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WO2025137264A1 - Substitut à petites molécules pour épitope de neutralisation sur la protéine d'enveloppe du virus zika - Google Patents

Substitut à petites molécules pour épitope de neutralisation sur la protéine d'enveloppe du virus zika Download PDF

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Publication number
WO2025137264A1
WO2025137264A1 PCT/US2024/061012 US2024061012W WO2025137264A1 WO 2025137264 A1 WO2025137264 A1 WO 2025137264A1 US 2024061012 W US2024061012 W US 2024061012W WO 2025137264 A1 WO2025137264 A1 WO 2025137264A1
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WIPO (PCT)
Prior art keywords
zikv
binding
binding reagent
czv1
antibody
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PCT/US2024/061012
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English (en)
Inventor
Ernesto Torres De Azeved Marques, Jr.
Priscila MAYRELLE DA SILVA CASTANHA
Thomas Kodadek
Donald S. Burke
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University of Florida
University of Pittsburgh
University of Florida Research Foundation Inc
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University of Florida
University of Pittsburgh
University of Florida Research Foundation Inc
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Publication of WO2025137264A1 publication Critical patent/WO2025137264A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/438—The ring being spiro-condensed with carbocyclic or heterocyclic ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
    • C07D491/10—Spiro-condensed systems
    • C07D491/113—Spiro-condensed systems with two or more oxygen atoms as ring hetero atoms in the oxygen-containing ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00
    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/18—Togaviridae; Flaviviridae
    • G01N2333/183—Flaviviridae, e.g. pestivirus, mucosal disease virus, bovine viral diarrhoea virus, classical swine fever virus (hog cholera virus) or border disease virus
    • G01N2333/185—Flaviviruses or Group B arboviruses, e.g. yellow fever virus, japanese encephalitis, tick-borne encephalitis, dengue
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the binding reagent of any of clauses 1-7 linked to a surface for use in an immunoassay.
  • 9. The binding reagent of any of clauses 1-8, linked to a nitrocellulose membrane, a cotton linter membrane, a glass fiber membrane, a silicon chip, a magnetic bead, a gold bead, a microelectrode, a microfluidic device, a flow cytometry bead, and/or a multi-well plate. 60M4845.DOCX 3 Attorney Docket No. 06527-2407085 [0022] 10. The binding reagent of any of clauses 1-9, linked to a bead or particle having a diameter of from about 10 nm to about 200 ⁇ m.
  • Figure 4 shows chemical structures of the building blocks used at the R1, R2, R3, R5 and R6 positions (related to Figure 1).
  • Figure 5 shows chemical structures of the aldehydes used to install the R4 unit (related to Figure 1).
  • Figure 6 shows a flowchart illustrating the down selection of Zika-specific reactive PICCOs identified during the screening procedure.
  • Figure 7 shows a distribution of PICCOs in terms of the number of sample hits.
  • Figure 8 shows a number of distinct PICCO compounds detected once, twice, or multiple times across Zika discovery runs.
  • Figure 9 shows a heatmap showing the chemical sequence similarity clustering of the screening candidate PICCOs. Five Zika-specific clusters encompassing 40 unique PICCO candidates were identified.
  • Figure 10 shows a comparison of antibody specificity profiles of Zika-specific PICCOs from within the same chemical sequence cluster versus distinct clusters.
  • Figure 11 shows the antibody binding profile against CZV1-1 and a “des-aldehyde” side product CZV1-1a. 60M4845.DOCX 5 Attorney Docket No.
  • Figure 12 shows a binding profile of Zika-specific antibodies to CZV1-1 derivatives.
  • Figure 13 shows validation of CZV1-1 as a Zika diagnostic marker. The receiver operating characteristics (ROC) curve for all samples and for the DENV-immune patients is shown. The calculated area under the curve (AUC) and 95% confidence intervals (95% CI) are depicted.
  • Figures 14A-14B show a binding profile of the purified IgG specific to CZV1-1 against (Panel A) ZIKV envelope protein (full-length and domain III) and (Panel B) DENV1- 4 envelope proteins.
  • ZIKV FL Full-length envelope protein
  • ZIKV DIII domain III of ZIKV envelope protein.
  • Figure 16 shows binding of purified IgG against CZV1-1 and several Zika-specific (ZV2, ZV48, ZV64, ZV67) or dengue-specific (DV10 and 2H2) monoclonal antibodies against DENV (Panel A) and ZIKV (Panel B) envelope proteins.
  • Figure 17 shows the binding free energy minima for the mAb/CZV1-1 complexes. The black arrow indicates the compound that obtained the most negative binding free energy, and thus the strongest association.
  • Figures 18A-C show that anti-Zika human sera reacts with BSA-CZV1-1 and CZV1-1-Cys in an ELISA format.
  • Figure 19 shows that mice immunized with CZV1-1-KLH and CZV1-1-Cys induce very high levels of antibodies anti-CZV1-1.
  • Figures 20A-C show that CZV1-1 immunization elicited very high titers of anti- CZV1-1 antibodies .
  • Figure 21 shows that mice immunized with CZV1-1-KLH induce anti-Zika envelope protein. 60M4845.DOCX 6 Attorney Docket No.
  • a “binding reagent” is a reagent, compound, or composition, e.g., a ligand, able to specifically bind a target compound, such as, for example and without limitation: anti-Zika virus (ZIKV) antibodies, such as anti-Zika virus (ZIKV) antibody paratope-containing molecules, for example to the exclusion of substantial binding to other antibodies, such as anti-dengue virus antibodies.
