WO2021214467A1 - Amélioration d'un outil de diagnostic comprenant une molécule de liaison à un agent pathogène - Google Patents

Amélioration d'un outil de diagnostic comprenant une molécule de liaison à un agent pathogène Download PDF

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Publication number
WO2021214467A1
WO2021214467A1 PCT/GB2021/050969 GB2021050969W WO2021214467A1 WO 2021214467 A1 WO2021214467 A1 WO 2021214467A1 GB 2021050969 W GB2021050969 W GB 2021050969W WO 2021214467 A1 WO2021214467 A1 WO 2021214467A1
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Prior art keywords
pathogen
binding
sars
diagnostic tool
cov
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PCT/GB2021/050969
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English (en)
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Hugh ILYINE
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Cellbio
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Priority claimed from GBGB2005965.5A external-priority patent/GB202005965D0/en
Priority claimed from GBGB2006146.1A external-priority patent/GB202006146D0/en
Application filed by Cellbio filed Critical Cellbio
Publication of WO2021214467A1 publication Critical patent/WO2021214467A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5029—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures using swabs
    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/16—Reagents, handling or storing thereof
    • 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
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • 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
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)

Definitions

  • the present disclosure provides novel uses of pathogen-binding molecules- that is, those molecules which are present on the surface of a cell and which bind certain pathogens. These molecules often facilitate pathogenesis providing a means by which a pathogen binds to, colonises and/or infects a cell.
  • pathogens face the problem of having to at least bind to a host cell - this initial binding event is often a crucial part of the pathogenesis of the organism. Once bound a pathogen can multiply and colonise the surface of that cell or a tissue comprising the same.
  • pathogens can infect the cell and replicate therein.
  • pathogens often express molecules which recognise and bind to host cell surface molecules (often referred to as host cell surface receptors (or pathogen binding molecules). These host cell surface receptors provide a convenient mechanism by which the pathogen can bind and subsequently infect a suitable or specific host cell.
  • SARS-CoV-2 is classified as a Betacoronavirus, belonging to the same genus as both SARS and MERS; it is in the same sub-genus grouping as SARS, sharing around 80% nucleotide identity across the whole genome.
  • SARS and SARS-CoV-2 use the Angiotensin converting enzyme 2 (ACE2) protein expressed on the host cell surface for cell entry. Infections with SARS-CoV-2 result in a disease referred to as COVID-19.
  • ACE2 Angiotensin converting enzyme 2
  • Angiotensin-converting enzyme 2 is a membrane-bound aminopeptidase that has a vital role in the cardiovascular and immune systems. SARS-CoV-2 infection is triggered by binding of the spike (S) protein of the virus to ACE2.
  • This molecule ACE2 is found in various human organs (oral and nasal mucosa, nasopharynx, lung, stomach, small intestine, colon, skin, lymph nodes, thymus, bone marrow, spleen, liver, kidney, and brain).
  • ACE2 protein is found on lung alveolar epithelial cells and enterocytes of the small intestine, as well as in arterial and venous endothelial cells as well as arterial smooth muscle cells.
  • ACE2 is abundantly present in humans in the epithelia of the lung and small intestine, providing possible routes of entry for the SARS-CoV-2.
  • the SARS-CoV-2 spike protein forms large protrusions on the viral surface (forming the “corona” or “crown”) and, as stated, is the molecule which interacts with the ACE2 receptor.
  • the S protein has a large ectodomain divided into the following domains/regions: S1 (receptor binding domain); S2 (membrane fusion domain); a transmembrane anchor; and a short intracellular tail.
  • the S1 domain is divided into 2 major domains: (i) N-terminal domain (S1- NTD) - responsible for binding sugar; and (ii) the C-terminal domain (S1 -CTD) - responsible for recognizing protein receptors ACE2, APN, and DPP4.
  • the present disclosure provides improved tools, kits, methods and equipment for use in diagnosis and protection.
  • the disclosure provides improved tools and methods for the diagnosis and/or detection of infectious diseases.
  • the disclosure also provides improved personal protective equipment (face masks, visors, respirators, gloves, gowns, aprons, body suits and the like) for use by health care workers, carers, medics (doctors, surgeons, paramedics and the like) and anyone having to interact with subjects potentially harbouring an infectious disease.
  • face masks, visors, respirators, gloves, gowns, aprons, body suits and the like for use by health care workers, carers, medics (doctors, surgeons, paramedics and the like) and anyone having to interact with subjects potentially harbouring an infectious disease.
