cycloalkyl, optionally substituted -C1-C4alkyl-phenyl, optionally substituted -C1-C4alkyl-4-6 membered heterocycloalkyl, optionally substituted -C1-C4alkyl-5-6 membered heteroaryl, or optionally substituted -C1-C4alkyl-9-10 membered heteroaryl is optionally substituted by 1-4 substituents each independently selected from halogen, hydroxy, -O-P(O)(OH)2, -O-P(O)(RIRII)2, amino, -(C1-C4alkyl)NH2, (C1-C4alkyl)amino-, (C1-C4alkyl)(C1-C4alkyl)amino-, C1-C4alkyl, halo(C1-C4alkyl), halo(C1-C4alkoxy)-, C1-C4alkoxy-, hydroxy-(C2-C4alkoxy)-, -(C2-C4alkoxy)-O-P(O)(OH)2, -(C2-C4alkoxy)-O-P(O)(RIRII)2, C1-C4alkoxy-(C1-C4alkoxy)-, -CORd, -CON(Rd)(Rf), and -CO2Rd; each Rd is independently H or C1-C4alkyl; each Re is independently H,
(Ci-C4alkyl), -CO(Ci-C4alkyl), -OCO(Ci-C4alkyl), -CO2(Ci-C4alkyl),
-(Ci-C4alkyl)NH2, -(Ci-C4alkyl) Ci-C4alkoxy, -CO-(optionally substituted 5-6 membered heterocycloalkyl), -CO(Ci-C4alkyl)-(optionally substituted 5-6 membered heterocycloalkyl), -CO(optionally substituted 5-6 membered heteroaryl), -CO(Ci-C4alkyl)-(optionally substituted 5-6 membered heteroaryl), wherein the optionally substituted 5-6 membered heterocycloalkyl or optionally substituted 5-6 membered heteroaryl is optionally substituted 1-4 substituents each independently selected from halogen, hydroxy, -O-P(O)(OH)2, -O-P(O)(RIR")2, amino, (Ci-C4alkyl)amino-, (Ci-C4alkyl)(Ci-C4alkyl)amino-, Ci-C4alkyl, halo(Ci-C4alkyl), halo(Ci-C4alkoxy)-, Ci-C4alkoxy-, hydroxy-(C2-C4alkoxy)-,
-(C2-C4alkoxy) O-P(O)(OH)2, -(C2-C4alkoxy)-O-P(O)(RIR")2, Ci-C4alkoxy-(Ci-C4alkoxy)-
, -CORd, -CON(Rd)(Rf), and -CO2Rd; each Rf is independently H or (Ci-C4alkyl);
Rg and Rh are each independently H or (Ci-C4alkyl) or Rg and Rh, taken together with the atom or atoms through which they are connected, form a 5-6 membered ring; and each occurrence of R1 and R" are independently (Ci-Cealkyl)oxy-; or a pharmaceutically acceptable salt thereof and/or a tautomer thereof.
Clause 52. The method, STING agonist, antigen or use of clause 33, wherein the STING agonist is selected from:
(E)-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1 H-pyrazole-5-carboxamido)-1 H- benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1 H-pyrazole-5-carboxamido)-7- (3-hydroxypropoxy)-1 H-benzo[d]imidazole-5-carboxamide
Figure imgf000185_0001
(E)-1-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-2,3- dihydro-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-7-(3-hydroxypropoxy)-2,3-dihydro-1 H-benzo[d]imidazole-5- carboxamide
Figure imgf000186_0001
(Z)-1-((E)-4-((Z)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-2,3- dihydro-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-7-(3-hydroxypropoxy)-2,3-dihydro-1 H-benzo[d]imidazole-5- carboxamide
Figure imgf000186_0002
(E)-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1 H-pyrazole-5-carboxamido)-7-(3- hydroxypropoxy)-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1 H- pyrazole-5-carboxamido)-7-methoxy-1 H-benzo[d]imidazole-5-carboxamide
Figure imgf000186_0003
(E)-1-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-7-(3- hydroxypropoxy)-2,3-dihydro-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3- methyl-1 H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1 H-benzo[d]imidazole-5- carboxamide
Figure imgf000187_0001
(Z)-1-((E)-4-((Z)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-7-(3- hydroxypropoxy)-2,3-dihydro-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3- methyl-1 H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1 H-benzo[d]imidazole-5- carboxamide
Figure imgf000187_0002
(E)-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1 H-pyrazole-5-carboxamido)-1 H- benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1 H-pyrazole-5-carboxamido)-7-
(3-morpholinopropoxy)-1 H-benzo[d]imidazole-5-carboxamide
Figure imgf000187_0003
(E)-1-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-2,3- dihydro-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-7-(3-morpholinopropoxy)-2,3-dihydro-1 H-benzo[d]imidazole-5- carboxamide
Figure imgf000188_0001
(Z)-1-((E)-4-((Z)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-2,3- dihydro-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-7-(3-morpholinopropoxy)-2,3-dihydro-1 H-benzo[d]imidazole-5- carboxamide
Figure imgf000188_0002
(E)-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1 H-pyrazole-5-carboxamido)-7-(3- morpholinopropoxy)-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1 H- pyrazole-5-carboxamido)-7-methoxy-1 H-benzo[d]imidazole-5-carboxamide
Figure imgf000188_0003
(E)-1-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-7-(3- morpholinopropoxy)-2,3-dihydro-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3- methyl-1 H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1 H-benzo[d]imidazole-5- carboxamide
Figure imgf000189_0001
(Z)-1-((E)-4-((Z)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-7-(3- morpholinopropoxy)-2,3-dihydro-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3- methyl-1 H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1 H-benzo[d]imidazole-5- carboxamide
Figure imgf000189_0002
3-(((Z)-6-carbamoyl-3-((E)-4-((Z)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-7-methoxy-2,3-dihydro-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1- ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1 H-benzo[d]imidazol-4- yl)oxy)propyldihydrogen phosphate
Figure imgf000189_0003
(E)-3-((5-carbamoyl-1 -(4-(5-carbamoyl-2-(1 -ethyl-3-methyl-1 H-pyrazole-5- carboxamido)-7-methoxy-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl- 1 H-pyrazole-5-carboxamido)-1 H-benzo[d]imidazol-7-yl)oxy)propyl dihydrogen phosphate
Figure imgf000190_0001
3-(((E)-6-carbamoyl-3-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-7-methoxy-2,3-dihydro-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1- ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1 H-benzo[d]imidazol-4- yl)oxy)propyl dihydrogen phosphate
Figure imgf000190_0002
(E)-4-((5-carbamoyl-1 -(4-(5-carbamoyl-2-(1 -ethyl-3-methyl-1 H-pyrazole-5- carboxamido)-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1 H-pyrazole-
5-carboxamido)-1 H-benzo[d]imidazol-7-yl)oxy)butanoic acid
Figure imgf000190_0003
(E)-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1 H-pyrazole-5-carboxamido)-7-methoxy-1 H- benzo[d]imidazol-1-yl)but-2-en-1-yl)-7-(3-(dimethylamino)propoxy)-2-(1-ethyl-3-methyl-
1 H-pyrazole-5-carboxamido)-1 H-benzo[d]imidazole-5-carboxamide
Figure imgf000191_0001
(E)-1-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-7-(3-
(4-(2-hydroxyethyl)piperazin-1-yl)propoxy)-2,3-dihydro-1 H-benzo[d]imidazol-1-yl)but-2- en-1-yl)-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1 H- benzo[d]imidazole-5-carboxamide
Figure imgf000191_0002
or a pharmaceutically acceptable salt thereof and/or a tautomer thereof.
Clause 53. The method, STING agonist, antigen or use of clause 33, wherein the STING agonist is 3-(((E)-6-carbamoyl-3-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H- pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2- ((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1 H-benzo[d]imidazol-4- yl) oxy) propyl
Figure imgf000192_0001
or a pharmaceutically acceptable salt thereof.
Clause 54. The method, STING agonist, antigen or use of any one of clauses 1 to 53 wherein the STING agonist of step (i) is the same as the STING agonist of step (ii).
Clause 55. The method, STING agonist, antigen or use of any one of clauses 1 to 53 wherein the STING agonist of step (i) is different to the STING agonist of step (ii).
Clause 56. The method, STING agonist, antigen or use of clause 55 wherein the STING agonist of step (i) is different to the STING agonist of step (ii) and wherein the STING agonist of step (i) is selected from the STING agonists defined in any one of clauses 27 to 53 and the STING agonist of step (ii) is selected from the STING agonists defined in any one of clauses 27 to 53.
Clause 57. The method, STING agonist, antigen or use of clause 55 wherein the STING agonist of step (i) is different to the STING agonist of step (ii) but the mode of action of both STING agonists is essentially identical.
Clause 58. The method, STING agonist, antigen or use of any one of clauses 1 to 57 wherein only one administration of the STING agonist is performed in step (i).
Clause 59. The method, STING agonist, antigen or use of any one of clauses 1 to 57 wherein at least 2 administrations of the STING agonist are performed in step (i).
Clause 60. The method, STING agonist, antigen or use of clause 59 wherein at least 3 administrations of the STING agonist are performed in step (i).
Clause 61. The method, STING agonist, antigen or use of clause 59 wherein 2 administrations of the STING agonist are performed in step (i).
Clause 62. The method, STING agonist, antigen or use of any one of clauses 1 to 61 wherein the STING agonist of step (i) is administered intradermally, intramuscularly, intraperitoneally or subcutaneously.
Clause 63. The method, STING agonist, antigen or use of clause 62 wherein the STING agonist of step (i) is administered intramuscularly. Clause 64. The method, STING agonist, antigen or use of either clause 62 or 63 wherein the volume of the STING agonist in step (i) is 0.05 ml to 1 ml, such as 0.1 to 0.6 ml.
Clause 65. The method, STING agonist, antigen or use of any one of clauses 1 to
64 wherein the pH of the STING agonist in step (i) is 4 to 9, especially 5 to 8.5, in particular 5.5 to 8, such as 6.5 to 7.4.
Clause 66. The method, STING agonist, antigen or use of any one of clauses 1 to
65 wherein the osmolarity of the STING agonist in step (i) is 250 to 750 mOsm/kg, especially 250 to 550 mOsm/kg, in particular 270 to 500 mOsm/kg, such as 270 to 400 mOsm/kg.
Clause 67. The method, STING agonist, antigen or use of any one of clauses 1 to
66 wherein the STING agonist of step (i) comprises buffer and/or tonicity modifying agents, such as modified phosphate buffered saline.
Clause 68. The method, STING agonist, antigen or use of any one of clauses 1 to
67 wherein in step (i) the period between administrations to the subject is 2 weeks to 12 months.
Clause 69. The method, STING agonist, antigen or use of clause 68 wherein in step (i) the period between administrations to the subject is 2 weeks to 6 months.
Clause 70. The method, STING agonist, antigen or use of clause 69 wherein in step (i) the period between administrations to the subject is 2 weeks to 3 months.
Clause 71. The method, STING agonist, antigen or use of clause 70 wherein in step
(i) the period between administrations to the subject is 2 weeks to 2 months, such as 3 to 5 weeks.
Clause 72. The method, STING agonist, antigen or use of any one of clauses 1 to 71 wherein step (ii) is initiated at an interval of 2 weeks to 2 years after step (i).
Clause 73. The method, STING agonist, antigen or use of clause 72 wherein step
(ii) is initiated at an interval of 2 weeks to 1 year after step (i).
Clause 74. The method, STING agonist, antigen or use of clause 73 wherein step (ii) is initiated at an interval of 3 weeks to 9 months after step (i).
Clause 75. The method, STING agonist, antigen or use of clause 74 wherein step (ii) is initiated at an interval of 4 weeks to 6 months after step (i).
Clause 76. The method, STING agonist, antigen or use of clause 72 wherein step (ii) is initiated at an interval of 2 weeks to 2 months after step (i).
Clause 77. The method, STING agonist, antigen or use of clause 72 wherein step (ii) is initiated at an interval of 1 month to 3 months after step (i).
Clause 78. The method, STING agonist, antigen or use of clause 72 wherein step (ii) is initiated at an interval of 2 months to 4 months after step (i).
Clause 79. The method, STING agonist, antigen or use of clause 73 wherein step (ii) is initiated at an interval of 3 months to 6 months after step (i). Clause 80. The method, STING agonist, antigen or use of clause 73 wherein step (ii) is initiated at an interval of 4 months to 12 months after step (i).
Clause 81. The method, STING agonist, antigen or use of any one of clauses 1 to 80 wherein only one administration of the STING agonist and antigen is performed in step (ii).
Clause 82. The method, STING agonist, antigen or use of any one of clauses 1 to 80 wherein at least 2 administrations of the STING agonist and antigen are performed in step (ii).
Clause 83. The method, STING agonist, antigen or use of clause 82 wherein at least 3 administrations of the STING agonist and antigen are performed in step (ii).
Clause 84. The method, STING agonist, antigen or use of clause 83 wherein 3 administrations of the STING agonist and antigen are performed in step (ii).
Clause 85. The method, STING agonist, antigen or use of any one of clauses 1 to 84 wherein in step (ii) the STING agonist is co-formulated with the antigen.
Clause 86. The method, STING agonist, antigen or use of clause 85 wherein the coformulated STING agonist and antigen of step (ii) is administered intradermally, intramuscularly, intraperitoneally or subcutaneously, in particular intramuscularly.
Clause 87. The method, STING agonist, antigen or use of clause 85 or 86 wherein the volume of the co-formulation of STING agonist and antigen in step (ii) is 0.05 ml to 1 ml, such as 0.1 to 0.6 ml.
Clause 88. The method, STING agonist, antigen or use of any one of clauses 85 to 87 wherein the pH of the co-formulation of STING agonist and antigen in step (ii) is 4 to 9, especially 5 to 8.5, in particular 5.5 to 8, such as 6.5 to 7.4.
Clause 89. The method, STING agonist, antigen or use of any one of clauses 85 to 87 wherein the osmolarity of the co-formulation of STING agonist and antigen in step (ii) is 250 to 750 mOsm/kg, especially 250 to 550 mOsm/kg, in particular 270 to 500 mOsm/kg, such as 270 to 400 mOsm/kg.
Clause 90. The method, STING agonist, antigen or use of any one of clauses 85 to 87 wherein the co-formulation of STING agonist and antigen of step (ii) comprises buffer and/or tonicity modifying agents, such as modified phosphate buffered saline.
Clause 91. The method, STING agonist, antigen or use of any one of clauses 1 to 84 wherein in step (ii) the STING agonist is formulated separately from the antigen.
Clause 92. The method, STING agonist, antigen or use of clause 91 wherein in step (ii) for at least one administration, the STING agonist is administered simultaneously with the antigen.
Clause 93. The method, STING agonist, antigen or use of clause 92 wherein in step (ii) for all administrations the STING agonist is administered simultaneously with the antigen. Clause 94. The method, STING agonist, antigen or use of clause 91 wherein in step (ii) for at least one administration, the STING agonist is administered separately from the antigen.
Clause 95. The method, STING agonist, antigen or use of clause 94 wherein in step (ii) for at least one administration, the STING agonist is administered before the antigen.
Clause 96. The method, STING agonist, antigen or use of clause 95 wherein in step (ii) for all administrations the STING agonist is administered before the antigen.
Clause 97. The method, STING agonist, antigen or use of clause 94 wherein in step (ii) for at least one administration, the antigen is administered before the STING agonist.
Clause 98. The method, STING agonist, antigen or use of clause 97 wherein in step (ii) for all administrations the antigen is administered before the STING agonist.
Clause 99. The method, STING agonist, antigen or use of any one of clauses 91 to 98 wherein in step (ii) for at least one administration, the STING agonist is administered to the same location on the subject as the antigen.
Clause 100. The method, STING agonist, antigen or use of clause 99 wherein in step (ii) for all administrations the STING agonist is administered to the same location on the subject as the antigen.
Clause 101. The method, STING agonist, antigen or use of any one of clauses 91 to 98 wherein in step (ii) for at least one administration, the STING agonist is administered to a different location on the subject as the antigen.
Clause 102. The method, STING agonist, antigen or use of clause 101 wherein in step (ii) for all administrations the STING agonist is administered to a different location on the subject as the antigen.
Clause 103. The method, STING agonist, antigen or use of any one of clauses 91 to 102 wherein in step (ii) for at least one administration, the STING agonist and the antigen are administered within 84 hours of each other.
Clause 104. The method, STING agonist, antigen or use of clause 103 wherein in step (ii) for at least one administration, the STING agonist and the antigen are administered within 30 minutes of each other.
Clause 105. The method, STING agonist, antigen or use of clause 103 wherein in step (ii) for all administrations the STING agonist and the antigen are administered within 84 hours of each other.
Clause 106. The method, STING agonist, antigen or use of clause 105 wherein in step (ii) for all administrations the STING agonist and the antigen are administered within 30 minutes of each other. Clause 107. The method, STING agonist, antigen or use of any one of clauses 91 to
106 wherein the STING agonist of step (ii) is administered intradermally, intramuscularly, intraperitoneally or subcutaneously, in particular intramuscularly.
Clause 108. The method, STING agonist, antigen or use of any one of clauses 91 to
107 wherein the antigen of step (ii) is administered intradermally, intramuscularly, intraperitoneally or subcutaneously, in particular intramuscularly.
Clause 109. The method, STING agonist, antigen or use of any one of clauses 91 to
108 wherein the volume of the separate formulation of STING agonist in step (ii) is 0.05 ml to 1 ml, such as 0.1 to 0.6 ml.
Clause 110. The method, STING agonist, antigen or use of any one of clauses 91 to
109 wherein the volume of the separate formulation of antigen in step (ii) is 0.05 ml to 1 ml, such as 0.1 to 0.6 ml.
Clause 111. The method, STING agonist, antigen or use of any one of clauses 91 to
110 wherein the pH of the separate formulation of STING agonist in step (ii) is 4 to 9, especially 5 to 8.5, in particular 5.5 to 8, such as 6.5 to 7.4.
Clause 112. The method, STING agonist, antigen or use of any one of clauses 91 to
111 wherein the pH of the separate formulation of antigen in step (ii) is 4 to 9, especially 5 to 8.5, in particular 5.5 to 8, such as 6.5 to 7.4.
Clause 113. The method, STING agonist, antigen or use of any one of clauses 91 to
112 wherein the osmolarity of the separate formulation of STING agonist in step (ii) is 250 to 750 mOsm/kg, especially 250 to 550 mOsm/kg, in particular 270 to 500 mOsm/kg, such as 270 to 400 mOsm/kg.
Clause 114. The method, STING agonist, antigen or use of any one of clauses 91 to
113 wherein the osmolarity of the separate formulation of antigen in step (ii) is 250 to 750 mOsm/kg, especially 250 to 550 mOsm/kg, in particular 270 to 500 mOsm/kg, such as 270 to 400 mOsm/kg.
Clause 115. The method, STING agonist, antigen or use of any one of clauses 91 to
114 wherein the separate formulation comprising the STING agonist of step (ii) comprises buffer and/or tonicity modifying agents, such as modified phosphate buffered saline.
Clause 116. The method, STING agonist, antigen or use of any one of clauses 91 to
115 wherein the separate formulation comprising the antigen of step (ii) comprises buffer and/or tonicity modifying agents, such as modified phosphate buffered saline.
Clause 117. The method, STING agonist, antigen or use of any one of clauses 1 to
116 wherein in step (ii) the period between administrations to the subject is 2 weeks to 12 months.
Clause 118. The method, STING agonist, antigen or use of clause 117 wherein in step (ii) the period between administrations to the subject is 2 weeks to 6 months. Clause 119. The method, STING agonist, antigen or use of clause 118 wherein in step (ii) the period between administrations to the subject is 2 weeks to 3 months.
Clause 120. The method, STING agonist, antigen or use of clause 119 wherein in step (ii) the period between administrations to the subject is 2 weeks to 2 months, such as 3 to 5 weeks.
Clause 121. The method, STING agonist, antigen or use of any one of clauses 1 to 120 wherein the combined duration of step (i), step (ii) and the interval between the steps is 1 month to 2 years.
Clause 122. The method, STING agonist, antigen or use of clause 121 wherein the combined duration of step (i), step (ii) and the interval between the steps is 6 weeks to 12 months.
Clause 123. The method, STING agonist, antigen or use of clause 122 wherein the combined duration of step (i), step (ii) and the interval between the steps is 2 months to 10 months.
Clause 124. The method, STING agonist, antigen or use of clause 123 wherein the combined duration of step (i), step (ii) and the interval between the steps is 10 weeks to 9 months.
Clause 125. The method, STING agonist, antigen or use of any one of any one of clauses 1 to 124 wherein the combined duration of step (i), step (ii) and the interval between the steps is at least 2 months, especially at least 3 months, in particular at least 4 months.
Clause 126. The method, STING agonist, antigen or use of clause 125 wherein the combined duration of step (i), step (ii) and the interval between the steps is at least 5 months, especially at least 6 months, in particular at least 7 months.
Clause 127. The method, STING agonist, antigen or use of any one of any one of clauses 1 to 126 wherein the combined duration of step (i), step (ii) and the interval between the steps is 5 years or less, especially 3 years or less, in particular 2 years or less.
Clause 128. The method, STING agonist, antigen or use of clause 127 wherein the combined duration of step (i), step (ii) and the interval between the steps is 18 months or less, especially 12 months or less, in particular 10 months or less.
Clause 129. The method, STING agonist, antigen or use of any one of clauses 1 to 128 wherein the subject is a mammal.
Clause 130. The method, STING agonist, antigen or use of clause 129 wherein the subject is a human.
Clause 131. The method, STING agonist, antigen or use of clause 130 wherein the subject is a human child.
Clause 132. The method, STING agonist, antigen or use of clause 130 wherein the subject is a human adult. Clause 133. The method, STING agonist, antigen or use of clause 130 wherein the subject is an older human.
Clause 134. The method, STING agonist, antigen or use of any one of clauses 1 to 133 wherein the administration route used for each administration of step (i) is the same as the administration route used for each administration of step (ii).
Clause 135. The method, STING agonist, antigen or use of clause 134 wherein the administration route used is intramuscular.
Clause 136. The method, STING agonist, antigen or use of any one of clauses 1 to 135 wherein the antigen is derived from bacteria, viruses or parasites (such as containing a portion of a polypeptide sequence obtainable from, such as at least 50, especially at least 100, in particular at least 150, such as at least 200 amino acids long).
Clause 137. The method, STING agonist, antigen or use of clause 136 wherein the antigen is derived from a bacterium.
Clause 138. The method, STING agonist, antigen or use of clause 136 wherein the antigen is derived from a virus.
Clause 139. The method, STING agonist, antigen or use of any one of clauses 1 to 138 wherein the antigen is a polypeptide, a polynucleotide or a polysaccharide.
Clause 140. The method, STING agonist, antigen or use of clause 139 wherein the antigen is a polypeptide.
Clause 141. The method, STING agonist, antigen or use of clause 140, wherein the polypeptide comprises at least one B or T cell epitope.
Clause 142. The method, STING agonist, antigen or use of clause 141 , wherein the polypeptide comprises B and T cell epitopes.
Clause 143. The method, STING agonist, antigen or use of any one of clauses 1 to 142 wherein the antigen is derived from a bacterium.
Clause 144. The method, STING agonist, antigen or use of clause 143 wherein the antigen is derived from Clostridia.
Clause 145. The method, STING agonist, antigen or use of clause 144 wherein the antigen is derived from C. difficile.
Clause 146. The method, STING agonist, antigen or use of clause 145 wherein the antigen is C. difficile toxin A and/or toxin B or fragments thereof.
Clause 147. The method, STING agonist, antigen or use of clause 146 wherein the antigen is the F2 antigen.
Clause 148. The method, STING agonist, antigen or use of clause 147 wherein the F2 antigen comprises an amino acid sequence having at least 80%, such as at least 90%, especially at least 95%, in particular at least 98% for example at least 99% or 100% identity to SEQ ID NO: 2. Clause 149. The method, STING agonist, antigen or use of any one of clauses 1 to 142 wherein the antigen is a cancer antigen, such as a tumour antigen and/or a neoantigen.
Clause 150. The method, STING agonist, antigen or use of clause 149 wherein the antigen is a human cancer antigen, such as a human tumour antigen and/or a neoantigen.
Clause 151. The method, STING agonist, antigen or use of any one of clauses 1 to 150 wherein the antigen is provided in the form of a polynucleotide encoding the antigen.
Clause 152. The method, STING agonist, antigen or use of clause 151 wherein the antigen is provided in the form of mRNA encoding the antigen.
Clause 153. The method, STING agonist, antigen or use of clause 152 wherein the mRNA is carrier-formulated, such as formulated in LNP, CNE or LION.
Clause 154. The method, STING agonist, antigen or use clause 153 wherein the carrier is LNP.
Clause 155. The method, STING agonist, antigen or use of any one of clauses 1 to 154, wherein the immune response to the antigen is a serum titer of IgG antibodies binding to the antigen.
Clause 156. The method, STING agonist, antigen or use of clause 155, wherein the IgG antibodies are neutralising antibodies.
Clause 157. The method, STING agonist, antigen or use of any one of clauses 1 to 156, wherein the immune response to the antigen is a T cell response specific to the antigen.
Clause 158. The method, STING agonist, antigen or use of clause 157, wherein the T cell response is a CD4+ T cell response.
Clause 159. The method, STING agonist, antigen or use of clause 157, wherein the T cell response is a CD8+ T cell response.
Clause 160. The method, STING agonist, antigen or use of clause 159, wherein the T cell response is a CD4+ and CD8+ T cell response.
Clause 161. The method, STING agonist, antigen or use of any one of clauses 1 to
160, wherein the immune response to the antigen reduces partially or completely the severity of one or more symptoms experienced by the subject due to infection by a pathogen.
Clause 162. The method, STING agonist, antigen or use of any one of clauses 1 to
161 , wherein the immune response to the antigen reduces partially or completely the time over which one or more symptoms are experienced by the subject due to infection by a pathogen.
Clause 163. The method, STING agonist, antigen or use of any one of clauses 1 to
162, wherein the immune response to the antigen reduces the likelihood of developing an established infection after challenge with the pathogen.
Clause 164. The method, STING agonist, antigen or use of any one of clauses 1 to
163, wherein the immune response to the antigen slows progression of illness (e.g. extends survival) due to infection by the pathogen. Clause 165. The method, STING agonist, antigen or use of any one of clauses 1 to 164, wherein the breadth of the immune response to the antigen in the subject is increased.
Clause 166. The method, STING agonist, antigen or use of clause 165, wherein serum neutralising antibodies bind to an increased range of variants of the antigen.
Clause 167. The method, STING agonist, antigen or use of either clause 165 or 166, wherein the immune response to the antigen is elicited in a wider variety of human HLA types.
Clause 168. The method, STING agonist, antigen or use of any one of clauses 1 to 167, wherein the level of the immune response is increased, such as by at least 50%, such as 100%, such as 150%.
Clause 169. The method, STING agonist, antigen or use of clause 168, wherein the level of the immune response to the antigen is increased, such as by at least 200%, such as 300%, such as 400%.
Clause 170. The method, STING agonist, antigen or use of any one of clauses 1 to
169, wherein the immune response to the antigen is maintained for at least 1 year, such as at least 3 years.
Clause 171. The method, STING agonist, antigen or use of any one of clauses 1 to
170, wherein the duration of the immune response to the antigen is increased, such as by at least 1 year, such as by at least 3 years.
Clause 172. The method, STING agonist, antigen or use of any one of clauses 1 to
171 , wherein the variability of the immune response to the antigen between subjects is decreased.
Clause 173. The method, STING agonist, antigen or use of clause 172, wherein the standard deviation of the immune response to the antigen is reduced.
Clause 174. The method, STING agonist, antigen or use of either clause 172 or 173, wherein the proportion of subjects meeting a specified threshold of immune response to the antigen is increased.
Clause 175. The method, STING agonist, antigen or use of any one of clauses 1 to 174, wherein the speed of the immune response to the antigen is increased.
Clause 176. The method, STING agonist, antigen or use of clause 175, wherein the time taken to achieve a protective immune response is reduced.
Clause 177. The method, STING agonist, antigen or use of either clause 175 or 176, wherein the time taken to reach peak immune response is reduced.
Clause 178. The method, STING agonist, antigen or use of any one of clauses 1 to 177, wherein the amount of antigen required to achieve a desired immune response, such as a protective immune response, is reduced. Clause 179. The method, STING agonist, antigen or use of any one of clauses 1 to 178, wherein the spacing between administrations of antigen may be reduced while maintaining immune response.
The invention is also illustrated by reference to the following further clauses.
These ‘B’ clauses relate to embodiments wherein a STING agonist and a first antigen are administered in step (i) and a STING agonist and a second antigen are administered in step (ii).
Clause 1 B. A method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a STING agonist and a second antigen which is associated with the pathogen.
Clause 2B. A method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist and a first antigen, prior to
(ii) administering to each of the subjects a STING agonist and the second antigen.
Clause 3B. A method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a STING agonist and the second antigen.
Clause 4B. A method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen and a STING agonist.
Clause 5B. A method for preparing the immune system of a subject for (ii) administration of a second antigen and a STING agonist, comprising (i) administering to the subject a STING agonist and a first antigen.
Clause 6B. A STING agonist for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a STING agonist and a second antigen which is associated with the pathogen.
Clause 7B. A first antigen for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to (ii) administering to the subject a STING agonist and a second antigen which is associated with the pathogen.
Clause 8B. A second antigen for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a STING agonist and a second antigen which is associated with the pathogen.
Clause 9B. A STING agonist for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist and a first antigen, prior to
(ii) administering to each of the subjects a STING agonist and the second antigen.
Clause 10B. A first antigen for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist and a first antigen, prior to
(ii) administering to each of the subjects a STING agonist and the second antigen.
Clause 11 B. A second antigen for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist and a first antigen, prior to
(ii) administering to each of the subjects a STING agonist and the second antigen.
Clause 12B. A STING agonist for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a STING agonist and the second antigen.
Clause 13B. A first antigen for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a STING agonist and the second antigen. Clause 14B. A second antigen for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a STING agonist and the second antigen.
Clause 15B. A STING agonist for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen and a STING agonist.
Clause 16B. A first antigen for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen and a STING agonist.
Clause 17B. A second antigen for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen and a STING agonist.
Clause 18B. A STING agonist for use in a method of preparing the immune system of a subject for (ii) administration of a second antigen and a STING agonist, wherein the method comprises (i) administering to the subject a STING agonist and a first antigen.
Clause 19B. A first antigen for use in a method of preparing the immune system of a subject for administration of a second antigen and a STING agonist, wherein the method comprises administering to the subject a STING agonist and a first antigen.
Clause 20B. A second antigen for use in a method of preparing the immune system of a subject for administration of a second antigen and a STING agonist, wherein the method comprises administering to the subject a STING agonist and a first antigen.
Clause 21 B. Use of a STING agonist for the manufacture of a medicament for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a STING agonist and a second antigen which is associated with the pathogen.
Clause 22B. Use of a first antigen for the manufacture of a medicament for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps: (i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a STING agonist and a second antigen which is associated with the pathogen.
Clause 23B. Use of a second antigen for the manufacture of a medicament for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a STING agonist and a second antigen which is associated with the pathogen.
Clause 24B. Use of a STING agonist for the manufacture of a medicament for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist and a first antigen, prior to
(ii) administering to each of the subjects a STING agonist and the second antigen.
Clause 25B. Use of a first antigen for the manufacture of a medicament for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist and a first antigen, prior to
(ii) administering to each of the subjects a STING agonist and the second antigen.
Clause 26B. Use of a second antigen for the manufacture of a medicament for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist and a first antigen, prior to
(ii) administering to each of the subjects a STING agonist and the second antigen.
Clause 27B. Use of a STING agonist in the manufacture of a medicament for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a STING agonist and the second antigen. Clause 28B. Use of a first antigen for the manufacture of a medicament for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a STING agonist and the second antigen.
Clause 29B. Use of a second antigen for the manufacture of a medicament for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a STING agonist and the second antigen.
Clause 30B. Use of a STING agonist for the manufacture of a medicament for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen and a STING agonist.
Clause 31 B. Use of a first antigen for the manufacture of a medicament for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen and a STING agonist.
Clause 32B. Use of a second antigen for the manufacture of a medicament for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen and a STING agonist.
Clause 33B. Use of a STING agonist for the manufacture of a medicament for use in a method of preparing the immune system of a subject for administration of a second antigen and a STING agonist, wherein the method comprises administering to the subject a STING agonist and a first antigen.
Clause 34B. Use of a STING agonist for the manufacture of a medicament for use in a method of preparing the immune system of a subject for administration of a second antigen and a STING agonist, wherein the method comprises administering to the subject a STING agonist and a first antigen.
Clause 35B. Use of a second antigen for the manufacture of a medicament for use in a method of preparing the immune system of a subject for (ii) administration of a second antigen and a STING agonist, wherein the method comprises (i) administering to the subject a STING agonist and a first antigen. Clause 36B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 1B to 35B, wherein the STING agonist binds to STING with an in vitro Kf of less than 0.750 µM, such as less than 0.500 µM. Clause 37B. The method, STING agonist, first antigen, second antigen or use of clause 36B, wherein the STING agonist binds to STING with an in vitro Kf of less than 0.250 µM, such as less than 0.100 µM. Clause 38B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 1B to 37B, wherein the STING agonist activates STING with an in vitro EC50 of 100 µM or less, such as 50 µM or less, when measured by monitoring phosphorylation of interferon regulatory factor-3 (IRF3). Clause 39B. The method, STING agonist, first antigen, second antigen or use of clause 38B, wherein the STING agonist activates STING with an in vitro EC50 of 20 µM or less, such as 10 µM or less, when measured by monitoring phosphorylation of interferon regulatory factor-3 (IRF3). Clause 40B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 1B to 39B, wherein the STING agonist activates STING with an in vitro EC50 of 100 µM or less, such as 50 µM or less, as measured by monitoring interferon-β induction. Clause 41B. The method, STING agonist, first antigen, second antigen or use of clause 40B, wherein the STING agonist activates STING with an in vitro EC50 of 20 µM or less, such as 10 µM or less, as measured by monitoring interferon-β induction. Clause 42B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 1B to 41B, wherein the STING agonist is a nucleic acid, a protein, a peptide, or a small molecule. Clause 43B. The method, STING agonist, first antigen, second antigen or use of clause 42B, wherein the STING agonist is a small molecule, such as a modified or unmodified cyclic dinucleotide. Clause 44B. The method, STING agonist, first antigen, second antigen or use of clause 42B, wherein the STING agonist is selected from a compound of Formulae (I)-(III):
Figure imgf000207_0002
wherein R1 and R2 are each independently selected from the following groups:
Figure imgf000207_0001
Figure imgf000208_0001
R3 and R4 are each independently -SH or -OH,
R5 and R6 are oxygen or sulphur,
R7 and R8 are each independently halogen, hydrogen, -OH, or OCH3; or a pharmaceutically acceptable salt thereof and/or a tautomer thereof.
Clause 45B. The method, STING agonist, first antigen, second antigen or use of clause 42B, wherein the STING agonist is the compound:
Figure imgf000208_0002
or a pharmaceutically acceptable salt thereof and/or a tautomer thereof.
Clause 46B. The method, STING agonist, first antigen, second antigen or use of clause 42B, wherein the STING agonist is selected from c-ditzGMP. c-di-thGMP, c-GtzGMP, c- GAMP, c-thGMP, c-tzGMP, c-di-thAMP, c-ditzAMP, c-di-AMP, c-di-GMP, c-diXGMP, c-GthXMP, c-GXMP, c-ACMP, c-AthXMP, c-AtzXMP, c-di-thXMP, or c-ditzXMP.
Clause 47B. The method, STING agonist, first antigen, second antigen or use of clause 42B, wherein the STING agonist is 2’,3’-cGAMP, or a pharmaceutically acceptable salt thereof.
Clause 48B. The method, STING agonist, first antigen, second antigen or use of clause 42B, wherein the STING agonist is 3’,3’-cGAMP, or a pharmaceutically acceptable salt thereof. Clause 49B. The method, STING agonist, first antigen, second antigen or use of clause 42B, wherein the STING agonist is the compound:
Figure imgf000209_0001
, or a pharmaceutically acceptable salt thereof.
Clause 50B. The method, STING agonist, first antigen, second antigen or use of clause 42B, wherein the STING agonist is 6-bromo-N-(naphthalen-1-yl)benzo[d][1 ,3]dioxole-5- carboxamide:
Figure imgf000209_0002
Clause 51 B. The method, STING agonist, first antigen, second antigen or use of any one of clause 42B, wherein the STING agonist is a compound of Formula A:
Figure imgf000209_0003
wherein
X is O or NR4A,
Y is O, NR4A, CH2, or absent, n is 0, 1 , 2, or 3, R1 and R2 are independently selected from OH, OR3, OR3A, SR3, and NR3R4,
R3, R4, and R4A are independently selected from hydrogen, C1-C10 alkyl optionally substituted with 1-6 halogen, C6-C10 aryl or 5-10 membered heteroaryl, or R3 and R4 together with the nitrogen atom to which they are attached form a 3 to 7 membered heterocycle or 5 to 10 membered heteroaryl,
Figure imgf000210_0001
represents the point of connection of R3A to the remainder of the molecule,
R5-R10 are independently selected from hydrogen, halogen, pseudohalogen, C1-C10 alkyl optionally substituted with 1-6 halogens, C6-C10 aryl, and 5 to 10 membered heteroaryl; or a pharmaceutically acceptable salt thereof and/or a tautomer thereof.
Clause 52B. The method, STING agonist, first antigen, second antigen or use of clause 42B, wherein the STING agonist is selected from:
Figure imgf000210_0002
acceptable salt thereof.
Clause 53B. The method, STING agonist, first antigen, second antigen or use of clause 42B, wherein the STING agonist is a flavonoid.
Clause 54B. The method, STING agonist, first antigen, second antigen or use of clause 53B, wherein the STING agonist is 10-(carboxymethyl)-9(10H)acridone (CMA), 5,6- Dimethylxanthenone-4-acetic acid (DMXAA), methoxyvone, 6,4'-dimethoxyflavone, 4'- methoxyflavone, 3',6'-dihydroxyflavone, 7,2'-dihydroxyflavone, daidzein, formononetin, retusin 7-methyl ether, xanthone, or any combination thereof.
Clause 55B. The method, STING agonist, first antigen, second antigen or use of clause 42B, wherein the STING agonist is a compound of Formula B:
Figure imgf000210_0003
wherein
Figure imgf000211_0001
represents two conjugated double bonds in a five-membered heteroaromatic ring and three conjugated double bonds in a six-membered aromatic or heteroaromatic ring, W1 is selected from CR11 and N; X1 is selected from CR1, C(R1)2, N, NR1, O and S; X2 is selected from CR2, C(R2)2, N, NR2, O and S; X3 is selected from CR3, C(R3)2, N, NR3, O and S; where two or three of X1, X2 and X3 are independently selected from N, NR1, NR2, NR3, O and S; and where at least one of X1, X2 and X3 is selected from N, NR1, NR2 and NR3; Y1 is selected from N, NR4, O, S, CR4 and C(R4)2; Y2 is selected from N, NR5, O, S, CR5 and C(R5)2; Y3 is selected from N, NR6, O, S, CR6 and C(R6)2; Y4 is selected from C and N; Y5 is selected from C and N; where at least one and not more than two of Y1, Y2 and Y3 are independently selected from N, NR4, NR5 and NR6; where when if one of Y4 or Y5 is N, the other one of Y4 or Y5 is C; Z1 is selected from C and N; Z2 is selected from N, NR8 and CR8; Z3 is selected from N, NR9 and CR9; Z4 is selected from N, NR10 and CR10; Z5 is selected from N, NR7 and CR7; where two or three of Z1, Z2, Z3, Z4 and Z5 are independently selected from N, NR7, NR8, NR9, and NR10; each R1 is independently selected from the group consisting of H, C1-C8 alkyl, C1-C8 alkylene-NRR and C1-C8 alkylene-C(O)OR; each R2 is independently selected from the group consisting of H, C1-C8 alkyl, C1-C8 alkylene-NRR, C1-C8 alkylene-C(O)OR, C1-C8 alkylene-OR and C1-C8 alkylene-O- P(O)(OH)2; each R3 is independently selected from the group consisting of H, C1-C8 alkyl, C1-C8 alkylene-NRR, C1-C8 alkylene-C(O)OR and C1-C8 alkylene-O-P(O)(OH)2; each R4 is independently selected from the group consisting of H, -OR, -NRR, C1-C8 alkyl optionally substituted with one or two -OR, C1-C8 alkylene-NRR, -C(O)OR, C1-C8 alkylene-C(O)OR, 3-10 membered heterocycle, C1-C8 alkylene-3-10 membered heterocycle optionally substituted with one 3-10 membered heterocycle, (C3 -C10)- cycloalkyl, and C1-C8 alkylene-(C3-C10)-cycloalkyl; each R5 is independently selected from the group consisting of H, OR, C1 -C8 alkyl, - NRR, C1-C8 alkylene-NRR, -C(O)OR, C1-C8 alkylene-C(O)OR, 3-10 membered heterocycle, C1-C8 alkylene-3-10 membered heterocycle optionally substituted with one 3-10 membered heterocycle, and C1-C8 alkylene-OR; each R6 is H; R7 is selected from the group consisting of H, halo, hydroxy or NH2; R8 is selected from the group consisting of H, C1-C8 alkyl optionally substituted with one or two -NRR or -OR, C1-C8 alkylene-C(O)OR and C1-C8 alkylene-SO2R; R9 is H; R10 is selected from the group consisting of H, C1-C8 alkyl optionally substituted with one or two -OR, and halo; R11 is selected from the group consisting of H, C1-C8 alkyl, -OR and halo; R12 is -C(O)N(R)2 or -C(O)NHR; R13 is H; each R is independently selected from the group consisting of H or C1-C8 alkyl, or C1-C8 haloalkyl, or two R join to form, together with the atom or atoms to which they are bound, a -C3-C10 cycloalkyl or 3-10 membered heterocycle, where said 3-10 membered heterocycle contains one, two or three atoms selected from N, O and S; and where, when two R join to form, together with the atom or atoms to which they are bound, a –(C3-C10) cycloalkyl or 3-10 membered heterocycle, said -C3-C10 cycloalkyl or 3-10 membered heterocycle is optionally substituted with one or more substituents each independently selected from C1-C8 alkyl, hydroxy, C1-C8 alkoxy, -(C3-C10) cycloalkyl, 3-10 membered heterocycle, halo and cyano; or a pharmaceutically acceptable salt thereof and/or a tautomer thereof. Clause 56B. The method, STING agonist, first antigen, second antigen or use of clause 42B, wherein the STING agonist is a compound selected from:
Figure imgf000213_0001
Figure imgf000214_0001
Figure imgf000215_0001
Figure imgf000216_0001
Figure imgf000217_0001
Figure imgf000218_0001
Figure imgf000219_0001
or a pharmaceutically acceptable salt thereof and/or a tautomer thereof. Clause 57B. The method, STING agonist, first antigen, second antigen or use of clause 42B, wherein the STING agonist is a compound of Formula C:
Figure imgf000220_0001
wherein G1 is independently selected from Ring A or , ring A is independently selected from optionally substituted heterocyclyl and optionally substituted heteroaryl, ring B is aromatic carbocyclic ring, ring C is optionally substituted five-membered heteroaryl, R1 is -CON(R3)2, R2 is independently selected from hydrogen, optionally substituted C1-C6 alkyl, and optionally substituted C3-C5 monocyclic cycloalkyl, R3 is independently selected from hydrogen, and optionally substituted C1-C6 alkyl; m is selected from 0, or 1; n is selected from 0, 1, or 2; o is 1; p is selected from 0, 1, or 2; when 'alkyl's substituted, it is substituted with 1 to 4 substituents independently selected from halogen, alkyl, perhaloalkyl, cycloalkyl, heterocyclyl, -N(R4)2, and -OR4; when ‘carbocycle’ or 'cycloalkyl' is substituted, it is substituted with 1 to 4 substituents independently selected from halogen, alkyl, perhaloalkyl, -N(R4)2, and -OR4; when 'heterocycle' or 'heterocyclyl' is substituted, it is substituted with 1 to 4 substituents independently selected from oxo (=0), halogen, cyano, alkyl, perhaloalkyl, -OR4, -C(=O)OH, -OP(O)(OR4)2, -P(O)(OR4)2, -P(O)(OR4)R4a, -SO2R4a, -SO2NH2, - C(=O)N(H)R4, -C(=O)N(alkyl)R4, -N(H)C(=O)R4a, -N(H)R4, and -N(alkyl)R4; when the 'heteroaryl' group is substituted, it is substituted with 1 to 4 substituents selected from halogen, cyano, alkyl, perhaloalkyl, -O-alkyl, -O-perhaloalkyl, - N(alkyl)alkyl, -N(H)R4, -SO2-alkyl, -N(alkyl)C(=O)alkyl, -N(H)C(=O)alkyl, - C(=O)N(alkyl)alkyl, -C(=O)N(H)alkyl, -C(=O)NH2, -SO2N(alkyl)alkyl, -SO2N(H)alkyl, - SO2NH2, -C(=O)OH, -OP(O)(OR4)2, -P(O)(OR4)2, and -P(O)(OR4)R4a; each R4 is independently selected from hydrogen, alkyl, and cycloalkyl; and each R4a is independently selected from alkyl, and cycloalkyl; or a pharmaceutically acceptable salt thereof and/or a tautomer thereof.