  • ZIKV anti-Zika virus
  • ZIKV anti-Zika virus
  • Binding reagents include, without limitation, antibodies (polyclonal, monoclonal, humanized, etc.), antibody fragments (e.g., a recombinant scFv), antibody mimetics such as affibodies, affilins, affimers, affitins, alphabodies, anticalins, avimers, DARPins, fynomers, monobodies, nucleic acid ligands (e.g., aptamers), engineered proteins, antigens, epitopes, epitope surrogates, haptens, or any target-specific binding reagent.
  • antibodies polyclonal, monoclonal, humanized, etc.
  • antibody fragments e.g., a recombinant scFv
  • antibody mimetics such as affibodies, affilins, affimers, affitins, alphabodies, anticalins, avimers, DA
  • binding reagents includes as a class: monoclonal antibodies, or derivatives or analogs thereof, including without limitation: Fv fragments, single chain Fv (scFv) fragments, Fab' fragments, F(ab')2 fragments, single domain antibodies, camelized antibodies and antibody fragments, humanized antibodies and antibody fragments, multivalent versions of the foregoing, and any paratope-containing compound or composition; multivalent activators including without limitation: monospecific or bispecific antibodies, such as disulfide stabilized Fv fragments, scFv tandems ((scFv)2 fragments), 60M4845.DOCX 7 Attorney Docket No.
  • a “non-peptide” molecule is a molecule that does not comprise a polypeptide moiety of two or more contiguous amino acids linked in a chain by conventional peptide bonds.
  • CZV1-1 is a non-peptide molecule.
  • CZV and “CZ” are used interchangeably (e.g., CVZ1-1 is the same as CZ1-1).
  • a non-protein molecule may be linked to a peptide-containing molecule, such as, without limitation, a protein or polypeptide chain or glycoprotein, such as in a hapten-carrier complex with a non- peptide hapten moiety, or in the case of a peptide-containing surface to which the non-peptide molecule is linked.
  • epitope surrogate refers to an artificial molecule or composition that replicates naturally occurring epitopes.
  • An epitope surrogate may bind anti- virus antibodies, for example, anti-zika virus antibodies, and in examples, does not bind anti- dengue virus antibodies to any significant extent.
  • An epitope surrogate may be a peptide molecule or composition that that specifically bind antibodies, e.g., antibodies relevant to a specific disease or condition.
  • An epitope surrogate may be a non-peptide molecule or composition that bind antibodies relevant to a specific disease or condition.
  • An epitope surrogate may be used as an immunogen or hapten for use in generating antibodies or an antibody response to a specific target antigen, such as, for example and without limitation, to a flavivirus or a zikavirus antigen as described herein.
  • hapten refers to any molecule that does not generate an immunogenic reaction on its own but can be made to if it is conjugated to a suitable carrier.
  • Carriers for haptens may be immunogenic on their own, or may produce no immune response without conjugation to a hapten.
  • Carriers for haptens (also called hapten carriers or hapten- carriers) may include, but are not limited to, gelatin, serum globulins, albumin, keyhole 60M4845.DOCX 8 Attorney Docket No.
  • KLH limpet hemocyanin
  • PPD purified protein derivative
  • Typhi Vi antigen, liposomes, polysaccharides (e.g., AECM-Ficoll, dextran, agar, carboxymethyl cellulose), synthetic polypeptides (e.g., poly-L-lysine and poly- L-glutamic acid), inorganic gold particles, dendrimers, and nanodiscs, outer membrane vesicles, generalized modules for membrane antigens (GMMA), glycoengineered proteins, virus-like particles, protein nanocages, and peptides (see, e.g., Lemus R, Karol MH. Conjugation of haptens. Methods Mol Med. 2008;138:167-82 and Van der Put RMF, Metz B, Pieters RJ.
  • polysaccharides e.g., AECM-Ficoll, dextran, agar, carboxymethyl cellulose
  • synthetic polypeptides e.g., poly-L-lysine and poly- L-glutamic acid
  • inorganic gold particles
  • An immune response is a response of a cell of the immune system, such as a B-cell, T-cell, macrophage or polymorphonucleocyte, to a stimulus, such as an antigen.
  • An immune response may include any cell of the body involved in a host defense response for example, an epithelial cell that secretes an interferon or a cytokine.
  • An immunogen refers to a compound, composition, or substance which is capable, under appropriate conditions, of eliciting, stimulating, or raising an immune response, such as the production of antibodies, such as neutralizing antibodies, or a T-cell response in a patient, including compositions that are injected or absorbed into a patient.
  • “to immunize” and/or “immunizing” refers to rendering a subject protected from an infectious disease, such as by vaccination.
  • a vaccine refers to a preparation of immunogenic material capable of stimulating or eliciting an immune response, administered for the prevention, inhibition, amelioration, or treatment of infectious, such as ZIKV infections, or other types of disease.
  • the immunogenic material may include antigenic proteins, peptides, non-peptides, or DNA derived from them. Vaccines may elicit both prophylactic (preventative or protective) and therapeutic responses. Methods of 60M4845.DOCX 9 Attorney Docket No.
  • 06527-2407085 administration vary according to the vaccine, but may include inoculation by topical, intramuscular, intravenous, subcutaneous, intradermal, subdermal, ingestion, inhalation, intraocular, intraperitoneal, and/or other routes of administration as known to those of skill in the art.
  • Vaccines may be administered with an adjuvant to boost the immune response raised in the patient.
  • a “moiety” refers to a portion of a larger molecule and may comprise a group that has a chemical or biological function, such as a reactive group such as an ethynyl group or an acetamide group, or a group that results in the binding of a surrogate epitope to an antibody, paratope, or binding partner.