  • a sampling device/tool for example a swab
  • respiratory pathogens both viral and bacterial
  • these are most often detected using procedures which involve sampling or swabbing a subject’s buccal cavity, throat and/or nasal passages.
  • a swab procedure is conducted too early in the disease process, there may not be enough pathogen present to be detected. Conversely, if the swab procedure is conducted later in the disease process, the pathogen may have moved to different or new tissues and therefore the amount of pathogen present in the tissue being swabbed is too low to be detected.
  • the present disclosure improves the sensitivity of the tools/devices which a user may use to conduct a diagnostic test on a subject.
  • diagnostic tool(s) ‘tools” or “tool”).
  • tools diagnostic tool(s)
  • tools embrace swabs and other devices that are used to sample tissues and/or cells for the presence of one or more pathogens.
  • diagnostic tool(s) or “tool(s)” may not embrace a microtitre plate, slide, histology section or chip.
  • subject refers to any human or animal subject either suffering from an infectious disease or suspected of having an infectious disease.
  • subject may also embrace human or animal subjects susceptible or predisposed to an infectious subject.
  • Subjects of this type may be subjected to diagnostic tests, which tests are designed to determine (a) whether or not the subject is infected; and (b) with what pathogen the subject is infected.
  • infectious disease relates to diseases caused by any type of pathogen - in particular those diseases which are contagious.
  • pathogen includes viral, bacterial, fungal, prion and/or protozoan type pathogens.
  • the disclosure provides a diagnostic tool comprising a pathogen-binding molecule.
  • pathogen-binding molecule may refer to any molecule which exhibits an affinity for or an ability to bind to a pathogen. Accordingly, the term ‘pathogen-binding molecule’ may refer to molecules (proteins, peptides and/or carbohydrates) which are derived from a host cell (i.e. a cell which is bound, colonised and/or infected by a pathogen). Alternatively, the term ‘pathogen-binding molecule’ may refer to extracellular molecules, for example carbohydrates, peptides and proteins. The term ‘pathogen-binding molecule’ may embrace extracellular matrix proteins and/or other proteins present in certain tissues, which extracellular proteins exhibit an affinity for or which bind to, a pathogen.
  • the pathogen binding molecule may bind to a molecule which is expressed on the surface of a pathogen.
  • the pathogen-binding molecule may be a molecule which is exploited or bound by a pathogen (via a molecule expressed by the pathogen) during pathogenesis and/or to bind, colonise and/or infect a cell.
  • a pathogen-binding molecule may be a cell surface molecule.
  • a pathogen-binding molecule may be a host cell surface molecule.
  • the term ‘host cell’ refers to cells which are bound, colonised and/or infected (entered) by pathogens.
  • a pathogen-binding molecule will usually be a molecule expressed on the surface of a (host) cell and which is complementary to (it ‘locks’ on to or binds to) a molecule expressed by the pathogen.
  • the molecule expressed by the pathogen may be referred to as a ligand - a ligand which binds to and/or associates with a pathogen-binding molecule expressed by a (host) cell.
  • the pathogen-binding molecule provides a moiety to which the pathogen’s counterpart molecule (or ligand) can bind.
  • the pathogen-binding molecule comes into contact with the counterpart molecule expressed by the pathogen, the two may bind to each other.
  • a pathogen that is present in or on a tissue may bind, for example preferentially bind, to any diagnostic tool which comprises the correct or corresponding pathogen-binding molecule - the binding occurring between the pathogen-binding molecule of the diagnostic tool and the pathogen’s ligand/molecule, which ligand/molecule has affinity or specificity for that receptor.
  • the diagnostic tool is not a microtitre plate, slide, histology section or chip.
  • the tool may be impregnated with the pathogen-binding molecule
  • one or more surfaces of the tool may be coated with the pathogen-binding molecule.
  • the diagnostic tool may be any type of sample collection tool.
  • the term ‘diagnostic tool’ may comprise a cyto-brush a perio-dental brush, a spatula and the like.
  • the diagnostic tool may be a swab.
  • a swab for use in the diagnosis or detection of a pathogen will usually comprise at least two parts - a handle part by which the user holds and manipulates/moves the swab across the surface of a tissue to be sampled and a sampling part, often a pad comprising a soft deformable/resilient material such as cotton wool or synthetic fabric such as polyester. This pad can be moved across a tissue without causing damage thereto.
  • At least the sampling pad part of a swab may comprise a pathogen-binding molecule.
  • at least the pathogen-binding molecule may be impregnated into or coated on to the surface of at least the sampling pad part of a swab.
  • impregnation or coating of at least the sampling pad part of the swab may occur at the point of swab manufacture and/or immediately prior to use of the swab (i.e. at the point of care) wherein the swab it dipped or placed into some solution or composition comprising the pathogen-binding molecule.
