A diagnostic method

Abstract

The invention relates to diagnostic methods, and particularly, although not exclusively, to the use of oligomeric protein traps in sample diagnostics. The invention extends to the use of oligomeric protein traps as binding agents, for example in methods for enriching samples of low-concentration proteins and / or genetic material, and methods for detecting the same in samples. The invention also encompasses high-affinity protein traps and their application in environmental monitoring, diagnosis and therapy.

Description

[0001] A Diagnostic Method

[0002] The present invention relates to diagnostic methods, and particularly, although not exclusively, to the use of oligomeric protein traps in sample diagnostics. The invention extends to the use of oligomeric protein traps as binding agents, for example in methods for enriching samples of low-concentration proteins and / or genetic material, and methods for detecting the same in samples. The invention also encompasses high-affinity protein traps and their application in environmental monitoring, diagnosis and therapy.

[0003] It is important to be able to accurately detect and quantify a target protein or nucleic acid in clinical or environmental samples to conduct an accurate diagnosis and / or treatment, or to monitor the environment. However, it is challenging to detect and quantify proteins and / or nucleic acids that are present in low concentrations, particularly when the target biomolecule is in the presence of other contaminating proteins and / or nucleic acids or sample matrices.

[0004] Mass spectrometry (MS) methodologies can be used to measure a target protein / nucleic acid within samples in order to detect it in the presence of all other proteins / nucleic acids present in the samples, particularly in the context of therapy and diagnostics. For instance, MS is gaining popularity as a fast, accurate, and quantitative diagnostic method for detecting SARS-CoV2 protein in biological samples, such as swabs, saliva and serum1-5. Additionally, it has the potential to be applied to other viruses. MS has significant advantages over other detection platforms, such as reverse transcription polymerase chain reaction, in that it can easily multiplex with other targets and provide absolute quantitation without analyte amplification. For instance, a single assay could quantify the immunoglobulin and complement response to infection proteins to establish a personalised response score6.

[0005] In order to detect low-abundance circulating proteins, such as viral antigens, with MS, it is important to carefully evaluate the background signals of matrix components to achieve a high level of sensitivity. Enrichment of the target virus, viral target protein, or post-digestive peptides from the target protein has been demonstrated to be effective in resolving this issue, thereby enhancing sensitivity7-9- Most enrichment methods involve the immunoaffinity capture of the target protein or peptides8'10'11. The process of generating and screening suitable monoclonal antibodies for target immunoaffinity enrichment is, however, time-consuming and relies on the use of animals. In addition, viral variants that contain mutations in or near the binding epitopes may result in a reduction or ablation of antibody binding. In enrichment and diagnostic MS, this could result in false negative results or underestimates of viral variant concentrations. Antibodies that target viral proteins / peptides that are most susceptible to mutation, such as the spike protein in SARS CoV-2, are therefore best avoided for immunoaffinity enrichment.

[0006] However, antibodies that target viral proteins that are less susceptible to mutations still pose the risk of failing to recover variants in which mutations have occurred in the antibody epitopes of these proteins.

[0007] The use of stable isotope standards and capture by anti-peptide antibodies (SISCAPA) antibody has been one solution to this issue for SARS-CoV-2, in which a CoV-2 peptide is bound and recovered from post-digest samples by the SISCAPA antibody. However, this method is of limited utility because it fails to retrieve the peptide from unknown variants that contain mutations in the target peptide that interfere with antibody binding. Variants may also be undetectable by other antibody approaches, as they may be similarly impacted by mutations.

[0008] Furthermore, antibodies are susceptible to mutations in the target protein, which can result in a partial or complete loss of binding activity. Mutations in the viral target in or near the antibody's epitope or in the epitope's access will decrease or prevent antibody binding, failing to detect variants with these mutations. This was observed with several therapeutic and diagnostic antibodies to SARS-CoV-2. It is also necessary to generate and screen antibodies for their capacity to bind target proteins, and subsequently characterise them for their use in detection assays. This is a resource-intensive and time-consuming process that may not necessarily yield the most optimal binding reagents. Additionally, the development of antibodies against specific targets is very challenging.

[0009] The inventors hypothesised that a high-affinity soluble version of a target protein, such as a viral receptor, could be oligomerised and then employed as an effective binding agent or "trap" to enrich a ligand (i.e. attachment protein), for example a pathogenic (e.g. viral or bacterial) protein in diagnostic MS, or a non-pathogenic endogenous protein (e.g. Tie2 for the diagnosis of other diseases). This approach would expedite the development of assays by eliminating the necessity for antibody generation and screening. In addition, it is expected that enrichment through a binding agent based on the host cell receptor would recover all variants that are infection-competent and capable of binding to the host receptor. To optimise recovery, such a binding agent or protein trap would benefit from having a high binding activity for the target attachment protein. One way to increase binding activity is via directed protein evolution. Although a variety of evolved Angiotensin Converting Enzyme 2 (ACE2) mutants with increased binding activity for SARS-CoV-2 spike protein have been reported12-14, evolved ACE2 with mutations in the binding site for the spike protein can be susceptible to certain mutations found in variant spike proteins14'15.

[0010] The high-affinity binding agent created by the inventors is a fusion protein and comprises a protein trap constructed by fusing the appropriate binding domain(s) of the receptor of the target protein to an oligomerisation domain. As described in the Examples, using the ACE2 ectodomain and SARS-CoV2 spike protein as an example, the inventors have created high binding activity versions of a host cell receptor by directed evolution (while retaining a non-mutated spike protein binding interface), and by oligomerizing the native ACE2 ectodomain. These binding agents were tested for binding and the oligomeric native receptor was found to be surprisingly more effective than the evolved receptor during the binding tests. The inventors subsequently demonstrated that this soluble receptor binding approach can be used to enrich target proteins for detection using conventional methods.

[0011] This approach, which involves the use of high-affinity receptors to target proteins for enrichment before MS and numerous other analytical methods, is applicable to a wide range of target proteins from any pathogen (and not just SARS CoV-2), or viral or bacterial proteins. Moreover, the oligomerised receptors can be used to bind to many non-viral proteins with known receptors or selective binding partners, such as circulating ligands, including growth factors and certain hormones in biological fluids (e.g. angiopoietin ligands). The inventors believe that this approach is compatible with diagnostic and detection methods other than MS.

[0012] Therefore, in one aspect of the invention, there is provided a method of detecting and / or quantifying a target molecule in a sample, the method comprising:

[0013] (i) providing an oligomeric protein trap for enriching the target molecule in the sample, the protein trap comprising: (i) a receptor or an extracellular binding domain thereof, wherein the receptor or an extracellular binding domain thereof is configured to specifically bind the target molecule; and (ii) an oligomerisation domain which is configured to oligomerise to thereby form the oligomeric protein trap; (ii) contacting the sample comprising the target molecule with the protein trap of step (i) to allow the protein trap to specifically bind to and thereby enrich the target molecule; and

[0014] (iii) detecting and / or quantifying the enriched target molecule.

[0015] Advantageously, combining the high binding activity of the oligomeric protein trap to capture the target molecule with a powerful detection and quantification tool significantly increases the level of specificity and selectivity for the detected target molecule.

[0016] In one embodiment, the target molecule may comprise a polypeptide. In another embodiment, the target molecule may comprise a whole microorganism, preferably a virus. In another embodiment, the target molecule may comprise nucleic acid. For example, the nucleic acid may be associated with or contained within a virus that is captured by the protein trap.

[0017] Advantageously, the protein trap of the invention facilitates the enrichment of the target polypeptide, which enables the detection of pathogens and other non-pathogenic targets at concentrations well below those normally possible without the protein trap of the invention. As such, the protein trap of the invention enables the detection of viruses and other targets that are present in biological fluids, wastewater, effluent, and other samples at very low concentrations and in the presence of other components / contaminants. The protein traps use a natural receptor or binding protein that is significantly enhanced in its ability to bind the target by its presentation as an oligomer or multimer. A unique advantage of employing the protein trap of the invention for capture of the target polypeptide or protein is that it does not require the use of antibodies, which therefore makes it significantly simpler and more efficient to construct and use. Additionally, the protein trap can be used to capture variant pathogens, such as mutated viruses or bacteria, that may typically evade conventional antibodies as their sequence evolves, and they can be used for targets that are refractory to antibody production, provided that a natural receptor or binding protein is present.

[0018] The protein traps of the invention are advantageous in that they concentrate the target polypeptide and eliminate the majority of other proteins when used to recover a target from a sample. A conventional detection method can then easily detect the recovered target with very high sensitivity and minimal background. A multimeric version of a native / wild-type, non-mutated protein trap, can recover all variants of a target that can bind cellular receptor (and therefore relevant to infection in the case of a viral target). Another advantage of the protein trap of the invention is that constructs based on the natural receptors for the target polypeptide already bind the target and only require cloning into or fusing with the oligomerization domain to create a high-affinity binding agent. In contrast, the development of antibodies against certain specific target proteins is often very challenging, whereas it is possible to produce the protein traps of the invention in the presence of natural receptors or binding proteins for these particular targets. Moreover, the protein trap based on the receptor for a target (such as a viral target) can bind to any variant or mutant that is capable of binding the same receptor and is therefore pathologically relevant, while most antibodies will be susceptible to mutations that could cause to loss of binding activity.

[0019] In one embodiment, the oligomeric protein trap of the invention may comprise a wild-type, native or natural receptor or an extracellular binding domain thereof.

[0020] In another embodiment, the oligomeric receptor protein trap may comprise a modified or mutated version of a wild-type, native or natural receptor or extracellular binding domain thereof.

[0021] Advantageously, the binding interaction of the protein traps described herein is mediated by a known receptor or extracellular binding domain thereof of a known receptor for the target polypeptide or protein, typically the full-length extracellular domain or relevant subdomains of the extracellular domain. Additionally, the receptor is presented in a multimeric format to enhance its binding activity by leveraging its avidity effects.

[0022] Typically, the protein traps described herein can be used to capture and enrich pathogens (e.g. viruses or bacteria) whose cell surface receptors or binding domains are known, i.e., have already been identified. Thus, in an embodiment, the target polypeptide for the protein trap is of a pathogen, which may be a virus or bacterium.

[0023] Therefore, in one embodiment, the receptor or extracellular binding domain thereof is configured to specifically bind to a target virus, which may be selected from a group of viruses consisting of: coronavirus, Nipah virus, Hendra virus, Cedar virus, HIV, HCV, Rhinovirus, EBV and / or Measles. Typically, the coronavirus is selected from SARS-CoV1, SARS-CoV2, MERS-CoV, and / or 229E.

[0024] In one embodiment, the receptor or extracellular binding domain thereof is configured to specifically bind to SARS-CoV2. More typically, the receptor or binding domain thereof comprises the ACE2 ectodomain or a modified ACE2 ectodomain, most typically a pentameric ACE2 ectodomain (ACE2-COMP) ACE2.

[0025] The inventors have exemplified several different embodiments of the receptor or an extracellular binding domain thereof as a proof of concept. However, as discussed above, the protein traps of the invention may comprise any receptor or an extracellular binding domain thereof capable of binding a selected target polypeptide.

[0026] In a first embodiment, the receptor or the extracellular binding domain thereof may comprise wild-type ACE2 ectodomain monomer (Wt-ACE2). The Wt-ACE2 ectodomain can have an amino acid sequence (including the CD5 secretory leader sequence underlined) referred to herein as SEQ ID No:l, as follows:

[0027] MPMGSLQPLATLYLLGMLVASCLGSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAF LKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNE IMANSLDYNERLWAWESWRSEVGKQLRPLYEEYWLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEH TFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQR IFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMA YAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWM VFKGEI PKDQWMKKWWEMKREIVGWEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLH KCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD

[0028] [SEQ ID No: 1]

[0029] Typically, therefore, the receptor or the extracellular binding domain thereof comprises a Wt-ACE2 ectodomain comprising an amino acid sequence substantially as set out in SEQ ID No:l, or a fragment or variant thereof.

