Conjugate molecules comprising carbohydrate binding molecule (CBM) and antibody

Abstract

Disclosed are molecules modified by the addition of a retention moiety (e.g. a CBM), the retention of such modified molecules may be increased or improved at a site (e.g. at or within a tissue) may be increased or improved.

Description

[0001] CONJUGATE MOLECULES

[0002] FIELD

[0003] The present disclosure relates to conjugate molecules which exhibit increased retention time at a target site, and uses thereof. The present disclosure also provides methods of increasing the retention time of a molecule of interest at a target site.

[0004] BACKGROUND

[0005] The demand for new drugs has never been higher but research and development is hampered by, for example, increasing costs and drug resistance. There is a need not only for new and effective drugs, but also for new treatment regimes, delivery mechanisms and other technologies which can be applied to the existing drug arsenal and to newly developed compounds to extend our treatment options and enhance clinical success. As new drugs and vaccines exploit alternate methods of administration - for example mucosal administration, there is a need for technology which improves the residence time in the host.

[0006] SUMMARY

[0007] The present disclosure provides means by which the retention time of molecules at a site (e.g. at or within a tissue) may be increased or improved.

[0008] . ) ) 3 “ 3 6 / ) ” “ 36 / / 4 ” 4( “ 36 / ) ” ) ) ( ( ) 4 ) 6) 4( ) 3 ( / 3 )4,. / / 4^)4 / 4. “ 36 / ) ” 6 / 2,) ),) ). be understood that these terms may also encompass aspects and / or embodiments B. / . “ 4 / ) )4 / 2,” “ 4 / ,”. ) ) A 4,) ),) )

[0009] In one teaching, the disclosure provides molecules which further comprise a retention moiety. In one teaching, the retention moiety comprises a carbohydrate binding module (CBM)

[0010] Additionally, the disclosure provides molecules which have been modified by the addition of a retention moiety (e.g. a CBM). Molecules of this type may be referred to as modified molecules. A modified molecule of this disclosure may be modified by the addition of a plurality of retention moieties (e.g. a plurality of CBMs), for example two, three, four, five, six, seven, eight or more retention moieties. Where the retention moiety comprises a CBM, a modified molecule of this disclosure may be modified by the addition of two, three, four, five, six, seven, eight or more CBMs.

[0011] 55740646-1 The disclosure also provides constructs, which constructs comprise a molecule for retention at a site and a retention moiety, wherein the retention moiety comprises a carbohydrate binding module (CBM). In one teaching, a construct of this disclosure may comprise a molecule for retention at a site and a plurality of retention moieties, two, three, four, five, six, seven, eight or more retention moieties. Where the retention moiety comprises a CBM, a construct of this disclosure may comprise a molecule for retention at a site and two, three, four, five, six, seven, eight or more CBMs (as retention moieties).

[0012] In a further teaching, a modified molecule or construct of this disclosure may comprise at least two parts; a first part which comprises the molecule which is to be retained at a specific site and a second part which facilitates that retention. The first part of the modified molecule may be fused (therefore the modified molecules disclosed herein may. ) B / ) ) ),) ) ( ‘, / 4 ’ ‘ 4 ’ ‘, / 4 4 ’, joined, bound or otherwise conjugated to the second part of the modified molecule. The molecule to be retained may be linked to the retention moiety, using a short amino acid / peptide linker. Such a linker may be of any suitable length (for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids in length) and sequence. A molecule to be retained may comprise more than one linker such that more than one retention moiety may be fused, joined, bound or conjugated thereto. By way of example, a molecule to be retained at a site may be modified by the addition to two linkers, each linker capable of being linked to a retention moiety. Of course, one of skill will appreciate that molecules to be retained could be modified by the addition of three, four, five, six, seven, eight or more linkers - the number depending, in part, on the specific chemistry of the molecule to be retained - with some molecules, one of skill will appreciate that the number of linkers that can be added to the molecule may be restricted.

[0013] . ) ) 3 “6) 6 / ( ) 24 ) ” 4( “ 40 / 4 6) 6 / ( ) ” 3 ) ) ( / 4 ). 4 ) 2 throughout the present disclosure.

[0014] Accordingly, this disclosure provides a molecule for retention (or increased residence time at) at a site, said molecule further comprising a retention moiety.

[0015] In a further teaching, the disclosure provides a modified molecule, wherein molecule is for retention at a site and it is modified by the addition of a retention moiety.

[0016] 55740646-1 In a yet further teaching, the disclosure provides the use of a retention moiety for achieving retention (or increased residence time) of a molecule at a site.

[0017] It should be noted that in this disclosure, the terms / 6. ) ‘ ) )4 Z 4’ 4( 74 ) ) ( ) / ( )4 ) / 3 ) ’ ) / 4 ). 4 ) 3

[0018] It should also be noted that whether it is referring to modified molecule or a construct, all of the text presented herein is relevant to any of the disclosed molecules, modified 3 3 3 4( 4 ( / 2 ) (. ) ) / 4 4^)4 / )4 ) 4 ),) )4 ) 3 3 3 ’. ) 4 ) ( ) / 4 4, / 3 3 3, ) ) 4 / 4 / ) 4( further comprising a retention moiety; (ii) a molecule modified by the addition of a retention moiety; and (iii) a construct comprising a molecule for retention at a site and a retention moiety.

[0019] The retention moiety of any of the disclosed molecules, constructs and modified molecules, facilitates retention of the molecule at the site. Indeed, as compared to a molecule (e.g. a naked molecule - that is a molecule not further comprising a retention moiety, an unmodified molecule (not comprising a retention moiety) or a molecule not provided in the form a construct of this disclosure), administered or contacted with a site of interest, the retention time of a molecule (administered to the same site of interest) which comprises any of the retention moieties described herein (e.g. a molecule provided in the form of a modified molecule or construct of this disclosure), is longer. Moreover, the molecules of this disclosure may be retained at sites which would not normally retain the molecule. One of skill will appreciate that where, for example, a site does not comprise or express anything to which a molecule might bind, then that molecule may not be retained by the site. In contrast, by modifying or augmenting a molecule to comprise a retention moiety which binds to a target present at (or in) the site, then administering the molecule to the site in this form, should result in retention of the molecule at the site - at least for as long as the retention moiety remains bound to its target.

[0020] Within the context of the various modified molecules, constructs and molecules for retention disclosed herein, it has been shown that the retention moiety may exhibit increased affinity for its target. For example, where the retention moiety comprises a CBM, the CBM may exhibit increased affinity for its carbohydrate, glycan and / or sialic

[0021] 55740646-1 acid target. This is especially true of modified molecules, constructs and molecules for retention comprising a plurality, for example two, three, four, five, six or more, retention moieties (e.g. CBMs). Any such improvement or increase in retention moiety target affinity may be as compared to the affinity of the retention moiety for its target when not fused, joined, bound or conjugated to a molecule for retention.

[0022] As explained in more detail below, the modified molecules (or the retention moieties) of this disclosure find particular application in medical uses and / or methods of treatment. Specifically, the retention moieties described herein may be used to increase the retention time of a drug at a specific site and / or to achieve the prolonged administration or dosing of a drug at a site. Indeed, using a retention moiety of this disclosure it may even be possible to administer drugs to sites which are not normally accessible using standard methods of administration and / or dosing.

[0023] In view of the above, a molecule to be retained (or for retention) at a site may be fused, joined, bound or otherwise conjugated to the retention moiety.

[0024] The retention moiety may bind to or have affinity for, a target molecule.

[0025] The retention moiety may comprise a peptide or protein which binds to or has affinity for a specific target.

[0026] Without wishing to be bound by theory, in the context of this disclosure, the retention of a molecule at a site is brought about through binding between the retention moiety (which has been added to the molecule e.g. as a modification) and its target. One of skill will appreciate that the choice of retention moiety may depend on the site at which the molecule is to be retained. The site (at which the molecule is to be retained) should comprise an appropriate target for the retention moiety - in that way, because of binding between the retention moiety and its target present at the site, the molecule, will become retained at that site.

[0027] In one teaching, the target of the retention moiety may be a carbohydrate (e.g. a polysaccharide or glycan). A retention moiety of this type may be referred to as a glycan binding molecule or carbohydrate binding molecule.

[0028] 55740646-1 In one teaching, the retention moiety may comprise a sialic acid binding molecule.

[0029] In a further teaching, the retention moiety may comprise a carbohydrate binding module (CBM). In some examples, the retention moiety may comprise two, three, four, five, six, seven, eight or more CBMs.

[0030] CBMs for use as retention moieties, are all characterised as being able to bind (or having an affinity for) a carbohydrate, a glycan and / or sialic acid. Examples of useful CBMs are 6 A( ) ( )2 B, 36 )4) / . S; ) 4( ) (.. ) ) 3 “ includes, for example, CBMs classified as belonging to any of CBM families 32, 40, 47, 67 and 70. One of skill will appreciate that there is a vast array of different CBMs and members of the abovementioned families can be derived from many different bacterial species. Further information regarding CBMs, in particular the family 32, 40, 47, 67 and 70 CBMs, can be found at the public CAZY database (available at: http: / / www.cazv.org: all CBMs available via this database are to be construed as included within the scope of. ) ) 3 ‘ ’ ) ( ■ ) ) / 4). Some particularly useful CBMs may be derived from bacterial species within the Genera: Streptococcus, Vibrio and Clostridium. It should also be understood (and as is set out in more detail below) that the disclosure embraces constructs which comprise one or more of the CBMs disclosed herein and / or any functional (e.g. carbohydrate-binding) fragment thereof or any (again functional) modified or variant forms thereof. By way of example a modified or variant CBM may comprise one or more mutated residues relative to the relevant wild-type CBM sequence. In this ) (. ) ) 3 ‘ ’ / 4 2 ( ) s not only members of any of the specific CBM families or CBM-types described herein but functional fragments or variants thereof.

[0031] A useful CBM32 (i.e. a CBM32 for use as a retention moiety described herein) may be derived from any suitable source. For example, a CBM32 for use as (or in) a retention moiety, may be obtained from a microorganism, including, for example, bacteria of the genera Cellvibrio, Yersinia, Micromonospora, Streptococcus, Bifidobacteria and Clostridium. For example, a useful CBM32 may be obtained or derived from, for example, Cellvibrio mixtus, Yersinia enterolitica, Clostridium perfringens, Clostridium thermocellum, Streptococcus pneumoniae, Bifidobacterium longum and Micromonospora viridifaciens. Further details concerning the source, structure and function of the CBM32 family can be found within the Carbohydrate Active Enzymes database (freely available on the internet at: http: / / www.cazy.org / CBM32.html).

[0032] 55740646-1 An exemplary CBM32 sequence is provided by SEQ ID NO: 1 below:

[0033] SEQ ID NO: 1 AIIETAIPQSEMTASATSEEGQDPASSAIDGNTNTMWHTKWNGSDALPQSLSVNLGSSRKVSSI AITPRTSGNNGFITKYEIHAINNGVETLVAEGTWEENNLVKTVTFDSPIDAEEIKITAIQGVGG FASIAELNVYE

[0034] Accordingly, a molecule for retention at a site may comprise a CBM having the sequence of SEQ ID NO: 1 or a functional (e.g. carbohydrate binding) fragment thereof.

[0035] A carbohydrate binding fragment of SEQ ID NO: 1 may comprise anywhere between about 5, 6, 7, 8, 9 or 10 (consecutive or contiguous) amino acids to about 138 (consecutive or contiguous) amino acids from SEQ ID NO: 1. Suitable fragments may comprise about 11, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130 or about 135 (consecutive or contiguous) amino acids from SEQ ID NO: 1.

[0036] Without wishing to be bound by theory, where the modified molecule comprises a CBM32, retention of the molecule is brought about by binding between CBM32 and its target. In this regard, CBM32 ora protein comprising, consisting essentially or consisting of SEQ ID NO: 1 may bind or target, for example, galactose, N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc) and / or lactose. Accordingly, any fragment for use may also bind galactose, N- acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc) and / or lactose. One of skill will appreciate that the binding affinity of any given CBM32 molecule may depend on the precise CBM32 subtype; by way of further examples, some CBM32s have shown affinity for a variety of ligands (examples include type II blood group H-trisaccharide (Fuca1-2Gal 1-4GlcNAc), N-acetyl-D-lactosamine (LacNAc), galactose, lacto-N-biose, disaccharide GlcNAc-α-1,4-Gal (which may be referred to as an N-acetylglucosamine linked alpha 1,4 to galactose), and / or GlcNAc). It should also be noted that multiple CBM32 subtypes may be derived from a single organism; these different CBM subtypes may exhibit the same, similar or different binding specificities. For example, Clostridium perfringens contains two sialidases NanJ and NanH; NanJ contains one galactose-specific CBM32; NanH contains four putative

[0037] 55740646-1 CBM32s with different binding selectivity - for example, the CBM32 encoded by NanH binds GlcNAc. As used herein, the term CBM32 embraces all CBM32 variants, derivatives and sub-types. Accordingly, CBM32 may be selected for use as (or in) a retention moiety, where the site at which the molecule of the construct is to be retained, comprises or expresses any of the abovementioned CBM32 targets or ligands.

