Receptor binding molecules

Abstract

Disclosed are molecules with an affinity for low-density lipoprotein receptor (LDLR) which may be used in the treatment or prevention of viral infections, for reducing the severity and / or number of symptoms of viral infections.

Description

[0001] Receptor binding molecules

[0002] FIELD

[0003] The present disclosure provides molecules for preventing viral infections, treating viral infections, reducing the severity and / or number of symptoms of viral infections.

[0004] BACKGROUND

[0005] Broad-acting antiviral strategies to prevent respiratory tract infections are urgently required. Emerging or re-emerging viral diseases, such as those caused by new or genetic variants of viruses (e.g. influenza viruses (IFVs), respiratory syncytial viruses (RSVs), human rhinoviruses (HRVs), parainfluenza viruses (PIVs) or coronaviruses (CoVs)), pose a severe threat to human health, particularly in the very young or old, or in those with pre-existing respiratory conditions such as asthma or chronic obstructive pulmonary disease (COPD). Although vaccines remain a key component in controlling and preventing viral infections, they are unable to provide broad-spectrum protection against recurring seasonal infections or newly emerging threats.

[0006] Existing strategies for broad-spectrum antivirals focus mainly on drugs such as nucleoside analogues, most notably remdesivir (1), ribavirin (2) and favipiravir (3), which modulate replication processes within the host cell (4). While these compounds have shown clinical promise, they carry a number of disadvantages, such as poor bioavailability or toxicity, and selection of antiviral drug resistance mutations.

[0007] Accordingly, there is currently a lack of broad-acting antiviral therapies that can be used to prevent infection by multiple potential respiratory viruses. It is amongst one of the objectives of the present disclosure to provide a receptor binding molecule to mitigate one or more of the above mentioned disadvantages of existing therapies.

[0008] SUMMARY

[0009] The present disclosure is based on the finding that molecules with an affinity for or which bind to, low-density lipoprotein receptor (LDLR), can be used to treat or prevent viral infections caused or contributed to by viruses which use or exploit LDLR as a means to gain entry into a host cell.

[0010] Molecules with an affinity for LDLR may be referred to as LDLR-binding molecules. An LDLR-binding molecule of this disclosure may comprise a ‘glycan-binding’ module, ‘glycoprotein binding’ molecule, a ‘sialic-acid binding’ molecule ora carbohydrate binding module (CBM) (which itself may be a component of glycan-binding / sialic acid-binding molecule). A LDLR-binding molecule may also comprise an LDLR-binding fragment of a sialic acid binding molecule, a glycan binding molecule or a CBM.

[0011] Throughout this disclosure, the terms ‘LDLR-binding molecule’ and ‘LDLR-binding CBM’ may be used interchangeably - but both terms describe molecules which bind to LDLR.

[0012] In one teaching, there is provided a LDLR-binding molecule for use in treating or preventing a viral infection and / or the symptoms of a viral infection caused or contributed to by a virus which uses or exploits LDLR for host cell entry.

[0013] It should be noted that the terms “comprise”, “comprising” and / or “comprises” is / are used to denote that the various aspects and embodiments of this disclosure “comprise” a particular feature or features. It should be understood that this / these terms may also encompass aspects and / or embodiments which “consist essentially of” or “consist of’ the relevant feature or features. Moreover, the entire contents of all references are to be regarded as incorporated herein by reference.

[0014] The term ‘viral infection’ embraces infections caused or contributed to by Rhinovirus. The term ‘Rhinovirus’ embraces those viruses classified as belonging to the genus Enterovirus in the family Picornaviridae. The term ‘Rhinovirus’ includes, for example Rhinovirus A, Rhinovirus B, Rhinovirus C and Enterovirus types A through to L. The various uses, methods and medicaments described herein may be used to treat, prevent or reduce the severity and / or number of symptoms of a Rhinovirus infection (e.g. the common cold) in a subject. The term ‘Rhinovirus’ also embraces those viruses more generally grouped as the Human Rhinoviruses. Rhinovirus lineages typically fall into major and minor groups - both of which are embraced by the general term ‘Rhinovirus’ as used herein. In one teaching, the various molecules of this disclosure may be used to treat or prevent infections caused or contributed to by the minor group Rhinovirus. Without wishing to be bound by theory, the minor group Rhinovirus use LDLR for cell entry and therefore a molecule of this invention (which molecule binds LDLR) may be used to prevent or inhibit Rhinovirus host cell entry (thereby also preventing Rhinovirus host cell infection).

[0015] Accordingly, there is provided a LDLR-binding molecule for use in treating or preventing a viral infection and / or the symptoms of a viral infection caused or contributed to by a:

[0016] (i) a Rhinovirus; or

[0017] (ii) a human Rhinovirus; or

[0018] (iii) a minor group Rhinovirus.

[0019] The terms ‘treating’ and ‘preventing’ may embrace full or partial resolution or clearance of a viral infection from a subject. These terms may also embrace the reduction in the number or severity of symptoms associated with a viral infection. In this regard, the term ‘symptom’ may include any of the symptoms commonly associated with an infection by or with the relevant virus. In the case of a Rhinovirus (e.g. minor group Rhinovirus) infection, the term ‘symptom’ may include any of the symptoms commonly associated with a disease or condition caused or contributed to by a Rhinovirus virus, including any of the symptoms that are associated with a common cold. Such symptoms may include, for example, a sore throat, a runny (or a ‘blocked’ or ‘stuffy’ nose), a cough, sneezing, watery eyes, headaches and body / muscle aches. As such, use of a LDLR-binding molecule according to this disclosure, to treat or prevent a viral infection (e.g. a Rhinoviral infection) in a subject, may reduce the number of symptoms or the severity of one or more of those symptoms in said subject. One of skill will appreciate that depending on the particular host, the viral strain causing the infection etc., the precise list of symptoms may vary and not all symptoms associated with the infection may be present at any given time

[0020] The term ‘subject’ may embrace any subject susceptible or vulnerable to a Rhinovirus infection (especially a minor group Rhinovirus infection). By way of example, the term ‘subject’ may include mammalian subjects, including, for example human or animal subjects. The subject may have an underlying disease or condition which is susceptible to Rhinovirus-induced exacerbation. The underlying disease or condition susceptible to Rhinovirus-induced exacerbation may be selected from chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis, non-cystic fibrosis bronchiectasis, pulmonary fibrosis and primary ciliary dyskinesia. A LDLR-binding molecule of this disclosure may comprise one or more modified carbohydrate binding modules (CBMs) or a LDLR-binding fragment thereof.

[0021] Carbohydrate binding modules are classified into families and CBMs classed as members of the Family 40 CBMs (CBM40) may be useful in the manufacture of the modified molecules for use in any of the LDLR-binding molecules described herein. The Family 40 CBMs embrace molecules of approximately 200 residues and are often found at the N-terminus of GH33 sialidases. They may also be found inserted in the p-propeller of GH33 sialidases.

[0022] As such, the LDLR-binding molecules described herein may comprise one or more modified Family 40 carbohydrate binding modules (CBM40s) or LDLR-binding fragments thereof.

[0023] This disclosure is, in part, based on the finding that as compared to a LDLR-binding molecule which comprises a naturally occurring CBM molecule, a LDLR-binding molecule which comprises a modified CBM exhibits a higher level of binding to LDLR. As such, a LDLR-binding molecule comprising a modified CBM (or a LDLR-binging fragment thereof) and for any of the uses, methods, medicaments and / or compositions described herein binds LDLR with a lower EC50 as compared to the EC50 of a binding event between a LDLR-binding molecule comprising an unmodified (or wild-type) CBM and LDLR. As such and without wishing to be bound by theory, a LDLR-binding molecule which comprises a modified CBM of this disclosure (or a LDLR-binding fragment thereof) represents a better therapeutic option.

[0024] It should be noted that ECso is the concentration of CBM that gives half-maximal binding determined by, for example, direct and saturable binding of a CBM dilution series to a target LDLR protein.

[0025] In view of the above, the term ‘LDLR-binding molecule’ embraces any LDLR-binding molecule which, as compared to Sp2CBMTD, binds to LDLR with an improved or better EC50.

[0026] A “modified CBM” for use in a LDLR-binding molecule of this disclosure may embrace a CBM which contains one or more mutations relative to a reference sequence. A “reference sequence” may be any wild type CBM sequence. For example, a reference sequence may comprise, consist essentially of or consist of a wild type family 40 CBM sequence, e.g. the wild type CBM sequences from Vibrio cholerae NanH sialidase or Streptococcus pneumoniae NanA sialidase (it should be appreciated that similar or homologous CBMs (including CBM40s) present in other organisms are to be encompassed within the scope of the term “CBM” and / or as CBM reference sequences).

[0027] Accordingly, a modified CBM may be derived from a specific or particular wild type CBM.

[0028] A modified CBM may comprise a wild type CBM sequence which includes, relative to the wild-type sequence) one or more mutations.

[0029] The one or more mutations may be functional - that is to say they may individually (and / or independently) or collectively (for example, synergistically) modulate (for example improve or increase) LDLR-binding, specifically for example, the level of binding between the modified CBM and LDLR. As stated, that the modulation may manifest as an improved LDLR-binding EC50 as compared to binding between LDLR and a LDLR- binding molecule which comprises a wild-type CBM and / or Sp2CBMTD.

