[go: up one dir, main page]

WO2025196262A1 - Dosage immunologique impliquant des anticorps se liant à des adjuvants vaccinaux - Google Patents

Dosage immunologique impliquant des anticorps se liant à des adjuvants vaccinaux

Info

Publication number
WO2025196262A1
WO2025196262A1 PCT/EP2025/057789 EP2025057789W WO2025196262A1 WO 2025196262 A1 WO2025196262 A1 WO 2025196262A1 EP 2025057789 W EP2025057789 W EP 2025057789W WO 2025196262 A1 WO2025196262 A1 WO 2025196262A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
antigen
antibody
amino acid
acid sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/EP2025/057789
Other languages
English (en)
Inventor
Andrej BAVDEK
Rémi FERTIN
Maria Pilar Lopez Deber
Inmaculada RENTERO REBOLLO
Marija Vukicevic Verhille
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
AC Immune SA
Original Assignee
AC Immune SA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by AC Immune SA filed Critical AC Immune SA
Publication of WO2025196262A1 publication Critical patent/WO2025196262A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/5432—Liposomes or microcapsules
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids

Definitions

  • the invention relates to methods of assessing liposomal vaccines.
  • Liposomes are artificial vesicles, mostly made of (phospho)lipids, that may be used inter alia as vehicles for the presentation of antigens.
  • Methods of preparing liposomes that display an antigen on their surface have been disclosed, for example, in WO2012/055933 or WO2012/020124.
  • liposomal vaccines are disclosed in W02007/068411 , which pertains to a liposomal vaccine composition comprising (i) an amyloid-beta (Abeta)-derived peptide antigen displayed on the surface of the liposome, and (ii) an adjuvant, and in WO2019/197414, which pertains to a liposomal vaccine composition comprising (i) an amyloid-beta (Abeta)-derived peptide antigen displayed on the surface of the liposome, (ii) a peptide comprising a T-cell epitope, and (iii) an adjuvant.
  • the vaccine compositions are disclosed for use in the treating, preventing, or alleviating the symptoms associated with an Abeta associated disease.
  • a detectable analyte-binding agent wherein either the capture agent is specific for the vaccine adjuvant and the detectable analyte-binding agent is specific for the antigen, or the capture agent is specific for the antigen and the detectable analyte-binding agent is specific for the vaccine adjuvant;
  • Detecting the detectable analyte-binding agent may involve the mere determination whether the detectable analyte-binding agent (and hence a complex comprising the same) is present or not.
  • detection includes measuring the intensity of a signal emitted or generated by a label linked to the detectable analyte-binding agent.
  • the label may be linked directly or indirectly to the detectable analyte-binding agent, and this linking may in some embodiments take place after step (b).
  • step (c) comprises measuring the intensity of a signal emitted or generated by a label linked to the detectable analyte-binding agent.
  • the signal strength/intensity may be compared to a reference and/or a control, or may be used to calculate the half maximal effective concentration (EC50) of the liposomal vaccine.
  • EC50 half maximal effective concentration
  • step (c) comprises measuring the intensity of a signal emitted or generated by a label linked to the detectable analyte-binding agent.
  • the method may further comprise a step (d) of comparing the measured signal intensity to a reference and/or a control.
  • the method may further comprise a step (e) of determining one or more properties of the liposomal vaccine on the basis of the result of step (c) and/or (d).
  • the signal intensity of a suitable control or reference may be known, or may have been predetermined, for example it may be a predetermined standard curve or a predetermined threshold.
  • the method may comprise assessing at least two liposomal populations, such that one liposomal population may serve as a reference or control for the other.
  • test vaccine the liposomal vaccine that is assessed via the provided methods (i.e. the liposomal vaccine of interest) is referred to as the “test vaccine”.
  • the signal intensity of the test vaccine may be compared to the signal intensity of a negative control, the signal intensity of a positive control, and/or the signal intensity of a reference.
  • control may be a negative control or a positive control.
  • a negative control should be a liposomal population that is not capable of forming a complex with the capture agent and the detectable analyte-binding agent, and consequently not capable of generating a significant signal intensity. Any signal intensity generated by such a negative control may be considered to be background signal intensity.
  • a negative control should typically lack the antigen and/or the vaccine adjuvant (i.e. lack the antigen and/or the vaccine adjuvant displayed on the surface of the liposome of the test vaccine). The negative control may thus, for example, be a liposome that
  • the liposome of the negative control may display on its surface the vaccine adjuvant, but lack the antigen.
  • it may display on its surface the antigen, but lack the vaccine adjuvant.
  • it may be a liposome lacking both the antigen and the vaccine adjuvant.
  • a positive control is meant a liposomal vaccine that is known to form a complex with the capture agent and the detectable analyte-binding agent, and consequently to generate a significant signal intensity.
  • the liposome of the positive control displays on its surface both the antigen and the vaccine adjuvant comprised by the test vaccine.
  • a positive control may, for example, be a first batch of a vaccine that has been determined to generate a significant signal, and this may be used to assess other batches of the vaccine.
  • reference is used to mean any liposomal vaccine that may be used as a comparator and thus a “positive control” is a specific example of a “reference”, but the term “reference” also encompasses other embodiments, as explained below.
  • the reference should be a liposomal population that is capable of forming a complex with a suitable capture agent and a suitable detectable analyte-binding agent, and consequently be capable of generating a significant signal intensity.
  • the reference comprises the same or a variant antigen compared to the test vaccine
  • the reference must be capable of forming a complex with the same capture agent and detectable analyte-binding agent as the test vaccine.
  • the reference may comprise a different antigen compared to the test vaccine and a suitable capture agent and a suitable detectable analyte-binding agent must be selected accordingly to allow complex formation of the reference liposome with these agents.
  • the reference should be a liposomal vaccine having known properties.
  • the reference may differ from the test vaccine by a feature of interest, but otherwise be similar or identical to the test vaccine.
  • the feature of interest may, for example, be the antigen, for example the sequence or orientation thereof.
  • the reference may display on its surface (instead of an antigen that is identical to the antigen of the test vaccine) a different antigen.
  • a different antigen is meant that the antigen is significantly different to the antigen of the test vaccine, for example if the antigen is an antigenic peptide, the antigenic peptide of the reference may have an amino acid sequence that shares no more than 20, or 10% sequence identity, or may share no sequence identity, with the amino acid sequence of the antigenic peptide of the test vaccine.
  • a “different” antigen would typically not exhibit any significant binding to an antibody that is specific for the surface-displayed antigen of the test vaccine.
  • the antigenspecific agent (which may be the capture agent or the detectable analyte-binding agent) may exhibit substantially no binding to a “different” antigen.
  • the antigen of the test vaccine is an Abeta- derived antigenic peptide
  • the “different” antigen may be an antigenic peptide derived from a protein that is unrelated to Abeta.
  • the reference may display on its surface (instead of an antigen that is identical to the antigen of the test vaccine) a variant antigen.
  • variant antigen is meant that the antigen is similar, but not identical, to the antigen of the test vaccine.
  • a “variant” antigen would typically exhibit at least some binding to an antibody that is specific for the antigen of the test vaccine.
  • the antigen-specific agent (which may be the capture agent or the detectable analyte-binding agent) may exhibit at least some binding to a “variant” antigen.
  • a variant antigen may, for example, comprise a variation in the antigenic moiety compared to the antigen of the test vaccine.
  • a variant may, for example, comprise up to 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid changes compared to the test vaccine.
  • a variant antigen that differs in the antigenic moiety may be referred to as an “antigenic moiety variant”.
  • a variant antigen may comprise a variation in a moiety that anchors it to the liposome.
  • it may differ in the type, location and/or number of hydrophobic moieties.
  • a variant antigen that differs in the moiety that anchors it to the liposome may be referred to as an “anchoring moiety variant”.
  • An anchoring moiety variant may impact on the presentation of the antigen on the liposomal surface. For example, it may impact on the orientation of the antigen, and/or its accessibility to an agent that is specific for the antigen.
  • the variant antigen is an antigenic moiety variant only, i.e. it does not vary in the anchoring moiety. In some embodiments, the variant antigen is an anchoring moiety variant only, i.e. it does not vary in the antigenic moiety. In some embodiments, the variant antigen is an antigenic moiety variant and anchoring moiety variant.
  • the reference or (positive) control is a liposomal vaccine having a known vaccine property, such as a known antibody response, a known in vivo immunogenicity and/or a known potency.
  • the reference or (positive) control is a liposomal vaccine that is not known to differ from the test vaccine.
  • the reference or (positive) control may have been prepared using the same components as the test vaccine, for example be a different batch of the same vaccine.
  • the method may be used to compare different batches of the same vaccine.
  • the method is typically carried out using a liposomal vaccine composition at a selected concentration (which is typically determined and expressed as the concentration of a surface-displayed antigenic peptide in the composition, using the molecule weight of the full antigen, which may for example be the molecule weight of the antigenic peptide plus its anchoring moiety).
  • concentration which is typically determined and expressed as the concentration of a surface-displayed antigenic peptide in the composition, using the molecule weight of the full antigen, which may for example be the molecule weight of the antigenic peptide plus its anchoring moiety.
  • the reference or control should typically be used at an equivalent concentration or equivalent dilution.
  • any of the methods provided herein may comprise a step of assessing the surface presentation of an antigen.
  • step (e) may comprise assessing or determining the surface presentation of an antigen.
  • step (e) assessing or determining the surface presentation of the antigen of the vaccine on the basis of the result of step (c) and/or (d).
  • the reference is preferably a liposomal vaccine comprising an antigen that is an anchoring moiety variant compared to the test vaccine.
  • the signal intensity obtained with a liposomal vaccine displaying an antigen may be compared to the signal intensity obtained with liposomal vaccines displaying an anchoring moiety variant of the antigen.
  • the anchoring moiety variants may have the same antigenic moiety (peptide sequence) but differ in the anchoring moiety.
  • the method may thus be used inter alia to obtain information regarding the orientation of an antigen on the surface of a liposome, and this information may, for example, be used to optimise the anchoring moiety and the like.
  • the method of assessing a vaccine may be used to compare antigenic moiety variants, to assess the impact of altering the antigenic moiety, to optimise the antigenic moiety and the like.
  • the signal intensity obtained with an antigen may be compared to the signal intensity obtained with one or more antigenic moiety variants. This information may, for example, be used to optimise the antigenic moiety.
  • any of the methods provided herein may comprise a step of assessing and/or predicting antibody response to a liposomal vaccine.
  • step (e) may comprise assessing and/or predicting antibody response to the liposomal vaccine.
  • the method may, for example, be a method of assessing and/or predicting antibody response to a liposomal vaccine.
  • a method of assessing and/or predicting antibody response to a vaccine wherein the vaccine comprises a liposome that comprises both a vaccine adjuvant and an antigen displayed on the surface of the liposome, the method comprising the steps of
  • a detectable analyte-binding agent wherein either the capture agent is specific for the vaccine adjuvant and the detectable analyte-binding agent is specific for the antigen, or the capture agent is specific for the antigen and the detectable analyte-binding agent is specific for the vaccine adjuvant;
  • step (c) detecting the complex by detecting the detectable analyte-binding agent; wherein step (c) preferably comprises measuring the intensity of a signal emitted or generated by a label linked (directly or indirectly) to the detectable analyte-binding agent.
  • the method further comprises
  • step (e) assessing and/or predicting antibody response to the vaccine on the basis of the result of step (c) and/or (d),
  • the reference or control may, for example, be a vaccine having a known antibody response.