[0030] In one embodiment, a nucleotide sequence encoding the Wt-ACE2 ectodomain is referred to herein as SEQ ID No:2, as follows:

[0031] ATGCCCATGGGATCTCTGCAGCCTCTGGCCACACTGTACCTGCTGGGAATGCTGGTGGCCAGCTGTCTGGGCTCTACA ATCGAGGAACAGGCCAAGACCTTCCTGGACAAGTTCAACCACGAGGCCGAGGACCTGTTCTACCAGTCTAGCCTGGCC TCCTGGAACTACAACACCAACATCACCGAAGAGAACGTGCAGAACATGAACAACGCCGGCGACAAGTGGTCCGCCTTC CTGAAAGAACAGAGCACACTGGCCCAGATGTACCCTCTGCAAGAGATCCAGAACCTGACCGTGAAACTCCAGCTGCAG GCCCTCCAGCAGAATGGCAGTTCTGTGCTGAGCGAGGACAAGAGCAAGCGGCTGAACACCATCCTGAATACCATGAGC ACCATCTACAGCACCGGCAAAGTGTGCAACCCCGACAATCCTCAAGAGTGCCTGCTGCTGGAACCCGGCCTGAACGAG ATCATGGCCAACAGCCTGGACTACAATGAGAGACTGTGGGCCTGGGAGTCTTGGAGATCTGAAGTGGGAAAGCAGCTG CGGCCCCTGTACGAGGAATACGTGGTGCTGAAGAACGAGATGGCCAGAGCCAACCACTACGAGGACTACGGCGACTAT TGGAGAGGCGACTACGAAGTGAATGGCGTGGACGGCTACGACTACAGCAGAGGCCAGCTGATCGAGGACGTGGAACAC ACCTTCGAGGAAATCAAGCCTCTGTACGAGCATCTGCACGCCTATGTGCGGGCCAAGCTGATGAACGCTTACCCCAGC TACATCAGCCCTATCGGCTGTCTGCCTGCTCATCTGCTGGGCGATATGTGGGGCAGATTCTGGACCAACCTGTACAGC CTGACAGTGCCCTTCGGCCAGAAACCTAACATCGACGTGACCGACGCCATGGTGGATCAGGCTTGGGATGCCCAGAGA ATCTTCAAAGAGGCCGAGAAGTTCTTCGTGTCCGTGGGCCTGCCTAATATGACCCAAGGCTTCTGGGAGAACTCCATG CTGACCGATCCTGGCAATGTGCAGAAAGCCGTGTGTCACCCTACAGCCTGGGATCTCGGAAAGGGCGACTTCAGAATC CTGATGTGCACCAAAGTGACCATGGACGACTTTCTGACAGCCCACCACGAGATGGGCCACATCCAGTACGATATGGCC TACGCCGCTCAGCCCTTCCTGCTGAGAAATGGCGCCAATGAGGGCTTTCACGAAGCCGTGGGAGAGATCATGAGCCTG TCTGCCGCCACACCTAAGCACCTGAAGTCTATCGGACTGCTGAGCCCCGACTTCCAAGAGGACAACGAGACAGAGATC AACTTTCTGCTGAAGCAGGCCCTGACCATCGTGGGCACACTGCCTTTCACCTACATGCTGGAAAAGTGGCGGTGGATG GTCTTTAAGGGCGAGATCCCCAAGGACCAGTGGATGAAGAAATGGTGGGAGATGAAGCGCGAGATCGTGGGCGTTGTG GAACCTGTGCCTCACGACGAGACATACTGTGACCCTGCCAGCCTGTTTCACGTGTCCAACGACTACTCCTTCATCCGG TACTACACACGGACCCTGTACCAGTTCCAGTTTCAAGAGGCTCTGTGCCAGGCCGCCAAACACGAAGGACCTCTGCAC AAGTGCGACATCAGCAATTCTACCGAGGCCGGACAGAAACTGTTCAACATGCTGAGACTGGGCAAGAGCGAGCCTTGG ACTCTGGCCCTGGAAAATGTCGTGGGCGCCAAGAATATGAACGTGCGGCCACTGCTGAACTACTTCGAGCCTCTGTTC ACCTGGCTGAAGGACCAGAACAAGAACAGCTTCGTCGGCTGGTCCACCGATTGGAGCCCTTACGCTGAT

[0032] [SEQ ID No: 2]

[0033] Typically, therefore, the receptor or the extracellular binding domain thereof is encoded by a nucleotide sequence substantially as set out in SEQ ID No: 2, or a fragment or variant thereof.

[0034] In a second embodiment, the receptor or the extracellular binding domain thereof may comprise a mutated or evolved ACE2 ectodomain monomer (Ev-ACE2-A386L). The Ev-ACE2-A386L ectodomain can have an amino acid sequence (including a secretory leader sequence underlined) referred to herein as SEQ ID No:3, as follows:

[0035] MPMGSLQPLATLYLLGMLVASCLGSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNWGDKWSAF LKKQSTIAQMYPLQEIQNLKVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTI YSTGKVCNPDNPQECLLLEPGLNE IMANSLDYNERLWAWESWRSEVGKQLRPLYEEYWLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEH TFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQR IFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMA YLAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWM VFKGEI PKDQWMKKWWEMKREIVGWEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLH KCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD

[0036] [SEQ ID No: 3] Typically, therefore, the receptor or the extracellular binding domain thereof comprises a Ev-ACE2-A386L ectodomain comprising an amino acid sequence substantially as set out in SEQ ID No:3, or a fragment or variant thereof.

[0037] In one embodiment, a nucleotide sequence encoding the Ev-ACE2-A386L ectodomain is referred to herein as SEQ ID No:4, as follows:

[0038] atgcccatgggatctctgcagcctctggccacactgtacctgctgggaatgctggtggccagctgtctgggctct acaatcgaggaacaggccaagaccttcctggacaagttcaaccacgaggccgaggacctgttctaccag'tctagc ctggcctcctggaactacaacaccaacatcaccgaagagaacgtgcagaacatgaacaactggggcgacaagtgg tccgccttcctgaaaaaacagagcacaattgcccagatgtaccctctgcaagagatccagaacctgaaagtgaaa ctccagctgcaggccctccagcagaatggcagttctgtgctgagcgaggacaagagcaagcggctgaacacca'tc ctgaataccatgagcaccatctacagcaccggcaaagtgtgcaaccccgacaatcctcaagagtgcctgctgctg gaacccggcctgaacgagatcatggccaacagcctggactacaatgagagactgtgggcctgggagtcttggaga tctgaagtgggaaagcagctgcggcccctgtacgaggaatacgtggtgctgaagaacgagatggccagagccaac cactacgaggactacggcgactattggagaggcgactacgaagtgaatggcgtggacggctacgactacagcaga ggccagctgatcgaggacgtggaacacaccttcgaggaaatcaagcctctgtacgagcatctgcacgcctatgtg cgggccaagctgatgaacgcttaccccagctacatcagccctatcggctgtctgcctgctcatctgctgggcgat atgtggggcagattctggaccaacctgtacagcctgacagtgcccttcggccagaaacctaacatcgacgtgacc gacgccatggtggatcaggcttgggatgcccagagaatcttcaaagaggccgagaagttcttcgtgtccgtgggc ctgcctaatatgacccaaggcttctgggagaactccatgctgaccgatcctggcaatgtgcagaaagccgtgtgt caccctacagcctgggatctcggaaagggcgacttcagaatcctgatgtgcaccaaagtgaccatggacgac't'tt ctgacagcccaccacgagatgggccacatccagtacgatatggcctacctggctcagcccttcctgctgagaaat ggcgccaatgagggctttcacgaagccgtgggagagatcatgagcctgtctgccgccacacctaagcacctgaag tctatcggactgctgagccccgacttccaagaggacaacgagacagagatcaactttctgctgaagcaggccctg accatcgtgggcacactgcctttcacctacatgctggaaaagtggcggtggatggtctttaagggcgagatcccc aaggaccagtggatgaagaaatggtgggagatgaagcgcgagatcgtgggcgttgtggaacctgtgcctcacgac gagacatactgtgaccctgccagcctgtttcacgtgtccaacgactactccttcatccggtactacacacggacc ctgtaccagttccagtttcaagaggctctgtgccaggccgccaaacacgaaggacctctgcacaagtgcgacatc agcaattctaccgaggccggacagaaactgttcaacatgctgagactgggcaagagcgagccttggactctggcc ctggaaaatgtcgtgggcgccaagaatatgaacgtgcggccactgctgaactacttcgagcctctgttcacctgg ctgaaggaccagaacaagaacagcttcgtcggctggtccaccgattggagcccttacgctgat

[0039] [SEQ ID No: 4]

[0040] Typically, therefore, the receptor or the extracellular binding domain thereof is encoded by a nucleotide sequence substantially as set out in SEQ ID No: 4, or a fragment or variant thereof.

[0041] In one embodiment, the receptor or the extracellular binding domain thereof may comprise the receptor or the extracellular binding domain thereof of the Nipah virus. More typically, the receptor or the extracellular binding domain thereof of the Nipah virus is a membrane-tethered protein of the Ephrin family, such as the Ephrin-B2. Advantageously, the Nipah virus receptor also binds to the Hendra virus and Cedar virus target proteins and can, therefore, enable the capture of these viruses. Therefore, in a third embodiment, the receptor or the extracellular binding domain thereof may comprise the Nipah virus receptor. The Ephrin-B2 receptor can have an amino acid sequence (including a CD5 secretory leader sequence underlined) referred to herein as SEQ ID No: 5, as follows:

[0042] MAVRRDSVWKYCWGVLMVLCRTAISKSIVLEPIYWNSSNSKFLPGQGLVLYPQIGDKLDIICPKVDSKTVGQYEYYKV YMVDKDQADRCTIKKENTPLLNCAKPDQDIKFTIKFQEFSPNLWGLEFQKNKDYYIISTSNGSLEGLDNQEGGVCQTR AMKILMKVGQDASSAGSTRNKDPTRRPELEAGTNGRSSTTSPFVKPNPGSSTDGNSAGHSGNNILGSEVALFA

[0043] [SEQ ID No: 5]

[0044] Typically, therefore, the receptor or an extracellular binding domain thereof comprises the Ephrin-B2 receptor comprising an amino acid sequence substantially as set out in SEQ ID No: 5, or a fragment or variant thereof.

[0045] In one embodiment, a nucleotide sequence encoding the Nipah virus receptor ectodomain is referred to herein as SEQ ID No: 6, as follows:

[0046] atggccgtgcgccgcgacagcgtgtggaagtactgctggggcgtgctgatggtgctgtgccgcaccgccatcagcaag agcatcgtgctggagcccatctactggaacagcagcaacagcaagttcctgcccggccagggcctggtgctgtacccc cagatcggcgacaagctggacatcatctgccccaaggtggacagcaagaccgtgggccagtacgagtactacaaggtg tacatggtggacaaggaccaggccgaccgctgcaccatcaagaaggagaacacccccctgctgaactgcgccaagccc gaccaggacatcaagttcaccatcaagttccaggagt'tcagccccaacctgtggggcctggag't'tccagaagaacaag gactactacatcatcagcaccagcaacggcagcctggagggcctggacaaccaggagggcggcgtgtgccagacccgc gccatgaagatcctgatgaaggtgggccaggacgccagcagcgccggcagcacccgcaacaaggaccccacccgccgc cccgagctggaggccggcaccaacggccgcagcagcaccaccagccccttcgtgaagcccaaccccggcagcagcacc gacggcaacagcgccggccacagcggcaacaacatcctgggcagcgaggtggccctgttcgcc

[0047] [SEQ ID No: 6]

[0048] Typically, therefore, the receptor or an extracellular binding domain thereof is encoded by a nucleotide sequence substantially as set out in SEQ ID No: 6, or a fragment or variant thereof.

[0049] Typically, the protein traps described herein can be used to capture and enrich non-pathogenic proteins with known receptors or selective binding partners. Thus, in another embodiment, the target polypeptide for the protein trap is a ligand, such as a growth factor and / or a hormone. In one embodiment, the receptor or the extracellular binding domain thereof may comprise the receptor or the extracellular binding domain thereof of angiopoietin. More typically, the receptor or the extracellular binding domain thereof of angiopoietin is the Tie2 receptor. Advantageously, the Tie2 receptor binds all three human angiopoietins.

[0050] Therefore, in a fourth embodiment, the receptor or the extracellular binding domain thereof may comprise the Tie2 receptor. The Tie2 receptor can have an amino acid sequence (including a CD5 secretory leader sequence underlined) referred to herein as SEQ ID No:7, as follows:

[0051] MDSLASLVLCGVSLLLSGTVEGAMDLILINSLPLVSDAETSLTCIASGWRPHEPITIGRDFEALMNQHQDPLEVTQDV TREWAKKWWKREKASKINGAYFCEGRVRGEAIRIRTMKMRQQASFLPATLTMTVDKGDNVNI SFKKVLIKEEDAVI Y KNGSFIHSVPRHEVPDILEVHLPHAQPQDAGVYSARYIGGNLFTSAFTRLIVRRCEAQKWGPECNHLCTACMNNGVCH EDTGECICPPGFMGRTCEKACELHTFGRTCKERCSGQEGCKSYVFCLPDPYGCSCATGWKGLQCNEACHPGFYGPDCK LRCSCNNGEMCDRFQGCLCSPGWQGLQCEREGIPRMTPKIVDLPDHIEVNSGKFNPICKASGWPLPTNEEMTLVKPDG TVLHPKDFNHTDHFSVAI FTIHRILPPDSGVWVCSVNTVAGMVEKPFNISVK

[0052] [SEQ ID No: 7]

[0053] Typically, therefore, the receptor or an extracellular binding domain thereof comprises a Tie2 receptor comprising an amino acid sequence substantially as set out in SEQ ID No:7, or a fragment or variant thereof.