[0038] SEQ ID NO: 1 is derived from the sequence deposited in the UniProt database under ID No: A0A2X2YJF2. This sequence is reproduced as SEQ ID NO: 2 below (SEQ ID NO: 1 appears as residues 42-180 - shaded in the sequence below).

[0039] SEQ ID NO: 2

[0040] MKSKKIIATL VASLVISNMG GYLVKANPNV NHKAVIIEDR;gg|fl|g|g||ggg ligillBiilf liBigillili sggggilliiiB BiiiiiBiii g

[0041]

[0042] lligigigl IgiiBiiBii ilg|g®|gB;lg|ggilggg;

[0043] IKGEVDEIAN YGNLKITKEE ERLNITRDLE KFSSLDEGTI VTRFNMNDTS IQSLIGLSDG NKANNYFSLY VSGGKVGYEL RRQEGNGDFN VHHSADVTFN KGINTLALKI EKGVGAKIFL NGSLVKTVSD PNIKFLNAIN LNSGFIGKTD RANGYNEYLF RGNIDFMNIY DKPVSDNYLL RKTGETKAPS EDSLLPDDVY KTQPVELFYP GYLESRGYRI PALETTKKGT VLASIDVRNN GDHDAPNNNI DVGIRRKEVN GEWEEGKVIL DYPGKSAAID TSLMSATIEE NGIEKERIFL IVTHFPEGYG FPNTEGGSGY KEIDGKYYFI LKDAQNNEYT VREDGIVYNS EGNETDYVMK NDKTLIQNGE EVGNALLSNS PLKAVGTAHI EMIYSDDDGN TWSEPEDLNP GLKKEWMKFF GTAPGKGIQI KNGEHKGRLV FPIYYTNQNN FQSSAVIYSD DFGETWKLGE SPIDTASVSS ETVSSGTQLT ECQVVEMPNG QLKLFMRNTG SYTRIATSFD GGATWHDEVP EDTSLREPYC QLSVINYSGK INGKDAIIFS NPDASSRVNG SVKVGLINEN GTYENGQPRY EFDWIYNKTV KPGSFAYSCL TELPDGNLGL FYEGEGAGRM AYTEFDLNYL KFNASEDSPA ATVQSIESLD EDLIYNAGDE VSIKVNFNQL VSLIGDRKIT LDIGGVDVPL NMVNYEGKSS AIFKGTIPEG INPGNYEIKL KENNALELNT VYNKVSTLNG LDNTGINVQI GELKTTVGNS TIKVNEEVQV GSAFEAILGI KGLNGDTEVY SAEYLFEYNA EAFKLNEITS FSDSLFVKSK EVEPGKVRIL VASLGNEIEK DSELVKVNLT PKISSELEVL GLTTALVGAG DGNTHDLELS SKEVKINEEA SGEIVVNPVQ NFEIPEINKK NVKLTWNAPI TTEGLEGYVI YKDGKKLSEV PAESTEFVVS KLNRHTIYNF KVAAKYSNGE LSAKESKTIR TAR

[0044] SEQ ID NOS 1 and 2 are derived from Clostridium perfringens.

[0045] A molecule for retention at a site may comprise one, two, three, four or more CBM32(s). A CBM for use as a retention moiety may comprise one, two, three, four or more proteins comprising SEQ ID NO: 1 or a carbohydrate binding fragment thereof. One of skill will appreciate that a useful CBM32 may comprise sequences which exhibit some degree

[0046] 55740646-1 (for example, 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65% or 60%) of sequence identity or homology with the CBM32 sequences of SEQ ID NOS: 1 and 2. All such variant or divergent sequences are to be embraced within the scope of this disclosure and by the term “ ” ( ) 4 / 2 4(. 3 2 ) 7 ) 4 ) s may have carbohydrate binding function.

[0047] CBM40

[0048] A useful CBM40 (i.e. a CBM40 for use as a retention moiety) may be derived from any suitable source. For example, CBM40s for use as (or in) a retention moiety, may be obtained from a microorganism, including, for example, bacteria of the genera Clostridium, Enterococcus, Staphylococcus, Streptococcus and Vibrio. For example, useful CBM40s may be obtained or derived from, for example Clostridium perfringens, Streptococcus pneumoniae and Vibrio cholerae. Further details concerning the source, structure and function of the CBM40 family can be found within the Carbohydrate Active Enzymes database (freely available on the internet at: htto: / / www.cazv.orq / CBM40.html).

[0049] Without wishing to be bound by theory, where the modified molecule comprises a a CBM40, retention of the molecule is brought about by binding between CBM40 and its target. In this regard, 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 β-propeller of GH33 sialidases. The family 40 CBMs may all be characterised by an ability to bind sialic acid and at least the CBM40 of Vibrio cholerae binds the alpha-anomer of sialic acid and, for example, α(2,3)-, α(2,6)-, and α(2,8)-linked sialosides. Accordingly, CBM40 may be selected for use as (or in) a retention moiety, where the site at which the molecule of the construct is to be retained comprises or expresses any of the abovementioned CBM40 targets or ligands - e.g. sialic acid.

[0050] Exemplary CBM40s for use may comprise the sialic acid binding domain of Vibrio cholerae NanH sialidase (\ / cCBM: a CBM40) and / or the equivalent (or homologous) domain from Streptococcus pneumoniae NanA sialidase (SpCBM: also a CBM40). Of course, similar or homologous sialic acid binding modules present in other organisms are to be encompassed within the scope o,. ) ) 3 “ ” 4( “

[0051] A retention moiety may comprise one, two, three, four or more CBM40(s).

[0052] 55740646-1 An exemplary Vibrio cholerae NanH sialidase amino acid sequence is deposited under accession number A5F7A4 and is reproduced below as SEQ ID NO: 3 (781 amino acids).

[0053] SEQ ID NO: 3

[0054] MRFKNVKKTA LMLAMFGMAT

[0055]

[0056] SSNAHglg|t|i Sill®®!!® Bggglliigg igggiiiiii iggiBBli iiggiiiiii Hliiggili Bgiigigigii iiigiiiiil ilfgiiiigii iitiiiili® iigggiiigi iigggigiii iiigiiilil ii iiiigigiB ilBiilil® iiiliiiiig g|i|giviFR GPDRIPSIVA SSVTPGVVTA FAEKRVGGGD PGALSNTNDI ITRTSRDGGI TWDTELNLTE QINVSDEFDF SDPRPIYDPS SNTVLVSYAR WPTDAAQNGD RIKPWMPNGI FYSVYDVASG NWQAPIDVTD QVKERSFQIA GWGGSELYRR NTSLNSQQDW QSNAKIRIVD GAANQIQVAD GSRKYVVTLS IDESGGLVAN LNGVSAPIIL QSEHAKVHSF HDYELQYSAL NHTTTLFVDG QQITTWAGEV SQENNIQFGN ADAQIDGRLH VQKIVLTQQG HNLVEFDAFY LAQQTPEVEK DLEKLGWTKI KTGNTMSLYG NASVNPGPGH GITLTRQQNI SGSQNGRLIY PAIVLDRFFL NVMSIYSDDG GSNWQTGSTL PIPFRWKSSS ILETLEPSEA DMVELQNGDL LLTARLDFNQ IVNGVNYSPR QQFLSKDGGI TWSLLEANNA NVFSNISTGT VDASITRFEQ SDGSHFLLFT NPQGNPAGTN GRQNLGLWFS FDEGVTWKGP IQLVNGASAY SDIYQLDSEN AIVIVETDNS NMRILRMPIT LLKQKLTLSQ N

[0057] The CBM region of SEQ ID NO: 3 is from amino acid residue 25 to 216 (grey shaded sequence) - this sequence may be SEQ ID NO: 4.

[0058] An exemplary Streptococcus pneumoniae NanA sialidase amino acid sequence has been deposited under accession number P62575 and is reproduced below as SEQ ID NO: 5 (1035 amino acids).

[0059] SEQ ID NO: 5

[0060] MSYFRNRDID IERNSMNRSV QERKCRYSIR KLSVGAVSMI VGAVVFGTSP VLAQEGASEQ PLANETQLSG ESSTLTDTEK SQPSSETELS GNKQEQERKD KQEEKIPRDY YARDLENVET

[0061]

[0062] iliiBOii igiggoii liiggiggii liitiiggii igiggigiii iiiiiiiiii iiiligigii illlllilli iiliiiggii ilBlililii iigiiigigi

[0063]

[0064] iHiiliiggi igiigggiiO iiilgiiiii gggggQLFKR SDLEKKLPEG AALTEKTDIF ESGRNGKPNK DGIKSYRIPA LLKTDKGTLI AGADERRLHS SDWGDIGMVI RRSEDNGKTW GDRVTITNLR DNPKASDPSI GSPVNIDMVL VQDPETKRIF SIYDMFPEGK GIFGMSSQKE EAYKKIDGKT YQILYREGEK GAYTIRENGT VYTPDGKATD YRVVVDPVKP AYSDKGDLYK GNQLLGNIYF TTNKTSPFRI AKDSYLWMSY SDDDGKTWSA PQDITPMVKA DWMKFLGVGP GTGIVLRNGP HKGRILIPVY TTNNVSHLNG

[0065] 55740646-1 SQSSRIIYSD DHGKTWHAGE AVNDNRQVDG QKIHSSTMNN RRAQNTESTV VQLNNGDVKL FMRGLTGDLQ VATSKDGGVT WEKDIKRYPQ VKDVYVQMSA IHTMHEGKEY IILSNAGGPK RENGMVHLAR VEENGELTWL KHNPIQKGEF AYNSLQELGN GEYGILYEHT EKGQNAYTLS FRKFNWDFLS KDLISPTEAK VKRTREMGKG VIGLEFDSEV LVNKAPTLQL ANGKTARFMT QYDTKTLLFT VDSEDMGQKV TGLAE GATES MHNLPVSVAG TKLSNGMNGS EAAVHEVPEY TGPLGTSGEE PAPTVEKPEY TGPLGTSGEE PAPTVEKPEY TGPLGTAGEE AAPTVEKPEF TGGVNGTEPA VHEIAEYKGS DSLVTLTTKE DYTYKAPLAQ QALPETGNKE SDLLASLGLT AFFLGLFTLG KKREQ

[0066] The CBM region of SEQ ID NO: 5 is from amino acid residue 121 to 305 (grey shaded sequence) - this sequence may be SEQ ID NO: 6.

[0067] In view of the above, CBMs for use as (or in) a retention moiety may comprise a protein or peptide having the sequence of SEQ ID NO: 3, 4, 5 or 6 or a carbohydrate binding fragment of any of these. For example, a useful molecule (i.e. a CBM40 for use as a retention moiety in any of the constructs described herein) may comprise a proteinaceous moiety encoded by the sialic acid binding domain of the nanH gene (encoding sialidase) of V. cholerae (as provided by SEQ ID NO: 3) or an equivalent or homologous gene present in another organism (for example the equivalent / homologous nanA sialidase gene of S. pneumoniae', see SEQ ID NO: 5).

[0068] A retention molecule may comprise from about residue 1, 5, 10, 15, 25 or 30 (i.e. from 1-30 or from any amino acid residue there between) to about residue 150, 175, 200, 210, 216, 220-781 (to any residue from 150 to 781 including any residue therebetween) of the V. cholerae sialidase molecule of SEQ ID NOS: 3 and 4.

[0069] A retention moiety may comprise a peptide having a sequence corresponding to residue 25 to about residue 216 of SEQ ID NO: 3.

[0070] A carbohydrate binding fragment of SEQ ID NO: 3 (which fragments can be used as (or in) a retention moiety may comprise anywhere between about 5, 6, 7, 8, 9 or 10 (consecutive or contiguous) amino acids to about 191 (consecutive or contiguous) amino acids from SEQ ID NO: 3. Suitable fragments may comprise about 11, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130 or about 135, about 140, about 150, about 155, about 160, about 165, about 170, about 180, about 185, about

[0071] 55740646-1 186, about 187, about 188, about 189 or about 190 (consecutive or contiguous) amino acids from SEQ ID NO: 3.

[0072] A retention moiety may comprise from about residue 1, 5, 10, 15, 25 or 30 (i.e. from 1-30 or from any amino acid residue there between) to about residue 150, 175, 200, 210, 216, 220-781 (to any residue from 150 to 781 including any residue therebetween) of the V. cholerae sialidase molecule of SEQ ID NOS: 3 and 4. For example, a sialic acid binding molecule for use may comprise a peptide having a sequence corresponding to residue 25 to about residue 216 of SEQ ID NO: 3 above.

[0073] A further molecule suitable for use as a retention moiety may comprise a protein or peptide having the sequence of SEQ ID NO: 5 or 6 or a carbohydrate binding fragment thereof. For example, a useful molecule may comprise a proteinaceous (sialic acid binding) moiety encoded by the sialic acid binding domain of the Streptococcus pneumoniae nanA gene (encoding sialidase).