  • a diagnostic tool which comprises a pathogen-binding molecule as set out herein might exhibit improved sensitivity.
  • the diagnostic tools described herein may be advantageous as they comprise a receptor which is specific for the pathogen to be detected. In this way, the tool exhibits an increased affinity for the pathogen and is more likely to bind the pathogen even when there are only low amounts of the pathogen present in a tissue.
  • pathogen-binding molecule embraces the full or complete pathogen-binding molecule - that is the wild-type form of any host cell surface protein or molecule exploited by a pathogen in order to gain access or entry to a cell and/or to bind or colonise a cell.
  • pathogen-binding molecule will also embrace fragments of wild- type pathogen-binding molecules, which fragments exhibit an ability to bind to some component of the pathogen - in particular, the usual counterpart molecule expressed by the pathogen which counterpart molecule (or ligand) binds the pathogen-binding molecule in the normal disease process - for example when binding, colonising and/or infecting a host cell.
  • a useful pathogen-binding molecule fragment will comprise at least the functional part of the pathogen-binding molecule - that is those parts which bind to or associate with the pathogen. Accordingly, the term “pathogen receptor” will include molecules which comprise only the pathogen binding part of the full or wild-type pathogen receptor. The pathogen binding part of a pathogen-binding molecule may otherwise be referred to as the ‘pathogen binding domain’. Accordingly, the term ‘pathogen receptor’ will include the ‘pathogen binding domain’ or any given pathogen receptor.
  • the diagnostic tools described herein may comprise any suitable pathogen-binding molecule or at least the pathogen binding domain part thereof.
  • the diagnostic tools of this disclosure may comprise the receptor which is specific for any or all of the pathogen(s) to be diagnosed and/or identified. Indeed the precise nature of the receptor used to make the tools described herein will vary depending on the type of pathogen(s) to be diagnosed.
  • the receptor will comprise a cell, for example a host cell, surface molecule or protein.
  • the tool for example swab
  • the tool may comprise a receptor specific for the type of bacteria to be diagnosed or detected.
  • the particular choice of receptor used will be guided by the pathology of the bacterial pathogen and in most cases will comprise a host cell surface expressed (bacterial) pathogen-binding molecule which is exploited by the bacterial pathogen when binding to, colonising and/or infecting a cell.
  • the tool may comprise one or more (bacterial) pathogen-binding molecules specific for one or more of the bacterial pathogens to be diagnosed or detected.
  • the tool for example swab
  • the tool may comprise a receptor specific for the type of virus to be diagnosed or detected.
  • the particular choice of receptor used will be guided by the pathology of the viral pathogen and in most cases will comprise a host cell surface expressed (viral) pathogen-binding molecule exploited by the viral pathogen when binding to, colonising and/or infecting a cell.
  • the tool may comprise one or more (viral) pathogen binding molecules specific for one or more of the viral pathogens to be diagnosed or detected.
  • the tool for example swab
  • the tool may comprise a receptor specific for the type of fungus to be diagnosed or detected.
  • the particular choice of receptor used will be guided by the pathology of the fungal pathogen and in most cases will comprise a host cell surface expressed (fungal) pathogen-binding molecule exploited by the fungal pathogen when binding to, colonising and/or infecting a cell.
  • the tool may comprise one or more (fungal) pathogen-binding molecules specific for one or more of the fungal pathogens to be diagnosed or detected.
  • a pathogen may exploit a plurality of different host cell molecules in order to colonise, bind and/or enter/infect a cell. Any one or more of these host cell molecules could be used in a diagnostic tool of this disclosure.
  • pathogen-binding molecule may include one or more different pathogen-binding molecules specific for one or more different pathogens.
  • a diagnostic tool of this disclosure may comprise, one, two or more pathogen-binding molecules.
  • a diagnostic tool of this disclosure may comprise those pathogen-binding molecules that are typically referred to as (host cell) secondary and/or co-receptors for a pathogen.
  • the tool may comprise the CD4 receptor and/or the CCR5 receptor and/or CXCR4 receptor (or an HIV bind portion of any of these).
  • the CD4 molecule may be regarded as the primary receptor with the CCR5 and CXCR4 molecules being referred to a co-receptors.
  • a diagnostic tool of this disclosure may comprise a plurality (for example two or more) pathogen-binding molecules. The pathogen-binding molecules may be exploited by one (the same) or two or more different pathogens.
  • the diagnostic tool may comprise pathogen-binding molecules which are able to bind a number, for example two, three or more, molecules present on the surface of a pathogen.