Clause 58B. The method, STING agonist, first antigen, second antigen or use of clause 42B, wherein the STING agonist is selected from:
Figure imgf000221_0001
Figure imgf000222_0001
Figure imgf000223_0001
Figure imgf000224_0001
or a pharmaceutically acceptable salt thereof and/or a tautomer thereof.
Clause 59B. The method, STING agonist, first antigen, second antigen or use of clause 42B, wherein the STING agonist is selected from IMSA101 , ADU-S100 (MIW815), BMS-986301 , CRD5500, CMA (IO-carboxymethyl-9-acridanone), diABZI STING agonist-1 (e.g., CAS No.: 2138299-34-8), DMXAA (ASA404/vadimezan), E7766 (Cas no. 2242635-02-3)
Figure imgf000224_0002
, MK-1454, MK-2118, SB-11285,
SRCB-0074, TAK-676, and TTI-10001 or a pharmaceutically acceptable salt thereof. Clause 60B. The method, STING agonist, first antigen, second antigen or use of clause 42B, wherein the STING agonist is a compound according to Formula (l-N):
Figure imgf000225_0001
wherein: q is 0 or 1; r is 0 or 1; s is 0 or 1; wherein q + r + s = 1 or 2; when q is 0, RA1 and RA2 are each independently H, halogen, hydroxy, –O-P(O)(OH)2, -O-(O)(RIRII)2, -N(Re)(Rf), -CO2Rf, -N(Rf)CORb, -N(Rg)SO2(C1-C4alkyl)-N(Re)(Rf), -N(Rg)CO(C1-C4alkyl)-N(Rh)(Rf), optionally substituted (C1-C6alkyl), optionally substituted (C1-C6alkyl)oxy-, optionally substituted (C1-C6alkyl)amino-, and optionally substituted (C1-C6alkyl)(C1-C4alkyl)amino-, wherein the (C1-C6alkyl) of said optionally substituted (C1-C6alkyl), optionally substituted (C1-C6alkyl)oxy-, optionally substituted (C1-C6alkyl)amino- and optionally substituted (C1-C6alkyl)(C1-C4alkyl)amino- is optionally substituted by 1-4 substituents each independently selected from hydroxy, -O-P(O)(OH)2, -O-P(O)(RIRII)2, C1-C4alkoxy-, -N(Re)(Rf), -CO2(Rf), -CON(Re)(Rf), optionally substituted phenyl, optionally substituted 5-6 membered heterocycloalkyl and optionally substituted 5-6 membered heteroaryl group, wherein said optionally substituted phenyl, 5-6 membered heterocycloalkyl or 5-6 membered heteroaryl is optionally substituted by 1-4 substituents each independently selected from halogen, hydroxy, -O-P(O)(OH)2, -O- P(O)(RIRII)2, amino, (C1-C6alkyl)amino-, (C1-C6alkyl)(C1-C6alkyl)amino-, -(C1-C6alkyl)- NH2, halo(C1-C6alkyl), hydroxy-(C1-C4alkyl)-, -(C1-C4alkyl)-O-P(O)(OH)2, - (C1-C4alkyl)-O-P(O)(RIRII)2, halo(C1-C4alkoxy)-, C1-C4alkoxy-, hydroxy-(C2-C4alkoxy)-, -(C2-C4alkoxy)-O-P(O)(OH)2, -(C2-C4alkoxy)-O-P(O)(RIRII)2, -C1-C4alkyl-(C1-C4alkoxy) or C1-C4alkoxy-(C1-C4alkoxy)-; when r is 0, RB1 and RB2 are each independently H, optionally substituted C1-C6alkyl, halo(C1-C6alkyl), optionally substituted C2-C6alkenyl, optionally substituted C2-C6alkynyl, optionally substituted C3-C6cycloalkyl, optionally substituted 4-6 membered heterocycloalkyl, optionally substituted phenyl, optionally substituted 5-6 membered heteroaryl, or optionally substituted 9-10 membered heteroaryl, wherein said optionally substituted C1-C6alkyl, optionally substituted C2-C6alkenyl, optionally substituted C2-C6alkynyl, optionally substituted C3-C6cycloalkyl, optionally substituted 4-6 membered heterocycloalkyl, optionally substituted phenyl, optionally substituted 5-6 membered heteroaryl, or optionally substituted 9-10 membered heteroaryl is optionally substituted by 1-4 substituents each independently selected from halogen, nitro, -Rc, -OH, -O-P(O)(OH)2, -O- P(O)(RIRII)2, -ORc, -NH2, -NRcRc, -NRcRd, -OCORc, -CO2H, -CO2Rc, -SORc, -SO2Rc, -CONH2, -CONRcRd, -SO2NH2, -SO2NRcRd, -OCONH2, -OCONRcRd, -NRdCORc, -NRdSORc, -NRdCO2Rc, and -NRdSO2Rc; when s is 0, RC1 is H, halogen, or C1-C4alkyl and RC2 is optionally substituted C1-C4alkyl, wherein said optionally substituted C1-C4alkyl group is optionally substituted by a substituent selected from -ORc, -NRcRd, -CO2Rc, -CONRcRd, -SO2NRcRd, and -OCONRcRd; when q is 1, RA1 and RA2 are each independently -CH2-, -NRe-, or -O-, and A, taken together with RA1 and RA2, forms a linking group, wherein A is -halo(C1-C12alkyl)-, optionally substituted -C1-C12alkyl-, optionally substituted -C2-C12alkenyl-, optionally substituted -C2-C12alkynyl-, optionally substituted -C1-C6alkyl-O-C1-C6alkyl-, optionally substituted -C1-C6alkyl-NRa-C1-C6alkyl-, optionally substituted -C1-C6alkyl-(C3-C6cycloalkyl)-C1-C6alkyl-, optionally substituted -C1-C6alkyl-phenyl-C1-C6alkyl-, optionally substituted -C1-C6alkyl-(4-6 membered heterocycloalkyl)-C1-C6alkyl-, or optionally substituted -C1-C6alkyl-(5-6 membered heteroaryl)-C1-C6alkyl-, wherein the alkyl moiety of said optionally substituted -C1-C12alkyl-, optionally substituted -C2-C12alkenyl-, optionally substituted -C2-C12alkynyl-, optionally substituted -C1-C6alkyl-O-C1-C6alkyl-, optionally substituted -C1-C6alkyl-NRa-C1-C6alkyl-, optionally substituted -C1-C6alkyl-(C3-C6cycloalkyl)-C1-C6alkyl-, optionally substituted -C1-C6alkyl-phenyl-C1-C6alkyl-, optionally substituted -C1-C6alkyl-(4-6 membered heterocycloalkyl)-C1-C6alkyl-, or optionally substituted -C1-C6alkyl-(5-6 membered heteroaryl)-C1-C6alkyl- is optionally substituted by 1-4 substituents each independently selected from halogen, halo(C1-C4alkyl), -OH, -O-P(O)(OH)2, -O-P(O)(RIRII)2, -ORc, -NH2, -NRcRd, -OCORc, -CO2H, -CO2Rc, -SORc, -SO2Rc, -CONH2, -CONRcRd, -SO2NH2, -SO2NRcRd, -OCONH2, -OCONRcRd, -NRdCORc, -NRdSORc, -NRdCO2Rc, and -NRdSO2Rc, and the C3-C6cycloalkyl, phenyl, 4-6 membered heterocycloalkyl, or 5-6 membered heteroaryl moiety of said optionally substituted -C1-C6alkyl-(C3-C6cycloalkyl)-C1-C6alkyl-, optionally substituted -C1-C6alkyl-phenyl-C1-C6alkyl-, optionally substituted -C1-C6alkyl-(4-6 membered heterocycloalkyl)-C1-C6alkyl-, or optionally substituted -C1-C6alkyl-(5-6 membered heteroaryl)-C1-C6alkyl- is optionally substituted by 1-4 substituents each independently selected from halogen, hydroxy, -O-P(O)(OH)2, -O-P(O)(RIRII)2, amino, (C1-C4alkyl)amino-, (C1-C4alkyl)(C1-C4alkyl)amino-, C1-C4alkyl, halo(C1-C4alkyl), halo(C1-C4alkoxy)-, C1-C4alkoxy-, hydroxy-(C1-C4alkoxy)-, -(C1-C4alkoxyl)-O-P(O)(OH)2, -(C1-C4alkoxyl)-O-P(O)(RIRII)2 and C1-C4alkoxy-(C1-C4alkoxy)-; when r is 1, RB1 and RB2 are each independently -CH -, and B, taken together wit B1 2 h R and RB2, forms a linking group, wherein B is a bond or B is -halo(C1-C10alkyl)-, optionally substituted -C1-C10alkyl-, optionally substituted -C2-C10alkenyl-, optionally substituted -C2-C10alkynyl-, optionally substituted -C1-C6alkyl-O-C1-C6alkyl-, optionally substituted -C1-C6alkyl-NRa-C1-C6alkyl-, optionally substituted C3-C6cycloalkyl, optionally substituted phenyl, optionally substituted 4-6 membered heterocycloalkyl, optionally substituted 5-6 membered heteroaryl, optionally substituted -C1-C4alkyl-(C3-C6cycloalkyl)-C1-C4alkyl-, optionally substituted -C1-C4alkyl-phenyl-C1-C4alkyl-, optionally substituted -C1-C4alkyl-(4-6 membered heterocycloalkyl)-C1-C4alkyl-, or optionally substituted -C1-C4alkyl-(5-6 membered heteroaryl)-C1-C4alkyl-, wherein the alkyl moiety of said optionally substituted -C1-C10alkyl-, optionally substituted -C2-C10alkenyl-, optionally substituted -C2-C10alkynyl-, optionally substituted -C1-C6alkyl-O-C1-C6alkyl-, optionally substituted -C1-C6alkyl-NRa-C1-C6alkyl-, optionally substituted -C1-C4alkyl-(C3-C6cycloalkyl)-C1-C4alkyl-, optionally substituted -C1-C4alkyl-phenyl-C1-C4alkyl-, optionally substituted -C1-C4alkyl-(4-6 membered heterocycloalkyl)-C1-C4alkyl-, or optionally substituted -C1-C4alkyl-(5-6 membered heteroaryl-C1-C4alkyl)- is optionally substituted by 1 or 2 substituents each independently selected from halogen, halo(C1-C4alkyl), -OH, -O-P(O)(OH)2, -O-P(O)(RIRII)2, -ORc, -NH2, -NRcRd, -OCORc, -CO2H, -CO2Rc, -SORc, -SO2Rc, -CONH2, -CONRcRd, -SO2NH2, -SO2NRcRd, -OCONH2, -OCONRcRd, -NRdCORc, -NRdSORc, -NRdCO2Rc, and -NRdSO2Rc, and the C3-C6cycloalkyl, phenyl, 4-6 membered heterocycloalkyl, or 5-6 membered heteroaryl moiety of said optionally substituted C3-C6cycloalkyl, optionally substituted phenyl, optionally substituted 4-6 membered heterocycloalkyl, optionally substituted 5-6 membered heteroaryl, optionally substituted -C1-C4alkyl-(C3-C6cycloalkyl)-C1-C4alkyl-, optionally substituted -C1-C4alkyl-phenyl-C1-C4alkyl-, optionally substituted -C1-C4alkyl-(4-6 membered heterocycloalkyl)-C1-C4alkyl-, or optionally substituted -C1-C4alkyl-(5-6 membered heteroaryl)-C1-C4alkyl- is optionally substituted by 1-4 substituents each independently selected from halogen, hydroxy, -O-P(O)(OH)2, -O- P(O)(RIRII)2, amino, (C1-C4alkyl)amino-, (C1-C4alkyl)(C1-C4alkyl)amino-, C1-C4alkyl, halo(C1-C4alkyl), halo(C1-C4alkoxy)-, C1-C4alkoxy-, hydroxy-(C2-C4alkoxy)-, -(C2-C4alkoxy) O-P(O)(OH)2, -(C2-C4alkoxy)-O-P(O)(RIRII)2, and C1-C4alkoxy-(C1-C4alkoxy)-; when s is 1, RC1 and RC2 are each independently -CH -, and C, taken toget C1 2 her with R and RC2, forms a linking group, wherein C is -halo(C1-C12alkyl)-, optionally substituted -C1-C12alkyl-, optionally substituted -C2-C12alkenyl-, optionally substituted -C2-C12alkynyl-, optionally substituted -C1-C6alkyl-O-C1-C6alkyl-, optionally substituted -C1-C6alkyl-NRa-C1-C6alkyl-, optionally substituted -C1-C6alkyl-(C3-C6cycloalkyl)-C1-C6alkyl-, optionally substituted -C1-C6alkyl-phenyl-C1-C6alkyl-, optionally substituted -C1-C6alkyl-(4-6 membered heterocycloalkyl)-C1-C6alkyl-, or optionally substituted -C1-C6alkyl-(5-6 membered heteroaryl)-C1-C6alkyl-, wherein the alkyl moiety of said optionally substituted -C1-C12alkyl-, optionally substituted -C2-C12alkenyl-, optionally substituted -C2-C12alkynyl-, optionally substituted -C1-C6alkyl-O-C1-C6alkyl-, optionally substituted -C1-C6alkyl-NRa-C1-C6alkyl-, optionally substituted -C1-C6alkyl-(C3-C6cycloalkyl)-C1-C6alkyl-, optionally substituted -C1-C6alkyl-phenyl-C1-C6alkyl-, optionally substituted -C1-C6alkyl-(4-6 membered heterocycloalkyl)-C1-C6alkyl-, or optionally substituted -C1-C6alkyl-(5-6 membered heteroaryl)-C1-C6alkyl- is optionally substituted by 1 or 2 substituents each independently selected from halogen, halo(C1-C4alkyl), -OH, -O-P(O)(OH)2, -O-P(O)(RIRII)2, -ORc, -NH2, -NRcRd, -OCORc, -CO2H, -CO2Rc, -SORc, -SO2Rc, -CONH2, -CONRcRd, -SO2NH2, -SO2NRcRd, -OCONH2, -OCONRcRd, -NRdCORc, -NRdSORc, -NRdCO2Rc, and -NRdSO2Rc, and the C3-C6cycloalkyl, phenyl, 4-6 membered heterocycloalkyl, or 5-6 membered heteroaryl moiety of said optionally substituted -C1-C6alkyl-(C3-C6cycloalkyl)-C1-C6alkyl-, optionally substituted -C1-C6alkyl-phenyl-C1-C6alkyl-, optionally substituted -C1-C6alkyl-(4-6 membered heterocycloalkyl)-C1-C6alkyl-, or optionally substituted -C1-C6alkyl-(5-6 membered heteroaryl)-C1-C6alkyl- is optionally substituted by 1-4 substituents each independently selected from halogen, hydroxy, -O-P(O)(OH)2, -O-P(O)(RIRII)2, amino, (C1-C4alkyl)amino-, (C1-C4alkyl)(C1-C4alkyl)amino-, C1-C4alkyl, halo(C1-C4alkyl), halo(C1-C4alkoxy)-, C1-C4alkoxy-, hydroxy-(C2-C4alkoxy)-, -(C2-C4alkoxy)-O-P(O)(OH)2, -(C2-C4alkoxy)-O-P(O)(RIRII)2,and C1-C4alkoxy-(C1-C4alkoxy)-; R3 and R5 are each independently -CON(Rd)(Rf), or one of R3 and R5 is -CON(Rd)(Rf), and the other of R3 and R5 is H, COOH or -CO2(Rc); R4 and R6 are each independently selected from H, halogen, halo(C1-C6alkyl), halo(C1-C6alkoxy)-, hydroxy, –O-P(O)(OH)2, –O-P(O)(RIRII)2, -NH2, -NRcRc, -NRcRd, -CORc, -CO2Rc, -N(Rd)CORc, -N(Rd)SO2Rc, -N(Rg)SO2(C1-C2alkyl)-N(Rh)(Rf), -N(Rg)CO(C1-C2alkyl)-N(Rh)(Rf), optionally substituted (C1-C6alkyl), optionally substituted (C1-C6alkyl)oxy-, optionally substituted (C1-C6alkyl)amino-, and optionally substituted (C1-C6alkyl)(C1-C4alkyl)amino-, wherein the (C1-C6alkyl) of said optionally substituted (C1-C6alkyl), optionally substituted (C1-C6alkyl)oxy-, optionally substituted (C1-C6alkyl)amino- and optionally substituted (C1-C6alkyl)(C1-C4alkyl)amino- is optionally substituted by 1-4 substituents each independently selected from -OH, -O-P(O)(OH)2, -O-P(O)(RIRII)2,-ORc, -NH2, -NRcRc, -NRcRd, -CO2H, -CO2Rc, -OCORc, -CO2H, -CO2Rc, -SORc, -SO2Rc, -CONH2, -CONRcRd, -SO2NH2, -SO2NRcRd, -OCONH2, -OCONRcRd, -NRdCORc, -NRdSORc, -NRdCO2Rc, -NRdSO2Rc, optionally substituted phenyl, optionally substituted 5-6 membered heterocycloalkyl and optionally substituted 5-6 membered heteroaryl group, wherein said optionally substituted phenyl, 5-6 membered heterocycloalkyl or 5-6 membered heteroaryl is optionally substituted by 1-4 substituents each independently selected from halogen, hydroxy, -O-P(O)(OH)2, -O- P(O)(RIRII)2, amino, (C1-C4alkyl)amino-, (C1-C4alkyl)(C1-C4alkyl)amino-, C1-C4alkyl, halo(C1-C4alkyl), hydroxy-(C1-C4alkyl)-, -(C1-C4alkyl)-O-P(O)(OH)2, -(C1-C4alkyl)-O-P(O)(RIRII)2, halo(C1-C4alkoxy)-, C1-C4alkoxy-, hydroxy-(C2-C4alkoxy)-, -(C2-C4alkoxy)-O-P(O)(OH)2, -(C2-C4alkoxy)-O-P(O)(RIRII)2, C1-C4alkoxy-(C1-C4alkoxy)-, -CORd, -CON(Rd)(Rf), and -CO2Rd; R14 is optionally substituted C1-C4alkyl, wherein said optionally substituted C1-C4alkyl is optionally substituted by a substituent selected from -ORc, -NRcRd, -CO2Rc, -CONRcRd, -SO2NRcRd, and -OCONRcRd; R16 is H, halogen, or C1-C4alkyl; R15 and R17 are each independently H, cyclopropyl, or C1-C4alkyl; Ra is H, -Rc, -CORc, -CO2H, -CO2Rc, -SORc, -SO2Rc, -CONH2, -CONRcRd, -SO2NH2, or -SO2NRcRd; each Rb is independently C1-C4alkyl, halo(C1-C4alkyl), -(C1-C4alkyl)-OH, -(C1-C4alkyl)-O-P(O)(OH)2, -(C1-C4alkyl)-O-P(O)(RIRII)2, -(C1-C4alkyl)-O-(C1-C4alkyl), -(C1-C4alkyl)-N(Re)(Rf), -(C1-C4alkyl)-O-CO(C1-C4alkyl), or -(C1-C4alkyl)-CO-O- (C1-C4alkyl); each Rc is independently C1-C4alkyl, halo(C1-C4alkyl), -(C1-C4alkyl)-OH, -(C1-C4alkyl)-O-P(O)(OH)2, -(C1-C4alkyl)-O-P(O)(RIRII)2,-(C1-C4alkyl)-O-(C1-C4alkyl), -(C1-C4alkyl)-N(Re)(Rf), -(C1-C4alkyl)-O-CO(C1-C4alkyl), -(C1-C4alkyl)-CO-O- (C1-C4alkyl), optionally substituted C3-C6cycloalkyl, optionally substituted phenyl, optionally substituted 4-6 membered heterocycloalkyl, optionally substituted 5-6 membered heteroaryl, optionally substituted 9-10 membered heteroaryl, optionally substituted -C1-C4alkyl-C3-C6cycloalkyl, optionally substituted -C1-C4alkyl-phenyl, optionally substituted -C1-C4alkyl-4-6 membered heterocycloalkyl, optionally substituted -C1-C4alkyl-5-6 membered heteroaryl, or optionally substituted -C1-C4alkyl-9-10 membered heteroaryl, wherein the C3-C6cycloalkyl, phenyl, 4-6 membered heterocycloalkyl, 5-6 membered heteroaryl or 9-10 membered heteroaryl moiety of said optionally substituted C3-C6cycloalkyl, optionally substituted phenyl, optionally substituted 4-6 membered heterocycloalkyl, optionally substituted 5-6 membered heteroaryl, optionally substituted 9-10 membered heteroaryl optionally substituted -C1-C4alkyl-C3-C6cycloalkyl, optionally substituted -C1-C4alkyl-phenyl, optionally substituted -C1-C4alkyl-4-6 membered heterocycloalkyl, optionally substituted -C1-C4alkyl-5-6 membered heteroaryl, or optionally substituted -C1-C4alkyl-9-10 membered heteroaryl is optionally substituted by 1-4 substituents each independently selected from halogen, hydroxy, -O-P(O)(OH)2, -O-P(O)(RIRII)2, amino, -(C1-C4alkyl)NH2, (C1-C4alkyl)amino-, (C1-C4alkyl)(C1-C4alkyl)amino-, C1-C4alkyl, halo(C1-C4alkyl), halo(C1-C4alkoxy)-, C1-C4alkoxy-, hydroxy-(C2-C4alkoxy)-, -(C2-C4alkoxy)-O-P(O)(OH)2, -(C2-C4alkoxy)-O-P(O)(RIRII)2, C1-C4alkoxy-(C1-C4alkoxy)-, -CORd, -CON(Rd)(Rf), and -CO2Rd; each Rd is independently H or C1-C4alkyl; each Re is independently H, (C1-C4alkyl), -CO(C1-C4alkyl), -OCO(C1-C4alkyl), -CO2(C1-C4alkyl), -(C1-C4alkyl)NH2, -(C1-C4alkyl) C1-C4alkoxy, -CO-(optionally substituted 5-6 membered heterocycloalkyl), -CO(C1-C4alkyl)-(optionally substituted 5-6 membered heterocycloalkyl), -CO(optionally substituted 5-6 membered heteroaryl), -CO(C1-C4alkyl)-(optionally substituted 5-6 membered heteroaryl), wherein the optionally substituted 5-6 membered heterocycloalkyl or optionally substituted 5-6 membered heteroaryl is optionally substituted 1-4 substituents each independently selected from halogen, hydroxy, -O-P(O)(OH)2, -O-P(O)(RIRII)2, amino, (C1-C4alkyl)amino-, (C1-C4alkyl)(C1-C4alkyl)amino-, C1-C4alkyl, halo(C1-C4alkyl), halo(C1-C4alkoxy)-, C1-C4alkoxy-, hydroxy-(C2-C4alkoxy)-, -(C2-C4alkoxy) O-P(O)(OH)2, -(C2-C4alkoxy)-O-P(O)(RIRII)2, C1-C4alkoxy-(C1-C4alkoxy)- , -CORd, -CON(Rd)(Rf), and -CO2Rd; each Rf is independently H or (C1-C4alkyl); Rg and Rh are each independently H or (C1-C4alkyl) or Rg and Rh, taken together with the atom or atoms through which they are connected, form a 5-6 membered ring; and each occurrence of RI and RII are independently (C1-C6alkyl)oxy-; or a pharmaceutically acceptable salt thereof and/or a tautomer thereof. Clause 61B. The method, STING agonist, first antigen, second antigen or use of clause 42B, wherein the STING agonist is selected from: (E)-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1H- benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-7- (3-hydroxypropoxy)-1H-benzo[d]imidazole-5-carboxamide
Figure imgf000231_0001
(E)-1-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-2,3- dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-5- carbonyl)imino)-7-(3-hydroxypropoxy)-2,3-dihydro-1H-benzo[d]imidazole-5- carboxamide
Figure imgf000232_0001
(Z)-1-((E)-4-((Z)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-2,3- dihydro-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-7-(3-hydroxypropoxy)-2,3-dihydro-1 H-benzo[d]imidazole-5- carboxamide
Figure imgf000232_0002
(E)-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1 H-pyrazole-5-carboxamido)-7-(3- hydroxypropoxy)-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1 H- pyrazole-5-carboxamido)-7-methoxy-1 H-benzo[d]imidazole-5-carboxamide
Figure imgf000232_0003
(E)-1-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-7-(3- hydroxypropoxy)-2,3-dihydro-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3- methyl-1 H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1 H-benzo[d]imidazole-5- carboxamide
Figure imgf000233_0001
(Z)-1-((E)-4-((Z)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-7-(3- hydroxypropoxy)-2,3-dihydro-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3- methyl-1 H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1 H-benzo[d]imidazole-5- carboxamide
Figure imgf000233_0002
(E)-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1 H-pyrazole-5-carboxamido)-1 H- benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1 H-pyrazole-5-carboxamido)-7-
(3-morpholinopropoxy)-1 H-benzo[d]imidazole-5-carboxamide
Figure imgf000233_0003
(E)-1-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-2,3- dihydro-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-7-(3-morpholinopropoxy)-2,3-dihydro-1 H-benzo[d]imidazole-5- carboxamide
Figure imgf000234_0001
(Z)-1-((E)-4-((Z)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-2,3- dihydro-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-7-(3-morpholinopropoxy)-2,3-dihydro-1 H-benzo[d]imidazole-5- carboxamide
Figure imgf000234_0002
(E)-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1 H-pyrazole-5-carboxamido)-7-(3- morpholinopropoxy)-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1 H- pyrazole-5-carboxamido)-7-methoxy-1 H-benzo[d]imidazole-5-carboxamide
Figure imgf000234_0003
(E)-1-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-7-(3- morpholinopropoxy)-2,3-dihydro-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3- methyl-1 H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1 H-benzo[d]imidazole-5- carboxamide
Figure imgf000235_0001
(Z)-1-((E)-4-((Z)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-7-(3- morpholinopropoxy)-2,3-dihydro-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3- methyl-1 H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1 H-benzo[d]imidazole-5- carboxamide
Figure imgf000235_0002
3-(((Z)-6-carbamoyl-3-((E)-4-((Z)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-7-methoxy-2,3-dihydro-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1- ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1 H-benzo[d]imidazol-4- yl)oxy)propyldihydrogen phosphate
Figure imgf000235_0003
(E)-3-((5-carbamoyl-1 -(4-(5-carbamoyl-2-(1 -ethyl-3-methyl-1 H-pyrazole-5- carboxamido)-7-methoxy-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl- 1 H-pyrazole-5-carboxamido)-1 H-benzo[d]imidazol-7-yl)oxy)propyl dihydrogen phosphate
Figure imgf000236_0001
3-(((E)-6-carbamoyl-3-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-7-methoxy-2,3-dihydro-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1- ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1 H-benzo[d]imidazol-4- yl)oxy)propyl dihydrogen phosphate
Figure imgf000236_0002
(E)-4-((5-carbamoyl-1 -(4-(5-carbamoyl-2-(1 -ethyl-3-methyl-1 H-pyrazole-5- carboxamido)-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1 H-pyrazole-
5-carboxamido)-1 H-benzo[d]imidazol-7-yl)oxy)butanoic acid
Figure imgf000236_0003
(E)-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1 H-pyrazole-5-carboxamido)-7-methoxy-1 H- benzo[d]imidazol-1-yl)but-2-en-1-yl)-7-(3-(dimethylamino)propoxy)-2-(1-ethyl-3-methyl-
1 H-pyrazole-5-carboxamido)-1 H-benzo[d]imidazole-5-carboxamide
Figure imgf000237_0001
(E)-1-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-7-(3-
(4-(2-hydroxyethyl)piperazin-1-yl)propoxy)-2,3-dihydro-1 H-benzo[d]imidazol-1-yl)but-2- en-1-yl)-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1 H- benzo[d]imidazole-5-carboxamide
Figure imgf000237_0002
or a pharmaceutically acceptable salt thereof and/or a tautomer thereof.
Clause 62B. The method, STING agonist, first antigen, second antigen or use of clause 42B, wherein the STING agonist is 3-(((E)-6-carbamoyl-3-((E)-4-((E)-5-carbamoyl-2-((1- ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1 H-benzo[d]imidazol-1- yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1 H- benzo[d]imidazol-4-yl)oxy)propyl
Figure imgf000238_0001
or a pharmaceutically acceptable salt thereof.
Clause 63B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 1B to 62B wherein the STING agonist of step (i) is the same as the STING agonist of step (ii).
Clause 64B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 1B to 62B wherein the STING agonist of step (i) is different to the STING agonist of step (ii).
Clause 65B. The method, STING agonist, first antigen, second antigen or use of clause 64B wherein the STING agonist of step (i) is different to the STING agonist of step (ii) and wherein the STING agonist of step (i) is selected from the STING agonists defined in any one of clauses 36B to 62B and the STING agonist of step (ii) is selected from the STING agonists defined in any one of clauses 36B to 62B.
Clause 66B. The method, STING agonist, first antigen, second antigen or use of clause 64B wherein the STING agonist of step (i) is different to the STING agonist of step (ii) but the mode of action of both STING agonists is essentially identical.
Clause 67B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 1B to 66B wherein only one administration of the STING agonist and first antigen is performed in step (i).
Clause 68B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 1B to 66B wherein at least 2 administrations of the STING agonist and first antigen are performed in step (i).
Clause 69B. The method, STING agonist, first antigen, second antigen or use of clause 68B wherein at least 3 administrations of the STING agonist and first antigen are performed in step (i).
Clause 70B. The method, STING agonist, first antigen, second antigen or use of clause 68B wherein 2 administrations of the STING agonist and first antigen are performed in step (i). Clause 71 B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 1B to 70B wherein in step (i) the STING agonist is co-formulated with the first antigen.
Clause 72B. The method, STING agonist, first antigen, second antigen or use of clause 71 B wherein the co-formulated STING agonist and first antigen of step (i) is administered intradermally, intramuscularly, intraperitoneally or subcutaneously, in particular intramuscularly.
Clause 73B. The method, STING agonist, first antigen, second antigen or use of clause 71 B or 72B wherein the volume of the co-formulation of STING agonist and first antigen in step (i) is 0.05 ml to 1 ml, such as 0.1 to 0.6 ml.
Clause 74B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 71 B to 73B wherein the pH of the co-formulation of STING agonist and first antigen in step (i) is 4 to 9, especially 5 to 8.5, in particular 5.5 to 8, such as 6.5 to 7.4.
Clause 75B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 71 B to 74B wherein the osmolarity of the co-formulation of STING agonist and first antigen in step (i) is 250 to 750 mOsm/kg, especially 250 to 550 mOsm/kg, in particular 270 to 500 mOsm/kg, such as 270 to 400 mOsm/kg.
Clause 76B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 71 B to 75B wherein the co-formulation of STING agonist and first antigen of step (i) comprises buffer and/or tonicity modifying agents, such as modified phosphate buffered saline.
Clause 77B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 1B to 70B wherein in step (i) the STING agonist is formulated separately from the first antigen.
Clause 78B. The method, STING agonist, first antigen, second antigen or use of clause 77B wherein in step (i) for at least one administration, the STING agonist is administered simultaneously with the first antigen.
Clause 79B. The method, STING agonist, first antigen, second antigen or use of clause 78B wherein in step (i) for all administrations the STING agonist is administered simultaneously with the first antigen.
Clause 80B. The method, STING agonist, first antigen, second antigen or use of clause 77B wherein in step (i) for at least one administration, the STING agonist is administered separately from the first antigen.
Clause 81 B. The method, STING agonist, first antigen, second antigen or use of clause 80B wherein in step (i) for at least one administration, the STING agonist is administered before the first antigen. Clause 82B. The method, STING agonist, first antigen, second antigen or use of clause 81 B wherein in step (i) for all administrations the STING agonist is administered before the first antigen.
Clause 83B. The method, STING agonist, first antigen, second antigen or use of clause 80B wherein in step (i) for at least one administration, the first antigen is administered before the STING agonist.
Clause 84B. The method, STING agonist, first antigen, second antigen or use of clause 83B wherein in step (i) for all administrations the first antigen is administered before the STING agonist.
Clause 85B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 77B to 84B wherein in step (i) for at least one administration, the STING agonist is administered to the same location on the subject as the first antigen.
Clause 86B. The method, STING agonist, first antigen, second antigen or use of clause 85B wherein in step (i) for all administrations the STING agonist is administered to the same location on the subject as the first antigen.
Clause 87B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 77B to 84B wherein in step (i) for at least one administration, the STING agonist is administered to a different location on the subject as the first antigen.
Clause 88B. The method, STING agonist, first antigen, second antigen or use of clause 87B wherein in step (i) for all administrations the STING agonist is administered to a different location on the subject as the first antigen.
Clause 89B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 77B to 88B wherein in step (i) for at least one administration, the STING agonist and the first antigen are administered within 84 hours of each other.
Clause 90B. The method, STING agonist, first antigen, second antigen or use of clause 89B wherein in step (i) for at least one administration, the STING agonist and the first antigen are administered within 30 minutes of each other.
Clause 91 B. The method, STING agonist, first antigen, second antigen or use of clause 89B wherein in step (i) for all administrations the STING agonist and the first antigen are administered within 84 hours of each other.
Clause 92B. The method, STING agonist, first antigen, second antigen or use of clause 90B wherein in step (i) for all administrations the STING agonist and the first antigen are administered within 30 minutes of each other.
Clause 93B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 77B to 92B wherein the STING agonist of step (i) is administered intradermally, intramuscularly, intraperitoneally or subcutaneously, in particular intramuscularly. Clause 94B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 77B to 93B wherein the first antigen of step (i) is administered intradermally, intramuscularly, intraperitoneally or subcutaneously, in particular intramuscularly.
Clause 95B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 77B to 94B wherein the volume of the separate formulation of STING agonist in step (i) is 0.05 ml to 1 ml, such as 0.1 to 0.6 ml.
Clause 96B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 77B to 95B wherein the volume of the separate formulation of first antigen in step (i) is 0.05 ml to 1 ml, such as 0.1 to 0.6 ml.
Clause 97B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 77B to 96B wherein the pH of the separate formulation of STING agonist in step (i) is 4 to 9, especially 5 to 8.5, in particular 5.5 to 8, such as 6.5 to 7.4.
Clause 98B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 77B to 97B wherein the pH of the separate formulation of first antigen in step (i) is 4 to 9, especially 5 to 8.5, in particular 5.5 to 8, such as 6.5 to 7.4.
Clause 99B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 77B to 98B wherein the osmolarity of the separate formulation of STING agonist in step (i) is 250 to 750 mOsm/kg, especially 250 to 550 mOsm/kg, in particular 270 to 500 mOsm/kg, such as 270 to 400 mOsm/kg.
Clause 100B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 77B to 99B wherein the osmolarity of the separate formulation of first antigen in step (i) is 250 to 750 mOsm/kg, especially 250 to 550 mOsm/kg, in particular 270 to 500 mOsm/kg, such as 270 to 400 mOsm/kg.
Clause 101 B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 77B to 100B wherein the separate formulation comprising the STING agonist of step (i) comprises buffer and/or tonicity modifying agents, such as modified phosphate buffered saline.
Clause 102B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 77B to 101 B wherein the separate formulation separate formulation of first antigen in step (i) comprises buffer and/or tonicity modifying agents, such as modified phosphate buffered saline.
Clause 103B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 1B to 102B wherein in step (i) the period between administrations to the subject is 2 weeks to 12 months.
Clause 104B. The method, STING agonist, first antigen, second antigen or use of clause 103B wherein in step (i) the period between administrations to the subject is 2 weeks to 6 months. Clause 105B. The method, STING agonist, first antigen, second antigen or use of clause 104B wherein in step (i) the period between administrations to the subject is 2 weeks to 3 months.
Clause 106B. The method, STING agonist, first antigen, second antigen or use of clause 105B wherein in step (i) the period between administrations to the subject is 2 weeks to 2 months, such as 3 to 5 weeks.
Clause 107B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 1 B to 106B wherein step (ii) is initiated at an interval of 2 weeks to 2 years after step (i).
Clause 108B. The method, STING agonist, first antigen, second antigen or use of clause 107B wherein step (ii) is initiated at an interval of 2 weeks to 1 year after step (i).
Clause 109B. The method, STING agonist, first antigen, second antigen or use of clause 108B wherein step (ii) is initiated at an interval of 3 weeks to 9 months after step (i).
Clause 110B. The method, STING agonist, first antigen, second antigen or use of clause 109B wherein step (ii) is initiated at an interval of 4 weeks to 6 months after step (i).
Clause 111 B. The method, STING agonist, first antigen, second antigen or use of clause 107B wherein step (ii) is initiated at an interval of 2 weeks to 2 months after step (i).
Clause 112B. The method, STING agonist, first antigen, second antigen or use of clause 107B wherein step (ii) is initiated at an interval of 1 month to 3 months after step (i).
Clause 113B. The method, STING agonist, first antigen, second antigen or use of clause 107B wherein step (ii) is initiated at an interval of 2 months to 4 months after step (i).
Clause 114B. The method, STING agonist, first antigen, second antigen or use of clause 108B wherein step (ii) is initiated at an interval of 3 months to 6 months after step (i).
Clause 115B. The method, STING agonist, first antigen, second antigen or use of clause 108B wherein step (ii) is initiated at an interval of 4 months to 12 months after step (i).
Clause 116B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 1 B to 115B wherein only one administration of the STI NG agonist and second antigen is performed in step (ii).
Clause 117B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 1 B to 115B wherein at least 2 administrations of the STI NG agonist and second antigen are performed in step (ii).
Clause 118B. The method, STING agonist, first antigen, second antigen or use of clause 117B wherein at least 3 administrations of the STING agonist and second antigen are performed in step (ii). Clause 119B. The method, STING agonist, first antigen, second antigen or use of clause 118B wherein 3 administrations of the STING agonist and second antigen are performed in step (ii).
Clause 120B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 1B to 119B wherein in step (ii) the STING agonist is co-formulated with the second antigen.
Clause 121 B. The method, STING agonist, first antigen, second antigen or use of clause 120B wherein the co-formulated STING agonist and second antigen of step (ii) is administered intradermally, intramuscularly, intraperitoneally or subcutaneously, in particular intramuscularly.
Clause 122B. The method, STING agonist, first antigen, second antigen or use of clause 120B or 121 B wherein the volume of the co-formulation of STING agonist and second antigen in step (ii) is 0.05 ml to 1 ml, such as 0.1 to 0.6 ml.
Clause 123B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 120B to 122B wherein the pH of the co-formulation of STING agonist and second antigen in step (ii) is 4 to 9, especially 5 to 8.5, in particular 5.5 to 8, such as 6.5 to 7.4.
Clause 124B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 120B to 122B wherein the osmolarity of the co-formulation of STING agonist and second antigen in step (ii) is 250 to 750 mOsm/kg, especially 250 to 550 mOsm/kg, in particular 270 to 500 mOsm/kg, such as 270 to 400 mOsm/kg.
Clause 125B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 120B to 122B wherein the co-formulation of STING agonist and second antigen of step (ii) comprises buffer and/or tonicity modifying agents, such as modified phosphate buffered saline.
Clause 126B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 1B to 119B wherein in step (ii) the STING agonist is formulated separately from the second antigen.
Clause 127B. The method, STING agonist, first antigen, second antigen or use of clause 126B wherein in step (ii) for at least one administration, the STING agonist is administered simultaneously with the second antigen.
Clause 128B. The method, STING agonist, first antigen, second antigen or use of clause 127B wherein in step (ii) for all administrations the STING agonist is administered simultaneously with the second antigen.
Clause 129B. The method, STING agonist, first antigen, second antigen or use of clause 126B wherein in step (ii) for at least one administration, the STING agonist is administered separately from the second antigen. Clause 130B. The method, STING agonist, first antigen, second antigen or use of clause 129B wherein in step (ii) for at least one administration, the STING agonist is administered before the second antigen.
Clause 131 B. The method, STING agonist, first antigen, second antigen or use of clause 130B wherein in step (ii) for all administrations the STING agonist is administered before the second antigen.
Clause 132B. The method, STING agonist, first antigen, second antigen or use of clause 129B wherein in step (ii) for at least one administration, the second antigen is administered before the STING agonist.
Clause 133B. The method, STING agonist, first antigen, second antigen or use of clause 132B wherein in step (ii) for all administrations the second antigen is administered before the STING agonist.
Clause 134B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 126B to 133B wherein in step (ii) for at least one administration, the STING agonist is administered to the same location on the subject as the second antigen.
Clause 135B. The method, STING agonist, first antigen, second antigen or use of clause 134B wherein in step (ii) for all administrations the STING agonist is administered to the same location on the subject as the second antigen.
Clause 136B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 126B to 133B wherein in step (ii) for at least one administration, the STING agonist is administered to a different location on the subject as the second antigen.
Clause 137B. The method, STING agonist, first antigen, second antigen or use of clause 136B wherein in step (ii) for all administrations the STING agonist is administered to a different location on the subject as the second antigen.
Clause 138B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 126B to 137B wherein in step (ii) for at least one administration, the STING agonist and the first antigen are administered within 84 hours of each other.
Clause 139B. The method, STING agonist, first antigen, second antigen or use of clause 138B wherein in step (ii) for at least one administration, the STING agonist and the second antigen are administered within 30 minutes of each other.
Clause 140B. The method, STING agonist, first antigen, second antigen or use of clause 138B wherein in step (ii) for all administrations the STING agonist and the second antigen are administered within 84 hours of each other.
Clause 141 B. The method, STING agonist, first antigen, second antigen or use of clause 140B wherein in step (ii) for all administrations the STING agonist and the second antigen are administered within 30 minutes of each other. Clause 142B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 126B to 141 B wherein the STING agonist of step (ii) is administered intradermally, intramuscularly, intraperitoneally or subcutaneously, in particular intramuscularly.
Clause 143B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 126B to 142B wherein the second antigen of step (ii) is administered intradermally, intramuscularly, intraperitoneally or subcutaneously, in particular intramuscularly.
Clause 144B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 126B to 143B wherein the volume of the separate formulation of STING agonist in step (ii) is 0.05 ml to 1 ml, such as 0.1 to 0.6 ml.
Clause 145B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 126B to 144B wherein the volume of the separate formulation of second antigen in step (ii) is 0.05 ml to 1 ml, such as 0.1 to 0.6 ml.
Clause 146B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 126B to 145B wherein the pH of the separate formulation of STING agonist in step (ii) is 4 to 9, especially 5 to 8.5, in particular 5.5 to 8, such as 6.5 to 7.4.
Clause 147B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 126B to 146B wherein the pH of the separate formulation of second antigen in step (ii) is 4 to 9, especially 5 to 8.5, in particular 5.5 to 8, such as 6.5 to 7.4.
Clause 148B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 126B to 147B wherein the osmolarity of the separate formulation of STING agonist in step (ii) is 250 to 750 mOsm/kg, especially 250 to 550 mOsm/kg, in particular 270 to 500 mOsm/kg, such as 270 to 400 mOsm/kg.
Clause 149B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 126B to 148B wherein the osmolarity of the separate formulation of second antigen in step (ii) is 250 to 750 mOsm/kg, especially 250 to 550 mOsm/kg, in particular 270 to 500 mOsm/kg, such as 270 to 400 mOsm/kg.
Clause 150B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 126B to 149B wherein the separate formulation comprising the STING agonist of step (ii) comprises buffer and/or tonicity modifying agents, such as modified phosphate buffered saline.
Clause 151 B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 126B to 150B wherein the separate formulation comprising the second antigen of step (ii) comprises buffer and/or tonicity modifying agents, such as modified phosphate buffered saline. Clause 152B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 1B to 151 B wherein in step (ii) the period between administrations to the subject is 2 weeks to 12 months.
Clause 153B. The method, STING agonist, first antigen, second antigen or use of clause 152B wherein in step (ii) the period between administrations to the subject is 2 weeks to 6 months.
Clause 154B. The method, STING agonist, first antigen, second antigen or use of clause 153B wherein in step (ii) the period between administrations to the subject is 2 weeks to 3 months.