[0030] A modified CBM for use in a LDLR-binding molecule of this invention may further comprise one or more other mutations which modulate (alter, improve or suppress / inhibit) one or more of the physiological, biological, immunological and / or pharmacological properties characteristic of a wild type CBM (for example, the wild type CBM from which the modified CBM is derived). In particular, the one or more mutations may:

[0031] (i) alter the immunogenicity (or antigenicity) of the CBM; and / or

[0032] (ii) alter (for example improve) the efficacy (of the CBM or of any multimeric molecule comprising a modified CBM)’ and / or

[0033] (iii) modulate (for example improve) the thermostability of the CBM; and / or

[0034] (iv) modulate (for example improve) the solubility of the CBM; and / or

[0035] (v) modulate (for example improve) the in vivo half-life of the molecule; and / or

[0036] A “mutation” may include any alteration to the wild-type CBM molecule. For example, the term “mutation” may embrace, for example: (i) one or more amino acid substitution(s) (where one or more of the wild type amino acid(s) is / are swapped or changed for another (different) amino acid - the term “substitutions” would include conservative amino acid substitutions); and / or

[0037] (ii) one or more amino acid deletion(s) (where one or more of the wild type amino acid residue(s) are removed); and / or

[0038] (iii) one or more amino acid addition(s) / insertion(s) (where additional amino acid residue(s) are added to a wild type (or reference) primary sequence); and / or

[0039] (iv) one or more amino acid / sequence inversions (usually where two or more consecutive amino acids in a primary sequence are reversed; and / or

[0040] (v) one or more amino acid / sequence duplications (where an amino acid or a part of the primary amino acid sequence (for example a stretch of 5-10 amino acids) is repeated)

[0041] As stated, a modified CBM for use in a LDLR-binding molecule according to this disclosure may comprise one or more of the mutations described herein.

[0042] An exemplary wild type CBM (in other words a reference sequence from which a useful modified CBM may be derived) is Streptococcus pneumoniae NanA sialidase - the amino acid sequence for which has been deposited under accession number P62575 and is reproduced below as SEQ ID NO: 1 (1035 amino acids).

[0043] SEQ ID NO: 1

[0044] MSYFRNRDID IERNSMNRSV QERKCRYSIR KLSVGAVSMI VGAWFGTSP VLAQEGASEQ PLANETQLSG ESSTLTDTEK SQPSSETELS GNKQEQERKD KQEEKI PRDY YARDLENVET VIEKEDVETN ASNGQRVDLS SELDKLKKLE NATVHMEFKP DAKAPAFYNL FSVSSATKKD EYFTMAVYNN TATLEGRGSD GKQFYNNYND APLKVKPGQW NSVTFTVEKP TAELPKGRVR LYVNGVLSRT SLRSGNFIKD MPDVTHVQIG ATKRANNTVW GSNLQIRNLT VYNRALTPEE VQKRSQLFKR SDLEKKLPEG AALTEKTDI F ESGRNGKPNK DGIKSYRI PA LLKTDKGTLI AGADERRLHS SDWGDIGMVI RRSEDNGKTW GDRVTITNLR DNPKASDPSI GSPVNIDMVL

[0045] VQDPETKRI F SIYDMFPEGK GI FGMSSQKE EAYKKIDGKT YQILYREGEK GAYTIRENGT VYTPDGKATD YRVWDPVKP AYSDKGDLYK GNQLLGNIYF TTNKTSPFRI AKDSYLWMSY SDDDGKTWSA PQDITPMVKA DWMKFLGVGP GTGIVLRNGP HKGRILI PVY TTNNVSHLNG

[0046] SQSSRI IYSD DHGKTWHAGE AVNDNRQVDG QKIHSSTMNN RRAQNTESTV VQLNNGDVKL FMRGLTGDLQ VATSKDGGVT WEKDIKRYPQ VKDVYVQMSA IHTMHEGKEY I ILSNAGGPK

[0047] RENGMVHLAR VEENGELTWL KHNPIQKGEF AYNSLQELGN GEYGILYEHT EKGQNAYTLS

[0048] FRKFNWDFLS KDLI SPTEAK VKRTREMGKG VIGLEFDSEV LVNKAPTLQL ANGKTARFMT

[0049] QYDTKTLLFT VDSEDMGQKV TGLAEGAIES MHNLPVSVAG TKLSNGMNGS EAAVHEVPEY

[0050] TGPLGTSGEE PAPTVEKPEY TGPLGTSGEE PAPTVEKPEY TGPLGTAGEE AAPTVEKPEF

[0051] TGGVNGTEPA VHEIAEYKGS DSLVTLTTKE DYTYKAPLAQ QALPETGNKE SDLLASLGLT

[0052] AFFLGLFTLG KKREQ

[0053] The CBM region of SEQ ID NO: 1 is from amino acid residue 121 to 305 - this sequence is designated SEQ ID NO: 2 (that sequence being: VIEKEDVETN ASNGQRVDLS SELDKLKKLE NATVHMEFKP DAKAPAFYNL FSVSSATKKD EYFTMAVYNN TATLEGRGSD

[0054] GKQFYNNYND APLKVKPGQW NSVTFTVEKP TAELPKGRVR LYVNGVLSRT SLRSGNFIKD

[0055] MPDVTHVQIG ATKRANNTVW GSNLQIRNLT VYNRALTPEE VQKRS).

[0056] Accordingly, this disclosure provides LDLR-binding molecules which comprise modified forms of SEQ ID NO: 1 or 2. A modified form of SEQ ID NO: 1 or 2 may comprise one or more mutated residues - the mutations being, for example, amino acid substitutions, additions / insertions, duplications, deletions and / or inversions made relative to the sequence of SEQ ID NO: 1 or 2. As stated, the mutations may be designed to modulate LDLR-binding.

[0057] An exemplary Vibrio cholerae NanH sialidase amino acid sequence is deposited under accession umber A5F7A4 and is reproduced below as SEQ ID NO: 3 (781 amino acids).

[0058] SEQ ID NO: 3

[0059] MRFKNVKKTA LMLAMFGMAT SSNAALFDYN ATGDTEFDSP AKQGWMQDNT NNGSGVLTNA DGMPAWLVQG IGGRAQWTYS LSTNQHAQAS SFGWRMTTEM KVLSGGMITN YYANGTQRVL PI I SLDSSGN LWEFEGQTG RTVLATGTAA TEYHKFELVF LPGSNPSASF YFDGKLIRDN

[0060] IQPTASKQNM IVWGNGSSNT DGVAAYRDIK FEIQGDVI FR GPDRI PSIVA SSVTPGWTA FAEKRVGGGD PGALSNTNDI ITRTSRDGGI TWDTELNLTE QINVSDEFDF SDPRPIYDPS

[0061] SNTVLVSYAR WPTDAAQNGD RIKPWMPNGI FYSVYDVASG NWQAPIDVTD QVKERSFQIA

[0062] GWGGSELYRR NTSLNSQQDW QSNAKIRIVD GAANQIQVAD GSRKYWTLS IDESGGLVAN

[0063] LNGVSAPI IL QSEHAKVHSF HDYELQYSAL NHTTTLFVDG QQITTWAGEV SQENNIQFGN

[0064] ADAQIDGRLH VQKIVLTQQG HNLVEFDAFY LAQQTPEVEK DLEKLGWTKI KTGNTMSLYG

[0065] NASVNPGPGH GITLTRQQNI SGSQNGRLIY PAIVLDRFFL NVMSIYSDDG GSNWQTGSTL

[0066] PI PFRWKSSS ILETLEPSEA DMVELQNGDL LLTARLDFNQ IVNGVNYSPR QQFLSKDGGI

[0067] TWSLLEANNA NVFSNI STGT VDASITRFEQ SDGSHFLLFT NPQGNPAGTN GRQNLGLWFS

[0068] FDEGVTWKGP IQLVNGASAY SDIYQLDSEN AIVIVETDNS NMRILRMPIT LLKQKLTLSQ N

[0069] The CBM region of SEQ ID NO: 3 is from amino acid residue 25 to 216 - this sequence may be SEQ ID NO: 4 (that sequence being: ALFDYNATGD TEFDSPAKQG WMQDNTNNGS

[0070] GVLTNADGMP AWLVQGIGGR AQWTYSLSTN QHAQASSFGW RMTTEMKVLS GGMITNYYAN

[0071] GTQRVLPI I S LDSSGNLWE FEGQTGRTVL ATGTAATEYH KFELVFLPGS NPSASFYFDG

[0072] KLIRDNIQPT ASKQNMIVWG NGSSNTDGVA AY )

[0073] Thus this disclosure provides LDLR- binding molecules which comprise modified forms of SEQ ID NO: 3 or 4. A modified form of SEQ ID NO: 3 or 4 may comprise one or more mutated residues - the mutations being, for example, amino acid substitutions, additions / insertions, duplications, deletions and / or inversions made relative to the sequence of SEQ ID NO: 3 or 4. As stated, the mutations may be designed to modulate LDLR-binding.

[0074] While a molecule which comprises SEQ ID NO: 1 , 2, 3 or 4 (or any LDLR-binding fragment thereof) binds LDLR, it has been shown that certain modifications to these sequences can modulate, for example, increase or improve, LDLR-binding.

[0075] As stated, the LDLR-binding molecules described herein may comprise one or more (for example two or more) modified CBMs (or a LDLR-binging fragment thereof: indeed, for convenience, the term ‘modified CBM’ should be taken to embrace any LDLR-binding fragment thereof). Where the LDLR- binding molecules comprise two or more modified CBMs, the molecule may be said to comprise a ‘multivalent CBM’. A LDLR-binding molecule comprising a multivalent CBM, may be prepared as a construct comprising the multiple (two or more) modified CBMs linked by amino acid / peptide linkers. Each modified CBM may be linked to another by, for example, peptides comprising 5, 10 or 15 amino acids. By way of example any one or more of the following peptides may be used to link two or more CBMs to produce a LDLR- binding molecule comprising a multivalent

[0076] CBM:

[0077] (i) 5 amino acid linkers: ALXGS ( SEQ ID NO : 5 )

[0078] LQALG ( SEQ ID NO : 6 )

[0079] GGXSG ( SEQ ID NO : 7 )

[0080] GGALG ( SEQ ID NO : 8 )

[0081] GGSLG ( SEQ ID NO : 9 )

[0082] (ii) 10 amino acid linkers: ALXGSGGGSG ( SEQ ID NO : 10 )

[0083] LQALGGGGSL ( SEQ ID NO : 11 )

[0084] (iii) 15 amino acid linkers: ALXGSGGGSGGGGSG ( SEQ ID NO : 12 ) where “X” is any amino acid.