  • any of the methods provided herein may comprise a step of predicting the in vivo immunogenicity of a vaccine.
  • step (e) may comprise prediction of the in vivo immunogenicity of the vaccine.
  • the vaccine comprises a liposome that comprises both a vaccine adjuvant and an antigen displayed on the surface of the liposome, the method comprising the steps of
  • a detectable analyte-binding agent wherein either the capture agent is specific for the vaccine adjuvant and the detectable analyte-binding agent is specific for the antigen, or the capture agent is specific for the antigen and the detectable analyte-binding agent is specific for the vaccine adjuvant;
  • step (b) incubation under conditions allowing the formation of a complex between the capture agent, the liposome and the detectable analyte-binding agent; and (c) detecting the complex by detecting the detectable analyte-binding agent; wherein step (c) preferably comprises measuring the intensity of a signal emitted or generated by a label linked (directly or indirectly) to the detectable analyte-binding agent.
  • the method further comprises
  • step (e) predicting the in vivo immunogenicity of the vaccine on the basis of the result of step (c) and/or (d),
  • the reference or control may, for example, be a liposomal vaccine having a known in vivo immunogenicity.
  • any of the methods provided herein may comprise a step of predicting the potency of a liposomal vaccine.
  • step (e) may comprise predicting the potency of the liposomal vaccine.
  • the method may, for example, be a method of predicting the potency of a liposomal vaccine.
  • the reference or control may, for example, be a liposomal vaccine having a known potency.
  • any of the methods provided herein may comprise assessing two or more different liposomal vaccines and ranking them according to the measured signal intensity.
  • the vaccines may be ranked according to their EC50 or according to their predicted in vivo immunogenicity.
  • any of the provided methods may allow the identification of a defective batch of liposomal vaccines.
  • any of the methods provided herein may comprise a step of assessing the degree of heterogeneity of a liposomal vaccine.
  • step (e) may comprise assessing or determining the degree of heterogeneity of the liposomal vaccine.
  • the antigen is a B-cell antigen, i.e. an antigen that is capable of inducing a B-cell response in the subject. Preferably, it is capable of specifically interacting with a BCR.
  • the antigen is a T-cell antigen, i.e. an antigen that is capable of inducing a T-cell response in the subject. Preferably, it is capable of specifically interacting with a TCR.
  • the antigen may, for example, be selected from a protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleic acid or any combination of any of the foregoing. Preferably, it comprises, consists essentially of, or consists of an (antigenic) peptide (which may optionally be linked to an anchoring moiety). In embodiments where the antigen comprises an antigenic peptide, the peptide will typically have a length of at least 8 amino acids and less than 100 amino acids. Preferably, the peptide comprises about 10-60 amino acids, for example about 10-50, 10-40, 10-30 or 10-20 amino acids.
  • An antigen may comprise an antigenic moiety and a further moiety, such as an anchoring moiety.
  • a further moiety such as an anchoring moiety.
  • peptide or “antigenic peptide” is used to denote the antigenic moiety portion of the antigen. This will typically correspond to the moiety that is displayed on the surface of the liposome and that is the target for the capture agent or detectable analyte binding agent.
  • the surface-displayed antigen may comprise a further moiety, such as a hydrophobic moiety, that inserts into the lipid layer. Such a moiety is also referred to herein as an anchoring moiety.
  • the expression that the antigen “is a peptide” or “is an antigenic peptide” or that the antigen “consists of” a particular amino acid sequence refers to the antigenic moiety of the antigen, and the antigen may optionally further comprise an additional moiety, such as an anchoring moiety.
  • the antigen comprises, consists essentially of, or consists of an antigenic peptide linked to one or more anchoring moieties. Details of suitable anchoring moieties are discussed elsewhere herein.
  • the antigenic peptide may be derived from a foreign (external) antigen, or may be derived from a self-antigen.
  • the antigenic peptide may be a protein characteristic of a diseased state, a fragment of such a protein, or a variant thereof.
  • the protein characteristic of a diseased state is a protein expressed by cancer cells, a protein expressed by a pathogen, or a protein involved in a proteinopathy.
  • self-antigen refers to any antigen (e.g. antigenic peptide) derived from an antigen naturally produced by an individual.
  • the immune system of the individual is tolerant to self-antigens and therefore no immune reaction occurs.
  • the immune system is not tolerant to the self-antigens and auto-immune diseases may occur.
  • the use of a self-antigen allows the targeting of a molecule to which the immune system is typically tolerant and therefore may help to induce an immune response that otherwise would not occur.
  • the self-antigen may be selected from cytokines such as, for example, IL-17, IL-27; or from a protein involved in a proteinopathy, such as Abeta, Tau, a- synuclein, huntingtin, prion, or an amylin protein.
  • cytokines such as, for example, IL-17, IL-27
  • a protein involved in a proteinopathy such as Abeta, Tau, a- synuclein, huntingtin, prion, or an amylin protein.
  • the proteinopathy may, for example be selected from Alzheimer's Disease (AD), mild cognitive impairment (MCI), Down syndrome (DS), including Down syndrome-related Alzheimer's disease, cardiac amyloidosis, cerebral amyloid angiopathy (CAA), multiple sclerosis, amyotrophic lateral sclerosis (ALS), Adult Onset Diabetes, inclusion body myositis (IBM), ocular amyloidosis, glaucoma, macular degeneration, lattice dystrophy, optic neuritis, Creutzfeldt-Jacob disease, Dementia pugilistica, Gerstmann Straussler-Scheinker disease, prion protein cerebral amyloid angiopathy, traumatic brain injury, Non-Guamanian motor neuron disease with neurofibrillary tangles, argyrophilic grain dementia, corticobasal degeneration, diffuse neurofibrillary tangles with AD and cognitive impairment
  • DS Down syndrome
  • D Down syndrome-related Alzheimer's disease
  • the antigen may be an amyloid-beta (Ap) peptide or a fragment thereof.
  • Ap amyloid-beta
  • it may comprise, consist essentially of, or consist of, amino acids 1-15 of amyloid-beta (SEQ ID NO: 9) (which may optionally be linked to an anchoring moiety).
  • the antigen may be an a-synuclein derived peptide.
  • suitable antigenic peptides derived from a-synuclein include those described in W02009/103105, WO2022/029181 and WO2023/152260.
  • it may comprise, consist essentially of or consist of an antigenic peptide of the sequence GGKESMPVDPDNEA (SEQ ID NO: 10) (which may optionally be linked to an anchoring moiety).
  • the self-antigen is a cancer antigen or a tumour antigen.
  • the antigen may be a foreign antigen, for example an antigen derived from a pathogen or an allergen.
  • pathogen refers to any organism that can cause disease.
  • the pathogen may for example be selected from viruses, fungi, parasites, yeast, bacteria, and protozoa.
  • viral antigens include, but are not limited to, antigens from rhinoviruses, coronaviruses, enteroviruses, adenoviruses, parainfluenza viruses and respiratory syncytial viruses.
  • the antigen may be a viral fusion protein in some embodiments.
  • the antigen may comprise or be a T-cell antigen, which may, for example, comprise, consist essentially or consist of, a peptide (which may optionally be linked to an anchoring moiety).
  • the T-cell antigen may, for example, comprise, consist essentially of, or consist of, one or more universal T-cell epitopes (which may optionally be linked to an anchoring moiety).
  • T-cell epitope an epitope that is specific to T-cells that are present in the majority of the human population. They commonly originate from antigens to which humans are normally exposed during their lifetime. Examples include antigens incorporated in routinely administered vaccines. Specific examples are T-cell epitopes included in tetanus, influenza and diphtheria, pan DR-binding epitope peptide (PADRE; WO2010/086294), and also Keyhole limpet hemocyanin (KLH) and Epstein Barr virus (EBV).
  • PADRE pan DR-binding epitope peptide
  • KLH Keyhole limpet hemocyanin
  • EBV Epstein Barr virus
  • the “universal” ability of a T-cell epitope to activate T cells is the result of at least two complementary properties: i) affinity of binding to the HLA groove, meaning the strength of the binding, as well as ii) its capacity to bind different HLA haplotypes in a promiscuous manner, meaning the ability to cover very diverse human populations, with regards to the differences in the expression of HLA molecules.
  • the universal T-cell epitopes may bind to a majority of MHC class II alleles present in the human population.
  • the universal T-cell epitopes which may be employed in the methods provided herein may thus be capable of stimulating a CD4 T-cell response.
  • the universal T-cell epitopes may thus be capable of stimulating a helper T-cell response that enhances antibody production by B-cells.
  • the minimum length of a T-cell epitope peptide to ensure a sufficient immunogenicity is typically around 10 amino acids.
  • the minimum length of the peptide is typically around 10 amino acids to ensure a sufficiently immunogenic T-cell epitope is generated.
  • the antigen may comprise two or more different T-cell epitopes.
  • an antigenic moiety of the antigen may in some embodiments be modified through at least one lipophilic or hydrophobic moiety to facilitate display on the surface of the lipid-based nanostructure.
  • a moiety may be referred to as an anchoring moiety.
  • the antigenic moiety e.g. peptide
  • the antigenic moiety may be modified through multiple lipophilic or hydrophobic moieties.
  • the antigenic moiety may comprise two, three or four lipophilic or hydrophobic moieties.
  • the lipophilic or hydrophobic moieties may connect the antigenic moiety to the lipid-based nanostructure.
  • the one or more lipophilic or hydrophobic moieties may insert at least partly into the outer surface of the lipid-based nanostructure, i.e. into the lipid bilayer of the lipid-based nanostructure.
  • the one or more lipophilic or hydrophobic moieties are preferably hydrophobic moieties for ease of insertion into the lipid bilayer.
  • the one or more moieties may be one or more of: a fatty acid, a triglyceride, diglyceride, steroid, sphingolipid, glycolipid, or a phospholipid.
  • the at least one lipophilic or hydrophobic moiety may facilitate connecting or anchoring of the antigenic moiety to/into the lipid bilayer of the liposome.
  • the nature and/or location of the anchoring moiety may impact on the location and orientation of the antigenic moiety of the antigen.
  • the at least one lipophilic or hydrophobic moiety is a fatty acid.
  • the fatty acid may comprise a carbon backbone of at least 3 carbon atoms.
  • the fatty acid may comprise a carbon backbone having at least or about 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 carbon atoms.
  • Hydrophobic moieties may include, but are not limited to: palmitic acid, stearic acid, myristic acid, lauric acid, oleic acid, linoleic acid, and linolenic acid, cholesterol or 1 ,2-distearoyl- sn- glycero-3-phosphatidylethanolamine (DSPE).
  • DSPE 1 ,2-distearoyl- sn- glycero-3-phosphatidylethanolamine
  • the anchoring moiety or moieties may comprise a palmitoyl residue.
  • the antigen may for example be an antigenic peptide that is palmitoylated, i.e. mono-palmitoylated or multi-palmitoylated.
  • the antigenic peptide may be modified by at least two palmitoyl residues, i.e. dipalmitoylated.
  • the antigenic peptide may be modified by at least four palmitoyl residues, i.e. tetrapalmitoylated.
  • the antigenic peptide can include additional residues, such as lysine residues to facilitate palmitoylation.
  • a preferred construction comprises the antigenic peptide attached to two palmitoyl residues in the N and/or C terminal regions of the peptide.
  • the antigenic peptide is dipalmitoylated or tetrapalmitoylated. This may be facilitated by incorporating two lysine residues in the N and/or C terminal regions of the peptide antigen, whereby the lysine residues are palmitoylated.
  • the antigen may not comprise an anchoring moiety.
  • the antigen may be able to stick to the surface of a liposome without being anchored into the lipid layer.
  • the antigen and vaccine adjuvant that are displayed on the surface of the liposome are different from one another.
  • the antigen and vaccine adjuvant differ such that an agent specific for the antigen is not specific for the vaccine adjuvant, and vice versa.
  • the vaccine comprises a liposome comprising at least one vaccine adjuvant displayed on the surface of the liposome (as well as at least one antigen displayed on the surface of the liposome) and the methods employ an agent specific for the vaccine adjuvant.
  • the agent specific for the vaccine adjuvant is the capture agent.
  • the agent specific for the vaccine adjuvant is the detectable analyte-binding agent.
  • the liposomal vaccines may comprise multiple adjuvants.
  • Adjuvants serve to enhance the immune response when used in pharmaceutical compositions, e.g. vaccines.
  • Adjuvants typically stimulate the immune system so as to induce a stronger and/or longer lasting immune response to an antigen.
  • the term “vaccine adjuvant” refers to any substance that acts to accelerate, prolong, or enhance an immune response induced by an antigen in the context of vaccination.
  • a vaccine adjuvant typically does not specifically interact with a B-cell receptor (BCR) or T cell antigen receptor (TCR); instead, it typically interacts with a Toll-like receptor.