[0074] A sialic acid binding molecule for use as a retention moiety may comprise from about residue 80, 90, 100, 110, 120, 121 to 130 (i.e. from any of about residues 80 to 130 including any residue therebetween) to about residue 250, 275, 300, 305, 310, 320-1035 (i.e. to any residue from about 250-1035 including to about any residue therebetween) of the S. pneumoniae sialidase molecule of SEQ ID NOS: 5 and 6.

[0075] For example, a sialic acid binding molecule for use as a retention moiety may comprise a peptide having a sequence corresponding to residue 121 to about residue 305 of SEQ ID NO: 5 above.

[0076] A carbohydrate binding fragment (also useful as a retention moiety) may comprise anywhere between about 5, 6, 7, 8, 9 or 10 (consecutive or contiguous) amino acids to about 185 (consecutive or contiguous) amino acids from SEQ ID NO: 5. Suitable fragments may comprise about 11, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 155, about 160, about 165, about 170, about 175, 180, 181, 182, 183 or 184 (consecutive or contiguous) amino acids from SEQ ID NO: 5.

[0077] 55740646-1 SEQ ID NOS 3 and 4 are derived from Vibrio cholerae and SEQ ID NOS 5 and 6 are derived from Streptococcus pneumoniae. A CBM for use as a retention may comprise one, two, three, four or more CBM40s. A molecule of this disclosure may comprise one, two, three, four or more proteins comprising SEQ ID NO: 3, 4, 5 or 6 or a carbohydrate binding fragment any of these. One of skill will appreciate that CBM40s useful as retention agents may comprise sequences which exhibit some degree (for example, 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65% or 60%) of sequence identity or homology with the CBM40 sequences of SEQ ID NOS: 3, 4, 5 and / or 6. All such variant or divergent sequences are to be embraced within the scope of this disclosure and by the term “ ” dentical and / or homologous CBM40 sequences may have carbohydrate / sialic acid binding function.

[0078] A retention moiety may comprise either or both of the sequences presented as SEQ ID NOS: 29 or 30.

[0079] CBM47

[0080] A useful CBM47 (i.e. a CBM47 for use as a retention moiety) may be obtained from microorganisms, including, for example, bacteria of the genera Acinetobacter, Bathymodiolus, Campylobacter, Planctomycetes, Streptococcus and Streptomyces. For example, useful CBM47s may be obtained or derived from, for example, Streptococcus mitis or Streptococcus pneumoniae. Further details concerning potential sources and the structure and function of the CBM47 family can be found within the Carbohydrate Active Enzymes database (freely available on the internet at: http: / / www.cazy.org / CBM47.html).

[0081] A retention moiety may comprise one, two, three, four or more CBM47(s).

[0082] An exemplary CBM47 sequence is provided by SEQ ID NO: 7 below:

[0083] SEQ ID NO: 7

[0084] TPDKFNDGNLNIAYAKPTTQSSVDYNGDPNRAVDGNRNGNFNSGSVTHTRADNPSWWEVDLKKM DKVGLVKIYNRTDAETQRLSNFDVILYDNNRNEVAKKHVNNLSGESVSLDFKEKGARYIKVKLL TSGVPLSLAEVEVFRES

[0085] 55740646-1 Accordingly, a CBM for use as a retention moiety may comprise, consist essentially or consist of a CBM having the sequence of SEQ ID NO: 7 or a carbohydrate binding portion thereof.

[0086] A carbohydrate binding fragment of SEQ ID NO: 7 may comprise anywhere between about 5, 6, 7, 8, 9 or 10 (consecutive or contiguous) amino acids to about 144 (consecutive or contiguous) amino acids from SEQ ID NO: 7. Suitable fragments may comprise about 11, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130 or about 135, about 140 or about 143 (consecutive or contiguous) amino acids from SEQ ID NO: 7.

[0087] CBM47 or a protein comprising, consisting essentially or consisting of SEQ ID NO: 7 may bind L-fucose, fucosyllactose, H-trisaccharide and / or Lewisyantigen. Accordingly, any fragment for use may also bind L-fucose, fucosyllactose, H-trisaccharide and / or Lewisyantigen.

[0088] SEQ ID NO: 7 is derived from the sequence deposited in the UniProt database under ID No: A0A1Q2T229. This sequence is reproduced as SEQ ID NO: 8 below (SEQ ID NO: 7 appears as residues 601-745 - shaded in the sequence below):

[0089] SEQ ID NO: 8

[0090] MNKEKIKRKL ITILFVCIGM LCFGLLAGVK ADNRVQMRTT INNESPLLLS PLYGNDNGNG LWWGNTLKGA WEAIPEDVKP YAAIELHPAK VCKPTSCIPR DTKELREWYV KMLEEAQSLN IPVFLVIMSA GERNTVPPEW LDEQFQKYSV LKGVLNIENY WIYNNQLAPH SAKYLEVCAK YGAHFIWHDH EKWFWETIMN DPTFFEASQK YHKNLVLATK NTPIRDDAGT DSIVSGFWLS GLCDNWGSST DTWKWWEKHY TNTFETGRAR DMRSYASEPE SMIAMEMMNV YTGGGTVYNF ECAAYTFMTN DVPTPAFTKG IIPFFRHAIQ NPAPSKEEVV NRTKAVFWNG EGRISSLNGF YQGLYSNDET MPLYNNGRYH ILPVIHEKID KEKISSIFPN AKILTKNSEE LSSKVNYLNS LYPKLYEGDG YAQRVGNSWY IYNSNANINK NQQVMLPMYT NNTKSLSLDL TPHTYAWKE NPNNLHILLN NYRTDKTAMW ALSGNFDASK SWKKEELELA NWISKNYSIN PVDNDFRTTT LTLKGHTGHK PQINISGDKN HYTYTENWDE NTHVYTITVN HNGMVEMSIN TEGTGPVSFP

[0091]

[0092] EEDIDKITED KWVSTNKVAT QSSTNYEGVA

[0093] 55740646-1 ALAVDGNKDG DYGHHSVTHT KEDSPSWWEI DLAQTEELEK LIIYNRTDAE IQRLSNFDII IYDSNDYEVF TQHIDSLESN NLSIDLKGLK GKKVRISLRN AGIPLSLAEV EVYTYK

[0094] SEQ ID NOS 7 and 8 are derived from Streptococcus pneumoniae.

[0095] A CBM for use as a retention moiety may comprise one, two, three, four or more CBM47s. A CBM for use as a retention moiety may comprise one, two, three, four or more proteins comprising SEQ ID NO: 8 or a carbohydrate binding fragment thereof.

[0096] One of skill will appreciate that useful CBM47s may comprise sequences which exhibit some degree (for example, 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65% or 60%) of sequence identity or homology with the CBM47 Sequences of SEQ ID NOS: 7 and 8 All such variant or divergent sequences are to be embraced within the scope of this disclosure and by the term “ ” ( )4 / 2 4(. 3 2 )7 )4 ) may have carbohydrate binding function.

[0097] CBM67

[0098] A useful CBM67 (i.e. a CBM67 for use as a retention moiety) may be derived from any suitable source. For example, CBM67s for use may be obtained from microorganisms, including, for example, bacteria of the genera Bacillus, Paenibacillus, Planctomycetes and Streptomyces. For example, a useful CBM67 may be obtained or derived from, for example, Streptomyces avermitilis. Further details concerning potential sources and the structure and function of the CBM67 family can be found within the

[0099] Carbohydrate Active Enzymes database (freely available on the internet at: http: / / www.cazy.org / CBM67.html).

[0100] A retention moiety may comprise one, two, three, four or more CBM67(s).

[0101] An exemplary CBM67 sequence is provided by SEQ ID NO: 9 below:

[0102] SEQ ID NO: 9

[0103] APSLEGSSWIWFPEGEPANSAPAATRWFRRTVDLPDDITGATLAISADNVYAVSVDGAEVARTD LEADNEGWRRPAVIDVLDHVHSGNNTLAVSASNASVGPAGWICVLVLTTASGEKKIFSDASWKS TDHEPADGWREPDFDDSGW P A AK VA AAWG AG P WGRV A

[0104] 55740646-1 Accordingly, a CBM for use as a retention moiety may comprise, consist essentially or consist of a CBM having the sequence of SEQ ID NO: 9 or a carbohydrate binding portion thereof.

[0105] A carbohydrate binding fragment of SEQ ID NO: 9 (for use as a retention moiety) may comprise anywhere between about 5, 6, 7, 8, 9 or 10 (consecutive or contiguous) amino acids to about 164 (consecutive or contiguous) amino acids from SEQ ID NO: 9. Suitable fragments may comprise about 11, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135 about 140, about 145, about 150, about 155, about 160 or about 163 (consecutive or contiguous) amino acids from SEQ ID NO: 9.

[0106] Without wishing to be bound by theory, where a modified molecule comprises a CBM67 as a retention moiety, retention of the molecule is brought about by binding between CBM67 and its target. As such, where a molecule is to be retained at a site known to express or comprise a CBM67 binding target, that molecule may comprise a CBM67. In this regard, CBM67 or a protein comprising, consisting essentially or consisting of SEQ ID NO: 9 may bind L-rhamnose. Accordingly, any fragment for use as a retention moiety may also bind L-rhamnose. SEQ ID NO: 9 is derived from the sequence deposited in the UniProt database under ID No: Q82PP4. This sequence is reproduced as SEQ ID NO: 10 below (SEQ ID NO: 9 appears as residues 132-296 - shaded in the sequence below):

[0107] SEQ ID NO: 10

[0108] MSALRVTSPS VEYVQRPLGL DAAHPRLSWP MASAAPGRRQ SAYQVRVASS AAGLSHPDVW DSGKVVSDDS VLVPYAGPPL KPRTRYFWSV RVWDADGGAS EWSAPSWWET GLMGASQWSA KWISAPAPLT E 111111111111®®^

[0109]

[0110] iiiiliiiiiliiiiiiM AANQLRHEFR LPHKKVSRAR LYATALGLYE AHLNGRRVGR DQLAPGWTDY RKRVQYQTYD VTSSVRPGAN ALAAYVAPGW YAGNVGMFGP HQYGERPALL AQLEVEYADG TSERITSGPD WRAASGPIVS ADLLSGETYD ARKETAGWTS PGFDDRAWLA VRGADNDVPE QIVAQVDGPV RIAKELPARK VTEPKPGVFV LDLGQNMVGS VRLRVSGDAG TTVRLRHAEV LNPDGTIYTA NLRSAAATDT YTLKGQGEET YEPRFTFHGF RYVEVTGFPG KPSTTSVTGR VMHTSAPFTF EFETNVPMLN KLHSNITWGQ RGNFLSVPTD TPARDERLGW TGDINVFAPT AAYTMESARF LTKWLVDLRD AQTSDGAFTD VAPAVGNLGN GVAGWGDAGV TVPWALYQAY GDRQVLADAL

[0111] 55740646-1 PSVHAWLRYL EKHSDGLLRP ADGYGDWLNV SDETPKDVIA TAYFAHSADL AARMATELGK DAAPYTDLFT RIRKAFQTAY VASDGKVKGD TQSAYVLTLS MNLVPDALRK AAADRLVALI EAKDWHLSTG FLGTPRLLPV LTDTGHTDVA YRLLHQRTFP SWGYPIDKGS TTMWERWDSI QPDGGFQTPE MNSFNHYAYG SVGEWMYANI AGIAPGRAGY RQVVIRPRPG GEVTSARATF ASLHGPVSTR WQQRSGGFVL TCSVPPNTTA EVWIPADHPD RVQHTHGTFV RAEDGCAVFE VGSGSHRFTV

[0112] SEQ ID NOS 9 and 10 are derived from Streptomyces avermitilis.

[0113] Accordingly, CBM67 may be selected for use as (or in) a retention moiety, where the site at which the molecule is to be retained, comprises or expresses any of the abovementioned CBM67 targets or ligands.

[0114] A CBM for use as a retention moiety may comprise one, two, three, four or more CBM67s.

[0115] A CBM for use as, or in, a retention moiety may comprise one, two, three, four or more proteins comprising SEQ ID NO: 9 or a carbohydrate binding fragment thereof.

[0116] One of skill will appreciate that CBM67s for use as retention moieties may comprise sequences which exhibit some degree (for example 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65% or 60%) of sequence identity or homology with the CBM67 Sequences of SEQ ID NOS: 9 and 10. All such variant or divergent sequences are to be embraced within. ) 6),. / ( / 2 ) 4(. ) ) 3 “ ” ( )4 / 2 4(. 3 2 CBM67 sequences may have carbohydrate binding function.