[0085] The LDLR-binding molecules for use may further comprise one or more oligomerisation domain(s). The oligomerisation domains may be derived from wild type oligomerisation domains and may comprise one or more mutations relative to a corresponding wild type oligomerisation domain sequence. Useful oligomerisation domains exhibit an ability to self-associate to form multimeric structures, for example, trimers. A modified oligomerisation domain for use may comprise any molecule with that same oligomerisation property. For the avoidance of doubt, the term “oligomerisation domain” as used herein embraces not only wild type oligomerisation domains, but also those that are modified (the “modified oligomerisation domains” disclosed herein). For example, a LDLR-binding molecule according to this disclosure may comprise one or more (for example, two) CBMs (for example, one, two or more modified CBMs as described herein) bound, coupled or fused to an oligomerisation domain. The resulting LDLR- binding molecule may therefore be a (multimeric) CBM:oligomerisation domain “fusion”.

[0086] Suitable oligomerisation domains may include those obtainable from the Pseudomonas aeruginosa pseudaminidase. An exemplary Pseudomonas aeruginosa pseudaminidase amino acid sequence has been deposited under accession number Q9L6G4 (derived from strain PAO579) and is reproduced below as SEQ ID NO: 13 (438 amino acids). It should be appreciated that similar, homologous or other useful oligomerisation domains may be encompassed within the scope of the general term “oligomerisation domain”.

[0087] SEQ ID NO: 13

[0088] MNTYFDI PHR LVGKALYESY YDHFGQMDIL SDGSLYLIYR RATEHVGGSD GRWFSKLEG

[0089] GIWSAPTIVA QAGGQDFRDV AGGTMPSGRI VAASTVYETG EVKVYVSDDS GVTWVHKFTL

[0090] ARGGADYNFA HGKSFQVGAR YVI PLYAATG VNYELKWLES SDGGETWGEG STIYSGNTPY

[0091] NETSYLPVGD GVILAVARVG SGAGGALRQF I SLDDGGTWT DQGNVTAQNG DSTDILVAPS

[0092] LSYIYSEGGT PHWLLYTNR TTHFCYYRTI LLAKAVAGSS GWTERVPVYS APAASGYTSQ

[0093] WLGGRRILG NLFRETSSTT SGAYQFEVYL GGVPDFESDW FSVSSNSLYT LSHGLQRSPR

[0094] RWVEFARSS SPSTWNIVMP SYFNDGGHKG SGAQVEVGSL NIRLGTGAAV WGTGYFGGID

[0095] NSATTRFATG YYRVRAWI

[0096] The oligomerisation domain of SEQ ID NO: 13 is from amino acid residue 333 to 438 - this sequence may be SEQ ID NO: 14 (that sequence being: VPDFESDWFS VSSNSLYTLS HGLQRSPRRV WEFARSSSP STWNIVMPSY FNDGGHKGSG AQVEVGSLNI RLGTGAAVWG

[0097] TGYFGGIDNS ATTRFATGYY RVRAWl). As stated, the invention may exploit modified oligomerisation domains and / or modified forms thereof. A modified oligomerisation domain may comprise an oligomerisation domain containing one or more mutations relative to a reference oligomerisation sequence.

[0098] A “reference oligomerisation sequence” may be any wild type oligomerisation domain sequence, including for example SEQ ID NOS: 13 and 14 above.

[0099] Accordingly, a modified oligomerisation domain may be derived from a specific or particular wild type oligomerisation domain.

[0100] A modified oligomerisation domain sequence may comprise a wild type oligomerisation domain sequence which includes one or more mutations.

[0101] The one or more mutations may be functional - that is to say they may individually (and / or independently) or collectively (for example synergistically) modulate (improve or suppress / inhibit) LDLR-binding (perhaps additively or synergistically with a CBM modification designed to achieve the same) and / or one or more of the physiological, immunological, biological and / or pharmacological properties characteristic of a wild type oligomerisation domain (for example the wild type oligomerisation domain from which the modified oligomerisation domain is derived).

[0102] As stated above, the mutation(s) may

[0103] (i) alter the immunogenicity (or antigenicity) of the oligomerisation domain; and / or (ii) improve efficacy (of, for example, multimeric molecules comprising one or more modified CBMs); and / or

[0104] (iii) modulate (for example improve) the thermostability of the oligomerisation domain; and / or

[0105] (iv) modulate (for example improve) the solubility of the oligomerisation domain; and / or

[0106] (v) modulate (for example improve) the in vivo half-life of the oligomerisation domain; and / or

[0107] (vi) modulate (increase) LDLR binding (of, for example, multimeric molecules comprising one or more modified CBMs).

[0108] In the context of a modified oligomerisation domain, the term “mutation” is as defined above.

[0109] There are a number of factors that influence the immunogenicity of a protein, for example a CBM or oligomerisation domain for use in a LDLR-binding molecule as described herein. One key determinant is the presence of intrinsic epitopes that can trigger T-cell dependent events that lead to antibody production. T-cells recognize short peptide fragments derived from the processing of the protein antigen that occurs within an antigen-presenting cell (APC). Some of these fragments will form complexes with human leukocyte antigen (HLA) / major histocompatability complex (MHC) class II molecules, and, if bound stably enough, will be carried to the surface of the APC and presented to T-cells. The recognition of non-self peptide by the T-cell receptor triggers the subsequent immune response. As such, in order to provide CBMs and / or oligomerisation domains which are less immunogenic, wild type CBM and / or oligomerisation domains may be analysed in order to identify those regions which are immunogenic / antigenic (i.e. regions or domains which are likely to harbour or contain immunological epitopes). Immunogenicity (or antigenicity) is a property that may be assessed relative to, for example a particular human or animal host - in other words a CBM and / or oligomerisation domain may be analysed in order to determine which regions or domains may be immunogenic / antigenic in a specific (for example human) host. Immunogenicity (or antigenicity) may be assessed using, for example, in silica in screening techniques (screening for, for example, T cell epitopes: including (as an example) ProPred in silico analysis), T-cell proliferation assays (including, Proimmune Human donor T-cell proliferation assay) and other immunological techniques. Further information regarding methods to “deimmunize” a protein may be derived from Jawa V, et al. ((2013) T-cell dependent immunogenicity of protein therapeutics: Preclinical assessment and mitigation. Clin Immunol. 149, 534-555) and Singh H, and Raghava GP ((2001) ProPred: prediction of HLA-DR binding sites. Bioinformatics. 17(12), 1236-7) the entire contents of which are incorporated by reference.

[0110] Using these techniques, it has been possible to identify immunogenic / antigenic regions of both the CBM and oligomerisation domains. Figure 1 shows a complete analysis (ProPred predictions) of the antigenic regions within a SpCBM sequence (Figure 1 A) and a PaTD sequence (Figure 1 B). Predicted binders are coloured blue, with the first residue of each binding region shown in red. Predicted antigenic peptides (green bars) and Proimmune (purple bars) are shown under the sequences.

[0111] For example, within the SpCBM molecule, various regions between residues 127 to 300 (as shown in Figure 1A) have been identified as harbouring immunogenic / antigenic domains. For example regions spanning residues 127-135, 146-154, 154-166, 158-166, 143-151 , 149-157, 167-176, 167-178, 182-196, 188-196, 185-193, 204-212, 213-221 , 220-228, 239-246, 239-254, 241-249, 242-249, 241-250, 241-251 , 239-251 , 239-249, 239-247, 246-254, 243-251 , 257-265, 267-275, 284-300, 284-299, 284-297, 286-284, 286-300 and 289-296 have been identified as potentially immunogenic (some of which are predicted to bind a small number of alleles, whereas others are regarded as moderately or highly immunogenic).

[0112] More specifically and using the above described in silico and immunologic (T cell proliferation) assays, the regions spanning residues:

[0113] 167 to 178 of SEQ ID NO: 1 (i.e. sequence: FYNLFSVSSATK)

[0114] 167 to 181 of SEQ ID NO: 1 (i.e. sequence: FYNLFSVSSATKKDE)

[0115] 239 to 251 of SEQ ID NO: 1 (i.e. sequence: VRLYVNGVLSRTS)

[0116] 236 to 250 of SEQ ID NO: 1 (i.e. sequence KGRVRLYVNGVLSRT)

[0117] 245 to 254 of SEQ ID NO: 1 (i.e. sequence: GVLSRTSLRS)

[0118] 286 to 294 of SEQ ID NO: 1 (i.e. sequence IRNLTVYNR) have been identified as immunogenic / antigenic. It should be noted that the region spanning residues 245 to 254 is identified as representing an area of significant immunogenicity.

[0119] Within the PaTD oligomerisation domain, regions between residues 336 to 424 has been identified as harbouring immunogenic / antigenic domains. For example, the regions spanning residues 336-344, 340-348, 341-349, 348-356, 353-363, 355-363, 355-370, 362-370, 362-370 / 371 , 375-383, 375-390, 400-407, 402-410, 397-405, 403-410 and 416-424 have been identified as potentially immunogenic (some of which are predicted to bind a small number of alleles, whereas others are regarded as moderately or highly immunogenic). By way of non-limiting example, residues 338 to 352 of SEQ ID NO: 13 have been identified as harbouring immunogenic / antigenic domains.