  • antibody fragment refers to any derivative of an antibody which is less than full-length. In exemplary embodiments, the antibody fragment retains at least a significant portion of the full-length antibody's specific binding ability. Examples of antibody fragments, but are not limited to, Fab, Fab′, F(ab′)2, Fv, Fd, dsFv, scFv, diabody, triabody, tetrabody, di-scFv (dimeric single-chain variable fragment), bi-specific T-cell engager (BiTE), single-domain antibody (sdAb), or antibody binding domain fragments.
  • the antibody fragment may be produced by any means.
  • the antibody fragment may be enzymatically or chemically produced by fragmentation of an intact antibody, or it may be recombinantly or synthetically produced.
  • the antibody fragment may optionally be a single chain antibody fragment.
  • the fragment may comprise multiple chains which are linked together, for instance, by disulfide linkages.
  • the fragment may also optionally be a multi-molecular complex.
  • a functional antibody fragment may consist of at least about 50 amino acids or at least about 200 amino acids.
  • Antibody fragments also include miniaturized antibodies or other engineered binding reagents, such as scFvs, that exploit the modular nature of antibody structure, comprising, often as a single chain, one or more antigen-binding or epitope-binding (e.g., paratope) sequences and, at a minimum, any other amino acid sequences needed to ensure appropriate specificity, delivery, and stability of the composition.
  • a binding reagent may be conjugated (covalently linked) or otherwise attached to a surface such as beads, e.g., magnetic beads or agarose beads (e.g., SEPHAROSE beads), or a membrane, e.g., a nitrocellulose membrane.
  • the binding reagent may be conjugated to a semi-conductor polymer such as polyaniline, poly-(3-amino-4- hydroxybenzoic acid)-modified pencil carbon graphite, and/or Electrocatalytic Prussian Blue Nanostructured Film.
  • a binding reagent may also be attached to another mobile compound, 60M4845.DOCX 10 Attorney Docket No. 06527-2407085 including but not limited to a carrier, e.g., a carrier protein. Attachment may be accomplished, among other methods, by adding a terminal cysteine to the binding reagent and conjugating it to the surface by forming a thioether bond.
  • the antibodies bound to the surface-conjugated binding reagent can be reconstituted by dissociating the target antibodies from the binding reagent by washing according to common practice.
  • the binding reagent may be an epitope surrogate, or any other suitable binding reagent.
  • the surface may be a magnetic bead, and the bound target may then be removed by magnetic removal.
  • the surface may be particles or beads retained in a column.
  • the surface may be a membrane that is part of, for example and without limitation, a lateral flow device.
  • methods of using binding reagents as described herein may include neutralization and/or purification, and a person of ordinary skill can readily envision a large variety of ways neutralization/affinity purification may be used to remove and/or neutralize a target.
  • a bead or particle to which the binding reagents described herein may be linked may be selected from a polystyrene bead, a magnetic bead, and/or a gold particle. Such beads and particles are known, and may be of any suitable size.
  • a bead and/or particle useful with the binding reagents described herein may have a diameter ranging from 10 nanometers (nm) to 200 micrometers ( ⁇ m), all values and subranges therebetween inclusive.
  • compositions including a binding reagent or any pharmaceutically-acceptable salt thereof for a Zika virus (ZIKV) antibody paratope pharmaceutical compositions including such binding reagents or any pharmaceutically- acceptable salt thereof, and methods of detecting ZIKV antibodies in a sample obtained from a patient, eliciting an immune response to ZIKV, identifying antibodies to ZIKV, and neutralizing ZIKV in patients.
  • the term “paratope” means a portion of an antibody that recognizes and binds to an epitope of an antigen, for example, an epitope of ZIKV.
  • the epitope of ZIKV is an envelope protein.
  • the envelope protein is in the Domain III (DIII) of the ZIKV envelope protein, more specifically a cryptic epitope in the C-C’ loop arranged within the ‘sandwich’ core of DIII.
  • Suitable compositions may have a structure according to one or more of the following depicted in FIG. 1.
  • a binding reagent as described herein has the following structure: 60M4845.DOCX 11 Attorney Docket No. 06527-2407085 .
  • Certain compounds described here may have asymmetric centers and therefore exist in different enantiomeric and diastereomeric forms.
  • a compound can be in the form of an optical isomer or a diastereomer. Accordingly, compounds described herein include their optical isomers, diastereoisomers and mixtures thereof, including a racemic mixture unless otherwise specified.
  • stereoisomer means one stereoisomer of a compound that is substantially free of other stereoisomers of that compound.
  • a stereomerically pure compound having one chiral center will be substantially free of the opposite enantiomer of the compound.
  • a stereomerically pure compound having two chiral centers will be substantially free of other diastereomers of the compound.
  • a typical stereomerically pure compound comprises greater than about 80% by weight of one stereoisomer of the compound and less than about 20% by weight of other stereoisomers of the compound, for example greater than about 90% by weight of one stereoisomer of the compound and less than about 10% by weight of the other stereoisomers of the compound, or greater than about 95% by weight of one stereoisomer of the compound and less than about 5% by weight of the other stereoisomers of the compound, or greater than about 97% by weight of one stereoisomer of the compound and less than about 3% by weight of the other stereoisomers of the compound.
  • all compounds and/or structures described herein comprise all possible stereoisomers, individually or mixtures thereof.
  • the compound and/or structure may be an enantiopure preparation consisting essentially of an (-) or (+) enantiomer of the compound, or may be a mixture of enantiomers in either equal (racemic) or unequal proportions. 60M4845.DOCX 12 Attorney Docket No. 06527-2407085 [0076] Binding reagents such as the compounds disclosed herein may include a fluorescent tag or other colorimetric indicator tag, such as a non-fluorescent dye or particle or enzyme, as are broadly-known, to allow for detection in binding assays and the like.