[0054] In one embodiment, a nucleotide sequence encoding the Tie2 receptor ectodomain is referred to herein as SEQ ID No:8, as follows:

[0055] ATGGACTCACTCGCATCTCTGGTTTTGTGTGGTGTCAGCCTCCTCCTGTCAGGGACCGTG GAGGGAGCTATGGACCTTATTCTGATCAACAGCCTGCCCCTCGTCAGTGATGCCGAGACC TCACTCACATGCATTGCCTCTGGCTGGAGGCCCCATGAACCTATAACCATCGGCCGGGAT TTCGAGGCACTCATGAATCAGCATCAAGACCCTCTGGAGGTGACACAAGACGTGACCAGG GAATGGGCAAAGAAGGTGGTGTGGAAGCGAGAAAAAGCCAGCAAGATTAACGGGGCTTAC TTCTGCGAGGGGCGAGTTAGAGGAGAGGCCATCAGGATTAGAACCATGAAAATGCGGCAA CAGGCGTCATTTCTCCCAGCTACTCTGACTATGACGGTCGATAAAGGAGACAATGTGAAC ATCTCCTTCAAAAAGGTTTTGATCAAAGAGGAGGATGCGGTCATTTACAAGAACGGATCC TTCATCCACTCAGTGCCCAGACATGAAGTACCTGATATACTCGAAGTGCATCTCCCGCAC GCACAGCCACAGGATGCAGGCGTCTACTCTGCACGATACATCGGCGGCAATCTTTTCACT TCTGCGTTTACGAGACTGATCGTGAGAAGATGCGAGGCCCAAAAGTGGGGGCCCGAATGC AATCATCTTTGTACCGCCTGTATGAATAATGGGGTGTGTCATGAGGACACGGGAGAGTGC ATATGCCCCCCTGGATTCATGGGACGGACTTGCGAAAAGGCATGCGAGCTTCACACCTTC GGGCGCACCTGTAAGGAACGCTGTTCTGGACAAGAAGGATGTAAGTCATACGTATTCTGC CTGCCAGATCCCTACGGGTGCTCTTGTGCCACCGGGTGGAAGGGTCTGCAATGTAACGAG GCCTGCCATCCGGGTTTCTATGGGCCTGACTGTAAACTGCGCTGCAGTTGCAATAACGGT GAGATGTGTGACCGATTTCAGGGATGCCTGTGCAGTCCAGGCTGGCAGGGTTTGCAGTGT GAACGGGAAGGAATCCCGAGAATGACACCTAAGATCGTTGACCTGCCAGACCACATTGAG GTGAACTCAGGCAAGTTTAATCCTATTTGCAAAGCGAGCGGCTGGCCCTTGCCTACAAAT GAGGAAATGACCCTCGTGAAACCGGACGGGACCGTGCTTCATCCAAAGGACTTCAACCAT ACGGATCACTTCTCAGTCGCTATCTTCACCATTCATCGGATTCTGCCCCCCGACAGTGGA GTTTGGGTGTGCAGTGTAAATACCGTCGCCGGCATGGTTGAAAAGCCCTTCAACATCTCC GTGAAA

[0056] [SEQ ID No: 8]

[0057] Typically, therefore, the receptor or an extracellular binding domain thereof is encoded by a nucleotide sequence substantially as set out in SEQ ID No: 8 or a fragment or variant thereof.

[0058] In one embodiment, the oligomeric protein trap of the invention may comprise an oligomerisation domain. As indicated by its name, the oligomerisation domain advantageously multimerises the receptor thereby increasing its binding activity by leveraging avidity effects. Any oligomerisation domain can be used, which is capable of creating an oligomer of the receptor or extracellular binding domain thereof. Examples of oligomerisation domains include but are not limited to the coiled:coil domain of GCN4, the IgM-Fc domain, the leucine zipper motifs, the coiled:coil domain from cartilage oligomeric matrix protein (COMP), and / or the vasodilator stimulated phosphoprotein (VASP).

[0059] The oligomerisation domain is configured to create a dimer, trimer, tetramer, or pentamer of the receptor or extracellular binding domain thereof.

[0060] However, typically, the oligomerisation domain is selected from the coiled:coil domain from cartilage oligomeric matrix protein (COMP) for pentamerisation

[0061] In one embodiment, oligomerisation domain may comprise a coiled:coil domain from cartilage oligomeric matrix protein (COMP). The COMP can have an amino acid sequence referred to herein as SEQ ID No:9, as follows:

[0062] DLAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMECDAC

[0063] [SEQ ID No: 9] Typically, therefore, the oligomerisation domain thereof comprises a COMP comprising an amino acid sequence substantially as set out in SEQ ID No:9, or a fragment or variant thereof.

[0064] In one embodiment, a nucleotide sequence encoding the COMP is referred to herein as SEQ ID No: 10, as follows:

[0065] gaccttgctcctcagatgctccgcgaactgcaggagactaatgctgcacttcaggatgttcgagagttgctg cggcaacaggtaaaagagattacgttcctcaagaatactgtcatggagtgcgatgcctgt

[0066] [SEQ ID No: 10]

[0067] Typically, therefore, the oligomerisation domain is encoded by a nucleotide sequence substantially as set out in SEQ ID No: 10, or a fragment or variant thereof.

[0068] In one embodiment, the oligomeric protein trap may comprise a first linker (e.g. flexible) disposed between the receptor or an extracellular binding domain thereof, and the oligomerisation domain. Advantageously, the flexible linker provided between the receptor or extracellular binding domain thereof and the oligomerisation domain allows for efficient oligomerisation by providing flexibility to the binding domain for maximising binding and minimizing spatial hinderance between binding domains and other parts of the fusion protein.

[0069] In an embodiment, the linker provided between the receptor or extracellular binding domain thereof and the oligomerisation domain can have an amino acid sequence referred to herein as SEQ ID No: 11, as follows:

[0070] GNSGGGGSGGGGSGGGGS

[0071] [SEQ ID No: 11]

[0072] Typically, therefore, the oligomeric protein trap comprises a linker comprising an amino acid sequence substantially as set out in SEQ ID No: 11, or a fragment or variant thereof.

[0073] In one embodiment, a nucleotide sequence encoding the flexible linker disposed between the receptor or extracellular binding domain thereof and the oligomerisation domain is referred to herein as SEQ ID No: 12, as follows: Gggaattctggaggcggcggatcaggcggaggaggttctggcggaggtgggagc

[0074] [SEQ ID No: 12] Typically, therefore, the linker is encoded by a nucleotide sequence substantially as set out in SEQ ID No: 12, or a fragment or variant thereof.

[0075] In some embodiments, the oligomeric protein trap may further comprise an epitope tag. The epitope tag may be disposed at the C-terminus of the oligomerisation domain. Typically, the epitope tag is a MDYKDDDK epitope tag or FLAG tag. The FLAG tag is a registered trademark for the MDYKDDDK epitope tag. It is used for antibody detection of proteins and sometimes for affinity purification of proteins.

[0076] Therefore, in one embodiment, the epitope tag may comprise a MDYKDDDK epitope tag. The MDYKDDDK epitope tag can have an amino acid sequence referred to herein as SEQ ID No: 13, as follows:

[0077] MDYKDDDDK

[0078] [SEQ ID No: 13]

[0079] Typically, therefore, the epitope tag comprises a MDYKDDDK epitope tag comprising an amino acid sequence substantially as set out in SEQ ID No: 13, or a fragment or variant thereof.

[0080] In one embodiment, a nucleotide sequence encoding the MDYKDDDK epitope tag is referred to herein as SEQ ID No: 14, as follows:

[0081] atggattacaaggacgacgacgacaaa

[0082] [SEQ ID No: 14]

[0083] Typically, therefore, the MDYKDDDK epitope tag is encoded by a nucleotide sequence substantially as set out in SEQ ID No: 14, or a fragment or variant thereof.

[0084] In another embodiment, the oligomeric protein trap may comprise a second linker (e.g. flexible) disposed at the C-terminus of the oligomerisation domain, and typically between the oligomerisation domain and the epitope tag if present. Advantageously, the second linker provided between the oligomerisation domain and the epitope tag minimises spatial hinderance between binding domains and between epitope tag and binding domain, and ensures access to the epitope tag.

[0085] Therefore, the second linker can have an amino acid sequence referred to herein as SEQ ID No: 15, as follows:

[0086] GGGGGS

[0087] [SEQ ID No: 15]

[0088] Typically, therefore, the oligomeric protein trap comprises a second linker comprising an amino acid sequence substantially as set out in SEQ ID No: 15, or a fragment or variant thereof.

[0089] In one embodiment, a nucleotide sequence encoding the second linker provided is referred to herein as SEQ ID No: 16, as follows:

[0090] ggaggaggaggcggctcc

[0091] [SEQ ID No: 16]

[0092] Typically, therefore, the second linker is encoded by a nucleotide sequence substantially as set out in SEQ ID No: 16, or a fragment or variant thereof.

[0093] In some embodiments, the oligomeric protein trap may further comprise an hexahistidine tag, typically disposed at the C-terminus of the oligomerisation domain, and ideally at the C-terminus of the epitope tag, if present. Typically, the hexahistidine tag is used for affinity purification and antibody detection of the target.

[0094] Therefore, in one embodiment, the hexahistidine tag can have an amino acid sequence referred to herein as SEQ ID No: 17, as follows:

[0095] HHHHHH [SEQ ID No: 17]

[0096] Typically, therefore, the hexahistidine tag comprises an amino acid sequence substantially as set out in SEQ ID No: 17, or a fragment or variant thereof.

[0097] In one embodiment, a nucleotide sequence encoding the hexahistidine tag is referred to herein as SEQ ID No: 18, as follows:

[0098] caccaccaccatcaccactga

[0099] [SEQ ID No: 18]

[0100] Typically, therefore, the hexahistidine tag is encoded by a nucleotide sequence substantially as set out in SEQ ID No: 18 or a fragment or variant thereof.

[0101] In yet another embodiment, the oligomeric protein trap may comprise a third linker (e.g. flexible), typically disposed at the C-terminus of the oligomerisation domain, ideally disposed between the epitope tag and the hexahistidine tag, if present. Advantageously, the third linker provided between the oligomerisation domain and the epitope tag minimises spatial hinderance between the epitope tag and hexahistidine tag to ensure access to both tags.

[0102] Therefore, the third linker disposed can have an amino acid sequence referred to herein as SEQ ID No: 19, as follows:

[0103] GSG

[0104] [SEQ ID No: 19]

[0105] Typically, therefore, the oligomeric protein trap comprises a third linker comprising an amino acid sequence substantially as set out in SEQ ID No: 19, or a fragment or variant thereof.

[0106] In one embodiment, a nucleotide sequence encoding the third linker is referred to herein as SEQ ID No:20, as follows:

[0107] ggcagcggc

[0108] [SEQ ID No: 20] Typically, therefore, the third linker is encoded by a nucleotide sequence substantially as set out in SEQ ID No: 20, or a fragment or variant thereof.

[0109] In one embodiment, the oligomerisation domain is disposed at the C-terminus of the oligomeric protein trap. However, in another embodiment, the oligomerisation domain may be disposed at the N-terminus of the oligomeric protein trap.

[0110] In one embodiment, the oligomeric protein trap comprises (in the following order from the N-terminus to the C-terminus): a receptor or extracellular binding domain thereof, a linker (which is typically flexible), and an oligomerisation domain.

[0111] However, in another embodiment, the oligomeric protein trap comprises (in the following order from the N-terminus to the C-terminus): a receptor or extracellular binding domain thereof, a linker, an oligomerisation domain, and optionally, a second linker, and an epitope tag and / or a hexahistidine tag. In another embodiment, the protein trap comprises a third linker disposed between the epitope tag and the hexahistidine tag. Most typically, the oligomerisation domain is the COMP. In yet another embodiment, the epitope tag is a MDYKDDDK epitope tag.

[0112] In one embodiment, the oligomeric protein trap may be based on the pentamerised SARS-COv2 receptor ACE2-COMP. Typically, therefore, the oligomeric protein trap comprises the ACE2-COMP receptor including a CD5 secretory leader sequence, the flexible linker having an amino acid sequence GNSGGGGSGGGGSGGGGS [SEQ ID No: 11], the COMP oligomerisation domain, the second flexible linker having an amino acid sequence GGGGGS, the MDYKDDDK epitope tag, the third flexible linker having an amino acid sequence GSG, and the hexahistidine tag.

[0113] Therefore, in one typical embodiment, the protein trap based on the pentamerised SARS-COv2 receptor ACE2-COMP can have an amino acid sequence referred to herein as SEQ ID No:21, as follows:

[0114] MPMGSLQPLATLYLLGMLVASCLGSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAF LKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNE IMANSLDYNERLWAWESWRSEVGKQLRPLYEEYWLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEH TFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQR IFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMA YAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWM VFKGEI PKDQWMKKWWEMKREIVGWEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLH KCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADGNS GGGGSGGGGSGGGGSDLAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMECDACGGGGGSMDYKDDDDKGSGH HHHHH [SEQ ID No: 21]

[0115] In one embodiment, therefore, the protein trap comprises an amino acid sequence substantially as set out in SEQ ID No:21, or a fragment or variant thereof.