[0120] More specifically and using the above described in silico and immunologic (T cell proliferation) assays) the regions spanning residues:

[0121] 340 to 349 of SEQ ID NO: 13 (i.e. sequence: WFSVSSNSLY)

[0122] 351 to 359 of SEQ ID NO: 13 (i.e. sequence: LSHGLQRSP)

[0123] 398 to 406 of SEQ ID NO: 13 (i.e. sequence: GSLNIRLGT)

[0124] 392 to 406 of SEQ ID NO: 13 (i.e. sequence: GAQVEVGSLNIRLGT)

[0125] 338 to 352 of SEQ ID NO: 13 (i.e. sequence: SDWFSVSSNSLYTLS) have been identified as immunogenic / antigenic. It should be noted that the regions spanning residues 340-349, 351-359 and 398-406 were identified as representing areas of significant immunogenicity.

[0126] Collectively, these regions (from both the CBM and oligomerisation domain) will be referred to as regions of “immunogenicity / antigenicity”.

[0127] Accordingly, a modified CBM and / or oligomerisation for use in a LDLR-binding molecule of this disclosure, can be generated by mutating (for example substituting, deleting, adding to, inverting and / or duplicating) one or more of the amino acid residues (or amino acid sequences) within, for example, the above identified regions of immunogenicity / antigenicity.

[0128] Thus, a LDLR-binding molecule of this disclosure may comprise a modified CBM or oligomerisation domain having a modified immunogenicity (or antigenicity) profile. In other words, as compared to a wild type CBM or oligomerisation domain, a modified CBM / oligomerisation domain of a LDLR-binding molecule of this disclosure, in additions to exhibiting improved LDLR-binging, may be less (or differently) immunogenic / antigenic in a human or animal host. One of skill will appreciate that mutations introduced to the primary amino acid sequence of a CBM or an oligomerisation domain, can modulate immunogenicity by rendering certain epitopes more or less immunogenic.

[0129] A CBM / oligomerisation domain for use in a LDLR-binding molecule of this disclosure may be subject to individual amino acid mutations. For example a specific amino acid residue may be replaced (substituted with another) or deleted. Within the context of this disclosure a mutation may be introduced to a CBM or oligomerisation domain sequence in order to modulate (for example improve) LDLR-binding. One or more other mutations may be introduced in order to modulate any one or more of the other factors listed herein (including for example the immunogenicity of the molecule). In order to predict the effect of any given mutation, a modified sequence (the sequence containing at least one mutation verses a reference sequence) may be fed into, for example, a program designed to identify immunogenic sequences (including for example MHC binding regions within an antigen sequence). One of skill will be familiar with suitable programs and systems - but ProPred is one example: the aim of this server is to predict MHC Class-I I binding regions in an antigen sequence, using quantitative matrices derived from published literature by Sturniolo et. al., 1999. The server will assist in locating promiscuous binding regions.

[0130] The user will aim to identify those mutations that might have an effect on LDLR-binding and / or the immunogenicity / antigenicity of the CBM / oligomerisation domain but at the same time not affect the protein structure (which may be crucial to the LDLR-binding property of the CBM and to the oligomerisation property of the oligomerisation domain). For example, the modified CBM disclosed herein may maintain binding affinity for sialyllactose as assessed by, for example, surface plasmon resonance (SPR).

[0131] Based on the above and using the SpCBM molecule as an example, one or more (for example (2-42, 3-41 , 4-40, 5-39, 6-38, 7-37, 8-36, 9-35, 10-34, 11-33, 12-32, 13-31 , 14-30, 15-29, 16-28, 17-27, 18-26, 19-25, 20-24, 21-23 or 22) of the following residues may be mutated:

[0132] Residue(s) 167 (F), 168 (Y) , 169 (N), 170 (L), 171 (F), 172 (S), 173 (V), 174 (S), 175

[0133] (S), 176 (A), 177 (T), 178 (K), 179 (K), 180 (D), 181 (E), 236 (K), 237 (G), 238 (R), 239 (V), 240 (R), 241 (L), 242 (Y), 243 (V), 244 (N), 245 (G), 246 (V), 247 (L), 248 (S), 249

[0134] (R), 250 (T), 251 (S), 252 (L), 253 (R), 254 (S), 286 (I), 287 (R), 288 (N), 289 (L), 290

[0135] (T), 291 (V), 292 (Y), 293 (N) and / or 294 (R)

[0136] Based on the above and using the PaTD as an example, one or more (for example 2- 37, 3-36, 4-35, 5-34, 6-33, 7-32, 8-31 , 9-30, 10-29, 11-28, 12-27, 13-26, 14-25, 15-24, 16-23, 17-22, 18-21 , 19-20) of the following residues may be mutated:

[0137] Residue(s) 338 (S), 339 (D), 340 (W), 341 (F), 342 (S), 343 (V), 344 (S), 345 (S), 346 (N), 347 (S), 348 (L), 349 (Y), 350 (T), 351 (L), 352 (S), 353 (H), 354 (G), 355 (L), 356 (Q), 357 (R), 358 (S), 359 (P), 392 (G), 393 (A), 394 (Q), 395 (V), 396 (E), 397 (V), 398 (G), 399 (S), 400 (L), 401 (N), 402 (I), 403 (R), 404 (L), 405 (G) and / or 406 (T).

[0138] It should be noted, that the term “mutation” includes the addition of further amino acids(s) to any of the regions (the “immunogenicity / antigenicity regions) containing these residues. Further, the term “mutation” may include the duplication of any one or more amino acids and / or the inversion of any sequence within these regions. For example, short sequences of two or more (for example 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 (or more)) amino acids may be inverted (and / or duplicated). Where the mutation is a substitution, the substituted amino acid may be any one of the other 19 naturally occurring amino acids or an artificial or synthetic amino acid. Further, the substituted amino acid may be a derivative or analogue of the wild type amino acid at the relevant position.

[0139] By way of non-limiting example and with reference to SpCBM, one or more the following mutations may be made (note, not all of these mutations may be directed at modulating the immunogenicity of a CBM) - for example, one or more of the mutations (including mutations M156F and M185I) may be used to modulate thermostability:

[0140] (i) M156F

[0141] (ii) Y168W (designated “Im 15”)

[0142] (iii) L170A (designated “Im16”)

[0143] (iv) L170T (designated “Im 17”)

[0144] (v) V173G (designated “Im 18”)

[0145] (vi) M185I

[0146] (vii) V239A (designated “Im 19”)

[0147] (viii) V239T (designated “lm20”)

[0148] (ix) V246G (designated “Im21”)

[0149] (x) I286A (designated “Im22”)

[0150] (xi) Y292E (designated “Im23”) Further, and again by way of non-limiting example and with reference to the PaTD molecule, one or more of the following mutations may be made:

[0151] (i) S342D (designated “Im24”)

[0152] (ii) S345D (designated “Im25”)

[0153] (iii) L348D (designated “Im26”)

[0154] (iv) R403K (designated “Im27”)

[0155] For the avoidance of doubt, any of CBM mutations listed as (i)-(xi) above and any of the PaTD mutations listed as (i)-(iv) may be made individually and / or in combination with one or more other of the listed mutations. For example, mutations at two or more of residues 156, 170 and 185 or 239, 246, 286 and 292 or 156, 170, 185, 239, 246, 286 and 292 may be combined. Additionally, or alternatively, mutations at residues 239 and 246 or 286 and 292 may be combined.

[0156] Further, the mutations listed above are examples only and it should be understood that any mutation at the noted positions which results in a CBM / oligomerisation domain having the desired properties (for example, improved LDLR-binding or reduced immunogenicity / antigenicity in a human host) is to be included within the scope of this invention.

[0157] In one teaching, a CBM for the various uses, methods, medicaments and compositions described herein may comprise a modified form of SEQ ID NO: 1 , wherein the modification comprises a mutation at position 162 (position 42 of SEQ ID NO: 2). By way of example the Alanine residue at position 162 may be substituted for another amino acid. In one teaching the LDLR-binding molecule of this disclosure comprises a sequence derived from SEQ ID NO: 1 or 2 and in which the residue corresponding to the alanine residue at position 162 of SEQ ID NO: 1 or position 42 of SEQ ID NO: 2 is substituted. In one teaching, that alanine residue may be substituted with (or for) a proline residue.

[0158] In view of the above, this disclosure provides a LDLR-binding molecule for use in treating or preventing a viral infection, wherein said LDLR-binding molecule comprises a sequence derived from SEQ ID NO: 1 or SEQ ID NO: 2 in which the residue corresponding to the alanine residue at position 162 of SEQ ID NO: 1 or position 42 of SEQ ID NO: 2 has been substituted with a proline residue and further wherein the viral infection is caused or contributed to by a virus which uses LDLR to enter the host cell, a human Rhinovirus or a human minor group Rhinovirus.

[0159] For convenience, a LDLR-binding molecule comprising a sequence derived from SEQ ID NO: 1 or SEQ ID NO: 2 in which the residue corresponding to the alanine residue at position 162 of SEQ ID NO: 1 or position 42 of SEQ ID NO: 2 has been substituted with a proline residue, shall be referred to hereinafter as a ‘LDLR162 molecule’. It should be noted that the term “LDLR162” may refer to multimeric or multivalent molecules comprising two or more copies of a LDLR162 molecule described herein. In such cases, each copy of the LDLR162 molecule may be linked by a linker peptide (see above) and / or further comprise an oligomerisation domain (again, see above).

[0160] In one teaching a LDLR162 molecule of this disclosure may further comprise one or more additional mutations (the term ‘mutation’, carrying the same definition as provided in the text above). Those additional mutations may comprise one or more conservative substitutions where one or more other residues have, relative to reference sequences SEQ ID NOS: 1 and 2, been replaced with another amino acid having the same or similar physico-chemical properties. Additionally or alternatively, a LDLR162 molecule of this disclosure may comprise one or more mutation(s) to modulate (for example increase or improve or (in the case of immunogenicity, reduce or decrease)):

[0161] (i) immunogenicity; and / or

[0162] (ii) efficacy; and / or

[0163] (iii) thermostability; and / or

[0164] (iv) solubility; and / or

[0165] (v) in vivo half-life; and / or

[0166] (vi) LDLR binding.