Clause 155B. The method, STING agonist, first antigen, second antigen or use of clause 154B wherein in step (ii) the period between administrations to the subject is 2 weeks to 2 months, such as 3 to 5 weeks.
Clause 156B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 1B to 155B wherein the combined duration of step (i), step (ii) and the interval between the steps is 1 month to 2 years.
Clause 157B. The method, STING agonist, first antigen, second antigen or use of clause 156B wherein the combined duration of step (i), step (ii) and the interval between the steps is 6 weeks to 12 months.
Clause 158B. The method, STING agonist, first antigen, second antigen or use of clause 157B wherein the combined duration of step (i), step (ii) and the interval between the steps is 2 months to 10 months.
Clause 159B. The method, STING agonist, first antigen, second antigen or use of clause 158B wherein the combined duration of step (i), step (ii) and the interval between the steps is 10 weeks to 9 months.
Clause 160B. The method, STING agonist, first antigen, second antigen or use of any one of any one of clauses 1 B to 159B wherein the combined duration of step (i), step (ii) and the interval between the steps is at least 2 months, especially at least 3 months, in particular at least 4 months.
Clause 161 B. The method, STING agonist, first antigen, second antigen or use of clause 160B wherein the combined duration of step (i), step (ii) and the interval between the steps is at least 5 months, especially at least 6 months, in particular at least 7 months.
Clause 162B. The method, STING agonist, first antigen, second antigen or use of any one of any one of clauses 1 B to 161 B wherein the combined duration of step (i), step (ii) and the interval between the steps is 5 years or less, especially 3 years or less, in particular 2 years or less. Clause 163B. The method, STING agonist, first antigen, second antigen or use of clause 162B wherein the combined duration of step (i), step (ii) and the interval between the steps is 18 months or less, especially 12 months or less, in particular 10 months or less.
Clause 164B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 1 B to 163B wherein the subject is a mammal.
Clause 165B. The method, STING agonist, first antigen, second antigen or use of clause 164B wherein the subject is a human.
Clause 166B. The method, STING agonist, first antigen, second antigen or use of clause 164B wherein the subject is a human child.
Clause 167B. The method, STING agonist, first antigen, second antigen or use of clause 164B wherein the subject is a human adult.
Clause 168B. The method, STING agonist, first antigen, second antigen or use of clause 164B wherein the subject is an older human.
Clause 169B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 1 B to 168B wherein the administration route used for each administration of step (i) is the same as the administration route used for each administration of step (ii).
Clause 170B. The method, STING agonist, first antigen, second antigen or use of clause 169B wherein the administration route used is intramuscular.
Clause 171 B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 1 B to 170B wherein the first antigen is derived from a pathogen.
Clause 172B. The method, STING agonist, first antigen, second antigen or use of clause 171 B wherein the first antigen is derived from a different pathogen to the second antigen.
Clause 173B. The method, STING agonist, first antigen, second antigen or use of clause 172B wherein the different pathogen is a pathogen which belongs to a different species.
Clause 174B. The method, STING agonist, first antigen, second antigen or use of clause 173B wherein the different pathogen is a pathogen which belongs to a different genus.
Clause 175B. The method, STING agonist, first antigen, second antigen or use of clause 174B wherein the different pathogen is a pathogen which belongs to a different family.
Clause 176B. The method, STING agonist, first antigen, second antigen or use of clause 175B wherein the different pathogen is a pathogen which belongs to a different order.
Clause 177B. The method, STING agonist, first antigen, second antigen or use of clause 176B wherein the different pathogen is a pathogen which belongs to a different class.
Clause 178B. The method, STING agonist, first antigen, second antigen or use of clause 177B wherein the different pathogen is a pathogen which belongs to a different phylum.
Clause 179B. The method, STING agonist, first antigen, second antigen or use of clause 178B wherein the different pathogen is a pathogen which belongs to a different domain. Clause 180B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 1 B to 179B wherein the first antigen is derived from (such as containing a portion of a polypeptide sequence obtainable from, such as at least 50, especially at least 100, in particular at least 150, such as at least 200 amino acids long) one of bacteria, viruses or parasites and the second antigen is derived from another of bacteria, viruses or parasites (such as containing a portion of a polypeptide sequence obtainable from, such as at least 50, especially at least 100, in particular at least 150, such as at least 200 amino acids long).
Clause 181 B. The method, STING agonist, first antigen, second antigen or use of clause 180B wherein the first antigen is derived from a virus and the second antigen is derived from a bacterium.
Clause 182B. The method, STING agonist, first antigen, second antigen or use of clause 180B wherein the first antigen is derived from a bacterium and the second antigen is derived from a virus.
Clause 183B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 1 B to 182B wherein the first antigen does not comprise any epitopes which are cross-reactive with epitopes of the second antigen in a CD4+ T cell response.
Clause 184B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 1 B to 183B wherein the first antigen does not comprise any epitopes which are cross-reactive with epitopes of the second antigen in a CD8+ T cell response.
Clause 185B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 1 B to 184B wherein the first antigen does not comprise any epitopes which are cross-reactive with epitopes of the second antigen in an antibody response.
Clause 186B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 1 B to 185B wherein the first antigen is a polypeptide, a polynucleotide or a polysaccharide.
Clause 187B. The method, STING agonist, first antigen, second antigen or use of clause 186B wherein the first antigen is a polypeptide.
Clause 188B. The method, STING agonist, first antigen, second antigen or use of clause 187B, wherein the polypeptide comprises at least one B or T cell epitope.
Clause 189B. The method, STING agonist, first antigen, second antigen or use of clause 188B, wherein the polypeptide comprises B and T cell epitopes.
Clause 190B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 183B to 189B wherein the first antigen is derived from a virus.
Clause 191 B. The method, STING agonist, first antigen, second antigen or use of clause 190B wherein the first antigen is derived from a herpesvirus.
Clause 192B. The method, STING agonist, first antigen, second antigen or use of clause 191 B wherein the herpesvirus is varicella-zoster virus (VZV). Clause 193B. The method, STING agonist, first antigen, second antigen or use of any one of clause 192B wherein the first antigen is a VZV-gE antigen.
Clause 194B. The method, STING agonist, first antigen, second antigen or use of clause 193B wherein the VZV-gE antigen comprises an amino acid sequence having at least 80%, such as at least 90%, especially at least 95%, in particular at least 98% for example at least 99% or 100% identity to SEQ ID NO: 1.
Clause 195B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 1B to 189B wherein the first antigen is a cancer antigen, such as a tumour antigen and/or a neoantigen.
Clause 196B. The method, STING agonist, first antigen, second antigen or use of clause 195B wherein the first antigen is a human cancer antigen, such as a human tumour antigen and/or a neoantigen.
Clause 197B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 1B to 196B wherein the first antigen is provided in the form of a polynucleotide encoding the antigen.
Clause 198B. The method, STING agonist, first antigen, second antigen or use of clause 197B wherein the first antigen is provided in the form of mRNA encoding the antigen.
Clause 199B. The method, STING agonist, first antigen, second antigen or use of clause 198B wherein the mRNA is carrier-formulated, such as formulated in LNP, CNE or LION.
Clause 200B. The method, STING agonist, first antigen, second antigen or use of clause 199B wherein the carrier is LNP.
Clause 201 B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 1B to 228B wherein the second antigen is a polypeptide, a polynucleotide or a polysaccharide.
Clause 202B. The method, STING agonist, first antigen, second antigen or use of clause 201 B wherein the second antigen is a polypeptide.
Clause 203B. The method, STING agonist, first antigen, second antigen or use of clause 202B, wherein the polypeptide comprises at least one B or T cell epitope.
Clause 204B. The method, STING agonist, first antigen, second antigen or use of clause 203B, wherein the polypeptide comprises B and T cell epitopes.
Clause 205B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 1B to 204B wherein the second antigen is derived from a bacterium.
Clause 206B. The method, STING agonist, first antigen, second antigen or use of clause 205B wherein the second antigen is derived from Clostridia.
Clause 207B. The method, STING agonist, first antigen, second antigen or use of clause 206B wherein the second antigen is derived from C. difficile. Clause 208B. The method, STING agonist, first antigen, second antigen or use of clause 207B wherein the second antigen is C. difficile toxin A and/or toxin B or fragments thereof.
Clause 209B. The method, STING agonist, first antigen, second antigen or use of clause 208B wherein the second antigen is the F2 antigen.
Clause 210B. The method, STING agonist, first antigen, second antigen or use of clause 209B wherein the F2 antigen comprises an amino acid sequence having at least 80%, such as at least 90%, especially at least 95%, in particular at least 98% for example at least 99% or 100% identity to SEQ ID NO: 2.
Clause 211B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 1B to 204B wherein the second antigen is a cancer antigen, such as a tumour antigen and/or a neoantigen.
Clause 212B. The method, STING agonist, first antigen, second antigen or use of clause 211 B wherein the second antigen is a human cancer antigen, such as a human tumour antigen and/or a neoantigen.
Clause 213B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 1B to 212B wherein the second antigen is provided in the form of a polynucleotide encoding the antigen.
Clause 214B. The method, STING agonist, first antigen, second antigen or use of clause 213B wherein the second antigen is provided in the form of mRNA encoding the antigen.
Clause 215B. The method, STING agonist, first antigen, second antigen or use of clause 214B wherein the mRNA is carrier-formulated, such as formulated in LNP, CNE or LION.
Clause 216B. The method, STING agonist, first antigen, second antigen or use clause 215B wherein the carrier is LNP.
Clause 217B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 1 B to 216B, wherein the immune response to the second antigen is a serum titer of IgG antibodies binding to the second antigen.
Clause 218B. The method, STING agonist, first antigen, second antigen or use of clause 217B, wherein the IgG antibodies are neutralising antibodies.
Clause 219B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 1B to 218B, wherein the immune response to the second antigen is a T cell response specific to the second antigen.
Clause 220B. The method, STING agonist, first antigen, second antigen or use of clause 219B, wherein the T cell response is a CD4+ T cell response. Clause 221 B. The method, STING agonist, first antigen, second antigen or use of clause 219B, wherein the T cell response is a CD8+ T cell response.
Clause 222B. The method, STING agonist, first antigen, second antigen or use of clause 221 B, wherein the T cell response is a CD4+ and CD8+ T cell response.
Clause 223B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 1B to 222B, wherein the immune response to the second antigen reduces partially or completely the severity of one or more symptoms experienced by the subject due to infection by a pathogen.
Clause 224B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 1B to 223B, wherein the immune response to the second antigen reduces partially or completely the time over which one or more symptoms are experienced by the subject due to infection by a pathogen.
Clause 225B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 1B to 224B, wherein the immune response to the second antigen reduces the likelihood of developing an established infection after challenge with the pathogen.
Clause 226B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 1B to 225B, wherein the immune response to the second antigen slows progression of illness (e.g. extends survival) due to infection by the pathogen.
Clause 227B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 1B to 226B, wherein the breadth of the immune response to the second antigen in the subject is increased.
Clause 228B. The method, STING agonist, first antigen, second antigen or use of clause 227B, wherein serum neutralising antibodies bind to an increased range of variants of the second antigen.
Clause 229B. The method, STING agonist, first antigen, second antigen or use of either clause 227B or 228B, wherein the immune response to the second antigen is elicited in a wider variety of human HLA types.
Clause 230B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 1B to 229B, wherein the level of the immune response is increased, such as by at least 50%, such as 100%, such as 150%.
Clause 231 B. The method, STING agonist, first antigen, second antigen or use of clause 230B, wherein the level of the immune response to the second antigen is increased, such as by at least 200%, such as 300%, such as 400%.
Clause 232B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 1 B to 231 B, wherein the immune response to the second antigen is maintained for at least 1 year, such as at least 3 years. Clause 233B. The method, STING agonist for use, first antigen for use, second antigen for use or use of any one of clauses 1 B to 232B, wherein the duration of the immune response to the second antigen is increased, such as by at least 1 year, such as by at least 3 years.
Clause 234B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 1 B to 233B, wherein the variability of the immune response to the second antigen between subjects is decreased.
Clause 235B. The method, STING agonist, first antigen, second antigen or use of clause 234B, wherein the standard deviation of the immune response to the second antigen is reduced.
Clause 236B. The method, STING agonist, first antigen, second antigen or use of either clause 234B or 235B, wherein the proportion of subjects meeting a specified threshold of immune response to the second antigen is increased.
Clause 237B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 1 B to 236B, wherein the speed of the immune response to the second antigen is increased.
Clause 238B. The method, STING agonist, first antigen, second antigen or use of clause 237B, wherein the time taken to achieve a protective immune response is reduced.
Clause 239B. The method, STING agonist, first antigen, second antigen or use of either clause 237B or 238B, wherein the time taken to reach peak immune response is reduced.
Clause 240B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 1B to 239B, wherein the amount of second antigen required to achieve a desired immune response, such as a protective immune response, is reduced.
Clause 241 B. The method, STING agonist, first antigen, second antigen or use of any one of clauses 1B to 240B, wherein the spacing between administrations of second antigen may be reduced while maintaining immune response.
The invention is also illustrated by reference to the following further clauses.
These ‘C’ clauses relate to embodiments wherein a STING agonist is administered in step (i), which may be followed by administration of an antigen and optionally a second adjuvant.
Clause 1C. A method for prophylaxis in a subject at risk of infection comprising administering a STING agonist to the subject.
Clause 2C. A STING agonist for use in a method for prophylaxis in a subject at risk of infection comprising administering a STING agonist to the subject.
Clause 3C. Use of a STING agonist for the manufacture of a medicament for use in a method for prophylaxis in a subject at risk of infection comprising administering a STING agonist to the subject.
Clause 4C. A method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject an antigen which is associated with the pathogen.
Clause 5C. A method of reducing the variability of the immune response to an antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist, prior to
(ii) administering to each of the subjects the antigen.
Clause 6C. A method of improving an immune response to an antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the antigen.
Clause 7C. A method of reducing the amount of an antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the antigen.
Clause 8C. A method of preparing the immune system of a subject for (ii) administration of an antigen, wherein the method comprises (i) administering to the subject a STING agonist.
Clause 9C. A STING agonist for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject an antigen which is associated with the pathogen.
Clause 10C. An antigen for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject an antigen which is associated with the pathogen. Clause 11 C. A STI NG agonist for use in a method of reducing the variability of the immune response to an antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist, prior to
(ii) administering to each of the subjects the antigen.
Clause 12C. An antigen for use in a method of reducing the variability of the immune response to an antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist, prior to
(ii) administering to each of the subjects the antigen.
Clause 13C. A STING agonist for use in a method of improving an immune response to an antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the antigen.
Clause 14C. An antigen for use in a method of improving an immune response to an antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the antigen.
Clause 15C. A STING agonist for use in a method of reducing the amount of an antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the antigen.
Clause 16C. An antigen for use in a method of reducing the amount of an antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the antigen.
Clause 17C. A STING agonist for use in a method of preparing the immune system of a subject for (ii) administration of an antigen, wherein the method comprises (i) administering to the subject a STING agonist.
Clause 18C. Use of a STING agonist for the manufacture of a medicament for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject an antigen which is associated with the pathogen.
Clause 19C. Use of an antigen for the manufacture of a medicament for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps: (i) administering to the subject a STING agonist, prior to
(ii) administering to the subject an antigen which is associated with the pathogen.
Clause 20C. Use of a STING agonist for the manufacture of a medicament for use in a method of reducing the variability of the immune response to an antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist, prior to
(ii) administering to each of the subjects the antigen.
Clause 21 C. Use of an antigen for the manufacture of a medicament for use in a method of reducing the variability of the immune response to an antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist, prior to
(ii) administering to each of the subjects the antigen.
Clause 22C. Use of a STING agonist in the manufacture of a medicament for use in a method of improving an immune response to an antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the antigen.
Clause 23C. Use of an antigen for the manufacture of a medicament for use in a method of improving an immune response to an antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the antigen.
Clause 24C. Use of a STING agonist for the manufacture of a medicament for use in a method of reducing the amount of an antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the antigen.
Clause 25C. Use of an antigen for the manufacture of a medicament for use in a method of reducing the amount of an antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the antigen.
Clause 26C. Use of a STING agonist for the manufacture of a medicament for use in a method of preparing the immune system of a subject for administration of an antigen, wherein the method comprises administering to the subject a STING agonist.
Clause 27C. A method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps: (i) administering to the subject a STING agonist, prior to
(ii) administering to the subject a second adjuvant and an antigen which is associated with the pathogen.
Clause 28C. A method of reducing the variability of the immune response to an antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist, prior to
(ii) administering to each of the subjects a second adjuvant and the antigen.
Clause 29C. A method of improving an immune response to an antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject a second adjuvant and the antigen.
Clause 30C. A method of reducing the amount of an antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the antigen and a second adjuvant.
Clause 31 C. A method of preparing the immune system of a subject for (ii) administration of an antigen and a second adjuvant, wherein the method comprises (i) administering to the subject a STING agonist.
Clause 32C. A STING agonist for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject a second adjuvant and an antigen which is associated with the pathogen.
Clause 33C. An antigen for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject a second adjuvant and an antigen which is associated with the pathogen.
Clause 34C. A second adjuvant for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the second adjuvant and an antigen which is associated with the pathogen.
Clause 35C. A STING agonist for use in a method of reducing the variability of the immune response to an antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist, prior to
(ii) administering to each of the subjects a second adjuvant and the antigen. Clause 36C. An antigen for use in a method of reducing the variability of the immune response to an antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist, prior to
(ii) administering to each of the subjects a STING agonist and the antigen.
Clause 37C. A second adjuvant for use in a method of reducing the variability of the immune response to an antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist, prior to
(ii) administering to each of the subjects the second adjuvant and an antigen.
Clause 38C. A STING agonist for use in a method of improving an immune response to an antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject a second adjuvant and the antigen.
Clause 39C. An antigen for use in a method of improving an immune response to an antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject a second adjuvant and the antigen.
Clause 40C. A second adjuvant for use in a method of improving an immune response to an antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the second adjuvant and the antigen.
Clause 41 C. A STING agonist for use in a method of reducing the amount of an antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the antigen and a second adjuvant.
Clause 42C. An antigen for use in a method of reducing the amount of an antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the antigen and a second adjuvant.
Clause 43C. A second adjuvant for use in a method of reducing the amount of an antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the antigen and the second adjuvant. Clause 44C. A STING agonist for use in a method of preparing the immune system of a subject for (ii) administration of an antigen and a second adjuvant, wherein the method comprises (i) administering to the subject a STING agonist.
Clause 45C. Use of a STING agonist for the manufacture of a medicament for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject a second adjuvant and an antigen which is associated with the pathogen.
Clause 46C. Use of an antigen for the manufacture of a medicament for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject a second adjuvant and an antigen which is associated with the pathogen.
Clause 47C. Use of a second adjuvant for the manufacture of a medicament for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the second adjuvant and an antigen which is associated with the pathogen.
Clause 48C. Use of a STING agonist for the manufacture of a medicament for use in a method of reducing the variability of the immune response to an antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist, prior to
(ii) administering to each of the subjects a second adjuvant and the antigen.
Clause 49C. Use of an antigen for the manufacture of a medicament for use in a method of reducing the variability of the immune response to an antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist, prior to
(ii) administering to each of the subjects a second adjuvant and the antigen.
Clause 50C. Use of a second adjuvant for the manufacture of a medicament for use in a method of reducing the variability of the immune response to an antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist, prior to
(ii) administering to each of the subjects the second adjuvant and the antigen. Clause 51 C. Use of a STING agonist in the manufacture of a medicament for use in a method of improving an immune response to an antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject a second adjuvant and the antigen.
Clause 52C. Use of an antigen for the manufacture of a medicament for use in a method of improving an immune response to an antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject a second adjuvant and the antigen.
Clause 53C. Use of a second adjuvant for the manufacture of a medicament for use in a method of improving an immune response to an antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the second adjuvant and the antigen.
Clause 54C. Use of a STING agonist for the manufacture of a medicament for use in a method of reducing the amount of an antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the antigen and a second adjuvant.
Clause 55C. Use of an antigen for the manufacture of a medicament for use in a method of reducing the amount of an antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the antigen and a second adjuvant.
Clause 56C. Use of a second adjuvant for the manufacture of a medicament for use in a method of reducing the amount of an antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject the antigen and the second adjuvant.
Clause 57C. Use of a STING agonist for the manufacture of a medicament for use in a method of preparing the immune system of a subject for administration of an antigen and a second adjuvant, wherein the method comprises administering to the subject a STING agonist.
Clause 58C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 1C to 57C, wherein the STING agonist binds to STING with an in vitro Kf of less than 0.750 pM, such as less than 0.500 pM. Clause 59C. The method, STING agonist, second adjuvant, antigen or use of clause 58C, wherein the STING agonist binds to STING with an in vitro Kf of less than 0.250 pM, such as less than 0.100 pM.
Clause 60C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 1C to 59C, wherein the STING agonist activates STING with an in vitro EC50 of 100 pM or less, such as 50 pM or less, when measured by monitoring phosphorylation of interferon regulatory factor-3 (I RF3).
Clause 61 C. The method, STING agonist, second adjuvant, antigen or use of clause 60C, wherein the STING agonist activates STING with an in vitro EC50 of 20 pM or less, such as 10 pM or less, when measured by monitoring phosphorylation of interferon regulatory factor-3 (IRF3).
Clause 62C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 1C to 61C, wherein the STING agonist activates STING with an in vitro EC50 of 100 pM or less, such as 50 pM or less, as measured by monitoring interferon-p induction.
Clause 63C. The method, STING agonist, second adjuvant, antigen or use of clause 62C, wherein the STING agonist activates STING with an in vitro EC50 of 20 pM or less, such as 10 pM or less, as measured by monitoring interferon-p induction.
Clause 64C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 1C to 63C, wherein the STING agonist is a nucleic acid, a protein, a peptide, or a small molecule.
Clause 65C. The method, STING agonist, second adjuvant, antigen or use of clause 64C, wherein the STING agonist is a small molecule, such as a modified or unmodified cyclic dinucleotide.
Clause 66C. The method, STING agonist, second adjuvant, antigen or use of clause 64C, wherein the STING agonist is selected from a compound of Formulae (l)-(lll):
Figure imgf000261_0002
wherein R1 and R2 are each independently selected from the following groups:
Figure imgf000261_0001
Figure imgf000262_0001
R3 and R4 are each independently -SH or -OH,
R5 and R6 are oxygen or sulphur,
R7 and R8 are each independently halogen, hydrogen, -OH, or OCH3; or a pharmaceutically acceptable salt thereof and/or a tautomer thereof.
Clause 67C. The method, STING agonist, second adjuvant, antigen or use of clause 64C, wherein the STING agonist is the compound:
Figure imgf000262_0002
or a pharmaceutically acceptable salt thereof and/or a tautomer thereof.
Clause 68C. The method, STING agonist, second adjuvant, antigen or use of clause 64, wherein the STING agonist is selected from c-ditzGMP. c-di-thGMP, c-GtzGMP, c-GAMP, c- thGMP, c-tzGMP, c-di-thAMP, c-ditzAMP, c-di-AMP, c-di-GMP, c-diXGMP, c-GthXMP, c-GXMP, c-ACMP, c-AthXMP, c-A^XMP, c-di-thXMP, or c-ditzXMP.
Clause 69C. The method, STING agonist, second adjuvant, antigen or use of clause 64C, wherein the STING agonist is 2’,3’-cGAMP, or a pharmaceutically acceptable salt thereof.
Clause 70C. The method, STING agonist, second adjuvant, antigen or use of clause 64C, wherein the STING agonist is 3’,3’-cGAMP, or a pharmaceutically acceptable salt thereof.
Clause 71 C. The method, STING agonist, second adjuvant, antigen or use of clause 64C, wherein the STING agonist is the compound:
Figure imgf000263_0001
, or a pharmaceutically acceptable salt thereof.
Clause 72C. The method, STING agonist, second adjuvant, antigen or use of clause 64C, wherein the STING agonist is 6-bromo-N-(naphthalen-1-yl)benzo[d][1 ,3]dioxole-5- carboxamide:
Figure imgf000263_0002
Clause 73C. The method, STING agonist, second adjuvant, antigen or use of clause 64C, wherein the STING agonist is a compound of Formula A:
Figure imgf000263_0003
wherein
X is O or NR4A,
Y is O, NR4A, CH2, or absent, n is 0, 1 , 2, or 3,
R1 and R2 are independently selected from OH, OR3, OR3A, SR3, and NR3R4, R3, R4, and R4A are independently selected from hydrogen, C1-C10 alkyl optionally substituted with 1-6 halogen, C6-C10 aryl or 5-10 membered heteroaryl, or R3 and R4 together with the nitrogen atom to which they are attached form a 3 to 7 membered heterocycle or 5 to 10 membered heteroaryl,
Figure imgf000264_0001
represents the point of connection of R3A to the remainder of the molecule,
R5-R10 are independently selected from hydrogen, halogen, pseudohalogen, C1-C10 alkyl optionally substituted with 1-6 halogens, C6-C10 aryl, and 5 to 10 membered heteroaryl; or a pharmaceutically acceptable salt thereof and/or a tautomer thereof.
Clause 74C. The method, STING agonist, second adjuvant, antigen or use of clause 64C, wherein the STING agonist is selected from:
Figure imgf000264_0002
, or a pharmaceutically acceptable salt thereof.
Clause 75C. The method, STING agonist, second adjuvant, antigen or use of clause 64C, wherein the STING agonist is a flavonoid.
Clause 76C. The method, STING agonist, second adjuvant, antigen or use of clause 75C, wherein the STING agonist is 10-(carboxymethyl)-9(10H)acridone (CMA), 5,6- Dimethylxanthenone-4-acetic acid (DMXAA), methoxyvone, 6,4'-dimethoxyflavone, 4'- methoxyflavone, 3',6'-dihydroxyflavone, 7,2'-dihydroxyflavone, daidzein, formononetin, retusin 7-methyl ether, xanthone, or any combination thereof.
Clause 77C. The method, STING agonist, second adjuvant, antigen or use of clause 64C, wherein the STING agonist is a compound of Formula B:
Figure imgf000264_0003
wherein
Figure imgf000265_0001
represents two conjugated double bonds in a five-membered heteroaromatic ring and three conjugated double bonds in a six-membered aromatic or heteroaromatic ring, W1 is selected from CR11 and N; X1 is selected from CR1, C(R1)2, N, NR1, O and S; X2 is selected from CR2, C(R2)2, N, NR2, O and S; X3 is selected from CR3, C(R3)2, N, NR3, O and S; where two or three of X1, X2 and X3 are independently selected from N, NR1, NR2, NR3, O and S; and where at least one of X1, X2 and X3 is selected from N, NR1, NR2 and NR3; Y1 is selected from N, NR4, O, S, CR4 and C(R4)2; Y2 is selected from N, NR5, O, S, CR5 and C(R5)2; Y3 is selected from N, NR6, O, S, CR6 and C(R6)2; Y4 is selected from C and N; Y5 is selected from C and N; where at least one and not more than two of Y1, Y2 and Y3 are independently selected from N, NR4, NR5 and NR6; where when if one of Y4 or Y5 is N, the other one of Y4 or Y5 is C; Z1 is selected from C and N; Z2 is selected from N, NR8 and CR8; Z3 is selected from N, NR9 and CR9; Z4 is selected from N, NR10 and CR10; Z5 is selected from N, NR7 and CR7; where two or three of Z1, Z2, Z3, Z4 and Z5 are independently selected from N, NR7, NR8, NR9, and NR10; each R1 is independently selected from the group consisting of H, C1-C8 alkyl, C1-C8 alkylene-NRR and C1-C8 alkylene-C(O)OR; each R2 is independently selected from the group consisting of H, C1-C8 alkyl, C1-C8 alkylene-NRR, C1-C8 alkylene-C(O)OR, C1-C8 alkylene-OR and C1-C8 alkylene-O- P(O)(OH)2; each R3 is independently selected from the group consisting of H, C1-C8 alkyl, C1-C8 alkylene-NRR, C1-C8 alkylene-C(O)OR and C1-C8 alkylene-O-P(O)(OH)2; each R4 is independently selected from the group consisting of H, -OR, -NRR, C1-C8 alkyl optionally substituted with one or two -OR, C1-C8 alkylene-NRR, -C(O)OR, C1-C8 alkylene-C(O)OR, 3-10 membered heterocycle, C1-C8 alkylene-3-10 membered heterocycle optionally substituted with one 3-10 membered heterocycle, (C3 -C10)- cycloalkyl, and C1-C8 alkylene-(C3-C10)-cycloalkyl; each R5 is independently selected from the group consisting of H, OR, C1 -C8 alkyl, - NRR, C1-C8 alkylene-NRR, -C(O)OR, C1-C8 alkylene-C(O)OR, 3-10 membered heterocycle, C1-C8 alkylene-3-10 membered heterocycle optionally substituted with one 3-10 membered heterocycle, and C1-C8 alkylene-OR; each R6 is H; R7 is selected from the group consisting of H, halo, hydroxy or NH2; R8 is selected from the group consisting of H, C1-C8 alkyl optionally substituted with one or two -NRR or -OR, C1-C8 alkylene-C(O)OR and C1-C8 alkylene-SO2R; R9 is H; R10 is selected from the group consisting of H, C1-C8 alkyl optionally substituted with one or two -OR, and halo; R11 is selected from the group consisting of H, C1-C8 alkyl, -OR and halo; R12 is -C(O)N(R)2 or -C(O)NHR; R13 is H; each R is independently selected from the group consisting of H or C1-C8 alkyl, or C1-C8 haloalkyl, or two R join to form, together with the atom or atoms to which they are bound, a -C3-C10 cycloalkyl or 3-10 membered heterocycle, where said 3-10 membered heterocycle contains one, two or three atoms selected from N, O and S; and where, when two R join to form, together with the atom or atoms to which they are bound, a –(C3-C10) cycloalkyl or 3-10 membered heterocycle, said -C3-C10 cycloalkyl or 3-10 membered heterocycle is optionally substituted with one or more substituents each independently selected from C1-C8 alkyl, hydroxy, C1-C8 alkoxy, -(C3-C10) cycloalkyl, 3-10 membered heterocycle, halo and cyano; or a pharmaceutically acceptable salt thereof and/or a tautomer thereof. Clause 78C. The method, STING agonist, second adjuvant, antigen or use of clause 64C, wherein the STING agonist is a compound selected from:
Figure imgf000267_0001
Figure imgf000268_0001
Figure imgf000269_0001
Figure imgf000270_0001
Figure imgf000271_0001
Figure imgf000272_0001
Figure imgf000273_0001
or a pharmaceutically acceptable salt thereof and/or a tautomer thereof.
Clause 79C. The method, STING agonist, second adjuvant, antigen or use of clauseC, wherein the STING agonist is a compound of Formula C:
Figure imgf000274_0001
wherein — CH-(CH2)n-ring A
Gi is independently selected from Ring A or * ring A is independently selected from optionally substituted heterocyclyl and optionally substituted heteroaryl, ring B is aromatic carbocyclic ring, ring C is optionally substituted five-membered heteroaryl,
R1 is -CON(R3)2,
R2 is independently selected from hydrogen, optionally substituted Ci-Ce alkyl, and optionally substituted C3-C5 monocyclic cycloalkyl,
R3 is independently selected from hydrogen, and optionally substituted Ci-Ce alkyl; m is selected from 0, or 1 ; n is selected from 0, 1 , or 2; o is 1 ; p is selected from 0, 1 , or 2; when 'alkyl's substituted, it is substituted with 1 to 4 substituents independently selected from halogen, alkyl, perhaloalkyl, cycloalkyl, heterocyclyl, -N(R4)2, and -OR4; when ‘carbocycle’ or 'cycloalkyl' is substituted, it is substituted with 1 to 4 substituents independently selected from halogen, alkyl, perhaloalkyl, -N(R4)2, and -OR4; when 'heterocycle' or 'heterocyclyl' is substituted, it is substituted with 1 to 4 substituents independently selected from oxo (=0), halogen, cyano, alkyl, perhaloalkyl, -OR4, -C(=O)OH, -OP(O)(OR4)2, -P(O)(OR4)2, -P(O)(OR4)R4a, -SO2R4a, -SO2NH2, - C(=O)N(H)R4, -C(=O)N(alkyl)R4, -N(H)C(=O)R4a, -N(H)R4, and -N(alkyl)R4; when the 'heteroaryl' group is substituted, it is substituted with 1 to 4 substituents selected from halogen, cyano, alkyl, perhaloalkyl, -O-alkyl, -O-perhaloalkyl, - N(alkyl)alkyl, -N(H)R4, -SO2-alkyl, -N(alkyl)C(=O)alkyl, -N(H)C(=O)alkyl, - C(=O)N(alkyl)alkyl, -C(=O)N(H)alkyl, -C(=O)NH2, -SO2N(alkyl)alkyl, -SO2N(H)alkyl, - SO2NH2, -C(=O)OH, -OP(O)(OR4)2, -P(O)(OR4)2, and -P(O)(OR4)R4a; each R4 is independently selected from hydrogen, alkyl, and cycloalkyl; and each R4a is independently selected from alkyl, and cycloalkyl; or a pharmaceutically acceptable salt thereof and/or a tautomer thereof.
Clause 80C. The method, STING agonist, second adjuvant, antigen or use of clause 64C, wherein the STING agonist is selected from:
Figure imgf000275_0001
Figure imgf000276_0001
Figure imgf000277_0001
Figure imgf000278_0001
or a pharmaceutically acceptable salt thereof and/or a tautomer thereof.