  • Target antibodies isolated using binding reagents such as the compounds disclosed herein may include a fluorescent tag or other colorimetric indicator tag, such as a non-fluorescent dye or particle or enzyme, as are broadly-known, to allow for detection in binding assays and the like.
  • suitable tags include, without limitation: fluorescent proteins (e.g. green fluorescent protein, GFP), cyanine based fluorescent dyes (e.g. cyanine-3, Cy3), triarylmethane dyes such as fluorescein based fluorescent dyes (e.g. fluorescein isothiocyanate, FITC) and rhodamine based fluorescent dyes (e.g.
  • tetramethylrhodamine isothiocyanate TRITC
  • coumarin based fluorescent dyes phthalocyanine
  • colloidal gold latex beads
  • quantum dots magnetic beads
  • horseradish peroxidase alkaline phosphatase
  • ⁇ - galactosidase alkaline phosphatase
  • acetylcholinesterase catalase
  • glucose oxidase glucose oxidase
  • Binding reagents or any pharmaceutically-acceptable salt thereof, such as the compounds disclosed herein, may also be formulated as pharmaceutical compositions for administration to a patient, for example to elicit an immune response and/or to neutralize ZIKV and/or to treat one or more conditions associated with infection with ZIKV, including at least Guillain-Barré syndrome, neuropathy, and/or myelitis.
  • Binding assays are assays that utilize at least one binding reagent to determine the presence or concentration of analyte(s) and/or the interactions between two molecules.
  • Immunoassays are highly selective bioanalytical methods that measure the presence or concentration of analytes in a solution using an antibody or an antigen as a biorecognition agent. They are based on the antibody-antigen immunoreaction and can achieve high specificity and sensitivity through signal transduction and amplification. Immunoassays may include, but are not limited, to enzyme-linked immunosorbent assays (ELISAs), lateral flow tests, magnetic immunoassays, western blots, dot blots, lateral flow tests, fluorescence activated cell sorting assays (FACS), antibody-binding assays, competitive binding assays, and immunoprecipitation.
  • ELISAs enzyme-linked immunosorbent assays
  • FACS fluorescence activated cell sorting assays
  • ELISAs are a plate-based assay technique designed for detecting and quantifying soluble substances such as peptides, proteins, antibodies, and hormones. Other names, such as enzyme immunoassay (EIA), are also used to describe the same technology.
  • EIA enzyme immunoassay
  • the antigen target macromolecule
  • microplate solid surface
  • Detection is accomplished by 60M4845.DOCX 13 Attorney Docket No.
  • Immunoassay devices are devices designed to implement highly selective bioanalytical methods that measure the presence or concentration of analytes in a solution using an antibody or an antigen as a biorecognition agent, including, but not limited to, lateral flow devices.
  • Lateral flow devices are devices in which binding reagents are immobilized on a strip of nitrocellulose or other suitable membrane, and a sample is applied at one end of the strip and any antibodies present diffuse along it until they reach the position where the appropriate binding reagent is immobilized.
  • Therapeutic compositions including those containing the binding reagents or any pharmaceutically-acceptable salt thereof disclosed herein, may comprise a pharmaceutically acceptable carrier, or excipient.
  • An excipient is an inactive substance used as a carrier for the active ingredients of a medication. Although "inactive,” excipients may facilitate and aid in increasing the delivery or bioavailability of an active ingredient in a drug product.
  • Non- limiting examples of useful excipients include: adjuvants, antiadherents, binders, rheology modifiers, carriers, coatings, disintegrants, emulsifiers, oils, buffers, salts, acids, bases, fillers, diluents, solvents, flavors, colorants, glidants, lubricants, preservatives, antioxidants, sorbents, vitamins, sweeteners, etc., as are available in the pharmaceutical/compounding arts.
  • the compositions, methods, and dosage forms disclosed herein may include one or more adjuvants or molecules with immune stimulant or adjuvant effect.
  • an adjuvant is not included in the composition, but is separately administered to a subject (for example, in combination with a composition disclosed herein) before, after, or substantially simultaneously with administration of one or more of the immunogen-containing compositions disclosed herein.
  • Adjuvants are agents that increase or enhance an immune response in a subject administered an antigen, compared to administration of the antigen in the absence of an adjuvant.
  • an adjuvant is an aluminum salt, such as aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate, or aluminum hydroxyphosphate.
  • adjuvants include biological adjuvants, such as cytokines (for example, IL-2, IL-6, IL-12, RANTES, GM-CSF, TNF- ⁇ , or IFN- ⁇ ), growth factors (for example, GM-CSF or G-CSF), one or more molecules such as OX-40L or 4-1 BBL, 60M4845.
  • cytokines for example, IL-2, IL-6, IL-12, RANTES, GM-CSF, TNF- ⁇ , or IFN- ⁇
  • growth factors for example, GM-CSF or G-CSF
  • one or more molecules such as OX-40L or 4-1 BBL, 60M4845.
  • DOCX 14 Attorney Docket No. 06527-2407085 immunostimulatory oligonucleotides (for example, CpG oligonucleotides, for example, see U.S. Pat. Nos.
  • An adjuvant may be a water-in-oil emulsion in which antigen solution is emulsified in mineral oil (for example, Freund's incomplete adjuvant), sometimes with the inclusion of killed mycobacteria (Freund's complete adjuvant) to further enhance antigenicity.
  • the adjuvant is a mixture of stabilizing detergents, micelle-forming agent, and oil available under the name PROVAX® (IDEC Pharmaceuticals, San Diego, Calif.).
  • One of skill in the art can select a suitable adjuvant or combination of adjuvants to be included in the compositions disclosed herein or administered to a subject in combination with the compositions disclosed herein.