[0167] A LDLR162 molecule may comprise a sequence derived from SEQ ID NO: 1 in which the amino acids at residues 162 and 239 have been mutated, e.g.. substituted. By way of example, the amino acids at residues 162 and 239 (of SEQ ID NO: 1) may have been substituted with proline and alanine respectively.

[0168] A LDLR162m molecule may comprise a sequence derived from SEQ ID NO: 1 in which the amino acids at residues 162, 239 and 246 have been mutated, e.g. substituted. By way of example, the amino acids at residues 162, 239 and 246 (of SEQ ID NO: 1) may have been substituted with proline, alanine and glycine respectively.

[0169] In all cases a LDLR162 molecule of this disclosure exhibits a higher level of binding to LDLR as compared to a wild-type CBM molecule. More specifically a LDLR162 molecule for any of the uses, methods, medicaments and / or compositions described herein may not only bind LDLR, but may bind LDLR with a lower EC50 as compared to the EC50 of a binding event between another LDLR-binding molecule comprising a sequence derived from SEQ ID NO: 1 or 2 and which lacks the alanine to proline substitution at position 162 / 42.

[0170] A LDLR162 molecule may comprise a sequence derived from SEQ ID NO: 15.

[0171] SEQ ID NO: 15

[0172] MSYFRNRDID IERNSMNRSV QERKCRYSIR KLSVGAVSMI VGAWFGTSP VLAQEGASEQ

[0173] PLANETQLSG ESSTLTDTEK SQPSSETELS GNKQEQERKD KQEEKI PRDY YARDLENVET

[0174] VIEKEDVETN ASNGQRVDLS SELDKLKKLE NATVHMEFKP DiKAPAFYNL FSVSSATKKD

[0175] EYFTMAVYNN TATLEGRGSD GKQFYNNYND APLKVKPGQW NSVTFTVEKP TAELPKG gR

[0176] LYVNGVLSRT SLRSGNFIKD MPDVTHVQIG ATKRANNTVW GSNLQIRNLT VYNRALTPEE

[0177] VQKRSQLFKR SDLEKKLPEG AALTEKTDI F ESGRNGKPNK DGIKSYRI PA LLKTDKGTLI

[0178] AGADERRLHS SDWGDIGMVI RRSEDNGKTW GDRVTITNLR DNPKASDPSI GSPVNIDMVL

[0179] VQDPETKRI F SIYDMFPEGK GI FGMSSQKE EAYKKIDGKT YQILYREGEK GAYTIRENGT

[0180] VYTPDGKATD YRVWDPVKP AYSDKGDLYK GNQLLGNIYF TTNKTSPFRI AKDSYLWMSY

[0181] SDDDGKTWSA PQDITPMVKA DWMKFLGVGP GTGIVLRNGP HKGRILI PVY TTNNVSHLNG

[0182] SQSSRI IYSD DHGKTWHAGE AVNDNRQVDG QKIHSSTMNN RRAQNTESTV VQLNNGDVKL

[0183] FMRGLTGDLQ VATSKDGGVT WEKDIKRYPQ VKDVYVQMSA IHTMHEGKEY I ILSNAGGPK

[0184] RENGMVHLAR VEENGELTWL KHNPIQKGEF AYNSLQELGN GEYGILYEHT EKGQNAYTLS

[0185] FRKFNWDFLS KDLI SPTEAK VKRTREMGKG VIGLEFDSEV LVNKAPTLQL ANGKTARFMT

[0186] QYDTKTLLFT VDSEDMGQKV TGLAEGAIES MHNLPVSVAG TKLSNGMNGS EAAVHEVPEY

[0187] TGPLGTSGEE PAPTVEKPEY TGPLGTSGEE PAPTVEKPEY TGPLGTAGEE AAPTVEKPEF

[0188] TGGVNGTEPA VHEIAEYKGS DSLVTLTTKE DYTYKAPLAQ QALPETGNKE SDLLASLGLT

[0189] AFFLGLFTLG KKREQ

[0190] A LDLR162 molecule may comprise a LDLR-binding fragment of SEQ ID NO: 15 (LDLR- binding being determined by the various methods described herein). SEQ ID NO: 15 may comprise one or more mutations, for example one or more conservative substitutions and / or mutations designed to alter or modulate (or further alter / modulate):

[0191] (i) immunogenicity; and / or

[0192] (ii) efficacy; and / or

[0193] (iii) thermostability; and / or

[0194] (iv) solubility; and / or

[0195] (v) in vivo half-life; and / or

[0196] (vi) LDLR binding.

[0197] A molecule for any of the uses, methods, medicaments and / or compositions of this disclosure may comprise a sequence which is about 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous or identical to the sequence of SEQ ID NO: 1. It should be noted that any such homologous or identical sequence should have at least a proline residue at the position corresponding to position 162 in SEQ ID NO: 15. Additionally any such homologous or identical sequence may have at least an alanine residue at the position corresponding to position 239 in SEQ ID NO: 15 and a glycine residue at the position corresponding to position 246 in SEQ ID NO: 15. In one teaching, a homologous or identical sequence for the various uses, methods, medicaments and / or compositions of this disclosure has an alanine residue at the position corresponding to position 162 in SEQ ID NO: 1 , an alanine residue at the position corresponding to position 239 in SEQ ID NO: 15 and a glycine residue at the position corresponding to position 246 in SEQ ID NO: 15.

[0198] In one teaching, a LDLR-binding molecule of this disclosure may be derived from SEQ ID NO: 15 and may comprise one or more mutations at positions other than at position 162.

[0199] In a further teaching, a LDLR-binding molecule of this disclosure may be derived from SEQ ID NO: 15 and may comprise one or more mutations at positions other than at positions 162 and position 239 and / or position 246. The term ‘one or more’ mutations may embrace any number of mutations (conservative or otherwise) that do not alter the fact that as compared to a molecule comprising a sequence derived from SEQ ID NO: 1 (having at residue 162 an alanine, at residue 239 a valine and at residue 246 a valine), a LDLR-binding molecule derived from SEQ ID NO: 15 (and having at residue 162 a proline and optionally an alanine at residue 239 and further optionally a glycine at residue 246). By way of example the term one or more mutations may embrace up to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 to up to about 100 additional mutations (the term ‘about meaning ± 1 , 2, 3 or 4 mutations).

[0200] Many techniques exist to allow one of skill in this field to introduce one or more amino acid mutations into a protein or peptide sequence, for example a CBM or oligomerisation domain sequence. Any of those techniques may be used here and they include, for example, mutagenesis techniques, including: (site-) directed mutagenesis, PCR mutagenesis, insertional mutagenesis, signature tagged mutagenesis, transposon mutagenesis and / or sequence saturation mutagenesis. Gene synthesis techniques may also be used to introduce any one or more of the mutations described herein.

[0201] Prior to any mutagenesis procedures, a wild type CBM and / or oligomerisation domain nucleic acid sequence may be subject to a codon-optimisation process in which the codons are optimised for expression in an expression system, such as, for example, a bacterial (e.g. an E.coli) expression system.

[0202] LDLR-binding molecules of this disclosure may comprise a plurality of CBMs, e.g. two or more modified CBMs wherein some but not all of the CBMs within the LDLR-binding molecule are modified. In other words, a LDLR- binding molecule, which comprises two or more CBMs, may comprise a mixture of modified and non-modified CBMs.

[0203] In one embodiment, the LDLR-binding molecules may comprise hexameric CBMs. A hexameric CBM comprises six CBM monomers. In most cases, a hexameric CBM comprises two fused CBMs which are further conjugated to themselves via a fused oligomerisation (trimerisation) domain.

[0204] By way of example, the generic structure of a useful hexameric CBM molecules may be represented as:

[0205] The schematic (and generic) structure above shows a LDLR-binding molecule comprising three repeat units each comprising 2 CBMs (CBM1 and CBM 2) and an oligomerisation domain (in this case, a trimerisation domain: TD).

[0206] As described herein, one or both of the CBM moieties may be a modified CBM as described herein (e.g. a sequence derived from SEQ ID NO: 15 or a LDLR-binding fragment thereof). Each repeat unit may be the same or different - in other words, while each of the repeat units may comprise 2 CBMs (modified or otherwise)., the type of CBM and / or the level or degree of modification, may vary. By way of non-limiting example, one repeat unit may contain two CBMs with wild type sequences. Another may comprise two different CBMs one with a wild type sequence and one with a modified sequence (comprising one or more mutations as described herein). The third repeat unit may comprise two modified CBMs (comprising one or more of the mutations described herein). One of skill will appreciate that other combinations of CBM type and / or sequence (wild type vs mutated sequence) may be prepared and tested.