Clause 81 C. The method, STING agonist, second adjuvant, antigen or use of clause 64C, wherein the STING agonist is selected from IMSA101 , ADU-S100 (MIW815), BMS-
986301, CRD5500, CMA (IO-carboxymethyl-9-acridanone), diABZI STING agonist-1 (e.g., CAS No.: 2138299-34-8), DMXAA (ASA404/vadimezan), E7766 (Cas no. 2242635-02-3)
Figure imgf000278_0002
, MK-1454, MK-2118, SB-11285,
SRCB-0074, TAK-676, and TTI-10001 or a pharmaceutically acceptable salt thereof. Clause 82C. The method, STING agonist, second adjuvant, antigen or use of clause
64C, wherein the STING agonist is a compound according to Formula (l-N):
Figure imgf000279_0001
wherein: q is 0 or 1; r is 0 or 1; s is 0 or 1; wherein q + r + s = 1 or 2; when q is 0, RA1 and RA2 are each independently H, halogen, hydroxy, –O-P(O)(OH)2, -O-(O)(RIRII)2, -N(Re)(Rf), -CO2Rf, -N(Rf)CORb, -N(Rg)SO2(C1-C4alkyl)-N(Re)(Rf), -N(Rg)CO(C1-C4alkyl)-N(Rh)(Rf), optionally substituted (C1-C6alkyl), optionally substituted (C1-C6alkyl)oxy-, optionally substituted (C1-C6alkyl)amino-, and optionally substituted (C1-C6alkyl)(C1-C4alkyl)amino-, wherein the (C1-C6alkyl) of said optionally substituted (C1-C6alkyl), optionally substituted (C1-C6alkyl)oxy-, optionally substituted (C1-C6alkyl)amino- and optionally substituted (C1-C6alkyl)(C1-C4alkyl)amino- is optionally substituted by 1-4 substituents each independently selected from hydroxy, -O-P(O)(OH)2, -O-P(O)(RIRII)2, C1-C4alkoxy-, -N(Re)(Rf), -CO2(Rf), -CON(Re)(Rf), optionally substituted phenyl, optionally substituted 5-6 membered heterocycloalkyl and optionally substituted 5-6 membered heteroaryl group, wherein said optionally substituted phenyl, 5-6 membered heterocycloalkyl or 5-6 membered heteroaryl is optionally substituted by 1-4 substituents each independently selected from halogen, hydroxy, -O-P(O)(OH)2, -O- P(O)(RIRII)2, amino, (C1-C6alkyl)amino-, (C1-C6alkyl)(C1-C6alkyl)amino-, -(C1-C6alkyl)- NH2, halo(C1-C6alkyl), hydroxy-(C1-C4alkyl)-, -(C1-C4alkyl)-O-P(O)(OH)2, - (C1-C4alkyl)-O-P(O)(RIRII)2, halo(C1-C4alkoxy)-, C1-C4alkoxy-, hydroxy-(C2-C4alkoxy)-, -(C2-C4alkoxy)-O-P(O)(OH)2, -(C2-C4alkoxy)-O-P(O)(RIRII)2, -C1-C4alkyl-(C1-C4alkoxy) or C1-C4alkoxy-(C1-C4alkoxy)-; when r is 0, RB1 and RB2 are each independently H, optionally substituted C1-C6alkyl, halo(C1-C6alkyl), optionally substituted C2-C6alkenyl, optionally substituted C2-C6alkynyl, optionally substituted C3-C6cycloalkyl, optionally substituted 4-6 membered heterocycloalkyl, optionally substituted phenyl, optionally substituted 5-6 membered heteroaryl, or optionally substituted 9-10 membered heteroaryl, wherein said optionally substituted C1-C6alkyl, optionally substituted C2-C6alkenyl, optionally substituted C2-C6alkynyl, optionally substituted C3-C6cycloalkyl, optionally substituted 4-6 membered heterocycloalkyl, optionally substituted phenyl, optionally substituted 5-6 membered heteroaryl, or optionally substituted 9-10 membered heteroaryl is optionally substituted by 1-4 substituents each independently selected from halogen, nitro, -Rc, -OH, -O-P(O)(OH)2, -O- P(O)(RIRII)2, -ORc, -NH2, -NRcRc, -NRcRd, -OCORc, -CO2H, -CO2Rc, -SORc, -SO2Rc, -CONH2, -CONRcRd, -SO2NH2, -SO2NRcRd, -OCONH2, -OCONRcRd, -NRdCORc, -NRdSORc, -NRdCO2Rc, and -NRdSO2Rc; when s is 0, RC1 is H, halogen, or C1-C4alkyl and RC2 is optionally substituted C1-C4alkyl, wherein said optionally substituted C1-C4alkyl group is optionally substituted by a substituent selected from -ORc, -NRcRd, -CO2Rc, -CONRcRd, -SO2NRcRd, and -OCONRcRd; when q is 1, RA1 and RA2 are each independently -CH2-, -NRe-, or -O-, and A, taken together with RA1 and RA2, forms a linking group, wherein A is -halo(C1-C12alkyl)-, optionally substituted -C1-C12alkyl-, optionally substituted -C2-C12alkenyl-, optionally substituted -C2-C12alkynyl-, optionally substituted -C1-C6alkyl-O-C1-C6alkyl-, optionally substituted -C1-C6alkyl-NRa-C1-C6alkyl-, optionally substituted -C1-C6alkyl-(C3-C6cycloalkyl)-C1-C6alkyl-, optionally substituted -C1-C6alkyl-phenyl-C1-C6alkyl-, optionally substituted -C1-C6alkyl-(4-6 membered heterocycloalkyl)-C1-C6alkyl-, or optionally substituted -C1-C6alkyl-(5-6 membered heteroaryl)-C1-C6alkyl-, wherein the alkyl moiety of said optionally substituted -C1-C12alkyl-, optionally substituted -C2-C12alkenyl-, optionally substituted -C2-C12alkynyl-, optionally substituted -C1-C6alkyl-O-C1-C6alkyl-, optionally substituted -C1-C6alkyl-NRa-C1-C6alkyl-, optionally substituted -C1-C6alkyl-(C3-C6cycloalkyl)-C1-C6alkyl-, optionally substituted -C1-C6alkyl-phenyl-C1-C6alkyl-, optionally substituted -C1-C6alkyl-(4-6 membered heterocycloalkyl)-C1-C6alkyl-, or optionally substituted -C1-C6alkyl-(5-6 membered heteroaryl)-C1-C6alkyl- is optionally substituted by 1-4 substituents each independently selected from halogen, halo(C1-C4alkyl), -OH, -O-P(O)(OH)2, -O-P(O)(RIRII)2, -ORc, -NH2, -NRcRd, -OCORc, -CO2H, -CO2Rc, -SORc, -SO2Rc, -CONH2, -CONRcRd, -SO2NH2, -SO2NRcRd, -OCONH2, -OCONRcRd, -NRdCORc, -NRdSORc, -NRdCO2Rc, and -NRdSO2Rc, and the C3-C6cycloalkyl, phenyl, 4-6 membered heterocycloalkyl, or 5-6 membered heteroaryl moiety of said optionally substituted -C1-C6alkyl-(C3-C6cycloalkyl)-C1-C6alkyl-, optionally substituted -C1-C6alkyl-phenyl-C1-C6alkyl-, optionally substituted -C1-C6alkyl-(4-6 membered heterocycloalkyl)-C1-C6alkyl-, or optionally substituted -C1-C6alkyl-(5-6 membered heteroaryl)-C1-C6alkyl- is optionally substituted by 1-4 substituents each independently selected from halogen, hydroxy, -O-P(O)(OH)2, -O-P(O)(RIRII)2, amino, (C1-C4alkyl)amino-, (C1-C4alkyl)(C1-C4alkyl)amino-, C1-C4alkyl, halo(C1-C4alkyl), halo(C1-C4alkoxy)-, C1-C4alkoxy-, hydroxy-(C1-C4alkoxy)-, -(C1-C4alkoxyl)-O-P(O)(OH)2, -(C1-C4alkoxyl)-O-P(O)(RIRII)2 and C1-C4alkoxy-(C1-C4alkoxy)-; when r is 1, RB1 and RB2 are each independently -CH -, and B, taken together wit B1 2 h R and RB2, forms a linking group, wherein B is a bond or B is -halo(C1-C10alkyl)-, optionally substituted -C1-C10alkyl-, optionally substituted -C2-C10alkenyl-, optionally substituted -C2-C10alkynyl-, optionally substituted -C1-C6alkyl-O-C1-C6alkyl-, optionally substituted -C1-C6alkyl-NRa-C1-C6alkyl-, optionally substituted C3-C6cycloalkyl, optionally substituted phenyl, optionally substituted 4-6 membered heterocycloalkyl, optionally substituted 5-6 membered heteroaryl, optionally substituted -C1-C4alkyl-(C3-C6cycloalkyl)-C1-C4alkyl-, optionally substituted -C1-C4alkyl-phenyl-C1-C4alkyl-, optionally substituted -C1-C4alkyl-(4-6 membered heterocycloalkyl)-C1-C4alkyl-, or optionally substituted -C1-C4alkyl-(5-6 membered heteroaryl)-C1-C4alkyl-, wherein the alkyl moiety of said optionally substituted -C1-C10alkyl-, optionally substituted -C2-C10alkenyl-, optionally substituted -C2-C10alkynyl-, optionally substituted -C1-C6alkyl-O-C1-C6alkyl-, optionally substituted -C1-C6alkyl-NRa-C1-C6alkyl-, optionally substituted -C1-C4alkyl-(C3-C6cycloalkyl)-C1-C4alkyl-, optionally substituted -C1-C4alkyl-phenyl-C1-C4alkyl-, optionally substituted -C1-C4alkyl-(4-6 membered heterocycloalkyl)-C1-C4alkyl-, or optionally substituted -C1-C4alkyl-(5-6 membered heteroaryl-C1-C4alkyl)- is optionally substituted by 1 or 2 substituents each independently selected from halogen, halo(C1-C4alkyl), -OH, -O-P(O)(OH)2, -O-P(O)(RIRII)2, -ORc, -NH2, -NRcRd, -OCORc, -CO2H, -CO2Rc, -SORc, -SO2Rc, -CONH2, -CONRcRd, -SO2NH2, -SO2NRcRd, -OCONH2, -OCONRcRd, -NRdCORc, -NRdSORc, -NRdCO2Rc, and -NRdSO2Rc, and the C3-C6cycloalkyl, phenyl, 4-6 membered heterocycloalkyl, or 5-6 membered heteroaryl moiety of said optionally substituted C3-C6cycloalkyl, optionally substituted phenyl, optionally substituted 4-6 membered heterocycloalkyl, optionally substituted 5-6 membered heteroaryl, optionally substituted -C1-C4alkyl-(C3-C6cycloalkyl)-C1-C4alkyl-, optionally substituted -C1-C4alkyl-phenyl-C1-C4alkyl-, optionally substituted -C1-C4alkyl-(4-6 membered heterocycloalkyl)-C1-C4alkyl-, or optionally substituted -C1-C4alkyl-(5-6 membered heteroaryl)-C1-C4alkyl- is optionally substituted by 1-4 substituents each independently selected from halogen, hydroxy, -O-P(O)(OH)2, -O- P(O)(RIRII)2, amino, (C1-C4alkyl)amino-, (C1-C4alkyl)(C1-C4alkyl)amino-, C1-C4alkyl, halo(C1-C4alkyl), halo(C1-C4alkoxy)-, C1-C4alkoxy-, hydroxy-(C2-C4alkoxy)-, -(C2-C4alkoxy) O-P(O)(OH)2, -(C2-C4alkoxy)-O-P(O)(RIRII)2, and C1-C4alkoxy-(C1-C4alkoxy)-; when s is 1, RC1 and RC2 are each independently -CH -, and C, taken toget C1 2 her with R and RC2, forms a linking group, wherein C is -halo(C1-C12alkyl)-, optionally substituted -C1-C12alkyl-, optionally substituted -C2-C12alkenyl-, optionally substituted -C2-C12alkynyl-, optionally substituted -C1-C6alkyl-O-C1-C6alkyl-, optionally substituted -C1-C6alkyl-NRa-C1-C6alkyl-, optionally substituted -C1-C6alkyl-(C3-C6cycloalkyl)-C1-C6alkyl-, optionally substituted -C1-C6alkyl-phenyl-C1-C6alkyl-, optionally substituted -C1-C6alkyl-(4-6 membered heterocycloalkyl)-C1-C6alkyl-, or optionally substituted -C1-C6alkyl-(5-6 membered heteroaryl)-C1-C6alkyl-, wherein the alkyl moiety of said optionally substituted -C1-C12alkyl-, optionally substituted -C2-C12alkenyl-, optionally substituted -C2-C12alkynyl-, optionally substituted -C1-C6alkyl-O-C1-C6alkyl-, optionally substituted -C1-C6alkyl-NRa-C1-C6alkyl-, optionally substituted -C1-C6alkyl-(C3-C6cycloalkyl)-C1-C6alkyl-, optionally substituted -C1-C6alkyl-phenyl-C1-C6alkyl-, optionally substituted -C1-C6alkyl-(4-6 membered heterocycloalkyl)-C1-C6alkyl-, or optionally substituted -C1-C6alkyl-(5-6 membered heteroaryl)-C1-C6alkyl- is optionally substituted by 1 or 2 substituents each independently selected from halogen, halo(C1-C4alkyl), -OH, -O-P(O)(OH)2, -O-P(O)(RIRII)2, -ORc, -NH2, -NRcRd, -OCORc, -CO2H, -CO2Rc, -SORc, -SO2Rc, -CONH2, -CONRcRd, -SO2NH2, -SO2NRcRd, -OCONH2, -OCONRcRd, -NRdCORc, -NRdSORc, -NRdCO2Rc, and -NRdSO2Rc, and the C3-C6cycloalkyl, phenyl, 4-6 membered heterocycloalkyl, or 5-6 membered heteroaryl moiety of said optionally substituted -C1-C6alkyl-(C3-C6cycloalkyl)-C1-C6alkyl-, optionally substituted -C1-C6alkyl-phenyl-C1-C6alkyl-, optionally substituted -C1-C6alkyl-(4-6 membered heterocycloalkyl)-C1-C6alkyl-, or optionally substituted -C1-C6alkyl-(5-6 membered heteroaryl)-C1-C6alkyl- is optionally substituted by 1-4 substituents each independently selected from halogen, hydroxy, -O-P(O)(OH)2, -O-P(O)(RIRII)2, amino, (C1-C4alkyl)amino-, (C1-C4alkyl)(C1-C4alkyl)amino-, C1-C4alkyl, halo(C1-C4alkyl), halo(C1-C4alkoxy)-, C1-C4alkoxy-, hydroxy-(C2-C4alkoxy)-, -(C2-C4alkoxy)-O-P(O)(OH)2, -(C2-C4alkoxy)-O-P(O)(RIRII)2,and C1-C4alkoxy-(C1-C4alkoxy)-; R3 and R5 are each independently -CON(Rd)(Rf), or one of R3 and R5 is -CON(Rd)(Rf), and the other of R3 and R5 is H, COOH or -CO2(Rc); R4 and R6 are each independently selected from H, halogen, halo(C1-C6alkyl), halo(C1-C6alkoxy)-, hydroxy, –O-P(O)(OH)2, –O-P(O)(RIRII)2, -NH2, -NRcRc, -NRcRd, -CORc, -CO2Rc, -N(Rd)CORc, -N(Rd)SO2Rc, -N(Rg)SO2(C1-C2alkyl)-N(Rh)(Rf), -N(Rg)CO(C1-C2alkyl)-N(Rh)(Rf), optionally substituted (C1-C6alkyl), optionally substituted (C1-C6alkyl)oxy-, optionally substituted (C1-C6alkyl)amino-, and optionally substituted (C1-C6alkyl)(C1-C4alkyl)amino-, wherein the (C1-C6alkyl) of said optionally substituted (C1-C6alkyl), optionally substituted (C1-C6alkyl)oxy-, optionally substituted (C1-C6alkyl)amino- and optionally substituted (C1-C6alkyl)(C1-C4alkyl)amino- is optionally substituted by 1-4 substituents each independently selected from -OH, -O-P(O)(OH)2, -O-P(O)(RIRII)2,-ORc, -NH2, -NRcRc, -NRcRd, -CO2H, -CO2Rc, -OCORc, -CO2H, -CO2Rc, -SORc, -SO2Rc, -CONH2, -CONRcRd, -SO2NH2, -SO2NRcRd, -OCONH2, -OCONRcRd, -NRdCORc, -NRdSORc, -NRdCO2Rc, -NRdSO2Rc, optionally substituted phenyl, optionally substituted 5-6 membered heterocycloalkyl and optionally substituted 5-6 membered heteroaryl group, wherein said optionally substituted phenyl, 5-6 membered heterocycloalkyl or 5-6 membered heteroaryl is optionally substituted by 1-4 substituents each independently selected from halogen, hydroxy, -O-P(O)(OH)2, -O- P(O)(RIRII)2, amino, (C1-C4alkyl)amino-, (C1-C4alkyl)(C1-C4alkyl)amino-, C1-C4alkyl, halo(C1-C4alkyl), hydroxy-(C1-C4alkyl)-, -(C1-C4alkyl)-O-P(O)(OH)2, -(C1-C4alkyl)-O-P(O)(RIRII)2, halo(C1-C4alkoxy)-, C1-C4alkoxy-, hydroxy-(C2-C4alkoxy)-, -(C2-C4alkoxy)-O-P(O)(OH)2, -(C2-C4alkoxy)-O-P(O)(RIRII)2, C1-C4alkoxy-(C1-C4alkoxy)-, -CORd, -CON(Rd)(Rf), and -CO2Rd; R14 is optionally substituted C1-C4alkyl, wherein said optionally substituted C1-C4alkyl is optionally substituted by a substituent selected from -ORc, -NRcRd, -CO2Rc, -CONRcRd, -SO2NRcRd, and -OCONRcRd; R16 is H, halogen, or C1-C4alkyl; R15 and R17 are each independently H, cyclopropyl, or C1-C4alkyl; Ra is H, -Rc, -CORc, -CO2H, -CO2Rc, -SORc, -SO2Rc, -CONH2, -CONRcRd, -SO2NH2, or -SO2NRcRd; each Rb is independently C1-C4alkyl, halo(C1-C4alkyl), -(C1-C4alkyl)-OH, -(C1-C4alkyl)-O-P(O)(OH)2, -(C1-C4alkyl)-O-P(O)(RIRII)2, -(C1-C4alkyl)-O-(C1-C4alkyl), -(C1-C4alkyl)-N(Re)(Rf), -(C1-C4alkyl)-O-CO(C1-C4alkyl), or -(C1-C4alkyl)-CO-O- (C1-C4alkyl); each Rc is independently C1-C4alkyl, halo(C1-C4alkyl), -(C1-C4alkyl)-OH, -(C1-C4alkyl)-O-P(O)(OH)2, -(C1-C4alkyl)-O-P(O)(RIRII)2,-(C1-C4alkyl)-O-(C1-C4alkyl), -(C1-C4alkyl)-N(Re)(Rf), -(C1-C4alkyl)-O-CO(C1-C4alkyl), -(C1-C4alkyl)-CO-O- (C1-C4alkyl), optionally substituted C3-C6cycloalkyl, optionally substituted phenyl, optionally substituted 4-6 membered heterocycloalkyl, optionally substituted 5-6 membered heteroaryl, optionally substituted 9-10 membered heteroaryl, optionally substituted -C1-C4alkyl-C3-C6cycloalkyl, optionally substituted -C1-C4alkyl-phenyl, optionally substituted -C1-C4alkyl-4-6 membered heterocycloalkyl, optionally substituted -C1-C4alkyl-5-6 membered heteroaryl, or optionally substituted -C1-C4alkyl-9-10 membered heteroaryl, wherein the C3-C6cycloalkyl, phenyl, 4-6 membered heterocycloalkyl, 5-6 membered heteroaryl or 9-10 membered heteroaryl moiety of said optionally substituted C3-C6cycloalkyl, optionally substituted phenyl, optionally substituted 4-6 membered heterocycloalkyl, optionally substituted 5-6 membered heteroaryl, optionally substituted 9-10 membered heteroaryl optionally substituted -C1-C4alkyl-C3-C6cycloalkyl, optionally substituted -C1-C4alkyl-phenyl, optionally substituted -C1-C4alkyl-4-6 membered heterocycloalkyl, optionally substituted -C1-C4alkyl-5-6 membered heteroaryl, or optionally substituted -C1-C4alkyl-9-10 membered heteroaryl is optionally substituted by 1-4 substituents each independently selected from halogen, hydroxy, -O-P(O)(OH)2, -O-P(O)(RIRII)2, amino, -(C1-C4alkyl)NH2, (C1-C4alkyl)amino-, (C1-C4alkyl)(C1-C4alkyl)amino-, C1-C4alkyl, halo(C1-C4alkyl), halo(C1-C4alkoxy)-, C1-C4alkoxy-, hydroxy-(C2-C4alkoxy)-, -(C2-C4alkoxy)-O-P(O)(OH)2, -(C2-C4alkoxy)-O-P(O)(RIRII)2, C1-C4alkoxy-(C1-C4alkoxy)-, -CORd, -CON(Rd)(Rf), and -CO2Rd; each Rd is independently H or C1-C4alkyl; each Re is independently H, (C1-C4alkyl), -CO(C1-C4alkyl), -OCO(C1-C4alkyl), -CO2(C1-C4alkyl), -(C1-C4alkyl)NH2, -(C1-C4alkyl) C1-C4alkoxy, -CO-(optionally substituted 5-6 membered heterocycloalkyl), -CO(C1-C4alkyl)-(optionally substituted 5-6 membered heterocycloalkyl), -CO(optionally substituted 5-6 membered heteroaryl), -CO(C1-C4alkyl)-(optionally substituted 5-6 membered heteroaryl), wherein the optionally substituted 5-6 membered heterocycloalkyl or optionally substituted 5-6 membered heteroaryl is optionally substituted 1-4 substituents each independently selected from halogen, hydroxy, -O-P(O)(OH)2, -O-P(O)(RIRII)2, amino, (C1-C4alkyl)amino-, (C1-C4alkyl)(C1-C4alkyl)amino-, C1-C4alkyl, halo(C1-C4alkyl), halo(C1-C4alkoxy)-, C1-C4alkoxy-, hydroxy-(C2-C4alkoxy)-, -(C2-C4alkoxy) O-P(O)(OH)2, -(C2-C4alkoxy)-O-P(O)(RIRII)2, C1-C4alkoxy-(C1-C4alkoxy)- , -CORd, -CON(Rd)(Rf), and -CO2Rd; each Rf is independently H or (C1-C4alkyl); Rg and Rh are each independently H or (C1-C4alkyl) or Rg and Rh, taken together with the atom or atoms through which they are connected, form a 5-6 membered ring; and each occurrence of RI and RII are independently (C1-C6alkyl)oxy-; or a pharmaceutically acceptable salt thereof and/or a tautomer thereof. Clause 83C. The method, STING agonist, second adjuvant, antigen or use of clause 64C, wherein the STING agonist is selected from: (E)-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1H- benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-7- (3-hydroxypropoxy)-1H-benzo[d]imidazole-5-carboxamide
Figure imgf000285_0001
(E)-1-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-2,3- dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-5- carbonyl)imino)-7-(3-hydroxypropoxy)-2,3-dihydro-1H-benzo[d]imidazole-5- carboxamide
Figure imgf000286_0001
(Z)-1-((E)-4-((Z)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-2,3- dihydro-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-7-(3-hydroxypropoxy)-2,3-dihydro-1 H-benzo[d]imidazole-5- carboxamide
Figure imgf000286_0002
(E)-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1 H-pyrazole-5-carboxamido)-7-(3- hydroxypropoxy)-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1 H- pyrazole-5-carboxamido)-7-methoxy-1 H-benzo[d]imidazole-5-carboxamide
Figure imgf000286_0003
(E)-1-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-7-(3- hydroxypropoxy)-2,3-dihydro-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3- methyl-1 H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1 H-benzo[d]imidazole-5- carboxamide
Figure imgf000287_0001
(Z)-1-((E)-4-((Z)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-7-(3- hydroxypropoxy)-2,3-dihydro-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3- methyl-1 H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1 H-benzo[d]imidazole-5- carboxamide
Figure imgf000287_0002
(E)-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1 H-pyrazole-5-carboxamido)-1 H- benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1 H-pyrazole-5-carboxamido)-7-
(3-morpholinopropoxy)-1 H-benzo[d]imidazole-5-carboxamide
Figure imgf000287_0003
(E)-1-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-2,3- dihydro-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-7-(3-morpholinopropoxy)-2,3-dihydro-1 H-benzo[d]imidazole-5- carboxamide
Figure imgf000288_0001
(Z)-1-((E)-4-((Z)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-2,3- dihydro-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-7-(3-morpholinopropoxy)-2,3-dihydro-1 H-benzo[d]imidazole-5- carboxamide
Figure imgf000288_0002
(E)-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1 H-pyrazole-5-carboxamido)-7-(3- morpholinopropoxy)-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1 H- pyrazole-5-carboxamido)-7-methoxy-1 H-benzo[d]imidazole-5-carboxamide
Figure imgf000288_0003
(E)-1-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-7-(3- morpholinopropoxy)-2,3-dihydro-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3- methyl-1 H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1 H-benzo[d]imidazole-5- carboxamide
Figure imgf000289_0001
(Z)-1-((E)-4-((Z)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-7-(3- morpholinopropoxy)-2,3-dihydro-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3- methyl-1 H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1 H-benzo[d]imidazole-5- carboxamide
Figure imgf000289_0002
3-(((Z)-6-carbamoyl-3-((E)-4-((Z)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-7-methoxy-2,3-dihydro-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1- ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1 H-benzo[d]imidazol-4- yl)oxy)propyldihydrogen phosphate
Figure imgf000289_0003
(E)-3-((5-carbamoyl-1 -(4-(5-carbamoyl-2-(1 -ethyl-3-methyl-1 H-pyrazole-5- carboxamido)-7-methoxy-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl- 1 H-pyrazole-5-carboxamido)-1 H-benzo[d]imidazol-7-yl)oxy)propyl dihydrogen phosphate
Figure imgf000290_0001
3-(((E)-6-carbamoyl-3-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-7-methoxy-2,3-dihydro-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1- ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1 H-benzo[d]imidazol-4- yl)oxy)propyl dihydrogen phosphate
Figure imgf000290_0002
(E)-4-((5-carbamoyl-1 -(4-(5-carbamoyl-2-(1 -ethyl-3-methyl-1 H-pyrazole-5- carboxamido)-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1 H-pyrazole-
5-carboxamido)-1 H-benzo[d]imidazol-7-yl)oxy)butanoic acid
Figure imgf000290_0003
(E)-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1 H-pyrazole-5-carboxamido)-7-methoxy-1 H- benzo[d]imidazol-1-yl)but-2-en-1-yl)-7-(3-(dimethylamino)propoxy)-2-(1-ethyl-3-methyl-
1 H-pyrazole-5-carboxamido)-1 H-benzo[d]imidazole-5-carboxamide
Figure imgf000291_0001
(E)-1-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-7-(3-
(4-(2-hydroxyethyl)piperazin-1-yl)propoxy)-2,3-dihydro-1 H-benzo[d]imidazol-1-yl)but-2- en-1-yl)-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1 H- benzo[d]imidazole-5-carboxamide
Figure imgf000291_0002
or a pharmaceutically acceptable salt thereof and/or a tautomer thereof.
Clause 84C. The method, STING agonist, second adjuvant, antigen or use of clause 64C, wherein the STING agonist is 3-(((E)-6-carbamoyl-3-((E)-4-((E)-5-carbamoyl-2-((1-ethyl- 3-methyl-1 H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1 H-benzo[d]imidazol-1-yl)but- 2-en-1-yl)-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1 H- benzo[d]imidazol-4-yl)oxy)propyl
Figure imgf000292_0001
or a pharmaceutically acceptable salt thereof.
Clause 85C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 1C to 84C, wherein a dose comprises 0.1 to 150 ug, especially 0.5 to 100 ug, such as 1 to 50 ug of STING agonist.
Clause 86C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 1C to 85C, wherein the STING agonist is formulated with a carrier.
Clause 87C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 1C to 86C, wherein the second adjuvant comprises a saponin.
Clause 88C. The method, STING agonist, second adjuvant, antigen or use of clause 87C, wherein the saponin is obtainable from Quillaja saponaria.
Clause 89C. The method, STING agonist, second adjuvant, antigen or use of clause 88C, wherein the saponin is obtained from Quillaja saponaria.
Clause 90C. The method, STING agonist, second adjuvant, antigen or use of either claim 87C or 88C, wherein the saponin is prepared synthetically.
Clause 91 C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 87C to 90C, wherein the saponin is Quil A or a fraction thereof, such as QS-7, QS- 17, QS-18 and/or QS-21.
Clause 92C. The method, STING agonist, second adjuvant, antigen or use of clause 91 C, wherein the saponin is QS-7.
Clause 93C. The method, STING agonist, second adjuvant, antigen or use of clause 91 C, wherein the saponin is QS-21.
Clause 94C. The method, STING agonist, second adjuvant, antigen or use of clause 93C, wherein the saponin comprises QS-21 1988 A component, such as at least 40%, such as at least 50%, in particular at least 60%, especially at least 65%, such as at least 70%, QS-21 1988 A component as determined by UV absorbance at 214 nm and by relative ion abundance. Clause 95C. The method, STING agonist, second adjuvant, antigen or use of clause 94C, wherein the saponin comprises QS-21 1988 A V1.
Clause 96C. The method, STING agonist, second adjuvant, antigen or use of either clause 94C or 95C, wherein the saponin comprises QS-21 1988 A V2.
Clause 97C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 93C to 96C, wherein the saponin comprises QS-21 1856 A component, such as at least 5%, such as at least 10% QS-21 1856 A by UV absorbance at 214 nm and by relative ion abundance.
Clause 98C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 93C to 97C wherein the saponin comprises QS-21 2002 A component, such as at least 0.5%, such as at least 1%, QS-21 2002 A component by UV absorbance at 214 nm and by relative ion abundance.
Clause 99C. The method, STING agonist, second adjuvant, antigen or use of clause 98C, wherein the saponin comprises QS-21 2002 A V1.
Clause 100C. The method, STING agonist, second adjuvant, antigen or use of either clause 98C or 99C, wherein the saponin comprises QS-21 2002 A V2.
Clause 101C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 91 C or 93C to 100C, wherein the saponin comprises at least 40%, such as at least 50%, suitably at least 60%, especially at least 70% and desirably at least 80%, for example at least 90% (as determined by UV absorbance at 214 nm and by relative ion abundance) QS-21 1988 A component, QS-21 1856 A component and/or QS-21 2002 A component.
Clause 102C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 87C to 101 C, wherein a dose comprises 1 to 100 ug of saponin.
Clause 103C. The method, STING agonist, second adjuvant, antigen or use of clause 102C, wherein a dose comprises 40 to 60 ug, such as 45 to 55 ug, especially 49 to 51 ug, in particular 50 ug of saponin.
Clause 104C. The method, STING agonist, second adjuvant, antigen or use of clause 102C, wherein a dose comprises 20 to 30 ug, such as 22 to 28 ug, especially 24 to 26 ug, in particular 25 ug of saponin.
Clause 105C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 87C to 104C, wherein the saponin is formulated with a carrier.
Clause 106C. The method, STING agonist, second adjuvant, antigen or use of clause 105C, wherein the saponin is formulated with ISCOMS.
Clause 107C. The method, STING agonist, second adjuvant, antigen or use of clause 105C, wherein the saponin is formulated with an emulsion.
Clause 108C. The method, STING agonist, second adjuvant, antigen or use of clause 105C, wherein the saponin is formulated with liposomes. Clause 109C. The method, STING agonist, second adjuvant, antigen or use of clause 108C, wherein the liposomes comprise DOPC.
Clause 110C. The method, STING agonist, second adjuvant, antigen or use of either clause 108C or 109C, wherein the liposomes comprise a sterol, such as cholesterol.
Clause 111C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 108C to 110C, wherein the liposome size is in the range of 50 nm to 200 nm, especially 60 nm to 180 nm.
Clause 112C. The method, STING agonist, second adjuvant, antigen or use of clause 111 C, wherein the liposome size is in the range of 70 to 165 nm, in particular about 100 nm.
Clause 113C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 108C to 112C, wherein the liposome polydispersity is not more than 0.3, such as not more than 0.2.
Clause 114C. The method, STING agonist, second adjuvant, antigen or use of clauses 87C to 113C, wherein the adjuvant comprising a saponin does not comprise non-saponin immunostimulants.
Clause 115C. The method, STING agonist, second adjuvant, antigen or use of clauses 87C to 113C, wherein the adjuvant comprising a saponin additionally comprises further immunostimulants.
Clause 116C. The method, STING agonist, second adjuvant, antigen or use of clause 115C, wherein the adjuvant comprising a saponin also comprises a TLR4 agonist.
Clause 117C. The method, STING agonist, second adjuvant, antigen or use of clause 116C, wherein the TLR4 agonist comprises, such as consists of, lipopolysaccharide.
Clause 118C. The method, STING agonist, second adjuvant, antigen or use of clause 117C, wherein the TLR4 agonist comprises, such as consists of, non-toxic derivatives of lipid A.
Clause 119C. The method, STING agonist, second adjuvant, antigen or use of clause 118C, wherein the TLR4 agonist comprises, such as consists of, monophosphoryl lipid A.
Clause 120C. The method, STING agonist, second adjuvant, antigen or use of clause 119C, wherein the TLR4 agonist comprises, such as consists of, 3-de-O-acylated monophosphoryl lipid A.
Clause 121C. The method, STING agonist, second adjuvant, antigen or use of clause 120C, wherein the TLR4 agonist comprises, such as consists of, 3-deacyl monophosphoryl hexa-acyl lipid A.
Clause 122C. The method, STING agonist, second adjuvant, antigen or use of clause 120C, wherein the TLR4 agonist comprises, such as consists of, 3-deacyl monophosphoryl lipid A. Clause 123C. The method, STING agonist, second adjuvant, antigen or use of clause 120C, wherein the TLR4 agonist comprises, such as consists of, aminoalkyl glucosaminide phosphate.
Clause 124C. The method, STING agonist, second adjuvant, antigen or use of clause 123C, wherein the TLR4 agonist comprises, such as consists of, CRX601 :
Figure imgf000295_0001
Clause 125C. The method, STING agonist, second adjuvant, antigen or use of clause 116C, wherein the TLR4 agonist comprises, such as consists of, dLOS.
Clause 126C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 116C to 125C, wherein a dose comprises 1 to 100 ug of TLR4 agonist.
Clause 127C. The method, STING agonist, second adjuvant, antigen or use of clause 126C, wherein a dose comprises 40 to 60 ug, such as 45 to 55 ug, especially 49 to 51 ug, in particular 50 ug of TLR4 agonist.
Clause 128C. The method, STING agonist, second adjuvant, antigen or use of clause 126C, wherein a dose comprises 20 to 30 ug, such as 22 to 28 ug, especially 24 to 26 ug, in particular 25 ug of TLR4 agonist.
Clause 129C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 116C to 128C, wherein the weight ratio of TLR4 agonist to saponin is between 1 :5 to 5: 1 , such as 1 :2 to 2: 1 , especially about 1:1.
Clause 130C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 116C to 129C, wherein the TLR4 agonist is formulated with a carrier. Clause 131C. The method, STING agonist, second adjuvant, antigen or use of clause 130C, wherein the TLR4 agonist is formulated with an emulsion.
Clause 132C. The method, STING agonist, second adjuvant, antigen or use of clause 130C, wherein the TLR4 agonist is formulated with liposomes.
Clause 133C. The method, STING agonist, second adjuvant, antigen or use of clause 132C, wherein the liposomes comprise DOPC.
Clause 134C. The method, STING agonist, second adjuvant, antigen or use of either clause 132C or 133C, wherein the liposomes comprise a sterol, such as cholesterol.
Clause 135C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 132C to 134C, wherein the liposome size is in the range of 50 nm to 200 nm, especially 60 nm to 180 nm.
Clause 136C. The method, STING agonist, second adjuvant, antigen or use of clause 135C, wherein the liposome size is in the range of 70 to 165 nm, in particular about 100 nm.
Clause 137C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 132C to 136C, wherein the liposome polydispersity is not more than 0.3, such as not more than 0.2.
Clause 138C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 130C to 137C, wherein the saponin and TLR4 agonist are formulated with the same carrier.
Clause 139C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 116C to 138C, wherein the saponin and TLR4 agonist are formulated together.
Clause 140C. The method, STING agonist, second adjuvant, antigen or use of clause 139C, wherein the saponin is QS-21, the TLR4 agonist is 3D-MPL and they are formulated in DOPC liposomes containing cholesterol.
Clause 141 C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 116C to 138C, wherein the saponin and TLR4 agonist are formulated separately.
Clause 142C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 27C to 141 C, wherein the second adjuvant does not comprise a STING agonist.
Clause 143C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 27C to 142C, wherein the second adjuvant comprises a TLR agonist.
Clause 144C. The method, STING agonist, second adjuvant, antigen or use of clause 143C, wherein the second adjuvant comprises a TLR1 agonist.
Clause 145C. The method, STING agonist, second adjuvant, antigen or use of clause 143C or 144C, wherein the second adjuvant comprises a TLR1/2 agonist, such as Pam3Cys.
Clause 146C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 143C to 145C, wherein the second adjuvant comprises a TLR2 agonist. Clause 147C. The method, STING agonist, second adjuvant, antigen or use of clause 146C, wherein the TLR2 agonist is CFA, MALP2, Pam2Cys, FSL-I or Hib-OMPC.
Clause 148C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 143C to 147C, wherein the second adjuvant comprises a TLR3 agonist.
Clause 149C. The method, STING agonist, second adjuvant, antigen or use of clause 148C, wherein the TLR3 agonist is polyinosinic: polycytidylic acid.
Clause 150C. The method, STING agonist, second adjuvant, antigen or use of clause 148C, wherein the TLR3 agonist is polyadenosine-polyuridylic acid.
Clause 151C. The method, STING agonist, second adjuvant, antigen or use of clause 148C, wherein the TLR3 agonist is polyinosinic-polycytidylic acid stabilized with poly-L-lysine and carboxymethylcellulose.
Clause 152C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 143C to 15C1 , wherein the second adjuvant comprises a TLR4 agonist.
Clause 153C. The method, STING agonist, second adjuvant, antigen or use of clause 152C, wherein the TLR4 agonist comprises, such as consists of, lipopolysaccharide.
Clause 154C. The method, STING agonist, second adjuvant, antigen or use of clause 153C, wherein the TLR4 agonist comprises, such as consists of, non-toxic derivatives of lipid A.
Clause 155C. The method, STING agonist, second adjuvant, antigen or use of clause 154C, wherein the TLR4 agonist comprises, such as consists of, monophosphoryl lipid A.
Clause 156C. The method, STING agonist, second adjuvant, antigen or use of clause 155C, wherein the TLR4 agonist comprises, such as consists of, 3-de-O-acylated monophosphoryl lipid A.
Clause 157C. The method, STING agonist, second adjuvant, antigen or use of clause 152C, wherein the TLR4 agonist comprises, such as consists of, 3-deacyl monophosphoryl hexa-acyl lipid A.
Clause 158C. The method, STING agonist, second adjuvant, antigen or use of clause 152C, wherein the TLR4 agonist comprises, such as consists of, 3-deacyl monophosphoryl lipid A.
Clause 159C. The method, STING agonist, second adjuvant, antigen or use of clause 152C, wherein the TLR4 agonist comprises, such as consists of, aminoalkyl glucosaminide phosphate.
Clause 160C. The method, STING agonist, second adjuvant, antigen or use of clause 159C, wherein the TLR4 agonist comprises, such as consists of, CRX601:
Figure imgf000298_0001
Clause 161C. The method, STING agonist, second adjuvant, antigen or use of clause 152C, wherein the TLR4 agonist comprises, such as consists of, dLOS.
Clause 162C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 152C to 161 C, wherein a dose comprises 1 to 100 ug of TLR4 agonist.
Clause 163C. The method, STING agonist, second adjuvant, antigen or use of clause 162C, wherein a dose comprises 40 to 60 ug, such as 45 to 55 ug, especially 49 to 51 ug, in particular 50 ug of TLR4 agonist.
Clause 164C. The method, STING agonist, second adjuvant, antigen or use of clause 162C, wherein a dose comprises 20 to 30 ug, such as 22 to 28 ug, especially 24 to 26 ug, in particular 25 ug of TLR4 agonist.
Clause 165C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 143C to 164C, wherein the second adjuvant comprises a TLR5 agonist, such as bacterial flagellin.
Clause 166C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 143C to 165C, wherein the second adjuvant comprises a TLR6 agonist.
Clause 167C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 143C to 166C, wherein the second adjuvant comprises a TLR7 and/or TLR8 agonist.
Clause 168C. The method, STING agonist, second adjuvant, antigen or use of clause 167C, wherein the second adjuvant comprises a TLR7 agonist.
Clause 169C. The method, STING agonist, second adjuvant, antigen or use of clause 167C, wherein the second adjuvant comprises a TLR8 agonist. Clause 170C. The method, STING agonist, second adjuvant, antigen or use of clauseC, wherein the second adjuvant comprises a TLR7/TLR8 agonist.
Clause 171C. The method, STING agonist, second adjuvant, antigen or use of clauseC, wherein the TLR7 and/or TLR8 agonist is imiquimod, resiquimod or loxoribine. Clause 172C. The method, STING agonist, second adjuvant, antigen or use of clauseC, wherein the TLR7 and/or TLR8 agonist is:
Figure imgf000299_0001
Figure imgf000300_0001
Figure imgf000301_0001
Clause 173C. The method, STING agonist, second adjuvant, antigen or use of clause 167C, wherein the TLR7 and/or TLR8 agonist is:
Figure imgf000301_0002
Clause 174C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 143C to 173C, wherein the second adjuvant comprises a TLR9 agonist, such as unmethylated CpG dinucleotide.
Clause 175C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 27C to 174C, wherein the second adjuvant comprises a squalene emulsion adjuvant.
Clause 176C. The method, STING agonist, second adjuvant, antigen or use of clause 175C, wherein the squalene emulsion adjuvant has an average droplet size of less than 1 urn, especially less than 500 nm and in particular less than 200 nm.
Clause 177C. The method, STING agonist, second adjuvant, antigen or use of clause 176C, wherein the squalene emulsion adjuvant has an average droplet size of 50 to 200 nm, especially 120 nm to 180 nm, in particular 140 nm to 180 nm.
Clause 178C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 175C to 177C, wherein the squalene emulsion adjuvant has a polydispersity of 0.5 or less, especially 0.3 or less, such as 0.2 or less.
Clause 179C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 175C to 178C, wherein the squalene emulsion adjuvant surfactant is selected from poloxamer 401, poloxamer 188, polysorbate 80, sorbitan trioleate, sorbitan monooleate and polyoxyethylene 12 cetyl/stearyl ether either alone, in combination with each other or in combination with other surfactants.
Clause 180C. The method, STING agonist, second adjuvant, antigen or use of clause 179C, wherein the squalene emulsion adjuvant surfactant is selected from polysorbate 80, sorbitan trioleate, sorbitan monooleate and polyoxyethylene 12 cetyl/stearyl ether either alone, or in combination with each other.
Clause 181C. The method, STING agonist, second adjuvant, antigen or use of clause 180C, wherein the squalene emulsion adjuvant surfactant includes polysorbate 80.
Clause 182C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 175C to 181 C, wherein the squalene emulsion adjuvant comprises one surfactant.
Clause 183C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 175C to 181 C, wherein the squalene emulsion adjuvant comprises two surfactants.
Clause 184C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 175C to 181 C, wherein the squalene emulsion adjuvant comprises three surfactants.
Clause 185C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 175C to 184C, wherein the amount of squalene in a single dose of the squalene emulsion adjuvant is 50 mg or less, especially 40 mg or less, in particular 30 mg or less.
Clause 186C. The method, STING agonist, second adjuvant, antigen or use of clause 185C, wherein the amount of squalene in a single dose of the squalene emulsion adjuvant is 20 mg or less, for example 15 mg or less.
Clause 187C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 175C to 186C, wherein the amount of squalene in a single dose of the squalene emulsion adjuvant is 0.5 mg or more, especially 1 mg or more, in particular 2 mg or more.
Clause 188C. The method, STING agonist, second adjuvant, antigen or use of clause 187C, wherein the amount of squalene in a single dose of the squalene emulsion adjuvant is 4 mg or more, for example, 8 mg or more.
Clause 189C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 185C to 188C, wherein the amount of squalene in a single dose of the squalene emulsion adjuvant is 2 to 4 mg.
Clause 190C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 185C to 188C, wherein the amount of squalene in a single dose of the squalene emulsion adjuvant is 4 to 8 mg.
Clause 191C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 185C to 188C, wherein the amount of squalene in a single dose of the squalene emulsion adjuvant is 8 to 12 mg.
Clause 192C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 175C to 188C, wherein the weight ratio of squalene to surfactant in the squalene emulsion adjuvant is 0.73 to 6.6, especially 1 to 5, in particular 1.5 to 4.5. Clause 193C. The method, STING agonist, second adjuvant, antigen or use of clause 192C, wherein the weight ratio of squalene to surfactant in the squalene emulsion adjuvant is
1.5 to 3, especially 1.71 to 2.8, such as 2.2 or 2.4.
Clause 194C. The method, STING agonist, second adjuvant, antigen or use of clause 192C, wherein the weight ratio of squalene to surfactant in the squalene emulsion adjuvant is
2.5 to 3.5, especially 3 or 3.1.
Clause 195C. The method, STING agonist, second adjuvant, antigen or use of clause 192C, wherein the weight ratio of squalene to surfactant in the squalene emulsion adjuvant is 3 to 4.5, especially 4 or 4.3.
Clause 196C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 175C to 195C, wherein the squalene emulsion adjuvant does not comprise tocopherol.
Clause 197C. The method, STING agonist, second adjuvant, antigen or use of clause 196C, wherein the squalene emulsion adjuvant consists essentially of squalene, surfactant and water.
Clause 198C. The method, STING agonist, second adjuvant, antigen or use of clause 196C, wherein the squalene emulsion adjuvant comprises squalene, polysorbate 80, sorbitan trioleate and water.
Clause 199C. The method, STING agonist, second adjuvant, antigen or use of clause 198C, wherein the squalene emulsion adjuvant consists essentially of squalene, polysorbate 80, sorbitan trioleate and water.
Clause 200C. The method, STING agonist, second adjuvant, antigen or use of either clause 198C or 199C, wherein squalene emulsion adjuvant comprises citrate ions e.g. 10mM sodium citrate buffer.
Clause 201 C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 198C to 200C, wherein the weight ratio of squalene to polysorbate 80 in the squalene emulsion adjuvant is 10 to 6.6, especially 9.1 to 7.5, in particular 8.7 to 7.9, such as 8.3.
Clause 202C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 198C to 201 C, wherein the weight ratio of squalene to sorbitan trioleate in the squalene emulsion adjuvant is 10 to 6.6, especially 9.1 to 7.5, in particular 8.7 to 7.9, such as 8.3.
Clause 203C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 198C to 202C, wherein a single dose of the squalene emulsion adjuvant comprises 0.9 to 11 mg of squalene. Clause 204C. The method, STING agonist, second adjuvant, antigen or use of clause 203C, wherein the single dose of the squalene emulsion adjuvant comprises 0.9 to 1.1 mg of squalene.
Clause 205C. The method, STING agonist, second adjuvant, antigen or use of clause 203C, wherein the single dose of the squalene emulsion adjuvant comprises 1.1 to 1.4 mg of squalene.
Clause 206C. The method, STING agonist, second adjuvant, antigen or use of clause 203C, wherein the single dose of the squalene emulsion adjuvant comprises 2.2 to 2.8 mg of squalene.
Clause 207C. The method, STING agonist, second adjuvant, antigen or use of clause 203C, wherein the single dose of the squalene emulsion adjuvant comprises 4.5 to 5.5 mg of squalene.
Clause 208C. The method, STING agonist, second adjuvant, antigen or use of clause 203C, wherein the single dose of the squalene emulsion adjuvant comprises 9 to 11 mg of squalene.
Clause 209C. The method, STING agonist, second adjuvant, antigen or use of clause 196C, wherein the squalene emulsion adjuvant comprises squalene, sorbitan monooleate, polyoxyethylene cetostearyl ether and water, optionally with mannitol.
Clause 210C. The method, STING agonist, second adjuvant, antigen or use of clause 209C, wherein the squalene emulsion adjuvant consists essentially of squalene, sorbitan monooleate, polyoxyethylene cetostearyl ether and water, optionally with mannitol.
Clause 211C. The method, STING agonist, second adjuvant, antigen or use of either clause 209C or 210C, wherein squalene emulsion adjuvant comprises phosphate buffered saline.
Clause 212C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 209C to 211 C, wherein the weight ratio of squalene to sorbitan monooleate in the squalene emulsion adjuvant is 7.8 to 5.2, especially 7.15 to 5.85, in particular 6.8 to 6.2, such as 6.5.
Clause 213C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 209C to 212C, wherein the weight ratio of squalene to polyoxyethylene cetostearyl ether in the squalene emulsion adjuvant is 6.2 to 4.1 , especially 5.7 to 4.7, in particular 5.4 to 4.9, such as 5.2.
Clause 214C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 209C to 213C, wherein the weight ratio of squalene to mannitol in the squalene emulsion adjuvant is 6.5 to 4.3, especially 5.9 to 4.9, in particular 5.7 to 5.1, such as 5.4. Clause 215C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 209C to 214C, wherein a single dose of the squalene emulsion adjuvant comprises 1.1 to 13.6 mg of squalene.
Clause 216C. The method, STING agonist, second adjuvant, antigen or use of clause 215C, wherein the single dose of the squalene emulsion adjuvant comprises 1.1 to 1.35 mg of squalene.
Clause 217C. The method, STING agonist, second adjuvant, antigen or use of clause 215C, wherein the single dose of the squalene emulsion adjuvant comprises 1.4 to 1.7 mg of squalene.
Clause 218C. The method, STING agonist, second adjuvant, antigen or use of clause 215C, wherein the single dose of the squalene emulsion adjuvant comprises 2.8 to 3.4 mg of squalene.
Clause 219C. The method, STING agonist, second adjuvant, antigen or use of clause 215C, wherein the single dose of the squalene emulsion adjuvant comprises 5.6 to 6.8 mg of squalene.
Clause 220C. The method, STING agonist, second adjuvant, antigen or use of clause 215C, wherein the single dose of the squalene emulsion adjuvant comprises 11.2 to 13.6 mg of squalene.
Clause 221C. The method, STING agonist, second adjuvant, antigen or use of clause 196C, wherein the squalene emulsion adjuvant comprises squalene, polysorbate 80, sorbitan trioleate and water.
Clause 222C. The method, STING agonist, second adjuvant, antigen or use of clause 221C, wherein the squalene emulsion adjuvant consists essentially of squalene, polysorbate 80, sorbitan trioleate and water.
Clause 223C. The method, STING agonist, second adjuvant, antigen or use of either clause 221 C or 222C, wherein squalene emulsion adjuvant comprises phosphate buffered saline, such as modified phosphate buffered saline.
Clause 224C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 221C to 223C, wherein the weight ratio of squalene to polysorbate 80 in the squalene emulsion adjuvant is 4.6 to 3.0, especially 4.2 to 3.4, in particular 4.0 to 3.6, such as 3.8.
Clause 225C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 221 C to 224C, wherein the weight ratio of squalene to sorbitan trioleate in the squalene emulsion adjuvant is 4.6 to 3.0, especially 4.2 to 3.4, in particular 4.0 to 3.6, such as 3.8. Clause 226C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 221 C to 225C, wherein a single dose of the squalene emulsion adjuvant comprises 0.68 to 8.4 mg of squalene.
Clause 227C. The method, STING agonist, second adjuvant, antigen or use of clause 226C, wherein the single dose of the squalene emulsion adjuvant comprises 0.68 to 0.84 mg of squalene.
Clause 228C. The method, STING agonist, second adjuvant, antigen or use of clause 226C, wherein the single dose of the squalene emulsion adjuvant comprises 0.85 to 1.1 mg of squalene.
Clause 229C. The method, STING agonist, second adjuvant, antigen or use of clause 226C, wherein the single dose of the squalene emulsion adjuvant comprises 1.7 to 2.1 mg of squalene.
Clause 230C. The method, STING agonist, second adjuvant, antigen or use of clause 226C, wherein the single dose of the squalene emulsion adjuvant comprises 3.4 to 4.2 mg of squalene.
Clause 231 C. The method, STING agonist, second adjuvant, antigen or use of clause 226C, wherein the single dose of the squalene emulsion adjuvant comprises 6.8 to 8.4 mg of squalene.
Clause 232C. The method, STING agonist, second adjuvant, antigen or use of clause 196C, wherein the squalene emulsion adjuvant comprises squalene, phosphatidyl choline, poloxamer 188 and water, optionally with glycerol.
Clause 233C. The method, STING agonist, second adjuvant, antigen or use of clause 232C, wherein the squalene emulsion adjuvant consists essentially of squalene, phosphatidyl choline, poloxamer 188 and water, optionally with glycerol.
Clause 234C. The method, STING agonist, second adjuvant, antigen or use of either clause 232C or 233C, wherein the squalene emulsion adjuvant comprises ammonium phosphate buffer and/or glycerol.
Clause 235C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 232C to 234C, wherein the weight ratio of squalene to phosphatidyl choline in the squalene emulsion adjuvant is 2.52 to 3.8, especially 2.85 to 3.5, in particular 3 to 3.3, such as 3.15
Clause 236C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 232C to 235C, wherein the weight ratio of squalene to poloxamer 188 in the squalene emulsion adjuvant is 55 to 83, especially 62 to 76, in particular 65.5 to 72.5, such as 69. Clause 237C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 232C to 236C, wherein a single dose of the squalene emulsion adjuvant comprises 0.77 to 9.5 mg of squalene.
Clause 238C. The method, STING agonist, second adjuvant, antigen or use of clause 237C, wherein the single dose of the squalene emulsion adjuvant comprises 0.77 to 0.95 mg of squalene.
Clause 239C. The method, STING agonist, second adjuvant, antigen or use of clause 237C, wherein the single dose of the squalene emulsion adjuvant comprises 0.96 to 1.2 mg of squalene.
Clause 240C. The method, STING agonist, second adjuvant, antigen or use of clause 237C, wherein the single dose of the squalene emulsion adjuvant comprises 1.9 to 2.4 mg of squalene.
Clause 241 C. The method, STING agonist, second adjuvant, antigen or use of clause 237C, wherein the single dose of the squalene emulsion adjuvant comprises 3.8 to 4.8 mg of squalene.
Clause 242C. The method, STING agonist, second adjuvant, antigen or use of clause 237C, wherein the single dose of the squalene emulsion adjuvant comprises 7.7 to 9.5 mg of squalene.
Clause 243C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 175C to 195C, wherein the squalene emulsion adjuvant comprises tocopherol.
Clause 244C. The method, STING agonist, second adjuvant, antigen or use of clause 243C, wherein the tocopherol is alpha-tocopherol.
Clause 245C. The method, STING agonist, second adjuvant, antigen or use of clause 244C, wherein the tocopherol is D/L-alpha-tocopherol.
Clause 246C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 243C to 24C5, wherein the squalene emulsion adjuvant consists essentially of squalene, tocopherol, surfactant and water.
Clause 247C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 243C to 246C, wherein the weight ratio of squalene to tocopherol in the squalene emulsion adjuvant is 20 or less, especially 10 or less.
Clause 248C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 243C to 247C, wherein the weight ratio of squalene to tocopherol in the squalene emulsion adjuvant is 0.1 or more.