[0117] CBM70

[0118] A useful CBM70 (i.e. a CBM70 for use as a retention moiety) may be derived from any suitable source. For example, CBM70s for use as a retention moiety may be obtained from microorganisms, including, for example, bacteria of the genera Bacillus, Paenibacillus, Planctomycetes and Streptococcus. For example, a useful CBM70s may be obtained or derived from, for example, Streptomyces pneumoniae. Further details concerning potential sources and the structure and function of the CBM70 family can be found within the Carbohydrate Active Enzymes database (freely available on the internet at: http: / / www.cazv.org / CBM70.html)

[0119] 55740646-1 A retention moiety may comprise one, two, three, four or more CBM70(s).

[0120] An exemplary CBM70 sequence is provided by SEQ ID NO: 11 below:

[0121] SEQ ID NO: 11

[0122] NLVENGDFGQTEDGSSPWTGSKAQGWSAWVDQKNSADASTRVIEAKDGAITISSHEKLRAALHR MVPIEAKKKYKLRFKIKTDNKIGIAKVRI IEESGKDKRLWNSATTSGTKDWQTIEADYSPTLDV DKIKLELFYETGTGTVSFKDIELVEVADQLS

[0123] A carbohydrate binding fragment of SEQ ID NO: 11 may comprise anywhere between about 5, 6, 7, 8, 9 or 10 (consecutive or contiguous) amino acids to about 158 (consecutive or contiguous) amino acids from SEQ ID NO: 11. Suitable fragments may comprise about 11, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 155 or about 157 (consecutive or contiguous) amino acids from

[0124] SEQ ID NO: 11.

[0125] Where a molecule is to be retained at a site known to express or comprise the CBM70 binding target, the molecule may comprise CBM70. CBM70 or a protein comprising, consisting essentially or consisting of SEQ ID NO: 11 may bind or target hyaluronan. Accordingly, any fragment for use as a retention moiety may also bind hyaluronan. Accordingly, CBM70 may be selected for use as or in a retention moiety, where the site at which the molecule of the construct is to be retained comprises or expresses any of the abovementioned CBM70 targets or ligands.

[0126] SEQ ID NO: 11 is derived from the sequence deposited in the UniProt database under ID No: Q54873. This sequence is reproduced as SEQ ID NO: 12 below (SEQ ID NO: 11 appears as residues 54-212 - shaded in the sequence below):

[0127] SEQ ID NO: 12

[0128] 55740646-1 MQTKTKKLIV SLSSLVLSGF LLNHYMTIGA EETTTNTIQQ SQKEVQYQQR

[0129]

[0130] iigiiiligi iiiiilgiiii lliliiilill iigiigiggi liiiiiligi liiiiitiii: lililiiiii iliiiiiiii Biiiiiiii iilililiii iilililiii iliiiiiiii iiiiiiiiii iiiiiiliiii igEDSQTDKQ LEEKIDLPIG KKHVFSLADY TYKVENPDVA SVKNGILEPL KEGTTNVIVS KDGKEVKKIP LKILASVKDA YTDRLDDWNG IIAGNQYYDS KNEQMAKLNQ ELEGKVADSL SSISSQADRT YLWEKFSNYK TSANLTATYR KLEEMAKQVT NPSSRYYQDE TVVRTVRDSM EWMHKHVYNS EKSIVGNWWD YEIGTPRAIN NTLSLMKEYF SDEEIKKYTD VIEKFVPDPE HFRKTTDNPF KALGGNLVDM GRVKVIAGLL RKDDQEISST IRSIEQVFKL VDQGEGFYQD GSYIDHTNVA YTGAYGNVLI DGLSQLLPVI QKTKNPIDKD KMQTMYHWID KSFAPLLVNG ELMDMSRGRS ISRANSEGHV AAVEVLRGIH RIADMSEGET KQCLQSLVKT IVQSDSYYDV FKNLKTYKDI SLMQSLLSDA GVASVPRPSY LSAFNKMDKT AMYNAEKGFG FGLSLFSSRT LNYEHMNKEN KRGWYTSDGM FYLYNGDLSH YSDGYWPTVN PYKMPGTTET DAKRADSDTG KVLPSAFVGT SKLDDANATA TMDFTNWNQT LTAHKSWFML KDKIAFLGSN IQNTSTDTAA TTIDQRKLES GNPYKVYVND KEASLTEQEK DYPETQSVFL ESFDSKKNIG YFFFKKSSIS MSKALQKGAW KDINEGQSDK EVENEFLTIS QAHKQNRDSY GYMLIPNVDR ATFNQMIKEL ESSLIENNET LQSVYDAKQG VWGIVKYDDS VSTISNQFQV LKRGVYTIRK EGDEYKIAYY NPETQESAPD QEVFKKLEQA AQPQVQNSKE KEKSEEEKNH SDQKNLPQTG EGQSILASLG FLLLGAFYLF RRGKNN

[0131] SEQ ID NOS 11 and 12 are derived from Streptococcus pneumoniae.

[0132] A CBM for use as a retention moiety may comprise one, two, three, four or more CBM70s and / or one, two, three, four or more proteins comprising SEQ ID NO: 11 or a carbohydrate binding fragment thereof.

[0133] One of skill will appreciate that CBM70s for use as, or in, retention moieties may comprise sequences which exhibit some degree (for example 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65% or 60%) of sequence identity or homology with the CBM70 Sequences of SEQ ID NOS: 11 and 12. All such variant or divergent sequences are to be embraced with / 4. ) 6),. / ( / 2 ) 4(. ) ) 3 “ ” ( )4 Z 2 and / or homologous CBM70 sequences may have carbohydrate binding function.

[0134] In view of the above, a retention moiety of a construct of this disclosure may comprise one or more CBMs selected from the group consisting of:

[0135] (i) CBM family 32 (CBM32);

[0136] (ii) CBM family 40 (CBM40);

[0137] 55740646-1 (iii) CBM family 47 (CBM47);

[0138] (iv) CBM family 67 (CBM67);

[0139] (v) CBM family 70 (CBM70); and

[0140] (vi) A target binding fragment of any of (i)-(v).

[0141] In one teaching, the retention moiety may comprise a CBM40.

[0142] Accordingly, the disclosure provides a molecule for retention at a specific site, which molecule further comprises a CBM40. The CBM40 may be fused, bound, joined or conjugated to the molecule.

[0143] The disclosure also provides a construct comprising a molecule for retention at a specific site and a CBM40. The CBM40 may be fused, bound, joined or conjugated to the molecule.

[0144] Also described is a modified molecule, wherein said molecule is modified by the addition of a CBM40. Again, the CBM40 may be fused, bound, joined or conjugated to the molecule.

[0145] . ) ) 3 ‘3 3 3 ’ 3 )3 ) 4, / 4. / ) ) / 4) ( 6 / 2 / ) 4( which can be modified, augmented or otherwise fused, bound, joined, linked or conjugated to any of the retention moieties described herein.

[0146] . ) ) 3 ‘3 3 3 ’ 3 / 4 2 ( ), )C 363 3 3 3 B. / . 3 6 / ) 6 ) / 4 peptides (including bicyclic peptides, small affinity ligand (e.g. affibody) type molecules and designed ankyrin repeat proteins (Darpin)-type molecules) amino acids, nucleic acids, RNA, DNA, carbohydrates, fatty acids, monomers, polymers (including natural poylmers) and the like.

[0147] . ) ) 3 ‘3 3 3 ’ 3 / 4 2 ( ) ( 4( 6 (

[0148] The term molecule may include (for example) anti-inflammatory drugs, chemotherapeutic drugs, antibiotics, immunotherapy compounds and the like.

[0149] 55740646-1 The term molecule may comprise detectable (for example optically or chemically detectable) markers, including fluorescent compounds (e.g. fluorophores), chemiluminescent compounds, gold / silver nanoparticles, latex beads, magnetic beads and the like.

[0150] .

[0151]

[0152] ) ) 3 ‘3 3 ’ 3 36 / ) 4 4 / )4

[0153] . ) ) 3 ‘3 3 3 ’ 3 36 / ) / 4). ) 3 / 4)

[0154] . ) ) 3 ‘3 3 3 ’ 3 embrace an antibody. The term antibody may be used to embrace all antigen binding fragments and variants, including, for example, a single chain antibody, a monoclonal antibody, a chimeric antibody, a domain antibody, a nanobody, a camelid antibody or an antigen-binding fragment of any of these. The term ‘ 4 / ( ’ 3,. ) )4 36 bispecific antibodies and fragments and variants known as: ’ 2, monospecific, bispecific Fab2, trispecific Faba, monovalent, scFv, Bispecific diabody, trispecific diabody, scFv-Fc, minibody and the like. There are likely too may antibody variants to be listed here - but the general rule is that if that can be joined, bound, fused, linked or conjugated to any of the retention moieties described herein,. ) 4. ). ) / 4 2 ( ) ( B / . / 4. ) 6),. ) ) 3 ‘ 4 / ( ’ For simplicity, all of these antibody types B / 2 ) 2 / A) 2 ),) ) ( ‘ 4 / ( / ) ’

[0155] The term ‘molecule’ may not embrace an oligomerisation domain; these being domains which exhibit an ability to self-associate to form multimer structures, for example trimers.

[0156] 4 4) ), / 4. ) ) 3 ‘3 3 3' 3 4 3 6 / ) 4 oligomerisation domain derived from Pseudomonas aeruginosa 6 ) ( 3 / 4 / ( ). ) ) 3 ‘3 3 3 ’ 3 4 ) 3 ) a molecule comprising an amino acid sequence deposited under accession number PAO579 and reproduced below as SEQ ID NO: 13 (438 amino acids).

[0157] MNTYFDIPHR LVGKALYESY YDHFGQMDIL SDGSLYLIYR RATEHVGGSD GRVVFSKLEG GIWSAPTIVA QAGGQDFRDV AGGTMPSGRI VAASTVYETG EVKVYVSDDS GVTWVHKFTL ARGGADYNFA HGKSFQVGAR YVIPLYAATG VNYELKWLES SDGGETWGEG STIYSGNTPY NETSYLPVGD GVILAVARVG SGAGGALRQF ISLDDGGTWT DQGNVTAQNG DSTDILVAPS LSYIYSEGGT PHVVLLYTNR TTHFCYYRTI LLAKAVAGSS GWTERVPVYS APAASGYTSQ VVLGGRRILG NLFRETSSTT SGAYQFEVYL GGVPDFESDW FSVSSNSLYT LSHGLQRSPR RVVVEFARSS SPSTWNIVMP SYFNDGGHKG SGAQVEVGSL NIRLGTGAAV WGTGYFGGID NSATTRFATG YYRVRAWI

[0158] 55740646-1 The oligomerisation domain of SEQ ID NO: 13 is from amino acid residue 333 to 438 -this sequence may be SEQ ID NO: 14.. ) ) 3 ‘3

[0159]

[0160] ’ 3 4 comprise an amino acid sequence comprising SEQ ID NO: 14.

[0161] In view of the above, this disclosure provides

[0162] an antibody for retention at a site of interest, said antibody comprising a retention moiety which facilitates retention of the antibody at the site.

[0163] The disclosure also provides a modified antibody, wherein said antibody has been modified by the addition of a retention moiety. In one teaching, the antibody is modified by the addition of a plurality of retention moieties.

[0164] The disclosure also provides constructs, which constructs comprise an antibody for retention at a site and a retention moiety.

[0165] In one teaching, the antibody for retention at a site, the modified antibody or construct comprising an antibody, may comprise a plurality of retention moieties - e.g. two, three, four, five, six, seven, eight or more retention moieties.

[0166] In one teaching, the retention moiety (or retention moieties) of an antibody for retention at a site, the modified antibody or construct comprising an antibody, may comprise a a CBM, for example a CBM40.

[0167] In one teaching the antibody for retention at a site, the modified antibody or construct comprising an antibody, may be fused, joined, bound, conjugated or linked to a retention moiety (for example a CBM) via or at the Fc region. Additionally, or alternatively, an antibody may comprise a CBM fused, bound, linked or conjugated to an exposed carboxy terminus. For example, and where the antibody comprises two or more heavy chains, a retention moiety (for example a CBM) may be fused, bound, linked or conjugated to the exposed carboxy terminus of one or both heavy chain(s).

[0168] In one example, the retention moiety may be joined, bound, fused, linked or conjugated to a CH2 or CH3 domain of a heavy chain of the antibody. In one example, each heavy chain of the antibody, such as the CH2 or CH3 domain of each heavy chain, may be joined, bound, fused, linked or conjugated to the retention moiety. The retention moiety

[0169] 55740646-1 and the antibody may be joined, bound, fused, linked or conjugated via one or more peptide linker(s) / conjugation peptide(s) detailed herein.

[0170] In a specific example, the retention moiety may be joined, bound, fused, linked or conjugated to the C-terminus of a heavy chain of the antibody, such as via one or more peptide linker(s) / conjugation peptide(s) detailed herein. The retention moiety may be joined, bound, fused, linked or conjugated to the CH3 domain of a heavy chain of the antibody. The C-terminus of the heavy chain of the antibody, such as the CH3 domain of the heavy chain of the antibody, may comprise one or more peptide linker(s) / conjugation peptide(s). The N-terminus of the retention moiety may comprise one or more peptide linker(s) / conjugation peptide(s).