  • BCR B-cell receptor
  • TCR T cell antigen receptor
  • the vaccine adjuvant(s) of the liposomal vaccine may be selected from one or more of: monophosphoryl lipid A (MPLA); diphosphoryl lipid A (DPLA); Alum; Pam2CSK4; Pam3CSK4; Pam3CAG; saponins; CpG; lipidated CpG, such as CpG-Cholesterol; cationic lipids; phosphorothioated PS-CpG-ODNs; CpG oligodeoxynucleotides (CpG-ODN); CpG-A; CpG-B or CpG-C.
  • MPLA monophosphoryl lipid A
  • DPLA diphosphoryl lipid A
  • Alum Alum
  • Pam2CSK4 Pam3CSK4
  • Pam3CAG saponins
  • CpG lipidated CpG, such as CpG-Cholesterol
  • cationic lipids phosphorothioated PS-CpG-ODNs
  • vaccine adjuvants include QuilA, QS-21 , trehalose dimycolate (TDM), lipoteichoic acid (purified from Staphylococcus aureus), DDAB (dimethyldioctadecylammonium (bromide salt)), L18-MDP & B30-MDP (hydrophobic muramyl-dipeptide derivatives), C12-iE-DAP (diamino-pimelic acid).
  • TDM trehalose dimycolate
  • TDM lipoteichoic acid
  • DDAB dimethyldioctadecylammonium (bromide salt)
  • L18-MDP & B30-MDP hydrophobic muramyl-dipeptide derivatives
  • C12-iE-DAP diamino-pimelic acid
  • the at least one vaccine adjuvant(s) may be associated with the liposome via any appropriate manner provided that the vaccine adjuvant is displayed, at least in part, on the surface of the liposome.
  • the vaccine adjuvant may form part of the liposome, for example, the vaccine adjuvant(s) may from part of the lipid bilayer of the liposome, or may be covalently linked to the lipid bilayer of the liposome through a covalent linkage
  • the adjuvant(s) may be at least in part in part integrated in the lipid bilayer of the liposome.
  • the at least one vaccine adjuvant is a lipid-based vaccine adjuvant.
  • the lipid-based vaccine adjuvant may be selected from, monophosphoryl lipid A (MPLA); diphosphoryl lipid A (DPLA); Pam2CSK4; Pam3CSK4; Pam3CAG; saponins; CpG; lipidated CpG, such as CpG-Cholesterol; cationic lipids; phosphorothioated PS-CpG-ODNs; CpG oligodeoxynucleotides (CpG-ODN); CpG-A; CpG-B or CpG-C.
  • MPLA monophosphoryl lipid A
  • DPLA diphosphoryl lipid A
  • Pam2CSK4 Pam3CSK4
  • Pam3CAG saponins
  • CpG lipidated CpG, such as CpG-Cholesterol
  • cationic lipids phosphorothioated PS-CpG-ODNs
  • the lipid-based vaccine adjuvant may be incorporated into a liposome during liposomal synthesis, for example by mixing the lipid-based vaccine adjuvant with the other components that form the lipid bilayer.
  • the liposome may comprise dimyristoylphosphatidyl-choline (DM PC), dimyristoylphosphatidyl-glycerol (DMPG), cholesterol and the lipid-based vaccine adjuvant.
  • DM PC dimyristoylphosphatidyl-choline
  • DMPG dimyristoylphosphatidyl-glycerol
  • cholesterol cholesterol
  • the molar ratios of these four components may be 9:1:7:0.05 in some embodiments.
  • the vaccine adjuvant may be a Toll-like receptor (TLR) agonist, in particular a TLR4 agonist or a TLR9 agonist.
  • TLR Toll-like receptor
  • TLR4 agonist refers to any compound that acts as an agonist of TLR4.
  • TLR4 agonist include, but not limited to, monophosphoryl lipid A (MPLA).
  • MPLA useful for the invention can be obtained using methods known in the art, or from a commercial source, such as 3D-(6-acyl) PHAD®, PHAD®, PHAD®-504, or 3D-PHAD® from Avanti Polar Lipids (Alabaster, Alabama, USA), or MPLTM from various commercial sources.
  • the vaccine adjuvant may for example, be derived from, or based on, a glycolipid and may thus be referred to as a “glycolipid-based” vaccine adjuvant.
  • the at least one vaccine adjuvant is a glycolipid-based vaccine adjuvant.
  • the at least one vaccine adjuvant is the glycolipid-based vaccine adjuvant MPLA.
  • the MPLA may be selected from Monophosphoryl Hexa-acyl Lipid A, 3-Deacyl (Synthetic) (3D-(6-acyl) PHAD®), PHAD® (Phosphorylated HexaAcyl Disaccharide), PHAD®-504, 3D-PHAD® from Avanti Polar Lipids (Alabaster, Alabama, USA)) or MPL.
  • the vaccine adjuvant may be 3D-(6-acyl) PHAD®.
  • the glycolipid-based vaccine adjuvant may for example, be derived from, or based on, the glycolipid Lipid A, or the glycolipid monophosphoryl lipid A (MPLA).
  • MPLA glycolipid monophosphoryl lipid A
  • the at least one vaccine adjuvant is the glycolipid- based vaccine adjuvant MPLA.
  • MPLA refers to a modified form of lipid A, which is the biologically active part of Gramnegative bacterial lipopolysaccharide (LPS) endotoxin. MPLA provides immunostimulatory activity, but is less toxic than LPS.
  • the term “MPLA” also encompasses MPLA-derivatives such as Monophosphoryl Hexa-acyl Lipid A, 3-Deacyl (Synthetic), PHAD® (Phosphorylated HexaAcyl Disaccharide), PHAD®-504, 3D-PHAD® from Avanti Polar Lipids (Alabaster, Alabama, USA)) or MPL. Monophosphoryl Hexa-acyl Lipid A, 3-Deacyl (Synthetic) is also known as (3D-(6-acyl) PHAD®), so these terms are used interchangeably herein.
  • MPLA is an agonist of Toll-like receptor 4 (TLR4). Accordingly, the glycolipid-based vaccine adjuvant is preferably a TLR4 agonist.
  • the glycolipid-based vaccine adjuvant may thus, for example, be the TLR4 agonist MPLA or an analog thereof that is a TLR4 agonist.
  • glycolipid-based vaccine adjuvant may, for example, comprise two glucosamine units, at least 1 acyl chain and one or more phosphate groups.
  • the glucosamine units are in a P(1 — >6) linkage.
  • the (glycolipid-based) vaccine adjuvant comprises only a single phosphate group.
  • each acyl chain may be about C10 to C16 (i.e. 10-16 carbons in length), preferably C12-C14. Six C14 acyl chains are especially preferred.
  • Monophosphoryl Hexa-acyl Lipid A, 3-Deacyl (Synthetic) which is also referred to herein as 3D-(6-acyl) PHAD®.
  • TLR9 agonist refers to any compound that acts as an agonist of TLR9.
  • suitable TLR9 agonist include, but not limited to, CpG oligonucleotides.
  • CpG oligonucleotide refers to an oligonucleotide comprising at least one CpG motif.
  • oligonucleotide refers to a polynucleotide formed from a plurality of linked nucleotide units.
  • CpG motif' refers to a nucleotide sequence which contains unmethylated cytosine-phosphateguanine (CpG) dinucleotides (i.e., a cytosine (C) followed by a guanine (G)) linked by a phosphate bond or a phosphodiester backbone or other internucleotide linkages.
  • CpG cytosine-phosphateguanine
  • G guanine
  • Examples of synthetic CpG oligonucleotides include, but are not limited to, CpG 2006 (also known as CpG 7909), CpG 1018, CpG 2395, CpG 2216 or CpG 2336.
  • the vaccine adjuvant(s) of the liposomal construct may comprise CpG, for example it may be a lipid-based vaccine adjuvant comprising CpG.
  • the CpG oligonucleotide may, for example, be covalently linked to the lipid bilayer of the liposome through a covalent linkage.
  • lipid-based adjuvant and “lipid-based vaccine adjuvant” are used interchangeably herein.
  • Agents employ an agent specific for the antigen and an agent specific for the vaccine adjuvant respectively.
  • One of these agents is the capture agent and the other is the detectable binding agent.
  • the antigen and vaccine adjuvant are targets of the capture agent and the detectable binding agent respectively.
  • capture agent and “detectable analyte-binding agent” are terms of art.
  • the capture agent binds to its target and captures it to a solid support (to which the capture agent is attached), thereby capturing the liposome comprising the target onto the solid support.
  • the detectable analyte-binding agent binds to its target.
  • a label may be linked directly or indirectly to the detectable analyte-binding agent, and this linking may in some embodiments take place after step (b).
  • the label emits or generates the signal that may be detected or measured in step (c).
  • the antigen and the vaccine adjuvant are different.
  • the antigen and vaccine adjuvant differ such that an agent specific for the antigen is not specific for the vaccine adjuvant, and vice versa.
  • the capture agent is specific for the antigen and the detectable analyte-binding agent is specific for the vaccine adjuvant.
  • the capture agent is specific for the vaccine adjuvant and the detectable analyte-binding agent is specific for the antigen.
  • agent used herein encompasses inter alia “capture agent” and “detectable analyte-binding agent”.
  • capture agent and “detectable analyte-binding agent”.
  • selective binding is meant that the agent preferentially binds to its specified target. Typically, it does not significantly bind, or does not bind, to any other unrelated species, particularly any other unrelated species present on the surface of the liposome or otherwise present in the assay.
  • an agent specific for the vaccine adjuvant preferentially binds to the vaccine adjuvant (i.e. its target) and does not significantly bind to the antigen, nor any other molecules at the surface of the liposome.
  • An agent specific for the antigen preferentially binds to the antigen (i.e. its target) and does not significantly bind to the vaccine adjuvant, nor any other molecules at the surface of the liposome.
  • any of the agents disclosed herein may, for example, be an antibody.
  • the term "antibody” is used herein in the broadest sense and encompasses any antibody structures that exhibit the desired binding activity, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific, biparatopic antibodies), fully-human antibodies and antigen-binding fragments.
  • the antibody is not polyclonal.
  • monoclonal antibodies or antigen-binding fragments thereof are preferred.
  • Antibodies which may be used according to the methods of present invention may be chimeric antibodies, recombinant antibodies, or humanized antibodies, or an antigen-binding fragment of any thereof.
  • an "antigen-binding fragment" of an antibody refers to a molecule other than an intact antibody that comprises a portion of an intact antibody and that binds the antigen to which the intact antibody binds. Accordingly, examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab' -SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments.
  • Monoclonal antibodies or antigen-binding fragments thereof are preferred.
  • the capture agent is preferably an antibody.
  • the detectable analyte-binding agent is preferably an antibody.
  • preferred capture agents may be selected from those that are specific for one of the preferred antigens described herein.
  • preferred detectable analyte-binding agents may be selected from those that are specific for one of the preferred antigens described herein.
  • preferred capture agents may be selected from those that are specific for one of the preferred vaccine adjuvants described herein.
  • preferred detectable analyte-binding agent may be selected from those that are specific for one of the preferred vaccine adjuvants described herein.
  • the vaccine adjuvant is preferably MPLA.
  • the agent specific for the vaccine adjuvant (which may be the capture agent or the detectable analyte-binding agent), is preferably specific for MPLA.
  • the MPLA may, for example, be 3D-(6-Acyl) PHAD®.
  • the agent specific for the vaccine adjuvant is an anti-MPLA antibody, for example an anti-MPLA antibody as disclosed herein by reference to any one of SEQ ID Nos 1-8.
  • the antigen is an antigenic peptide.
  • the agent specific for the antigen (which may be the capture agent or the detectable analyte-binding agent) is preferably specific for such an antigenic peptide.
  • the agent may be an antibody specific for an antigenic peptide. Suitable antibodies are well known.
  • the antigenic peptide is an amyloid-beta (Ap) peptide or a fragment thereof
  • the agent specific for the antigen (which may be the capture agent or the detectable analyte-binding agent) is an A binding antibody that can specifically bind that peptide or to an epitope in that peptide, for example an epitope within SEQ ID NO: 9.
  • the agent specific for the antigen is a commercially available Ap binding antibody.
  • the antibody is selected from: NAB228 (Invitrogen), 6E10 (Covance), 2C8 (Invitrogen), 4G8 (Chemicon), WO-2 (Merck) and DE2 (Chemicon).
  • the antibody is 6E10.
  • the antigenic peptide is a Tau peptide or fragment thereof
  • the agent specific for the antigen (which may be the capture agent or the detectable analytebinding agent) is a Tau binding antibody that can specifically bind that peptide or to an epitope in that peptide.
  • the agent specific for the antigen is a commercially available Tau binding antibody.
  • the antibody is selected from: Tau12 (Covance), Tau13 (Covance), 43D (Covance), 3H6.H7 (Covance), 77E9 (Covance), Tau1 (Chemicon), BT-2 (Pierce), Tau46 (Abeam), Tau2 (Covance), AT270 (ThermoFisher), HT7 (ThermoFisher), Tau5 (ThermoFisher), 77G7 (Covance), AT100 (ThermoFisher), AT180 (ThermoFisher), PHF13 (ThermoFisher) and AT8 (ThermoFisher).
  • the antigenic peptide is an alpha-synuclein peptide or fragment thereof
  • the agent specific for the antigen (which may be the capture agent or the detectable analyte-binding agent) is an alpha-synuclein binding antibody that can specifically bind that peptide or to an epitope in that peptide, for example an epitope within SEQ ID NO: 10.
  • the agent specific for the antigen is a commercially available alpha-synuclein binding antibody.
  • the antibody is selected from MJFR1 (Abeam), Syn211 (ThermoFisher) and LB-509 (BioLegend). In preferred embodiments, the antibody is MJFR1.
  • the capture agent is immobilized on a solid support.
  • Methods for immobilising agents on a solid support such as coupling antibodies to solid support, are well known to those of ordinary skill in the art.
  • Suitable solid supports are well known in the field of radioimmunoassay and enzyme immunoassay.
  • Exemplary solid support substances include, but are not limited to, microtiter plates, multiwell plates, test tubes, beads and slides.
  • the solid support may be made of a suitable material such as plastic or glass; in the case of e.g. beads, it may alternatively be made of polystyrene or be magnetic.
  • the solid support may comprise a porous material such as nylon, nitrocellulose, cellulose acetate, glass fibers and other porous polymers.