[0207] By way of non-limiting example, the following represent individual units (referred to as “HEX” units) which may be used to make hexameric LDLR-binding molecules. In each case, the HEX unit comprises two modified CBMs (denoted CBM1 and CBM 2) with the specific mutations introduced to each CBM being identified in parenthesis. It should be noted that a -” symbol indicates an amino acid linker (linking one CBM to another or a CBM to an oligomerisation domain). As such, a hexameric LDLR-binding molecule may be made up of several (for example 3) HEX units. In each case, the oligomerisation domain (denoted “TD”) conjugates the units together as a trimer. While any given hexamer may comprise identical copies of the units described above (and below) under the headings HEX1 , HEX2, HEX3, HEX4, HEX5, HEX6 and HEX17, one of skill will appreciate that further options are available. For example, a HEX unit may be made up of two CBMs, each having different mutations (the mutations being one or more selected from the options detailed herein). (i) HEX1

[0208] CBMX1 (L170T V239A V246G I286A Y2 92 E) - CBMX2 (L170T V239A V246G

[0209] I 28 6A Y2 92 E) - TD (S342D L348 D R403K)

[0210] (ii) HEX2

[0211] CBMX1 (V239A V246G I 28 6A Y292 E) - CBMX2 (V239A V246G I 28 6A Y292 E)—

[0212] -TD (S342D R403K)

[0213] (iii) HEX3

[0214] CBMX1 (V239A V24 6G I286A)- — CBMX2 (V239A V246G I286A)- — TD (S342D R403K)

[0215] (iv) HEX4

[0216] CBMX1 (V239A V246G)- — CBMX2 (V239A V246G)- — TD (S342D)

[0217] (v) HEX5

[0218] CBMX1 (V239A V246G)- — CBMX2 (V239A V246G)- — TD (R403K)

[0219] (vi) HEX6

[0220] CBMX1 (V239A V246G) - CBMX2 (V239A V246G) - TD (S342D R403K)

[0221] (vii) HEX17

[0222] CBMX1 (V239A V24 6G A162 P) - CBMX2 (V239A V246G A162 P) - TD (S342 D

[0223] R403K)

[0224] A suitable HEX unit may comprise two modified CBMs (denoted CBMX1 and CBMX2 above) with the specific mutations introduced to each CBM being identified in parenthesis. In one teaching the units CBMX1 and CBMX2 may comprise (consist of, or consist essentially of) any type of CBM (for example any member of the CBM40 group / class). It should be noted that a symbol indicates an amino acid linker (linking one modified CBM to another modified CBM or a modified CBM to an oligomerisation domain).

[0225] In each case, the oligomerisation domain (denoted “TD”) present in each HEX unit conjugates the units together as a trimer. While any given hexamer may comprise three identical copies of one of the units described above, one of skill will appreciate that further options are available. For example, a HEX unit may be made up of two CBMs, each having different mutations (the mutations being one or more selected from the options detailed herein). It will be noted that HEX6 and HEX17 are identical except for the additional A162P mutation.

[0226] This proline mutation (a substitution for the wild type alanine at residue 162) has been shown to improve thermostability (the single CBM Tm by 3-4°C). Further information regarding the use of proline mutations may be derived from Fu 2009, 'Increasing protein stability by improving beta-turns' (DOI 10.1002 / prot.22509) which describes the general approach. The proline mutation does not affect (increase or decrease) the predicted immunogenicity of the CBM molecule, is not located near the other mutations, the N- or C-termini or the ligand binding site.

[0227] Relative to the amino acid sequences of Sp2CBMTD (aka “SpOrig”) the amino acid sequence of the HEX6 and HEX17 molecules is:

[0228] SpOrig GAMVIEKEDVETNASNGQRVDLSSELDKLKKLENATVHMEFKPDAKAPAFYNLFSVSSAT

[0229] HEX 6 GAMVIEKEDVETNASNGQRVDLSSELDKLKKLENATVHMEFKPDAKAPAFYNLFSVSSAT

[0230] HEX17 GAMVIEKEDVETNASNGQRVDLSSELDKLKKLENATVHMEFKPDPKAPAFYNLFSVSSAT

[0231] SpOrig KKDEYFTMAVYNNTATLEGRGSDGKQFYNNYNDAPLKVKPGQWNSVTFTVEKPTAELPKG

[0232] HEX6 KKDEYFTMAVYNNTATLEGRGSDGKQFYNNYNDAPLKVKPGQWNSVTFTVEKPTAELPKG

[0233] HEX17 KKDEYFTMAVYNNTATLEGRGSDGKQFYNNYNDAPLKVKPGQWNSVTFTVEKPTAELPKG

[0234] SpOrig RVRLYVNGVLSRTSLRSGNFIKDMPDVTHVQIGATKRANNTVWGSNLQIRNLTVYNRALT

[0235] HEX6 RARLYVNGGLSRTSLRSGNFIKDMPDVTHVQIGATKRANNTVWGSNLQIRNLTVYNRALT

[0236] HEX17 RARLYVNGGLSRTSLRSGNFIKDMPDVTHVQIGATKRANNTVWGSNLQIRNLTVYNRALT

[0237] SpOrig PEEVQKRSGGGSGVIEKEDVETNASNGQRVDLSSELDKLKKLENATVHMEFKPDAKAPAF

[0238] HEX6 PEEVQKRSGGGSGVIEKEDVETNASNGQRVDLSSELDKLKKLENATVHMEFKPDAKAPAF

[0239] HEX17 PEEVQKRSGGGSGVIEKEDVETNASNGQRVDLSSELDKLKKLENATVHMEFKPDPKAPAF

[0240] SpOrig YNLFSVSSATKKDEYFTMAVYNNTATLEGRGSDGKQFYNNYNDAPLKVKPGQWNSVTFTV

[0241] HEX6 YNLFSVSSATKKDEYFTMAVYNNTATLEGRGSDGKQFYNNYNDAPLKVKPGQWNSVTFTV

[0242] HEX17 YNLFSVSSATKKDEYFTMAVYNNTATLEGRGSDGKQFYNNYNDAPLKVKPGQWNSVTFTV

[0243] SpOrig EKPTAELPKGRVRLYVNGVLSRTSLRSGNFIKDMPDVTHVQIGATKRANNTVWGSNLQIR

[0244] HEX6 EKPTAELPKGRARLYVNGGLSRTSLRSGNFIKDMPDVTHVQIGATKRANNTVWGSNLQIR

[0245] HEX17 EKPTAELPKGRARLYVNGGLSRTSLRSGNFIKDMPDVTHVQIGATKRANNTVWGSNLQIR SpOrig NLTVYNRALTPEEVQKRSGGALGVPDFESDWFSVSSNSLYTLSHGLQRSPRRVWEFARS HEX 6 NLTVYNRALTPEEVQKRSGGSLGVPDFESDWFDVSSNSLYTLSHGLQRSPRRVWEFARS HEX17 NLTVYNRALTPEEVQKRSGGSLGVPDFESDWFDVSSNSLYTLSHGLQRSPRRVWEFARS

[0246] SpOrig SSPSTWNIVMPSYFNDGGHKGSGAQVEVGSLNIRLGTGAAVWGTGYFGGIDNSATTRFAT HEX6 SSPSTWNIVMPSYFNDGGHKGSGAQVEVGSLNIKLGTGAAVWGTGYFGGIDNSATTRFAT HEX17 SSPSTWNIVMPSYFNDGGHKGSGAQVEVGSLNIKLGTGAAVWGTGYFGGIDNSATTRFAT

[0247] SpOrig GYYRVRAWI

[0248] HEX 6 GYYRVRAWI

[0249] HEX 17 GYYRVRAWI

[0250] [SpOrig (SEQ ID NO: 16); HEX6 (SEQ ID NO: 17) and HEX17 (SEQ ID NO: 18)]

[0251] Rather unexpectedly, it was noted that as compared to SpOrig, HEX17 exhibits a marked improvement in LDLR binding.

[0252] SpOrig may be modified to include the following mutation (R274Q). This mutation modulates the binding affinity of the CBM for sialic acid.

[0253] A molecule comprising or consisting essentially of SEQ ID NO:18 may find particular application in the methods and uses described herein. The present disclosure further extends to variants, including LDLR-binding fragments, thereof. In some examples, a variant of SEQ ID NO: 18 may encompass molecules comprising the general structure as shown below:

[0254] HEX17

[0255] CBMX1 (V239A V246G A162P)- — CBMX2 (V239A V246G A162P)- — TD (S342D R403K);

[0256] And which further comprise one or more additional amino acid mutations (as described herein) relative to the wild-type sequence SpOrig (SEQ ID NO: 16). By way of example the variant may comprise between 1 and 30, between 1 and 20, between 1 and 10, or between 1 and 5 additional amino acid mutations (such as conservative amino acid substitutions) relative to SpOrig. The present disclosure will now be described, by way of example only, with reference to the following figures which show:

[0257] Figure 1. Building blocks of the multivalent CBM forms and their affinities for LDLR a, VcCBM, residues 25-216 of the V. cholerae sialidase (PDB:1w0p) with a-2,3- sialyllactose drawn as spheres, b, SpCBM, residues 121-305 of S. pneumoniae NanA sialidase with a-2,3-sialyllactose (PDB:4c1w). c, TD, the trimerisation domain, residues 333-438, of the P. aeruginosa pseudaminidase (PDB:2w38) in rainbow colours; the other two monomers in single colours, d, Multivalent forms: their molecular weights, valencies and binding affinities for a2,3-sialyllactose as determined by surface plasmon resonance (SPR) at 25°C (KD values for VcCBM, Vc2CBM and Vc3CBM had been reported previously (Connaris et al., 2009)). Tandem repeat CBMs, and oligomeric CBMs fused to TD are linked by a 5-amino linker (details in Connaris, H. et al., (2014). PNAS 111 :6401-6406).

[0258] Figure 2. Detection of CBM binding to human LDLR: non-linear regression plots of Absorbance vs CBM concentration, testing both Sp2CBMTD and Null / Sp2R274QTD as a negative control. Each point is the mean of the absorbances at that concentration (n=3, error bars represent SEM), and three independent assays are shown for LDLR, a)-c).

[0259] Figure 3. Detection of CBM binding to human LDLR: non-linear regression plots of Absorbance vs CBM concentration, testing both HEX17 and Null / Sp2R274QTD as a negative control, to test binding of the mCBM to immobilised minor group receptor LDLR. Each point is the mean of the absorbances at that concentration (n=3, error is SEM), and three independent assays are shown for a)-c).