Clause 249C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 243C to 248C, wherein the weight ratio of squalene to tocopherol in the squalene emulsion adjuvant is 0.2 to 5. Clause 250C. The method, STING agonist, second adjuvant, antigen or use of clause 249C, wherein the weight ratio of squalene to tocopherol in the squalene emulsion adjuvant is 0.72 to 1.136, especially 0.8 to 1 , in particular 0.85 to 0.95, such as 0.9.
Clause 251 C. The method, STING agonist, second adjuvant, antigen or use of clause 249C, wherein the weight ratio of squalene to tocopherol in the squalene emulsion adjuvant is 3.4 to 4.6, especially 3.6 to 4.4, in particular 3.8 to 4.2, such as 4.
Clause 252C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 243C to 251 C, wherein the squalene emulsion adjuvant comprises squalene, tocopherol, polysorbate 80 and water.
Clause 253C. The method, STING agonist, second adjuvant, antigen or use of clause 274C, wherein the squalene emulsion adjuvant consists essentially of squalene, tocopherol, polysorbate 80 and water.
Clause 254C. The method, STING agonist, second adjuvant, antigen or use of either clause 252C or 253C, wherein squalene emulsion adjuvant comprises a phosphate buffered saline, such as modified phosphate buffered saline.
Clause 255C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 252C to 254C, wherein the weight ratio of squalene to tocopherol in the squalene emulsion adjuvant is 0.5 to 1.5, especially 0.6 to 1.35, in particular 0.7 to 1.1 , such as 0.85 to 0.95 e.g. 0.9.
Clause 256C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 252C to 255C, wherein the weight ratio of squalene to polysorbate 80 in the squalene emulsion adjuvant is 1.2 to 3.6, especially 1.46 to 3.3, in particular 1.9 to 2.5 such as 2.1 to 2.3 e.g. 2.2.
Clause 257C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 252C to 256C, wherein a single dose of the squalene emulsion adjuvant comprises 0.9 to 12.1 mg of squalene.
Clause 258C. The method, STING agonist, second adjuvant, antigen or use of clause 257C, wherein the single dose of the squalene emulsion adjuvant comprises 0.9 to 1.3 mg of squalene, typically with 1 to 1.4 mg tocopherol and 0.43 to 0.57 mg polysorbate 80.
Clause 259C. The method, STING agonist, second adjuvant, antigen or use of clause 257C, wherein the single dose of the squalene emulsion adjuvant comprises 1.2 to 1.6 mg of squalene, typically with 1.3 to 1.7 mg tocopherol and 0.54 to 0.71 mg polysorbate 80.
Clause 260C. The method, STING agonist, second adjuvant, antigen or use of clause 257C, wherein the single dose of the squalene emulsion adjuvant comprises 2.4 to 3 mg of squalene, typically with 2.6 to 3.4 mg tocopherol and 1 to 1.5 mg polysorbate 80. Clause 261 C. The method, STING agonist, second adjuvant, antigen or use of clause 257C, wherein the single dose of the squalene emulsion adjuvant comprises 4.8 to 6.1 mg of squalene, typically with 5.3 to 6.8 mg tocopherol and 2.1 to 2.9 mg polysorbate 80.
Clause 262C. The method, STING agonist, second adjuvant, antigen or use of clause 257C, wherein the single dose of the squalene emulsion adjuvant comprises 9.7 to 12.1 mg of squalene, typically with 10.6 to 13.6 mg tocopherol and 4.3 to 5.7 mg polysorbate 80.
Clause 26C3. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 252C to 254C, wherein the weight ratio of squalene to tocopherol in the squalene emulsion adjuvant is 2.6 to 4.5, especially 2.8 to 4.3, in particular 3.25 to 4, such as 3.4 to 3.8 e.g. 3.6.
Clause 264C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 252C to 254C or 263C, wherein the weight ratio of squalene to polysorbate 80 in the squalene emulsion adjuvant is 11.3 to 2.5, especially 1.56 to 2.3, in particular 1.75 to 2.15 such as 1.85 to 2 e.g. 1.94.
Clause 265C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 252C to 254C, 263C or 264C, wherein a single dose of the squalene emulsion adjuvant comprises 1.1 to 14.3 mg of squalene.
Clause 266C. The method, STING agonist, second adjuvant, antigen or use of clause 265C, wherein the single dose of the squalene emulsion adjuvant comprises 1.1 to 1.5 mg of squalene.
Clause 267C. The method, STING agonist, second adjuvant, antigen or use of clause 265C, wherein the single dose of the squalene emulsion adjuvant comprises 1.4 to 1.8 mg of squalene.
Clause 268. The method, STING agonist, second adjuvant, antigen or use of clause 265C, wherein the single dose of the squalene emulsion adjuvant comprises 2.9 to 3.6 mg of squalene.
Clause 269C. The method, STING agonist, second adjuvant, antigen or use of clause 265C, wherein the single dose of the squalene emulsion adjuvant comprises 5.8 to 7.2 mg of squalene.
Clause 270C. The method, STING agonist, second adjuvant, antigen or use of clause 265C, wherein the single dose of the squalene emulsion adjuvant comprises 11.7 to 14.3 mg of squalene.
Clause 271 C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 243C to 292C, wherein the squalene emulsion adjuvant comprises squalene, tocopherol, phosphatidyl choline, poloxamer 188 and water, optionally with glycerol. Clause 272C. The method, STING agonist, second adjuvant, antigen or use of clause 271C, wherein the squalene emulsion adjuvant consists essentially of squalene, tocopherol, phosphatidyl choline, poloxamer 188 and water, optionally with glycerol.
Clause 273C. The method, STING agonist, second adjuvant, antigen or use of either clause 271 C or 272C, wherein the squalene emulsion adjuvant comprises ammonium phosphate buffer and/or glycerol.
Clause 274C. The method, STING agonist, second adjuvant, antigen or use of clauses 271 C to 273C, wherein the weight ratio of squalene to tocopherol in the squalene emulsion adjuvant is at least 50, especially 137 to 207, in particular 154 to 190, such as 163 to 181, for example 172.
Clause 275C. The method, STING agonist, second adjuvant, antigen or use of clauses 271 C to 274C, wherein the weight ratio of squalene to phosphatidyl choline in the squalene emulsion adjuvant is 2.52 to 3.8, especially 2.85 to 3.5, in particular 3 to 3.3, such as 3.15
Clause 276C. The method, STING agonist, second adjuvant, antigen or use of clauses 271 C to 275C, wherein the weight ratio of squalene to poloxamer 188 in the squalene emulsion adjuvant is 55 to 83, especially 62 to 76, in particular 65.5 to 72.5, such as 69.
Clause 277C. The method, STING agonist, second adjuvant, antigen or use of clauses 271 C to 276C, wherein a single dose of the squalene emulsion adjuvant comprises 0.77 to 9.5 mg of squalene.
Clause 278C. The method, STING agonist, second adjuvant, antigen or use of clause 277C, wherein the single dose of the squalene emulsion adjuvant comprises 0.77 to 0.95 mg of squalene.
Clause 279C. The method, STING agonist, second adjuvant, antigen or use of clause 277C, wherein the single dose of the squalene emulsion adjuvant comprises 0.96 to 1.2 mg of squalene.
Clause 280C. The method, STING agonist, second adjuvant, antigen or use of clause 277C, wherein the single dose of the squalene emulsion adjuvant comprises 1.9 to 2.4 mg of squalene.
Clause 281 C. The method, STING agonist, second adjuvant, antigen or use of clause 277C, wherein the single dose of the squalene emulsion adjuvant comprises 3.8 to 4.8 mg of squalene.
Clause 282C. The method, STING agonist, second adjuvant, antigen or use of clause 277C, wherein the single dose of the squalene emulsion adjuvant comprises 7.7 to 9.5 mg of squalene.
Clause 283C. The method, STING agonist, second adjuvant, antigen or use of clause 277C, wherein the single dose of the squalene emulsion adjuvant comprises 7.7 to 9.5 mg of squalene. Clause 284C. The method, STING agonist, second adjuvant, antigen or use of clauses 27C to 283C, wherein the second adjuvant comprises one immunostimulant.
Clause 285C. The method, STING agonist, second adjuvant, antigen or use of clauses 27C to 284C, wherein the second adjuvant comprises two immunostimulants.
Clause 286C. The method, STING agonist, second adjuvant, antigen or use of clauses 1C to 285C wherein only one administration of the STING agonist is performed in step (i).
Clause 287C. The method, STING agonist, second adjuvant, antigen or use of clauses 1C to 285C wherein at least 2 administrations of the STING agonist are performed in step (i).
Clause 288C. The method, STING agonist, second adjuvant, antigen or use of clause 287C wherein at least 3 administrations of the STING agonist are performed in step (i).
Clause 289C. The method, STING agonist, second adjuvant, antigen or use of clause 287C wherein 2 administrations of the STING agonist are performed in step (i).
Clause 290C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 1C to 289C wherein in step (i) the STING agonist is administered intradermally, intramuscularly, intraperitoneally or subcutaneously.
Clause 291 C. The method, STING agonist, second adjuvant, antigen or use of clause 290C wherein the STING agonist is administered intramuscularly.
Clause 292C. The method, STING agonist, second adjuvant, antigen or use of clause 290C or 291 C wherein the volume of the STING agonist in step (i) is 0.05 ml to 1 ml, such as 0.1 to 0.6 ml.
Clause 293C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 290C to 292C wherein the pH of the STING agonist in step (i) is 4 to 9, especially 5 to 8.5, in particular 5.5 to 8, such as 6.5 to 7.4.
Clause 294C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 290C to 293C wherein the osmolarity of the STING agonist in step (i) is 250 to 750 mOsm/kg, especially 250 to 550 mOsm/kg, in particular 270 to 500 mOsm/kg, such as 270 to 400 mOsm/kg.
Clause 295C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 290C to 294C wherein the STING agonist of step (i) comprises buffer and/or tonicity modifying agents, such as modified phosphate buffered saline.
Clause 296C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 1C to 295C wherein in step (i) the period between administrations to the subject is 2 weeks to 12 months.
Clause 297C. The method, STING agonist, second adjuvant, antigen or use of clause 296C wherein in step (i) the period between administrations to the subject is 2 weeks to 6 months. Clause 298C. The method, STING agonist, second adjuvant, antigen or use of clause 297C wherein in step (i) the period between administrations to the subject is 2 weeks to 3 months.
Clause 299C. The method, STING agonist, second adjuvant, antigen or use of clause 298C wherein in step (i) the period between administrations to the subject is 2 weeks to 2 months, such as 3 to 5 weeks.
Clause 300C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 1C to 299C wherein step (ii) is initiated at an interval of 2 weeks to 2 years after step (i).
Clause 301 C The method, STING agonist, second adjuvant, antigen or use of clause 300C wherein step (ii) is initiated at an interval of 2 weeks to 1 year after step (i).
Clause 302C. The method, STING agonist, second adjuvant, antigen or use of clause 301 C wherein step (ii) is initiated at an interval of 3 weeks to 9 months after step (i).
Clause 303C. The method, STING agonist, second adjuvant, antigen or use of clause 302C wherein step (ii) is initiated at an interval of 4 weeks to 6 months after step (i).
Clause 304C. The method, STING agonist, second adjuvant, antigen or use of clause 303C wherein step (ii) is initiated at an interval of 2 weeks to 2 months after step (i).
Clause 305C. The method, STING agonist, second adjuvant, antigen or use of clause 304C wherein step (ii) is initiated at an interval of 1 month to 3 months after step (i).
Clause 306C. The method, STING agonist, second adjuvant, antigen or use of clause 305C wherein step (ii) is initiated at an interval of 2 months to 4 months after step (i).
Clause 307C. The method, STING agonist, second adjuvant, antigen or use of clause 306C wherein step (ii) is initiated at an interval of 3 months to 6 months after step (i).
Clause 308C. The method, STING agonist, second adjuvant, antigen or use of clause 307C wherein step (ii) is initiated at an interval of 4 months to 12 months after step (i).
Clause 309C. The method, STING agonist or use of any one of clauses 1C to 3C, 58C to 141 C or 286C to 299C wherein the risk of infection occurs 2 weeks to 2 years after administering a STING agonist to the subject.
Clause 310C The method, STING agonist or use of clause 309C wherein the risk of infection occurs 2 weeks to 1 year after administering a STING agonist to the subject.
Clause 311C. The method, STING agonist or use of clause 310C wherein the risk of infection occurs 3 weeks to 9 months after administering a STING agonist to the subject.
Clause 312C. The method, STING agonist or use of clause 311C wherein the risk of infection occurs 4 weeks to 6 months after administering a STING agonist to the subject.
Clause 313C. The method, STING agonist or use of clause 309C wherein the risk of infection occurs 2 weeks to 2 months after administering a STING agonist to the subject. Clause 314C. The method, STING agonist or use of clause 309C wherein the risk of infection occurs 1 month to 3 months after administering a STING agonist to the subject.
Clause 315C. The method, STING agonist or use of clause 309C wherein the risk of infection occurs 2 months to 4 months after administering a STING agonist to the subject.
Clause 316C. The method, STING agonist or use of clause 310C wherein the risk of infection occurs 3 months to 6 months after administering a STING agonist to the subject.
Clause 317C. The method, STING agonist or use of clause 310C wherein the risk of infection occurs 4 months to 12 months after administering a STING agonist to the subject.
Clause 318C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 4C to 317C wherein only one administration is performed in step (ii).
Clause 319C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 4C to 317C wherein at least 2 administrations are performed in step (ii).
Clause 320C. The method, STING agonist, second adjuvant, antigen or use of clause 319C wherein at least 3 administrations are performed in step (ii).
Clause 321C. The method, STING agonist, second adjuvant, antigen or use of clause 319C wherein 2 administrations are performed in step (ii).
Clause 322C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 4C to 308C or 318C to 321 C wherein in step (ii) the second adjuvant, if present, is co-formulated with the antigen.
Clause 323C. The method, STING agonist, second adjuvant, antigen or use of clause 322C wherein the antigen of step (ii) is administered intradermally, intramuscularly, intraperitoneally or subcutaneously, in particular intramuscularly.
Clause 324C. The method, STING agonist, second adjuvant, antigen or use of clause 322C or 323C wherein the volume of the formulation of antigen in step (ii) is 0.05 ml to 1 ml, such as 0.1 to 0.6 ml.
Clause 325C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 322C to 324C wherein the pH of the formulation of antigen in step (ii) is 4 to 9, especially 5 to 8.5, in particular 5.5 to 8, such as 6.5 to 7.4.
Clause 326C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 322C to 325C wherein the osmolarity of the formulation of antigen in step (ii) is 250 to 750 mOsm/kg, especially 250 to 550 mOsm/kg, in particular 270 to 500 mOsm/kg, such as 270 to 400 mOsm/kg.
Clause 327C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 322C to 326C wherein the formulation of antigen of step (ii) comprises buffer and/or tonicity modifying agents, such as modified phosphate buffered saline. Clause 328C. The method, STING agonist, second adjuvant, antigen or use of clauses 4C to 308C or 318C to 321 C wherein in step (ii) the second adjuvant, if present, is formulated separately from the antigen.
Clause 329C. The method, STING agonist, second adjuvant, antigen or use of clause 328C wherein in step (ii) for at least one administration, the second adjuvant is administered simultaneously with the antigen.
Clause 330C. The method, STING agonist, second adjuvant, antigen or use of clause 328C wherein in step (ii) for all administrations the second adjuvant is administered simultaneously with the antigen.
Clause 331C. The method, STING agonist, second adjuvant, antigen or use of clause 328C wherein in step (ii) for at least one administration, the second adjuvant is administered separately from the antigen.
Clause 332C. The method, STING agonist, second adjuvant, antigen or use of clause 331 C wherein in step (ii) for at least one administration, the second adjuvant is administered before the antigen.
Clause 333C. The method, STING agonist, second adjuvant, antigen or use of clause 332C wherein in step (ii) for all administrations the second adjuvant is administered before the antigen.
Clause 334C. The method, STING agonist, second adjuvant, antigen or use of clause 332C wherein in step (ii) for at least one administration, the antigen is administered before the second adjuvant.
Clause 335C. The method, STING agonist, second adjuvant, antigen or use of clause 334C wherein in step (ii) for all administrations the antigen is administered before the second adjuvant.
Clause 336C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 331 C to 335C wherein in step (ii) for at least one administration, the second adjuvant is administered to the same location on the subject as the antigen.
Clause 337C. The method, STING agonist, second adjuvant, antigen or use of clause 336C wherein in step (ii) for all administrations the second adjuvant is administered to the same location on the subject as the antigen.
Clause 338C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 328C to 336C wherein in step (ii) for at least one administration, the second adjuvant is administered to a different location on the subject as the antigen.
Clause 339C. The method, STING agonist, second adjuvant, antigen or use of clause 338C wherein in step (ii) for all administrations the second adjuvant is administered to a different location on the subject as the antigen. Clause 340C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 328C to 339C wherein in step (ii) for at least one administration, the second adjuvant and the antigen are administered within 84 hours of each other.
Clause 341C. The method, STING agonist, second adjuvant, antigen or use of clause 340C wherein in step (ii) for at least one administration, the second adjuvant and the antigen are administered within 30 minutes of each other.
Clause 342C. The method, STING agonist, second adjuvant, antigen or use of clause 340C wherein in step (ii) for all administrations the second adjuvant and the antigen are administered within 84 hours of each other.
Clause 343C. The method, STING agonist, second adjuvant, antigen or use of clause 342C wherein in step (ii) for all administrations the second adjuvant and the antigen are administered within 30 minutes of each other.
Clause 344C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 328C to 343C wherein the STING agonist of step (ii) is administered intradermally, intramuscularly, intraperitoneally or subcutaneously, in particular intramuscularly.
Clause 345C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 328C to 344C wherein the antigen of step (ii) is administered intradermally, intramuscularly, intraperitoneally or subcutaneously, in particular intramuscularly.
Clause 346C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 328C to 345C wherein the volume of the separate formulation of second adjuvant in step (ii) is 0.05 ml to 1 ml, such as 0.1 to 0.6 ml.
Clause 347C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 328C to 346C wherein the volume of the separate formulation of antigen in step (ii) is 0.05 ml to 1 ml, such as 0.1 to 0.6 ml.
Clause 348C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 328C to 347C wherein the pH of the separate formulation of second adjuvant in step (ii) is 4 to 9, especially 5 to 8.5, in particular 5.5 to 8, such as 6.5 to 7.4.
Clause 349C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 328C to 348C wherein the pH of the separate formulation of antigen in step (ii) is 4 to 9, especially 5 to 8.5, in particular 5.5 to 8, such as 6.5 to 7.4.
Clause 350C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 328C to 349C wherein the osmolarity of the separate formulation of second adjuvant in step (ii) is 250 to 750 mOsm/kg, especially 250 to 550 mOsm/kg, in particular 270 to 500 mOsm/kg, such as 270 to 400 mOsm/kg.
Clause 351 C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 328C to 350C wherein the osmolarity of the separate formulation of antigen in step (ii) is 250 to 750 mOsm/kg, especially 250 to 550 mOsm/kg, in particular 270 to 500 mOsm/kg, such as 270 to 400 mOsm/kg.
Clause 352C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 328C to 351 C wherein the separate formulation comprising the second adjuvant of step (ii) comprises buffer and/or tonicity modifying agents, such as modified phosphate buffered saline.
Clause 353C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 328C to 352C wherein the separate formulation comprising the antigen of step (ii) comprises buffer and/or tonicity modifying agents, such as modified phosphate buffered saline.
Clause 354C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 4C to 353C wherein in step (ii) the period between administrations to the subject is 2 weeks to 12 months.
Clause 355C. The method, STING agonist, second adjuvant, antigen or use of clause 354C wherein in step (ii) the period between administrations to the subject is 2 weeks to 6 months.
Clause 356C. The method, STING agonist, second adjuvant, antigen or use of clause 354C wherein in step (ii) the period between administrations to the subject is 2 weeks to 3 months.
Clause 357C. The method, STING agonist, second adjuvant, antigen or use of clause 354C wherein in step (ii) the period between administrations to the subject is 2 weeks to 2 months, such as 3 to 5 weeks.
Clause 358C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 4C to 357C wherein the combined duration of step (i), step (ii) and the interval between the steps is 1 month to 2 years.
Clause 359C. The method, STING agonist, second adjuvant, antigen or use of clause 358C wherein the combined duration of step (i), step (ii) and the interval between the steps is 6 weeks to 12 months.
Clause 360C. The method, STING agonist, second adjuvant, antigen or use of clause 359C wherein the combined duration of step (i), step (ii) and the interval between the steps is 2 months to 10 months.
Clause 361C. The method, STING agonist, second adjuvant, antigen or use of clause 360C wherein the combined duration of step (i), step (ii) and the interval between the steps is 10 weeks to 9 months.
Clause 362C. The method, STING agonist, second adjuvant, antigen or use of any one of any one of clauses 1C to 361 C wherein the combined duration of step (i), step (ii) and the interval between the steps is at least 2 months, especially at least 3 months, in particular at least 4 months. Clause 363C. The method, STING agonist, second adjuvant, antigen or use of clause 362C wherein the combined duration of step (i), step (ii) and the interval between the steps is at least 5 months, especially at least 6 months, in particular at least 7 months.
Clause 364C. The method, STING agonist, second adjuvant, antigen or use of any one of any one of clauses 1C to 363C wherein the combined duration of step (i), step (ii) and the interval between the steps is 5 years or less, especially 3 years or less, in particular 2 years or less.
Clause 365C. The method, STING agonist, second adjuvant, antigen or use of clause 364C wherein the combined duration of step (i), step (ii) and the interval between the steps is 18 months or less, especially 12 months or less, in particular 10 months or less.
Clause 366C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 1C to 365C wherein the subject is a mammal.
Clause 367C. The method, STING agonist, second adjuvant, antigen or use of clause 366C wherein the subject is a human.
Clause 368C. The method, STING agonist, second adjuvant, antigen or use of clause 367C wherein the subject is a human child.
Clause 369C. The method, STING agonist, second adjuvant, antigen or use of clause 367C wherein the subject is a human adult.
Clause 370C. The method, STING agonist, second adjuvant, antigen or use of clause 367C wherein the subject is an older human.
Clause 371 C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 4C to 370C wherein the administration route used for each administration of step (i) is the same as the administration route used for each administration of step (ii).
Clause 372C. The method, STING agonist, second adjuvant, antigen or use of clause 371 C wherein the administration route used is intramuscular.
Clause 373C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 1C to 372C wherein the antigen is derived from bacteria, viruses or parasites (such as containing a portion of a polypeptide sequence obtainable from, such as at least 50, especially at least 100, in particular at least 150, such as at least 200 amino acids long).
Clause 374C. The method, STING agonist, second adjuvant, antigen or use of clause 373C wherein the antigen is derived from a bacterium.
Clause 375C. The method, STING agonist, second adjuvant, antigen or use of clause 373C wherein the antigen is derived from a virus.
Clause 376C. The method, STING agonist, or use of any one of clauses 1C to 375C wherein the pathogen presenting the risk of infection is a bacterium.
Clause 377C. The method, STING agonist or use of any one of clauses 1C to 375C wherein the pathogen presenting the risk of infection is a virus. Clause 378C The method, STING agonist, second adjuvant, antigen or use of any one of clauses 4C to 377C wherein the antigen is a polypeptide, a polynucleotide or a polysaccharide.
Clause 379C. The method, STING agonist, second adjuvant, antigen or use clause 378C wherein the antigen is a polypeptide.
Clause 380C. The method, STING agonist, second adjuvant, antigen or use of clause 379C, wherein the polypeptide comprises at least one B or T cell epitope.
Clause 381C. The method, STING agonist, second adjuvant, antigen or use of clause 380C, wherein the polypeptide comprises B and T cell epitopes.
Clause 382C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 4C to 381 C wherein the antigen is derived from a virus.
Clause 383C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 4C to 381 C wherein the antigen is derived from a bacterium.
Clause 384C. The method, STING agonist, second adjuvant, antigen or use of clause 383C wherein the antigen is derived from Clostridia.
Clause 385C. The method, STING agonist, second adjuvant, antigen or use of clause 384C wherein the antigen is derived from C. difficile.
Clause 386C. The method, STING agonist, second adjuvant, antigen or use of clause 385C wherein the antigen is C. difficile toxin A and/or toxin B or fragments thereof.
Clause 387C. The method, STING agonist, second adjuvant, antigen or use of clause 386C wherein the antigen is the F2 antigen.
Clause 388C. The method, STING agonist, second adjuvant, antigen or use of clause 387C wherein the F2 antigen comprises an amino acid sequence having at least 80%, such as at least 90%, especially at least 95%, in particular at least 98% for example at least 99% or 100% identity to SEQ ID NO: 2.
Clause 389C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 4C to 381 C wherein the antigen is a cancer antigen, such as a tumour antigen and/or a neoantigen.
Clause 390C. The method, STING agonist, second adjuvant, antigen or use of clause 389C wherein the antigen is a human cancer antigen, such as a human tumour antigen and/or a neoantigen.
Clause 391 C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 4C to 378C wherein the antigen is provided in the form of a polynucleotide encoding the antigen.
Clause 392C. The method, STING agonist, second adjuvant, antigen or use of clause 391 C wherein the antigen is provided in the form of mRNA encoding the antigen. Clause 393C. The method, STING agonist, second adjuvant, antigen or use of clause 392C wherein the mRNA is carrier-formulated, such as formulated in LNP, CNE or LION.
Clause 394C. The method, STING agonist, second adjuvant, antigen or use of clause 393C wherein the carrier is LNP.
Clause 395C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 4C to 394C, wherein the immune response to the antigen is a serum titer of IgG antibodies binding to the antigen.
Clause 396C. The method, STING agonist, second adjuvant, antigen or use of 395C, wherein the IgG antibodies are neutralising antibodies.
Clause 397C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 4C to 396C, wherein the immune response to the antigen is a T cell response specific to the antigen.
Clause 398C. The method, STING agonist, second adjuvant, antigen or use of clause 397C, wherein the T cell response is a CD4+ T cell response.
Clause 399C. The method, STING agonist, second adjuvant, antigen or use of clause 397C, wherein the T cell response is a CD8+ T cell response.
Clause 400C. The method, STING agonist, second adjuvant, antigen or use of clause 397C, wherein the T cell response is a CD4+ and CD8+ T cell response.
Clause 401 C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 4C to 400C, wherein the immune response to the antigen reduces partially or completely the severity of one or more symptoms experienced by the subject due to infection by a pathogen.
Clause 402C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 4C to 401 C, wherein the immune response to the antigen reduces partially or completely the time over which one or more symptoms are experienced by the subject due to infection by a pathogen.
Clause 403C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 4C to 402C, wherein the immune response to the antigen reduces the likelihood of developing an established infection after challenge with the pathogen.
Clause 404C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 4C to 403C, wherein the immune response to the antigen slows progression of illness (e.g. extends survival) due to infection by the pathogen.
Clause 405C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 4C to 404C, wherein the breadth of the immune response to the antigen in the subject is increased. Clause 406C. The method, STING agonist, second adjuvant, antigen or use of clause 405C, wherein serum neutralising antibodies bind to an increased range of variants of the antigen.
Clause 407C. The method, STING agonist, second adjuvant, antigen or use of either clause 405C or 406C, wherein the immune response to the antigen is elicited in a wider variety of human HLA types.
Clause 408C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 4C to 407C, wherein the level of the immune response is increased, such as by at least 25%, such as 50%, such as 100%.
Clause 409C. The method, STING agonist, second adjuvant, antigen or use of clause 408C, wherein the level of the immune response to the antigen is increased, such as by at least 200%, such as 300%, such as 400%, such as 900%.
Clause 410C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 4C to 409C, wherein the immune response to the antigen is maintained for at least 1 year, such as at least 3 years.
Clause 411C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 4C to 410C, wherein the duration of the immune response to the antigen is increased, such as by at least 1 year, such as by at least 3 years.
Clause 412C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 4C to 411 C, wherein the variability of the immune response to the antigen between subjects is decreased.
Clause 413C. The method, STING agonist, second adjuvant, antigen or use of clause 412C, wherein the standard deviation of the immune response to the antigen is reduced.
Clause 414C. The method, STING agonist, second adjuvant, antigen or use of either clause 412C or 413C, wherein the proportion of subjects meeting a specified threshold of immune response to the antigen is increased.
Clause 415C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 4C to 414C, wherein the speed of the immune response to the antigen is increased.
Clause 416C. The method, STING agonist, second adjuvant, antigen or use of clause 415C, wherein the time taken to achieve a protective immune response is reduced.
Clause 417C. The method, STING agonist, second adjuvant, antigen or use of either clause 415C or 416C, wherein the time taken to reach peak immune response is reduced.
Clause 418C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 4C to 417C, wherein the amount of antigen required to achieve a desired immune response, such as a protective immune response, is reduced. Clause 419C. The method, STING agonist, second adjuvant, antigen or use of any one of clauses 4C to 418C, wherein the spacing between administrations of antigen may be reduced while maintaining immune response.
Clause 420C. The method, STING agonist, or use of any one of clauses 1C to 419C, wherein the immune response reduces partially or completely the severity of one or more symptoms experienced by the subject due to infection by the pathogen presenting the risk of infection.
Clause 421C. The method, STING agonist, or use of any one of clauses 1C to 420C, wherein the immune response reduces partially or completely the time over which one or more symptoms are experienced by the subject due to infection by the pathogen presenting the risk of infection.
Clause 422C. The method, STING agonist, or use of any one of clauses 1C to 421 C, wherein the immune response reduces the likelihood of developing an established infection after challenge with the pathogen presenting the risk of infection.
Clause 423C. The method, STING agonist, or use of any one of clauses 1C to 422C, wherein the immune response slows progression of illness (e.g. extends survival) due to infection by the pathogen presenting the risk of infection.
Clause 424C. The method, STING agonist, or use of any one of clauses 1C to 423C, wherein the risk of infection is 2 weeks to 2 years, especially 2 weeks to 1 year, in particular 3 weeks to 9 months, such as 4 weeks to 6 months from completion of administration of STING agonist (i.e. step (i)).
Clause 425C. The method, STING agonist, or use of any one of clauses 1C to 423C, wherein the risk of infection is 2 weeks to 2 months, 1 month to 3 months, 2 months to 4 months, 3 months to 6 months or 4 months to 12 months from completion of administration of STING agonist.
Clause 426C. The method, STING agonist, or use of any one of clauses 1C to 425C, wherein the subject at risk of infection is be a diabetic who has experienced an abrasion or injury.
Clause 427C. The method, STING agonist, or use of any one of clauses 1C to 426C, wherein the subject at risk of infection is an individual expected to undergo hospital treatment.
Clause 428C. The method, STING agonist, or use of any one of clauses 1C to 427C, wherein the subject is at risk of infection from Staphylococcus aureus.
Clause 429C. The method, STING agonist, or use of any one of clauses 1C to 427C, wherein the pathogen presenting a risk to the subject at risk of infection may be a pathogen causing a pandemic. Clause 430C. The method, STING agonist, or use of any one of clauses 1C to 427C or 429C, wherein the pathogen presenting a risk to the subject at risk of infection is an influenza virus.
Clause 431 C. The method, STING agonist, or use of any one of clauses 1C to 427C or 429C, wherein the pathogen presenting a risk to the subject at risk of infection is a coronavirus.
Clause 432C. The method, STING agonist, or use of clause 431 C, wherein the pathogen presenting the risk of infection is SARS-Cov-2.
Clause 433C. A kit of parts comprising: (i) a STING agonist;
(ii) an antigen; and
(iii) a second adjuvant as described in any one of clauses 1C to 432C.
The invention is also illustrated by reference to the following further clauses.
These ‘D’ clauses relate to embodiments wherein a STING agonist and an antigen are administered in step (i), which may be followed by administration of a second antigen and optionally a second adjuvant.
Clause 1 D. A method for prophylaxis in a subject at risk of infection comprising administering a STING agonist and a first antigen to the subject.
Clause 2D. A STING agonist for use in a method for prophylaxis in a subject at risk of infection comprising administering a STING agonist and a first antigen to the subject.
Clause 3D. A first antigen for use in a method for prophylaxis in a subject at risk of infection comprising administering a STING agonist and a first antigen to the subject.
Clause 4D. Use of a STING agonist for the manufacture of a medicament for use in a method for prophylaxis in a subject at risk of infection comprising administering a STING agonist and a first antigen to the subject.
Clause 5D. Use of a first antigen for the manufacture of a medicament for use in a method for prophylaxis in a subject at risk of infection comprising administering a STING agonist and a first antigen to the subject.
Clause 6D. A method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a second antigen which is associated with the pathogen.
Clause 7D. A method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist and a first antigen, prior to
(ii) administering to each of the subjects the second antigen.
Clause 8D. A method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen.
Clause 9D. A method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen.
Clause 10D. A method of preparing the immune system of a subject for (ii) administration of a second antigen, wherein the method comprises (i) administering to the subject a STING agonist and a first antigen.
Clause 11 D. A STING agonist for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps: (i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a second antigen which is associated with the pathogen.
Clause 12D. A first antigen for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a second antigen which is associated with the pathogen.
Clause 13D. A second antigen for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a second antigen which is associated with the pathogen.
Clause 14D. A STING agonist for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist and a first antigen, prior to
(ii) administering to each of the subjects the second antigen.
Clause 15D. A first antigen for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist and a first antigen, prior to
(ii) administering to each of the subjects the second antigen.
Clause 16D. A second antigen for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist and a first antigen, prior to
(ii) administering to each of the subjects the second antigen.
Clause 17D. A STING agonist for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen.
Clause 18D. A first antigen for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen.
Clause 19D. A second antigen for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen. Clause 20D. A STING agonist for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen.
Clause 21 D. A first antigen for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen.
Clause 22D. A second antigen for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen.
Clause 23D. A STING agonist for use in a method of preparing the immune system of a subject for (ii) administration of a second antigen, wherein the method comprises (i) administering to the subject a STING agonist and a first antigen.
Clause 24D. A first antigen for use in a method of preparing the immune system of a subject for administration of a second antigen, wherein the method comprises administering to the subject a STING agonist and a first antigen.
Clause 25D. Use of a STING agonist for the manufacture of a medicament for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a second antigen which is associated with the pathogen.
Clause 26D. Use of a first antigen for the manufacture of a medicament for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a second antigen which is associated with the pathogen.
Clause 27D. Use of a second antigen for the manufacture of a medicament for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a second antigen which is associated with the pathogen. Clause 28D. Use of a STING agonist for the manufacture of a medicament for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist and a first antigen, prior to
(ii) administering to each of the subjects the second antigen.
Clause 29D. Use of a first antigen for the manufacture of a medicament for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist and a first antigen, prior to
(ii) administering to each of the subjects the second antigen.
Clause 30D. Use of a second antigen for the manufacture of a medicament for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist and a first antigen, prior to
(ii) administering to each of the subjects the second antigen.
Clause 31 D. Use of a STING agonist in the manufacture of a medicament for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen.
Clause 32D. Use of a first antigen for the manufacture of a medicament for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen.
Clause 33D. Use of a second antigen for the manufacture of a medicament for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen.
Clause 34D. Use of a STING agonist for the manufacture of a medicament for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen. Clause 35D. Use of a first antigen for the manufacture of a medicament for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen.
Clause 36D. Use of a second antigen for the manufacture of a medicament for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen.
Clause 37D. Use of a STING agonist for the manufacture of a medicament for use in a method of preparing the immune system of a subject for administration of a second antigen, wherein the method comprises administering to the subject a STING agonist and a first antigen.
Clause 38D. Use of a first antigen for the manufacture of a medicament for use in a method of preparing the immune system of a subject for administration of a second antigen, wherein the method comprises administering to the subject a STING agonist and a first antigen.
Clause 39D. A method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a second adjuvant and a second antigen which is associated with the pathogen.
Clause 40D. A method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist and a first antigen, prior to
(ii) administering to each of the subjects a second adjuvant and the second antigen.
Clause 41 D. A method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a second adjuvant and the second antigen.
Clause 42D. A method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen and a second adjuvant. Clause 43D. A method of preparing the immune system of a subject for (ii) administration of a second antigen and a second adjuvant, wherein the method comprises (i) administering to the subject a STING agonist and a first antigen.
Clause 44D. A STING agonist for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a second adjuvant and a second antigen which is associated with the pathogen.
Clause 45D. A first antigen for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a second adjuvant and a second antigen which is associated with the pathogen.
Clause 46D. A second antigen for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a second adjuvant and a second antigen which is associated with the pathogen.
Clause 47D. A second adjuvant for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second adjuvant and a second antigen which is associated with the pathogen.
Clause 48D. A STING agonist for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist and a first antigen, prior to
(ii) administering to each of the subjects a second adjuvant and the second antigen.
Clause 49D. A first antigen for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist and a first antigen, prior to
(ii) administering to each of the subjects a second adjuvant and the second antigen.
Clause 50D. A second antigen for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist and a first antigen, prior to (ii) administering to each of the subjects a STING agonist and the second antigen.
Clause 51 D. A second adjuvant for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist and a first antigen, prior to
(ii) administering to each of the subjects the second adjuvant and a second antigen.
Clause 52D. A STING agonist for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a second adjuvant and the second antigen.
Clause 53D. A first antigen for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a second adjuvant and the second antigen.
Clause 54D. A second antigen for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a second adjuvant and the second antigen.
Clause 55D. A second adjuvant for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second adjuvant and a second antigen.
Clause 56D. A STING agonist for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen and a second adjuvant.
Clause 57D. A first antigen for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen and a second adjuvant.
Clause 58D. A second antigen for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen and a second adjuvant. Clause 59D. A second adjuvant for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen and the second adjuvant.
Clause 60D. A STING agonist for use in a method of preparing the immune system of a subject for (ii) administration of a second antigen and a second adjuvant, wherein the method comprises (i) administering to the subject a STING agonist and a first antigen.
Clause 61 D. A first antigen for use in a method of preparing the immune system of a subject for administration of a second antigen and a second adjuvant, wherein the method comprises administering to the subject a STING agonist and a first antigen.
Clause 62D. Use of a STING agonist for the manufacture of a medicament for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a second adjuvant and a second antigen which is associated with the pathogen.
Clause 63D. Use of a first antigen for the manufacture of a medicament for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a second adjuvant and a second antigen which is associated with the pathogen.
Clause 64D. Use of a second antigen for the manufacture of a medicament for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a second adjuvant and a second antigen which is associated with the pathogen.
Clause 65D. Use of a second adjuvant for the manufacture of a medicament for use in a method of prophylaxis of infection by a pathogen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second adjuvant and a second antigen which is associated with the pathogen. Clause 66D. Use of a STING agonist for the manufacture of a medicament for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist and a first antigen, prior to
(ii) administering to each of the subjects a second adjuvant and the second antigen.
Clause 67D. Use of a first antigen for the manufacture of a medicament for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist and a first antigen, prior to
(ii) administering to each of the subjects a second adjuvant and the second antigen.
Clause 68D. Use of a second antigen for the manufacture of a medicament for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist and a first antigen, prior to
(ii) administering to each of the subjects a second adjuvant and the second antigen.
Clause 69D. Use of a second adjuvant for the manufacture of a medicament for use in a method of reducing the variability of the immune response to a second antigen in subjects, wherein the method comprises the following steps:
(i) administering to each of the subjects a STING agonist and a first antigen, prior to
(ii) administering to each of the subjects the second adjuvant and the second antigen.
Clause 70D. Use of a STING agonist in the manufacture of a medicament for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a second adjuvant and the second antigen.
Clause 71 D. Use of a first antigen for the manufacture of a medicament for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a second adjuvant and the second antigen.
Clause 72D. Use of a second antigen for the manufacture of a medicament for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject a second adjuvant and the second antigen. Clause 73D. Use of a second adjuvant for the manufacture of a medicament for use in a method of improving an immune response to a second antigen in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second adjuvant and the second antigen.
Clause 74D. Use of a STING agonist for the manufacture of a medicament for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen and a second adjuvant.
Clause 75D. Use of a first antigen for the manufacture of a medicament for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen and a second adjuvant.
Clause 76D. Use of a second antigen for the manufacture of a medicament for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen and a second adjuvant.
Clause 77D. Use of a second adjuvant for the manufacture of a medicament for use in a method of reducing the amount of a second antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen, prior to
(ii) administering to the subject the second antigen and the second adjuvant.
Clause 78D. Use of a STING agonist for the manufacture of a medicament for use in a method of preparing the immune system of a subject for administration of a second antigen and a second adjuvant, wherein the method comprises administering to the subject a STING agonist and a first antigen.
Clause 79D. Use of a first antigen for the manufacture of a medicament for use in a method of preparing the immune system of a subject for administration of a second antigen and a second adjuvant, wherein the method comprises administering to the subject a STING agonist and a first antigen.
Clause 80D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 1 D to 79D, wherein the STING agonist binds to STING with an in vitro Kf of less than 0.750 pM, such as less than 0.500 pM. Clause 81 D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 80D, wherein the STING agonist binds to STING with an in vitro Kf of less than 0.250 pM, such as less than 0.100 pM.
Clause 82D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 1 D to 81 D, wherein the STING agonist activates STING with an in vitro EC50 of 100 pM or less, such as 50 pM or less, when measured by monitoring phosphorylation of interferon regulatory factor-3 (I RF3).
Clause 83D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 82D, wherein the STING agonist activates STING with an in vitro EC50 of 20 pM or less, such as 10 pM or less, when measured by monitoring phosphorylation of interferon regulatory factor-3 (I RF3).
Clause 84D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 1 D to 83D, wherein the STING agonist activates STING with an in vitro EC50 of 100 pM or less, such as 50 pM or less, as measured by monitoring interferon-p induction.
Clause 85D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 84D, wherein the STING agonist activates STING with an in vitro EC50 of 20 pM or less, such as 10 pM or less, as measured by monitoring interferon-p induction.
Clause 86D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 1 D to 85D, wherein the STING agonist is a nucleic acid, a protein, a peptide, or a small molecule.
Clause 87D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 86D, wherein the STING agonist is a small molecule, such as a modified or unmodified cyclic dinucleotide.
Clause 88D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 86D, wherein the STING agonist is selected from a compound of Formulae (l)-(lll):
Figure imgf000334_0002
wherein R1 and R2 are each independently selected from the following groups:
Figure imgf000334_0001
Figure imgf000335_0001
R3 and R4 are each independently -SH or -OH,
R5 and R6 are oxygen or sulphur,
R7 and R8 are each independently halogen, hydrogen, -OH, or OCH3; or a pharmaceutically acceptable salt thereof and/or a tautomer thereof.
Clause 89D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 86D, wherein the STING agonist is the compound:
Figure imgf000335_0002
or a pharmaceutically acceptable salt thereof and/or a tautomer thereof.
Clause 90D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 86D, wherein the STING agonist is selected from c-ditzGMP. c-di- thGMP, c-GtzGMP, c-GAMP, c-thGMP, c-tzGMP, c-di-thAMP, c-ditzAMP, c-di-AMP, c-di-GMP, c- diXGMP, c-GthXMP, c-GXMP, c-ACMP, c-AthXMP, c-AtzXMP, c-di-thXMP, or c-ditzXMP.
Clause 91 D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 86D, wherein the STING agonist is 2’,3’-cGAMP, or a pharmaceutically acceptable salt thereof.
Clause 92D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 86D, wherein the STING agonist is 3’,3’-cGAMP, or a pharmaceutically acceptable salt thereof. Clause 93D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 86D, wherein the STING agonist is the compound:
Figure imgf000336_0001
, or a pharmaceutically acceptable salt thereof.
Clause 94D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 86D, wherein the STING agonist is 6-bromo-N-(naphthalen-1- yl)benzo[d][1 ,3]dioxole-5-carboxamide:
Figure imgf000336_0002
Clause 95D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 86D, wherein the STING agonist is a compound of Formula A:
Figure imgf000336_0003
wherein
X is O or NR4A,
Y is O, NR4A, CH2, or absent, n is 0, 1 , 2, or 3,
R1 and R2 are independently selected from OH, OR3, OR3A, SR3, and NR3R4,
R3, R4, and R4A are independently selected from hydrogen, C1-C10 alkyl optionally substituted with 1-6 halogen, C6-C10 aryl or 5-10 membered heteroaryl, or R3 and R4 together with the nitrogen atom to which they are attached form a 3 to 7 membered heterocycle or 5 to 10 membered heteroaryl,
Figure imgf000337_0001
represents the point of connection of R3A to the remainder of the molecule,
R5-R10 are independently selected from hydrogen, halogen, pseudohalogen, C1-C10 alkyl optionally substituted with 1-6 halogens, C6-C10 aryl, and 5 to 10 membered heteroaryl; or a pharmaceutically acceptable salt thereof and/or a tautomer thereof.