[0171] Within the context of any of the disclosed antibodies for retention at a site, modified antibodies or constructs comprising an antibody, the fused, joined, bound or conjugated to one or more (for example two) the retention moiety / moieties (each retention moiety comprising, for example, a CBM) may exhibit improved or increased affinity for its target. For example, where the retention moiety / moieties comprise(s) a CBM, the (or each) CBM may exhibit increased affinity for its carbohydrate, glycan and / or sialic acid target. Any such improvement or increase in retention moiety target affinity may be as compared to the affinity of the retention moiety for its target when not fused, joined, bound or conjugated to an antibody.

[0172] In one teaching, the antibody may not be an anti-CBM antibody.

[0173] As stated, a useful retention moiety may comprise one or more CBMs, such as one, two, three, four, five or six CBMs, for example. The retention moiety may comprise two or more CBMs. In one example, the retention moiety may comprise two CBMs. In one example, the retention moiety may comprise four CBMs. When there are two or more CBMs, the CBMs may be joined, linked or fused to each other by a linker, such as a short peptide sequence. Linkers which may be used to join, link or fuse two or more CBMs will be known to the skilled person in the art. By way of example only, a linker may comprise or may be selected from the group consisting of:

[0174] GSSGSA (SEQ ID NO: 15);

[0175] GGGS (SEQ ID NO: 16);

[0176] LQALG (SEQ ID NO: 17);

[0177] 55740646-1 GGNSG (SEQ ID NO: 18); or

[0178] GGGSG (SEQ ID NO: 19).

[0179] In one teaching, the retention moiety of any of the aspects, examples or teachings disclosed herein may not comprise an oligomerisation domain.

[0180] In one teaching, the antibody may be an anti-viral antibody, such as an antihemagglutinin antibody, for example.

[0181] . ) ) 3 ‘ / ) ’ / 4 ‘ / ), / 4 ) ) ’ 3 ) 3 ) 4 / ) ) 4 which, or in which, a molecule of this disclosure (e.g. an antibody) may be (or is to be) retained.. ) ) 3 ‘ / )’ 3 )3 ) 4 6), / ) ) 4 B. / . construct of this disclosure can be administered or applied.

[0182] In one teaching, the site may comprise epithelial cells.

[0183] In one teaching, the site may comprise mucosal epithelial cells, a mucosal surface or tissue.

[0184] In one teaching, the site may comprise human nasal epithelium tissue.

[0185] In a further teaching, the site may comprise a mucosal surface, for example the nasal passages or lung respiratory airways.

[0186] The site may be present in a sample which has been provided or obtained from a subject - for example a human or animal subject. The sample may take the form of a biopsy comprising any of the sites of interest described herein.

[0187] The present disclosure also provides a composition, for example a pharmaceutical composition, comprising any of the molecules for retention, modified molecules or constructs disclosed herein. In one example, a pharmaceutical composition may comprise a molecule for retention, modified molecule or construct of this disclosure and a pharmaceutically acceptable carrier, excipient or diluent. A pharmaceutical composition of this disclosure may be sterile.

[0188] 55740646-1 Also provided is a method of increasing the retention or residence time of a molecule (for example a drug or an antibody) at (or in) a site of interest, wherein said molecule is either (i) modified to comprise any of the retention moieties described herein or (ii) bound, linked, conjugated, fused or joined to any of the retention moieties described herein; the resulting molecule for retention, modified molecule or construct may then be administered to (or contacted with) the site of interest. A method of this type may be performed as an in vitro method, whereby a molecule for retention, modified molecule or construct of this disclosure is administered, contacted or applied to / with a sample, for example a biopsy, which sample represents or comprises the site of interest. By way of example, the sample may comprise a sample of mucosal tissue - but it may comprise or represent any site of interest described herein.

[0189] In a further aspect, the disclosure provides the use of any of the retention moieties described herein (for example the various described CBMs, including members of CBM40) for retaining a molecule at a site (of interest) or in a method (including in vivo methods) of retaining a molecule at a site of interest.

[0190] In another aspect, this disclosure provides a variety of uses for the molecules for retention, modified molecules and / or constructs described herein. One of skill will appreciate that the specific use will depend on the nature of the molecule; for example, where the molecule to be retained is a drug, modification to include a retention moiety (as described herein) may result in a drug for retention, a modified drug or a drug construct which may be used in the treatment or prevention of those conditions / diseases and / or syndromes that the drug is intended for. Where the molecule comprises an antigen, the may be for use in raising an immune response in a subject and / or for modulating a host immune response. Of course (as stated), an advantage associated with the technical detail of this disclosure is that it enables the retention of molecules at a site of interest, wherein the retention time (is longer as compared to the retention time of the same molecule when administered to the same site of interest without use of a retention moiety.

[0191] In this regard, the disclosure provides molecules comprising retention moieties (as described herein):

[0192] (i) for use in therapy or for use as a medicament;

[0193] 55740646-1 (ii) for use in (a method of) modulating an immune response (in human or animal subjects);

[0194] (iii) for use in (a method of) raising an immune response (for example a protective immune response) in a subject or host (for example a human or animal subject / host);

[0195] (iv) for use in modulating cell growth, proliferation and / or differentiation; (v) for use as an adjuvants and / or to promote or augment an immune response to an antigen;

[0196] (vi) for use as an anti-infective;

[0197] (v) for use in treating or preventing a viral infection (e.g. an influenza infection); and

[0198] (vi) in the treatment of cancer.

[0199] DETAILED DESCRIPTION

[0200] The present disclosure will now be further described by way of example and with reference to the Figures, which show:

[0201] Figure 1. Schematic illustration of PTH22 sdAb and PTH22-SpCBM Constructs with 6xHIS tag at N-terminus.

[0202] Figure 2. Small-scale test expression of soluble fractions. Lane 1 / 7: Protein standard; Lane 2: PTH22 sdAb; Lane 6: PTH22-SpCBM; Lane 8: Sp2 control; Please ignore the rest lanes.

[0203] Figure 3. A. SDS-Gel stained with Coomassie Blue. Lane 1: Protein Marker; Lane 2: PTH22 sdAb; Lane 3. PTH22-SpCBM; Lane 4. Sp2CBMTD. B. Western Blot detected using anti-HIS-HRP antibody.

[0204] Figure 4. Results of ELISA experiments. A. PTH22-SpCBM bindings to SA similarly as Sp2CBMTD; B. PTH22-SpCBM binding to SA while PTH22 sdAb is not binding to SA; C. PTH-SpCBM and PTH22 sdAb bind to peptide target.

[0205] Figure 5. Titration of SP-CBM-Nanobody fusion on A549 Cells

[0206] Figure 6. Titration of SP-CBM, SP-CBM--Nanobody or Nanobody alone on A549 cells (anti-SP-CBM).

[0207] Figure 7. Schematic illustration of PTH22 sdAb and PTH22-VcCBM Constructs with 6xHIS tag at N-terminus.

[0208] Figure 8. Purification of new Nb-SVC-2c construct.

[0209] 55740646-1 Figure 9. Results of ELISA experiments. Binding of purified VcCBM conjugates were evaluated using (A) streptavidin-coated plate and anti-Vc2 antibody; (B) high-binding plate and anti-Vc2 antibody; (C) high-binding plate and anti-His-HRP antibody.

[0210] Figure 10. Titration of Vc2, Nano-Vc2 Fusion and Nanobody Alone on A549 cells (detection by anti-Vc2 antibody).

[0211] Figure 11. Titration of Vc2, Nano-Vc2 Fusion and Nanobody Alone on A549 cells (detection by anti-6His-HRP antibody).

[0212] Figure 12. Conjugation of anti-HA antibody to Vc2 or Vc4. Conjugated proteins analysed pre and post gel filtration under reducing conditions (in the presence of DTT) by SDS PAGE. Gels stained with Coomasie and purity estimated by densitometry.

[0213] Figure 13. Binding of SVc2 and SVc4 monoclonal antibody conjugates to cells. (A) Titration of SVc2-mAB conjugate on MDCK cells (detection with anti-Vc). (B) Titration of SVc2-mAB conjugate on MDCK cells (detection with anti-IgG). (C) Titration of SVc4-mAB conjugate on MDCK cells (detection with anti-Vc). (D) Titration on SVc4-mAB conjugate on MDCK cells (detection with anti-IgG).

[0214] Figure 14. Binding of SVc2 and SVc4 monoclonal antibody conjugates to sialic acid. (A) Titration of SVc2-3 40 ) 4 ’ ( ) ) / 4 BZ. 4 Z-Vc). (B) Titration of SVc4- 3 _ 40 ) 4 ’ ( ) ) Z 4 BZ. 4 Z-Vc). (C) Titration fo SVc4-mAB conjugate on ( ) ) Z 4 BZ. 4 Z-IgG).

[0215] EXAMPLES

[0216] Design of CBM conjugate molecules

[0217] PTH22 is a parathyroid hormone (PTH) binding single domain antibody (sdAb); PTH22 was isolated via bio-panning of a naive llama VHH phage display library [1] against a peptide corresponding to the N-terminal 31 PTH amino acids (referred as BioBAST) [2], The seguence of PTH22 and BioBAST peptide was adapted from previous publications and expired patents [3-5],

[0218] The strategies to generate sdAb and sdAb-SpCBM molecules:

[0219] • Synthesized DNA fragments encoding PTH22 or PTH22-SpCBM were ordered commercially

[0220] • The synthesized fragments were cloned into pEHISTEV vector to create a construct designated as PTH22 sdAb or PTH22-SpCBM

[0221] DNA_PTH22_sdAb (SEQ ID NO: 20) ACAGCCATGGCTGAAGTTCAGCTGCAGGCAAGCGGTGGTGGTCTGGTTCAGCCTGGTGGT AGCCTGCGTCTGAGCTGTGCAGCCAGCGGTAGCCTGAGCCGTATTACCGTTATGGGTTGG TATCGTCAGGCACCGGGTAAACAGCGTGAACTGGTTGCAATTATTACCAGCAGCGGTGGC

[0222] 55740646-1 ACCGATTATGCAGATAGCGTTAAAGGTCGTTTTACCATCAGCAAAGATAATGCAAAAGCC CTGATGTATCTGCAAATGACCAGTCTGCGTCCGGAAGATACCGCAGTGTATTATTGTGTT GGTAAAAGCCGTGATAGCGCAGGTCTGAGTTGGGATTATTGGGGTCAGGGCACCCAGGTT ACCGTTAGCAGCTAACTCGAGTCGTA

[0223] Protein_PTH22_sdAb (SEQ ID NO: 21) TAilgillilligl^

[0224]

[0225] iiiiiiiiiiiitiiiiiiiii® LES DNA_PTH22-SpCBM (SEQ ID NO: 22) CCTGGGGGAAATTCCCTCTAGAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGAGCT ATTATCATCATCACCATCATCATGGTGGTGGCGGTAGCGAAGTTCAGCTGCAGGCAAGCGGTGG TGGTCTGGTTCAGCCTGGTGGTAGCCTGCGTCTGAGCTGTGCAGCCAGCGGTAGCCTGAGCCGT ATTACCGTTATGGGTTGGTATCGTCAGGCACCGGGTAAACAGCGTGAACTGGTTGCAATTATTA CCAGCAGCGGTGGCACCGATTATGCAGATAGCGTTAAAGGTCGTTTTACCATCAGCAAAGATAA TGCAAAAGCCCTGATGTATCTGCAAATGACCAGTCTGCGTCCGGAAGATACCGCAGTGTATTAT TGTGTTGGTAAAAGCCGTGATAGCGCAGGTCTGAGTTGGGATTATTGGGGTCAGGGCACCCAGG TTACCGTTAGCAGCGAAGGTAAAAGCAGCGGTAGTGGTAGCGAAAGCAAAAGCACCGTTATTGA AAAAGAAGATGTTGAAACCAACGCCAGCAATGGTCAGCGTGTTGATCTGAGCAGCGAACTGGAT AAACTGAAAAAGCTGGAAAATGCCACCGTGCACATGGAATTTAAACCGGATGCCAAAGCACCAG CCTTTTATAACCTGTTTAGCGTGAGCAGCGCAACCAAAAAGGATGAATATTTCACAATGGCCGT GTATAATAACACCGCAACACTGGAAGGTCGTGGTTCAGATGGTAAACAGTTCTATAACAACTAT AACGACGCACCGCTGAAAGTTAAACCTGGTCAGTGGAATAGCGTTACCTTTACCGTTGAAAAAC CGACCGCAGAACTGCCGAAAGGTCGTGTTCGTCTGTATGTTAATGGTGTTCTGAGTCGTACCAG CCTGCGTAGCGGCAATTTCATTAAAGATATGCCGGATGTTACCCATGTTCAGATTGGTGCAACC AAACGTGCAAATAATACCGTTTGGGGTAGCAACCTGCAGATTCGTAATCTGACCGTTTATAATC GTGCACTGACACCGGAAGAAGTTCAGAAACGTAGCTAACTCGAGCACCACCCCAC

[0226] Protein_PTH22-SpCBM (SEQ ID NO: 23)

[0227] PGGNSL-NNFV-L- E G D I H|ggg||||||g^^ iiiiiiiiiiiiiM

[0228]

[0229] liiill|l|iili|ilil|iiiiBiii|if|iiii|ii|||GSDGKQFYNNYNDAPLKVKPGQWNS VTFTVEKPTAELPKGRVRLYVNGVLSRTSLRSGNFIKDMPDVTHVQIGATKRANNTVWGSNLQI RNLTVYNRALTPEEVQKRS-LEHHP

[0230] • 6xHIS tag was introduced at the N-term of PTH22 sdAb or PTH22-SpCBM to facilitate purification and characterization (Figure 1)

[0231] Cloning, Expression and Purification

[0232] 55740646-1 • Synthesized DNA fragments encoding PTH22 or PTH22-SpCBM was ordered commercially;

[0233] • The synthesized fragments were cloned into pEHISTEV vector to created construct designated as PTH22 sdAb or PTH22-SpCBM (Table 1).