  • pathogen-binding molecules which are able to bind a number, for example two, three or more, molecules present on the surface of a pathogen.
  • Table 1 viral pathogen ligands and (host) cell receptors (pathogen-binding molecules).
  • Table 2 bacterial pathogen ligands and (host) cell receptors (pathogen-binding molecules)
  • Table 3 fungal pathogen ligands and (host) cell receptors (pathogen-binding molecules)
  • Table 4 protozoal pathogen ligands and (host) cell receptors (pathogen-binding molecules)
  • This disclosure therefore provides a diagnostic tool comprising one or more of the pathogen binding molecule(s) identified in Tables 1-4 above.
  • the disclosure provides a swab comprising one or more the pathogen-binding molecule(s) identified in Tables 1-4 above.
  • the disclosure provides a swab in which the part for sampling a tissue comprises a pathogen-binding molecule and/or one or more of the pathogen-binding molecule(s) identified in Tables 1-4.
  • the part for sampling a tissue is impregnated and/or coated with one or more pathogen-binding molecule(s) and/or one or more of the molecules identified in Tables 1-4 above.
  • the pathogen-binding molecule may comprise a CD cell marker.
  • the pathogen-binding molecule may comprise a Toll-like receptor - for example any one or more of the Toll-like receptors (TLR) identified as TLR1-9.
  • a diagnostic tool of this disclosure may comprise a pathogen-binding fragment of a TLR.
  • TLR1 , TLR2, TLR4, TLR5 and TLR9 (or pathogen binding fragments thereof) have affinity for or are able to bind to bacterial pathogens - as such they may be used in diagnostic tools that are intended for use in the diagnosis of a bacterial infection.
  • TLR3 has affinity for or binds to double stranded RNA and is therefore able to bind to viral pathogens.
  • the disclosure provides the use of a diagnostic tool of this disclosure in the diagnosis and/or detection of a disease, condition and/or infection.
  • the disclosure also provides a method of diagnosing or detecting a disease, condition and/or infection, said method comprising using a tool, for example a swab, of this disclosure to sample a tissue for the presence of a pathogen associated with the disease, condition or infection, wherein the tool comprises a pathogen-binding molecule specific for the pathogen to be diagnosed or detected.
  • the method may further comprise the step of processing the tool so as to determine whether or not a particular pathogen or pathogen is/are present on or within the sampled tissue.
  • a pathogen-binding molecule specific for the pathogen to be diagnosed or detected is a pathogen-binding molecule which binds to or associates with (or is bound by) a molecule expressed by the pathogen.
  • SARS-CoV and SARS-Cov-2 exploit Angiotensin-Converting Enzyme 2 (ACE-2) as their cellular entry receptor.
  • ACE-2 is an enzyme attached to the outer surface (cell membranes) of a number of different cells types. For example, ACE-2 is present on the surface of cells in the buccal cavity, upper and lower respiratory tracts (including the throat and lung). ACE-2 is bound by the S (spike) protein expressed on the surface of SARS-CoV and SARS-CoV-2.
  • a diagnostic tool for example a swab, which comprises the ACE2 receptor, or a SARS-CoV/SARS-CoV-2 binding portion thereof, may facilitate the diagnosis and detection of SARS-CoV and SARS-CoV-2 infections (and associated disease such as COVID-19).
  • the presence of the ACE2 receptor in or on the diagnostic tool will mean that even when the quantity, titre, amount or level of the virus is low in the tissue being sampled, the available viral particles will preferentially bind the diagnostic tool (via the incorporated, impregnated or coated pathogen receptor) during the sampling process. This enhances the sensitivity of the diagnostic tool and improvise the sensitivity and accuracy of the diagnostic/detection test.
  • the disclosure provides a diagnostic tool comprising the ACE2 inhibitor and/or a SARS-CoV/SARS-CoV-2 binding portion thereof.
  • the diagnostic tool is not a microtitre plate or chip.
  • the diagnostic tool comprising the ACE2 inhibitor and/or a SARS- CoV/SARS-CoV-2 binding portion thereof, is a swab.
  • the disclosure provides a swab comprising the ACE2 inhibitor and/or a SARS-CoV/SARS- CoV-2 binding portion thereof.
  • the disclosure provides a swab comprising the ACE2 inhibitor and/or a SARS-CoV/SARS- CoV-2 binding portion thereof, wherein the swab is for sampling the tissues of the buccal cavity and/or throat and/or nasal passages.