Clause 96D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 86D, wherein the STING agonist is selected from:
Figure imgf000337_0002
acceptable salt thereof.
Clause 97D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 86D, wherein the STING agonist is a flavonoid.
Clause 98D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 97D, wherein the STING agonist is 10-(carboxymethyl)- 9(10H)acridone (CMA), 5,6-Dimethylxanthenone-4-acetic acid (DMXAA), methoxyvone, 6,4'- dimethoxyflavone, 4'-methoxyflavone, 3',6'-dihydroxyflavone, 7,2'-dihydroxyflavone, daidzein, formononetin, retusin 7-methyl ether, xanthone, or any combination thereof.
Clause 99D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 86D, wherein the STING agonist is a compound of Formula B:
Figure imgf000337_0003
wherein
Figure imgf000338_0001
represents two conjugated double bonds in a five-membered heteroaromatic ring and three conjugated double bonds in a six-membered aromatic or heteroaromatic ring,
W1 is selected from CR11 and N;
X1 is selected from CR1, C(R1)2, N, NR1, O and S;
X2 is selected from CR2, C(R2)2, N, NR2, O and S;
X3 is selected from CR3, C(R3)2, N, NR3, O and S; where two or three of X1, X2 and X3 are independently selected from N, NR1, NR2, NR3,
O and S; and where at least one of X1, X2 and X3 is selected from N, NR1, NR2 and NR3;
Y1 is selected from N, NR4, O, S, CR4 and C(R4)2;
Y2 is selected from N, NR5, O, S, CR5 and C(R5)2;
Y3 is selected from N, NR6, O, S, CR6 and C(R6)2;
Y4 is selected from C and N;
Y5 is selected from C and N; where at least one and not more than two of Y1, Y2 and Y3 are independently selected from N, NR4, NR5 and NR6; where when if one of Y4 or Y5 is N, the other one of Y4 or Y5 is C;
Z1 is selected from C and N;
Z2 is selected from N, NR8 and CR8;
Z3 is selected from N, NR9 and CR9;
Z4 is selected from N, NR10 and CR10;
Z5 is selected from N, NR7 and CR7; where two or three of Z1, Z2, Z3, Z4 and Z5 are independently selected from N, NR7, NR8, NR9, and NR10; each R1 is independently selected from the group consisting of H, Ci-Cs alkyl, Ci-Cs alkylene-NRR and Ci-Cs alkylene-C(O)OR; each R2 is independently selected from the group consisting of H, Ci-Cs alkyl, Ci-Cs alkylene-NRR, Ci-Cs alkylene-C(O)OR, Ci-Cs alkylene-OR and Ci-Cs alkylene-O- P(O)(OH)2; each R3 is independently selected from the group consisting of H, Ci-Cs alkyl, Ci-Cs alkylene-NRR, Ci-Cs alkylene-C(O)OR and Ci-Cs alkylene-O-P(O)(OH)2; each R4 is independently selected from the group consisting of H, -OR, -NRR, Ci-Cs alkyl optionally substituted with one or two -OR, Ci-Cs alkylene-NRR, -C(O)OR, Ci-Cs alkylene-C(O)OR, 3-10 membered heterocycle, Ci-Cs alkylene-3-10 membered heterocycle optionally substituted with one 3-10 membered heterocycle, (C3 -C10)- cycloalkyl, and C1-C8 alkylene-(C3-C10)-cycloalkyl; each R5 is independently selected from the group consisting of H, OR, C1 -C8 alkyl, - NRR, C1-C8 alkylene-NRR, -C(O)OR, C1-C8 alkylene-C(O)OR, 3-10 membered heterocycle, C1-C8 alkylene-3-10 membered heterocycle optionally substituted with one 3-10 membered heterocycle, and C1-C8 alkylene-OR; each R6 is H; R7 is selected from the group consisting of H, halo, hydroxy or NH2; R8 is selected from the group consisting of H, C1-C8 alkyl optionally substituted with one or two -NRR or -OR, C1-C8 alkylene-C(O)OR and C1-C8 alkylene-SO2R; R9 is H; R10 is selected from the group consisting of H, C1-C8 alkyl optionally substituted with one or two -OR, and halo; R11 is selected from the group consisting of H, C1-C8 alkyl, -OR and halo; R12 is -C(O)N(R)2 or -C(O)NHR; R13 is H; each R is independently selected from the group consisting of H or C1-C8 alkyl, or C1-C8 haloalkyl, or two R join to form, together with the atom or atoms to which they are bound, a -C3-C10 cycloalkyl or 3-10 membered heterocycle, where said 3-10 membered heterocycle contains one, two or three atoms selected from N, O and S; and where, when two R join to form, together with the atom or atoms to which they are bound, a –(C3-C10) cycloalkyl or 3-10 membered heterocycle, said -C3-C10 cycloalkyl or 3-10 membered heterocycle is optionally substituted with one or more substituents each independently selected from C1-C8 alkyl, hydroxy, C1-C8 alkoxy, -(C3-C10) cycloalkyl, 3-10 membered heterocycle, halo and cyano; or a pharmaceutically acceptable salt thereof and/or a tautomer thereof. Clause 100D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 86D, wherein the STING agonist is a compound selected from:
Figure imgf000340_0001
Figure imgf000341_0001
Figure imgf000342_0001
Figure imgf000343_0001
Figure imgf000344_0001
Figure imgf000345_0001
Figure imgf000346_0001
or a pharmaceutically acceptable salt thereof and/or a tautomer thereof.
Clause 101 D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 86D, wherein the STING agonist is a compound of Formula C:
Figure imgf000347_0001
wherein
Gi is independently selected from Ring A or
Figure imgf000347_0002
ring A is independently selected from optionally substituted heterocyclyl and optionally substituted heteroaryl, ring B is aromatic carbocyclic ring, ring C is optionally substituted five-membered heteroaryl,
R1 is -CON(R3)2,
R2 is independently selected from hydrogen, optionally substituted Ci-Ce alkyl, and optionally substituted C3-C5 monocyclic cycloalkyl,
R3 is independently selected from hydrogen, and optionally substituted Ci-Ce alkyl; m is selected from 0, or 1 ; n is selected from 0, 1 , or 2; o is 1 ; p is selected from 0, 1 , or 2; when 'alkyl's substituted, it is substituted with 1 to 4 substituents independently selected from halogen, alkyl, perhaloalkyl, cycloalkyl, heterocyclyl, -N(R4)2, and -OR4; when ‘carbocycle’ or 'cycloalkyl' is substituted, it is substituted with 1 to 4 substituents independently selected from halogen, alkyl, perhaloalkyl, -N(R4)2, and -OR4; when 'heterocycle' or 'heterocyclyl' is substituted, it is substituted with 1 to 4 substituents independently selected from oxo (=0), halogen, cyano, alkyl, perhaloalkyl, -OR4, -C(=O)OH, -OP(O)(OR4)2, -P(O)(OR4)2, -P(O)(OR4)R4a, -SO2R4a, -SO2NH2, - C(=O)N(H)R4, -C(=O)N(alkyl)R4, -N(H)C(=O)R4a, -N(H)R4, and -N(alkyl)R4; when the 'heteroaryl' group is substituted, it is substituted with 1 to 4 substituents selected from halogen, cyano, alkyl, perhaloalkyl, -O-alkyl, -O-perhaloalkyl, - N(alkyl)alkyl, -N(H)R4, -SO2-alkyl, -N(alkyl)C(=O)alkyl, -N(H)C(=O)alkyl, - C(=O)N(alkyl)alkyl, -C(=O)N(H)alkyl, -C(=O)NH2, -SO2N(alkyl)alkyl, -SO2N(H)alkyl, - SO2NH2, -C(=O)OH, -OP(O)(OR4)2, -P(O)(OR4)2, and -P(O)(OR4)R4a; each R4 is independently selected from hydrogen, alkyl, and cycloalkyl; and each R4a is independently selected from alkyl, and cycloalkyl; or a pharmaceutically acceptable salt thereof and/or a tautomer thereof.
Clause 102D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 86D, wherein the STING agonist is selected from:
Figure imgf000348_0001
Figure imgf000349_0001
Figure imgf000350_0001
Figure imgf000351_0001
or a pharmaceutically acceptable salt thereof and/or a tautomer thereof.
Clause 103D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 86D, wherein the STING agonist is selected from IMSA101 , ADU- S100 (MIW815), BMS-986301 , CRD5500, CMA (IO-carboxymethyl-9-acridanone), diABZI STING agonist-1 (e.g., CAS No.: 2138299-34-8), DMXAA (ASA404/vadimezan), E7766 (Cas no.
Figure imgf000351_0002
, MK-1454, MK-
2118, SB-11285, SRCB-0074, TAK-676, and TTI-10001 or a pharmaceutically acceptable salt thereof.
Clause 104D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 86D, wherein the STING agonist is a compound according to Formula (l-N):
Figure imgf000352_0001
wherein: q is 0 or 1; r is 0 or 1; s is 0 or 1; wherein q + r + s = 1 or 2; when q is 0, RA1 and RA2 are each independently H, halogen, hydroxy, –O-P(O)(OH)2, -O-(O)(RIRII)2, -N(Re)(Rf), -CO2Rf, -N(Rf)CORb, -N(Rg)SO2(C1-C4alkyl)-N(Re)(Rf), -N(Rg)CO(C1-C4alkyl)-N(Rh)(Rf), optionally substituted (C1-C6alkyl), optionally substituted (C1-C6alkyl)oxy-, optionally substituted (C1-C6alkyl)amino-, and optionally substituted (C1-C6alkyl)(C1-C4alkyl)amino-, wherein the (C1-C6alkyl) of said optionally substituted (C1-C6alkyl), optionally substituted (C1-C6alkyl)oxy-, optionally substituted (C1-C6alkyl)amino- and optionally substituted (C1-C6alkyl)(C1-C4alkyl)amino- is optionally substituted by 1-4 substituents each independently selected from hydroxy, -O-P(O)(OH)2, -O-P(O)(RIRII)2, C1-C4alkoxy-, -N(Re)(Rf), -CO2(Rf), -CON(Re)(Rf), optionally substituted phenyl, optionally substituted 5-6 membered heterocycloalkyl and optionally substituted 5-6 membered heteroaryl group, wherein said optionally substituted phenyl, 5-6 membered heterocycloalkyl or 5-6 membered heteroaryl is optionally substituted by 1-4 substituents each independently selected from halogen, hydroxy, -O-P(O)(OH)2, -O- P(O)(RIRII)2, amino, (C1-C6alkyl)amino-, (C1-C6alkyl)(C1-C6alkyl)amino-, -(C1-C6alkyl)- NH2, halo(C1-C6alkyl), hydroxy-(C1-C4alkyl)-, -(C1-C4alkyl)-O-P(O)(OH)2, - (C1-C4alkyl)-O-P(O)(RIRII)2, halo(C1-C4alkoxy)-, C1-C4alkoxy-, hydroxy-(C2-C4alkoxy)-, -(C2-C4alkoxy)-O-P(O)(OH)2, -(C2-C4alkoxy)-O-P(O)(RIRII)2, -C1-C4alkyl-(C1-C4alkoxy) or C1-C4alkoxy-(C1-C4alkoxy)-; when r is 0, RB1 and RB2 are each independently H, optionally substituted C1-C6alkyl, halo(C1-C6alkyl), optionally substituted C2-C6alkenyl, optionally substituted C2-C6alkynyl, optionally substituted C3-C6cycloalkyl, optionally substituted 4-6 membered heterocycloalkyl, optionally substituted phenyl, optionally substituted 5-6 membered heteroaryl, or optionally substituted 9-10 membered heteroaryl, wherein said optionally substituted C1-C6alkyl, optionally substituted C2-C6alkenyl, optionally substituted C2-C6alkynyl, optionally substituted C3-C6cycloalkyl, optionally substituted 4-6 membered heterocycloalkyl, optionally substituted phenyl, optionally substituted 5-6 membered heteroaryl, or optionally substituted 9-10 membered heteroaryl is optionally substituted by 1-4 substituents each independently selected from halogen, nitro, -Rc, -OH, -O-P(O)(OH)2, -O- P(O)(RIRII)2, -ORc, -NH2, -NRcRc, -NRcRd, -OCORc, -CO2H, -CO2Rc, -SORc, -SO2Rc, -CONH2, -CONRcRd, -SO2NH2, -SO2NRcRd, -OCONH2, -OCONRcRd, -NRdCORc, -NRdSORc, -NRdCO2Rc, and -NRdSO2Rc; when s is 0, RC1 is H, halogen, or C1-C4alkyl and RC2 is optionally substituted C1-C4alkyl, wherein said optionally substituted C1-C4alkyl group is optionally substituted by a substituent selected from -ORc, -NRcRd, -CO2Rc, -CONRcRd, -SO2NRcRd, and -OCONRcRd; when q is 1, RA1 and RA2 are each independently -CH2-, -NRe-, or -O-, and A, taken together with RA1 and RA2, forms a linking group, wherein A is -halo(C1-C12alkyl)-, optionally substituted -C1-C12alkyl-, optionally substituted -C2-C12alkenyl-, optionally substituted -C2-C12alkynyl-, optionally substituted -C1-C6alkyl-O-C1-C6alkyl-, optionally substituted -C1-C6alkyl-NRa-C1-C6alkyl-, optionally substituted -C1-C6alkyl-(C3-C6cycloalkyl)-C1-C6alkyl-, optionally substituted -C1-C6alkyl-phenyl-C1-C6alkyl-, optionally substituted -C1-C6alkyl-(4-6 membered heterocycloalkyl)-C1-C6alkyl-, or optionally substituted -C1-C6alkyl-(5-6 membered heteroaryl)-C1-C6alkyl-, wherein the alkyl moiety of said optionally substituted -C1-C12alkyl-, optionally substituted -C2-C12alkenyl-, optionally substituted -C2-C12alkynyl-, optionally substituted -C1-C6alkyl-O-C1-C6alkyl-, optionally substituted -C1-C6alkyl-NRa-C1-C6alkyl-, optionally substituted -C1-C6alkyl-(C3-C6cycloalkyl)-C1-C6alkyl-, optionally substituted -C1-C6alkyl-phenyl-C1-C6alkyl-, optionally substituted -C1-C6alkyl-(4-6 membered heterocycloalkyl)-C1-C6alkyl-, or optionally substituted -C1-C6alkyl-(5-6 membered heteroaryl)-C1-C6alkyl- is optionally substituted by 1-4 substituents each independently selected from halogen, halo(C1-C4alkyl), -OH, -O-P(O)(OH)2, -O-P(O)(RIRII)2, -ORc, -NH2, -NRcRd, -OCORc, -CO2H, -CO2Rc, -SORc, -SO2Rc, -CONH2, -CONRcRd, -SO2NH2, -SO2NRcRd, -OCONH2, -OCONRcRd, -NRdCORc, -NRdSORc, -NRdCO2Rc, and -NRdSO2Rc, and the C3-C6cycloalkyl, phenyl, 4-6 membered heterocycloalkyl, or 5-6 membered heteroaryl moiety of said optionally substituted -C1-C6alkyl-(C3-C6cycloalkyl)-C1-C6alkyl-, optionally substituted -C1-C6alkyl-phenyl-C1-C6alkyl-, optionally substituted -C1-C6alkyl-(4-6 membered heterocycloalkyl)-C1-C6alkyl-, or optionally substituted -C1-C6alkyl-(5-6 membered heteroaryl)-C1-C6alkyl- is optionally substituted by 1-4 substituents each independently selected from halogen, hydroxy, -O-P(O)(OH)2, -O-P(O)(RIRII)2, amino, (C1-C4alkyl)amino-, (C1-C4alkyl)(C1-C4alkyl)amino-, C1-C4alkyl, halo(C1-C4alkyl), halo(C1-C4alkoxy)-, C1-C4alkoxy-, hydroxy-(C1-C4alkoxy)-, -(C1-C4alkoxyl)-O-P(O)(OH)2, -(C1-C4alkoxyl)-O-P(O)(RIRII)2 and C1-C4alkoxy-(C1-C4alkoxy)-; when r is 1, RB1 and RB2 are each independently -CH -, and B, taken together wit B1 2 h R and RB2, forms a linking group, wherein B is a bond or B is -halo(C1-C10alkyl)-, optionally substituted -C1-C10alkyl-, optionally substituted -C2-C10alkenyl-, optionally substituted -C2-C10alkynyl-, optionally substituted -C1-C6alkyl-O-C1-C6alkyl-, optionally substituted -C1-C6alkyl-NRa-C1-C6alkyl-, optionally substituted C3-C6cycloalkyl, optionally substituted phenyl, optionally substituted 4-6 membered heterocycloalkyl, optionally substituted 5-6 membered heteroaryl, optionally substituted -C1-C4alkyl-(C3-C6cycloalkyl)-C1-C4alkyl-, optionally substituted -C1-C4alkyl-phenyl-C1-C4alkyl-, optionally substituted -C1-C4alkyl-(4-6 membered heterocycloalkyl)-C1-C4alkyl-, or optionally substituted -C1-C4alkyl-(5-6 membered heteroaryl)-C1-C4alkyl-, wherein the alkyl moiety of said optionally substituted -C1-C10alkyl-, optionally substituted -C2-C10alkenyl-, optionally substituted -C2-C10alkynyl-, optionally substituted -C1-C6alkyl-O-C1-C6alkyl-, optionally substituted -C1-C6alkyl-NRa-C1-C6alkyl-, optionally substituted -C1-C4alkyl-(C3-C6cycloalkyl)-C1-C4alkyl-, optionally substituted -C1-C4alkyl-phenyl-C1-C4alkyl-, optionally substituted -C1-C4alkyl-(4-6 membered heterocycloalkyl)-C1-C4alkyl-, or optionally substituted -C1-C4alkyl-(5-6 membered heteroaryl-C1-C4alkyl)- is optionally substituted by 1 or 2 substituents each independently selected from halogen, halo(C1-C4alkyl), -OH, -O-P(O)(OH)2, -O-P(O)(RIRII)2, -ORc, -NH2, -NRcRd, -OCORc, -CO2H, -CO2Rc, -SORc, -SO2Rc, -CONH2, -CONRcRd, -SO2NH2, -SO2NRcRd, -OCONH2, -OCONRcRd, -NRdCORc, -NRdSORc, -NRdCO2Rc, and -NRdSO2Rc, and the C3-C6cycloalkyl, phenyl, 4-6 membered heterocycloalkyl, or 5-6 membered heteroaryl moiety of said optionally substituted C3-C6cycloalkyl, optionally substituted phenyl, optionally substituted 4-6 membered heterocycloalkyl, optionally substituted 5-6 membered heteroaryl, optionally substituted -C1-C4alkyl-(C3-C6cycloalkyl)-C1-C4alkyl-, optionally substituted -C1-C4alkyl-phenyl-C1-C4alkyl-, optionally substituted -C1-C4alkyl-(4-6 membered heterocycloalkyl)-C1-C4alkyl-, or optionally substituted -C1-C4alkyl-(5-6 membered heteroaryl)-C1-C4alkyl- is optionally substituted by 1-4 substituents each independently selected from halogen, hydroxy, -O-P(O)(OH)2, -O- P(O)(RIRII)2, amino, (C1-C4alkyl)amino-, (C1-C4alkyl)(C1-C4alkyl)amino-, C1-C4alkyl, halo(C1-C4alkyl), halo(C1-C4alkoxy)-, C1-C4alkoxy-, hydroxy-(C2-C4alkoxy)-, -(C2-C4alkoxy) O-P(O)(OH)2, -(C2-C4alkoxy)-O-P(O)(RIRII)2, and C1-C4alkoxy-(C1-C4alkoxy)-; when s is 1, RC1 and RC2 are each independently -CH -, and C, taken toget C1 2 her with R and RC2, forms a linking group, wherein C is -halo(C1-C12alkyl)-, optionally substituted -C1-C12alkyl-, optionally substituted -C2-C12alkenyl-, optionally substituted -C2-C12alkynyl-, optionally substituted -C1-C6alkyl-O-C1-C6alkyl-, optionally substituted -C1-C6alkyl-NRa-C1-C6alkyl-, optionally substituted -C1-C6alkyl-(C3-C6cycloalkyl)-C1-C6alkyl-, optionally substituted -C1-C6alkyl-phenyl-C1-C6alkyl-, optionally substituted -C1-C6alkyl-(4-6 membered heterocycloalkyl)-C1-C6alkyl-, or optionally substituted -C1-C6alkyl-(5-6 membered heteroaryl)-C1-C6alkyl-, wherein the alkyl moiety of said optionally substituted -C1-C12alkyl-, optionally substituted -C2-C12alkenyl-, optionally substituted -C2-C12alkynyl-, optionally substituted -C1-C6alkyl-O-C1-C6alkyl-, optionally substituted -C1-C6alkyl-NRa-C1-C6alkyl-, optionally substituted -C1-C6alkyl-(C3-C6cycloalkyl)-C1-C6alkyl-, optionally substituted -C1-C6alkyl-phenyl-C1-C6alkyl-, optionally substituted -C1-C6alkyl-(4-6 membered heterocycloalkyl)-C1-C6alkyl-, or optionally substituted -C1-C6alkyl-(5-6 membered heteroaryl)-C1-C6alkyl- is optionally substituted by 1 or 2 substituents each independently selected from halogen, halo(C1-C4alkyl), -OH, -O-P(O)(OH)2, -O-P(O)(RIRII)2, -ORc, -NH2, -NRcRd, -OCORc, -CO2H, -CO2Rc, -SORc, -SO2Rc, -CONH2, -CONRcRd, -SO2NH2, -SO2NRcRd, -OCONH2, -OCONRcRd, -NRdCORc, -NRdSORc, -NRdCO2Rc, and -NRdSO2Rc, and the C3-C6cycloalkyl, phenyl, 4-6 membered heterocycloalkyl, or 5-6 membered heteroaryl moiety of said optionally substituted -C1-C6alkyl-(C3-C6cycloalkyl)-C1-C6alkyl-, optionally substituted -C1-C6alkyl-phenyl-C1-C6alkyl-, optionally substituted -C1-C6alkyl-(4-6 membered heterocycloalkyl)-C1-C6alkyl-, or optionally substituted -C1-C6alkyl-(5-6 membered heteroaryl)-C1-C6alkyl- is optionally substituted by 1-4 substituents each independently selected from halogen, hydroxy, -O-P(O)(OH)2, -O-P(O)(RIRII)2, amino, (C1-C4alkyl)amino-, (C1-C4alkyl)(C1-C4alkyl)amino-, C1-C4alkyl, halo(C1-C4alkyl), halo(C1-C4alkoxy)-, C1-C4alkoxy-, hydroxy-(C2-C4alkoxy)-, -(C2-C4alkoxy)-O-P(O)(OH)2, -(C2-C4alkoxy)-O-P(O)(RIRII)2,and C1-C4alkoxy-(C1-C4alkoxy)-; R3 and R5 are each independently -CON(Rd)(Rf), or one of R3 and R5 is -CON(Rd)(Rf), and the other of R3 and R5 is H, COOH or -CO2(Rc); R4 and R6 are each independently selected from H, halogen, halo(C1-C6alkyl), halo(C1-C6alkoxy)-, hydroxy, –O-P(O)(OH)2, –O-P(O)(RIRII)2, -NH2, -NRcRc, -NRcRd, -CORc, -CO2Rc, -N(Rd)CORc, -N(Rd)SO2Rc, -N(Rg)SO2(C1-C2alkyl)-N(Rh)(Rf), -N(Rg)CO(C1-C2alkyl)-N(Rh)(Rf), optionally substituted (C1-C6alkyl), optionally substituted (C1-C6alkyl)oxy-, optionally substituted (C1-C6alkyl)amino-, and optionally substituted (C1-C6alkyl)(C1-C4alkyl)amino-, wherein the (C1-C6alkyl) of said optionally substituted (C1-C6alkyl), optionally substituted (C1-C6alkyl)oxy-, optionally substituted (C1-C6alkyl)amino- and optionally substituted (C1-C6alkyl)(C1-C4alkyl)amino- is optionally substituted by 1-4 substituents each independently selected from -OH, -O-P(O)(OH)2, -O-P(O)(RIRII)2,-ORc, -NH2, -NRcRc, -NRcRd, -CO2H, -CO2Rc, -OCORc, -CO2H, -CO2Rc, -SORc, -SO2Rc, -CONH2, -CONRcRd, -SO2NH2, -SO2NRcRd, -OCONH2, -OCONRcRd, -NRdCORc, -NRdSORc, -NRdCO2Rc, -NRdSO2Rc, optionally substituted phenyl, optionally substituted 5-6 membered heterocycloalkyl and optionally substituted 5-6 membered heteroaryl group, wherein said optionally substituted phenyl, 5-6 membered heterocycloalkyl or 5-6 membered heteroaryl is optionally substituted by 1-4 substituents each independently selected from halogen, hydroxy, -O-P(O)(OH)2, -O- P(O)(RIRII)2, amino, (C1-C4alkyl)amino-, (C1-C4alkyl)(C1-C4alkyl)amino-, C1-C4alkyl, halo(C1-C4alkyl), hydroxy-(C1-C4alkyl)-, -(C1-C4alkyl)-O-P(O)(OH)2, -(C1-C4alkyl)-O-P(O)(RIRII)2, halo(C1-C4alkoxy)-, C1-C4alkoxy-, hydroxy-(C2-C4alkoxy)-, -(C2-C4alkoxy)-O-P(O)(OH)2, -(C2-C4alkoxy)-O-P(O)(RIRII)2, C1-C4alkoxy-(C1-C4alkoxy)-, -CORd, -CON(Rd)(Rf), and -CO2Rd; R14 is optionally substituted C1-C4alkyl, wherein said optionally substituted C1-C4alkyl is optionally substituted by a substituent selected from -ORc, -NRcRd, -CO2Rc, -CONRcRd, -SO2NRcRd, and -OCONRcRd; R16 is H, halogen, or C1-C4alkyl; R15 and R17 are each independently H, cyclopropyl, or C1-C4alkyl; Ra is H, -Rc, -CORc, -CO2H, -CO2Rc, -SORc, -SO2Rc, -CONH2, -CONRcRd, -SO2NH2, or -SO2NRcRd; each Rb is independently C1-C4alkyl, halo(C1-C4alkyl), -(C1-C4alkyl)-OH, -(C1-C4alkyl)-O-P(O)(OH)2, -(C1-C4alkyl)-O-P(O)(RIRII)2, -(C1-C4alkyl)-O-(C1-C4alkyl), -(C1-C4alkyl)-N(Re)(Rf), -(C1-C4alkyl)-O-CO(C1-C4alkyl), or -(C1-C4alkyl)-CO-O- (C1-C4alkyl); each Rc is independently C1-C4alkyl, halo(C1-C4alkyl), -(C1-C4alkyl)-OH, -(C1-C4alkyl)-O-P(O)(OH)2, -(C1-C4alkyl)-O-P(O)(RIRII)2,-(C1-C4alkyl)-O-(C1-C4alkyl), -(C1-C4alkyl)-N(Re)(Rf), -(C1-C4alkyl)-O-CO(C1-C4alkyl), -(C1-C4alkyl)-CO-O- (C1-C4alkyl), optionally substituted C3-C6cycloalkyl, optionally substituted phenyl, optionally substituted 4-6 membered heterocycloalkyl, optionally substituted 5-6 membered heteroaryl, optionally substituted 9-10 membered heteroaryl, optionally substituted -C1-C4alkyl-C3-C6cycloalkyl, optionally substituted -C1-C4alkyl-phenyl, optionally substituted -C1-C4alkyl-4-6 membered heterocycloalkyl, optionally substituted -C1-C4alkyl-5-6 membered heteroaryl, or optionally substituted -C1-C4alkyl-9-10 membered heteroaryl, wherein the C3-C6cycloalkyl, phenyl, 4-6 membered heterocycloalkyl, 5-6 membered heteroaryl or 9-10 membered heteroaryl moiety of said optionally substituted C3-C6cycloalkyl, optionally substituted phenyl, optionally substituted 4-6 membered heterocycloalkyl, optionally substituted 5-6 membered heteroaryl, optionally substituted 9-10 membered heteroaryl optionally substituted -C1-C4alkyl-C3-C6cycloalkyl, optionally substituted -C1-C4alkyl-phenyl, optionally substituted -C1-C4alkyl-4-6 membered heterocycloalkyl, optionally substituted -C1-C4alkyl-5-6 membered heteroaryl, or optionally substituted -C1-C4alkyl-9-10 membered heteroaryl is optionally substituted by 1-4 substituents each independently selected from halogen, hydroxy, -O-P(O)(OH)2, -O-P(O)(RIRII)2, amino, -(C1-C4alkyl)NH2, (C1-C4alkyl)amino-, (C1-C4alkyl)(C1-C4alkyl)amino-, C1-C4alkyl, halo(C1-C4alkyl), halo(C1-C4alkoxy)-, C1-C4alkoxy-, hydroxy-(C2-C4alkoxy)-, -(C2-C4alkoxy)-O-P(O)(OH)2, -(C2-C4alkoxy)-O-P(O)(RIRII)2, C1-C4alkoxy-(C1-C4alkoxy)-, -CORd, -CON(Rd)(Rf), and -CO2Rd; each Rd is independently H or C1-C4alkyl; each Re is independently H, (C1-C4alkyl), -CO(C1-C4alkyl), -OCO(C1-C4alkyl), -CO2(C1-C4alkyl), -(C1-C4alkyl)NH2, -(C1-C4alkyl) C1-C4alkoxy, -CO-(optionally substituted 5-6 membered heterocycloalkyl), -CO(C1-C4alkyl)-(optionally substituted 5-6 membered heterocycloalkyl), -CO(optionally substituted 5-6 membered heteroaryl), -CO(C1-C4alkyl)-(optionally substituted 5-6 membered heteroaryl), wherein the optionally substituted 5-6 membered heterocycloalkyl or optionally substituted 5-6 membered heteroaryl is optionally substituted 1-4 substituents each independently selected from halogen, hydroxy, -O-P(O)(OH)2, -O-P(O)(RIRII)2, amino, (C1-C4alkyl)amino-, (C1-C4alkyl)(C1-C4alkyl)amino-, C1-C4alkyl, halo(C1-C4alkyl), halo(C1-C4alkoxy)-, C1-C4alkoxy-, hydroxy-(C2-C4alkoxy)-, -(C2-C4alkoxy) O-P(O)(OH)2, -(C2-C4alkoxy)-O-P(O)(RIRII)2, C1-C4alkoxy-(C1-C4alkoxy)- , -CORd, -CON(Rd)(Rf), and -CO2Rd; each Rf is independently H or (C1-C4alkyl); Rg and Rh are each independently H or (C1-C4alkyl) or Rg and Rh, taken together with the atom or atoms through which they are connected, form a 5-6 membered ring; and each occurrence of RI and RII are independently (C1-C6alkyl)oxy-; or a pharmaceutically acceptable salt thereof and/or a tautomer thereof. Clause 105D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 86D, wherein the STING agonist is selected from: (E)-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1H- benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-7- (3-hydroxypropoxy)-1H-benzo[d]imidazole-5-carboxamide
Figure imgf000358_0001
(E)-1-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-2,3- dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-5- carbonyl)imino)-7-(3-hydroxypropoxy)-2,3-dihydro-1H-benzo[d]imidazole-5- carboxamide
Figure imgf000359_0001
(Z)-1-((E)-4-((Z)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-2,3- dihydro-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-7-(3-hydroxypropoxy)-2,3-dihydro-1 H-benzo[d]imidazole-5- carboxamide
Figure imgf000359_0002
(E)-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1 H-pyrazole-5-carboxamido)-7-(3- hydroxypropoxy)-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1 H- pyrazole-5-carboxamido)-7-methoxy-1 H-benzo[d]imidazole-5-carboxamide
Figure imgf000359_0003
(E)-1-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-7-(3- hydroxypropoxy)-2,3-dihydro-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3- methyl-1 H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1 H-benzo[d]imidazole-5- carboxamide
Figure imgf000360_0001
(Z)-1-((E)-4-((Z)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-7-(3- hydroxypropoxy)-2,3-dihydro-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3- methyl-1 H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1 H-benzo[d]imidazole-5- carboxamide
Figure imgf000360_0002
(E)-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1 H-pyrazole-5-carboxamido)-1 H- benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1 H-pyrazole-5-carboxamido)-7-
(3-morpholinopropoxy)-1 H-benzo[d]imidazole-5-carboxamide
Figure imgf000360_0003
(E)-1-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-2,3- dihydro-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-7-(3-morpholinopropoxy)-2,3-dihydro-1 H-benzo[d]imidazole-5- carboxamide
Figure imgf000361_0001
(Z)-1-((E)-4-((Z)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-2,3- dihydro-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-7-(3-morpholinopropoxy)-2,3-dihydro-1 H-benzo[d]imidazole-5- carboxamide
Figure imgf000361_0002
(E)-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1 H-pyrazole-5-carboxamido)-7-(3- morpholinopropoxy)-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1 H- pyrazole-5-carboxamido)-7-methoxy-1 H-benzo[d]imidazole-5-carboxamide
Figure imgf000361_0003
(E)-1-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-7-(3- morpholinopropoxy)-2,3-dihydro-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3- methyl-1 H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1 H-benzo[d]imidazole-5- carboxamide
Figure imgf000362_0001
(Z)-1-((E)-4-((Z)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-7-(3- morpholinopropoxy)-2,3-dihydro-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3- methyl-1 H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1 H-benzo[d]imidazole-5- carboxamide
Figure imgf000362_0002
3-(((Z)-6-carbamoyl-3-((E)-4-((Z)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-7-methoxy-2,3-dihydro-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1- ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1 H-benzo[d]imidazol-4- yl)oxy)propyldihydrogen phosphate
Figure imgf000362_0003
(E)-3-((5-carbamoyl-1 -(4-(5-carbamoyl-2-(1 -ethyl-3-methyl-1 H-pyrazole-5- carboxamido)-7-methoxy-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl- 1 H-pyrazole-5-carboxamido)-1 H-benzo[d]imidazol-7-yl)oxy)propyl dihydrogen phosphate
Figure imgf000363_0001
3-(((E)-6-carbamoyl-3-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-7-methoxy-2,3-dihydro-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1- ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1 H-benzo[d]imidazol-4- yl)oxy)propyl dihydrogen phosphate
Figure imgf000363_0002
(E)-4-((5-carbamoyl-1 -(4-(5-carbamoyl-2-(1 -ethyl-3-methyl-1 H-pyrazole-5- carboxamido)-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1 H-pyrazole-
5-carboxamido)-1 H-benzo[d]imidazol-7-yl)oxy)butanoic acid
Figure imgf000363_0003
(E)-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1 H-pyrazole-5-carboxamido)-7-methoxy-1 H- benzo[d]imidazol-1-yl)but-2-en-1-yl)-7-(3-(dimethylamino)propoxy)-2-(1-ethyl-3-methyl-
1 H-pyrazole-5-carboxamido)-1 H-benzo[d]imidazole-5-carboxamide
Figure imgf000364_0001
(E)-1-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-7-(3-
(4-(2-hydroxyethyl)piperazin-1-yl)propoxy)-2,3-dihydro-1 H-benzo[d]imidazol-1-yl)but-2- en-1-yl)-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1 H- benzo[d]imidazole-5-carboxamide
Figure imgf000364_0002
or a pharmaceutically acceptable salt thereof and/or a tautomer thereof.
Clause 106D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 86D, wherein the STING agonist is 3-(((E)-6-carbamoyl-3-((E)-4-((E)- 5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1 H- benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-2,3- dihydro-1 H-benzo[d]imidazol-4-yl)oxy)propyl
Figure imgf000365_0001
or a pharmaceutically acceptable salt thereof.
Clause 107D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 1 D to 106D, wherein a dose comprises 0.1 to 150 ug, especially 0.5 to 100 ug, such as 1 to 50 ug of STING agonist.
Clause 108D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 1 D to 107D, wherein the STI NG agonist is formulated with a carrier.
Clause 109D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 1 D to 108D, wherein the second adjuvant comprises a saponin.
Clause 110D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 109D, wherein the saponin is obtainable from Quillaja saponaria.
Clause 111 D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 110D, wherein the saponin is obtained from Quillaja saponaria.
Clause 112D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of either clause 109D or 110D, wherein the saponin is prepared synthetically.
Clause 113D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 1 D to 112D, wherein the saponin is Quil A or a fraction thereof, such as QS-7, QS-17, QS-18 and/or QS-21.
Clause 114D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 113D, wherein the saponin is QS-7.
Clause 115D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 113D, wherein the saponin is QS-21.
Clause 116D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 115D, wherein the saponin comprises QS-21 1988 A component, such as at least 40%, such as at least 50%, in particular at least 60%, especially at least 65%, such as at least 70%, QS-21 1988 A component as determined by UV absorbance at 214 nm and by relative ion abundance.
Clause 117D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 116D, wherein the saponin comprises QS-21 1988 A V1.
Clause 118D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of either clause 116D or 117D, wherein the saponin comprises QS-21 1988 A V2.
Clause 119D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 115D to 118D, wherein the saponin comprises QS-21 1856 A component, such as at least 5%, such as at least 10% QS-21 1856 A by UV absorbance at 214 nm and by relative ion abundance.
Clause 120D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 115D to 119D wherein the saponin comprises QS-21 2002 A component, such as at least 0.5%, such as at least 1%, QS-21 2002 A component by UV absorbance at 214 nm and by relative ion abundance.
Clause 121 D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 120D, wherein the saponin comprises QS-21 2002 A V1.
Clause 122D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of either clause 120D or 121 D, wherein the saponin comprises QS-21 2002 A V2.
Clause 123D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 113D or 115D to 122D, wherein the saponin comprises at least 40%, such as at least 50%, suitably at least 60%, especially at least 70% and desirably at least 80%, for example at least 90% (as determined by UV absorbance at 214 nm and by relative ion abundance) QS-21 1988 A component, QS-21 1856 A component and/or QS-21 2002 A component.
Clause 124D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 1 D to 123D, wherein a dose comprises 1 to 100 ug of saponin.
Clause 125D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 124D, wherein a dose comprises 40 to 60 ug, such as 45 to 55 ug, especially 49 to 51 ug, in particular 50 ug of saponin.
Clause 126D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 124D, wherein a dose comprises 20 to 30 ug, such as 22 to 28 ug, especially 24 to 26 ug, in particular 25 ug of saponin. Clause 127D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 109D to 126D, wherein the saponin is formulated with a carrier.
Clause 128D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 127D, wherein the saponin is formulated with ISCOMS.
Clause 129D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 127D, wherein the saponin is formulated with an emulsion.
Clause 130D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 127D, wherein the saponin is formulated with liposomes.
Clause 131 D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 130D, wherein the liposomes comprise DOPC.
Clause 132D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of either clause 130D or 131 D, wherein the liposomes comprise a sterol, such as cholesterol.
Clause 133D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 130D to 132D, wherein the liposome size is in the range of 50 nm to 200 nm, especially 60 nm to 180 nm.
Clause 134D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 133D, wherein the liposome size is in the range of 70 to 165 nm, in particular about 100 nm.
Clause 135D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 130D to 134D, wherein the liposome polydispersity is not more than 0.3, such as not more than 0.2.
Clause 136D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clauses 109D to 135D, wherein the adjuvant comprising a saponin does not comprise non-saponin immunostimulants.
Clause 137D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clauses 109D to 135D, wherein the adjuvant comprising a saponin additionally comprises further immunostimulants.
Clause 138D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 137D, wherein the adjuvant comprising a saponin also comprises a TLR4 agonist.
Clause 139D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 138D, wherein the TLR4 agonist comprises, such as consists of, lipopolysaccharide. Clause 140D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 139, wherein the TLR4 agonist comprises, such as consists of, nontoxic derivatives of lipid AD.
Clause 141 D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 140D, wherein the TLR4 agonist comprises, such as consists of, monophosphoryl lipid A.
Clause 142D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 141 D, wherein the TLR4 agonist comprises, such as consists of, 3-de- O-acylated monophosphoryl lipid A.
Clause 143D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 142D, wherein the TLR4 agonist comprises, such as consists of, 3- deacyl monophosphoryl hexa-acyl lipid A.
Clause 144D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 142D, wherein the TLR4 agonist comprises, such as consists of, 3- deacyl monophosphoryl lipid A.
Clause 145D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 142D, wherein the TLR4 agonist comprises, such as consists of, aminoalkyl glucosaminide phosphate.
Clause 146D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 145D, wherein the TLR4 agonist comprises, such as consists of, CRX601 :
Figure imgf000368_0001
Clause 147D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 138D, wherein the TLR4 agonist comprises, such as consists of, dLOS.
Clause 148D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 138D to 147D, wherein a dose comprises 1 to 100 ug of TLR4 agonist.
Clause 149D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 148D, wherein a dose comprises 40 to 60 ug, such as 45 to 55 ug, especially 49 to 51 ug, in particular 50 ug of TLR4 agonist.
Clause 150D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 148D, wherein a dose comprises 20 to 30 ug, such as 22 to 28 ug, especially 24 to 26 ug, in particular 25 ug of TLR4 agonist.
Clause 151 D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 138D to 150D, wherein the weight ratio of TLR4 agonist to saponin is between 1 :5 to 5:1 , such as 1:2 to 2:1, especially about 1 :1.
Clause 152D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 138D to 151 D, wherein the TLR4 agonist is formulated with a carrier.
Clause 153D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 152D, wherein the TLR4 agonist is formulated with an emulsion.
Clause 154D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 152D, wherein the TLR4 agonist is formulated with liposomes.
Clause 155D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 154D, wherein the liposomes comprise DOPC.
Clause 156D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of either clause 154D or 155D, wherein the liposomes comprise a sterol, such as cholesterol.
Clause 157D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 154D to 156D, wherein the liposome size is in the range of 50 nm to 200 nm, especially 60 nm to 180 nm.
Clause 158D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 157D, wherein the liposome size is in the range of 70 to 165 nm, in particular about 100 nm.
Clause 159D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 154D to 158D, wherein the liposome polydispersity is not more than 0.3, such as not more than 0.2. Clause 160D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 152D to 159D, wherein the saponin and TLR4 agonist are formulated with the same carrier.
Clause 161 D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 138D to 160D, wherein the saponin and TLR4 agonist are formulated together.
Clause 162D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 161 D, wherein the saponin is QS-21 , the TLR4 agonist is 3D-MPL and they are formulated in DOPC liposomes containing cholesterol.
Clause 163D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 138D to 160D, wherein the saponin and TLR4 agonist are formulated separately.
Clause 164D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 39D to 163D, wherein the second adjuvant does not comprise a STING agonist.
Clause 165D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 39D to 164D, wherein the second adjuvant comprises a TLR agonist.
Clause 166D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 165D, wherein the second adjuvant comprises a TLR1 agonist.
Clause 167D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 165D or 166D, wherein the second adjuvant comprises a TLR1/2 agonist, such as Pam3Cys.
Clause 168D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 165D to 167D, wherein the second adjuvant comprises a TLR2 agonist.
Clause 169D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 168D, wherein the TLR2 agonist is CFA, MALP2, Pam2Cys, FSL-I or Hib-OMPC.
Clause 170D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 165D to 169D, wherein the second adjuvant comprises a TLR3 agonist.
Clause 171 D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 170D, wherein the TLR3 agonist is polyinosinic:polycytidylic acid.
Clause 172D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 170D, wherein the TLR3 agonist is polyadenosine-polyuridylic acid. Clause 173D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 170D, wherein the TLR3 agonist is polyinosinic-polycytidylic acid stabilized with poly-L-lysine and carboxymethylcellulose.
Clause 174D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 165D to 173D, wherein the second adjuvant comprises a TLR4 agonist.
Clause 175D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 174D, wherein the TLR4 agonist comprises, such as consists of, lipopolysaccharide.
Clause 176D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 175D, wherein the TLR4 agonist comprises, such as consists of, nontoxic derivatives of lipid A.
Clause 177D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 176D, wherein the TLR4 agonist comprises, such as consists of, monophosphoryl lipid A.
Clause 178D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 177D, wherein the TLR4 agonist comprises, such as consists of, 3-de- O-acylated monophosphoryl lipid A.
Clause 179D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 174D, wherein the TLR4 agonist comprises, such as consists of, 3- deacyl monophosphoryl hexa-acyl lipid A.
Clause 180D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 174D, wherein the TLR4 agonist comprises, such as consists of, 3- deacyl monophosphoryl lipid A.
Clause 181 D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 174D, wherein the TLR4 agonist comprises, such as consists of, aminoalkyl glucosaminide phosphate.