[0234] • All constructs were propagated in E. coli cells, and constructs were verified by DNA sequencing, before transforming the expression host E. coli (DE3) for protein production.

[0235] • The crude soluble fraction was directly separated by SDS-PAGE. 16kDa overexpressed band in Lane 2 and 36.5kDa overexpressed band in Lane 4 was corresponding to the expected MW of PTH22 sdAb or PTH22-SpCBM (Figure 2);

[0236] SDS-PAGE and Western blot

[0237] • The expressed proteins were purified as previously described [6], Briefly, E. coli cells containing either His-tagged PTH22 or PTH22-SpCBM were lysed, and then clarified lysates were applied onto a HisTrap HP column, precharged with nickel (GE Healthcare) before elution of histidine-tagged CBMs using PBS containing 0.3 M NaCI and 250 mM imidazole. Both proteins were purified further using size-exclusion chromatography in PBS.

[0238] • Protein purity and size were verified by 12% (wt / vol) SDS / PAGE and then Western Blot using anti-HIS-HRP antibody for detection (Figure 3).

[0239] • Protein purity and size were verified by 12% (wt / vol) SDS / PAGE and then Western Blot using anti-HIS-HRP antibody for detection (Figure 3).

[0240] ELISA

[0241] The aim of characterization is to demonstrate

[0242] • After conjugated with SpCBM, PTH22 retains its target specificity (Figure 4. C) • Conjugation PTH22 with SpCBM, allows PTH22 binding to sialic acids (Figure 4.

[0243] B)

[0244] • Conjugation PTH22 with SpCBM is not affecting the binding property of SpCBM to sialic acids (Figure 4. A)

[0245] The experiment used ELISA to evaluate binding, using:

[0246] • anti-Sp2 antibody - to demonstrate Sp2 retain its integrity after conjugation with PTH22 (Figure 4. A)

[0247] • anti-His HRP-conjugated antibody - to allow comparison the binding properties of PTH22 sdAb and PTH22-SpCBM to sialic acids (Figure 4. B)

[0248] • Streptavidin-HRP conjugated to detect the biotin labelled BAST17-31 peptide (target antigen for PTH22 sdAb) (Figure 4. C).

[0249] 55740646-1 Cell

[0250]

[0251] Performed to determine ability of PTH22-SpCBM fusion to bind to cells

[0252] • A549 cells (Lung epithelium) seeded onto 2 x 96 well tissue culture plates (Greiner 655-180) and grown overnight at 37°C, 5% CO2 to approximately 95% confluence, and after washing in 1x PBS (GIBCO 70013-016) and fixed in 10% formalin solution

[0253] • Plates blocked with 5% non-fat dried milk (Marvel) in PBS (200pL / well, 1 hour at room temp)

[0254] • Cells washed 2x with PBS (230pL / well)

[0255] • Cells incubated with proteins below, diluted in PBS from 100pg / mL to 0.16pg / mL (5-fold dilutions) (50pL / well, 1 hour at room temp)

[0256] o Purified PTH22 sdAb only (contains 6 His tag) (AT20240208) o Purified PTH22-SpCBM fusion (contains 6His tag) (AT20231221) o Purified SPCBM only (JP20170113)

[0257] • Cells washed 3x with PBS (230pL / well)

[0258] • Cells incubated with anti-His-HRP (Merck, A7058) or anti-SP-CBM (Pneumagen ZGB23011) both diluted 1 / 10000 in 0.5% NFDM-PBS (50pL / well, 1 hour at room temp)

[0259] • Cells washed 3x with PBS (230pL / well)

[0260] • Anti-SP-CBM antibody followed by anti-rabbit IgG Fc-HRP (Merck AP156P) diluted 1 / 20000 in 0.5% NFDM-PBS (50pL / well, 1 hour at room temp)

[0261] • Cells washed 3x with PBS (230pL / well)

[0262] • TMB (Thermo, 34028) used for colorimetric detection (50pL / well, 6 minutes) on the Spectramax M2 plate reader (Molecular Devices) at 450 / 620nm Results

[0263] • Anti-His antibody detected SP-CBM bound to A549 cells, but not nanobody or SP-CBM alone (Figure 5). This indicates that nanobody itself does not bind to the cells and that the SP-CBM is not recognised by the anti-His antibody, so the titrated signal has been generated by binding of the fusion protein to the cells.

[0264] • Anti-SP-CBM antibody detected both the SP-CBM and the SP-CBM-Nanobody fusion bound to the A549 cells (Figure 6), but not the Nanobody alone EXAMPLE 2

[0265] Design of CBM conjugate molecules

[0266] The strategies to generate sdAb and sdAb-VcCBM molecules were as follows:

[0267] 55740646-1 • Synthesized DNA fragments encoding PTH22 or PTH22-VcCBM was ordered commercially.

[0268] • The synthesized fragments were cloned into pEHISTEV vector to created construct designated as PTH22 sdAb or PTH22-VcCBM

[0269] • 6xHIS tag was introduced at the N-terminus of PTH22 sdAb or PTH22-VcCBM to facilitate purification and characterization (Figure 7).

[0270] Cloning the new Nb-sVc constructs

[0271] • Synthesized DNA fragments encoding Nb-sVcs was ordered commercially.

[0272] • Synthesized fragments were cloned into pEHis-TEV vector to created constructs Nb-sVc-2c and Nb-sVc-3.

[0273] Nb-sVc-2c

[0274] (SEQ ID NO: 24) M

[0275]

[0276] SYYiiil|iGGGGS|iiiiiiiiGGLVQPGGSLRLSCAASGSLSRITVMGWYRQAPGK QRELVAI ITSSGGTDYADSVKGRFTISKDNAKALMYLQMTSLRPEDTAVYYCVGKSRD SAGLSWDYWGQGTOVTVSSEGKSSGSGSESKSTALFDYNATGDTEFDSPAKQGWMQDN TNNGSGVLTNADGMPAWLVQGIGGRAQWTYSLSTNQHAQASSFGWRMTTEMKVLSGGM ITNYYANGTQRVLPI ISLDSSGNLVVEFEGQTGRTVLATGTAATEYHKFELVFLPGSN PSASFYFDGKLIRDNIQPTASKQNMIVWGNGSSNTDGVAAYRDIKFEIQGD

[0277] • Constructs were verified by DNA sequencing.

[0278] • Expression cells have been transformed with constructs of interests.

[0279] Construct Construct Ligation Seq Expression Soluble Purification ID Name Positive fraction

[0280] 2c Nb-sVc-2c

[0281] 3 Nb-sVc-3 -

[0282]

[0283] ELISA

[0284] The aim of characterization was to demonstrate:

[0285] • Conjugation PTH22 with VcCBM is not affecting the integrity of both proteins. • Conjugation PTH22 with VcCBM, allows PTH22 binding to sialic acids.

[0286] • Conjugation PTH22 with VcCBM is not affecting the binding property of VcCBM to sialic acids.

[0287] The experiment used ELISA to evaluate binding, using:

[0288] • anti-Vc antibody - to demonstrate Vc retain its integrity after conjugation with PTH22 (Figure 9A and 9B).

[0289] • anti-His HRP-conjugated antibody - to allow comparison the binding properties of PTH22 sdAb and PTH22-VcCBM to sialic acids (Figure 9C).

[0290] 55740646-1 Titration of Nano-Vc2 fusion on A549 Cells

[0291] Titration assay was performed to determine ability of Nano-Vc2 fusion to bind to cells:

[0292] • A549 cells seeded at 2.5x105 cells / mL and grown overnight to confluence in 2 x 96 well plates.

[0293] • Cells fixed with 10% formalin solution.

[0294] • Plates block with 5% NFDM in PBS.

[0295] • Cells treated with each antigen diluted in PBS

[0296] • Nano only;

[0297] • Nano-Vc2;

[0298] • Vc2 protein only.

[0299] • Plate 1: anti-Vc 500nM-0.16 nM (5 fold dilutions).

[0300] • Plate 2: anti-6His 5000nM- 1,6nM (5 fold dilution)

[0301] • Bound protein detected using:

[0302] • Plate 1: anti-Vc2 (1 / 1000 in 0.5% NFDM-PBS) followed by anti-rabbit- HRP (1 / 10000);

[0303] • Plate 2: anti-His-HRP (1 / 1000 in in 0.5% NFDM-PBS);

[0304] • Plates developed with TMB for 5 minutes then stopped with 1M HCL • Note: all constructs contain a 6His tag

[0305] Results

[0306] • Using an anti-Vc2 antibody, the Nb-Vc2 and Vc2 were detected to similar degrees, but not the Nb alone, indicating that the fusion does not affect the ability of the Vc2 to bind to cells (Figure 10).

[0307] • Using an anti-His antibody, only the Vc2 was recognised, not the Nb-Vc2 fusion or the Nb alone. Unclear why the Nb-Vc2 not recognised (Figure 11 ).

[0308] EXAMPLE 3

[0309] Exemplary CBM-antibodv coniuqates

[0310] The sialic acid binding domain (CBM) derived from Vibrio cholerae sialidase was engineered such that either two or four domains were linked tandemly and conjugated to another protein (SVc2 and SVc4). An A / -terminal 6His tag was also included in the constructs to aid purification.

[0311] SVc2 and SVc4 described above are shown using a schematic representation below (6His tag is omitted from the representation):

[0312] SVc2

[0313] [Antibody]— [Vc1]-[Vc2]

[0314] 55740646-1 )6 ) )4 24 ) which may comprise any of the following short peptide sequences and which may be present between the domains.

[0315] Linker: GGTAGCAGTGGTAGCGCC (SEQ ID NO: 25)

[0316] Vcl ATGGCACTTTTTGACTATAACGCAACGGGTGACACTGAGTTTGACAGTCCAGCCAAACAGGGAT GGATGCAAGACAACACGAATAATGGCAGCGGCGTTTTAACCAATGCAGATGGAATGCCCGCTTG GTTGGTGCAAGGTATTGGAGGGAGAGCTCAATGGACATATTCTCTCTCTACTAATCAACATGCC CAAGCATCAAGTTTCGGTTGGCGAATGACGACAGAAATGAAAGTGCTCAGTGGTGGAATGATCA CAAACTACTACGCCAACGGCACTCAGCGTGTCTTACCCATCATTTCATTAGATAGCAGTGGTAA CTTAGTTGTTGAGTTTGAAGGGCAAACTGGACGCACCGTTTTGGCAACCGGCACAGCAGCAACG GAATATCATAAATTTGAATTGGTATTCCTTCCTGGAAGTAACCCATCCGCTAGCTTTTACTTCG ATGGCAAACTCATTCGTGACAACATCCAGCCGACTGCATCAAAACAAAATATGATCGTATGGGG GAATGGCTCATCAAATACGGATGGTGTCGCCGCTTATCGTGATATTAAGTTTGAAATTCAAGGC GAC (SEQ ID NO: 26)

[0317] Linker: GGTGGTGGATCCGGT (SEQ ID NO: 27 )

[0318] Vc2 GCACTTTTTGACTATAACGCAACGGGTGACACTGAGTTTGACAGTCCAGCCAAACAGGGATGGA TGCAAGACAACACGAATAATGGCAGCGGCGTTTTAACCAATGCAGATGGAATGCCCGCTTGGTT GGTGCAAGGTATTGGAGGGAGAGCTCAATGGACATATTCTCTCTCTACTAATCAACATGCCCAA GCATCAAGTTTCGGTTGGCGAATGACGACAGAAATGAAAGTGCTCAGTGGTGGAATGATCACAA ACTACTACGCCAACGGCACTCAGCGTGTCTTACCCATCATTTCATTAGATAGCAGTGGTAACTT AGTTGTTGAGTTTGAAGGGCAAACTGGACGCACCGTTTTGGCAACCGGCACAGCAGCAACGGAA TATCATAAATTTGAATTGGTATTCCTTCCTGGAAGTAACCCATCCGCTAGCTTTTACTTCGATG GCAAACTCATTCGTGACAACATCCAGCCGACTGCATCAAAACAAAATATGATCGTATGGGGGAA TGGCTCATCAAATACGGATGGTGTCGCCGCTTATCGTGATATTAAGTTTGAAATTCAAGGCGAC