  • a swab comprising the ACE2 inhibitor and/or a SARS-CoV/SARS-CoV-2 binding portion thereof in the diagnosis and/or detection of SARS-CoV/SARS-CoV-2 infections and/or respiratory diseases such as SARS or COVID-19
  • a diagnostic tool or swab for sampling the buccal cavity and/or throat and/or nasal passages; or (ii) a diagnostic tool or swab for use in the diagnosis or detection of a SARS-CoV and/or SARS-Cov-2 infection and/or SARS and/or COVID-19.
  • the method of detecting and/or diagnosing a SAR-CoV and/or SARS-CoV-2 infection further comprises processing the diagnostic tool or swab for to determine whether or not the sampled tissues comprises SARS-CoV and/or SARS-CoV-2.
  • the pathogen-binding molecule for use may be harvested from the relevant host cell or made recombinantly or synthesised.
  • cells may be processed and useful pathogen-binding molecules extracted from cell membrane preparations.
  • Methods for the extraction of specific proteins from complex mixtures and/or cell membrane preparations are known and may include size exclusion and affinity chromatography methods.
  • a diagnostic tool of this disclosure may comprise a recombinant pathogen receptor.
  • a recombinant form of ACE2 may be used.
  • a diagnostic tool of this disclosure may comprise a recombinant ACE2 receptor, and/or a pathogen binding fragment thereof.
  • SARS-CoV-2 binding interface The region spanning residue 24-393 may be referred to as defining the SARS-CoV-2 binding interface. Indeed this region of the ACE2 receptor may bind to and recognise the receptor (ACE2) binding domain (RBD) of the SARS-CoV-2 Spike protein.
  • ACE2 binding domain The sequence of this SARS-CoV-2 RBD is provided below as SEQ ID NO: 1a
  • RVQPTESIVR FPNITNLCPF GEVFNATRFA SVYAWNRKRI SNCVADYSVL YNSASFSTFK CYGVSPTKLN
  • DLCFTNVYAD SFVIRGDEVR QIAPGQTGKI ADYNYKLPDD FTGCVIAWN
  • the Grey highlighted domain represents the receptor binding motif (spanning residues 438- 506 of the S1 protein).
  • the associated (human ACE2) nucleic acid sequence is available as NCBI Reference Sequence: NM 001371415.1 ; that sequence (which encodes SEQ ID NO: 1 above) is reproduced below as SEQ ID NO: 2.
  • a diagnostic tool of this disclosure may comprise a peptide having or comprising a sequence of SEQ ID NO: 1 or a SARS-CoV or SARS-CoV-2 binding fragment thereof.
  • the disclosure may provide a swab comprising a peptide having or comprising a sequence of SEQ ID NO: 1 or a SARS-CoV or SARS-CoV-2 binding fragment thereof.
  • ACE2 proteins for use may include orthologous sequence derived from other species. Some examples of potentially useful ACE2 proteins may include those listed below.
  • ACE2 sequence is deposited as Q56H28 (UniProtKB: NCBI Reference Sequence: XM 023248796.1); that sequence is reproduced below as SEQ ID NO: 3:
  • the associated (feline ACE2) nucleic acid sequence is available as NCBI Reference Sequence: XM 023248796.1 ; that sequence (which encodes SEQ ID NO: 3 above) is reproduced below as SEQ ID NO: 4.
  • SEQ ID NO: 5 An exemplary (Amur Tiger: Panthera tigris altaica) ACE2 sequence is available as NCBI Reference Sequence: XP 007090142.1 ; that sequence is reproduced below as SEQ ID NO: 5.
  • SEQ ID NO: 5 An exemplary (Amur Tiger: Panthera tigris altaica) ACE2 sequence is available as NCBI Reference Sequence: XP 007090142.1 ; that sequence is reproduced below as SEQ ID NO: 5.
  • the associated (Amur tiger ACE2) nucleic acid sequence is available as NCBI Reference Sequence: XM 007090080.2; that sequence (which encodes SEQ ID NO: 5 above) is reproduced below as SEQ ID NO: 6.
  • An ACE2 molecule for use in a diagnostic tool of this disclosure may comprise or be derived from any one of the sequences provided above - in particular SEQ ID NOS: 1 , 3 and 5.
  • SEQ ID NO: 2, 4 and 6 are nucleic acid sequences that encode various forms of ACE2 and each one may be used as a template sequence from which a recombinant ACE2 receptor or ACE2 fragment (for example a SARS-CoV/SARS-CoV-2 binding fragment), can be generated.
  • a recombinant ACE2 receptor or ACE2 fragment for example a SARS-CoV/SARS-CoV-2 binding fragment
  • ACE2 molecules for use may comprise functional fragments of any of SEQ ID NOS: 1 , 3 and 5.