[0116] In one embodiment, a nucleotide sequence encoding the protein trap based on the pentamerised SARS-COv2 receptor ACE2-COMP is referred to herein as SEQ ID No:22, as follows:

[0117] ATGCCCATGGGATCTCTGCAGCCTCTGGCCACACTGTACCTGCTGGGAATGCTGGTGGCCAGCTGTCTGGGCTCTACAATCGAGG AACAGGCCAAGACCTTCCTGGACAAGTTCAACCACGAGGCCGAGGACCTGTTCTACCAGTCTAGCCTGGCCTCCTGGAACTACAA CACCAACATCACCGAAGAGAACGTGCAGAACATGAACAACGCCGGCGACAAGTGGTCCGCCTTCCTGAAAGAACAGAGCACACTG GCCCAGATGTACCCTCTGCAAGAGATCCAGAACCTGACCGTGAAACTCCAGCTGCAGGCCCTCCAGCAGAATGGCAGTTCTGTGC TGAGCGAGGACAAGAGCAAGCGGCTGAACACCATCCTGAATACCATGAGCACCATCTACAGCACCGGCAAAGTGTGCAACCCCGA CAATCCTCAAGAGTGCCTGCTGCTGGAACCCGGCCTGAACGAGATCATGGCCAACAGCCTGGACTACAATGAGAGACTGTGGGCC TGGGAGTCTTGGAGATCTGAAGTGGGAAAGCAGCTGCGGCCCCTGTACGAGGAATACGTGGTGCTGAAGAACGAGATGGCCAGAG CCAACCACTACGAGGACTACGGCGACTATTGGAGAGGCGACTACGAAGTGAATGGCGTGGACGGCTACGACTACAGCAGAGGCCA GCTGATCGAGGACGTGGAACACACCTTCGAGGAAATCAAGCCTCTGTACGAGCATCTGCACGCCTATGTGCGGGCCAAGCTGATG AACGCTTACCCCAGCTACATCAGCCCTATCGGCTGTCTGCCTGCTCATCTGCTGGGCGATATGTGGGGCAGATTCTGGACCAACC TGTACAGCCTGACAGTGCCCTTCGGCCAGAAACCTAACATCGACGTGACCGACGCCATGGTGGATCAGGCTTGGGATGCCCAGAG AATCTTCAAAGAGGCCGAGAAGTTCTTCGTGTCCGTGGGCCTGCCTAATATGACCCAAGGCTTCTGGGAGAACTCCATGCTGACC GATCCTGGCAATGTGCAGAAAGCCGTGTGTCACCCTACAGCCTGGGATCTCGGAAAGGGCGACTTCAGAATCCTGATGTGCACCA AAGTGACCATGGACGACTTTCTGACAGCCCACCACGAGATGGGCCACATCCAGTACGATATGGCCTACGCCGCTCAGCCCTTCCT GCTGAGAAATGGCGCCAATGAGGGCTTTCACGAAGCCGTGGGAGAGATCATGAGCCTGTCTGCCGCCACACCTAAGCACCTGAAG TCTATCGGACTGCTGAGCCCCGACTTCCAAGAGGACAACGAGACAGAGATCAACTTTCTGCTGAAGCAGGCCCTGACCATCGTGG GCACACTGCCTTTCACCTACATGCTGGAAAAGTGGCGGTGGATGGTCTTTAAGGGCGAGATCCCCAAGGACCAGTGGATGAAGAA ATGGTGGGAGATGAAGCGCGAGATCGTGGGCGTTGTGGAACCTGTGCCTCACGACGAGACATACTGTGACCCTGCCAGCCTGTTT CACGTGTCCAACGACTACTCCTTCATCCGGTACTACACACGGACCCTGTACCAGTTCCAGTTTCAAGAGGCTCTGTGCCAGGCCG CCAAACACGAAGGACCTCTGCACAAGTGCGACATCAGCAATTCTACCGAGGCCGGACAGAAACTGTTCAACATGCTGAGACTGGG CAAGAGCGAGCCTTGGACTCTGGCCCTGGAAAATGTCGTGGGCGCCAAGAATATGAACGTGCGGCCACTGCTGAACTACTTCGAG CCTCTGTTCACCTGGCTGAAGGACCAGAACAAGAACAGCTTCGTCGGCTGGTCCACCGATTGGAGCCCTTACGCTGATGGGAATT CTGGAGGCGGCGGATCAGGCGGAGGAGGTTCTGGCGGAGGTGGGAGCGACCTTGCTCCTCAGATGCTCCGCGAACTGCAGGAGAC TAATGCTGCACTTCAGGATGTTCGAGAGTTGCTGCGGCAACAGGTAAAAGAGATTACGTTCCTCAAGAATACTGTCATGGAGTGC GATGCCTGTGGAGGAGGAGGCGGCTCCATGGATTACAAGGACGACGACGACAAAGGCAGCGGCCACCACCACCATCACCACTGAT

[0118] [SEQ ID No: 22]

[0119] Typically, therefore, the protein trap is encoded by a nucleotide sequence substantially as set out in SEQ ID No: 22, or a fragment or variant thereof.

[0120] In another embodiment, the oligomeric protein trap may be based on the pentamerised Nipah virus receptor Ephrin-B2. Typically, therefore, the oligomeric protein trap comprises the COMP-Ephrin-B2 receptor including a CD5 secretory

[0121] leader sequence, the flexible linker having an amino acid sequence GNSGGGGSGGGGSGGGGS [SEQ ID No: 11, the COMP oligomerisation domain, the second flexible linker having an amino acid sequence GGGGGS [SEQ ID No: 15], the MDYKDDDK [SEQ ID No: 13] epitope tag, the third flexible linker having an amino acid sequence GSG, and the hexahistidine tag.

[0122] Therefore, in one embodiment, the protein trap based on the pentamerised Nipah virus receptor can have an amino acid sequence referred to herein as SEQ ID No:23, as follows:

[0123] MAVRRDSVWKYCWGVLMVLCRTAISKSIVLEPIYWNSSNSKFLPGQGLVLYPQIGDKLDIICPKVDSKTVGQYEYYKV YMVDKDQADRCTIKKENTPLLNCAKPDQDIKFTIKFQEFSPNLWGLEFQKNKDYYIISTSNGSLEGLDNQEGGVCQTR AMKILMKVGQDASSAGSTRNKDPTRRPELEAGTNGRSSTTSPFVKPNPGSSTDGNSAGHSGNNILGSEVALFAGNSGG GGSGGGGSGGGGSDLAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMECDACGGGGGSMDYKDDDDKGSGHHH HHH

[0124] [SEQ ID No: 23]

[0125] In one embodiment, therefore, the protein trap comprises an amino acid sequence substantially as set out in in SEQ ID No:23, or a fragment or variant thereof.

[0126] In one embodiment, a nucleotide sequence encoding the protein trap based on the pentamerised Nipah virus receptor Ephrin-B2 is referred to herein as SEQ ID No: 24, as follows:

[0127] Atggccgtgcgccgcgacagcgtgtggaagtactgctggggcgtgctgatggtgctgtgccgcaccgccatcagcaag agcatcgtgctggagcccatctactggaacagcagcaacagcaagttcctgcccggccagggcctggtgctgtacccc cagatcggcgacaagctggacatcatctgccccaaggtggacagcaagaccgtgggccagtacgagtactacaaggtg tacatggtggacaaggaccaggccgaccgctgcaccatcaagaaggagaacacccccctgctgaactgcgccaagccc gaccaggacatcaagttcaccatcaagttccaggagttcagccccaacctgtggggcctggagttccagaagaacaag gactactacatcatcagcaccagcaacggcagcctggagggcctggacaaccaggagggcggcgtgtgccagacccgc gccatgaagatcctgatgaaggtgggccaggacgccagcagcgccggcagcacccgcaacaaggaccccacccgccgc cccgagctggaggccggcaccaacggccgcagcagcaccaccagccccttcgtgaagcccaaccccggcagcagcacc gacggcaacagcgccggccacagcggcaacaacatcctgggcagcgaggtggccctgttcgccgggaattctggaggc ggcggatcaggcggaggaggttctggcggaggtgggagcgaccttgctcctcagatgctccgcgaactgcaggagact aatgctgcacttcaggatgttcgagagttgctgcggcaacaggtaaaagagattacgttcctcaagaatactgtcatg gagtgcgatgcctgtggaggaggaggcggctccatggattacaaggacgacgacgacaaaggcagcggccaccaccac catcaccactgataa

[0128] [SEQ ID No: 24]

[0129] Typically, therefore, the protein trap is encoded by a nucleotide sequence substantially as set out in SEQ ID No: 24, or a fragment or variant thereof.

[0130] In another embodiment, the oligomeric protein trap may be based on the pentamerised angiopoietin receptor Tie2. Typically, therefore, the oligomeric protein trap comprises the COMP-Tie2 receptor including a CD5 secretory leader sequence, the flexible linker having an amino acid sequence - I -

[0131] GNSGGGGSGGGGSGGGGS [SEQ ID No: 11], the COMP oligomerisation domain, the second flexible linker having an amino acid sequence GGGGGS [SEQ ID No: 15], the MDYKDDDK [SEQ ID No: 13] epitope tag, the third flexible linker having an amino acid sequence GSG, and the hexahistidine tag.

[0132] Therefore, in one embodiment, the protein trap based on the pentamerised angiopoietin receptor Tie2 can have an amino acid sequence referred to herein as SEQ ID No:25, as follows:

[0133] MDSLASLVLCGVSLLLSGTVEGAMDLILINSLPLVSDAETSLTCIASGWRPHEPITIGRDFEALMNQHQDPLEVTQDV TREWAKKWWKREKASKINGAYFCEGRVRGEAIRIRTMKMRQQASFLPATLTMTVDKGDNVNI SFKKVLIKEEDAVI Y KNGSFIHSVPRHEVPDILEVHLPHAQPQDAGVYSARYIGGNLFTSAFTRLIVRRCEAQKWGPECNHLCTACMNNGVCH EDTGECICPPGFMGRTCEKACELHTFGRTCKERCSGQEGCKSYVFCLPDPYGCSCATGWKGLQCNEACHPGFYGPDCK LRCSCNNGEMCDRFQGCLCSPGWQGLQCEREGIPRMTPKIVDLPDHIEVNSGKFNPICKASGWPLPTNEEMTLVKPDG TVLHPKDFNHTDHFSVAIFTIHRILPPDSGVWVCSVNTVAGMVEKPFNISVKGNSGGGGSGGGGSGGGGSDLAPQMLR ELQETNAALQDVRELLRQQVKEITFLKNTVMECDACGGGGGSMDYKDDDDKGSGHHHHHH

[0134] [SEQ ID No: 25]

[0135] In one embodiment, therefore, the protein trap comprises an amino acid sequence substantially as set out in SEQ ID No:25, or a fragment or variant thereof.

[0136] In one embodiment, a nucleotide sequence encoding the protein trap based on the pentamerised angiopoietin receptor Tie2 is referred to herein as SEQ ID No:26, as follows:

[0137] ATGGACTCACTCGCATCTCTGGTTTTGTGTGGTGTCAGCCTCCTCCTGTCAGGGACCGTG GAGGGAGCTATGGACCTTATTCTGATCAACAGCCTGCCCCTCGTCAGTGATGCCGAGACC TCACTCACATGCATTGCCTCTGGCTGGAGGCCCCATGAACCTATAACCATCGGCCGGGAT TTCGAGGCACTCATGAATCAGCATCAAGACCCTCTGGAGGTGACACAAGACGTGACCAGG GAATGGGCAAAGAAGGTGGTGTGGAAGCGAGAAAAAGCCAGCAAGATTAACGGGGCTTAC TTCTGCGAGGGGCGAGTTAGAGGAGAGGCCATCAGGATTAGAACCATGAAAATGCGGCAA CAGGCGTCATTTCTCCCAGCTACTCTGACTATGACGGTCGATAAAGGAGACAATGTGAAC ATCTCCTTCAAAAAGGTTTTGATCAAAGAGGAGGATGCGGTCATTTACAAGAACGGATCC TTCATCCACTCAGTGCCCAGACATGAAGTACCTGATATACTCGAAGTGCATCTCCCGCAC GCACAGCCACAGGATGCAGGCGTCTACTCTGCACGATACATCGGCGGCAATCTTTTCACT TCTGCGTTTACGAGACTGATCGTGAGAAGATGCGAGGCCCAAAAGTGGGGGCCCGAATGC AATCATCTTTGTACCGCCTGTATGAATAATGGGGTGTGTCATGAGGACACGGGAGAGTGC ATATGCCCCCCTGGATTCATGGGACGGACTTGCGAAAAGGCATGCGAGCTTCACACCTTC GGGCGCACCTGTAAGGAACGCTGTTCTGGACAAGAAGGATGTAAGTCATACGTATTCTGC CTGCCAGATCCCTACGGGTGCTCTTGTGCCACCGGGTGGAAGGGTCTGCAATGTAACGAG GCCTGCCATCCGGGTTTCTATGGGCCTGACTGTAAACTGCGCTGCAGTTGCAATAACGGT GAGATGTGTGACCGATTTCAGGGATGCCTGTGCAGTCCAGGCTGGCAGGGTTTGCAGTGT GAACGGGAAGGAATCCCGAGAATGACACCTAAGATCGTTGACCTGCCAGACCACATTGAG GTGAACTCAGGCAAGTTTAATCCTATTTGCAAAGCGAGCGGCTGGCCCTTGCCTACAAAT GAGGAAATGACCCTCGTGAAACCGGACGGGACCGTGCTTCATCCAAAGGACTTCAACCAT ACGGATCACTTCTCAGTCGCTATCTTCACCATTCATCGGATTCTGCCCCCCGACAGTGGA GTTTGGGTGTGCAGTGTAAATACCGTCGCCGGCATGGTTGAAAAGCCCTTCAACATCTCC GTGAAAGGGAATTCTGGAGGCGGCGGATCAGGCGGAGGAGGTTCTGGCGGAGGTGGGAGC GACCTTGCTCCTCAGATGCTCCGCGAACTGCAGGAGACTAATGCTGCACTTCAGGATGTT CGAGAGTTGCTGCGGCAACAGGTAAAAGAGATTACGTTCCTCAAGAATACTGTCATGGAG TGCGATGCCTGTGGAGGAGGAGGCGGCTCCATGGATTACAAGGACGACGACGACAAAGGC AGCGGCCACCACCACCATCACCACTGATAA

[0138] [SEQ ID No: 26]

[0139] Typically, therefore, the protein trap is encoded by a nucleotide sequence substantially as set out in SEQ ID No: 26, or a fragment or variant thereof.