[0319] (SEQ ID NO: 28 )

[0320] The corresponding amino acid sequence may comprise:

[0321] Linker: GSSGSA (SEQ ID NO: 15 )

[0322] VC1 MALFDYNATGDTEFDSPAKQGWMQDNTNNGSGVLTNADGMPAWLVQGIGGRAQWTYSLSTNQHA QASSFGWRMTTEMKVLSGGMITNYYANGTQRVLPIISLDSSGNLWEFEGQTGRTVLATGTAAT EYHKFELVFLPGSNPSASFYFDGKLIRDNIQPTASKQNMIVWGNGSSNTDGVAAYRDIKFEIQG

[0323] D (SEQ ID NO: 29)

[0324] Linker: GGGSG (SEQ ID NO: 19

[0325] VC2

[0326] 55740646-1 ALFDYNATGDTEFDSPAKQGWMQDNTNNGSGVLTNADGMPAWLVQGIGGRAQWTYSLSTNQHAQ ASSFGWRMTTEMKVLSGGMITNYYANGTQRVLPI ISLDSSGNLVVEFEGQTGRTVLATGTAATE YHKFELVFLPGSNPSASFYFDGKLIRDNIQPTASKQNMIVWGNGSSNTDGVAAYRDIKFEIQGD

[0327] (SEQ ID NO: 30 )

[0328] SVc4

[0329] [Antibody]— [Vc1]-[Vc2]-[Vc3]-[Vc4]“-” )6 ) )4 24 ) B. / . 3 36 / ) 4, the following short peptide sequences and which may be present between the domains. Linker: GGTAGCAGTGGTAGCGCC (SEQ ID NO: 25)

[0330] VC1:

[0331] ATGGCACTTTTTGACTATAACGCAACGGGTGACACTGAGTTTGACAGTCCAGCCAAACAGGGAT GGATGCAAGACAACACGAATAATGGCAGCGGCGTTTTAACCAATGCAGATGGAATGCCCGCTTG GTTGGTGCAAGGTATTGGAGGGAGAGCTCAATGGACATATTCTCTCTCTACTAATCAACATGCC CAAGCATCAAGTTTCGGTTGGCGAATGACGACAGAAATGAAAGTGCTCAGTGGTGGAATGATCA CAAACTACTACGCCAACGGCACTCAGCGTGTCTTACCCATCATTTCATTAGATAGCAGTGGTAA CTTAGTTGTTGAGTTTGAAGGGCAAACTGGACGCACCGTTTTGGCAACCGGCACAGCAGCAACG GAATATCATAAATTTGAATTGGTATTCCTTCCTGGAAGTAACCCATCCGCTAGCTTTTACTTCG ATGGCAAACTCATTCGTGACAACATCCAGCCGACTGCATCAAAACAAAATATGATCGTATGGGG GAATGGCTCATCAAATACGGATGGTGTCGCCGCTTATCGTGATATTAAGTTTGAAATTCAAGGC GAC (SEQ ID NO: 26)

[0332] Linker: GGTGGTGGATCC (SEQ ID NO: 32 )

[0333] VC2:

[0334] ATGGCACTTTTTGACTATAACGCAACGGGTGACACTGAGTTTGACAGTCCAGCCAAACAGGGAT GGATGCAAGACAACACGAATAATGGCAGCGGCGTTTTAACCAATGCAGATGGAATGCCCGCTTG GTTGGTGCAAGGTATTGGAGGGAGAGCTCAATGGACATATTCTCTCTCTACTAATCAACATGCC CAAGCATCAAGTTTCGGTTGGCGAATGACGACAGAAATGAAAGTGCTCAGTGGTGGAATGATCA CAAACTACTACGCCAACGGCACTCAGCGTGTCTTACCCATCATTTCATTAGATAGCAGTGGTAA CTTAGTTGTTGAGTTTGAAGGGCAAACTGGACGCACCGTTTTGGCAACCGGCACAGCAGCAACG GAATATCATAAATTTGAATTGGTATTCCTTCCTGGAAGTAACCCATCCGCTAGCTTTTACTTCG ATGGCAAACTCATTCGTGACAACATCCAGCCGACTGCATCAAAACAAAATATGATCGTATGGGG GAATGGCTCATCAAATACGGATGGTGTCGCCGCTTATCGTGATATTAAGTTTGAAATTCAAGGC GAC (SEQ ID NO: 26)

[0335] Linker: CTGCAAGCTTTGGGA (SEQ ID NO: 32 )

[0336] VC3 ATGGCACTTTTTGACTATAACGCAACGGGTGACACTGAGTTTGACAGTCCAGCCAAACAGGGAT GGATGCAAGACAACACGAATAATGGCAGCGGCGTTTTAACCAATGCAGATGGAATGCCCGCTTG GTTGGTGCAAGGTATTGGAGGGAGAGCTCAATGGACATATTCTCTCTCTACTAATCAACATGCC

[0337] 55740646-1 CAAGCATCAAGTTTCGGTTGGCGAATGACGACAGAAATGAAAGTGCTCAGTGGTGGAATGATCA CAAACTACTACGCCAACGGCACTCAGCGTGTCTTACCCATCATTTCATTAGATAGCAGTGGTAA CTTAGTTGTTGAGTTTGAAGGGCAAACTGGACGCACCGTTTTGGCAACCGGCACAGCAGCAACG GAATATCATAAATTTGAATTGGTATTCCTTCCTGGAAGTAACCCATCCGCTAGCTTTTACTTCG ATGGCAAACTCATTCGTGACAACATCCAGCCGACTGCATCAAAACAAAATATGATCGTATGGGG GAATGGCTCATCAAATACGGATGGTGTCGCCGCTTATCGTGATATTAAGTTTGAAATTCAAGGC GAC (SEQ ID NO: 26)

[0338] Linker: GGAGGGAATTCGGGA (SEQ ID NO: 33)

[0339] VC 4 ATGGCACTTTTTGACTATAACGCAACGGGTGACACTGAGTTTGACAGTCCAGCCAAACAGGGAT GGATGCAAGACAACACGAATAATGGCAGCGGCGTTTTAACCAATGCAGATGGAATGCCCGCTTG GTTGGTGCAAGGTATTGGAGGGAGAGCTCAATGGACATATTCTCTCTCTACTAATCAACATGCC CAAGCATCAAGTTTCGGTTGGCGAATGACGACAGAAATGAAAGTGCTCAGTGGTGGAATGATCA CAAACTACTACGCCAACGGCACTCAGCGTGTCTTACCCATCATTTCATTAGATAGCAGTGGTAA CTTAGTTGTTGAGTTTGAAGGGCAAACTGGACGCACCGTTTTGGCAACCGGCACAGCAGCAACG GAATATCATAAATTTGAATTGGTATTCCTTCCTGGAAGTAACCCATCCGCTAGCTTTTACTTCG ATGGCAAACTCATTCGTGACAACATCCAGCCGACTGCATCAAAACAAAATATGATCGTATGGGG GAATGGCTCATCAAATACGGATGGTGTCGCCGCTTATCGTGATATTAAGTTTGAAATTCAAGGC GAC (SEQ ID NO: 25)

[0340] The corresponding amino acid sequence may comprise:

[0341] Linker: GSSGSA (SEQ ID NO: 15 )

[0342] VC1 MALFDYNATGDTEFDSPAKQGWMQDNTNNGSGVLTNADGMPAWLVQGIGGRAQWTYSLSTNQHA QASSFGWRMTTEMKVLSGGMITNYYANGTQRVLPIISLDSSGNLWEFEGQTGRTVLATGTAAT EYHKFELVFLPGSNPSASFYFDGKLIRDNIQPTASKQNMIVWGNGSSNTDGVAAYRDIKFEIQG

[0343] D (SEQ ID NO: 29 )

[0344] Linker: GGGS (SEQ ID NO: 16)

[0345] VC2 MALFDYNATGDTEFDSPAKQGWMQDNTNNGSGVLTNADGMPAWLVQGIGGRAQWTYSLSTNQHA QASSFGWRMTTEMKVLSGGMITNYYANGTQRVLPIISLDSSGNLWEFEGQTGRTVLATGTAAT EYHKFELVFLPGSNPSASFYFDGKLIRDNIQPTASKQNMIVWGNGSSNTDGVAAYRDIKFEIQG

[0346] D (SEQ ID NO: 29 )

[0347] Linker: LQALG (SEQ ID NO: 17 )

[0348] VC3 MALFDYNATGDTEFDSPAKQGWMQDNTNNGSGVLTNADGMPAWLVQGIGGRAQWTYSLSTNQHA QASSFGWRMTTEMKVLSGGMITNYYANGTQRVLPIISLDSSGNLWEFEGQTGRTVLATGTAAT

[0349] 55740646-1 EYHKFELVFLPGSNPSASFYFDGKLIRDNIQPTASKQNMIVWGNGSSNTDGVAAYRDIKFEIQG D (SEQ ID NO: 29 )

[0350] Linker: GGNSG (SEQ ID NO: 18 )

[0351] VC 4 MALFDYNATGDTEFDSPAKQGWMQDNTNNGSGVLTNADGMPAWLVQGIGGRAQWTYSLSTNQHA QASSFGWRMTTEMKVLSGGMITNYYANGTQRVLPIISLDSSGNLWEFEGQTGRTVLATGTAAT EYHKFELVFLPGSNPSASFYFDGKLIRDNIQPTASKQNMIVWGNGSSNTDGVAAYRDIKFEIQG

[0352] D (SEQ ID NO: 29 )

[0353] The proteins were expressed from a pET-based vector, overnight at 16°C in E coli BL21 (DE3) following induction by IPTG. Recombinant proteins were purified from the cell cytoplasm over nickel resin, eluted with a gradient of imidazole (20-500mM). The eluted material was dialysed overnight to remove the imidazole and during this step TEV protease was added to cleave the 6His tag. Cleaved protein was passed back over nickel resin and the unbound material collected and concentrated using centrifugal devices. Proteins were further purified using size exclusion chromatography (Superdex S200) with PBS as the mobile phase. SEC fractions were analysed by SDS PAGE and appropriate samples pooled and concentrated before storage at -80°C. Concentration of final products determined by nanodrop using predicted extinction coefficients calculated from protein sequences.

[0354] Plasmids containing light and heavy chains of an anti-haemagglutinin (HA) antibody (mAB) were used to transfect HEK293 cells. Antibody expressed as a secreted protein and purified over protein A resin (eluted with 0.1M Glycine pH 3.5, and dialysed into PBS). Conjugation of the antibody with SVc2 or SVc4 performed at 1:1 molar ratios at room temperature for 2 hours or overnight at 4-8°C.

[0355] Conjugated protein separated from non-complexed constituents by size exclusion chromatography. Tween-20 added to the conjugate at a final concentration of 0.05% to help prevent non-specific binding to the column resin. 40-60pL of conjugate loaded onto Agilent Advance Bio SEC 300, 2.7pM, 7.8 x 300mm column and run at 0.5mL / min using PBS as a mobile phase. Fractions collected manually (approx 200pL / fraction) and analysed by SDS PAGE. Complex containing fractions pooled and concentrated using centrifugal devices. Purified protein analysed by SDS PAGE and ELISA, concentrations determined by nanodrop using predicted extinction coefficients calculated from protein sequences.

[0356] 55740646-1 Conjugated proteins analysed pre and post gel filtration under reducing conditions (in the presence of DTT) by SDS PAGE. Gels stained with Coomasie and purity estimated by densitometry.

[0357] SVc2 / SVc4-mAB complexes can successfully be separated from non-complexed components by size exclusion chromatography (SEC) (Figure 12).

[0358] Binding of SVc2 and SVc4 mAB conjugates to cells

[0359] Assays were performed to determine the ability of SVc2 and SVc4 mAB conjugates to bind to cells (Figure 13).

[0360] MDCK cells seeded and grown to confluence in 96 well plates and cells were fixed with 10% formamide. Plates were blocked with 5% non-fat dried milk in PBS-0.05% Tween-20 and cells were treated with proteins (23-0.01 nM):

[0361] o SVc2 (unconjugated);

[0362] o SVc4 (unconjugated);

[0363] o SVc2-mAB conjugate;

[0364] o SVc4-mAB conjugate;

[0365] o mAB (unconjugated).

[0366] Bound protein was detected using rabbit anti-Vc polyclonal antibody followed by anti-rabbit- H RP (detects Vc part of complex) or anti-human IgG-HRP (detects antibody part of complex).

[0367] Results

[0368] Anti-Vc detects both the SVc2 / SVc4 proteins alone and the conjugates (see Figure 13A and Figure 13C) as both bind to the cells.