  • the capture agent may be immobilized on the surface of a well of the solid support, such as a multiwell plate.
  • Detection of the detectable analyte-binding agent involves detection of a signal emitted or generated by a label linked to the detectable analyte-binding agent.
  • the label may be linked directly or indirectly to the detectable analyte-binding agent and indirect linking may in some embodiments take place after step (b).
  • the detectable analyte-binding agent comprises a label that is directly linked to the detectable analyte-binding agent, for example through a covalent bond.
  • a further agent is used to (indirectly) label the detectable analytebinding agent, for example a labelled secondary antibody may be used to bind to, and thereby label, the detectable analyte-binding agent.
  • the detectable analyte-binding agent may comprise a label.
  • the methods of the invention may further comprise a step of (indirectly) linking a label to the detectable analyte-binding agent.
  • Suitable labels are known in the art and include enzymes; radioisotopes; and fluorescent, luminescent or chromogenic substances.
  • the label may emit a signal, for example a fluorescent signal.
  • the label may generate a signal, for example enzymes generate a signal by converting a substrate into a detectable product.
  • the label may be selected from horseradish peroxidase, alkaline phosphatase, a coloured particle or a fluorescent moiety, preferably horseradish peroxidase or alkaline phosphatase.
  • any suitable method may be used to (indirectly) label the detectable analyte-binding agent by contacting the detectable analyte-binding agent with a secondary agent that comprises a label, i.e. a labelled secondary agent.
  • the labelled secondary agent is specific for a moiety comprised by the detectable analyte-binding agent.
  • the labelled secondary agent may be specific for the Fc domain, for example it may comprise protein A or G, or a secondary antibody.
  • the detectable analyte-binding agent comprises biotin
  • the labelled secondary agent may comprise avidin or streptavidin; or in embodiments where the detectable analyte-binding agent comprises a hapten, the labelled secondary agent may comprise an anti-hapten binding peptide.
  • the detectable analyte-binding agent is a primary antibody and the secondary agent is a labelled secondary antibody, most preferably labelled with horseradish peroxidase or alkaline phosphatase.
  • Conjugation methods are well known in the art and several technologies are commercially available for conjugating antibodies to a label or other moiety. Conjugation is typically through amino acid residues contained within the binding agent (such as lysine, histidine or cysteine). They may rely upon methods such as the NHS (Succinimidyl) ester method, isothiocyanate method, carbodiimide method and periodate method. Conjugation may be achieved through creation of fusion proteins for example. This is appropriate where the detectable analyte-binding agent is conjugated with another protein molecule. Thus, suitable genetic constructs may be formed that permit the expression of a fusion of the detectable analyte-binding agent of the invention with the label or other molecule. Conjugation may be via a suitable linker moiety to ensure suitable spatial separation of the antibody and conjugated molecule, such as detectable label. However, a linker may not be required in all instances.
  • the label is capable of emitting a signal, for example it may be radioactive or fluorescent.
  • the label is capable of generating a signal. This typically requires a suitable substrate to be present. Accordingly, in some embodiments, the method further comprises adding a suitable substrate for signal generation by the label.
  • the label may be an enzyme requiring a suitable substrate to generate a signal. The enzyme may interact with a substrate to induce a quantifiable colour change or fluorescence.
  • the detectable analyte-binding agent may be labelled with horseradish peroxidase (HRP). Upon incubation with a HRP substrate, HRP catalyses a reaction causing a detectable colour change.
  • HRP substrates include O- Phenylenediamine (OPD), tetramethyl benzidine (TMB) or ABTS (2,2'-azino-bis(3- ethylbenzothiazoline-6-sulphonic acid).
  • OPD O- Phenylenediamine
  • TMB tetramethyl benzidine
  • ABTS 2,2'-azino-bis(3- ethylbenzothiazoline-6-sulphonic acid
  • the methods of the invention comprise a step of allowing the formation of a complex between the capture agent, the liposome and the detectable analyte-binding agent.
  • the complex is formed through specific binding of the agents to their targets such that the resulting complex comprises the capture agent bound to its target on the surface of the liposome and the detectable analyte-binding agent bound to its target on the surface of the liposome.
  • complex is meant herein a complex that comprises the capture agent bound to its target on the surface of the liposome and the detectable analyte-binding agent bound to its target on the surface of the liposome.
  • the “complex” is a liposome having specifically bound to it (more specifically to its antigen and its vaccine adjuvant) a capture agent and a detectable analyte-binding agent respectively. Accordingly, a liposome having bound thereto no capture agent and/or no detectable analyte-binding agent is not a complex as defined herein.
  • complex formation relies inter alia on the antigen being accessible to take part in the complex formation. Accordingly, liposomes that lack the antigen will substantially not take part in the complex formation. Liposomes that display the antigen on their surface may take part in complex formation, but their ability to take part in the complex formation may be affected by factors such as the affinity of the antigen-specific agent (capture agent or detectable analyte-binding agent, as the case may be) for the antigen; and how accessible the antigen is to the antigen-specific agent.
  • the affinity of the antigen-specific agent capture agent or detectable analyte-binding agent, as the case may be
  • the capture agent is specific for the antigen of the liposome and the detectable analyte-binding agent is specific for the vaccine adjuvant of the liposome. As such, the capture agent binds the antigen of the liposome and the detectable analyte-binding agent binds the vaccine adjuvant so as to form a complex.
  • the capture agent is specific for the vaccine adjuvant of the liposome and the detectable analyte-binding agent is specific for the antigen of the liposome. As such, the capture agent binds the vaccine adjuvant of the liposome and the detectable analytebinding agent binds the antigen so as to form a complex.
  • the liposome, the capture agent and the detectable analyte-binding agent may be simultaneous or sequential.
  • the liposome, the capture agent and the detectable analyte-binding agent are contacted simultaneously, for example by simultaneously, or substantially simultaneously adding each of these components to an assay compartment, or by adding two of these components to an assay compartment already containing the remaining component.
  • the liposome, the capture agent and the detectable analyte-binding agent are contacted sequentially.
  • the liposome may in a first step be contacted with the capture agent. During this first step, the capture agent may be allowed to bind to its target on the liposome (if its target is present).
  • the liposome (and any capture agent bound thereto) may be contacted with the detectable analyte-binding agent.
  • the detectable analyte-binding agent may be allowed to bind to its target on the liposome (if its target is present).
  • the liposome may in a first step be contacted with the detectable analytebinding agent. During this first step, the detectable analyte-binding agent may be allowed to bind to its target on the liposome (if its target is present).
  • the liposome (and any detectable analyte-binding agent bound thereto) may be contacted with the capture agent. During this second step, the capture agent may be allowed to bind to its target on the liposome (if its target is present).
  • the method may comprise one or more steps of removing free assay components.
  • free is meant that the assay component is not bound to its target.
  • the “assay component” may be the capture agent, the liposome, the detectable analyte-binding agent, or any other component used in the method, such as any component used to detect the detectable analyte-binding agent.
  • a convenient example of such a step is a wash step, in which a free component is removed using a suitable wash solution.
  • a step of removing free assay components, such as a wash step may, e.g., be carried out after the step of contacting the liposome with the capture agent and/or after the step of contacting the liposome with the detectable analyte-binding agent.
  • the method may comprise a wash step after complex formation has been allowed to take place.
  • a wash step may remove any liposomes that are not bound to a capture agent (including liposome bound solely to a detectable analyte-binding agent), and any detectable analyte-binding agent that is not bound to a liposome.
  • an appropriate wash step is carried out to remove any free detectable analyte-binding agent, such that detection of the detectable analyte-binding agent detects substantially only detectable analyte-binding agent that forms part of a complex as defined herein.
  • a liposomal vaccine comprises a population of liposomes, so any reference herein to a “liposomal vaccine” should be understood to encompass a reference to a population of liposomes.
  • liposomal vaccine is meant a vaccine comprising a liposome that comprises both a vaccine adjuvant and an antigen displayed on its surface.
  • the vaccine adjuvant is displayed on the liposome’s surface and the antigen is displayed on the liposome’s surface.
  • at least a proportion of the liposomes comprise the antigen displayed on the surface.
  • all, substantially all, or at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the liposomes in the liposomal vaccine comprise the antigen displayed on the surface of the liposome.
  • the method is carried out on a liposomal vaccine comprising a significant proportion of liposomes that do not comprise the antigen displayed on the surface, for example at least 20%, at least 30%, at least 40% or at least 50% of liposomes may not comprise the antigen displayed on the surface of the liposome.
  • the liposomes comprise the vaccine adjuvant displayed on the surface of the liposome.
  • all, substantially all, or at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the liposomes in the liposomal vaccine comprise the vaccine adjuvant displayed on the surface of the liposome.
  • a vaccine comprising a liposome that comprises both a vaccine adjuvant and an antigen displayed on the surface of the liposome is meant that at least a proportion of the liposomes of the vaccine (liposomal population) comprise both a vaccine adjuvant and an antigen displayed on the surface of the liposome.
  • at least a proportion of the liposomes of the liposomal vaccine comprise a vaccine adjuvant displayed on the surface of the liposome but do not comprise an antigen displayed on the surface of the liposome.
  • the liposomal population may be considered heterogeneous with respect to the presence or absence of the antigen.
  • the provided methods may be used to assess the degree of heterogeneity of the liposomal vaccine with regard to the presence or absence of a target.
  • the target is typically the antigen, but may alternatively be the vaccine adjuvant or a difference surface molecule.
  • heterogeneity or “homogeneity” is used herein with respect to the presence or absence of a target.
  • the term “heterogeneity” as used here does not refer to the inherent polydispersity of liposomal populations.
  • a population of liposomes in which all or substantially all of the liposomes comprise the target may be considered to be “homogeneous” (with regard to the presence of that target, meaning that there are substantially no liposomes in the population that lack the target).
  • the term “homogeneous” as used herein does not necessarily mean that there are no differences between the liposomes in the population.
  • the “degree of heterogeneity” as used herein refers to the proportion of liposomes in the population that lack the target.
  • a “low degree of heterogeneity” is meant that at least 80%, at least 85%, at least 90%, or at least 95% of the liposomes comprise the target.
  • a “high degree of heterogeneity” is meant that at least at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% of liposomes may not comprise the target.
  • the methods provided herein have significant utility in vaccine characterization, development and production, inter alia in assessing and/or predicting the antibody response to a liposomal vaccine; predicting the in vivo immunogenicity of a liposomal vaccine; or predicting the potency of a liposomal vaccine.
  • Antibody response refers to the production of circulating antibodies by B- cells and their progeny in response to an antigen.
  • “In vivo immunogenicity” as used herein refers to a measure of the ability of the liposomal vaccine to elicit an immune response (humoral and cellular) when administered to a recipient.
  • “Potency” refers to the biological activity of a vaccine required in the treatment or prevention of diseases, disorders, or conditions.
  • Liposomes are widely used as model systems for cell membrane and as drug carriers in drug delivery systems. Liposomes that can be used in the methods of the present invention include those known to one skilled in the art. Any of the standard lipids useful for making liposomes may be used. Any method of making liposomes known to one skilled in the art may be used, for example, the method disclosed in Alving et al., Infect. Immun. 60:2438- 2444, 1992.
  • Liposomes are vesicles composed of (phospho)lipid molecules comprising a hydrophilic head group and a hydrophobic tail.