[0140] In one embodiment, the sample is a biological sample, which may be taken from a subject. In one embodiment, the biological sample is selected from a swab, saliva, sputum, serum, blood, urine, faeces, cerebrospinal fluid, semen, vaginal discharge, tissue biopsy and / or mucus.

[0141] In another embodiment, the sample is environmental sample. The environmental sample may be selected from water, wastewater, effluent, sand, and / or soil.

[0142] In one embodiment, the protein trap is incubated in a sample or solution comprising the target molecule, which may be a protein or virus. In an alternative embodiment, the protein trap is immobilised to capture the target molecule and / or protein and / or virus.

[0143] Typically, when contacting the sample comprising the target molecule with the protein trap, the protein trap can be incubated or immobilised for up to 5 minutes, up to 10 minutes, up to 15 minutes, up to 20 minutes, up to 25 minutes, up to 30 minutes, up to 35 minutes, up to 40 minutes, up to 45 minutes, up to 50 minutes, up to 55 minutes, up to 60 minutes, up to 75 minutes, or up to 90 minutes.

[0144] In one embodiment, the step of contacting the sample comprising the target molecule with the protein trap of step (i) may further comprise the step of isolating the target molecule bound to the trap. In one embodiment, the isolating step is performed using microbeads. Typically, the microbeads are beads that bind to the hexahistidine tag of the oligomeric protein trap. Most typically, the microbeads are Nickel-IMAC (immobilised metal affinity chromatography) beads.

[0145] Typically, in one embodiment, the target-selective detection and / or quantification method is Mass Spectrometry. Advantageously, the method according to the invention enables the accurate quantification of the target polypeptide that has been enriched / captured by the protein trap, regardless of the presence of other contaminants in the sample. The accurate quantification of the target protein from complex mixtures is enabled by the combination of target-specific analysis (using, for example, MS and / or PCR) and the protein trap's ability to recover / enrich the target protein, even when the target protein is at low concentration.

[0146] Moreover, the protein trap can also be used to capture whole virus as described in the Examples. This allows the detection of other viral components and other proteins that are part of the virus, in addition to the protein that binds the trap. This is exemplified in the detection of the nucleocapsid protein or the enrichment of viruses for PCR detection. This enables the quantification of the intact virus instead of the free spike protein.

[0147] Advantageously, the method of the invention can be used to decontaminate a biological fluid prior to transfusion or transplant, wherein the protein trap captures a target contaminant.

[0148] In addition, the method according to the invention can be used in monitoring pathogen contamination of an environmental sample.

[0149] Advantageously, the protein traps disclosed herein can also bind and block pathogenic agents, rendering them suitable for therapeutic applications.

[0150] It will be appreciated that the invention extends to any nucleic acid or peptide or variant, derivative or analogue thereof, which comprises substantially the amino acid or nucleic acid sequences of any of the sequences referred to herein, including variants or fragments thereof. The terms "substantially the amino acid / nucleotide / peptide sequence", "variant" and "fragment", can be a sequence that has at least 40% sequence identity with the amino acid / nucleotide / peptide sequences of any one of the sequences referred to herein, for example 40% identity with the sequence identified as SEQ ID Nos: 1-26 and so on.

[0151] Amino acid / polynucleotide / polypeptide sequences with a sequence identity which is greater than 65%, more typically greater than 70%, even more typically greater than 75%, and still more typically greater than 80% sequence identity to any of the sequences referred to are also envisaged. Typically, the amino acid / polynucleotide / polypeptide sequence has at least 85% identity with any of the sequences referred to, more typically at least 90% identity, even more typically at least 92% identity, even more typically at least 95% identity, even more typically at least 97% identity, even more typically at least 98% identity and, most typically at least 99% identity with any of the sequences referred to herein.

[0152] The skilled technician will appreciate how to calculate the percentage identity between two amino acid / polynucleotide / polypeptide sequences. In order to calculate the percentage identity between two amino acid / polynucleotide / polypeptide sequences, an alignment of the two sequences must first be prepared, followed by calculation of the sequence identity value. The percentage identity for two sequences may take different values depending on:- (i) the method used to align the sequences, for example, ClustalW, BLAST, FASTA, Smith-Waterman (implemented in different programs), or structural alignment from 3D comparison; and (ii) the parameters used by the alignment method, for example, local vs global alignment, the pair-score matrix used (e.g. BLOSUM62, PAM250, Gonnet etc.), and gap-penalty, e.g. functional form and constants.

[0153] Having made the alignment, there are many different ways of calculating percentage identity between the two sequences. For example, one may divide the number of identities by: (i) the length of shortest sequence; (ii) the length of alignment; (iii) the mean length of sequence; (iv) the number of non-gap positions; or (v) the number of equivalenced positions excluding overhangs. Furthermore, it will be appreciated that percentage identity is also strongly length dependent.

[0154] Therefore, the shorter a pair of sequences is, the higher the sequence identity one may expect to occur by chance.

[0155] Hence, it will be appreciated that the accurate alignment of protein or DNA sequences is a complex process. The popular multiple alignment program ClustalW (Thompson et al., 1994, Nucleic Acids Research, 22, 4673-4680; Thompson et al., 1997, Nucleic Acids Research, 24, 4876-4882) is a typical way for generating multiple alignments of proteins or DNA in accordance with the invention. Suitable parameters for ClustalW may be as follows: For DNA alignments: Gap Open Penalty = 15.0, Gap Extension Penalty = 6.66, and Matrix = Identity. For protein alignments: Gap Open Penalty = 10.0, Gap Extension Penalty = 0.2, and Matrix = Gonnet. For DNA and Protein alignments: ENDGAP = -1, and GAPDIST = 4. Those skilled in the art will be aware that it may be necessary to vary these and other parameters for optimal sequence alignment.

[0156] Typically, calculation of percentage identities between two amino acid / polynucleotide / polypeptide sequences may then be calculated from such an alignment as (N / T)*100, where N is the number of positions at which the sequences share an identical residue, and T is the total number of positions compared including gaps and either including or excluding overhangs. Typically, overhangs are included in the calculation. Hence, a typical method for calculating percentage identity between two sequences comprises (i) preparing a sequence alignment using the ClustalW program using a suitable set of parameters, for example, as set out above; and (ii) inserting the values of N and T into the following formula:- Sequence Identity = (N / T)*100.

[0157] Alternative methods for identifying similar sequences will be known to those skilled in the art. For example, a substantially similar nucleotide sequence will be encoded by a sequence which hybridizes to DNA sequences or their complements under stringent conditions. By stringent conditions, the inventors mean the nucleotide hybridises to filter-bound DNA or RNA in 3x sodium chloride / sodium citrate (SSC) at approximately 45°C followed by at least one wash in 0.2x SSC / 0.1% SDS at approximately 20-65°C. Alternatively, a substantially similar polypeptide may differ by at least 1, but less than 5, 10, 20, 50 or 100 amino acids from the sequences shown in, for example, in those of SEQ ID Nos: 1 to 26 that are amino acid sequences.

[0158] Due to the degeneracy of the genetic code, it is clear that any nucleic acid sequence described herein could be varied or changed without substantially affecting the sequence of the protein encoded thereby, to provide a functional variant thereof. Suitable nucleotide variants are those having a sequence altered by the substitution of different codons that encode the same amino acid within the sequence, thus producing a silent (synonymous) change. Other suitable variants are those having homologous nucleotide sequences but comprising all, or portions of, sequence, which are altered by the substitution of different codons that encode an amino acid with a side chain of similar biophysical properties to the amino acid it substitutes, to produce a conservative change. For example, small non-polar, hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine. Large non-polar, hydrophobic amino acids include phenylalanine, tryptophan and tyrosine. The polar neutral amino acids include serine, threonine, cysteine, asparagine and glutamine. The positively charged (basic) amino acids include lysine, arginine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. It will therefore be appreciated which amino acids may be replaced with an amino acid having similar biophysical properties, and the skilled technician will know the nucleotide sequences encoding these amino acids.

[0159] All of the features described herein (including any accompanying claims, abstracts and drawings), and / or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some features and / or steps are mutually exclusive.

[0160] For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying Figures, in which:-

[0161] Figure 1 provides an overview of the directed evolution of the SARS CoV-2 spike protein receptor, ACE2, ectodomain, to produce an embodiment of the protein trap of the invention. In particular, Figure 1A shows a schematic representation of cell surface expressed ACE2 ectodomain, i.e. the receptor domain that extends into the extracellular space, and which initiates contact with the target protein on a pathogen (e.g. SARS-CoV2 spike protein). A full-length ACE2 was expressed with an amino-terminal MDYKDDDK epitope tag, which is used for antibody detection of proteins and for affinity purification of proteins, and short linker region. Figure IB shows the selection of ACE2 for increased affinity. Flow cytometry plots are shown for cells incubated with the receptor biding domain (RBD) of the SARS-CoV2 spike protein and stained for bound RBD using anti-His-phycoerythrin, and ACE2 expression using anti-FLAG-allophycocyanin. Three rounds of selection, at the indicated RBD concentrations, and diversification were performed. The sort windows are indicated for each round of selection. Figure 1C shows the amino acid substitutions present in evolved ACE2 in red. Figure ID shows the position of substitutions in ACE2 structure (PDB entry ID: 6M0J18. The left panel shows part of ACE2 structure (blue) in complex with RBD (yellow) with substitutions in evolved ACE2 shown in red. The position of A368L is shown in orange. The right panel shows structure of RBD binding interface of ACE2 and the position of substitutions. Position of RBD-interacting residues in Wt-ACE2 are indicated in green. Figure IE shows the kinetic binding constants for RBD binding by Wt-ACE2, evolved ACE2 and evolved ACE2 with A386L, measured by biolayer interferometry with soluble ACE2 (19-615) and immobilised monomeric RBD. Data are shown as means and SEM for at least two experiments.

[0162] Figure 2 shows that ACE2 multimerisation increases binding to RBD. In particular, Figure 2A shows a schematic representation of monomeric (ACE2-615), dimeric (ACE2-740) and pentameric (ACE2-COMP) ACE2. Figure 2B shows the negative stain electron microscopy of monomeric, dimeric and pentameric ACE2 (left to right). Scale bar, 50nm. Figure 2C shows the kinetic binding constants for RBD binding by Wt-ACE2 dimer and pentamer measured by biolayer interferometry with soluble ACE2 and immobilised RBD. Data are shown as means and SEM for two experiments. Figure 2D shows the kinetic association and dissociation curves for monomeric, dimeric and pentameric ACE2 demonstrating decreased dissociation kinetics for dimer and pentamer, and retention of bound RBD for more than 8 h for pentamer. Figure 2E shows the negative stain electron microscopy image of ACE2-COMP bound to SARS-CoV-2 virus. Scale bar, lOOnm.

[0163] Figure 3 shows the binding of ACE2-COMP to RBD variants. In particular, Figure 3A shows the kinetic binding constants for RBD binding by ACE2-COMP measured by biolayer interferometry with soluble ACE2 and immobilised monomeric RBD. Data are shown as means and SEM for two experiments. Figure 3B shows the kinetic association and dissociation curves for binding of ACE2-COMP to

[0164] B.1.617.1 / 3 and B.1.351 variant RBD demonstrating retention of bound RBD for more than 8 h.