[0260] Figure 4. a-c) Non-linear regression plots of plaque number vs Sp2CBMTD concentration against challenge with HRV-14. The points represent the mean number of plaques per concentration, and the error bars the standard error of the mean (SEM). Three replicate experiments were performed (n=2 in each) and plotted (n= 6 in total).

[0261] Figure 5. a-c) Non-linear regression plots of plaque number vs HEX17 concentration against challenge with HRV-14. The points represent the mean number of plaques per concentration, and the error bars the standard error of the mean (SEM). Three replicate experiments were performed (n=2 in each) and plotted (n= 6 in total). Figure 6. Non-linear regression plots of plaque number vs Sp2CBMTD concentration against challenge with HRV-1A. The points represent the mean number of plaques per concentration, and the error bars the standard error of the mean (SEM). Three replicate experiments were performed (n=2 in each) and plotted (n= 6 in total).

[0262] Figure 7. Representative non-linear regression plots of plaque number vs CBM concentration against challenge with human rhinovirus (HRV). The points represent the mean number of plaques per concentration and the error bars indicate the standard error of the mean (SEM). Three replicate experiments were performed (n=2 in each; n= 6 in total). The plots show dose-dependent protective effects of (a) HEX17 against HRV-14, (b) Sp2CBMTD against HRV-14 and (c) Sp2CBMTD against HRV-1A.

[0263] EXAMPLES

[0264] Example 1. ELISA assay: Interaction of Sp2CBMTD and HRV Receptors

[0265] The plots of three independent assays of Sp2CBMTD binding to LDLR coated plates are presented in Figure 2. From these plots, a non-linear regression was performed and ECso values calculated.

[0266] The plots of Sp2CBMTD and LDLR demonstrate a relationship between the absorbance and the mCBM concentration, indicating that the mCBM is binding to the receptor. The null binder, Sp2R274QTD, also showed significant absorbance, and therefore binding to the immobilised protein. The EC50 produced here was approximately 10-fold greater than that of Sp2CBMTD.

[0267] The increase in the EC50 and shift of the curve to the right of the plot (signifying a lower level of binding) demonstrated that Sp2R274QTD could still be utilised as a control.

[0268] Example 2. ELISA assay: Interaction of HEX17 with HRV Receptors

[0269] The plots of three independent assays of Sp2CBMTD binding to LDLR coated plates are presented in Figure 3. From these plots, a non-linear regression was performed as described above, and EC50 values calculated.

[0270] The LDLR showed a higher level of binding to HEX17 than to Sp2CBMTD, with the mean EC50 decreasing by roughly half (from 94.7 ng / mL for Sp2CBMTD to 49.4 ng / mL for HEX17). The overall trend showed that HEX17 bound to LDLR in a dose-dependent manner.

[0271] Example 3. Plaque assay: HRV-14 treated with Sp2CBMTD and HEX17

[0272] The efficacy of HEX17 against HRV-14 infection was tested (Figures 4-5). In all experiments, the data generated indicated that the application of Sp2CBMTD or HEX17 reduces the formation of plaques by HRV-14 in a dose-dependent manner. The range of ECso values is 0.024 - 0.099 mg / mL, which demonstrate the efficacy of HEX17 in reducing the formation of plaques by two strains of influenza virus H1 N1 and H3N2.

[0273] The variation in the EC50 values between replicates could be explained by the natural variation inherent to cell-based assays; however, it should be noted that all experiments utilised the same batch of Sp2CBMTD and were performed on the same batch of H1 HeLa cells from the working cell bank.

[0274] Example 4. HRV-1 A treated with Sp2CBMTD

[0275] A plot of the 3 independent experiments reported below in Figure 6. The EC50 values range from 0.063 to 0.073 mg / mL, and all three assays demonstrate a dose-dependent reduction in plaque formation by HRV-1A when cells were pre-treated with Sp2CBMTD (Figure 6). The SEM variation is extremely low (± 0.003) indicating good reproducibility of the experiment. These experiments were all performed on the same batch of HI HeLa cells from the working cell bank.

[0276] Example 5. HEX17 reduces infection against HRVs.

[0277] HRVs are the most prevalent respiratory viruses in humans, causing more than 50% of cold-like illnesses and posing a serious risk to vulnerable patients (5,6). With over 100 recognised serotypes that induce little or no cross-protection to infected individuals, control of HRV by vaccination is not feasible (7). Their lineages fall into major and minor groups that use different host receptors (ICAM-1 and LDLR, respectively) to infect cells (8,9).

[0278] In one teaching, prevention of HRV cell recognition using CBMs may require targeting the host cell receptors, rather than the virus. Initial ELISA binding tests demonstrated that HEX17 was able to interact with the receptor protein Low-Density Lipoprotein (LDLR) of HRVs (Examples 1 and 2). In vitro efficacy against HRV was tested using an H1-HeLa cell infection model. HEX17 and Sp2CBMTD reduced the formation of plaques by HRV-14 (a major group HRV) in a dose-dependent manner, with mean EC50s of 0.051 ± 0.014 mg / mL and 0.065 ± 0.019 mg / mL, respectively (Figures 7a and 7b). A further experiment, testing Sp2CBMTD against a minor group HRV strain (HRV-1A) showed similar efficacy (EC50 = 0.067 ± 0.003), indicating that the effect is not HRV strain-specific (Figure 7c).

[0279] Conclusion

[0280] All mCBMs tested bound to the cellular receptors utilised by major and minor group HRVs in a dose-dependent manner. This indicates a potential mechanism for preventing viral infection through blocking viral attachment, which was further investigated in the plaque assay aspect of this work.

[0281] Pre-treatment of cells with mCBMs reduced plaque formation of both major and minor group HRVs for Sp2CBMTD and of major group viruses for HEX17. While some variability between replicates was observed (which is not unexpected given the inherent variation in assays that combine both live cells and live virus), the assays show strong evidence of protection against HRV infection by Sp2CBMTD and HEX17.

[0282] Methodology

[0283] Method: ELISA

[0284] 1 . Day 1. Coat the plate: Add 100 pL of 1 pg / mL of receptor to each well, seal the plate with parafilm and incubate overnight at 4C.

[0285] 2. Day 2. Wash the plate 3x with PBST, 250 pL per well.

[0286] 3. Block the plate: Add Blocking solution, 200 pL per well. Incubate at RT for 1 h.

[0287] 4. Wash the plate 3x with PBST, 250 pL per well.

[0288] 5. Add the mCBM dilutions, 100 pL per well. Incubate at RT for 90 min.

[0289] 6. Wash the plate 3x with PBST, 250 pL per well.

[0290] 7. Add the 1 ° antibody (rabbit anti-HEX17), 100 pL per well. Incubate at RT for 1h.

[0291] 8. Wash the plate 3x with PBST, 250 pL per well.

[0292] 9. Add the 2° antibody (goat anti-rabbit-IgG HRP), 100 pL per well. Incubate at RT for 1h.

[0293] 10. Wash the plate 3x with PBST, 250 pL per well.

[0294] 11. Add the substrate TMB, 100 pL per well. Incubate at RT until colour development is visible (approx 5 minutes). 12. Stop the reaction: Add 1M HCI, 100 pL per well.

[0295] 13. Measure the OD at 450 nm and the reference wavelength 620 nm.

[0296] 14. Data Analysis: The reference wavelength is subtracted from the 450 nm value, and the resulting data plotted against the mCBM concentration in GraphPad Prism. A non-linear regression is then performed (Analyse > Non-Linear Regression (Curve Fit) > [Agonist] vs. Response- Variable slope (Four Parameters)). Data with an R2above 0.9 is accepted for analysis.

[0297] Method: Plaque assay

[0298] Dav 1 :

[0299] 1. Trypsinise T-175 Flasks of HI HeLa cells and quantify the cell density as described in PGN ROP 0134. Only proceed if cell viability is above 90%.

[0300] 2. Generate a 3.2x105cells / mL solution of cells, diluting using complete MEM solution.

[0301] 3. Seed the required number of 6-well plates with 2 mL of this solution and incubate overnight at 37°C, 5% CO2. Each experiment is performed in duplicate, and across two 6-well plates, for a total of 4 plates.

[0302] Dav 2:

[0303] 4. Confirm that all wells have a near confluent monolayer (90%) of cells.

[0304] 5. Prepare 50:50 MEM / PBS as in section 3.1 and warm to room temperature.

[0305] 6. Prepare CBM Dilutions as in section 3.3 and store on ice.

[0306] 7. Place Viral Stock on ice to defrost, as described in section 3.5.

[0307] 8. Incubate the agarose at 65°C as described in Section 3.6

[0308] 9. Working one plate at a time, remove media from wells using the aspirator.

[0309] 10. Wash wells 3x with 2 mL PBS.

[0310] 11 . Working one plate at a time, remove PBS from wells using the aspirator.

[0311] 12. Pipette 400 pL of CBM dilution into the appropriate wells (Fig 3.1), and incubate the plate at 37°C, 5% CO2for 1 hour. Gently rock the plate every 10-15 minutes.

[0312] 13. Whilst incubating the plates, prepare the Virus Solution (section 3.3.5), and keep on ice.

[0313] 14. Pre-warm 2X MEM in the 37°C water bath.

[0314] 15. Working one plate at a time, remove the CBM from the wells by aspiration, changing tip after each well.

[0315] 16. Wash each well gently with 2 mL PBS before aspirating the PBS. 17. Add 400 pL of viral solution or serum free media to the appropriate wells (Fig 5.1), and incubate the plates as in step 4.12, with gentle rocking every 10-15 min.

[0316] 18. Remove viral solution by pipette and dispense into 2-5% (w / v) Virkon solution.

[0317] 19. Apply 2 mL of agarose overlay onto each well and allow to set, using the remaining agarose as a guide to avoid disturbing the plates. Apply 2ml of Complete MEM on top of the set agarose.