  • the (phospho)lipid molecules assemble in an aqueous solution such that the hydrophobic parts get oriented toward each other to avoid contact with the aqueous phase, whereas the hydrophilic head groups are oriented such that they make maximal contact with the aqueous surrounding. This leads to spontaneous self-assembly into spherical structures that contain an inner aqueous compartment surrounded by a lipid bilayer.
  • a liposome can be used as a carrier for presenting an antigen on its outer surface; as well as comprising an adjuvant to increase or stimulate the immune response against the antigen within the target animal or human.
  • surface of a liposome is meant the external surface, i.e. the surface which is oriented towards the aqueous solution surrounding the liposome.
  • surface molecule surface antigen
  • surface-displayed antigen surface-displayed antigen
  • dispenser on the surface of the liposome and the like mean that the molecule (e.g. the antigen or the vaccine adjuvant respectively) is presented, at least partially, on the external surface of the (intact) liposome, as would be understood by one skilled in the art (see e.g. Muhs, 2007, Pihlgren, 2013). This is typically by insertion of the molecule (e.g. the antigen or the vaccine adjuvant respectively) into, or otherwise anchoring of the molecule to, the outer surface of the liposome, for example via a hydrophobic moiety.
  • the vaccine adjuvant is lipid-based, it intrinsically comprises a suitable hydrophobic moiety.
  • the vaccine adjuvant may for example comprise a vaccine adjuvant moiety that has been covalently linked to one or more anchoring moieties.
  • the antigen may for example comprise an antigenic moiety that has been covalently linked to one or more anchoring moieties.
  • Suitable anchoring moieties are discussed elsewhere herein.
  • the liposomes may also comprise additional components that are not targets for any of the agents used in the methods provided herein.
  • additional components if present, are neither a target for the capture agent nor a target for the detectable analytebinding agent.
  • the capture agent nor the detectable analyte-binding agent are specific for such additional components.
  • Such an additional component may, for example be a further adjuvant, provided that the further adjuvant is sufficiently different from the vaccine adjuvant that is the target of the capture agent or the detectable binding agent respectively so as to not interfere with the method.
  • a suitable example is CpG, but other adjuvants are well known such as alum (e.g.
  • aluminium phosphate or aluminium hydroxide calcium phosphate
  • calcium phosphate cytokines (e.g. interleukin-1), muramyl peptides (e.g., N-acetylmuramyl-L- threonyl-D-isoglutamine (thrMDP) or N-acetylglucosaminyl-N-acetylinuramyl-L-Ala-D-isoGlu- L-Ala-dipalmitoxy propylamide (DTP-DPP) TheramideTM), oil (e.g.
  • cytokines e.g. interleukin-1
  • muramyl peptides e.g., N-acetylmuramyl-L- threonyl-D-isoglutamine (thrMDP) or N-acetylglucosaminyl-N-acetylinuramyl-L-Ala-D-isoGlu- L
  • an additional component may, for example be a further antigen, provided that the further antigen is sufficiently different from the antigen that is the target of the capture agent or the detectable binding agent respectively so as to not interfere with the method.
  • the liposome may comprise one or more additional antigenic peptides and/or adjuvants. Any such additional component may be encapsulated by the liposome or be partially or fully displayed on its surface.
  • the liposome may comprise a vaccine adjuvant and an antigen displayed on its surface, and may further comprises one or more additional components, optionally selected from one or more encapsulated universal T-cell epitopes; one or more different adjuvants; and/or one or more different surface-displayed antigens.
  • the vaccine comprises at least a B-cell antigen displayed on the surface of the liposome and at least one T-cell antigen displayed, at least partially, on the surface of the liposome.
  • the inventors have generated an anti-MPLA antibody. To our knowledge, this is the first report of an anti-MPLA antibody.
  • the antibody is capable of binding to 3D-(6-acyl) PHAD® and may thus also be referred to as an anti-3D-(6- acyl) PHAD® antibody.
  • an antigen binding molecule capable of binding MPLA i.e. is an anti-MPLA binding molecule.
  • the anti-MPLA binding molecule is an anti-MPLA antibody. It may be referred to as an anti-3D-(6-acyl) PHAD® antibody.
  • the anti-MPLA binding molecule comprises a Heavy Chain Variable Region comprising:
  • VH variable heavy
  • VH CDR2 that has the amino acid sequence of SEQ ID NO: 2, or a sequence having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity thereto;
  • VH CDR3 that has the amino acid sequence of SEQ ID NO: 3, or a sequence having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity thereto; and/or a Light Chain Variable Region comprising:
  • VL variable light
  • VL CDR2 that has the amino acid sequence of SEQ ID NO: 5, or a sequence having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity thereto;
  • VL CDR3 that has the amino acid sequence of SEQ ID NO: 6, or a sequence having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity thereto.
  • the anti-MPLA binding molecule may, for example, comprise a Heavy Chain Variable Region comprising:
  • VH CDR1 that has the amino acid sequence of SEQ ID NO: 1;
  • VH CDR2 that has the amino acid sequence of SEQ ID NO: 2
  • VH CDR3 that has the amino acid sequence of SEQ ID NO: 3
  • a Light Chain Variable Region comprising:
  • VL CDR1 that has the amino acid sequence of SEQ IDNO: 4;
  • VL CDR3 that has the amino acid sequence of SEQ ID NO: 6.
  • a Heavy Chain Variable Region comprising:
  • VH CDR1 that has the amino acid sequence of SEQ ID NO: 1;
  • VH CDR2 that has the amino acid sequence of SEQ ID NO: 2;
  • VH CDR3 that has the amino acid sequence of SEQ ID NO: 3; and a Light Chain Variable Region comprising:
  • VL CDR1 that has the amino acid sequence of SEQ ID NO: 4;
  • VL CDR3 that has the amino acid sequence of SEQ ID NO: 6.
  • the anti-MPLA binding molecule binding molecule may, for example, comprise a Heavy Chain Variable Region comprising the amino acid sequence of SEQ ID NO: 7 or a sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity thereto; and/or a Light Chain Variable Region comprising the amino acid sequence of SEQ ID NO: 8 or a sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity thereto.
  • it may comprise a Heavy Chain Variable Region comprising the amino acid sequence of SEQ ID NO: 7 or a sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity thereto, and a Light Chain Variable Region comprising the amino acid sequence of SEQ ID NO: 8 or a sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity thereto.
  • it may comprise a Heavy Chain Variable Region comprising the amino acid sequence of SEQ ID NO: 7 and a Light Chain Variable Region comprising the amino acid sequence of SEQ ID NO: 8
  • an “antigen binding molecule,” as used herein, is any molecule that can specifically or selectively bind to an antigen.
  • a binding molecule may include or be an antibody or an antigen-binding fragment thereof.
  • An anti-MPLA binding molecule is a molecule that binds to a region of MPLA that is exposed on the outer surface of a liposome that comprises MPLA.
  • CDR as employed herein relates to “complementary determining region”, which is well known in the art.
  • the CDRs are parts of immunoglobulins that determine the specificity of said molecules and make contact with a specific ligand.
  • the CDRs are the most variable part of the molecule and contribute to the diversity of these molecules.
  • CDR-H depicts a CDR region of a variable heavy chain and CDR-L relates to a CDR region of a variable light chain.
  • VH means the variable heavy chain and VL means the variable light chain.
  • the CDR regions of an Ig- derived region may be determined as described in Kabat “Sequences of Proteins of Immunological Interest”, 5th edit. NIH Publication no. 91-3242 U.S. Department of Health and Human Services (1991). CDR sequences provided herein are defined according to Kabat.
  • the invention is intended to encompass anti-MPLA binding molecules in which the CDR sequences are defined according to any useful identification/numbering scheme.
  • Chothia Canonical structures for the hypervariable regions of immunoglobulins. Chothia C, LeskAM. J Mol Biol. 1987 Aug 20; 196(4):901-17
  • IMGT IMGT
  • Giudicelli V Chaume D, Bodmer J, Muller W, Busin C, Marsh S, Bontrop R, Marc L, Malik A, Lefranc MP. Nucleic Acids Res. 1997 Jan 1; 25(l):206-l I and Unique database numbering system for immunogenetic analysis. Lefranc MP.
  • kits comprising: a) a capture agent; and b) detectable analyte-binding agent; wherein the capture agent or detectable analyte-binding agent specifically binds to a vaccine adjuvant.
  • the vaccine adjuvant is a glycolipid-based vaccine adjuvant, more preferably MPLA, for example 3D-(6-acyl) PHAD®.
  • the capture agent or detectable analyte-binding agent may be an anti- MPLA binding agent, such as an anti-MPLA binding agent as described herein by reference to one or more of SEQ ID Nos 1-8.
  • the kit may further comprise a solid support.
  • a solid support may comprise a solid support having the capture agent immobilised thereon.
  • any relevant components particularly capture agents, detectable analyte-binding agents and solid supports, set out herein in connection with the provided methods or anti-MPLA binding agent apply mutatis mutandis to the corresponding components of the kit.
  • the kit may in some embodiments comprise all necessary components for performing the herein provided methods, such as, for example, buffers, detectable labels, substrates, reaction containers, and the like.
  • the kit may optionally be provided with suitable instructions for use.
  • the instructions for use may further explain the storage conditions for the compositions therein.
  • liposomal vaccine This term may be used interchangeably with “liposomal population” or “vaccine comprising a liposome”.
  • liposomal population or “vaccine comprising a liposome”.
  • vaccine should be understood to refer to a vaccine comprising a liposome unless explicitly stated otherwise.
  • Liposomes are a type of lipid-based nanovesicles, and the provided methods are equally applicable to other types of lipid-based nanovesicles or lipid-based nanoparticles, collectively referred to herein as lipid-based nanostructures.
  • any reference herein to a “liposome” may, where appropriate, be used interchangeably with “lipid-based nanostructure” and thus any reference to a “liposomal vaccine” may, where appropriate, be used interchangeably with “population of lipid-based nanostructures”.
  • lipid-based nanostructure is meant a sphere-shaped structure comprising a lipid outer layer that encapsulates an inner compartment.
  • the lipid outer layer may be a monolayer or may be a bilayer.
  • the structure may have a single monolayer, a single bilayer, or more than one mono- and/or bi-layer.
  • the inner compartment may be aqueous or non-aqueous.
  • the nanostructure may be a vesicle comprising an aqueous inner compartment.
  • the nanostructure may be a self-assembled spherical structure that contains an inner aqueous compartment surrounded by a lipid bilayer typically composed of phospholipids and sterols, such as a liposome.
  • Standard bilayer and multi-layer lipid-based nanostructures may be used in the methods of the present invention.
  • an antigen is an example of a surface molecule
  • a vaccine adjuvant is another example of a surface molecule.
  • lipid adjuvant and “lipid-based adjuvant” are used interchangeably herein.
  • a method of assessing a population of lipid-based nanostructures wherein the population of lipid-based nanostructures preferably comprises a lipid-based nanostructure comprising a first surface molecule and a second surface molecule, the method comprising
  • detecting the complex by detecting the detectable analyte-binding agent, which may preferably comprise measuring the signal intensity of a signal emitted or generated by a label linked directly or indirectly to the detectable analyte-binding agent.
  • the first surface molecule is a vaccine adjuvant and the capture agent is specific for the vaccine adjuvant;
  • the second surface molecule is a vaccine adjuvant and the detectable analyte-binding agent is specific for the vaccine adjuvant.
  • the vaccine adjuvant comprises, consists essentially of, or consists of, Monophosphoryl Hexa-acyl Lipid A, 3- Deacyl (3D-(6-acyl) PHAD®). 8. The method according to any one of the preceding clauses, wherein the first surface molecule or the second surface molecule is an antigen.
  • the antigen is an antigenic peptide, wherein the antigenic peptide is optionally linked to an anchoring moiety.
  • the antigenic peptide is derived from a protein derived from a pathogen or a protein characteristic of a diseased state, for example a protein derived from a viral pathogen, a protein expressed by cancer cells, or a protein involved in a proteinopathy.
  • the self-antigen is selected from p-amyloid (Ap), Tau, a-synuclein, huntingtin, prion, an amylin protein, IL-17 or IL-27.
  • the antigen is an antigenic peptide derived from p-amyloid (Ap), preferably wherein the antigenic peptide comprises, consists essentially of, or consists of, amino acids 1-15 of p-amyloid (Ap) (DAEFRHDSGYEVHHQ, SEQ ID NO: 9).
  • the antigen is an antigenic peptide derived from a-synuclein, preferably wherein the antigenic peptide comprises, consists essentially of, or consists of, SEQ ID NO: 10 (GGKESMPVDPDNEA).
  • the first surface molecule is a lipid-based vaccine adjuvant and the capture agent is specific for the lipid- based vaccine adjuvant
  • the second surface molecule is an antigen and the detectable analyte-binding agent is specific for the antigen
  • the lipid-based adjuvant is preferably a glycolipid-based vaccine adjuvant.