  • Molecules with immune stimulant or adjuvant effects include, without limitation: TLR3 agonists such as Poly(I:C) or Poly-ICLC; TLR 4 agonists such as LPS or monophosphoryl lipid derivatives; TLR 5 agonists such as flagellin derivatives; TLR 7/8 agonists such as imiquimod or R848; TLR 9 agonists such as CpG sequences; Stimulator of Interferon Genes (STING) pathway agonists such as ADU-S100; stimulatory neuroimmune mediators such as calcitonin gene-related peptide (CGRP); neurokinin 1 (NK1) receptor agonists such as Hemokinin 1 and Substance P; saponin related adjuvants such as QS-21 (Quillaja saponaria); purinoergic receptor agonists such as ATP; or oil-in-water emulsion adjuvants such as MF59.
  • TLR3 agonists such as Poly(I:C) or Poly-IC
  • Useful dosage forms for the binding reagents or any pharmaceutically-acceptable salt thereof disclosed herein include, for example and without limitation: parenteral, intravenous, intramuscular, intraocular, or intraperitoneal solutions, oral tablets or liquids, topical drops, ointments, or creams, and transdermal devices (e.g., patches).
  • the compound may be a sterile solution comprising the active ingredient (drug or compound), and a solvent, such as water, saline, lactated Ringer's solution, or phosphate-buffered saline (PBS). Additional excipients, such as polyethylene glycol, emulsifiers, salts and buffers may be included in the solution.
  • compositions may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use. Extemporaneous solutions and suspensions may be prepared from sterile powders, granules and tablets.
  • Therapeutic/pharmaceutical compositions as described herein may be prepared in accordance with acceptable pharmaceutical procedures, such as described in Remington: The Science and Practice of Pharmacy, 21 st edition, ed. Paul Beringer et al., Lippincott, Williams & Wilkins, Baltimore, MD Easton, Pa.
  • Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage.
  • sterile injectable solutions can be prepared by incorporating the active agent in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • binding reagents and any pharmaceutically-acceptable salt thereof and/or pharmaceutical compositions including the same as disclosed herein for treatment of individuals at risk of being infected with, or who are infected with, ZIKV.
  • a pharmaceutical composition including binding reagent and any pharmaceutically-acceptable salt thereof as disclosed herein may be administered to a patient at risk of being infected with ZIKV, thereby eliciting an immune 60M4845.
  • DOCX 16 Attorney Docket No. 06527-2407085 response to ZIKV.
  • Lateral flow device 10 may include a housing 12 comprising a first hole 14 into which a sample is placed according to common usage, and a second hole 16, permitting visualization of a membrane 18, such as a nitrocellulose membrane, internal to the housing 12 and spanning the first 14 and second 16 holes, the extent of which is shown in phantom in the bottom schematic.
  • a test line 20 and a control line 21 (e.g., positive control), where, for example, the test line comprises, affixed to the membrane 18, a bound binding reagent, such as membrane-bound CZV1-1, and the control line 21 comprises a surface-bound reagent that provides confirmation that an indicator used in the assay is functioning.
  • the indicator such as a colorimetric indicator
  • the indicator is typically absorbed into the membrane 18 between the first 14 and second 16 holes, and when liquid is placed in the first hole 14, it may migrate along the membrane 18 past all testing lines, carrying the indicator past all test lines.
  • Test line 20 is not visible unless any test sample placed in the first hole comprises the analyte to be tested, such as an anti-zikavirus antibody. Additional test lines may be added for binding different antibodies or other analytes.
  • FIG. 2 is illustrative of one example of a lateral flow device.
  • 06527-2407085 control patient populations with different colored fluorescent dyes by pre-incubation with dye-conjugated single-chain anti-IgG antibodies.
  • the serum samples are then mixed and incubated with a one-bead-one-compound (OBOC) DNA-encoded library (DEL) of synthetic, nonpeptidic molecules.
  • OBOC one-bead-one-compound
  • DEL DNA-encoded library
  • the beads are passed through a fluorescence-activated cell sorter (FACS) gated to collect beads that display a high level of fluorescence in the “case channel” but a low level of fluorescence in the “control channel”. The assumption is that these beads display a ligand for the antigen-binding site of antibodies that are present at much higher levels in the case sera than the control sera.
  • FACS fluorescence-activated cell sorter
  • epitope surrogates are called epitope surrogates.
  • epitope surrogate technology has the additional advantage of providing a pathway to identify the native antigen recognized by the differentiating antibodies. This involves using an immobilized epitope surrogate as an affinity reagent to enrich from the serum the IgGs to which it binds. These antibodies can then be used to identify the native antigen they recognize by either testing purified candidate antigens in ELISA assays or by carrying out immunoprecipitation from extracts that contain the native antigen, then identifying the retained proteins by mass spectrometry.
  • PICCOs peptoid-inspired conformationally constrained oligomers
  • the dengue panel consisted of serial samples from patients with confirmed (by PCR, IgM, and IgG and neutralization assays) primary dengue infections and divided into two groups: a) acute samples collected no more than 7 days from the start of the symptoms and confirmed as dengue-IgG negative by ELISA, and b) convalescent samples collected after 23 days from the start of the symptoms and confirmed as anti-dengue IgG positive.
  • Dengue samples were from two natural history cohorts carried out in Northeast Brazil before Zika introduction in the area (2004-2009) (Table 2). 60M4845.DOCX 20 Attorney Docket No. 06527-2407085 Table 2 60M4845.DOCX 21 Attorney Docket No.
  • Hierarchical clustering analysis [0097] This analysis was carried out to determine if two or more highhit PICCOs were similar in structure. We assigned a probability of similarity for each of the six chemical components based on the number of alternative components at that site. For positions one through six, the number of chemical component synthesis options at each position were respectively 2, 6, 6, 81, 7, and 13.