[0165] Figure 4 shows the poor sensitivity of detection of SARS-CoV-2 spike protein without sample enrichment. In particular, Figure 4A shows the detection of a coeluting, interfering peak for the ASA peptide in spike protein naive saliva, highlighting the challenges in discerning specific signals amidst background noise, which can lead to false positives or inaccurate quantification. Figure 4B shows the absence of signal for the SFI peptide in saliva fortified with 100 fmol / pL spike protein, indicating limitations in sensitivity and detection capability without employing capture techniques, which can hinder reliable identification and quantification of target analytes. Figure 5 shows the ACE2-COMP protein trap capture-based mass spectrometric assay for the resolution and detection of SARS-Cov-2 spike peptides. In particular, Figure 5A displays an overlayed chromatogram of spike fortified saliva (157 fmol / pL) extracted with the protein trap assay but with plates that were absent of ACE2-COMP capture protein. Figure 5B shows an overlayed chromatogram of fortified saliva (157 fmol / pL) extracted using plates treated with the ACE2-COMP capture protein. Figure 5C shows the binding saturation series using the ACE-24 protein trap assay with variably fortified saliva and buffer (PBS) (1.2 - 157 fmol / pL) and constant ACE2-COMP (125 ng). Examples

[0166] The inventors aimed to develop a high affinity receptor-based capture platform for viral target protein enrichment applicable to diagnostic mass spectrometry. Directed protein evolution is a powerful method for engineering protein functionality, including affinity. However, mutations introduced in the evolution into the RBD binding site on ACE2 can make such proteins sensitive to mutations in spike protein, potentially decreasing their ability to bind variants14'15. Here the inventors focussed on ACE2 in which they evolved increased affinity for RBD but retained the Wt binding interface, Ev-ACE2-A386L. Nevertheless, even with this evolved ACE2 they found that even though all mutations were peripheral to the RBD binding site, Ev-ACE-A386L displayed differences in binding ability across the limited number of RBD variants tested by the inventors. As an alternative to evolving ACE2, the inventors therefore examined non-mutated Wt-ACE2, and sought to leverage avidity effects of multimerization to increase binding to viral spike. Pentameric ACE2, ACE2-COMP, was found to bind very strongly to RBD and retain the bound viral protein over several hours. Furthermore, ACE2-COMP successfully quantitatively enriched for viral spike protein from biological samples, and increased sensitivity of detection of viral spike by MS.

[0167] Materials and methods

[0168] Materials

[0169] DNA encoding full-length human ACE2 was a gift from Hyeryun Choe25(Addgene plasmid # 1786; http: / / n2t.net / addgene: 1786; RRID: Addgene_1786). Codon optimised DNA encoding CD5 secretory leader (residues 1-24) followed by human ACE2 (residues 19-615), or human ACE2 (residues 19-740), a short linker then the FLAG epitope tag and C-terminus Histidine6 was synthesized by GeneArt. DNA encoding codon optimised ACE2-COMP was also synthesized by GeneArt. This ACE2-COMP fusion protein comprised of a CD5 secretory leader, human ACE2 (residues 19-615) a short linker followed by residues 29-73 of rat cartilage oligomeric matrix protein then another linker before a FLAG epitope tag and C-terminus Histidine6.

[0170] Water, acetonitrile and formic acid, all Optima LC-MS grade, were sourced from Fisher Chemical (Geel, Belgium). Phosphate buffered saline (PBS) tablets were purchased from Thermo Fisher (Rockford, IL, USA). Trypsin from bovine pancreas, 1,4-Dithiothreitol (DTT), iodoacetamide (IAA), ammonium bicarbonate (ABC), and Tris-buffered saline (TBS), and acetone were obtained from Sigma-Aldrich (St. Louis, MO, USA). The trimeric SARS-CoV-2 spike protein was purchased from BioServUK (Rotherham, UK). Stable isotope standard peptides, GWIFGTTLDSK(*) and SFIEDLLFNK(*) (*modified using13Ce15N2 K), were produced by PepScan (Lelystad, Netherlands). For peptide preparation, a stable isotope labelled peptide mix was made (500 fmol / pL each of GWIFGTTLDSK(*) and SFIEDLLFNK(*)).

[0171] Reagents were made up in Protein LoBind® Tubes (Eppendorf, Stevenage, UK). Nunc™ MicroWell™ 96-Well Microplates (Fisher Scientific, Loughborough, UK) and QuanRecovery with MaxPeak plates (Waters, Milford, MA, USA) were sourced for protein enrichment and LC-MS analysis, respectively. An Eppendorf ThermoMixer® C, fitted with a PCR 96 SmartBlock and a ThermoTop®, was used throughout for each incubation step (Eppendorf, Stevenage, UK).

[0172] All other reagents were as described previously in the art17.

[0173] Methods

[0174] Directed protein evolution

[0175] Directed evolution of ACE2 was performed using the cell surface display DT40 system that was previously described16. Cells were transfected with DNA encoding a fusion protein comprising of an N-terminal CD5 secretory leader sequence (residues 1-24) followed by FLAG-epitope tag, flanked on both sides by short linkers, and human ACE2. Stable transfectants were obtained by selection in puromycin and DT40 cell clones with ACE2 integrated into the rearranged Ig locus were identified by PCR, and cell surface expression of the FLAG-ACE2 fusion protein was confirmed by anti-FLAG immunostaining as previously described16. Cells were cultured in RPMI containing 7% (v / v) fetal bovine serum (FBS) plus 3% (v / v) chicken serum at 37°C and 5% CO2.

[0176] For evolving ACE2 with enhanced affinity, approximately 40 million cells were incubated with Hise-tagged SARS-CoV-2 receptor binding domain (RBD) at concentrations between 2nM and lOOpM (as indicated in Results and Discussion) in PBS with 10% (v / v) FBS at room temperature for 30 mins. Bound RBD was detected, following cell washing, with anti-Hise-tag, and surface expressed ACE2 with anti-FLAG, antibodies. Cells with RBD bound were selected by fluorescence activated cell sorting with sort windows indicated in Results and Discussion. DNA encoding ACE2 was recovered from genomic DNA prepared from sorted cells and sequenced as described previously17.

[0177] Site directed mutagenesis and expression and purification of soluble proteins were performed as described previously17.

[0178] Biolaver interferometry

[0179] Biolayer interferometry using the Octet R8 platform was performed to analyse binding. Assays were performed in tris-buffered saline with 0.05% (v / v) Tween-20 and Img / ml BSA. Monomeric RBD was covalently immobilised onto AR2G biosensors as described by the manufacturer. After washing, ACE2 binding was performed over the concentration ranges indicated in Results and Discussion, with an association time of 60 sec, followed by immersion in assay buffer to measure dissociation. Data was analysed using Octet Analysis software with a 1:1 binding model for monomeric interactions and 1:2 binding model for dimeric and pentameric ACE2 binding.

[0180] Electron microscopy

[0181] Negative stain grids were prepared by glow discharging 200 square continuous carbon copper grids (Agar Scientific) for 30 seconds at 30mA (Gloqube, Quorum Technologies). 20pl of sample was applied to clean parafilm, and the freshly glow discharged grid was floated carbon side down on the drop for 1 minute. Excess sample was blotted from the grid using filter paper, and the grid transferred to a 20pl drop of 2% uranyl acetate (w / v) for 30 seconds, followed by blotting and transferring to a second drop of uranyl acetate for 30 seconds, before finally blotting with filter paper and leaving to dry. Samples were viewed on a JEOL JEM-1400 electron microscope with an accelerating voltage of 120kV. Digital micrographs were collected with an EMSIS Xarosa digital camera with Radius software (see Figure 2E).

[0182] Viral capture

[0183] SARS-CoV-2 virus (variant B.1.1.369; isolated in Leicester, May 2020) stock was propagated in Dulbecco's Modified Eagle Medium (DMEM; Gibco) on T25 flasks of confluent Vero E6 cells for 72 hours. Tissue culture flask supernatants were harvested, cell debris removed by centrifugation at 500 x g for 10 minutes, and virus stocks aliquoted and stored at -80°C; typical titres were 106pfu / ml. All live SARS-CoV-2 virus work was performed in the Containment Level 3 (CL3) Laboratories (University of Leicester).

[0184] ACE2-COMP immobilized to nickel beads via the Hise-tag was added to a 250 pL aliquot of freshly defrosted stock SARS-CoV-2 and incubated at 37°C for 20 minutes. Beads were harvested by centrifugation at 1700 x g for 5 minutes and washed twice with 1 mL of TBSG (?). The beads were re-suspended in 200 pL of 300 mM imidazole (in TBSG) for 2 minutes to elute ACE2-COMP from beads and then collected again by centrifugation. Supernatant, containing released COMP-ACE2 (and any bound virus), was made up to 4% (w / v) formaldehyde and stored for 24 hours before removal from the CL3 lab and analysis by electron microscopy.

[0185] Sample preparation for mass spectrometry without enrichment

[0186] Saliva was fortified to the following levels and analysed: 100, 50, 20, 5, 1, 0.5 fmol / pL. In duplicate, 40 pL of sample was precipitated with 160 pL ice cold acetone and centrifuged at 13,000 rpm for 15 minutes using a Labnet Prism™ R Refrigerated Microcentrifuge (Labnet International, Edison, NJ USA). The supernatant was removed, and the pellet was dried under vacuum using a Thermo Savant ISS110 SpeedVac System (Thermo Fisher, Rockford, IL, USA) before reconstitution with 40 pL ABC (50 mM). Samples were then digested with 2.8 pL trypsin (1 mg / mL) for 15 minutes at 37 °C. Formic acid was added to a final 1% v / v, and samples were transferred to a QuanRecovery 96-Well plate for LC-MS analysis.

[0187] Sample preparation for mass spectrometry with enrichment

[0188] Nunc™ MicroWell™ 96-Well Microplates Were pre-wet and incubated for 1 hour with 150 pL TBS (per well). The TBS was then removed, before coating each well with 25 pL ACE2-COMP or BSA control (5 pg / mL in TBS). The protein was allowed to bind to the plate overnight at 4 °C. After, the unbound fraction was discarded, and the plate was blocked with the addition of 5% powdered milk (in IxTBS) for 1 hour at room temperature. The blocking buffer was then removed, and the plate was washed with 150 pL TBS.

[0189] To the ACE2-COMP functionalised plate, 25 pL of SARS-CoV2 spike protein (added into PBS or saliva) was added at varying concentrations (1.2 - 156.8 fmol / pL), before a 1-hour incubation at room temperature. The unbound fraction was removed, and the plate was washed thrice with 150 pL TBS. ABC was added to the plate (47.5 pL, 50 mM) along with the stable isotope labelled peptide mix (2.5 pL, 500 fmol / μL). The bound protein complex was then subject to reduction with 5.5 μL DTT (15.4 mg / mL) for 30 minutes at 60 °C, then alkylation with 6.1 μL IAA (35.04 mg / mL) for 30 minutes at room temperature and under the absence of light. Proteins were then digested with 5 μL trypsin (1 mg / mL) overnight at 37 °C. Digestion was stopped with the addition of formic acid to a final 1% v / v. Samples were then transferred to a clean QuanRecovery 96-Well plate for LC-MS analysis. For the saturation series experiment, buffer or saliva were fortified with the spike protein in triplicate at the following levels before enrichment as described above: 156.8, 78.4, 39.2, 19.6, 9.8, 4.88, 2.44, 1.2 fmol / μL. To assess non-specific binding, saliva was fortified with spike at 156.8 fmol / μL in triplicate and processed with the enrichment protocol, but with plates not treated with ACE2-COMP.

[0190] LC-MS operation and analysis

[0191] An ACQUITY UPLC I-Class PLUS system coupled to a Xevo TQ-XS Tandem Quadrupole (LC-MS / MS), and ACQUITY UPLC Peptide BEH C18 1.7 pm, 2.1 mm x 50 mm column from Waters Corporation (Milford, MA, USA) were used. The Xevo TQ-XS was equipped with a Z-spray electrospray ionisation (ESI) source set to positive mode.

[0192] Enriched peptide eluates were analysed as 10 pL injections and chromatographic separation was performed at a constant flow rate of 0.6 mL / min with water / 0.1% formic acid (A) and acetonitrile / 0.1% formic acid (B) mobile phases. A 3.5-minute gradient was used: 95% mobile phase A initial to 0.25-minutes, to 55% A at 2.00-minutes, to 30% A at 2.20-minutes, to 5% A at 2.21-minutes until 2.68-minutes, and to 95% A at 2.70-minutes until 3.00-minutes. Column temperature was set to 40 °C. For the ESI source parameters, desolvation temperature (650 °C), capillary voltage (0.6 kV), desolvation flow (1000 L / hour) and cone flow (150 L / hour) were set. Simultaneous multiple reaction monitoring (MRM) was performed for the ACE2-COMP peptides (EITFLK (EIT), LFNMLR (LFN), AVCHPTAWDLGK (AVC), LWAWESWR (LWA), SEPWTLALENVVGAK (SEP)), SARS-CoV-2 peptides (SFIEDLLFNK (SFI), ASANLAATK (ASA), GWIFGTTLDSK (GWI), GVYYPDK (GVY)), and the stable isotope labelled analogue peptides (GWIFGTTLDSK(*) (GWI*) and SFIEDLLFNK(*) (SFI*)). MRM transitions for each of the peptides are provided in (Table 1). Table 1: LC-MS / MS MRM transitions for the SARS-CoV2, stable isotope label, and ACE2-COMP peptides. Stable isotope labels were modified using13C615N2K (*). Carbamidomethyl (C) modifications are shown.