  • the lipid-based adjuvant is a glycolipid-based vaccine adjuvant and wherein the antigen is an antigenic peptide.
  • one of the surface molecules is a glycolipid-based vaccine adjuvant that is an agonist of Toll-like receptor 4 (TLR4), wherein the TLR4 agonist is preferably MPLA or an analog thereof, most preferably Monophosphoryl Hexa-acyl Lipid A, 3-Deacyl (3D-(6-acyl) PHAD®).
  • TLR4 agonist is preferably MPLA or an analog thereof, most preferably Monophosphoryl Hexa-acyl Lipid A, 3-Deacyl (3D-(6-acyl) PHAD®).
  • the capture agent and/or the analyte-binding agent is an antibody, preferably a monoclonal antibody.
  • the antibody comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 1 , a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 5 and/or a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 6, wherein the antibody preferably comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, a VH- CDR3 comprising the amino acid sequence of SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO:
  • one agent out of the detectable analyte-binding agent and the capture agent is an antibody that is specific for a surface molecule that is an antigenic peptide and wherein the antibody that is specific for the antigenic peptide is preferably an antibody that is specific for an antigenic peptide derived from p-amyloid (Ap), such as an antibody specific for an antigenic peptide comprising, consisting essentially of, or consisting of, amino acids 1-15 of p-amyloid (AP) (DAEFRHDSGYEVHHQ, SEQ ID NO: 9).
  • Ap p-amyloid
  • lipid-based nanostructure is a liposome, preferably wherein the population of lipid-based nanostructures is a liposomal vaccine.
  • the antigen is a T-cell antigen
  • the antigen preferably comprises, consists essentially of, or consists of, one or more universal T- cell epitopes.
  • the first surface molecule is a glycolipid- based vaccine adjuvant that is an agonist of Toll-like receptor 4 (TLR4), wherein the TLR4 agonist is preferably MPLA and most preferably 3D-(6-acyl) PHAD® and wherein the capture agent is specific for said glycolipid-based vaccine adjuvant and is preferably an anti-MPLA antibody, such as an anti-MPLA antibody as defined in any one of clauses 21-23, and wherein the second surface molecule is an antigenic peptide and the detectable analyte-binding agent is specific for that peptide, wherein the antigenic-peptide is preferably derived from a self-antigen.
  • TLR4 agonist is preferably MPLA and most preferably 3D-(6-acyl) PHAD®
  • the capture agent is specific for said glycolipid-based vaccine adjuvant and is preferably an anti-MPLA antibody, such as an anti-MPLA antibody as defined in any one of clauses 21-23
  • the second surface molecule is
  • the first surface molecule is an antigenic peptide and the capture agent is specific for said antigenic peptide, wherein the antigenic peptide is preferably derived from a self-antigen, and wherein the second surface molecule is a glycolipid-based vaccine adjuvant that is an agonist of Toll-like receptor 4 (TLR4), wherein the TLR4 agonist is preferably MPLA and most preferably 3D-(6-acyl) PHAD® and the detectable analyte-binding agent is specific for that glycolipid-based vaccine adjuvant and is preferably an anti-MPLA antibody, such as an anti-MPLA antibody as defined in any one of clauses 21-23.
  • TLR4 agonist is preferably MPLA and most preferably 3D-(6-acyl) PHAD®
  • the detectable analyte-binding agent is specific for that glycolipid-based vaccine adjuvant and is preferably an anti-MPLA antibody, such as an anti-MPLA antibody as defined in any one of clauses 21-23
  • detecting the complex by detecting the detectable analyte-binding agent, which may preferably comprise measuring the signal intensity of a signal emitted or generated by a label linked directly or indirectly to the detectable analyte-binding agent, wherein preferably
  • the antigenic peptide is derived from p-amyloid (Ap), preferably wherein the antigenic peptide comprises, consists essentially of, or consists of, amino acids 1-15 of p-amyloid (Ap) (DAEFRHDSGYEVHHQ, SEQ ID NO: 9) (which may optionally be linked to an anchoring moiety) and the detectable analyte-binding agent is an antibody that binds thereto, such as antibody 6E10; or
  • the antigenic peptide is derived from a-synuclein, preferably wherein the antigenic peptide comprises, consists essentially of, or consists of, SEQ ID NO: 10 (GGKESMPVDPDNEA) (which may optionally be linked to an anchoring moiety) and the detectable analyte-binding agent is an antibody that binds thereto, such as antibody MJFR1.
  • a method of assessing a population of lipid-based nanostructures, which may be a liposomal vaccine comprising:
  • a detectable analyte-binding agent that is an anti-MPLA antibody, such as an anti-MPLA antibody as defined in any one of clauses 21-23;
  • detecting the complex by detecting the detectable analyte-binding agent, which may preferably comprise measuring the signal intensity of a signal emitted or generated by a label linked directly or indirectly to the detectable analyte-binding agent, wherein preferably
  • the antigenic peptide is derived from p-amyloid (A ), preferably wherein the antigenic peptide comprises, consists essentially of, or consists of, amino acids 1-15 of p-amyloid (Ap) (DAEFRHDSGYEVHHQ, SEQ ID NO: 9) (which may optionally be linked to an anchoring moiety) and the detectable analyte-binding agent is an antibody that binds thereto, such as antibody 6E10; or (ii) the antigenic peptide is derived from a-synuclein, preferably wherein the antigenic peptide comprises, consists essentially of, or consists of, SEQ ID NO: 10 (GGKESMPVDPDNEA) (which may optionally be linked to an anchoring moiety) and the detectable analyte-binding agent is an antibody that binds thereto, such as antibody MJFR1.
  • A p-amyloid
  • the antigenic peptide comprises, consists essentially of,
  • step (a) the nanostructure is contacted with the capture agent prior to, simultaneously with, or after it is contacted with the detectable analyte-binding agent.
  • step (c) the method further comprises a step of contacting the detectable analyte-binding agent with a labelled secondary agent, thereby linking the label indirectly to the detectable analyte-binding agent.
  • the label is selected from horseradish peroxidase, alkaline phosphatase, a coloured particle or a fluorescent moiety, preferably horseradish peroxidase or alkaline phosphatase.
  • the method is a method of assessing a vaccine comprising a liposome that comprises both a vaccine adjuvant and an antigen displayed on the surface of the liposome, the method comprising the steps of
  • a detectable analyte-binding agent wherein either the capture agent is specific for the vaccine adjuvant and the detectable analyte-binding agent is specific for the antigen, or the capture agent is specific for the antigen and the detectable analyte-binding agent is specific for the vaccine adjuvant;
  • step (c) comprises measuring the intensity of a signal emitted or generated by a label linked directly or indirectly to the detectable analyte-binding agent, and the method further comprises
  • step (e) determining one or more properties of the test vaccine on the basis of the result of step (c) and/or (d).
  • (ii) comprises a variant antigen displayed on the surface of the liposome
  • (iii) comprises a different antigen displayed on the surface of the liposome
  • (iv) has a known vaccine property, such as a known ability to induce an antibody response, a known in vivo immunogenicity, or a known potency.
  • control is the signal intensity of a control liposomal population that is not capable of forming a complex with the capture agent and the detectable analyte-binding agent because the liposome of the control population
  • step (e) comprises assessing the antibody response to the liposomal vaccine; predicting the in vivo immunogenicity of the liposomal vaccine; and/or predicting the potency of the liposomal vaccine.
  • Fig. 1 Diagram of sandwich ELISA in an exemplary setup wherein A is an antigen and capture of the complex onto a solid support is via B, a vaccine adjuvant.
  • This setup may be referred to as “adjuvant-based capture”.
  • the detectable analyte-binding agent is shown lacking a covalently linked label; and use of a labelled secondary agent is shown to indirectly link a label to the detectable analyte-binding agent, but alternatively the detectable analytebinding agent may comprise a covalently linked label.
  • Fig. 2 Diagram of sandwich ELISA in an exemplary setup wherein A is a vaccine adjuvant and B is an antigen and capture of the complex onto a solid support is via this antigen.
  • This setup may be referred to as “antigen-based capture”.
  • the detectable analyte-binding agent is shown lacking a covalently linked label; and use of a labelled secondary agent is shown to indirectly link a label to the detectable analyte-binding agent, but alternatively the detectable analyte-binding agent may comprise a covalently linked label.
  • the graph shows resulting OD values for sandwich ELISA analysis. Samples were tested in duplicates. Optical density at 405 nm (average ⁇ standard deviation) is plotted against peptide concentration.
  • ACI-IL.062 is a negative control having the same composition as ACI- 7110.062, ACI-7111.062 and ACI-7108.062 but which does not comprise the alpha-synuclein antigenic peptide.
  • FIG. 4 Graph showing IgG titers against an a-synuclein antigenic peptide in mice immunized with ACI-7108.062, ACI-7110.062 or ACI-7111.062 liposomal preparations (vaccines).
  • the Y axis shows anti-a-synuclein antigenic peptide IgG titers in All/mL and the X axis shows days post immunization.
  • ACI-7108.062 liposomal vaccine treated group left
  • ACI-7110.62 treated group middle
  • ACI-7111.062 treated group right
  • at theoretical a-synuclein antigenic peptide dose 0.2 pg dose (squares), 2 pg (circles) or 80 pg (triangles).
  • FIG. 5 Sandwich ELISA results for liposomal formulations “ACI-24.060200 pg/mL” and “ACI-24.060400 pg/mL: ACI-24E.060 (1:1)”. Optical density at 405 nm (average ⁇ standard deviation) is plotted against liposome dilution factor. Samples were tested in duplicates. The ELISA set up was as shown in Figure 1.
  • FIG. 6 Sandwich ELISA results for liposomal formulations “ACI-24.060200 pg/mL” and “ACI- 24.060400 pg/mL: ACI-24E.060 (1:1)”. Optical density at 405 nm (average ⁇ standard deviation) is plotted against liposome dilution factor. Samples were tested in duplicates. The ELISA set up was as shown in Figure 2.
  • FIG. 8 Sandwich ELISA results for liposomal formulations “ACI-24.060” (control sample, stored at 2-8 °C) and “ACI-24.0606M 25C” (stressed sample, stored 6 months at 23-27 °C). Optical density at 405 nm (average ⁇ standard deviation) is plotted against liposome dilution factor. Samples were tested in duplicates. The ELISA set up was as shown in Figure 1.
  • FIG. 9 Sandwich ELISA results for liposomal formulations “ACI-24.060” (positive control sample, stored at 2-8 °C) and “ACI-24.0606M 25C” (stressed sample, stored 6 months at 23-27 °C). Optical density at 405 nm (average ⁇ standard deviation) is plotted against liposome dilution factor. Samples were tested in duplicates. The ELISA set up was as shown in Figure 2.
  • FIG. 11A and 11B Graphical representation of ACI-8039.044-918.14C2-Ab1 and 6E10 titration on ELISA plates coated with 3D-(6-acyl) PHAD® or biotinylated liposomes captured onto Neutravidin-coated ELISA plates. 6E10 was used as a control for liposomes displaying Abeta1-15 antigenic peptide. The following sequences are disclosed herein:
  • SEQ ID Nos 1-8 are sequences of the anti-MPLA antibody as shown in Example 6.
  • SEQ ID NO: 9 is amino acids 1-15 of -amyloid (A ) (DAEFRHDSGYEVHHQ).
  • SEQ ID NO: 10 is GGKESMPVDPDNEA.
  • a sandwich enzyme-linked immunosorbent assay was developed to assess antibody binding to an antigen displayed on the surface of a liposome.
  • a schematic of the ELISA set up is shown in Figure 1.
  • Three liposomal formulations were prepared, each displaying a surface molecule that is a peptide antigen derived from alpha-synuclein.
  • the three peptide antigens shared the same peptide sequence, GGKESMPVDPDNEA (SEQ ID NO: 10), and all comprised a hydrophobic moiety, comprising palmitic acid chains, but the location at which the palmitic acid chains were attached to the peptide differed between the three liposomal formulations.
  • ACI-7110.062 the peptide was tetrapalmitoylated via its N and C terminus; in ACI-7111.062, the peptide was dipalmitoylated via its N and C terminus; and in ACI-7108.062, the peptide was dipalmitoylated via its N terminus
  • the three formulations contained comparable peptide concentrations (464 to 491 pg/mL) and comparable lipid concentrations (16.2 to 16.8 mg/mL).
  • any indication provided herein of the weight of a peptide should be understood to refer to the weight of the peptide including any covalently attached moieties, such as lipid chains.
  • the reference to 464 to 491 pg/mL of the peptide should be understood to refer to the weight of the palmitoylated peptide (peptide sequence + palmitic chains).
  • the Mw of the three palmitoylated peptides are in the same range (2236.71 to 2969.87).
  • the liposomes all comprised the vaccine adjuvant MPLA, specifically 3D-(6-acyl) PHAD®.
  • the anti-MPLA antibody provided in Example 6 was used as a capture agent to capture the liposomes onto a solid support.
  • an ELISA was carried out to assess the capacity of the antibody to bind the antigens when displayed on the surface of a liposome.
  • Any anti-alpha-synuclein antibody having an epitope within the antigenic region of the antigen may suitably be used.
  • Suitable antibodies include any antialpha synuclein antibody having an epitope binding the antigenic peptide SEQ ID NO: 10 .
  • ACI-IL.062 was used as a negative control (ACI-IL.062 has the same composition as ACI-7110.062, ACI-7111.062 and ACI-7108.062 but does not comprise the alpha-synuclein antigenic peptide).