[0318] 20. Incubate the plates at 35°C, 5% CO2 for 48 to 72 hours, depending on the virus, as noted in Section 3.3.5.

[0319] Day 3:

[0320] 21. In a fume cupboard, apply 2 mL of 10% (v / v) formalin to each well. Incubate at room temperature in the fume cupboard for 15 min to fix the cells.

[0321] 22. Remove the formalin and dispense into special waste (with 2-5% (w / v) Virkon in the bottle). Tap off the agarose plug, and wash wells gently with 2 mL PBS before aspirating the PBS.

[0322] 23. Apply 2 mL of 1 % (w / v) crystal violet solution to each well and incubate at room temperature for 10-15 minutes.

[0323] 24. Remove the staining solution to special waste and rinse the plate with dFW which is also removed to special waste. Tap dry on white roll.

[0324] References

[0325] (1). Lo MK, Jordan R, Arvey A, Sudhamsu J, Shrivastava-Ranjan P, Hotard AL, Flint M, McMullan LK, Siegel D, Clarke MO, et al. GS-5734 and its parent nucleoside analog inhibit Filo-, Pneumo-, and Paramyxoviruses. Scientific Reports. 2017;7(1):43395. doi:10.1038 / srep43395

[0326] (2). Sidwell RW, Robins RK, Hillyard IW. Ribavirin: An antiviral agent. Pharmacology & Therapeutics. 1979;6(1 ): 123-146. doi: 10.1016 / 0163-7258(79)90058-5

[0327] (3). Furuta Y, Takahashi K, Fukuda Y, Kuno M, Kamiyama T, Kozaki K, Nomura N, Egawa H, Minami S, Watanabe Y, et al. In Vitro and In Vivo Activities of Anti-Influenza Virus Compound T-705. Antimicrobial Agents and Chemotherapy. 2002;46(4):977-981. doi: 10.1128 / AAC.46.4.977-981.2002 (4). Geraghty R, Aliota M, Bonnac L. Broad-Spectrum Antiviral Strategies and Nucleoside Analogues. Viruses. 2021;13(4):667. doi:10.3390 / v13040667

[0328] (5) Makela MJ, Puhakka T, Ruuskanen O, Leinonen M, Saikku P, Kimpimaki M, Blomqvist S, Hyypia T, Arstila P. Viruses and Bacteria in the Etiology of the Common

[0329] Cold. Journal of Clinical Microbiology. 1998;36(2):539-542. doi:10.1128 / JCM.36.2.539- 542.1998

[0330] (6) Thibaut HJ, Lacroix C, De Palma AM, Franco D, Decramer M, Neyts J. Toward antiviral therapy / prophylaxis for rhinovirus-induced exacerbations of chronic obstructive pulmonary disease: challenges, opportunities, and strategies: Treatment of COPD exacerbations. Reviews in Medical Virology. 2016;26(1):21-33. doi:10.1002 / rmv.1856

Claims

CLAIMS:1 . A low-density lipoprotein receptor (LDLR)-binding CBM, for use in the prevention and / or treatment of a viral infection.

2. The LDLR-binding CBM for use according to claim 1 , wherein the viral infection is caused by a virus that exploits LDLR for host cell binding and / or entry.

3. The LDLR-binding CBM for use according to any preceding claim, wherein the viral infection is a Human Rhinovirus (HRV) infection.

4. The LDLR-binding CBM for use according to any preceding claim, wherein the viral infection is a minor group Human Rhinovirus (HRV) infection.

5. The LDLR-binding CBM for use according to any preceding claim, wherein the compound comprises at least two carbohydrate binding modules.

6. The LDLR-binding CBM for use according to any preceding claim, wherein the compound does not exhibit sialidase activity.

7. The LDLR-binding CBM for use according to any preceding claim, wherein the compound does not bind heparin or heparin sulfate and / or does not comprise the GAG-binding domain of a protein that binds heparin or heparin sulfate.

8. The LDLR-binding CBM for use according to any preceding claim, wherein said compound comprises a Family 40 CBM (CBM40).

9. The LDLR-binding CBM for use according to any preceding claim, wherein the LDLR-binding CBM comprises a modified CBM40.

10. The LDLR-binding CBM for use of claim 9, wherein the modified CBM40 contain(s) one or more mutations relative to a reference sequence.11 . The LDLR-binding CBM for use of claim 9 or 10, wherein the reference sequence is selected from the group consisting of:(i) a wild type family 40 CBM sequence;(ii) a wild type CBM40 sequences from Vibrio cholerae’,(iii) the NanH sialidase sequence of Vibrio cholerae’,(iv) a wild type CBM40 sequences from Streptococcus pneumoniae’,(v) the NanA sialidase sequence of Streptococcus pneumoniae’,(vi) The sequence of SEQ ID NO: 1;(vii) The sequence of SEQ ID NO: 2;(viii) The sequence of SEQ ID NO: 3; and(ix) The sequence of SEQ ID NO: 4.

12. The LDLR-binding CBM for use of claim 9, 10 or 11, wherein the mutation is selected from the group consisting of:(i) one or more amino acid substitution(s);(ii) one or more amino acid deletion(s);(iii) one or more amino acid addition(s) / insertion(s);(iv) one or more amino acid / sequence inversions; and(v) one or more amino acid / sequence duplications.

13. The LDLR-binding CBM for use of any preceding claim, wherein the LDLR- binding CBM comprises a oligomerisation domain.

14. The LDLR-binding CBM for use of any preceding claim, wherein the LDLR- binding CBM comprises a modified oligomerisation domain.

15. The LDLR-binding CBM for use of claim 14, wherein the modified oligomerisation domain contains one or more mutations relative to a reference sequence.

16. The LDLR-binding CBM for use of claim 15, wherein the reference sequence is selected from the group consisting of:(i) a wild type Pseudomonas aeruginosa pseudaminidase sequence;(ii) the Pseudomonas aeruginosa pseudaminidase amino acid sequence deposited under accession number Q9L6G4;(iii) the sequence of SEQ ID NO: 13; and(iv) the sequence of SEQ ID NO: 14.

17. The LDLR-binding CBM for use of any preceding claim, wherein the LDLR- binding CBM comprises one or more family 40 carbohydrate binding modules (CBM40(s)), wherein relative to SEQ ID NO: 1 , the one or more CBM40(s) comprises an A162P mutation and relative to Sp2CBMTD the (LDLR)-binding CBM binds LDLR with a reduced ECso.

18. The LDLR-binding CBM for use of claim 17, wherein relative to SEQ ID NO: 1 , the one or more CBM40(s) contain(s) one or more additional mutations.

19. The LDLR-binding CBM for use of claim 16 or 17, wherein relative to SEQ ID NO:

1. the one or more CBM40(s) comprises additional mutations at one or more residues selected from the group consisting of: residue 167; residue 168; residue 169; residue 170; residue 171 ; residue 172; residue 173; residue 174; residue175; residue 176; residue 177; residue 178; residue 179; residue 180; residue181 ; residue 236; residue 237; residue 238; residue 239; residue 240; residue241 ; residue 242; residue 243; residue 244; residue 245; residue 246; residue247; residue 248; residue 249; residue 250; residue 251 ; residue 252; residue253; residue 254; residue 286; residue 287; residue 288; residue 289; residue290; residue 291 ; residue 292; residue 293; and residue 294.

20. The LDLR-binding CBM for use of any one of claims 16-18, wherein with reference to SEQ ID NO: 1 , the one or more CBM40(s) includes one or more additional mutations selected from the group consisting of: M156F; Y168W; L170A; L170T; V173G; M185I; V239A; V239T; V246G; I286A; Y292E.

21. The LDLR-binding CBM for use of any preceding claim, wherein the LDLR- binding CBM comprises the following structure:CBM1 (V239A V246G A162P ) — CBM2 (V239A V246G A162P ) — TD ( S342D R403K) wherein CBM 1 and CBM 2 are derived from CBM40 sequences and TD is derived from a trimerisation domain.

22. The LDLR-binding CBM for use of any preceding claim, wherein the LDLR- binding CBM comprises the following sequence:GAMVIEKEDVETNASNGQRVDLSSELDKLKKLENATVHMEFKPDPKAPAFYNLFSVSS ATKKDEYFTMAVYNNTATLEGRGSDGKQFYNNYNDAPLKVKPGQWNSVTFTVEKPTAE LPKGRARLYVNGGLSRTSLRSGNFIKDMPDVTHVQIGATKRANNTVWGSNLQIRNLTV YNRALTPEEVQKRSGGGSGVIEKEDVETNASNGQRVDLSSELDKLKKLENATVHMEFK PDPKAPAFYNLFSVSSATKKDEYFTMAVYNNTATLEGRGSDGKQFYNNYNDAPLKVKP GQWNSVTFTVEKPTAELPKGRARLYVNGGLSRTSLRSGNFIKDMPDVTHVQIGATKRA NNTVWGSNLQIRNLTVYNRALTPEEVQKRSGGSLGVPDFESDWFDVSSNSLYTLSHGL QRSPRRVVVEFARSSSPSTWNIVMPSYFNDGGHKGSGAQVEVGSLNIKLGTGAAVWGT GYFGGIDNSATTRFATGYYRVRAWI23. A method of identifying an agent for use in the treatment of a minor group Rhinovirus, said method comprising:(i) contacting a cell expressing LDLR with a test agent and a LDLR- binding CBM under conditions which permit binding between the LDLR and the LDLR-binding CBM; and(ii) detecting binding between the LDLR expressed by the cell and the test agent; wherein a test agent which binds the LDLR expressed by the cell may be for use in the treatment of a minor group rhiniovirus.

24. An in vitro method of rendering cells non-permissive to a viral infection, said method comprising contacting or incubating cells susceptible or vulnerable to a viral infection with the compound according to any one of claims 1 - 22 under conditions which permit binding between said compound and cell surface LDLR.

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