Clause 182D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 181 D, wherein the TLR4 agonist comprises, such as consists of, CRX601 :
Figure imgf000372_0001
Clause 183D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 174D, wherein the TLR4 agonist comprises, such as consists of, dLOS.
Clause 184D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 174D to 183D, wherein a dose comprises 1 to 100 ug of TLR4 agonist.
Clause 185D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 184D, wherein a dose comprises 40 to 60 ug, such as 45 to 55 ug, especially 49 to 51 ug, in particular 50 ug of TLR4 agonist.
Clause 186D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 184D, wherein a dose comprises 20 to 30 ug, such as 22 to 28 ug, especially 24 to 26 ug, in particular 25 ug of TLR4 agonist.
Clause 187D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 165D to 186D, wherein the second adjuvant comprises a TLR5 agonist, such as bacterial flagellin.
Clause 188D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 165D to 187D, wherein the second adjuvant comprises a TLR6 agonist.
Clause 189D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 165D to 188D, wherein the second adjuvant comprises a TLR7 and/or TLR8 agonist. Clause 190D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 189D, wherein the second adjuvant comprises a TLR7 agonist.
Clause 191 D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 189D, wherein the second adjuvant comprises a TLR8 agonist. Clause 192D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 189D, wherein the second adjuvant comprises a TLR7/TLR8 agonist.
Clause 193D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 189D, wherein the TLR7 and/or TLR8 agonist is imiquimod, resiquimod or loxoribine. Clause 194D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 189D, wherein the TLR7 and/or TLR8 agonist is:
Figure imgf000373_0001
Figure imgf000374_0001
Figure imgf000375_0001
Clause 195D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 189D, wherein the TLR7 and/or TLR8 agonist is:
Figure imgf000375_0002
Clause 196D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 165D to 195D, wherein the second adjuvant comprises a TLR9 agonist, such as unmethylated CpG dinucleotide.
Clause 197D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 39D to 196D, wherein the second adjuvant comprises a squalene emulsion adjuvant.
Clause 198D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 197D, wherein the squalene emulsion adjuvant has an average droplet size of less than 1 urn, especially less than 500 nm and in particular less than 200 nm.
Clause 199D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 198D, wherein the squalene emulsion adjuvant has an average droplet size of 50 to 200 nm, especially 120 nm to 180 nm, in particular 140 nm to 180 nm.
Clause 200D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 197D to 199D, wherein the squalene emulsion adjuvant has a polydispersity of 0.5 or less, especially 0.3 or less, such as 0.2 or less.
Clause 201 D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 197D to 200D, wherein the squalene emulsion adjuvant surfactant is selected from poloxamer 401, poloxamer 188, polysorbate 80, sorbitan trioleate, sorbitan monooleate and polyoxyethylene 12 cetyl/stearyl ether either alone, in combination with each other or in combination with other surfactants.
Clause 202D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 201 D, wherein the squalene emulsion adjuvant surfactant is selected from polysorbate 80, sorbitan trioleate, sorbitan monooleate and polyoxyethylene 12 cetyl/stearyl ether either alone, or in combination with each other.
Clause 203D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 202D, wherein the squalene emulsion adjuvant surfactant includes polysorbate 80.
Clause 204D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 197D to 203D, wherein the squalene emulsion adjuvant comprises one surfactant.
Clause 205D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 197D to 203D, wherein the squalene emulsion adjuvant comprises two surfactants.
Clause 206D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 197D to 203D, wherein the squalene emulsion adjuvant comprises three surfactants.
Clause 207D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 197D to 206D, wherein the amount of squalene in a single dose of the squalene emulsion adjuvant is 50 mg or less, especially 40 mg or less, in particular 30 mg or less.
Clause 208D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 207D, wherein the amount of squalene in a single dose of the squalene emulsion adjuvant is 20 mg or less, for example 15 mg or less.
Clause 209D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 197D to 208D, wherein the amount of squalene in a single dose of the squalene emulsion adjuvant is 0.5 mg or more, especially 1 mg or more, in particular 2 mg or more.
Clause 210D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 209D, wherein the amount of squalene in a single dose of the squalene emulsion adjuvant is 4 mg or more, for example, 8 mg or more.
Clause 211 D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 207D to 210D, wherein the amount of squalene in a single dose of the squalene emulsion adjuvant is 2 to 4 mg. Clause 212D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 207D to 210D, wherein the amount of squalene in a single dose of the squalene emulsion adjuvant is 4 to 8 mg.
Clause 213D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 207D to 210D, wherein the amount of squalene in a single dose of the squalene emulsion adjuvant is 8 to 12 mg.
Clause 214D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 197 to 210D, wherein the weight ratio of squalene to surfactant in the squalene emulsion adjuvant is 0.73 to 6.6, especially 1 to 5, in particular 1.5 to 4.5.
Clause 215D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 214D, wherein the weight ratio of squalene to surfactant in the squalene emulsion adjuvant is 1.5 to 3, especially 1.71 to 2.8, such as 2.2 or 2.4.
Clause 216D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 214D, wherein the weight ratio of squalene to surfactant in the squalene emulsion adjuvant is 2.5 to 3.5, especially 3 or 3.1.
Clause 217D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 214D, wherein the weight ratio of squalene to surfactant in the squalene emulsion adjuvant is 3 to 4.5, especially 4 or 4.3.
Clause 218D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 197D to 217D, wherein the squalene emulsion adjuvant does not comprise tocopherol.
Clause 219D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 218D, wherein the squalene emulsion adjuvant consists essentially of squalene, surfactant and water.
Clause 220D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 218D, wherein the squalene emulsion adjuvant comprises squalene, polysorbate 80, sorbitan trioleate and water.
Clause 221 D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 220D, wherein the squalene emulsion adjuvant consists essentially of squalene, polysorbate 80, sorbitan trioleate and water.
Clause 222D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of either clause 220D or 221 D, wherein squalene emulsion adjuvant comprises citrate ions e.g. 10mM sodium citrate buffer.
Clause 223D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 220D to 222D, wherein the weight ratio of squalene to polysorbate 80 in the squalene emulsion adjuvant is 10 to 6.6, especially 9.1 to 7.5, in particular 8.7 to 7.9, such as 8.3.
Clause 224D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 220 to 223, wherein the weight ratio of squalene to sorbitan trioleate in the squalene emulsion adjuvant is 10 to 6.6, especially 9.1 to 7.5, in particular 8.7 to 7.9, such as 8.3.
Clause 225D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 220D to 224D, wherein a single dose of the squalene emulsion adjuvant comprises 0.9 to 11 mg of squalene.
Clause 226D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 225D, wherein the single dose of the squalene emulsion adjuvant comprises 0.9 to 1.1 mg of squalene.
Clause 227D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 225D, wherein the single dose of the squalene emulsion adjuvant comprises 1.1 to 1.4 mg of squalene.
Clause 228D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 225D, wherein the single dose of the squalene emulsion adjuvant comprises 2.2 to 2.8 mg of squalene.
Clause 229D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 225D, wherein the single dose of the squalene emulsion adjuvant comprises 4.5 to 5.5 mg of squalene.
Clause 230D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 225D, wherein the single dose of the squalene emulsion adjuvant comprises 9 to 11 mg of squalene.
Clause 231 D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 218D, wherein the squalene emulsion adjuvant comprises squalene, sorbitan monooleate, polyoxyethylene cetostearyl ether and water, optionally with mannitol.
Clause 232D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 231 D, wherein the squalene emulsion adjuvant consists essentially of squalene, sorbitan monooleate, polyoxyethylene cetostearyl ether and water, optionally with mannitol.
Clause 233D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of either clause 231 D or 232D, wherein squalene emulsion adjuvant comprises phosphate buffered saline.
Clause 234D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 231 D to 233D, wherein the weight ratio of squalene to sorbitan monooleate in the squalene emulsion adjuvant is 7.8 to 5.2, especially 7.15 to 5.85, in particular 6.8 to 6.2, such as 6.5.
Clause 235D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 231 D to 234D, wherein the weight ratio of squalene to polyoxyethylene cetostearyl ether in the squalene emulsion adjuvant is 6.2 to 4.1 , especially 5.7 to 4.7, in particular 5.4 to 4.9, such as 5.2.
Clause 236D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 231 D to 235D, wherein the weight ratio of squalene to mannitol in the squalene emulsion adjuvant is 6.5 to 4.3, especially 5.9 to 4.9, in particular 5.7 to 5.1 , such as 5.4.
Clause 237D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 231 D to 236D, wherein a single dose of the squalene emulsion adjuvant comprises 1.1 to 13.6 mg of squalene.
Clause 238D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 237D, wherein the single dose of the squalene emulsion adjuvant comprises 1.1 to 1.35 mg of squalene.
Clause 239D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 237D, wherein the single dose of the squalene emulsion adjuvant comprises 1.4 to 1.7 mg of squalene.
Clause 240D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 237D, wherein the single dose of the squalene emulsion adjuvant comprises 2.8 to 3.4 mg of squalene.
Clause 241 D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 237D, wherein the single dose of the squalene emulsion adjuvant comprises 5.6 to 6.8 mg of squalene.
Clause 242D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 237D, wherein the single dose of the squalene emulsion adjuvant comprises 11.2 to 13.6 mg of squalene.
Clause 243D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 218D, wherein the squalene emulsion adjuvant comprises squalene, polysorbate 80, sorbitan trioleate and water.
Clause 244D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 243D, wherein the squalene emulsion adjuvant consists essentially of squalene, polysorbate 80, sorbitan trioleate and water.
Clause 245D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of either clause 243D or 244D, wherein squalene emulsion adjuvant comprises phosphate buffered saline, such as modified phosphate buffered saline. Clause 246D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 243D to 245D, wherein the weight ratio of squalene to polysorbate 80 in the squalene emulsion adjuvant is 4.6 to 3.0, especially 4.2 to 3.4, in particular 4.0 to 3.6, such as 3.8.
Clause 247D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 243D to 246D, wherein the weight ratio of squalene to sorbitan trioleate in the squalene emulsion adjuvant is 4.6 to 3.0, especially 4.2 to 3.4, in particular 4.0 to 3.6, such as 3.8.
Clause 248D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 243D to 247D, wherein a single dose of the squalene emulsion adjuvant comprises 0.68 to 8.4 mg of squalene.
Clause 249D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 248D, wherein the single dose of the squalene emulsion adjuvant comprises 0.68 to 0.84 mg of squalene.
Clause 250D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 248D, wherein the single dose of the squalene emulsion adjuvant comprises 0.85 to 1.1 mg of squalene.
Clause 251 D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 248D, wherein the single dose of the squalene emulsion adjuvant comprises 1.7 to 2.1 mg of squalene.
Clause 252D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 248D, wherein the single dose of the squalene emulsion adjuvant comprises 3.4 to 4.2 mg of squalene.
Clause 253D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 248D, wherein the single dose of the squalene emulsion adjuvant comprises 6.8 to 8.4 mg of squalene.
Clause 254D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 218D, wherein the squalene emulsion adjuvant comprises squalene, phosphatidyl choline, poloxamer 188 and water, optionally with glycerol.
Clause 255D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 254D, wherein the squalene emulsion adjuvant consists essentially of squalene, phosphatidyl choline, poloxamer 188 and water, optionally with glycerol.
Clause 256D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of either clause 254D or 255D, wherein the squalene emulsion adjuvant comprises ammonium phosphate buffer and/or glycerol.
Clause 257D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 254D to 256D, wherein the weight ratio of squalene to phosphatidyl choline in the squalene emulsion adjuvant is 2.52 to 3.8, especially 2.85 to 3.5, in particular 3 to 3.3, such as 3.15
Clause 258D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 254D to 257D, wherein the weight ratio of squalene to poloxamer 188 in the squalene emulsion adjuvant is 55 to 83, especially 62 to 76, in particular 65.5 to 72.5, such as 69.
Clause 259D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 254D to 258D, wherein a single dose of the squalene emulsion adjuvant comprises 0.77 to 9.5 mg of squalene.
Clause 260D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 259D, wherein the single dose of the squalene emulsion adjuvant comprises 0.77 to 0.95 mg of squalene.
Clause 261 D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 259D, wherein the single dose of the squalene emulsion adjuvant comprises 0.96 to 1.2 mg of squalene.
Clause 262D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 259D, wherein the single dose of the squalene emulsion adjuvant comprises 1.9 to 2.4 mg of squalene.
Clause 263D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 259D, wherein the single dose of the squalene emulsion adjuvant comprises 3.8 to 4.8 mg of squalene.
Clause 264D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 259D, wherein the single dose of the squalene emulsion adjuvant comprises 7.7 to 9.5 mg of squalene.
Clause 265D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 197D to 217D, wherein the squalene emulsion adjuvant comprises tocopherol.
Clause 266D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 265D, wherein the tocopherol is alpha-tocopherol.
Clause 267D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 266D, wherein the tocopherol is D/L-alpha-tocopherol.
Clause 268D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 265D to 267D, wherein the squalene emulsion adjuvant consists essentially of squalene, tocopherol, surfactant and water.
Clause 269D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 265D to 268D, wherein the weight ratio of squalene to tocopherol in the squalene emulsion adjuvant is 20 or less, especially 10 or less. Clause 270D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 265D to 269D, wherein the weight ratio of squalene to tocopherol in the squalene emulsion adjuvant is 0.1 or more.
Clause 271 D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 265D to 270D, wherein the weight ratio of squalene to tocopherol in the squalene emulsion adjuvant is 0.2 to 5.
Clause 272D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 271 D, wherein the weight ratio of squalene to tocopherol in the squalene emulsion adjuvant is 0.72 to 1.136, especially 0.8 to 1, in particular 0.85 to 0.95, such as 0.9.
Clause 273D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 271 D, wherein the weight ratio of squalene to tocopherol in the squalene emulsion adjuvant is 3.4 to 4.6, especially 3.6 to 4.4, in particular 3.8 to 4.2, such as 4.
Clause 274D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 265D to 273D, wherein the squalene emulsion adjuvant comprises squalene, tocopherol, polysorbate 80 and water.
Clause 275D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 274D, wherein the squalene emulsion adjuvant consists essentially of squalene, tocopherol, polysorbate 80 and water.
Clause 276D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of either clause 274D or 275D, wherein squalene emulsion adjuvant comprises a phosphate buffered saline, such as modified phosphate buffered saline.
Clause 277D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 274D to 276D, wherein the weight ratio of squalene to tocopherol in the squalene emulsion adjuvant is 0.5 to 1.5, especially 0.6 to 1.35, in particular 0.7 to 1.1, such as 0.85 to 0.95 e.g. 0.9.
Clause 278D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 274D to 277D, wherein the weight ratio of squalene to polysorbate 80 in the squalene emulsion adjuvant is 1.2 to 3.6, especially 1.46 to 3.3, in particular 1.9 to 2.5 such as 2.1 to 2.3 e.g. 2.2.
Clause 279D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 274D to 278D, wherein a single dose of the squalene emulsion adjuvant comprises 0.9 to 12.1 mg of squalene.
Clause 280D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 279D, wherein the single dose of the squalene emulsion adjuvant comprises 0.9 to 1.3 mg of squalene, typically with 1 to 1.4 mg tocopherol and 0.43 to 0.57 mg polysorbate 80.
Clause 281 D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 279D, wherein the single dose of the squalene emulsion adjuvant comprises 1.2 to 1.6 mg of squalene, typically with 1.3 to 1.7 mg tocopherol and 0.54 to 0.71 mg polysorbate 80.
Clause 282D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 279D, wherein the single dose of the squalene emulsion adjuvant comprises 2.4 to 3 mg of squalene, typically with 2.6 to 3.4 mg tocopherol and 1 to 1.5 mg polysorbate 80.
Clause 283D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 279D, wherein the single dose of the squalene emulsion adjuvant comprises 4.8 to 6.1 mg of squalene, typically with 5.3 to 6.8 mg tocopherol and 2.1 to 2.9 mg polysorbate 80.
Clause 284D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 279D, wherein the single dose of the squalene emulsion adjuvant comprises 9.7 to 12.1 mg of squalene, typically with 10.6 to 13.6 mg tocopherol and 4.3 to 5.7 mg polysorbate 80.
Clause 285D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 274D to 276D, wherein the weight ratio of squalene to tocopherol in the squalene emulsion adjuvant is 2.6 to 4.5, especially 2.8 to 4.3, in particular 3.25 to 4, such as 3.4 to 3.8 e.g. 3.6.
Clause 286D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 274D to 276D or 285D, wherein the weight ratio of squalene to polysorbate 80 in the squalene emulsion adjuvant is 11.3 to 2.5, especially 1.56 to 2.3, in particular 1.75 to 2.15 such as 1.85 to 2 e.g. 1.94.
Clause 287D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 274D to 276D, 285D or 286D, wherein a single dose of the squalene emulsion adjuvant comprises 1.1 to 14.3 mg of squalene.
Clause 288D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 287D, wherein the single dose of the squalene emulsion adjuvant comprises 1.1 to 1.5 mg of squalene.
Clause 289D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 287D, wherein the single dose of the squalene emulsion adjuvant comprises 1.4 to 1.8 mg of squalene. Clause 290D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 287D, wherein the single dose of the squalene emulsion adjuvant comprises 2.9 to 3.6 mg of squalene.
Clause 291 D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 287D, wherein the single dose of the squalene emulsion adjuvant comprises 5.8 to 7.2 mg of squalene.
Clause 292D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 287D, wherein the single dose of the squalene emulsion adjuvant comprises 11.7 to 14.3 mg of squalene.
Clause 293D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 265D to 270D, wherein the squalene emulsion adjuvant comprises squalene, tocopherol, phosphatidyl choline, poloxamer 188 and water, optionally with glycerol.
Clause 294D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 293D, wherein the squalene emulsion adjuvant consists essentially of squalene, tocopherol, phosphatidyl choline, poloxamer 188 and water, optionally with glycerol.
Clause 295D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of either clause 293D or 294D, wherein the squalene emulsion adjuvant comprises ammonium phosphate buffer and/or glycerol.
Clause 296D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clauses 293D to 295D, wherein the weight ratio of squalene to tocopherol in the squalene emulsion adjuvant is at least 50, especially 137 to 207, in particular 154 to 190, such as 163 to 181 , for example 172.
Clause 297D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clauses 293D to 296D, wherein the weight ratio of squalene to phosphatidyl choline in the squalene emulsion adjuvant is 2.52 to 3.8, especially 2.85 to 3.5, in particular 3 to 3.3, such as 3.15
Clause 298D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clauses 293D to 297D, wherein the weight ratio of squalene to poloxamer 188 in the squalene emulsion adjuvant is 55 to 83, especially 62 to 76, in particular 65.5 to 72.5, such as 69.
Clause 299D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clauses 293D to 298D, wherein a single dose of the squalene emulsion adjuvant comprises 0.77 to 9.5 mg of squalene.
Clause 300D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 299D, wherein the single dose of the squalene emulsion adjuvant comprises 0.77 to 0.95 mg of squalene. Clause 301 D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 299D, wherein the single dose of the squalene emulsion adjuvant comprises 0.96 to 1.2 mg of squalene.
Clause 302D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 299D, wherein the single dose of the squalene emulsion adjuvant comprises 1.9 to 2.4 mg of squalene.
Clause 303D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 299D, wherein the single dose of the squalene emulsion adjuvant comprises 3.8 to 4.8 mg of squalene.
Clause 304D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 299D, wherein the single dose of the squalene emulsion adjuvant comprises 7.7 to 9.5 mg of squalene.
Clause 304D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 299D, wherein the single dose of the squalene emulsion adjuvant comprises 7.7 to 9.5 mg of squalene.
Clause 305D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clauses 39D to 305D, wherein the second adjuvant comprises one immunostimulant.
Clause 306D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clauses 39D to 305D, wherein the second adjuvant comprises two immunostimulants.
Clause 307D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clauses 1 D to 306D wherein only one administration of the STING agonist and first antigen is performed in step (i).
Clause 308D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clauses 1 D to 306D wherein at least 2 administrations of the STING agonist and first antigen are performed in step (i).
Clause 309D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 308D wherein at least 3 administrations of the STING agonist and first antigen are performed in step (i).
Clause 310D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 308D wherein 2 administrations of the STING agonist and first antigen are performed in step (i).
Clause 311 D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 1 D to 310D wherein in step (i) the STING agonist is coformulated with the first antigen. Clause 312D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 311 D wherein the co-formulated STING agonist and first antigen of step (i) is administered intradermally, intramuscularly, intraperitoneally or subcutaneously, in particular intramuscularly.
Clause 313D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 311 D or 312D wherein the volume of the co-formulation of STING agonist and first antigen in step (i) is 0.05 ml to 1 ml, such as 0.1 to 0.6 ml.
Clause 314D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 311 D to 313D wherein the pH of the co-formulation of STING agonist and first antigen in step (i) is 4 to 9, especially 5 to 8.5, in particular 5.5 to 8, such as 6.5 to 7.4.
Clause 315D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 311 D to 314D wherein the osmolarity of the co- formulation of STING agonist and first antigen in step (i) is 250 to 750 mOsm/kg, especially 250 to 550 mOsm/kg, in particular 270 to 500 mOsm/kg, such as 270 to 400 mOsm/kg.
Clause 316D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 311 D to 315D wherein the co-formulation of STING agonist and first antigen of step (i) comprises buffer and/or tonicity modifying agents, such as modified phosphate buffered saline.
Clause 317D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 1 D to 310D wherein in step (i) the STING agonist is formulated separately from the first antigen.
Clause 318D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 317D wherein in step (i) for at least one administration, the STING agonist is administered simultaneously with the first antigen.
Clause 319D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 318D wherein in step (i) for all administrations the STING agonist is administered simultaneously with the first antigen.
Clause 320D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 318D wherein in step (i) for at least one administration, the STING agonist is administered separately from the first antigen.
Clause 321 D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 320D wherein in step (i) for at least one administration, the STING agonist is administered before the first antigen.
Clause 322D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 321 D wherein in step (i) for all administrations the STING agonist is administered before the first antigen. Clause 323D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 321 D wherein in step (i) for at least one administration, the first antigen is administered before the STING agonist.
Clause 324D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 323D wherein in step (i) for all administrations the first antigen is administered before the STING agonist.
Clause 325D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 317D to 324D wherein in step (i) for at least one administration, the STING agonist is administered to the same location on the subject as the first antigen.
Clause 326D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 325D wherein in step (i) for all administrations the STING agonist is administered to the same location on the subject as the first antigen.
Clause 327D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 317D to 325D wherein in step (i) for at least one administration, the STING agonist is administered to a different location on the subject as the first antigen.
Clause 328D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 327D wherein in step (i) for all administrations the STING agonist is administered to a different location on the subject as the first antigen.
Clause 329D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 307D to 328D wherein in step (i) for at least one administration, the STING agonist and the first antigen are administered within 84 hours of each other.
Clause 330D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 329D wherein in step (i) for at least one administration, the STING agonist and the first antigen are administered within 30 minutes of each other.
Clause 331 D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 329D wherein in step (i) for all administrations the STING agonist and the first antigen are administered within 84 hours of each other.
Clause 332D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 330D wherein in step (i) for all administrations the STING agonist and the first antigen are administered within 30 minutes of each other.
Clause 333D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 317D to 332D wherein the STING agonist of step (i) is administered intradermally, intramuscularly, intraperitoneally or subcutaneously, in particular intramuscularly. Clause 334D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 317D to 333D wherein the first antigen of step (i) is administered intradermally, intramuscularly, intraperitoneally or subcutaneously, in particular intramuscularly.
Clause 335D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 317D to 334D wherein the volume of the separate formulation of STING agonist in step (i) is 0.05 ml to 1 ml, such as 0.1 to 0.6 ml.
Clause 336D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 317D to 335D wherein the volume of the separate formulation of first antigen in step (i) is 0.05 ml to 1 ml, such as 0.1 to 0.6 ml.
Clause 337D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 317D to 336D wherein the pH of the separate formulation of STING agonist in step (i) is 4 to 9, especially 5 to 8.5, in particular 5.5 to 8, such as 6.5 to 7.4.
Clause 338D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 317D to 337D wherein the pH of the separate formulation of first antigen in step (i) is 4 to 9, especially 5 to 8.5, in particular 5.5 to 8, such as 6.5 to 7.4.
Clause 339D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 317D to 338D wherein the osmolarity of the separate formulation of STING agonist in step (i) is 250 to 750 mOsm/kg, especially 250 to 550 mOsm/kg, in particular 270 to 500 mOsm/kg, such as 270 to 400 mOsm/kg.
Clause 340D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 317D to 339D wherein the osmolarity of the separate formulation of first antigen in step (i) is 250 to 750 mOsm/kg, especially 250 to 550 mOsm/kg, in particular 270 to 500 mOsm/kg, such as 270 to 400 mOsm/kg.
Clause 341 D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 317D to 340D wherein the separate formulation comprising the STING agonist of step (i) comprises buffer and/or tonicity modifying agents, such as modified phosphate buffered saline.
Clause 342D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 317D to 341 D wherein the separate formulation separate formulation of first antigen in step (i) comprises buffer and/or tonicity modifying agents, such as modified phosphate buffered saline.
Clause 343D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 1 D to 342D wherein in step (i) the period between administrations to the subject is 2 weeks to 12 months. Clause 344D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 343D wherein in step (i) the period between administrations to the subject is 2 weeks to 6 months.
Clause 345D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 344D wherein in step (i) the period between administrations to the subject is 2 weeks to 3 months.
Clause 346D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 345D wherein in step (i) the period between administrations to the subject is 2 weeks to 2 months, such as 3 to 5 weeks.
Clause 347D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 1 D to 346D wherein step (ii) is initiated at an interval of 2 weeks to 2 years after step (i).
Clause 348D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 347D wherein step (ii) is initiated at an interval of 2 weeks to 1 year after step (i).
Clause 349D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 348D wherein step (ii) is initiated at an interval of 3 weeks to 9 months after step (i).
Clause 350D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 349D wherein step (ii) is initiated at an interval of 4 weeks to 6 months after step (i).
Clause 351 D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 347D wherein step (ii) is initiated at an interval of 2 weeks to 2 months after step (i).
Clause 352D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 347D wherein step (ii) is initiated at an interval of 1 month to 3 months after step (i).
Clause 353D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 347D wherein step (ii) is initiated at an interval of 2 months to 4 months after step (i).
Clause 354D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 348D wherein step (ii) is initiated at an interval of 3 months to 6 months after step (i).
Clause 355D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 348D wherein step (ii) is initiated at an interval of 4 months to 12 months after step (i). Clause 356D. The method, STING agonist, first antigen or use of any one of clauses 1 D to 5D, 80D to 163D or 307D to 346D wherein the risk of infection occurs 2 weeks to 2 years after administering a STING agonist and a first antigen to the subject.
Clause 357D. The method, STING agonist, first antigen or use of clause 356D wherein the risk of infection occurs 2 weeks to 1 year after administering a STING agonist and a first antigen to the subject.
Clause 358D. The method, STING agonist, first antigen or use of clause 357D wherein the risk of infection occurs 3 weeks to 9 months after administering a STING agonist and a first antigen to the subject.
Clause 359D. The method, STING agonist, first antigen or use of clause 358D wherein the risk of infection occurs 4 weeks to 6 months after administering a STING agonist and a first antigen to the subject.
Clause 360D. The method, STING agonist, first antigen or use of clause 356D wherein the risk of infection occurs 2 weeks to 2 months after administering a STING agonist and a first antigen to the subject.
Clause 361 D. The method, STING agonist, first antigen or use of clause 356D wherein the risk of infection occurs 1 month to 3 months after administering a STING agonist and a first antigen to the subject.
Clause 362D. The method, STING agonist, first antigen or use of clause 356D wherein the risk of infection occurs 2 months to 4 months after administering a STING agonist and a first antigen to the subject.
Clause 363D. The method, STING agonist, first antigen or use of clause 357D wherein the risk of infection occurs 3 months to 6 months after administering a STING agonist and a first antigen to the subject.
Clause 364D. The method, STING agonist, first antigen or use of clause 357D wherein the risk of infection occurs 4 months to 12 months after administering a STING agonist and a first antigen to the subject.
Clause 365D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 6D to 364D wherein only one administration is performed in step (ii).
Clause 366D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 6D to 364D wherein at least 2 administrations are performed in step (ii).
Clause 367D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 366D wherein at least 3 administrations are performed in step (ii).
Clause 368D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 366D wherein 2 administrations are performed in step (ii). Clause 369D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 6D to 355D or 365D to 368D wherein in step (ii) the second adjuvant, if present, is co-formulated with the second antigen.
Clause 370D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 369D wherein the second antigen of step (ii) is administered intradermally, intramuscularly, intraperitoneally or subcutaneously, in particular intramuscularly.
Clause 371 D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 369D or 370D wherein the volume of the formulation of second antigen in step (ii) is 0.05 ml to 1 ml, such as 0.1 to 0.6 ml.
Clause 372D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 369D to 371 D wherein the pH of the formulation of second antigen in step (ii) is 4 to 9, especially 5 to 8.5, in particular 5.5 to 8, such as 6.5 to 7.4.
Clause 373D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 369D to 372D wherein the osmolarity of the formulation of second antigen in step (ii) is 250 to 750 mOsm/kg, especially 250 to 550 mOsm/kg, in particular 270 to 500 mOsm/kg, such as 270 to 400 mOsm/kg.
Clause 374D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 369D to 373D wherein the formulation of second antigen of step (ii) comprises buffer and/or tonicity modifying agents, such as modified phosphate buffered saline.
Clause 375D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clauses 6D to 355D or 365D to 368D wherein in step (ii) the second adjuvant, if present, is formulated separately from the second antigen.
Clause 376D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 375D wherein in step (ii) for at least one administration, the second adjuvant is administered simultaneously with the second antigen.
Clause 377D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 375D wherein in step (ii) for all administrations the second adjuvant is administered simultaneously with the second antigen.
Clause 378D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 375D wherein in step (ii) for at least one administration, the second adjuvant is administered separately from the second antigen.
Clause 379D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 378D wherein in step (ii) for at least one administration, the second adjuvant is administered before the second antigen. Clause 380D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 379D wherein in step (ii) for all administrations the second adjuvant is administered before the second antigen.
Clause 381 D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 379D wherein in step (ii) for at least one administration, the second antigen is administered before the second adjuvant.
Clause 382D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 381 D wherein in step (ii) for all administrations the second antigen is administered before the second adjuvant.
Clause 383D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 375D to 382D wherein in step (ii) for at least one administration, the second adjuvant is administered to the same location on the subject as the second antigen.
Clause 384D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 383D wherein in step (ii) for all administrations the second adjuvant is administered to the same location on the subject as the second antigen.
Clause 385D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 375D to 383D wherein in step (ii) for at least one administration, the second adjuvant is administered to a different location on the subject as the second antigen.
Clause 386D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 385D wherein in step (ii) for all administrations the second adjuvant is administered to a different location on the subject as the second antigen.
Clause 387D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 375D to 386D wherein in step (ii) for at least one administration, the second adjuvant and the second antigen are administered within 84 hours of each other.
Clause 388D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 387D wherein in step (ii) for at least one administration, the second adjuvant and the second antigen are administered within 30 minutes of each other.
Clause 389D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 387D wherein in step (ii) for all administrations the second adjuvant and the second antigen are administered within 84 hours of each other.
Clause 390D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 389D wherein in step (ii) for all administrations the second adjuvant and the second antigen are administered within 30 minutes of each other. Clause 391 D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 375D to 390D wherein the STING agonist of step (ii) is administered intradermally, intramuscularly, intraperitoneally or subcutaneously, in particular intramuscularly.
Clause 392D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 375D to 391 D wherein the second antigen of step (ii) is administered intradermally, intramuscularly, intraperitoneally or subcutaneously, in particular intramuscularly.
Clause 393D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 375D to 392D wherein the volume of the separate formulation of second adjuvant in step (ii) is 0.05 ml to 1 ml, such as 0.1 to 0.6 ml.
Clause 394D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 375D to 393D wherein the volume of the separate formulation of second antigen in step (ii) is 0.05 ml to 1 ml, such as 0.1 to 0.6 ml.
Clause 395D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 375D to 394D wherein the pH of the separate formulation of second adjuvant in step (ii) is 4 to 9, especially 5 to 8.5, in particular 5.5 to 8, such as 6.5 to 7.4.
Clause 396D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 375D to 395D wherein the pH of the separate formulation of second antigen in step (ii) is 4 to 9, especially 5 to 8.5, in particular 5.5 to 8, such as 6.5 to 7.4.
Clause 397D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 375D to 396D wherein the osmolarity of the separate formulation of second adjuvant in step (ii) is 250 to 750 mOsm/kg, especially 250 to 550 mOsm/kg, in particular 270 to 500 mOsm/kg, such as 270 to 400 mOsm/kg.
Clause 398D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 375D to 397D wherein the osmolarity of the separate formulation of second antigen in step (ii) is 250 to 750 mOsm/kg, especially 250 to 550 mOsm/kg, in particular 270 to 500 mOsm/kg, such as 270 to 400 mOsm/kg.
Clause 399D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 375D to 398D wherein the separate formulation comprising the second adjuvant of step (ii) comprises buffer and/or tonicity modifying agents, such as modified phosphate buffered saline.
Clause 400D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 375D to 399D wherein the separate formulation comprising the second antigen of step (ii) comprises buffer and/or tonicity modifying agents, such as modified phosphate buffered saline.
Clause 401 D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 6D to 400D wherein in step (ii) the period between administrations to the subject is 2 weeks to 12 months.
Clause 402D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 401 D wherein in step (ii) the period between administrations to the subject is 2 weeks to 6 months.
Clause 403D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 401 D wherein in step (ii) the period between administrations to the subject is 2 weeks to 3 months.
Clause 404D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 401 D wherein in step (ii) the period between administrations to the subject is 2 weeks to 2 months, such as 3 to 5 weeks.
Clause 405D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 6D to 404D wherein the combined duration of step (i), step (ii) and the interval between the steps is 1 month to 2 years.
Clause 406D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 405D wherein the combined duration of step (i), step (ii) and the interval between the steps is 6 weeks to 12 months.
Clause 407D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 406D wherein the combined duration of step (i), step (ii) and the interval between the steps is 2 months to 10 months.
Clause 408D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 407D wherein the combined duration of step (i), step (ii) and the interval between the steps is 10 weeks to 9 months.
Clause 409D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of any one of clauses 1D to 408D wherein the combined duration of step (i), step (ii) and the interval between the steps is at least 2 months, especially at least 3 months, in particular at least 4 months.
Clause 410D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 409D wherein the combined duration of step (i), step (ii) and the interval between the steps is at least 5 months, especially at least 6 months, in particular at least 7 months.
Clause 411 D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of any one of clauses 1D to 410D wherein the combined duration of step (i), step (ii) and the interval between the steps is 5 years or less, especially 3 years or less, in particular 2 years or less.
Clause 412D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 411 D wherein the combined duration of step (i), step (ii) and the interval between the steps is 18 months or less, especially 12 months or less, in particular 10 months or less.
Clause 413D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 1 D to 412D wherein the subject is a mammal.
Clause 414D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 412D wherein the subject is a human.
Clause 415D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 414D wherein the subject is a human child.
Clause 416D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 414D wherein the subject is a human adult.
Clause 417D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 414D wherein the subject is an older human.
Clause 418D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 6D to 417D wherein the administration route used for each administration of step (i) is the same as the administration route used for each administration of step (ii).
Clause 419D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 418D wherein the administration route used is intramuscular.
Clause 420D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 1 D to 419D wherein the first antigen is derived from a pathogen.
Clause 421 D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 420D wherein the first antigen is derived from a different pathogen to the second antigen.
Clause 422D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 420D wherein the first antigen is derived from a different pathogen from the pathogen presenting the risk of infection.
Clause 423D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of either clause 421 D or 422D wherein the different pathogen is a pathogen which belongs to a different species.
Clause 424D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 423D wherein the different pathogen is a pathogen which belongs to a different genus. Clause 425D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 424D wherein the different pathogen is a pathogen which belongs to a different family.
Clause 426D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 425D wherein the different pathogen is a pathogen which belongs to a different order.
Clause 427D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 426D wherein the different pathogen is a pathogen which belongs to a different class.
Clause 428D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 427D wherein the different pathogen is a pathogen which belongs to a different phylum.
Clause 429D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 428D wherein the different pathogen is a pathogen which belongs to a different domain.
Clause 430D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 1 D to 429D wherein the first antigen is derived from (such as containing a portion of a polypeptide sequence obtainable from, such as at least 50, especially at least 100, in particular at least 150, such as at least 200 amino acids long) one of bacteria, viruses or parasites and the second antigen is derived from another of bacteria, viruses or parasites (such as containing a portion of a polypeptide sequence obtainable from, such as at least 50, especially at least 100, in particular at least 150, such as at least 200 amino acids long).
Clause 431 D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 430D wherein the first antigen is derived from a virus and the second antigen is derived from a bacterium.
Clause 432D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 430D wherein the first antigen is derived from a bacterium and the second antigen is derived from a virus.
Clause 433D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 6D to 432D wherein the first antigen does not comprise any epitopes which are cross-reactive with epitopes of the second antigen in a CD4+ T cell response.
Clause 434D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 6D to 433D wherein the first antigen does not comprise any epitopes which are cross-reactive with epitopes of the second antigen in a CD8+ T cell response. Clause 435D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 6D to 434D wherein the first antigen does not comprise any epitopes which are cross-reactive with epitopes of the second antigen in an antibody response.
Clause 436D. The method, STING agonist, first antigen, or use of any one of clauses 1 D to 429D wherein the first antigen is derived from (such as containing a portion of a polypeptide sequence obtainable from, such as at least 50, especially at least 100, in particular at least 150, such as at least 200 amino acids long) one of bacteria, viruses or parasites and the pathogen presenting the risk of infection is a bacteria, viruses or parasites.
Clause 437D. The method, STING agonist, first antigen, or use of clause 436D wherein the first antigen is derived from (such as containing a portion of a polypeptide sequence obtainable from, such as at least 50, especially at least 100, in particular at least 150, such as at least 200 amino acids long) one of bacteria, viruses or parasites and the pathogen presenting the risk of infection is another of bacteria, viruses or parasites.
Clause 438D. The method, STING agonist, first antigen, or use of clause 436D wherein the first antigen is derived from a virus and the pathogen presenting the risk of infection is a bacterium.
Clause 439D. The method, STING agonist, first antigen or use of clause 436D wherein the first antigen is derived from a bacterium and the pathogen presenting the risk of infection is a virus.
Clause 440D. The method, STING agonist, first antigen or use of clause 436D wherein the first antigen is derived from a virus and the pathogen presenting the risk of infection is a virus.
Clause 441 D. The method, STING agonist, first antigen, or use of any one of clauses 1 D to 429D or 436D to 440D wherein the first antigen does not comprise any epitopes which are cross-reactive with epitopes of pathogen presenting the risk of infection in a CD4+ T cell response.
Clause 442D. The method, STING agonist, first antigen, or use of any one of clauses 1 D to 429D or 436D to 441 D wherein the first antigen does not comprise any epitopes which are cross-reactive with epitopes of the pathogen presenting the risk of infection in a CD8+ T cell response.
Clause 443D. The method, STING agonist, first antigen, or use of any one of clauses 1 D to 429D or 436D to 442D wherein the first antigen does not comprise any epitopes which are cross-reactive with epitopes of the pathogen presenting the risk of infection in an antibody response. Clause 444D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 1 D to 443D wherein the first antigen is a polypeptide, a polynucleotide or a polysaccharide.
Clause 445D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 444D wherein the first antigen is a polypeptide.
Clause 446D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 445D, wherein the polypeptide comprises at least one B or T cell epitope.
Clause 447D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 446D, wherein the polypeptide comprises B and T cell epitopes.
Clause 448D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 444D to 447D wherein the first antigen is derived from a virus.
Clause 449D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 448D wherein the first antigen is derived from a herpesvirus.
Clause 450D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 449D wherein the herpesvirus is varicella-zoster virus (VZV).
Clause 451 D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 450D wherein the first antigen is a VZV-gE antigen.
Clause 452D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 451 D wherein the VZV-gE antigen comprises an amino acid sequence having at least 80%, such as at least 90%, especially at least 95%, in particular at least 98% for example at least 99% or 100% identity to SEQ ID NO: 1.
Clause 453D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 444D to 447D wherein the first antigen is a cancer antigen, such as a tumour antigen and/or a neoantigen.
Clause 454D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 453D wherein the first antigen is a human cancer antigen, such as a human tumour antigen and/or a neoantigen.
Clause 455D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 1 D to 444D wherein the first antigen is provided in the form of a polynucleotide encoding the antigen.
Clause 456D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 455D wherein the first antigen is provided in the form of mRNA encoding the antigen. Clause 457D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 456D wherein the mRNA is carrier-formulated, such as formulated in LNP, CNE or LION.
Clause 458D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 457D wherein the carrier is LNP.
Clause 459D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 6D to 458D wherein the second antigen is a polypeptide, a polynucleotide or a polysaccharide.
Clause 460D. The method, STING agonist, second adjuvant, first antigen, second antigen or use clause 459D wherein the second antigen is a polypeptide.
Clause 461 D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 460D, wherein the polypeptide comprises at least one B or T cell epitope.
Clause 462D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 461 D, wherein the polypeptide comprises B and T cell epitopes.
Clause 463D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 6D to 462D wherein the second antigen is derived from a virus.
Clause 464D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 6D to 462D wherein the second antigen is derived from a bacterium.
Clause 465D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 464D wherein the second antigen is derived from Clostridia.
Clause 466D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 465D wherein the second antigen is derived from C. difficile.
Clause 467D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 466D wherein the second antigen is C. difficile toxin A and/or toxin B or fragments thereof.
Clause 468D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of 467D wherein the second antigen is the F2 antigen.
Clause 469D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 468D wherein the F2 antigen comprises an amino acid sequence having at least 80%, such as at least 90%, especially at least 95%, in particular at least 98% for example at least 99% or 100% identity to SEQ ID NO: 2.
Clause 470D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 6D to 462D wherein the second antigen is a cancer antigen, such as a tumour antigen and/or a neoantigen. Clause 471 D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 470D wherein the second antigen is a human cancer antigen, such as a human tumour antigen and/or a neoantigen.
Clause 472D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 6D to 459D wherein the second antigen is provided in the form of a polynucleotide encoding the antigen.
Clause 473D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 472D wherein the second antigen is provided in the form of mRNA encoding the antigen.
Clause 474D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 473D wherein the mRNA is carrier-formulated, such as formulated in LNP, CNE or LION.
Clause 475D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 474D wherein the carrier is LNP.
Clause 476D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 6D to 475D, wherein the immune response to the second antigen is a serum titer of IgG antibodies binding to the second antigen.
Clause 477D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of 476D, wherein the IgG antibodies are neutralising antibodies.
Clause 478D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 6D to 477D, wherein the immune response to the second antigen is a T cell response specific to the second antigen.
Clause 479D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 478D, wherein the T cell response is a CD4+ T cell response.
Clause 480D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 478D, wherein the T cell response is a CD8+ T cell response.
Clause 481 D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 478D, wherein the T cell response is a CD4+ and CD8+ T cell response.
Clause 482D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 6D to 481 D, wherein the immune response to the second antigen reduces partially or completely the severity of one or more symptoms experienced by the subject due to infection by a pathogen.
Clause 483D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 6D to 482D, wherein the immune response to the second antigen reduces partially or completely the time over which one or more symptoms are experienced by the subject due to infection by a pathogen. Clause 484D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 6D to 483D, wherein the immune response to the second antigen reduces the likelihood of developing an established infection after challenge with the pathogen.
Clause 485D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 6D to 484D, wherein the immune response to the second antigen slows progression of illness (e.g. extends survival) due to infection by the pathogen.
Clause 486D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 6D to 485D, wherein the breadth of the immune response to the second antigen in the subject is increased.
Clause 487D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 486D, wherein serum neutralising antibodies bind to an increased range of variants of the second antigen.
Clause 488D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of either clause 486D or 487D, wherein the immune response to the second antigen is elicited in a wider variety of human HLA types.
Clause 489D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 6D to 488D, wherein the level of the immune response is increased, such as by at least 25%, such as 50%, such as 100%.
Clause 490D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 489D, wherein the level of the immune response to the second antigen is increased, such as by at least 200%, such as 300%, such as 400%, such as 900%.
Clause 491 D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 6D to 490D, wherein the immune response to the second antigen is maintained for at least 1 year, such as at least 3 years.
Clause 492D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 6D to 491 D, wherein the duration of the immune response to the second antigen is increased, such as by at least 1 year, such as by at least 3 years.
Clause 493D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 6D to 492D, wherein the variability of the immune response to the second antigen between subjects is decreased.
Clause 494D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 493D, wherein the standard deviation of the immune response to the second antigen is reduced. Clause 495D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of either clause 493D or 494D, wherein the proportion of subjects meeting a specified threshold of immune response to the second antigen is increased.