  • ELISA plates (Nunc MicroWell 96-Well, Non-Treated, Flat-Bottom Microplate, ThermoFisher Scientific) were coated with 5 pg/mL of ACI-8039.044-918.14C2-Ab1 (anti-MPLA murine lgG2a recombinant antibody) in PBS, overnight at 4 °C. Plates were then washed with 0.0025% Tween-20/PBS and blocked with 1% bovine serum albumin (BSA) in 0.0025% Tween-20/PBS for 1 hour at 37 °C. The blocking buffer was removed by flipping the plate and patting on a paper napkin.
  • BSA bovine serum albumin
  • ab209420 was then added to the wells at 0.5 pg/mL (ACI-7110.062, ACI-7108.062) or 5 pg/mL (ACI-7111.062) and incubated for 1 hour at 37 °C, after which the plates were washed.
  • Anti-rabbit IgG-AP an antibody that binds to the rabbit Fc of Anti-alpha-synuclein [MJFR1] antibody and is labelled with alkaline phosphatase; available from Abeam, catalogue ref. ab6722
  • MJFR1 Anti-alpha-synuclein
  • pNPP p-Nitrophenyl Phosphate
  • mice A total of 75 C57BL/6J female mice, approximately 10 weeks old at 1 st immunization, were allocated to nine groups (with 10 or 5 animals/group) as indicated in Table 2.
  • the nine groups were immunized three times by subcutaneous (s.c) injection into the subcutis of the dorsum on Days 1, 15 and 29 with either ACI-7108.062 (Groups 1 to 3), ACI- 7110.062 (Groups 4 to 6) or ACI-7111.062 (Groups 7 to 9) at 80, 2 or 0.2 pg of alpha- synuclein antigenic peptide/injection.
  • Immunogenicity against an a-synuclein-derived antigenic peptide was assessed in plasma samples collected at pre-dose and one week after each immunization (on Days 8, 22 and 36).
  • the anti-a-synuclein-derived antigenic peptide IgG titers were analyzed at each timepoint by an enzyme-linked immune sorbent assay (ELISA). Briefly, BSA conjugated to SEQ ID NO: 10 peptide was immobilized on 96-well micro titers plates overnight. After washing and blocking, plates were incubated with the plasma samples for two hours at 37°C, allowing the antibodies present in plasma to bind the peptide. After incubation, the plates were washed to remove non-reactive plasma components.
  • ELISA enzyme-linked immune sorbent assay
  • the antibody/antigen complex was detected via a secondary anti-mouse IgG antibody conjugated to alkaline phosphatase.
  • pNPP p-Nitrophenyl Phosphate
  • the anti-a- synuclein-derived antigenic peptide IgG titers were back-calculated against a calibration curve in eight two-fold serial dilution, using an unweighted four-parameter logistic regression model using the Gen5 software (BioTek, Switzerland). Results are expressed as AU/mL.
  • FIG. 4 shows that animals immunized with ACI-7108.062 and ACI-7110.062 vaccines developed a robust antibody response against the peptide of SEQ ID NO: 10 after one immunization, at all doses tested and in a dose dependent manner, with higher IgG titers observed in animals immunized at highest dose (80 pg dose), whereas animals immunized with ACI-7111.062 showed only low IgG titers at the highest dose tested (80 pg dose), with no IgG titers induced in 0.2 or 2 pg dose immunized animals.
  • an assay as exemplified in Example 1 provides an effective method for predicting antibody response to a liposomal vaccine or predicting the in vivo immunogenicity of a liposomal vaccine.
  • the Sandwich ELISA was also shown to be effective for determining the distribution of surface molecules, such as antigens, in a liposomal population.
  • Two liposomal formulations were prepared containing the same concentration of the same surface-displayed peptide antigen (Abeta1-15 (SEQ ID NO: 9)) but the peptide antigen was differently distributed in the liposomal population.
  • Concentrations are expressed as the concentration of the tetrapalm itoylated Abeta1-15 peptide antigen.
  • the peptide antigen was added to all liposomes in the liposome population to reach a final concentration of 200 pg/ml.
  • the peptide antigen was in a first step added to all liposomes in the liposome population to reach a final concentration of 400 pg/mL.
  • the resulting (intermediate) liposome population was subsequently mixed 1:1 with a liposome population lacking the peptide antigen to yield a heterogeneous liposomal vaccine preparation (i.e. a liposomal vaccine preparation comprising a substantial proportion of liposomes lacking the peptide antigen) denoted ACI- 24.060400 pg/mL+ACI-24E.O6O.
  • all (or substantially all) of the liposomes comprised 3D-(6-acyl) PHAD®, which served as a vaccine adjuvant targeted by a capture or analyte-binding agent.
  • ELISA plates were coated with an antigen comprising an antigenic peptide derived from Ap.
  • the liposomal formulation comprising the antigenic peptide derived from Ap as a surface-displayed antigen were used to compete with the ELISA plates for binding to an anti-Abeta antibody (antibody 6E10).
  • the difference in antigen distribution could be detected by sandwich ELISA using an “adjuvant-based capture” set-up (see Figure 1 for illustration), with results shown in Fig. 5.
  • the difference could also be detected using an “antigen-based capture” set up (see Figure 2 for illustration), with results shown in Fig. 6.
  • ELISA plates (Nunc MicroWell 96-Well, Non-Treated, Flat-Bottom Microplate, ThermoFisher Scientific) were coated with 5 pg/mL of ACI-8039.044-918.14C2-Ab1 (anti-MPLA human I gG 1 recombinant antibody) in PBS, overnight at 4 °C. Plates were then washed with 0.0025% Tween-20/PBS and blocked with 1% bovine serum albumin (BSA) in 0.0025% Tween-20/PBS for 1 hour at 37 °C. The blocking buffer was removed by flipping the plate and patting on paper napkin.
  • BSA bovine serum albumin
  • the plate was read at 405 nm using an ELISA plate reader (Tecan, Switzerland).
  • Emax The maximum effect (Emax) was calculated by plotting the optical density (OD) at 405 nm against the logarithms of concentrations of peptide using a four-parameter logistics (4-PL) with shared bottom and hill slope, using the GraphPad Prism (version 10.0.2) application. Results are summarized in Table 3.
  • ELISA plates (Nunc MicroWell 96-Well, Non-Treated, Flat-Bottom Microplate, ThermoFisher Scientific) were coated with 8 pg/mL of 6E10 (BioLegend Inc., ref. 803002) in PBS, overnight at 4 °C. Plates were then washed with 0.0025% Tween-20/PBS and blocked with 1% bovine serum albumin (BSA) in 0.0025% Tween-20/PBS for 1 hour at 37 °C. The blocking buffer was removed by flipping the plate and patting on a paper napkin.
  • BSA bovine serum albumin
  • EC50 half maximal effective concentration
  • Figure 5 and Table 3 show that a heterogeneous liposomal vaccine preparation (i.e. a liposomal vaccine preparation comprising a substantial proportion of liposomes lacking the peptide antigen) presents a decreased Emax OD compared to a homogeneous liposomal vaccine preparation (i.e. liposomal vaccine preparation wherein the peptide antigen was added to all liposomes in the liposome population) in the adjuvant-capture set-up.
  • Figure 6 and Table 3 show that a heterogeneous liposomal vaccine preparation presents a decreased EC50 compared to homogeneous liposomal vaccine preparations in the antigen-capture set-up.
  • Two liposomal vaccine preparations were produced containing comparable concentration of the tetrapalmitoylated Abeta1-15 antigenic peptide, which comprises SEQ ID NO: 9 (400- 460 pg/mL range), this concentration serving as a proxy for the liposome concentration.
  • the reference liposomal vaccine preparation was stored at long-term storage temperature (2- 8°C) and the stressed liposomal vaccine preparation was stored at 23-27 °C for 6 months. No change in peptide concentration was observed.
  • the capacity of antibodies to bind the vaccine adjuvant 3D-(6-acyl) PHAD® and the antigen Abeta1-15 antigenic peptide (SEQ ID NO:9), which is derived from Ap, displayed on the surface of the liposomes was determined using a sandwich enzyme-linked immunosorbent assay (ELISA) using an adjuvant-capture set up.
  • ELISA sandwich enzyme-linked immunosorbent assay
  • ELISA plates (Nunc MicroWell 96-Well, Non-Treated, Flat-Bottom Microplate, ThermoFisher Scientific) were coated with 5 pg/mL of ACI-8039.044-918.14C2-Ab1 (anti-MPLA human I gG 1 recombinant antibody) in PBS, overnight at 4 °C. Plates were then washed with 0.0025% Tween-20/PBS and blocked with 1% bovine serum albumin (BSA) in 0.0025% Tween-20/PBS for 1 hour at 37 °C. The blocking buffer was removed by flipping the plate and patting on a paper napkin.
  • BSA bovine serum albumin
  • pNPP p-Nitrophenyl Phosphate
  • Emax The maximum effect (Emax) was calculated by plotting the optical density at 405 nm against the logarithms of concentrations of peptide using a four-parameter logistics (4-PL) with shared bottom and hill slope, using the GraphPad Prism (version 10.0.2) application. Results are shown in Figure 8 and summarized in Table 4.
  • the capacity of antibodies to bind 3D-(6-acyl) PHAD® vaccine adjuvant and the antigen Abeta1-15 antigenic peptide (SEQ ID NO:9), which is derived from Ap, displayed on the surface of the liposomes was also determined using a sandwich enzyme-linked immunosorbent assay (ELISA) using an antigen capture set up.
  • ELISA sandwich enzyme-linked immunosorbent assay
  • ELISA plates (Nunc MicroWell 96-Well, Non-Treated, Flat-Bottom Microplate, ThermoFisher Scientific) were coated with 4 pg/mL of 6E10 (BioLegend Inc., ref. 803002) in PBS, overnight at 4 °C. Plates were then washed with 0.0025% Tween-20/PBS and blocked with 1% bovine serum albumin (BSA) in 0.0025% Tween-20/PBS for 1 hour at 37 °C. The blocking buffer was removed by flipping the plate and patting on a paper napkin.
  • 6E10 BioLegend Inc., ref. 803002
  • Emax The maximum effect (Emax) was calculated by plotting the optical density at 405 nm against the logarithms of concentrations of peptide using a four-parameter logistics (4-PL) with shared bottom, top and hill slope, using the GraphPad Prism (version 10.0.2) application. Results are shown in Figure 9 and summarized in Table 4.
  • the two groups were immunized three time by intramuscular immunization in the lateral surface of left quadriceps muscle on Days 1 , 15 and 29 with “ACI-24.060” (reference sample, stored at 2-8 °C) or “ACI-24.0606M 25C” (stressed sample, stored 6 months at 23-27 °C) at 0.02 pg of Pal1-15/injection.
  • a fixed volume of 0.05 mL/animal was administered for each injection, diluting the vaccine 1000-times in formulation buffer (10 mM histidine, 250 mM sucrose, pH 6.5) before injection.
  • Immunogenicity against Abetal -42 was assessed in plasma samples collected at pre-dose and one week after each immunization (on Days 8, 22 and 36).
  • the anti-Abeta1-42 IgG titers were analyzed at each timepoint by an enzyme-linked immune sorbent assay (ELISA). Briefly, Abeta1-42 peptide film was immobilized on 96-well micro titers plates overnight. After washing and blocking, plates were incubated with the plasma samples for two hours at 37°C, allowing the antibodies present in plasma to bind the Abetal- 42. After incubation, the plates were washed to remove non-reactive plasma components.
  • ELISA enzyme-linked immune sorbent assay
  • the antibody/antigen complex was detected via a secondary anti-mouse IgG antibody conjugated to alkaline phosphatase.
  • pNPP p-Nitrophenyl Phosphate
  • the anti- Abeta1-42 IgG titers were back-calculated against a calibration curve in eight two-fold serial dilution, using a four-parameter logistic regression model using the SoftMax Pro. Results are expressed as AU/mL. Results are shown in Figure 10 and Table 6. Data are expressed as individual values. Geometric mean ⁇ 95% confidence interval (Cl) is also indicated. Table 6
  • the stressed liposomal vaccine preparation induced a lower antibody response in-vivo than the reference liposomal vaccine.
  • the liposome-based vaccines were prepared according to the protocols published in W02012/055933.
  • Liposomal vaccine preparations comprising the Monophosphoryl Hexa-acyl Lipid A, 3-Deacyl (Synthetic) (3D-(6-acyl) PHAD®) adjuvant, hereinafter 3D-(6-acyl) PHAD®, were used for antibody generation.
  • mice Female C57BL/6JOIaHsd (C57BL/6) and BALB/c OlaHsd (BALB/c) wild-type mice (Harlan, USA) were received at 9 weeks of age. Vaccinations started at 10 weeks. Mice were vaccinated with the liposomal vaccine preparations by subcutaneous injection (s.c.) on days 0, 7, 28, 49. Mice were bled and heparinized plasma prepared 2 days before immunization (pre-immune plasma) and on days 14, 35 and 56 after first immunization. The presence of antibodies binding to 3D-(6-acyl) PHAD® was evaluated in mice plasma collected at day 56.
  • biotinylated control liposomal vaccine comprising only the 3D-(6-acyl) PHAD® adjuvant was captured on pre-coated Neutravidin plates to evaluate the specific response to the adjuvant by ELISA.
  • the dose response of mice presenting an anti-3D-(6-acyl) PHAD® titer was determined and the two mice showing the highest response were selected for the generation of hybridomas.
  • mice were euthanized and fusion with myeloma cells was performed using cells from lymph nodes and spleen. Screening for antibodies from the successfully fused hybridoma cell lines was performed by ELISA. The evaluation of the antibody response in hybridoma supernatant was carried out at multiple timepoints of sequential cell cloning steps leading to the final isolation of single cells defining a monoclonal hybridoma and the identification of ACI- 8039.044-918.14C2-Ab1 antibody.
  • ACI-8039.044-918.14C2-Ab1 antibody was further characterized by ELISA against various liposomal vaccine preparations comprising either MPLA, 3D-(6-acyl) PHAD® and/or Abetal- 15 antigenic peptide, as well as liposome controls without adjuvant to assess the specificity (Figure 11 A).
  • the different liposomal vaccine preparations were biotinylated and captured on pre-coated Neutravidin plates, or plates directly coated with 3D-(6-acyl) PHAD®.
  • the 6E10 antibody was used as a positive control for liposome displaying Abeta 1- 15 antigenic peptide ( Figure 11 B).
  • ACI-8039.044-918.14C2-Ab1 antibody binds to both liposome- displayed MPLA and 3D-(6-acyl) PHAD® as well as isolated 3D-(6-acyl) PHAD® and does not exhibit any cross-reactivity to Abeta1-15 antigenic peptide or any other components of the liposome.
  • VH (SEQ ID NO: 7)
  • VL (SEQ ID NO: 8)