  • the hierarchical clustering method used was Ward's minimum variance method, to generate compact, spherical clusters.
  • Chemical structure clusters were defined as contiguous groups with similarity probability scores of -4 or lower.
  • Affinity-purification of IgG antibody from serum [0098]
  • Antibodies that bind CZV1-1 were enriched from a pool of ZIKV immune sera by affinity purification.
  • CZV1-1 was covalently immobilized to a SulfoLinkTM affinity column (ThermoFisher) following the manufacturer’s protocol. Undiluted, pooled ZIKV immune sera was incubated to the column overnight at 4°C. The column was then washed, and the bound IgG was eluted using Gentle Ag/Ab elution buffer pH 6.6 (ThermoFisher). The IgG was dialyzed overnight, and the sample was concentrated using centrifugal concentrators. Total IgG levels were quantified using a sandwich ELISA.
  • Binding IgG ELISA Assays [0099] To assess the binding of the purified IgG-CZV1 to different ZIKV and DENV antigens, high-binding, half-area 96-well flat-bottom polystyrene plates (Corning) were coated with the following recombinant proteins (at 2 ⁇ g/ml) or cell extracts in carbonate/bicarbonate buffer overnight at 4°C: ZIKV or DENV envelope protein (Sino Biologicals), ZIKV or DENV1-4 NS1 proteins (Native Antigen), ZIKV or DENV1-4 cell extracts, and ZIKV or DENV1-4 cell lysates.
  • NFDM non-fat dry milk
  • Bio-Rad non-fat dry milk
  • purified IgG- CZV1 diluted in 5% (w/v) of NFDM in PBS were added to the plate at different concentrations and incubated for 1 hour at room temperature. Plates were washed six times with PBS-T and incubated for 1 hour with horseradish peroxidase (HRP)-linked goat antihuman IgG (Jackson ImmunoResearch).
  • HRP horseradish peroxidase
  • BOB assay measures the level of purified IgG specific to CZV1-1 that blocks the binding of highly specific monoclonal antibodies (mAbs) to the ZIKV envelope. Briefly, high-binding, half-area 96-well flat-bottom polystyrene plates (Corning) were coated with 2 ⁇ g/ml of ZIKV or DENV recombinant envelope protein (Sino Biologicals) in carbonate/bicarbonate buffer overnight at 4°C. Plates were blocked with non-fat dry milk (NFDM; Bio-Rad) at 5% (w/v) in PBS for 1 hour at room temperature.
  • NFDM non-fat dry milk
  • IgG- CZV1 purified IgG- CZV1 diluted in 5% (w/v) of NFDM in PBS were added to the plate at different concentrations (600, 300, 100, and 33ng/mL). Plates were then incubated with IgG-CZV1 for 30, 45, and 55 minutes. A solution containing 200ng/mL of ZIKV or DENV-specific mouse mAbs (ZV-2, ZV-67, ZV-64, ZV-48, DV-10, Absolute antibody) was immediately pipetted on top of the IgG-CZV1 sample and incubated for 30, 15, and 5 minutes for a total incubation period of 60 minutes.
  • ZIKV or DENV-specific mouse mAbs ZV-2, ZV-67, ZV-64, ZV-48, DV-10, Absolute antibody
  • HRP horseradish peroxidase
  • mAbs diluted in 5% (w/v) of NFDM in PBS were added to the plate at different concentrations (800, 400, 200, 100, and 50ng/mL) and were incubated for 30 minutes.
  • a solution containing 200ng/mL of purified IgG-CZV1 was immediately pipetted on top of the mAbs and incubated for additional 30 minutes.
  • HRP horseradish peroxidase
  • HRP substrate 3, 3′, 5, 5′′- tetramethylbenzidine
  • Optical densities at a wavelength of 450nm were measured using SpectraMax Plus PC380 microplate spectrophotometer using SoftMax Pro software version 6.4 (Molecular Devices, USA). Optical densities from blank wells were subtracted from all measurements before analysis.
  • the plaque reduction neutralization test was used to assess the neutralization activity of the purified IgG-CZV1 following a protocol described in detail elsewhere.
  • the assay was carried out using the following virus strains: ZIKV strain H. sapiens/PE243/2015 (Brazil 2015), ZIKV strain H. sapiens/PF/2013 (French Polynesia, 2013), and DENV-3 H. sapiens/PE/02-95016 (Brazil 2002).
  • the PRNT positivity was 60M4845.DOCX 24 Attorney Docket No.
  • 06527-2407085 defined based on a 50% reduction in plaque counts (PRNT50) and neutralizing antibody titers were estimated using a four-parameter-non-linear regression.
  • Molecular and metadynamic simulations [00102] The binding pose prediction of the CZV1-1 adducts was performed using a semi- flexible approach coupled to the genetic algorithm of the GOLD software (34), considering the following x-ray structures as receptors: 5KVE, 5KVG, and 5VIC.
  • the x-ray structures 5KVE and 5KVG correspond to the ZIKV envelope protein complexed to the mAbs ZV48 and ZV67, respectively; and 5VIC corresponds to the envelope protein of DENV-1 complexed to neutralizing mAb Z004.
  • Positions R1 and R5 were derived from diverse chloroacids. Positions R2 and R6 were derived from amine building blocks. Position R3 was derived from Fmoc-protected amino acids that also contained an azide functionality and position R4 was derived from diverse aldehydes (FIGS. 4 and 5).
  • the PICCOs library was screened in a stepwise manner using two well-characterized serum panels from documented ZIKV infected patients (Table 1, above) and DENV-infected patients (Table 2, above).
  • matched pre- and post- infection sera were available from 16 dengue-infected patients. Matched pre- and post- infection sera were pooled to form five matched pools of 2-4 patients each for flow cytometry testing.