[0193] Qualifier ion

[0194] Peptides Precursor ion (charge) Quantifier ion (charge, ion position) Qualifier ion (charge, ion position) Qualifier ion (charge, ion position)

[0195] position) position) position)

[0196]

[0197] To process the LC-MS / MS data, MRM data was exported into Skyline (version 23.1). Peaks with matching expected and found retention times were integrated. All peaks were manually checked. Only data with both quantifier and qualifier ion presence were used. Results were imported into RStudio (version 1.4.1106), where the peak area for the quantifying peptide (SFI), was normalised against the peak area of the corresponding stable isotope labelled peptide (SFI(*)). Normalised data were then imported into GraphPad Prism (version 4.07) for analysis and processing.

[0198] Example 1: Directed evolution of ACE2 for enhanced binding to CoV2 RBD

[0199] In order to create a form of ACE2 with increased affinity for SARS-CoV2 RBD the inventors first performed directed protein evolution. This was done using a cell surface display and mutagenesis system that was previously described16'17. The cell surface display consisted of the secretory leader sequence from CD5, a FLAG-epitope tag and linker region followed by full-length human ACE2, which includes the CoV2 RBD binding site, and the ACE2 transmembrane and intracellular domains (Fig. 1A). To evolve enhanced RBD binding, cells were incubated with 2nM monomeric SARS-CoV2 RBD (residues 319 - 541) containing a HA-epitope tag, and bound RBD detected with anti-HA, along with ACE2 expression level detected using anti-FLAG. Diagonal sort windows were used, to correct binding for ACE2 expression level, and cells displaying the highest levels of RBD binding selected (Fig. IB). Three rounds of selection and expansion were performed, two at 2nM and one at lOOpM RBD (Fig. IB). DNA encoding evolved ACE2 was recovered from cells selected at round 3 and sequenced. In the pool of higher affinity ACE2 mutants was a form in which all mutations were outside the binding interface for SARS-CoV2 RBD. This evolved ACE2 has four substitutions Ala65Val, Glu75Lys, Leu79Ile, Thr92Lys (Fig. 1C). The position of the mutations relative to the RBD binding site in the previously reported crystal structure of ACE2 in complex with CoV2 RBD is shown in Figure ID. The peripheral localization of the mutations means that the binding interface and primary binding residues of Wt-ACE2 remain unchanged in this mutant ACE2. Thus, it is possible that RBD variants capable of binding Wt host cell ACE2 should also bind this evolved ACE2, minimizing the risk of loss of binding of relevant RBD variants. In order to directly test whether the mutant ACE2 binds CoV-2 RBD with increased affinity, wild-type ACE2 (Wt-ACE2) and the mutant receptor (residues 19-615), with C-terminal epitope tags, were expressed as soluble proteins and their binding to RBD determined by biolayer interferometry. In this mutant receptor the Ala65Val mutation was changed to Ala65Trp in order to maximize improvement of affinity as others had reported Ala65Trp to have higher affinity gain than Ala65Val12. Consistent with the evolution strategy, the mutant ACE2 bound with higher affinity than Wt-ACE2, by more than 10-fold (Fig. IE).

[0200] To gain insight into potential mechanisms for the increase in affinity of the evolved ACE2 the inventors examined the published structure of Wt-ACE2 in complex with RBD18. The ACE2 substitutions Glu75Lys and Leu75Ile are located close together and opposite residues Glu484 and Phe486 in RBD (Fig 1. D). These RBD residues are located on a highly flexible loop, which would allow Glu484 and Phe465 to come close enough to Lys75 and Ile79, respectively, in the mutated ACE2 to facilitate new bonding interactions. Trp65 in mutant ACE2 is located opposite Val445 and Tyr449 on another flexible loop in RBD, providing the possibility that loop flexibility could enable a new hydrophobic interaction between RBD and mutant ACE2. The Thr92Lys substitution occurs within the consensus site required for N-linked glycosylation of Asn90 and would result in loss of Asn90 glycosylation. Loss of this glycosylation has previously been shown to increase ACE2 binding affinity for RBD19-

[0201] CoV-2 RBD interacts with two distinct sites in ACE2, a patch of residues in the al helix of ACE2, along with a more limited interaction of the a2 helix and the loop between b3 and b4 sheet18'20. The inventors hypothesized that the binding affinity of the mutant receptor could be increased further by providing additional binding options on the al helix in ACE2 or around the a2 and b3 / 4 loop region. As the al helix lies in the core binding interface and the inventors wanted to retain this intact without mutations, the inventors focussed on potential mutations that could be introduced outside this region. The inventors therefore tested the effects of adding Ala386Leu, a mutation localized close to the b3 / 4 loop, but like the mutations in evolved ACE2 also outside the core binding interface, and already shown to increase binding affinity to RBD12. Combining the mutations in the evolved ACE2 with Ala386Leu, designated Ev-ACE2-A386L, resulted in a further 2.5-fold affinity gain, causing overall a 28-fold increase in affinity compared with Wt-ACE2 (Fig IF).

[0202] Example 2: Binding of evolved ACE2 to COV-2 variant RBD

[0203] The inventors then examined the ability of Wt and Ev-ACE2-A386L to bind two CoV-2 variant RBDs, an RBD containing the Leu452Arg and Glu484Gln mutations common to variants B.1.617.1 and B.1.617.3, and an RBD containing Lys417Asn, Glu484Lys and AsnN501Tyr mutations found in variant B.1.35 (Table 2). Similar to the situation with binding to Wuhan-Hu-1 RBD (WH-RBD), Ev-ACE2-A386L bound both RBD variants with higher affinity than Wt-ACE2 bound the RBD variants.

[0204] Interestingly however, the affinity gain of Ev-ACE2-A386L is lower for the variants than it is for WH-RBD (Table 2). Thus, whilst evolved ACE2 still shows higher affinity than Wt-ACE2 for the variants tested, the binding ability of the evolved receptor is sensitive to mutations in spike protein, even though the mutations in evolved ACE2 are outside the Wt binding interface. Together, these data highlight that evolved ACE2 can be sensitive to mutations that arise in spike variants, irrespective of whether the evolved ACE2 has residue substitutions in the core binding interface or peripheral to it. This potentially limits the utility of such an evolved ACE2 for quantitative enrichment for MS detection of variants.

[0205] Table 2: Binding of Wt and evolved ACE2 to RBD variants. Kinetic binding constants for RBD binding by Wt-ACE2 dimer and Ev-ACE2-A368L were measured by biolayer interferometry with soluble ACE2 and immobilised RBD. Data are shown as means and SEM for at least three experiments.

[0206] B.1.617.1 / 3 (L452R, E484Q) B1.351 (K417N, E484K, N501Y)

[0207]

[0208] Wt

[0209]

[0210] E75K, L79I,

[0211] Y92K, A65W,

[0212]

[0213] A386L

[0214] Example 3: Enhancing binding by oligomerization

[0215] As an alternative to enhancing RBD binding by evolving ACE2, the inventors sought to leverage avidity effects to increase binding ability of Wt-ACE by presenting the receptor in multimeric format. Specifically, the inventors examined dimeric and pentameric forms of Wt-ACE2. Dimeric ACE2 was created by extending the sequence beyond residue 615 to residue 740, to incorporate the naturally occurring homodimerization motif in ACE220. To create pentameric ACE2 the inventors used the coiled:coil domain peptide from cartilage oligomeric matrix protein and fused this to ACE2 (19-615). This domain forms a very stable pentamer21which the inventors and others have previously used as a pentameric protein trap for presenting proteins22'23. These ACE2 forms are shown schematically in Figure 2A. The soluble proteins were directly visualized with negative staining electron microscopy (Fig. 2B). Wt-ACE2 (19-615) was revealed as almost spherical structures measuring around 9.5nm across, whereas Wt-ACE2 (19-740) showed as clear dimeric structures (Fig. 2B). ACE2-COMP was evident in negative staining electron microscopy as doughnut shaped structures of approximately 18.5nm diameter in which the globular protein monomers are arranged around a central hole, consistent with a pentameric structure.

[0216] Relative binding of the Wt-ACE2 (19-615), Wt-ACE2 (19-740) and ACE2-COMP (19-615) to RBD were assessed using biolayer interferometry. The dimeric Wt-ACE2 (19-740) bound CoV-2 RBD with a KD of 1.5nm (Fig. 2C), demonstrating a considerable increase in binding ability compared with the monomeric ACE2 (Fig. IE). Others have reported that dimerizing ACE2 increases its ability to bind RBD13. The pentameric Wt-ACE2-COMP (19-615) bound CoV-2 RBD extremely tightly, with a KD of less than 25pM as determined by biolayer interferometry, representing several thousand-fold apparent improvement compared with monomeric ACE2. However, it should be noted that it is not possible to derive a true KD from the association and dissociation curves due to the multimeric nature of Wt-ACE2-COMP. Nevertheless, it is clear that compared to Wt-ACE2 (19-615) or Wt-ACE2 (19-740), this ACE2 pentamer has very substantially increased binding to RBD (Fig. 2C, D). Oligomerization of binding proteins increases binding by avidity effects and these are largely due to a decrease in the ability of the protein to dissociate from its partner24. Examination of the kinetics of monomeric, dimeric and pentameric ACE2 interacting with monomeric CoV-2 RBD clearly shows a moderately increased association rate of the dimer and pentamer forms and a very substantial decrease in dissociation rates (Fig. 2C). Indeed, observation of ACE2 dissociation from RBD over an extended time period reveals around 90% of bound pentameric ACE2 remains attached to RBD even after more than 8 hours (Fig. 2D). These data show pentamerization, in particular, leads to a major increase in binding ability of ACE2, and allows markedly extended retention of bound RBD. Incubating ACE2-COMP with SARS-CoV-2 virus and recovering the His6-tagged pentameric receptor, followed by negative staining electron microscopy shows ACE2-COMP still bound to the surface of captured virus (Fig. 2E).

[0217] Binding of pentameric ACE2 to B.1.617.1 / 3 and B.1.35 variant RBD was performed (Fig 3). As with WH-RBD, the pentameric ACE2-COMP bound very tightly to the variant RBD, with apparent KD's in the pM range (Fig. 3A). Again, the variant RBD's remained bound to ACE2-COMP for several hours in dissociation buffer with approximately 90% of the bound ACE2-COMP still retained after 8 hours (Fig. 3B). The very high binding activity of ACE2-COMP, and its retention of RBD, make this fusion protein a good candidate for capture of spike protein for MS detection.

[0218] Furthermore, at this high level of binding activity engineered ACE2 would be expected to capture all spike protein variants capable of binding the cellular receptor, and therefore relevant to infectious virus.

[0219] Example 4: Enrichment with ACE2-COMP increases sensitivity of spike protein detection bv mass spectrometry

[0220] Prior to our enrichment strategy, the inventors began the development of a noncapture based MS assay in 2021. Saliva (40 pL) was fortified with the SARS-CoV2 spike protein over a series (0.5 - 100 fmol / pL), before precipitation with ice cold acetone (160 pL), and tryptic digestion (1 mg / mL, 37 °C) of the resolved proteins. Issues were noted with both interference and sensitivity. Figure 4A shows an interfering component in saliva that lacked spike protein, where an intense MS signal was seen at the expected retention time (0.6 min) of one of the spike peptides (ASA). Monitoring multiple peptides with mass spectrometry provides the opportunity to simply target other candidates when there are issues with particular peptides. However, even for peptides without interfering peaks (SFI), sensitivity was not sufficient to detect at 100 fmol / pL fortified saliva (Figure 4B).

[0221] The high RBD binding ability of ACE2-COMP, together with its retention of bound RBD, would be expected to make this protein a good candidate for enrichment of spike protein from samples for MS. To test this, the inventors examined the ability of ACE2-COMP to recover SARS-CoV2 spike protein for detection by LC-MS, firstly in matrix naive samples, and subsequently in saliva. The inventors therefore developed an enrichment assay that functionalised 96-well plates with the ACE2-COMP protein trap protein (125 ng per well) before the addition of a sample (PBS or saliva) fortified with SARS-CoV2 spike protein. Following incubation and washing steps, bound spike and ACE2-COMP were subject to tryptic digestion (1 mg / mL, 37 °C, overnight) after the reduction (DTT, 60 °C, 30 minutes) and alkylation (IAA, 21 °C, 30 minutes) of the cysteine residues. A stable isotope labelled peptide mix (500 fmol / pL) was added prior the digestion to allow for peptide normalisation of the spike tryptic peptides. Digested samples were injected (10 pL) onto the LC-MS system, where peptides were chromatographically resolved using a binary pump, reversed phase (C18) gradient elution with acidified water and acetonitrile. Peptides were detected following the observation of multiple precursors to product ion transitions within the MS operating in MRM mode. LC-MS detection required only 3 minutes per sample.