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Pathology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Endocrinology (AREA)
  • Biophysics (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne des essais et des méthodes d'évaluation d'un vaccin comprenant un liposome, ainsi que des anticorps et des trousses utilisés dans ces essais et méthodes.
PCT/EP2025/057789 2024-03-22 2025-03-21 Dosage immunologique impliquant des anticorps se liant à des adjuvants vaccinaux Pending WO2025196262A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP24165620.6 2024-03-22
EP24165620 2024-03-22

Publications (1)

Publication Number Publication Date
WO2025196262A1 true WO2025196262A1 (fr) 2025-09-25

Family

ID=95064424

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2025/057789 Pending WO2025196262A1 (fr) 2024-03-22 2025-03-21 Dosage immunologique impliquant des anticorps se liant à des adjuvants vaccinaux

Country Status (1)

Country Link
WO (1) WO2025196262A1 (fr)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007068411A2 (fr) 2005-12-12 2007-06-21 Ac Immune S.A. Vaccin therapeutique
WO2010086294A2 (fr) 2009-01-28 2010-08-05 Epimmune Inc. Polypeptides de liaison de pan-dr et leurs utilisations
WO2012020124A1 (fr) 2010-08-12 2012-02-16 Ac Immune S.A. Vaccin obtenu par génie génétique
WO2012055933A1 (fr) 2010-10-26 2012-05-03 Ac Immune S.A. Préparation d'un produit de recombinaison antigénique
WO2019197414A1 (fr) 2018-04-10 2019-10-17 Ac Immune Sa Vaccins thérapeutiques anti-abêta

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007068411A2 (fr) 2005-12-12 2007-06-21 Ac Immune S.A. Vaccin therapeutique
WO2010086294A2 (fr) 2009-01-28 2010-08-05 Epimmune Inc. Polypeptides de liaison de pan-dr et leurs utilisations
WO2012020124A1 (fr) 2010-08-12 2012-02-16 Ac Immune S.A. Vaccin obtenu par génie génétique
WO2012055933A1 (fr) 2010-10-26 2012-05-03 Ac Immune S.A. Préparation d'un produit de recombinaison antigénique
WO2019197414A1 (fr) 2018-04-10 2019-10-17 Ac Immune Sa Vaccins thérapeutiques anti-abêta

Non-Patent Citations (13)

* Cited by examiner, † Cited by third party
Title
ABHINANDAN KRMARTIN ACR: "Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains", MOL IMMUNOL., vol. 45, 2008, pages 3832 - 9, XP023437109, DOI: 10.1016/j.molimm.2008.05.022
ALVING ET AL., INFECT. IMMUN., vol. 60, 1992, pages 2438 - 2444
BOGARD W C ET AL: "Human monoclonal antibody HA-1A binds to endotoxin via an epitope in the lipid A domain of lipopolysaccharide", JOURNAL OF IMMUNOLOGY, vol. 150, no. 10, 15 May 1993 (1993-05-15), pages 4438 - 4449, XP093276263, ISSN: 0022-1767, Retrieved from the Internet <URL:https://watermark.silverchair.com/4438.pdf?token=AQECAHi208BE49Ooan9kkhW_Ercy7Dm3ZL_9Cf3qfKAc485ysgAABeIwggXeBgkqhkiG9w0BBwagggXPMIIFywIBADCCBcQGCSqGSIb3DQEHATAeBglghkgBZQMEAS4wEQQMcXNFW_pQWQcPuuxxAgEQgIIFleZzbMDMfyJBBHotc0eEqIr70jAmSbVOmgpHTkvRcqoArEJe0H0g3TlMOGIeEP9M7j1OAM5-oPO6aiZekN_cQet1eU1zuFD> DOI: 10.4049/jimmunol.150.10.4438 *
CHOTHIA CLESKAM: "Canonical structures for the hypervariable regions of immunoglobulins", J MOL BIOL., vol. 196, no. 4, 20 August 1987 (1987-08-20), pages 901 - 17, XP024010426, DOI: 10.1016/0022-2836(87)90412-8
ESTRADA MARCUS ET AL: "Development of a quantitative ELISA for SARS-CoV-2 vaccine candidate, NDV-HXP-S, with CpG 1018 adjuvant", HUMAN VACCINES & IMMUNOTHERAPEUTICS, vol. 20, no. 1, 19 February 2024 (2024-02-19), US, XP093276239, ISSN: 2164-5515, Retrieved from the Internet <URL:https://pmc.ncbi.nlm.nih.gov/articles/PMC10877971/pdf/KHVI_20_2315709.pdf> DOI: 10.1080/21645515.2024.2315709 *
GIUDICELLI VCHAUME DBODMER JMULLER WBUSIN CMARSH SBONTROP RMARC LMALIK ALEFRANC MP, NUCLEIC ACIDS RES., vol. 25, 1 January 1997 (1997-01-01), pages 206 - 1
KABAT: "Sequences of Proteins of Immunological Interest", 1991, NIH PUBLICATION
KATZ J B ET AL: "In vitro assessment of viral antigen content in inactivated aluminum hydroxide adjuvanted vaccines", JOURNAL OF VIROLOGICAL METHODS, ELSEVIER BV, NL, vol. 25, no. 1, 1 July 1989 (1989-07-01), pages 101 - 108, XP023696300, ISSN: 0166-0934, [retrieved on 19890701], DOI: 10.1016/0166-0934(89)90104-3 *
KENNEDY, J. H. ET AL., CLIN. CHIM. ACTA, vol. 70, 1976, pages 1 - 31
KOHLER, G.MILSTEIN, C.: "Continuous cultures of fused cells secreting antibody of predefined specificity", NATURE, vol. 256, 1975, pages 495 - 497, XP037052082, DOI: 10.1038/256495a0
LEFRANC MP: "Unique database numbering system for immunogenetic analysis", IMMUNOL TODAY., vol. 18, no. 11, November 1997 (1997-11-01), pages 509, XP004093509, DOI: 10.1016/S0167-5699(97)01163-8
MACCALLUM RMMARTIN ACTHORNTON JM, J MOL BIOL., vol. 262, no. 5, 11 October 1996 (1996-10-11), pages 732 - 45
SCHURS, A. H. W. M. ET AL., CLIN. CHIM ACTA, vol. 57, 1977, pages 1 - 40

Similar Documents

Publication Publication Date Title
US10696739B2 (en) Anti-tau antibodies
DK2368907T3 (en) Anti-Abeta antibodies and their use
TWI827557B (zh) Ｔａｕ胜肽之組成物及其用途
TW202045543A (zh) 抗ｔｒｅｍ２抗體及其使用方法
RU2011144307A (ru) Фармацевтическая композиция
KR20030066695A (ko) 베타 아밀로이드 펩티드를 인식하는 인간화된 항체
JP7018885B2 (ja) 食道癌の検出および治療のための組成物および方法
WO2019098763A9 (fr) ANTICORPS ANTI-α-SYNUCLÉINE ET LEURS UTILISATIONS
JP2022526528A (ja) ピログルタメートアミロイド－βに対する抗体及びその使用
JP2022009080A (ja) 卵巣癌の検出および治療のための組成物および方法
TW202110478A (zh) Tau疫苗之異種投予
US20130302831A1 (en) Methods and kits for detecting and diagnosing neurotrauma
JP2021100936A (ja) ハンチントン病の予防および／または処置における使用のための物質および方法
WO2025196262A1 (fr) Dosage immunologique impliquant des anticorps se liant à des adjuvants vaccinaux
TW202423962A (zh) 製造細胞外囊泡的方法及細胞外囊泡的用途
JP6030650B2 (ja) ポリオーマウイルス関連病態を阻害するための方法および組成物
TW201623329A (zh) 針對骨調素截斷變異體的疫苗及單株抗體暨其用途
CN113424060B (zh) 抗纳洛酮和抗纳曲酮单克隆抗体及其生产和使用方法
US20190275125A1 (en) Abeta variants, assay, method and treatment of alzheimer&#39;s disease
US20220221455A1 (en) Antigen binding proteins and assays
Varnado Measuring Activation of the Cytosolic DNA Sensing Pathway
EA048027B1 (ru) АНТИТЕЛА К α-СИНУКЛЕИНУ И ИХ ПРИМЕНЕНИЯ
HK40039870B (zh) 磷酸化Tau肽的组合物及其用途
AU2017204635A1 (en) Methods and kits for detecting and diagnosing neurotrauma
JP2017223636A (ja) 神経自己免疫疾患の診断

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 25712980

Country of ref document: EP

Kind code of ref document: A1