  • Zika-specific IgG antibody detection no pre-infection sera were available, so highly characterized ZIKV-specific sera from five patients were tested against flavivirus- negative sera (negative for ZIKV and DENV antibodies) (Table 3). 60M4845.DOCX 25 Attorney Docket No. 06527-2407085 Table 3 [00104]
  • ZIKV screens ten pools of two ZIKV-infected patients each were tested against three pools of flavivirus-negative sera.
  • a PICCO cluster was defined as multiple PICCOs that shared identical sub-units at four or five of the six variable positions (see FIG. 3). Five PICCO clusters were identified that encompassed 40 of the 50 PICCOs of interest (FIG. 6). Over 4,000 distinct Zika and dengue cross-reactive PICCOs were detected in more than 7 runs, while 2,220 distinct Zika and dengue cross-reactive PICCOs were detected in more than 15 runs (FIG. 8). For the top 50 Zika-specific PICCO candidates, only one unique PICCO compound was detected in a 60M4845.DOCX 27 Attorney Docket No.
  • the validation serum panels included: (1) two pools of 3-4 serum each from Zika IgG positive, dengue IgG negative patients; (2) four pools of 3 serum each 60M4845.DOCX 28 Attorney Docket No. 06527-2407085 from dengue IgG positive, Zika IgG negative patients; and (3) two pools of 3 serum each from patients positive for both Zika and dengue IgG antibodies (Table 6).
  • Table 6 [00110] The three pools of 3 samples each from Flavivirus na ⁇ ve patients (IgG negative for Zika and dengue) were the same used for the screening step (Table 4, above). Single-color FACS-based validation experiments were performed by incubating each pooled sera group with the selected Zika candidate PICCOs.
  • PICCOs and pooled sera were tested randomly to blind operator from ligand and sera specificity. Histograms were plotted and PICCOs were confirmed as positive by a shift to the right of distributions of the serum pools in comparison to blank control (bead without sera) and the percentage of positive beads was then calculated. All sixteen Zika PICCOs candidates were seroreactive: six showed strong Zika specificity and no reactivity with dengue samples, five showed marginal reactivity, and five showed some level of unwanted cross-reactivity with dengue samples. The comparison of the antibody specificity profiles of PICCOs from within the same chemical sequence cluster versus different clusters reveal that PICCOs with similar chemical sequence structures have 60M4845.DOCX 29 Attorney Docket No.
  • CZV1-1 was re-synthesized on-resin without the DNA encoding tag on TentaGel beads containing a RAM linker, allowing the compound to be released from the resin and characterized by liquid chromatography/mass spectrometry (LC-MS). This is important because in the high-throughput library synthesis, unexpected events sometimes occur, and it is possible that a “side product” may be the active compound rather than the structure predicted by the DNA sequence.
  • CZV1-1 discriminates between Zika and other flaviviruses infections
  • the antibody binding properties of CZV1-1 were further examined using a larger set of serum samples with a broad repertoire of flavivirus immune reactivity. Binding to CZV1-1 was assessed for each serum individually using a single-color FACS-based assay and each serum was tested blindly without previous knowledge of the patient serostatus. The percentage of positive CZV1-1 beads was used for discriminating between Zika-positive and negative samples.
  • CZV1- 1-specific IgG and two of the four ZIKV mAbs (ZV48 and ZV67), were able to bind with high potency to the ZIKV envelope DIII protein used in our assays (FIG. 16).
  • ZV48 and ZV67 were able to bind with high potency to the ZIKV envelope DIII protein used in our assays.
  • CZV1-1-specific IgG exhibited substantial competition for binding with ZV48 and ZV67, as 100ng of these mAbs blocked 50% binding of 100 ng/ml of the IgG specific to CZV1-1 (FIGS. 14A-14B, panel C).
  • CZV1-1-specific IgG neutralizes the Brazilian ZIKV strain (PE243) with a potency similar to that of monoclonal antibody ZV67 (IC50: 2.05 versus 1.27 ug/ml) and better than ZV48 (IC50: 2.05 versus 3.8 ug/ml).
  • CZV1-1- specific IgG also neutralized the ZIKV French Polynesia strain (H/PF2013) but with lower potency than ZV48 and ZV67 (FIGS. 14A-14B, panel E).
  • CZV1-1 exhibits a high rate of native contacts and hydrogen bonds with mAb ZV48 than other mAbs (data not shown). Furthermore, CZV1-1 shares a greater number of common interactions with the ZIKV DIII envelope subunit when bound to mAb ZV48 (data not shown), which includes ZV48 residues Y31, Y38, W56, Y97, Y100, Y102, W161, M178, and M231 (data not shown).
  • CZV1-1-based assays continue to show favorable diagnostic properties, they could have considerable value in high-risk situations like ZIKV infections in pregnancy.
  • This approach for the discovery of specific small molecule biomarkers of viral infection and immunity shown here in the specific case of Zika, can potentially be applied to any virus.
  • the general strategy of discovery of antibody biomarkers by comparing the PICCO binding profile of IgG in serum specimens from patients with, versus those without, a disease might also be applicable to non-infectious diseases such as cancers, autoimmune diseases, and other chronic conditions.
  • a critical component of this study was our ability to construct “gold-plated” panels of Zika-immune and dengue-immune human sera that were carefully characterized by conventional ELISA and neutralization assays. Although we were unable to determine the timing of sample collection after infection for the Zika-immune individuals used for our initial screens, all serum samples were confirmed to show no reactivity with dengue antigens in ELISA and neutralization assays, confirming that samples were highly ZIKV immune specific. Then, in our validation and evaluation experiments, we showed that samples from 60M4845.DOCX 34 Attorney Docket No. 06527-2407085 individuals with immunity to both Zika and dengue infections still retained IgG that bound specifically to CZV1-1.