[0222] Recovery of the spike protein in both matrix naive samples and biological matrices (saliva) was successful when using the enrichment assay detailed above, as demonstrated in Figure 5. ACE2-COMP functionalised plates allowed clear detection of spike peptides by MS following spike protein capture from saliva (Figures 5A and B). Spike peptides where undetectable in control wells that lacked ACE2-COMP. Testing recovery and quantitation of spike over a range of different concentrations (1.2 - 156.8 fmol / pL) confirmed MS peptide signals scale with spike concentrations up until saturation of the binding sites was reached (Figure 5C). Our system appeared to work better in biological matrices than in fortified buffer. Given the high binding capacity of our ACE2-COMP, even when immobilised at relatively low levels (125 ng per enrichment), it would be possible to readily decrease or increase the amount of protein trap used to accommodate sufficient antigen binding in different matrices and uses. Conclusions

[0223] The inventors have successfully developed a method for high affinity receptorbased capture combined with MS for detection and quantitation of target proteins. SARS-CoV-2 spike protein was used as an example of an important target protein. The data presented herein show that combining capture by engineered high binding activity receptors with MS provides a method for rapid detection and quantitation of target protein. This provides an elegant alternative to immunoaffinity enrichment methods. This approach could be used for detection of viral attachment proteins other than SARS-CoV-2 spike protein, where host cell receptors are tractable to engineering. Furthermore, the method could be for any target protein for which a suitable receptor or binding protein is known. References

[0224] 1. Kollhoff, L. et al. Development of a rapid and specific MALDI-TOF mass spectrometric assay for SARS-CoV-2 detection. Clin Proteomics 20, 26 (2023).

[0225] 2. Hallqvist, J. et al. Operation Moonshot: rapid translation of a SARS-CoV-2 targeted peptide immunoaffinity liquid chromatography-tandem mass spectrometry test from research into routine clinical use. Clinical Chemistry and Laboratory Medicine (CCLM) 61, 302-310 (2023).

[0226] 3. Kipping, M., Tanzler, D. & Sinz, A. A rapid and reliable liquid chromatography / mass spectrometry method for SARS-CoV-2 analysis from gargle solutions and saliva. Anal Bioanal Chem 413, 6503-6511 (2021).

[0227] 4. Lane, D. et al. A high throughput immuno-affinity mass spectrometry method for detection and quantitation of SARS-CoV-2 nucleoprotein in human saliva and its comparison with RT-PCR, RT-LAMP, and lateral flow rapid antigen test. Clinical Chemistry and Laboratory Medicine (CCLM) (2024) doi:10.1515 / cclm-2023-0243.

[0228] 5. Suvarna, K. et al. A Multi-omics Longitudinal Study Reveals Alteration of the Leukocyte Activation Pathway in COVID-19 Patients. J. Proteome Res. 20, 4667-4680 (2021).

[0229] 6. Doykov, I. et al. Quantitative, multiplexed, targeted proteomics for ascertaining variant specific SARS-CoV-2 antibody response. Cell Reports Methods 2, 100279 (2022).

[0230] 7. Yoshinari, T. et al. Matrix-Assisted Laser Desorption and Ionization Time-of-Flight Mass Spectrometry Analysis for the Direct Detection of SARS-CoV-2 in Nasopharyngeal Swabs. Anal. Chem.

[0231] 94, 4218-4226 (2022).

[0232] 8. Renuse, S. et al. A mass spectrometry-based targeted assay for detection of SARS-CoV-2 antigen from clinical specimens. eBioMedicine 69, 103465 (2021).

[0233] 9. Hober, A. et al. Rapid and sensitive detection of SARS-CoV-2 infection using quantitative peptide enrichment LC-MS analysis. eLife 10, e70843 (2021).

[0234] 10. Van Puyvelde, B. et al. Cov2MS: An Automated and Quantitative Matrix-Independent Assay for Mass Spectrometric Measurement of SARS-CoV-2 Nucleocapsid Protein. Anal. Chem. 94, 17379-17387 (2022).

[0235] 11. Mangalaparthi, K. K. et al. A SISCAPA-based approach for detection of SARS-CoV-2 viral antigens from clinical samples. Clinical Proteomics 18, 25 (2021).

[0236] 12. Chan, K. K. et al. Engineering human ACE2 to optimize binding to the spike protein of SARS coronavirus 2. Science 369, 1261 (2020).

[0237] 13. Glasgow, A. et al. Engineered ACE2 receptor traps potently neutralize SARS-CoV-2. Proc. Natl. Acad. Sci. U. S. A. (2020) doi:10.1073 / pnas.2016093117.

[0238] 14. Tanaka, S. et al. An ACE2 Triple Decoy that neutralizes SARS-CoV-2 shows enhanced affinity for virus variants. Sci. Rep. 11, 12740 (2021).

[0239] 15. Chan, K. K., Tan, T. J. C., Narayanan, K. K. & Procko, E. An engineered decoy receptor for SARS-CoV-2 broadly binds protein S sequence variants. Science Advances 7, eabfl738 (2021).

[0240] 16. Brindle, N. P. et al. Directed Evolution of an Angiopoietin-2 Ligand Trap by Somatic Hypermutation and Cell Surface Display. J. Biol. Chem. 288, 33205-33212 (2013).

[0241] 17. Bate, N. et al. In vitro evolution predicts emerging SARS-CoV-2 mutations with high affinity for ACE2 and cross-species binding. PLOS Pathogens 18, el010733 (2022).

[0242] 18. Lan, J. et al. Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor. Nature 581, 215-220 (2020).

[0243] 19. Isobe, A. et al. ACE2 N-glycosylation modulates interactions with SARS-CoV-2 spike protein in a site-specific manner. Common Biol 5, 1-11 (2022). 20. Yan, R. et al. Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2. Science 367, 1444 (2020).

[0244] 21. Malashkevich, V. N., Kammerer, R. A., Efimov, V. P., Schulthess, T. & Engel, J. The crystal structure of a five-stranded coiled coil in COMP: a prototype ion channel? Science 274, 761-5 (1996).

[0245] 22. Terskikh, A. V. et al. "Peptabody": A new type of high avidity binding protein. Proceedings of the National Academy of Sciences 94, 1663-1668 (1997).

[0246] 23. Issa, E. et al. Development of an Orthogonal Tie2 Ligand Resistant to Inhibition by Ang2. Mol. Pharm. 15, 3962-3968 (2018).

[0247] 24. Vauquelin, G. & Charlton, S. J. Exploring avidity: understanding the potential gains in functional affinity and target residence time of bivalent and heterobivalent ligands. Br. J. Pharmacol. 168, 1771-85 (2013).

[0248] 25. Li, W. et al. Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature 426, 450-4 (2003).

Claims

Claims1. A method of detecting and / or quantifying a target molecule in a sample, the method comprising:(i) providing an oligomeric protein trap for enriching the target molecule in the sample, the protein trap comprising: (i) a receptor or an extracellular binding domain thereof, wherein the receptor or an extracellular binding domain thereof is configured to specifically bind the target molecule; and (ii) an oligomerisation domain which is configured to oligomerise to thereby form the oligomeric protein trap;(ii) contacting the sample comprising the target molecule with the protein trap of step (i) to allow the protein trap to specifically bind to and thereby enrich the target molecule; and(iii) detecting and / or quantifying the enriched target molecule.

2. The method according to claim 1, wherein the target molecule is a polypeptide, a whole microorganism or a nucleic acid.

3. The method according to claim 2, wherein the target polypeptide is a pathogenic polypeptide or a non-pathogenic polypeptide ligand selected from a growth factor and / or a hormone.

4. The method according to claim 2, wherein the whole microorganism is a bacterium, a virus, or a fungus, optionally wherein the whole microorganism is a virus.

5. The method according to claim 4, wherein the virus is selected from a group consisting of: coronavirus, Nipah virus, Hendra virus, Cedar virus, HIV, HCV, Rhinovirus, EBV and Measles; optionally wherein the coronavirus is selected from SARS-CoV1, SARS-CoV2, MERS-CoV, and / or 229E.

6. The method according to any preceding claim, wherein the sample is a biological sample or an environmental sample.

7. The method according to claim 6, wherein the biological sample is selected from a swab, saliva, sputum, serum, blood, urine, faeces, cerebrospinal fluid, semen, vaginal discharge, tissue biopsy and / or mucus.

8. The method according to claim 6, wherein the environmental sample is selected from water, wastewater, effluent, sand, and / or soil.

9. The method according to any preceding claim, wherein the receptor or the extracellular binding domain thereof is:(i) a wild-type, native or natural receptor or extracellular binding domain thereof; or(ii) a modified or mutated version of a wild-type, native or natural receptor or extracellular binding domain thereof.

10. The method according to any preceding claim, wherein the receptor or the extracellular binding domain thereof comprises a wild-type ACE2 (Wt-ACE2) ectodomain.

11. The method according to any one of claim 1-9, wherein the receptor or the extracellular binding domain thereof comprises an Ephrin-B2 receptor.

12. The method according to any one of claim 1-9, wherein the receptor or the extracellular binding domain thereof comprises a mutated or evolved ACE2 ectodomain, optionally wherein the mutated or evolved ACE2 ectodomain is Ev-ACE2-A386L.

13. The method according to any one of claim 1-9, wherein the receptor or the extracellular binding domain thereof comprises a Tie2 receptor.

14. The method according to any preceding claim, wherein the oligomerisation domain comprises the coiled:coil domain of GCN4, the IgM-Fc domain, the leucine zipper motifs, the coiled:coil domain from cartilage oligomeric matrix protein (COMP), and / or the vasodilator stimulated phosphoprotein (VASP).

15. The method according to claim 14, wherein the oligomerisation domain comprises the coiled:coil domain from cartilage oligomeric matrix protein (COMP).

16. The method according to any preceding claim, wherein the oligomeric protein trap further comprises a first linker disposed between the receptor or an extracellular binding domain thereof, and the oligomerisation domain.

17. The method according to any preceding claim, wherein the oligomeric protein trap further comprises an epitope tag, optionally wherein the epitope tag is disposed at the C-terminus of the oligomerisation domain.

18. The method according to claim 17, wherein the oligomeric protein trap further comprises a second linker, optionally wherein the second linker is disposed at the C-terminus of the oligomerisation domain, and more optionally wherein the second linker is disposed between the oligomerisation domain and the epitope tag.

19. The method according to claim 17, wherein the oligomeric protein trap further comprises an hexahistidine tag, optionally wherein the hexahistidine tag is disposed at the C-terminus of the oligomerisation domain, more optionally wherein hexahistidine tag is disposed at C-terminus of the epitope tag.

20. The method according to claim 19, wherein the oligomeric protein trap further comprises a third linker, optionally wherein the third linker is disposed at the C-terminus of the oligomerisation domain, more optionally wherein the third linker is disposed between the epitope tag and the hexahistidine tag.

21. The method according to any preceding claim, wherein:(i) the oligomerisation domain is disposed at the C-terminus of the oligomeric protein trap, or(ii) the oligomerisation domain is disposed at the N-terminus of the oligomeric protein trap.

22. The method according to any preceding claim, wherein contacting the sample comprising the target molecule with the protein trap of step (i) comprises: (i) incubating the protein trap in a solution comprising the target molecule, or (ii) immobilising the protein trap to capture the target molecule.

23. The method according to claim 22, wherein the protein trap is incubated or immobilised for up to 5 minutes, up to 10 minutes, up to 15 minutes, up to 20 minutes, up to 25 minutes, up to 30 minutes, up to 35 minutes, up to 40 minutes,up to 45 minutes, up to 50 minutes, up to 55 minutes, up to 60 minutes, up to 75 minutes, or up to 90 minutes.

24. The method according to any preceding claim, wherein contacting the sample comprising the target molecule with the protein trap of step (i) further comprises the step of isolating the target molecule bound to the trap.

25. The method according to claim 24, wherein isolating the target molecule bound to the trap is performed using microbeads; optionally wherein the microbeads are Nickel-IMAC (immobilised metal affinity chromatography) beads.

26. The method according to any preceding claim, wherein detecting and / or quantifying the enriched target molecule is performed using Mass Spectrometry.

27. Use of the method according to any one of claims 1 to 26, in decontaminating a biological sample.

28. The use according to claim 27, wherein the biological sample is decontaminated prior to transfusion or transplant and wherein the oligomeric protein trap captures a target contaminant.

29. Use of the method according to any one of claim 1 to 26, in monitoring pathogen contamination of an environmental sample.

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