  • Useful fragments may retain one or more of the functions of a wild-type ACE2 molecule (enzyme).
  • a useful ACE2 fragment may retain an affinity for SARS-CoV and/or SARS-CoV-2.
  • useful ACE2 fragments bind to the S-protein of the SARS or SARS- CoV-2 virus.
  • Useful fragments may comprise or be derived from residues 24-393 of SEQ ID NOS: 1 , 3 or 5.
  • Useful fragments may comprise one or more of the following residues: Q24; D30; K31 ; H34; L79; M82 and/or Y83. It should be noted that I some sequences the precise identity of the residue may vary and it may be substituted conservatively with another amino acid having the same or similar properties.
  • a useful fragment may comprise a sequence having at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65% or at least 60% identity or homology to the sequence spanning residues 24-293 of SEQ ID NOS: 1 , 3 or 5.
  • a useful fragment may comprise anywhere between about 5 and about 369 residues of the sequence spanning residues 24-393 of SEQ ID NOS: 1 , 3 and 5.
  • a useful fragment may comprise about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140, about 150,
  • Useful fragments may comprise moieties which bind to the SARS-CoV-2 binding domain or binding motif (see SEQ ID NO: 1 a).
  • an ACE2 fragment for use may comprise about 20, about 30, about 40, about 50, about 100, about 150, about 200 , about 250, about 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 361 , about 362, about 363, about 364, about 365, about 366, about 367, about 368, about 369, about 370, about 371 , about 372, about 373, about 374, about 375, about 376, about 377, about 378, about 379, about 380, about 385, about 390, about 395, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 760, about 770, about 780 , about 790, about 791 , about 792, about 793, about 794, about 795, about 796, about 797, about 798, about 799.
  • ACE2 fragments about 800, about 801 , about 802, about 803 or about 804 residues of any of SEQ ID NOS: 1 , 3 or 5. All such fragments may be referred to as ACE2 fragments and any useful ACE2 fragments may retain an ability to bind (i) SARS and/or (ii) SARS-CoV-2 and/or (iii) the S-protein of SARS and/or (iv) the S-protein of SARS-CoV-2.
  • a useful ACE2 fragment may comprise a SARS and/or SARS-CoV-2 binding fragment of any of SEQ ID NOS: 1 , 3 or 5.
  • the disclosure may embrace other sequences with identity and/or homology thereto or to any of the useful ACE2 fragments described above.
  • the disclosure may relate to sequences which have at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94% at least 93% at least 92% at least 91% at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65% or at least 60% sequence identity or sequence homology to (or with) SEQ ID NO: 1 , 3 or 5 or any of the ACE2 fragments (including the SARS and SARS- CoV-2 binding fragments) described herein.
  • the disclosure also relates to the nucleic acid sequences encoding and identical or homologus ACE2 sequence, which nucleic acid sequences can be used to generate recombinant ACE2 molecules for use.
  • the disclosure provides a diagnostic tool providing a protein or peptide comprising a sequence according to any one of SEQ ID NOS: 1 , 3 or 5 or a SARS/SARS- Cov-2 binding fragment thereof.
  • the disclosure also provides a swab comprising a protein or peptide comprising a sequence according to any one of SEQ ID NOS: 1 , 3 or 5 or a SARS/SARS-Cov-2 binding fragment thereof.
  • the disclosure further provides a swab comprising a handle part and a tissue sampling part, wherein at least the tissue sampling part of the swab comprises a protein or peptide comprising a sequence according to any one of SEQ ID NOS: 1 , 3 or 5 or a SARS/SARS- Cov-2 binding fragment thereof.
  • the protein or peptide comprising a sequence according to any one of SEQ ID NOS: 1 , 3 or 5 or a SARS/SARS-Cov-2 binding fragment thereof may be immobilised on/within, bound to, impregnated within and/or coated onto at least the tissue sampling part of the swab.
  • a diagnostic tool for use in the detection of a disease, condition or infection may be provided in the form of a kit.
  • the kit may comprise a diagnostic tool as described herein - wherein said tool comprises a pathogen receptor.
  • kits may contain or comprise one or more swabs, which swab(s) comprise a pathogen receptor.
  • the kit may contain blank or sterile diagnostic tool(s) (for example swab(s)) which is/are to be prepared for use.
  • the step of preparing a diagnostic tool for use may comprise impregnating or coating the tool with/in the appropriate pathogen receptor(s) prior to use.