  • DNA-encoding of the PICCO-displaying beads enabled NGS-based analysis which provided rapid elucidation of the chemical structures identified during the screening steps.
  • Our clustering analysis provided insights into the structural similarities and features important for Zika-specific IgG binding to these molecules.
  • Our data showing similar IgG binding profiles within hit candidates selected from the ZIKV-specific clusters revealed that a larger set of potential, analogous small molecule candidates could also be used for improving diagnostic accuracy.
  • Affinity purification experiments yielded concentrated CZV1-1-specific IgG antibodies.
  • Purified CZV1-1-specific IgG has neutralization activity against at least two ZIKV strains, which suggests the potential for CZV1-1 to be used as a vaccine immunogen.
  • CZV1-1 is not a high-affinity ligand for IgG interaction, this limitation might be circumvented by further structural optimizations of the PICCO molecule, synthesis of vaccines with multiple copies, conjugation of CZV1-1 to carriers, or use of adjuvants. Regardless, our results further indicate that the tools and approaches used here might be useful in the rational design of small molecule vaccines to stimulate high potency Zika IgG neutralizing antibodies.
  • CZV1-1 small nonbiological synthetic molecular mimic
  • the small molecule CZV1-1 corresponds to a conformational epitope that would be difficult to synthesize from knowledge of the envelope protein structure.
  • the CZV1-1 PICCO molecule can serve as a biomarker on which to base diagnostic assays for evidence of prior Zika infection. Because it is a mimic of a neutralizing epitope, it may also serve as a biomarker of immunity 60M4845.DOCX 35 Attorney Docket No.
  • CZV1-1 can be used in an ELISA as a specific marker for Zika, that CZV1-1 is immunogenic in mice, and that immunized mouse serum has anti-CZV1-1 that reacts specifically with Zika and does not cross-react with dengue.
  • CZV1-1 cysteine was conjugated to a carrier protein BSA using commercially available kits (ImjectTM Maleimide-Activated BSA, ThermoFisher Scientific, Catalog number 77116) and used for ELISA assays.
  • CZV1-1:mcBSA ELISA shows reactivity of human sera immune to ZIKV and no reactivity of human sera negative for ZIKV with the conjugated CZV1-1 (FIG. 18A).
  • CZV1-1 cysteine (unconjugated) does not discriminate between ZIKV+DENV+ and ZIKV- DENV+ (FIG. 18B) likely due to unspecific binding to the CZV1-1 cysteine groups.
  • CZV1-1 cysteine was conjugated to a carrier protein KLH using commercially available kits (ImjectTM Maleimide-Activated mcKLH, ThermoFisher Scientific, Catalog number 77605).
  • Complete Freund’s adjuvant used for the first injection incomplete Freund’s adjuvant for subsequent injections.
  • Mice were immunized on day 0, day 21, day 42, day 49, and day 63. Mice were bled at pre-injection, day 42, day 70, and final exsanguination at day 111.

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Abstract

L'invention concerne des compositions contenant un réactif de liaison pour un épitope du virus Zika (ZIKV), des compositions pharmaceutiques contenant de tels réactifs de liaison et des procédés de détection d'anticorps de liaison à ZIKV et d'anticorps neutralisant ZIKV chez des patients.
PCT/US2024/061012 2023-12-20 2024-12-19 Substitut à petites molécules pour épitope de neutralisation sur la protéine d'enveloppe du virus zika Pending WO2025137264A1 (fr)

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CASTANHA PRISCILA M.S., PATRICK J. MCENANEY , YONGSEOK PARK, ANTHEA BOUWER, ELTON J. F. CHAVES , ROBERTO D. LINS, NICHOLAS G. PACI: "Identification and characterization of a nonbiological small-molecular mimic of a Zika virus conformational neutralizing epitope", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, vol. 121, no. 21, 14 May 2024 (2024-05-14), pages e2312755121, XP093325617, DOI: 10.1073/pnas.2312755121 *
KODADEK THOMAS, MCENANEY PATRICK J.: "Towards vast libraries of scaffold-diverse, conformationally constrained oligomers", CHEMICAL COMMUNICATIONS, ROYAL SOCIETY OF CHEMISTRY, UK, vol. 52, no. 36, 1 January 2016 (2016-01-01), UK , pages 6038 - 6059, XP093325597, ISSN: 1359-7345, DOI: 10.1039/C6CC00617E *
MENDES KIMBERLY R., MALONE MARIE LYNNE, NDUNGU JOHN MAINA, SUPONITSKY-KROYTER IRENA, CAVETT VALERIE J., MCENANEY PATRICK J., MACCO: "High-throughput Identification of DNA-Encoded IgG Ligands that Distinguish Active and Latent Mycobacterium tuberculosis Infections", ACS CHEMICAL BIOLOGY, AMERICAN CHEMICAL SOCIETY, vol. 12, no. 1, 20 January 2017 (2017-01-20), pages 234 - 243, XP055802825, ISSN: 1554-8929, DOI: 10.1021/acschembio.6b00855 *
NGUYEN ANH THI, DUONG BAO TUAN, PARK HYUN, YEO SEON-JU: "Development of a peptide aptamer pair-linked rapid fluorescent diagnostic system for Zika virus detection", BIOSENSORS AND BIOELECTRONICS, ELSEVIER SCIENCE LTD, UK, AMSTERDAM , NL, vol. 197, 1 February 2022 (2022-02-01), Amsterdam , NL , pages 113768, XP093325601, ISSN: 0956-5663, DOI: 10.1016/j.bios.2021.113768 *
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