[0369] Anti-IgG detects only the conjugate as the monoclonal antibody alone does not bind to MDCK cells and the anti IgG does not recognise the bound SVc2 / SVc4 (see Figure 13B and Figure 13D). This confirms that the monoclonal antibody is not contributing to the signal from the conjugate in this assay.

[0370] The slope of the curves generated by the SVc2 and SVc4 mAB conjugates indicates that these constructs generate a stronger signal than the SVc2 / SVc4 alone at the same molar concentration - this exemplary data suggests that the act of conjugating the two proteins together affects the manner in which the carbohydrate binding motif binds to its substrate and thus the amount of signal generated.

[0371] Binding of SVc2 and SVc4 mAB conjugates to sialic acid

[0372] Assays were performed to determine ability of SVc2 and SVc4 mAB conjugates to bind to sialic acid (3'SL) (Figure 14).

[0373] 55740646-1 Plates were coated with suitable sialic acid antigen and plates were blocked with 5% BSA in PBS-0.05% Tween-20. Plates treated with test proteins (between 18-0.02nM):

[0374] o SVc2 (unconjugated);

[0375] o SVc4 (unconjugated);

[0376] o SVc2-mAB conjugate;

[0377] o SVc4-mAB conjugate;

[0378] o mAB (unconjugated).

[0379] Bound protein detected using rabbit anti-Vc polyclonal antibody followed by anti-rabbit— HRP (detects Vc part of complex) or anti-human IgG-HRP (detects antibody part of complex).

[0380] Results

[0381] • Anti-Vc detects both the SVc2 / SVc4 proteins alone and the conjugates (see Figure 14A and Figure 14B) as both bind to the sialic acid.

[0382] • Anti-IgG detects only the conjugate as the monoclonal antibody alone does not bind to the sialic acid and the anti IgG does not recognise the bound SVc4 (see Figure 14C). This confirms that the monoclonal antibody is not contributing to the signal from the conjugate in this assay.

[0383] • The slope of the curves generated by the SVc2 and SVc4 mAB conjugates indicates that these constructs generate a stronger signal than the SVc2 / SVc4 alone at the same molar concentration - this exemplary data suggests the act of conjugating the two proteins together affects the manner in which the carbohydrate binding motif binds to its substrate and thus the amount of signal generated.

[0384] REFERENCES

[0385] [1] Tanha, J., et aL, (2002). Selection by phage display of llama conventional V(H) fragments with heavy chain antibody V(H)H properties. J. Immunol. Methods 263, 97.

[0386] [2] Zhang, J., et aL, (2004). Pentamerization of single-domain antibodies from phage libraries: a novel strategy for the rapid generation of high-avidity antibody reagents. J. Mol. Biol. 335, 49.

[0387] [3] Kerrm Y. F. Yau., (2003). Isolation and Affinity Maturation of Recombinant Antibody Fragments Using Phage- and Ribosome-Display Technologies. [Doctoral dissertation, The University of Guelph],

[0388] [4] Kerrm Y. F. Yau., Ginette Dubuc, Shenghua Li, Tomoko Hirama, C. Roger Mac enzie, Lutz Jermutus, J. Christopher Hall, Jamshid Tanha, Affinity maturation of a VHH by mutational hotspot randomization, Journal of Immunological Methods,

[0389] Volume 297, Issues 1-2, 2005, Pages 213-224.

[0390] 55740646-1 [5] Tanha J., et aL, National Research Council of Canada. Single-domain Antigenbinding Antibody Fragments Derived from Llama Antibodies. US 8,257,705.

[0391] [6] Connaris H, et aL, Prevention of influenza by targeting host receptors using engineered proteins. Proc Natl Acad Sci U S A. 2014 Apr 29; 111 (17):6401 -6.

[0392] 55740646-1

Claims

1. CLAIMS1. A molecule for retention at a site, said molecule further comprising a retention moiety, wherein the retention moiety comprises a carbohydrate binding module (CBM).

2. A modified molecule for retention at a site, wherein said molecule is modified by the addition of a retention moiety, wherein the retention moiety comprises a carbohydrate binding module (CBM).

3. A construct comprising a molecule for retention at a site and a retention moiety, wherein the retention moiety comprises a carbohydrate binding module (CBM).

4. The molecule, modified molecule or construct of any preceding claim, wherein the molecule for retention at a site is conjugated, joined, fused, linked or bound to the retention moiety.

5. The molecule, modified molecule or construct of any preceding claim, wherein the molecule for retention at a site comprises an antibody.

6. The molecule, modified molecule or construct of claim 5, wherein the antibody is conjugated, joined, fused, linked or bound to two retention moieties, optionally wherein each retention moiety comprises a CBM.

7. The molecule, modified molecule or construct of claim 6, wherein the, or each retention moiety comprises 2, 3 or 4 CBMs.

8. The molecule, modified molecule or construct of claim 5-7, wherein the, or each retention moiety is conjugated, joined, fused, linked or bound to a heavy chain of the antibody.

9. The molecule, modified molecule or construct according to any preceding claim, wherein the, or each retention moiety comprises a family 40 carbohydrate binding module (CBM40).11.55740646-1 10. The molecule, modified molecule or construct according to claim 6, wherein the CBM40 comprises a sequence of SEQ ID NOS: 3, 4, 5 or 6 or a carbohydrate binding fragment of any of these.

11. The molecule, modified molecule or construct according to claim 6, wherein the CBM40 comprises a sequence having one or more mutations relative to a reference sequence.

12. The molecule, modified molecule or construct according to claim 8, wherein the mutation is selected from the group consisting of:14.(i) one or more amino acid substitution(s);15.(ii) one or more amino acid deletion(s);16.(iii) one or more amino acid addition(s) / insertion(s);17.(iv) one or more amino acid / sequence inversions; and18.(v) one or more amino acid / sequence duplications; and19.the reference sequence is any of SEQ ID NOS: 3, 4, 5 or 6.

13. The molecule, modified molecule or construct according to any one of claims 1-6, 8 or 9, wherein the retention moiety comprises the following sequence:21.GAMVIEKEDVETNASNGQRVDLSSELDKLKKLENATVHMEFKPDPKAPAFYNLFSVS SATKKDEYFTMAVYNNTATLEGRGSDGKQFYNNYNDAPLKVKPGQWNSVTFTVEKPT AELPKGRARLYVNGGLSRTSLRSGNFIKDMPDVTHVQIGATKRANNTVWGSNLQIRN LTVYNRALTPEEVQKRSGGGSGVIEKEDVETNASNGQRVDLSSELDKLKKLENATVH MEFKPDPKAPAFYNLFSVSSATKKDEYFTMAVYNNTATLEGRGSDGKQFYNNYNDAP LKVKPGQWNSVTFTVEKPTAELPKGRARLYVNGGLSRTSLRSGNFIKDMPDVTHVQI GATKRANNTVWGSNLQIRNLTVYNRALTPEEVQKRSGGSLGVPDFESDWFDVSSNSL YTLSHGLQRSPRRVVVEFARSSSPSTWNIVMPSYFNDGGHKGSGAQVEVGSLNIKLG TGAAVWGTGYFGGIDNSATTRFATGYYRVRAWI14. The molecule, modified molecule or construct according to any one of claims 1-6, 8 or 9, wherein the retention moiety comprises the following sequence: ALFDYNATGDTEFDSPAKQGWMQDNTNNGSGVLTNADGMPAWLVQGIGGRAQWTYSLSTN QHAQASSFGWRMTTEMKVLSGGMITNYYANGTQRVLPI ISLDSSGNLVVEFEGQTGRTVL ATGTAATEYHKFELVFLPGSNPSASFYFDGKLIRDNIQPTASKQNMIVWGNGSSNTDGVA AYRDIKFEIQGD23.55740646-1 15. The molecule, modified molecule or construct according to claim 14, wherein the retention moiety comprises 1, 2, 3 or 4 copies of the sequence.

16. The molecule, modified molecule or construct according to claim 14, wherein the molecule for retention at a site comprises an antibody.

17. The molecule, modified molecule or construct according to any one of claims 14- 16, wherein the molecule for retention at a site comprises a plurality, for example two, three, four or more retention moieties.

18. The molecule, modified molecule or construct according to any preceding claim wherein the molecule further comprises:27.(i) a purification tag; and / or28.(ii) a linker; and / or29.(iii) a solubilising moiety.

19. A composition or pharmaceutical composition comprising the molecule, modified molecule or construct of any preceding claim.

20. The molecule, modified molecule or construct of any one of claims 1-18, wherein the site comprises a retention moiety binder.

21. The molecule, modified molecule or construct of claim 20, wherein the retention moiety binder exhibits an ability to bind a CBM.

22. The molecule, modified molecule or construct of claim 20, wherein the site comprises a mucosal surface or tissue, epithelial cells, mucosal epithelial cells, human nasal epithelium tissue, a mucosal surface of the nasal passage or a mucosal surface of the lung respiratory airway.

23. A method or in vitro method of increasing the residence or retention time of a molecule at a site of interest, said method comprising administering or contacting the molecule to / with the site of interest, wherein the molecule is as defined in any one of claims 1-18, comprised within a modified molecule or construct according to35.55740646-1 claims 1-18 or administered or contacted in the form of a composition according to claim 19.

24. A molecule, modified molecule or construct according to any one of claims 1-18 or composition or pharmaceutical composition of claim 19, for use:37.(i) in therapy or as a medicament;38.(ii) in the treatment and / or prevention of diseases in a subject in need thereof; (iii) in modulating immune responses (in human or animal subjects);39.(iv) in modulating cell growth, proliferation and / or differentiation;40.(v) as an adjuvant;41.(vi) as a medicament;42.(vii) as an anti-infective; and43.(viii) in treating or preventing a viral infection (e.g. an influenza infection); and (ix) in the treatment of cancer.

25. Use of:45.(i) a carbohydrate binding module (CBM); or46.(ii) a family 40 carbohydrate binding module (CBM40); or47.(iii) a CBM40 comprising a sequence of SEQ ID NOS: 3, 4, 5 or 6 or a carbohydrate binding fragment of any of these; or48.(iv) a CBM40 comprising a sequence having one or more mutations relative to a reference sequence; or49.(v) a CBM comprising the following sequence:50.ALFDYNATGDTEFDSPAKQGWMQDNTNNGSGVLTNADGMPAWLVQGIGGRAQWTYSLSTN QHAQASSFGWRMTTEMKVLSGGMITNYYANGTQRVLPI ISLDSSGNLVVEFEGQTGRTVL ATGTAATEYHKFELVFLPGSNPSASFYFDGKLIRDNIQPTASKQNMIVWGNGSSNTDGVA AYRDIKFEIQGD51.(vi) a CBM comprising the following sequence:52.GAMVIEKEDVETNASNGQRVDLSSELDKLKKLENATVHMEFKPDPKAPAFYNLFSVS SATKKDEYFTMAVYNNTATLEGRGSDGKQFYNNYNDAPLKVKPGQWNSVTFTVEKPT AELPKGRARLYVNGGLSRTSLRSGNFIKDMPDVTHVQIGATKRANNTVWGSNLQIRN53.55740646-1 LTVYNRALTPEEVQKRSGGGSGVIEKEDVETNASNGQRVDLSSELDKLKKLENATVH MEFKPDPKAPAFYNLFSVSSATKKDEYFTMAVYNNTATLEGRGSDGKQFYNNYNDAP LKVKPGQWNSVTFTVEKPTAELPKGRARLYVNGGLSRTSLRSGNFIKDMPDVTHVQI GATKRANNTVWGSNLQIRNLTVYNRALTPEEVQKRSGGSLGVPDFESDWFDVSSNSL YTLSHGLQRSPRRVVVEFARSSSPSTWNIVMPSYFNDGGHKGSGAQVEVGSLNIKLG TGAAVWGTGYFGGIDNSATTRFATGYYRVRAWI;54.for retaining a molecule at a site in a method of retaining a molecule at a site.

26. An antibody fused, bound, conjugated or linked to a retention moiety, said retention moiety comprising the sequence:55.ALFDYNATGDTEFDSPAKQGWMQDNTNNGSGVLTNADGMPAWLVQGIGGRAQWTYSLSTN QHAQASSFGWRMTTEMKVLSGGMITNYYANGTQRVLPI ISLDSSGNLVVEFEGQTGRTVL ATGTAATEYHKFELVFLPGSNPSASFYFDGKLIRDNIQPTASKQNMIVWGNGSSNTDGVA AYRDIKFEIQGD27. The antibody of claim 26, wherein the retention moiety comprises multiple copies of the sequence.

28. The antibody of claim 27, wherein the retention moiety, comprises 2, 34 or more copies of the sequence.

29. The antibody of claim 26, wherein the retention moiety is bound, conjugated or linked to a heavy chain of the antibody.

30. The antibody of claim 29, wherein the antibody comprises two heavy chains and each heavy chain is bound, conjugated or linked to a retention moiety.

31. The antibody of any preceding claims, wherein the antibody is an antiinfluenza or anti-HA antibody61.55740646-1

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