Clause 496D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 6D to 495D, wherein the speed of the immune response to the second antigen is increased.
Clause 497D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of clause 496D, wherein the time taken to achieve a protective immune response is reduced.
Clause 498D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of either clause 496D or 497D, wherein the time taken to reach peak immune response is reduced.
Clause 499D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 6D to 498D, wherein the amount of second antigen required to achieve a desired immune response, such as a protective immune response, is reduced.
Clause 500D. The method, STING agonist, second adjuvant, first antigen, second antigen or use of any one of clauses 6D to 499D, wherein the spacing between administrations of second antigen may be reduced while maintaining immune response.
Clause 501 D. The method, STING agonist, first antigen, or use of any one of clauses 1 D to 500D, wherein the immune response reduces partially or completely the severity of one or more symptoms experienced by the subject due to infection by the pathogen presenting the risk of infection.
Clause 502D. The method, STING agonist, first antigen, or use of any one of clauses 1 D to 501 D, wherein the immune response reduces partially or completely the time over which one or more symptoms are experienced by the subject due to infection by the pathogen presenting the risk of infection.
Clause 503D. The method, STING agonist, first antigen, or use of any one of clauses 1 D to 502D, wherein the immune response reduces the likelihood of developing an established infection after challenge with the pathogen presenting the risk of infection.
Clause 504D. The method, STING agonist, first antigen, or use of any one of clauses 1 D to 503D, wherein the immune response slows progression of illness (e.g. extends survival) due to infection by the pathogen presenting the risk of infection.
Clause 505D. The method, STING agonist, first antigen, or use of any one of clauses 1 D to 504D, wherein the risk of infection is 2 weeks to 2 years, especially 2 weeks to 1 year, in particular 3 weeks to 9 months, such as 4 weeks to 6 months from completion of administration of STING agonist and the first antigen (i.e. step (i)). Clause 506D. The method, STING agonist, first antigen, or use of any one of clauses 1 D to 504D, wherein the risk of infection is 2 weeks to 2 months, 1 month to 3 months, 2 months to 4 months, 3 months to 6 months or 4 months to 12 months from completion of administration of STING agonist and the first antigen.
Clause 507D. The method, STING agonist, first antigen, or use of any one of clauses 1 D to 506D, wherein the subject at risk of infection is be a diabetic who has experienced an abrasion or injury.
Clause 508D. The method, STING agonist, first antigen, or use of any one of clauses 1 D to 507D, wherein the subject at risk of infection is an individual expected to undergo hospital treatment.
Clause 509D. The method, STING agonist, first antigen, or use of any one of clauses 1 D to 508D, wherein the subject is at risk of infection from Staphylococcus aureus.
Clause 510D. The method, STING agonist, first antigen, or use of any one of clauses 1 D to 508D, wherein the pathogen presenting a risk to the subject at risk of infection may be a pathogen causing a pandemic.
Clause 511 D. The method, STING agonist, first antigen, or use of any one of clauses 1 D to 508D or 510D, wherein the pathogen presenting a risk to the subject at risk of infection is an influenza virus.
Clause 512D. The method, STING agonist, first antigen, or use of any one of clauses 1 D to 508D or 510D, wherein the pathogen presenting a risk to the subject at risk of infection is a coronavirus.
Clause 513D. The method, STING agonist, first antigen, or use of clause 512D, wherein the pathogen presenting the risk of infection is SARS-Cov-2.
Clause 514D. A kit of parts comprising:
(iv) a STING agonist;
(v) a first antigen; and
(vi) a second antigen as described in any one of clauses 1 D to 513D.
Clause 515D. The kit of parts according to clause 506D, further comprising (iv) a second adjuvant as described in any one of clauses 1 D to 514D. EXAMPLES
Example 1 - Mouse administration of STING agonist in advance of Clostridium difficile
F2 antigen with adjuvant comprising a saponin (Study 1)
Methods
Clostridium difficile is an anaerobic, Gram+ and spore-forming bacteria which is the main cause of nosocomial antibiotic-associated diarrhea and colitis. Two exotoxins, TcdA (called Toxin A) and especially TcdB (called Toxin B), are the main causal agents of the C. difficile disease (CDi). The fusion protein F2 is composed of the C terminus (Ct) sequences of the Combined Repetitive OligoPeptide (CROP) domain of Toxin A and Toxin B which contains multiple carbohydrate-binding sites known to be receptor binding sites in similar toxins (Orrell et al., 2017).
The polypeptide sequence of VZV gE antigen is provided in SEQ ID NO: 1.
AS01 B was prepared by analogous methods to those set out in WO2013041572 - containing approximately 50 ug QS-21 and 50 ug 3D-MPL with cholesterol containing DOPC liposomes per 500 ul dose.
The STING agonist used was:
(a) 3-(((E)-6-carbamoyl-3-((E)-4-((E)-5-carbamoyl-2-((1 -ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-7-methoxy-2,3-dihydro-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3- methyl-1 H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1 H-benzo[d]imidazol-4-yl)oxy)propyl
Figure imgf000404_0001
referred to below as “STINGa", delivered with or without
AI(OH)3. AI(OH)3 is referred to as ‘Alum’ in the figures. The STING agonist was prepared analogously to the manner set out in WO2017175147. Naive C57BL/6 mice (6-8 weeks at study start) received five immunizations through intramuscular injection (50ul) in the same gastrocnemius muscle (left). Mice were randomly allocated to each group (n=8) and identified individually.
Table 1 Summary of the study design and formulation tested (Study 1)
Figure imgf000405_0001
Sample collection D14,D28,D42,D56,D70(sera) D85 (spleen/sera)
IgG antibodies directed against Toxin A and Toxin B (ELISA)
The Toxin A C-terminus (Ct) fragment or Toxin B Ct fragment were coated respectively at 1 pg/mL each in PBS on high-binding microtiter plates overnight at 4°C. The plates were blocked with PBS-Bovine Serum Albumin (BSA) 1% for 30 minutes at room temperature (RT) under shaking. Then serial two-fold dilutions of mouse sera in PBS-BSA 0.2%-TWEEN 0.05% were incubated at RT for 60 minutes under shaking. After washing, peroxidase-conjugated antimouse IgG antibodies diluted 1/5000 in PBS-BSA 0.2%-TWEEN 0.05% were added for 60 minutes at RT under shaking. After an additional washing step, the bound mouse antibodies were detected by adding a solution containing 4 mg O-phenylenediamine and 5 uL H2O2 per 10 mL of 0.1M citrate buffer pH 4.5 for 15 minutes in the dark at RT. The colorimetric reaction was stopped by adding HCI 1 N and the optical densities (OD), which are directly proportional to the amounts of antibodies present in the serum, were measured using a spectrophotometer. The level of specific anti-Toxin A Ct or anti-Toxin B Ct IgG antibodies present in mouse sera was calculated by the four-parameter method in comparison to the curve of a calibrated standard serum using the SoftMaxPro software. Results were expressed as ug/mL of serum igG.
Neutralizing antibodies directed against Toxin A and Toxin B (neutralization assays) These assays aimed at showing the functionality of elicited antibodies. Two human colonic epithelial cell lines were used: HT29 for Toxin A and HCT-116 cells for Toxin B neutralization assay. HT29 were cultured at 37°C with 5% CO2 in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum, 1% glutamine and 1% of an antibiotic cocktail containing penicillin, streptomycin and amphotericin. HCT-116 cells were cultured at 37°C with 5% CO2 in Eagle's minimum essential medium supplemented with 10% fetal bovine serum, 1% glutamine and 1% of an antibiotic cocktail containing penicillin, streptomycin and amphotericin. Cells were seeded in 96-well black tissue culture plates (Greiner Bio-one) at a density of 4x103 cells/well for HT29 and 1x103 cells/well for HCT 116. After 24 hours, the medium was removed from the wells. Then 50 pL of three-fold serial dilutions of mouse sera followed by 50 pL of native Toxin A (67 ng/mL) or native Toxin B (200 ng/mL) depending on the assay were added per well and plates were incubated at 37°C with 5% CO2 for 6 days. After removal of antisera I toxin mixture, 100 pL of DNA-staining Hoechst fluorescent reagent (BD Pharmingen, Ref:561908) diluted 1/500 in PBS were added for 2 hours in the dark at room temperature.
After removal of Hoechst dye solution, the fluorescence-covered surface of each well (corresponding to the well surface still covered by living cells, which is minimal in case of full cytotoxicity and maximal in absence of any cytotoxicity) was determined using the AxioVision microscope software. Neutralization titres were defined as the reciprocal dilution of serum inducing a 50% inhibition of the Toxin A or B cytotoxic effect.
In order to avoid any plate bias for group comparisons, the individual mouse samples were randomized across the plates. As the number of samples that can be tested by plate was limited, samples for groups to be compared were allocated to the same plates (e.g. samples groups 1 , 2 and 3 were allocated to plates 1 , 2, 3 and 4 for testing). As in some cases the PBS control group was used as reference for several research questions, the group was repeated on different plates.
Evaluation of F2 specific T cell responses
Spleen collection & sample processing for cell suspension
Spleens were collected in Roswell Park Memorial Institute Medium (RPMI) medium and dissociated using a potter tissue grinder (homogenizer) using two up and down strokes. Homogenized samples were transferred to 50 mL polypropylene tubes. Fibrous material was removed by filtration through a 100 uM nylon cell strainer. Cells were then washed, counted and re-suspended at 107 cells per mL.
Intracellular Cytokine Staining (ICS)
Intracellular cytokine detection by flow cytometry allowing the evaluation of cytokine production at the single-cell level. It detects the production and accumulation of cytokines within the endoplasmic reticulum after cell stimulation, allowing direct TH1 versus TH2 determination.
ICS is the technology which allows the quantification of antigen-specific T lymphocytes via the cytokine production. Lymphoid cells were re-stimulated overnight with F2-protein or medium in the presence of a protein transport inhibitor added (brefeldin A). These cells were then processed by conventional immunofluorescent procedure using fluorescent antibodies (extracellular staining: CD4, CD8; intracellular staining: Tumour Necrosis Factor-a (TNF-a), Interferon-g (IFN-g) - and IL2). Results are expressed as a frequency of cytokine positive cells within CD4 cell populations after subtraction of the medium condition for each mouse. The statistical analysis was done on the population that showed expression of at least one cytokine (IFN-g and/or IL2 and/or TNF-a).
Results
The results are shown in Fig. 1 to Fig. 9.
Example 2 - Mouse administration of STING agonist in advance of Clostridium difficile F2 antigen with (a) adjuvant comprising a saponin, (b) squalene emulsion adjuvant or (c) STING agonist (Study 2)
Following the general procedures laid out in Example 1 , a further study was completed. ELISA analysis of samples utilised a new lot for toxin B Ct fragment, with lower toxicity than that used in Example 1.
AS03 emulsion was prepared by analogous methods to those set out in Example 1 of W02022002783. Briefly, oil phase composed of squalene and D/L-alpha tocopherol was formulated under a nitrogen atmosphere. Aqueous phase, composed of modified phosphate buffered saline and polysorbate 80, was prepared separately. Oil and aqueous phases were combined at a ratio of 1:9 (volume of oil phase to volume of aqueous phase) before homogenisation and microfluidisation (three passes through a microfluidiser at around 15000 psi). The resulting emulsion was sterile filtered through two trains of two 0.5/0.2 urn filters in series (i.e. 0.5/0.2/0.5/0.2). The final emulsion contained approximately 42.76 mg/ml squalene, 47.44 mg/ml tocopherol and 19.44 mg/ml polysorbate 80, i.e. double strength AS03A based on a 500 ul dose volume. Particle size and polydispersity was determined by DLS to be within the range 140 to 180 nm and less than 0.2 respectively.
Table 2 Summary of the study design and formulation tested (Study 2)
Figure imgf000408_0001
Figure imgf000409_0001
Results
The results are shown in Fig. 10 to Fig. 39.
REFERENCES
Arias MA et al. (2012) Glucopyranosyl Lipid Adjuvant (GLA), a Synthetic TLR4 Agonist, Promotes Potent Systemic and Mucosal Responses to Intranasal Immunization with HIVgp140. PLoS ONE 7(7): e41144. doi:10.1371/journal.pone.0041144.
Black, S., E. De Gregorio, and R. Rappuoli. 2015. Developing vaccines for an aging population. Science translational medicine. 7:281 ps288
Bosch et al., The Coronavirus Spike Protein Is a Class I Virus Fusion Protein: Structural and Functional Characterization of the Fusion Core Complex J. Virol. 77:8801-8811 (2003) Brito, L.A et al. A cationic nanoemulsion for the delivery of next-generation RNA vaccines, Molecular Therapy, 2014, 22,12, 2118-2129
Carmona Martinez A, et al. A phase 1, open-label safety and immunogenicity study of an AS03-adjuvanted trivalent inactivated influenza vaccine in children aged 6 to 35 months. Hum Vaccin Immunother. 2014 10(7): 1959- 1968
Carter C, et al. A structure-function approach to optimizing TLR4 ligands for human vaccines, Clinical & Translation Immunology, 2016, 5, e108.
Cohet C, et al. Safety of AS03-adjuvanted Influenza Vaccines: A Review of the Evidence Vaccine 2019 37(23) :3006-3021
Dayhoff M, O et al. A model of evolutionary change in proteins, Atlas of Protein Sequence and Structure, 1978 vol 5, supp. 3
Dayhoff M, O et al. A model of evolutionary change in proteins, Atlas of Protein Sequence and Structure, 1978 vol 5, supp. 3.
Dubensky et al. Rationale, progress and development of vaccines utilizing STING-activating cyclic dinucleotide adjuvants, Ther Adv Vaccines 2013 1(4): 131 -43
Dubensky, Thomas W Jr et al. “Rationale, progress and development of vaccines utilizing STING-activating cyclic dinucleotide adjuvants.” Therapeutic advances in vaccines vol. 1 ,4 (2013): 131-43. doi:10.1177/2051013613501988
Fox C. Squalene Emulsions for Parenteral Vaccine and Drug Delivery Molecules 2009 14:3286-3312
Gargon N, et al. Development and Evaluation of AS03, an Adjuvant System Containing a- Tocopherol and Squalene in an Oil-In-Water Emulsion Expert Rev Vaccines 2012 11:349-366 Geall et al. (2012) PNAS USA. Sep 4; 109(36): 14604-9 (LNP delivery system)
Hamid, M. M. A et al. Vaccination with Plasmodium knowlesi AMA1 formulated in the novel adjuvant co-vaccine HT™ protects against blood-stage challenge in rhesus macaques. PLoS One. 2011;6(5):e20547. Hilgers, L. A. et al. Sucrose fatty acid sulphate esters as novel vaccine adjuvant, Vaccine, 2006, 24 Suppl 2:S2-81-2. Handbook of Pharmaceutical Excipients (eds. Rowe, Sheskey, & Quinn) 6th edition, 2009 ISBN 978-0-85369-792-3 https://www.who.int/emergencies/diseases/novel-coronavirus-2019; accessed 16 Jun 2020 Humenza™ Summary of Product Characteristics
Jackson L, et al. Effect of Varying Doses of a Monovalent H7N9 Influenza Vaccine With and Without AS03 and MF59 Adjuvants on Immune Response: A Randomized Clinical Trial JAMA 2015 314(3):237-246
Jiang S, et al., Lipidoid-Coated Iron Oxide Nanoparticles for Efficient DNA and siRNA delivery Nano Lett., 13:1059-1064 (2013).
Kirchdoerfer et al., Stabilized coronavirus spikes are resistant to conformational changes induced by receptor recognition or proteolysis Sci. Rep. 8:15701 (2018)
Kite G.C. et al. Metabolomic analysis of saponins in crude extracts of Quillaja saponaria by liquid chromatography/mass spectrometry for product authentication Rapid Communications in Mass Spectrometry 2004 18: 2859-2870
Klucker M-F, et al. AF03, an Alternative Squalene Emulsion-Based Vaccine Adjuvant Prepared by a Phase Inversion Temperature Method J Pharm Sci. 2012 101(12):4490-4500
Li, Structure, Function, and Evolution of Coronavirus Spike Proteins Annu. Rev. Virol. 3:237- 261 (2016)
Light Scattering from Polymer Solutions and Nanoparticle Dispersions Schartl W. 2007 ISBN 978-3-540-71950-2
Liu et al., Improvement of Pharmacokinetic Profile of TRAIL via Trimer-Tag Enhances Its Antitumor Activity in Vivo Sci Rep. 7:8953 (2017)
Lodaya R, et al. Formulation Design, Optimization and In Vivo Evaluations of an a-Tocopherol- Containing Self-Emulsified Adjuvant System using Inactivated Influenza Vaccine J Control Release 2019 316:12-21
Moderna Announces Positive Interim Phase 1 Data for its mRNA Vaccine (mRNA-1273) Against Novel Coronavirus. Press Release May 18, 2020.
Morel S, et al. Adjuvant System AS03 Containing a-Tocopherol Modulates Innate Immune Response and Leads to Improved Adaptive Immunity Vaccine 2011 29:2461-2473 New Generation Vaccines (eds. Levine et al.). 3rd edition, 2004. ISBN 0-8247-4071-8 Norbert Pardi, Michael J. Hogan, Frederick W. Porter and Drew Weissman Nature Reviews Drug Discovery 2018 17:261-279
Nyberg N. et al. Separation and structural analysis of some saponins from Quillaja saponaria Molina Carbohydrate Research 2000 323:87-97
Nyberg N. et al. Solid-Phase Extraction NMR Studies of Chromatographic Fractions of Saponins from Quillaja Saponaria Anal. Chem. 2003 752:268-274 O’Hagan D, et al. The History of MF59(®) Adjuvant: A Phoenix That Arose From the Ashes Expert Rev Vaccines 2013 12(1):13-30
O’Hagan D. MF59 Is a Safe and Potent Vaccine Adjuvant That Enhances Protection Against Influenza Virus Infection Expert Rev Vaccines 20076(5):699-710
Pallesen et al., Immunogenicity and structures of a rationally designed prefusion MERS-CoV spike antigen Proc. Natl. Acad. Sci. U.S.A. 114:E7348-E7357 (2017)
Podda A and Giudice D. MF59-adjuvanted vaccines: increased immunogenicity with an optimal safety profile Expert Rev Vaccines 20032:197-203
Podda A. The Adjuvanted Influenza Vaccines With Novel Adjuvants: Experience With the MF59-adjuvanted Vaccine Vaccine 2001 19:2673-2680
Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, 1985, p.1418
Shah R, et al. The development of self-emulsifying oil-in-water emulsion adjuvant and an evaluation of the impact of droplet size on performance. J Pharm Sci. 2015 104:1352-1361 Shah R, et al. The droplet size of emulsion adjuvants has significant impact on their potency, due to differences in immune cell-recruitment and -activation Scientific Reports 2019 9:11520 Shah R, et al. The impact of size on particulate vaccine adjuvants. Nanomedicine (Lond) 2014 9:2671-2681
Sun, J et al. Analytical characterization of an oil-in-water adjuvant emulsion, AAPS PharmSciTech, 2017 18, 5, 1595-1604.
Vaccine Adjuvants: Preparation Methods and Research Protocols (chapter 12; Ott G, et al.) 2000 ISBN 0-89603-735-5.
Vaccine Design: The Subunit and Adjuvant Approach (chapter 10; Ott G, et al.) 1995 ISBN 978-1-4613-5737-7.
Wrapp et al., Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation Science 367:1260-1263 (2020)
Younis, S et al. Identification of adjuvants for clinical trials performed with Plasmodium falciparum AMA1 in rabbits. BMC Immunol, 20, 25 (2019).
Younis, S et al. Down selecting adjuvanted vaccine formulations: a comparative method for harmonized evaluation, BMC Immunol, 19, 6 (2018).
Zhang, C et al. Advances in mRNA vaccines for infectious diseases, Front. Immunol. 2019, 10:594.
EP0868918B1
GB2220211
PCT/US2020/15565
US2007/0014805 US2021/0101924
US4373071
US4458066
US4500707
US4668777
US4973679
US5047524
US5132418
US5153319
US5262530
US5700642
WO 2017/175147
W02005/113782
W02006/100109
W02008/043774
WO20 11/076807
WO20 11/154443
WO20 11/154444
WO20 12/006376
WO20 12/006378
WO20 12/006380
W02012/030901
WO20 12/031043
WO20 12/031046
WO20 13/006825
WO2013/006834
WO20 13/006837
WO20 13/033563
WO2013/041572
WO20 14/136086
WO20 15/095340
WO20 15/095346
W02015/140138
WO20 16/037053
WO2016/135154
WO20 16/172396
WO20 17/070620 WO2017/175147
WO20 18/114892
W02018/176103 W02022/002783
WO90/14837

Claims

Claims
1. A method of:
(a) prophylaxis of infection by a pathogen in a subject,
(b) reducing the variability of the immune response to an antigen in subjects,
(c) improving an immune response to an antigen in a subject, and/or
(d) reducing the amount of an antigen required to elicit a desired immune response in a subject, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist, prior to
(ii) administering to the subject an antigen which is associated with the pathogen.
2. The method of claim 1 , for the prophylaxis of infection by a pathogen in a subject.
3. The method of claim 1 , for reducing the variability of the immune response to an antigen in subjects.
4. The method of claim 1, for improving an immune response to an antigen in a subject.
5. The method of claim 1 , for reducing the amount of an antigen required to elicit a desired immune response in a subject.
6. A method of preparing the immune system of a subject for (ii) administration of an antigen, wherein the method comprises (i) administering to the subject a STING agonist.
7. The method of any one of claims 1 to 6 wherein the antigen is derived from a virus.
8. The method of any one of claims 1 to 6 wherein the antigen is derived from a bacterium.
9. The method of any one of claims 1 to 8 wherein step (i) comprises administering the
STING agonist and a first antigen.
10. The method of claim 9, wherein the first antigen is derived from a different pathogen to the second antigen.
11. The method of claim 10, wherein the first antigen is derived from a virus and the second antigen is derived from a bacterium.
12. The method of claim 10, wherein the first antigen is derived from a bacterium and the second antigen is derived from a virus.
13. The method of any one of claims 1 to 12 wherein step (ii) comprises administering a STING agonist and the second antigen.
14. The method of any one of claims 1 to 12 wherein step (ii) comprises administering a second adjuvant and the second antigen.
15. The method of claim 14 wherein the second adjuvant comprises a squalene emulsion adjuvant.
16. The method of any one of claims 1 to 12 wherein step (ii) comprises administering the second antigen without a second adjuvant.
17. A method for prophylaxis in a subject at risk of infection comprising administering a STING agonist to the subject.
18. The method of any one of claims 1 to 17, wherein the STING agonist binds to STING with an in vitro Kf of less than 0.750 pM.
19. The method of any one of claims 1 to 18, wherein the STING agonist activates STING with an in vitro EC50 of 100 pM or less when measured by monitoring phosphorylation of interferon regulatory factor-3 (IRF3).
20. The method of any one of claims 1 to 19, wherein the STING agonist activates STING with an in vitro EC50 of 100 pM or less as measured by monitoring interferon-p induction.
21. The method of any one of claims 1 to 20, wherein the STING agonist is 3-(((E)-6- carbamoyl-3-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-7- methoxy-2,3-dihydro-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1 H-pyrazole- 5-carbonyl)imino)-2,3-dihydro-1 H-benzo[d]imidazol-4-yl)oxy)propyl
Figure imgf000417_0001
or a pharmaceutically acceptable salt thereof.
22. The method of claim 1, wherein the method comprises the following steps: (i) administering to the subject a STING agonist, prior to
(ii) administering to the subject an antigen which is associated with the pathogen and a squalene emulsion adjuvant, wherein the antigen is derived from a bacterium and the STING agonist is 3-(((E)-6-carbamoyl-3-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-7-methoxy-2,3-dihydro-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3- methyl-1 H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1 H-benzo[d]imidazol-4-yl)oxy)propyl
Figure imgf000417_0002
pharmaceutically acceptable salt thereof.
23. The method of claim 1, wherein the method comprises the following steps:
(i) administering to the subject a STING agonist and a first antigen derived from a virus, prior to
(ii) administering to the subject a second antigen which is associated with the pathogen and a second adjuvant comprising a squalene emulsion adjuvant, and wherein the second antigen is derived from a bacterium and the STING agonist is 3-(((E)-6- carbamoyl-3-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-7- methoxy-2,3-dihydro-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1 H-pyrazole-
5-carbonyl)imino)-2,3-dihydro-1 H-benzo[d]imidazol-4-yl)oxy)propyl
Figure imgf000418_0001
pharmaceutically acceptable salt thereof.
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Citations (56)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4373071A (en) 1981-04-30 1983-02-08 City Of Hope Research Institute Solid-phase synthesis of polynucleotides
US4436727A (en) 1982-05-26 1984-03-13 Ribi Immunochem Research, Inc. Refined detoxified endotoxin product
US4458066A (en) 1980-02-29 1984-07-03 University Patents, Inc. Process for preparing polynucleotides
US4500707A (en) 1980-02-29 1985-02-19 University Patents, Inc. Nucleosides useful in the preparation of polynucleotides
US4668777A (en) 1981-03-27 1987-05-26 University Patents, Inc. Phosphoramidite nucleoside compounds
US4866034A (en) 1982-05-26 1989-09-12 Ribi Immunochem Research Inc. Refined detoxified endotoxin
US4877611A (en) 1986-04-15 1989-10-31 Ribi Immunochem Research Inc. Vaccine containing tumor antigens and adjuvants
GB2220211A (en) 1988-06-29 1990-01-04 Ribi Immunochem Research Inc Modified lipopolysaccharides
EP0362278A1 (en) 1987-06-05 1990-04-11 THE UNITED STATES OF AMERICA as represented by the Secretary United States Department of Commerce Autocrine motility factors in cancer diagnosis and management
US4973679A (en) 1981-03-27 1990-11-27 University Patents, Inc. Process for oligonucleo tide synthesis using phosphormidite intermediates
WO1990014837A1 (en) 1989-05-25 1990-12-13 Chiron Corporation Adjuvant formulation comprising a submicron oil droplet emulsion
US5047524A (en) 1988-12-21 1991-09-10 Applied Biosystems, Inc. Automated system for polynucleotide synthesis and purification
US5132418A (en) 1980-02-29 1992-07-21 University Patents, Inc. Process for preparing polynucleotides
US5153319A (en) 1986-03-31 1992-10-06 University Patents, Inc. Process for preparing polynucleotides
US5262530A (en) 1988-12-21 1993-11-16 Applied Biosystems, Inc. Automated system for polynucleotide synthesis and purification
WO1994021292A1 (en) 1993-03-23 1994-09-29 Smithkline Beecham Biologicals (S.A.) Vaccine compositions containing 3-o deacylated monophosphoryl lipid a
US5700642A (en) 1995-05-22 1997-12-23 Sri International Oligonucleotide sizing using immobilized cleavable primers
WO1998050399A1 (en) 1997-05-08 1998-11-12 Corixa Corporation Aminoalkyl glucosamine phosphate compounds and their use as adjuvants and immunoeffectors
US6303347B1 (en) 1997-05-08 2001-10-16 Corixa Corporation Aminoalkyl glucosaminide phosphate compounds and their use as adjuvants and immunoeffectors
EP0868918B1 (en) 1993-12-23 2004-04-28 GlaxoSmithKline Biologicals S.A. Vaccines comprising oil/water emulsion with tocopherol and squalene
WO2005113782A1 (en) 2004-05-18 2005-12-01 Alphavax, Inc. Tc-83-derived alphavirus vectors, particles and methods
WO2006100109A1 (en) 2005-03-23 2006-09-28 Glaxosmithkline Biologicals S.A. Use of an influenza virus an oil-in-water emulsion adjuvant to induce cd4 t-cell and/or improved b-memory cell response
US20070014805A1 (en) 2005-07-07 2007-01-18 Sanofi Pasteur Immuno-adjuvant emulsion
WO2008043774A1 (en) 2006-10-12 2008-04-17 Glaxosmithkline Biologicals S.A. Vaccine comprising an oil in water emulsion adjuvant
WO2008153541A1 (en) 2006-09-26 2008-12-18 Infectious Disease Research Institute Vaccine composition containing synthetic adjuvant
WO2009143457A2 (en) 2008-05-22 2009-11-26 Infectious Disease Research Institute Vaccine composition containing synthetic adjuvant
WO2011076807A2 (en) 2009-12-23 2011-06-30 Novartis Ag Lipids, lipid compositions, and methods of using them
WO2011154443A1 (en) 2010-06-10 2011-12-15 Glaxosmithkline Biologicals S.A. Novel process
WO2011154444A1 (en) 2010-06-10 2011-12-15 Glaxosmithkline Biologicals S.A. Novel process
WO2012006380A2 (en) 2010-07-06 2012-01-12 Novartis Ag Cationic oil-in-water emulsions
WO2012006378A1 (en) 2010-07-06 2012-01-12 Novartis Ag Liposomes with lipids having an advantageous pka- value for rna delivery
WO2012006376A2 (en) 2010-07-06 2012-01-12 Novartis Ag Virion-like delivery particles for self-replicating rna molecules
WO2012031043A1 (en) 2010-08-31 2012-03-08 Novartis Ag Pegylated liposomes for delivery of immunogen-encoding rna
WO2012030901A1 (en) 2010-08-31 2012-03-08 Novartis Ag Small liposomes for delivery of immunogen-encoding rna
WO2012031046A2 (en) 2010-08-31 2012-03-08 Novartis Ag Lipids suitable for liposomal delivery of protein-coding rna
WO2012163811A1 (en) 2011-05-27 2012-12-06 Glaxosmithkline Biologicals S.A. Immunogenic composition
WO2013006837A1 (en) 2011-07-06 2013-01-10 Novartis Ag Cationic oil-in-water emulsions
WO2013006825A1 (en) 2011-07-06 2013-01-10 Novartis Ag Liposomes having useful n:p ratio for delivery of rna molecules
WO2013006834A1 (en) 2011-07-06 2013-01-10 Novartis Ag Oil-in-water emulsions that contain nucleic acids
WO2013033563A1 (en) 2011-08-31 2013-03-07 Novartis Ag Pegylated liposomes for delivery of immunogen-encoding rna
WO2014136086A1 (en) 2013-03-08 2014-09-12 Novartis Ag Lipids and lipid compositions for the delivery of active agents
WO2015095340A1 (en) 2013-12-19 2015-06-25 Novartis Ag Lipids and lipid compositions for the delivery of active agents
WO2015095346A1 (en) 2013-12-19 2015-06-25 Novartis Ag Lipids and lipid compositions for the delivery of active agents
WO2015140138A1 (en) 2014-03-17 2015-09-24 Glaxosmithkline Biologicals Sa Oil/surfactant mixtures for self-emulsification
WO2016037053A1 (en) 2014-09-05 2016-03-10 Novartis Ag Lipids and lipid compositions for the delivery of active agents
WO2016135154A1 (en) 2015-02-23 2016-09-01 Novartis Ag Oil/surfactant mixtures for self-emulsification
WO2016172396A2 (en) 2015-04-22 2016-10-27 Saab Ihab Multi-chamber sequential delivery syringe
WO2017070620A2 (en) 2015-10-22 2017-04-27 Modernatx, Inc. Broad spectrum influenza virus vaccine
WO2017075477A1 (en) * 2015-10-28 2017-05-04 Aduro Biotech, Inc. Compositions and methods for activating "stimulator of interferon gene"-dependent signalling
WO2017162265A1 (en) 2016-03-21 2017-09-28 Biontech Rna Pharmaceuticals Gmbh Trans-replicating rna
WO2017175147A1 (en) 2016-04-07 2017-10-12 Glaxosmithkline Intellectual Property Development Limited Heterocyclic amides useful as protein modulators
WO2018114892A1 (en) 2016-12-20 2018-06-28 Glaxosmithkline Biologicals S.A. Novel methods for inducing an immune response
WO2018156625A1 (en) * 2017-02-21 2018-08-30 Board Of Regents,The University Of Texas System Cyclic dinucleotides as agonists of stimulator of interferon gene dependent signalling
WO2019106192A1 (en) 2017-12-01 2019-06-06 Glaxosmithkline Biologicals Sa Saponin purification
WO2020160080A1 (en) 2019-01-30 2020-08-06 Glaxosmithkline Llc Oil/surfactant mixtures for self-emulsification
US20210101924A1 (en) 2019-08-27 2021-04-08 The Regents Of The University Of California Cyclic dinucleotide compounds as sting agonists

Patent Citations (58)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4458066A (en) 1980-02-29 1984-07-03 University Patents, Inc. Process for preparing polynucleotides
US4500707A (en) 1980-02-29 1985-02-19 University Patents, Inc. Nucleosides useful in the preparation of polynucleotides
US5132418A (en) 1980-02-29 1992-07-21 University Patents, Inc. Process for preparing polynucleotides
US4668777A (en) 1981-03-27 1987-05-26 University Patents, Inc. Phosphoramidite nucleoside compounds
US4973679A (en) 1981-03-27 1990-11-27 University Patents, Inc. Process for oligonucleo tide synthesis using phosphormidite intermediates
US4373071A (en) 1981-04-30 1983-02-08 City Of Hope Research Institute Solid-phase synthesis of polynucleotides
US4866034A (en) 1982-05-26 1989-09-12 Ribi Immunochem Research Inc. Refined detoxified endotoxin
US4436727A (en) 1982-05-26 1984-03-13 Ribi Immunochem Research, Inc. Refined detoxified endotoxin product
US5153319A (en) 1986-03-31 1992-10-06 University Patents, Inc. Process for preparing polynucleotides
US4877611A (en) 1986-04-15 1989-10-31 Ribi Immunochem Research Inc. Vaccine containing tumor antigens and adjuvants
EP0362278A1 (en) 1987-06-05 1990-04-11 THE UNITED STATES OF AMERICA as represented by the Secretary United States Department of Commerce Autocrine motility factors in cancer diagnosis and management
GB2220211A (en) 1988-06-29 1990-01-04 Ribi Immunochem Research Inc Modified lipopolysaccharides
US4912094A (en) 1988-06-29 1990-03-27 Ribi Immunochem Research, Inc. Modified lipopolysaccharides and process of preparation
US4912094B1 (en) 1988-06-29 1994-02-15 Ribi Immunochem Research Inc. Modified lipopolysaccharides and process of preparation
US5262530A (en) 1988-12-21 1993-11-16 Applied Biosystems, Inc. Automated system for polynucleotide synthesis and purification
US5047524A (en) 1988-12-21 1991-09-10 Applied Biosystems, Inc. Automated system for polynucleotide synthesis and purification
WO1990014837A1 (en) 1989-05-25 1990-12-13 Chiron Corporation Adjuvant formulation comprising a submicron oil droplet emulsion
WO1994021292A1 (en) 1993-03-23 1994-09-29 Smithkline Beecham Biologicals (S.A.) Vaccine compositions containing 3-o deacylated monophosphoryl lipid a
EP0868918B1 (en) 1993-12-23 2004-04-28 GlaxoSmithKline Biologicals S.A. Vaccines comprising oil/water emulsion with tocopherol and squalene
US5700642A (en) 1995-05-22 1997-12-23 Sri International Oligonucleotide sizing using immobilized cleavable primers
US6303347B1 (en) 1997-05-08 2001-10-16 Corixa Corporation Aminoalkyl glucosaminide phosphate compounds and their use as adjuvants and immunoeffectors
WO1998050399A1 (en) 1997-05-08 1998-11-12 Corixa Corporation Aminoalkyl glucosamine phosphate compounds and their use as adjuvants and immunoeffectors
WO2005113782A1 (en) 2004-05-18 2005-12-01 Alphavax, Inc. Tc-83-derived alphavirus vectors, particles and methods
WO2006100109A1 (en) 2005-03-23 2006-09-28 Glaxosmithkline Biologicals S.A. Use of an influenza virus an oil-in-water emulsion adjuvant to induce cd4 t-cell and/or improved b-memory cell response
US20070014805A1 (en) 2005-07-07 2007-01-18 Sanofi Pasteur Immuno-adjuvant emulsion
WO2008153541A1 (en) 2006-09-26 2008-12-18 Infectious Disease Research Institute Vaccine composition containing synthetic adjuvant
WO2008043774A1 (en) 2006-10-12 2008-04-17 Glaxosmithkline Biologicals S.A. Vaccine comprising an oil in water emulsion adjuvant
WO2009143457A2 (en) 2008-05-22 2009-11-26 Infectious Disease Research Institute Vaccine composition containing synthetic adjuvant
WO2011076807A2 (en) 2009-12-23 2011-06-30 Novartis Ag Lipids, lipid compositions, and methods of using them
WO2011154443A1 (en) 2010-06-10 2011-12-15 Glaxosmithkline Biologicals S.A. Novel process
WO2011154444A1 (en) 2010-06-10 2011-12-15 Glaxosmithkline Biologicals S.A. Novel process
WO2012006380A2 (en) 2010-07-06 2012-01-12 Novartis Ag Cationic oil-in-water emulsions
WO2012006378A1 (en) 2010-07-06 2012-01-12 Novartis Ag Liposomes with lipids having an advantageous pka- value for rna delivery
WO2012006376A2 (en) 2010-07-06 2012-01-12 Novartis Ag Virion-like delivery particles for self-replicating rna molecules
WO2012031043A1 (en) 2010-08-31 2012-03-08 Novartis Ag Pegylated liposomes for delivery of immunogen-encoding rna
WO2012030901A1 (en) 2010-08-31 2012-03-08 Novartis Ag Small liposomes for delivery of immunogen-encoding rna
WO2012031046A2 (en) 2010-08-31 2012-03-08 Novartis Ag Lipids suitable for liposomal delivery of protein-coding rna
WO2012163811A1 (en) 2011-05-27 2012-12-06 Glaxosmithkline Biologicals S.A. Immunogenic composition
WO2013006837A1 (en) 2011-07-06 2013-01-10 Novartis Ag Cationic oil-in-water emulsions
WO2013006825A1 (en) 2011-07-06 2013-01-10 Novartis Ag Liposomes having useful n:p ratio for delivery of rna molecules
WO2013006834A1 (en) 2011-07-06 2013-01-10 Novartis Ag Oil-in-water emulsions that contain nucleic acids
WO2013033563A1 (en) 2011-08-31 2013-03-07 Novartis Ag Pegylated liposomes for delivery of immunogen-encoding rna
WO2014136086A1 (en) 2013-03-08 2014-09-12 Novartis Ag Lipids and lipid compositions for the delivery of active agents
WO2015095340A1 (en) 2013-12-19 2015-06-25 Novartis Ag Lipids and lipid compositions for the delivery of active agents
WO2015095346A1 (en) 2013-12-19 2015-06-25 Novartis Ag Lipids and lipid compositions for the delivery of active agents
WO2015140138A1 (en) 2014-03-17 2015-09-24 Glaxosmithkline Biologicals Sa Oil/surfactant mixtures for self-emulsification
WO2016037053A1 (en) 2014-09-05 2016-03-10 Novartis Ag Lipids and lipid compositions for the delivery of active agents
WO2016135154A1 (en) 2015-02-23 2016-09-01 Novartis Ag Oil/surfactant mixtures for self-emulsification
WO2016172396A2 (en) 2015-04-22 2016-10-27 Saab Ihab Multi-chamber sequential delivery syringe
WO2017070620A2 (en) 2015-10-22 2017-04-27 Modernatx, Inc. Broad spectrum influenza virus vaccine
WO2017075477A1 (en) * 2015-10-28 2017-05-04 Aduro Biotech, Inc. Compositions and methods for activating "stimulator of interferon gene"-dependent signalling
WO2017162265A1 (en) 2016-03-21 2017-09-28 Biontech Rna Pharmaceuticals Gmbh Trans-replicating rna
WO2017175147A1 (en) 2016-04-07 2017-10-12 Glaxosmithkline Intellectual Property Development Limited Heterocyclic amides useful as protein modulators
WO2018114892A1 (en) 2016-12-20 2018-06-28 Glaxosmithkline Biologicals S.A. Novel methods for inducing an immune response
WO2018156625A1 (en) * 2017-02-21 2018-08-30 Board Of Regents,The University Of Texas System Cyclic dinucleotides as agonists of stimulator of interferon gene dependent signalling
WO2019106192A1 (en) 2017-12-01 2019-06-06 Glaxosmithkline Biologicals Sa Saponin purification
WO2020160080A1 (en) 2019-01-30 2020-08-06 Glaxosmithkline Llc Oil/surfactant mixtures for self-emulsification
US20210101924A1 (en) 2019-08-27 2021-04-08 The Regents Of The University Of California Cyclic dinucleotide compounds as sting agonists

Non-Patent Citations (17)

* Cited by examiner, † Cited by third party
Title
"Handbook of Pharmaceutical Excipients", 2009
"Pharmaceutical Sciences", 1985, MACK PUBLISHING COMPANY, pages: 1418
ARIAS MA ET AL.: "Glucopyranosyl Lipid Adjuvant (GLA), a Synthetic TLR4 Agonist, Promotes Potent Systemic and Mucosal Responses to Intranasal Immunization with HIVgp140", PLOS ONE, vol. 7, no. 7, 2012, pages e41144, XP055047355, DOI: 10.1371/journal.pone.0041144
ARTS ROB J.W. ET AL: "BCG Vaccination Protects against Experimental Viral Infection in Humans through the Induction of Cytokines Associated with Trained Immunity", CELL HOST & MICROBE, vol. 23, no. 1, 1 January 2018 (2018-01-01), NL, pages 89 - 100.e5, XP055983235, ISSN: 1931-3128, DOI: 10.1016/j.chom.2017.12.010 *
CAS, no. 2138299-34-8
CAS, no. 2242635-02-3
COLER RN ET AL.: "Development and Characterization of Synthetic Glucopyranosyl Lipid Adjuvant System as a Vaccine Adjuvant", PLOS ONE, vol. 6, no. 1, 2011, pages e16333, XP055030257, DOI: 10.1371/journal.pone.0016333
DALSGAARD ET AL.: "Archiv. fur die gesamte Virusforschung", vol. 44, 1974, SPRINGER VERLAG, article "Saponin adjuvants", pages: 243 - 254
DAYHOFF ET AL., ATLAS OF PROTEIN SEQUENCE AND STRUCTURE, vol. 5, 1978
DUBENSKY: "Rationale, progress and development of vaccines utilizing STING-activating cyclic dinucleotide adjuvants", THERAPEUTIC ADVANCES IN VACCINES, vol. 1, no. 4, 5 September 2013 (2013-09-05), GB, pages 131 - 143, XP055177403, ISSN: 2051-0136, DOI: 10.1177/2051013613501988 *
ERIK VAN DIS ET AL: "STING-Activating Adjuvants Elicit a Th17 Immune Response and Protect against Mycobacterium tuberculosis Infection", CELL REPORTS, vol. 23, no. 5, 1 May 2018 (2018-05-01), US, pages 1435 - 1447, XP055627339, ISSN: 2211-1247, DOI: 10.1016/j.celrep.2018.04.003 *
GEALL ET AL., PNAS USA, vol. 109, no. 36, 2012, pages 14604 - 9
GEALL ET AL., PNAS USA., vol. 109, no. 36, 2012, pages 14604 - 9
KRAMSKAYA T. ET AL: "Combined immunization with attenuated live influenza vaccine and chimeric pneumococcal recombinant protein improves the outcome of virus-bacterial infection in mice", PLOS ONE, vol. 14, no. 9, 12 September 2019 (2019-09-12), pages e0222148, XP093000161, DOI: 10.1371/journal.pone.0222148 *
LEENTJENS JENNEKE ET AL: "BCG Vaccination Enhances the Immunogenicity of Subsequent Influenza Vaccination in Healthy Volunteers: A Randomized, Placebo-Controlled Pilot Study", JOURNAL OF INFECTIOUS DISEASES, vol. 212, no. 12, 12 June 2015 (2015-06-12), US, pages 1930 - 1938, XP055959469, ISSN: 0022-1899, DOI: 10.1093/infdis/jiv332 *
SARKAR INDRANIL ET AL: "Selection of adjuvants for vaccines targeting specific pathogens", EXPERT REVIEW OF VACCINES, vol. 18, no. 5, 4 May 2019 (2019-05-04), GB, pages 505 - 521, XP055851002, ISSN: 1476-0584, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7103699/pdf/IERV_18_1604231.pdf> DOI: 10.1080/14760584.2019.1604231 *
VASSILIEVA ELENA V. ET AL: "Combination of STING Pathway Agonist With Saponin Is an Effective Adjuvant in Immunosenescent Mice", FRONTIERS IN IMMUNOLOGY, vol. 10, 23 December 2019 (2019-12-23), XP093000517, DOI: 10.3389/fimmu.2019.03006 *

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