  • a kit of this type may contain, for example, a preparation of the pathogen binding molecule into which the diagnostic tool is inserted or dipped. In this way, at least a part of the tool, for example a tissue sampling part of the tool, can be impregnated with or coated in the pathogen-binding molecule prior to use.
  • the kit may comprise a preparation or composition comprising one or more of the pathogen binding molecules identified in Tables 1-4.
  • the Kit may comprise a preparation or composition comprising an ACE2 protein or a SARS/SARS-CoV-2 binding fragment thereof.
  • the kit may comprise a preparation or composition comprising a protein or peptide comprising a sequence according to any one of SEQ ID NOS: 1 , 3 or 5 or a SARS/SARS- Cov-2 binding fragment thereof.
  • the present disclosure may also provide articles, for example receptacles, at least a surface of which is coated in a pathogen-binding molecule as described herein.
  • tubes, bottles and other receptacles may comprise surfaces (for example inside surfaces) that comprise a pathogen-binding molecule.
  • the pathogen-binding molecule may be bound to or immobilised/coated onto the surface (or example at least part of an inside surface) of a receptacle.
  • the receptacle may be a tube for collecting a sample.
  • the sample may comprise a biological material or biological fluid such as a tissue biopsy, a tissue scraping, blood, serum, plasma, saliva, tears or sweat.
  • the receptacle may comprise an inside surface that comprises an ACE2 molecule or a SARS-CoV-2 binding part thereof.
  • the disclosure provides a tube for collecting a sample of saliva, wherein at least an inside surface of the tube is coated with or has immobilised thereto, an ACE2 molecule and/or a SARS-CoV-2 binding fragment thereof.
  • any virus present in saliva may bind to the ACE2 derived component bound to or immobilised onto the inside surface of the collection tube.
  • the tube could then be sent for processing as part of a diagnostic process to determine the presence (or absence) of SARS- CoV-2 in a saliva sample.
  • the disclosure also provides the use of a receptacle (for example a tube) comprising a pathogen-binding molecule (for example ACE2 or a SARS-CoV-2 binding fragment thereof) in a method of diagnosis (for example a method of diagnosing a SARS-CoV-2 infection).
  • a receptacle for example a tube
  • a pathogen-binding molecule for example ACE2 or a SARS-CoV-2 binding fragment thereof
  • the present disclosure further provides personal protective equipment (PPE) which comprises a pathogen-binding molecule.
  • PPE personal protective equipment
  • PPE may embrace face visors, goggles, aprons, respirators, body suits, masks (half and/or full for the face and/or mouth and nose), facepieces, gloves and the like. All of these items may comprise a surface and/or component which comprises a pathogen-binding molecule.
  • the PPE item comprises a filter - for example to filter air as it is breathed in by a user
  • that filter may comprise a pathogen-binding molecule.
  • filters in PPE devices may work at least by size exclusion - i.e. the filter contains a substrate which retains particles above a certain size, preventing them from being breathed in or ingested.
  • the presence of a pathogen-binding molecule in or on these filter component supplements the existing filtration system with one which can bind a specific pathogen present in the sir.
  • Visors, aprons and/or other protective clothing equipment may comprise a pathogen-binding molecule as an impregnated component or as a surface coating or layer.
  • the disclosure provides a face mask comprising a pathogen-binding molecule.
  • the face mask comprises a filter, for example a particulate, gas and/or vapour filter and the filter comprises the pathogen-binding molecule.
  • the pathogen-binding molecule may be impregnated within or coated onto one or more of the filter substrate(s) and/or a surface thereof.
  • a substrate for use in a filter comprising a pathogen-binding molecule.
  • the pathogen-binding molecule may be impregnated within and/or coated onto one or more of the substrate surface(s).
  • the filter substrate may be for use in PPE or for use in a face mask.
  • a filter for use in PPE and/or for use in a face mask comprising a pathogen binding molecule.
  • the pathogen-binding molecule may be impregnated within and/or coated onto one or more of the filter substrate surface(s).
  • PPE (as described above: including face masks and the like) comprising one or more selected from the group consisting of:
  • a molecule comprising a sequence according to SEQ ID NOS: 1 , 3 or 5 or a SARS/SARS-CoV-2 binding fragment thereof.
  • diagnostic tool may extend to substrates and other materials to be placed into the buccal cavity and manipulated by the subject therein in order to collect tissue, cell, fluid samples.
  • a tool of this disclosure may comprise a chewable substrate comprising a pathogen-binding molecule.
  • the chewable substrate may comprise a rubber, a plastic, a polymer, a fibrous material, a fabric and/or a gum.
  • the gum may comprise any natural and/or synthetic gum base ingredient; without wishing to be bound by example, suitable gum-base(s) may be selected from the group consisting of: chicle, natural rubber, massaranduba balata, nispero, butyl rubber, petroleum wax (synthetic), polyethylene, microbial gum-type polysaccharises and polyisobutylene polyvinyl acetate.
  • suitable gum-base(s) may be selected from the group consisting of: chicle, natural rubber, massaranduba balata, nispero, butyl rubber, petroleum wax (synthetic), polyethylene, microbial gum-type polysaccharises and polyisobutylene polyvinyl acetate.
  • the chewable substrate may comprise one or more pathogen binding molecules - including any of those pathogen-binding molecules described herein.
  • the pathogen-binding molecule may be coated, impregnated in or with the pathogen-binding molecule.
  • the pathogen-binding molecule may be immobilised onto a surface or material of the chewable substrate.
  • the chewable substrate may be designed not to be swallowed.
  • a gum of this disclosure may be administered to any subject for the purpose of collecting a sample to be processed for the presence or absence of a particular pathogen.
  • a gum of this disclosure may comprise a molecule capable of binding a pathogen that is to be detected or diagnosed.
  • a gum of this disclosure is of particular use where the pathogen to be detected or diagnosed is present in the tissues and/or cells of the buccal cavity, the throat and//or respiratory airways and/or in saliva and/or mucus produced by the subject.
  • the gum may be chewed or masticated by the subject so that any pathogen present in the cells, tissues or fluids (saliva and/or mucus) present in the buccal cavity (throat and respiratory airways) of the subject, is exposed to the pathogen binding receptor of the gum and is able to bind thereto.
  • a gum (or chewable substrate) of the type described herein is of particular use where the amount of any given pathogen to be detected or diagnosed is low; a gum which includes a pathogen-binding molecule, will have a high affinity for the pathogen (because of the included pathogen-binding receptor) and thus even low numbers of the pathogen will be bound by the gum.
  • the gum can be sent for processing.
  • the gum may be processed through some form of procedure or protocol that permits the detection of any of the pathogen present within the gum.
  • the gum may be processed so as to extract nucleic acid and the extracted nucleic acid subjected to a process which uses PCR and other molecule techniques to detect the presence or absence of nucleic acid from the pathogen.
  • a chewable substrate for example a gum, comprising a pathogen-binding molecule.
  • the chewable substrate may be a gum.
  • a chewable substrate or gum for use in the diagnosis of a disease, for example an infectious disease, wherein the chewable substrate or gum comprises a pathogen-binding molecule.
  • the pathogen-binding molecule may bind a pathogen associated with the disease or infectious disease to be diagnosed.
  • a chewable substrate or gum in the diagnosis of a disease, for example an infectious disease.
  • the chewable substrate or gum may comprise a pathogen-binding molecule which binds a pathogen associated with the disease or infectious disease to be diagnosed.
  • a method of detecting a pathogen associated with a disease or infectious disease comprising providing a subject with a chewable substrate or gum, comprising a pathogen-binding molecule, allowing the subject to chew the substrate or gum for a time period suitable to allow any pathogen present in the cells, tissues and/or fluids of the subject to bind to the pathogen-binding molecule of the chewable substrate or gum and processing the chewable substrate or gum to confirm the presence or absence of the pathogen.
  • the pathogen-binding molecule may bind to the pathogen to be detected.
  • a gum comprising an ACE2 molecule or SARS-CoV-2 binding part thereof.
  • a chewable substrate or gum for use in the diagnosis of COVID-19 said chewable substrate or gum comprising an ACE2 molecule or a SARS-CoV-2 binding fragment thereof.
  • a chewable substrate or gum in the diagnosis of COVID-19, said chewable substrate or gum comprising an ACE2 molecule or a SARS-CoV-2 binding fragment thereof.

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Abstract

La présente invention concerne des outils, des kits, des procédés et un équipement améliorés destinés à être utilisés dans le diagnostic et la protection. Les outils/dispositifs de l'invention comprennent une molécule de liaison aux agents pathogènes et présentent une sensibilité améliorée et peuvent être utilisés pour réaliser un test de diagnostic sur un sujet.
PCT/GB2021/050969 2020-04-23 2021-04-22 Amélioration d'un outil de diagnostic comprenant une molécule de liaison à un agent pathogène WO2021214467A1 (fr)

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GB2005965.5 2020-04-23
GBGB2005965.5A GB202005965D0 (en) 2020-04-23 2020-04-23 Tool improvement
GBGB2006146.1A GB202006146D0 (en) 2020-04-27 2020-04-27 Toll Improvement
GB2006146.1 2020-04-27

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